A novel combination therapy of anti miR-21 and baculoviral TGFβ1 Gene via PLGA-gelatin-genipin nanocomposite hydrogel for arterial plaque stabilization and angiogenesis

A novel combination therapy of anti miR-21 and baculoviral TGFβ1 Gene via PLGA-gelatin-genipin nanocomposite hydrogel for arterial plaque stabilization and angiogenesis | 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 Article A novel combination therapy of anti miR-21 and baculoviral TGFβ1 Gene via PLGA-gelatin-genipin nanocomposite hydrogel for arterial plaque stabilization and angiogenesis Paromita Islam, Ahmed Abosalha, Sabrina Schaly, Jacqueline L. Boyajian, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6770483/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Atherosclerosis is the primary cause of most cases of coronary artery disease, peripheral arterial disease, and many strokes. It is characterized by pathological vascular smooth muscle cell hyperplasia. Current treatment regimens are associated with several adverse effects including hepatotoxicity, hemorrhagic complications, and non-selective cellular inhibition. Plaque stabilization and angiogenesis are critical for mitigating adverse cardiovascular outcomes. Stabilized plaques exhibit reduced vulnerability to rupture, thereby lowering the risk of thrombus formation, myocardial infarction, and ischemic stroke. Transforming Growth Factor Beta 1 (TGF-β1cells) is instrumental in promoting angiogenesis, facilitating the regrowth of endothelial cells, and contributing to the stabilization of atherosclerotic plaques. Anti-miRNA 21 can lead to plaque stabilization by decreasing inflammation and limiting the growth of smooth muscle cells while encouraging cell death, which helps prevent plaque rupture. PLGA nanoparticles can ensure high encapsulation and effective delivery of genes and viral vectors over time and can offer superior protection for their encapsulated contents, which is particularly valuable for delicate substances such as proteins and nucleic acids. This research investigates a novel combination therapy utilizing baculovirus expressing TGF-β1 gene and anti-miR-21, incorporated into gelatin-genipin polymeric nanocomposite hydrogels. The therapy demonstrates synergistic effects through dual mechanisms: promoting neo-vascularization via selective endothelial cell proliferation while inducing smooth muscle cell apoptosis to control extracellular matrix secretion and stabilize plaque. The therapeutic efficacy is evidenced by significant reduction in PTEN expression (251.1 ± 16 pg/ml compared to 375.2 ± 5.29 pg/ml in control) and enhanced angiogenic responses in the CAM assay, showing a 126.46 ± 16.62% increase in vessel length. Biological sciences/Biotechnology Health sciences/Cardiology Health sciences/Medical research Physical sciences/Materials science Angiogenesis tissue regeneration gene therapy viral vector atherosclerosis baculovirus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Vascular proliferative disorders, including atherosclerosis, in-stent restenosis, and vein graft disease, are characterized by pathological vascular smooth muscle cell hyperplasia initially forming plaques followed by plaque destabilization and endothelial dysfunction, leading to significant morbidity and mortality(1, 2). Current therapeutic interventions, such as systemic administration of HMG-CoA reductase inhibitors and antiplatelet agents, while effective, are associated with considerable adverse effects including hepatotoxicity, hemorrhagic complications, and non-selective cellular inhibition. Additionally, drug-eluting stents, while reducing restenosis rates, may impair re-endothelialization and increase the risk of late thrombotic events(3, 4). Plaque stabilization and angiogenesis are critical for mitigating these adverse cardiovascular outcomes (5). Stabilized plaques exhibit reduced vulnerability to rupture, thereby lowering the risk of thrombus formation, myocardial infarction, and ischemic stroke (6). Angiogenesis facilitates neovascularization and ischemic tissue repair, restoring perfusion in compromised areas. These processes are integral to halting the progression of atherosclerotic lesions, preventing plaque destabilization, and promoting vascular homeostasis (7). Collectively, they play essential roles in reducing morbidity and mortality associated with advanced atherosclerotic disease. TGF-β1 (Transforming Growth Factor Beta 1) is essential in angiogenesis, re-endothelialization, and plaque stabilization (8). It promotes new blood vessel formation and supports endothelial repair after vascular injury, enhancing healing and reducing thrombosis. In plaque stabilization, TGF-β1 encourages the deposition of extracellular matrix, increases smooth muscle cell proliferation, and modulates inflammation, making plaques less prone to rupture (9). Its regulatory role in these processes is key to maintaining vascular integrity and preventing atherosclerosis progression and complications. On the other hand, miR-21 contributes to plaque destabilization by promoting inflammation, smooth muscle cell proliferation, and reducing apoptosis, making plaques more prone to rupture (10). In angiogenesis, miR-21 enhances vascular growth by regulating endothelial cell behavior (11). However, it impairs re-endothelialization by inhibiting endothelial cell migration, hindering vascular repair and healing processes (12). By inhibiting miR-21, this therapy could potentially reduce inflammation and fibrosis in atherosclerotic plaques, promoting a more stable phenotype (13). Antagonism of miR-21 can stabilize plaques by reducing inflammation and smooth muscle cell proliferation while promoting apoptosis, thereby preventing plaque rupture (14). By inhibiting miR-21, endothelial cell migration and proliferation are enhanced, facilitating better re-endothelialization and vascular repair after injury (15). Additionally, miR-21 antagonism can help normalize angiogenesis, ensuring controlled vessel formation and reducing pathological vascular growth. Overall, targeting miR-21 offers a therapeutic approach to improve vascular stability and repair, reducing atherosclerosis progression and related complications (16). Anti-miR-21 therapy has shown clear benefits in reducing neointimal hyperplasia (17). Combining this with TGFβ1 modulation could potentially enhance this effect by regulating smooth muscle cell proliferation and migration. Baculoviral gene therapy offers a versatile, safe, and efficient system for wound healing and revascularization due to its high gene loading capacity, low toxicity, and targeted delivery (18). With a large 130 kb genome, baculoviruses (BVs) can deliver large therapeutic genes, and their rapid, scalable production in insect cells makes them cost-effective for regenerative medicine and other therapies (19).According to our previously published study, Poly (d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) encapsulating BV carrying the TGF-β1 gene enhances endothelial wound healing in human umbilical vein endothelial cells (HUVECs) by providing sustained, efficient gene delivery and protecting the virus from degradation (20). This system improves cell migration, proliferation, and wound closure compared to free BV. Hydrogels consist of crosslinked polymer chains arranged in a three-dimensional network, allowing them to absorb substantial amounts of liquid. Their high-water content, soft texture, and porous nature make them similar to living tissues. It is becoming a promising and effective option for drug delivery because their adjustable functional properties can be tailored to enhance healing. These properties include biodegradability, adhesiveness, antimicrobial effects, anti-inflammatory capabilities, and pre-angiogenic bioactivities (21). Together, these features can significantly accelerate the healing of chronic wounds. Genipin is a natural crosslinker frequently used in drug administration due to its outstanding properties. It is recognized as a biocompatible, biodegradable, non-toxic, and stable crosslinking agent, making it highly valuable in pharmaceutical and biomedical applications (22). Thus, this study investigates the fabrication and efficacy of a combination therapy of TGF-β1 gene using BV and anti miR-21 encapsulated in these PLGA NPs to formulate the polymeric nanocomposite hydrogels with gelatin-genipin to deliver a combination therapy of TGF-β1 gene using BV and anti miR-21 to simultaneously induce smooth muscle cell apoptosis while promoting endothelial cell proliferation, minimize systemic exposure while maintaining therapeutic efficacy, enhance vascular homeostasis by modulating cell-specific responses, potentially reducing the incidence of vulnerable plaque formation and late thrombotic complications(23). Materials & Methods Virus generation and titration. The virus generation and titration were performed following a previously discussed method by this group.(20) Briefly, Sf21, Spodoptera frugiperda, insect cells (Invitrogen Life Technologies, Carlsbad, CA, USA) were cultured in Sf-900™ III SFM medium (Gibco) at 27°C and sub-cultured 2–3 times weekly. The TGF-β1 gene (GenScript Biotech, USA) was cloned into the pACEBac1 vector (Geneva Biotec, Switzerland) and recombined with a DH10EMBacVSV™ bacmid via transposition in E. coli. Then, the recombinant bacmid was transfected into Sf21 cells using TransIT-Insect Reagent (Mirus Bio LLC, USA) to produce P 0 BV stock. This was amplified in Sf21 cells to generate P 1 and P2 stocks. Viral titers were determined via flow cytometry using mCherry fluorescence and confirmed by cytopathic endpoint dilution.(24) Design and characterization of nanocomposite hydrogels carrying TGF-β1 expressing baculovirus and anti-miR-21 Preparation of dual combination therapy PLGA nanoparticles. Bac-anti-miR-PLGA-NPs loaded with TGF-β1 gene carrying BV and FAM-tagged locked nucleic acid (LNA) anti-miR21 (5′-ACGGCAACACCAGUCGAUGGGCUGU-3, Creative Biogene Inc., USA) were prepared using the double emulsion solvent evaporation technique previously described (20, 25). Briefly, PLGA (Millipore Sigma, Germany) was dissolved in dichloromethane (DCM- Millipore Sigma, Germany), and 100 MOI recombinant BV and 0.2 nmol anti miR-21 was coated with 1% polyvinyl alcohol (PVA- Millipore Sigma, Germany) to protect it during emulsification and to facilitate NPs solidification. The resultant W/O emulsion was further emulsified in 1 % aqueous PVA solution to form the final W/O/W nanoemulion. This emulsion is then stirred for 6 h to solidify the NPs, which were then collected by ultracentrifugation, washed, and freeze-dried for further embedding. Formulation of Bac-anti-miR-PLGA nanocomposite gelatin-genipin hydrogel. The prepared PLGA NPs is then embedded in gelatin-genipin hydrogels by a previously used method.(26) The gelatin from porcine skin (Millipore Sigma, Germany) was dissolved in distilled water by stirring the mixture for 30 minutes at 50°C. After cooling down upto 25°C, previously dissolved genipin solution (Fisher Scientific, USA) was slowly added to the gelatin solution to make 4% w/v gelatin and 0.25% w/v genipin concentration in the final formulation under continuous mechanical stirring. To further formulate the nanocomposite hydrogel, amount of previously prepared Bac-anti-miR-PLGA-NPs dose adjusted according to entrapment efficiency were dispersed into 10 ml gelatin–genipin solution.(27) This dispersion process was carried out under constant stirring at 50°C for 30 minutes. The nanoparticle-incorporated gelatin-genipin gels were finally left to gel overnight at 40°C for further crosslinking. The crosslinked Bac-anti-miR-PLGA nanocomposite hydrogel was then vacuum freeze-dried for 24 hours, and the resulting samples were stored for further analysis. In vitro characterization of nanoparticles and nanocomposite hydrogel Physicochemical characterization. The physicochemical characterization of the NPs was performed using dynamic light scattering (DLS) and electrophoretic laser Doppler anemometry (Brookhaven Instruments, Holtsville, NY, USA). Mean particle size and polydispersity index were measured at 25°C, employing a 90° scattering angle and 1.33 refractive index. Zeta potential was determined using the same instrument. All measurements were conducted in triplicate using disposable cells. Zeta Potential Analyzer v3.57 software facilitated zeta potential calculations. Atomic Force Microscope: For particle size quantification, the substrate with the dried nanoparticle was mounted onto the stage of the Atomic Force Microscope (AFM) equipped with a molecular force probe controller (Asylum Research - Oxford Instruments, Santa Barbara, CA) in AC mode in the air using ACTA probes (Applied Nanostructures, Inc., Mountain View, CA). Data is processed using MountainsSPIP v. 10.1.10606 (Digital Surf, Besançon, France). Scanning Electron Microscope. The prepared NPs and nanocomposite hydrogel surface morphology, particle size and polymer crosslinking were examined using scanning electron microscopy (SEM) with a FEI 450 Quanta SEM at McGill University, operated at 10 kV. The hydrogel was freeze dried and deposited on carbon tape. All the samples were then sputter coated with platinum before imaging. Fourier transform infrared (FTIR): The FTIR spectra of PLGA, lyophilized PLGA NPs, hydrogel, genipin and gelatin were generated using a computerized FTIR spectroscopy, Perkin Elmer Spectrum, Waltham, Massachusetts, USA (IR Version, 10.7.2) that operated in the 400–4000 cm –1 scanning wave number range at a resolution of 1 cm -1 . Encapsulation Efficiency of the nanocomposite hydrogel. After preparation, the nanocomposite hydrogel was washed in PBS. The rinsing PBS supernatant was collected and subsequently, subjected to analysis using UV spectroscopy by a Nanodrop 2000 (Thermofischer, Massachusetts, USA). Encapsulation efficiency (EE) was calculated by determining the percentage of the drug encapsulated into the hydrogel, employing the standard formula. All measurements were conducted in triplicate, and the results were reported as the mean ± standard deviation (SD). Cell line studies Mammalian Cell Culture. Human umbilical vein endothelial cells (HUVECs), procured from Sigma Aldrich, were cultured using complete endothelial growth medium from Sigma Aldrich with 10% Fetal Bovine Serum and Human Aortal Smooth Muscle Cells (HASMCs) were cultured with Medium 199 with 0.02 mg/mL endothelial growth supplement. These cells were incubated in T-25 flasks within a 37 °C, 5% CO2 incubator and were employed within five passages upon receipt. Cellular Adhesion Study . To determine if the nanocomposite hydrogels promoted cellular adhesion, thin film of the hydrogel was created and seeded on a cell culture plate. The hydrogel was dried overnight and then swollen in cell media for one hour. After one hour, the cell media was removed and HUVECs were seeded onto the hydrogel. Empty tissue treated wells were used as a control. The seeded hydrogel was incubated at 37 °C for two hours. After two hours, the plate was washed with HBSS to remove any unattached cells. The hydrogel was then fixed in 3.7 % (v/v) formaldehyde for 15 minutes and washed with HBSS. Finally, the hydrogel was stained with 0.1 % (m/v) crystal violet to visualize the cells under a brightfield microscope. The cells were counted using ImageJ software. Assessment of Hemocompatibility. Blood samples were collected commercially and tested independently in triplicate. The potential hemolysis of the virus itself, placebo and the gene carrying NPs were evaluated. Briefly, all tested samples were immersed into 5 mL of PBS in a 15 mL centrifuge tube. Next, 4 mL of citrated blood were mixed with 5 mL PBS and 0.1 mL of the diluted blood were added to each sample. The samples were incubated at 37 C for 1 h and then centrifuged at 1000 rpm for 10 min. The supernatant containing the lysed hemoglobin were placed into a 96-well plate, and the absorbance were read at 545 nm. The negative and positive control were PBS and deionized water, respectively. The following equation is used to determine the % hemolysis (28). Live/Dead Cell Viability Assay. A Live/Dead Assay was employed to estimate the safety profile of the combination therapy delivered via nanocomposite hydrogel using Calcein AM and propidium iodide. HUVECs and HASMCs were seeded (2 × 10⁴ cells per well) in 48-well plates and incubated overnight. Free BVs, anti miR-21s, their free combination and the combination therapy were added to different wells, with media alone as control, and incubated for 12 hours before replacing the transduction media with fresh media. After 48 hours, live and dead cells were stained with Calcein AM (green, live) and propidium iodide (red, dead). Cells were imaged using the Leica DMIL microscope, and ImageJ software was used to quantify cell viability based on fluorescence images. C-Reactive Protein (CRP) Assay. To quantify serum CRP levels, HUVECs and HASMCs were cultured and treated with free BVs, anti miR-21s and hydrogel elutions for 12 hours. Cell culture supernatants were centrifuged and assayed for CRP levels using an ELISA kit (Abcam, USA). ELISA was performed according to the manufacturer's protocol, with absorbance measured at 450 nm using a multi-mode plate reader (EnSpire Multimode, Perkin Elmer, USA). All assays were performed in triplicate. MTT Cell Proliferation Assay. Cell proliferation and viability were evaluated using an MTT assay. HUVECs and HASMCs (10,000 cells/well) were seeded in 96-well plates and treated with free BVs, free anti miR-21s and supernatant containing the combination therapy eluted from the nanocomposite hydrogel. After a 12-hour incubation, 20 µL of MTT solution (5 mg/mL) was added, and cells were incubated for 4 hours at 37°C. The resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and absorbance was measured at 570 nm using an EnSpire Multimode plate reader (Perkin Elmer, USA). Cytotoxicity was calculated as % Cytotoxicity = (OD of control - OD of test) × 100 / OD of control, and all experiments were performed in triplicate. Evaluation of PTEN expression in HUVECs. The cells were treated with the free BVs, free anti-miR21, free combination, combination therapy nanocomposite supernatant, PBS for control. After 24 hours incubation, the conditioned medium was collected and their Phospatase and tensin homolog (PTEN) protein expression was quantified by a sandwich Human PTEN enzyme-linked immunosorbent Kit (AB206979- 1002, Abcam, MA, USA) with 39.9 pg/ml sensitivity. Measurement of sample absorbance was carried out using a EnSpire Multimode plate reader (Perkin Elmer, USA) at a wavelength of 450 nm. Evaluation of Apoptosis via Flow Cytometry . The pro-apoptotic effects of combination of free BVs and anti-miR-21 and the combination NPs were evaluated through flow cytometry. HASMC cells (1 × 10⁶ cells/ml) were cultured in 24-well plates (Corning, NY, USA) and treated with control, free treatments, or combination therapy. The cells were incubated for 24 hours in a mammalian cell incubator. Following the incubation, the cells were washed three times with PBS (0.1 M, pH 7.4) and resuspended in 100 µl of 1× binding buffer (eBioscience, Inc., San Diego, USA). Next, 5 µl of Annexin V-FITC (final concentration, 1 µg/ml; eBioscience, Inc.) and 5 µl of propidium iodide (10 µg/µl; eBioscience, Inc.) were added to the suspension, and the mixture was incubated for 15 minutes in the dark at room temperature. Before analysis, 200 µl of 1× binding buffer was added, and the extent of apoptosis was determined using a FACScan flow cytometer with Cell Quest software (BDFACSAria Fusion Flow Cytometer, New Jersey, USA). Evaluation of angiogenesis on the chorioallantoic membrane (CAM) in-ovo. Fertilized chicken eggs were incubated at 37°C and 60% humidity. On embryonic development day 6 (EDD6) a window was made into the shell of the egg, to expose the CAM. The chorion was carefully removed, and the eggs were treated with 100 µL of either Combination therapy, placebo and PBS (control) directly onto the CAM surface. The window is sealed with transparent tape to prevent dehydration. The eggs were examined throughout the week for mortality, no eggs perished throughout the week. After 7 days, the CAM is photographed to observe and quantify vascular changes. Key parameters measured include vessel density, branching, and diameter. Angiogenic response is typically assessed by counting new blood vessel branch points within a defined area around the application site with the help of angiotool software. Statistical Analysis. The data are expressed as mean ± standard deviation or mean ± standard error of the mean, as specified. One-way ANOVA followed by Tukey’s multiple comparisons test was performed using GraphPad Prism version 10.2.2 for Windows, GraphPad Software, Boston, Massachusetts USA, www.graphpad.com. Significance levels are denoted as **** for p < 0.0001, *** for p < 0.001, ** for p ≤ 0.01 and * for p ≤ 0.01. Results In-vitro characterization TGFβ1 and Anti-miR-21 combination therapy nanocomposite hydrogel The anionic BVs and mir21 antagomirs underwent surface coating using polymers to generate Bac-anti-miR-polymer NPs. Bac-anti-miR-PLGA-NPs were successfully formulated using the double emulsion method(25), and showed spherically shaped particles distinctively prepared (n = 3). The surface morphology of the NPs and the nanocomposite hydrogel were analyzed to confirm the formation of NPs via AFM. The AFM image in figure 1A reveal spherical Bac-anti-miR-PLGA-NPs with heterogeneous size distribution, predominantly clustered in specific regions. The particles exhibit varying heights up to 800 nm, with most particles showing diameters between 200-500 nm. The surface topography indicates partial aggregation of particles, suggesting potential interactions between individual nanostructures. These results demonstrate the effectiveness of the double-emulsion solvent evaporation method used for nanoparticle preparation. TEM images of the formulated Bac-anti-miR-PLGA-NPs showed small, spherical, uniformly distributed, and non-aggregated particles as demonstrated in Figure 1B. The size values obtained from TEM agree with size measurements resulted from DLS i.e. 372.4±20.45. It is well known that spherical shaped NPs exhibit higher surface area, drug loading, controlled release of the cargo and good mobility within biological environments. The surface charge of these NPs is - 26.39±2.79 that is typical for PLGA, a negatively charged polymer, encapsulating baculovirus that is also negatively charged on its surface which is beneficial for controlled drug release systems(29, 30). Additionally, SEM imaging of the Bac-anti-miR-PLGA-NPs alone as well as embedded within the nanocomposite gelatin- genipin hydrogels, confirmed the presence of pores within the gel matrix and the crosslinking necessary for nanoparticle encapsulation, as shown in Figure 1C. These findings highlight genipin's ability to crosslink with gelatin, facilitating the formation of pores that can effectively load BV-carrying PLGA NPs. The FTIR spectra in figure 3 reveals characteristic peaks for each component and their interactions in the final nanocomposite hydrogel system. The genipin spectrum shows distinct peaks in the 1750-1000 cm⁻¹ region, while PLGA and PLGA NPs exhibit similar patterns with characteristic ester bonds at 1750 cm⁻¹. The gelatin spectrum shows typical amide bands at 3300-3500 cm⁻¹. In the genipin-gelatin hydrogel, peak shifts indicate successful crosslinking. The nanocomposite hydrogel spectrum combines features from all components, confirming successful incorporation of PLGA NPs into the genipin-crosslinked gelatin matrix. Figure 1D displays the FTIR spectra of the individual polymers used along with the final Bac-anti-miR-PLGA-NPs investigated in this study. The FTIR characterization data for gelatin-genipin hydrogels reveals distinct spectral features indicative of their compositions. The FTIR spectra reveals successful crosslinking. The gen-gelatin shows broader peaks, especially in the 1600-1700 cm -1 range (amide I band), indicating crosslinked gelatin. The presence of genipin is confirmed by unique peaks at the region 1500-1700 cm -1 (C=O and C=C stretching), which are absent in the gelatin alone. Both hydrogel types exhibit broad peaks at 3400 cm -1 (O-H stretching), typical of hydroxyl groups in gelatin. The distinct shifts and new peaks in the gelatin-genipin hydrogel spectra highlight the crosslinking effects of genipin, demonstrating successful chemical modifications as shown in Figure 2. The percentage of entrapment efficiency (% EE) is one of the most important physicochemical characterizations as it represents the number of active molecules encapsulated within the prepared NPs. It is always favorable to achieve high % EE to deliver the cargo with high concentration to the desired cells. Hydrophilic molecules such as miRNAs exhibit low % EE due to the possibility of their leakage to the external aqueous phase during the fabrication of NPs. The adopted double emulsion solvent evaporation technique is well known by its ability to enhance the loading of hydrophilic drugs into NPs. The % EE of the formulated Bac-anti-miR-PLGA-NPs is 73.69% +- 0.006. TGFβ1 and Anti-miR-21 combination therapy nanocomposite hydrogel promotes cellular adhesion and hemocompatibility The hemolysis assay in figure 2A demonstrated that the gelatin-genipin hydrogel had a low hemolysis percentage, comparable to PBS and placebo, and significantly lower than the DI control (p < 0.0001), indicating its biocompatibility. In the adhesion study with HUVECs, microscopy images (figure 2A) revealed a higher density of adherent cells on surfaces treated with combination therapy compared to the control. Quantitative analysis (figure 2B) further confirmed significantly enhanced HUVEC adhesion to the gelatin-genipin hydrogel (p < 0.0001), supporting its potential as a favorable scaffold for vascular tissue engineering. 2C The gelatin-genipin hydrogel provides a biocompatible and supportive environment for endothelial cell adhesion, making it promising for therapeutic and tissue engineering applications. TGFβ1 and Anti-miR-21 Combination Therapy Nanocomposite Hydrogel is safe and reduces C-reactive protein in HUVECs and HASMCs The effect of the combination therapy on CRP concentration in HASMCs and HUVECs was evaluated and is presented in Figure 3A and 3B, respectively. In HASMCs, treatment with the combination therapy of TGFβ1 and anti-miR-21 resulted in a significant decrease in CRP concentration compared to all other conditions, including the placebo and individual treatments. This suggests a potential synergistic anti-inflammatory effect of TGFβ1 and anti-miR-21 in HASMCs. In HUVECs, a similar trend was observed, where the combination therapy again led to the least CRP concentration, significantly lower than the levels observed with individual treatments, the placebo, and the control group. Both Free TGFβ1 and Free anti-miR-21 on their own showed minimal effects on CRP levels in HUVECs, indicating that the observed reduction in CRP concentration is primarily a result of the combination rather than the individual effects of each agent. Treatment with LPS & IFN-γ again showed an increase in CRP levels compared to the control as expected as they were the positive control of the experiment, though this increase was less substantial compared to the combination therapy (*p < 0.001). In the viability assay as depicted in figure 3C and 3D, HASMCs and HUVECs were treated with placebo and combination therapy exhibited significantly reduced cell viability compared to the control group, with combination therapy showing a stronger effect. Additionally, a significant difference was observed between the placebo and combination therapy in HASMCs. TGFβ1 and Anti-miR-21 combination therapy nanocomposite hydrogel selectively promotes HUVECs proliferation and suppresses HASMCs to control ECM secretion The impact of the treatments on cell proliferation was assessed in both HASMCs and HUVECs, as shown in Figure 4A and 4B, respectively. In HASMCs (Figure 4A), free TGFβ1 had a proliferative effect on the HASMCs, whereas Free anti-miR-21 alone also had a suppressive impact on their proliferation, which is seen in previous studies as well.(31, 32) However, the free combination of these two genes had no effect on the cells, whereas the nanocomposite combination therapy significantly decreased cell proliferation compared to the control, placebo, and individual treatments which indicates a synergistic anti- proliferative effect when TGFβ1 is combined with anti-miR-21. This can be explained by the inherent tendency of free miRNA getting degraded in cellular environment as well as the increased cellular uptake of nanoencapsulated BVs and anti-miR-2 in mammalian cells.(20, 33) The placebo group exhibited relatively low proliferation rates, further highlighting the safety profile of the nanocomposite hydrogel delivery system in HASMC. Similarly, in HUVECs (Figure 4B), free TGFβ1, had the highest proliferative effect along with the other treatments having a positive effect on proliferation. It is noteworthy that the free anti miR-21 has no significant effect in the HUVECs proliferation which is a unique property of miR-21 antagonism that is specific to HASMCs. Every other treatment had the combination therapy induced the highest level of cell proliferation, significantly surpassing the proliferation rates observed in all other treatment groups (**p < 0.0001). Neither Free TGFβ1 nor Free anti-miR-21 alone yielded a comparable increase in proliferation, underscoring the additive effect of the combined therapy. This suggests a robust synergistic interaction between TGFβ1 and anti-miR-21, making this combination therapy a promising candidate for further investigation in therapeutic strategies aimed at modulating inflammation and promoting controlled cell growth in vascular cells. Nanocomposite hydrogel-mediated delivery of TGFβ1 and anti-miR-21 downregulates PTEN expression in HUVECs to promote angiogenesis In the PTEN protein quantification study for angiogenesis in HUVECs demonstrated in figure 4C, the control group established a baseline PTEN concentration of 375.2 ± 5.29 pg/ml. Free anti-miR-21 treatment demonstrated the highest PTEN expression at 542.53 ± 14.05 pg/ml, showing a significant increase compared to control (p < 0.05). The free combination treatment maintained elevated PTEN levels at 440 ± 5.77 pg/ml, while free TGFβ1 and placebo treatments showed moderate PTEN expression at 422.53 ± 23.35 pg/ml and 429.2 ± 76.4 pg/ml respectively. Most notably, the combination therapy group exhibited the lowest PTEN concentration at 251.1 ± 16 pg/ml, significantly lower than both the free combination and control groups (p < 0.0001). This substantial reduction in PTEN levels through combination therapy suggests a potential mechanism for enhanced angiogenic signaling, as decreased PTEN expression typically correlates with increased activation of the PI3K/Akt pathway, a crucial mediator of angiogenesis i.e. enhanced angiogenic activity. (34) TGFβ1 and Anti-miR-21 combination therapy nanocomposite hydrogel significantly enhances apoptosis in HASMCs as Demonstrated by Annexin V-FITC/PI Staining Flow cytometric analysis of HASMC apoptosis revealed significant differences between treatment groups as demonstrated by Annexin V-FITC/PI staining as illustrated in Figure 5D. The control group exhibited minimal apoptotic activity, while both free combination and combination therapy groups showed marked increases in apoptotic cell populations. The combination therapy demonstrated the highest apoptotic rate, showing 22.27 ± 1.2% apoptotic cells, which was significantly higher (p < 0.001) compared to the free combination (16.06 ± 0.46%) and control groups. The scatter plots and corresponding histograms, in figure 5A-C, clearly illustrate the shift in cell populations towards the apoptotic quadrants, with the combination therapy group showing the most pronounced effect. The statistical analysis confirms the enhanced pro-apoptotic efficacy of the combination therapy approach, showing significant differences between all treatment groups (****p < 0.0001, ***p < 0.001). TGFβ1 and Anti-miR-21 combination therapy nanocomposite hydrogel (CAM) enhances angiogenesis in chicken embryo chorioallantoic membrane (CAM) model: Quantitative analysis of vascular development In this study, Figure 6 illustrates the effect of combination therapy on vascular development i.e angiogenesis in a chicken embryo model (in-ovo), specifically through the chorioallantoic membrane (CAM) assay. Figure 6A details the process, showing that on day 6 of fertilization, precision hydrogels carrying BV and miRNA antagomir NPs were applied to the CAM. By day 13, notable neovascularization is observed around the embryo. Figure 6B compares vascular morphology across treatments: the placebo, combination therapy, and control groups. The combination therapy shows a more extensive vascular network, with increased branching and vessel complexity. Quantitative analysis in figure 6C-E reveals significantly greater vessel length (126.46± 16.62 %), junction count (63.265 ± 12.75 %), and moderately higher vessel area (16.368 ± 8.38%) in the combination therapy group compared to the control group (p < 0.05). These findings suggest that the combination therapy promotes angiogenesis more effectively, likely due to enhanced targeting, protection from the cellular environment for the BV as well as the miR-21 antagomir, sustained release properties of the hydrogel system, supporting its potential in therapeutic applications. Previous studies have showed that, miR-21 acts as a negative regulator of angiogenesis by reducing endothelial cell proliferation, migration, and tube formation when overexpressed, while inhibition of miR-21 using a locked nucleic acid (LNA) anti-miR enhances these angiogenic processes in endothelial cells.(35, 36) Discussion The experimental findings demonstrate the successful development and characterization of a novel nanocomposite hydrogel system for combination therapy. The AFM and SEM analyses confirmed the successful formation of Bac-anti-miR-PLGA-NPs with uniform morphology and their effective incorporation into the gelatin-genipin hydrogel matrix. The FTIR spectral analysis validated the crosslinking between gelatin and genipin, particularly evident in the 1600–1700 cm- 1 range, indicating successful hydrogel formation. The system achieved a notable entrapment efficiency of 73.69% ± 0.006, demonstrating effective cargo loading. Biocompatibility studies revealed favorable characteristics, with low hemolysis percentages comparable to PBS controls and enhanced HUVEC adhesion to the gelatin-genipin hydrogel. The combination therapy showed a dual mechanism of action: reducing HASMC proliferation while promoting HUVEC proliferation, suggesting targeted vascular remodeling potential. A key finding was the significant reduction in PTEN expression (250 pg/ml) in the combination therapy group compared to control (375.2 pg/ml) and free treatments. This reduction in PTEN, a known negative regulator of angiogenesis, correlates with enhanced PI3K/Akt pathway activation, crucial for angiogenic responses. The CAM assay provided compelling evidence of enhanced angiogenesis, with significant increases in vessel length (126.46 ± 16.62%), junction count (63.265 ± 12.75%), and vessel area (16.368 ± 8.38%). The reduced CRP concentrations in both HASMCs and HUVECs under combination therapy suggest potent anti-inflammatory effects, while maintaining acceptable cell viability profiles. These results align with previous studies showing miR-21 inhibition enhances angiogenic processes in endothelial cells through modulation of the PTEN/PI3K/Akt pathway. The synergistic effects observed between TGFβ1 and anti-miR-21 in the nanocomposite hydrogel system present a promising therapeutic strategy for applications requiring controlled angiogenic responses and vascular regeneration.( 37 ) TGF-β1 plays a dual role in angiogenesis by promoting endothelial cell proliferation and differentiation in the early stages but inhibiting excessive endothelial growth in the later stages, facilitating vessel maturation( 38 ). When an additional dose of TGF-β1 is introduced, it enhances SMAD signaling, promoting endothelial-to-mesenchymal transition (EndMT) and encouraging the stabilization of nascent blood vessels by recruiting smooth muscle cells and pericytes.( 39 ) In contrast, miR-21 antagonist inhibits miR-21 activity, which normally promotes angiogenesis by targeting PTEN and increasing AKT signaling.( 35 ) By inhibiting miR-21, endothelial cell migration and tube formation are reduced, leading to decreased angiogenic sprouting.( 40 ) This antagonism shifts the balance toward vessel stability rather than excessive proliferation. The combined effect of TGF-β1 and miR-21 antagonist promotes the formation of stable, mature vessels through vasculogenesis while limiting uncontrolled angiogenesis. The combination therapy demonstrates promising potential for plaque stabilization through multiple synergistic mechanisms.( 41 , 42 ) The significant reduction in PTEN expression (250 pg/ml compared to 375 pg/ml control) enhances PI3K/Akt pathway activation, promoting endothelial cell survival. TGF-β1's dual functionality supports endothelial-to-mesenchymal transition and vessel stabilization while stimulating matrix deposition.( 37 , 43 ) The therapy exhibits selective action by reducing HASMC proliferation while enhancing HUVEC growth, coupled with decreased CRP levels indicating anti-inflammatory effects.( 44 ) This balanced approach results in controlled angiogenesis and stable vessel formation rather than excessive proliferation, suggesting effective plaque stabilization through coordinated vascular remodeling and inflammatory modulation.( 10 ) This dual-action approach promoting selective HASMC apoptosis while enhancing HUVEC proliferation can be particularly beneficial in treating atherosclerosis and in-stent restenosis, where excessive HASMC proliferation leads to vessel narrowing and reduced blood flow( 45 , 46 ) It can provide advantages over current treatments like drug-eluting stents and balloon angioplasty, which often lack cell-type specificity and can impair endothelial healing while targeting smooth muscle cells( 46 , 47 ). The selective targeting mechanism could potentially reduce complications associated with delayed re-endothelialization and late stent thrombosis, while maintaining vascular patency through controlled HASMC reduction and healthy endothelial regeneration( 1 , 48 ). Conclusion In conclusion, this study demonstrates the successful development of a novel dual-therapy approach combining TGF-β1 and anti-miR-21 in a PLGA nanocomposite gelatin-genipin hydrogels that achieved high entrapment efficiency (73.69%) and demonstrated excellent biocompatibility. The therapy demonstrates synergistic effects through dual mechanisms: promoting neo-vascularization via selective endothelial cell proliferation while inducing smooth muscle cell apoptosis to control ECM secretion and stabilize plaque. This could offer improved treatment of atherosclerosis and in-stent restenosis by addressing both pathological vessel narrowing and endothelial regeneration, offering advantages over current non-selective approaches like drug-eluting stents. Abbreviations Serial Abbreviation Full form 1 AFM Atomic force microscopy 2 BV Baculovirus 3 CAM Chorioallantoic Membrane 4 CRP C- Reactive Protein 5 DCM Dichloromethane 6 DI Deionized water 7 DLS Dynamic Light Scattering 8 DMSO Dimethyl sulfoxide 9 ECM Extracellular Matrix 10 EDD Embryonic Development Day 11 EE Entrapment Efficiency 12 ELISA Enzyme-linked immunosorbent assay 13 ERK Extracellular signal-regulated kinase 14 FACS Fluorescence-activated cell sorting 15 FAM Fluorescein amidite 16 FITC Fluorescein isothiocyanate 17 FTIR Fourier Transform Infrared Spectroscopy 18 HASMC Human Aortic Smooth Muscle Cell 19 HBSS Hanks' Balanced Salt Solution 20 HUVEC Human umbilical vein endothelial cell 21 IFN Interferon 22 LNA Locked Nucleic Acids 23 LPS Lipopolysaccharides 24 MOI Multiplicity of infection 25 MTT Thiazolyl blue tetrazolium bromide 26 OD Optical Density 27 P0 Parental Generation 0 28 PBS Phosphate Buffer Saline 29 PI Propidium Iodide 30 PI3K Phosphatidylinositol 3-kinases 31 PTEN Phosphatase and tensin homolog 32 SFM Serum-Free Media 33 SMAD Suppressor of Mothers against Decapentaplegic 34 TEM Transmission Electron Microscope 35 TGF Transforming growth factor 36 VEGF Vascular endothelial growth factor 37 VSV Vesicular stomatitis virus Declarations Acknowledgements We thank the Facility for Electron Microscopy Research of McGill University (SEM), Mohini Ramkaran of Microscopy & Imaging Lab, McGill Chemistry Characterization (MC 2 ) Facility (AFM), Biomat’X lab (DLS and multiplate reader), Julien Sirois of The Montreal Neurological Institute - McGill University (flow cytometry) for their equipment, training, and services. Funding This work was supported by a research grant from the Canadian Institute of Health Research (CIHR) to Dominique Shum-Tim, Arghya Paul and Satya Prakash (CIHR 252743). P.I. is funded by the Islamic Development Bank Scholarship (2020-245622). A.A. is fully funded by a scholarship from the Ministry of Higher Education of the Arab Republic of Egypt. S.S. is fully funded by the Canadian Graduate Scholarship-Doctoral Award from the Natural Sciences and Engineering Research Council (NSERC, 569661-2022). E.R is fully funded by the Indonesia Endowment Fund for Education from the Ministry of Finance of the Republic of Indonesia. Availability of materials and data All data generated or analysed during this study are included in this published article. Authors’ Contributions Conceptualization: PI, SP, AP, DST Methodology: PI, SP, AA Investigation: PI, JLB, AK Visualization: PI, SP, SM Supervision: SP, DST Writing- original draft: PI, SP Writing- review & editing: PI, AA, MS, SM, ER, CST, DST Ethics declarations Conflict of interest The authors of this manuscript have no conflict of interest to disclose. References Li M, Qian M, Kyler K, Xu J. Endothelial-Vascular Smooth Muscle Cells Interactions in Atherosclerosis. Front Cardiovasc Med. 2018;5:151. Cao G, Xuan X, Zhang R, Hu J, Dong H. Gene Therapy for Cardiovascular Disease: Basic Research and Clinical Prospects. Frontiers in Cardiovascular Medicine. 2021;8. Bennett MR. Apoptosis of vascular smooth muscle cells in vascular remodelling and atherosclerotic plaque rupture. Cardiovascular Research. 1999;41(2):361-8. McVey DG, Andreadi C, Gong P, Stanczyk PJ, Solomon CU, Turner L, et al. Genetic influence on vascular smooth muscle cell apoptosis. Cell Death & Disease. 2024;15(6):402. 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BMC Molecular and Cell Biology. 2022;23(1):31. Sabatel C, Malvaux L, Bovy N, Deroanne CF, Lambert V, Gonzalez M-LA, et al. MicroRNA-21 Exhibits Antiangiogenic Function by Targeting RhoB Expression in Endothelial Cells. PLoS ONE. 2011;6. Liao Z, Chen Y, Duan C, Zhu K, Huang R, Zhao H, et al. Cardiac telocytes inhibit cardiac microvascular endothelial cell apoptosis through exosomal miRNA-21-5p-targeted cdip1 silencing to improve angiogenesis following myocardial infarction. Theranostics. 2021;11(1):268-91. Redondo S, Navarro-Dorado J, Ramajo M, Medina Ú, Tejerina T. The complex regulation of TGF-β in cardiovascular disease. Vasc Health Risk Manag. 2012;8:533-9. Pardali E, Goumans MJ, ten Dijke P. Signaling by members of the TGF-beta family in vascular morphogenesis and disease. Trends Cell Biol. 2010;20(9):556-67. Goumans M-J, Liu Z, ten Dijke P. TGF-β signaling in vascular biology and dysfunction. Cell Research. 2009;19(1):116-27. Wang S, Olson EN. AngiomiRs--key regulators of angiogenesis. Curr Opin Genet Dev. 2009;19(3):205-11. Rodriguez S, Huynh-Do U. The Role of PTEN in Tumor Angiogenesis. J Oncol. 2012;2012:141236. Jiang BH, Liu LZ. PI3K/PTEN signaling in angiogenesis and tumorigenesis. Adv Cancer Res. 2009;102:19-65. Alvandi Z, Bischoff J. Endothelial-Mesenchymal Transition in Cardiovascular Disease. Arteriosclerosis, Thrombosis, and Vascular Biology. 2021;41(9):2357-69. Huang S, Xu T, Huang X, Li S, Qin W, Chen W, et al. miR-21 regulates vascular smooth muscle cell function in arteriosclerosis obliterans of lower extremities through AKT and ERK1/2 pathways. Archives of Medical Science. 2019;15(6):1490-7. Pickett JR, Wu Y, Zacchi LF, Ta HT. Targeting endothelial vascular cell adhesion molecule-1 in atherosclerosis: drug discovery and development of vascular cell adhesion molecule-1–directed novel therapeutics. Cardiovascular Research. 2023;119(13):2278-93. Her AY, Shin ES. Current Management of In-Stent Restenosis. Korean Circ J. 2018;48(5):337-49. Buccheri D, Piraino D, Andolina G, Cortese B. Understanding and managing in-stent restenosis: a review of clinical data, from pathogenesis to treatment. Journal of Thoracic Disease. 2016;8(10):E1150-E62. Méndez-Barbero N, Gutiérrez-Muñoz C, Blanco-Colio LM. Cellular Crosstalk between Endothelial and Smooth Muscle Cells in Vascular Wall Remodeling. Int J Mol Sci. 2021;22(14). Additional Declarations No competing interests reported. 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A) Atomic Force Microscopy (AFM) topographical analysis of nanoparticles. The 2D height map showing particle distribution over 10 μm × 10 μm scan area and the 3D surface reconstruction revealing spherical NPs with heights ranging from 0-800 nm. Scale bar represents height in nm. B) TEM image of the prepared Bac-anti-miR-PLGA-NPs showing small, uniform and spherical nanoparticles. C) SEM image of the formulated Bac-anti-miR-PLGA-NPs embedded in gelatin- genipin hydrogels illustrating the efficient cross linking of genipin with gelatin nanocomposite hydrogel. D) FTIR spectra of individual components (genipin, PVA, PLGA, PLGA NPs, gelatin) and composite materials (freeze-dried hydrogel, genipin-gelatin hydrogel, and final nanocomposite hydrogel) showing characteristic peaks and molecular interactions in the range of 4000-400 cm⁻¹.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6770483/v1/204d3680f1dc554ff4323b55.png"},{"id":84451843,"identity":"edb6a7f9-7eb0-4132-9da6-8289c41f9a32","added_by":"auto","created_at":"2025-06-12 06:57:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1001663,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Representative microscopic images of cell adhesion in the combination therapy of baculovirus expressing TGF-β1 gene and anti-miR-21 in nanocomposite hydrogel and control groups. (B) Quantification of adhered HUVECs in the control group compared to the gelatin-genipin hydrogel group, with statistical significance highlighted. (C) Hemolysis assay results display the hemolytic percentage for water (DI), PBS, hydrogel, and placebo treatments.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6770483/v1/3e9c0e10b7d92cdfa714898c.png"},{"id":84450979,"identity":"1eb3fd3d-42f5-4b2b-a6f8-caa72f1f0d2d","added_by":"auto","created_at":"2025-06-12 06:49:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":236741,"visible":true,"origin":"","legend":"\u003cp\u003eA) Evaluation c-reactive protein level following treatments with different groups on HASMCs cells (n=5). B) The level of C-reactive protein in HUVECs (n=5). C) The percentage of cell viability as determined by live/dead assay across the placebo, combination therapy of baculovirus expressing TGF-β1 gene and anti-miR-21 in nanocomposite hydrogel, and control groups in HASMCs (n=5). (D) The percentage of cell viability as determined by live/dead assay across the placebo, combination therapy, and control groups in HUVECs (n=5).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6770483/v1/209281b053da6abee33919fa.png"},{"id":84450231,"identity":"4e93a056-4ec1-42b1-8645-be7e62cb7a7b","added_by":"auto","created_at":"2025-06-12 06:41:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":325105,"visible":true,"origin":"","legend":"\u003cp\u003eA) Schematic diagram of baculovirus expressing TGF-β1 gene and anti-miR-21 eluted from combination therapy nanocomposite hydrogel selectively suppressing HASMC proliferation to control extracellular matrix (ECM) production. B) Evaluation of the percentage proliferation of HASMCs after the treatment with control, placebo, free TGFβ1, free anti miR-21, free combinatorial therapy, and TGFβ1 and anti-miR-21 combination therapy (n=5). C) The % proliferation of HUVECs treated with same groups (n=5). D) The PTEN (pg/ml) expression analysis in HUVECs across six distinct treatment groups, revealing significant variations in protein expression levels (n=5).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6770483/v1/e73f548857abb6c45dc7bda4.png"},{"id":84450228,"identity":"dae20a9e-7cab-4471-ac78-28c5995ea4c3","added_by":"auto","created_at":"2025-06-12 06:41:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":381688,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometric analysis of HASMC apoptosis using Annexin V-FITC/PI staining. (A) Control group showing minimal apoptotic activity. (B) Free combination treatment demonstrating increased apoptotic population. (C) Combination therapy of baculovirus expressing TGF-β1 gene and anti-miR-21 in nanocomposite hydrogel showing enhanced apoptotic effect. Representative histograms and scatter plots are shown for each condition. (D) Quantitative analysis of apoptotic rates across treatment groups (n=3).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6770483/v1/89f7b7c4c2ab20ed0cf44362.png"},{"id":84450246,"identity":"fcb2f162-73c2-47da-81a1-4e9177c75f86","added_by":"auto","created_at":"2025-06-12 06:41:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":693216,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of angiogenesis in chicken embryo CAM in-ovo assay. (A) Schematic showing experimental setup: Precision hydrogel loaded with baculovirus, and miRNA antagomir NPs, administered onto the chorioallantoic membrane (CAM) of the embryo on day 6 post-fertilization, with neovascularization observed on day 13. (B) Representative images of CAM vasculature from placebo, combination therapy of baculovirus expressing TGF-β1 gene and anti-miR-21 in nanocomposite hydrogel, and control groups. (C) Quantification of % vessel length (D) Quantification of % vessel junctions. (E) Quantification of % vessel area in the CAM, n=5.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6770483/v1/b87e8b845e3200560a4c0d07.png"},{"id":101151842,"identity":"0018d3c6-20a1-4c3b-aa4b-f73df32fc443","added_by":"auto","created_at":"2026-01-26 16:06:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5147448,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6770483/v1/d306aac4-79e2-47b5-b94f-701fe2e78104.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A novel combination therapy of anti miR-21 and baculoviral TGFβ1 Gene via PLGA-gelatin-genipin nanocomposite hydrogel for arterial plaque stabilization and angiogenesis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVascular proliferative disorders, including atherosclerosis, in-stent restenosis, and vein graft disease, are characterized by pathological vascular smooth muscle cell hyperplasia initially forming plaques followed by plaque destabilization and endothelial dysfunction, leading to significant morbidity and mortality(1, 2). Current therapeutic interventions, such as systemic administration of HMG-CoA reductase inhibitors and antiplatelet agents, while effective, are associated with considerable adverse effects including hepatotoxicity, hemorrhagic complications, and non-selective cellular inhibition. Additionally, drug-eluting stents, while reducing restenosis rates, may impair re-endothelialization and increase the risk of late thrombotic events(3, 4). Plaque stabilization and angiogenesis are critical for mitigating these adverse cardiovascular outcomes (5). Stabilized plaques exhibit reduced vulnerability to rupture, thereby lowering the risk of thrombus formation, myocardial infarction, and ischemic stroke (6). Angiogenesis facilitates neovascularization and ischemic tissue repair, restoring perfusion in compromised areas. These processes are integral to halting the progression of atherosclerotic lesions, preventing plaque destabilization, and promoting vascular homeostasis (7). Collectively, they play essential roles in reducing morbidity and mortality associated with advanced atherosclerotic disease.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTGF-\u0026beta;1 (Transforming Growth Factor Beta 1) is essential in angiogenesis, re-endothelialization, and plaque stabilization (8). It promotes new blood vessel formation and supports endothelial repair after vascular injury, enhancing healing and reducing thrombosis. In plaque stabilization, TGF-\u0026beta;1 encourages the deposition of extracellular matrix, increases smooth muscle cell proliferation, and modulates inflammation, making plaques less prone to rupture (9). Its regulatory role in these processes is key to maintaining vascular integrity and preventing atherosclerosis progression and complications. On the other hand, miR-21 contributes to plaque destabilization by promoting inflammation, smooth muscle cell proliferation, and reducing apoptosis, making plaques more prone to rupture (10). In angiogenesis, miR-21 enhances vascular growth by regulating endothelial cell behavior (11). However, it impairs re-endothelialization by inhibiting endothelial cell migration, hindering vascular repair and healing processes (12). By inhibiting miR-21, this therapy could potentially reduce inflammation and fibrosis in atherosclerotic plaques, promoting a more stable phenotype (13). Antagonism of miR-21 can stabilize plaques by reducing inflammation and smooth muscle cell proliferation while promoting apoptosis, thereby preventing plaque rupture (14). By inhibiting miR-21, endothelial cell migration and proliferation are enhanced, facilitating better re-endothelialization and vascular repair after injury (15). Additionally, miR-21 antagonism can help normalize angiogenesis, ensuring controlled vessel formation and reducing pathological vascular growth. Overall, targeting miR-21 offers a therapeutic approach to improve vascular stability and repair, reducing atherosclerosis progression and related complications (16). Anti-miR-21 therapy has shown clear benefits in reducing neointimal hyperplasia (17). Combining this with TGF\u0026beta;1 modulation could potentially enhance this effect by regulating smooth muscle cell proliferation and migration.\u003c/p\u003e\n\u003cp\u003eBaculoviral gene therapy offers a versatile, safe, and efficient system for wound healing and revascularization due to its high gene loading capacity, low toxicity, and targeted delivery (18). With a large 130 kb genome, baculoviruses (BVs) can deliver large therapeutic genes, and their rapid, scalable production in insect cells makes them cost-effective for regenerative medicine and other therapies (19).According to our previously published study, Poly (d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) encapsulating BV carrying the TGF-\u0026beta;1 gene enhances endothelial wound healing in human umbilical vein endothelial cells (HUVECs) by providing sustained, efficient gene delivery and protecting the virus from degradation (20). This system improves cell migration, proliferation, and wound closure compared to free BV. \u0026nbsp;Hydrogels consist of crosslinked polymer chains arranged in a three-dimensional network, allowing them to absorb substantial amounts of liquid. Their high-water content, soft texture, and porous nature make them similar to living tissues. It is becoming a promising and effective option for drug delivery because their adjustable functional properties can be tailored to enhance healing. These properties include biodegradability, adhesiveness, antimicrobial effects, anti-inflammatory capabilities, and pre-angiogenic bioactivities (21). Together, these features can significantly accelerate the healing of chronic wounds. Genipin is a natural crosslinker frequently used in drug administration due to its outstanding properties. It is recognized as a biocompatible, biodegradable, non-toxic, and stable crosslinking agent, making it highly valuable in pharmaceutical and biomedical applications (22).\u003c/p\u003e\n\u003cp\u003eThus, this study investigates the fabrication and efficacy of a combination therapy of TGF-\u0026beta;1 gene using BV and anti miR-21 encapsulated in these PLGA NPs to formulate the polymeric nanocomposite hydrogels with gelatin-genipin to deliver a combination therapy of TGF-\u0026beta;1 gene using BV and anti miR-21 to simultaneously induce smooth muscle cell apoptosis while promoting endothelial cell proliferation, minimize systemic exposure while maintaining therapeutic efficacy, enhance vascular homeostasis by modulating cell-specific responses, potentially reducing the incidence of vulnerable plaque formation and late thrombotic complications(23).\u003c/p\u003e"},{"header":"Materials \u0026 Methods","content":"\u003cp\u003e\u003cstrong\u003eVirus generation and titration.\u0026nbsp;\u003c/strong\u003eThe virus generation and titration were performed following a previously discussed method by this group.(20) Briefly, Sf21, \u003cem\u003eSpodoptera\u003c/em\u003e \u003cem\u003efrugiperda,\u0026nbsp;\u003c/em\u003einsect cells (Invitrogen Life Technologies, Carlsbad, CA, USA) were cultured in Sf-900\u0026trade; III SFM medium (Gibco) at 27\u0026deg;C and sub-cultured 2\u0026ndash;3 times weekly. The TGF-\u0026beta;1 gene (GenScript Biotech, USA) was cloned into the pACEBac1 vector (Geneva Biotec, Switzerland) and recombined with a DH10EMBacVSV\u0026trade; bacmid via transposition in \u003cem\u003eE. coli.\u003c/em\u003e Then, the recombinant bacmid was transfected into Sf21 cells using TransIT-Insect Reagent (Mirus Bio LLC, USA) to produce P\u003csub\u003e0\u003c/sub\u003e BV stock. This was amplified in Sf21 cells to generate P\u003csub\u003e1\u003c/sub\u003e and P2 stocks. Viral titers were determined via flow cytometry using mCherry fluorescence and confirmed by cytopathic endpoint dilution.(24)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDesign and characterization of nanocomposite hydrogels carrying TGF-\u0026beta;1 expressing baculovirus and anti-miR-21\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of dual combination therapy PLGA nanoparticles.\u0026nbsp;\u003c/strong\u003eBac-anti-miR-PLGA-NPs loaded with TGF-\u0026beta;1\u0026nbsp;gene carrying BV and FAM-tagged locked nucleic acid (LNA) anti-miR21 (5\u0026prime;-ACGGCAACACCAGUCGAUGGGCUGU-3, Creative Biogene Inc., USA)\u0026nbsp;were prepared using the double emulsion solvent evaporation technique previously\u0026nbsp;described\u0026nbsp;(20, 25). Briefly,\u0026nbsp;PLGA (Millipore Sigma, Germany) was dissolved in dichloromethane (DCM- Millipore Sigma, Germany), and\u0026nbsp;100 MOI\u0026nbsp;recombinant BV and 0.2 nmol anti miR-21 was coated with 1% polyvinyl alcohol (PVA- Millipore Sigma, Germany) to protect it during emulsification and to facilitate NPs solidification. The resultant W/O emulsion was further emulsified in 1 % aqueous PVA solution to form the final W/O/W nanoemulion. This emulsion is then stirred for 6 h to solidify the NPs, which were then collected by ultracentrifugation, washed, and freeze-dried for further embedding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFormulation of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eBac-anti-miR-PLGA\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;nanocomposite gelatin-genipin hydrogel.\u0026nbsp;\u003c/strong\u003eThe prepared PLGA NPs is then embedded\u0026nbsp;in gelatin-genipin hydrogels by a previously used method.(26) The gelatin from porcine skin\u0026nbsp;(Millipore Sigma, Germany)\u0026nbsp;was dissolved in distilled water by stirring the mixture for 30 minutes at 50\u0026deg;C. After cooling down upto 25\u0026deg;C, previously dissolved genipin solution (Fisher Scientific, USA) was slowly added to the gelatin solution to make 4% w/v gelatin and 0.25% w/v genipin concentration in the final formulation under continuous mechanical stirring. To further \u0026nbsp; formulate the nanocomposite hydrogel, amount of previously prepared\u0026nbsp;Bac-anti-miR-PLGA-NPs\u0026nbsp;dose adjusted according to entrapment efficiency were dispersed into 10 ml gelatin\u0026ndash;genipin solution.(27)\u0026nbsp;This dispersion process was carried out under constant stirring at 50\u0026deg;C for 30 minutes. The nanoparticle-incorporated gelatin-genipin gels were finally left to gel overnight at 40\u0026deg;C for further crosslinking. The crosslinked\u0026nbsp;Bac-anti-miR-PLGA nanocomposite hydrogel was then vacuum freeze-dried for 24 hours, and the resulting samples were stored for further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro characterization of nanoparticles and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003enanocomposite hydrogel\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysicochemical characterization.\u0026nbsp;\u003c/strong\u003eThe physicochemical characterization of the NPs was performed using dynamic light scattering (DLS) and electrophoretic laser Doppler anemometry (Brookhaven Instruments, Holtsville, NY, USA). Mean particle size and polydispersity index were measured at 25\u0026deg;C, employing a 90\u0026deg; scattering angle and 1.33 refractive index. Zeta potential was determined using the same instrument. All measurements were conducted in triplicate using disposable cells. Zeta Potential Analyzer v3.57 software facilitated zeta potential calculations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAtomic Force Microscope:\u003c/strong\u003e For particle size quantification, the substrate with the dried nanoparticle was mounted onto the stage of the Atomic Force Microscope (AFM) equipped with a molecular force probe controller (Asylum Research - Oxford Instruments, Santa Barbara, CA) in AC mode in the air using ACTA probes (Applied Nanostructures, Inc., Mountain View, CA). Data is processed using MountainsSPIP v. 10.1.10606 (Digital Surf, Besan\u0026ccedil;on, France).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScanning Electron Microscope.\u003c/strong\u003e The prepared NPs and nanocomposite hydrogel surface morphology, particle size and polymer crosslinking were examined using scanning electron microscopy (SEM) with a FEI 450 Quanta SEM at McGill University, operated at 10 kV. The hydrogel was freeze dried and deposited on carbon tape. All the samples were then sputter coated with platinum before imaging.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFourier transform infrared (FTIR):\u003c/strong\u003e The FTIR spectra of PLGA, lyophilized PLGA NPs, hydrogel, genipin and gelatin were generated using a computerized FTIR spectroscopy, Perkin Elmer Spectrum, Waltham, Massachusetts, USA (IR Version, 10.7.2) that operated in the 400\u0026ndash;4000 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e scanning wave number range at a resolution of 1 cm\u003csup\u003e-1\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEncapsulation Efficiency of the nanocomposite hydrogel.\u0026nbsp;\u003c/strong\u003eAfter preparation, the nanocomposite hydrogel was washed in PBS. The rinsing PBS supernatant was collected and subsequently, subjected to analysis using UV spectroscopy by a Nanodrop 2000 (Thermofischer, Massachusetts, USA). Encapsulation efficiency (EE) was calculated by determining the percentage of the drug encapsulated into the hydrogel, employing the standard formula. All measurements were conducted in triplicate, and the results were reported as the mean \u0026plusmn; standard deviation (SD).\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" width=\"735\" height=\"79\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell line studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMammalian Cell Culture.\u0026nbsp;\u003c/strong\u003eHuman umbilical vein endothelial cells (HUVECs), procured from Sigma Aldrich, were cultured using complete endothelial growth medium from Sigma Aldrich with 10% Fetal Bovine Serum and Human Aortal Smooth Muscle Cells (HASMCs) were cultured with Medium 199 with 0.02 mg/mL endothelial growth supplement. These cells were incubated in T-25 flasks within a 37 \u0026deg;C, 5% CO2 incubator and were employed within five passages upon receipt. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCellular Adhesion Study\u003c/strong\u003e. To determine if the nanocomposite hydrogels promoted cellular adhesion, thin film of the hydrogel was created and seeded on a cell culture plate. The hydrogel was dried overnight and then swollen in cell media for one hour. After one hour, the cell media was removed and HUVECs were seeded onto the hydrogel. Empty tissue treated wells were used as a control. The seeded hydrogel was incubated at 37 \u0026deg;C for two hours. After two hours, the plate was washed with HBSS to remove any unattached cells. The hydrogel was then fixed in 3.7 % (v/v) formaldehyde for 15 minutes and washed with HBSS. Finally, the hydrogel was stained with 0.1 % (m/v) crystal violet to visualize the cells under a brightfield microscope. The cells were counted\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eusing ImageJ software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of Hemocompatibility.\u0026nbsp;\u003c/strong\u003eBlood samples were collected commercially and tested independently in triplicate. The potential hemolysis of the virus itself, placebo and the gene carrying NPs were evaluated. Briefly, all tested samples were immersed into 5 mL of PBS in a 15 mL centrifuge tube. Next, 4 mL of citrated blood were mixed with 5 mL PBS and 0.1 mL of the diluted blood were added to each sample. The samples were incubated at 37 C for 1 h and then centrifuged at 1000 rpm for 10 min. The supernatant containing the lysed hemoglobin were placed into a 96-well plate, and the absorbance were read at 545 nm. The negative and positive control were PBS and deionized water, respectively. The following equation is used to determine the % hemolysis (28).\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" width=\"746\" height=\"60\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLive/Dead Cell Viability Assay.\u0026nbsp;\u003c/strong\u003eA Live/Dead Assay was employed to estimate the safety profile of the combination therapy delivered via nanocomposite hydrogel using Calcein AM and propidium iodide. HUVECs and HASMCs were seeded (2 \u0026times; 10⁴ cells per well) in 48-well plates and incubated overnight. Free BVs, anti miR-21s, their free combination and the combination therapy were added to different wells, with media alone as control, and incubated for 12 hours before replacing the transduction media with fresh media. After 48 hours, live and dead cells were stained with Calcein AM (green, live) and propidium iodide (red, dead). Cells were imaged using the Leica DMIL microscope, and ImageJ software was used to quantify cell viability based on fluorescence images.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC-Reactive Protein (CRP) Assay. To quantify serum CRP levels, HUVECs and HASMCs were cultured and treated with free BVs, anti miR-21s and hydrogel elutions for 12 hours. Cell culture supernatants were centrifuged and assayed for CRP levels using an ELISA kit (Abcam, USA). ELISA was performed according to the manufacturer\u0026apos;s protocol, with absorbance measured at 450 nm using a multi-mode plate reader (EnSpire Multimode, Perkin Elmer, USA). All assays were performed in triplicate. \u003c/p\u003e\n\u003cp\u003eMTT Cell Proliferation Assay. Cell proliferation and viability were evaluated using an MTT assay. HUVECs and HASMCs (10,000 cells/well) were seeded in 96-well plates and treated with free BVs, free anti miR-21s and supernatant containing the combination therapy eluted from the nanocomposite hydrogel. After a 12-hour incubation, 20 \u0026micro;L of MTT solution (5 mg/mL) was added, and cells were incubated for 4 hours at 37\u0026deg;C. The resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and absorbance was measured at 570 nm using an EnSpire Multimode plate reader (Perkin Elmer, USA). Cytotoxicity was calculated as % Cytotoxicity = (OD of control - OD of test) \u0026times; 100 / OD of control, and all experiments were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003e\u003cstrong\u003eEvaluation of PTEN expression in HUVECs.\u0026nbsp;\u003c/strong\u003eThe cells were treated with the free BVs, free anti-miR21, free combination, combination therapy nanocomposite supernatant, PBS for control. After 24 hours incubation, the conditioned medium was collected and their Phospatase and tensin homolog (PTEN) protein expression was quantified by a sandwich Human PTEN enzyme-linked immunosorbent Kit (AB206979- 1002, Abcam, MA, USA) with 39.9 pg/ml sensitivity. Measurement of sample absorbance was carried out using a EnSpire Multimode plate reader (Perkin Elmer, USA) at a wavelength of 450 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of Apoptosis via Flow Cytometry\u003c/strong\u003e. The pro-apoptotic effects of combination of free BVs and anti-miR-21 and the combination NPs were evaluated through flow cytometry.\u0026nbsp;\u003cstrong\u003eHASMC\u0026nbsp;\u003c/strong\u003ecells (1 \u0026times; 10⁶ cells/ml) were cultured in 24-well plates (Corning, NY, USA) and treated with control, free treatments, or combination therapy. The cells were incubated for 24 hours in a mammalian cell incubator. Following the incubation, the cells were washed three times with PBS (0.1 M, pH 7.4) and resuspended in 100 \u0026micro;l of 1\u0026times; binding buffer (eBioscience, Inc., San Diego, USA). Next, 5 \u0026micro;l of Annexin V-FITC (final concentration, 1 \u0026micro;g/ml; eBioscience, Inc.) and 5 \u0026micro;l of propidium iodide (10 \u0026micro;g/\u0026micro;l; eBioscience, Inc.) were added to the suspension, and the mixture was incubated for 15 minutes in the dark at room temperature. Before analysis, 200 \u0026micro;l of 1\u0026times; binding buffer was added, and the extent of apoptosis was determined using a FACScan flow cytometer with Cell Quest software (BDFACSAria Fusion Flow Cytometer, New Jersey, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of angiogenesis on the chorioallantoic membrane (CAM) in-ovo.\u0026nbsp;\u003c/strong\u003eFertilized chicken eggs were incubated at 37\u0026deg;C and 60% humidity. On embryonic development day 6 (EDD6) a window was made into the shell of the egg, to expose the CAM. The chorion was carefully removed, and the eggs were treated with 100 \u0026micro;L of either Combination therapy, placebo and PBS (control) directly onto the CAM surface. The window is sealed with transparent tape to prevent dehydration. The eggs were examined throughout the week for mortality, no eggs perished throughout the week. After 7 days, the CAM is photographed to observe and quantify vascular changes. Key parameters measured include vessel density, branching, and diameter. Angiogenic response is typically assessed by counting new blood vessel branch points within a defined area around the application site with the help of angiotool software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis.\u0026nbsp;\u003c/strong\u003eThe data are expressed as mean \u0026plusmn; standard deviation or mean \u0026plusmn; standard error of the mean, as specified. One-way ANOVA followed by Tukey\u0026rsquo;s multiple comparisons test was performed using GraphPad Prism version 10.2.2 for Windows, GraphPad Software, Boston, Massachusetts USA, www.graphpad.com. Significance levels are denoted as **** for p \u0026lt; 0.0001, *** for p \u0026lt; 0.001, ** for p \u0026le; 0.01 and * for p \u0026le; 0.01.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIn-vitro characterization\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTGF\u0026beta;1 and Anti-miR-21 combination therapy\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003enanocomposite hydrogel\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe anionic BVs and mir21 antagomirs underwent surface coating using polymers to generate Bac-anti-miR-polymer NPs. Bac-anti-miR-PLGA-NPs were successfully formulated using the double emulsion method(25), and showed spherically shaped particles distinctively prepared (n = 3). \u0026nbsp;The surface morphology of the NPs and the nanocomposite hydrogel were analyzed to confirm the formation of NPs via AFM. The AFM image in figure 1A reveal spherical Bac-anti-miR-PLGA-NPs with heterogeneous size distribution, predominantly clustered in specific regions. The particles exhibit varying heights up to 800 nm, with most particles showing diameters between 200-500 nm. The surface topography indicates partial aggregation of particles, suggesting potential interactions between individual nanostructures. These results demonstrate the effectiveness of the double-emulsion solvent evaporation method used for nanoparticle preparation. TEM images of the formulated Bac-anti-miR-PLGA-NPs showed small, spherical, uniformly distributed, and non-aggregated particles as demonstrated in Figure 1B. The size values obtained from TEM agree with size measurements resulted from DLS i.e. 372.4\u0026plusmn;20.45. It is well known that spherical shaped NPs exhibit higher surface area, drug loading, controlled release of the cargo and good mobility within biological environments. The surface charge of these NPs is - 26.39\u0026plusmn;2.79 that is typical for PLGA, a negatively charged polymer, encapsulating baculovirus that is also negatively charged on its surface which is beneficial for controlled drug release systems(29, 30). Additionally, SEM imaging of the Bac-anti-miR-PLGA-NPs alone as well as embedded within the nanocomposite gelatin- genipin hydrogels, confirmed the presence of pores within the gel matrix and the crosslinking necessary for nanoparticle encapsulation, as shown in Figure 1C. These findings highlight genipin\u0026apos;s ability to crosslink with gelatin, facilitating the formation of pores that can effectively load BV-carrying PLGA NPs. The FTIR spectra in figure 3 reveals characteristic peaks for each component and their interactions in the final nanocomposite hydrogel system. The genipin spectrum shows distinct peaks in the 1750-1000 cm⁻\u0026sup1; region, while PLGA and PLGA NPs exhibit similar patterns with characteristic ester bonds at 1750 cm⁻\u0026sup1;. The gelatin spectrum shows typical amide bands at 3300-3500 cm⁻\u0026sup1;. In the genipin-gelatin hydrogel, peak shifts indicate successful crosslinking. The nanocomposite hydrogel spectrum combines features from all components, confirming successful incorporation of PLGA NPs into the genipin-crosslinked gelatin matrix.\u003c/p\u003e\n\u003cp\u003eFigure 1D displays the FTIR spectra of the individual polymers used along with the final Bac-anti-miR-PLGA-NPs investigated in this study. The FTIR characterization data for gelatin-genipin hydrogels reveals distinct spectral features indicative of their compositions. The FTIR spectra reveals successful crosslinking. The gen-gelatin shows broader peaks, especially in the 1600-1700 cm\u003csup\u003e-1\u003c/sup\u003e range (amide I band), indicating crosslinked gelatin. The presence of genipin is confirmed by unique peaks at the region 1500-1700 cm\u003csup\u003e-1\u003c/sup\u003e (C=O and C=C stretching), which are absent in the gelatin alone. Both hydrogel types exhibit broad peaks at 3400 cm\u003csup\u003e-1\u003c/sup\u003e (O-H stretching), typical of hydroxyl groups in gelatin. The distinct shifts and new peaks in the gelatin-genipin hydrogel spectra highlight the crosslinking effects of genipin, demonstrating successful chemical modifications as shown in Figure 2. The percentage of entrapment efficiency (% EE) is one of the most important physicochemical characterizations as it represents the number of active molecules encapsulated within the prepared NPs. It is always favorable to achieve high % EE to deliver the cargo with high concentration to the desired cells. Hydrophilic molecules such as miRNAs exhibit low % EE due to the possibility of their leakage to the external aqueous phase during the fabrication of NPs. The adopted double emulsion solvent evaporation technique is well known by its ability to enhance the loading of hydrophilic drugs into NPs. The % EE of the formulated Bac-anti-miR-PLGA-NPs is 73.69% +- 0.006.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTGF\u0026beta;1 and Anti-miR-21 combination therapy\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003enanocomposite hydrogel promotes cellular adhesion and hemocompatibility\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe hemolysis assay in figure 2A demonstrated that the gelatin-genipin hydrogel had a low hemolysis percentage, comparable to PBS and placebo, and significantly lower than the DI control (p \u0026lt; 0.0001), indicating its biocompatibility. In the adhesion study with HUVECs, microscopy images (figure 2A) revealed a higher density of adherent cells on surfaces treated with combination therapy compared to the control. Quantitative analysis (figure 2B) further confirmed significantly enhanced HUVEC adhesion to the gelatin-genipin hydrogel (p \u0026lt; 0.0001), supporting its potential as a favorable scaffold for vascular tissue engineering. 2C\u003c/p\u003e\n\u003cp\u003eThe gelatin-genipin hydrogel provides a biocompatible and supportive environment for endothelial cell adhesion, making it promising for therapeutic and tissue engineering applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTGF\u0026beta;1 and Anti-miR-21 Combination Therapy\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eNanocomposite Hydrogel\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;is safe and reduces C-reactive protein in HUVECs and HASMCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effect of the combination therapy on CRP concentration in HASMCs and HUVECs was evaluated and is presented in Figure 3A and 3B, respectively. In HASMCs, treatment with the combination therapy of TGF\u0026beta;1 and anti-miR-21 resulted in a significant decrease in CRP concentration compared to all other conditions, including the placebo and individual treatments. This suggests a potential synergistic anti-inflammatory effect of TGF\u0026beta;1 and anti-miR-21 in HASMCs. In HUVECs, a similar trend was observed, where the combination therapy again led to the least CRP concentration, significantly lower than the levels observed with individual treatments, the placebo, and the control group. Both Free TGF\u0026beta;1 and Free anti-miR-21 on their own showed minimal effects on CRP levels in HUVECs, indicating that the observed reduction in CRP concentration is primarily a result of the combination rather than the individual effects of each agent. Treatment with LPS \u0026amp; IFN-\u0026gamma; again showed an increase in CRP levels compared to the control as expected as they were the positive control of the experiment, though this increase was less substantial compared to the combination therapy (*p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eIn the viability assay as depicted in figure 3C and 3D, HASMCs and HUVECs were treated with placebo and combination therapy exhibited significantly reduced cell viability compared to the control group, with combination therapy showing a stronger effect. Additionally, a significant difference was observed between the placebo and combination therapy in HASMCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTGF\u0026beta;1 and Anti-miR-21 combination therapy\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003enanocomposite hydrogel\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;selectively promotes HUVECs proliferation and suppresses HASMCs to control ECM secretion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe impact of the treatments on cell proliferation was assessed in both HASMCs and HUVECs, as shown in Figure 4A and 4B, respectively. In HASMCs (Figure 4A), free TGF\u0026beta;1 had a proliferative effect on the HASMCs, whereas Free anti-miR-21 alone also had a suppressive impact on their proliferation, which is seen in previous studies as well.(31, 32) However, the free combination of these two genes had no effect on the cells, whereas the nanocomposite combination therapy significantly decreased cell proliferation compared to the control, placebo, and individual treatments which indicates a synergistic anti- proliferative effect when TGF\u0026beta;1 is combined with anti-miR-21. This can be explained by the inherent tendency of free miRNA getting degraded in cellular environment as well as the increased cellular uptake of nanoencapsulated BVs and anti-miR-2 in mammalian cells.(20, 33) The placebo group exhibited relatively low proliferation rates, further highlighting the safety profile of the nanocomposite hydrogel delivery system in HASMC.\u003c/p\u003e\n\u003cp\u003eSimilarly, in HUVECs (Figure 4B), free TGF\u0026beta;1, had the highest proliferative effect along with the other treatments having a positive effect on proliferation. It is noteworthy that the free anti miR-21 has no significant effect in the HUVECs proliferation which is a unique property of miR-21 antagonism that is specific to HASMCs. Every other treatment had the combination therapy induced the highest level of cell proliferation, significantly surpassing the proliferation rates observed in all other treatment groups (**p \u0026lt; 0.0001). Neither Free TGF\u0026beta;1 nor Free anti-miR-21 alone yielded a comparable increase in proliferation, underscoring the additive effect of the combined therapy. This suggests a robust synergistic interaction between TGF\u0026beta;1 and anti-miR-21, making this combination therapy a promising candidate for further investigation in therapeutic strategies aimed at modulating inflammation and promoting controlled cell growth in vascular cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNanocomposite hydrogel-mediated delivery of TGF\u0026beta;1 and anti-miR-21 downregulates PTEN expression in HUVECs to promote angiogenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the PTEN protein quantification study for angiogenesis in HUVECs demonstrated in figure 4C, the control group established a baseline PTEN concentration of 375.2 \u0026plusmn; 5.29 pg/ml. Free anti-miR-21 treatment demonstrated the highest PTEN expression at 542.53 \u0026plusmn; 14.05 pg/ml, showing a significant increase compared to control (p \u0026lt; 0.05). The free combination treatment maintained elevated PTEN levels at 440 \u0026plusmn; 5.77 pg/ml, while free TGF\u0026beta;1 and placebo treatments showed moderate PTEN expression at 422.53 \u0026plusmn; 23.35 pg/ml and 429.2 \u0026plusmn; 76.4 pg/ml respectively. Most notably, the combination therapy group exhibited the lowest PTEN concentration at 251.1 \u0026plusmn; 16 pg/ml, significantly lower than both the free combination and control groups (p \u0026lt; 0.0001). This substantial reduction in PTEN levels through combination therapy suggests a potential mechanism for enhanced angiogenic signaling, as decreased PTEN expression typically correlates with increased activation of the PI3K/Akt pathway, a crucial mediator of angiogenesis i.e. enhanced angiogenic activity. (34)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTGF\u0026beta;1 and Anti-miR-21 combination therapy\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003enanocomposite hydrogel\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esignificantly enhances apoptosis in HASMCs as Demonstrated by Annexin V-FITC/PI Staining\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlow cytometric analysis of HASMC apoptosis revealed significant differences between treatment groups as demonstrated by Annexin V-FITC/PI staining as illustrated in Figure 5D. The control group exhibited minimal apoptotic activity, while both free combination and combination therapy groups showed marked increases in apoptotic cell populations. The combination therapy demonstrated the highest apoptotic rate, showing 22.27 \u0026plusmn; 1.2% apoptotic cells, which was significantly higher (p \u0026lt; 0.001) compared to the free combination (16.06 \u0026plusmn; 0.46%) and control groups. The scatter plots and corresponding histograms, in figure 5A-C, clearly illustrate the shift in cell populations towards the apoptotic quadrants, with the combination therapy group showing the most pronounced effect. The statistical analysis confirms the enhanced pro-apoptotic efficacy of the combination therapy approach, showing significant differences between all treatment groups (****p \u0026lt; 0.0001, ***p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTGF\u0026beta;1 and Anti-miR-21 combination therapy\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003enanocomposite hydrogel\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(CAM) enhances angiogenesis in chicken embryo chorioallantoic membrane (CAM) model: Quantitative analysis of vascular development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, Figure 6 illustrates the effect of combination therapy on vascular development i.e angiogenesis in a chicken embryo model (in-ovo), specifically through the chorioallantoic membrane (CAM) assay. Figure 6A details the process, showing that on day 6 of fertilization, precision hydrogels carrying BV and miRNA antagomir NPs were applied to the CAM. By day 13, notable neovascularization is observed around the embryo. Figure 6B compares vascular morphology across treatments: the placebo, combination therapy, and control groups. The combination therapy shows a more extensive vascular network, with increased branching and vessel complexity. Quantitative analysis in figure 6C-E reveals significantly greater vessel length (126.46\u0026plusmn; 16.62 %), junction count (63.265 \u0026plusmn; 12.75 %), and moderately higher vessel area (16.368 \u0026plusmn; 8.38%) in the combination therapy group compared to the control group (p \u0026lt; 0.05). These findings suggest that the combination therapy promotes angiogenesis more effectively, likely due to enhanced targeting, protection from the cellular environment for the BV as well as the miR-21 antagomir, sustained release properties of the hydrogel system, supporting its potential in therapeutic applications. Previous studies have showed that, miR-21 acts as a negative regulator of angiogenesis by reducing endothelial cell proliferation, migration, and tube formation when overexpressed, while inhibition of miR-21 using a locked nucleic acid (LNA) anti-miR enhances these angiogenic processes in endothelial cells.(35, 36)\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe experimental findings demonstrate the successful development and characterization of a novel nanocomposite hydrogel system for combination therapy. The AFM and SEM analyses confirmed the successful formation of Bac-anti-miR-PLGA-NPs with uniform morphology and their effective incorporation into the gelatin-genipin hydrogel matrix. The FTIR spectral analysis validated the crosslinking between gelatin and genipin, particularly evident in the 1600\u0026ndash;1700 cm-\u003csup\u003e1\u003c/sup\u003e range, indicating successful hydrogel formation. The system achieved a notable entrapment efficiency of 73.69% \u0026plusmn; 0.006, demonstrating effective cargo loading. Biocompatibility studies revealed favorable characteristics, with low hemolysis percentages comparable to PBS controls and enhanced HUVEC adhesion to the gelatin-genipin hydrogel. The combination therapy showed a dual mechanism of action: reducing HASMC proliferation while promoting HUVEC proliferation, suggesting targeted vascular remodeling potential.\u003c/p\u003e \u003cp\u003eA key finding was the significant reduction in PTEN expression (250 pg/ml) in the combination therapy group compared to control (375.2 pg/ml) and free treatments. This reduction in PTEN, a known negative regulator of angiogenesis, correlates with enhanced PI3K/Akt pathway activation, crucial for angiogenic responses. The CAM assay provided compelling evidence of enhanced angiogenesis, with significant increases in vessel length (126.46\u0026thinsp;\u0026plusmn;\u0026thinsp;16.62%), junction count (63.265\u0026thinsp;\u0026plusmn;\u0026thinsp;12.75%), and vessel area (16.368\u0026thinsp;\u0026plusmn;\u0026thinsp;8.38%). The reduced CRP concentrations in both HASMCs and HUVECs under combination therapy suggest potent anti-inflammatory effects, while maintaining acceptable cell viability profiles. These results align with previous studies showing miR-21 inhibition enhances angiogenic processes in endothelial cells through modulation of the PTEN/PI3K/Akt pathway. The synergistic effects observed between TGFβ1 and anti-miR-21 in the nanocomposite hydrogel system present a promising therapeutic strategy for applications requiring controlled angiogenic responses and vascular regeneration.(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) TGF-β1 plays a dual role in angiogenesis by promoting endothelial cell proliferation and differentiation in the early stages but inhibiting excessive endothelial growth in the later stages, facilitating vessel maturation(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). When an additional dose of TGF-β1 is introduced, it enhances SMAD signaling, promoting endothelial-to-mesenchymal transition (EndMT) and encouraging the stabilization of nascent blood vessels by recruiting smooth muscle cells and pericytes.(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) In contrast, miR-21 antagonist inhibits miR-21 activity, which normally promotes angiogenesis by targeting PTEN and increasing AKT signaling.(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) By inhibiting miR-21, endothelial cell migration and tube formation are reduced, leading to decreased angiogenic sprouting.(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) This antagonism shifts the balance toward vessel stability rather than excessive proliferation. The combined effect of TGF-β1 and miR-21 antagonist promotes the formation of stable, mature vessels through vasculogenesis while limiting uncontrolled angiogenesis.\u003c/p\u003e \u003cp\u003eThe combination therapy demonstrates promising potential for plaque stabilization through multiple synergistic mechanisms.(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) The significant reduction in PTEN expression (250 pg/ml compared to 375 pg/ml control) enhances PI3K/Akt pathway activation, promoting endothelial cell survival. TGF-β1's dual functionality supports endothelial-to-mesenchymal transition and vessel stabilization while stimulating matrix deposition.(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e) The therapy exhibits selective action by reducing HASMC proliferation while enhancing HUVEC growth, coupled with decreased CRP levels indicating anti-inflammatory effects.(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) This balanced approach results in controlled angiogenesis and stable vessel formation rather than excessive proliferation, suggesting effective plaque stabilization through coordinated vascular remodeling and inflammatory modulation.(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThis dual-action approach promoting selective HASMC apoptosis while enhancing HUVEC proliferation can be particularly beneficial in treating atherosclerosis and in-stent restenosis, where excessive HASMC proliferation leads to vessel narrowing and reduced blood flow(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) It can provide advantages over current treatments like drug-eluting stents and balloon angioplasty, which often lack cell-type specificity and can impair endothelial healing while targeting smooth muscle cells(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). The selective targeting mechanism could potentially reduce complications associated with delayed re-endothelialization and late stent thrombosis, while maintaining vascular patency through controlled HASMC reduction and healthy endothelial regeneration(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study demonstrates the successful development of a novel dual-therapy approach combining TGF-β1 and anti-miR-21 in a PLGA nanocomposite gelatin-genipin hydrogels that achieved high entrapment efficiency (73.69%) and demonstrated excellent biocompatibility. The therapy demonstrates synergistic effects through dual mechanisms: promoting neo-vascularization via selective endothelial cell proliferation while inducing smooth muscle cell apoptosis to control ECM secretion and stabilize plaque. This could offer improved treatment of atherosclerosis and in-stent restenosis by addressing both pathological vessel narrowing and endothelial regeneration, offering advantages over current non-selective approaches like drug-eluting stents.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSerial\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbbreviation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFull form\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eAFM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eAtomic force microscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eBV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eBaculovirus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eCAM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eChorioallantoic Membrane\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eCRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eC- Reactive Protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eDCM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eDichloromethane\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eDI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eDeionized water\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eDLS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eDynamic Light Scattering\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eDMSO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eDimethyl sulfoxide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eECM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eExtracellular Matrix\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eEDD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eEmbryonic Development Day\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eEE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eEntrapment Efficiency\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eELISA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eEnzyme-linked immunosorbent assay\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eERK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eExtracellular signal-regulated kinase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eFACS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eFluorescence-activated\u0026nbsp;cell sorting\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eFAM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eFluorescein\u0026nbsp;amidite\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eFITC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eFluorescein\u0026nbsp;isothiocyanate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eFTIR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eFourier Transform Infrared Spectroscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eHASMC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eHuman Aortic Smooth Muscle Cell\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eHBSS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eHanks\u0026apos; Balanced Salt Solution\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eHUVEC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eHuman\u0026nbsp;umbilical vein\u0026nbsp;endothelial cell\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eIFN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eInterferon\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eLNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eLocked Nucleic Acids\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eLPS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eLipopolysaccharides\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eMOI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eMultiplicity of infection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eMTT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eThiazolyl blue tetrazolium bromide\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eOD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eOptical Density\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eP0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eParental Generation 0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003ePhosphate Buffer Saline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003ePI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003ePropidium Iodide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003ePI3K\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003ePhosphatidylinositol 3-kinases\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003ePTEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003ePhosphatase and tensin homolog\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eSFM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eSerum-Free Media\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eSMAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eSuppressor of Mothers against Decapentaplegic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eTEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eTransmission Electron Microscope\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eTGF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eTransforming growth factor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eVEGF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eVascular endothelial growth factor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eVSV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 321px;\"\u003e\n \u003cp\u003eVesicular stomatitis virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Facility for Electron Microscopy Research of McGill University (SEM), Mohini Ramkaran of Microscopy \u0026amp; Imaging Lab, McGill Chemistry Characterization (MC\u003csup\u003e2\u003c/sup\u003e) Facility (AFM), Biomat\u0026rsquo;X lab (DLS and multiplate reader), Julien Sirois of The Montreal Neurological Institute - McGill University (flow cytometry) for their equipment, training, and services.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a research grant from the Canadian Institute of Health Research (CIHR) to Dominique Shum-Tim, Arghya Paul and Satya Prakash (CIHR 252743).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eP.I. is funded by the Islamic Development Bank Scholarship (2020-245622).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA.A. is fully funded by a scholarship from the Ministry of Higher Education of the Arab Republic of Egypt.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eS.S. is fully funded by the Canadian Graduate Scholarship-Doctoral Award from the Natural Sciences and Engineering Research Council (NSERC, 569661-2022).\u003c/p\u003e\n\u003cp\u003eE.R is fully funded by the Indonesia Endowment Fund for Education from the Ministry of Finance of the Republic of Indonesia.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of materials and data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConceptualization: PI, SP, AP, DST\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMethodology: PI, SP, AA\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInvestigation: PI, JLB, AK\u003c/p\u003e\n\u003cp\u003eVisualization: PI, SP, SM\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSupervision: SP, DST\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWriting- original draft: PI, SP\u003c/p\u003e\n\u003cp\u003eWriting- review \u0026amp; editing: PI, AA, MS, SM, ER, CST, DST\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConflict of interest\u003c/p\u003e\n\u003cp\u003eThe authors of this manuscript have no conflict of interest to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLi M, Qian M, Kyler K, Xu J. 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Int J Mol Sci. 2021;22(14).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Angiogenesis, tissue regeneration, gene therapy, viral vector, atherosclerosis, baculovirus","lastPublishedDoi":"10.21203/rs.3.rs-6770483/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6770483/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAtherosclerosis is the primary cause of most cases of coronary artery disease, peripheral arterial disease, and many strokes. It is characterized by pathological vascular smooth muscle cell hyperplasia. Current treatment regimens are associated with several adverse effects including hepatotoxicity, hemorrhagic complications, and non-selective cellular inhibition. Plaque stabilization and angiogenesis are critical for mitigating adverse cardiovascular outcomes. Stabilized plaques exhibit reduced vulnerability to rupture, thereby lowering the risk of thrombus formation, myocardial infarction, and ischemic stroke. Transforming Growth Factor Beta 1 (TGF-β1cells) is instrumental in promoting angiogenesis, facilitating the regrowth of endothelial cells, and contributing to the stabilization of atherosclerotic plaques. Anti-miRNA 21 can lead to plaque stabilization by decreasing inflammation and limiting the growth of smooth muscle cells while encouraging cell death, which helps prevent plaque rupture. PLGA nanoparticles can ensure high encapsulation and effective delivery of genes and viral vectors over time and can offer superior protection for their encapsulated contents, which is particularly valuable for delicate substances such as proteins and nucleic acids. This research investigates a novel combination therapy utilizing baculovirus expressing TGF-β1 gene and anti-miR-21, incorporated into gelatin-genipin polymeric nanocomposite hydrogels. The therapy demonstrates synergistic effects through dual mechanisms: promoting neo-vascularization via selective endothelial cell proliferation while inducing smooth muscle cell apoptosis to control extracellular matrix secretion and stabilize plaque. The therapeutic efficacy is evidenced by significant reduction in PTEN expression (251.1\u0026thinsp;\u0026plusmn;\u0026thinsp;16 pg/ml compared to 375.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.29 pg/ml in control) and enhanced angiogenic responses in the CAM assay, showing a 126.46\u0026thinsp;\u0026plusmn;\u0026thinsp;16.62% increase in vessel length.\u003c/p\u003e","manuscriptTitle":"A novel combination therapy of anti miR-21 and baculoviral TGFβ1 Gene via PLGA-gelatin-genipin nanocomposite hydrogel for arterial plaque stabilization and angiogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-12 06:41:05","doi":"10.21203/rs.3.rs-6770483/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-17T07:52:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-16T13:08:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-14T12:31:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"255015655715857318609212681050863834648","date":"2025-06-12T05:58:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-10T09:30:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"91696083183004302407063942037322406104","date":"2025-06-10T08:10:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"229423484746807476761183822628517889029","date":"2025-06-10T06:28:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-10T03:06:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-09T03:27:55+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-06T02:49:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-05T05:48:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-28T18:18:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9fd51a1c-6c76-424b-9d7a-e36d8047f6d8","owner":[],"postedDate":"June 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":49806455,"name":"Biological sciences/Biotechnology"},{"id":49806456,"name":"Health sciences/Cardiology"},{"id":49806457,"name":"Health sciences/Medical research"},{"id":49806458,"name":"Physical sciences/Materials science"}],"tags":[],"updatedAt":"2026-01-26T16:03:03+00:00","versionOfRecord":{"articleIdentity":"rs-6770483","link":"https://doi.org/10.1038/s41598-025-32264-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-23 15:58:14","publishedOnDateReadable":"January 23rd, 2026"},"versionCreatedAt":"2025-06-12 06:41:05","video":"","vorDoi":"10.1038/s41598-025-32264-8","vorDoiUrl":"https://doi.org/10.1038/s41598-025-32264-8","workflowStages":[]},"version":"v1","identity":"rs-6770483","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6770483","identity":"rs-6770483","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}