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• Pulmonary arterial hypertension (PAH) is an inflammatory disease clinically characterized by abnormally high pressures in the pulmonary arteries (PAs) and PA remodeling. PAH patients often demonstrate a resting mean pulmonary artery pressure (mPAP) of 25 mmHg or above [1, 2]. PAH has a 0.3%–6% prevalence and a 3-year survival rate of only 49% [3-5]. Resultantly, the PA remodeling provokes pulmonary hemodynamics abnormalities of pulmonary blood flow, pulmonary vascular resistance and pulmonary venous pressures, and therefore, results in the overload and hypertrophy of the right ventricle (RV), heart failure and death. PAH-treated drugs always include five classes involved in three pathways: endothelin receptor antagonists, nitric oxide (NO) pathway and prostaglandin analogs [6]. However, they consistently demonstrate a modest effect on PA remodeling. PA remodeling is mainly associated with the overproliferation of PA smooth muscle cells (PASMCs) and an increased release of inflammatory cytokines, such as interleukin-6 (IL-6), IL-12 and tumor necrosis factor-α (TNF-α); and frequently, the inflammation aggravates the oxidative stress and PASMC proliferation [1, 7, 8]. As a result, the inhibition of PASMC hyperproliferation and inflammation is promising to combat PA remodeling and improve pulmonary hemodynamics.

  Gene therapy refers to delivering genetic materials to mend faulty or mutant genes or leverage existing cellular processes to treat disease. Increasing evidence indicates that gene mutations are closely linked to the development of PAH. microRNAs (miRNAs) with 19–25 nucleotides can regulate the post-transcriptional suppression of target genes. A previous report indicated that the upregulation of miR138 in PASMCs could significantly drive PA remodeling [9]. miR138 in PASMCs suppresses their apoptosis by preventing caspase-3 activation and down-regulating Bcl-2 signaling transduction. Baicalein is a water-insoluble anti-inflammatory drug, widely used to treat hypertension, atherosclerosis and other respiratory disorders [10, 11]. Moreover, baicalein can downregulate the inflammatory mediators, i.e., TNF-α and IL-12, thereby inhibiting the PASMC hyperproliferation is more appropriate [12]. Accordingly, the gene therapy of antisense miR138 (anti-miR138) combined with baicalein can potentially inhibit PA remodeling.

  Nevertheless, the membrane diffusion of miRNA and the intracellular delivery efficacy are tremendously poor due to its negative charges, high molecular weight and hydrophilicity [13, 14]. Three challenges are involved in intracellular delivery: extracellular barrier, endosome escape, and intracellular liberation [15]. Previous reports indicated that nanocrystals of insoluble drugs could utilized as a "carrier" for protein delivery through anchoring the protein onto the particle surface [16, 17]. Moreover, the pure drug nanocrystals always have a nonspherical shape, such as rod- and needle-like morphology [18], and improve PA accumulation and intracellular delivery [17, 19].

  In this study, rod-shaped baicalein nanocrystals (baicalein nanorods, BNRs) were utilized as a carrier for targeted delivery of anti-miR138 to PASMCs, inhibiting PASMC proliferation and PA remodeling and improving pulmonary hemodynamics. The baicalein-assisted anti-miR138 delivery system (GA-BNRplex) for gene therapy was designed as follows. Anti-miR138 was anchored onto the nanorods (NRs) via electrostatic interaction, followed by coating using glucuronic acid (GA) for targeting the glucose transport-1 (GLUT-1) on PASMCs. First, BNRs with a diameter of 132 nm (hydrodynamic diameter) in length were prepared via the precipitation-sonication method using cationic β-lactoglobulin (CLG) as a stabilizer (Fig. 1A). The CLG coating allowed BNRs to have a positive charge and interact with the miRNA. Next, BNRplex was prepared by the electrostatic interaction between BNRs and anti-miR138. The optimal compression ratio of CLG and anti-miR138 in BNRplex was investigated by agarose gel electrophoresis. As the ratio changed to 1:32, the miRNA band disappeared, indicating complete compression (Fig. 1B). BNRplex at the ratio has a particle size of 178.1 ± 3.2 nm and a potential of 23.14 ± 0.20 mV (Figs. 1C and D). Finally, the GLUT-1 ligand (GA) was coated on BNRplex to prepare the GA-BNRplex. The GA-concentration elevation increased the diameter from 161.3 nm to 201.2 nm (Fig. 1E) and reduced the potential charge from 24.02 mV to −7.73 mV (Fig. 1F). GA-BNRplex with a formulation of 1:32:2 (anti-miR138: CLG: GA) was selected for subsequent study due to the smallest size (161.3 ± 3.5). GA-BNRplex displayed a rod-like shape with a diameter of 180 nm in length (Fig. 1G). GA-BNRplex demonstrated an EE/DL of 87.67%/65.24% for baicalein and 77.4%/0.6% for anti-miR138. A 12-h incubation with 10% fetal bovine serum (FBS) did not significantly change the nanoparticle size and indicated their promising stability after intravenous injection (Fig. 1H). GA-BNRplex released baicalein < 25% at pH values of 6.8 and 7.4 at 24 h, whereas it released over 80% at pH 5.0 because the drug has a weak-basic feature allowing solubility increase in weak-acid conditions (Fig. 1I). In contrast, the nanoparticles released anti-miR138 approximately 60% at 24 h (Fig. 1J). The asynchronous dissociation of the small molecular drug and the biopharmaceutical from the codelivery preparation might benefit the synergy that the biopharmaceuticals often take longer to exert their activity [19].

  Figure 1

  

  Figure 1.  Preparation and Characterization. (A) Schematic illustration of GA-BNRplex preparation. GA-BNRplex was prepared by mixing BNRs with an anti-miR138 solution, followed by GA coating. (B) Agarose gel electrophoresis of anti-miR138 loaded on BNRplex. Effect of mass ratio of anti-miR138/CLG on (C) particle size and (D) zeta potential of BNRplex. Effect of GA loading on (E) particle size and (F) zeta potential of GA-BNRplex. Particle size and PDI were measured by dynamic light scattering (DLS). (G) Transmission electron microscopy (TEM) image of GA-BNRplex. (H) Serum stability in 10% FBS of GA-BNRplex was determined by monitoring particle size changes. In vitro release of (I) baicalein and (J) anti-miR138 from GA-BNRplex. The data are expressed as mean ± SD (n = 3).

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  To study the intracellular delivery of anti-miR138 and transfection, we first investigated the targeted uptake of GA-BNRplex by PASMCs. Strong fluorescence was exhibited in the cells dosed with BNRplex and GA-BNRplex, 3–4 fold higher than the free anti-miR138 group (Figs. 2A–C). The fluorescence intensity in the GA-BNRplex group is significantly stronger than that of the BNRplex group, indicating the targeted delivery. The GLUT-1 receptor is highly expressed on the surface of PASMCs [20]. Subsequently, GA-BNRplex can specifically bind to the GLUT-1 receptors via the ligand-receptor affinity. Previous reports indicated rod-like nanoparticles entered cells mainly through the caveolar pathway [16]. The pre-incubation with the caveolin inhibitors, M-CD or nystatin, declined the uptake by 60% (Figs. 2D–F). indicating the significant involvement of the caveolar pathway. Confocal imaging revealed the co-localization of GA-BNRplex with the caveolin-related markers, Cave-1 and F-actin, further confirming the caveolae-mediated uptake (Fig. 2G). Always, caveolae-mediated endocytosis allows material uptake without entrapment by the lysosomes and enhances intracellular delivery [21, 22]. Then, the intracellular delivery of anti-miR138 and transfection was evaluated. The miR138 level from the groups treated with BNRplex and GA-BNRplex decreased by 78% compared with the saline group (P  <  0.05, Fig. 3A). The over-expression of miR138 causes the abnormal proliferation of PASMCs by disrupting the pathway Bcl-2 associated X protein/B-cell lymphoma-2/caspase 3 (Bax/Bcl-2/Cas-3) [23-25]. As a result, we further assayed the transfection efficacy of preparations by determining these proteins' expression. Dosing free anti-miR138 allowed little effect on the protein expression of Bax, Bcl-2 and Cas-3. Dosing with BNRplex and GA-BNRplex significantly upregulated Bax and Cas-3 and down-regulated Bcl-2 compared with the saline (P  <  0.05, Figs. 3B–E); meanwhile, GA-BNRplex demonstrated a more profound modulation on the protein expression. In summary, GA-BNRplex can target PASMCs and improve the transfection of anti-miR138.

  Figure 2

  

  Figure 2.  PASMC-targeting. Cellular uptake of GA-BNRplex (Cy5-labeled anti-miR138, red fluorescence) at 37 ℃ for 4 h. (A) Confocal laser scanning microscope (CLSM) observation. (B) Flow chart and (C) quantitative analysis using flow cytometry. ^***P  <  0.001 vs. anti-miR138 group. ^#P  <  0.05. Cellular uptake of Cy5-labeled GA-BNRplex (red fluorescence) in PASMCs at 37 ℃ for 4 h after pretreated with endocytosis inhibitors (Nystatin, M-CD) for 0.5 h, determined by (D) CLSM, (E) flow chart and (F) quantitative analysis using flow cytometry. ^***P  <  0.001 vs. control group. (G) Co-localization of Cy5-labeled GA-BNRplex (red fluorescence) with caveolae marker (Alexa flour 488 labeled Cave-1 and F-actin, green fluorescence) after incubation for 4 h at 37 ℃. The concentration of Cy5 is 100 nmol/L. Scale bar: 10 µm. The data are expressed as mean ± SD (n = 3).

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  Figure 3

  

  Figure 3.  Intracellular delivery of anti-miR138 and Bax-Bcl-2-Cas-3 signal axis in PASMCs. Expression of miR138 examined by real-time PCR. The final concentration was 200 nmol/L for anti-miR138 and 60 µg/mL for baicalein. ^***P  <  0.001 vs. the saline group. (B) The protein levels of apoptotic proteins, including (C) Bax, (D) Bcl-2 and (E) Cas-3 were determined by Western blot (WB) analysis. PASMCs were harvested 12 h post-transfection for protein extraction. The final concentration was 200 nmol/L for anti-miR138 and 60 µg/mL for baicalein. The internal control for normalizing protein expression was β-actin. ^*P  <  0.05, ^**P  <  0.01 vs. the anti-miR138 group. ^#P  <  0.05. ns, no significance. The data are expressed as mean ± SD (n = 3).

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  Next, the anti-PAH activities in vitro of preparations were studied. The overproliferation of PASMCs can be driven by the over-expression of miR138 [9]. Accordingly, we first studied the PASMC apoptosis after preparation administration. The incubation with BNRs or anti-miR138 displayed modest apoptosis (Figs. S1A and B in Supporting information). In contrast, the apoptosis rate in the BNRplex and GA-BNRplex groups was over 75%, 13-fold higher than in those treated with free anti-miR138 and BNRs. As expected, GA-BNRplex elevated the apoptosis compared to BNRplex (P  <  0.05). The cytotoxicity of each preparation group towards the PASMCs was further examined by the MTT (Fig. S2 in Supporting information), confirming the apoptosis results. Peripheral vascular inflammation aggravates PAH progression [28], while PASMCs also contribute to inflammation [26]. The treatment with the baicalein-containing preparations significantly suppressed the IL-12 expression compared to the saline group (P  <  0.01, Fig. S3 in Supporting information), indicating effective inflammation inhibition.

  To examine the PA-targeting, we first studied the pharmacokinetics of the nanoparticles by intravenous injection of DiR-labeled nanoparticles in the PAH rats. The plasma concentration of the nanoparticles (BNRplex and GA-BNRplex) was measured by detecting the fluorescence of DiR at different time intervals. The nanoparticles had higher fluorescence intensity for 48 h over free DiR (Fig. 4A). As shown in pharmacokinetic parameters calculated according to the plasma concentration (Table S1 in Supporting information), BNRplex and GA-BNRplex demonstrated 3.6- and 6.4-fold increase of blood half-life and 6.8- and 8.2-fold increase of area under the curve (AUC) compared with free DiR. Then, we studied the biodistribution and lung targeting of the IR783-labeled preparations in MCT-induced PAH rats. The preparation predominantly accumulated in the liver, heart, and lung at 2, 8, and 12 h post-injection (Fig. S4 in Supporting information, Figs. 4B–D). The two nanoparticles had significantly increased lung accumulation than the free dye (Fig. 4E), indicating lung targeting. In addition, the GA-BNRplex showed increased lung accumulation over BNRplex, suggesting that GA modification allowed lung-targeting enhancement. Lastly, we investigated the IR783-labeled preparation ability to target the PAs by assaying the co-localization with the specific PA marker (α-smooth muscle actin (α-SMA)) after 2-h administration [27]. The free dye had little co-localization with PA (purple fluorescence spots). In contrast, BNRplex and GA-BNRplex displayed significant purple fluorescence in the merged images compared to the free dye (P  <  0.01, Figs. 4F and G). Also, GA-BNRplex had a more vigorous fluorescence intensity than BNRplex (P  <  0.05). Collectively, GA-BNRplex could significantly target the lung and PAs.

  Figure 4

  

  Figure 4.  Pharmacokinetic study and PA-targeting. (A) Plasma concentration of GA-BNRplex over time. Plasma concentration was expressed as the plasma fluorescence directly measured after injection. DiR-labeled preparations were injected via the tail vein at the DiR dose of 0.5 mg/kg, according to the body weight (n = 4). (B–D) Biodistribution. The tissues were collected at 2, 8, and 12 h post-injection of at an IR783 dose of 2.5 mg/kg, according to the animal's body weight (n = 5). ^*P  <  0.05, ^**P  <  0.01, ^***P  <  0.001 vs. the 2 h-lung group. (E) Lung distribution at 12 h post-injection. ^*P  <  0.05, ^**P  <  0.01, ^***P  <  0.001 vs. the GA-BNRplex group. (F) Quantitative analysis and (G) CLSM observation for the co-localization of IR783-labeled preparation (red fluorescence) with PA α-SMA marked with Alexa flour 488 (green fluorescence) at 2 h after injection (n = 3). Scale bar: 50 µm. ^*P  <  0.05, ^***P  <  0.001 vs. the GA-BNRplex group. The data are expressed as mean ± SD. DAPI, 4′, 6-diamidino-2-phenylindole.

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  Finally, we studied the treatment efficacy of the preparations against PAH in the model by determining pulmonary hemodynamics, histology analysis of the RV and PAs, and examining inflammation, cell proliferation and apoptosis. The animals were intravenously injected with the 0.5 mL preparations every 3 days 5 times at a dose of 5 mg/kg baicalein and 0.4 mg/kg anti-miR138. The animals used in all experiments were cared for in accordance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals. The animal experiments followed a protocol approved by the China Pharmaceutical University Institutional Animal Care and Use Committee.

  We first investigated the pulmonary hemodynamics at the end of treatment. As shown in Fig. 5A, dosing the preparations, except the naked anti-miR138, reduced the mPAP by > 40% (P  <  0.001) compared to the saline. The treatment with BNRplex or GA-BNRplex decreased the mPAP by 50%. The echocardiographic assessment indicated that GA-BNRplex administration significantly improved RV functions by elevating pulmonary artery acceleration time (PAAT) (P  <  0.05, Figs. 5B and C, Fig. S5 in Supporting information), reducing the RV hypertrophy index (right ventricle internal diameter (RVID), P  <  0.01, Figs. 5D and F, Fig. S6 in Supporting information) and increasing tricuspid annular plane systolic excursion (TAPSE) (P  <  0.001, Figs. 5E and G, Fig. S7 in Supporting information) and cardiac output (CO) (P  <  0.05, Fig. 5H) compared to the saline. Treatment with the pure baicalein nanocrystals (BNRs) also declined mPAP reduction (P  <  0.001) and, however, allowed modest improvement of RV function. In contrast, integrating the miRNA onto the BNRs (GA-BNRplex) could both effectively decrease mPAP and enhance cardiopulmonary functions.

  Figure 5

  

  Figure 5.  Improved pulmonary hemodynamics. (A) mPAP was measured by open-chest hemodynamic measurements. (C–E) Representative images of echocardiography and Doppler echocardiography. Quantified hemodynamics: (B) PAAT, (F) RVID, (G) TAPSE and (H) CO. White arrows point to systolic notching in Doppler flow waves (C). (D, F) RVID morphologically indicates the alleviation of right ventricular hypertrophy after treatment. (B, C) PAAT and (E, G) TAPSE indicate significant improvement in right ventricular function after treatment. CO indicates cardio ejection function. The animals were intravenously injected with 0.5 mL of these preparations every 3 days for 5 times at a baicalein dose of 5 mg/kg and anti-miR138 dose of 0.4 mg/kg, according to the animal's body weight. The data are expressed as mean ± SD (n = 5). ^*P  <  0.05, ^**P  <  0.01, ^***P  <  0.001 vs. the anti-miR138 group. ^#P  <  0.05, ^###P  <  0.001.

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  Then, we studied the RV hypertrophy by measuring the RV cardiomyocyte proliferation and Fulton's index (Fig. S8 in Supporting information). Dosing BNRplex and GA-BNRplex inhibited the cardiomyocyte proliferation by approximately 60% (P  <  0.05) while other preparations did not show significant inhibition, as compared to the saline group (Figs. S8A and B). Also, the two nanoparticles reduced the RVHI (RV/(LV+S) = right ventricle/left ventricle plus septum) by 40% (vs. saline group) (Fig. S8C).

  To investigate the inhibition of PA remodeling after treatment, we initially investigated the lung inflammation by assaying the IL-12 expression. Dosing the baicalein-containing preparations downregulated the cytokine by 50% compared to the saline treatment, indicating inflammation inhibition (Fig. S9 in Supporting information). Then, the PA remodeling was assayed by testing the medial wall thickening (%) and expression of marker α-SMA at the end of treatment. Dosing free anti-miR138 demonstrated little effect on the PA thickening compared to the saline (Figs. 6A and C). The PA thickening from the groups treated with BNRs, BNRs+anti-miR138, BNRplex, and GA-BNRplex was alleviated. Moreover, BNRplex and GA-BNRplex reduced the thickness significantly (P  <  0.05). PA remodeling was further confirmed by examining the overexpression of α-SMA. As shown in Figs. 6B and D, α-SMA was down-regulated after treatment, especially in the BNRplex and GA-BNRplex groups. Additionally, the proliferation and apoptosis of PASMCs in PAs were investigated. The treatment with BNRplex or GA-BNRplex allowed an apoptosis rate of 70% and 80% and a proliferation rate of 7% and 13%, respectively (Figs. 6E and F). The results indicated that GA-BNRplex effectively inhibited PA remodeling by promoting pro-apoptosis and anti-proliferation of PASMCs. In addition, the hematoxylin and eosin (H & E) assay showed that GA-BNRplex induced little toxicity to the major organs compared with the saline group (Fig. S10 in Supporting information).

  Figure 6

  

  Figure 6.  Inhibited PA-remodeling in the MCT-induced pH. (A) Representative changes and (C) quantitative analysis on the medial wall thickness of PAs. (B) Representative changes and (D) quantitative analysis on the α-SMA expression in PAs. Quantitative assay of (E) terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and (F) Ki67 staining. The animals were intravenously injected with 0.5 mL of these preparations every 3 days 5 times at a fixed baicalein dose of 5 mg/kg and anti-miR138 dose of 0.4 mg/kg, according to the animal's body weight. The data are expressed as mean ± SD (n = 5). Scale bar: 50 µm. ^*P  <  0.05, ^**P  <  0.01, ^***P  <  0.001 vs. the anti-miR138 group. ^#P  <  0.05, ^#P  <  0.01, ^#P  <  0.001.

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  To study the mechanism of PASMC-proliferation inhibition, we also determined the miR-138 expression in the lung at the end of treatment using the preparations. The administration of free anti-miR-138 demonstrated little effect on the miR-138 level compared to saline. Dosing BNRplex or GA-BNRplex declined miR-138 expression (Fig. S11 in Supporting information, P  <  0.001), and GA-BNRplex showed additional inhibition vs. BNRplex (P  <  0.05). The data implied that the transfection efficacy of nanoparticles is closely linked to their PA-targeted ability. Then, we assayed the signal pathway Bax/Bcl-2/Cas-3 in the lung at the end of treatment. Treatment with BNRplex or GA-BNRplex reduced the Bcl-2 expression by 50% (P  <  0.001 vs. free anti-miR138, Figs. 7A and C) or 20% (P  <  0.01), increased the expression of Bax and Cas-3 by 2–3-fold (P  <  0.01, Figs. 7B and D). The results indicated that GA-BNRplex could effectively enhance the transfection efficiency of anti-miR138 in vivo and modulate the Bax/Bcl-2/Cas-3 pathway, consistent with the in vitro transfection experiment.

  Figure 7

  

  Figure 7.  Bax-Bcl-2-Cas-3 signal axis in vivo. (A) The protein levels of (B) Bax, (C) Bcl-2, and (D) Cas-3 in the lung were determined by WB analysis at the end of treatment. The animals were intravenously injected with 0.5 mL of these preparations every 3 days 5 times at a fixed baicalein dose of 5 mg/kg and anti-miR138 dose of 0.4 mg/kg, according to the animal's body weight. The internal control for normalizing protein expression was β-actin. The data are expressed as mean ± SD (n = 3). ^*P  <  0.05, ^**P  <  0.01, ^***P  <  0.001 vs. the anti-miR138 group. ^#P  <  0.05.

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  Few reports indicated that miRNA-based gene therapy could be against PAH [28, 29]. miR145 allows PASMC development and contractility. McLendonad et al. investigated the efficacy of anti-miR145-loaded liposomes in a PAH-induced model [30]. The results indicated that the intravenous injections lowered the mPAP, the level of cardiac hypertrophy and the intensity of pulmonary arteriopathy. In this study, we also proved that targeted delivery of anti-miR138 using BNRs could effectively inhibit PA remodeling and alleviate the MCT-PAH model. However, our injection dose is significantly lower than the previous report (0.4 vs. 2 mg/kg) because BNRs had improved PA-targeted ability over the spherical liposomes [17]. Our study indicated that PA-targeted miRNA gene therapy is promising to treat PAH.

  Combination therapies are increasingly used to treat PAH in clinics due to the complex pathology. Several combination regimens have been tested; treprostinil inhalations + tadalafil (NCT01305252) and beraprost + sildenafil (NCT03431649) reached phase 4 of clinical trials. In this study, dosing BNRs decreased mPAP but did not significantly improve RV function. However, the codelivery of anti-miR138 and the anti-inflammatory drug baicalein could significantly inhibit PA remodeling and elevate cardiopulmonary functions. The results demonstrated that combining PASMC-proliferation inhibitor and anti-inflammatory drug represents a potential treatment approach against PAH.

  In conclusion, rod-shaped nanomedicine (BNRs) could efficiently target PAs. BNRs-assisted anti-miR138 gene therapy inhibited PA remodeling and alleviated PAH. Integrating microRNA gene therapy and anti-inflammation is promising for treating PAH.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Chenshi Lin: Methodology, Investigation. Chao Teng: Writing – original draft, Methodology, Investigation. Bingbing Li: Writing – review & editing, Supervision. Wei He: Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition, Conceptualization.

  Acknowledgments

  This study was supported by the National Natural Science Foundation of China (Nos. 81872823, 82073782, and 82241002) and the Shanghai Science and Technology Committee (No. 19430741500).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110450.

  
  
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• 

  Figure 1  Preparation and Characterization. (A) Schematic illustration of GA-BNRplex preparation. GA-BNRplex was prepared by mixing BNRs with an anti-miR138 solution, followed by GA coating. (B) Agarose gel electrophoresis of anti-miR138 loaded on BNRplex. Effect of mass ratio of anti-miR138/CLG on (C) particle size and (D) zeta potential of BNRplex. Effect of GA loading on (E) particle size and (F) zeta potential of GA-BNRplex. Particle size and PDI were measured by dynamic light scattering (DLS). (G) Transmission electron microscopy (TEM) image of GA-BNRplex. (H) Serum stability in 10% FBS of GA-BNRplex was determined by monitoring particle size changes. In vitro release of (I) baicalein and (J) anti-miR138 from GA-BNRplex. The data are expressed as mean ± SD (n = 3).

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  Figure 2  PASMC-targeting. Cellular uptake of GA-BNRplex (Cy5-labeled anti-miR138, red fluorescence) at 37 ℃ for 4 h. (A) Confocal laser scanning microscope (CLSM) observation. (B) Flow chart and (C) quantitative analysis using flow cytometry. ^***P  <  0.001 vs. anti-miR138 group. ^#P  <  0.05. Cellular uptake of Cy5-labeled GA-BNRplex (red fluorescence) in PASMCs at 37 ℃ for 4 h after pretreated with endocytosis inhibitors (Nystatin, M-CD) for 0.5 h, determined by (D) CLSM, (E) flow chart and (F) quantitative analysis using flow cytometry. ^***P  <  0.001 vs. control group. (G) Co-localization of Cy5-labeled GA-BNRplex (red fluorescence) with caveolae marker (Alexa flour 488 labeled Cave-1 and F-actin, green fluorescence) after incubation for 4 h at 37 ℃. The concentration of Cy5 is 100 nmol/L. Scale bar: 10 µm. The data are expressed as mean ± SD (n = 3).

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  Figure 3  Intracellular delivery of anti-miR138 and Bax-Bcl-2-Cas-3 signal axis in PASMCs. Expression of miR138 examined by real-time PCR. The final concentration was 200 nmol/L for anti-miR138 and 60 µg/mL for baicalein. ^***P  <  0.001 vs. the saline group. (B) The protein levels of apoptotic proteins, including (C) Bax, (D) Bcl-2 and (E) Cas-3 were determined by Western blot (WB) analysis. PASMCs were harvested 12 h post-transfection for protein extraction. The final concentration was 200 nmol/L for anti-miR138 and 60 µg/mL for baicalein. The internal control for normalizing protein expression was β-actin. ^*P  <  0.05, ^**P  <  0.01 vs. the anti-miR138 group. ^#P  <  0.05. ns, no significance. The data are expressed as mean ± SD (n = 3).

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  Figure 4  Pharmacokinetic study and PA-targeting. (A) Plasma concentration of GA-BNRplex over time. Plasma concentration was expressed as the plasma fluorescence directly measured after injection. DiR-labeled preparations were injected via the tail vein at the DiR dose of 0.5 mg/kg, according to the body weight (n = 4). (B–D) Biodistribution. The tissues were collected at 2, 8, and 12 h post-injection of at an IR783 dose of 2.5 mg/kg, according to the animal's body weight (n = 5). ^*P  <  0.05, ^**P  <  0.01, ^***P  <  0.001 vs. the 2 h-lung group. (E) Lung distribution at 12 h post-injection. ^*P  <  0.05, ^**P  <  0.01, ^***P  <  0.001 vs. the GA-BNRplex group. (F) Quantitative analysis and (G) CLSM observation for the co-localization of IR783-labeled preparation (red fluorescence) with PA α-SMA marked with Alexa flour 488 (green fluorescence) at 2 h after injection (n = 3). Scale bar: 50 µm. ^*P  <  0.05, ^***P  <  0.001 vs. the GA-BNRplex group. The data are expressed as mean ± SD. DAPI, 4′, 6-diamidino-2-phenylindole.

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  Figure 5  Improved pulmonary hemodynamics. (A) mPAP was measured by open-chest hemodynamic measurements. (C–E) Representative images of echocardiography and Doppler echocardiography. Quantified hemodynamics: (B) PAAT, (F) RVID, (G) TAPSE and (H) CO. White arrows point to systolic notching in Doppler flow waves (C). (D, F) RVID morphologically indicates the alleviation of right ventricular hypertrophy after treatment. (B, C) PAAT and (E, G) TAPSE indicate significant improvement in right ventricular function after treatment. CO indicates cardio ejection function. The animals were intravenously injected with 0.5 mL of these preparations every 3 days for 5 times at a baicalein dose of 5 mg/kg and anti-miR138 dose of 0.4 mg/kg, according to the animal's body weight. The data are expressed as mean ± SD (n = 5). ^*P  <  0.05, ^**P  <  0.01, ^***P  <  0.001 vs. the anti-miR138 group. ^#P  <  0.05, ^###P  <  0.001.

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  Figure 6  Inhibited PA-remodeling in the MCT-induced pH. (A) Representative changes and (C) quantitative analysis on the medial wall thickness of PAs. (B) Representative changes and (D) quantitative analysis on the α-SMA expression in PAs. Quantitative assay of (E) terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and (F) Ki67 staining. The animals were intravenously injected with 0.5 mL of these preparations every 3 days 5 times at a fixed baicalein dose of 5 mg/kg and anti-miR138 dose of 0.4 mg/kg, according to the animal's body weight. The data are expressed as mean ± SD (n = 5). Scale bar: 50 µm. ^*P  <  0.05, ^**P  <  0.01, ^***P  <  0.001 vs. the anti-miR138 group. ^#P  <  0.05, ^#P  <  0.01, ^#P  <  0.001.

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  Figure 7  Bax-Bcl-2-Cas-3 signal axis in vivo. (A) The protein levels of (B) Bax, (C) Bcl-2, and (D) Cas-3 in the lung were determined by WB analysis at the end of treatment. The animals were intravenously injected with 0.5 mL of these preparations every 3 days 5 times at a fixed baicalein dose of 5 mg/kg and anti-miR138 dose of 0.4 mg/kg, according to the animal's body weight. The internal control for normalizing protein expression was β-actin. The data are expressed as mean ± SD (n = 3). ^*P  <  0.05, ^**P  <  0.01, ^***P  <  0.001 vs. the anti-miR138 group. ^#P  <  0.05.

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