Folic acid grafted amphiphilic dextran-b-poly(ε-benzyloxycarbonyl-L-lysine) micelles for drug’s pH-responsive release

Folic acid grafted amphiphilic dextran-b-poly(ε-benzyloxycarbonyl-L-lysine) micelles for drug’s pH-responsive release | 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 Folic acid grafted amphiphilic dextran-b-poly(ε-benzyloxycarbonyl-L-lysine) micelles for drug’s pH-responsive release Ruoxi Huang, Yaxin Yi, Shiyu Zhang, Jiaxin Wang, Haikuan Yuan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7201841/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Dec, 2025 Read the published version in Polymer Bulletin → Version 1 posted 10 You are reading this latest preprint version Abstract A stimulus-responsive drug delivery system holds promise for reducing adverse effects and enhancing the bioavailability of chemotherapeutic agents. In this study, amino-terminated dextran was utilized as a macromolecular initiator to prepare a range of amphiphilic dextran-b-poly(ε-benzyloxycarbonyl- l -lysine) (Dex-b-PZLL) copolymers with varying hydrophobic segment lengths via ring-opening polymerization (ROP). Folic acid (FA) was further grafted onto the dextran side chains via esterification (FA-Dex-b-PZLL), offering the potential for tumor-specific recognition. The resulting copolymers were able to self-assemble into stable spherical micelles via dialysis and efficiently encapsulate the hydrophobic drug letrozole (LTZ). A systematic assessment was conducted on the drug encapsulation efficiency/loading capacity, drug release performance in vitro, and biocompatibility of the copolymers under aqueous conditions, indicating that these properties of the FA-Dex-b-PZLL micelles mainly depended on the copolymer composition. The optimized micelles, with a balanced hydrophilic/hydrophobic ratio, achieved a drug loading capacity of approximately 8.49% and an encapsulation efficiency of about 46.39%. Moreover, LTZ/FA-Dex-b-PZLL micelles exhibited pH-responsive release, with cumulative release rates within 72 h of 20.74%, 27.14% and 62.96% in PBS at pH 7.4, 6.5 and 4.5, respectively, indicating accelerated release under acidic conditions. In addition, the micelles showed good cytocompatibility according to the CCK-8 assay. Amphiphilic polymer Micelles Drug delivery pH-responsive Biocompatibility Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Drug delivery systems encompass diverse pharmaceutical formulations designed to transport therapeutic agents to specific pathological sites, enabling accurate diagnosis and disease management through controlled pharmacological action [ 1 ]. In recent years, the emergence of numerous drug candidates with low aqueous solubility has exposed limitations in conventional oral dosage forms, such as tablets and capsules, which often fail to achieve optimal therapeutic outcomes. To address these challenges, nanotechnology-driven delivery platforms have gained prominence as innovative solutions. Distinguished from traditional systems, nano-enabled carriers demonstrate superior capabilities in enhancing drug solubility, minimizing systemic toxicity, improving bioavailability, and enabling site-specific targeting through tailored physicochemical properties [ 2 ]. Current advances in this field include liposomes [ 3 ], dendrimers [ 4 ], nanogels [ 5 ], polymeric nanoparticles and micellar systems [ 6 ]. Notably, polymeric micelles have emerged as versatile vehicles in anticancer drug delivery, given their ability to increase drug solubility, extend blood circulation and lower toxicity [ 7 ]. Polymeric micelles, which self-assemble from amphiphilic polymers in water, possess a distinctive core-shell structure. The core with hydrophobic properties serves as a reservoir for encapsulating lipophilic therapeutics, effectively enhancing their aqueous solubility, while the hydrophilic corona stabilizes the nanostructure via hydration layer formation, prolonging systemic circulation [ 8 ]. Leveraging their exceptional drug-loading capacity, micelles have emerged as pivotal carriers for hydrophobic chemotherapeutic agents, addressing challenges such as low bioavailability and off-target toxicity. Recent advancements integrate tumor microenvironment-specific stimuli (pH, enzymes, or redox gradients) into micelle design, enabling intelligent drug release and active targeting [ 9 , 10 ]. These strategies synergistically enhance tumor-specific accumulation, minimize premature leakage, and optimize therapeutic efficacy [ 11 ]. Over the past few years, considerable study has concentrated on exploring novel materials or optimizing established ones to engineer tailored polymeric micelles for advanced drug delivery applications. Dextran, a non-immunogenic and biocompatible polysaccharide, consists primarily of α-(1,6)-linked D-glucopyranose chains with minor (1,3)-branched segments [ 12 ]. Due to these features, it has been extensively employed as a plasma expander, drug delivery vehicle, and protein stabilizer [ 13 ]. The dextran molecular backbone contains abundant hydroxyl groups, conferring exceptional water solubility and modifiability. Selective functionalization of these hydroxyl sites enables the construction of dextran derivatives with amphiphilic structures [ 14 ]. Polypeptides, polymers built up from amino acids joined by peptide bonds, inherently possess biodegradability, biocompatibility, and protein-mimetic properties [ 15 ]. Amino acids can be arranged in various sequences and unique structures, and can also be incorporated into block or graft copolymers to prepare core-shell micelles [ 16 , 17 ]. This research presents an innovative polymeric micellar nanocarrier engineered with dual-functional capabilities of tumor-targeting and pH microenvironment- triggered drug release, aiming to optimize treatment accuracy and therapeutic outcomes in malignant neoplasms management (Fig. 1 ). To construct the polymeric micelles, biocompatible dextran and poly(ε-benzyloxycarbonyl- l -lysine) were employed as hydrophilic along with hydrophobic segments. The pH-responsive characteristics of the micelle were achieved by conjugating dextran to poly(ε-benzyloxycarbonyl- l -lysine) through ring opening polymerization reactions, enabling the system to release the drug specifically in the acidic tumor microenvironment. Additionally, folic acid (FA) was conjugated to dextran via esterification to confer tumor-targeting ability by binding selectively to folate receptors highly expressed on tumor cells. Comprehensive characterization of the carrier’s structure and properties using Fourier-transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance spectroscopy ( 1 H NMR), dynamic light scattering (DLS), gel permeation chromatography (GPC) and scanning electron microscopy (SEM) confirmed the successful synthesis and desired characteristics of the micelle. Using letrozole (LTZ) as a model, the micelle’s drug-loading efficiency and release profile were examined, while cytotoxicity assays verified its biocompatibility, suggesting potential for clinical translation. 2. Experimental 2.1. Materials Dextran (Leuconostoc mesenteroides origin, nominal M r = 6 kDa, Sigma-Aldrich) underwent SEC analysis with dextran calibration standards, revealing M w = 6385 Da and M n = 4500 Da. Sodium cyanoborohydride (NaBH 3 CN, ≥ 95.0%, Adamas). N6-carbobenzoxy- l -lysine N-carboxyanhydride (Lys(Z)-NCA, ≥ 95%), dimethyl sulfoxide (DMSO), methanol, letrozole (LTZ, ≥ 98%), folic acid (FA), N,N′-dicyclohexylcarbodiimide (DCC) as well as 4-dimethylaminopyridine (DMAP) were all obtained from Aladdin. Ethylenediamine was obtained from Sinopharm Chemical Reagent (China). 1,6-Diphenyl-1,3,5-hexatriene (DPH, ≥ 98%) was obtained from Sigma-Aldrich. 2.2. Characterization FTIR spectra were recorded ranging from 4000 to 500 cm − 1 by means of a Vertex 70 spectrometer (Bruker, Karlsruhe, Germany), operating at a resolution of 4 cm − 1 and eight scans. 1 H NMR spectra were acquired on a Bruker Avance 400 spectrometer (Bruker, Karlsruhe, Germany) in DMSO-d6 containing 0.03% tetramethylsilane (TMS). DLS analyses were conducted with a Nano ZS90 (Malvern Panalytical, UK) at 25°C and the scattering angle of 90°. GPC was conducted on a PL-GPC50 (Agilent, California, USA) to determine the polydispersity index (PDI) and molecular weight of copolymers at 45°C using DMSO as the eluent (1.0 mL/min) and polymethyl methacrylate (PMMA) for standard calibration. SEM analysis was carried out using a Sigma 300 (Zeiss, Oberkochen, Germany) to observe the morphology of the micelles. The cell viability assay was performed on a SPARK multimode microplate reader (Tecan, Männedorf, Switzerland). 2.3. Synthesis of FA-Dex-b-PCL block copolymer 2.3.1. Synthesis of amino-terminated dextran (Dex-NH 2 ) Dextran (3 g, 0.67 mmol) and 30 mL DMSO were charged into a 100 mL round-bottom flask and stirred vigorously until fully dissolved, after which excess ethylenediamine (1.32 g, 21.96 mmol) was supplemented. The system was held at 60°C with constant stirring for 48 h. Subsequently, NaBH 3 CN (0.3 g, 4.77 mmol) was introduced as a reducing agent and the system was allowed to react for an additional 48 h. The resultant solution was precipitated with methanol, and the solid was collected via vacuum filtration. Purification was performed through solvent/anti-solvent precipitation cycles using water (solvent) and methanol (anti-solvent). The purified product was redissolved in deionized water and dialyzed against distilled water for 24 h utilizing a 3500 Da molecular weight cutoff (MWCO) membrane. The dialyzed solution was lyophilized to yield a fluffy white solid designated as Dex-NH 2 [ 18 , 19 ]. 2.3.2. Synthesis of Dex-b-PZLL copolymer The amphiphilic Dex-b-PZLL was obtained via ROP of Lys(Z)-NCA initiated by the macromolecular initiator Dex-NH 2 [ 20 ]. Under an inert nitrogen atmosphere, Dex-NH 2 (0.45 g, 0.1 mmol) along with Lys(Z)-NCA (0.61 g, 2 mmol) were solubilized in 5 mL anhydrous DMSO, followed by continuous polymerization at ambient temperature for 72 h. The resulting solution was precipitated in methanol, with subsequent isolation via centrifugation (8,000 rpm, 10 min). The collected precipitate underwent three washing cycles with deionized water before lyophilization to yield a white solid product (Dex-b-PZLL). By systematically varying the molar ratios between the macroinitiator and Lys(Z)-NCA (1:10, 1:20, 1:30), a series of Dex-b-PZLL copolymers with tailored hydrophobic chain compositions were obtained. These derivatives were designated as Dex-b-PZLL 10 , Dex-b-PZLL 20 and Dex-b-PZLL 30 , respectively, based on their feed molar ratios. 2.3.3. Synthesis of folic acid-decorated Dex-b-PZLL block copolymer Folic acid (0.03 g, 0.068 mmol), DMAP (0.017 g, 0.136 mmol) as well as DCC (0.028 g, 0.136 mmol) were solubilized in 20 mL DMSO. To activate the carboxyl groups, the reaction system was shielded from light and stirred for 30 minutes amid nitrogen at 30°C. Subsequently, 0.3 g of Dex-b-PZLL was introduced, and the coupling reaction proceeded for 24 h. The crude product underwent dialysis against PBS (pH 7.4) to eliminate residual FA, followed by further dialysis in deionized water. After centrifugation (10,000 rpm, 15 min), the dialysate was lyophilized to obtain FA-Dex-b-PZLL as a yellowish solid [ 21 ]. The degree of substitution ( DS ) of FA was quantified by 1 H NMR and calculated using the following equation [ 22 ]: here, I A represents the integral of the pyrazine ring protons (δ = 8.6–8.8 ppm) from FA, and I 2 − 6 corresponds to the integral of dextran backbone protons (δ = 3.0–3.7 ppm). 2.4. Preparation of blank and drug-loaded micelles The FA-Dex-b-PZLL 10 (FDP-1) blank micelles and letrozole-loaded FA-Dex-b-PZLL 10 (LTZ/FDP-1) micelles were both prepared using the dialysis method [ 23 ]. In brief, FDP-1 micelles (50 mg) were solubilized in 500 µL DMSO under ultrasonication to achieve homogeneous dispersion. Subsequently, the prepared solution was gradually added dropwise into deionized water (20 mL) under stirring to ensure complete mixing. Following transfer to a dialysis bag (MWCO 3500), the mixture underwent dialysis in 2000 mL deionized water for 48 h. The resulting dialysate was subsequently filtered through a 0.22 µm syringe filter to obtain a clear filtrate. Lastly, the filtrate was lyophilized to yield blank micelle solids. To prepare LTZ/FDP-1 micelles, 10 mg LTZ was solubilized into DMSO with 50 mg FDP-1 micelles. Other amphiphilic copolymers with different ratios of hydrophobic chains FA-Dex-b-PZLL 20 (FDP-2) and FA-Dex-b-PZLL 30 (FDP-3) blank micelles and corresponding LTZ-loaded (LTZ/FDP-2, LTZ/FDP-3) micelles were synthesized using a similar method. 2.5. Determination of the drug-loading performance of micelles A UV-Vis spectrophotometer was utilized to determine the actual encapsulation and drug-loading status of LTZ-loaded micelles [ 24 ]. A standard curve (Fig. S1 ) was constructed by plotting the absorbance values at λ = 240 nm of letrozole standard solutions against their known concentrations ( C , µg·mL − 1 ). 10 mg of LTZ-loaded micelle was ultrasonically dissolved in 100 µL of DMSO to disrupt micellar structures and liberate encapsulated drug. Acetonitrile was then introduced at a volumetric ratio (DMSO:acetonitrile = 1:9) to solubilize LTZ while precipitating FA-Dex-b-PZLL carriers. Following centrifugation (12,000 rpm, 10 min) and filtration through a 0.22 µm syringe filter, the UV absorbance of the filtrate was recorded. Triplicate measurements per sample ensured statistical validity, with data expressed as mean ± SD. The measured absorbance values were substituted into the standard curve to determine the amount of LTZ encapsulated within micelles. The encapsulation efficiency ( EE %) as well as drug loading ( DL %) were then calculated [ 25 ]: 2.6. Measuring critical micellar concentration The dye solubilization technique reported by Alexandridis et al. [ 26 ] was utilized to measure the critical micelle concentration (CMC) of FDP micelles in water. A blank micelle dispersion was prepared in water at an initial concentration of 1 mg·mL − 1 . Sequential dilutions were performed to generate 12 concentration gradients (0.001–0.5 mg·mL − 1 ). To each 4 mL micellar dispersion (with increasing concentrations), 40 µL of a 0.4 mmol·L − 1 DPH in methanol was added, yielding a final DPH concentration of 4×10 − 6 mol·L − 1 . The samples were then subjected to 24 h incubation under light-protected conditions at ambient temperature to ensure complete partitioning of DPH into the hydrophobic micellar cores. UV-visible spectra of DPH (300–500 nm) were acquired using a TU-1901 spectrophotometer (Puxi Inc., Beijing, China). Absorbance maxima at λ = 350 nm were correlated with polymeric concentrations, with CMC determined via intersection point analysis of the biphasic curve in absorbance-concentration plots. 2.7. In vitro drug release The pH-dependent release profile of LTZ/FDP micelles was examined utilizing dialysis. In a typical procedure, 3 mL of the micellar dispersion was loaded into dialysis bags and dialyzed against 47 mL of PBS solutions with pH values of 4.5, 6.5 and 7.4. The systems were incubated at 37°C with shaking (200 rpm) for 72 h. At predetermined time intervals (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 24, 30, 36, 48, 54, 60, and 72 h), 3 mL samples were collected and replaced with fresh buffer of equal volume to maintain sink conditions. Quantitative analysis was performed using UV spectrophotometry: sample absorbance was recorded at 240 nm (characteristic wavelength of letrozole), with drug concentrations subsequently determined by the pre-established letrozole standard curve. The cumulative release percentage, CR (%), was determined by Eq. ( 4 ) [ 27 ]. where V e is the replacement volume (3 mL), V 0 is the total volume (50 mL), C i represents the concentration of letrozole at the i -th sampling point, and m drug refers to the total mass of letrozole encapsulated in the micelles. 2.8. Biocompatibility experiment L-929 and MDA-MB-231 cells were cocultured with FDP blank micelles at different concentrations to evaluate their biocompatibility. In brief, when the monolayer coverage rate of cells reached 80%, they were digested with an appropriate amount of trypsin. The digested cells were then seeded into 96-well plates (1×10 4 cells/well) and incubated for 24 h under standard conditions (37°C, 5% CO 2 ). FDP blank micelle solutions at different concentrations were introduced and followed by an incubation for 48 h. Each sample has five parallel experiments. Finally, 100 µL of CCK-8 solution was introduced per well followed by incubation for 1 h. The optical density at 450 nm in each well was then recorded with a microplate reader in order to determine the cell viability [ 28 ]: where OD sample , OD blank and OD control are the optical density of the sample wells, blank wells and control group, respectively. 3. Results and Discussion 3.1. Characterization of copolymer FA-Dex-b-PZLL 3.1.1. FTIR analysis The FTIR spectra of the raw materials and copolymers were acquired using the KBr pellet method, as shown in Fig. 3 . Compared with native dextran (Dex), the typical peaks of Dex-NH 2 did not exhibit substantial changes, potentially due to the masking effect of polysaccharide’s stronger absorption bands over the signals from ethylenediamine [ 29 ]. In the spectrum of Lys(Z)-NCA precursor, the absorption bands at 1856 and 1775 cm − 1 correspond to the stretching vibrations of the dual carbonyl (C = O) in the cyclic anhydride structure, while the peaks observed at 749 and 695 cm − 1 are assigned to the out-of-plane bending vibrations of the benzene ring’s C-H bonds [ 30 ]. The FTIR spectrum of Dex-b-PZLL exhibits three notable changes compared to the precursors: (1) The disappearance of cyclic anhydride peaks at 1856 and 1775 cm − 1 indicates successful ring-opening polymerization initiated by amino groups on dextran; (2) The absorption bands of benzene ring emerge in the 900 − 650 cm-1 region; (3) New absorption peaks appear at 1627 and 1535 cm − 1 , corresponding to peptide bond vibrations in polyamino acid segments. Together, these spectral changes confirm the effective formation of Dex-b-PZLL block copolymer [ 31 ]. Furthermore, FA-Dex-b-PZLL displays additional characteristic peaks at 1570, 1311, 1193, 840 and 640 cm − 1 , which matches the distinctive vibrational modes of folic acid, thereby verifying its successful conjugation [ 32 ]. 3.1.2. 1 H NMR analysis The 1 H NMR spectra of Dex, Dex-NH 2 , Dex-b-PZLL and FA-Dex-b-PZLL are shown in Fig. 4 . Comparative analysis of 1 H NMR spectra between dextran (Dex) and its amine-functionalized derivative (Dex-NH 2 ) reveals the entire loss of the characteristic anomeric proton signal (δ = 6–7 ppm) associated with the terminal glucopyranose unit of Dex, confirming successful end-group amination via ethylenediamine modification [ 33 ]. Notably, the methylene (-CH 2 -) proton signals of ethylenediamine are not independently resolved in the spectrum due to resonance overlap with dextran backbone protons in the δ = 3–5 ppm region. For Dex-b-PZLL block copolymer, the peak at 7.31 ppm indicates the presence of aromatic protons from the benzene ring, while the methylene protons on the benzyloxycarbonyl group are located at 4.96 ppm. The peaks at 3.76 ppm, 1-1.68 ppm and 2.94 ppm are the proton absorption peaks of the secondary and methylene groups of the hydrophobic block polylysine [ 20 ]. The spectrum of FA-Dex-b-PZLL exhibit two new proton signals at 8.62 ppm and 6.63 ppm, corresponding to the pyrazine and aromatic protons of folic acid, respectively [ 34 ]. The degree of folate substitution is determined by integrating the characteristic folate proton signals relative to the dextran backbone protons using Eq. ( 1 ), yielding a substitution degree of 7.39 wt%, equivalent to approximately 1.08 folate molecules per dextran. These 1 H NMR analyses conclusively demonstrate the successful synthesis of the target folate-conjugated block copolymer FA-Dex-b-PZLL. 3.1.3. GPC analysis Three block copolymers with varying molecular weights (Dex-b-PZLL 10 , Dex-b-PZLL 20 as well as Dex-b-PZLL 30 ) were synthesized via ROP by adjusting the molar ratios of initiator to Lys(Z)-NCA (1:10, 1:20 and 1:30, respectively). The molecular weights and their distributions of the Dex-b-PZLL copolymers are characterized by GPC, as summarized in Table S1 and illustrated in Fig. 5 . Three GPC curves exhibit monomodal distributions with narrow polydispersity indices, indicating the absence of unreacted precursors and suggesting efficient polymerization processes. According to GPC separation principles, higher molecular weight components elute earlier, thus the left-shifted peaks in the chromatograms correspond to larger molecular weights. The combined analysis of GPC data and elution profiles confirms the successful preparation of these three distinct copolymers with controlled molecular weights. 3.2. Characterization of FDP and LTZ/FDP micelles Under aqueous conditions, the copolymers form micelles once their concentrations exceed the CMC values, which are 0.04, 0.017 and 0.011 mg·mL − 1 for FDP-1, FDP-2 and FDP-3, respectively (Fig. S2). The low CMC favors stable micelles suitable for prolonged circulation [ 35 ]. The DLS analysis reveals both FDP-1 and LTZ/FDP-1 micelles have low PDI and narrow size distributions (Fig. 6 ), with sizes of 97.9 nm (PDI = 0.119) as well as 113.9 nm (PDI = 0.125), respectively. Similar trends were observed for FDP-2, FDP-3 and their corresponding LTZ-loaded micelles (Fig. S3). Interestingly, the LTZ/FDP-2 and FDP-2 micelles are similar in size, while the size of LTZ/FDP-3 micelles is considerably lower than its blank micelles. This may arise from the hydrophobic segments of the polymer have a larger molecular weight, which can generate higher intermolecular forces, thereby forming a more compact hydrophobic core. This tight structure may make the shell of the micelle more stable, which to some extent limits the expansion of the micelle and even causes a slight reduction in particle size [ 36 ]. The microstructure of FDP and LTZ/FDP micelles was examined by SEM. As shown in Fig. 7 , both FDP and LTZ/FDP micelles exhibit uniform spherical structures with well-defined boundaries and good dispersibility in aqueous solutions, with particle sizes below 150 nm. The slightly smaller particle sizes observed by SEM compared to DLS measurements could be attributed to the different measurement conditions: DLS analysis is performed in aqueous solutions while SEM samples are examined under vacuum-dried conditions [ 37 ]. These nano-sized FDP and LTZ/FDP micelles offer prospect of enhancing tissue permeability and systemic bioavailability, thereby promoting the antitumor therapeutic effectiveness [ 38 ]. The ideal drug delivery system should exhibit high stability to prevent drug leakage during circulation. DLS was employed to monitor the variations in micelle size under normal physiological conditions (pH 7.4) (Fig. 8 ). Taking FDP-3 micelle as an example, after 96 h of incubation in PBS buffer (pH 7.4), the blank micelles retained an average hydrodynamic diameter of 136.08 nm. The micelles demonstrate good stability without significant disintegration in physiological environments, confirming their suitability as drug carriers for blood transport. 3.3. Drug-loading analysis The drug loading capacities of LTZ/FDP-1, LTZ/FDP-2 and LTZ/FDP-3 micelles are approximately 7.12%, 8.49% and 7.81%, respectively, with encapsulation efficiencies of 38.33%, 46.39% and 42.36% (Table 1 ). Experimental results reveal a nonlinear correlation between the drug encapsulation efficiency/loading capacity of FDP micelles as well as the molecular weight of hydrophobic segment (PZLL). Increasing the polymerization degree of PZLL chains enhance both the hydrophobic core volume expansion and hydrophobic association effects, thereby significantly improving the micelles’ letrozole encapsulation capability. However, when the PZLL molecular weight exceed a critical threshold (e.g., LTZ/FDP-3 micelles), a slight decline in drug loading efficiency is observed. This phenomenon might be attributed to excessive molecular entanglement within elongated hydrophobic chains, which promoted structural densification of micelles and restricted drug diffusion into the core [ 35 ]. Additionally, overly strong hydrophobic interactions could induce premature micellar aggregation, potentially compromising the stability of drug-loaded structures. Table 1 Drug-loading performance of the micelles. Drug-loaded micelles DL (%) EE (%) LTZ/FDP-1 7.12% 38.33% LTZ/FDP-2 8.49% 46.39% LTZ/FDP-3 7.81% 42.36% 3.4. In vitro drug release In vitro release profiles of LTZ/FDP micelles under different pH values are shown in Fig. 9 and Table 2 . As illustrated in Fig. 9 , all LTZ/FDP micelles exhibit a sustained slow-release behavior, which gradually accelerate with decreasing pH. For instance, the cumulative amounts of letrozole released from LTZ/FDP-1 micelles at 72 h were 15.14%, 18.02% and 50.46% at pH 7.4, 6.5 along with 4.5, respectively. This gradient release phenomenon is attributed to the pH-responsive chemical bond (amide bond) introduced in the micelle molecular design. Under acidic conditions, hydrolysis and amide bond cleavage induce polymer backbone dissociation, which disrupts the micellar core-shell structure and accelerates drug release [ 39 , 40 ]. The detailed cumulative release data for LTZ/FDP-2 and LTZ/FDP-3 micelles, which display similar pH-dependent release trends to LTZ/FDP-1 micelles, are summarized in Table 2 . A vertical comparison under the same pH conditions indicates that the release order is consistent with the micelles’ drug loading capacity, both being strongly influenced by the PZLL chain length. A longer PZLL chain promotes hydrophobic core expansion and enhances drug release, but excessive chain length may cause core densification and restrict diffusion. Moreover, the FA-Dex-b-PZLL micelles achieve dual-functional drug delivery: maintaining structural stability in physiological conditions to prolong circulation while enabling pH-triggered drug release in acidic tumor microenvironments. Combined with the EPR effect, this system enhances tumor drug accumulation while minimizing systemic toxicity. Its sustained release further reduces administration frequency, demonstrating promising translational potential for targeted cancer therapy. Table 2 Comparison of in vitro release profiles of LTZ/FDP micelles. Drug-loaded micelles pH = 7.4 pH = 6.5 pH = 4.5 LTZ/FDP-1 15.14% 18.02% 50.46% LTZ/FDP-2 20.74% 27.14% 62.96% LTZ/FDP-3 17.57% 23.67% 56.36% 3.5. In vitro biocompatibility Biocompatibility evaluation is essential for assessing the biosafety of biomaterials intended for in vivo applications, providing fundamental data for subsequent drug delivery system development or biomedical material utilization to ensure their stability and safety in biological environments. As illustrated in Fig. 10 , the biocompatibility test results reveal that the cell viability slightly decreased with increasing micelle concentration, it remained above 90% even at the maximum micelle concentration (200 µg·mL − 1 ). These findings demonstrate that FDP blank micelles exerted no inhibitory effects on cellular proliferation, indicating the absence of cytotoxic effects from blank micelles and confirming their excellent biocompatibility. 4. Conclusions Herein, a series of folic acid modified amphiphilic dextran-b-poly(ε- benzyloxycarbonyl- l -lysine) (FA-Dex-b-PZLL) copolymers with varying hydrophobic segment lengths are successfully synthesized by reduction amidation, ring-opening polymerization and esterification reaction. All copolymers can self-assemble via dialysis into uniformly dispersed spherical micelles in an aqueous medium with a low critical micelle concentration. The polymer micelles possess tumor targeting properties and pH responsiveness. By adjusting the hydrophilic-to-hydrophobic ratio of the amphiphilic FA-Dex-b-PZLL carrier, precise modulation of drug encapsulation efficiency and release efficiency is achieved. The micelles maintain good stability in normal physiological conditions, while showing rapid payload release in acidic tumor microenvironment. Cytocompatibility assessments reveal negligible cytotoxicity (cell viability > 90%) and excellent biocompatibility of blank carriers. These findings validate this pH-responsive nanosystem as a versatile nanoplatform for hydrophobic chemotherapeutics delivery with enhanced therapeutic outcomes. Declarations Conflicts of interest There are no conflicts to declare. Supporting Information Molecular weight of block copolymers Dex-b-PZLL (Table S1 ). UV-vis absorption spectra and standard curve of letrozole (Fig. S1 ). CMC of FDP micelles (Fig. S2). Particle size distribution of FDP and LTZ/FDP micelles (Fig. S3). Author Contribution Ruoxi Huang: Project administration, Writing - review & editing. Yaxin Yi: Data curation, Writing - original draft. Shiyu Zhang: Visualization, Methodology. Jiaxin Wang: Formal analysis, Validation. Haikuan Yuan: Conceptualization, Investigation. Jie Lu: Supervision, Funding acquisition. All authors reviewed the manuscript. Acknowledgement The authors appreciated the National Natural Science Foundation of China (Nos. 21978165 and 92156020) and the Natural Science Foundation of Shanghai Municipality (Grant No. 25ZR1401151) to support this work. Data availability The authors confirm that the data supporting the findings of this study are available within the article. References Wang G, Zhang X, Kapilevich L, Hu M (2023) Recent advances in polymeric microparticle-based drug delivery systems for knee osteoarthritis treatment. Front Bioeng Biotechnol 11:1290870. https://doi.org/10.3389/fbioe.2023.1290870 Duan X, Wang Z, Liu H, Ma H, Zhang X, Liu L, Shang H, Qiao N (2025) Folate-modified drug-carrying micelles with pH-responsiveness for curcumin-targeted delivery. 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Colloids Surf, B 159:484-492. https://doi.org/10.1016/j.colsurfb.2017.08.018 Almeida A, Araujo M, Novoa-Carballal R, Andrade F, Goncalves H, Reis RL, Lucio M, Schwartz Jr S, Sarmento B (2020) Novel amphiphilic chitosan micelles as carriers for hydrophobic anticancer drugs. Mater Sci Eng C 112:110920. https://doi.org/10.1016/j.msec.2020.110920 Yang C, Huang S, Wang X, Wang M (2016) Theranostic unimolecular micelles of highly fluorescent conjugated polymer bottlebrushes for far red/near infrared bioimaging and efficient anticancer drug delivery. Polym Chem 7:7455-7468. https://doi.org/10.1039/c6py01838f Li M, Zhang Le, Xuan Y, Zhi D, Wang W, Zhang W, Zhao Y, Zhang S, Zhang S (2022) pH-sensitive hyaluronic acid-targeted prodrug micelles constructed via a one-step reaction for enhanced chemotherapy. Int J Biol Macromol 206:489-500. https://doi.org/10.1016/j.ijbiomac.2022.02.131 Zhang J, Lou B, Qin X, Li Y, Yuan H, Zhang L, Liu X, Zhang Y, Lu J (2022) Using amphiphilic polymer micelles as the templates of antisolvent crystallization to produce drug nanocrystals. ACS Omega 7:21000-21013. https://doi.org/10.1021/acsomega.2c01792 Additional Declarations No competing interests reported. Supplementary Files Supportinginformation.docx Supporting Information Molecular weight of block copolymers Dex-b-PZLL (Table S1). UV-vis absorption spectra and standard curve of letrozole (Fig. S1). CMC of FDP micelles (Fig. S2). Particle size distribution of FDP and LTZ/FDP micelles (Fig. S3). Cite Share Download PDF Status: Published Journal Publication published 04 Dec, 2025 Read the published version in Polymer Bulletin → Version 1 posted Editorial decision: Revision requested 22 Oct, 2025 Reviews received at journal 14 Oct, 2025 Reviewers agreed at journal 10 Oct, 2025 Reviews received at journal 10 Oct, 2025 Reviewers agreed at journal 07 Oct, 2025 Reviewers agreed at journal 06 Oct, 2025 Reviewers invited by journal 24 Aug, 2025 Editor assigned by journal 29 Jul, 2025 Submission checks completed at journal 28 Jul, 2025 First submitted to journal 24 Jul, 2025 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7201841","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":508404181,"identity":"e1b36cc8-6963-40f3-9ccf-07671b84edad","order_by":0,"name":"Ruoxi Huang","email":"","orcid":"","institution":"Shanghai University of Engineering Science","correspondingAuthor":false,"prefix":"","firstName":"Ruoxi","middleName":"","lastName":"Huang","suffix":""},{"id":508404189,"identity":"27987388-2789-4196-b00c-686578581abe","order_by":1,"name":"Yaxin Yi","email":"","orcid":"","institution":"Shanghai University of Engineering 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05:53:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7201841/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7201841/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00289-025-06092-9","type":"published","date":"2025-12-04T15:57:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90379152,"identity":"cf010b71-bc21-4149-a5d7-9df947f894ba","added_by":"auto","created_at":"2025-09-02 06:27:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":261089,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of pH-sensitive polymeric micelles and the drug delivery route.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7201841/v1/8b496736380fdd38fe5a0d3e.png"},{"id":90379154,"identity":"e6b553b9-6e93-4d03-a5ba-bbcc64d68ff8","added_by":"auto","created_at":"2025-09-02 06:27:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":77070,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis schema for FA-Dex-b-PZLL block copolymer.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7201841/v1/da38b076dd81319ab6a6bc97.png"},{"id":90379156,"identity":"8ec47a40-25fc-47ea-9a12-49d4fd000002","added_by":"auto","created_at":"2025-09-02 06:27:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":134520,"visible":true,"origin":"","legend":"\u003cp\u003eIR spectra of Dex, Dex-NH\u003csub\u003e2\u003c/sub\u003e, Lys(Z)-NCA, Dex-b-PZLL (a), FA and FA-Dex-b-PZLL (b,c).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7201841/v1/3bbe0ee196327e338e76b70f.png"},{"id":90379430,"identity":"544ea099-b577-4136-bfdf-beb6eb8a9ac2","added_by":"auto","created_at":"2025-09-02 06:35:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":120456,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH-NMR spectra of Dex (a), Dex-NH\u003csub\u003e2\u003c/sub\u003e (b), Dex-b-PZLL (c) and FA-Dex-b-PZLL (d).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7201841/v1/c86091ea4e1464fbac26dc67.png"},{"id":90379158,"identity":"c472baec-b54d-46c3-a7e2-656198cc1e13","added_by":"auto","created_at":"2025-09-02 06:27:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":35862,"visible":true,"origin":"","legend":"\u003cp\u003eGPC elution curves of Dex-b-PZLL.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7201841/v1/07829c0919385c6f7c313227.png"},{"id":90379431,"identity":"c02e447e-68d0-4c6c-9be9-274c6d996823","added_by":"auto","created_at":"2025-09-02 06:35:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":36331,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size distribution of FDP-1 and LTZ/FDP-1 micelles.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7201841/v1/043a87224577509f339d869c.png"},{"id":90382120,"identity":"489247ed-8fba-4a07-9b67-1909741b5510","added_by":"auto","created_at":"2025-09-02 06:51:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":169042,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of blank micelles FDP-1 (a), FDP-2 (b), FDP-3 (c) and LTZ-loaded micelles LTZ/FDP-1 (d), LTZ/FDP-2 (e), LTZ/FDP-3 (f).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7201841/v1/46bb27de64a44b7ee75dd9ac.png"},{"id":90379436,"identity":"878fffcf-2be6-4cbd-8e61-4fc43b0b3a98","added_by":"auto","created_at":"2025-09-02 06:35:54","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":41739,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size changes (a) and line (b) graph of FDP blank micelles incubated at PBS (pH = 7.4) for 96 h.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7201841/v1/a3517967cc75a2a3c7151928.png"},{"id":90380533,"identity":"65e3ad53-e0f0-4e22-9e6a-cfed65d0345a","added_by":"auto","created_at":"2025-09-02 06:43:54","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":45749,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro release behavior of LTZ/FDP micelles at different pH levels (7.4, 6.5, 4.5) at 37 °C.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7201841/v1/12bbe9b67bfe2821a7611db1.png"},{"id":90380534,"identity":"260260e0-19e3-4deb-b1a7-31bd6b4c353c","added_by":"auto","created_at":"2025-09-02 06:43:54","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":40826,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of L929 cells (a) along with MDA-MB-231 cells (b) treated with blank micelles at varying concentrations.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7201841/v1/f71dd600c2adf716a4088585.png"},{"id":97723905,"identity":"0914e1fe-dd7b-4218-b4c3-b08e9700cc6d","added_by":"auto","created_at":"2025-12-08 16:09:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1662092,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7201841/v1/4f5ca35e-fee8-4c72-85e5-fcf247c5b656.pdf"},{"id":90379175,"identity":"cc78c554-b817-493f-aa1d-8523dd9ee9c6","added_by":"auto","created_at":"2025-09-02 06:27:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7164179,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupporting Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular weight of block copolymers Dex-b-PZLL (Table S1). UV-vis absorption spectra and standard curve of letrozole (Fig. S1). CMC of FDP micelles (Fig. S2). Particle size distribution of FDP and LTZ/FDP micelles (Fig. S3).\u003c/p\u003e","description":"","filename":"Supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7201841/v1/843515c602a04519c9107f96.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Folic acid grafted amphiphilic dextran-b-poly(ε-benzyloxycarbonyl-L-lysine) micelles for drug’s pH-responsive release","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDrug delivery systems encompass diverse pharmaceutical formulations designed to transport therapeutic agents to specific pathological sites, enabling accurate diagnosis and disease management through controlled pharmacological action [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In recent years, the emergence of numerous drug candidates with low aqueous solubility has exposed limitations in conventional oral dosage forms, such as tablets and capsules, which often fail to achieve optimal therapeutic outcomes. To address these challenges, nanotechnology-driven delivery platforms have gained prominence as innovative solutions. Distinguished from traditional systems, nano-enabled carriers demonstrate superior capabilities in enhancing drug solubility, minimizing systemic toxicity, improving bioavailability, and enabling site-specific targeting through tailored physicochemical properties [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Current advances in this field include liposomes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], dendrimers [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], nanogels [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], polymeric nanoparticles and micellar systems [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Notably, polymeric micelles have emerged as versatile vehicles in anticancer drug delivery, given their ability to increase drug solubility, extend blood circulation and lower toxicity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePolymeric micelles, which self-assemble from amphiphilic polymers in water, possess a distinctive core-shell structure. The core with hydrophobic properties serves as a reservoir for encapsulating lipophilic therapeutics, effectively enhancing their aqueous solubility, while the hydrophilic corona stabilizes the nanostructure via hydration layer formation, prolonging systemic circulation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Leveraging their exceptional drug-loading capacity, micelles have emerged as pivotal carriers for hydrophobic chemotherapeutic agents, addressing challenges such as low bioavailability and off-target toxicity. Recent advancements integrate tumor microenvironment-specific stimuli (pH, enzymes, or redox gradients) into micelle design, enabling intelligent drug release and active targeting [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These strategies synergistically enhance tumor-specific accumulation, minimize premature leakage, and optimize therapeutic efficacy [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOver the past few years, considerable study has concentrated on exploring novel materials or optimizing established ones to engineer tailored polymeric micelles for advanced drug delivery applications. Dextran, a non-immunogenic and biocompatible polysaccharide, consists primarily of α-(1,6)-linked D-glucopyranose chains with minor (1,3)-branched segments [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Due to these features, it has been extensively employed as a plasma expander, drug delivery vehicle, and protein stabilizer [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The dextran molecular backbone contains abundant hydroxyl groups, conferring exceptional water solubility and modifiability. Selective functionalization of these hydroxyl sites enables the construction of dextran derivatives with amphiphilic structures [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Polypeptides, polymers built up from amino acids joined by peptide bonds, inherently possess biodegradability, biocompatibility, and protein-mimetic properties [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Amino acids can be arranged in various sequences and unique structures, and can also be incorporated into block or graft copolymers to prepare core-shell micelles [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis research presents an innovative polymeric micellar nanocarrier engineered with dual-functional capabilities of tumor-targeting and pH microenvironment- triggered drug release, aiming to optimize treatment accuracy and therapeutic outcomes in malignant neoplasms management (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To construct the polymeric micelles, biocompatible dextran and poly(ε-benzyloxycarbonyl-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-lysine) were employed as hydrophilic along with hydrophobic segments. The pH-responsive characteristics of the micelle were achieved by conjugating dextran to poly(ε-benzyloxycarbonyl-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-lysine) through ring opening polymerization reactions, enabling the system to release the drug specifically in the acidic tumor microenvironment. Additionally, folic acid (FA) was conjugated to dextran via esterification to confer tumor-targeting ability by binding selectively to folate receptors highly expressed on tumor cells. Comprehensive characterization of the carrier\u0026rsquo;s structure and properties using Fourier-transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance spectroscopy (\u003csup\u003e1\u003c/sup\u003eH NMR), dynamic light scattering (DLS), gel permeation chromatography (GPC) and scanning electron microscopy (SEM) confirmed the successful synthesis and desired characteristics of the micelle. Using letrozole (LTZ) as a model, the micelle\u0026rsquo;s drug-loading efficiency and release profile were examined, while cytotoxicity assays verified its biocompatibility, suggesting potential for clinical translation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Materials\u003c/h2\u003e\n \u003cp\u003eDextran (Leuconostoc mesenteroides origin, nominal \u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003er\u003c/em\u003e\u003c/sub\u003e = 6 kDa, Sigma-Aldrich) underwent SEC analysis with dextran calibration standards, revealing \u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e = 6385 Da and \u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003en\u003c/em\u003e\u003c/sub\u003e = 4500 Da. Sodium cyanoborohydride (NaBH\u003csub\u003e3\u003c/sub\u003eCN, \u0026ge; 95.0%, Adamas). N6-carbobenzoxy-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-lysine N-carboxyanhydride (Lys(Z)-NCA, \u0026ge; 95%), dimethyl sulfoxide (DMSO), methanol, letrozole (LTZ, \u0026ge; 98%), folic acid (FA), N,N\u0026prime;-dicyclohexylcarbodiimide (DCC) as well as 4-dimethylaminopyridine (DMAP) were all obtained from Aladdin. Ethylenediamine was obtained from Sinopharm Chemical Reagent (China). 1,6-Diphenyl-1,3,5-hexatriene (DPH, \u0026ge; 98%) was obtained from Sigma-Aldrich.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Characterization\u003c/h2\u003e\n \u003cp\u003eFTIR spectra were recorded ranging from 4000 to 500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e by means of a Vertex 70 spectrometer (Bruker, Karlsruhe, Germany), operating at a resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and eight scans. \u003csup\u003e1\u003c/sup\u003eH NMR spectra were acquired on a Bruker Avance 400 spectrometer (Bruker, Karlsruhe, Germany) in DMSO-d6 containing 0.03% tetramethylsilane (TMS). DLS analyses were conducted with a Nano ZS90 (Malvern Panalytical, UK) at 25\u0026deg;C and the scattering angle of 90\u0026deg;. GPC was conducted on a PL-GPC50 (Agilent, California, USA) to determine the polydispersity index (PDI) and molecular weight of copolymers at 45\u0026deg;C using DMSO as the eluent (1.0 mL/min) and polymethyl methacrylate (PMMA) for standard calibration. SEM analysis was carried out using a Sigma 300 (Zeiss, Oberkochen, Germany) to observe the morphology of the micelles. The cell viability assay was performed on a SPARK multimode microplate reader (Tecan, M\u0026auml;nnedorf, Switzerland).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Synthesis of FA-Dex-b-PCL block copolymer\u003c/h2\u003e\n \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003ch2\u003e2.3.1. Synthesis of amino-terminated dextran (Dex-NH\u003csub\u003e2\u003c/sub\u003e)\u003c/h2\u003e\n \u003cp\u003eDextran (3 g, 0.67 mmol) and 30 mL DMSO were charged into a 100 mL round-bottom flask and stirred vigorously until fully dissolved, after which excess ethylenediamine (1.32 g, 21.96 mmol) was supplemented. The system was held at 60\u0026deg;C with constant stirring for 48 h. Subsequently, NaBH\u003csub\u003e3\u003c/sub\u003eCN (0.3 g, 4.77 mmol) was introduced as a reducing agent and the system was allowed to react for an additional 48 h. The resultant solution was precipitated with methanol, and the solid was collected via vacuum filtration. Purification was performed through solvent/anti-solvent precipitation cycles using water (solvent) and methanol (anti-solvent). The purified product was redissolved in deionized water and dialyzed against distilled water for 24 h utilizing a 3500 Da molecular weight cutoff (MWCO) membrane. The dialyzed solution was lyophilized to yield a fluffy white solid designated as Dex-NH\u003csub\u003e2\u003c/sub\u003e [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n \u003ch2\u003e2.3.2. Synthesis of Dex-b-PZLL copolymer\u003c/h2\u003e\n \u003cp\u003eThe amphiphilic Dex-b-PZLL was obtained via ROP of Lys(Z)-NCA initiated by the macromolecular initiator Dex-NH\u003csub\u003e2\u003c/sub\u003e [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. Under an inert nitrogen atmosphere, Dex-NH\u003csub\u003e2\u003c/sub\u003e (0.45 g, 0.1 mmol) along with Lys(Z)-NCA (0.61 g, 2 mmol) were solubilized in 5 mL anhydrous DMSO, followed by continuous polymerization at ambient temperature for 72 h. The resulting solution was precipitated in methanol, with subsequent isolation via centrifugation (8,000 rpm, 10 min). The collected precipitate underwent three washing cycles with deionized water before lyophilization to yield a white solid product (Dex-b-PZLL). By systematically varying the molar ratios between the macroinitiator and Lys(Z)-NCA (1:10, 1:20, 1:30), a series of Dex-b-PZLL copolymers with tailored hydrophobic chain compositions were obtained. These derivatives were designated as Dex-b-PZLL\u003csub\u003e10\u003c/sub\u003e, Dex-b-PZLL\u003csub\u003e20\u003c/sub\u003e and Dex-b-PZLL\u003csub\u003e30\u003c/sub\u003e, respectively, based on their feed molar ratios.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n \u003ch2\u003e2.3.3. Synthesis of folic acid-decorated Dex-b-PZLL block copolymer\u003c/h2\u003e\n \u003cp\u003eFolic acid (0.03 g, 0.068 mmol), DMAP (0.017 g, 0.136 mmol) as well as DCC (0.028 g, 0.136 mmol) were solubilized in 20 mL DMSO. To activate the carboxyl groups, the reaction system was shielded from light and stirred for 30 minutes amid nitrogen at 30\u0026deg;C. Subsequently, 0.3 g of Dex-b-PZLL was introduced, and the coupling reaction proceeded for 24 h. The crude product underwent dialysis against PBS (pH 7.4) to eliminate residual FA, followed by further dialysis in deionized water. After centrifugation (10,000 rpm, 15 min), the dialysate was lyophilized to obtain FA-Dex-b-PZLL as a yellowish solid [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. The degree of substitution (\u003cem\u003eDS\u003c/em\u003e) of FA was quantified by \u003csup\u003e1\u003c/sup\u003eH NMR and calculated using the following equation [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]:\u003c/p\u003e\n \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\n \u003cdiv class=\"EquationNumber\"\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAlUAAABCCAYAAAB+SiO4AAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAAA1lSURBVHhe7d1fSFvnHwbwJz96GedFhBmzjjXBetctlbFdBFv/0DGWQAvdBl7EgujQG9sqm4OV6nqxdktodtOBtDCz4qTeDDydpdV2aG6TFO9sphs4c3Kxm3LOZeHdxTznl/Pm5J8eV63PB85F3vdNNHLAh/d9z/d1CSEEiIiIiGhX/ic3EBEREVH9GKqIiIiIHMBQRUREROQAhioiIiIiBzBUERERETmAoYqIiIjIAQxVRERERA5gqCIiIiJyAEMVERERkQMYqoiIiIgcwFBFRERE5ACGKiIiIiIHMFQREREROYChioiIiMgBDFVEREREDmCoqlNfXx9cLpd5xeNxecieUBQFPT090HVd7gIq9Ou6jp6eHtu+YpOTk1BVVW6GqqoIBALm91UURR5CREREDFX1m56eRiwWg8fjQSaTwejoqDzEcaqqYmRkRG42letXVRVvv/02lpaW5C4LI0x5vV5Lu67ruHr1KlKpFIQQiMViiEQiDFZEREQ2GKp2YHV1FR999BGCwaDctSempqZw7tw5udlUrt/r9WJ9fR2xWEzuskin02hvb5ebkclkMDQ0ZIatzz77DN3d3Zibm5OHEhERHXoMVXXSdR1bW1v4+OOP5a6qdF3H4OBgyTJbNpstO+OlKAra29tLZpEM1fprUS5UdXR0WIKj2+2Gz+ezjCEiIqJ/MVTVKZfLYXNzc0fhwu12Y3JyEpcuXTKDVTabxfXr1zE5OSkPh6qqSKfTCIfDchdQQ38tyi392dlNoCQiInrVMVTV6fHjxzh69ChaW1vlrpp4vV5cu3YNly5dwr179zAzM4M7d+7A7XbLQzE1NYXBwUG52VStvxblZqns5HI5eDwenD59Wu4iIiI69Biq6qDrOhYWFvDhhx/ahqBatba24osvvsCVK1fQ29tr+1nZbBadnZ1lZ5Cq9deqnlB19+5djI+P2/6+REREhx1DVR2Mpb+uri65qy65XA43btzAtWvXMDMzY1vqIJFI4NSpU2Ypg7GxMSwtLaGhoQGKolTtr0U9S3/xeBydnZ3/2eZ8IiKig4ahqg6Vlv6y2Sza2tqQzWblLgtVVXHlyhXcvHkTn3zyCXp7e9Hf318SrKanpyGEMK9YLIbu7m5omoZwOFy1vxaFQgGdnZ1ycwkjpBmfm81msby8LI0iIiI63BiqapTL5XDr1q2KS39vvvkmHj9+LDebjLpPN2/eNGeHgsEgxsfHcfXqVXm4Y1ZXV+UmYPs72QXEYoqiIBKJYGxszJwVO3/+fNX3ERERHTZ7EqrkquMulwttbW0YHR0tmZExygy4XC40NTVhdnYWqqri3r17lnEvUzwex/Hjx7GxsYGxsTHb6uSFQgEjIyNYWFgo6TO43W5MTU2VLLcFg8E9qcxuVENPJpNYWlrCW2+9Zc6k6bqOtbU1NDQ0yG8zxeNxRCIRuRmhUKjkO+xUtYrv8Xi8YjX3WvubmppsZxEVRdmTvz0REf2fy+WyvJ6dnYXL5UIqlbK0DQ8PW8YdOGKPxGIx4ff7RT6fF0IIsby8LPx+v+ju7haappnjotGoiEajQtM0oWmamJiYEADE/Px80aftb/l8XmQyGZHP50VPT4/5namyfD4v/H6/AFByXwghxPz8vHkPGWOL74tq/ZlMRhw/flxkMhkRi8VKfkY+nxcTExPmayIictbKyooAINbX1822Dz74QAAQAMTKyopl/I0bN0QgELC0HSR7FqqMsFRsfn7eEpjy+bx49913RSaTMcdomiYGBgYOVDAxvpdxHaRAuB/YBR5N00R3d3dJiDLGVesXUrCfn58XHo/Hcq9NTEwcqPuMiOigkQOVwQhbcqgS28FqaGhIbj4Q9mT5T1VVpFKpkiKR7e3t8Pv95jEnDQ0NeO2115BIJMwxbrcb7733XsVlqf2msbERmqZBCIH5+flDf4zLTirHy+yKrPp8PmxubiKXy1Xtr8aJSvRERFTe8PAwhoaG4Pf75a6KPv/8czx8+NCyNHhQ7EmoSqfTeP78eUnV8YaGBhw7dsx87Xa7cfHiRdy/fx99fX3mnpr+/v6ym8H3E2M/UEdHB3777TcAwNzcHJLJZNk9QodBvZXj7WxtbeHFixdobm4225qbm/HixQtsbW1V7QeArq4uHDlyBIVCAWtra3jnnXfQ2trqSCV6IiKq7IcffkBvb6/cXJMzZ85gZmZGbt739iRUFf8DK6ZpGv744w9LWzgcxqNHj3D//n2cPXvW8SBit2m++Orr65PfUjO3243FxUUIIcx/0Eapg8XFxQMRDPdKPZXj90owGMTg4CBOnjyJb775Bt999535sIAxkxYIBHZ9HxARkZUxy9TS0iJ31aSjowMPHz6Um/c9x0NVparjhUIBz58/x4kTJyztwWAQjx49wtOnTx0PVnI9J/manp6W30IOqaVyfD2M+6ccu/7R0VEIIfD3338jGAxalv3Gx8cRCoWgaRq2trb4FCARkUP++usvAKh76a/Y+vq63LTvOR6qKlUdTyQSaGxsRG9vLxRFsTwCHwwG8eWXX+Lp06c17YnZS/JsFq/aLlktlePL8fl85tJdMY/HA5/PV7XfTjabxZMnTxAOh6FvHw594sQJuN1u+Hy+svW8iIiIauF4qCpXdVxRFCSTSXz//ffwer1YW1uz9ANAW1sbGhsb0dzcbFmaMa7ifUq1VjDfyfKfPJvFq7arWK2V48tpbW3F0aNHzf1RkO6tav0yXdehKErNe7qIiGjn3njjDQDAxsaG3FWzQCAgN+17joYqu6rj2WwWg4ODuHDhAn7++Wdz79Hq6ipGRkbMWalcLodEImEWlvR6vUilUpiYmIAQApqm4dSpU5afV62CObj891LodVaOt5shMh5iSCQS0HUdqqri1q1buHjxItxud9V+2ezsLMLhsNlXPDtlPK0qL0sTEdHOhEIhAEA+n5e7arK8vIwzZ87IzfufXGNhp2KxmKVWk3F5PB4xMDAgnj17Zo7N5/Pi9u3b4tmzZ2JgYMAce/nyZdvijJqmidu3b5vtQgjx66+/ltQlooOluPinca8U15ES0n1lV/+rWr/Yrl8Vi8XkZrPWFYCSmmpERLQ7Q0NDtvWmhoaGLDnBbkwgELCtYbXfuYS8brOPqKqKUCiEjY0NRKNRc1ZJVVUUCgU0NzcjGo0imUyy3hAREdE+43K5sL6+XteG9dnZWfz444948OCB3LXvObr8txei0Sg0TUNHR4fZlk6ncfLkSbS0tGBxcRHpdNryHiIiInr51tfXEQgEai7k+e233+Krr746kIEKByFUYXv/S39/v/n6v65gLm92f1mP3hvFRqsVFjUOEQ4EAmbxTXnjv93hw8UmJydLKqIbstksmpqaKm72JyIi8vv9EELgwoULcleJ2dlZ/Pnnn/j999/lroNDXg/cL4r32xj7YYr3wBj7Z6LRqECZA3mdFIvFbPf8/FeqHT5cPKb4IGthc56isQ+p3B6kagcNy/vnyn0OERHRYbKv91TtJ8ZszMt+WjAej2NhYQG//PKL5Sk3Y/9ZKBQq+R2Xl5fR0NCAYDAIbM94nT17Fj6fr2QststfYLvavUxVVUxNTdk+xUdERHSYHYjlv5fNKBQpHxC9W7oDBw8bxsfHAQDXr1+Xu9DR0WEGKhSVEygnnU6jvb1dbga2+yYmJmpaQiQiIjpMGKpqYFSJrxREdsLtwMHD2J49SqVSOHfuHEKhUMl+KlmlkGi8x+5pSl3XkUgkzNeRSIT7qYiIiLYxVNWgUqXu3XLi4OF0Oo2NjQ1sbm4ilUpB0zQcO3YMoVDINljlcjl4PB6cPn1a7qo4S+UuOkBa0zR0d3cjmUxyxoqIiIihqjq9wgHRTnHi4GG/349EIgGv12tWG9/Y2LAtN3H37l2Mj4/b/pxKoaqYEbCi0eieP31JRER0EDBUVVHpgGin7Obg4XLa29tti63F43F0dnZa9lgZKi39lWO3hEhERHQYMVRVYbf0V1zzqZaaVUbdKLux6i4PHsZ2gML2LJOhUCjgyJEjln1g8lN92WwWy8vLlvd0dnaar2vFYEVERMRQVZHdAdEAsLKyglQqhXw+jwcPHtjuWzIYQUoIUfJEX70HD6PM4cNerxfDw8MYGRkxf5dEIoH333/fnJFSFAWRSARjY2Nm0c7z589bwmIul6tr31g2m8VPP/1kuzeLiIjo0JELV9G/5AKX5QpuXr58uWxB0OKCnXJBznrVcviwUQgV0gHB8nexG6Npmvj6669tv6Mhk8kIj8dT9W9CRER0GLH45y7duXMHn376qe2m7+LlNmPGSp6tIiIiolcDl/92QVEUvP766wCAnp4ey/mA8vl8XV1daGxsLHo3ERERvUoYqnZIURTMzc0hHA5b6jcZ1+LiItra2vDkyRMAwNbWFpqbm+WPISIiolcEQ9UOxONxRCIRJJPJise1BINBtLS0wOVymQGMiIiIXk3cU0VERETkAM5UERERETmAoYqIiIjIAQxVRERERA5gqCIiIiJyAEMVERERkQMYqoiIiIgcwFBFRERE5ACGKiIiIiIHMFQREREROYChioiIiMgBDFVEREREDvgHCqeT/bKrYrwAAAAASUVORK5CYII=\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003ehere, \u003cem\u003eI\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e represents the integral of the pyrazine ring protons (\u0026delta;\u0026thinsp;=\u0026thinsp;8.6\u0026ndash;8.8 ppm) from FA, and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e2\u0026thinsp;\u0026minus;\u0026thinsp;6\u003c/sub\u003e corresponds to the integral of dextran backbone protons (\u0026delta;\u0026thinsp;=\u0026thinsp;3.0\u0026ndash;3.7 ppm).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Preparation of blank and drug-loaded micelles\u003c/h2\u003e\n \u003cp\u003eThe FA-Dex-b-PZLL\u003csub\u003e10\u003c/sub\u003e (FDP-1) blank micelles and letrozole-loaded FA-Dex-b-PZLL\u003csub\u003e10\u003c/sub\u003e (LTZ/FDP-1) micelles were both prepared using the dialysis method [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. In brief, FDP-1 micelles (50 mg) were solubilized in 500 \u0026micro;L DMSO under ultrasonication to achieve homogeneous dispersion. Subsequently, the prepared solution was gradually added dropwise into deionized water (20 mL) under stirring to ensure complete mixing. Following transfer to a dialysis bag (MWCO 3500), the mixture underwent dialysis in 2000 mL deionized water for 48 h. The resulting dialysate was subsequently filtered through a 0.22 \u0026micro;m syringe filter to obtain a clear filtrate. Lastly, the filtrate was lyophilized to yield blank micelle solids. To prepare LTZ/FDP-1 micelles, 10 mg LTZ was solubilized into DMSO with 50 mg FDP-1 micelles. Other amphiphilic copolymers with different ratios of hydrophobic chains FA-Dex-b-PZLL\u003csub\u003e20\u003c/sub\u003e (FDP-2) and FA-Dex-b-PZLL\u003csub\u003e30\u003c/sub\u003e (FDP-3) blank micelles and corresponding LTZ-loaded (LTZ/FDP-2, LTZ/FDP-3) micelles were synthesized using a similar method.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Determination of the drug-loading performance of micelles\u003c/h2\u003e\n \u003cp\u003eA UV-Vis spectrophotometer was utilized to determine the actual encapsulation and drug-loading status of LTZ-loaded micelles [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. A standard curve (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e) was constructed by plotting the absorbance values at \u003cem\u003e\u0026lambda;\u003c/em\u003e\u0026thinsp;=\u0026thinsp;240 nm of letrozole standard solutions against their known concentrations (\u003cem\u003eC\u003c/em\u003e, \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e\n \u003cp\u003e10 mg of LTZ-loaded micelle was ultrasonically dissolved in 100 \u0026micro;L of DMSO to disrupt micellar structures and liberate encapsulated drug. Acetonitrile was then introduced at a volumetric ratio (DMSO:acetonitrile\u0026thinsp;=\u0026thinsp;1:9) to solubilize LTZ while precipitating FA-Dex-b-PZLL carriers. Following centrifugation (12,000 rpm, 10 min) and filtration through a 0.22 \u0026micro;m syringe filter, the UV absorbance of the filtrate was recorded. Triplicate measurements per sample ensured statistical validity, with data expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The measured absorbance values were substituted into the standard curve to determine the amount of LTZ encapsulated within micelles. The encapsulation efficiency (\u003cem\u003eEE\u003c/em\u003e%) as well as drug loading (\u003cem\u003eDL\u003c/em\u003e%) were then calculated [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]:\u003c/p\u003e\n \u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\n \u003cdiv class=\"EquationNumber\"\u003e\u003cimg 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\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6. Measuring critical micellar concentration\u003c/h2\u003e\n \u003cp\u003eThe dye solubilization technique reported by Alexandridis et al. [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e] was utilized to measure the critical micelle concentration (CMC) of FDP micelles in water. A blank micelle dispersion was prepared in water at an initial concentration of 1 mg\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Sequential dilutions were performed to generate 12 concentration gradients (0.001\u0026ndash;0.5 mg\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). To each 4 mL micellar dispersion (with increasing concentrations), 40 \u0026micro;L of a 0.4 mmol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DPH in methanol was added, yielding a final DPH concentration of 4\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The samples were then subjected to 24 h incubation under light-protected conditions at ambient temperature to ensure complete partitioning of DPH into the hydrophobic micellar cores. UV-visible spectra of DPH (300\u0026ndash;500 nm) were acquired using a TU-1901 spectrophotometer (Puxi Inc., Beijing, China). Absorbance maxima at \u003cem\u003e\u0026lambda;\u003c/em\u003e\u0026thinsp;=\u0026thinsp;350 nm were correlated with polymeric concentrations, with CMC determined via intersection point analysis of the biphasic curve in absorbance-concentration plots.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7. In vitro drug release\u003c/h2\u003e\n \u003cp\u003eThe pH-dependent release profile of LTZ/FDP micelles was examined utilizing dialysis. In a typical procedure, 3 mL of the micellar dispersion was loaded into dialysis bags and dialyzed against 47 mL of PBS solutions with pH values of 4.5, 6.5 and 7.4. The systems were incubated at 37\u0026deg;C with shaking (200 rpm) for 72 h. At predetermined time intervals (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 24, 30, 36, 48, 54, 60, and 72 h), 3 mL samples were collected and replaced with fresh buffer of equal volume to maintain sink conditions. Quantitative analysis was performed using UV spectrophotometry: sample absorbance was recorded at 240 nm (characteristic wavelength of letrozole), with drug concentrations subsequently determined by the pre-established letrozole standard curve. The cumulative release percentage, \u003cem\u003eCR\u003c/em\u003e (%), was determined by Eq. (\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003e\u003cimg 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nE4nqqurMTMzAwAIBAJwuVwoLy9XZ8QGAgGUl5djeHhYnSmr0+nQ2tqa9m8TERFRcXDR4DWqv78fmBti7e7uxr59+9DX1wcAGBoawu3btxGJRNT6F198Ec8//7x6/u677y5p71wiIiIqDPbMrVHxeBz19fUAgM2bN0Ov16OzsxN79uzJODmiqakJ9+7dg06nw927d2Gz2bRNiIiIqAgY5tYok8mEYDAIAJidncWJEyegKIpabzabsX79egDA48ePUV1djaqqKgghMDIykjHwERER0bPHYdY1SpZlOBwOPHjwAADg9/tx/fp1hEIhhEIhAEir/+yzz/D111+r79e53W5cvXo15RuJiIioGBjmKCuSJKGiogJGoxGKouCnn37C4cOHtc2IiIjoGeMwK2UlGAzCZDJBp9Nh48aNqK6u1jYhIiKiImDPHBEREVEJY88cERERUQljmCMiIiIqYQxzRERERCWMYY6IiIiohDHMEREREZUwhjkiIiKiEsYwR0RERFTCGOaIiIiIShjDHBEREVEJY5gjIiIiKmEMc0REREQljGGOiIiIqIQxzBERERGVMIY5IiIiohL2PyRk53BCmUkvAAAAAElFTkSuQmCC\"\u003e\u003c/p\u003e\n \u003cp\u003ewhere \u003cem\u003eV\u003c/em\u003e\u003csub\u003ee\u003c/sub\u003e is the replacement volume (3 mL), \u003cem\u003eV\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e is the total volume (50 mL), \u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e represents the concentration of letrozole at the \u003cem\u003ei\u003c/em\u003e-th sampling point, and \u003cem\u003em\u003c/em\u003e\u003csub\u003edrug\u003c/sub\u003e refers to the total mass of letrozole encapsulated in the micelles.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8. Biocompatibility experiment\u003c/h2\u003e\n \u003cp\u003eL-929 and MDA-MB-231 cells were cocultured with FDP blank micelles at different concentrations to evaluate their biocompatibility. In brief, when the monolayer coverage rate of cells reached 80%, they were digested with an appropriate amount of trypsin. The digested cells were then seeded into 96-well plates (1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well) and incubated for 24 h under standard conditions (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e). FDP blank micelle solutions at different concentrations were introduced and followed by an incubation for 48 h. Each sample has five parallel experiments. Finally, 100 \u0026micro;L of CCK-8 solution was introduced per well followed by incubation for 1 h. The optical density at 450 nm in each well was then recorded with a microplate reader in order to determine the cell viability [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]:\u003c/p\u003e\n \u003cdiv id=\"Equ5\" class=\"Equation\"\u003e\n \u003cdiv class=\"EquationNumber\"\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003ewhere \u003cem\u003eOD\u003c/em\u003e\u003csub\u003esample\u003c/sub\u003e, \u003cem\u003eOD\u003c/em\u003e\u003csub\u003eblank\u003c/sub\u003e and \u003cem\u003eOD\u003c/em\u003e\u003csub\u003econtrol\u003c/sub\u003e are the optical density of the sample wells, blank wells and control group, respectively.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Characterization of copolymer FA-Dex-b-PZLL\u003c/h2\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e3.1.1. FTIR analysis\u003c/h2\u003e\u003cp\u003eThe FTIR spectra of the raw materials and copolymers were acquired using the KBr pellet method, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Compared with native dextran (Dex), the typical peaks of Dex-NH\u003csub\u003e2\u003c/sub\u003e did not exhibit substantial changes, potentially due to the masking effect of polysaccharide\u0026rsquo;s stronger absorption bands over the signals from ethylenediamine [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In the spectrum of Lys(Z)-NCA precursor, the absorption bands at 1856 and 1775 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e correspond to the stretching vibrations of the dual carbonyl (C\u0026thinsp;=\u0026thinsp;O) in the cyclic anhydride structure, while the peaks observed at 749 and 695 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are assigned to the out-of-plane bending vibrations of the benzene ring\u0026rsquo;s C-H bonds [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The FTIR spectrum of Dex-b-PZLL exhibits three notable changes compared to the precursors: (1) The disappearance of cyclic anhydride peaks at 1856 and 1775 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicates successful ring-opening polymerization initiated by amino groups on dextran; (2) The absorption bands of benzene ring emerge in the 900\u0026thinsp;\u0026minus;\u0026thinsp;650 cm-1 region; (3) New absorption peaks appear at 1627 and 1535 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, corresponding to peptide bond vibrations in polyamino acid segments. Together, these spectral changes confirm the effective formation of Dex-b-PZLL block copolymer [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Furthermore, FA-Dex-b-PZLL displays additional characteristic peaks at 1570, 1311, 1193, 840 and 640 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which matches the distinctive vibrational modes of folic acid, thereby verifying its successful conjugation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.1.2. \u003csup\u003e1\u003c/sup\u003eH NMR analysis\u003c/h2\u003e\u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH NMR spectra of Dex, Dex-NH\u003csub\u003e2\u003c/sub\u003e, Dex-b-PZLL and FA-Dex-b-PZLL are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Comparative analysis of \u003csup\u003e1\u003c/sup\u003eH NMR spectra between dextran (Dex) and its amine-functionalized derivative (Dex-NH\u003csub\u003e2\u003c/sub\u003e) reveals the entire loss of the characteristic anomeric proton signal (δ\u0026thinsp;=\u0026thinsp;6\u0026ndash;7 ppm) associated with the terminal glucopyranose unit of Dex, confirming successful end-group amination via ethylenediamine modification [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Notably, the methylene (-CH\u003csub\u003e2\u003c/sub\u003e-) proton signals of ethylenediamine are not independently resolved in the spectrum due to resonance overlap with dextran backbone protons in the δ\u0026thinsp;=\u0026thinsp;3\u0026ndash;5 ppm region. For Dex-b-PZLL block copolymer, the peak at 7.31 ppm indicates the presence of aromatic protons from the benzene ring, while the methylene protons on the benzyloxycarbonyl group are located at 4.96 ppm. The peaks at 3.76 ppm, 1-1.68 ppm and 2.94 ppm are the proton absorption peaks of the secondary and methylene groups of the hydrophobic block polylysine [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The spectrum of FA-Dex-b-PZLL exhibit two new proton signals at 8.62 ppm and 6.63 ppm, corresponding to the pyrazine and aromatic protons of folic acid, respectively [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The degree of folate substitution is determined by integrating the characteristic folate proton signals relative to the dextran backbone protons using Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), yielding a substitution degree of 7.39 wt%, equivalent to approximately 1.08 folate molecules per dextran. These \u003csup\u003e1\u003c/sup\u003eH NMR analyses conclusively demonstrate the successful synthesis of the target folate-conjugated block copolymer FA-Dex-b-PZLL.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e3.1.3. GPC analysis\u003c/h2\u003e\u003cp\u003eThree block copolymers with varying molecular weights (Dex-b-PZLL\u003csub\u003e10\u003c/sub\u003e, Dex-b-PZLL\u003csub\u003e20\u003c/sub\u003e as well as Dex-b-PZLL\u003csub\u003e30\u003c/sub\u003e) were synthesized via ROP by adjusting the molar ratios of initiator to Lys(Z)-NCA (1:10, 1:20 and 1:30, respectively). The molecular weights and their distributions of the Dex-b-PZLL copolymers are characterized by GPC, as summarized in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Three GPC curves exhibit monomodal distributions with narrow polydispersity indices, indicating the absence of unreacted precursors and suggesting efficient polymerization processes. According to GPC separation principles, higher molecular weight components elute earlier, thus the left-shifted peaks in the chromatograms correspond to larger molecular weights. The combined analysis of GPC data and elution profiles confirms the successful preparation of these three distinct copolymers with controlled molecular weights.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Characterization of FDP and LTZ/FDP micelles\u003c/h2\u003e\u003cp\u003eUnder aqueous conditions, the copolymers form micelles once their concentrations exceed the CMC values, which are 0.04, 0.017 and 0.011 mg\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for FDP-1, FDP-2 and FDP-3, respectively (Fig. S2). The low CMC favors stable micelles suitable for prolonged circulation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The DLS analysis reveals both FDP-1 and LTZ/FDP-1 micelles have low PDI and narrow size distributions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), with sizes of 97.9 nm (PDI\u0026thinsp;=\u0026thinsp;0.119) as well as 113.9 nm (PDI\u0026thinsp;=\u0026thinsp;0.125), respectively. Similar trends were observed for FDP-2, FDP-3 and their corresponding LTZ-loaded micelles (Fig. S3). Interestingly, the LTZ/FDP-2 and FDP-2 micelles are similar in size, while the size of LTZ/FDP-3 micelles is considerably lower than its blank micelles. This may arise from the hydrophobic segments of the polymer have a larger molecular weight, which can generate higher intermolecular forces, thereby forming a more compact hydrophobic core. This tight structure may make the shell of the micelle more stable, which to some extent limits the expansion of the micelle and even causes a slight reduction in particle size [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe microstructure of FDP and LTZ/FDP micelles was examined by SEM. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, both FDP and LTZ/FDP micelles exhibit uniform spherical structures with well-defined boundaries and good dispersibility in aqueous solutions, with particle sizes below 150 nm. The slightly smaller particle sizes observed by SEM compared to DLS measurements could be attributed to the different measurement conditions: DLS analysis is performed in aqueous solutions while SEM samples are examined under vacuum-dried conditions [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. These nano-sized FDP and LTZ/FDP micelles offer prospect of enhancing tissue permeability and systemic bioavailability, thereby promoting the antitumor therapeutic effectiveness [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The ideal drug delivery system should exhibit high stability to prevent drug leakage during circulation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDLS was employed to monitor the variations in micelle size under normal physiological conditions (pH 7.4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Taking FDP-3 micelle as an example, after 96 h of incubation in PBS buffer (pH 7.4), the blank micelles retained an average hydrodynamic diameter of 136.08 nm. The micelles demonstrate good stability without significant disintegration in physiological environments, confirming their suitability as drug carriers for blood transport.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Drug-loading analysis\u003c/h2\u003e\u003cp\u003eThe drug loading capacities of LTZ/FDP-1, LTZ/FDP-2 and LTZ/FDP-3 micelles are approximately 7.12%, 8.49% and 7.81%, respectively, with encapsulation efficiencies of 38.33%, 46.39% and 42.36% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Experimental results reveal a nonlinear correlation between the drug encapsulation efficiency/loading capacity of FDP micelles as well as the molecular weight of hydrophobic segment (PZLL). Increasing the polymerization degree of PZLL chains enhance both the hydrophobic core volume expansion and hydrophobic association effects, thereby significantly improving the micelles\u0026rsquo; letrozole encapsulation capability. However, when the PZLL molecular weight exceed a critical threshold (e.g., LTZ/FDP-3 micelles), a slight decline in drug loading efficiency is observed. This phenomenon might be attributed to excessive molecular entanglement within elongated hydrophobic chains, which promoted structural densification of micelles and restricted drug diffusion into the core [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Additionally, overly strong hydrophobic interactions could induce premature micellar aggregation, potentially compromising the stability of drug-loaded structures.\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\u003eDrug-loading performance of the 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=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDrug-loaded micelles\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eDL\u003c/em\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eEE\u003c/em\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLTZ/FDP-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7.12%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e38.33%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLTZ/FDP-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8.49%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e46.39%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLTZ/FDP-3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7.81%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e42.36%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.4. In vitro drug release\u003c/h2\u003e\u003cp\u003eIn vitro release profiles of LTZ/FDP micelles under different pH values are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, all LTZ/FDP micelles exhibit a sustained slow-release behavior, which gradually accelerate with decreasing pH. For instance, the cumulative amounts of letrozole released from LTZ/FDP-1 micelles at 72 h were 15.14%, 18.02% and 50.46% at pH 7.4, 6.5 along with 4.5, respectively. This gradient release phenomenon is attributed to the pH-responsive chemical bond (amide bond) introduced in the micelle molecular design. Under acidic conditions, hydrolysis and amide bond cleavage induce polymer backbone dissociation, which disrupts the micellar core-shell structure and accelerates drug release [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The detailed cumulative release data for LTZ/FDP-2 and LTZ/FDP-3 micelles, which display similar pH-dependent release trends to LTZ/FDP-1 micelles, are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. A vertical comparison under the same pH conditions indicates that the release order is consistent with the micelles\u0026rsquo; drug loading capacity, both being strongly influenced by the PZLL chain length. A longer PZLL chain promotes hydrophobic core expansion and enhances drug release, but excessive chain length may cause core densification and restrict diffusion.\u003c/p\u003e\u003cp\u003eMoreover, the FA-Dex-b-PZLL micelles achieve dual-functional drug delivery: maintaining structural stability in physiological conditions to prolong circulation while enabling pH-triggered drug release in acidic tumor microenvironments. Combined with the EPR effect, this system enhances tumor drug accumulation while minimizing systemic toxicity. Its sustained release further reduces administration frequency, demonstrating promising translational potential for targeted cancer therapy.\u003c/p\u003e\u003cp\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\u003eComparison of in vitro release profiles of LTZ/FDP micelles.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" 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=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDrug-loaded micelles\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003epH\u0026thinsp;=\u0026thinsp;7.4\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003epH\u0026thinsp;=\u0026thinsp;6.5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003epH\u0026thinsp;=\u0026thinsp;4.5\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLTZ/FDP-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e15.14%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e18.02%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e50.46%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLTZ/FDP-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20.74%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e27.14%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e62.96%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLTZ/FDP-3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e17.57%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e23.67%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e56.36%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.5. In vitro biocompatibility\u003c/h2\u003e\u003cp\u003eBiocompatibility evaluation is essential for assessing the biosafety of biomaterials intended for in vivo applications, providing fundamental data for subsequent drug delivery system development or biomedical material utilization to ensure their stability and safety in biological environments. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, the biocompatibility test results reveal that the cell viability slightly decreased with increasing micelle concentration, it remained above 90% even at the maximum micelle concentration (200 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). These findings demonstrate that FDP blank micelles exerted no inhibitory effects on cellular proliferation, indicating the absence of cytotoxic effects from blank micelles and confirming their excellent biocompatibility.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eHerein, a series of folic acid modified amphiphilic dextran-b-poly(ε- benzyloxycarbonyl-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-lysine) (FA-Dex-b-PZLL) copolymers with varying hydrophobic segment lengths are successfully synthesized by reduction amidation, ring-opening polymerization and esterification reaction. All copolymers can self-assemble via dialysis into uniformly dispersed spherical micelles in an aqueous medium with a low critical micelle concentration. The polymer micelles possess tumor targeting properties and pH responsiveness. By adjusting the hydrophilic-to-hydrophobic ratio of the amphiphilic FA-Dex-b-PZLL carrier, precise modulation of drug encapsulation efficiency and release efficiency is achieved. The micelles maintain good stability in normal physiological conditions, while showing rapid payload release in acidic tumor microenvironment. Cytocompatibility assessments reveal negligible cytotoxicity (cell viability\u0026thinsp;\u0026gt;\u0026thinsp;90%) and excellent biocompatibility of blank carriers. These findings validate this pH-responsive nanosystem as a versatile nanoplatform for hydrophobic chemotherapeutics delivery with enhanced therapeutic outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflicts of interest\u003c/h2\u003e\u003cp\u003eThere are no conflicts to declare.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eSupporting Information\u003c/h2\u003e\u003cp\u003eMolecular weight of block copolymers Dex-b-PZLL (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). UV-vis absorption spectra and standard curve of letrozole (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). CMC of FDP micelles (Fig. S2). Particle size distribution of FDP and LTZ/FDP micelles (Fig. S3).\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRuoxi Huang: Project administration, Writing - review \u0026amp; editing. Yaxin Yi: Data curation, Writing - original draft. Shiyu Zhang: Visualization, Methodology. Jiaxin Wang: Formal analysis, Validation. Haikuan Yuan: Conceptualization, Investigation. Jie Lu: Supervision, Funding acquisition. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors appreciated the National Natural Science Foundation of China (Nos. 21978165 and 92156020) and the Natural Science Foundation of Shanghai Municipality (Grant No. 25ZR1401151) to support this work.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang G, Zhang X, Kapilevich L, Hu M (2023) Recent advances in polymeric microparticle-based drug delivery systems for knee osteoarthritis treatment. 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ACS Omega 7:21000-21013. https://doi.org/10.1021/acsomega.2c01792\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"polymer-bulletin","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pobu","sideBox":"Learn more about [Polymer Bulletin](http://link.springer.com/journal/289)","snPcode":"289","submissionUrl":"https://submission.nature.com/new-submission/289/3","title":"Polymer Bulletin","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Amphiphilic polymer, Micelles, Drug delivery, pH-responsive, Biocompatibility","lastPublishedDoi":"10.21203/rs.3.rs-7201841/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7201841/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA stimulus-responsive drug delivery system holds promise for reducing adverse effects and enhancing the bioavailability of chemotherapeutic agents. In this study, amino-terminated dextran was utilized as a macromolecular initiator to prepare a range of amphiphilic dextran-b-poly(ε-benzyloxycarbonyl-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-lysine) (Dex-b-PZLL) copolymers with varying hydrophobic segment lengths via ring-opening polymerization (ROP). Folic acid (FA) was further grafted onto the dextran side chains via esterification (FA-Dex-b-PZLL), offering the potential for tumor-specific recognition. The resulting copolymers were able to self-assemble into stable spherical micelles via dialysis and efficiently encapsulate the hydrophobic drug letrozole (LTZ). A systematic assessment was conducted on the drug encapsulation efficiency/loading capacity, drug release performance in vitro, and biocompatibility of the copolymers under aqueous conditions, indicating that these properties of the FA-Dex-b-PZLL micelles mainly depended on the copolymer composition. The optimized micelles, with a balanced hydrophilic/hydrophobic ratio, achieved a drug loading capacity of approximately 8.49% and an encapsulation efficiency of about 46.39%. Moreover, LTZ/FA-Dex-b-PZLL micelles exhibited pH-responsive release, with cumulative release rates within 72 h of 20.74%, 27.14% and 62.96% in PBS at pH 7.4, 6.5 and 4.5, respectively, indicating accelerated release under acidic conditions. In addition, the micelles showed good cytocompatibility according to the CCK-8 assay.\u003c/p\u003e","manuscriptTitle":"Folic acid grafted amphiphilic dextran-b-poly(ε-benzyloxycarbonyl-L-lysine) micelles for drug’s pH-responsive release","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-02 06:27:49","doi":"10.21203/rs.3.rs-7201841/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-22T11:33:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-14T14:38:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210220619477425601865294647824681356666","date":"2025-10-10T13:27:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-10T08:24:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"165798025913031829190065306359347223147","date":"2025-10-07T05:02:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"274302198594157580460834219925554208732","date":"2025-10-06T13:06:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-24T14:38:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-29T12:19:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-28T15:06:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Polymer Bulletin","date":"2025-07-24T05:42:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"polymer-bulletin","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pobu","sideBox":"Learn more about [Polymer Bulletin](http://link.springer.com/journal/289)","snPcode":"289","submissionUrl":"https://submission.nature.com/new-submission/289/3","title":"Polymer Bulletin","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b289bafe-5ac1-4b02-98a9-5f56c75bc479","owner":[],"postedDate":"September 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:02:35+00:00","versionOfRecord":{"articleIdentity":"rs-7201841","link":"https://doi.org/10.1007/s00289-025-06092-9","journal":{"identity":"polymer-bulletin","isVorOnly":false,"title":"Polymer Bulletin"},"publishedOn":"2025-12-04 15:57:13","publishedOnDateReadable":"December 4th, 2025"},"versionCreatedAt":"2025-09-02 06:27:49","video":"","vorDoi":"10.1007/s00289-025-06092-9","vorDoiUrl":"https://doi.org/10.1007/s00289-025-06092-9","workflowStages":[]},"version":"v1","identity":"rs-7201841","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7201841","identity":"rs-7201841","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}