Method for promoting heart valve endothelialization and artificial heart valve

Abstract

The present invention discloses a method for promoting heart valve endothelialization and an artificial heart valve. The method is based on modifying circular DNA on the surface of a decellularized heart valve (DHV). A DNA hydrogel targeting thrombin is obtained on the surface of the DHV by rolling circle amplification. The DNA hydrogel not only exerts an anticoagulant function, but also acts as a heme capturer, recruiting heme from the blood. The 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 immune regulation and reactive oxygen scavenging, thereby creating a regenerative immune microenvironment and promoting valve endothelialization.

Description

Technical Field

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

[0002] Valvular heart disease is a cardiovascular disease with high morbidity and mortality. Currently, the most effective treatment for valvular heart disease is valve replacement surgery. It is estimated that by 2050, 850,000 valve replacements will be performed annually worldwide. Bioprosthetic valves are one of the most commonly used valve replacements in clinical practice. They have good blood compatibility and do not require long-term anticoagulation therapy. However, their lifespan is limited, and patients often require reoperation due to bioprosthetic valve degradation and calcification.

[0003] Currently, the primary bioprosthetic valve processing technique involves glutaraldehyde cross-linking. While this cross-linking method effectively improves bioprosthetic valve stability, it exhibits significant cytotoxicity, making it difficult to meet the requirements for cell adhesion and proliferation, and hindering the endothelialization of the stent material. However, valve endothelialization is generally considered a prerequisite for achieving long-term anticoagulation and anticalcification, and maintaining long-term valve functional stability.

[0004] Therefore, developing and exploring new valve cross-linking strategies to avoid the toxic side effects of glutaraldehyde and at the same time improve 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 industries. Summary of the Invention

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

[0006] In view of this, the solution 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, comprising the following steps:

[0008] Decellularization of heart valves;

[0009] 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;

[0010] The circular DNA is modified with a maleimide group, and then reacted with the sulfhydryl group on the chemically modified decellularized valve to obtain a circular DNA-modified decellularized valve; the cDNA includes circular DNA-1 and circular DNA-2; circular DNA-1 is a circular DNA encoded by an antithrombin active nucleotide sequence, and circular DNA-2 contains a DNA fragment complementary to 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 HO1-loaded DNA hydrogel-modified heart valve, that is, a heart valve that promotes endothelialization.

[0012] Furthermore, the chemical modification process of the acellular valve surface includes the following steps:

[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 decellularized valve with a polymer to obtain a chemically modified decellularized 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 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, azobisisoheptanonitrile, 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 targeted to thrombin.

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

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

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

[0024] Furthermore, 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.

[0025] The second aspect of the present invention is to provide an artificial bioprosthetic 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. A thrombin-targeting DNA hydrogel is obtained on the surface of DHV through rolling circle amplification. This DNA hydrogel not only exerts an anti-coagulant function, but also acts as a heme capturer, recruiting heme from the blood. Zwitterions are used to modify DHV 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 described in the present invention, heme is further converted into biliverdin (BV) through the heme oxygenase (HO1) loaded in the DNA, ensuring the continuous production of biliverdin, promoting anti-inflammatory immune regulation and reactive oxygen species 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 These are the gel imaging analysis results of cDNA-1 and cDNA-2 synthesized in the examples of the present invention.

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

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

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

[0035] Figure 6 These are the histological staining results 28 days after DNA / HO1-DHV was transplanted into the abdominal aorta in the examples 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 the abdominal aorta in the example of the present invention.

[0037] Figure 8 28 days after DNA / HO1-DHV was transplanted into the abdominal aorta, the results of staining and analysis of type I collagen, endothelial cells, and interstitial cells were obtained. DETAILED DESCRIPTION

[0038] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts 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 replacements, the present invention proposes a strategy for constructing a biosynthetic factory on the valve surface, which can continuously convert the abundant heme in the blood into an immunomodulator (biliverdin), thereby supporting long-term immunoregulation and tissue regeneration. To achieve this goal, circular DNA (cDNA) is modified to the DHV surface, and a DNA hydrogel loaded with heme oxygenase (HO1) is obtained on the DHV surface by rolling circle amplification. The anticoagulant NU172 aptamer is encoded in the DNA hydrogel, which not only exerts an anticoagulant function, but also plays the role of a heme capturer, recruiting heme from the blood. In addition, the heme oxygenase (HO1) loaded in the DNA further converts heme into biliverdin (BV), ensuring the continuous production of biliverdin, promoting anti-inflammatory immune regulation and reactive oxygen scavenging, and thus creating a regenerative immune microenvironment.

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

[0041] 1. Decellularize the heart valve;

[0042] 2. Chemically modifying the surface of the decellularized valve by introducing zwitterions and thiol groups through free radical polymerization of unsaturated monomers; 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 decellularized valve surface with a polymer to obtain a chemically modified decellularized valve; the polymer polymerization system comprises a first monomer, a second monomer, and a water-soluble initiator; the first monomer is an unsaturated zwitterionic compound monomer, a dipolar ion monomer with both positive and negative charges on the same molecule; and the second monomer is an unsaturated thiol. The polymer is obtained by free radical polymerization and then cross-linked with a cross-linker to modify the zwitterion and allylthiol on the DHV surface, respectively forming a hydrogel layer and providing thiol groups.

[0045] 3. Modifying the circular DNA with a maleimide group and reacting the resulting reaction with the sulfhydryl groups on the chemically modified decellularized valve to obtain a circular DNA-modified decellularized valve; the cDNA comprises circular DNA-1 and circular DNA-2; circular DNA-1 is a circular DNA encoded by an antithrombin active nucleotide sequence that targets a thrombin-specific aptamer, and circular DNA-2 contains a DNA fragment complementary to circular DNA-1;

[0046] 4. After circular DNA modification, the decellularized valve was placed in an RCA reaction solution or an RCA reaction solution supplemented with HO1 for rolling circle amplification to produce a DNA-modified heart valve. Using a rolling circle amplification strategy, starting with the cDNA modified onto the DHV surface, a DNA hydrogel layer was constructed in situ on the DHV surface, enriching a large number of thrombin-targeting aptamers and HO1 payloads.

[0047] In the above examples, a circular DNA aptamer targeting thrombin-specific aptamers was introduced through chemical modification of the decellularized valve surface. The G-quadruplex structure of the DNA aptamer allowed it to bind to circulating heme, enabling autonomous heme capture in vivo and providing a continuous supply of raw material for the HO1-based enzymatic reaction. The HO1-based enzymatic reaction catalyzed the production of biliverdin from heme, which exerted anti-inflammatory effects and promoted macrophage polarization toward the M2 phenotype, promoting valve endothelialization.

[0048] In the above embodiment, DHV is modified with a zwitterion sandwich 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 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, azobisisoheptanonitrile, dimethyl azobisisobutyrate, 2,2'-azobisisobutylamidine dihydrochloride, Vazo TM at least one of a free radical initiator;

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

[0054] The ratio of each monomer in the free radical polymerization reaction of the above monomers and the reaction addition 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, which is a single-stranded DNA enriched with a large amount of the thrombin-specific DNA aptamer NU172. The circular DNA-1 synthesis process includes the use of primers and ligase. The ssDNA is a linear ssDNA NU172 template targeting thrombin, which is used to inhibit thrombin activity and has a nucleotide sequence as shown in SEQ ID NO: 1. 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 partially complementary to cDNA-1, so that the RCA products of cDNA-1 and cDNA-2 can form a DNA hydrogel-loaded HO1 through complementary base pairing.

[0056] In a preferred embodiment, the maleimide group of the circular DNA is modified by base pairing with a primer having 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, comprising the following steps:

[0059] 1. Obtaining the porcine aortic valve:

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

[0061] 2. Preparation of Decellularized Valves:

[0062] Porcine aortic valves were 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 subjected to decellularization with continuous shaking at room temperature for 24 hours. The valves were then rinsed six times with sterile water for 10 minutes each. The valves were then 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 subjected to further decellularization with shaking at room temperature for 24 hours.

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

[0064] 1) Synthesis of cDNA-1: Mix the linear ssDNA NU172 template 1 and A15-primer 1 in a 1:1 ratio. During annealing, both ends of the NU172 template 1 hybridize with the A15-primer 1. Add 2 U of T4 DNA ligase and 1× T4 DNA ligase buffer to seal the gaps and generate cDNA-1.

[0065] 2) Using the random sequence Random template2 as a template and hybridizing with A15-primer2, cDNA-2 was obtained using the same method as in 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 a 12% native PAGE gel at 6 V / cm on ice for 2 hours. The gel was then stained with GelRed for 20 minutes in a three-dimensional shaker and observed under UV 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 between aldehyde and amino groups to obtain acrolein-modified DHV (MA-DHV).

[0068] 2) Prepare a stock solution of N'N-methylenebisacrylamide (MBA, 3 mg / ml), a stock solution of 2,2-azobis(2-methylpropylimidazole) dihydrochloride (AIBA; 120 mg / mL), a stock solution of N-(4-aminobutyl)-N-(methacryloyloxyethyl)-N,N,N-trimethylammonium chloride (SBMA; 2 M in water), and a stock solution of allyl mercaptan (2 M in 50% ethanol). Maintaining a constant total molar concentration of SBMA and allyl mercaptan at 1 M, immerse the MA-DHV in a 1:1 volume ratio of SBMA to allyl mercaptan. Add 200 μL of MBA and 100 μL of the AIBA stock solution to the mixture, and stir in a 37°C water bath 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 with maleimide modified at the 5' end (Mal T20 primer) 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 via a click chemistry reaction between maleimide and thiol groups. The cDNA-modified DHV was immersed in an RCA reaction solution containing 2× BSA, dNTPs (1 mM), NaCl (40 mM), 1× phi29 buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4), and 4 mM DTT, and adjusted to pH 7.5 with phi29 polymerase (0.002 U / L). The DHV was then incubated at 400 rpm at 37°C for 24 hours to obtain DNA-DHV. For the DNA / HO1-DHV preparation, 40 ng of HO1 protein was evenly dispersed into the RCA reaction solution following the same preparation protocol.

[0072] The relevant DNA and primer sequences used in the above modifications 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 3Figure A shows a macroscopic photograph. Cross-linking the valve via free radical polymerization and DNA modification do not significantly alter the original appearance of the DHV. Figure B shows SEM images and EDS of the surface and cross-section of the decellularized heart valve. The surface and cross-sectional morphologies demonstrate that cross-linking and modification do not disrupt the original loose porous structure of the DHV, satisfying the requirements for cell infiltration. EDS also shows a significant increase in the phosphorus content of the DNA-DHV and DNA / HO1-DHV, indicating successful DNA modification.

[0076] The characterization results of DNA / HO1-DHV components are as follows Figure 4 shown. Figure 4 Figure A shows imaging of DNA on the surfaces of DNA-DHV and DNA / HO1-DHV using a FAM-labeled DNA probe, demonstrating uniform DNA modification on the DHV surface. Figure B shows the results of sectioning DNA / HO1-DHV and labeling HO1 on the DNA / HO1-DHV surface with an HO1 antibody, demonstrating successful HO1 modification on the DHV surface.

[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 hemodynamic conditions. After 2 weeks, the stents were immunostained for CD31 and DAPI. After 4 weeks, Doppler ultrasound was performed to assess stent patency, followed by Masson and Von Kossa staining to visualize ECM structure and calcification. Macrophage markers (CD68, iNOS, and CD206), endothelial cell markers (CD31 and vWF), and interstitial cell markers (vimentin) and type I collagen staining were used to assess 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 2. Figure 5 shown. Figure 5 Figure 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 DHV surface, and DNA-DHV and DNA / HO1-DHV exhibited excellent anti-thrombotic properties. Figure C shows SEM analysis of the sample surface after 2 hours of circulation in the carotid artery, demonstrating that DNA-DHV and DNA / HO1-DHV exhibited excellent anti-thrombotic properties. Figure D also shows that DNA-DHV effectively inhibited platelet adhesion and activation in an in vitro platelet adhesion assay.

[0080] Figure 6 This is the histological staining result 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 28 days after abdominal aorta transplantation, the results of staining and analysis of type I collagen, endothelial cells and interstitial cells were obtained. 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] While 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 these embodiments without departing from the principles and spirit of the invention, and that the scope of the 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; 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; Circular DNA is modified with a maleimide group, and then reacted with the sulfhydryl group on the chemically modified decellularized valve to obtain a circular DNA-modified decellularized valve; the circular DNA includes circular DNA-1 and circular DNA-2; circular DNA-1 is a circular DNA encoded by an antithrombin active nucleotide sequence, and circular DNA-2 contains a DNA fragment complementary to circular DNA-1, so as to form a DNA hydrogel with the RCA product of circular DNA-1 through complementary base pairing; After circular DNA modification, the decellularized valve is placed in an RCA reaction solution containing HO1 for rolling circle amplification to obtain a HO1-loaded DNA hydrogel-modified heart valve, that is, 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 decellularized valve with a polymer to obtain a chemically modified decellularized 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 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, azobisisoheptanonitrile, dimethyl azobisisobutyrate, 2,2'-azobisisobutylamidine dihydrochloride, Vazo TM At least one free radical initiator.

4. The method according to claim 1, wherein 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 based on the synthesis of a random sequence as a template.

8. The method according to claim 1, characterized in that The maleimide group of the circular DNA is modified by base pairing of a 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 bioheart valve according to claim 9 in preparing biological scaffold materials.

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