A high-performance hydrogel platform enabling dual anti-miR-21 and TGF-β1 delivery to improve arterial plaque stability and enhance therapeutic angiogenesis outcomes

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. This research investigates a novel combination therapy utilizing anti-miR-21 and baculovirus expressing TGF-β1 gene for vascular tissue regeneration. A hemocompatible nanocomposite hydrogel with remarkable cellular adhesion profile was prepared by encapsulating anti-miR-21 and baculovirus expressing TGF-β1 gene in PLGA nanoparticles, followed by embedding them in a gelatin-genipin crosslinked nanocomposite hydrogel. Chorioallantoic membrane assay in chicken embryo and PTEN quantification study was used for angiogenesis. MTT assay followed by Annexin V-FITC/PI stained flowcytometry was used for HASMCs apoptosis study. The combination therapy demonstrates synergistic effects through dual mechanisms: promoting neo-vascularization via selective endothelial cell proliferation while inducing arterial smooth muscle cell apoptosis (22.27 ± 1.2%) 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.

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 outcomes⁵. Stabilized plaques exhibit reduced vulnerability to rupture, thereby lowering the risk of thrombus formation, myocardial infarction, and ischemic stroke⁶. 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 homeostasis⁷. 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 stabilization⁸. 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 rupture⁹. 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 rupture¹⁰. In angiogenesis, miR-21 enhances vascular growth by regulating endothelial cell behavior¹¹. However, it impairs re-endothelialization by inhibiting endothelial cell migration, hindering vascular repair and healing processes¹². By inhibiting miR-21, this therapy could potentially reduce inflammation and fibrosis in atherosclerotic plaques, promoting a more stable phenotype¹¹. Antagonism of miR-21 can stabilize plaques by reducing inflammation and smooth muscle cell proliferation while promoting apoptosis, thereby preventing plaque rupture¹³. By inhibiting miR-21, endothelial cell migration and proliferation are enhanced, facilitating better re-endothelialization and vascular repair after injury¹⁴. 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 complications¹³. Anti-miR-21 therapy has shown clear benefits in reducing neointimal hyperplasia¹⁵. 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 delivery¹⁶. 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 therapies¹⁷.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 degradation¹⁸. 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 bioactivities¹⁹. 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 applications²⁰.

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 complications²¹.

Materials & methods

Virus generation and titration

The virus generation and titration were performed following a previously discussed method by this group¹⁸ 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 P₀ BV stock. This was amplified in Sf21 cells to generate P₁ and P2 stocks. Viral titers were determined via flow cytometry using mCherry fluorescence and confirmed by cytopathic endpoint dilution²².

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^18,23. 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 method²⁴. The gelatin from porcine skin (Millipore Sigma, Germany) was dissolved in distilled water by stirring the mixture for 30 min 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 solution²⁵. This dispersion process was carried out under constant stirring at 50 °C for 30 min. 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 h, 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 min 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

Citrated Single Donor Human Whole Blood were collected commercially from Innovative Research, Inc, MI, USA 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 % hemolysis²⁶.

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-21 s, their free combination and the combination therapy were added to different wells, with media alone as control, and incubated for 12 h before replacing the transduction media with fresh media. After 48 h, 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-21 s and hydrogel elutions for 12 h. 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-21 s and supernatant containing the combination therapy eluted from the nanocomposite hydrogel. After a 12-h incubation, 20 µL of MTT solution (5 mg/mL) was added, and cells were incubated for 4 h 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 h 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 h 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 min 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 method²³, 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 Fig. 1A and 1B 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 Fig. 1C. 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^27,28. In a previous study by this group, the release profile of TGF-β1 from PLGA nanoparticles was characterized and demonstrated a sustained release pattern. Building on these findings, the current study focuses on the co-delivery approach, and individual release profiling was not repeated to maintain alignment with the combined therapeutic strategy¹⁸. 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 Fig. 1D. 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 Fig. 1E 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.

Fig. 1.

Physicochemical characterization of TGFβ1 and Anti-miR-21 combination therapy nanocomposite hydrogel. (A–B) 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. (C) TEM image of the prepared Bac-anti-miR-PLGA-NPs showing small, uniform and spherical nanoparticles. (D) 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. (E) 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.

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 Fig. 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.

Fig. 2.

A (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.

TGFβ1 and Anti-miR-21 combination therapy nanocomposite hydrogel promotes cellular adhesion and hemocompatibility

In the adhesion study with HUVECs, microscopy images (Fig. 2A) revealed a higher density of adherent cells on surfaces treated with combination therapy compared to the control. Quantitative analysis (Fig. 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. The hemolysis assay in Fig. 2C 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. 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 Fig. 3A, B, 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).

Fig. 3.

A (A) 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).

In the viability assay as depicted in Fig. 3C, D, 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 (Fig. 4) in both HASMCs and HUVECs, as shown in Fig. 4A, B, respectively. In HASMCs (Fig. 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^29,30. 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^18,31. The placebo group exhibited relatively low proliferation rates, further highlighting the safety profile of the nanocomposite hydrogel delivery system in HASMC.

Fig. 4.

(A) 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).

Similarly, in HUVECs (Fig. 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 (Fig. 4D). 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 Fig. 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 activity³².

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 Fig. 5A. 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 Fig. 5B, 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).

Fig. 5.

Flow cytometric analysis of HASMC apoptosis using Annexin V-FITC/PI staining. (A) Control group showing minimal apoptotic activity, free combination treatment demonstrating. increased apoptotic population, 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. (B) Quantitative analysis of apoptotic rates across treatment groups (n = 3).

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, Fig. 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 Fig. 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^33,34.

Fig. 6.

Evaluation 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.

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 stability and shelf-life of the formulated PLGA nanoparticles and gelatin-genipin hydrogel composite were not extensively evaluated in this study, representing a limitation. Although PLGA nanoparticles are generally known to retain stability under refrigerated or lyophilized conditions, factors such as temperature fluctuations, moisture exposure, and the nature of the encapsulated cargo (e.g., miRNA and baculovirus) can significantly impact degradation rates and release kinetics. Similarly, gelatin-genipin hydrogels may experience structural changes over time, affecting mechanical integrity and therapeutic diffusion profiles. Batch-to-batch variations in nanoparticle size, loading efficiency, and crosslinking density may also influence reproducibility. Future work will incorporate systematic stability testing under varied storage conditions to ensure long-term formulation consistency and translational readiness. 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. While parameters like, mechanical strength, porosity, or degradation profile of the gelatin-genipin hydrogels are essential for evaluating suitability for vascular implantation, the primary focus of this preliminary work was to establish biocompatibility and therapeutic efficacy. Future studies will incorporate detailed mechanical and structural characterization to better support the hydrogel’s translational potential in vascular applications. 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 regeneration³⁵. 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 maturation³⁶. 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 pericytes³⁷. In contrast, miR-21 antagonist inhibits miR-21 activity, which normally promotes angiogenesis by targeting PTEN and increasing AKT signaling³³. By inhibiting miR-21, endothelial cell migration and tube formation are reduced, leading to decreased angiogenic sprouting³⁸. 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. While PTEN protein levels were quantified via ELISA, this study did not include RT-qPCR analysis for gene expression of key markers such as VEGF, SMADs, miR-21, PTEN, or other TGF-β1 targets. Future studies will incorporate RT-qPCR to more directly validate molecular changes and further elucidate the underlying mechanisms of action.

The combination therapy demonstrates promising potential for plaque stabilization through multiple synergistic mechanisms^39,40. 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^35,41. The therapy exhibits selective action by reducing HASMC proliferation while enhancing HUVEC growth, coupled with decreased CRP levels indicating anti-inflammatory effects⁴². 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 modulation¹⁰.

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^43,44. 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^44,45. The nanocomposite hydrogel, composed of gelatin-genipin crosslinked matrix embedded with anti-miR-21 and baculovirus-expressing TGF-β1 PLGA nanoparticles are also suitable for localized, injectable delivery. Its mechanical integrity and uniform particle incorporation suggest injectability for targeted application. Clinically, this system offers potential in localized drug delivery for atherosclerotic plaque stabilization, particularly in coronary or peripheral arteries. By selectively inducing HASMC apoptosis and promoting endothelial proliferation, the formulation may enhance vascular remodeling post-angioplasty or reduce in-stent restenosis, offering a more precise alternative to systemic or non-selective stent-based therapies. 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^1,46. This preliminary study requires further validation in higher mammalian models, such as rodents or rabbits to confirm therapeutic efficacy, pharmacokinetics, and long-term vascular remodeling.

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. This study serves as a preliminary investigation demonstrating proof-of-concept using cellular models and the CAM assay. The results highlight a promising platform for selective vascular remodeling and plaque stabilization, with clear translational potential for localized therapy in atherosclerotic disease. To advance toward clinical application, future studies should include in vivo testing in atherosclerotic mouse or large animal models to evaluate therapeutic efficacy, pharmacokinetics, biocompatibility, and long-term vascular integration while having VEGF-treated group as a positive control in future experiments to enhance the interpretability and robustness of the data.

Abbreviations

AFM

Atomic force microscopy

BV

Baculovirus

CAM

Chorioallantoic membrane

CRP

C- reactive protein

DCM

Dichloromethane

DI

Deionized water

DLS

Dynamic light scattering

DMSO

Dimethyl sulfoxide

ECM

Extracellular matrix

EDD

Embryonic development day

EE

Entrapment efficiency

ELISA

Enzyme-linked immunosorbent assay

ERK

Extracellular signal-regulated kinase

FACS

Fluorescence-activated cell sorting

FAM

Fluorescein amidite

FITC

Fluorescein isothiocyanate

FTIR

Fourier transform infrared spectroscopy

HASMC

Human aortic smooth muscle cell

HBSS

Hanks’ balanced salt solution

HUVEC

Human umbilical vein endothelial cell

IFN

Interferon

LNA

Locked nucleic acids

LPS

Lipopolysaccharides

MOI

Multiplicity of infection

MTT

Thiazolyl blue tetrazolium bromide

OD

Optical density

P0

Parental generation 0

PBS

Phosphate buffer saline

PI

Propidium iodide

PI3K

Phosphatidylinositol 3-kinases

PTEN

Phosphatase and tensin homolog

SFM

Serum-free Media

SMAD

Suppressor of mothers against decapentaplegic

TEM

Transmission electron microscope

TGF

Transforming growth factor

VEGF

Vascular endothelial growth factor

VSV

Vesicular stomatitis virus

Author contributions

Conceptualization: PI, SP, AP, DST Methodology: PI, SP, AA, SS Investigation: PI, JLB, AK,SS Visualization: PI, SP, SM Supervision: SP, DST Writing- original draft: PI, SP Writing- review & editing: PI, AA, MS, SM, ER, SS,CST, DST.

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.

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.