Inhalable Porous PLGA Microspheres Enable Lung-Targeted Dual-Drug Delivery for the Treatment of High-Altitude Pulmonary Hypertension

Inhalable Porous PLGA Microspheres Enable Lung-Targeted Dual-Drug Delivery for the Treatment of High-Altitude Pulmonary Hypertension | 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 Inhalable Porous PLGA Microspheres Enable Lung-Targeted Dual-Drug Delivery for the Treatment of High-Altitude Pulmonary Hypertension Na Li, Xiangbo Zheng, Qing He, Yuan Cheng, Tao Jin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8275947/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background High-altitude pulmonary hypertension (HAPH), driven by hypobaric hypoxia–induced vasoconstriction and vascular remodeling, remains a therapeutic challenge due to the limited efficacy and systemic side effects of current treatments. This study aimed to develop a dual-drug–loaded microsphere system for targeted pulmonary delivery. Methods Porous PLGA microspheres co-loaded with hydrophilic L-arginine and hydrophobic tadalafil (PLGA-LA/Tada MSs) were fabricated using a gas-foamed double-emulsion technique. The microspheres were characterized for morphology, porosity, aerodynamic properties, and in vitro release profiles. In vivo pharmacokinetics, safety, and therapeutic efficacy were evaluated following a single intratracheal administration in a chronic hypobaric hypoxia–induced HAPH rat model. Results The PLGA-LA/Tada MSs displayed uniform spherical morphology, well-developed porosity, and favorable aerodynamic behavior (MMAD ~ 4.7 µm), supporting deep-lung deposition. In vitro, L-arginine exhibited rapid release, whereas tadalafil showed sustained release. A single intratracheal dose achieved efficient pulmonary deposition, prolonged lung retention, and reduced systemic exposure. Therapeutically, PLGA-LA/Tada MSs improved RVSP, mPAP, and Fulton’s index; mitigated fibrosis and vascular muscularization; and preserved alveolar structure. Mechanistic studies indicated restored NO/cGMP signaling, reduced oxidative stress and inflammation, and normalization of erythropoietic markers. Conclusion PLGA-LA/Tada MSs enable spatially and temporally coordinated pulmonary delivery of hydrophilic and hydrophobic agents, allowing synchronized modulation of the NO–cGMP axis. This dual-drug microsphere platform provides durable pulmonary vascular protection with minimal systemic exposure and demonstrates strong translational potential for treating HAPH and other hypoxia-driven pulmonary diseases. High-altitude pulmonary hypertension Porous PLGA microspheres Dual-drug delivery L-arginine and Tadalafil Pulmonary targeting Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights Localized dual-drug pulmonary delivery : Porous PLGA microspheres enabled intratracheal co-delivery of hydrophilic L-arginine and hydrophobic tadalafil, achieving high lung specificity and reduced systemic exposure. Spatiotemporally coordinated release : The formulation provided rapid release of L-arginine and sustained tadalafil delivery, synergistically restoring the NO/cGMP pathway. Mechanism-driven vascular protection : Microspheres significantly attenuated oxidative stress, suppressed inflammation, and improved endothelial signaling in high-altitude pulmonary hypertension. Functional and structural reversal : A single intratracheal dose effectively improved hemodynamics, reduced right ventricular hypertrophy, and reversed pulmonary vascular remodeling. Therapeutic potential for HAPH : PLGA-LA/Tada MSs represent a simplified and efficient inhalation platform for targeted, sustained treatment of high-altitude pulmonary hypertension. 1. Introduction Pulmonary hypertension (PH) is a progressive cardiopulmonary disorder characterized by elevated pulmonary arterial pressure and vascular remodeling, ultimately leading to right ventricular failure and high mortality [ 1 , 2 ]. Among its subtypes, high-altitude pulmonary hypertension (HAPH) is a distinct form induced by chronic exposure to hypobaric hypoxia, which triggers sustained pulmonary vasoconstriction, inflammation, oxidative stress, and excessive erythropoiesis [ 3 , 4 ]. These pathological changes collectively contribute to increased pulmonary vascular resistance and maladaptive right heart remodeling [ 5 , 6 ]. Despite its global relevance in populations residing or working at high-altitudes, effective therapeutic options for HAPH remain limited [ 4 ]. Nitric oxide (NO) signaling plays a central role in maintaining pulmonary vascular tone and endothelial homeostasis [ 7 , 8 ]. In HAPH, chronic hypobaric hypoxia suppresses endothelial nitric oxide synthase (eNOS) activity and accelerates cyclic guanosine monophosphate (cGMP) degradation through phosphodiesterase-5 (PDE5), ultimately leading to sustained vasoconstriction and pulmonary vascular remodeling [ 8 – 10 ]. Although several pharmacological agents—including oxygen therapy, calcium channel blockers, prostacyclins, endothelin receptor antagonists, and phosphodiesterase inhibitors—have shown potential in mitigating pulmonary hypertension, their clinical efficacy in HAPH remains limited due to unclear mechanisms of action in the context of hypobaric hypoxia, poor lung selectivity, rapid systemic clearance, and the need for frequent administration [ 4 , 11 , 12 ]. Descent to lower altitudes remains the most effective intervention; however, for patients who continue to reside at high-altitude, there is a pressing need for therapeutic strategies that offer improved pulmonary targeting and prolonged efficacy under hypoxic stress. To address these challenges, pulmonary drug delivery has emerged as a promising approach, enabling localized, sustained release of vasodilatory agents directly to the lungs, thereby enhancing therapeutic precision while minimizing systemic side effects [ 13 – 16 ]. Recent studies on inhalable formulations of phosphodiesterase inhibitors—such as tadalafil nanocomposites and spray-dried sildenafil microparticles—have demonstrated enhanced pulmonary targeting and prolonged therapeutic action in the treatment of pulmonary arterial hypertension [ 17 , 18 ]. Microsphere-based carriers, particularly those formulated with biodegradable polymers like poly(lactic-co-glycolic acid) (PLGA), offer unique advantages in terms of controlled release, biocompatibility, and lung deposition [ 12 , 19 , 20 ]. Recent studies—for instance, Rashid et al.—have demonstrated that inhaled PLGA-encapsulated sildenafil can effectively treat pulmonary arterial hypertension at reduced doses and with extended dosing intervals compared to the oral formulation, highlighting the potential of PLGA microspheres as an alternative to systemic administration [ 12 ]. However, the co-encapsulation and coordinated delivery of hydrophilic and hydrophobic agents with distinct pharmacokinetics remain technically challenging [ 21 – 24 ]. In this study, we developed a dual-drug-loaded porous PLGA microsphere system co-encapsulating hydrophilic L-arginine and hydrophobic tadalafil (PLGA-LA/Tada MSs) using a gas-foamed double emulsion method. The microspheres were engineered to achieve favorable aerodynamic properties for deep-lung delivery and to enable a biphasic release profile for synchronized pharmacological action. We hypothesized that this system could locally restore NO/cGMP signaling, mitigate oxidative and inflammatory injury, and reverse vascular remodeling in HAPH. Through comprehensive physicochemical characterization, pharmacokinetic analysis, and in vivo efficacy evaluation in a hypobaric hypoxia-induced HAPH rat model, we aimed to establish the therapeutic potential of PLGA-LA/Tada MSs as a multifunctional, inhalable platform for the treatment of HAPH. 2. Materials and Methods 2.1 Materials and Reagents Poly(D,L-lactide-co-glycolide) (PLGA, RG503H; lactide:glycolide = 50:50; acid-terminated; inherent viscosity ~ 0.32 dL/g; Cat# RG503H, Sigma-Aldrich, USA) was used at 200 mg per batch for microsphere preparation. Dichloromethane (DCM, Cat# 270997, Sigma-Aldrich) and polyvinyl alcohol (PVA, 87–89% hydrolyzed, Mw 30,000–70,000; Cat# 363065, Sigma-Aldrich) were used as received. Ammonium bicarbonate (ABC, Cat# 09830, Sigma-Aldrich) was used at 40 mg/mL as the porogen solution. L-arginine (≥ 98%, Cat# HY-N0455, MedChemExpress, China) and tadalafil (≥ 98%, Cat# HY-90009A, MedChemExpress) were incorporated into the internal and external phases, respectively. Alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) activities were quantified using commercial kits from Leidu Biotechnology (ALP: Cat# S03038, Lot# 20240412; LDH: Cat# S03034, Lot# 20240329), following the manufacturer’s instructions. eNOS protein expression was analyzed using a Western Blot detection kit (Sevier Biotechnology, China; Cat# WB-eNOS-01, used according to the manufacturer’s protocol). Cyclic GMP (cGMP) and nitrate (NO₃⁻) levels were measured using ELISA kits from Jinmei Biotech (Cat# JM-01434R1 and JM-11652R2, Lot# 202501). Nitrite (NO₂⁻) levels were quantified with a Nitrite Assay Kit (Nanjing Jiancheng Bioengineering Institute; Cat# A038-1, Lot# 20250414). Inflammatory and oxidative stress markers were assessed using ELISA kits. Rat TNF-α and IL-6 were measured with uncoated ELISA kits from Thermo Fisher Scientific (Cat# 88-7340-88 and 88-50625-88; Lot# 378167-000 and 382209-000). Catalase (CAT) and reactive oxygen species (ROS) were examined using research-use-only ELISA kits from Jinmei Biotech (Cat# JM-10334R2, JM-10531R2; Lot# 202501). Malondialdehyde (MDA) and total/oxidized glutathione (GSH/GSSG) levels were quantified using corresponding kits from Servicebio (MDA: Cat# G4302, Lot# MPC2412127; GSH/GSSG: Cat# G4304, Lot# MPC2412126). Protein concentrations were determined using a BCA assay kit (Servicebio; Cat# G2026-1000T, Lot# CR2503100-1). Sprague–Dawley (SD) healthy rats (6–8 weeks old, 200–250 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China; License No. H0.1100 11241106222542). All experimental procedures were approved by the Ethics Committee of Southwest Jiaotong University (Approval No. 2024110). Animal studies were conducted in accordance with the ARRIVE guidelines and institutional regulations governing the care and use of laboratory animals. Rats were housed under standard specific pathogen-free (SPF) conditions with free access to food and water. Group allocation was performed using a random number table. After randomization, investigators responsible for conducting the experiments, assessing outcomes, and performing data analysis were aware of the group assignments. Anaesthesia was induced by intraperitoneal injection of tribromoethanol, and all possible measures were taken to minimize animal discomfort and suffering. Animals that died during the experiments were excluded from analysis, resulting in the removal of four rats in total. 2.2 Preparation of Dual-Drug Loaded Porous PLGA PLGA-LA/Tada MSs Porous PLGA microspheres co-encapsulating a hydrophilic agent (L-arginine) and a hydrophobic agent (tadalafil) were prepared via a water-in-oil-in-water (W/O/W) double emulsion solvent evaporation method and are herein referred to as PLGA-LA/Tada MSs. This formulation strategy leverages the physicochemical complementarity of both drugs to achieve spatial compartmentalization within the polymer matrix [ 25 , 26 ]. Briefly, 200 mg of PLGA was dissolved in 7 mL of DCM to form the oil phase. Tadalafil, due to its lipophilic nature, was co-dissolved in the DCM with PLGA, facilitating uniform dispersion within the hydrophobic polymer matrix. In parallel, the hydrophilic drug L-arginine was fully dissolved in 1 mL deionized water containing 4% (w/v) ABC, which served both as the internal aqueous phase and as a gas-forming porogen to generate internal pores. The primary W/O emulsion was formed by homogenizing the L-arginine/ABC solution with the PLGA/DCM solution at 3,000 rpm for 2 minutes. This emulsion was then added to 50 mL of 1% (w/v) PVA and homogenized again at 3,000 rpm for 2 minutes to yield the W/O/W double emulsion. Subsequently, the emulsion was transferred to 100 mL of 1% PVA solution and stirred magnetically at room temperature for 5 hours to allow complete DCM evaporation and microsphere solidification. The resulting microspheres were collected by centrifugation, washed four times with deionized water to remove residual PVA and unencapsulated drugs, and lyophilized for 16 hours. The ABC concentration was adjustable to modulate porosity and density, thereby tuning both aerodynamic properties and biphasic drug release behavior. 2.3 Characterization of Porous PLGA-LA/Tada MSs The surface morphology, pore structure, and particle integrity of PLGA-LA/Tada MSs were examined using a field-emission scanning electron microscope (FE-SEM, S-4800, HITACHI, Japan) equipped with energy-dispersive X-ray spectroscopy (EDX, HORIBA EMAX mics2). Samples were diluted (1:5) with ultrapure water, and 20 µL of the diluted suspension was dropped onto a clean silicon wafer and air-dried for 2 h. Before imaging, samples were sputter-coated with gold using an MC1000 ion sputter coater (HITACHI, Japan) and dried with a critical point dryer (Tousimis Autosamdri-815). Images were acquired under accelerating voltages of 3 or 5 kV, with a resolution of 50 nm. Particle size was quantitatively assessed by analyzing SEM images using ImageJ software, with measurements taken from over 200 microspheres per group. 2.3.1 Specific Surface Area and Porosity Specific surface area and porosity of PLGA-LA/Tada MSs were determined by nitrogen adsorption–desorption analysis using the Brunauer–Emmett–Teller (BET) method with a TriStar 3000 analyzer (Micromeritics, USA) [ 27 , 28 ]. Freeze-dried samples were pretreated by vacuum degassing and analyzed under nitrogen atmosphere to determine specific surface area, pore volume, and pore size distribution. Pore size distribution was calculated based on the Barrett–Joyner–Halenda (BJH) model. 2.3.2 Aerodynamic Properties The mass median aerodynamic diameter (MMAD) and fine particle fraction (FPF) were measured using an aerodynamic particle sizer (APS 3321, TSI, USA) [ 29 , 30 ]. Samples were aerosolized with high-purity argon and passed through the spectrometer under set conditions (particle size range: 1–100 µm; sampling rate: 1000/s). The MMAD and FPF were calculated from the cumulative mass distribution provided by APS 3321 software, with FPF defined as the percentage of particles ≤ 5 µm in aerodynamic diameter [ 31 , 32 ]. To assess repeatability, three independent batches were tested under identical conditions. 2.3.3 Surface Charge The surface charge (zeta potential) of the microspheres was measured using a Malvern Zetasizer Nano ZS90 (Malvern Instruments, UK) with a He-Ne laser (633 nm) at a 90° scattering angle. Measurements were conducted at 25°C with 100 µL of nanoparticle dispersion in a cuvette. Each sample was analyzed in triplicate, with 20 measurement cycles per run and 10 s delay between cycles. Data were analyzed using Malvern Dispersion Technology Software 4.2, and average values were reported. 2.3.4 Crystallinity and Thermal Properties X-ray diffraction (XRD) analysis was performed using a Bruker D8 DISCOVER diffractometer. Freeze-dried microsphere powders (10 mg) were scanned over a 2θ range of 10°–30° at room temperature to evaluate the crystalline or amorphous nature of the encapsulated drugs and polymer matrix. Differential scanning calorimetry (DSC) was conducted using a DSC 3500 Sirius system. Approximately 20 mg of freeze-dried sample was sealed in an aluminum pan and analyzed under argon flow. The sample was cooled to 20°C, then heated to 250°C at a rate of 10°C/min to evaluate thermal transitions such as glass transition temperature (Tg) and melting behavior. 2.3.4 Drug Loading and Encapsulation Efficiency Drug loading (LE) and encapsulation efficiency (EE) of L-Arg and Tadalafil were quantified using high-performance liquid chromatography (HPLC, 1260 Infinity II, Agilent Technologies, USA). L-Arg and Tadalafil were detected at 220 nm and 280 nm, respectively. After centrifugation (8000 rpm, 4°C, 20 min), the supernatant was collected for quantification. LE and EE were calculated using the following equations: $$\:EE\left(\%\right)=\frac{{C}_{1}{V}_{1}-{C}_{2}{V}_{2}}{{C}_{1}{C}_{1}}\times\:100$$ $$\:LE\left(\%\right)=\frac{{C}_{1}{V}_{1}-{C}_{2}{V}_{2}}{W}\times\:100$$ where C 1 and V 1 represent the initial drug concentration and volume, C 2 and V 2 refer to the drug concentration and volume in the filtrate, and W is the mass of the freeze-dried microspheres. 2.4 In vitro drug release The in vitro release profiles of L-arginine and tadalafil from PLGA-LA/Tada MSs were evaluated in simulated lung fluid (SLF). Drug-loaded microspheres were placed in dialysis bags with a molecular weight cut-off (MWCO) of 1 kDa and incubated at room temperature in SLF under gentle agitation. At predetermined time points (0, 0.5, 0.75, 1, 2, 3, 4, 5, 6, and 17 h), aliquots of the external SLF medium were collected, centrifuged to remove any particulate matter, and analyzed using high-performance liquid chromatography (HPLC) to determine the concentrations of released L-arginine and tadalafil. After each sampling, the withdrawn volume was replaced with an equal volume of fresh SLF to maintain sink conditions. All experiments were conducted in triplicate using independently prepared batches of drug-loaded microspheres. 2.5 Pharmacokinetic Studies Sprague–Dawley rats were used for all pharmacokinetic studies and were acclimated for one week under SPF conditions prior to experimentation. Two separate studies were conducted to evaluate plasma and pulmonary pharmacokinetics of tadalafil. Pharmacokinetic analysis was limited to tadalafil, as L-arginine is an endogenous molecule with high and variable baseline levels in biological matrices, making accurate differentiation of exogenous levels challenging. Moreover, since L-arginine primarily exerts a local pulmonary effect via NO signaling, systemic pharmacokinetics are less relevant to the study aims. For plasma pharmacokinetics, six rats were randomly divided into two groups (n = 3 each) receiving a single dose of either free tadalafil via oral gavage (10 mg/kg) or PLGA-LA/Tada MSs via intratracheal instillation (10 mg/kg). Blood samples were collected at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 30, 36, and 48 hours post-administration. For pulmonary pharmacokinetics, thirty rats were divided into two groups (n = 15 each) receiving the same treatments. At predetermined time points (1, 3, 6, 10, and 24 hours), animals (n = 3 per time point) were sacrificed to collect bronchoalveolar lavage fluid (BALF) and lung tissues for pharmacokinetic analysis. Tadalafil concentrations in all biological matrices were quantified using a validated LC–MS/MS method. Everolimus-d₄ served as the internal standard to correct for variations in sample preparation and instrument response. Data were processed using Xcalibur software (Thermo) with linear regression and 1/X² weighting. 2.6 Safety Profile of PLGA-LA/Tada MSs in rats To preliminarily evaluate the pulmonary safety and tolerability of PLGA-LA/Tada MSs, Sprague–Dawley rats were used. Animals were housed under SPF conditions and acclimatized for one week. Following ethical approval and verification of animal health certification, rats were randomly assigned to three groups (n = 3/group): (1) negative control (intratracheal saline), (2) positive control (0.1% sodium dodecyl sulfate, SDS), and (3) microsphere group (PLGA-LA/Tada MSs, 10 mg/kg, intratracheal). At 12 h post-administration, blood was collected via the abdominal aorta, and serum was analyzed for LDH-L and ALP, which are commonly used markers for acute pulmonary epithelial injury. In addition, pharmacokinetic profiles in BALF, lung tissue, and serum were referenced to support the safety assessment. 2.7 In Vivo Hypobaric Hypoxia-Induced HAPH Model and Drug Administration Sprague–Dawley rats were housed under SPF conditions with free access to standard chow and water, maintained on a 12-hour light/dark cycle at an ambient temperature of 22 ± 2°C and relative humidity of 35 ± 5%. After one week of acclimatization, animals were randomly assigned to five groups (n = 4–6 per group): normoxic control, hypobaric hypoxia-induced pulmonary hypertension model (hypoxia-PH model), oral tadalafil treatment (hypoxia-TAD), oral tadalafil plus intratracheal L-arginine (hypoxia-TAD + L-Arg), and PLGA-LA/Tada microspheres group (hypoxia-MSs). Except for the normoxic control group, all rats were fasted for 10 hours (with free access to water) prior to exposure to simulated high-altitude hypobaric hypoxia (equivalent to 5000 m, 10% oxygen) in a decompression chamber for four consecutive weeks. During the hypobaric hypoxia exposure period, animals were monitored daily for general appearance and behavior. Upon completion of the four-week modeling phase, a single dose of the assigned treatment (10 mg/kg) was administered immediately. Twelve hours after dosing, all animals were evaluated for physiological, biochemical, and histopathological parameters. Hemodynamic parameters including mean pulmonary arterial pressure (mPAP) and right ventricular systolic pressure (RVSP) were assessed. RVSP was directly measured via right heart catheterization, and mPAP was estimated using the validated formula mPAP ≈ 0.61 × RVSP + 2 for rats [ 33 , 34 ]. The RVSP trend was used as an indirect indicator of pulmonary vascular resistance. Anticoagulated whole blood was collected and separated into two aliquots: one for plasma-based ELISA quantification of nitrate (NO₃ ⁻ ) and nitrite (NO₂ ⁻ ), and the other for hematological analysis including red blood cell count (RBC), hemoglobin (Hb), and hematocrit (Hct). The heart was excised, and the right ventricle (RV), left ventricle (LV), and septum (S) were weighed to calculate Fulton’s index [RV/(LV + S)] as a measure of right ventricular hypertrophy. The RV tissue was also frozen for subsequent ELISA analysis of cyclic GMP (cGMP). The right lung was divided for multiple analyses: one portion was used for Western blotting to assess endothelial nitric oxide synthase (eNOS) monomer and dimer expression; another was used for biochemical assays of oxidative stress markers including malondialdehyde (MDA), glutathione/glutathione disulfide (GSH/GSSG), and catalase (CAT); and a third was processed for ELISA detection of reactive oxygen species (ROS), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and cGMP. The left lung was fixed in 4% paraformaldehyde and embedded for histological analysis, including hematoxylin and eosin (H&E) staining, Masson’s trichrome staining, and immunohistochemical (IHC) detection of α-smooth muscle actin (α-SMA). 2.8 Statistical analysis All continuous data are presented as mean ± standard deviation (SD). Statistical comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A two-tailed P-value < 0.05 was considered statistically significant. All statistical analyses were conducted using Stata 17.0 software. 3. Results 3.1 Fabrication and Characterization of Porous PLGA-LA/Tada MSs Dual-drug-loaded porous PLGA microspheres were successfully fabricated using a double emulsion method with ammonium bicarbonate as a porogen. The resulting microspheres exhibited an internal porous structure suitable for inhalation-based administration. Representative SEM images (Fig. 1 A) show that the microspheres possessed a spherical morphology with smooth outlines, no visible aggregation, and well-maintained structural integrity. At higher magnification, the microspheres displayed uniformly distributed nanoscale pores. The particle size distribution (Fig. 1 B) revealed a narrow range of 11–14 µm, with an average diameter of approximately 12.0 µm, consistent with SEM observations. Nitrogen adsorption–desorption isotherms and BJH analysis (Fig. 1 C) showed a Type IV isotherm with a pronounced hysteresis loop at P/P₀ >0.8, and a pore size distribution centered around 55 nm (Fig. 1 D). The BET surface area was 360.43 m²/g, and the total porosity reached 71.2%. Aerodynamic characterization showed MMAD values of 4.2, 5.1, and 4.7 µm, with corresponding FPF values of 42%, 40%, and 45% across three batches, indicating suitable aerosol properties. The zeta potential values were − 18.9 mV, − 18.6 mV, and − 17.9 mV, suggesting negatively charged, moderately stable particles. XRD analysis (Fig. 1 E) revealed no distinct diffraction peaks, and DSC thermograms (Fig. 1 F) showed a broad glass transition temperature near 59°C, consistent with an amorphous PLGA matrix. The encapsulation efficiency (EE) and drug loading (LE) of tadalafil were 50.7% and 29.6%, respectively, while those of L-arginine were 75.5% and 22.0%. 3.2 In Vitro Drug Release Behavior of L-Arginine and Tadalafil from PLGA-LA/Tada MSs The release profiles of L-arginine and tadalafil from PLGA-LA/Tada MSs were determined in simulated lung fluid (Fig. 2 A). L-arginine exhibited a pronounced burst release, reaching over 26% within the first hour and nearly complete release (~ 88%) by 48 h. In contrast, tadalafil displayed a slower, sustained release, with ~ 14.5% released at 0.75 h and a cumulative release of 87.0% at 48 h. 3.3 Pharmacokinetic Behavior in Plasma, BALF, and Lung Tissue of PLGA-LA/Tada MSs Following a single intratracheal administration, the pharmacokinetics of tadalafil were assessed in plasma, BALF, and lung tissue, with oral free tadalafil serving as the control. In plasma (Fig. 2 B), oral tadalafil achieved a Cmax of 1464.8 ± 181.6 ng/mL at 4 h, whereas PLGA-LA/Tada MSs reached a lower Cmax of 714.4 ± 53.5 ng/mL at 0.5 h, with sustained levels up to 20 h. The AUC₀–∞ for the microsphere group (3620.6 ± 830.9 h·ng/mL) was markedly reduced compared to the oral group (14408.6 ± 2337.9 h·ng/mL). In BALF (Fig. 2 C), PLGA-LA/Tada MSs showed an earlier Cmax (1077.6 ± 152.3 ng/mL at 1 h) than the oral formulation (1156.7 ± 390.4 ng/mL at 3 h). Drug concentrations were maintained for up to 24 h, with an MRTlast of 5.6 ± 0.5 h. In lung tissue (Fig. 2 D), the microsphere group exhibited a higher Cmax (6580.2 ± 683.7 ng/g at 1 h) than the free tadalafil group (6135.7 ± 780.3 ng/g at 3 h), along with prolonged retention and an extended half-life. 3.4 Preliminary Safety Evaluation of PLGA-LA/Tada MSs At 12 h post intratracheal administration, serum LDH-L levels in the PLGA-LA/Tada MSs group were significantly lower than in the saline and SDS groups (p < 0.05, Fig. 2 E), suggesting minimal cytotoxicity. ALP levels showed no significant differences among groups. The reduced ALP value in the microsphere group compared to the SDS group indicates a mild irritative response. 3.5 Hemodynamic Improvement by PLGA-LA/Tada MSs In the hypobaric hypoxia model, both RVSP (Fig. 3 A) and mPAP (Fig. 3 B) were significantly elevated compared with normoxic controls (p < 0.05), confirming the successful establishment of HAPH. Oral tadalafil monotherapy produced slight but non-significant reductions in RVSP and mPAP. In contrast, combined oral tadalafil and intratracheal L-arginine treatment significantly lowered both parameters (#p < 0.05). Notably, PLGA-LA/Tada MSs markedly reduced RVSP and mPAP (#p < 0.05), achieving therapeutic effects comparable to the combination therapy through a single intratracheal dose. 3.6 Attenuation of Right Ventricular Hypertrophy by PLGA-LA/Tada MSs Right ventricular hypertrophy was assessed by Fulton’s index [RV/(LV + S)] (Fig. 3 C). The hypoxia model group exhibited a significant increase compared with the normoxic group (p < 0.05). Both oral tadalafil and the combination therapy significantly reduced the index (#p < 0.05). The PLGA-LA/Tada MSs group also showed a significant reduction (#p < 0.05), restoring the index to levels comparable with the normoxic control. 3.7 Histological Preservation of Pulmonary Architecture by PLGA-LA/Tada MSs Histological examination (Fig. 3 D) revealed distinct pathological changes in the hypoxia model, including arterial wall thickening, luminal narrowing, and perivascular inflammation. Oral tadalafil produced mild improvement, while combination therapy achieved more apparent restoration of vascular morphology. In contrast, the PLGA-LA/Tada MSs group exhibited near-normal histology with thin vascular walls, minimal inflammation, and well-preserved alveolar structures. 3.8 Suppression of Fibrosis and Vascular Remodeling by PLGA-LA/Tada MSs Masson’s trichrome staining (Fig. 4 A) demonstrated prominent perivascular collagen deposition in the model group. Tadalafil monotherapy slightly reduced fibrosis, whereas combination therapy further alleviated collagen accumulation. The PLGA-LA/Tada MSs group showed minimal perivascular collagen, indicating strong antifibrotic efficacy. Immunohistochemical staining of α-SMA (Fig. 4 B) showed pronounced muscularization in the model group. Both tadalafil and combination groups exhibited moderate reductions in α-SMA expression, while the PLGA-LA/Tada MSs group displayed markedly decreased staining, indicating effective suppression of smooth muscle proliferation. 3.9 Modulation of the NO/cGMP Signaling Axis by PLGA-LA/Tada MSs Under chronic hypoxia, lung tissue showed reduced eNOS expression (Fig. 5 A) and decreased cGMP levels in lung and right ventricular tissues (Fig. 5 B) (p < 0.05), confirming inhibition of the NO/cGMP pathway. Plasma NO metabolites (NO₃⁻ and NO₂⁻; Figs. 5 C, 5 D) were also diminished. Treatment with oral tadalafil, combination therapy, and PLGA-LA/Tada MSs significantly upregulated eNOS expression (#p < 0.05). NO₃⁻ and NO₂⁻ levels increased significantly in most treatment groups, with the microsphere group showing robust recovery. cGMP levels in lung tissue were significantly elevated by both oral tadalafil and PLGA-LA/Tada MSs (p < 0.05), while right ventricular cGMP levels showed an upward trend. 3.10 Attenuation of Inflammatory Response by PLGA-LA/Tada MSs Chronic hypoxia elevated pulmonary TNF-α (Fig. 5 E) and IL-6 (Fig. 5 F) expression (p < 0.05). All treatments significantly reduced TNF-α (#p < 0.05). IL-6 was significantly decreased in the tadalafil and combination groups, with the microsphere group showing a non-significant downward trend. 3.11 Reduction of Oxidative Stress by PLGA-LA/Tada MSs Chronic hypobaric hypoxia markedly increased oxidative stress in lung tissue, evidenced by elevated ROS levels (Fig. 6 A) and reduced CAT activity (Fig. 6 B) (p < 0.05). MDA levels (Fig. 6 C) were higher, and the GSH/GSSG ratio (Fig. 6 D) lower, compared with normoxic controls, though these changes did not reach statistical significance. All treatment groups—oral tadalafil, combination therapy, and PLGA-LA/Tada MSs—significantly reduced ROS levels (Fig. 6 A) and restored CAT activity (#p < 0.05, Fig. 6 B). Tadalafil monotherapy significantly improved the GSH/GSSG ratio (#p < 0.05), while combination and microsphere groups showed mild, non-significant increases (Fig. 6 D). MDA levels trended downward across all treatments (Fig. 6 C). 3.12 Hematological Alterations and Therapeutic Modulation by PLGA-LA/Tada MSs Chronic hypoxia increased RBC count (Fig. 6 E), HGB (Fig. 6 F), and HCT (Fig. 6 G) (p < 0.05), indicating compensatory erythropoiesis. MCV (Fig. 6 H) was unchanged between control and hypoxia groups. Treatment with oral tadalafil and PLGA-LA/Tada MSs significantly decreased MCV (#p < 0.05), and all therapies significantly reduced RBC, HGB, and HCT compared to the model group (#p < 0.05). Notably, the PLGA-LA/Tada MSs group normalized hematological parameters after a single intratracheal administration. 3.13 Integrated Therapeutic Efficacy of PLGA-LA/Tada MSs in HAPH In summary, intratracheal PLGA-LA/Tada MSs achieved localized, sustained therapeutic effects in HAPH. The microspheres exhibited high porosity, optimal aerodynamic behavior, and dual-drug encapsulation, enabling synchronized pulmonary delivery: rapid L-arginine release and sustained tadalafil release. The formulation restored NO/cGMP signaling, reduced oxidative stress, suppressed inflammation, and improved hemodynamics, while histology showed reversal of vascular remodeling. Systemically, hematologic abnormalities induced by chronic hypoxia were normalized, indicating improved oxygenation and reduced blood viscosity. 4. Discussion Porous dual-drug-loaded PLGA microspheres were successfully engineered for localized pulmonary co-delivery of hydrophilic L-arginine and hydrophobic tadalafil. Ammonium bicarbonate acted as an efficient gas-forming porogen, generating a well-defined mesoporous architecture (pore size ≈ 55 nm, surface area 360.43 m²/g) that enhanced drug loading and diffusion. The spherical morphology and uniform pore distribution confirmed stable emulsification and controlled gas foaming during fabrication. This structure enabled rapid diffusion of L-arginine through aqueous channels and sustained tadalafil release from the hydrophobic PLGA matrix [ 35 ]. The aerodynamic properties (MMAD ≈ 4–5 µm; FPF ≈ 40–45%) place the PLGA-LA/Tada microspheres within the optimal respirable range for efficient deep-lung deposition [ 36 , 37 ]. Their high porosity and low particle density effectively reduce the aerodynamic diameter relative to the geometric size (~ 12 µm), facilitating alveolar delivery [ 38 – 40 ]. Consistent with previous findings by Kadota et al. , Alipour et al. , and Tse et al. , porous microspheres with MMADs near 5 µm achieve efficient alveolar deposition and markedly enhance pulmonary drug bioavailability in vivo [ 41 – 43 ]. The moderately negative surface charge (≈ − 18 mV) and steric stabilization by residual PVA contributed to colloidal stability [ 44 – 46 ]. XRD and DSC analyses confirmed the amorphous dispersion of both drugs within PLGA, which prevents recrystallization and supports consistent release behavior [ 47 , 48 ]. The distinct release kinetics of the two agents reflect their physicochemical properties: L-arginine exhibited a rapid burst release owing to its hydrophilicity and surface localization, while tadalafil displayed sustained, diffusion-controlled release through the polymer matrix [ 49 , 50 ]. This biphasic pattern enables temporally coordinated pharmacological action—early enhancement of nitric oxide signaling by L-arginine followed by prolonged vasodilation via tadalafil [ 23 , 51 ]. Pharmacokinetic analysis further confirmed targeted pulmonary delivery with rapid lung deposition, extended retention, and markedly reduced systemic exposure compared to oral tadalafil. The absence of significant changes in serum LDH-L and ALP indicates minimal pulmonary irritation and excellent biocompatibility [ 38 , 52 ] . In vivo, the PLGA-LA/Tada microspheres effectively attenuated pulmonary hypertension and right ventricular remodeling in the hypoxic model. Decreases in RVSP and mPAP demonstrated alleviation of pulmonary vasoconstriction, while normalization of Fulton’s index indicated improved right ventricular unloading. Histological and immunohistochemical analyses revealed thinner vascular walls, reduced collagen deposition, and decreased α-SMA expression, confirming reversal of vascular remodeling. Mechanistically, the microspheres restored the NO/cGMP signaling axis by simultaneously promoting eNOS-mediated NO synthesis (via L-arginine) and inhibiting PDE5-mediated cGMP degradation (via tadalafil) [ 10 , 53 ]. The treatment also mitigated oxidative stress and inflammation, two key contributors to hypoxia-induced endothelial dysfunction. Decreased ROS levels and restored CAT activity reflected improved redox homeostasis, while downregulation of TNF-α and IL-6 indicated suppression of inflammatory activation [ 5 , 6 , 10 ]. Moreover, normalization of hematologic parameters (RBC, HGB, and HCT) suggested improved oxygenation and reduced erythropoietin-driven polycythemia [ 54 ]. Collectively, these results demonstrate that PLGA-LA/Tada microspheres integrate favorable physicochemical characteristics, optimized aerodynamic behavior, and coordinated dual-drug pharmacodynamics to achieve synchronized release and comprehensive therapeutic benefits. By reactivating the NO/cGMP signaling pathway and concurrently alleviating oxidative, inflammatory, and hemodynamic abnormalities, this localized pulmonary delivery platform provides a promising strategy for treating hypoxia-induced pulmonary hypertension. 5. Conclusions In this study, a dual-drug-loaded porous PLGA microsphere system (PLGA-LA/Tada MSs) was successfully developed for intratracheal administration to achieve targeted pulmonary therapy for HAPH. The microspheres exhibited favorable physicochemical characteristics, including high porosity, optimized aerodynamic diameter, and stable co-encapsulation of hydrophilic and hydrophobic agents, supporting efficient deep-lung deposition and synchronized dual-drug release. A single pulmonary dose achieved rapid local accumulation in lung tissue, prolonged retention, and markedly reduced systemic exposure. Therapeutically, PLGA-LA/Tada MSs alleviated pulmonary hypertension, attenuated right ventricular hypertrophy, preserved alveolar architecture, and reversed vascular remodeling. Mechanistically, the formulation reactivated NO/cGMP signaling, mitigated oxidative stress and inflammation, and normalized erythropoietic activity, demonstrating multimodal synergy. Overall, this localized, sustained-release microsphere platform provides a promising strategy to simultaneously address the functional, structural, and molecular pathologies of HAPH. The robust efficacy achieved through a single-dose pulmonary administration highlights its potential for clinical translation in the treatment of hypoxia-driven pulmonary vascular diseases. Declarations Author Contributions: Na Li: Conceptualization, Methodology, Data curation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing. Xiangbo Zheng and Qing He: Conceptualization, Methodology, Writing – review & editing. Yuan Cheng: Data curation, Investigation, Formal analysis. Tao Jin: Data curation, Investigation, Formal analysis. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Acknowledgements None. Data Availability Statement: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Competing Interests: The authors have no relevant financial or non-financial interests to disclose. 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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-8275947","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":559017423,"identity":"cd868b1c-42fc-4d0e-beb0-9e295a1b7f5a","order_by":0,"name":"Na Li","email":"","orcid":"","institution":"Southwest Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Na","middleName":"","lastName":"Li","suffix":""},{"id":559017428,"identity":"ce88d178-6bd3-4f36-8e1c-e8d16934e252","order_by":1,"name":"Xiangbo Zheng","email":"","orcid":"","institution":"Southwest Jiaotong 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12:24:23","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":691204,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineGraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-8275947/v1/af4efc11e5b920033a6cb2d4.png"},{"id":98068454,"identity":"0ee11f2b-3904-400c-aa24-3837e3152621","added_by":"auto","created_at":"2025-12-12 12:24:24","extension":"xml","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":132740,"visible":true,"origin":"","legend":"","description":"","filename":"42be097d26354331baf981dbd59d6b621structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8275947/v1/402f01ae58a4ed95c1d4b841.xml"},{"id":98068448,"identity":"f3008568-b79d-44eb-b63f-f21d075b11fe","added_by":"auto","created_at":"2025-12-12 12:24:24","extension":"html","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":142905,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8275947/v1/7bfa0ee5d0a428798f08513a.html"},{"id":98068428,"identity":"797606f4-7250-401d-aebf-23bdc5812b5b","added_by":"auto","created_at":"2025-12-12 12:24:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3015639,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComprehensive morphological and porosity characterization of PLGA-LA/Tada MSs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Low-magnification SEM image showing overall spherical morphology and uniform size distribution (scale bar = 50 μm). High-magnification SEM image highlighting porous surface structure (scale bar = 5 μm). (B) Particle size distribution of PLGA-LA/Tada MSs determined by ImageJ-based analysis (n \u0026gt; 200). (C) Nitrogen adsorption–desorption isotherms indicating mesoporous structure. (D) Pore size distribution curve showing a dominant pore diameter of approximately 55 nm. (E) Full-range (3°–100° 2θ) XRD pattern of PLGA-LA/Tada MSs. (F) DSC thermogram of PLGA-LA/Tada MSs.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8275947/v1/076f0b523c99e3a8a8245b64.png"},{"id":98426292,"identity":"8912f0db-90a8-4e1d-9e20-aa0b717cef0e","added_by":"auto","created_at":"2025-12-17 16:36:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1709564,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDrug release profile, pharmacokinetics, pulmonary distribution, and biocompatibility evaluation of PLGA-LA/Tada MSs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) In vitro cumulative release profiles of L-arginine (green) and tadalafil (yellow) from PLGA-LA/Tada MSs over 48 hours. L-arginine exhibited rapid release within the first few hours, while tadalafil showed a sustained release over 48 h (n = 3). (B-D) Compared to oral free tadalafil, PLGA-LA/Tada MSs demonstrated earlier Tmax, lower systemic exposure, and enhanced, sustained pulmonary drug levels in plasma, BALF, and lung tissue. (E) Serum biochemical indicators of pulmonary injury at 12 h post intratracheal administration (n = 3 per group). *P \u0026lt; 0.05 vs. PLGA-LA/Tada MSs group.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8275947/v1/ab4cbbd3c9f83aa3fe80c2d5.png"},{"id":98068432,"identity":"9b445483-34d0-480c-b3ac-2b92d02a3e0e","added_by":"auto","created_at":"2025-12-12 12:24:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9062456,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHemodynamic parameters (RVSP, mPAP), right ventricular hypertrophy (Fulton’s index), and lung histology in different groups.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) RVSP levels in normoxic control, hypobaric hypoxia model, oral tadalafil, combined tadalafil and L-arginine, and PLGA-LA/Tada microsphere groups (n = 4–6 per group). (B) mPAP levels in normoxic control, hypobaric hypoxia model, oral tadalafil, combined tadalafil and L-arginine, and PLGA-LA/Tada microsphere groups (n = 4–6 per group). (C) Fulton’s index [RV/(LV+S)] as an indicator of right ventricular hypertrophy in different treatment groups (n = 4–6 per group). (D) Representative H\u0026amp;E-stained sections of rat lung tissue from different treatment groups (×200). Scale bar = 20 μm. *P \u0026lt; 0.05 vs. normoxic control; \u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05 vs. hypoxia-PH model group.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8275947/v1/491b3fd72a0e7255121c07aa.png"},{"id":98068445,"identity":"a97ca392-2ff0-4c80-968c-7049eda7a467","added_by":"auto","created_at":"2025-12-12 12:24:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":25150279,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMasson's trichrome staining and Immunohistochemical staining of α-SMA in lung tissue.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Masson's trichrome staining showing perivascular collagen deposition in lung tissues from hypoxia-induced PH model and treatment groups. Scale bar = 50 μm. (B) Representative images showing α-SMA expression (brown) in pulmonary vasculature from normoxic control, hypoxia-PH model, and treatment groups. Scale bar = 50 μm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8275947/v1/1bfa69d872052ddad8b9830b.png"},{"id":98068438,"identity":"9ccb0418-87a7-4e7c-ba37-76b3a36876c3","added_by":"auto","created_at":"2025-12-12 12:24:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2083696,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eeNOS expression and cGMP content; nitrate (NO₃⁻) and nitrite (NO₂⁻) levels; TNF-α and IL-6 expression in lung tissue across different experimental groups.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Comparison of eNOS expression and cGMP content in lung tissue across different experimental groups. (C-D) Levels of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e in lung tissue across different groups. (E-F) Expression levels of inflammatory cytokines TNF-α and IL-6 in lung tissue across different groups. *p \u0026lt; 0.05 vs. normoxic control; \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05 vs. hypoxia-PH model group.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8275947/v1/3c8450b80d786ac127ee5bdd.png"},{"id":98428843,"identity":"de41be51-01f4-4e1e-aec2-db7c5eb112f5","added_by":"auto","created_at":"2025-12-17 16:42:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1794430,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOxidative stress markers in lung tissue and hematological parameters in peripheral blood across different experimental groups.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-D) Oxidative stress markers in lung tissue across different groups, including ROS levels, CAT activity, MDA levels and GSH/GSSG ratio.\u003c/p\u003e\n\u003cp\u003e(E-H) Hematological parameters in peripheral blood. Bar graphs HCT, HGB, RBC and MCV in each group. *p \u0026lt; 0.05 vs. normoxic control; \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05 vs. hypoxia-PH model.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8275947/v1/52cd17685e6baaad2f69418e.png"},{"id":100949838,"identity":"0e0cf64a-a93f-4c3c-a939-08c1c5fbca73","added_by":"auto","created_at":"2026-01-23 07:05:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":39899120,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8275947/v1/70fad890-0f47-463c-82fd-a2967d8b8847.pdf"},{"id":98068436,"identity":"c038ed28-b050-4962-8708-b3f87a7d2a82","added_by":"auto","created_at":"2025-12-12 12:24:23","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5040592,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-8275947/v1/e87579d9991235964bfbfa44.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Inhalable Porous PLGA Microspheres Enable Lung-Targeted Dual-Drug Delivery for the Treatment of High-Altitude Pulmonary Hypertension","fulltext":[{"header":"Highlights","content":"\u003cp\u003e\u003cstrong\u003eLocalized dual-drug pulmonary delivery\u003c/strong\u003e: Porous PLGA microspheres enabled intratracheal co-delivery of hydrophilic L-arginine and hydrophobic tadalafil, achieving high lung specificity and reduced systemic exposure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpatiotemporally coordinated release\u003c/strong\u003e: The formulation provided rapid release of L-arginine and sustained tadalafil delivery, synergistically restoring the NO/cGMP pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMechanism-driven vascular protection\u003c/strong\u003e: Microspheres significantly attenuated oxidative stress, suppressed inflammation, and improved endothelial signaling in high-altitude pulmonary hypertension.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional and structural reversal\u003c/strong\u003e: A single intratracheal dose effectively improved hemodynamics, reduced right ventricular hypertrophy, and reversed pulmonary vascular remodeling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTherapeutic potential for HAPH\u003c/strong\u003e: PLGA-LA/Tada MSs represent a simplified and efficient inhalation platform for targeted, sustained treatment of high-altitude pulmonary hypertension.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003ePulmonary hypertension (PH) is a progressive cardiopulmonary disorder characterized by elevated pulmonary arterial pressure and vascular remodeling, ultimately leading to right ventricular failure and high mortality [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among its subtypes, high-altitude pulmonary hypertension (HAPH) is a distinct form induced by chronic exposure to hypobaric hypoxia, which triggers sustained pulmonary vasoconstriction, inflammation, oxidative stress, and excessive erythropoiesis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These pathological changes collectively contribute to increased pulmonary vascular resistance and maladaptive right heart remodeling [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Despite its global relevance in populations residing or working at high-altitudes, effective therapeutic options for HAPH remain limited [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNitric oxide (NO) signaling plays a central role in maintaining pulmonary vascular tone and endothelial homeostasis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In HAPH, chronic hypobaric hypoxia suppresses endothelial nitric oxide synthase (eNOS) activity and accelerates cyclic guanosine monophosphate (cGMP) degradation through phosphodiesterase-5 (PDE5), ultimately leading to sustained vasoconstriction and pulmonary vascular remodeling [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Although several pharmacological agents\u0026mdash;including oxygen therapy, calcium channel blockers, prostacyclins, endothelin receptor antagonists, and phosphodiesterase inhibitors\u0026mdash;have shown potential in mitigating pulmonary hypertension, their clinical efficacy in HAPH remains limited due to unclear mechanisms of action in the context of hypobaric hypoxia, poor lung selectivity, rapid systemic clearance, and the need for frequent administration [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Descent to lower altitudes remains the most effective intervention; however, for patients who continue to reside at high-altitude, there is a pressing need for therapeutic strategies that offer improved pulmonary targeting and prolonged efficacy under hypoxic stress. To address these challenges, pulmonary drug delivery has emerged as a promising approach, enabling localized, sustained release of vasodilatory agents directly to the lungs, thereby enhancing therapeutic precision while minimizing systemic side effects [\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Recent studies on inhalable formulations of phosphodiesterase inhibitors\u0026mdash;such as tadalafil nanocomposites and spray-dried sildenafil microparticles\u0026mdash;have demonstrated enhanced pulmonary targeting and prolonged therapeutic action in the treatment of pulmonary arterial hypertension [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMicrosphere-based carriers, particularly those formulated with biodegradable polymers like poly(lactic-co-glycolic acid) (PLGA), offer unique advantages in terms of controlled release, biocompatibility, and lung deposition [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Recent studies\u0026mdash;for instance, Rashid et al.\u0026mdash;have demonstrated that inhaled PLGA-encapsulated sildenafil can effectively treat pulmonary arterial hypertension at reduced doses and with extended dosing intervals compared to the oral formulation, highlighting the potential of PLGA microspheres as an alternative to systemic administration [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the co-encapsulation and coordinated delivery of hydrophilic and hydrophobic agents with distinct pharmacokinetics remain technically challenging [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we developed a dual-drug-loaded porous PLGA microsphere system co-encapsulating hydrophilic L-arginine and hydrophobic tadalafil (PLGA-LA/Tada MSs) using a gas-foamed double emulsion method. The microspheres were engineered to achieve favorable aerodynamic properties for deep-lung delivery and to enable a biphasic release profile for synchronized pharmacological action. We hypothesized that this system could locally restore NO/cGMP signaling, mitigate oxidative and inflammatory injury, and reverse vascular remodeling in HAPH. Through comprehensive physicochemical characterization, pharmacokinetic analysis, and in vivo efficacy evaluation in a hypobaric hypoxia-induced HAPH rat model, we aimed to establish the therapeutic potential of PLGA-LA/Tada MSs as a multifunctional, inhalable platform for the treatment of HAPH.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials and Reagents\u003c/h2\u003e\u003cp\u003ePoly(D,L-lactide-co-glycolide) (PLGA, RG503H; lactide:glycolide\u0026thinsp;=\u0026thinsp;50:50; acid-terminated; inherent viscosity\u0026thinsp;~\u0026thinsp;0.32 dL/g; Cat# RG503H, Sigma-Aldrich, USA) was used at 200 mg per batch for microsphere preparation. Dichloromethane (DCM, Cat# 270997, Sigma-Aldrich) and polyvinyl alcohol (PVA, 87\u0026ndash;89% hydrolyzed, Mw 30,000\u0026ndash;70,000; Cat# 363065, Sigma-Aldrich) were used as received. Ammonium bicarbonate (ABC, Cat# 09830, Sigma-Aldrich) was used at 40 mg/mL as the porogen solution. L-arginine (\u0026ge;\u0026thinsp;98%, Cat# HY-N0455, MedChemExpress, China) and tadalafil (\u0026ge;\u0026thinsp;98%, Cat# HY-90009A, MedChemExpress) were incorporated into the internal and external phases, respectively. Alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) activities were quantified using commercial kits from Leidu Biotechnology (ALP: Cat# S03038, Lot# 20240412; LDH: Cat# S03034, Lot# 20240329), following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003eeNOS protein expression was analyzed using a Western Blot detection kit (Sevier Biotechnology, China; Cat# WB-eNOS-01, used according to the manufacturer\u0026rsquo;s protocol). Cyclic GMP (cGMP) and nitrate (NO₃⁻) levels were measured using ELISA kits from Jinmei Biotech (Cat# JM-01434R1 and JM-11652R2, Lot# 202501). Nitrite (NO₂⁻) levels were quantified with a Nitrite Assay Kit (Nanjing Jiancheng Bioengineering Institute; Cat# A038-1, Lot# 20250414). Inflammatory and oxidative stress markers were assessed using ELISA kits. Rat TNF-α and IL-6 were measured with uncoated ELISA kits from Thermo Fisher Scientific (Cat# 88-7340-88 and 88-50625-88; Lot# 378167-000 and 382209-000). Catalase (CAT) and reactive oxygen species (ROS) were examined using research-use-only ELISA kits from Jinmei Biotech (Cat# JM-10334R2, JM-10531R2; Lot# 202501). Malondialdehyde (MDA) and total/oxidized glutathione (GSH/GSSG) levels were quantified using corresponding kits from Servicebio (MDA: Cat# G4302, Lot# MPC2412127; GSH/GSSG: Cat# G4304, Lot# MPC2412126). Protein concentrations were determined using a BCA assay kit (Servicebio; Cat# G2026-1000T, Lot# CR2503100-1).\u003c/p\u003e\u003cp\u003eSprague\u0026ndash;Dawley (SD) healthy rats (6\u0026ndash;8 weeks old, 200\u0026ndash;250 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China; License No. H0.1100 11241106222542). All experimental procedures were approved by the Ethics Committee of Southwest Jiaotong University (Approval No. 2024110). Animal studies were conducted in accordance with the ARRIVE guidelines and institutional regulations governing the care and use of laboratory animals. Rats were housed under standard specific pathogen-free (SPF) conditions with free access to food and water. Group allocation was performed using a random number table. After randomization, investigators responsible for conducting the experiments, assessing outcomes, and performing data analysis were aware of the group assignments. Anaesthesia was induced by intraperitoneal injection of tribromoethanol, and all possible measures were taken to minimize animal discomfort and suffering. Animals that died during the experiments were excluded from analysis, resulting in the removal of four rats in total.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Preparation of Dual-Drug Loaded Porous PLGA PLGA-LA/Tada MSs\u003c/h2\u003e\u003cp\u003ePorous PLGA microspheres co-encapsulating a hydrophilic agent (L-arginine) and a hydrophobic agent (tadalafil) were prepared via a water-in-oil-in-water (W/O/W) double emulsion solvent evaporation method and are herein referred to as PLGA-LA/Tada MSs. This formulation strategy leverages the physicochemical complementarity of both drugs to achieve spatial compartmentalization within the polymer matrix [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Briefly, 200 mg of PLGA was dissolved in 7 mL of DCM to form the oil phase. Tadalafil, due to its lipophilic nature, was co-dissolved in the DCM with PLGA, facilitating uniform dispersion within the hydrophobic polymer matrix. In parallel, the hydrophilic drug L-arginine was fully dissolved in 1 mL deionized water containing 4% (w/v) ABC, which served both as the internal aqueous phase and as a gas-forming porogen to generate internal pores.\u003c/p\u003e\u003cp\u003eThe primary W/O emulsion was formed by homogenizing the L-arginine/ABC solution with the PLGA/DCM solution at 3,000 rpm for 2 minutes. This emulsion was then added to 50 mL of 1% (w/v) PVA and homogenized again at 3,000 rpm for 2 minutes to yield the W/O/W double emulsion. Subsequently, the emulsion was transferred to 100 mL of 1% PVA solution and stirred magnetically at room temperature for 5 hours to allow complete DCM evaporation and microsphere solidification. The resulting microspheres were collected by centrifugation, washed four times with deionized water to remove residual PVA and unencapsulated drugs, and lyophilized for 16 hours. The ABC concentration was adjustable to modulate porosity and density, thereby tuning both aerodynamic properties and biphasic drug release behavior.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Characterization of Porous PLGA-LA/Tada MSs\u003c/h2\u003e\u003cp\u003eThe surface morphology, pore structure, and particle integrity of PLGA-LA/Tada MSs were examined using a field-emission scanning electron microscope (FE-SEM, S-4800, HITACHI, Japan) equipped with energy-dispersive X-ray spectroscopy (EDX, HORIBA EMAX mics2). Samples were diluted (1:5) with ultrapure water, and 20 \u0026micro;L of the diluted suspension was dropped onto a clean silicon wafer and air-dried for 2 h. Before imaging, samples were sputter-coated with gold using an MC1000 ion sputter coater (HITACHI, Japan) and dried with a critical point dryer (Tousimis Autosamdri-815). Images were acquired under accelerating voltages of 3 or 5 kV, with a resolution of 50 nm. Particle size was quantitatively assessed by analyzing SEM images using ImageJ software, with measurements taken from over 200 microspheres per group.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1 Specific Surface Area and Porosity\u003c/h2\u003e\u003cp\u003eSpecific surface area and porosity of PLGA-LA/Tada MSs were determined by nitrogen adsorption\u0026ndash;desorption analysis using the Brunauer\u0026ndash;Emmett\u0026ndash;Teller (BET) method with a TriStar 3000 analyzer (Micromeritics, USA) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Freeze-dried samples were pretreated by vacuum degassing and analyzed under nitrogen atmosphere to determine specific surface area, pore volume, and pore size distribution. Pore size distribution was calculated based on the Barrett\u0026ndash;Joyner\u0026ndash;Halenda (BJH) model.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2 Aerodynamic Properties\u003c/h2\u003e\u003cp\u003eThe mass median aerodynamic diameter (MMAD) and fine particle fraction (FPF) were measured using an aerodynamic particle sizer (APS 3321, TSI, USA) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Samples were aerosolized with high-purity argon and passed through the spectrometer under set conditions (particle size range: 1\u0026ndash;100 \u0026micro;m; sampling rate: 1000/s). The MMAD and FPF were calculated from the cumulative mass distribution provided by APS 3321 software, with FPF defined as the percentage of particles\u0026thinsp;\u0026le;\u0026thinsp;5 \u0026micro;m in aerodynamic diameter [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. To assess repeatability, three independent batches were tested under identical conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3 Surface Charge\u003c/h2\u003e\u003cp\u003eThe surface charge (zeta potential) of the microspheres was measured using a Malvern Zetasizer Nano ZS90 (Malvern Instruments, UK) with a He-Ne laser (633 nm) at a 90\u0026deg; scattering angle. Measurements were conducted at 25\u0026deg;C with 100 \u0026micro;L of nanoparticle dispersion in a cuvette. Each sample was analyzed in triplicate, with 20 measurement cycles per run and 10 s delay between cycles. Data were analyzed using Malvern Dispersion Technology Software 4.2, and average values were reported.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.3.4 Crystallinity and Thermal Properties\u003c/h2\u003e\u003cp\u003eX-ray diffraction (XRD) analysis was performed using a Bruker D8 DISCOVER diffractometer. Freeze-dried microsphere powders (10 mg) were scanned over a 2θ range of 10\u0026deg;\u0026ndash;30\u0026deg; at room temperature to evaluate the crystalline or amorphous nature of the encapsulated drugs and polymer matrix.\u003c/p\u003e\u003cp\u003eDifferential scanning calorimetry (DSC) was conducted using a DSC 3500 Sirius system. Approximately 20 mg of freeze-dried sample was sealed in an aluminum pan and analyzed under argon flow. The sample was cooled to 20\u0026deg;C, then heated to 250\u0026deg;C at a rate of 10\u0026deg;C/min to evaluate thermal transitions such as glass transition temperature (Tg) and melting behavior.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.3.4 Drug Loading and Encapsulation Efficiency\u003c/h2\u003e\u003cp\u003eDrug loading (LE) and encapsulation efficiency (EE) of L-Arg and Tadalafil were quantified using high-performance liquid chromatography (HPLC, 1260 Infinity II, Agilent Technologies, USA). L-Arg and Tadalafil were detected at 220 nm and 280 nm, respectively. After centrifugation (8000 rpm, 4\u0026deg;C, 20 min), the supernatant was collected for quantification.\u003c/p\u003e\u003cp\u003eLE and EE were calculated using the following equations:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:EE\\left(\\%\\right)=\\frac{{C}_{1}{V}_{1}-{C}_{2}{V}_{2}}{{C}_{1}{C}_{1}}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:LE\\left(\\%\\right)=\\frac{{C}_{1}{V}_{1}-{C}_{2}{V}_{2}}{W}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere \u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e represent the initial drug concentration and volume, \u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e refer to the drug concentration and volume in the filtrate, and \u003cem\u003eW\u003c/em\u003e is the mass of the freeze-dried microspheres.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.4 In vitro drug release\u003c/h2\u003e\u003cp\u003eThe in vitro release profiles of L-arginine and tadalafil from PLGA-LA/Tada MSs were evaluated in simulated lung fluid (SLF). Drug-loaded microspheres were placed in dialysis bags with a molecular weight cut-off (MWCO) of 1 kDa and incubated at room temperature in SLF under gentle agitation. At predetermined time points (0, 0.5, 0.75, 1, 2, 3, 4, 5, 6, and 17 h), aliquots of the external SLF medium were collected, centrifuged to remove any particulate matter, and analyzed using high-performance liquid chromatography (HPLC) to determine the concentrations of released L-arginine and tadalafil. After each sampling, the withdrawn volume was replaced with an equal volume of fresh SLF to maintain sink conditions. All experiments were conducted in triplicate using independently prepared batches of drug-loaded microspheres.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Pharmacokinetic Studies\u003c/h2\u003e\u003cp\u003eSprague\u0026ndash;Dawley rats were used for all pharmacokinetic studies and were acclimated for one week under SPF conditions prior to experimentation. Two separate studies were conducted to evaluate plasma and pulmonary pharmacokinetics of tadalafil. Pharmacokinetic analysis was limited to tadalafil, as L-arginine is an endogenous molecule with high and variable baseline levels in biological matrices, making accurate differentiation of exogenous levels challenging. Moreover, since L-arginine primarily exerts a local pulmonary effect via NO signaling, systemic pharmacokinetics are less relevant to the study aims.\u003c/p\u003e\u003cp\u003eFor plasma pharmacokinetics, six rats were randomly divided into two groups (n\u0026thinsp;=\u0026thinsp;3 each) receiving a single dose of either free tadalafil via oral gavage (10 mg/kg) or PLGA-LA/Tada MSs via intratracheal instillation (10 mg/kg). Blood samples were collected at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 30, 36, and 48 hours post-administration. For pulmonary pharmacokinetics, thirty rats were divided into two groups (n\u0026thinsp;=\u0026thinsp;15 each) receiving the same treatments. At predetermined time points (1, 3, 6, 10, and 24 hours), animals (n\u0026thinsp;=\u0026thinsp;3 per time point) were sacrificed to collect bronchoalveolar lavage fluid (BALF) and lung tissues for pharmacokinetic analysis. Tadalafil concentrations in all biological matrices were quantified using a validated LC\u0026ndash;MS/MS method. Everolimus-d₄ served as the internal standard to correct for variations in sample preparation and instrument response. Data were processed using Xcalibur software (Thermo) with linear regression and 1/X\u0026sup2; weighting.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Safety Profile of PLGA-LA/Tada MSs in rats\u003c/h2\u003e\u003cp\u003eTo preliminarily evaluate the pulmonary safety and tolerability of PLGA-LA/Tada MSs, Sprague\u0026ndash;Dawley rats were used. Animals were housed under SPF conditions and acclimatized for one week. Following ethical approval and verification of animal health certification, rats were randomly assigned to three groups (n\u0026thinsp;=\u0026thinsp;3/group): (1) negative control (intratracheal saline), (2) positive control (0.1% sodium dodecyl sulfate, SDS), and (3) microsphere group (PLGA-LA/Tada MSs, 10 mg/kg, intratracheal). At 12 h post-administration, blood was collected via the abdominal aorta, and serum was analyzed for LDH-L and ALP, which are commonly used markers for acute pulmonary epithelial injury. In addition, pharmacokinetic profiles in BALF, lung tissue, and serum were referenced to support the safety assessment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.7 In Vivo Hypobaric Hypoxia-Induced HAPH Model and Drug Administration\u003c/h2\u003e\u003cp\u003eSprague\u0026ndash;Dawley rats were housed under SPF conditions with free access to standard chow and water, maintained on a 12-hour light/dark cycle at an ambient temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and relative humidity of 35\u0026thinsp;\u0026plusmn;\u0026thinsp;5%. After one week of acclimatization, animals were randomly assigned to five groups (n\u0026thinsp;=\u0026thinsp;4\u0026ndash;6 per group): normoxic control, hypobaric hypoxia-induced pulmonary hypertension model (hypoxia-PH model), oral tadalafil treatment (hypoxia-TAD), oral tadalafil plus intratracheal L-arginine (hypoxia-TAD\u0026thinsp;+\u0026thinsp;L-Arg), and PLGA-LA/Tada microspheres group (hypoxia-MSs). Except for the normoxic control group, all rats were fasted for 10 hours (with free access to water) prior to exposure to simulated high-altitude hypobaric hypoxia (equivalent to 5000 m, 10% oxygen) in a decompression chamber for four consecutive weeks. During the hypobaric hypoxia exposure period, animals were monitored daily for general appearance and behavior. Upon completion of the four-week modeling phase, a single dose of the assigned treatment (10 mg/kg) was administered immediately. Twelve hours after dosing, all animals were evaluated for physiological, biochemical, and histopathological parameters.\u003c/p\u003e\u003cp\u003eHemodynamic parameters including mean pulmonary arterial pressure (mPAP) and right ventricular systolic pressure (RVSP) were assessed. RVSP was directly measured via right heart catheterization, and mPAP was estimated using the validated formula mPAP\u0026thinsp;\u0026asymp;\u0026thinsp;0.61 \u0026times; RVSP\u0026thinsp;+\u0026thinsp;2 for rats [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The RVSP trend was used as an indirect indicator of pulmonary vascular resistance. Anticoagulated whole blood was collected and separated into two aliquots: one for plasma-based ELISA quantification of nitrate (NO₃\u003csup\u003e⁻\u003c/sup\u003e) and nitrite (NO₂\u003csup\u003e⁻\u003c/sup\u003e), and the other for hematological analysis including red blood cell count (RBC), hemoglobin (Hb), and hematocrit (Hct). The heart was excised, and the right ventricle (RV), left ventricle (LV), and septum (S) were weighed to calculate Fulton\u0026rsquo;s index [RV/(LV\u0026thinsp;+\u0026thinsp;S)] as a measure of right ventricular hypertrophy. The RV tissue was also frozen for subsequent ELISA analysis of cyclic GMP (cGMP).\u003c/p\u003e\u003cp\u003eThe right lung was divided for multiple analyses: one portion was used for Western blotting to assess endothelial nitric oxide synthase (eNOS) monomer and dimer expression; another was used for biochemical assays of oxidative stress markers including malondialdehyde (MDA), glutathione/glutathione disulfide (GSH/GSSG), and catalase (CAT); and a third was processed for ELISA detection of reactive oxygen species (ROS), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and cGMP. The left lung was fixed in 4% paraformaldehyde and embedded for histological analysis, including hematoxylin and eosin (H\u0026amp;E) staining, Masson\u0026rsquo;s trichrome staining, and immunohistochemical (IHC) detection of α-smooth muscle actin (α-SMA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e\u003cp\u003eAll continuous data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test. A two-tailed P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. All statistical analyses were conducted using Stata 17.0 software.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Fabrication and Characterization of Porous PLGA-LA/Tada MSs\u003c/h2\u003e\u003cp\u003eDual-drug-loaded porous PLGA microspheres were successfully fabricated using a double emulsion method with ammonium bicarbonate as a porogen. The resulting microspheres exhibited an internal porous structure suitable for inhalation-based administration.\u003c/p\u003e\u003cp\u003eRepresentative SEM images (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) show that the microspheres possessed a spherical morphology with smooth outlines, no visible aggregation, and well-maintained structural integrity. At higher magnification, the microspheres displayed uniformly distributed nanoscale pores. The particle size distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) revealed a narrow range of 11\u0026ndash;14 \u0026micro;m, with an average diameter of approximately 12.0 \u0026micro;m, consistent with SEM observations. Nitrogen adsorption\u0026ndash;desorption isotherms and BJH analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) showed a Type IV isotherm with a pronounced hysteresis loop at P/P₀ \u0026gt;0.8, and a pore size distribution centered around 55 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The BET surface area was 360.43 m\u0026sup2;/g, and the total porosity reached 71.2%.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAerodynamic characterization showed MMAD values of 4.2, 5.1, and 4.7 \u0026micro;m, with corresponding FPF values of 42%, 40%, and 45% across three batches, indicating suitable aerosol properties. The zeta potential values were \u0026minus;\u0026thinsp;18.9 mV, \u0026minus;\u0026thinsp;18.6 mV, and \u0026minus;\u0026thinsp;17.9 mV, suggesting negatively charged, moderately stable particles. XRD analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) revealed no distinct diffraction peaks, and DSC thermograms (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) showed a broad glass transition temperature near 59\u0026deg;C, consistent with an amorphous PLGA matrix. The encapsulation efficiency (EE) and drug loading (LE) of tadalafil were 50.7% and 29.6%, respectively, while those of L-arginine were 75.5% and 22.0%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.2 In Vitro Drug Release Behavior of L-Arginine and Tadalafil from PLGA-LA/Tada MSs\u003c/h2\u003e\u003cp\u003eThe release profiles of L-arginine and tadalafil from PLGA-LA/Tada MSs were determined in simulated lung fluid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). L-arginine exhibited a pronounced burst release, reaching over 26% within the first hour and nearly complete release (~\u0026thinsp;88%) by 48 h. In contrast, tadalafil displayed a slower, sustained release, with ~\u0026thinsp;14.5% released at 0.75 h and a cumulative release of 87.0% at 48 h.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Pharmacokinetic Behavior in Plasma, BALF, and Lung Tissue of PLGA-LA/Tada MSs\u003c/h2\u003e\u003cp\u003eFollowing a single intratracheal administration, the pharmacokinetics of tadalafil were assessed in plasma, BALF, and lung tissue, with oral free tadalafil serving as the control. In plasma (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), oral tadalafil achieved a Cmax of 1464.8\u0026thinsp;\u0026plusmn;\u0026thinsp;181.6 ng/mL at 4 h, whereas PLGA-LA/Tada MSs reached a lower Cmax of 714.4\u0026thinsp;\u0026plusmn;\u0026thinsp;53.5 ng/mL at 0.5 h, with sustained levels up to 20 h. The AUC₀\u0026ndash;\u0026infin; for the microsphere group (3620.6\u0026thinsp;\u0026plusmn;\u0026thinsp;830.9 h\u0026middot;ng/mL) was markedly reduced compared to the oral group (14408.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2337.9 h\u0026middot;ng/mL).\u003c/p\u003e\u003cp\u003eIn BALF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), PLGA-LA/Tada MSs showed an earlier Cmax (1077.6\u0026thinsp;\u0026plusmn;\u0026thinsp;152.3 ng/mL at 1 h) than the oral formulation (1156.7\u0026thinsp;\u0026plusmn;\u0026thinsp;390.4 ng/mL at 3 h). Drug concentrations were maintained for up to 24 h, with an MRTlast of 5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 h. In lung tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), the microsphere group exhibited a higher Cmax (6580.2\u0026thinsp;\u0026plusmn;\u0026thinsp;683.7 ng/g at 1 h) than the free tadalafil group (6135.7\u0026thinsp;\u0026plusmn;\u0026thinsp;780.3 ng/g at 3 h), along with prolonged retention and an extended half-life.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Preliminary Safety Evaluation of PLGA-LA/Tada MSs\u003c/h2\u003e\u003cp\u003eAt 12 h post intratracheal administration, serum LDH-L levels in the PLGA-LA/Tada MSs group were significantly lower than in the saline and SDS groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), suggesting minimal cytotoxicity. ALP levels showed no significant differences among groups. The reduced ALP value in the microsphere group compared to the SDS group indicates a mild irritative response.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Hemodynamic Improvement by PLGA-LA/Tada MSs\u003c/h2\u003e\u003cp\u003eIn the hypobaric hypoxia model, both RVSP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and mPAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) were significantly elevated compared with normoxic controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), confirming the successful establishment of HAPH. Oral tadalafil monotherapy produced slight but non-significant reductions in RVSP and mPAP. In contrast, combined oral tadalafil and intratracheal L-arginine treatment significantly lowered both parameters (#p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Notably, PLGA-LA/Tada MSs markedly reduced RVSP and mPAP (#p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), achieving therapeutic effects comparable to the combination therapy through a single intratracheal dose.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Attenuation of Right Ventricular Hypertrophy by PLGA-LA/Tada MSs\u003c/h2\u003e\u003cp\u003eRight ventricular hypertrophy was assessed by Fulton\u0026rsquo;s index [RV/(LV\u0026thinsp;+\u0026thinsp;S)] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The hypoxia model group exhibited a significant increase compared with the normoxic group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Both oral tadalafil and the combination therapy significantly reduced the index (#p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The PLGA-LA/Tada MSs group also showed a significant reduction (#p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), restoring the index to levels comparable with the normoxic control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Histological Preservation of Pulmonary Architecture by PLGA-LA/Tada MSs\u003c/h2\u003e\u003cp\u003eHistological examination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) revealed distinct pathological changes in the hypoxia model, including arterial wall thickening, luminal narrowing, and perivascular inflammation. Oral tadalafil produced mild improvement, while combination therapy achieved more apparent restoration of vascular morphology. In contrast, the PLGA-LA/Tada MSs group exhibited near-normal histology with thin vascular walls, minimal inflammation, and well-preserved alveolar structures.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.8 Suppression of Fibrosis and Vascular Remodeling by PLGA-LA/Tada MSs\u003c/h2\u003e\u003cp\u003eMasson\u0026rsquo;s trichrome staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) demonstrated prominent perivascular collagen deposition in the model group. Tadalafil monotherapy slightly reduced fibrosis, whereas combination therapy further alleviated collagen accumulation. The PLGA-LA/Tada MSs group showed minimal perivascular collagen, indicating strong antifibrotic efficacy. Immunohistochemical staining of α-SMA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) showed pronounced muscularization in the model group. Both tadalafil and combination groups exhibited moderate reductions in α-SMA expression, while the PLGA-LA/Tada MSs group displayed markedly decreased staining, indicating effective suppression of smooth muscle proliferation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.9 Modulation of the NO/cGMP Signaling Axis by PLGA-LA/Tada MSs\u003c/h2\u003e\u003cp\u003eUnder chronic hypoxia, lung tissue showed reduced eNOS expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and decreased cGMP levels in lung and right ventricular tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), confirming inhibition of the NO/cGMP pathway. Plasma NO metabolites (NO₃⁻ and NO₂⁻; Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) were also diminished. Treatment with oral tadalafil, combination therapy, and PLGA-LA/Tada MSs significantly upregulated eNOS expression (#p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). NO₃⁻ and NO₂⁻ levels increased significantly in most treatment groups, with the microsphere group showing robust recovery. cGMP levels in lung tissue were significantly elevated by both oral tadalafil and PLGA-LA/Tada MSs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while right ventricular cGMP levels showed an upward trend.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.10 Attenuation of Inflammatory Response by PLGA-LA/Tada MSs\u003c/h2\u003e\u003cp\u003eChronic hypoxia elevated pulmonary TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) and IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) expression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). All treatments significantly reduced TNF-α (#p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). IL-6 was significantly decreased in the tadalafil and combination groups, with the microsphere group showing a non-significant downward trend.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.11 Reduction of Oxidative Stress by PLGA-LA/Tada MSs\u003c/h2\u003e\u003cp\u003eChronic hypobaric hypoxia markedly increased oxidative stress in lung tissue, evidenced by elevated ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) and reduced CAT activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). MDA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) were higher, and the GSH/GSSG ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) lower, compared with normoxic controls, though these changes did not reach statistical significance. All treatment groups\u0026mdash;oral tadalafil, combination therapy, and PLGA-LA/Tada MSs\u0026mdash;significantly reduced ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) and restored CAT activity (#p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Tadalafil monotherapy significantly improved the GSH/GSSG ratio (#p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while combination and microsphere groups showed mild, non-significant increases (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). MDA levels trended downward across all treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.12 Hematological Alterations and Therapeutic Modulation by PLGA-LA/Tada MSs\u003c/h2\u003e\u003cp\u003eChronic hypoxia increased RBC count (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), HGB (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), and HCT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating compensatory erythropoiesis. MCV (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH) was unchanged between control and hypoxia groups.\u003c/p\u003e\u003cp\u003eTreatment with oral tadalafil and PLGA-LA/Tada MSs significantly decreased MCV (#p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and all therapies significantly reduced RBC, HGB, and HCT compared to the model group (#p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Notably, the PLGA-LA/Tada MSs group normalized hematological parameters after a single intratracheal administration.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.13 Integrated Therapeutic Efficacy of PLGA-LA/Tada MSs in HAPH\u003c/h2\u003e\u003cp\u003eIn summary, intratracheal PLGA-LA/Tada MSs achieved localized, sustained therapeutic effects in HAPH. The microspheres exhibited high porosity, optimal aerodynamic behavior, and dual-drug encapsulation, enabling synchronized pulmonary delivery: rapid L-arginine release and sustained tadalafil release. The formulation restored NO/cGMP signaling, reduced oxidative stress, suppressed inflammation, and improved hemodynamics, while histology showed reversal of vascular remodeling. Systemically, hematologic abnormalities induced by chronic hypoxia were normalized, indicating improved oxygenation and reduced blood viscosity.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePorous dual-drug-loaded PLGA microspheres were successfully engineered for localized pulmonary co-delivery of hydrophilic L-arginine and hydrophobic tadalafil. Ammonium bicarbonate acted as an efficient gas-forming porogen, generating a well-defined mesoporous architecture (pore size\u0026thinsp;\u0026asymp;\u0026thinsp;55 nm, surface area 360.43 m\u0026sup2;/g) that enhanced drug loading and diffusion. The spherical morphology and uniform pore distribution confirmed stable emulsification and controlled gas foaming during fabrication. This structure enabled rapid diffusion of L-arginine through aqueous channels and sustained tadalafil release from the hydrophobic PLGA matrix [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe aerodynamic properties (MMAD\u0026thinsp;\u0026asymp;\u0026thinsp;4\u0026ndash;5 \u0026micro;m; FPF\u0026thinsp;\u0026asymp;\u0026thinsp;40\u0026ndash;45%) place the PLGA-LA/Tada microspheres within the optimal respirable range for efficient deep-lung deposition [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Their high porosity and low particle density effectively reduce the aerodynamic diameter relative to the geometric size (~\u0026thinsp;12 \u0026micro;m), facilitating alveolar delivery [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Consistent with previous findings by Kadota \u003cem\u003eet al.\u003c/em\u003e, Alipour \u003cem\u003eet al.\u003c/em\u003e, and Tse \u003cem\u003eet al.\u003c/em\u003e, porous microspheres with MMADs near 5 \u0026micro;m achieve efficient alveolar deposition and markedly enhance pulmonary drug bioavailability \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The moderately negative surface charge (\u0026asymp; \u0026minus;\u0026thinsp;18 mV) and steric stabilization by residual PVA contributed to colloidal stability [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. XRD and DSC analyses confirmed the amorphous dispersion of both drugs within PLGA, which prevents recrystallization and supports consistent release behavior [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe distinct release kinetics of the two agents reflect their physicochemical properties: L-arginine exhibited a rapid burst release owing to its hydrophilicity and surface localization, while tadalafil displayed sustained, diffusion-controlled release through the polymer matrix [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. This biphasic pattern enables temporally coordinated pharmacological action\u0026mdash;early enhancement of nitric oxide signaling by L-arginine followed by prolonged vasodilation via tadalafil [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Pharmacokinetic analysis further confirmed targeted pulmonary delivery with rapid lung deposition, extended retention, and markedly reduced systemic exposure compared to oral tadalafil. The absence of significant changes in serum LDH-L and ALP indicates minimal pulmonary irritation and excellent biocompatibility [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] .\u003c/p\u003e\u003cp\u003eIn vivo, the PLGA-LA/Tada microspheres effectively attenuated pulmonary hypertension and right ventricular remodeling in the hypoxic model. Decreases in RVSP and mPAP demonstrated alleviation of pulmonary vasoconstriction, while normalization of Fulton\u0026rsquo;s index indicated improved right ventricular unloading. Histological and immunohistochemical analyses revealed thinner vascular walls, reduced collagen deposition, and decreased α-SMA expression, confirming reversal of vascular remodeling. Mechanistically, the microspheres restored the NO/cGMP signaling axis by simultaneously promoting eNOS-mediated NO synthesis (via L-arginine) and inhibiting PDE5-mediated cGMP degradation (via tadalafil) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The treatment also mitigated oxidative stress and inflammation, two key contributors to hypoxia-induced endothelial dysfunction. Decreased ROS levels and restored CAT activity reflected improved redox homeostasis, while downregulation of TNF-α and IL-6 indicated suppression of inflammatory activation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Moreover, normalization of hematologic parameters (RBC, HGB, and HCT) suggested improved oxygenation and reduced erythropoietin-driven polycythemia [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCollectively, these results demonstrate that PLGA-LA/Tada microspheres integrate favorable physicochemical characteristics, optimized aerodynamic behavior, and coordinated dual-drug pharmacodynamics to achieve synchronized release and comprehensive therapeutic benefits. By reactivating the NO/cGMP signaling pathway and concurrently alleviating oxidative, inflammatory, and hemodynamic abnormalities, this localized pulmonary delivery platform provides a promising strategy for treating hypoxia-induced pulmonary hypertension.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn this study, a dual-drug-loaded porous PLGA microsphere system (PLGA-LA/Tada MSs) was successfully developed for intratracheal administration to achieve targeted pulmonary therapy for HAPH. The microspheres exhibited favorable physicochemical characteristics, including high porosity, optimized aerodynamic diameter, and stable co-encapsulation of hydrophilic and hydrophobic agents, supporting efficient deep-lung deposition and synchronized dual-drug release. A single pulmonary dose achieved rapid local accumulation in lung tissue, prolonged retention, and markedly reduced systemic exposure. Therapeutically, PLGA-LA/Tada MSs alleviated pulmonary hypertension, attenuated right ventricular hypertrophy, preserved alveolar architecture, and reversed vascular remodeling. Mechanistically, the formulation reactivated NO/cGMP signaling, mitigated oxidative stress and inflammation, and normalized erythropoietic activity, demonstrating multimodal synergy.\u003c/p\u003e\u003cp\u003eOverall, this localized, sustained-release microsphere platform provides a promising strategy to simultaneously address the functional, structural, and molecular pathologies of HAPH. The robust efficacy achieved through a single-dose pulmonary administration highlights its potential for clinical translation in the treatment of hypoxia-driven pulmonary vascular diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNa Li: Conceptualization, Methodology, Data curation, Formal analysis, Investigation, Writing – original draft, Writing – review \u0026amp; editing. Xiangbo Zheng and Qing He: Conceptualization, Methodology, Writing – review \u0026amp; editing. Yuan Cheng: Data curation, Investigation, Formal analysis. Tao Jin: Data curation, Investigation, Formal analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMocumbi A, Humbert M, Saxena A, Jing ZC, Sliwa K, Thienemann F, Archer SL, Stewart S: Pulmonary hypertension. 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Semin Respir Crit Care Med. 2023;44\u003cstrong\u003e:\u003c/strong\u003e681-95.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"High-altitude pulmonary hypertension, Porous PLGA microspheres, Dual-drug delivery, L-arginine and Tadalafil, Pulmonary targeting","lastPublishedDoi":"10.21203/rs.3.rs-8275947/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8275947/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eHigh-altitude pulmonary hypertension (HAPH), driven by hypobaric hypoxia\u0026ndash;induced vasoconstriction and vascular remodeling, remains a therapeutic challenge due to the limited efficacy and systemic side effects of current treatments. This study aimed to develop a dual-drug\u0026ndash;loaded microsphere system for targeted pulmonary delivery.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003ePorous PLGA microspheres co-loaded with hydrophilic L-arginine and hydrophobic tadalafil (PLGA-LA/Tada MSs) were fabricated using a gas-foamed double-emulsion technique. The microspheres were characterized for morphology, porosity, aerodynamic properties, and in vitro release profiles. In vivo pharmacokinetics, safety, and therapeutic efficacy were evaluated following a single intratracheal administration in a chronic hypobaric hypoxia\u0026ndash;induced HAPH rat model.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe PLGA-LA/Tada MSs displayed uniform spherical morphology, well-developed porosity, and favorable aerodynamic behavior (MMAD\u0026thinsp;~\u0026thinsp;4.7 \u0026micro;m), supporting deep-lung deposition. In vitro, L-arginine exhibited rapid release, whereas tadalafil showed sustained release. A single intratracheal dose achieved efficient pulmonary deposition, prolonged lung retention, and reduced systemic exposure. Therapeutically, PLGA-LA/Tada MSs improved RVSP, mPAP, and Fulton\u0026rsquo;s index; mitigated fibrosis and vascular muscularization; and preserved alveolar structure. Mechanistic studies indicated restored NO/cGMP signaling, reduced oxidative stress and inflammation, and normalization of erythropoietic markers.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003ePLGA-LA/Tada MSs enable spatially and temporally coordinated pulmonary delivery of hydrophilic and hydrophobic agents, allowing synchronized modulation of the NO\u0026ndash;cGMP axis. This dual-drug microsphere platform provides durable pulmonary vascular protection with minimal systemic exposure and demonstrates strong translational potential for treating HAPH and other hypoxia-driven pulmonary diseases.\u003c/p\u003e","manuscriptTitle":"Inhalable Porous PLGA Microspheres Enable Lung-Targeted Dual-Drug Delivery for the Treatment of High-Altitude Pulmonary Hypertension","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-12 12:24:15","doi":"10.21203/rs.3.rs-8275947/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e56d55b6-632a-44fa-b253-fe61a7841df6","owner":[],"postedDate":"December 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-22T07:11:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-12 12:24:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8275947","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8275947","identity":"rs-8275947","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}