Engineered Spray-Dried PLGA-PEG Dual-Coated Micelles with Lactose/Trehalose Matrices for Pulmonary Doxorubicin Delivery: Achieving Sustained Release and Improved Aerosol Performance in NSCLC Therapy

Engineered Spray-Dried PLGA-PEG Dual-Coated Micelles with Lactose/Trehalose Matrices for Pulmonary Doxorubicin Delivery: Achieving Sustained Release and Improved Aerosol Performance in NSCLC Therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Engineered Spray-Dried PLGA-PEG Dual-Coated Micelles with Lactose/Trehalose Matrices for Pulmonary Doxorubicin Delivery: Achieving Sustained Release and Improved Aerosol Performance in NSCLC Therapy Randa Mohammed Zaki, Alaa Ayman, Eman Mostafa Samy, Omiya A Hasan, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7640535/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Feb, 2026 Read the published version in Journal of Pharmaceutical Innovation → Version 1 posted You are reading this latest preprint version Abstract Background Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality. Intravenous doxorubicin (DOX) is effective but limited by severe cardiotoxicity and poor lung tumor selectivity. Objective To develop an inhalable dry powder inhaler (DPI) of DOX-loaded PLGA-PEG dual-coated micelles embedded in a lactose/trehalose matrix for targeted pulmonary delivery and reduced systemic toxicity. Methods Micelles were prepared via nanoprecipitation and engineered into respirable powders via spray-drying. Formulations were characterized for size, stability, aerosol performance, drug release, and cytotoxicity (SLC cells). In vivo pharmacokinetics, lung deposition, antitumor efficacy, and systemic safety were assessed in rat models. Results Optimized micelles showed high encapsulation efficiency (91.3%), desirable aerodynamic properties (MMAD 2.9 µm, FPF 62.4%), and sustained biphasic release. They significantly enhanced cytotoxicity (IC₅₀ 1.82 vs. 3.96 µg/mL) and apoptosis (48.6% vs. 29.7%) compared to free DOX. In vivo, pulmonary delivery achieved 46.8% lung retention at 24 h (vs. 12.4%), prolonged systemic residence time (MRT 14.7 h vs. 6.1 h), and superior tumor growth inhibition (68.5% vs. 25.3%). Critically, micellar DOX markedly reduced cardiotoxicity (CK-MB ↓55%, LDH ↓48%) with minimal histopathological cardiac damage. Conclusion The spray-dried micellar DPI platform enables efficient lung-targeted delivery of DOX, enhancing antitumor efficacy while mitigating systemic cardiotoxicity, presenting a promising therapeutic strategy for NSCLC. Pulmonary drug delivery PLGA-PEG micelles Doxorubicin Dry powder inhaler Non-small cell lung cancer Spray-drying Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Lung cancer is the leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 80–85% of cases and presenting a critical need for more effective and safer therapies [ 1 ]. Doxorubicin (DOX) remains a clinically effective chemotherapeutic agent; however, its intravenous administration is severely limited by nonspecific biodistribution, inadequate tumor selectivity, and dose-dependent toxicities, most notably life-threatening cardiotoxicity and myelosuppression [ 2 ]. Inhalable nanocarrier systems represent a promising strategy to overcome these limitations by leveraging the lungs as both the primary disease site and a directly accessible portal for localized therapy. This approach aims to maximize intratumoral drug deposition while minimizing systemic exposure and associated toxicity [ 3 ]. Among inhalation modalities, dry powder inhalers (DPIs) offer a particularly attractive option for delivering chemotherapeutics, enabling advanced particle engineering to optimize aerodynamic diameter, dispersibility, and deep-lung deposition [ 4 ]. Despite this potential, current inhalable nanocarrier platforms—including liposomes and conventional polymeric micelles—face significant translational barriers. These systems are often plagued by (1) poor aerosolization stability and powder aggregation, leading to low fine-particle fractions (FPF); (2) rapid clearance from the lung environment via mucociliary clearance and alveolar macrophage uptake; and (3) an inability to prevent systemic drug leakage, which undermines the goal of reducing off-target toxicity [ 5 , 6 ]. Consequently, there is a pressing need for rationally engineered inhalable formulations that integrate enhanced aerodynamic performance with sustained pulmonary release and reduced systemic leakage. Poly (lactic-co-glycolic acid) (PLGA) and poly (ethylene glycol) (PEG) are well-established biomaterials that offer complementary advantages for drug delivery. PLGA provides biodegradability and facilitates sustained release through diffusion-erosion mechanisms, while PEG confers "stealth" properties by reducing protein adsorption and macrophage uptake [ 7 ]. The integration of these polymers into a PLGA-PEG architecture can enhance hydrophilicity, biocompatibility, and controlled release behavior. Furthermore, spray drying presents a scalable method to transform nano-dispersions into respirable dry powders. The inclusion of glass-forming stabilizers like lactose and trehalose is critical to protect nanostructures during atomization and drying, enhance powder flowability, and improve aerosolization efficiency [ 8 ]. However, the application of a PLGA-PEG dual-coated micellar system, optimized within a lactose/trehalose matrix for pulmonary DOX delivery, remains largely unexplored. We hypothesized that a spray-dried DPI comprising PLGA-PEG dual-coated micelles stabilized within a lactose/trehalose glassy matrix would overcome these limitations, providing superior aerosol performance, sustained pulmonary release, and a markedly improved therapeutic index for DOX in NSCLC. Accordingly, this study aimed to develop, characterize, and evaluate this novel micellar DPI platform through comprehensive assessment of its physicochemical properties, aerosol performance, in vitro and in vivo efficacy, and systemic safety profile. 2. Materials and Methods 2.1 Materials Doxorubicin hydrochloride was obtained from Sigma-Aldrich (St. Louis, MO, USA). The block copolymer poly(ethylene glycol)-poly(D,L-lactide-co-glycolide) (PLGA-PEG; PEG average Mn 5000 g·mol⁻¹, PLGA Mn 5000 g·mol⁻¹, lactide:glycolide feed ratio 50:50) was purchased from Sigma-Aldrich (Darmstadt, Germany). Lactose monohydrate (inhalation grade) and trehalose dihydrate were supplied by DFE Pharma (Goch, Germany) and Hayashibara Co., Ltd. (Okayama, Japan), respectively. Acetonitrile, methanol, and ethanol (HPLC grade) were obtained from Fisher Scientific (Waltham, MA, USA), while dichloromethane and other analytical-grade reagents were procured from Merck (Darmstadt, Germany). Deionized water was prepared using a Milli-Q purification system (Millipore, Billerica, MA, USA). All materials were used as received, without further modification or purification. 2.2 Preparation of PLGA-PEG dual-coated micelles Doxorubicin-loaded PLGA-PEG dual-coated micelles were prepared using the nanoprecipitation method for fabricating polymeric nanocarriers with a narrow size distribution and high drug entrapment efficiency [ 12 , 13 ]. Briefly, predetermined amounts of PLGA and PEG were dissolved in acetone containing doxorubicin (previously neutralized with triethylamine to enhance lipophilicity) and added dropwise to an aqueous phase under continuous magnetic stirring. The resulting colloidal suspension was maintained under agitation to allow complete solvent evaporation, thereby yielding self-assembled micelles. Unencapsulated drug was removed by centrifugation and dialysis against deionized water. The drug-to-polymer ratio was optimized by varying the weight ratio of doxorubicin to PLGA-PEG (1:5, 1:10, and 1:20, w/w). The formulations were evaluated for particle size, polydispersity index (PDI), zeta potential, and encapsulation efficiency, with the optimal ratio selected based on a balance between high drug loading and desirable physicochemical stability [ 14 , 15 ]. The optimized micellar suspension was subsequently subjected to spray-drying with lactose and trehalose excipients for conversion into a respirable dry powder suitable for pulmonary administration. 2.3 Spray-drying with excipients The optimized PLGA-PEG micellar suspension was converted into a dry powder inhalation (DPI) formulation using a laboratory-scale spray dryer (Büchi B-290, Büchi Labortechnik AG, Flawil, Switzerland). Prior to spray-drying, lactose monohydrate and trehalose dihydrate were dissolved in the micellar suspension at a 1:1 ratio (w/w, total 10% w/v) and used as stabilizing excipients to protect nanostructures during atomization and drying. Spray-drying was performed under the following conditions: inlet temperature 120 ± 2°C, feed rate 5 mL/min, aspirator setting 90%, and atomizing airflow 600 L/h. The outlet temperature was maintained at 65 ± 3°C. The resulting spray-dried formulations were collected using a high-performance cyclone separator, sealed in airtight amber glass vials, and stored at 4°C until further use. The incorporation of lactose and trehalose was intended to enhance powder dispersibility, improve aerosolization performance, and prevent aggregation during long-term storage, as previously reported in inhalable nanocarrier formulations [ 16 , 17 , 18 ]. 2.4 Physicochemical characterization The hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the micelles were measured using dynamic light scattering (DLS; Zetasizer Nano ZS, Malvern Instruments, UK) at 25°C. All measurements were performed in triplicate on freshly prepared dispersions [ 19 , 20 ] Drug loading capacity and encapsulation efficiency were determined using high-performance liquid chromatography (HPLC; Agilent 1260 Infinity, USA) equipped with a C18 column (4.6 × 150 mm, 5 µm). Samples were dissolved in acetonitrile-water (70:30 v/v), filtered through 0.22 µm membranes, and quantified at 480 nm. For comparison, UV-visible spectrophotometry (UV-2600, Shimadzu, Japan) was employed in preliminary analyses [ 14 , 21 ]. The solid-state properties were characterized by Fourier-transform infrared spectroscopy (FTIR; Nicolet iS50, Thermo Fisher Scientific, USA), X-ray powder diffraction (XRPD; D8 Advance, Bruker, Germany), and differential scanning calorimetry (DSC; DSC 214 Polyma, Netzsch, Germany) to evaluate potential interactions between the drug, polymer, and excipients, and to assess the crystalline or amorphous state of the formulations [ 16 , 22 ] 2.5 Aerosol performance The aerosolization properties of the spray-dried formulations were evaluated using a Next Generation Impactor (NGI; Copley Scientific, Nottingham, UK) equipped with a standard dry powder inhaler device. Approximately 10 mg of the formulation was loaded into size 3 hydroxypropyl methylcellulose capsules and dispersed at a flow rate of 60 L/min for 4 s, corresponding to a 4 L inhalation volume, in accordance with the United States Pharmacopeia (USP) guidelines. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated from the drug deposition profile across the NGI stages. Fine particle fraction (FPF) was determined as the percentage of the emitted dose with an aerodynamic diameter below 5 µm. Drug content on each stage was quantified by washing the collection plates with acetonitrile-water (70:30 v/v) and analyzing by HPLC at 480 nm. All measurements were performed in triplicate, and results were expressed as mean ± SD. The assessment of MMAD, GSD, and FPF provides critical insights into the respirable dose and deposition efficiency of dry powder formulations, parameters that strongly influence clinical efficacy of inhalable chemotherapeutics [ 23 – 25 ]. 2.6 In vitro release and stability The release profile of doxorubicin from the spray-dried PLGA-PEG dual-coated micelles was evaluated in simulated lung fluid (SLF, pH 7.4), prepared according to the composition described by May et al. [ 26 ]. Briefly, 10 mg of spray-dried powder was dispersed in 10 mL of SLF and incubated at 37 ± 0.5°C under gentle shaking (100 rpm). At predetermined intervals (0.5, 1, 2, 4, 8, 12, 24, and 48 h), samples were withdrawn and centrifuged at 15,000 rpm for 15 min. The supernatant was analyzed by HPLC at 480 nm to determine drug release, while an equivalent volume of fresh medium was replenished to maintain sink conditions. Cumulative release was expressed as the percentage of drug released relative to the total encapsulated amount. Stability studies were conducted following ICH Q1A(R2) guidelines. Optimized formulations were stored in sealed amber glass vials at 4°C (refrigerated), 25°C/60% RH (ambient), and 40°C/75% RH (accelerated) for three months. At monthly intervals, samples were evaluated for changes in particle size, PDI, zeta potential, drug content, and aerosol performance. Stability was expressed as percentage retention compared with initial values. These assays were designed to assess both the controlled-release behavior of the micelles in the pulmonary environment and the robustness of the formulation during long-term storage [ 27 , 28 ]. 2.7 Cell culture and cytotoxicity studies The rat lung carcinoma SLC cell line (RIKEN BioResource Research Center, Tsukuba, Japan; Cell No. RCB2862) was cultured in RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin, according to the supplier’s recommendations. Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂ and harvested at ~ 80% confluence using Accutase™ (Nacalai Tesque, Kyoto, Japan). For cytotoxicity testing, cells were seeded into 96-well plates (1 × 10⁴ cells/well) and treated with free doxorubicin, blank PLGA-PEG dual-coated micelles, or DOX-micelles (0.1–20 µg/mL) for 24 and 48 h. Cell viability was assessed by the MTT assay, while apoptosis was quantified using Annexin V-FITC/PI staining and flow cytometry, as described previously [ 29 – 31 ]. These assays provided complementary information regarding the cytotoxic potential and apoptotic mechanisms of micellar formulations compared with free doxorubicin, thereby supporting the therapeutic potential of DOX-loaded PLGA-PEG dual-coated micelles. [ 29 – 31 ]. 2.8 In vivo pharmacokinetics and lung distribution All animal experiments were performed in accordance with institutional ethical guidelines and approved by the Faculty of Pharmacy, Deraya University Animal Care and Use Committee (ethical approval no. 7/2024, issued February 14, 2024). Male Wistar rats (200–250 g) were obtained from the Animal House, Faculty of Pharmacy, Deraya University (Minya, Egypt) and acclimatized for one week under controlled environmental conditions (22 ± 2°C, 55 ± 10% relative humidity, 12 h light/dark cycle) with free access to food and water.” Pharmacokinetic and biodistribution studies were conducted in healthy male Wistar rats after intratracheal administration of either free doxorubicin solution or DOX-micelles (2 mg/kg equivalent DOX). Animals were anesthetized with isoflurane (2–3% in oxygen) and positioned supine. Formulations were delivered intratracheally using a PennCentury™ MicroSprayer® Aerosolizer (Penn-Century Inc., Philadelphia, PA, USA). Blood samples were collected via retro-orbital puncture into heparinized tubes at predetermined time points (0.25, 0.5, 1, 2, 4, 8, 12, and 24 h) and centrifuged at 4000 rpm for 10 min to separate plasma. For lung distribution studies, animals were sacrificed at 1, 4, 8, and 24 h post-dose. Lungs were excised, rinsed with saline, blotted dry, weighed, and homogenized in ice-cold PBS. Plasma, lung homogenates, and tissue samples from the heart, kidney, liver, and spleen were collected for doxorubicin quantification. All samples were subjected to protein precipitation with acetonitrile, vortex-mixed, and centrifuged at 12,000 rpm for 15 min. The supernatants were then transferred to HPLC vials for analysis. Doxorubicin concentrations were determined using a validated HPLC method (Agilent 1260 Infinity system, Agilent Technologies, Santa Clara, CA, USA) equipped with a fluorescence detector (λ_ex = 480 nm, λ_em = 590 nm). Chromatographic separation was performed on a C18 column (4.6 × 150 mm, 5 µm) with a mobile phase of acetonitrile:water (30:70, v/v) containing 0.1% trifluoroacetic acid at a flow rate of 1.0 mL/min. The method was validated according to bioanalytical guidelines, showing linear calibration curves over the range of 10 − 5,000 ng/mL (r² >0.99), with limits of detection and quantification of 5 ng/mL and 10 ng/mL, respectively. Intra- and inter-day precision values were within 10%, and recovery exceeded 85% across all matrices (plasma, lung, heart, kidney, liver, and spleen), confirming the suitability of the assay for pharmacokinetic and biodistribution analysis. Pharmacokinetic parameters, including maximum plasma concentration (C max ), time to reach C max (T max ), area under the plasma concentration-time curve from 0–24 h (AUC₀-₂₄h), mean residence time (MRT) and elimination half-life (t½), were calculated using non-compartmental analysis (Phoenix WinNonlin 10, Certara, USA). Lung tissue concentrations were expressed as ng of drug per g of wet tissue. These studies enabled a direct comparison of systemic exposure and pulmonary retention between free doxorubicin and micellar formulations, highlighting the advantages of localized, sustained drug delivery in the lung environment [ 28 , 32 , 33 ]. 2.9 In vivo antitumor efficacy in orthotopic tumor-bearing rats The antitumor efficacy of the experimental treatment was evaluated in an orthotopic rat model of SLC. Male Wistar rats (200–250 g) were obtained from the Animal House, Faculty of Pharmacy, Deraya University (Minya, Egypt). Animals were acclimatized for one week under controlled environmental conditions (22 ± 2°C, 55 ± 10% relative humidity, 12 h light/dark cycle) with free access to food and water [ 34 , 35 ]. To avoid overlap, pharmacokinetic evaluations were conducted exclusively in healthy rats, whereas efficacy studies were performed only in tumor-bearing rats. Tumor-bearing rats were generated via the intratracheal inoculation of 1 × 10⁶ luciferase-expressing SLC cells in 50 µL of PBS under anesthesia. Tumor establishment and growth were confirmed and monitored weekly via non-invasive ultrasound imaging and bioluminescence imaging (BLI) prior to the initiation of the study. Rats with confirmed tumors were then randomly allocated into treatment and control groups (n = 6 per group: (i) untreated control, (ii) free doxorubicin (DOX), and (iii) DOX-loaded PLGA-PEG micelles. Tumor progression was monitored at defined intervals, and at the experimental endpoint animals were humanely sacrificed for histopathological evaluation. Tumor volume was measured by ultrasound imaging and calculated using the following formula: V = 0.5 * length * (width) 2 where length is the longest diameter and width is the perpendicular measurement. Tumor volume was recorded throughout the study. At endpoint, tumors were excised, weighed, and processed for histological evaluation (H&E staining). Antitumor efficacy was expressed as tumor growth inhibition (TGI%): TGI% \(\:=\:\frac{\left(\varvec{\Delta\:}\mathbf{V}\mathbf{c}\mathbf{o}\mathbf{n}\mathbf{t}\mathbf{r}\mathbf{o}\mathbf{l}\mathbf{}\right)-\:\left(\varvec{\Delta\:}\mathbf{V}\mathbf{t}\mathbf{r}\mathbf{e}\mathbf{a}\mathbf{t}\mathbf{e}\mathbf{d}\mathbf{}\right)\:}{\left(\varvec{\Delta\:}\mathbf{V}\mathbf{c}\mathbf{o}\mathbf{n}\mathbf{t}\mathbf{r}\mathbf{o}\mathbf{l}\mathbf{}\right)}\:\) ×100 where ΔV is the change in tumor volume from baseline to endpoint. 2.10 Cardiotoxicity and systemic toxicity assessment At the end of the efficacy study, blood samples were collected from the retro-orbital plexus under light anesthesia. Serum was separated and analyzed for creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) using commercial ELISA kits (Sigma-Aldrich, USA), following manufacturer’s instructions [ 36 ]. The hearts were excised, rinsed with saline, and fixed in 10% neutral-buffered formalin for 24 h. Tissues were subsequently dehydrated, embedded in paraffin, and sectioned at a thickness of 5 µm. Sections were stained with hematoxylin-eosin (H&E) and examined under a light microscope for structural alterations, including myocyte degeneration, cytoplasmic vacuolization, interstitial edema, and necrosis, to evaluate doxorubicin-induced cardiotoxicity [ 37 ]. 2.11 Statistical analysis All experiments were performed in triplicate unless otherwise specified, and results are expressed as mean ± standard deviation (SD). Statistical comparisons between groups were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. For two-group comparisons, an unpaired Student’s t -test was applied. A p value < 0.05 was considered statistically significant. Data analysis and graph generation were performed using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA). This approach is consistent with previous studies evaluating spray-dried nanocarriers and pulmonary formulations [ 32 , 38 ], ensuring robust interpretation of physicochemical and biological outcomes. 3. Results 3.1 Micelle Formation and Characterization Doxorubicin-loaded PLGA-PEG dual-coated micelles were fabricated by nanoprecipitation, yielding self-assembled micelles that were stable and monodisperse. DLS analysis showed an average hydrodynamic diameter of 145.4 ± 6.0 nm. The polydispersity index was 0.19 ± 0.02, confirming a narrow size distribution. The predominant population ranged from 100 to 160 nm, suitable for pulmonary delivery (Table 1 ). Transmission electron microscopy (TEM) further revealed well-defined spherical to near-spherical nanostructures with smooth contours, uniform dispersion, and negligible aggregation (Fig. 1 ). The particle dimensions observed by TEM were consistent with DLS results, confirming the uniform self-assembly and structural stability of the micelles. Incorporation of lactose or trehalose as stabilizing excipients preserved discrete nanostructures, whereas excipient-free formulations showed partial fusion and irregular morphology. Zeta potential measurements were − 18.6 ± 1.2 mV, suggesting moderate colloidal stability and sufficient repulsive surface interactions to maintain dispersion in aqueous media over extended periods [ 12 , 14 ]. Optimization of the drug-to-polymer ratio demonstrated that a 1:10 (w/w) doxorubicin:PLGA-PEG ratio provided the highest encapsulation efficiency ( 91.3 ± 2.5% ) and drug loading ( 8.4 ± 0.3%). Lower polymer ratios resulted in reduced encapsulation, whereas higher polymer ratios did not significantly improve drug loading but increased particle size, highlighting the importance of balancing polymer content for optimal micellar performance [ 12 , 15 ]. Overall, these results confirm that PLGA-PEG dual-coated micelles prepared under optimized conditions form well-defined, stable nanoparticles capable of efficiently encapsulating doxorubicin, providing a promising platform for inhalable anticancer therapy (Table 2 ). Table 1 Optimization of drug-to-polymer ratio for PLGA-PEG dual-coated micelles prepared by nanoprecipitation. Drug: Polymer Ratio Particle Size (nm) PDI Zeta Potential (mV) Encapsulation Efficiency (%EE) Drug Loading (%DL) 1:5 165 ± 8 0.21 -15.1 ± 1.0 72.4 ± 3.1 6.7 ± 0.7 1:10 145.4 ± 6 nm 0.19 -18.6 ± 1.2 91.3 ± 2.5 8.4 ± 0.3 1:20 132 ± 5 0.16 -22.5 ± 1.3 88.9 ± 2.5 4.5 ± 0.3 Note: Higher polymer content (1:10 to 1:20) yielded smaller particles and lower PDI but did not further improve EE and resulted in decreased DL Table 2 Effect of PEGylation on physicochemical properties of PLGA micelles. Parameter Non-PEGylated PLGA Micelles PLGA-PEG dual-coated micelles Particle Size (nm) 155 ± 7 145.4 ± 6 Colloidal Stability (48 h, % aggregation) 28% 5% Mucoadhesion Resistance (%) 32% 78% Drug Release (t½, hours) 6.5 h 14.2 h PEGylation enhances stability, reduces mucoadhesion, and prolongs release kinetics. 3.2 Spray-Dried Powder Properties and Effect of Lactose/Trehalose Solid-state characterization confirmed the successful amorphization of doxorubicin within the micellar matrix. X-ray powder diffraction (XRPD) patterns showed the absence of crystalline peaks corresponding to native doxorubicin, indicating complete encapsulation in an amorphous state. Differential scanning calorimetry (DSC) analysis supported this finding, showing no distinct melting endotherm for doxorubicin, while Fourier-transform infrared (FTIR) spectra revealed no evidence of strong chemical interactions between drug, polymer, and excipients, suggesting physical stabilization rather than covalent modification (Fig. 2 ). Aerosol performance testing using a Next Generation Impactor demonstrated excellent respirable properties. The optimized formulations displayed a mass median aerodynamic diameter (MMAD) of 2.86 ± 0.15 µm with a geometric standard deviation (GSD) of 1.78 ± 0.06, values well within the optimal range for deep lung deposition. The fine particle fraction (FPF, < 5 µm) reached 62.4 ± 3.1%, significantly higher than excipient-free spray-dried formulations (41.7 ± 2.8%, p < 0.01), confirming the role of lactose and trehalose in enhancing aerodynamic performance (Fig. 3 ). Overall, inclusion of hydrophilic sugars preserved micellar integrity during atomization and drying, reduced particle aggregation, and markedly improved aerosolization efficiency. These findings are consistent with previous studies highlighting the utility of lactose and trehalose as protective excipients in inhalable nanocarrier formulations [ 16 , 38 ]. (A) Differential scanning calorimetry (DSC): Pure doxorubicin displayed a distinct melting endotherm around 210°C, absent in PLGA-PEG dual-coated micelles, indicating successful amorphization. (B) Fourier-transform infrared spectroscopy (FTIR): PLGA-PEG dual-coated micelles with lactose/trehalose excipients exhibited spectra comparable to pure components, with no evidence of new covalent bonds, suggesting physical stabilization without chemical interaction. (C) X-ray powder diffraction (XRPD): Pure doxorubicin exhibited sharp crystalline peaks, while PLGA-PEG dual-coated micelles showed a halo pattern, confirming amorphous encapsulation of the drug. 3.3 In Vitro Drug Release and Stability The release profile of doxorubicin-loaded PLGA-PEG dual-coated micelles in simulated lung fluid (SLF, pH 7.4) exhibited a biphasic release profile, typical of micellar drug delivery systems (Fig. 4 ). Initially, a burst release of 21.4 ± 2.3% occurs within the first 2 hours, primarily due to the drug molecules that are surface-associated or loosely entrapped within the micellar structure. These surface-bound drug molecules are readily available for diffusion when exposed to the SLF, and the hydrophilic PEG shell facilitates the initial release by enhancing drug diffusion through its interaction with water. This rapid release phase was followed by a more sustained release, with the cumulative release reaching 76.8 ± 3.5% at 48 hours. The sustained release phase is largely driven by the hydrophobic PLGA core, which holds the drug within its matrix. As PLGA degrades via hydrolysis, the drug is gradually released over time, offering a prolonged therapeutic effect [ 38 , 39 ]. Release kinetics. Kinetic modeling revealed that drug release was best described by the Higuchi model ( k_H = 0.115, R² = 0.998), confirming a diffusion-driven process (Table 3). The Korsmeyer-Peppas model (n = 0.74, R²= 0.976, Fₜ ≤ 0.6) indicated anomalous transport, where release is governed by a combination of Fickian diffusion and polymer relaxation/erosion. The first-order model ( k₁ = 0.0922, R ² = 0.949) also showed a good fit, suggesting concentration-dependent release dynamics, while the zero-order model ( k₀ = 0.0140, R²= 0.817) provided the weakest correlation, confirming that drug liberation was not constant over time. Mechanistically, the hydrophilic PEG shell functioned as a diffusion barrier, whereas the gradual degradation of the PLGA core modulated sustained release. This dual mechanism aligns with prior reports of micellar and polymeric carriers, where drug liberation is mediated by both Fickian diffusion and polymer erosion [ 14 , 40 ]. Table (3). Kinetic modeling parameters of drug release Model Parameter(s) R² Fit range Higuchi k H = 0.115 0.998 t > 0 h Zero-order k0 = 0.0140 0.817 t > 0 h First-order k1 = 0.0922 0. 949 Ft < 0.995 Korsmeyer-Peppas n = 0.74 0.976 Ft ≤ 0.6 Note: Fit ranges reflect the theoretical applicability of each model: the Higuchi equation is valid during the diffusion-dominated phase (typically up to ~ 60–70% release); the zero-order model can be applied across all time points but rarely fits well; the first-order model is appropriate until near-complete release (Ft < 0.995) to avoid asymptotic deviation; and the Korsmeyer-Peppas model is semi-empirical and only valid for the initial 60% of drug release [ 41 ]. PLGA-PEG dual-coated micelles showed favorable stability profiles under both refrigerated and ambient storage conditions (Table 4). After three months at 4°C, formulations retained 96.2 ± 1.4% of their initial drug content, with negligible changes in particle size (145.4 ± 6.0 nm → 149.2 ± 5.1 nm) and PDI (0.182 → 0.190). At 25°C/60% RH, drug retention was slightly reduced (92.4 ± 2.0%), accompanied by modest particle growth (to 154.8 ± 5.6 nm) and a minor increase in PDI (0.182 → 0.196); however, aerosolization properties, including MMAD (~ 2.84 µm) and FPF (~ 61.7%), remained unchanged. Under accelerated stress (40°C/75% RH), drug retention declined further to 85.6 ± 3.2%, with greater particle enlargement (to 162.3 ± 6.2 nm), elevated PDI (0.221), and early signs of aggregation, though aerodynamic performance was largely preserved (MMAD 2.82 µm; FPF 60.9%). In contrast, excipient-free formulations were highly unstable, showing marked aggregation (> 183 nm), substantial drug loss (72.3 ± 4.1% retention), and a decline in respirable fraction (FPF 55.8 ± 4.2%). These findings highlight the protective role of trehalose and lactose as sugar-glass stabilizers, consistent with previous reports on the cryo- and lyoprotective effects of disaccharides in maintaining nanocarrier integrity during storage [ 42 , 43 ]. Disaccharides such as trehalose and lactose serve as cryo- and lyoprotectants , stabilizing nanocarriers during spray-drying and storage by replacing hydrogen bonds with water molecules and forming a protective amorphous glassy matrix [ 11 , 44 ]. This explains the superior retention of drug content and maintenance of micelle integrity in formulations containing sugars compared with excipient-free formulations. Table (4). Stability of Doxorubicin-Loaded PLGA-PEG dual-coated micelles Under Different Storage Conditions (3 Months) Storage Condition Drug Retention (%) Particle Size (nm) PDI MMAD (µm) FPF (%) Observations 4°C (Refrigerated) 96.2 ± 1.4 145.4 ± 6 nm → 149.2 ± 5.1 0.182 → 0.190 2.86 ± 0.15 62.1 ± 2.9 Excellent stability; minimal changes 25°C / 60% RH (Ambient) 92.4 ± 2.0 145.4 ± 6 nm → 154.8 ± 5.6 0.182 → 0.196 2.84 ± 0.17 61.7 ± 3.0 Slight decline in drug retention; aerosolization preserved 40°C / 75% RH (Accelerated) 85.6 ± 3.2 145.4 ± 6 nm → 162.3 ± 6.2 0.182 → 0.221 2.82 ± 0.18 60.9 ± 3.5 Reduced drug retention, increased PDI, early aggregation Excipient-free formulations 72.3 ± 4.1 145.4 ± 6 nm → >183 nm 0.182 → 0.265 2.78 ± 0.20 55.8 ± 4.2 Marked instability; aggregation and drug loss The combination of PEGylation and PLGA encapsulation contributed to both sustained drug release and enhanced stability. PEG provided steric stabilization against aggregation and reduced protein/surfactant adsorption onto micelle surfaces, thereby helping to maintain colloidal stability in simulated lung fluids [ 45 ]. The hydrophobic PLGA core reduced burst release and protected doxorubicin from hydrolytic/degradative pathways, producing more predictable, prolonged release profiles typical of PLGA matrix or core-shell systems [ 46 ]. Lactose and trehalose acted as stabilizing excipients during spray drying and storage by forming hydrogen bonds with the micellar surface and by vitrifying into an amorphous glass that prevents particle fusion or collapse [ 47 ]. Collectively, these mechanisms underpin the long-term physicochemical stability and predictable release kinetics needed for pulmonary dry-powder systems designed to deliver localized, sustained anticancer activity with reduced systemic exposure. 3.4 In Vitro Cytotoxicity and Apoptosis Cytotoxicity of doxorubicin-loaded PLGA-PEG dual-coated micelles was assessed in SLC lung carcinoma cells using the MTT assay. As shown in Table 5, the micellar formulation produced a significantly lower IC₅₀ (1.82 ± 0.14 µg/mL) compared with free doxorubicin (3.96 ± 0.21 µg/mL, p < 0.01), demonstrating enhanced inhibition of cell proliferation, (Fig. 5 ). Blank micelles showed negligible cytotoxicity, confirming that the antitumor effects were due to doxorubicin encapsulation rather than carrier toxicity. These results are consistent with earlier reports that PLGA-PEG dual-coated micelles increase drug potency through improved cellular uptake and reduced efflux [ 48 , 49 ]. While the SLC cell line offers a convenient and reproducible rat lung carcinoma model, it does not capture the full biological heterogeneity of human NSCLC. Consequently, cytotoxicity results obtained here should be interpreted with caution when considering clinical translation. Human-derived NSCLC cell lines (e.g., H460, H1975) often differ in resistance mechanisms and uptake pathways, while non-malignant bronchial epithelial cells (e.g., BEAS-2B) provide essential insights into off-target safety. Broader testing across malignant and normal human cell models will therefore be necessary to validate the selectivity, safety, and translational relevance of the PLGA-PEG micellar formulation. Flow cytometry-based apoptosis analysis further reinforced the superior efficacy of micellar doxorubicin. Treatment with doxorubicin-loaded micelles induced a significantly higher proportion of early and late apoptotic cells (48.6 ± 3.8%) compared to free doxorubicin (29.7 ± 2.9%, p < 0.001). This enhanced apoptotic response is consistent with prior reports demonstrating that nanoparticle-mediated drug delivery can potentiate mitochondrial membrane depolarization, caspase activation, and reactive oxygen species (ROS) generation, all of which play key roles in amplifying apoptosis[ 50 ]. Mechanistically, the sustained intracellular accumulation of micellar doxorubicin facilitates efficient nuclear delivery, which promotes increased DNA intercalation and topoisomerase II inhibition [ 51 ]. The higher levels of apoptosis observed in micelle-treated cells are likely due to a combination of enhanced drug delivery to the nucleus and the increased generation of ROS, which acts as a secondary trigger for mitochondrial dysfunction and caspase-mediated cell death. Another key aspect of the micellar formulation is its potential to overcome multidrug resistance (MDR), a major challenge in cancer chemotherapy. By facilitating endocytosis-mediated uptake and bypassing P-glycoprotein (P-gp) efflux pumps, micelles improve the intracellular retention and therapeutic bioavailability of doxorubicin in resistant tumor cells [ 52 ]. This is particularly significant for tumors that exhibit P-gp overexpression, which is commonly associated with resistance to doxorubicin and other chemotherapy agents. The micellar system's ability to overcome these resistance mechanisms could substantially improve the efficacy of doxorubicin, particularly in patients with tumors that are refractory to conventional treatments. PLGA-PEG micelles markedly enhance doxorubicin's anticancer efficacy. However, establishing a complete safety and efficacy profile will require comprehensive evaluation across different cancer types and normal cell lines. The safety of blank micelles, which showed negligible cytotoxicity (< 5% apoptosis), highlights the biocompatibility of the carrier system and confirms that therapeutic efficacy arises exclusively from the encapsulated doxorubicin [ 7 , 53 ]. Collectively, these findings suggest that PLGA-PEG dual-coated micelles significantly enhance the anticancer potency of doxorubicin by improving drug delivery, promoting apoptosis, and bypassing multidrug resistance, all while maintaining excellent carrier safety. However, to fully translate these promising in vitro results into clinical applications, further studies evaluating the formulation in a broader range of cell types, as well as in vivo models, are necessary to confirm its therapeutic potential and to evaluate the long-term safety of micellar-based therapies in clinical settings. Table (5). Comparative Cytotoxicity and Apoptosis Induction in SLC cell line Treatment IC₅₀ (µg/mL) % Viability at 5 µg/mL Early + Late Apoptosis (%) Notes Free Doxorubicin 3.96 ± 0.21 54.2 ± 4.3 29.7 ± 2.9 Rapid uptake, faster efflux Dox-Loaded PLGA-PEG dual-coated micelles 1.82 ± 0.14 31.8 ± 3.6 48.6 ± 3.8 Sustained uptake, enhanced apoptosis Blank Micelles > 100 ~ 100 < 5 Negligible cytotoxicity Several studies have consistently demonstrated that nanoformulation of doxorubicin, particularly with PLGA-PEG dual-coated micelles, enhances cytotoxicity and apoptosis induction in lung carcinoma and other tumor models compared to free doxorubicin. PLGA-PEG dual-coated micelles significantly enhanced the cytotoxic potency of doxorubicin in SLC cells compared with the free drug (Fig. 5 ). This effect is consistent with previous findings that PEG-b-PLGA nanopolymersomes improve intracellular accumulation and retention of doxorubicin in tumor cells, thereby increasing therapeutic efficacy [ 54 ]. The nanoscale size (~ 100 nm) and PEGylation of the micelles facilitate endocytosis while limiting premature drug efflux. These features collectively improve nuclear delivery of doxorubicin, contributing to greater drug-induced cytotoxicity. Furthermore, blank micelles were shown to be biocompatible, exhibiting negligible cytotoxicity, which confirms that the observed effects are exclusively due to the encapsulated drug. Flow cytometry analysis further confirms the superior apoptotic activity of doxorubicin-loaded micelles, as they significantly increase both early and late apoptosis (48.6% vs. 29.7% for free drug). These results are consistent with Jin et al. (2014), who demonstrated that polymeric micelle formulations enhance mitochondrial membrane depolarization and activate the caspase cascade, key steps in apoptosis induction [ 55 ]. The amplified apoptotic response is primarily attributed to sustained intracellular retention, efficient nuclear delivery, and inhibition of topoisomerase II, which represent the core mechanisms of doxorubicin-mediated cytotoxicity. Nanocarrier-mediated delivery of doxorubicin can effectively overcome multidrug resistance (MDR) by bypassing P-glycoprotein (P-gp)-mediated efflux, thereby prolonging intracellular drug retention and enhancing therapeutic efficacy [ 56 ]. P-gp overexpression is a major contributor to reduced doxorubicin accumulation in tumor cells, and strategies that evade its activity have been shown to restore drug sensitivity and improve cytotoxic outcomes. In parallel, micellar formulations can induce intracellular reactive oxygen species (ROS) overproduction, which potentiates apoptotic signaling through mitochondrial membrane depolarization and caspase activation [ 57 ]. Together, these mechanistic advantages, enhanced intracellular retention via P-gp evasion and ROS-mediated amplification of apoptosis, likely underpin the marked increase in cytotoxicity and apoptotic response observed in SLC lung carcinoma cells following micelle-mediated doxorubicin delivery. Collectively, these mechanistic advantages explain the observed increase in cytotoxicity and apoptotic response in SLC lung carcinoma cells. Importantly, blank micelles remain non-toxic to SLC cells and normal fibroblasts, underscoring their biocompatibility and highlighting that the therapeutic efficacy arises from encapsulated doxorubicin rather than the carrier system itself. Collectively, these results indicate that PLGA-PEG dual-coated micelles enhance the anticancer activity of doxorubicin by improving intracellular delivery, inducing apoptosis, and overcoming multidrug resistance, while exhibiting minimal carrier-related toxicity. Nevertheless, the current in vitro evaluation was limited to a single rat-derived lung carcinoma cell line (SLC, RCB2862). Although SLC provides a useful syngeneic model for adenocarcinoma, it does not capture the full heterogeneity of human NSCLC. Validation in additional human lung carcinoma cell lines (e.g., H460, H1975) and in normal bronchial epithelial cells (e.g., BEAS-2B) will be required to establish the generalizability, selectivity, and translational relevance of these findings. 3.5 In Vivo Pharmacokinetics, Lung Distribution, and Antitumor Efficacy Plasma pharmacokinetic evaluation following intratracheal administration of free DOX and DOX-micelles in rats is presented in Fig. 6 . Free DOX exhibited a sharp rise in plasma levels followed by a rapid decline, reaching a peak concentration (C max ) of 2.8 µg/mL within the first hour. In contrast, DOX-micelles displayed a slower absorption profile with a markedly reduced C max (1.1 µg/mL) and a more gradual elimination phase. The calculated pharmacokinetic parameters demonstrated lower systemic exposure (AUC₀-₂₄h: 14.3 vs 22.5 µg·h/mL) but a significantly prolonged mean residence time (MRT: 14.7 vs 6.1 h) for DOX-micelles compared with free DOX. These findings confirm that micellar encapsulation attenuates acute plasma spikes and reduces total systemic exposure (AUC), which is advantageous for minimizing systemic toxicity, while simultaneously prolonging therapeutic coverage (MRT) (Table 6). Biodistribution analysis confirmed efficient lung targeting, with DOX-micelles exhibiting significantly enhanced pulmonary deposition compared to free doxorubicin in an orthotopic tumor model. At 24 h post-administration, 46.8 ± 5.2% of the micellar dose was retained in pulmonary tissue, compared with only 12.4 ± 3.7% for free drug (p < 0.01) (Table 7, Fig. 7 ). This prolonged retention, likely attributable to micelle-surfactant interactions and reduced mucociliary clearance; these results align with those reported by Hu et al. (2014) [ 58 ]), conferred a marked therapeutic advantage: DOX-micelles reduced tumor volume by 68.5% compared with untreated controls, whereas free DOX achieved only a 25.3% reduction (Figs. 8 and 9 ). Notably, micelle treatment yielded an additional 43.2% reduction in tumor burden relative to free DOX (Table 7). These findings are consistent with previous studies showing that self-assembled polymeric micelles improve the antitumor activity of doxorubicin while reducing systemic toxicity, owing to their ability to sustain drug accumulation within tumors and enhance penetration across the extracellular matrix [ 59 , 60 ]. To ensure consistent interpretation of these outcomes, aligning the dosing regimen (every 3 days for 2 weeks) with the observed tumor growth inhibition further highlights the temporal relationship between drug exposure and therapeutic efficacy. Similar observations have been reported in lung cancer models, where composite micelle encapsulation of doxorubicin not only enhanced radiosensitivity but also provided prolonged retention and therapeutic benefit compared with the free drug [ 61 ]. Beyond tumor volume reductions, a deeper understanding of the pharmacodynamics of the micellar formulation is critical to elucidate its mechanism of action and clinical potential. The sustained intratumoral drug accumulation observed here suggests that the micelle-encapsulated doxorubicin remains localized within the tumor microenvironment for extended periods, thereby maintaining effective drug concentrations at the site of action. In contrast, free doxorubicin typically undergoes rapid distribution and clearance, limiting its duration of therapeutic effect. Prolonged drug presence within the tumor may thus enable continuous exposure of cancer cells to cytotoxic levels of doxorubicin, resulting in more efficient tumor cell killing and enhanced tumor regression. Moreover, micelles demonstrated the ability to penetrate more deeply into the extracellular matrix. This feature suggests that the formulation can overcome physical barriers within the tumor microenvironment. Such barriers often limit drug delivery, especially for larger molecules or conventional formulations. Enhanced penetration is particularly important in solid tumors. The dense extracellular matrix often restricts drug diffusion and reduces the efficacy of many chemotherapeutics. In addition, the efficient cellular uptake of the micelles facilitates higher intracellular accumulation of doxorubicin in malignant cells, thereby amplifying its cytotoxic effects. Collectively, these pharmacodynamic advantages indicate that the micellar formulation not only improves localized drug delivery but also sustains elevated concentrations of doxorubicin at the tumor site over extended periods. This prolonged retention may reduce the need for frequent dosing while enhancing overall therapeutic efficacy. Importantly, by localizing drug exposure within the tumor and limiting systemic distribution, the micellar system has the potential to mitigate the dose-limiting toxicities typically associated with conventional chemotherapy, positioning it as a promising strategy for targeted cancer therapy. Systemic safety evaluation revealed that micellar delivery attenuated cardiotoxicity. Serum CK-MB and LDH levels were reduced by approximately 55% and 48%, respectively, compared with free doxorubicin. Histopathological analysis demonstrated minimal myocardial degeneration in micelle-treated rats, consistent with the reduced systemic C max and preferential pulmonary retention [ 58 ], (Table 8). The reduction in doxorubicin-induced cardiotoxicity observed in this study can be explained by altered biodistribution and controlled drug release. Following intrapulmonary administration, micelles deposit directly in the lung, leading to high local concentrations at the tumor site while minimizing systemic exposure and cardiac accumulation, consistent with previous biodistribution studies of pulmonary micellar delivery [ 58 ]. PEGylation contributes to colloidal stability and steric shielding, limiting protein adsorption and premature clearance, whereas the PLGA core supports a biphasic release profile, an initial burst phase that ensures rapid cytotoxic activity followed by a sustained release that prolongs exposure without sharp systemic peaks. Such a spatiotemporal release pattern markedly reduced acute cardiac exposure to doxorubicin. As shown in Table 8, serum CK-MB levels were significantly lower in the DOX-micelle group (145 ± 20 U/L) compared with free DOX (320 ± 30 U/L), representing a ~ 55% reduction. Similarly, LDH levels decreased from 480 ± 40 U/L in free DOX-treated rats to 250 ± 28 U/L with micelles, approaching control values (220 ± 25 U/L). Histopathological analysis further supported these findings, revealing severe myocardial degeneration in free DOX-treated hearts, whereas micelle-treated animals exhibited only minimal changes, comparable to controls. Collectively, these results confirm that micellar delivery mitigates doxorubicin-induced cardiotoxicity, consistent with prior evidence that nanocarrier systems, including liposomal DOX, confer cardioprotection through reduced cardiac uptake and controlled release [ 62 ]. Table (6). Pharmacokinetic Parameters of Free DOX and DOX-Micelles After Pulmonary Administration in Rats Parameter Free DOX DOX-Micelles Fold Change C_max (µg/mL) 2.8 ± 0.3 1.1 ± 0.2 ↓ 2.5-fold AUC₀-₂₄h (µg·h/mL) 22.5 ± 2.1 14.3 ± 1.6 ↓ 1.6-fold MRT (h) 6.1 ± 0.8 14.7 ± 1.3 ↑ 2.4-fold Note: Values are expressed as mean ± SD (n = 6). DOX-micelles reduced peak exposure while prolonging systemic residence time. Table (7). Lung deposition and tumor volume reduction following administration of free DOX and DOX-micelles. Parameter Free DOX DOX-Micelles Lung retention at 24 h (%) 12.4 ± 3.7 46.8 ± 5.2 Tumor volume reduction vs. control 25.3% 68.5% Tumor volume reduction vs. free DOX - 43.2% DOX-micelles achieved higher pulmonary retention and superior tumor suppression compared with free drug. Table (8). Cardiotoxicity Biomarkers Marker Control Free DOX DOX-Micelles CK-MB (U/L) 120 ± 15 320 ± 30 145 ± 20 LDH (U/L) 220 ± 25 480 ± 40 250 ± 28 Cardiac histopathology Normal Severe degeneration Minimal degeneration Micellar delivery reduced cardiotoxicity markers by ~ 55% compared with free doxorubicin. Percentage of drug retained in lung tissue 24 h post-instillation for free doxorubicin (12.4 ± 3.7%) versus doxorubicin-loaded micelles (46.8 ± 5.2%). Micellar delivery achieved approximately four-fold higher pulmonary retention, confirming efficient localized deposition and reduced clearance. Data represent mean ± SD (n = 6). Biodistribution in Major Organs Time-concentration analysis demonstrated marked differences in organ distribution between free DOX and DOX-micelles (Table 9 ). Free DOX exhibited rapid distribution into systemic organs, with high concentrations detected in the liver and heart within the first 2 h post-administration, followed by a steep decline. In contrast, DOX-micelles showed significantly lower accumulation in systemic organs at all time points, while maintaining sustained presence in the lungs. At 24 h, heart DOX levels in the free drug group remained at 480 ± 55 ng/g, whereas DOX-micelles showed only 210 ± 35 ng/g, representing a ~ 2.3-fold reduction. Similarly, hepatic exposure was markedly reduced (2,150 ± 210 ng/g vs. 1,050 ± 140 ng/g for free DOX and micelles, respectively). Splenic uptake was also lower for DOX-micelles, consistent with reduced RES sequestration. Renal distribution was comparable between groups, reflecting similar clearance mechanisms. These results confirm that micellar encapsulation not only enhances pulmonary retention but also minimizes systemic exposure to off-target organs, particularly the heart, thereby reducing cardiotoxic risk. Consistent with the pharmacokinetic parameters summarized in Table 9 , the biodistribution profiles clearly distinguished the two formulations. Free DOX exhibited markedly higher Cmax and AUC values in systemic organs, particularly the liver and heart, indicating rapid uptake and greater systemic exposure. For example, hepatic accumulation of free DOX peaked early (T max = 2 h) with a substantially higher AUC compared with the micellar formulation. In contrast, DOX-micelles demonstrated a lower C max and reduced overall AUC , reflecting attenuated systemic exposure, while simultaneously exhibiting a prolonged t 1/2 and MRT . This pharmacokinetic signature suggests that micellar encapsulation dampens acute systemic peaks and lowers cumulative exposure, yet maintains extended drug residence through sustained release and slower clearance. Together, these features indicate improved safety by limiting off-target accumulation without compromising therapeutic persistence [ 60 ]. Cardiac exposure, as reflected in both C max and AUC, was substantially lower for DOX-micelles, supporting their cardioprotective potential. Similarly, splenic distribution was reduced in the micellar group, consistent with diminished RES sequestration. Renal pharmacokinetics did not differ markedly between the two groups, with comparable T max and elimination profiles, indicating that micellar encapsulation did not alter renal excretion. Collectively, these PK findings confirm that DOX-micelles minimize systemic accumulation while sustaining targeted pulmonary delivery. Representative H&E-stained myocardial sections are shown in Fig. 10 . Control animals exhibited normal cardiac architecture with well-aligned myocardial fibers and intact nuclei. In contrast, free DOX treatment caused marked cardiomyocyte degeneration, nuclear condensation, and disruption of myocardial fibers, consistent with classical features of anthracycline-induced cardiotoxicity. Importantly, animals receiving DOX-loaded PLGA-PEG micelles displayed largely preserved myocardial structure with only minimal alterations, comparable to control. These histological observations are in strong agreement with the cardiac biochemistry profiles, confirming reduced oxidative stress and myocardial injury in the micelle-treated group. Table 9 Pharmacokinetic Parameters of DOX in Systemic Organs After Pulmonary Administration in Rats Organ Parameter Free DOX DOX-Micelles Fold Change Heart Cmax (µg/g) 1.25 ± 0.15 0.48 ± 0.08 ↓ 2.6-fold AUC₀-₂₄h (µg·h/g) 14.8 ± 1.9 6.2 ± 0.9 ↓ 2.4-fold MRT (h) 5.4 ± 0.7 10.1 ± 1.1 ↑ 1.9-fold Liver C max (µg/g) 3.85 ± 0.32 1.96 ± 0.25 ↓ 2.0-fold AUC₀-₂₄h (µg·h/g) 45.2 ± 3.6 22.1 ± 2.5 ↓ 2.0-fold MRT (h) 4.8 ± 0.6 9.5 ± 1.2 ↑ 2.0-fold Kidney C max (µg/g) 1.65 ± 0.20 1.40 ± 0.18 ↓ 1.2-fold AUC₀-₂₄h (µg·h/g) 18.5 ± 2.2 15.7 ± 2.0 ↓ 1.2-fold MRT (h) 6.2 ± 0.9 8.7 ± 1.0 ↑ 1.4-fold Spleen C max (µg/g) 2.35 ± 0.28 1.10 ± 0.15 ↓ 2.1-fold AUC₀-₂₄h (µg·h/g) 28.9 ± 3.4 12.5 ± 1.7 ↓ 2.3-fold MRT (h) 5.0 ± 0.8 9.2 ± 1.1 ↑ 1.8-fold Values are mean ± SD, n = 6. DOX-micelles significantly reduced cardiac and hepatic exposure while maintaining longer residence times, consistent with preferential pulmonary retention and improved safety profile. The observed reduction in cardiotoxicity markers (CK-MB, LDH) and minimal histopathological damage in the DOX-micelle group can be mechanistically attributed to the significantly lower systemic exposure and reduced cardiac accumulation of doxorubicin. As shown in Table 9 , DOX-micelles exhibited a 2.6-fold lower C max and 2.4-fold lower AUC in heart tissue compared to free DOX. This attenuated cardiac exposure directly correlates with the diminished release of biomarkers associated with myocardial injury. The sustained release profile and pulmonary retention of micellar DOX effectively limit peak plasma concentrations and systemic distribution, thereby reducing off-target toxicity in cardiac tissue—a well-established dose-limiting effect of doxorubicin. These findings confirm the favorable cardiac safety profile of micellar pulmonary delivery (Fig. 10 ). Previous studies have highlighted the potential of inhalable nanocarriers for the treatment of lung cancer. Gupta et al. (2022) provided a comprehensive review of inhalable formulations for non-small cell lung cancer, emphasizing that micelles, liposomes, and dendrimers can improve pulmonary residence and limit systemic toxicity [ 60 ]. However, their work was primarily conceptual and did not present direct pharmacokinetic or safety data. Wang et al. (2018) reported that matrix metalloproteinase (MMP-2/9)-responsive micelles for paclitaxel inhalation achieved approximately 38% pulmonary deposition and superior tumor suppression compared with intravenous administration [ 63 ]. This study confirmed the benefit of enzyme-triggered release and local lung retention but did not investigate doxorubicin or address systemic cardiotoxicity. Similarly, Hu et al. (2014) demonstrated that polymeric micelles delivered intratracheally in rats prolonged pulmonary retention and extended systemic circulation, yet they did not assess therapeutic efficacy in tumor models or evaluate safety endpoints [ 58 ]. The present work enhances previous findings by integrating pharmacokinetics, biodistribution, antitumor efficacy, and systemic safety of pulmonary doxorubicin-loaded micelles. Compared to free doxorubicin, micelles significantly reduced plasma C max (1.1 ± 0.2 µg/mL vs. 2.8 ± 0.3 µg/mL) and AUC (14.3 ± 1.6 µg·h/mL vs. 22.5 ± 2.1 µg·h/mL), while markedly prolonging mean residence time (MRT of 14.7 ± 1.3 h vs. 6.1 ± 0.8 h). This pharmacokinetic profile reflects controlled drug release from the micelles in the alveolar microenvironment, leading to reduced systemic peaks and prolonged circulation. Biodistribution studies further demonstrated that nearly half of the micellar dose (46.8 ± 5.2%) remained in lung tissue at 24 hours, compared with only 12.4 ± 3.7% for free doxorubicin. This enhanced pulmonary retention is attributed to micelle-surfactant interactions, which promote spreading over the alveolar surface, and reduced clearance through mucociliary and phagocytic pathways, consistent with previous nanoparticle studies. To clarify the impact of pulmonary retention on therapeutic outcomes, a detailed comparison of lung tissue concentrations with systemic plasma levels would be useful. This comparison would highlight how sustained drug release in the lungs directly contributes to the observed therapeutic benefits, particularly in terms of tumor targeting and reduced systemic toxicity. The enhanced deposition of the micelles in the lungs ensures a higher localized drug concentration at the tumor site, maximizing the therapeutic effect while minimizing systemic exposure and associated side effects. Systemic safety assessments revealed that micelle treatment significantly attenuated doxorubicin-induced cardiotoxicity. Specifically, serum CK-MB and LDH levels were reduced by approximately 50–55% in micelle-treated animals, compared to free doxorubicin treatment. Histopathological analysis of myocardial tissue further demonstrated minimal degeneration in the hearts of micelle-treated rats, in contrast to the severe myocardial damage observed in the free doxorubicin group. Mechanistically, this safety benefit is directly linked to the lower systemic C max (1.1 ± 0.2 µg/mL vs. 2.8 ± 0.3 µg/mL) and AUC (14.3 ± 1.6 µg·h/mL vs. 22.5 ± 2.1 µg·h/mL) of micellar doxorubicin, as well as the pulmonary "drug depot effect," which together result in reduced cardiac exposure. These findings provide the first integrated evidence that pulmonary micellar delivery not only enhances therapeutic efficacy but also mitigates one of the most significant dose-limiting toxicities of doxorubicin, namely cardiotoxicity. The PLGA-PEG dual-coated micelles outperformed liposomal formulations in aerosolization efficiency, achieving an MMAD of 2.9 µm and an FPF of 62%, both of which are critical for effective deep lung deposition. In contrast, liposomes, although valuable in enhancing the solubility and stability of anticancer drugs, often encounter limitations in aerosolization due to their larger particle size and propensity to aggregate, resulting in reduced fine particle fractions [ 64 ]. Polymeric micelles generally provide superior performance in this regard, owing to their smaller size, uniform dispersion, and improved stability during aerosolization [ 65 ]. Furthermore, the excipient-assisted spray-drying approach employed in this study effectively preserved the structural integrity of the micelles during aerosolization, while simultaneously optimizing fine particle deposition in the lungs. This represents a key advantage over liposomal dry powder inhalers and is consistent with previous reports demonstrating the ability of micelle-based and PLGA nanoparticle systems to achieve favorable aerosolization and lung deposition profiles [ 38 , 66 ]. The therapeutic outcomes clearly indicate that PLGA-PEG dual-coated micelles enhance the antitumor efficacy of doxorubicin compared with both untreated controls and the free drug. This improvement can be attributed to several complementary mechanisms. Sustained pulmonary release ensures prolonged intratumoral exposure, while the nanoscale architecture of the micelles enables more efficient penetration through the tumor extracellular matrix. Additionally, preferential endocytic uptake by malignant cells increases intracellular drug accumulation, thereby amplifying cytotoxic activity. Notably, compared with conventional liposomal formulations, the dual-coated micelles showed superior tumor penetration, likely due to their smaller size and more uniform distribution, which facilitated deeper and more consistent tissue permeation [ 67 ]. In addition to enhanced efficacy, the micellar formulation markedly reduced systemic toxicity. Serum CK-MB and LDH levels decreased by approximately 50–55%, while histopathological evaluation revealed only minimal myocardial degeneration. These results demonstrate that the micellar system not only improves the therapeutic index but also mitigates cardiotoxicity, one of the major dose-limiting adverse effects of doxorubicin [ 64 ]. Overall, the PLGA-PEG dual-coated micellar dry powder formulation offers multiple advantages over liposomes and conventional polymeric micelles. These include controlled drug release, superior aerosolization efficiency, and improved therapeutic outcomes. The enhanced stability, fine particle deposition, and sustained pulmonary drug release highlight its potential as a promising strategy for inhalation-based chemotherapy [ 38 ]. From a translational perspective, the combination of enhanced antitumor efficacy and reduced systemic toxicity highlights the clinical potential of pulmonary micelle-based delivery systems for achieving effective tumor suppression at lower cumulative systemic doses of doxorubicin. By directly targeting the lungs and minimizing systemic exposure, this approach may reduce chemotherapy-associated cardiotoxicity, improve patient compliance through reduced adverse effects, and enable the development of outpatient inhalation regimens that enhance quality of life for lung cancer patients. 4. Discussion Conventional intravenous doxorubicin is limited by rapid distribution, high peak plasma concentrations, and cumulative cardiotoxicity. Liposomal formulations such as Doxil® extend circulation and partially reduce acute cardiac toxicity, yet myocardial accumulation remains significant and long-term cardiac safety is not ensured. Systemic administration also limits local drug exposure in pulmonary malignancies, necessitating higher doses to achieve therapeutic lung concentrations. The selection of appropriate inlet and outlet temperatures was paramount to achieving a powder with optimal properties for pulmonary delivery. While high inlet temperatures (> 150°C) can maximize yield, they pose a significant risk of degrading our thermolabile API. Furthermore, such temperatures could potentially induce crystallization of our crystalline matrix former, lactose, or cause melting and degradation of our amorphous stabilizer, trehalose, which possesses a critical glass transition temperature (Tg). Conversely, excessively low temperatures can produce powders with high residual moisture, promoting instability, crystallization of lactose, and poor aerosolization. Our chosen parameters, an inlet temperature of 120°C resulting in an outlet temperature of ~ 65°C, were strategically optimized for this specific formulation. This gentle thermal profile successfully prevented API degradation, as confirmed by the HPLC analysis (Table 2 ). For the excipients, the outlet temperature was maintained low enough to prevent the crystallization of lactose and, most critically, was kept safely below the Tg of the amorphous trehalose phase. This prevented sticky-wall deposition and ensured a high process yield. Most critically, these conditions produced particles with an ideal morphology and density, as vividly illustrated in the SEM micrographs of Fig. 3 A. The resulting powder exhibited excellent aerodynamic performance (Fig. 3 B), with a high fine particle fraction (FPF) of > 75%, directly attributable to the low moisture content and near-spherical, hollow particle structure enabled by this precise thermal control. Therefore, the excellent results in Fig. 3 are a direct consequence of the carefully calibrated spray-drying temperatures that balanced product stability with powder functionality. Spray drying is widely used in pharmaceutical and nutraceutical formulations, with inlet and outlet temperatures consistently identified as key parameters determining product quality. These thermal conditions govern drying kinetics, particle morphology, and the retention of bioactive compounds, ultimately shaping essential physicochemical attributes such as solubility, dispersibility, and storage stability. The inlet temperature is the primary driver of solvent evaporation, providing the thermal energy required for drying. Higher inlet temperatures generally accelerate drying, which can be advantageous in limiting the exposure of heat-sensitive compounds to prolonged thermal stress [ 68 ]. However, excessively high inlet temperatures can cause particle brittleness, morphological changes, or chemical degradation. Tay et al. (2021) reported that temperatures above 150°C led to the breakdown of bioactive compounds, reducing both stability and therapeutic efficacy [ 69 ]. These findings emphasize the importance of balancing sufficient thermal input for efficient drying with the risk of thermally induced degradation. While outlet temperature is generally recognized as a key parameter influencing particle size and surface area during spray drying [ 70 ], its role appeared limited in this study, as the outlet was maintained at ~ 65°C. Under these fixed thermal conditions, the observed aerodynamic performance (MMAD ~ 2.9 µm, high FPF) was more convincingly linked to excipient effects. The inclusion of lactose and trehalose not only stabilized the nanostructures during drying but also enhanced powder dispersibility upon aerosolization. These findings suggest that, in systems where thermal variability is minimized, excipient composition rather than outlet temperature becomes the dominant determinant of aerosol behavior [ 70 ]. Yet, insufficient outlet temperatures may result in higher residual moisture, predisposing the product to instability from hydrolytic degradation or microbial growth. Conversely, higher outlet temperatures often generate larger, denser particles with lower surface areas, which may enhance physical stability but at the expense of slower reconstitution [ 68 ]. Recent evidence further highlights the interplay between inlet and outlet temperatures in shaping product performance. Akbarbaglu et al. (2021) demonstrated that carefully optimized thermal conditions can significantly improve the retention of labile compounds such as proteins and antioxidants. In their study, an inlet temperature of 130°C combined with an outlet temperature of 70°C produced formulations with enhanced stability and extended shelf-life compared to those dried under more extreme conditions [ 71 ]. This underscores the importance of considering both parameters simultaneously rather than in isolation. These studies demonstrate that inlet and outlet temperatures exert complementary but distinct effects on spray-dried products. Optimal conditions must strike a balance between drying efficiency, particle integrity, and active compound stability. A deeper mechanistic understanding of temperature effects on particle morphology and degradation is essential. Such knowledge will help refine spray-drying protocols and support the development of more robust pharmaceutical and nutraceutical formulations. Inhalable doxorubicin formulations have long been explored to enhance pulmonary deposition and limit systemic toxicity, but early platforms such as liposomes, polymeric nanoparticles, and microspheres were constrained by aerosolization-induced aggregation, modest fine-particle fractions (35–45%), and poor dispersibility after spray drying [ 72 , 73 ]. Liposomes, despite the clinical success of Doxil®, remain particularly problematic in inhalable forms due to phospholipid oxidation, particle aggregation, and dependence on cryoprotectants, with fine-particle fractions rarely exceeding ~ 45% and drug retention often limited to 70–75% after spray drying [ 23 , 74 ]. The PLGA-PEG micellar dry powder inhaler developed here overcomes these barriers. It achieved a fine-particle fraction > 60%, reflecting markedly improved aerosol performance, and retained > 90% drug content after spray drying and one-month storage at room temperature, well above values reported for liposomal formulations [ 75 ]. In addition, the micelles displayed rapid redispersibility (> 95%), reduced macrophage uptake by nearly 50%, and sustained release with > 70% cumulative doxorubicin release over 48 hours, compared with ~ 40% for liposomal formulations [ 14 , 76 ]. This study provides the first demonstration of a PLGA-PEG micellar dry powder inhaler for doxorubicin that unites high aerosol efficiency, long-term stability without cryoprotectants, and enhanced in vivo efficacy. These attributes represent a decisive advance over prior inhalable liposomal systems and position PLGA-PEG micelles as a next-generation platform for pulmonary cancer therapy. However, the aggregation of micelles or crystallization of doxorubicin within the micelles could significantly impact the drug release kinetics and overall delivery performance. Micellar aggregation may occur during storage or within the physiological environment, compromising the structural integrity of the micelles. When micelles aggregate, their surface area available for drug release decreases, potentially slowing the release rate. Additionally, aggregation could hinder the ability of the micelles to effectively reach deep lung regions during aerosolization, as larger aggregates may not be as easily inhaled or deposited in the distal lung areas [ 77 ]. Furthermore, crystallization of doxorubicin within the micelles could negatively affect the release profile. Crystalline forms of doxorubicin are less soluble than their amorphous counterparts, resulting in slower and potentially erratic release. If doxorubicin crystallizes within the micelles, it could slow down the release rate, disrupting the intended biphasic release and diminishing the therapeutic effect. Crystallization could also alter the micelles' size and surface properties, which may impact their dispersibility and aerosol performance. Larger, aggregated, or crystalline micelles may exhibit reduced aerodynamic properties, leading to a decreased fine particle fraction (FPF), which is critical for drug deposition in the small airways and alveoli [ 78 ]. These changes (aggregation or crystallization) could severely impair lung delivery performance. A reduced FPF means that fewer drug particles will reach the target sites in the lungs, potentially compromising the effectiveness of localized therapy for conditions such as lung cancer. Moreover, an uncontrolled release profile resulting from aggregation or crystallization could cause premature drug release or delayed delivery, both of which would undermine the goal of achieving a sustained and controlled therapeutic effect. Consequently, these factors could negatively impact both therapeutic outcomes and patient safety. In conclusion, while the biphasic release behavior of doxorubicin-loaded PLGA-PEG dual-coated micelles holds promise for sustained pulmonary delivery, the potential for micellar aggregation and crystallization poses significant challenges. These factors must be carefully monitored during formulation and storage to ensure that the system maintains its stability, performance, and the desired therapeutic effect in lung cancer treatment. The Korsmeyer-Peppas model is commonly used to describe the release kinetics of drug delivery systems, particularly when the release mechanism is complex and involves multiple processes. In the context of the present study, the Korsmeyer-Peppas model (with an exponent n = 0.74, R2 = 0.976, and Ft ​≤ 0.6) indicated anomalous transport, suggesting that drug release is governed by a combination of Fickian diffusion and polymer relaxation/erosion. This is significant because the value of n in the Korsmeyer-Peppas model provides insight into the release mechanism: values between 0.5 and 1.0 are indicative of a combination of diffusion and polymer relaxation, which is consistent with the characteristics of the delivery system. The specific combination of PLGA (poly(lactic-co-glycolic acid)) and PEG (polyethylene glycol) in this system plays a critical role in modulating the drug release profile. PLGA, a biodegradable polymer, undergoes hydrolytic degradation over time, which leads to erosion and the controlled release of the encapsulated drug. On the other hand, PEG, known for its hydrophilicity, forms a shell around the drug, acting as a diffusion barrier. This dual mechanism, diffusion through the PEG shell and gradual erosion of the PLGA core, leads to sustained and controlled drug release, a feature commonly observed in micellar and polymeric drug carriers [ 14 , 42 ]. In more complex in vivo environments, this release profile is likely to change due to factors such as pH, enzymatic activity, and the presence of biological fluids, which can alter the rate of polymer degradation and diffusion. For example, the local pH of the tissue or the action of specific enzymes may accelerate the degradation of the PLGA core, leading to a faster release of the drug compared to in vitro conditions. Moreover, the hydrophilic nature of PEG can be influenced by the surrounding biological medium, potentially altering its effectiveness as a diffusion barrier. These factors make the release behavior in vivo more dynamic and less predictable, but also potentially more adaptable to the needs of targeted drug delivery. The PEG corona stabilizes micelles and suppresses premature leakage, while the PLGA core sustains intracellular release [ 58 ]. Lactose and trehalose act as lyoprotectants, preventing aggregation during lyophilization and preserving aerosol performance [ 79 ]. In contrast to liposomal and nanoparticle-based inhalable formulations, which frequently exhibit limited fine particle fractions, aggregation, and inconsistent lung deposition, the dual-coated micellar dry powder demonstrates reproducible aerodynamic performance with significantly higher deposition efficiency. Mechanistically, pulmonary micelle reservoirs modulate systemic exposure by lowering C_max and extending residence time [ 58 ], thereby attenuating cardiotoxicity relative to free drug and conventional intravenous dosing, as corroborated by suppressed CK-MB, LDH, and negligible histopathological changes. Unlike earlier liposomal or polymeric nanoparticle systems, which offered only partial solutions to pulmonary delivery challenges, this platform integrates pulmonary targeting, structural stability, and systemic safety into a single formulation, representing a decisive advancement in inhalable doxorubicin therapy. Surface modification of PLGA carriers with polyethylene glycol (PEG) has proven to be a pivotal strategy for optimizing pulmonary drug delivery. The dual contribution of the PLGA matrix and PEG corona establishes a synergistic framework that balances controlled release with extended lung retention. At the structural level, PLGA provides the degradable hydrophobic depot, encapsulating doxorubicin and releasing it in a time-dependent manner through hydrolytic erosion into lactic and glycolic acids. This gradual degradation avoids the burst release characteristic of unmodified polymers, maintaining therapeutic concentrations in the pulmonary microenvironment and supporting prolonged local bioavailability. Superimposed on this depot effect, PEG functionalization imparts steric stabilization, preventing aggregation during nebulization and ensuring reproducible aerosolization. Once deposited, the PEG corona limits protein adsorption and opsonization, thereby reducing phagocytic clearance and systemic leakage. This stealth behavior not only extends pulmonary residence but also enhances epithelial uptake, establishing a localized drug reservoir with minimized off-target exposure. Evidence from preclinical studies validates this mechanistic synergy. Li et al. (2021) showed that PEGylated PLGA microspheres achieve tunable lung retention and reduced systemic distribution compared with unmodified PLGA carriers [ 80 ]. Similarly, Omidian and Wilson (2025) highlighted that PLGA-PEG formulations optimize aerosol performance and mucosal interactions, strengthening their value for both pulmonary and intranasal delivery [ 81 ]. Beyond respiratory applications, Kesharwani et al. (2025) demonstrated that PEGylated PLGA nanoparticles improve tumor accumulation and intracellular uptake, confirming PEG’s role in enhancing therapeutic precision across oncological models [ 48 ]. Broader reviews further consolidate PEGylation as a key translational modification for PLGA-based drug delivery [ 82 ]. These data position PLGA-PEG nanocarriers as a versatile and clinically relevant platform. By combining PLGA’s sustained release profile with PEG’s pharmacokinetic and stability advantages, this dual-coated system maximizes lung retention, improves epithelial transport, and reduces systemic cardiotoxicity, an especially favorable profile for localized chemotherapy in lung malignancies. The cardioprotective effect observed with micellar DOX is mechanistically grounded in its altered pharmacokinetic profile. The significantly lower Cmax and AUC in heart tissue directly explain the reduction in cardiotoxicity biomarkers and histopathological damage (Table 9 ). By minimizing peak systemic exposure and prolonging pulmonary retention, the micellar formulation reduces the cumulative dose delivered to the heart, thereby mitigating doxorubicin-induced oxidative stress and mitochondrial dysfunction. This is consistent with prior reports that nanocarrier systems reduce cardiotoxicity through controlled release and reduced cardiac uptake (e.g., liposomal doxorubicin). Thus, the dual mechanisms of localized lung deposition and sustained systemic release collectively contribute to an improved safety profile without compromising antitumor efficacy. Unmodified PLGA carriers have been extensively applied in inhalable and injectable drug delivery owing to their biodegradability and predictable hydrolysis into lactic and glycolic acids, which supports depot-like sustained release of therapeutics [ 82 ]. However, their hydrophobic surface chemistry promotes aggregation during aerosolization, impairing aerodynamic performance and reproducibility of lung deposition. Furthermore, once deposited in the alveoli, PLGA microspheres and nanoparticles are rapidly opsonized and cleared by macrophages, leading to short pulmonary residence times and reduced therapeutic efficiency [ 81 ]. Thus, while PLGA alone provides reliable controlled release, its clinical utility in pulmonary delivery is constrained by instability and premature clearance. PEGylation strategies, by contrast, are designed to enhance colloidal stability and biological persistence. The hydrophilic PEG corona provides a steric barrier that minimizes aggregation, reduces protein adsorption, and confers “stealth” characteristics against phagocytic clearance (50). In pulmonary and intranasal contexts, PEGylation has been shown to improve epithelial permeability and extend retention time within mucosal and alveolar compartments [ 81 ]. Nonetheless, PEG-only systems lack a depot effect: encapsulation efficiency is relatively low, and drug leakage is common due to PEG’s hydrophilic nature. As a result, while PEG formulations extend persistence, they cannot ensure sustained and localized drug release. The integration of PLGA cores with PEG shells offers a complementary solution. PLGA provides degradation-mediated, time-dependent release, while PEG improves colloidal stability during aerosolization, reduces immune recognition, and prolongs alveolar residence. This synergy has been validated in vivo, where PEG-modified PLGA microspheres achieved tunable lung retention and reduced systemic exposure compared with unmodified PLGA formulations (80). Beyond pulmonary delivery, PEGylated PLGA nanoparticles have also improved tumor accumulation and reduced systemic toxicity of doxorubicin in oncology models, highlighting the broad translational relevance of this dual strategy [ 48 ]. Despite these advances, polymeric carriers alone remain vulnerable to stresses encountered during spray drying, nebulization, and pulmonary deposition. Here, lactose and trehalose serve as critical stabilizing excipients. These disaccharides enhance dispersibility, prevent nanoparticle aggregation, and improve powder flowability. Trehalose, in particular, stabilizes labile polymeric matrices via hydrogen bonding and elevation of glass transition temperature, while lactose enhances aerodynamic uniformity and promotes homogeneous deposition within the lower respiratory tract [ 81 , 82 ]. When co-formulated with PLGA-PEG systems, lactose and trehalose improve emitted dose, fine particle fraction, and delivery efficiency, while mitigating moisture-induced instability [ 79 ]. Cumulatively, PLGA-PEG nanostructures complemented by lactose and trehalose represent a rational, translationally relevant platform. By establishing a localized pulmonary reservoir with sustained release, enhanced retention, and reduced systemic exposure, this strategy directly addresses key clinical challenges such as systemic cardiotoxicity and frequent dosing. Moreover, the regulatory familiarity of PLGA, PEG, lactose, and trehalose, all FDA-approved or GRAS, facilitates a clear pathway toward clinical adoption. Importantly, the modularity of this approach supports broader applicability across chemotherapeutics, biologics, and gene therapies, extending its utility beyond oncology to chronic respiratory diseases. The literature collectively highlights the complementary strengths of polymeric systems in pulmonary delivery. PLGA-only carriers reliably provide controlled release via gradual hydrolytic degradation but are limited by aggregation during aerosolization and rapid clearance by alveolar macrophages. PEG-only systems, in contrast, prolong pulmonary retention through steric stabilization and reduced opsonization, yet lack depot capacity and often exhibit premature drug leakage. PLGA-PEG dual systems uniquely merge these functionalities: PLGA offers sustained, degradation-mediated release, while PEG enhances colloidal stability, reduces immune clearance, and extends alveolar residence. Preclinical evidence confirms this synergy, with PEG-modified PLGA microspheres showing tunable lung retention and reduced systemic exposure relative to unmodified PLGA (80). Similarly, oncology studies demonstrate enhanced tumor accumulation and reduced cardiotoxicity with PEGylated PLGA nanoparticles, reinforcing their translational potential [ 48 ]. Excipients such as lactose and trehalose address practical bottlenecks in inhalation formulations by stabilizing particles during spray drying, reducing aggregation, and enhancing dispersibility and aerodynamic performance [ 82 ]. By safeguarding powder integrity, these sugars preserve the pharmacokinetic advantages of PLGA-PEG systems, ensuring reproducible lung deposition and sustained therapeutic benefit. This integrated design paradigm establishes a localized pulmonary reservoir with prolonged drug exposure and reduced systemic toxicity, positioning PLGA-PEG-sugar systems as one of the most clinically promising inhalation platforms [ 48 , 80 , 82 ]. Despite our findings, this study has limitations that must be considered. A primary constraint is the reliance on the rat-derived SLC cell line (RCB2862) as a model for human NSCLC. While this model provided valuable insights into the role of SLCs in metabolic reprogramming, it does not capture the full biological and genetic heterogeneity of human lung cancers. Consequently, the direct translational relevance of our results may be limited. Future studies are essential to validate these mechanisms in a panel of well-characterized human NSCLC cell lines and primary patient-derived models to strengthen the clinical applicability of our conclusions. Translational Relevance Pulmonary delivery of doxorubicin-loaded micelles achieved high lung retention, sustained pharmacokinetics, and enhanced antitumor efficacy while markedly reducing systemic cardiotoxicity. By lowering peak plasma exposure and acting as a localized drug depot, this approach enabled effective tumor suppression at reduced systemic doses. These findings provide preclinical evidence that inhalable micellar formulations hold translational potential for further investigation as an alternative to conventional systemic chemotherapy in lung cancer. Nonetheless, key limitations must be acknowledged. Long-term in vivo safety within the pulmonary environment remains incompletely understood. Repeated dosing raises the possibility of anti-PEG antibody formation and immunogenic responses [ 83 , 84 ], or low-grade inflammation from persistent polymeric fragments. Moreover, scaling PLGA-PEG formulations to industrial production poses significant challenges: ensuring particle size uniformity, PEG surface density, and excipient crystallinity during spray drying or lyophilization requires stringent process control. Regulatory approval will further demand extensive pharmacokinetic, toxicological, and stability data, which may slow translation despite encouraging preclinical outcomes. Addressing these limitations through long-term pulmonary safety studies and scalable manufacturing innovations will be essential to bridge the gap from proof-of-concept to clinical implementation. 5. Conclusion This study establishes a novel inhalable dry powder platform based on spray-dried, PLGA-PEG dual-coated micelles embedded in a lactose/trehalose matrix, designed to overcome the limitations of conventional doxorubicin formulations and earlier pulmonary delivery systems. The formulation exhibits three key innovations: (i) a dual polymeric coating that enhances micellar stability, prevents premature drug leakage, and prolongs lung retention; (ii) excipient-mediated aerosol stabilization that ensures high fine particle fraction and reproducible lung deposition, mitigating aggregation common in nanoparticulate dry powder inhalers; and (iii) pulmonary pharmacokinetic modulation, enabling localized drug reservoirs that reduce systemic Cmax, extend residence time, and significantly attenuate cardiotoxicity without sacrificing therapeutic efficacy. Mechanistically, this approach achieves an optimal balance between antitumor potency and cardioprotection, as demonstrated by robust tumor suppression, reduced serum CK-MB and LDH levels, and preserved myocardial architecture. The platform leverages clinically accepted excipients and scalable spray-drying, underscoring its translational feasibility. In conclusion, this spray-dried micellar system represents a comprehensive preclinical validation of an inhalable doxorubicin formulation with enhanced lung targeting, sustained release, and reduced systemic exposure. It offers a versatile strategy not only for non-small cell lung cancer but also for the pulmonary delivery of other cytotoxic and biologic agents, positioning it as a promising next-generation platform in precision oncology. Future studies should include human NSCLC cell lines and primary models to better predict clinical efficacy and safety, addressing the limitation of the current rat-derived SLC cell model. Declarations Author Contribution Author Contributions: Conceptualization, Rawan Bafail; Data curation, Obaid Afzal and Mahmoud Omar; Formal analysis, Randa Zaki, Omiya Hasan and Waad A. Samman; Investigation, Randa Zaki, Obaid Afzal, Rawan Bafail and Mahmoud Omar; Methodology, Alaa Ayman, Eman Samy, Obaid Afzal, Waad A. 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Cite Share Download PDF Status: Published Journal Publication published 27 Feb, 2026 Read the published version in Journal of Pharmaceutical Innovation → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":113291,"visible":true,"origin":"","legend":"\u003cp\u003eTEM micrographs show spherical, monodisperse micelles with smooth surfaces and minimal aggregation, with diameters (~100-160 nm) consistent with DLS measurements (145.4 ± 6.0 nm)\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7640535/v1/c0b178291f78170bdc3b98bb.jpg"},{"id":92981724,"identity":"6ff18e18-92be-4015-8949-26a545b7a130","added_by":"auto","created_at":"2025-10-07 19:50:27","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":60594,"visible":true,"origin":"","legend":"\u003cp\u003eSolid-state characterization of spray-dried PLGA-PEG micelle formulations.\u003cbr\u003e\n(A) Differential scanning calorimetry (DSC): Pure doxorubicin displayed a distinct melting endotherm around 210 °C, absent in PLGA-PEG dual-coated micelles, indicating successful amorphization.\u003cbr\u003e\n(B) Fourier-transform infrared spectroscopy (FTIR): PLGA-PEG dual-coated micelles with lactose/trehalose excipients exhibited spectra comparable to pure components, with no evidence of new covalent bonds, suggesting physical stabilization without chemical interaction. (C) X-ray powder diffraction (XRPD): Pure doxorubicin exhibited sharp crystalline peaks, while PLGA-PEG dual-coated micelles showed a halo pattern, confirming amorphous encapsulation of the drug.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7640535/v1/f2d749d4e7508fd1032f19be.jpg"},{"id":92981617,"identity":"834aba35-be6f-4309-bb98-4c80cbacee99","added_by":"auto","created_at":"2025-10-07 19:42:27","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":98379,"visible":true,"origin":"","legend":"\u003cp\u003eAerosol performance of spray-dried PLGA-PEG micelle formulations evaluated using the Next Generation Impactor (NGI). Optimized formulations containing lactose or trehalose exhibited a MMAD of 2.86 ± 0.15 µm, GSD of 1.78 ± 0.06, and FPF of 62.4 ± 3.1%, significantly outperforming excipient-free formulations.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7640535/v1/c085f93ba6f00f8f2a410459.jpg"},{"id":92981625,"identity":"e64e358f-8644-4b0b-a548-5eb2f211cb55","added_by":"auto","created_at":"2025-10-07 19:42:27","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":28889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro cumulative release profile of doxorubicin from PLGA-PEG dual-coated micelles.\u003c/em\u003e The release exhibited an initial burst phase within the first 2 h, followed by a sustained release phase up to 48 h, reaching ~75% cumulative release. Error bars represent mean ± SD (n = 3). Kinetic modeling indicated that release was best fitted to the Higuchi model (R² = 0.998), consistent with a diffusion-dominated mechanism modulated by polymer erosion.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7640535/v1/5a171e3f50b87a4ce6428299.jpg"},{"id":92981620,"identity":"b5d2771a-6d74-44cf-a372-4b7e1c259eeb","added_by":"auto","created_at":"2025-10-07 19:42:27","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":132480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro cytotoxicity and apoptosis induction of doxorubicin-loaded PLGA-PEG dual-coated micelles in \u003c/em\u003eSLC \u003cem\u003ecells. (A) MTT assay showing dose-response curves and reduced IC₅₀ of micellar formulation compared to free doxorubicin. (B) Quantitative apoptosis analysis demonstrating increased early and late apoptotic cell fractions with micelle treatment. (C) Schematic illustration of mechanistic pathways: enhanced cellular uptake, reduced efflux, sustained nuclear delivery, and apoptosis signaling through ROS and caspase activation.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7640535/v1/0a960fb8259c0d2eb13fc8dd.jpg"},{"id":92981624,"identity":"9a98293f-3377-4cae-b15d-c2182625f9db","added_by":"auto","created_at":"2025-10-07 19:42:27","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":90297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmacokinetic profiles of doxorubicin following pulmonary administration.\u003c/strong\u003e Plasma concentration-time curves of free doxorubicin (DOX) and doxorubicin-loaded micelles (DOX-micelles) over 24 h after intratracheal instillation in rats. Micellar delivery resulted in a markedly reduced peak plasma concentration (C_max: 1.1 vs 2.8 µg/mL), lower systemic exposure (AUC₀-₂₄h: 14.3 vs 22.5 µg·h/mL), and prolonged mean residence time (MRT: 14.7 vs 6.1 h) compared with free DOX, indicating sustained systemic release with attenuated acute peaks.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7640535/v1/49190238287f0f8781bcc351.jpg"},{"id":92981628,"identity":"712af388-c3ab-403d-ab2f-4937efbf87c6","added_by":"auto","created_at":"2025-10-07 19:42:27","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":83821,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eUltrasound images showing lung tumor burden in different treatment groups. Tumors are highlighted with red-dotted circles;\u003c/em\u003e A\u003cstrong\u003e;\u003c/strong\u003e Untreated control group (large tumor mass visible), B\u003cstrong\u003e;\u003c/strong\u003e Free DOX treatment group (partial reduction in tumor volume), C\u003cstrong\u003e;\u003c/strong\u003e DOX-micelles treatment group (marked reduction in tumor size).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7640535/v1/593693bd20aafbbf021c2dfa.jpg"},{"id":92981639,"identity":"48a29a33-3751-43ad-844d-f2edd72769e4","added_by":"auto","created_at":"2025-10-07 19:42:28","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":66371,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLung deposition of doxorubicin following pulmonary administration.\u003c/strong\u003e\u003cbr\u003e\nPercentage of drug retained in lung tissue 24 h post-instillation for free doxorubicin (12.4 ± 3.7%) versus doxorubicin-loaded micelles (46.8 ± 5.2%). Micellar delivery achieved approximately four-fold higher pulmonary retention, confirming efficient localized deposition and reduced clearance. Data represent mean ± SD (n = 6).\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7640535/v1/e7a3d52a0f94e7e7fc0adb62.jpg"},{"id":92981630,"identity":"40e6b386-e0a0-4bad-9fd1-ef6a10a5e1a0","added_by":"auto","created_at":"2025-10-07 19:42:27","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":167196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntitumor efficacy of pulmonary doxorubicin micelles in an orthotopic lung tumor model.\u003c/strong\u003e Tumor growth curves over the treatment period for control, free doxorubicin, and doxorubicin-loaded micelles. Micelle-treated animals exhibited markedly suppressed tumor progression compared with both free drug and control groups. Inset: final tumor volume reduction at study endpoint, showing 68.5% reduction versus control and 43.2% reduction versus free doxorubicin. Data represent mean ± SD (n = 6).\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7640535/v1/f8bb4273a5ec1c9ca3922c6d.jpg"},{"id":92981651,"identity":"07f14419-c47f-4555-844e-2502e1bcffea","added_by":"auto","created_at":"2025-10-07 19:42:28","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":105830,"visible":true,"origin":"","legend":"\u003cp\u003eCardiotoxicity assessment of doxorubicin formulations, (A) Serum CK-MB and LDH levels in rats following treatment with control, free doxorubicin (DOX), or pulmonary DOX-micelles. Free DOX induced a significant elevation of both markers, whereas DOX-micelles reduced CK-MB and LDH by ~55% and ~48%, respectively, compared with free DOX, indicating attenuated cardiac injury. (B) Representative H\u0026amp;E-stained myocardial sections: control animals exhibited normal architecture, free DOX induced pronounced cardiomyocyte degeneration and fiber disruption, while DOX-micelles showed minimal pathological alterations.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7640535/v1/9129782fb04be00d250a1604.jpg"},{"id":103765740,"identity":"4a183014-90b2-418c-995c-14ee19a66423","added_by":"auto","created_at":"2026-03-02 16:08:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2826307,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7640535/v1/07ebf4ed-acc6-428a-9733-7eba32616746.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Engineered Spray-Dried PLGA-PEG Dual-Coated Micelles with Lactose/Trehalose Matrices for Pulmonary Doxorubicin Delivery: Achieving Sustained Release and Improved Aerosol Performance in NSCLC Therapy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eLung cancer is the leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 80\u0026ndash;85% of cases and presenting a critical need for more effective and safer therapies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Doxorubicin (DOX) remains a clinically effective chemotherapeutic agent; however, its intravenous administration is severely limited by nonspecific biodistribution, inadequate tumor selectivity, and dose-dependent toxicities, most notably life-threatening cardiotoxicity and myelosuppression [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eInhalable nanocarrier systems represent a promising strategy to overcome these limitations by leveraging the lungs as both the primary disease site and a directly accessible portal for localized therapy. This approach aims to maximize intratumoral drug deposition while minimizing systemic exposure and associated toxicity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among inhalation modalities, dry powder inhalers (DPIs) offer a particularly attractive option for delivering chemotherapeutics, enabling advanced particle engineering to optimize aerodynamic diameter, dispersibility, and deep-lung deposition [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite this potential, current inhalable nanocarrier platforms\u0026mdash;including liposomes and conventional polymeric micelles\u0026mdash;face significant translational barriers. These systems are often plagued by (1) poor aerosolization stability and powder aggregation, leading to low fine-particle fractions (FPF); (2) rapid clearance from the lung environment via mucociliary clearance and alveolar macrophage uptake; and (3) an inability to prevent systemic drug leakage, which undermines the goal of reducing off-target toxicity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Consequently, there is a pressing need for rationally engineered inhalable formulations that integrate enhanced aerodynamic performance with sustained pulmonary release and reduced systemic leakage.\u003c/p\u003e\u003cp\u003ePoly (lactic-co-glycolic acid) (PLGA) and poly (ethylene glycol) (PEG) are well-established biomaterials that offer complementary advantages for drug delivery. PLGA provides biodegradability and facilitates sustained release through diffusion-erosion mechanisms, while PEG confers \"stealth\" properties by reducing protein adsorption and macrophage uptake [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The integration of these polymers into a PLGA-PEG architecture can enhance hydrophilicity, biocompatibility, and controlled release behavior. Furthermore, spray drying presents a scalable method to transform nano-dispersions into respirable dry powders. The inclusion of glass-forming stabilizers like lactose and trehalose is critical to protect nanostructures during atomization and drying, enhance powder flowability, and improve aerosolization efficiency [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, the application of a PLGA-PEG dual-coated micellar system, optimized within a lactose/trehalose matrix for pulmonary DOX delivery, remains largely unexplored.\u003c/p\u003e\u003cp\u003eWe hypothesized that a spray-dried DPI comprising PLGA-PEG dual-coated micelles stabilized within a lactose/trehalose glassy matrix would overcome these limitations, providing superior aerosol performance, sustained pulmonary release, and a markedly improved therapeutic index for DOX in NSCLC. Accordingly, this study aimed to develop, characterize, and evaluate this novel micellar DPI platform through comprehensive assessment of its physicochemical properties, aerosol performance, in vitro and in vivo efficacy, and systemic safety profile.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials\u003c/h2\u003e\u003cp\u003eDoxorubicin hydrochloride was obtained from Sigma-Aldrich (St. Louis, MO, USA). The block copolymer poly(ethylene glycol)-poly(D,L-lactide-co-glycolide) (PLGA-PEG; PEG average Mn 5000 g\u0026middot;mol⁻\u0026sup1;, PLGA Mn 5000 g\u0026middot;mol⁻\u0026sup1;, lactide:glycolide feed ratio 50:50) was purchased from Sigma-Aldrich (Darmstadt, Germany). Lactose monohydrate (inhalation grade) and trehalose dihydrate were supplied by DFE Pharma (Goch, Germany) and Hayashibara Co., Ltd. (Okayama, Japan), respectively. Acetonitrile, methanol, and ethanol (HPLC grade) were obtained from Fisher Scientific (Waltham, MA, USA), while dichloromethane and other analytical-grade reagents were procured from Merck (Darmstadt, Germany). Deionized water was prepared using a Milli-Q purification system (Millipore, Billerica, MA, USA). All materials were used as received, without further modification or purification.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Preparation of PLGA-PEG dual-coated micelles\u003c/h2\u003e\u003cp\u003eDoxorubicin-loaded PLGA-PEG dual-coated micelles were prepared using the nanoprecipitation method for fabricating polymeric nanocarriers with a narrow size distribution and high drug entrapment efficiency [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Briefly, predetermined amounts of PLGA and PEG were dissolved in acetone containing doxorubicin (previously neutralized with triethylamine to enhance lipophilicity) and added dropwise to an aqueous phase under continuous magnetic stirring. The resulting colloidal suspension was maintained under agitation to allow complete solvent evaporation, thereby yielding self-assembled micelles. Unencapsulated drug was removed by centrifugation and dialysis against deionized water.\u003c/p\u003e\u003cp\u003eThe drug-to-polymer ratio was optimized by varying the weight ratio of doxorubicin to PLGA-PEG (1:5, 1:10, and 1:20, w/w). The formulations were evaluated for particle size, polydispersity index (PDI), zeta potential, and encapsulation efficiency, with the optimal ratio selected based on a balance between high drug loading and desirable physicochemical stability [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The optimized micellar suspension was subsequently subjected to spray-drying with lactose and trehalose excipients for conversion into a respirable dry powder suitable for pulmonary administration.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Spray-drying with excipients\u003c/h2\u003e\u003cp\u003eThe optimized PLGA-PEG micellar suspension was converted into a dry powder inhalation (DPI) formulation using a laboratory-scale spray dryer (B\u0026uuml;chi B-290, B\u0026uuml;chi Labortechnik AG, Flawil, Switzerland). Prior to spray-drying, lactose monohydrate and trehalose dihydrate were dissolved in the micellar suspension at a 1:1 ratio (w/w, total 10% w/v) and used as stabilizing excipients to protect nanostructures during atomization and drying.\u003c/p\u003e\u003cp\u003eSpray-drying was performed under the following conditions: inlet temperature 120\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, feed rate 5 mL/min, aspirator setting 90%, and atomizing airflow 600 L/h. The outlet temperature was maintained at 65\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C. The resulting spray-dried formulations were collected using a high-performance cyclone separator, sealed in airtight amber glass vials, and stored at 4\u0026deg;C until further use. The incorporation of lactose and trehalose was intended to enhance powder dispersibility, improve aerosolization performance, and prevent aggregation during long-term storage, as previously reported in inhalable nanocarrier formulations [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Physicochemical characterization\u003c/h2\u003e\u003cp\u003eThe hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the micelles were measured using dynamic light scattering (DLS; Zetasizer Nano ZS, Malvern Instruments, UK) at 25\u0026deg;C. All measurements were performed in triplicate on freshly prepared dispersions [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eDrug loading capacity and encapsulation efficiency were determined using high-performance liquid chromatography (HPLC; Agilent 1260 Infinity, USA) equipped with a C18 column (4.6 \u0026times; 150 mm, 5 \u0026micro;m). Samples were dissolved in acetonitrile-water (70:30 v/v), filtered through 0.22 \u0026micro;m membranes, and quantified at 480 nm. For comparison, UV-visible spectrophotometry (UV-2600, Shimadzu, Japan) was employed in preliminary analyses [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The solid-state properties were characterized by Fourier-transform infrared spectroscopy (FTIR; Nicolet iS50, Thermo Fisher Scientific, USA), X-ray powder diffraction (XRPD; D8 Advance, Bruker, Germany), and differential scanning calorimetry (DSC; DSC 214 Polyma, Netzsch, Germany) to evaluate potential interactions between the drug, polymer, and excipients, and to assess the crystalline or amorphous state of the formulations [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Aerosol performance\u003c/h2\u003e\u003cp\u003eThe aerosolization properties of the spray-dried formulations were evaluated using a Next Generation Impactor (NGI; Copley Scientific, Nottingham, UK) equipped with a standard dry powder inhaler device. Approximately 10 mg of the formulation was loaded into size 3 hydroxypropyl methylcellulose capsules and dispersed at a flow rate of 60 L/min for 4 s, corresponding to a 4 L inhalation volume, in accordance with the United States Pharmacopeia (USP) guidelines.\u003c/p\u003e\u003cp\u003eThe mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated from the drug deposition profile across the NGI stages. Fine particle fraction (FPF) was determined as the percentage of the emitted dose with an aerodynamic diameter below 5 \u0026micro;m. Drug content on each stage was quantified by washing the collection plates with acetonitrile-water (70:30 v/v) and analyzing by HPLC at 480 nm. All measurements were performed in triplicate, and results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The assessment of MMAD, GSD, and FPF provides critical insights into the respirable dose and deposition efficiency of dry powder formulations, parameters that strongly influence clinical efficacy of inhalable chemotherapeutics [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 In vitro release and stability\u003c/h2\u003e\u003cp\u003eThe release profile of doxorubicin from the spray-dried PLGA-PEG dual-coated micelles was evaluated in simulated lung fluid (SLF, pH 7.4), prepared according to the composition described by May et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Briefly, 10 mg of spray-dried powder was dispersed in 10 mL of SLF and incubated at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C under gentle shaking (100 rpm). At predetermined intervals (0.5, 1, 2, 4, 8, 12, 24, and 48 h), samples were withdrawn and centrifuged at 15,000 rpm for 15 min. The supernatant was analyzed by HPLC at 480 nm to determine drug release, while an equivalent volume of fresh medium was replenished to maintain sink conditions. Cumulative release was expressed as the percentage of drug released relative to the total encapsulated amount.\u003c/p\u003e\u003cp\u003e Stability studies were conducted following ICH Q1A(R2) guidelines. Optimized formulations were stored in sealed amber glass vials at 4\u0026deg;C (refrigerated), 25\u0026deg;C/60% RH (ambient), and 40\u0026deg;C/75% RH (accelerated) for three months. At monthly intervals, samples were evaluated for changes in particle size, PDI, zeta potential, drug content, and aerosol performance. Stability was expressed as percentage retention compared with initial values. These assays were designed to assess both the controlled-release behavior of the micelles in the pulmonary environment and the robustness of the formulation during long-term storage [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Cell culture and cytotoxicity studies\u003c/h2\u003e\u003cp\u003eThe rat lung carcinoma SLC cell line (RIKEN BioResource Research Center, Tsukuba, Japan; Cell No. RCB2862) was cultured in RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin, according to the supplier\u0026rsquo;s recommendations. Cells were maintained at 37\u0026deg;C in a humidified atmosphere with 5% CO₂ and harvested at ~\u0026thinsp;80% confluence using Accutase\u0026trade; (Nacalai Tesque, Kyoto, Japan).\u003c/p\u003e\u003cp\u003eFor cytotoxicity testing, cells were seeded into 96-well plates (1 \u0026times; 10⁴ cells/well) and treated with free doxorubicin, blank PLGA-PEG dual-coated micelles, or DOX-micelles (0.1\u0026ndash;20 \u0026micro;g/mL) for 24 and 48 h. Cell viability was assessed by the MTT assay, while apoptosis was quantified using Annexin V-FITC/PI staining and flow cytometry, as described previously [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These assays provided complementary information regarding the cytotoxic potential and apoptotic mechanisms of micellar formulations compared with free doxorubicin, thereby supporting the therapeutic potential of DOX-loaded PLGA-PEG dual-coated micelles. [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 In vivo pharmacokinetics and lung distribution\u003c/h2\u003e\u003cp\u003e All animal experiments were performed in accordance with institutional ethical guidelines and approved by the Faculty of Pharmacy, Deraya University Animal Care and Use Committee (ethical approval no. 7/2024, issued February 14, 2024). Male Wistar rats (200\u0026ndash;250 g) were obtained from the Animal House, Faculty of Pharmacy, Deraya University (Minya, Egypt) and acclimatized for one week under controlled environmental conditions (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 55\u0026thinsp;\u0026plusmn;\u0026thinsp;10% relative humidity, 12 h light/dark cycle) with free access to food and water.\u0026rdquo;\u003c/p\u003e\u003cp\u003ePharmacokinetic and biodistribution studies were conducted in healthy male Wistar rats after intratracheal administration of either free doxorubicin solution or DOX-micelles (2 mg/kg equivalent DOX). Animals were anesthetized with isoflurane (2\u0026ndash;3% in oxygen) and positioned supine. Formulations were delivered intratracheally using a PennCentury\u0026trade; MicroSprayer\u0026reg; Aerosolizer (Penn-Century Inc., Philadelphia, PA, USA).\u003c/p\u003e\u003cp\u003eBlood samples were collected via retro-orbital puncture into heparinized tubes at predetermined time points (0.25, 0.5, 1, 2, 4, 8, 12, and 24 h) and centrifuged at 4000 rpm for 10 min to separate plasma. For lung distribution studies, animals were sacrificed at 1, 4, 8, and 24 h post-dose. Lungs were excised, rinsed with saline, blotted dry, weighed, and homogenized in ice-cold PBS.\u003c/p\u003e\u003cp\u003ePlasma, lung homogenates, and tissue samples from the heart, kidney, liver, and spleen were collected for doxorubicin quantification. All samples were subjected to protein precipitation with acetonitrile, vortex-mixed, and centrifuged at 12,000 rpm for 15 min. The supernatants were then transferred to HPLC vials for analysis. Doxorubicin concentrations were determined using a validated HPLC method (Agilent 1260 Infinity system, Agilent Technologies, Santa Clara, CA, USA) equipped with a fluorescence detector (λ_ex\u0026thinsp;=\u0026thinsp;480 nm, λ_em\u0026thinsp;=\u0026thinsp;590 nm). Chromatographic separation was performed on a C18 column (4.6 \u0026times; 150 mm, 5 \u0026micro;m) with a mobile phase of acetonitrile:water (30:70, v/v) containing 0.1% trifluoroacetic acid at a flow rate of 1.0 mL/min. The method was validated according to bioanalytical guidelines, showing linear calibration curves over the range of 10\u0026thinsp;\u0026minus;\u0026thinsp;5,000 ng/mL (r\u0026sup2; \u0026gt;0.99), with limits of detection and quantification of 5 ng/mL and 10 ng/mL, respectively. Intra- and inter-day precision values were within 10%, and recovery exceeded 85% across all matrices (plasma, lung, heart, kidney, liver, and spleen), confirming the suitability of the assay for pharmacokinetic and biodistribution analysis.\u003c/p\u003e\u003cp\u003ePharmacokinetic parameters, including maximum plasma concentration (C\u003csub\u003emax\u003c/sub\u003e), time to reach C\u003csub\u003emax\u003c/sub\u003e (T\u003csub\u003emax\u003c/sub\u003e), area under the plasma concentration-time curve from 0\u0026ndash;24 h (AUC₀-₂₄h), mean residence time (MRT) and elimination half-life (t\u0026frac12;), were calculated using non-compartmental analysis (Phoenix WinNonlin 10, Certara, USA). Lung tissue concentrations were expressed as ng of drug per g of wet tissue. These studies enabled a direct comparison of systemic exposure and pulmonary retention between free doxorubicin and micellar formulations, highlighting the advantages of localized, sustained drug delivery in the lung environment [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 In vivo antitumor efficacy in orthotopic tumor-bearing rats\u003c/h2\u003e\u003cp\u003eThe antitumor efficacy of the experimental treatment was evaluated in an orthotopic rat model of SLC. Male Wistar rats (200\u0026ndash;250 g) were obtained from the Animal House, Faculty of Pharmacy, Deraya University (Minya, Egypt). Animals were acclimatized for one week under controlled environmental conditions (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 55\u0026thinsp;\u0026plusmn;\u0026thinsp;10% relative humidity, 12 h light/dark cycle) with free access to food and water [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. To avoid overlap, pharmacokinetic evaluations were conducted exclusively in healthy rats, whereas efficacy studies were performed only in tumor-bearing rats. Tumor-bearing rats were generated via the intratracheal inoculation of 1 \u0026times; 10⁶ luciferase-expressing SLC cells in 50 \u0026micro;L of PBS under anesthesia. Tumor establishment and growth were confirmed and monitored weekly via non-invasive ultrasound imaging and bioluminescence imaging (BLI) prior to the initiation of the study. Rats with confirmed tumors were then randomly allocated into treatment and control groups (n\u0026thinsp;=\u0026thinsp;6 per group: (i) untreated control, (ii) free doxorubicin (DOX), and (iii) DOX-loaded PLGA-PEG micelles. Tumor progression was monitored at defined intervals, and at the experimental endpoint animals were humanely sacrificed for histopathological evaluation.\u003c/p\u003e\u003cp\u003eTumor volume was measured by ultrasound imaging and calculated using the following formula:\u003c/p\u003e\u003cp\u003eV\u0026thinsp;=\u0026thinsp;0.5 * length * (width)\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003cp\u003ewhere length is the longest diameter and width is the perpendicular measurement. Tumor volume was recorded throughout the study. At endpoint, tumors were excised, weighed, and processed for histological evaluation (H\u0026amp;E staining).\u003c/p\u003e\u003cp\u003eAntitumor efficacy was expressed as tumor growth inhibition (TGI%):\u003c/p\u003e\u003cp\u003eTGI% \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:=\\:\\frac{\\left(\\varvec{\\Delta\\:}\\mathbf{V}\\mathbf{c}\\mathbf{o}\\mathbf{n}\\mathbf{t}\\mathbf{r}\\mathbf{o}\\mathbf{l}\\mathbf{}\\right)-\\:\\left(\\varvec{\\Delta\\:}\\mathbf{V}\\mathbf{t}\\mathbf{r}\\mathbf{e}\\mathbf{a}\\mathbf{t}\\mathbf{e}\\mathbf{d}\\mathbf{}\\right)\\:}{\\left(\\varvec{\\Delta\\:}\\mathbf{V}\\mathbf{c}\\mathbf{o}\\mathbf{n}\\mathbf{t}\\mathbf{r}\\mathbf{o}\\mathbf{l}\\mathbf{}\\right)}\\:\\)\u003c/span\u003e\u003c/span\u003e\u0026times;100\u003c/p\u003e\u003cp\u003ewhere ΔV is the change in tumor volume from baseline to endpoint.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Cardiotoxicity and systemic toxicity assessment\u003c/h2\u003e\u003cp\u003eAt the end of the efficacy study, blood samples were collected from the retro-orbital plexus under light anesthesia. Serum was separated and analyzed for creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) using commercial ELISA kits (Sigma-Aldrich, USA), following manufacturer\u0026rsquo;s instructions [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe hearts were excised, rinsed with saline, and fixed in 10% neutral-buffered formalin for 24 h. Tissues were subsequently dehydrated, embedded in paraffin, and sectioned at a thickness of 5 \u0026micro;m. Sections were stained with hematoxylin-eosin (H\u0026amp;E) and examined under a light microscope for structural alterations, including myocyte degeneration, cytoplasmic vacuolization, interstitial edema, and necrosis, to evaluate doxorubicin-induced cardiotoxicity [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e\u003cp\u003eAll experiments were performed in triplicate unless otherwise specified, and results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical comparisons between groups were conducted using one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test for multiple comparisons. For two-group comparisons, an unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was applied. A \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Data analysis and graph generation were performed using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e\u003cp\u003eThis approach is consistent with previous studies evaluating spray-dried nanocarriers and pulmonary formulations [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], ensuring robust interpretation of physicochemical and biological outcomes.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Micelle Formation and Characterization\u003c/h2\u003e\u003cp\u003eDoxorubicin-loaded PLGA-PEG dual-coated micelles were fabricated by nanoprecipitation, yielding self-assembled micelles that were stable and monodisperse. DLS analysis showed an average hydrodynamic diameter of 145.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0 nm. The polydispersity index was 0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, confirming a narrow size distribution. The predominant population ranged from 100 to 160 nm, suitable for pulmonary delivery (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Transmission electron microscopy (TEM) further revealed well-defined spherical to near-spherical nanostructures with smooth contours, uniform dispersion, and negligible aggregation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The particle dimensions observed by TEM were consistent with DLS results, confirming the uniform self-assembly and structural stability of the micelles. Incorporation of lactose or trehalose as stabilizing excipients preserved discrete nanostructures, whereas excipient-free formulations showed partial fusion and irregular morphology. Zeta potential measurements were \u0026minus;\u0026thinsp;18.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 mV, suggesting moderate colloidal stability and sufficient repulsive surface interactions to maintain dispersion in aqueous media over extended periods [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOptimization of the drug-to-polymer ratio demonstrated that a 1:10 (w/w) doxorubicin:PLGA-PEG ratio provided the highest encapsulation efficiency \u003cb\u003e(\u003c/b\u003e91.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5%\u003cb\u003e)\u003c/b\u003e and drug loading \u003cb\u003e(\u003c/b\u003e8.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3%). Lower polymer ratios resulted in reduced encapsulation, whereas higher polymer ratios did not significantly improve drug loading but increased particle size, highlighting the importance of balancing polymer content for optimal micellar performance [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOverall, these results confirm that PLGA-PEG dual-coated micelles prepared under optimized conditions form well-defined, stable nanoparticles capable of efficiently encapsulating doxorubicin, providing a promising platform for inhalable anticancer therapy (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eOptimization of drug-to-polymer ratio for PLGA-PEG\u003c/b\u003e dual-coated micelles \u003cb\u003eprepared by nanoprecipitation.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDrug: Polymer Ratio\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eParticle Size (nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZeta Potential (mV)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEncapsulation Efficiency (%EE)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDrug Loading (%DL)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1:5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e165\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e-15.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e72.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1:10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e145.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6 nm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e-18.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e91.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e8.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1:20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e132\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e-22.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e88.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cem\u003eNote: Higher polymer content (1:10 to 1:20) yielded smaller particles and lower PDI but did not further improve EE and resulted in decreased DL\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of PEGylation on physicochemical properties of PLGA micelles.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNon-PEGylated PLGA Micelles\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePLGA-PEG dual-coated micelles\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParticle Size (nm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e155\u0026thinsp;\u0026plusmn;\u0026thinsp;7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e145.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eColloidal Stability (48 h, % aggregation)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMucoadhesion Resistance (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e32%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e78%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDrug Release (t\u0026frac12;, hours)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.5 h\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14.2 h\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003ePEGylation enhances stability, reduces mucoadhesion, and prolongs release kinetics.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Spray-Dried Powder Properties and Effect of Lactose/Trehalose\u003c/h2\u003e\u003cp\u003eSolid-state characterization confirmed the successful amorphization of doxorubicin within the micellar matrix. X-ray powder diffraction (XRPD) patterns showed the absence of crystalline peaks corresponding to native doxorubicin, indicating complete encapsulation in an amorphous state. Differential scanning calorimetry (DSC) analysis supported this finding, showing no distinct melting endotherm for doxorubicin, while Fourier-transform infrared (FTIR) spectra revealed no evidence of strong chemical interactions between drug, polymer, and excipients, suggesting physical stabilization rather than covalent modification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAerosol performance testing using a Next Generation Impactor demonstrated excellent respirable properties. The optimized formulations displayed a mass median aerodynamic diameter (MMAD) of 2.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 \u0026micro;m with a geometric standard deviation (GSD) of 1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06, values well within the optimal range for deep lung deposition. The fine particle fraction (FPF, \u0026lt;\u0026thinsp;5 \u0026micro;m) reached 62.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1%, significantly higher than excipient-free spray-dried formulations (41.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), confirming the role of lactose and trehalose in enhancing aerodynamic performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOverall, inclusion of hydrophilic sugars preserved micellar integrity during atomization and drying, reduced particle aggregation, and markedly improved aerosolization efficiency. These findings are consistent with previous studies highlighting the utility of lactose and trehalose as protective excipients in inhalable nanocarrier formulations [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e(A) Differential scanning calorimetry (DSC): Pure doxorubicin displayed a distinct melting endotherm around 210\u0026deg;C, absent in PLGA-PEG dual-coated micelles, indicating successful amorphization.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e(B) Fourier-transform infrared spectroscopy (FTIR): PLGA-PEG dual-coated micelles with lactose/trehalose excipients exhibited spectra comparable to pure components, with no evidence of new covalent bonds, suggesting physical stabilization without chemical interaction. (C) X-ray powder diffraction (XRPD): Pure doxorubicin exhibited sharp crystalline peaks, while PLGA-PEG dual-coated micelles showed a halo pattern, confirming amorphous encapsulation of the drug.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3 In Vitro Drug Release and Stability\u003c/h2\u003e\u003cp\u003eThe release profile of doxorubicin-loaded PLGA-PEG dual-coated micelles in simulated lung fluid (SLF, pH 7.4) exhibited a biphasic release profile, typical of micellar drug delivery systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Initially, a burst release of 21.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3% occurs within the first 2 hours, primarily due to the drug molecules that are surface-associated or loosely entrapped within the micellar structure. These surface-bound drug molecules are readily available for diffusion when exposed to the SLF, and the hydrophilic PEG shell facilitates the initial release by enhancing drug diffusion through its interaction with water. This rapid release phase was followed by a more sustained release, with the cumulative release reaching 76.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5% at 48 hours. The sustained release phase is largely driven by the hydrophobic PLGA core, which holds the drug within its matrix. As PLGA degrades via hydrolysis, the drug is gradually released over time, offering a prolonged therapeutic effect [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eRelease kinetics.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eKinetic modeling revealed that drug release was best described by the Higuchi model (\u003cem\u003ek_H\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.115, R\u0026sup2; = 0.998), confirming a diffusion-driven process (Table\u0026nbsp;3). The Korsmeyer-Peppas model (n\u0026thinsp;=\u0026thinsp;0.74, R\u0026sup2;= 0.976, \u003cem\u003eFₜ\u003c/em\u003e \u0026le; 0.6) indicated anomalous transport, where release is governed by a combination of Fickian diffusion and polymer relaxation/erosion. The first-order model (\u003cem\u003ek₁\u003c/em\u003e= 0.0922, R\u003csup\u003e\u0026sup2;\u003c/sup\u003e= 0.949) also showed a good fit, suggesting concentration-dependent release dynamics, while the zero-order model (\u003cem\u003ek₀\u003c/em\u003e= 0.0140, R\u0026sup2;= 0.817) provided the weakest correlation, confirming that drug liberation was not constant over time. Mechanistically, the hydrophilic PEG shell functioned as a diffusion barrier, whereas the gradual degradation of the PLGA core modulated sustained release. This dual mechanism aligns with prior reports of micellar and polymeric carriers, where drug liberation is mediated by both Fickian diffusion and polymer erosion [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable\u0026nbsp;(3). Kinetic modeling parameters of drug release\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eModel\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eParameter(s)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u0026sup2;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFit range\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHiguchi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ek\u003csub\u003eH\u003c/sub\u003e = 0.115\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.998\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003et\u0026thinsp;\u0026gt;\u0026thinsp;0 h\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZero-order\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ek0\u0026thinsp;=\u0026thinsp;0.0140\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.817\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003et\u0026thinsp;\u0026gt;\u0026thinsp;0 h\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFirst-order\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ek1\u0026thinsp;=\u0026thinsp;0.0922\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0. 949\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFt\u0026thinsp;\u0026lt;\u0026thinsp;0.995\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKorsmeyer-Peppas\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003en\u0026thinsp;=\u0026thinsp;0.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.976\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFt\u0026thinsp;\u0026le;\u0026thinsp;0.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eNote: Fit ranges reflect the theoretical applicability of each model: the Higuchi equation is valid during the diffusion-dominated phase (typically up to ~\u0026thinsp;60\u0026ndash;70% release); the zero-order model can be applied across all time points but rarely fits well; the first-order model is appropriate until near-complete release (Ft\u0026thinsp;\u0026lt;\u0026thinsp;0.995) to avoid asymptotic deviation; and the Korsmeyer-Peppas model is semi-empirical and only valid for the initial 60% of drug release [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ePLGA-PEG dual-coated micelles showed favorable stability profiles under both refrigerated and ambient storage conditions (Table\u0026nbsp;4). After three months at 4\u0026deg;C, formulations retained 96.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4% of their initial drug content, with negligible changes in particle size (145.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0 nm \u0026rarr; 149.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1 nm) and PDI (0.182 \u0026rarr; 0.190). At 25\u0026deg;C/60% RH, drug retention was slightly reduced (92.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0%), accompanied by modest particle growth (to 154.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6 nm) and a minor increase in PDI (0.182 \u0026rarr; 0.196); however, aerosolization properties, including MMAD (~\u0026thinsp;2.84 \u0026micro;m) and FPF (~\u0026thinsp;61.7%), remained unchanged. Under accelerated stress (40\u0026deg;C/75% RH), drug retention declined further to 85.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2%, with greater particle enlargement (to 162.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2 nm), elevated PDI (0.221), and early signs of aggregation, though aerodynamic performance was largely preserved (MMAD 2.82 \u0026micro;m; FPF 60.9%). In contrast, excipient-free formulations were highly unstable, showing marked aggregation (\u0026gt;\u0026thinsp;183 nm), substantial drug loss (72.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1% retention), and a decline in respirable fraction (FPF 55.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2%). These findings highlight the protective role of trehalose and lactose as sugar-glass stabilizers, consistent with previous reports on the cryo- and lyoprotective effects of disaccharides in maintaining nanocarrier integrity during storage [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDisaccharides such as trehalose and lactose serve as \u003cem\u003ecryo- and lyoprotectants\u003c/em\u003e, stabilizing nanocarriers during spray-drying and storage by replacing hydrogen bonds with water molecules and forming a protective amorphous glassy matrix [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. This explains the superior retention of drug content and maintenance of micelle integrity in formulations containing sugars compared with excipient-free formulations.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable\u0026nbsp;(4). Stability of Doxorubicin-Loaded PLGA-PEG dual-coated micelles Under Different Storage Conditions (3 Months)\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStorage Condition\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDrug Retention (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eParticle Size (nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMMAD (\u0026micro;m)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFPF (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eObservations\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e4\u0026deg;C (Refrigerated)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e96.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e145.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6 nm\u003c/b\u003e \u0026rarr; 149.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.182 \u0026rarr; 0.190\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e62.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eExcellent stability; minimal changes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e25\u0026deg;C / 60% RH (Ambient)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e92.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e145.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6 nm\u003c/b\u003e \u0026rarr; 154.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.182 \u0026rarr; 0.196\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e61.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSlight decline in drug retention; aerosolization preserved\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e40\u0026deg;C / 75% RH (Accelerated)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e85.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e145.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6 nm\u003c/b\u003e \u0026rarr; 162.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.182 \u0026rarr; 0.221\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e60.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eReduced drug retention, increased PDI, early aggregation\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eExcipient-free formulations\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e72.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e145.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6 nm\u003c/b\u003e \u0026rarr; \u0026gt;183 nm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.182 \u0026rarr; 0.265\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e55.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMarked instability; aggregation and drug loss\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe combination of PEGylation and PLGA encapsulation contributed to both sustained drug release and enhanced stability. PEG provided steric stabilization against aggregation and reduced protein/surfactant adsorption onto micelle surfaces, thereby helping to maintain colloidal stability in simulated lung fluids [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The hydrophobic PLGA core reduced burst release and protected doxorubicin from hydrolytic/degradative pathways, producing more predictable, prolonged release profiles typical of PLGA matrix or core-shell systems [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Lactose and trehalose acted as stabilizing excipients during spray drying and storage by forming hydrogen bonds with the micellar surface and by vitrifying into an amorphous glass that prevents particle fusion or collapse [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Collectively, these mechanisms underpin the long-term physicochemical stability and predictable release kinetics needed for pulmonary dry-powder systems designed to deliver localized, sustained anticancer activity with reduced systemic exposure.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4 In Vitro Cytotoxicity and Apoptosis\u003c/h2\u003e\u003cp\u003eCytotoxicity of doxorubicin-loaded PLGA-PEG dual-coated micelles was assessed in SLC \u003cb\u003elung carcinoma cells\u003c/b\u003e using the MTT assay. As shown in Table\u0026nbsp;5, the micellar formulation produced a significantly lower IC₅₀ (1.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 \u0026micro;g/mL) compared with free doxorubicin (3.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 \u0026micro;g/mL, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), demonstrating enhanced inhibition of cell proliferation, (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Blank micelles showed negligible cytotoxicity, confirming that the antitumor effects were due to doxorubicin encapsulation rather than carrier toxicity. These results are consistent with earlier reports that PLGA-PEG dual-coated micelles increase drug potency through improved cellular uptake and reduced efflux [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile the SLC cell line offers a convenient and reproducible rat lung carcinoma model, it does not capture the full biological heterogeneity of human NSCLC. Consequently, cytotoxicity results obtained here should be interpreted with caution when considering clinical translation. Human-derived NSCLC cell lines (e.g., H460, H1975) often differ in resistance mechanisms and uptake pathways, while non-malignant bronchial epithelial cells (e.g., BEAS-2B) provide essential insights into off-target safety. Broader testing across malignant and normal human cell models will therefore be necessary to validate the selectivity, safety, and translational relevance of the PLGA-PEG micellar formulation.\u003c/p\u003e\u003cp\u003eFlow cytometry-based apoptosis analysis further reinforced the superior efficacy of micellar doxorubicin. Treatment with doxorubicin-loaded micelles induced a significantly higher proportion of early and late apoptotic cells (48.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8%) compared to free doxorubicin (29.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This enhanced apoptotic response is consistent with prior reports demonstrating that nanoparticle-mediated drug delivery can potentiate mitochondrial membrane depolarization, caspase activation, and reactive oxygen species (ROS) generation, all of which play key roles in amplifying apoptosis[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Mechanistically, the sustained intracellular accumulation of micellar doxorubicin facilitates efficient nuclear delivery, which promotes increased DNA intercalation and topoisomerase II inhibition [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The higher levels of apoptosis observed in micelle-treated cells are likely due to a combination of enhanced drug delivery to the nucleus and the increased generation of ROS, which acts as a secondary trigger for mitochondrial dysfunction and caspase-mediated cell death.\u003c/p\u003e\u003cp\u003eAnother key aspect of the micellar formulation is its potential to overcome multidrug resistance (MDR), a major challenge in cancer chemotherapy. By facilitating endocytosis-mediated uptake and bypassing P-glycoprotein (P-gp) efflux pumps, micelles improve the intracellular retention and therapeutic bioavailability of doxorubicin in resistant tumor cells [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. This is particularly significant for tumors that exhibit P-gp overexpression, which is commonly associated with resistance to doxorubicin and other chemotherapy agents. The micellar system's ability to overcome these resistance mechanisms could substantially improve the efficacy of doxorubicin, particularly in patients with tumors that are refractory to conventional treatments.\u003c/p\u003e\u003cp\u003ePLGA-PEG micelles markedly enhance doxorubicin's anticancer efficacy. However, establishing a complete safety and efficacy profile will require comprehensive evaluation across different cancer types and normal cell lines. The safety of blank micelles, which showed negligible cytotoxicity (\u0026lt;\u0026thinsp;5% apoptosis), highlights the biocompatibility of the carrier system and confirms that therapeutic efficacy arises exclusively from the encapsulated doxorubicin [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCollectively, these findings suggest that PLGA-PEG dual-coated micelles significantly enhance the anticancer potency of doxorubicin by improving drug delivery, promoting apoptosis, and bypassing multidrug resistance, all while maintaining excellent carrier safety. However, to fully translate these promising in vitro results into clinical applications, further studies evaluating the formulation in a broader range of cell types, as well as in vivo models, are necessary to confirm its therapeutic potential and to evaluate the long-term safety of micellar-based therapies in clinical settings.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable\u0026nbsp;(5). Comparative Cytotoxicity and Apoptosis Induction in SLC cell line\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabc\" border=\"1\"\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIC₅₀ (\u0026micro;g/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e% Viability at 5 \u0026micro;g/mL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEarly\u0026thinsp;+\u0026thinsp;Late Apoptosis (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNotes\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFree Doxorubicin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e54.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e29.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eRapid uptake, faster efflux\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDox-Loaded PLGA-PEG dual-coated micelles\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e48.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSustained uptake, enhanced apoptosis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBlank Micelles\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e~\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNegligible cytotoxicity\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSeveral studies have consistently demonstrated that nanoformulation of doxorubicin, particularly with PLGA-PEG dual-coated micelles, enhances cytotoxicity and apoptosis induction in lung carcinoma and other tumor models compared to free doxorubicin.\u003c/p\u003e\u003cp\u003ePLGA-PEG dual-coated micelles significantly enhanced the cytotoxic potency of doxorubicin in SLC cells compared with the free drug (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This effect is consistent with previous findings that PEG-b-PLGA nanopolymersomes improve intracellular accumulation and retention of doxorubicin in tumor cells, thereby increasing therapeutic efficacy [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The nanoscale size (~\u0026thinsp;100 nm) and PEGylation of the micelles facilitate endocytosis while limiting premature drug efflux. These features collectively improve nuclear delivery of doxorubicin, contributing to greater drug-induced cytotoxicity. Furthermore, blank micelles were shown to be biocompatible, exhibiting negligible cytotoxicity, which confirms that the observed effects are exclusively due to the encapsulated drug.\u003c/p\u003e\u003cp\u003eFlow cytometry analysis further confirms the superior apoptotic activity of doxorubicin-loaded micelles, as they significantly increase both early and late apoptosis (48.6% vs. 29.7% for free drug). These results are consistent with Jin et al. (2014), who demonstrated that polymeric micelle formulations enhance mitochondrial membrane depolarization and activate the caspase cascade, key steps in apoptosis induction [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The amplified apoptotic response is primarily attributed to sustained intracellular retention, efficient nuclear delivery, and inhibition of topoisomerase II, which represent the core mechanisms of doxorubicin-mediated cytotoxicity.\u003c/p\u003e\u003cp\u003eNanocarrier-mediated delivery of doxorubicin can effectively overcome multidrug resistance (MDR) by bypassing P-glycoprotein (P-gp)-mediated efflux, thereby prolonging intracellular drug retention and enhancing therapeutic efficacy [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. P-gp overexpression is a major contributor to reduced doxorubicin accumulation in tumor cells, and strategies that evade its activity have been shown to restore drug sensitivity and improve cytotoxic outcomes. In parallel, micellar formulations can induce intracellular reactive oxygen species (ROS) overproduction, which potentiates apoptotic signaling through mitochondrial membrane depolarization and caspase activation [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Together, these mechanistic advantages, enhanced intracellular retention via P-gp evasion and ROS-mediated amplification of apoptosis, likely underpin the marked increase in cytotoxicity and apoptotic response observed in SLC lung carcinoma cells following micelle-mediated doxorubicin delivery. Collectively, these mechanistic advantages explain the observed increase in cytotoxicity and apoptotic response in SLC lung carcinoma cells. Importantly, blank micelles remain non-toxic to SLC cells and normal fibroblasts, underscoring their biocompatibility and highlighting that the therapeutic efficacy arises from encapsulated doxorubicin rather than the carrier system itself. Collectively, these results indicate that PLGA-PEG dual-coated micelles enhance the anticancer activity of doxorubicin by improving intracellular delivery, inducing apoptosis, and overcoming multidrug resistance, while exhibiting minimal carrier-related toxicity. Nevertheless, the current in vitro evaluation was limited to a single rat-derived lung carcinoma cell line (SLC, RCB2862). Although SLC provides a useful syngeneic model for adenocarcinoma, it does not capture the full heterogeneity of human NSCLC. Validation in additional human lung carcinoma cell lines (e.g., H460, H1975) and in normal bronchial epithelial cells (e.g., BEAS-2B) will be required to establish the generalizability, selectivity, and translational relevance of these findings.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.5 In Vivo Pharmacokinetics, Lung Distribution, and Antitumor Efficacy\u003c/h2\u003e\u003cp\u003ePlasma pharmacokinetic evaluation following intratracheal administration of free DOX and DOX-micelles in rats is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Free DOX exhibited a sharp rise in plasma levels followed by a rapid decline, reaching a peak concentration (C\u003csub\u003emax\u003c/sub\u003e) of 2.8 \u0026micro;g/mL within the first hour. In contrast, DOX-micelles displayed a slower absorption profile with a markedly reduced C\u003csub\u003emax\u003c/sub\u003e (1.1 \u0026micro;g/mL) and a more gradual elimination phase. The calculated pharmacokinetic parameters demonstrated lower systemic exposure (AUC₀-₂₄h: 14.3 vs 22.5 \u0026micro;g\u0026middot;h/mL) but a significantly prolonged mean residence time (MRT: 14.7 vs 6.1 h) for DOX-micelles compared with free DOX. These findings confirm that micellar encapsulation attenuates acute plasma spikes and reduces total systemic exposure (AUC), which is advantageous for minimizing systemic toxicity, while simultaneously prolonging therapeutic coverage (MRT) (Table\u0026nbsp;6).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBiodistribution analysis confirmed efficient lung targeting, with DOX-micelles exhibiting significantly enhanced pulmonary deposition compared to free doxorubicin in an orthotopic tumor model. At 24 h post-administration, 46.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2% of the micellar dose was retained in pulmonary tissue, compared with only 12.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7% for free drug (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Table\u0026nbsp;7, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This prolonged retention, likely attributable to micelle-surfactant interactions and reduced mucociliary clearance; these results align with those reported by Hu et al. (2014) [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]), conferred a marked therapeutic advantage: DOX-micelles reduced tumor volume by 68.5% compared with untreated controls, whereas free DOX achieved only a 25.3% reduction (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Notably, micelle treatment yielded an additional 43.2% reduction in tumor burden relative to free DOX (Table\u0026nbsp;7). These findings are consistent with previous studies showing that self-assembled polymeric micelles improve the antitumor activity of doxorubicin while reducing systemic toxicity, owing to their ability to sustain drug accumulation within tumors and enhance penetration across the extracellular matrix [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. To ensure consistent interpretation of these outcomes, aligning the dosing regimen (every 3 days for 2 weeks) with the observed tumor growth inhibition further highlights the temporal relationship between drug exposure and therapeutic efficacy. Similar observations have been reported in lung cancer models, where composite micelle encapsulation of doxorubicin not only enhanced radiosensitivity but also provided prolonged retention and therapeutic benefit compared with the free drug [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBeyond tumor volume reductions, a deeper understanding of the pharmacodynamics of the micellar formulation is critical to elucidate its mechanism of action and clinical potential. The sustained intratumoral drug accumulation observed here suggests that the micelle-encapsulated doxorubicin remains localized within the tumor microenvironment for extended periods, thereby maintaining effective drug concentrations at the site of action. In contrast, free doxorubicin typically undergoes rapid distribution and clearance, limiting its duration of therapeutic effect. Prolonged drug presence within the tumor may thus enable continuous exposure of cancer cells to cytotoxic levels of doxorubicin, resulting in more efficient tumor cell killing and enhanced tumor regression.\u003c/p\u003e\u003cp\u003eMoreover, micelles demonstrated the ability to penetrate more deeply into the extracellular matrix. This feature suggests that the formulation can overcome physical barriers within the tumor microenvironment. Such barriers often limit drug delivery, especially for larger molecules or conventional formulations. Enhanced penetration is particularly important in solid tumors. The dense extracellular matrix often restricts drug diffusion and reduces the efficacy of many chemotherapeutics. In addition, the efficient cellular uptake of the micelles facilitates higher intracellular accumulation of doxorubicin in malignant cells, thereby amplifying its cytotoxic effects. Collectively, these pharmacodynamic advantages indicate that the micellar formulation not only improves localized drug delivery but also sustains elevated concentrations of doxorubicin at the tumor site over extended periods. This prolonged retention may reduce the need for frequent dosing while enhancing overall therapeutic efficacy. Importantly, by localizing drug exposure within the tumor and limiting systemic distribution, the micellar system has the potential to mitigate the dose-limiting toxicities typically associated with conventional chemotherapy, positioning it as a promising strategy for targeted cancer therapy.\u003c/p\u003e\u003cp\u003eSystemic safety evaluation revealed that micellar delivery attenuated cardiotoxicity. Serum CK-MB and LDH levels were reduced by approximately 55% and 48%, respectively, compared with free doxorubicin. Histopathological analysis demonstrated minimal myocardial degeneration in micelle-treated rats, consistent with the reduced systemic C\u003csub\u003emax\u003c/sub\u003e and preferential pulmonary retention [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], (Table\u0026nbsp;8).\u003c/p\u003e\u003cp\u003eThe reduction in doxorubicin-induced cardiotoxicity observed in this study can be explained by altered biodistribution and controlled drug release. Following intrapulmonary administration, micelles deposit directly in the lung, leading to high local concentrations at the tumor site while minimizing systemic exposure and cardiac accumulation, consistent with previous biodistribution studies of pulmonary micellar delivery [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. PEGylation contributes to colloidal stability and steric shielding, limiting protein adsorption and premature clearance, whereas the PLGA core supports a biphasic release profile, an initial burst phase that ensures rapid cytotoxic activity followed by a sustained release that prolongs exposure without sharp systemic peaks. Such a spatiotemporal release pattern markedly reduced acute cardiac exposure to doxorubicin. As shown in Table\u0026nbsp;8, serum CK-MB levels were significantly lower in the DOX-micelle group (145\u0026thinsp;\u0026plusmn;\u0026thinsp;20 U/L) compared with free DOX (320\u0026thinsp;\u0026plusmn;\u0026thinsp;30 U/L), representing a\u0026thinsp;~\u0026thinsp;55% reduction. Similarly, LDH levels decreased from 480\u0026thinsp;\u0026plusmn;\u0026thinsp;40 U/L in free DOX-treated rats to 250\u0026thinsp;\u0026plusmn;\u0026thinsp;28 U/L with micelles, approaching control values (220\u0026thinsp;\u0026plusmn;\u0026thinsp;25 U/L). Histopathological analysis further supported these findings, revealing severe myocardial degeneration in free DOX-treated hearts, whereas micelle-treated animals exhibited only minimal changes, comparable to controls. Collectively, these results confirm that micellar delivery mitigates doxorubicin-induced cardiotoxicity, consistent with prior evidence that nanocarrier systems, including liposomal DOX, confer cardioprotection through reduced cardiac uptake and controlled release [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable\u0026nbsp;(6). Pharmacokinetic Parameters of Free DOX and DOX-Micelles After Pulmonary Administration in Rats\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabd\" border=\"1\"\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFree DOX\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDOX-Micelles\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFold Change\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC_max (\u0026micro;g/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026darr; 2.5-fold\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAUC₀-₂₄h (\u0026micro;g\u0026middot;h/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e22.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026darr; 1.6-fold\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMRT (h)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e6.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e14.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026uarr; 2.4-fold\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003cb\u003eNote: Values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;6). DOX-micelles reduced peak exposure while prolonging systemic residence time.\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable\u0026nbsp;(7).\u003c/b\u003e Lung deposition and tumor volume reduction following administration of free DOX and DOX-micelles.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabe\" border=\"1\"\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFree DOX\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDOX-Micelles\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLung retention at 24 h (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTumor volume reduction vs. control\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25.3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e68.5%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTumor volume reduction vs. free DOX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e43.2%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eDOX-micelles achieved higher pulmonary retention and superior tumor suppression compared with free drug.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable\u0026nbsp;(8). Cardiotoxicity Biomarkers\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabf\" border=\"1\"\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMarker\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFree DOX\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDOX-Micelles\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCK-MB (U/L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e120\u0026thinsp;\u0026plusmn;\u0026thinsp;15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e320\u0026thinsp;\u0026plusmn;\u0026thinsp;30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e145\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLDH (U/L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e220\u0026thinsp;\u0026plusmn;\u0026thinsp;25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e480\u0026thinsp;\u0026plusmn;\u0026thinsp;40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e250\u0026thinsp;\u0026plusmn;\u0026thinsp;28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCardiac histopathology\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNormal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSevere degeneration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMinimal degeneration\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eMicellar delivery reduced cardiotoxicity markers by ~\u0026thinsp;55% compared with free doxorubicin.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePercentage of drug retained in lung tissue 24 h post-instillation for free doxorubicin (12.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7%) versus doxorubicin-loaded micelles (46.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2%). Micellar delivery achieved approximately four-fold higher pulmonary retention, confirming efficient localized deposition and reduced clearance. Data represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBiodistribution in Major Organs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTime-concentration analysis demonstrated marked differences in organ distribution between free DOX and DOX-micelles (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Free DOX exhibited rapid distribution into systemic organs, with high concentrations detected in the liver and heart within the first 2 h post-administration, followed by a steep decline. In contrast, DOX-micelles showed significantly lower accumulation in systemic organs at all time points, while maintaining sustained presence in the lungs.\u003c/p\u003e\u003cp\u003eAt 24 h, heart DOX levels in the free drug group remained at 480\u0026thinsp;\u0026plusmn;\u0026thinsp;55 ng/g, whereas DOX-micelles showed only 210\u0026thinsp;\u0026plusmn;\u0026thinsp;35 ng/g, representing a\u0026thinsp;~\u0026thinsp;2.3-fold reduction. Similarly, hepatic exposure was markedly reduced (2,150\u0026thinsp;\u0026plusmn;\u0026thinsp;210 ng/g vs. 1,050\u0026thinsp;\u0026plusmn;\u0026thinsp;140 ng/g for free DOX and micelles, respectively). Splenic uptake was also lower for DOX-micelles, consistent with reduced RES sequestration. Renal distribution was comparable between groups, reflecting similar clearance mechanisms.\u003c/p\u003e\u003cp\u003eThese results confirm that micellar encapsulation not only enhances pulmonary retention but also minimizes systemic exposure to off-target organs, particularly the heart, thereby reducing cardiotoxic risk.\u003c/p\u003e\u003cp\u003eConsistent with the pharmacokinetic parameters summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e9\u003c/span\u003e, the biodistribution profiles clearly distinguished the two formulations. Free DOX exhibited markedly higher \u003cb\u003eCmax\u003c/b\u003e and \u003cb\u003eAUC\u003c/b\u003e values in systemic organs, particularly the liver and heart, indicating rapid uptake and greater systemic exposure. For example, hepatic accumulation of free DOX peaked early (T\u003csub\u003emax\u003c/sub\u003e = 2 h) with a substantially higher AUC compared with the micellar formulation. In contrast, DOX-micelles demonstrated a lower \u003cb\u003eC\u003c/b\u003e\u003csub\u003e\u003cb\u003emax\u003c/b\u003e\u003c/sub\u003e and reduced overall \u003cb\u003eAUC\u003c/b\u003e, reflecting attenuated systemic exposure, while simultaneously exhibiting a prolonged \u003cb\u003et\u003c/b\u003e\u003csub\u003e\u003cb\u003e1/2\u003c/b\u003e\u003c/sub\u003e and \u003cb\u003eMRT\u003c/b\u003e. This pharmacokinetic signature suggests that micellar encapsulation dampens acute systemic peaks and lowers cumulative exposure, yet maintains extended drug residence through sustained release and slower clearance. Together, these features indicate improved safety by limiting off-target accumulation without compromising therapeutic persistence [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Cardiac exposure, as reflected in both C\u003csub\u003emax\u003c/sub\u003e and AUC, was substantially lower for DOX-micelles, supporting their cardioprotective potential. Similarly, splenic distribution was reduced in the micellar group, consistent with diminished RES sequestration. Renal pharmacokinetics did not differ markedly between the two groups, with comparable T\u003csub\u003emax\u003c/sub\u003e and elimination profiles, indicating that micellar encapsulation did not alter renal excretion. Collectively, these PK findings confirm that DOX-micelles minimize systemic accumulation while sustaining targeted pulmonary delivery. Representative H\u0026amp;E-stained myocardial sections are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e. Control animals exhibited normal cardiac architecture with well-aligned myocardial fibers and intact nuclei. In contrast, free DOX treatment caused marked cardiomyocyte degeneration, nuclear condensation, and disruption of myocardial fibers, consistent with classical features of anthracycline-induced cardiotoxicity. Importantly, animals receiving DOX-loaded PLGA-PEG micelles displayed largely preserved myocardial structure with only minimal alterations, comparable to control. These histological observations are in strong agreement with the cardiac biochemistry profiles, confirming reduced oxidative stress and myocardial injury in the micelle-treated group.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 9\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePharmacokinetic Parameters of DOX in Systemic Organs After Pulmonary Administration in Rats\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOrgan\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFree DOX\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDOX-Micelles\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFold Change\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eHeart\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCmax (\u0026micro;g/g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026darr; 2.6-fold\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAUC₀-₂₄h (\u0026micro;g\u0026middot;h/g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e14.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026darr; 2.4-fold\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMRT (h)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e10.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026uarr; 1.9-fold\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eLiver\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e (\u0026micro;g/g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e3.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026darr; 2.0-fold\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAUC₀-₂₄h (\u0026micro;g\u0026middot;h/g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e45.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e22.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026darr; 2.0-fold\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMRT (h)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e9.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026uarr; 2.0-fold\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eKidney\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e (\u0026micro;g/g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026darr; 1.2-fold\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAUC₀-₂₄h (\u0026micro;g\u0026middot;h/g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e18.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026darr; 1.2-fold\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMRT (h)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e8.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026uarr; 1.4-fold\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eSpleen\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e (\u0026micro;g/g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026darr; 2.1-fold\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAUC₀-₂₄h (\u0026micro;g\u0026middot;h/g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e28.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026darr; 2.3-fold\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMRT (h)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026uarr; 1.8-fold\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eValues are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;6. DOX-micelles significantly reduced cardiac and hepatic exposure while maintaining longer residence times, consistent with preferential pulmonary retention and improved safety profile.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe observed reduction in cardiotoxicity markers (CK-MB, LDH) and minimal histopathological damage in the DOX-micelle group can be mechanistically attributed to the significantly lower systemic exposure and reduced cardiac accumulation of doxorubicin. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e9\u003c/span\u003e, DOX-micelles exhibited a 2.6-fold lower C\u003csub\u003emax\u003c/sub\u003e and 2.4-fold lower AUC in heart tissue compared to free DOX. This attenuated cardiac exposure directly correlates with the diminished release of biomarkers associated with myocardial injury. The sustained release profile and pulmonary retention of micellar DOX effectively limit peak plasma concentrations and systemic distribution, thereby reducing off-target toxicity in cardiac tissue\u0026mdash;a well-established dose-limiting effect of doxorubicin.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese findings confirm the favorable cardiac safety profile of micellar pulmonary delivery (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). Previous studies have highlighted the potential of inhalable nanocarriers for the treatment of lung cancer. Gupta et al. (2022) provided a comprehensive review of inhalable formulations for non-small cell lung cancer, emphasizing that micelles, liposomes, and dendrimers can improve pulmonary residence and limit systemic toxicity [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. However, their work was primarily conceptual and did not present direct pharmacokinetic or safety data. Wang et al. (2018) reported that matrix metalloproteinase (MMP-2/9)-responsive micelles for paclitaxel inhalation achieved approximately 38% pulmonary deposition and superior tumor suppression compared with intravenous administration [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. This study confirmed the benefit of enzyme-triggered release and local lung retention but did not investigate doxorubicin or address systemic cardiotoxicity. Similarly, Hu et al. (2014) demonstrated that polymeric micelles delivered intratracheally in rats prolonged pulmonary retention and extended systemic circulation, yet they did not assess therapeutic efficacy in tumor models or evaluate safety endpoints [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe present work enhances previous findings by integrating pharmacokinetics, biodistribution, antitumor efficacy, and systemic safety of pulmonary doxorubicin-loaded micelles. Compared to free doxorubicin, micelles significantly reduced plasma C\u003csub\u003emax\u003c/sub\u003e (1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u0026micro;g/mL vs. 2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 \u0026micro;g/mL) and AUC (14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 \u0026micro;g\u0026middot;h/mL vs. 22.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 \u0026micro;g\u0026middot;h/mL), while markedly prolonging mean residence time (MRT of 14.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 h vs. 6.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 h). This pharmacokinetic profile reflects controlled drug release from the micelles in the alveolar microenvironment, leading to reduced systemic peaks and prolonged circulation. Biodistribution studies further demonstrated that nearly half of the micellar dose (46.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2%) remained in lung tissue at 24 hours, compared with only 12.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7% for free doxorubicin. This enhanced pulmonary retention is attributed to micelle-surfactant interactions, which promote spreading over the alveolar surface, and reduced clearance through mucociliary and phagocytic pathways, consistent with previous nanoparticle studies.\u003c/p\u003e\u003cp\u003eTo clarify the impact of pulmonary retention on therapeutic outcomes, a detailed comparison of lung tissue concentrations with systemic plasma levels would be useful. This comparison would highlight how sustained drug release in the lungs directly contributes to the observed therapeutic benefits, particularly in terms of tumor targeting and reduced systemic toxicity. The enhanced deposition of the micelles in the lungs ensures a higher localized drug concentration at the tumor site, maximizing the therapeutic effect while minimizing systemic exposure and associated side effects.\u003c/p\u003e\u003cp\u003eSystemic safety assessments revealed that micelle treatment significantly attenuated doxorubicin-induced cardiotoxicity. Specifically, serum CK-MB and LDH levels were reduced by approximately 50\u0026ndash;55% in micelle-treated animals, compared to free doxorubicin treatment. Histopathological analysis of myocardial tissue further demonstrated minimal degeneration in the hearts of micelle-treated rats, in contrast to the severe myocardial damage observed in the free doxorubicin group. Mechanistically, this safety benefit is directly linked to the lower systemic C\u003csub\u003emax\u003c/sub\u003e (1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u0026micro;g/mL vs. 2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 \u0026micro;g/mL) and AUC (14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 \u0026micro;g\u0026middot;h/mL vs. 22.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 \u0026micro;g\u0026middot;h/mL) of micellar doxorubicin, as well as the pulmonary \"drug depot effect,\" which together result in reduced cardiac exposure. These findings provide the first integrated evidence that pulmonary micellar delivery not only enhances therapeutic efficacy but also mitigates one of the most significant dose-limiting toxicities of doxorubicin, namely cardiotoxicity.\u003c/p\u003e\u003cp\u003eThe PLGA-PEG dual-coated micelles outperformed liposomal formulations in aerosolization efficiency, achieving an MMAD of 2.9 \u0026micro;m and an FPF of 62%, both of which are critical for effective deep lung deposition. In contrast, liposomes, although valuable in enhancing the solubility and stability of anticancer drugs, often encounter limitations in aerosolization due to their larger particle size and propensity to aggregate, resulting in reduced fine particle fractions [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Polymeric micelles generally provide superior performance in this regard, owing to their smaller size, uniform dispersion, and improved stability during aerosolization [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Furthermore, the excipient-assisted spray-drying approach employed in this study effectively preserved the structural integrity of the micelles during aerosolization, while simultaneously optimizing fine particle deposition in the lungs. This represents a key advantage over liposomal dry powder inhalers and is consistent with previous reports demonstrating the ability of micelle-based and PLGA nanoparticle systems to achieve favorable aerosolization and lung deposition profiles [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe therapeutic outcomes clearly indicate that PLGA-PEG dual-coated micelles enhance the antitumor efficacy of doxorubicin compared with both untreated controls and the free drug. This improvement can be attributed to several complementary mechanisms. Sustained pulmonary release ensures prolonged intratumoral exposure, while the nanoscale architecture of the micelles enables more efficient penetration through the tumor extracellular matrix. Additionally, preferential endocytic uptake by malignant cells increases intracellular drug accumulation, thereby amplifying cytotoxic activity. Notably, compared with conventional liposomal formulations, the dual-coated micelles showed superior tumor penetration, likely due to their smaller size and more uniform distribution, which facilitated deeper and more consistent tissue permeation [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition to enhanced efficacy, the micellar formulation markedly reduced systemic toxicity. Serum CK-MB and LDH levels decreased by approximately 50\u0026ndash;55%, while histopathological evaluation revealed only minimal myocardial degeneration. These results demonstrate that the micellar system not only improves the therapeutic index but also mitigates cardiotoxicity, one of the major dose-limiting adverse effects of doxorubicin [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOverall, the PLGA-PEG dual-coated micellar dry powder formulation offers multiple advantages over liposomes and conventional polymeric micelles. These include controlled drug release, superior aerosolization efficiency, and improved therapeutic outcomes. The enhanced stability, fine particle deposition, and sustained pulmonary drug release highlight its potential as a promising strategy for inhalation-based chemotherapy [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFrom a translational perspective, the combination of enhanced antitumor efficacy and reduced systemic toxicity highlights the clinical potential of pulmonary micelle-based delivery systems for achieving effective tumor suppression at lower cumulative systemic doses of doxorubicin. By directly targeting the lungs and minimizing systemic exposure, this approach may reduce chemotherapy-associated cardiotoxicity, improve patient compliance through reduced adverse effects, and enable the development of outpatient inhalation regimens that enhance quality of life for lung cancer patients.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eConventional intravenous doxorubicin is limited by rapid distribution, high peak plasma concentrations, and cumulative cardiotoxicity. Liposomal formulations such as Doxil\u0026reg; extend circulation and partially reduce acute cardiac toxicity, yet myocardial accumulation remains significant and long-term cardiac safety is not ensured. Systemic administration also limits local drug exposure in pulmonary malignancies, necessitating higher doses to achieve therapeutic lung concentrations.\u003c/p\u003e\u003cp\u003eThe selection of appropriate inlet and outlet temperatures was paramount to achieving a powder with optimal properties for pulmonary delivery. While high inlet temperatures (\u0026gt;\u0026thinsp;150\u0026deg;C) can maximize yield, they pose a significant risk of degrading our thermolabile API. Furthermore, such temperatures could potentially induce crystallization of our crystalline matrix former, lactose, or cause melting and degradation of our amorphous stabilizer, trehalose, which possesses a critical glass transition temperature (Tg). Conversely, excessively low temperatures can produce powders with high residual moisture, promoting instability, crystallization of lactose, and poor aerosolization.\u003c/p\u003e\u003cp\u003eOur chosen parameters, an inlet temperature of 120\u0026deg;C resulting in an outlet temperature of ~\u0026thinsp;65\u0026deg;C, were strategically optimized for this specific formulation. This gentle thermal profile successfully prevented API degradation, as confirmed by the HPLC analysis (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For the excipients, the outlet temperature was maintained low enough to prevent the crystallization of lactose and, most critically, was kept safely below the Tg of the amorphous trehalose phase. This prevented sticky-wall deposition and ensured a high process yield. Most critically, these conditions produced particles with an ideal morphology and density, as vividly illustrated in the SEM micrographs of Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. The resulting powder exhibited excellent aerodynamic performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), with a high fine particle fraction (FPF) of \u0026gt;\u0026thinsp;75%, directly attributable to the low moisture content and near-spherical, hollow particle structure enabled by this precise thermal control. Therefore, the excellent results in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e are a direct consequence of the carefully calibrated spray-drying temperatures that balanced product stability with powder functionality.\u003c/p\u003e\u003cp\u003eSpray drying is widely used in pharmaceutical and nutraceutical formulations, with inlet and outlet temperatures consistently identified as key parameters determining product quality. These thermal conditions govern drying kinetics, particle morphology, and the retention of bioactive compounds, ultimately shaping essential physicochemical attributes such as solubility, dispersibility, and storage stability. The inlet temperature is the primary driver of solvent evaporation, providing the thermal energy required for drying. Higher inlet temperatures generally accelerate drying, which can be advantageous in limiting the exposure of heat-sensitive compounds to prolonged thermal stress [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. However, excessively high inlet temperatures can cause particle brittleness, morphological changes, or chemical degradation. Tay et al. (2021) reported that temperatures above 150\u0026deg;C led to the breakdown of bioactive compounds, reducing both stability and therapeutic efficacy [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. These findings emphasize the importance of balancing sufficient thermal input for efficient drying with the risk of thermally induced degradation.\u003c/p\u003e\u003cp\u003eWhile outlet temperature is generally recognized as a key parameter influencing particle size and surface area during spray drying [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e], its role appeared limited in this study, as the outlet was maintained at ~\u0026thinsp;65\u0026deg;C. Under these fixed thermal conditions, the observed aerodynamic performance (MMAD\u0026thinsp;~\u0026thinsp;2.9 \u0026micro;m, high FPF) was more convincingly linked to excipient effects. The inclusion of lactose and trehalose not only stabilized the nanostructures during drying but also enhanced powder dispersibility upon aerosolization. These findings suggest that, in systems where thermal variability is minimized, excipient composition rather than outlet temperature becomes the dominant determinant of aerosol behavior [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Yet, insufficient outlet temperatures may result in higher residual moisture, predisposing the product to instability from hydrolytic degradation or microbial growth. Conversely, higher outlet temperatures often generate larger, denser particles with lower surface areas, which may enhance physical stability but at the expense of slower reconstitution [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecent evidence further highlights the interplay between inlet and outlet temperatures in shaping product performance. Akbarbaglu et al. (2021) demonstrated that carefully optimized thermal conditions can significantly improve the retention of labile compounds such as proteins and antioxidants. In their study, an inlet temperature of 130\u0026deg;C combined with an outlet temperature of 70\u0026deg;C produced formulations with enhanced stability and extended shelf-life compared to those dried under more extreme conditions [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. This underscores the importance of considering both parameters simultaneously rather than in isolation. These studies demonstrate that inlet and outlet temperatures exert complementary but distinct effects on spray-dried products. Optimal conditions must strike a balance between drying efficiency, particle integrity, and active compound stability. A deeper mechanistic understanding of temperature effects on particle morphology and degradation is essential. Such knowledge will help refine spray-drying protocols and support the development of more robust pharmaceutical and nutraceutical formulations.\u003c/p\u003e\u003cp\u003eInhalable doxorubicin formulations have long been explored to enhance pulmonary deposition and limit systemic toxicity, but early platforms such as liposomes, polymeric nanoparticles, and microspheres were constrained by aerosolization-induced aggregation, modest fine-particle fractions (35\u0026ndash;45%), and poor dispersibility after spray drying [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Liposomes, despite the clinical success of Doxil\u0026reg;, remain particularly problematic in inhalable forms due to phospholipid oxidation, particle aggregation, and dependence on cryoprotectants, with fine-particle fractions rarely exceeding\u0026thinsp;~\u0026thinsp;45% and drug retention often limited to 70\u0026ndash;75% after spray drying [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe PLGA-PEG micellar dry powder inhaler developed here overcomes these barriers. It achieved a fine-particle fraction\u0026thinsp;\u0026gt;\u0026thinsp;60%, reflecting markedly improved aerosol performance, and retained\u0026thinsp;\u0026gt;\u0026thinsp;90% drug content after spray drying and one-month storage at room temperature, well above values reported for liposomal formulations [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. In addition, the micelles displayed rapid redispersibility (\u0026gt;\u0026thinsp;95%), reduced macrophage uptake by nearly 50%, and sustained release with \u0026gt;\u0026thinsp;70% cumulative doxorubicin release over 48 hours, compared with ~\u0026thinsp;40% for liposomal formulations [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study provides the first demonstration of a PLGA-PEG micellar dry powder inhaler for doxorubicin that unites high aerosol efficiency, long-term stability without cryoprotectants, and enhanced in vivo efficacy. These attributes represent a decisive advance over prior inhalable liposomal systems and position PLGA-PEG micelles as a next-generation platform for pulmonary cancer therapy.\u003c/p\u003e\u003cp\u003eHowever, the aggregation of micelles or crystallization of doxorubicin within the micelles could significantly impact the drug release kinetics and overall delivery performance. Micellar aggregation may occur during storage or within the physiological environment, compromising the structural integrity of the micelles. When micelles aggregate, their surface area available for drug release decreases, potentially slowing the release rate. Additionally, aggregation could hinder the ability of the micelles to effectively reach deep lung regions during aerosolization, as larger aggregates may not be as easily inhaled or deposited in the distal lung areas [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFurthermore, crystallization of doxorubicin within the micelles could negatively affect the release profile. Crystalline forms of doxorubicin are less soluble than their amorphous counterparts, resulting in slower and potentially erratic release. If doxorubicin crystallizes within the micelles, it could slow down the release rate, disrupting the intended biphasic release and diminishing the therapeutic effect. Crystallization could also alter the micelles' size and surface properties, which may impact their dispersibility and aerosol performance. Larger, aggregated, or crystalline micelles may exhibit reduced aerodynamic properties, leading to a decreased fine particle fraction (FPF), which is critical for drug deposition in the small airways and alveoli [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese changes (aggregation or crystallization) could severely impair lung delivery performance. A reduced FPF means that fewer drug particles will reach the target sites in the lungs, potentially compromising the effectiveness of localized therapy for conditions such as lung cancer. Moreover, an uncontrolled release profile resulting from aggregation or crystallization could cause premature drug release or delayed delivery, both of which would undermine the goal of achieving a sustained and controlled therapeutic effect. Consequently, these factors could negatively impact both therapeutic outcomes and patient safety.\u003c/p\u003e\u003cp\u003eIn conclusion, while the biphasic release behavior of doxorubicin-loaded PLGA-PEG dual-coated micelles holds promise for sustained pulmonary delivery, the potential for micellar aggregation and crystallization poses significant challenges. These factors must be carefully monitored during formulation and storage to ensure that the system maintains its stability, performance, and the desired therapeutic effect in lung cancer treatment.\u003c/p\u003e\u003cp\u003eThe Korsmeyer-Peppas model is commonly used to describe the release kinetics of drug delivery systems, particularly when the release mechanism is complex and involves multiple processes. In the context of the present study, the Korsmeyer-Peppas model (with an exponent n\u0026thinsp;=\u0026thinsp;0.74, R2\u0026thinsp;=\u0026thinsp;0.976, and Ft ​\u0026le; 0.6) indicated anomalous transport, suggesting that drug release is governed by a combination of Fickian diffusion and polymer relaxation/erosion. This is significant because the value of n in the Korsmeyer-Peppas model provides insight into the release mechanism: values between 0.5 and 1.0 are indicative of a combination of diffusion and polymer relaxation, which is consistent with the characteristics of the delivery system.\u003c/p\u003e\u003cp\u003eThe specific combination of PLGA (poly(lactic-co-glycolic acid)) and PEG (polyethylene glycol) in this system plays a critical role in modulating the drug release profile. PLGA, a biodegradable polymer, undergoes hydrolytic degradation over time, which leads to erosion and the controlled release of the encapsulated drug. On the other hand, PEG, known for its hydrophilicity, forms a shell around the drug, acting as a diffusion barrier. This dual mechanism, diffusion through the PEG shell and gradual erosion of the PLGA core, leads to sustained and controlled drug release, a feature commonly observed in micellar and polymeric drug carriers [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn more complex in vivo environments, this release profile is likely to change due to factors such as pH, enzymatic activity, and the presence of biological fluids, which can alter the rate of polymer degradation and diffusion. For example, the local pH of the tissue or the action of specific enzymes may accelerate the degradation of the PLGA core, leading to a faster release of the drug compared to in vitro conditions. Moreover, the hydrophilic nature of PEG can be influenced by the surrounding biological medium, potentially altering its effectiveness as a diffusion barrier. These factors make the release behavior in vivo more dynamic and less predictable, but also potentially more adaptable to the needs of targeted drug delivery.\u003c/p\u003e\u003cp\u003eThe PEG corona stabilizes micelles and suppresses premature leakage, while the PLGA core sustains intracellular release [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Lactose and trehalose act as lyoprotectants, preventing aggregation during lyophilization and preserving aerosol performance [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. In contrast to liposomal and nanoparticle-based inhalable formulations, which frequently exhibit limited fine particle fractions, aggregation, and inconsistent lung deposition, the dual-coated micellar dry powder demonstrates reproducible aerodynamic performance with significantly higher deposition efficiency. Mechanistically, pulmonary micelle reservoirs modulate systemic exposure by lowering C_max and extending residence time [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], thereby attenuating cardiotoxicity relative to free drug and conventional intravenous dosing, as corroborated by suppressed CK-MB, LDH, and negligible histopathological changes.\u003c/p\u003e\u003cp\u003eUnlike earlier liposomal or polymeric nanoparticle systems, which offered only partial solutions to pulmonary delivery challenges, this platform integrates pulmonary targeting, structural stability, and systemic safety into a single formulation, representing a decisive advancement in inhalable doxorubicin therapy.\u003c/p\u003e\u003cp\u003eSurface modification of PLGA carriers with polyethylene glycol (PEG) has proven to be a pivotal strategy for optimizing pulmonary drug delivery. The dual contribution of the PLGA matrix and PEG corona establishes a synergistic framework that balances controlled release with extended lung retention.\u003c/p\u003e\u003cp\u003eAt the structural level, PLGA provides the degradable hydrophobic depot, encapsulating doxorubicin and releasing it in a time-dependent manner through hydrolytic erosion into lactic and glycolic acids. This gradual degradation avoids the burst release characteristic of unmodified polymers, maintaining therapeutic concentrations in the pulmonary microenvironment and supporting prolonged local bioavailability.\u003c/p\u003e\u003cp\u003eSuperimposed on this depot effect, PEG functionalization imparts steric stabilization, preventing aggregation during nebulization and ensuring reproducible aerosolization. Once deposited, the PEG corona limits protein adsorption and opsonization, thereby reducing phagocytic clearance and systemic leakage. This stealth behavior not only extends pulmonary residence but also enhances epithelial uptake, establishing a localized drug reservoir with minimized off-target exposure.\u003c/p\u003e\u003cp\u003eEvidence from preclinical studies validates this mechanistic synergy. Li et al. (2021) showed that PEGylated PLGA microspheres achieve tunable lung retention and reduced systemic distribution compared with unmodified PLGA carriers [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Similarly, Omidian and Wilson (2025) highlighted that PLGA-PEG formulations optimize aerosol performance and mucosal interactions, strengthening their value for both pulmonary and intranasal delivery [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. Beyond respiratory applications, Kesharwani et al. (2025) demonstrated that PEGylated PLGA nanoparticles improve tumor accumulation and intracellular uptake, confirming PEG\u0026rsquo;s role in enhancing therapeutic precision across oncological models [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Broader reviews further consolidate PEGylation as a key translational modification for PLGA-based drug delivery [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. These data position PLGA-PEG nanocarriers as a versatile and clinically relevant platform. By combining PLGA\u0026rsquo;s sustained release profile with PEG\u0026rsquo;s pharmacokinetic and stability advantages, this dual-coated system maximizes lung retention, improves epithelial transport, and reduces systemic cardiotoxicity, an especially favorable profile for localized chemotherapy in lung malignancies.\u003c/p\u003e\u003cp\u003eThe cardioprotective effect observed with micellar DOX is mechanistically grounded in its altered pharmacokinetic profile. The significantly lower Cmax and AUC in heart tissue directly explain the reduction in cardiotoxicity biomarkers and histopathological damage (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e9\u003c/span\u003e). By minimizing peak systemic exposure and prolonging pulmonary retention, the micellar formulation reduces the cumulative dose delivered to the heart, thereby mitigating doxorubicin-induced oxidative stress and mitochondrial dysfunction. This is consistent with prior reports that nanocarrier systems reduce cardiotoxicity through controlled release and reduced cardiac uptake (e.g., liposomal doxorubicin). Thus, the dual mechanisms of localized lung deposition and sustained systemic release collectively contribute to an improved safety profile without compromising antitumor efficacy.\u003c/p\u003e\u003cp\u003eUnmodified PLGA carriers have been extensively applied in inhalable and injectable drug delivery owing to their biodegradability and predictable hydrolysis into lactic and glycolic acids, which supports depot-like sustained release of therapeutics [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. However, their hydrophobic surface chemistry promotes aggregation during aerosolization, impairing aerodynamic performance and reproducibility of lung deposition. Furthermore, once deposited in the alveoli, PLGA microspheres and nanoparticles are rapidly opsonized and cleared by macrophages, leading to short pulmonary residence times and reduced therapeutic efficiency [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. Thus, while PLGA alone provides reliable controlled release, its clinical utility in pulmonary delivery is constrained by instability and premature clearance.\u003c/p\u003e\u003cp\u003ePEGylation strategies, by contrast, are designed to enhance colloidal stability and biological persistence. The hydrophilic PEG corona provides a steric barrier that minimizes aggregation, reduces protein adsorption, and confers \u0026ldquo;stealth\u0026rdquo; characteristics against phagocytic clearance (50). In pulmonary and intranasal contexts, PEGylation has been shown to improve epithelial permeability and extend retention time within mucosal and alveolar compartments [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. Nonetheless, PEG-only systems lack a depot effect: encapsulation efficiency is relatively low, and drug leakage is common due to PEG\u0026rsquo;s hydrophilic nature. As a result, while PEG formulations extend persistence, they cannot ensure sustained and localized drug release.\u003c/p\u003e\u003cp\u003eThe integration of PLGA cores with PEG shells offers a complementary solution. PLGA provides degradation-mediated, time-dependent release, while PEG improves colloidal stability during aerosolization, reduces immune recognition, and prolongs alveolar residence. This synergy has been validated in vivo, where PEG-modified PLGA microspheres achieved tunable lung retention and reduced systemic exposure compared with unmodified PLGA formulations (80). Beyond pulmonary delivery, PEGylated PLGA nanoparticles have also improved tumor accumulation and reduced systemic toxicity of doxorubicin in oncology models, highlighting the broad translational relevance of this dual strategy [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite these advances, polymeric carriers alone remain vulnerable to stresses encountered during spray drying, nebulization, and pulmonary deposition. Here, lactose and trehalose serve as critical stabilizing excipients. These disaccharides enhance dispersibility, prevent nanoparticle aggregation, and improve powder flowability. Trehalose, in particular, stabilizes labile polymeric matrices via hydrogen bonding and elevation of glass transition temperature, while lactose enhances aerodynamic uniformity and promotes homogeneous deposition within the lower respiratory tract [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. When co-formulated with PLGA-PEG systems, lactose and trehalose improve emitted dose, fine particle fraction, and delivery efficiency, while mitigating moisture-induced instability [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Cumulatively, PLGA-PEG nanostructures complemented by lactose and trehalose represent a rational, translationally relevant platform. By establishing a localized pulmonary reservoir with sustained release, enhanced retention, and reduced systemic exposure, this strategy directly addresses key clinical challenges such as systemic cardiotoxicity and frequent dosing. Moreover, the regulatory familiarity of PLGA, PEG, lactose, and trehalose, all FDA-approved or GRAS, facilitates a clear pathway toward clinical adoption. Importantly, the modularity of this approach supports broader applicability across chemotherapeutics, biologics, and gene therapies, extending its utility beyond oncology to chronic respiratory diseases.\u003c/p\u003e\u003cp\u003eThe literature collectively highlights the complementary strengths of polymeric systems in pulmonary delivery. PLGA-only carriers reliably provide controlled release via gradual hydrolytic degradation but are limited by aggregation during aerosolization and rapid clearance by alveolar macrophages. PEG-only systems, in contrast, prolong pulmonary retention through steric stabilization and reduced opsonization, yet lack depot capacity and often exhibit premature drug leakage. PLGA-PEG dual systems uniquely merge these functionalities: PLGA offers sustained, degradation-mediated release, while PEG enhances colloidal stability, reduces immune clearance, and extends alveolar residence. Preclinical evidence confirms this synergy, with PEG-modified PLGA microspheres showing tunable lung retention and reduced systemic exposure relative to unmodified PLGA (80). Similarly, oncology studies demonstrate enhanced tumor accumulation and reduced cardiotoxicity with PEGylated PLGA nanoparticles, reinforcing their translational potential [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eExcipients such as lactose and trehalose address practical bottlenecks in inhalation formulations by stabilizing particles during spray drying, reducing aggregation, and enhancing dispersibility and aerodynamic performance [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. By safeguarding powder integrity, these sugars preserve the pharmacokinetic advantages of PLGA-PEG systems, ensuring reproducible lung deposition and sustained therapeutic benefit. This integrated design paradigm establishes a localized pulmonary reservoir with prolonged drug exposure and reduced systemic toxicity, positioning PLGA-PEG-sugar systems as one of the most clinically promising inhalation platforms [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite our findings, this study has limitations that must be considered. A primary constraint is the reliance on the rat-derived SLC cell line (RCB2862) as a model for human NSCLC. While this model provided valuable insights into the role of SLCs in metabolic reprogramming, it does not capture the full biological and genetic heterogeneity of human lung cancers. Consequently, the direct translational relevance of our results may be limited. Future studies are essential to validate these mechanisms in a panel of well-characterized human NSCLC cell lines and primary patient-derived models to strengthen the clinical applicability of our conclusions.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTranslational Relevance\u003c/strong\u003e\u003cp\u003ePulmonary delivery of doxorubicin-loaded micelles achieved high lung retention, sustained pharmacokinetics, and enhanced antitumor efficacy while markedly reducing systemic cardiotoxicity. By lowering peak plasma exposure and acting as a localized drug depot, this approach enabled effective tumor suppression at reduced systemic doses. These findings provide preclinical evidence that inhalable micellar formulations hold translational potential for further investigation as an alternative to conventional systemic chemotherapy in lung cancer.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eNonetheless, key limitations must be acknowledged. Long-term in vivo safety within the pulmonary environment remains incompletely understood. Repeated dosing raises the possibility of anti-PEG antibody formation and immunogenic responses [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e], or low-grade inflammation from persistent polymeric fragments. Moreover, scaling PLGA-PEG formulations to industrial production poses significant challenges: ensuring particle size uniformity, PEG surface density, and excipient crystallinity during spray drying or lyophilization requires stringent process control. Regulatory approval will further demand extensive pharmacokinetic, toxicological, and stability data, which may slow translation despite encouraging preclinical outcomes. Addressing these limitations through long-term pulmonary safety studies and scalable manufacturing innovations will be essential to bridge the gap from proof-of-concept to clinical implementation.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study establishes a novel inhalable dry powder platform based on spray-dried, PLGA-PEG dual-coated micelles embedded in a lactose/trehalose matrix, designed to overcome the limitations of conventional doxorubicin formulations and earlier pulmonary delivery systems. The formulation exhibits three key innovations: (i) a dual polymeric coating that enhances micellar stability, prevents premature drug leakage, and prolongs lung retention; (ii) excipient-mediated aerosol stabilization that ensures high fine particle fraction and reproducible lung deposition, mitigating aggregation common in nanoparticulate dry powder inhalers; and (iii) pulmonary pharmacokinetic modulation, enabling localized drug reservoirs that reduce systemic Cmax, extend residence time, and significantly attenuate cardiotoxicity without sacrificing therapeutic efficacy.\u003c/p\u003e\u003cp\u003eMechanistically, this approach achieves an optimal balance between antitumor potency and cardioprotection, as demonstrated by robust tumor suppression, reduced serum CK-MB and LDH levels, and preserved myocardial architecture. The platform leverages clinically accepted excipients and scalable spray-drying, underscoring its translational feasibility.\u003c/p\u003e\u003cp\u003eIn conclusion, this spray-dried micellar system represents a comprehensive preclinical validation of an inhalable doxorubicin formulation with enhanced lung targeting, sustained release, and reduced systemic exposure. It offers a versatile strategy not only for non-small cell lung cancer but also for the pulmonary delivery of other cytotoxic and biologic agents, positioning it as a promising next-generation platform in precision oncology. Future studies should include human NSCLC cell lines and primary models to better predict clinical efficacy and safety, addressing the limitation of the current rat-derived SLC cell model.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor Contributions: Conceptualization, Rawan Bafail; Data curation, Obaid Afzal and Mahmoud Omar; Formal analysis, Randa Zaki, Omiya Hasan and Waad A. Samman; Investigation, Randa Zaki, Obaid Afzal, Rawan Bafail and Mahmoud Omar; Methodology, Alaa Ayman, Eman Samy, Obaid Afzal, Waad A. Samman and Mahmoud Omar; Software, Alaa Ayman, Omiya Hasan and Obaid Afzal; Supervision, Omiya Hasan and Mahmoud Omar; Validation, Alaa Ayman, Eman Samy and Mahmoud Omar; Writing - original draft, Randa Zaki, Alaa Ayman, Eman Samy, Omiya Hasan, Obaid Afzal, Rawan Bafail, Waad A. Samman and Mahmoud Omar; Writing - review \u0026amp; editing, Alaa Ayman, Eman Samy, Omiya Hasan, Rawan Bafail, Waad A. Samman and Mahmoud Omar.\u003c/p\u003e\u003ch2\u003eAcknowledgement:\u003c/h2\u003e\u003cp\u003eThis study is supported via funding from Prince Sattam Bin Abdulaziz University Project number (PSAU/2025/R/1446).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSharma R. Mapping of global, regional and national incidence, mortality and mortality-to-incidence ratio of lung cancer in 2020 and 2050. Int J Clin Oncol. 2022;27(4):665\u0026ndash;75. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10147-021-02108-2\u003c/span\u003e\u003cspan address=\"10.1007/s10147-021-02108-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBhadran A, Polara H, Babanyinah GK, Baburaj S, Stefan MC. Advances in doxorubicin chemotherapy: emerging polymeric nanocarriers for drug loading and delivery. 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Pharmaceutics. 2025;17(8):1074. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/pharmaceutics17081074\u003c/span\u003e\u003cspan address=\"10.3390/pharmaceutics17081074\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pulmonary drug delivery, PLGA-PEG micelles, Doxorubicin, Dry powder inhaler, Non-small cell lung cancer, Spray-drying","lastPublishedDoi":"10.21203/rs.3.rs-7640535/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7640535/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eNon-small cell lung cancer (NSCLC) is a leading cause of cancer mortality. Intravenous doxorubicin (DOX) is effective but limited by severe cardiotoxicity and poor lung tumor selectivity.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eTo develop an inhalable dry powder inhaler (DPI) of DOX-loaded PLGA-PEG dual-coated micelles embedded in a lactose/trehalose matrix for targeted pulmonary delivery and reduced systemic toxicity.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eMicelles were prepared via nanoprecipitation and engineered into respirable powders via spray-drying. Formulations were characterized for size, stability, aerosol performance, drug release, and cytotoxicity (SLC cells). In vivo pharmacokinetics, lung deposition, antitumor efficacy, and systemic safety were assessed in rat models.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eOptimized micelles showed high encapsulation efficiency (91.3%), desirable aerodynamic properties (MMAD 2.9 \u0026micro;m, FPF 62.4%), and sustained biphasic release. They significantly enhanced cytotoxicity (IC₅₀ 1.82 vs. 3.96 \u0026micro;g/mL) and apoptosis (48.6% vs. 29.7%) compared to free DOX. In vivo, pulmonary delivery achieved 46.8% lung retention at 24 h (vs. 12.4%), prolonged systemic residence time (MRT 14.7 h vs. 6.1 h), and superior tumor growth inhibition (68.5% vs. 25.3%). Critically, micellar DOX markedly reduced cardiotoxicity (CK-MB \u0026darr;55%, LDH \u0026darr;48%) with minimal histopathological cardiac damage.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThe spray-dried micellar DPI platform enables efficient lung-targeted delivery of DOX, enhancing antitumor efficacy while mitigating systemic cardiotoxicity, presenting a promising therapeutic strategy for NSCLC.\u003c/p\u003e","manuscriptTitle":"Engineered Spray-Dried PLGA-PEG Dual-Coated Micelles with Lactose/Trehalose Matrices for Pulmonary Doxorubicin Delivery: Achieving Sustained Release and Improved Aerosol Performance in NSCLC Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-07 19:42:22","doi":"10.21203/rs.3.rs-7640535/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"52947425-caa6-42f0-9d3c-b832aff989fa","owner":[],"postedDate":"October 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:04:41+00:00","versionOfRecord":{"articleIdentity":"rs-7640535","link":"https://doi.org/10.1007/s12247-026-10408-1","journal":{"identity":"journal-of-pharmaceutical-innovation","isVorOnly":false,"title":"Journal of Pharmaceutical Innovation"},"publishedOn":"2026-02-27 15:58:29","publishedOnDateReadable":"February 27th, 2026"},"versionCreatedAt":"2025-10-07 19:42:22","video":"","vorDoi":"10.1007/s12247-026-10408-1","vorDoiUrl":"https://doi.org/10.1007/s12247-026-10408-1","workflowStages":[]},"version":"v1","identity":"rs-7640535","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7640535","identity":"rs-7640535","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}