Method for promoting endothelialization of heart valve and artificial heart valve

Abstract

The invention discloses a method for promoting endothelialization of a heart valve and an artificial heart valve.The method is characterized in that circular DNA is modified on the surface of a decellularized valve (DHV), DNA hydrogel of targeted thrombin is obtained on the surface of the DHV through rolling circle amplification, the DNA hydrogel plays a role of a heme catcher while playing a role of the heme catcher, and heme is recruited from blood; heme is further converted into biliverdin (BV) through heme oxygenase (HO1) loaded in DNA, so that continuous production of biliverdin is ensured, anti-inflammatory immune regulation and active oxygen removal are promoted, a regenerative immune microenvironment is created, and endothelialization of the valve is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of artificial heart valves, in particular to a method for promoting endothelialization of heart valves and an artificial heart valve. Background Art

[0002] Heart valve disease is a cardiovascular disease with high morbidity and mortality. Currently, the most effective treatment for heart valve disease is valve replacement surgery. It is estimated that 850,000 valve replacements will be required worldwide each year by 2050. Bioprosthetic valves are one of the most commonly used valve substitutes in clinical practice. They have good blood compatibility and do not require long-term anticoagulation therapy, but their service life is limited. Patients often need reoperation due to bioprosthetic valve degradation and calcification.

[0003] At present, the main processing technology for biological valves is glutaraldehyde cross-linking. Although this cross-linking method effectively improves the stability of biological valves, it shows obvious cytotoxicity, which is difficult to meet the needs of cell adhesion and proliferation, and is not conducive to the endothelialization of stent materials. However, valve endothelialization is generally considered to be a prerequisite for achieving long-term anticoagulation, anti-calcification, and maintaining long-term functional stability of the valve.

[0004] Therefore, developing and exploring new valve cross-linking strategies to avoid the toxic side effects of glutaraldehyde while improving the structural stability, blood compatibility and long-term functional stability of heart valves is of great significance to scientific research and the development of related industrial fields. Summary of the invention

[0005] The present invention aims to provide a method for promoting endothelialization of heart valves, which is used to promote endothelialization of heart valves.

[0006] In view of this, the scheme of the present invention is:

[0007] The first aspect of the present invention is to provide a method for modifying a heart valve to promote endothelialization, the steps comprising:

[0008] Decellularization of heart valves;

[0009] The surface of the decellularized valve is chemically modified, wherein the modification process is based on the free radical polymerization reaction of unsaturated monomers to introduce zwitterions and thiol groups;

[0010] The circular DNA is modified by a maleimide group, and then reacted with the thiol group on the chemically modified decellularized valve to obtain the decellularized valve modified by the circular DNA; the cDNA includes circular DNA-1 and circular DNA-2; the circular DNA-1 is a circular DNA encoded by an antithrombin active nucleotide sequence, and the circular DNA-2 contains a DNA fragment complementary to the circular DNA-1;

[0011] After circular DNA modification, the decellularized valve is placed in an RCA reaction solution containing HO1 for rolling circle amplification to obtain a heart valve modified with a DNA hydrogel loaded with HO1, i.e., a heart valve that promotes endothelialization.

[0012] Furthermore, the steps of chemical modification of the surface of the decellularized valve include:

[0013] A1) using unsaturated aldehyde to react with amino groups on the surface of the decellularized valve to obtain a decellularized valve with unsaturated surface modification;

[0014] A2) cross-linking the surface of the unsaturated modified decellular valve with a polymer to obtain a chemically modified decellular valve; the polymer is obtained by free radical polymerization based on monomer one, monomer two and a water-soluble initiator; monomer one is an unsaturated zwitterionic compound monomer, and monomer two is an unsaturated thiol.

[0015] Furthermore, during the free radical polymerization process, the polymer:

[0016] The monomer 1 is selected from one of N-(4-aminobutyl)-N-(methacryloyloxyethyl)-N,N,N-trimethylammonium chloride, sulfobetaine methacrylate, 2-methacryloyloxyethyl phosphorylcholine, and carboxybetaine methacrylate;

[0017] The second monomer is selected from one of allyl mercaptan and α-acrylate-ω-mercaptopolyethylene glycol;

[0018] The water-soluble initiator is selected from persulfate, azobisisoheptanenitrile, dimethyl azobisisobutyrate, 2,2'-azobisisobutylamidine dihydrochloride, Vazo TM at least one of a free radical initiator;

[0019] The crosslinking agent used in the polymer crosslinking process is N'N-methylenebisacrylamide.

[0020] Furthermore, the circular DNA-1 is synthesized from ssDNA targeting thrombin.

[0021] Preferably, the ssDNA is a single-stranded DNA containing a thrombin-specific DNA aptamer NU172.

[0022] Preferably, the nucleotide sequence of the ssDNA is as shown in SEQ ID NO: 1; the synthesis process comprises using a primer and a ligase, and the nucleotide sequence of the primer is as shown in SEQ ID NO: 2.

[0023] Furthermore, the circular DNA-2 synthesis process is synthesized based on a random sequence as a template.

[0024] Furthermore, the maleimide group of the circular DNA is modified by base pairing between the primer with maleimide modified at the 5' end and the circular DNA-1 and circular DNA-2.

[0025] The second aspect of the present invention is to provide an artificial biological heart valve constructed by the method described in the first aspect.

[0026] The third aspect of the present invention is to propose the use of the artificial biological heart valve described in the second aspect in the preparation of biological scaffold materials.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The method described in the present invention is based on modifying circular DNA on the surface of DHV, and obtaining a DNA hydrogel targeting thrombin on the surface of DHV through rolling circle amplification. The DNA hydrogel not only exerts an anti-coagulation function, but also plays the role of a heme capturer to recruit heme from the blood; zwitterions are used for DHV modification through a sandwich-like modification strategy, and also give DHV anti-calcification and anti-adhesion properties, which helps to improve the structural stability, blood compatibility and long-term functional stability of the heart valve.

[0029] In the method of the present invention, heme is further converted into biliverdin (BV) by the heme oxygenase (HO1) loaded in DNA, thereby ensuring the continuous production of biliverdin, promoting anti-inflammatory immune regulation and active oxygen scavenging, thereby creating a regenerative immune microenvironment and promoting valve endothelialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the process of enzymatic cross-linking treatment of heart valves described in an embodiment of the present invention.

[0031] Figure 2 The gel imaging analysis results of cDNA-1 and cDNA-2 synthesized in the examples of the present invention are shown.

[0032] Figure 3 The results of DNA / HO1-DHV structure and morphology characterization in the examples of the present invention are shown.

[0033] Figure 4 This is the characterization result of DNA / HO1-DHV components in the examples of the present invention.

[0034] Figure 5 The results of the in vivo blood compatibility characterization of DNA / HO1-DHV in the examples of the present invention are shown.

[0035] Figure 6 This is the histological staining result 28 days after the DNA / HO1-DHV was transplanted into the abdominal aorta in the example of the present invention.

[0036] Figure 7 This is the result of staining analysis of macrophage phenotype 28 days after DNA / HO1-DHV was transplanted into abdominal aorta in the example of the present invention.

[0037] Figure 8 This is the staining analysis result of type I collagen, endothelial cells and interstitial cells 28 days after the DNA / HO1-DHV was transplanted into the abdominal aorta in the example of the present invention. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] In one embodiment, in order to address the needs of anticoagulation and endothelialization of existing valve substitutes, in the present invention, a strategy for constructing a biosynthetic factory on the valve surface is proposed, which can continuously convert the abundant heme in the blood into an immunomodulator (bililiverdin), thereby supporting long-term immunoregulation and tissue regeneration. To achieve this goal, circular DNA (cDNA) is modified to the surface of DHV, and a DNA hydrogel loaded with heme oxygenase (HO1) is obtained on the surface of DHV by rolling circle amplification. The anticoagulant NU172 aptamer encoded in the DNA hydrogel not only plays an anticoagulant function, but also plays the role of a heme capturer to recruit heme from the blood. In addition, heme is further converted into biliverdin (BV) by the heme oxygenase (HO1) loaded in the DNA, ensuring the continuous production of biliverdin, promoting anti-inflammatory immunoregulation and reactive oxygen scavenging, thereby creating a regenerative immune microenvironment.

[0040] Specifically, the flow chart is as follows Figure 1 As shown, the steps include:

[0041] 1. Remove tissue cells from the heart valve;

[0042] 2. Chemically modifying the surface of the decellularized valve, wherein the modification process is based on the free radical polymerization reaction of unsaturated monomers to introduce zwitterions and thiol groups; specifically:

[0043] a) utilizing the bioorthogonal reaction between aldehyde groups and amino groups, using unsaturated aldehydes to react with amino groups on the surface of the decellularized valve to obtain a decellularized valve with unsaturated surface modification;

[0044] b) Cross-linking the unsaturated modified decellular valve surface with a polymer to obtain a chemically modified decellular valve; the polymer system includes monomer 1, monomer 2 and a water-soluble initiator; monomer 1 is an unsaturated zwitterionic compound monomer, which is a dipole ion monomer with both positive and negative charges on the same molecule; monomer 2 is an unsaturated thiol. The polymer is obtained by free radical polymerization, and then cross-linked by a cross-linking agent to modify the zwitterion and allyl mercaptan on the DHV surface, which are used to construct a hydrogel layer and provide a thiol group, respectively.

[0045] 3. The circular DNA is modified by a maleimide group, and then reacted with the thiol group on the chemically modified decellularized valve to obtain a decellularized valve modified by the circular DNA; the cDNA includes circular DNA-1 and circular DNA-2; the circular DNA-1 is a circular DNA encoded by an antithrombin active nucleotide sequence, targeting a thrombin-specific aptamer, and the circular DNA-2 contains a DNA fragment complementary to the circular DNA-1;

[0046] 4. After circular DNA modification, the decellularized valve was placed in an RCA reaction solution or an RCA reaction solution with HO1 added for rolling circle amplification to obtain a DNA-modified heart valve. Through the rolling circle amplification strategy, the cDNA modified on the DHV surface was used as the starting point for rolling circle amplification to achieve the construction of an in situ DNA hydrogel layer on the DHV surface, enriching a large number of thrombin-specific aptamers and HO1 loads.

[0047] In the above embodiment, the DNA of the ring-shaped targeted thrombin-specific aptamer is introduced by chemical modification of the decellularized valve surface, and the G-quadruplex structure of the DNA aptamer is used to bind to heme in the blood circulation, thereby achieving autonomous capture of heme in the body and continuously supplying raw materials for the HO1-based enzyme catalytic reaction. The HO1-based enzyme catalytic reaction is used to catalyze heme to produce biliverdin, which exerts anti-inflammatory effects and promotes the polarization of macrophages to the M2 phenotype, thereby promoting valve endothelialization.

[0048] In the above embodiments, DHV is modified by a zwitterion sandwich-like structure to impart anti-calcification and anti-adhesion properties to DHV, thereby helping to improve the stability of DNA.

[0049] In a preferred embodiment, in step b):

[0050] The monomer 1 is selected from one of N-(4-aminobutyl)-N-(methacryloyloxyethyl)-N,N,N-trimethylammonium chloride, sulfobetaine methacrylate, 2-methacryloyloxyethyl phosphorylcholine, and carboxybetaine methacrylate;

[0051] The second monomer is selected from one of allyl mercaptan and α-acrylate-ω-mercaptopolyethylene glycol;

[0052] The water-soluble initiator is selected from persulfate, azobisisoheptanenitrile, dimethyl azobisisobutyrate, 2,2'-azobisisobutylamidine dihydrochloride, Vazo TM at least one of free radical initiators;

[0053] The crosslinking agent used in the polymer crosslinking process is N'N-methylenebisacrylamide.

[0054] The ratio of each monomer and the reaction addition of the free radical polymerization reaction based on the above monomers can be set according to the reaction principle of the free radical polymerization reaction.

[0055] In a preferred embodiment, the circular DNA-1 is synthesized from ssDNA targeting thrombin, and the ssDNA is a single-stranded DNA rich in a large amount of thrombin-specific DNA aptamer NU172; the circular DNA-1 synthesis process includes the use of primers and ligases, the ssDNA is a linear ssDNA NU172 template targeting thrombin specificity, used to inhibit thrombin activity, the nucleotide sequence is shown in SEQ ID NO: 1, and the nucleotide sequence of the primer is shown in SEQ ID NO: 2. The circular DNA-2 synthesis process is synthesized based on a random sequence as a template, which contains a base sequence that is partially complementary to cDNA-1, so that the RCA products of cDNA-1 and cDNA-2 form a DNA hydrogel loaded HO1 through base complementary pairing.

[0056] In a preferred embodiment, the maleimide group of the circular DNA is modified by base pairing between a primer with maleimide modified at the 5' end and the circular DNA-1 and circular DNA-2.

[0057] Example

[0058] A method for promoting heart valve endothelialization is proposed, the steps comprising:

[0059] 1. Obtaining porcine aortic valve:

[0060] Fresh porcine aortic valves were cut off in a clean environment, rinsed with heparin saline for 3 times, placed in saline solution containing double antibiotics (100 U / ml penicillin, 100 mg / ml streptomycin), and stored at 4°C for later use.

[0061] 2. Preparation of decellularized valves:

[0062] The porcine aortic valve was placed in TRIS-HCl buffer (40 mM, pH 7.8) containing 2% 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (CHAPS) and 2 mmol / l tributylphosphine (TnBP), and the cells were continuously shaken for 24 h at room temperature. Rinse with sterile water for 6 times, 10 minutes each time. Then, it was placed in TRIS-HCl buffer (40 mM, pH 7.8) containing 2% CHAPS, 2 mmol / l TnBP, 1% amidinosulfobetaine (ASB-14) and 2% sulfobetaine 10 (SB 3-10), and the cells were continuously shaken for 24 h at room temperature.

[0063] 3. Synthesis of cDNA-1 and cDNA-2:

[0064] 1) Synthesis of cDNA-1: Linear ssDNA NU172 template1 and A15-primer1 were mixed at a ratio of 1:1. During the annealing process, both ends of NU172 template1 hybridized with A15-primer1. T4 DNA ligase (2U) and 1×T4 DNA ligase buffer were added to seal the gap and obtain cDNA-1.

[0065] 2) Using the random sequence Random template2 as a template and A15-primer2 for hybridization, cDNA-2 was obtained by the same method as step 1). To prove the successful synthesis of cDNA-1 and cDNA-2, template-NU172, cDNA-1, template-Random2 and cDNA-2 samples were run on ice at 6V / cm for 2 hours in a 12% native PAGE gel. Then, the gel was stained with GelRed for 20 minutes in a three-dimensional shaker and observed under ultraviolet light using a gel imaging analysis system. Figure 2 shown.

[0066] 4. Cross-linking DHV based on free radical polymerization strategy:

[0067] 1) Acrolein was modified onto the valve surface by using the bioorthogonal reaction of aldehyde and amino groups to obtain acrolein-modified DHV (MA-DHV).

[0068] 2) Prepare N'N-methylenebisacrylamide mother solution (MBA, 3 mg / ml), 2.2-azobis(2-methylpropylimidamide) dihydrochloride mother solution (AIBA; 120 mg / mL), N-(4-aminobutyl)-N-(methacryloyloxyethyl)-N,N,N-trimethylammonium chloride mother solution (SBMA; 2 M in water) and allyl mercaptan mother solution (2 M in 50% ethanol). Keeping the total molar concentration of SBMA and allyl mercaptan constant at 1 M, immerse MA-DHV in a mixed solution of SBMA and allyl mercaptan in a volume ratio of 1:1. Add 200 μL of MBA and 100 μL of AIBA mother solution to the mixture, and stir in a water bath at 37°C to initiate free radical polymerization.

[0069] 5. Construction of DNA-DHV and DNA / HO1-DHV:

[0070] 1) cDNA-1 and cDNA-2 were modified with maleimide by using a primer (Mal T20 primer) with maleimide modified at the 5' end to achieve base complementary pairing with the redundant base sequence of A15-primer1 in cDNA-1 and cDNA-2.

[0071] 2) cDNA was modified onto the DHV surface by a click chemistry reaction between maleimide groups and thiol groups. The cDNA-modified DHV was immersed in a solution containing 2×BSA, dNTPs (1mM), NaCl (40mM), 1×phi29 buffer (50mM Tris-HCl, 10mM MgCl 2 、10mM(NH4) 2 SO 4 ), 4 mM DTT in the RCA reaction solution, and adjusted to pH 7.5 with phi29 polymerase (0.002 U / L). Then DHV was incubated at 400 rpm at 37 °C for 24 h to obtain DNA-DHV. For DNA / HO1-DHV preparation, 40 ng of HO1 protein was evenly dispersed into the RCA reaction solution according to the same preparation protocol.

[0072] The relevant DNA and primer sequences used in the above modification and subsequent characterization processes are shown in Table 1.

[0073] Table 1.: Sequence information

[0074]

[0075] The decellularized heart valve (DHV group), glutaraldehyde cross-linked decellularized heart valve (GV group), linear ssDNA modified decellularized heart valve (SS-DHV group), DNA-DHV and DNA / HO1-DHV groups constructed in step 5 were characterized for their structures and morphologies. Figure 3 shown. Figure 3 Figure A is a general photo. The original appearance of DHV will not be significantly changed after cross-linking the valve through free radical polymerization and DNA modification. Figure B is the surface and cross-sectional SEM image and EDS of the decellularized heart valve. The surface and cross-sectional morphology can be observed that cross-linking and modification will not destroy the original loose pore structure of DHV, meeting the needs of cell infiltration. At the same time, EDS shows that the phosphorus content of DNA-DHV and DNA / HO1-DHV is significantly increased, indicating the successful modification of DNA.

[0076] DNA / HO1-DHV component characterization results are as follows Figure 4 shown. Figure 4 In the figure, Figure A shows the imaging of DNA on the surface of DNA-DHV and DNA / HO1-DHV by DNA probe labeled with FAM, indicating that DNA was uniformly modified on the surface of DHV. Figure B shows the result of slicing DNA / HO1-DHV and labeling HO1 on the surface of DNA / HO1-DHV with HO1 antibody, indicating that HO1 was successfully modified on the surface of DHV.

[0077] 6. Verification of heart valve endothelialization and anti-calcification effects in vivo:

[0078] A rabbit carotid artery implantation model was established to evaluate the performance of the stent under a hemodynamic environment. After 2 weeks, the stents were stained with CD31 and DAPI immunofluorescence. After 4 weeks, Doppler ultrasound was performed to examine the patency of the stent, and then the stents were stained with Masson and Von Kossa to show the ECM structure and calcification. Macrophage markers (CD68, iNOS, and CD206), endothelial cell markers (CD31 and vWF), interstitial cell markers (vimentin), and type I collagen staining were used to evaluate the inflammation, cellularization, and remodeling of the implanted stents.

[0079] The blood compatibility characterization results of decellularized heart valves (DHV group), glutaraldehyde cross-linked decellularized heart valves (GV group), linear ssDNA modified decellularized heart valves (SS-DHV group), DNA-DHV constructed in step 5, and DNA / HO1-DHV group are shown in Figure 5 shown. Figure 5Figure A is a schematic diagram of the rabbit carotid artery model. Figure B shows that after 2 hours of circulation in the rabbit carotid artery, a large number of thrombi appeared on the surface of DHV, and DNA-DHV and DNA / HO1-DHV showed excellent anti-thrombotic ability. Figure C shows the SEM analysis results of the sample surface after 2 hours of circulation in the carotid artery. The results show that DNA-DHV and DNA / HO1-DHV show excellent anti-thrombotic ability. Figure D shows that the in vitro platelet adhesion experiment also shows that DNA-DHV can effectively inhibit platelet adhesion and activation.

[0080] Figure 6 This is the result of histological staining 28 days after abdominal aorta transplantation. Figure 6 The results show that DNA-DHV and DNA / HO1-DHV can effectively inhibit valve calcification, and compared with DHV, they can also effectively inhibit valve degradation while meeting the requirements of cell infiltration.

[0081] Figure 7 The results of staining analysis of macrophage phenotype 28 days after abdominal aorta transplantation. Figure 7 The results show that DNA / HO1-DHV can significantly induce macrophage polarization to M2 phenotype, demonstrating excellent immune regulatory ability.

[0082] Figure 8 The results of staining and analysis of type I collagen, endothelial cells, and interstitial cells were obtained 28 days after abdominal aorta transplantation. Figure 8 The results show that DNA / HO1-DHV can effectively promote the regeneration of type I collagen. At the same time, partial endothelialization occurred 14 days after implantation, and complete endothelialization was achieved 28 days after implantation, showing excellent regenerative ability.

[0083] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for modifying a heart valve to promote endothelialization, characterized in that the steps include: Decellularization of heart valves; The surface of the decellularized valve is chemically modified, wherein the modification process is based on the free radical polymerization reaction of unsaturated monomers to introduce zwitterions and thiol groups; The circular DNA is modified by a maleimide group, and then reacted with the thiol group on the chemically modified decellularized valve to obtain the decellularized valve modified by the circular DNA; the circular DNA includes circular DNA-1 and circular DNA-2; the circular DNA-1 is a circular DNA encoded by an antithrombin active nucleotide sequence, and the circular DNA-2 contains a DNA fragment complementary to the circular DNA-1, so as to form a DNA hydrogel with the RCA product of the circular DNA-1 through base complementary pairing; After circular DNA modification, the decellularized valve is placed in an RCA reaction solution containing HO1 for rolling circle amplification to obtain a heart valve modified with a DNA hydrogel loaded with HO1, i.e., a heart valve that promotes endothelialization.

2. The method according to claim 1, characterized in that The steps of the chemical modification process of the decellularized valve surface include: A1) using unsaturated aldehyde to react with amino groups on the surface of the decellularized valve to obtain a decellularized valve with unsaturated surface modification; A2) cross-linking the surface of the unsaturated modified decellular valve with a polymer to obtain a chemically modified decellular valve; the polymer is obtained by free radical polymerization based on monomer one, monomer two and a water-soluble initiator; monomer one is an unsaturated zwitterionic compound monomer, and monomer two is an unsaturated thiol.

3. The method according to claim 2, characterized in that The monomer 1 is selected from one of N-(4-aminobutyl)-N-(methacryloyloxyethyl)-N,N,N-trimethylammonium chloride, sulfobetaine methacrylate, 2-methacryloyloxyethyl phosphorylcholine, and carboxybetaine methacrylate; And / or, the second monomer is selected from one of allyl mercaptan and α-acrylate-ω-mercaptopolyethylene glycol; And / or, the water-soluble initiator is selected from persulfate, azobisisoheptanenitrile, dimethyl azobisisobutyrate, 2,2'-azobisisobutylamidine dihydrochloride, Vazo TM At least one free radical initiator.

4. The method according to claim 1, characterized in that: The circular DNA-1 is synthesized from ssDNA targeted to thrombin.

5. The method according to claim 4, characterized in that The ssDNA is a single-stranded DNA containing a thrombin-specific DNA aptamer NU172.

6. The method according to claim 4, characterized in that The nucleotide sequence of the ssDNA is shown in SEQ ID NO: 1; the synthesis process includes using a primer and a ligase, and the nucleotide sequence of the primer is shown in SEQ ID NO:

2.

7. The method according to claim 1, characterized in that The circular DNA-2 synthesis process is synthesized based on a random sequence as a template.

8. The method according to claim 1, characterized in that The maleimide group modification of the circular DNA is carried out by base pairing of the primer with maleimide modified at the 5' end with the circular DNA-1 and the circular DNA-2.

9. A bioprosthetic heart valve, characterized in that: The method is constructed by any one of claims 1 to 8.

10. Use of the artificial biological heart valve according to claim 9 in preparing biological scaffold materials.

Images