Artificial blood vessel with functions of resisting inflammation and promoting endothelialization as well as preparation method and application of artificial blood vessel

Abstract

The invention belongs to the field of artificial blood vessels, and particularly relates to an artificial blood vessel with functions of resisting inflammation and promoting endothelialization as well as a preparation method and application of the artificial blood vessel. The artificial blood vessel comprises a matrix material and FTY720. The FTY720-loaded artificial blood vessel provided by the technical scheme of the invention needs to have good anti-inflammatory performance and good endothelialization promotion performance after being implanted in vivo, and long-term smoothness can be realized through combination of the anti-inflammatory performance and the endothelialization promotion performance; according to the technical scheme, the artificial blood vessel does not need to be processed in multiple steps, operation steps are simple, and preparation time and economic cost are reduced.

Description

Technical Field

[0001] The present invention belongs to the field of artificial blood vessels, and in particular relates to an artificial blood vessel having both anti-inflammatory and endothelialization promoting functions, and a preparation method and application thereof. Background Art

[0002] The number of deaths from cardiovascular diseases worldwide has reached about 30% of the total number of deaths, and cardiovascular diseases have become the number one killer of human health. The occurrence of cardiovascular diseases is often due to stenosis or blockage of blood vessels, which leads to reduced blood flow and lack of nutrients, thus damaging tissues or organs, usually manifested as coronary heart disease, cerebrovascular disease, and peripheral arterial disease. Autologous blood vessels are the most ideal vascular substitutes, but the use of autologous blood vessels is limited by limited sources, risk of infection at the donor site, and size mismatch. Therefore, in most cases, artificial vascular substitute materials are needed for vascular transplantation.

[0003] For large-caliber blood vessels (caliber ≥ 6 mm), although polyethylene terephthalate has been used clinically Expanded polytetrafluoroethylene (ePTFE, ) and other materials, but the current products do not have regenerative ability and cannot achieve endothelial and smooth muscle regeneration. There is always a problem of long-term chronic inflammation after graft implantation. Clinically, operations such as coronary artery bypass grafting, peripheral vascular replacement below the knee, and liver / kidney organ transplantation all require the use of small-caliber (caliber <6mm) blood vessels, but there are currently no small-caliber artificial blood vessel products for medical use. After small-caliber artificial blood vessels are implanted in the body, problems such as thrombosis, intimal hyperplasia, and calcification often occur, leading to transplant failure.

[0004] Current artificial blood vessels can achieve inflammatory regulation in the artificial blood vessel regeneration process by loading anti-inflammatory drugs such as rapamycin and aspirin, but they do not have the ability to promote tissue regeneration such as artificial blood vessel endothelialization. Some artificial blood vessels can promote endothelialization by loading CD133, VEGF, TPS, etc., but these molecules do not have the function of regulating inflammation. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an artificial blood vessel having both anti-inflammatory and endothelialization promoting functions and a preparation method and application thereof.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] An artificial blood vessel with both anti-inflammatory and endothelialization promoting functions, comprising a matrix material and FTY720.

[0008] The mass ratio of the matrix material to FTY720 is 1:(2.5×10 -3-1×10 -2 ); preferably 1:5×10 -3 .

[0009] The matrix material is a polymer material for artificial blood vessels; preferably at least one of polycaprolactone, polyglycolic acid, polylactic acid-glycolic acid copolymer, poly (3-hydroxybutyrate-co-4-hydroxybutyrate), polylactic acid, poly L-lactide-caprolactone, and polydioxanone, or a mixture of any proportions of several of them.

[0010] The present invention also includes a method for preparing an artificial blood vessel having both anti-inflammatory and endothelialization promoting functions, comprising the following steps: dissolving a matrix material in an organic solution to form a matrix solution, adding FTY720 powder to the matrix solution to form a mixed solution; and then using a textile method to prepare the mixed solution into an artificial blood vessel loaded with FTY720.

[0011] The organic solvent is a mixture of chloroform and methanol in any proportion; preferably, it is a mixture of chloroform and methanol; preferably, the volume ratio of chloroform to methanol is 5:1.

[0012] The mass concentration of the matrix material in the matrix solution is 0.10 mg / mL-0.30 mg / mL, preferably 0.15 mg / mL.

[0013] The present invention also includes an application of the artificial blood vessel having both anti-inflammatory and endothelialization promoting functions.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The artificial blood vessel loaded with FTY720 provided by the technical solution of the present invention needs to have both good anti-inflammatory properties and good endothelialization-promoting properties after implantation in vivo. The combination of the two properties can achieve long-term patency. The technical solution of the present application does not require multi-step processing of the artificial blood vessel, the operation steps are simple, and the preparation time and economic cost are reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a structural characterization diagram of artificial blood vessels electrospun from PCL loaded with FTY720.

[0017] Figure 2 The cumulative release of FTY720 from electrospun PCL artificial blood vessels loaded with FTY720 within 30 days in vitro;

[0018] Figure 3 The graph shows the blood compatibility of electrospun PCL artificial blood vessels loaded with FTY720 and unloaded with FTY720;

[0019] Figure 4This is an analysis chart of the regeneration of the electrospun PCL artificial blood vessels of Comparative Example 1 and Example 2 implanted in the mouse carotid artery 4 weeks later. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and the best embodiments.

[0021] Example 1

[0022] An artificial blood vessel with both anti-inflammatory and endothelialization functions, comprising a matrix material and FTY720. In the present application, polycaprolactone (PCL) is used as the matrix material for exemplary description. PCL is dissolved in a mixed organic solution of chloroform and methanol, wherein the volume ratio of chloroform to methanol is 5:1. After PCL is completely dissolved, FTY720 powder is added. The final concentration of PCL dissolved in the mixed organic solution is 0.15 mg / mL, and the final concentration of FTY720 dissolved in the mixed organic solution is 4×10 - 6 mol / L, the mass ratio of PCL to FTY720 is 1:1×10 -2 The above solution was used for electrospinning to prepare electrospun PCL artificial blood vessels or electrospun membranes loaded with FTY720, named PFTY100, which were sterilized overnight under ultraviolet light for later use.

[0023] Example 2

[0024] The preparation method is the same as that of Example 1, but the final concentration of FTY720 dissolved in the mixed organic solution is 2×10 -6 mol / L, the mass ratio of PCL to FTY720 is 1:5×10 -3 The prepared electrospun PCL artificial blood vessel or electrospun membrane loaded with FTY720 was named PFTY200.

[0025] Example 3

[0026] The preparation method is the same as that of Example 1, but the final concentration of FTY720 dissolved in the mixed organic solution is 1×10 -6 mol / L, the mass ratio of PCL to FTY720 is 1:2.5×10 -3 The prepared electrospun PCL artificial blood vessel or electrospun membrane loaded with FTY720 was named PFTY400.

[0027] Comparative Example 1

[0028] Polycaprolactone (PCL) was used as raw material, and PCL was dissolved in a mixed organic solution of chloroform and methanol, the volume ratio of chloroform to methanol was 5:1, and the final concentration of PCL dissolved in the above mixed organic solution was 0.15 mg / mL; the above PCL solution was used for electrospinning to prepare PCL electrospun artificial blood vessels or PCL electrospun membranes, which were sterilized under UV overnight for use.

[0029] In order to further illustrate the beneficial effects of the present invention, the following tests are performed:

[0030] 1. Structural characterization:

[0031] The artificial blood vessels prepared in Examples 1-3 and Comparative Example 1 were characterized for their structures. The results are shown in Table 1 and Figure 1 As shown, Figure 1 A is a scanning electron microscope (SEM) image, B is a statistical graph of fiber wall thickness, C is a statistical graph of fiber diameter; and D is a statistical graph of pore size.

[0032] Scanning electron microscope (SEM) observations showed that the four groups of vascular materials in Comparative Example 1, Example 1, Example 2 and Example 3 all had the same tubular structure, and there was no statistical difference in the inner diameter and wall thickness of each group of vascular stents (Table 1). SEM of the inner cavity surface of the vascular material showed that the fiber morphology of the four artificial blood vessels in Comparative Example 1, Example 1, Example 2 and Example 3 was good and basically the same, and there was no statistical difference in fiber diameter and pore size (Table 1). These results show that the addition of FTY720 did not affect the electrospinning fiber process.

[0033] Table 1 Structural characterization of electrospun PCL artificial blood vessels loaded with FTY720 and unloaded FTY720

[0034]

[0035]

[0036] 2. FTY720 release detection:

[0037] The ultraviolet-visible spectrophotometer (UV-3101pc spectrometer) showed that the vascular grafts of Examples 1-3 exhibited sustained release of FTY720, which was evaluated in vitro for up to 12 days. The cumulative release of FTY720 of Examples 1-3 on the 12th day was 111.31±2.33μg, 67.13±2.69μg and 28.64±3.90μg, respectively. The release of FTY720 increased slightly from 12 days to 30 days ( Figure 2 ).

[0038] 3. Blood compatibility performance test

[0039] The blood compatibility of the artificial blood vessels prepared in Examples 1-3 and Comparative Example 1 was characterized. The results are shown in the attached figure. Figure 3 shown.

[0040] Figure 3 (AC) Statistical analysis of PT (A), APTT (B) and TT (C) of each group of vascular materials; (D) Statistical analysis of platelet activation based on P-selectin ELISA of each group of vascular materials; (E) Statistical analysis of hemolysis rate of each group of vascular materials.

[0041] Coagulation test analysis showed that PT, APTT, TT and fibrinogen adsorption were not affected by FTY720 loading ( Figure 3 AC). Based on the p-selectin ELISA, it was confirmed that compared with Comparative Example 1, the three groups of Example 1, Example 2 and Example 3 did not affect the activation of platelets ( Figure 3 D). The analysis of the hemolysis rate showed that there was no significant difference in the hemolysis rate among the four groups of Comparative Example 1, Example 1, Example 2 and Example 3 ( Figure 3 E). These results indicate that the loading of FTY720 does not affect the in vitro blood compatibility of PCL scaffolds.

[0042] 4. Regulation of endothelial cell function

[0043] In order to evaluate the proliferation of endothelial cells, the proliferation of HUVECs was studied using the Cell Counting Kit-8 (CCK-8, Beyotime Biotechnology, Shanghai, China). The four groups of transplants from Comparative Example 1, Example 1, Example 2, and Example 3 were placed in a 48-well cell culture plate, and HUVECs (about 300 μL, passage 7-9, 3.0×10 4 Cells / mL) were suspended in endothelial cell culture medium and cultured on PCL membranes at 37°C and 5% CO2. The culture medium was replaced every 24 hours. On days 1, 3, and 5, CCK-8 reagent (the ratio of culture medium to CCK-8 was 1:10) was used and incubated for 4 hours. At the predetermined time point, about 100 μL of supernatant was collected from each well and transferred to a 96-well plate. The absorbance was measured at 450 nm using a Bio-Rad microplate reader (iMark, Bio-Rad Laboratories, US). The results are shown in Appendix 2.

[0044] The CCK8 experiment showed that the proliferation of HUVECs in the three groups of transplants in Example 1, Example 2 and Example 3 was improved compared with that in Comparative Example 1, especially on the 3rd and 5th days of culture (Table 2). These results indicate that loading FTY720 can positively regulate the proliferation function of HUVECs, among which the effect of Example 2 group was the best (Table 2).

[0045] In order to evaluate the effect on the migration ability of endothelial cells, the four groups of transplants in Comparative Example 1, Example 1, Example 2 and Example 3 were immersed in 5 mL endothelial cell medium (ECM) at 37°C for 24 hours. The extract was stored at 4°C for later use. HUVECs were inoculated into a 24-well cell culture plate and then covered with the wall. A scratch test was performed using a 1 mL gun tip. After the cells were washed with PBS after scratching, an inverted microscope (Nikon ECLIPSE Ti-U, Tokyo, Japan) was used to collect 0h images immediately. The extracts of the four groups of transplants in Comparative Example 1, Example 1, Example 2 and Example 3 were added to the culture wells. When incubated for 6h and 24h, an inverted microscope was used to photograph the cells that had migrated to the scratch area. The migration rate was calculated using Image-Pro Plus (IPP) software, and the results are shown in Appendix 2. In addition, a Transwell cell migration experiment was performed, and HUVECs were first grown in serum-free ECM for 12 hours and the cells were collected. Subsequently, the membrane materials of Comparative Example 1, Example 1, Example 2 and Example 3 were placed in the lower chamber of the Transwell chamber. 5 Starved HUVECs were resuspended in 200 μL ECM. After 12 h of incubation, the non-migrated cells on the upper chamber side of the chamber were removed with a cotton swab. The cells that migrated to the lower side of the membrane were fixed with 4% paraformaldehyde (PFA) and then stained with 0.1% crystal violet. The stained cells were counted in five different fields of view under a light microscope. Three samples were used for each group, and 5 images were used for each sample to count the number of migrated cells in each group. The results are shown in Supplementary Table 2.

[0046] Through the scratch test and Transwell cell migration test, the quantitative analysis of the HUVECs migration rate and the number of migrating cells of the four groups of transplants in Comparative Example 1, Example 1, Example 2 and Example 3 showed that the three groups of transplants in Example 1, Example 2 and Example 3 all showed a promoting effect on the migration of HUVECs, among which the scaffold in Example 2 had the strongest effect on promoting the migration of HUVECs (Table 2).

[0047] In order to evaluate the in vitro angiogenesis performance of the four groups of grafts in Comparative Example 1, Example 1, Example 2 and Example 3, 100 μL of Matrigel was coated on each well of a 96-well culture plate and incubated at 37°C for 30 min. 4HUVECs were seeded on Matrigel at a density of 10 cells / well, and the ECM extracts of the four groups of transplants from Comparative Example 1, Example 1, Example 2, and Example 3 were added to the wells. After incubation for 6 hours, the formation of tubular structures was observed under an inverted microscope (Nikon ECLIPSE Ti-U, Tokyo, Japan). The number of tubule branches in each high-power field (HPF) was counted using Image J software. The results are shown in Appendix 2.

[0048] Through the tube formation experiment, we conducted a quantitative analysis of the number of HUVECs formed tubes in 6 hours and showed that Example 1, Example 2, and Example 3 all enhanced the tube formation ability of HUVECs compared with Comparative Example 1, among which the scaffold tube formation effect in Example 2 was the most obvious, and the number of small tube branches was the largest (Table 2).

[0049] Table 2 Quantitative analysis of HUVECs proliferation, migration and tube formation

[0050]

[0051]

[0052] 5. Analysis of the regulatory effect of electrospun PCL membrane materials on T cells

[0053] The membrane materials prepared in Examples 1-3 and Comparative Example 1 were analyzed for their regulating effects on lymphocytes. The results are shown in Appendix 3.

[0054] The results showed that by targeting FOXP3 in lymphocytes + Tregs and TBET + Quantitative analysis of the percentage of Th1 cells showed that compared with Comparative Example 1, the FTY720 released by Examples 1, 2 and 3 increased the expression of FOXP3 + TBET + Expression of Th1 cells, in which FTY720 released by Example 2 increased FOXP3 + The expression of Tregs was the most obvious, while the decrease of TBET + The expression of Th1 cells was also the most obvious (Table 3).

[0055] Table 3 Quantitative analysis of the regulation of T cell population by electrospun PCL membrane materials

[0056]

[0057] 6. Analysis of the regulatory effect of electrospun PCL artificial blood vessels on T cells 2 weeks after implantation into the mouse carotid artery

[0058] The artificial blood vessels prepared in Examples 1-3 and Comparative Example 1 were implanted into the carotid arteries of mice for 2 weeks and then T cell regulation was analyzed. The results are shown in Table 4.

[0059] Statistical analysis based on macroscopic images after vascular sampling showed that 2 weeks after implantation, the patency rate of comparative example 1 was 0%, the patency rate of example 1 was 75%, the patency rate of example 2 was 100%, and the patency rate of example 3 was 50%, among which the patency rate of the graft in the example 2 group was the highest (Table 4). To evaluate the effect of FTY720 loading on T cell infiltration, we performed immunofluorescence staining on the cross-section of the graft. Using anti-CD4, anti-TBET and anti-FOXP3 + Antibodies detect CD4 + T cells, TBET + T helper 1 cells (TH1) and FOXP3 + The experimental results show that compared with the comparative example 1, the CD4 + T cell infiltration was reduced, among which CD4 + On the other hand, compared with Comparative Example 1, the TBET observed in the three groups of transplants in Example 1, Example 2 and Example 3 was significantly reduced (Table 4). + The TH1 cell density was reduced, among which the TBET + The density of TH1 cells was significantly reduced (Table 4). At the same time, compared with Comparative Example 1, the FOXP3 + The density of Tregs cells increased, and the FOXP3 + The increase in Tregs1 cell density was more obvious (Table 4).

[0060] Table 4 Patency and cell quantitative analysis of artificial blood vessels implanted in mouse carotid arteries for 2 weeks

[0061]

[0062] 7. Macrophage analysis of electrospun PCL artificial blood vessels implanted in mouse carotid arteries 2 weeks later

[0063] The artificial blood vessels prepared in Examples 1-3 and Comparative Example 1 were implanted into the carotid arteries of mice for 2 weeks and then macrophage analysis was performed. The results are shown in Appendix 5.

[0064] The infiltration and polarization of macrophages were further evaluated to understand the regulatory effect of FTY720 loading on inflammatory response. Immunofluorescence staining was used to detect the expression of CD68, iNOS and CD206, and the total infiltration of macrophages, pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages was evaluated. The experimental results showed that compared with Comparative Example 1, the CD68 in the vascular grafts of Example 1, Example 2 and Example 3 was significantly higher than that of the control group. + The number of macrophages was low, among which CD68 + The number of macrophages was lower, but there was no significant difference among the four groups (Table 5). + The percentage of macrophages was lower than that of comparative example 1, and iNOS + The percentage of macrophages in the three groups of vascular grafts in Example 1, Example 2 and Example 3 was more obvious than that in Comparative Example 1 (Table 5). + The percentage of macrophages was higher, among which CD206 + The percentage of macrophages was significantly higher (Table 5). These results indicate that the Example 2 graft effectively modulates the inflammatory response by promoting the polarization of M2 macrophages, leading them to a regenerative state.

[0065] Table 5 Quantitative analysis of macrophages in mice carotid arteries 2 weeks after implantation of artificial blood vessels

[0066]

[0067] 8. Evaluation of implanted blood vessel regeneration in mice

[0068] The vascular materials prepared in Comparative Example 1 and Examples 1-3 were implanted into the carotid arteries of mice. One month after the transplantation, the graft of Comparative Example 1 was occluded and had no blood flow, while the blood vessels of Examples 1, 2 and 3 were all unobstructed.

[0069] To evaluate the effect of loading FTY720 on endothelial cell regeneration, we performed statistical analysis on the endothelial cell coverage of the grafts, and the results are shown in Supplementary Table 6.

[0070] The results showed that the comparative example 1 had no endothelial coverage, while the grafts of examples 1 and 3 had endothelial coverage, but the coverage was not as high as that of the graft of example 2 (Table 6).

[0071] Table 6 Analysis of endothelial cell coverage

[0072]

[0073] In addition, we also focused on evaluating the endothelial and smooth muscle regeneration of the grafts of Comparative Example 1 and Example 2. The results are shown in the attached Figure 4 shown.

[0074] Figure 4 (A) SEM micrographs of the lumen of the grafts of Comparative Example 1 and Example 2; (B) CD31 antibody immunofluorescence staining analysis of endothelial cell regeneration; (C) CD31 antibody immunofluorescence staining analysis of endothelial cell capillary formation; (D) Representative α-SMA of PCL and Example 2 grafts 4 weeks after transplantation + Cross-sectional analysis of SMCs antibody immunofluorescence staining.

[0075] In order to evaluate the effect of loading FTY720 on endothelial cell regeneration, we performed scanning electron microscopy on the grafts. SEM observation showed that the lumen of the comparative example 1 was almost not covered by endothelial cells, but presented a large amount of coagulation matrix. The lumen of the graft of Example 2 was covered by a layer of fused endothelial cells, which were arranged along the direction of blood flow and had a cobblestone-like morphology ( Figure 4 A) CD31 + Immunofluorescence staining further confirmed that compared with Comparative Example 1, the endothelial coverage rate of the vascular material lumen in Example 2 was higher ( Figure 4 B). At the same time, compared with Comparative Example 1, the graft of Example 2 showed a higher capillary formation density ( Figure 4 C) In addition, compared with Comparative Example 1, the graft of Example 2 showed complete and continuous α-SMA + The lack of SMCs regeneration is more obvious ( Figure 4 D) The results show that the graft of Example 2 can effectively promote endothelialization.

[0076] Based on the above in vivo / in vitro results, the FTY720-loaded artificial blood vessels prepared by the present invention have both anti-inflammatory and endothelial regeneration-promoting properties.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An artificial blood vessel with both anti-inflammatory and endothelialization promoting functions, characterized in that: Includes matrix material and FTY720.

2. The artificial blood vessel having both anti-inflammatory and endothelialization promoting functions according to claim 1, characterized in that: The mass ratio of the matrix material to FTY720 is 1:(2.5×10 -3 -1×10 -2 ); preferably 1:5×10 -3 .

3. The artificial blood vessel having both anti-inflammatory and endothelialization promoting functions according to claim 1, characterized in that: The matrix material is a polymer material for artificial blood vessels; preferably at least one of polycaprolactone, polyglycolic acid, polylactic acid-glycolic acid copolymer, poly (3-hydroxybutyrate-co-4-hydroxybutyrate), polylactic acid, poly L-lactide-caprolactone, and polydioxanone, or a mixture of any proportions of several of them.

4. A method for preparing an artificial blood vessel having both anti-inflammatory and endothelialization promoting functions according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: dissolving the matrix material in an organic solution to form a matrix solution, adding FTY720 powder into the matrix solution to form a mixed solution; and then using a weaving method to prepare the mixed solution into an artificial blood vessel loaded with FTY720.

5. The preparation method according to claim 4, characterized in that: The organic solvent is a mixture of chloroform and methanol in any proportion; preferably, it is a mixture of chloroform and methanol; preferably, the volume ratio of chloroform to methanol is 5:

1.

6. The preparation method according to claim 4, characterized in that: The mass concentration of the matrix material in the matrix solution is 0.10 mg / mL-0.30 mg / mL, preferably 0.15 mg / mL.

7. Use of an artificial blood vessel having both anti-inflammatory and endothelialization promoting functions as claimed in any one of claims 1 to 3.

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