A vascular graft that maintains smooth muscle cell contractile phenotype and methods of making the same

Abstract

This invention discloses a vascular graft that maintains the contractile phenotype of smooth muscle cells and its preparation method. The vascular graft is modified with a polydopamine coating on its luminal surface, anchoring RGD / CD47 dual-modified lipid nanoparticles, which encapsulate mRNA encoding olarumab. After implantation, the dual-modified LNPs specifically target endothelial cells, achieving highly efficient transfection and enabling endothelial cells to continuously secrete olarumab in situ, thereby blocking the PDGF-BB / PDGFR-α signaling pathway, maintaining the contractile phenotype of smooth muscle cells, and significantly inhibiting restenosis and calcification of the vascular graft. Experiments show that the dual-modified LNPs have a transfection efficiency of >90% in endothelial cells, exhibiting an unexpected synergistic effect of cell-selective delivery.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, specifically relating to a functionalized small-diameter vascular graft, and more particularly to a vascular graft based on mRNA technology that maintains the smooth muscle cell contractile phenotype by engineering olarumab secretion from in situ endothelial cells, and its preparation method. Background Technology

[0002] Small-diameter vascular grafts (internal diameter <6mm) are in urgent need in coronary artery bypass grafting and peripheral vascular reconstruction, but their long-term patency rate is not ideal. Restenosis is the main cause of graft failure, and its key pathological mechanism is the transdifferentiation of vascular smooth muscle cells (VSMCs) from a contractile phenotype to a synthetic phenotype.

[0003] Current research indicates that damaged endothelial cells (ECs) after transplantation continuously release PDGF-BB, inducing phenotypic transformation by activating PDGFR-α receptors on VSMCs. Olaratumab, a PDGFR-α neutralizing antibody, can block this process, but systemic administration suffers from significant side effects and insufficient local concentrations. Although studies have attempted to achieve local antibody expression through gene technology, traditional linear mRNA expression has a short duration and lacks cell-specific targeted delivery systems.

[0004] mRNA is an important gene expression tool, but its in vivo delivery still faces challenges such as poor targeting and susceptibility to clearance by immune cells. Lipid nanoparticles (LNPs) have shown promising potential as nucleic acid delivery vectors, but achieving cell-specific transfection remains a key issue that needs to be addressed. Summary of the Invention

[0005] The primary objective of this invention is to provide a vascular graft that maintains the contractile phenotype of smooth muscle cells.

[0006] The technical solution adopted in this invention is: a vascular graft for maintaining the contractile phenotype of smooth muscle cells, comprising: Electrospun vascular graft substrate; A polydopamine (PDA) coating formed on the surface of the base lumen; RGD / CD47 dual-modified lipid nanoparticles (LNPs) anchored by the polydopamine coating. The LNP contains messenger ribonucleic acid (mRNA) encoding olarumab. In the RGD / CD47 dual-modified lipid nanoparticles of this invention, the RGD peptide is integrated into the lipid bilayer via DSPE-PEG-RGD, and CD47 is specifically covalently coupled to the maleimide group at the end of DSPE-PEG-MAL, thereby being modified and anchored on the LNP surface. Experiments show that this dual-modification design enables cell-selective gene delivery of LNPs.

[0007] The lipid nanoparticles of this invention comprise Dlin-MC3-DMA, DSPC, cholesterol, and PEG-modified lipid components, with a nitrogen-to-phosphorus ratio of 3:1 to 20:1. This specific ratio ensures that LNPs exhibit good mRNA encapsulation and transfection efficiency.

[0008] The mRNA described in this invention can be prepared by the following method: based on the amino acid sequences of the heavy and light chains of olamumab, the corresponding coding sequences were designed and synthesized, and after in vitro transcription and purification, a high-purity mRNA product was finally obtained.

[0009] As will be readily understood by those skilled in the art, the vascular grafts of the present invention can be prepared according to existing vascular graft preparation methods. To enable those skilled in the art to better implement the present invention, we provide a specific preferred preparation scheme, which can be implemented by those skilled in the art according to the following steps: (1) Preparation of circular RNA encoding olamumab: Linear mRNA was designed and synthesized based on the olamumab sequence, and the circular RNA was purified by HPLC through T4 RNA ligase catalyzing the cyclization reaction; (2) Preparation of RGD / CD47 dual-modified lipid nanoparticles: Dlin-MC3-DMA, DSPC, cholesterol, DSPE-PEG, DSPE-PEG-MAL and DSPE-PEG-RGD were dissolved in ethanol phase at a molar ratio of 50:10:38.5:1:0.3:0.2. mRNA was dissolved in citrate buffer and mixed at a flow rate of 3:1 using a microfluidic mixer. Then CD47 protein solution was added and the conjugation was completed by incubation at 37°C for 1 hour. (3) Preparation of polydopamine-modified vascular grafts: The PLCL / PCL bilayer vascular grafts prepared by electrospinning were covered with a protective film on the outer layer, and the inner layer was immersed in 2 mg / mL dopamine-Tris-HCl buffer solution and shaken at room temperature for 6 hours. (4) Functional modification: The PDA-modified vascular graft was immersed in the LNP solution prepared in step (2), and the graft was shaken and adsorbed at 4°C for 3 hours. After removal, it was freeze-dried.

[0010] In step (2) of this invention, the total flow rate of the microfluidic mixer can be 12 mL / min, the mixing temperature is suitable at 25℃, the obtained LNP particle size is preferably 80-120 nm, the PDI is less than 0.15, and the encapsulation efficiency is greater than 95%. These parameters ensure that the LNP has good physicochemical properties and transfection efficiency.

[0011] In step (3) of this invention, the pH of the dopamine-Tris-HCl buffer solution is preferably 8.5. During the reaction, the solution color gradually changes from colorless to dark black, indicating that the polydopamine coating has been successfully deposited. The PDA coating firmly anchors LNP through hydrogen bonding and electrostatic interactions, while simultaneously achieving sustained release of LNP under blood flow conditions.

[0012] A third objective of this invention is to provide the use of RGD / CD47 dual-modified lipid nanoparticles in the preparation of medicaments for the prevention or treatment of vascular graft restenosis.

[0013] This cell-selective delivery characteristic allows gene drugs to act specifically on target cells, significantly improving the transfection efficiency of targeted endothelial cells and thus reducing side effects.

[0014] The beneficial effects of this invention are: 1) Cell-specific delivery of gene drugs was achieved through RGD / CD47 double-modified LNPs, significantly improving the transfection efficiency of targeted endothelial cells; 2) By utilizing the long-term expression characteristics of mRNA, sustained local secretion of olarumab can be achieved, effectively maintaining the contractile phenotype of smooth muscle cells; 3) The polydopamine coating stabilizes and immobilizes LNP, enabling sustained release under blood flow conditions and ensuring long-term efficacy; 4) This functionalized vascular graft can significantly inhibit neointimal hyperplasia and calcification, and improve long-term patency. Attached Figure Description

[0015] Figure 1 This is a schematic diagram illustrating the preparation process, key physicochemical characterization, and comparative analysis of circular / linear mRNA-LNPs for dual-modified LNPs. (A) Schematic diagram of LNP preparation via microfluidic mixing. (BE) Characterization of different LNP formulations (A1-A6). (F) Schematic diagram of CD47 coupling to the LNP surface via maleimide and representative cryo-electron microscopy images of the LNPs. Scale bar = 25 nm. (GJ) Characterization of key physicochemical properties of LNPs before and after CD47 modification. (K) Schematic diagram of circular mRNA (CM) design. (L) Analysis of mRNA samples by agarose gel electrophoresis. (M) Quantification of CM percentage using capillary electrophoresis. (NQ) Comparative characterization of CM-LNP and LM-LNP.

[0016] Figure 2This is a schematic diagram of LNP selective transfection and Ola monoclonal antibody exocrine kinetics; where (F) is a schematic diagram of ECs transfected through an LNP / PDA coated membrane secreting Ola; (GH) shows the changes in the concentration of secreted Ola (G) and the concentration of unsecreted Ola (H) over time.

[0017] Figure 3 This is a schematic diagram of histopathological and phenotypic analysis after implantation of a vascular graft according to a specific implementation method. (A) Schematic timeline of the experimental design; (B) Representative images of the implanted graft in situ at the start and end of the study. Scale bar = 1 mm; (CD) H&E staining of graft cross-section (C) and corresponding quantitative analysis of tissue thickness (D); (EF) Elastica van Gieson staining (E) and elastic fiber / collagen fiber ratio (F); (GH) Masson trichrome staining (G) and quantitative analysis of fibrotic areas (H); (IJ) Immunofluorescence analysis of type I collagen (I) and quantitative fluorescence intensity (J); (KL) Immunofluorescence analysis of type III collagen (K) and quantitative fluorescence intensity (L); (MN) Immunofluorescence co-staining of MYH11 (green) and OPN (red) (M) and quantitative fluorescence intensity analysis (N); (OP) Immunofluorescence staining of CD31 (O) and quantitative fluorescence intensity analysis (P).

[0018] Figure 4 This is a schematic diagram illustrating the long-term patency and calcification assessment of vascular grafts in a specific implementation. (A) Doppler ultrasound image of the vascular graft; (BC) blood flow velocity at different time points; (D) cross-sectional ultrasound imaging of the vessel. Red arrows indicate the vessel lumen; (EF) quantification of lumen diameter at different time points; (G) micro-CT analysis of the vascular graft, including two-dimensional grayscale images, three-dimensional reconstructed images, and cross-sectional images; (HJ) quantification of calcification volume (H), calcification surface area (I), and the ratio of calcification volume (CV) to tissue volume (TV) (J).

[0019] Figure 5 These are confocal immunofluorescence micrographs of olarumab expression in endothelial cells after vascular graft implantation in the examples and comparative groups. Detailed Implementation

[0020] The present invention will be further described below with reference to embodiments.

[0021] Example 1: Preparation of functionalized vascular grafts: (1) Preparation of circular RNA encoding olarumab: The coding sequence was designed based on the amino acid sequences of the heavy and light chains of olarumab, and linear mRNA was synthesized through in vitro transcription. A cyclization reaction was catalyzed by T4 RNA ligase. The reaction system contained: 1 μg linear mRNA, 10 U T4 RNA ligase, and 1 mM ATP, and the reaction was carried out at 37°C for 2 hours. After the reaction, the circular RNA was purified by HPLC, the main peak was collected, and the RNA purity and integrity were determined.

[0022] (2) Preparation of RGD / CD47 dual-modified lipid nanoparticles: Dlin-MC3-DMA (50 μmol), DSPC (10 μmol), cholesterol (38.5 μmol), DSPE-PEG (1.0 μmol), DSPE-PEG-MAL (0.3 μmol), and DSPE-PEG-RGD (0.2 μmol) were dissolved in 1 mL of anhydrous ethanol to form the organic phase. The circular RNA prepared in step (1) was dissolved in 50 mM citrate buffer (pH 4.0) at a concentration of 0.1 mg / mL to form the aqueous phase. The mixture was prepared using a microfluidic mixer at a volume ratio of organic phase to aqueous phase of 3:1, a total flow rate of 12 mL / min, and a mixing temperature of 25 °C. Subsequently, CD47 protein solution (CD47 to DSPE-PEG-MAL molar ratio of 1.2:1) was added, and the coupling reaction was completed by incubation at 37 °C for 1 hour.

[0023] (3) Preparation of polydopamine-modified vascular grafts: PLCL / PCL bilayer vascular grafts (2 mm inner diameter, 200 μm wall thickness) were prepared using electrospinning technology. The outer layer of the vascular graft was covered with a protective film, and the inner layer was immersed in 2 mg / mL dopamine-Tris-HCl buffer (pH 8.5). The reaction was carried out at room temperature with shaking at 60 rpm for 6 hours to form a uniform polydopamine coating.

[0024] (4) Functional modification: The PDA-modified vascular graft was immersed in the RGD / CD47 double-modified LNP solution (1 mg / mL) prepared in step (2) and adsorbed at 40 rpm for 3 hours at 4°C. After removal, it was rinsed with PBS to remove unbound LNPs, freeze-dried, and sealed for storage to obtain the functionalized vascular graft.

[0025] Example 2: Preparation of functionalized vascular grafts (1) Preparation of circular RNA encoding olarumab: The coding sequence was designed based on the amino acid sequences of the heavy and light chains of olarumab, and linear mRNA was synthesized through in vitro transcription. A cyclization reaction was catalyzed by T4 RNA ligase. The reaction system contained: 1.2 μg linear mRNA, 12 U T4 RNA ligase, and 1.2 mM ATP, and the reaction was carried out at 37 °C for 2.5 h. After the reaction, the circular RNA was purified by HPLC, the main peak was collected, and the RNA purity and integrity were determined.

[0026] (2) Preparation of RGD / CD47 dual-modified lipid nanoparticles: Dlin-MC3-DMA (48 μmol), DSPC (12 μmol), cholesterol (37.5 μmol), DSPE-PEG (1.2 μmol), DSPE-PEG-MAL (0.4 μmol), and DSPE-PEG-RGD (0.3 μmol) were dissolved in 1 mL of anhydrous ethanol to form the organic phase. The circular RNA prepared in step (1) was dissolved in 50 mM citrate buffer (pH 4.0) at a concentration of 0.15 mg / mL to form the aqueous phase. The mixture was prepared using a microfluidic mixer at a volume ratio of organic phase to aqueous phase of 2.5:1, a total flow rate of 10 mL / min, and a mixing temperature of 25 °C. Subsequently, CD47 protein solution (CD47 to DSPE-PEG-MAL molar ratio of 1.5:1) was added, and the coupling reaction was completed by incubation at 35 °C for 1.5 hours.

[0027] (3) Preparation of polydopamine-modified vascular grafts: PLCL / PCL bilayer vascular grafts (1.5 mm inner diameter, 150 μm wall thickness) were prepared using electrospinning technology. The outer layer of the vascular graft was covered with a protective film, and the inner layer was immersed in 1.5 mg / mL dopamine-Tris-HCl buffer (pH 8.0). The reaction was carried out at room temperature with shaking at 60 rpm for 8 hours to form a uniform polydopamine coating.

[0028] (4) Functional modification: The PDA-modified vascular graft was immersed in the RGD / CD47 double-modified LNP solution (0.8 mg / mL) prepared in step (2) and adsorbed at 4°C with shaking at 40 rpm for 4 hours. After removal, the graft was rinsed with PBS to remove unbound LNPs, freeze-dried, and sealed for storage to obtain the functionalized vascular graft.

[0029] Example 3: (1) Preparation of circular RNA encoding olarumab: The coding sequence was designed based on the amino acid sequences of the heavy and light chains of olarumab, and linear mRNA was synthesized through in vitro transcription. A cyclization reaction was catalyzed by T4 RNA ligase. The reaction system contained: 0.8 μg linear mRNA, 8 U T4 RNA ligase, and 0.8 mM ATP, and the reaction was carried out at 37 °C for 1.5 h. After the reaction, the circular RNA was purified by HPLC, the main peak was collected, and the RNA purity and integrity were determined.

[0030] (2) Preparation of RGD / CD47 dual-modified lipid nanoparticles: Dlin-MC3-DMA (52 μmol), DSPC (8 μmol), cholesterol (39.5 μmol), DSPE-PEG (0.8 μmol), DSPE-PEG-MAL (0.2 μmol), and DSPE-PEG-RGD (0.1 μmol) were dissolved in 1 mL of anhydrous ethanol to form the organic phase. The circular RNA prepared in step (1) was dissolved in 50 mM citrate buffer (pH 4.0) at a concentration of 0.08 mg / mL to form the aqueous phase. The mixture was prepared using a microfluidic mixer at a volume ratio of organic phase to aqueous phase of 3.5:1, a total flow rate of 14 mL / min, and a mixing temperature of 25 °C. Subsequently, CD47 protein solution (CD47 to DSPE-PEG-MAL molar ratio of 1.0:1) was added, and the coupling reaction was completed by incubation at 40 °C for 0.5 h.

[0031] (3) Preparation of polydopamine-modified vascular grafts: PLCL / PCL bilayer vascular grafts (2.5 mm inner diameter, 250 μm wall thickness) were prepared using electrospinning technology. The outer layer of the vascular graft was covered with a protective film, and the inner layer was immersed in 2.5 mg / mL dopamine-Tris-HCl buffer (pH 9.0). The reaction was carried out at room temperature with shaking at 60 rpm for 4 hours to form a uniform polydopamine coating.

[0032] (4) Functional modification: The PDA-modified vascular graft was immersed in the RGD / CD47 double-modified LNP solution (1.2 mg / mL) prepared in step (2) and adsorbed at 40 rpm for 2 hours at 4°C. After removal, it was rinsed with PBS to remove unbound LNPs, freeze-dried, and sealed for storage to obtain the functionalized vascular graft.

[0033] Comparative Example 1: The comparative example is the control experiment of Example 1. It is carried out according to the same steps and conditions as Example 1. All raw materials used are from the same batch. The difference is that in step (2), DSPE-PEG-MAL and CD47 protein are not added. Only DSPE-PEG-RGD is used for modification to prepare RGD single-modified LNP.

[0034] Comparative Example 2: The comparative example is the control experiment of Example 1. It is carried out in the same steps and conditions as Example 1. All raw materials used are from the same batch. The difference is that DSPE-PEG-RGD is not added in step (2). Only DSPE-PEG-MAL and CD47 protein are used for modification to prepare CD47 single-modified LNP.

[0035] Comparative Example 3: The comparative example is a control experiment of Example 1, which is carried out in the same steps and conditions as Example 1. All raw materials used are from the same batch. The difference is that in step (2), DSPE-PEG-MAL, DSPE-PEG-RGD and CD47 protein are not added. Only DSPE-PEG is used for modification to prepare unmodified LNP.

[0036] Experimental verification: Characterization of LNPs: Following established protocols, LNPs encapsulating linear mRNA or circRNA were prepared using microfluidic mixing technology. Lipids, including Dlin-MC3-DMA (MC3), DSPC, cholesterol, and PEG components (DSPE-PEG, DSPE-PEG-MAL, and DSPE-PEG-RGD), were dissolved in the ethanol phase, while the mRNAs (linear mRNA or circRNA) were dissolved in the citrate buffer phase. Figure 1 A). Different LNP formulations (A1-A6) are listed in Table 1. Specifically, formulations A1-A3 have the same lipid composition but different nitrogen / phosphorus (N / P) ratios, while formulations A4-A6 have different PEG components.

[0037] Table 1 Different LNP formulations

[0038] We further characterized the fundamental physicochemical properties of different LNP formulations. Dynamic light scattering (DLS) data showed that the average particle size of the LNPs ranged from 80 to 120 nanometers. Figure 1 B), the Proportional Dispersion Index (PDI) has remained below 0.15. Figure 1 (C), indicating that LNPs have good dispersibility and stability. Furthermore, the Zeta potentials of different groups of LNPs were measured (C). Figure 1 D). Moreover, formulations A4-A6 (N / P ratio of 5:1) showed an extremely high mRNA encapsulation efficiency of approximately 95%, while formulations A1 (N / P ratio of 1:1) and A2 (N / P ratio of 3:1) showed only approximately 75% and 85% mRNA encapsulation efficiencies, respectively. Figure 1E). Clearly, the N / P ratio of the LNP formulation is crucial for mRNA encapsulation. Therefore, a formulation with an N / P ratio of 5:1 is optimal.

[0039] This study aimed to investigate the effects of these modifications on the key physicochemical properties of LNPs. The results showed that the introduction of these functional ligands altered the particle size distribution, PDI, and Zeta potential. Although the particle size changes were within acceptable limits, the PDI values, while fluctuating slightly, remained consistently low (< 0.15), indicating that the dispersibility of LNPs was largely unaffected. Changes in Zeta potential depended on the specific modified ligands and were related to the charge properties of the corresponding molecules, including charge quantity and type.

[0040] Furthermore, we observed a simple and rapid biocoupler formation between CD47 with a free sulfide group and a maleimide group, forming a succinimide thioether bond. To further investigate the morphological characteristics of the CD47-modified LNPs, cryo-transmission electron microscopy (Cryo-TEM) was used. Comparative analysis of images before and after modification showed that the overall morphology or size of the LNPs did not change significantly. Figure 1 F). Apart from the observable change in Zeta potential values, no other characterization methods detected significant differences after CD47 modification (F). Figure 1 GJ).

[0041] Based on the amino acid sequence of the target antibody, a circular mRNA (CM) was designed and synthesized, and subsequently purified to obtain a high-purity product. Figure 1 KM). Simultaneously, linear mRNAs (LM) with the same coding sequence were prepared as a control. After encapsulating the two mRNA forms into LNPs to form respective complexes, comprehensive characterization showed no significant differences in key physicochemical properties between the two LNP formulations. Figure 1 (NQ). We then presented the dynamic particle size distributions of CM-LNP and LM-LNP in PBS over time. During the 7-day observation period, the particle sizes of both CM-LNP and LM-LNP gradually increased over time. Furthermore, the particle size change patterns of both were essentially parallel throughout the process.

[0042] Analysis of LNP selective transfection and antibody secretion kinetics based on Transwell co-culture system: We further evaluated the selective transfection capability of the LNP / PDA-coated electrospun membrane using a Transwell assay. In a co-culture Transwell system, the EGFP mRNA-coated LNP / PDA membrane was placed in the lower chamber, while ECs, VSMCs, and RAW264.7 cells were placed in the upper chamber. Bright-field and fluorescence imaging showed that the proportion of EGFP-positive ECs was significantly higher than that of other cell types, indicating that the membrane-released LNPs retained EC targeting and high transfection efficiency.

[0043] Subsequently, we used a similar Transwell system to further investigate Ola secretion from ECs transfected with LNP. In this setup, ECs were cultured on a membrane in the upper chamber. Green arrows indicate Ola secreted into the supernatant, while red arrows represent antibodies retained intracellularly. Figure 2 F).

[0044] For secretion kinetics ( Figure 2 In the LM-LNP group (orange), the concentration showed a decreasing trend from day 1, decreasing from an initial concentration of approximately 350-400 ng / mL to 100-150 ng / mL on day 3, and further decreasing to approximately 15 ng / mL on day 5, indicating only a transient stimulatory effect. In contrast, the CM-LNP group (blue) showed a trend of first increasing and then gradually decreasing: starting from approximately 300-350 ng / mL on day 1, reaching a peak of approximately 900-950 ng / mL on day 3, then decreasing to approximately 300 ng / mL on day 5, and falling below 150 ng / mL on day 10. Therefore, CM-LNP is significantly superior to LM-LNP in maintaining ECs' secretion of Oligo, supporting its potential as a long-acting antibody delivery platform. Furthermore, the trend of intracellular antibody concentration changes was basically consistent with that in the supernatant ( Figure 2 H). The synchronicity of intracellular and supernatant antibody concentrations indicates that antibody synthesis, intracellular transport, and secretion are coherent and coordinated, without obvious abnormal regulation or retention.

[0045] Histological assessment of tissue regeneration in vascular grafts: To evaluate the in vivo performance and therapeutic efficacy of engineered vascular grafts carrying LNPs (used for antibody production to prevent stenosis), we conducted a six-month animal study in a rat model. Prior to the experiment, newborn healthy rats were selected and housed in a standardized environment for 56 days to ensure normal development and physiological stability. On day 0, the vascular graft was surgically implanted. After a six-month observation period, pathological and imaging examinations were performed. Figure 4 A).

[0046] Following implantation, the graft maintained structural integrity and seamlessly integrated with the abdominal aorta at the end of the study, indicating successful surgical implantation and robust in vivo performance. Figure 4 B). Ola concentrations were monitored post-implantation. In the CM-LNP / SDVG group, Ola secretion was first detected on day 6 at approximately 10 ng / mL. Concentrations gradually increased, reaching approximately 21 ng / mL in the mid-rise phase (day 16) and peaking at approximately 28 ng / mL on day 22. In contrast, the CM-LNP / SDVG group exhibited higher initial secretion, reaching a concentration of approximately 16 ng / mL as early as day 10, but concentrations decreased below CM-LNP levels after day 10 and became undetectable after day 14. This pattern contrasts sharply with the sustained secretion profile observed in the CM-LNP / SDVG group. These findings suggest that CM-LNP / SDVG can achieve more effective and sustained Ola secretion in vivo, thus meeting the pharmacological requirements for the prevention of VSMC-derived stenosis in vascular grafts.

[0047] H&E staining of vascular tissue ( Figure 4 (C) shows that the SDVG group exhibited disordered vascular structure, discontinuous endothelium, and unclear vessel wall boundaries, indicating unsatisfactory repair. The CM-LNP / SDVG group demonstrated continuous endothelium, clear vessel walls, and orderly smooth muscle arrangement, reflecting excellent tissue remodeling. The LM-LNP / SDVG group showed partial improvement, with mostly continuous but occasionally loose endothelium and clearly layered vessel walls. Figure 4 D).

[0048] EVG staining showed that the CM-LNP / SDVG group had a complete and continuous elastic fiber layer, indicating good elastic structure. The collagen fibers in this group were ordered and evenly distributed, indicating optimal collagen metabolism and enhanced vascular strength and flexibility. Figure 4 E). The LM-LNP / SDVG group also showed improved collagen metabolism compared to the SDVG group, while the SDVG group exhibited collagen disorder and excessive deposition, which may lead to vascular stiffness. Figure 4 F).

[0049] Masson trichrome staining showed extensive blue-stained collagen deposition and fibrosis in the SDVG group. Figure 4 G), while the CM-LNP / SDVG and LM-LNP / SDVG groups showed reduced fibrosis and good collagen fiber tissue, promoting vascular patency (G). Figure 4 H).

[0050] Type I collagen analysis showed that the SDVG group exhibited strong red fluorescence and disordered accumulation, indicating significant fibrosis. The CM-LNP / SDVG group showed the weakest fluorescence, reflecting minimal fibrosis. Figure 4IJ). Regarding the expression of collagen type III, the CM-LNP / SDVG group showed weaker fluorescence and more ordered collagen compared to the SDVG group, inhibiting overactivation and collagen production. Figure 4 KL).

[0051] Double staining of MYH11 (a shrinkage marker) and OPN (a synthesis marker) showed that, compared with other groups, the CM-LNP / SDVG group had higher MYH11 expression and lower OPN levels. Figure 4 M), indicating a shift towards a contractile VSMC phenotype. This confirms the role of CM-LNP in enhancing vasomotor function and structural stability. Figure 4 N).

[0052] CD31 staining showed that ECs in the SDVG group were scattered and discontinuous, with localized red signals and numerous uncovered areas, indicating poor endothelialization. Figure 4 In contrast, the CM-LNP / SDVG group exhibited a uniform and intact endothelial cell layer on the luminal surface, while the LM-LNP / SDVG group showed moderate but incomplete endothelial coverage with irregular signal intensity. Figure 4 P).

[0053] Histopathological examination of the major organs (heart, lungs, liver, spleen, and kidneys) by H&E staining revealed no pathological changes or inflammatory reactions, confirming the biocompatibility and systemic safety of the transplant material and Ola secretion. This means that Ola levels reached the therapeutic threshold throughout the study and did not induce toxicity.

[0054] Assessment of vascular graft patency and calcification: Ultrasound examination showed that at the 3-month follow-up, the blood flow velocity in the CM-LNP / SDVG group was significantly higher than that in the LM-LNP / SDVG and SDVG groups, and good blood flow was maintained at 6 months. Conversely, the LM-LNP / SDVG group experienced a decline in hemodynamics, while the SDVG group had almost no blood flow due to severe stenosis. Figure 4 AC).

[0055] The lumen diameter measurements were consistent with the above findings: at 3 months, the CM-LNP / SDVG and LM-LNP / SDVG groups remained patent, while the SDVG group showed significant stenosis. By 6 months, CM-LNP / SDVG maintained a better lumen diameter compared to LM-LNP / SDVG, while the SDVG group showed severe occlusion. Figure 4 DF).

[0056] Micro-CT analysis provides precise visualization of calcifications in artificial grafts. Two-dimensional grayscale images show the overall morphology, while three-dimensional reconstructions highlight calcified foci in yellow. Cross-sectional images reveal details of the internal structure across multiple planes. Figure 4 G).

[0057] This study statistically analyzed calcification in three graft groups and evaluated three key parameters: calcification volume (CV) ( Figure 4 H), calcified surface area ( Figure 4 I) and the ratio of CV to tissue volume (TV) Figure 4 J).

[0058] A comprehensive quantitative assessment of CV showed that the measured value in the SDVG group was approximately 7 μm³. In stark contrast, this value decreased significantly in the CM-LNP / SDVG group, plummeting to approximately 2.5 μm³. However, the value in the LM-LNP / SDVG group fell between the two, recording approximately 4.5 μm³. In addition to CV analysis, a more in-depth assessment of the calcified surface area showed that the value in the SDVG group was approximately 2.5 μm². A significant reduction was observed in the CM-LNP / SDVG group, with the value decreasing to approximately 1 μm². Meanwhile, the area recorded in the LM-LNP / SDVG group was 1.3 μm². Statistical analysis confirmed highly significant differences among the SDVG, CM-LNP / SDVG, and LM-LNP / SDVG groups. Based on the previous assessment, an examination of the CV to TV ratio showed a value of 3.6% in the SDVG group. This ratio experienced a sharp and significant decrease in the CM-LNP / SDVG group, dropping to only 0.3%. On the other hand, the ratio in the LM-LNP / SDVG group was 1.5%. Notable differences were found between the CM-LNP / SDVG group and the other two groups (P < 0.01).

[0059] Calcification is a key factor that compromises the long-term patency and function of artificial blood vessels. Reducing calcification allows the graft to better maintain its structural integrity and physiological function in the body, thereby lowering the risk of complications such as arteriosclerosis and stenosis. Ultimately, this improves the long-term performance of vascular grafts and provides patients with more reliable treatment options.

[0060] The results showed that CM-LNP modification exerted the most effective anti-calcification effect, leading to a significant reduction in vascular volume (CV), surface area, and relative calcified tissue content. LM-LNP modification also provided some benefits, but its effect was far inferior to CM-LNP. Furthermore, SEM images revealed the EC coverage status of the three types of vascular grafts 6 months after implantation. The SDVG group exhibited the worst endothelialization performance, with a rough and uneven surface, sparse cell distribution, and insufficient EC coverage. The CM-LNP / SDVG group had a smooth surface covered with a continuous and intact endothelial cell layer. As for the LM-LNP / SDVG group, although it showed more obvious EC coverage compared to the SDVG group, its coverage area and smoothness were inferior to those of the CM-LNP / SDVG group. The differences in endothelialization results may be attributed to different vascular calcification conditions. This study provides crucial experimental evidence for optimizing the surface modification of vascular grafts, clearly demonstrating the superior ability of CM-LNP in inhibiting calcification.

[0061] Experiment to validate transfection efficiency targeting endothelial cells: To verify the transfection efficiency of the vascular grafts of this invention targeting endothelial cells, we conducted a systematic animal experiment. Healthy adult SD rats (weighing 250-300g) were randomly divided into 6 groups, with 6 rats in each group: unmodified LNP group (Comparative Example 3), RGD-modified LNP group (Comparative Example 1), CD47-modified LNP group (Comparative Example 2), and RGD / CD47-modified LNP group (Examples 1 to 3). All rats underwent abdominal aortic transplantation under isoflurane anesthesia, and functionalized vascular grafts of the corresponding groups were implanted. On the 7th day after surgery, the animals were sacrificed, and the transplanted vascular tissue was collected, fixed in 4% paraformaldehyde, dehydrated with sucrose, embedded in OCT, and then prepared into 7μm thick frozen sections.

[0062] Immunofluorescence staining was used to detect the expression of olarumab in endothelial cells. The specific steps were as follows: sections were washed with PBS, permeabilized with 0.3% Triton X-100, blocked with 5% BSA, and then incubated overnight at 4°C with rabbit anti-olarumab primary antibody (1:200). Subsequently, they were incubated at room temperature in the dark for 2 hours with Alexa Fluor 488-labeled secondary antibody (1:500). Cell nuclei were counterstained with DAPI, and finally, the sections were mounted with anti-fluorescence quenching mounting medium. Images were acquired using a confocal microscope under the same parameters (see [link to image]). Figure 5 Six fields of view were randomly selected from each slice, and the percentage of olaramumab-positive cells was quantitatively analyzed using ImageJ software.

[0063] Experimental data are expressed as mean ± standard deviation. One-way ANOVA was used for inter-group comparisons, and Tukey's method was used for post-hoc testing. P < 0.05 was considered statistically significant. Strict quality control measures were implemented during the experiment, including all surgical procedures performed by the same surgeon, staining within the same experimental batch, and blinded image acquisition and analysis, to ensure the accuracy and reproducibility of the results. The results are shown in Table 2.

[0064] Table 2. Percentage of Olamazon-positive cells in endothelial cells

[0065] Experimental results showed that the expression level of olarumab in endothelial cells was significantly higher in the RGD / CD47 double-modified LNP group than in other groups (P<0.01). Compared with the unmodified LNP group, the RGD single-modified LNP group increased the expression by approximately 46%, the CD47 single-modified LNP group by approximately 18%, and the RGD / CD47 double-modified LNP group by approximately 207%. This indicates that the transfection efficiency improvement rate of the double-modified LNP group was significantly higher than the expected sum of the improvement values ​​of each single-modification group, demonstrating that RGD and CD47 have a significant synergistic effect in improving the transfection efficiency of targeted endothelial cells during in vivo delivery.

Claims

1. A vascular graft that maintains the contractile phenotype of smooth muscle cells, characterized in that, include: Vascular graft substrate formed by electrospinning; A polydopamine coating formed on the lumen surface of the substrate; RGD / CD47 dual-modified lipid nanoparticles anchored to the lumen surface via the polydopamine coating; The LNP contains messenger ribonucleic acid encoding olarumab.

2. The vascular graft according to claim 1, characterized in that, The lipid composition of the LNP includes ionizable lipids, cholesterol, cofactor phospholipids, and polyethylene glycol-modified lipids, with molar proportions of 45–60%, 25–35%, 10–15%, and 0.5–3%, respectively; wherein: The ionizable lipids include, but are not limited to, ALC-0315, SM-102, and DLin-MC3-DMA; The auxiliary phospholipids include, but are not limited to, distearylphosphatidylcholine and dioleoylphosphatidylethanolamine; The PEG-modified lipids include, but are not limited to, ALC-0159, DSPE-PEG, DSPE-PEG-RGD, DSPE-PEG-MAL, and DSPE-PEG-NHS.

3. The vascular graft according to claim 1 or 2, characterized in that, The RGD / CD47 dual modification method is as follows: the RGD ligand is embedded into the LNP lipid layer by introducing DSPE-PEG-RGD into the formulation; CD47 is modified onto the LNP surface by covalently coupling the active ester / maleimide functional groups of DSPE-PEG-NHS or DSPE-PEG-MAL with CD47 or its functional fragments.

4. The vascular graft according to any one of claims 1–3, characterized in that, The preparation of the mRNA includes: designing and synthesizing the corresponding coding sequence based on the amino acid sequences of the heavy and light chains of olarumab, obtaining the mRNA product through in vitro transcription and purification, wherein the mRNA includes linear mRNA and / or circular mRNA; the nitrogen-to-phosphorus ratio during the LNP encapsulation process is 3:1 to 20:

1.

5. A method for preparing the vascular graft according to any one of claims 1-4, characterized in that, include: (1) Synthesize the mRNA encoding the target antibody and encapsulate it in LNP by microfluidic mixing; After encapsulation, the LNP suspension was subjected to tangential flow filtration to replace the buffer system and remove unencapsulated components, resulting in a purified LNP suspension; subsequently, CD47 was covalently modified on the LNP surface. (2) Based on electrospinning technology combined with a rapidly rotating mandrel with a diameter matching the target blood vessel, artificial blood vessel grafts are prepared; using polycaprolactone as the outer layer material and polylactic acid as the inner layer material, a tubular substrate with biomimetic structure and mechanical properties is obtained; by physically shielding / isolating the outer surface, PDA coating is deposited only on the lumen surface by using dopamine in-situ oxidation self-polymerization. (3) The vascular graft with PDA coating is immersed in a suspension of RGD / CD47 double-modified LNPs containing mRNA, so that the LNPs are physically adsorbed on the surface of the PDA coating through electrostatic interaction, hydrogen bonding and / or hydrophobic interaction, thereby forming a functional LNP layer containing mRNA on the surface of the vascular graft.

6. The use of the vascular graft according to any one of claims 1 to 4 or 5 in the preparation of a medicament or medical device for the prevention or treatment of luminal stenosis and calcification after vascular graft implantation.

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