Application of VEGF (vascular endothelial growth factor) mimic peptide in small-diameter intravascular stent

Abstract

The invention relates to the technical field of medical instruments, discloses application of VEGF (vascular endothelial growth factor) mimic peptide in a small-diameter intravascular stent, and particularly designs a fast endothelialization SDVG prototype stent adopting a QKCMP (quick K chemical mechanical polishing) technology. The QKCMP technology shows high affinity to the synthetic stent by negatively adjusting a Hippo-YAP / TAZ signal channel, so that the stability and removal resistance of the stent are ensured; in addition, the binding force of the stent is remarkably enhanced by using the VEGF mimic peptide QKCMP, and continuous and effective release of the VEGF is realized; the innovative technology is expected to obviously improve the effect of the small-diameter intravascular stent in clinical application.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and in particular to the application of a VEGF mimetic peptide in a small-diameter vascular stent. Background Art

[0002] Vascular endothelial growth factor (VEGF) plays a crucial role in promoting the attachment, differentiation and proliferation of endothelial cells (ECs). VEGF activates signal transduction by binding to VEGF receptors (VEGFRs) and participates in multiple signaling pathways such as PLCγ / PKC, PI3K / Akt, and p38 MAPK. Recent studies have highlighted the importance of VEGF in the application of vascular stents, especially in small-diameter vascular grafts (SDVGs) with poor clinical efficacy. Han successfully used physical encapsulation technology to achieve rapid release of VEGF from SDVGs in rabbits and maintained the effect for up to 8 weeks. In contrast, half of Jang's stents failed in the absence of VEGF in dogs after only 2 weeks. The lack of endothelial cells leads to direct exposure of synthetic materials to the blood and activation of coagulation mechanisms, which is the main reason for acute thrombosis in the early stage of SDVG implantation. Although anticoagulants or antiplatelet drugs can reduce the risk of thrombosis, they all have limitations and side effects. Therefore, early or rapid endothelialization is the preferred and effective strategy to prevent SDVG thrombosis.

[0003] However, bioactive VEGF is easily eluted due to its water-soluble characteristics. Therefore, the use of physical encapsulation technology to achieve sustained release of VEGF is not stable enough, especially for the early rapid endothelialization process. Studies have shown that by changing the chemical structure of VEGF, its binding ability to the stent can be significantly enhanced. Therefore, finding a new method to stabilize the binding of VEGF on the stent surface, thereby achieving its sustained and effective release, is of great significance for improving the clinical effect of small-diameter vascular stents. Summary of the invention

[0004] The VEGF mimetic peptide QKCMP shows great potential. QKCMP is a bifunctional peptide that combines the QK peptide and CMP structure. The former retains the core function of VEGF, while the latter gives the QK peptide the ability to bind tightly to collagen. Collagen and its hydrolysate gelatin, due to their good hydrophilicity, biocompatibility, degradability and low antigenicity, help promote the chemotaxis and adhesion properties of vascular endothelial cells during angiogenesis.

[0005] In view of the advantages of QKCMP, it shows significant potential value in the application field of SDVG. Therefore, the present invention has formulated a plan to use QKCMP to construct an SDVG prototype in order to quickly promote the growth of endothelial cells and evaluate its application performance. In the implementation of the plan, removable dimethyl sulfoxide (DMSO) was used as a solvent to prepare poly (D, L-lactide-co-glycolide) (PLGA) to enhance the fluidity and mechanical properties of the model. By encapsulating platelet-derived growth factor (PDGF) in PLGA, slow proliferation stimulation of vascular smooth muscle cells (VSMCs) can be achieved, and signals can be provided for mature endothelial cells (ECs). In addition, the use of DMSO is intended to prevent the hydrolysis and inactivation of QKCMP. Finally, the gel film is used to cover the two surfaces of PLGA, and QKCMP is bound to the cavity.

[0006] In view of this, the present invention proposes a method for preparing a SDVG prototype bracket, comprising:

[0007] The PDGF and PLGA solutions are mixed in DMSO to obtain a mixture, the mixture is filled into a tubular mold and then solidified, then rinsed with cold deionized water, and finally transferred into a vacuum chamber to naturally evaporate the DMSO to obtain a PLGA scaffold;

[0008] Premixing a gelatin solution dissolved in acetic acid with a collagen solution to form a collagen-gelatin mixed solution; uniformly coating the collagen-gelatin mixed solution on the lumen surface and the outer surface of the PLGA scaffold to obtain a PLGA scaffold with a dry protein film;

[0009] A QKCMP stock solution was prepared, heated to 80±5°C and maintained for at least 30 minutes, and then cooled to 37±2°C to obtain a peptide solution; the peptide solution was completely injected into the lumen of the PLGA scaffold with a dried protein film and sealed at both ends for incubation, followed by incubation again in PBS to obtain the SDVG prototype scaffold.

[0010] Preferably, the temperature at which the mixture is cured after being filled into the tubular mold is -20°C.

[0011] Preferably, the collagen solution is rat tail type I collagen solution.

[0012] Preferably, the collagen-gelatin mixed solution is uniformly coated on the lumen surface and the outer surface of the PLGA scaffold, comprising:

[0013] directly coating the collagen-gelatin mixed solution on the outer surface of the PLGA scaffold and performing a drying process;

[0014] The collagen-gelatin mixed solution was completely injected into the lumen of the PLGA scaffold and dried, and this was repeated at least twice.

[0015] Preferably, the collagen-gelatin mixed solution is uniformly coated on the luminal surface and the outer surface of the PLGA scaffold and then dried, the PLGA scaffold with the dried protein film is immersed in PBS for neutralization, and the neutralized film is air-dried to obtain a PLGA scaffold with a dried protein film.

[0016] Preferably, the method for preparing the QKCMP stock solution comprises:

[0017] After the resin is soaked and expanded in DMF or NMP, it is treated with a 20% piperidine solution to remove the N-terminal Fmoc protecting group;

[0018] The amino acids with Fmoc protection groups were mixed with HBTU and DIPEA to activate the carboxyl groups;

[0019] In the direction of synthesis from the N-terminus to the C-terminus, the activated amino acid reacts with the amino group on the resin to form a peptide bond; repeating the steps to couple the next amino acid according to the sequence described in claim 1 until the synthesis is completed;

[0020] After the synthesis is completed, all side chain protecting groups are removed and the peptide is cleaved from the resin to obtain the collagen-gelatin-bound VEGF mimetic peptide, namely, QKCMP stock solution.

[0021] Preferably, the incubation temperature is 37±2°C.

[0022] The present invention also proposes a SDVG prototype bracket, and the SDVG prototype bracket is prepared according to the above-mentioned preparation method.

[0023] The present invention also proposes an application of the SDVG prototype stent in a vascular stent.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention relates to a method for preparing a SDVG prototype scaffold, the steps of which include mixing PDGF and PLGA solution in DMSO to form a mixture, then injecting the mixture into a tubular mold and performing a curing treatment. After curing, the scaffold is rinsed with cold deionized water and transferred to a vacuum chamber, and a PLGA scaffold is prepared by naturally evaporating DMSO. The present invention utilizes VEGF mimetic peptide QKCMP to significantly improve its binding force with the scaffold, thereby achieving a sustained and effective release of VEGF. QKCMP, as a bifunctional peptide, combines QK peptide and CMP structure, wherein QK peptide retains the core function of VEGF, and CMP structure gives QK peptide a strong binding ability with collagen. Collagen and its hydrolyzate gelatin, due to its excellent hydrophilicity, biocompatibility, degradability and low antigenicity, help promote the chemotaxis and adhesion of vascular endothelial cells in the process of angiogenesis. In addition, the method of the present invention can effectively improve the clinical application effect of small-diameter vascular scaffolds. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0027] Figure 1 Prototype preparation process for SDVG;

[0028] Figure 2 are the results of the tensile properties and DMSO evaporation tests, where A is the morphology of SDVG and its diameter, B is the appearance and SEM micrograph of the cross section of the SDVG prototype, C is the mass percentage of DMSO evaporated, and D is the typical tensile stress-strain curve of the SDVG prototype;

[0029] Figure 3 is the stability test result, where A is the cumulative release of PDGF from the PQ and PB components, B is the cumulative release rate of PDGF from the PQ and PB components, C is the protein remaining in the Col-Gel membrane in the SDVG prototype, and D is the rate at which the bound peptide remains on the Col-Gel membrane;

[0030] Figure 4 The growth results of HUVECs on the SDVG model, where A is the dynamic changes of cell growth observed by phase contrast microscopy, B is the evolution of cell growth monitored by fluorescence microscopy, and C is the proliferation of HUVECs in different experimental groups evaluated by CCK-8 method;

[0031] Figure 5The results of cell cycle and apoptosis rate of HUVECs on the SDVG prototype, wherein A and B are the apoptosis rates of HUVECs on the SDVG prototype under the action of four different compositions at a predetermined time point; C and D are the cell cycle conditions of HUVECs on the SDVG prototype under the action of four compositions at the same time point;

[0032] Figure 6 Figure 3 is the differential expression and functional enrichment analysis, where A is the analysis of BB / PB, BB / PQ and PB / PQ differences; B is the histogram of the number of differentially expressed genes (DEGs) in the three groups of differential analysis results; C is the biological pathway enrichment result of differentially expressed genes; D is a pie chart of the number of differentially expressed genes enriched in VEGFA-VEGFR2 signaling pathway and Hippo-Merlin signaling pathway in each group; E is the expression distribution of genes in BB, PQ and PB samples, where blue indicates downregulated expression and red indicates upregulated expression; F is the expression level of genes in BB, PQ and PB groups. DETAILED DESCRIPTION

[0033] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features described in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0034] Example 1

[0035] 1. Experimental Materials

[0036] Poly(lactic-co-glycolic acid) (PLGA, model P1941), gelatin (model 1288485), acetic acid (model W200603), live / dead cell double staining kit (model 04511-1KT-F), insulin (model 1342106), transferrin (model 10652202001), and sodium selenite (model 214485) were purchased from Sigma-Aldrich, Missouri, USA. Animal-free recombinant human platelet-derived growth factor-BB (model ab184860) and its enzyme-linked immunosorbent assay (ELISA) kit (model ab184860), recombinant human vascular endothelial growth factor-165A (model ab9571) and its ELISA kit (model ab273164), BCA protein detection kit (model ab102536), anti-von Willebrand factor antibody (anti-VWF, model ab212657), anti-CD31 antibody (anti-CD31, model ab9498), Phalloidin (model ab176753), CCK-8 kit (model ab228554), and goat anti-mouse IgG H&L (model ab97035) were purchased from Abcam, Cambridge, UK. Dimethyl sulfoxide (DMSO, model D8371), phosphate buffered saline (1×PBS, model P1020), penicillin / streptomycin (P / S, models IP0150 and SS8800), 2-(4-aminophenyl)-6-indoleamidoamine dihydrochloride (DAPI, model C0065), heparin (model YA2490), ascorbic acid (model A8101), N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES, model H1070), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES, model T8350) and fluorescein isothiocyanate (FITC, model F8070) were purchased from Solarbio, Beijing, China. Fetal bovine serum (FBS, model 10100) and trypsin (model R001100) were purchased from Gibco, USA, which is affiliated to Thermo Fisher Scientific Group. Endothelial cell growth medium 2 (EGM-2, model 1001), endothelial cell growth supplement (ECGs, model 1052) and Ham's F-12K (model PM150910) were purchased from Louis Sciencell, USA and Procell, Wuhan, China, respectively. Rat tail collagen type I (model 40125ES50) and annexin V-FITC / PI apoptosis detection kit (model 40302ES60) were purchased from Yease, Shanghai, China. Human umbilical vein endothelial cells (HUVEC, model CTCC-004-0062) and rat aortic smooth muscle cells (T / G HA-VSMC, model CTCC-001-0065) were provided by CTCC, Zhejiang, China. Except for FITC, amino acid and peptide synthesis reagents such as trifluoroacetic acid (TFA), N -methylpyrrolidone (NMP), and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were prepared by the laboratory.

[0037] Preparation of collagen-gelatin combined with VEGF mimetic peptide QKCMP:

[0038] A1) The synthetic sequence of the collagen-gelatin combined VEGF mimetic peptide QKCMP is: "Ac-KLTWQELYQLKYKGI-(GPO) 5 -(KR) 4 -(RGD) 4 -(KR) 4 -GGG-(Gly) 3 -NH 2"Ac represents the N-terminal acetylation protection to prevent the N-terminal degradation of the peptide chain during synthesis. KLTWQELYQLKYKGI is the QK domain, which mimics the biological activity of VEGF and helps promote angiogenesis. (GPO) 5 Mimics the triple helix structure of collagen to enhance the binding force with gelatin. (KR) 4 A repeating sequence of arginine and lysine that helps enhance electrostatic interactions with gelatin. (RGD) 4 Rich in cell adhesion sequences, promoting cell-material interactions. (KR) 4 -GGG reintroduces an arginine- and lysine-rich sequence that enhances binding to gelatin and may provide additional structural stability. (Gly) 3 It is a flexible linker sequence that increases the flexibility of the peptide chain and makes it easier to adapt to the structure of gelatin. 2 It represents C-terminal amidation to improve the stability and biological activity of the peptide chain.

[0039] A2) In the preparation stage, the solid phase peptide synthesis (SPPS) method is selected to synthesize from the N-terminus to the C-terminus. Fmoc-protected amino acids are prepared and an appropriate resin (polystyrene resin) is selected.

[0040] A3) During the synthesis, the resin is first soaked in DMF or NMP to fully swell it. The resin is then treated with a 20% piperidine solution to remove the N-terminal Fmoc protecting group. The amino acid with the Fmoc protecting group is mixed with HBTU and DIPEA to activate the carboxyl group. The activated amino acid is reacted with the amino group on the resin to form a peptide bond. The next amino acid is coupled repeatedly in the sequence until the synthesis is complete. All side chain protecting groups are removed using TFA reagent. The peptide is cleaved from the resin using TFA reagent.

[0041] A4) During the purification process, the crude polypeptide obtained by cleavage is first freeze-dried. Then, a C18 column is used to remove impurities by gradient elution with water and acetonitrile. Further purification is performed using an ion exchange chromatography column to ensure purity.

[0042] A5) During the identification and storage stage, use MALDI-TOF MS and other techniques to identify the molecular weight of the peptide. Analyze the amino acid composition of the peptide. Store the purified peptide in aliquots at -80°C to avoid repeated freezing and thawing.

[0043] 2. SDVG Prototype Production

[0044] according to Figure 1 The fabrication process shown is to prepare the SDVG prototype.

[0045] First, PDGF was mixed with 12 wt% PLGA solution in DMSO at a ratio of 3 ng / mg, and then the mixture was filled into a tubular mold with dimensions of 3.0 mm (inner diameter) × 1.0 mm (thickness) × 60.0 mm (length). Next, PLGA was cured at -20°C for one hour, followed by rinsing with cold deionized water for 30 minutes. Finally, the scaffold was removed from the mold and transferred to a vacuum chamber to allow DMSO to evaporate naturally for 7 days.

[0046] Rat tail type I collagen solution (concentration of 3.34 mg / ml) and 0.02N acetic acid dissolved gelatin solution (concentration of 4 mg / ml) were pre-mixed to form a mixed solution of 50wt% collagen and 50wt% gelatin. Then, the mixed solution was evenly coated on both surfaces of the PLGA scaffold (about 165μl per square centimeter, protein mass 600μg / cm 2 ). For the outer surface, the protein solution was directly coated and dried; for the lumen surface, 350 μl of protein solution was completely injected into the lumen and dried twice to maintain the hollow structure of the scaffold. After drying overnight, the scaffold with the dried protein film was immersed in 1× PBS and neutralized for at least 5 minutes. Afterwards, the neutralized film was air-dried for the subsequent peptide binding step.

[0047] Next, a QKCMP stock solution (molecular weight 4530.01 g / mol, purity 92%) with a concentration of 340 μM was prepared, heated to 80°C and maintained for at least 30 min, and then rapidly cooled to 37°C. The peptide solution was completely injected into the lumen of the stent (approximately 350 μl, concentration 25 nmol / cm 2 , equivalent to 100 μM in vitro), and sealed at both ends. Incubate overnight at 37°C to promote peptide binding. Subsequently, the scaffold was incubated again in 1× PBS at 37°C for 30 minutes to remove unbound peptide. Finally, the finished product was cut into 50.0 mm long sections and replaced with 1× PBS every two days for temporary storage.

[0048] For comparative analysis, we divided the scaffold samples into four groups: the blank control group (BB group) did not add any growth factors; the PDGF group (PB group) only added PDGF; the QKCMP group (BQ group) only added QKCMP; and the PDGF / QKCMP group (PQ group) added PDGF and QKCMP at the same time.

[0049] 3. SDVG prototype performance test

[0050] 1. Morphological examination

[0051] The surfaces and cross-sections of the four groups of SDVG prototypes were examined using a scanning electron microscope (SEM, model Phenom Pro PW-100-516).

[0052] The final PLGA scaffold morphology is as follows Figure 2 As shown in A, its dimensions are 3.0 mm inner diameter, 1.0 mm thickness, and 50.0 mm length. QKCMP is evenly distributed in the lumen, and the stent presents a hollow structure with intact walls and no obvious collapse in the lumen. Through SEM micrographs, we can observe its microstructure, including surface morphology, cross-sectional characteristics, and porosity (see Figure 2 B).

[0053] 2. Tensile properties and DMSO evaporation test

[0054] The tubular samples (average outer diameter of 5.0 mm, length of 50.0 mm, thickness of 1.0 mm, n = 3) were cut into flat plates along the axial direction and immersed in phosphate buffer solution (PBS) at room temperature for 12 hours before testing. Subsequently, the uniaxial tensile test was performed in a wet environment. The moving speed of the crosshead was set to 5.0 mm per minute and the gauge length was 10.0 mm. The maximum stress value before the sample broke was identified as the tensile strength of the material. The tensile stress-strain curve was plotted based on the experimental results to demonstrate its tensile properties (see Figure 2 D). The measured results of tensile strength, Young's modulus and elongation were 0.44±0.06MPa, 11.47±2.56MPa and 10.30±1.82%, respectively (see Table 1).

[0055] Table 1

[0056] Project Parameter Tensile strength(MPa) 0.44±0.06 Young's modulus (MPa) 11.47±2.56 Elongation(%) 10.30±1.82

[0057] To evaluate the residual mass of dimethyl sulfoxide (DMSO) in the scaffolds (BB group, n = 3), we monitored its mass loss over time in the vacuum chamber. At set time points (i.e., the initial mass of the 12% poly(lactic-co-glycolic acid) (PLGA) / DMSO solution after rinsing with cold deionized water, and on days 1, 3, 5, and 7 in the vacuum chamber), we measured the remaining mass of each scaffold. The mass loss curve after DMSO evaporation revealed that the evaporation amount of DMSO stabilized at about 10% on days 5 and 7 (see Figure 2 C).

[0058] 3. Stability test

[0059] 3.1 In vitro encapsulation efficiency of PDGF

[0060] The actual loading mass of PDGF in the vascular samples (n=3) was determined by the Grinberg method (37), and the encapsulation efficiency was calculated as follows:

[0061] Packaging efficiency (%) = ma / m0 × 100%;

[0062] Where ma and m0 represent the actual loading mass and theoretical loading mass of PDGF in PLGA scaffolds, respectively.

[0063] 3.2 In vitro release behavior of PDGF

[0064] 20 mg of scaffold samples (PB and PQ, n = 3 each) were immersed in 2.0 mL of 1× PBS and incubated in a shaking water bath at 37°C. 1.0 mL of 1× PBS was taken out for detection at the predetermined time points (1, 2, 4, 7, 10, 14, 21, 28, 35, 42, 49 and 56 days) and supplemented with 1.0 mL of 1× PBS. The release of PDGF in the samples was detected using an ELISA kit. The cumulative release rate was calculated as follows:

[0065] Cumulative release rate (%) = ∑mx / ma × 100%;

[0066] Wherein, ∑mx and ma are the cumulative and actual loading amounts of PDGF in the PLGA scaffold, respectively.

[0067] 3.3 Stability of Col-Gel membrane

[0068] To evaluate the stability of the gel membrane, we examined the stability of the Col-Gel membrane by measuring the loss of protein mass on the scaffold over time. First, the BB scaffold was cut into a disc shape with a diameter of 1.5 cm (n=3), and then 165 μl of protein solution (equivalent to the amount on 1.0 square centimeter of the scaffold) was applied to its surface and allowed to air dry. Next, 1500 μl of 1× PBS was used in each sample well for neutralization, and all PBS washes were collected. After that, 500 μl of deionized water was added and incubated at 37°C overnight. The next day, after removing the deionized water, 500 μl of 1× PBS was added and incubated at 37°C for another 30 minutes. This process is designed to simulate the steps taken during the QKCMP binding process. Subsequently, the PBS solution in the well was collected and replaced every other day. The mass loss of protein in each collected solution was detected using the BCA protein quantification kit, and the calculation formula is as follows:

[0069] Residual mass (%) = (m0-∑md) / m0×100%;

[0070] Where m0 is the initial mass of the Col-Gel membrane, and ∑md is the cumulative mass loss after degradation for a predetermined time.

[0071] 3.4 Comparison of the stability of QKCMP / VEGF binding to Col-Gel membrane

[0072] In the field of fluorescence detection, this study used FITC-labeled QKCMP (molecular weight 5161.67 g / mol, purity 90%), i.e., FITC-QKCMP, which has fluorescein isothiocyanate (FITC) linked to the N-terminus. First, 330 μl of gel solution (n=3) was added to each well of a 24-well plate to form a gel membrane, followed by air drying and neutralization. Next, FITC-QKCMP was added to the Col-Gel membrane at 500 μl / well (about 25 nmol / cm2) and incubated overnight at 37°C. On the second day, the peptide solution was collected from each well and replaced with 500 μl of 1× PBS, and then incubated at 37°C for 30 minutes. Thereafter, the PBS solution in the well was replaced every other day, and the old solution was collected and fresh PBS was added each time it was replaced. The concentration of FITC-QKCMP was determined by a simulated ELISA method. Meanwhile, separate wells containing known concentrations of FITC-QKCMP (128, 64, 32, 16, 8, 4, 2, 0 μm, respectively) were set up as standards for the calibration curve. The excitation wavelength used in the experiment was 490 nm.

[0073] 3.5 Experimental Results

[0074] Table 2 shows the actual loading mass and packaging efficiency of PDGF in PB and PQ. Figure 3 A and B reveal the slow and sustained release characteristics of PDGF. Over a time span of 8 weeks, the average cumulative release of PDGF in PB and PQ reached 6.15±0.41ng and 4.70±0.60ng, respectively, and the corresponding cumulative release rates were 52.86±3.55% and 53.88±6.84%, respectively. In the SDVG prototype, the Col-Gel membrane experienced the greatest mass loss in the initial stage (first day), and then maintained a relatively stable state ( Figure 3 C). VEGF-165A cultured in Col-Gel membranes was rapidly cleared within the first three days. In contrast, FITC-QKCMP was preserved longer, persisting until day 5, and almost ceased release ( Figure 3 D).

[0075] Table 2

[0076] Actual loading PB PQ Theoretical loading(ng) 11.64±1.05 8.72±0.3 Encapsulation efficiency (%) 60.00 60.00 Cumulative release in 8 weeks (ng) 19.39±1.74 14.53±0.50 Cumulativereleaseratein8weeks(%) 6.15±0.41 4.70±0.60 Actual loading 52.86±3.55 53.88±6.84

[0077] 4. Experimental study on the effect of vascular stents on HUVEC cell proliferation and adhesion

[0078] 4.1 Cell culture

[0079] HUVEC cells were purchased from CTCC in Zhejiang, China (CTCC-004-0062). Four types of vascular stents (BB, BQ, PB, PQ) were cut into 6 mm diameter discs, sterilized by radiation, and placed in Transwell plates according to the grouping principle, with three copies per group, and the lumen of the stent facing down. HUVECs were cultured at 1.0×10 per square centimeter. 4 The cells were seeded at a density of 100 cells / mL in the lower chamber of a Transwell plate, which is a 24-well plate with a pore size of 0.4 μm (Corning, product number CLS3379, located in New York, USA). After the cells were seeded, they were cultured in EGM-2 medium containing 5% fetal bovine serum (FBS), 0.1 mg / mL heparin, 0.03 mg / mL endothelial cell growth factor (ECGs) and 1% antibiotic / antimycotic (P / S). At the same time, VSMCs were seeded in the corresponding Transwell plate at the same cell density and cultured in Ham's F12K medium containing 5% FBS, 0.05 mg / mL ascorbic acid, 0.03 mg / mL insulin, 0.01 mg / mL transferrin, 10 ng / mL sodium selenite, 0.03 mg / mL ECGs, 10 mM HEPES, 10 mM TES and 1% P / S. After the two cells were seeded, a 1:1 ratio of mixed culture medium was used to establish a HUVEC / VSMC co-culture system. Since the pore size of the insert is 0.4 μm, cells cannot pass through. The Transwell co-culture system is maintained in a 5% CO 2 , cultured in a humid environment at 37°C.

[0080] 4.2 Detection of HUVECs proliferation by CCK-8 method

[0081] 1.0 ml of EGM-2 medium containing 5% fetal bovine serum (FBS), 0.1 mg / ml heparin, 0.03 mg / ml endothelial cell growth supplement (ECGs) and 1% antibiotic / antimycotic (P / S) was added to each well of a 24-well plate. As a control, a test group (n=3) was set up in this experiment, in which 100 micromol / L (QKCMP) was added. Subsequently, 100 umbilical vein endothelial cells (HUVEC) were seeded in each well and incubated at 5% CO. 2 The cells were cultured in a humidified environment at 37°C. At predetermined time points (days 1, 3, 5, 7, 10, and 14), the CCK-8 assay was performed. The cell proliferation rate of HUVECs was determined by measuring the optical density (OD) value at a wavelength of 450 nm.

[0082] 4.3 Observation of HUVECs cell number under microscope

[0083] At predetermined time intervals (1 day, 2 days, 3 days, 5 days, 7 days, and 14 days), human umbilical vein endothelial cells (HUVECs) were observed by phase contrast microscopy and fluorescence microscopy. Anti-CD31 and anti-vWF antibodies were used for immunofluorescence staining to identify cells. In the photos taken by fluorescence microscopy, cell nuclei were stained with DAPI for cell counting.

[0084] 4.4 Flow cytometry

[0085] By flow cytometry, we were able to determine the apoptotic rate and the distribution of each phase of the cell cycle (G1, S, and G2). At set time points (1, 2, 3, 5, 7, and 14 days, respectively), we collected appropriate amounts of HUVECs from the corresponding culture wells and analyzed the HUVECs using a FACScan flow cytometer (Sysmex Partec Cube 6, from Kobe, Japan) for analysis.

[0086] 4.5 Cell Adhesion Assay

[0087] Fibronectin was added to the 24-well plate at a concentration of 10 μg / ml for coating for 1 hour, and then the coating solution was removed. Next, 3% BSA solution was added and sealed at 37°C for 2 hours. 4 The cells were seeded into 24-well plates at a density of 10 cells, and scaffolds of different treatment groups (BB, PB, BQ and PQ groups) were added. The cells were placed in an incubator at 37°C and 5% CO2 for 1 day, 2 days, 3 days, 5 days, 7 days and 14 days. After the culture, the cells were washed three times with PBS, and the cell number was finally detected by CCK-8 method.

[0088] 4.6 Experimental Results

[0089] From the phase contrast micrographs, it can be observed that the density of HUVECs in various compositions increased (Figure 4A). This trend was further confirmed by DAPI nuclei counting and quantitative growth curves in fluorescence micrographs (Figure 4B). Compared with BB and PB components, BQ and PQ had a faster growth trend in the early stage, especially on days 3, 5, and 7. The phenotypic expression of endothelial differentiation markers CD31 and vWF was detected by immunofluorescence, and the results showed that both CD31 and vWF showed strong fluorescence intensity ( Figure 4C). However, at subsequent stages, there was no significant difference in growth between the two components. Throughout the two-week experiment, HUVECs in both the experimental and control groups continued to grow and eventually reached comparable OD values. Nevertheless, QKCMP-stimulated HUVECs showed a significantly greater and faster increase in OD values, especially from day 3 to day 7 ( Figure 4 D). In addition, the results of the cell adhesion experiment showed that under the stimulation of QKCMP, the cell adhesion of HUVEC increased significantly, and the cell adhesion of HUVEC in the PQ group was significantly better than that in the PB and BQ groups ( Figure 4 E).

[0090] In terms of apoptosis rate, the PQ component maintained the lowest level in all intervals. In contrast, QKCMP or PDGF alone did not seem to show a more significant effect than the blank control on the BQ and PB components (see Figure 5 A, B). As for the cell cycle distribution, it was observed that in the early stages of the PQ and BQ compositions, the number of cells in the G2 and S phases was higher (see Figure 5 C,D).

[0091] 5. RNA-seq analysis

[0092] On the third day, we collected an appropriate amount of HUVECs from the lower layer of the Transwell plate. These cells were divided into three groups: BB, PB, and PQ, each with three replicates. Next, we performed RNA-seq analysis with the goal of revealing potential biological differences through the identification of differentially expressed genes (DEGs). To further explore the biological functions or signaling pathways involved in these DEGs, we utilized the web-based analysis tool Metascape (access URL: https: / / Metascape.org / gp / index.html# / main / step1). In addition, using the ggplot2 package in R language, we compared the mRNA expression levels of key genes involved in relevant signaling pathways in the samples.

[0093] In the differential analysis of the three groups of samples, BB / PB, BB / PQ, and PB / PQ, we identified 176, 1075, and 1086 differentially expressed genes (DEGs), respectively. Figure 6 A and B. Through enrichment analysis, we further determined the biological functions or pathways involved in these DEGs. The results are shown in Figure 6C. Among these signaling pathways, the VEGFA-VEGFR2 signaling pathway and the Hippo-Merlin signaling pathway attracted our special attention. The pie chart reveals the number of genes enriched in the VEGFA-VEGFR2 signaling pathway or the Hippo-Merlin signaling pathway in each group of DEGs. Overall, the number of DEGs enriched in the PB / PQ and BB / PQ groups is relatively large (see Figure 6 D). In BB, PQ, and PB samples, we compared the expression levels of 24 DEGs associated with the Hippo-Merlin signaling pathway. Of particular note, HRAS and RBX1 showed significant upregulation in the PQ group (see Figure 6 E). Next, we compared the expression levels of HRAS and RBX1 among the three sample groups, and the results revealed significant differential expression between BB and PQ, whereas no such difference was observed between BB and PB (see Figure 6 F).

[0094] IV. Conclusion and Discussion

[0095] The rapid endothelialization process mediated by VEGF has the potential to significantly reduce the risk of early thrombosis after SDVG implantation. However, the current connection stability of VEGF on the stent is insufficient, which limits its potential in SDVG applications. Therefore, it becomes crucial to chemically modify VEGF to maintain its biological function and achieve a tight connection with the stent. In order to meet these two conditions at the same time, this study designed an SDVG prototype using QKCMP for the first time and tested its performance through multiple methods.

[0096] The VEGF-mimicking QK domain was originally synthesized by D'Andrea and subsequently characterized by Chan. The domain contains a repeating CMP domain, a proline-glycine peptide structure that shows a strong affinity for collagen binding. Their results showed that QKCMP was able to activate endothelial cell (ECs) proliferation and angiogenesis in two-dimensional (2D) and three-dimensional (3D) wound healing models. However, due to the short and intermittent nature of their experiments, it was necessary to revalidate the function of QKCMP in rapid endothelialization through continuous and full-time evaluation. Therefore, we used 3D printing technology to fabricate a SDVG prototype. 3D printing technology can effectively predict arterial deformation caused by the insertion of endovascular materials, which can help improve surgical planning, prevent complications, and reduce material waste. Based on this technology, the SDVG prototype we designed received good evaluation in terms of performance. Next, this study established a transwell co-culture system for two weeks in vitro culture and performed cell proliferation and apoptosis assays. The results showed that over time, the growth rate of human umbilical vein endothelial cells (HUVECs) in the PQ group was significantly higher than that in the other three groups, while the apoptosis rate was significantly lower than that in the other three groups. This trend was particularly evident from day 3 to day 7. These results confirm that QKCMP plays a beneficial role in promoting rapid endothelialization of HUVECs. This evidence suggests that inserting SDVG containing QKCMP into blood vessels can prevent the formation of thrombi and reduce the risk of treatment complications in patients.

[0097] To further explore how the QKCMP-designed SDVG prototype specifically promotes the endothelialization of HUVECs, we performed RNA-seq analysis, aiming to reveal the underlying signaling mechanisms at the RNA level. The analysis revealed two significant signaling pathways: the VEGFA-VEGFR2 signaling pathway and the Hippo signaling pathway. In fact, the proliferation and differentiation of HUVECs are in response to a diversity of extracellular signals, which mainly include chemical signals such as growth factors, as well as mechanical signals related to cell contact inhibition. Among the many growth factors, VEGF is a key factor and is involved in the formation of a new PI3K-AKT signaling axis. This signaling axis plays a vital role in regulating a variety of biological processes, involving cell proliferation, growth, DNA repair, angiogenesis, and self-renewal of stem cells. As for mechanical signals, the Hippo signaling pathway is believed to integrate signals from spatial organization and the physical state of neighboring cells. When activated, the Hippo signaling pathway phosphorylates YAP / TAZ proteins through MST / LATS kinases, triggering the recruitment of 14-3-3 proteins, which in turn leads to protein retention or degradation in the cytoplasm. Typically, these extracellular signals maintain the balance of cellular processes through crosstalk, and any imbalance may cause cell overgrowth or even malignant transformation. For example, in endothelial cells, YAP / TAZ activation is closely associated with the VEGF-PI3K-AKT and MEK-ERK signaling pathways, leading to the inhibition of MST and LATS kinases. Conversely, the loss of YAP / TAZ significantly attenuated the effect of VEGF.

[0098] RNA sequencing provides an opportunity to fundamentally reveal the molecular mechanisms by which QKCMP promotes HUVECs. In our results, both the VEGFA-VEGFR2 and Hippo signaling pathways were found to be responsive to QKCMP in our HUVECs, indicating that it works in two key ways: I) stimulating the VEGFA-VEGFR2 signaling pathway; II) activating YAP / TAZ by inhibiting the Hippo-MST / LATS pathway and enhancing the PI3K-AKT pathway. Our study confirmed the promotion of HUVECs growth by OKCMP, especially in the early stages of HUVEC growth. In addition, two important genes associated with dysregulation of Hippo-Merlin signaling were identified: HRAS and RBX1. They are mutated or deleted in a variety of cancers and are significantly associated with poor cancer prognosis.

[0099] It is particularly important that QKCMP exhibits compelling potential and unique advantages in SDVG scaffolds. First, it is able to stimulate the growth of HUVECs in a manner similar to VEGF. Second, it exerts a negative regulatory effect on the Hippo-YAP / TAZ signaling pathway. Most importantly, it shows extremely high affinity for synthetic scaffolds, ensuring their stability and resistance to easy removal. Although QKCMP showed excellent performance under our experimental conditions, we must recognize that the microenvironment of SDVGs implanted in vivo is undoubtedly more complex. This complexity involves potential interactions and interferences with other signals. Therefore, in order to provide a reliable reference for clinical application, a large amount of data needs to be obtained through animal experiments. However, before such evaluations can be carried out, improvements must be made to the SDVG prototype, including standardization of mechanical manufacturing, optimization of PDGF encapsulation, and degradation of PLGA. In addition, in addition to studying the effects of QKCMP on the endothelial function of HUVECs, other cell types, such as smooth muscle cells and fibroblasts, should also be considered to evaluate the biocompatibility of QKCMP. Ultimately, the scalability, cost-effectiveness, and legal regulations of QKCMP-coated stents should be considered during the research process to address the challenges that may be encountered in practical applications.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a SDVG prototype scaffold, characterized in that: include: The PDGF and PLGA solutions are mixed in DMSO to obtain a mixture, the mixture is filled into a tubular mold and then solidified, then rinsed with cold deionized water, and finally transferred into a vacuum chamber to naturally evaporate the DMSO to obtain a PLGA scaffold; Premixing a gelatin solution dissolved in acetic acid with a collagen solution to form a collagen-gelatin mixed solution; uniformly coating the collagen-gelatin mixed solution on the lumen surface and the outer surface of the PLGA scaffold to obtain a PLGA scaffold with a dry protein film; A QKCMP stock solution was prepared, heated to 80±5°C and maintained for at least 30 minutes, and then cooled to 37±2°C to obtain a peptide solution; the peptide solution was completely injected into the lumen of the PLGA scaffold with a dried protein film and sealed at both ends for incubation, followed by incubation again in PBS to obtain the SDVG prototype scaffold.

2. The method for preparing the SDVG prototype bracket according to claim 1, characterized in that: The temperature for curing after the mixture was filled into the tubular mold was -20°C.

3. The method for preparing the SDVG prototype bracket according to claim 1, characterized in that: The collagen solution is rat tail type I collagen solution.

4. The method for preparing the SDVG prototype bracket according to claim 1, characterized in that: The method comprises: uniformly coating the collagen-gelatin mixed solution on the lumen surface and the outer surface of the PLGA scaffold; directly coating the collagen-gelatin mixed solution on the outer surface of the PLGA scaffold and performing a drying process; The collagen-gelatin mixed solution was completely injected into the lumen of the PLGA scaffold and dried, and this was repeated at least twice.

5. The method for preparing the SDVG prototype bracket according to claim 1, characterized in that: The collagen-gelatin mixed solution is uniformly coated on the lumen surface and the outer surface of the PLGA scaffold and then dried. The PLGA scaffold with the dried protein film is immersed in PBS for neutralization, and the neutralized film is air-dried to obtain a PLGA scaffold with a dried protein film.

6. The method for preparing the SDVG prototype bracket according to claim 1, characterized in that: The preparation method of the QKCMP stock solution comprises: After the resin is soaked and expanded in DMF or NMP, it is treated with a 20% piperidine solution to remove the N-terminal Fmoc protecting group; The amino acids with Fmoc protection groups were mixed with HBTU and DIPEA to activate the carboxyl groups; In the direction of synthesis from the N-terminus to the C-terminus, the activated amino acid reacts with the amino group on the resin to form a peptide bond; repeating the steps to couple the next amino acid according to the sequence described in claim 1 until the synthesis is completed; After the synthesis is completed, all side chain protecting groups are removed and the peptide is cleaved from the resin to obtain the collagen-gelatin-bound VEGF mimetic peptide, namely, QKCMP stock solution.

7. The method for preparing the SDVG prototype bracket according to claim 1, characterized in that: The incubation temperature is 37±2°C.

8. A SDVG prototype bracket, characterized in that: The SDVG prototype bracket is prepared according to the preparation method according to any one of claims 1-7.

9. Use of the SDVG prototype stent according to claim 8 in a vascular stent.

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