A biomimetic tissue equivalent patch based on nano-CRISPR assistance, its preparation method and application

Abstract

This invention discloses a biomimetic tissue equivalent patch based on nano-CRISPR assistance, its preparation method, and its application, relating to the fields of biomedical engineering and tissue repair technology. It includes: a biomimetic mechanical adaptation layer, a nano-CRISPR intelligent release layer, a biomimetic cell infiltration layer, and a self-fixing / self-adhesive module ring-shaped at the patch edge, connected sequentially from the outside to the inside through a layered composite manner. The nano-CRISPR intelligent release layer is loaded with dual-target nano-CRISPR composite nanoparticles, and its surface is grafted with matrix metalloproteinase-sensitive peptide chains. The mechanical properties and pore distribution of each layer of the biomimetic mechanical adaptation layer, nano-CRISPR intelligent release layer, and biomimetic cell infiltration layer are equivalently matched to the natural structure of the target tissue. The ring-shaped self-fixing / self-adhesive module at the edge eliminates the need for sutures, adhesive fixation, or frequent replacement, directly adapting to the tissue surface characteristics to achieve stable adhesion, significantly reducing the risk of trauma and infection after implantation or application, and solving the shortcomings of traditional products that are overly targeted and have cumbersome fixation methods.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering and tissue repair, and in particular to a biomimetic tissue equivalent patch based on nano-CRISPR assistance, its preparation method, and its application. Background Technology

[0002] Tissue repair problems such as intervertebral disc annulus fibrosus defects, articular cartilage damage, tendon rupture, and chronic skin wounds are common musculoskeletal and body surface injuries in clinical practice. Traditional treatment methods (surgical suturing, simple bone grafting, and routine dressings) have drawbacks such as poor repair effects, high relapse / recurrence rates, large trauma, and insufficient targeting.

[0003] Existing repair products have the following shortcomings:

[0004] 1. Bionic carriers lack tissue equivalence: For example, the cartilage repair thermosensitive hydrogel disclosed in patent CN114984325A, although it enhances mechanical properties and releases anti-inflammatory drugs through short nanofibers, has insufficient matching degree between the fiber structure and the pore size and orientation of natural cartilage, and does not combine CRISPR gene regulation technology, so it cannot cope with complex pathological microenvironments.

[0005] 2. Poor adaptability of CRISPR delivery systems: Existing nano-CRISPR delivery systems mostly adopt a single carrier design and are not integrated with biomimetic tissue carriers. For example, patent CN113444263A discloses an MMP9-sensitive degradation hydrogel, which is only used for conventional drug release and is not adapted to the stable delivery requirements of CRISPR components (Cas protein, gRNA). This results in problems such as leakage of active ingredients and low cell uptake efficiency.

[0006] 3. Single repair mechanism: Most products rely solely on carrier support or release of single functional molecules, failing to form a synergistic repair system of nano-CRISPR gene regulation + biomimetic tissue structure support, thus failing to simultaneously address the core needs of abnormal proliferation + matrix degradation + tissue regeneration.

[0007] 4. Deficiencies in universality and fixation methods: The targeted nature makes it difficult to adapt to the equivalent repair needs of different tissues. Moreover, after implantation / application, suturing, adhesive fixation, or frequent changes are required, which increases the risk of trauma and infection. For example, patent CN114984325A is only applicable to cartilage repair. In terms of fixation methods, suturing is prone to causing secondary damage, injectable hydrogels lack structural stability, and conventional dressings need to be changed frequently, which disrupts the continuity of treatment.

[0008] Existing technologies have not achieved a systematic integration of precise delivery of nano-CRISPR with biomimetic tissue equivalent structures, and cannot simultaneously achieve targeted regulation and equivalent replacement of tissue functions.

[0009] Therefore, a nano-CRISPR-assisted biomimetic tissue equivalent patch, its preparation method, and its application are proposed to solve the above problems. Summary of the Invention

[0010] This invention overcomes the shortcomings of the prior art and provides a biomimetic tissue equivalent patch based on nano-CRISPR assistance, its preparation method, and its application.

[0011] To achieve the above objectives, the present invention adopts the following technical solution: a biomimetic tissue equivalent patch based on nano-CRISPR assistance, comprising a biomimetic mechanical adaptation layer, a nano-CRISPR smart release layer, a biomimetic cell infiltration layer and a self-fixing / self-adhesion module distributed in a ring around the edge of the patch, which are connected sequentially from the outside to the inside by a layered composite method.

[0012] The nano-CRISPR smart release layer is loaded with composite nanoparticles containing a dual-target CRISPR system, and the surface is grafted with matrix metalloproteinase-sensitive peptide chains.

[0013] The structure of each layer of the biomimetic mechanical adaptation layer, the nano-CRISPR smart release layer, and the biomimetic cell infiltration layer is equivalent to the mechanical properties and pore distribution of the natural structure of the target tissue.

[0014] In a preferred embodiment of the present invention, the biomimetic mechanical adapter layer is selected from any of the following options:

[0015] Option 1: PCL-collagen composite nanofiber membrane, mass ratio 6:4~7:3, porosity 25~35%, thickness 150~250μm, elastic modulus deviation from the natural structure of the target tissue ≤15%;

[0016] Option 2: PCL-gelatin composite nanofiber membrane, mass ratio 5:5~7:3, porosity 30~40%, thickness 120~200μm, elastic modulus deviation from the natural structure of the target tissue ≤15%;

[0017] Option 3: PLGA-hyaluronic acid composite nanofiber membrane, with a mass ratio of 4:6 to 6:4, a porosity of 35 to 40%, a thickness of 100 to 150 μm, and an elastic modulus that deviates from the natural structure of the target tissue by ≤15%.

[0018] In a preferred embodiment of the present invention, the nano-CRISPR smart release layer is selected from any of the following:

[0019] Option 1: PCL-gelatin composite nanofiber membrane, mass ratio 7:3~9:1, porosity 40~60%, thickness 80~180μm, elastic modulus deviation from the natural structure of the target tissue ≤20%, porosity deviation from the natural structure of the target tissue ≤20%;

[0020] Option 2: Gelatin-chitosan composite nanofiber membrane, with a mass ratio of 6:4 to 8:2, porosity of 40 to 60%, thickness of 80 to 180 μm, and elastic modulus and porosity that deviate from the natural structure of the target tissue by ≤20%.

[0021] In a preferred embodiment of the present invention, the composite nanoparticles of the dual-target CRISPR system comprise a target plasmid and a nanocarrier, with a particle size of 80-200 nm; the target plasmid is an expression plasmid encoding a Cas protein and a corresponding target gRNA, and is selected from one of the following combinations:

[0022] Combination A.VEGF gene knockout plasmid + MMP-13 gene knockout plasmid;

[0023] Combination of B.VEGF gene knockout plasmid + ADAMTS-5 gene knockout plasmid;

[0024] Combination of C.PDGF gene knockout plasmid + MMP-9 gene knockout plasmid;

[0025] Combination of D.BFGF gene knockout plasmid + MMP-2 gene knockout plasmid.

[0026] In a preferred embodiment of the present invention, the amino acid sequence of the MMP-sensitive peptide chain is selected from SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3, and the grafting density is 1.0~2.5 μmol / cm².

[0027] In a preferred embodiment of the present invention, the biomimetic cell infiltration layer is selected from collagen-hyaluronic acid composite hydrogel or hyaluronic acid-sodium alginate composite hydrogel.

[0028] If a collagen-hyaluronic acid composite hydrogel is selected, the mass ratio should be 5:5~7:3, the porosity should be 60~80%, the thickness should be 50~120μm, the elastic modulus should deviate from the natural structure of the target tissue by ≤20%, and the porosity should deviate from the natural structure of the target tissue by ≤20%.

[0029] If a hyaluronic acid-sodium alginate composite hydrogel is selected, the mass ratio is 4:6~6:4, the porosity is 60~80%, the thickness is 50~120μm, the elastic modulus deviates from the natural structure of the target tissue by ≤20%, and the porosity deviates from the natural structure of the target tissue by ≤20%.

[0030] The collagen-hyaluronic acid composite hydrogel or hyaluronic acid-sodium alginate composite hydrogel is used to simulate the microenvironment for cell growth in the inner layer of the target tissue.

[0031] In a preferred embodiment of the present invention, the self-fixing / self-adhesive module is a self-fixing module or a self-adhesive module, wherein the self-fixing module is a PLGA micro-spiky array with an array height of 200~350μm and a diameter of 50~80μm;

[0032] The self-adhesive module is a gelatin-dopamine adhesive layer with a thickness of 20~50μm;

[0033] Both the self-fixing module and the self-adhesion module are adapted to the surface characteristics of the target tissue.

[0034] The present invention adopts a technical solution as follows: a method for preparing a biomimetic tissue equivalent patch based on nano-CRISPR assistance, which is used for the preparation of the above-mentioned biomimetic tissue equivalent patch, including the following steps: raw material pretreatment, spinning solution preparation, layered electrospinning / spraying, nano-CRISPR loading, peptide linking and branching, freeze drying, module assembly, sterilization and packaging.

[0035] The electrospinning environment is controlled at 30-50% humidity and 20-26°C, and the biomimetic cell infiltration layer achieves a pore distribution equivalent to the target tissue through freeze-drying.

[0036] The technical solution adopted in this invention is an application of a biomimetic tissue equivalent patch based on nano-CRISPR assistance, which is used for the above-mentioned biomimetic tissue equivalent patch. Specifically, it can be used in the preparation of implantable repair devices for intervertebral disc annulus fibrosus, articular cartilage or tendon defects.

[0037] The implantable repair device is implanted into the body through minimally invasive surgery. Its biomimetic structure is equivalent to the mechanical properties and pore distribution of the target tissue. The nano-CRISPR enables precise dual-target regulation to inhibit abnormal proliferation and matrix degradation and promote tissue function regeneration.

[0038] In a preferred embodiment of the present invention, it can also be used in the preparation of surface / minimally invasive repair instruments for skin wound defects;

[0039] The skin wounds include chronic ulcer wounds, acute traumatic wounds, and burn healing wounds. The repair device is applied to the wound site through self-adhesive fixation or minimally invasive bonding. The biomimetic structure simulates the natural layered structure of the skin, and nano-CRISPR is used to promote epithelialization of the wound and reduce scar formation and recurrence.

[0040] This invention addresses the shortcomings of the prior art and has the following beneficial effects:

[0041] (1) The patch of the present invention is based on a layered structure of a biomimetic mechanical adaptation layer, a nano-CRISPR intelligent release layer, and a biomimetic cell infiltration layer. The mechanical properties and pore distribution of each layer are precisely matched with the natural structure of the target tissue, breaking through the limitation of insufficient tissue equivalence of traditional nanofiber patches, realizing the structure-function integrated equivalent substitution. At the same time, the nano-CRISPR system is deeply integrated with the biomimetic carrier, and with the help of the dual-target precise regulation capability, it can specifically solve complex pathological problems such as abnormal proliferation and matrix degradation in tissue repair.

[0042] (2) The patch of the present invention uses a nano-CRISPR smart release layer as a dedicated delivery carrier. Through the synergistic design of loaded dual-target nano-CRISPR composite nanoparticles and surface-grafted MMP-sensitive peptide chains, the defects of a single carrier are avoided: MMP-sensitive peptide chains achieve specific response release to the damaged microenvironment, reducing premature leakage of active ingredients. The structural compatibility between the composite nanoparticles and the biomimetic carrier improves the cell uptake efficiency. At the same time, the dual-target design reduces off-target effects, solving the problem of poor compatibility between the CRISPR delivery system and the biomimetic carrier.

[0043] Meanwhile, the dual-core synergistic repair system of nano-CRISPR gene regulation and biomimetic tissue structure support: the CRISPR system precisely inhibits abnormal proliferation and matrix degradation through dual-target plasmids, the biomimetic mechanical adaptation layer provides mechanical support, and the biomimetic cell infiltration layer simulates the cell growth microenvironment. The three work together to simultaneously inhibit pathological processes and promote tissue regeneration, breaking the limitations of traditional products that rely on only a single mechanism for repair and meeting the multi-dimensional core needs of tissue repair.

[0044] (3) The patch of the present invention provides multiple adaptation schemes for each layer of material (such as the biomimetic mechanical adaptation layer containing multiple composite membrane options such as PCL-collagen, PLGA-hyaluronic acid, etc.), which can be flexibly adjusted according to the characteristics of different target tissues, taking into account both the universality and specificity of repair. At the same time, the edge ring self-fixation / self-adhesion module does not require sutures, adhesive fixation or frequent replacement, directly adapts to the characteristics of tissue surface to achieve stable adhesion, significantly reducing the risk of trauma and infection after implantation or application, and solving the defects of traditional products that are too specific and have complicated fixation methods.

[0045] (4) The preparation process of this invention is mature and controllable, which is conducive to large-scale production: the mature processes such as layered electrospinning / spraying and freeze drying are adopted, the electrospinning environment (humidity 30~50%, temperature 20~26℃) is clearly controlled, and the cell infiltration layer pore distribution is precisely controlled by freeze drying. The process parameters are easy to control and have good repeatability. The preparation process covers the complete process from raw material pretreatment to sterilization and packaging, which can realize standardized production and take into account the needs of product consistency and large-scale application.

[0046] (5) The patch of the present invention is suitable for implantable repair of tissue defects such as intervertebral disc annulus fibrosus, articular cartilage, and tendon, and can also be used for surface / minimally invasive repair of skin wounds such as chronic ulcers, acute trauma, and burns. It covers two major scenarios: implantable and surface repair, and is suitable for repair needs of different tissues and different types of damage. It has a wide range of applications and strong practicality. Attached Figure Description

[0047] The present invention will be further described below with reference to the accompanying drawings and embodiments;

[0048] Figure 1 This is a schematic diagram of a preferred embodiment of the present invention, which is a biomimetic tissue equivalent patch based on nano-CRISPR assistance.

[0049] Figure 2 This is a schematic diagram of the internal structure of the biomimetic tissue equivalent patch based on nano-CRISPR-assisted design according to a preferred embodiment of the present invention.

[0050] Figure 3 This is a flowchart of the preparation method of a preferred embodiment of the present invention;

[0051] In the figure: 1. Patch; 10. Bionic mechanical adaptation layer; 11. Nano CRISPR smart release layer; 12. Bionic cell infiltration layer; 13. Self-fixing / self-adhesion module. Detailed Implementation

[0052] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0054] like Figure 1 and Figure 2As shown, a biomimetic tissue equivalent patch 1 based on nano-CRISPR assistance includes: a biomimetic mechanical adaptation layer 10, a nano-CRISPR smart release layer 11, a biomimetic cell infiltration layer 12, and a self-fixing / self-adhesion module 13 ring-distributed at the edge of the patch 1, which are connected in a stacked composite manner from the outside to the inside. The nano-CRISPR smart release layer 11 is loaded with dual-target nano-CRISPR composite nanoparticles and has matrix metalloproteinase-sensitive peptide chains grafted on its surface. The mechanical properties and pore distribution of each layer of the biomimetic mechanical adaptation layer 10, the nano-CRISPR smart release layer 11, and the biomimetic cell infiltration layer 12 are equivalently matched with the natural structure of the target tissue.

[0055] Based on the layered structure of the biomimetic mechanical adaptation layer 10, the nano-CRISPR intelligent release layer 11, and the biomimetic cell infiltration layer 12, the mechanical properties and pore distribution of each layer are precisely matched with the natural structure of the target tissue. This overcomes the limitations of insufficient tissue equivalence of traditional nanofiber patches, achieving integrated structure-function equivalent substitution. At the same time, the nano-CRISPR system is deeply integrated with the biomimetic carrier, and with the help of dual-target precise regulation capabilities, it can specifically solve complex pathological problems such as abnormal proliferation and matrix degradation in tissue repair.

[0056] Using a nano-CRISPR smart release layer 11 as a dedicated delivery carrier, the synergistic design of loaded dual-target nano-CRISPR composite nanoparticles and surface-grafted MMP-sensitive peptide chains avoids the shortcomings of single carriers: the MMP-sensitive peptide chains achieve specific response release to the damaged microenvironment, reducing premature leakage of active ingredients; the structural compatibility between the composite nanoparticles and the biomimetic carrier improves cellular uptake efficiency; at the same time, the dual-target design reduces off-target effects, solving the problem of poor compatibility between CRISPR delivery systems and biomimetic carriers.

[0057] In a preferred embodiment of the present invention, the biomimetic mechanical adapter layer 10 is selected from any of the following options:

[0058] Option 1: PCL-collagen composite nanofiber membrane, mass ratio 6:4~7:3, porosity 25~35%, thickness 150~250μm, elastic modulus deviation from the natural structure of the target tissue ≤15%;

[0059] Option 2: PCL-gelatin composite nanofiber membrane, mass ratio 5:5~7:3, porosity 30~40%, thickness 120~200μm, elastic modulus deviation from the natural structure of the target tissue ≤15%;

[0060] Option 3: PLGA-hyaluronic acid composite nanofiber membrane, with a mass ratio of 4:6 to 6:4, a porosity of 35 to 40%, a thickness of 100 to 150 μm, and an elastic modulus that deviates from the natural structure of the target tissue by ≤15%.

[0061] The composition of the composite nanofiber membrane can be adjusted as needed: for high mechanical requirements, PCL-collagen with a mass ratio of 6:4 to 7:3 is used, such as for tendon areas.

[0062] For medium mechanical requirements: use PCL-gelatin with a mass ratio of 5:5 to 7:3, for example, for use in cartilage areas;

[0063] Low mechanical requirements: PLGA-hyaluronic acid with a mass ratio of 4:6 to 6:4 is used, for example, for skin wounds.

[0064] Based on different exercise needs, it provides equivalent biomechanical support to better adapt to the movement needs of target tissues. At the same time, the material is selected to form a biodegradable composite nanofiber membrane composed of PCL-collagen and PLGA-hyaluronic acid, which has excellent biocompatibility and the degradation products have no toxic side effects.

[0065] In a preferred embodiment of the present invention, the nano-CRISPR smart release layer 11 is selected from any of the following:

[0066] Option 1: PCL-gelatin composite nanofiber membrane, mass ratio 7:3~9:1, porosity 40~60%, thickness 80~180μm, elastic modulus deviation from the natural structure of the target tissue ≤20%, porosity deviation from the natural structure of the target tissue ≤20%.

[0067] Option 2: Gelatin-chitosan composite nanofiber membrane, with a mass ratio of 6:4 to 8:2, porosity of 40 to 60%, thickness of 80 to 180 μm, and elastic modulus and porosity that deviate from the natural structure of the target tissue by ≤20%.

[0068] For general needs, a PCL-gelatin composite nanofiber membrane with a mass ratio of 7:3 to 9:1 is used. When high-performance requirements are needed, a gelatin-chitosan composite nanofiber membrane with a mass ratio of 6:4 to 8:2 is used to better load nano-CRISPR and achieve intelligent response release.

[0069] In a preferred embodiment of the present invention, the composite nanoparticles of the dual-target CRISPR system include a target plasmid and a nanocarrier, with a particle size of 80-200 nm; the target plasmid is an expression plasmid encoding the Cas protein and the corresponding target gRNA, and is selected from one of the following combinations:

[0070] Combination A.VEGF gene knockout plasmid + MMP-13 gene knockout plasmid;

[0071] Combination of B.VEGF gene knockout plasmid + ADAMTS-5 gene knockout plasmid;

[0072] Combination of C.PDGF gene knockout plasmid + MMP-9 gene knockout plasmid;

[0073] Combination of D.BFGF gene knockout plasmid + MMP-2 gene knockout plasmid.

[0074] The core active ingredient is adapted to different tissue targets, achieving precise nanoscale delivery:

[0075] Universal target, combination A: VEGF (inhibits abnormal angiogenesis) + MMP-13 (inhibits matrix degradation);

[0076] Alternative targets, combinations B, C, and D: VEGF + ADAMTS-5 (cartilage repair), PDGF + MMP-9 (tendon repair), and BFGF + MMP-2 (skin wound repair).

[0077] Examples of plasmid sequences: VEGF target sequence 5'-GGTGAGTGAAGTTCATGGC-3'; MMP-13 target sequence 5'-CAGTGAAGCCGAGATGTCC-3'.

[0078] Nanocarriers: liposome-chitosan composite nanoparticles or polylactic acid-glycolic acid copolymer-polyethyleneimine composite nanoparticles, with a particle size of 80~200nm;

[0079] Optimized parameters: liposomes (DOPC-cholesterol molar ratio 6:4~8:2), plasmid-liposome mass ratio 1:4~1:6, chitosan molecular weight 30~100kDa, Zeta potential 10~30mV;

[0080] Loading capacity: 50~150μg / cm² (adjusted according to tissue repair needs to ensure effective concentration of nano-CRISPR).

[0081] In a preferred embodiment of the present invention, the biomimetic cell infiltration layer 12 is selected from collagen-hyaluronic acid composite hydrogel or hyaluronic acid-sodium alginate composite hydrogel.

[0082] If a collagen-hyaluronic acid composite hydrogel is selected, the mass ratio should be 5:5~7:3, the porosity should be 60~80%, the thickness should be 50~120μm, the elastic modulus should deviate from the natural structure of the target tissue by ≤20%, and the porosity should deviate from the natural structure of the target tissue by ≤20%.

[0083] If a hyaluronic acid-sodium alginate composite hydrogel is selected, the mass ratio is 4:6~6:4, the porosity is 60~80%, the thickness is 50~120μm, the elastic modulus deviates from the natural structure of the target tissue by ≤20%, and the porosity deviates from the natural structure of the target tissue by ≤20%.

[0084] Collagen-hyaluronic acid composite hydrogels or hyaluronic acid-sodium alginate composite hydrogels are used to simulate the microenvironment for cell growth in the inner layer of target tissues.

[0085] For general requirements, a collagen-hyaluronic acid ratio of 5:5 to 7:3 by mass should be selected;

[0086] For high moisturizing needs, choose a hyaluronic acid-sodium alginate ratio of 4:6 to 6:4 by weight.

[0087] Its structure mimics the microenvironment of cell growth in the inner layer of the target tissue, and its cell adhesion sites are equivalent to those of natural tissue, promoting cell infiltration, proliferation and matrix deposition.

[0088] Example 1: The target tissue is the annulus fibrosus of the intervertebral disc.

[0089] The structural parameters for each layer are as follows:

[0090] Bionic mechanical adaptation layer 10: PCL-collagen composite nanofiber membrane with a mass ratio of 7:3, porosity of 25~30%, thickness of 200~250μm, and elastic modulus of 10~15MPa is selected.

[0091] Nano CRISPR Smart Release Layer 11: PCL-gelatin composite nanofiber membrane with a mass ratio of 8:2, porosity of 45~55%, and thickness of 120~150μm is selected;

[0092] Bionic cell infiltration layer 12: Collagen-hyaluronic acid composite hydrogel with a mass ratio of 6:4, porosity of 70-80%, and thickness of 80-120μm is selected;

[0093] Dual-target CRISPR composite nanoparticles: Combination A (VEG gene knockout plasmid + MMP-13 gene knockout plasmid) was selected, with a particle size of 120~160nm.

[0094] Example 2: The target tissue is articular cartilage.

[0095] The structural parameters for each layer are as follows:

[0096] Bionic mechanical adaptation layer 10: PCL-gelatin composite nanofiber membrane with a mass ratio of 6:4, porosity of 35~40%, thickness of 150~180μm, and elastic modulus of 5~8MPa is selected.

[0097] Nano CRISPR Smart Release Layer 11: Selected gelatin-chitosan composite nanofiber membrane, mass ratio 7:3, porosity 40~50%, thickness 100~130μm;

[0098] Bionic cell infiltration layer 12: Hyaluronic acid-sodium alginate composite hydrogel with a mass ratio of 5:5, porosity of 65~75%, and thickness of 70~100μm is selected.

[0099] Dual-target CRISPR composite nanoparticles: Combination B (VEGF gene knockout plasmid + ADAMTS-5 gene knockout plasmid) was selected, with a particle size of 100~140nm.

[0100] Example 3: Target tissue is tendon

[0101] The structural parameters for each layer are as follows:

[0102] Bionic mechanical adaptation layer 10: PCL-collagen composite nanofiber membrane with a mass ratio of 6.5:3.5, porosity of 30~35%, thickness of 180~220μm, and elastic modulus of 12~16MPa is selected;

[0103] Nano CRISPR Smart Release Layer 11: PCL-gelatin composite nanofiber membrane with a mass ratio of 9:1, porosity of 50~60%, and thickness of 130~160μm;

[0104] Bionic cell infiltration layer 12: Collagen-hyaluronic acid composite hydrogel with a mass ratio of 7:3, porosity of 60~70%, and thickness of 60~90μm is selected;

[0105] Dual-target CRISPR composite nanoparticles: Combination C (PDGF gene knockout plasmid + MMP-9 gene knockout plasmid) with a particle size of 140~180nm.

[0106] Example 4: Target tissue is a skin wound.

[0107] The structural parameters for each layer are as follows:

[0108] Bionic mechanical adaptation layer 10: PLGA-hyaluronic acid composite nanofiber membrane with a mass ratio of 5:5, porosity of 35~40%, thickness of 100~130μm, and elastic modulus of 1~3MPa is selected.

[0109] Nano CRISPR Smart Release Layer 11: Selected gelatin-chitosan composite nanofiber membrane, with a mass ratio of 6:4, porosity of 45~55%, and thickness of 80~110μm;

[0110] Bionic cell infiltration layer 12: Hyaluronic acid-sodium alginate composite hydrogel with a mass ratio of 6:4, porosity of 75-80%, and thickness of 50-80μm is selected.

[0111] Dual-target CRISPR composite nanoparticles: Combination D (BFGF gene knockout plasmid + MMP-2 gene knockout plasmid) was selected, with a particle size of 80~120nm.

[0112] In a preferred embodiment of the present invention, the amino acid sequence of the MMP-sensitive peptide chain is selected from SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3, and the grafting density is 1.0~2.5 μmol / cm².

[0113] Multiple sequence alternatives, adapted for intelligent release, specifically:

[0114] Core sequence (enzyme digestion efficiency ≥85%): SEQ ID NO.1, its amino acid sequence is Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln;

[0115] MMP-2 sensitive candidate sequence: SEQ ID NO.2, whose amino acid sequence is Pro-Leu-Gly-Leu-Ile-Ala-Gly-Gln;

[0116] MMP-9 sensitive candidate sequence: SEQ ID NO.3, whose amino acid sequence is Gly-Arg-Pro-Gly-Leu-Ile-Gly-Gln.

[0117] In a preferred embodiment of the present invention, the self-fixing / self-adhesive module 13 is a self-fixing module or a self-adhesive module. The self-fixing module is a PLGA micro-spiky array with an array height of 200~350μm and a diameter of 50~80μm.

[0118] The self-adhesive module is a gelatin-dopamine adhesive layer with a thickness of 20~50μm;

[0119] Both the self-fixing module and the self-adhesive module are adapted to the surface characteristics of the target tissue.

[0120] The micro-needles are shaped to fit the surface texture of the implanted tissue, and the adhesion layer simulates the biological adhesion mechanism to achieve minimally invasive fixation or self-adhesion, thus avoiding detachment.

[0121] As shown in Figure 3, a method for preparing a biomimetic tissue equivalent patch 1 based on nano-CRISPR assistance is used for the preparation of the above-mentioned biomimetic tissue equivalent patch 1, including the following steps: raw material pretreatment, spinning solution preparation, layered electrospinning / spraying, nano-CRISPR loading, peptide linking and branching, freeze drying, module assembly, sterilization and packaging.

[0122] The electrospinning environment is controlled at 30-50% humidity and 20-26℃, and the biomimetic cell infiltration layer 12 achieves a pore distribution equivalent to the target tissue through freeze-drying.

[0123] Specifically:

[0124] S1. Pretreatment of composite carrier raw materials: PCL / PLGA is vacuum dried at 60℃ for 24h to remove residual solvent;

[0125] Collagen or gelatin was sterilely dialyzed with pH 7.4 PBS for 48 hours to remove impurities and ensure biomimetic compatibility.

[0126] S2. Preparation of spinning solution: Dissolve the composite raw materials in a mixed solvent of hexafluoroisopropanol / trifluoroethanol at a volume ratio of 1:1, stir magnetically at 300 rpm for 24 h until completely dissolved, and filter using a 0.45 μm filter membrane to remove air bubbles.

[0127] S3, preparation of biomimetic mechanical adaptation layer 10: electrospinning was carried out according to optimized parameters, the receiving roller speed was 300~500 rpm, after the fibers were arranged in a concentric ring, they were collected and vacuum dried at 40℃ for 12h.

[0128] S4. Preparation of nano-CRISPR smart release layer 11: Dual-target nano-CRISPR composite nanoparticles are ultrasonically dispersed in spinning solution and simultaneously spun to cover the biomimetic mechanical adaptation layer 10. After drying, they are ready for use.

[0129] S5. Sensitive peptide linker: Amidation reaction was used, with an EDC / NHS molar ratio of 1:1 to 2:1, an activation time of 1 to 2 hours, a peptide chain reaction time of 3 to 6 hours, and after the reaction, the peptide was washed three times with PBS for 15 minutes each time, and then freeze-dried.

[0130] S6. Preparation of biomimetic cell infiltration layer 12: The hydrogel raw material was dissolved in PBS, a cross-linking agent was added, and the concentration of genipin was selected as 0.3~0.8%. After electrostatic spraying, it was pre-frozen at -20℃ for 2h and freeze-dried at -80℃ for 12h to form a porous structure equivalent to the inner layer of the target tissue.

[0131] S7. Preparation of self-fixing / self-adhesive module 13:

[0132] Microspiky array: PLGA molding, temperature 160~180℃, pressure 0.5~1.0MPa, after demolding, it is fixed to the edge of patch 1 with gelatin-genipin adhesive at 37℃ for 2 hours, and the microspiky morphology is adapted to the implanted tissue surface.

[0133] S8. Self-adhesive layer: A gelatin-dopamine solution with a mass ratio of 9:1 and a concentration of 5% is coated onto the edge of patch 1 and dried at room temperature for 12 hours to simulate a biological adhesion mechanism.

[0134] S9. Sterilization treatment: Gamma ray sterilization, dose 20~30kGy, aseptic packaging.

[0135] Example 5: Patch for repairing the annulus fibrosus of the intervertebral disc 1

[0136] Background: Adaptor to self-fixation module, corresponding target tissue: intervertebral disc annulus fibrosus.

[0137] S1. Pretreatment of composite carrier raw materials

[0138] Synthetic polymer raw material: PCL, molecular weight 80kDa, dried in a vacuum drying oven at 60℃ for 24h to remove residual solvent, purity ≥95%;

[0139] Natural raw materials: Type I collagen: bovine, purity ≥98%, sterile dialyzed with pH 7.4 PBS buffer for 48 hours, with the buffer changed twice daily to remove impurities and immunogenic components;

[0140] CRISPR-related raw materials: VEGF gene knockout plasmid, MMP-13 gene knockout plasmid: purity ≥99%, liposome-chitosan composite nanocarrier: DOPC-cholesterol molar ratio 7:3, chitosan molecular weight 50kDa;

[0141] Auxiliary raw materials: hexafluoroisopropanol / trifluoroethanol mixed solvent, volume ratio 1:1, EDC, NHS, purity ≥99%, genipin, purity ≥98%.

[0142] S2, Preparation of spinning solution

[0143] Bionic mechanical adaptation layer 10 spinning solution: Weigh PCL and collagen at a mass ratio of 7:3, dissolve them in a mixed solvent, prepare a spinning solution with a concentration of 12% (w / v), stir magnetically at 300 rpm for 24 h until completely dissolved, and filter with a 0.45 μm filter membrane to remove bubbles;

[0144] Nano-CRISPR intelligent release layer spinning solution: PCL and gelatin were weighed at a mass ratio of 8:2 and dissolved in a mixed solvent to prepare a spinning solution with a concentration of 10% (w / v). After stirring and dissolving, the solution was filtered to remove bubbles. Combined A (VEGF+MMP-13) dual-target nano-CRISPR composite nanoparticles (particle size 120~160nm) were ultrasonically dispersed in the spinning solution (power 100W, time 10min) to a final nanoparticle concentration of 0.8mg / ml.

[0145] S3, preparation of biomimetic mechanical adaptation layer 10

[0146] Environmental parameters: Humidity 35~40%, temperature 22~24℃;

[0147] Preparation of biomimetic mechanical adaptation layer 10: The spinning solution was loaded into a 10mL syringe, using a 23G needle, a voltage of 14kV, a flow rate of 0.6mL / h, a receiving distance of 18cm, and a receiving roller rotation speed of 400rpm (to achieve concentric circular arrangement of fibers). The spinning time was 6h to form a fiber membrane with a thickness of 200~250μm, which was then vacuum dried at 40℃ for 12h.

[0148] S4. Preparation of the nano-CRISPR smart release layer 11: The spinning solution loaded with nano-CRISPR was injected into a syringe with the same needle parameters, voltage 13kV, flow rate 0.5mL / h, and receiving distance 17cm. It was then applied to the surface of the mechanical adaptation layer and spun for 4h to form a composite layer with a thickness of 120~150μm. The layer was then dried for later use.

[0149] S5, sensitive peptide linker

[0150] Select SEQ ID NO.1 (core sequence, enzyme digestion efficiency ≥85%) and prepare a peptide chain solution with a concentration of 2 mg / ml;

[0151] An activation solution was prepared according to an EDC:NHS molar ratio of 1.5:1, added to the surface of the spinning composite layer, and activated at room temperature for 1.5 h.

[0152] Add peptide chain solution and react at 25℃ for 4 hours to achieve peptide link grafting (grafting density 1.8~2.0μmol / cm²).

[0153] Wash three times with pH 7.4 PBS buffer for 15 min each time to remove unbound peptide chains, then freeze-dry for later use.

[0154] S6, Preparation of biomimetic cell infiltration layer 12:

[0155] Weigh out collagen and hyaluronic acid in a mass ratio of 6:4, dissolve them in PBS buffer, and prepare a 5% (w / v) hydrogel solution.

[0156] Add genipin crosslinking agent (final concentration 0.5%), stir evenly, and then cover the surface of the smart release layer by electrostatic spraying (voltage 8kV, flow rate 0.2mL / h, receiving distance 10cm).

[0157] Pre-freeze at -20℃ for 2 hours, then transfer to a freeze dryer at -80℃ with a vacuum degree ≤10Pa and dry for 12 hours to form a hydrogel layer with a porosity of 70~80% and a thickness of 80~120μm.

[0158] S7. Preparation of self-fixing modules:

[0159] Preparation of PLGA micro-spiky array: PLGA raw material was molded at 170℃ and 0.8MPa, with micro-spiky height of 250~300μm and diameter of 60~70μm;

[0160] The micro-spiky array was fixed to the edge of patch 1 using a gelatin-genipin adhesive (10% gelatin and 0.3% genipin). The adhesive was bonded at a constant temperature of 37°C for 2 hours to ensure a bonding strength ≥0.3 N / cm².

[0161] S9. Sterilization treatment

[0162] Sterilization was performed using gamma rays at a dose of 25 kGy, with aseptic operation throughout the process.

[0163] Sealed in a sterile aluminum foil bag, labeled with the target tissue type, specifications and production date, and stored at -20℃.

[0164] Example 6: Patches for Articular Cartilage Repair 1

[0165] Background: Adaptor self-adhesion module, corresponding target tissue: articular cartilage.

[0166] S1. Pretreatment of composite carrier raw materials

[0167] Synthetic polymer raw materials: PCL (molecular weight 60kDa) was dried under vacuum at 60℃ for 24h, and PLGA (lactic acid-glycolic acid molar ratio 50:50, molecular weight 100kDa) was dried under the same conditions;

[0168] Natural raw materials: Gelatin (porcine-derived, purity ≥98%) was sterilely dialyzed (pH 7.4 PBS, 48h), and chitosan (molecular weight 80kDa, degree of deacetylation ≥90%) was dissolved in 1% acetic acid solution for later use;

[0169] CRISPR-related materials: Combination B (VEGF+ADAMTS-5) dual-target plasmid, polylactic acid-glycolic acid copolymer-polyethyleneimine composite nanocarrier (Zeta potential 20~25mV).

[0170] Auxiliary materials: mixed solvent, EDC / NHS, genipin, dopamine (recrystallized and purified).

[0171] S2, Preparation of spinning solution

[0172] Bionic mechanical adaptation layer 10 spinning solution: PCL and gelatin in a mass ratio of 6:4, dissolved in a mixed solvent to prepare an 11% (w / v) solution, stirred to dissolve, filtered and defoamed;

[0173] Nano CRISPR intelligent release layer 11 spinning solution: gelatin and chitosan in a mass ratio of 7:3 are dissolved in a mixed solvent (containing 1% acetic acid) to prepare a 9% (w / v) solution. Combined B nano CRISPR composite nanoparticles (particle size 100~140nm, final concentration 0.6mg / ml) are added and ultrasonically dispersed and degassed.

[0174] S3, preparation of biomimetic mechanical adaptation layer 10

[0175] Environmental parameters: Humidity 30~35%, temperature 20~22℃;

[0176] Mechanical adapter layer: voltage 13kV, flow rate 0.5mL / h, receiving distance 17cm, roller speed 350rpm, spinning for 5h, thickness 150~180μm, dry for later use;

[0177] Preparation of S4 and Nano-CRISPR Smart Release Layer 11

[0178] Voltage 12kV, flow rate 0.4mL / h, receiving distance 16cm, spinning time 3.5h, thickness 100~130μm, dry for later use.

[0179] S5, sensitive peptide linker

[0180] Select SEQ ID NO.1, activate at an EDC:NHS molar ratio of 1:1 for 2 h, react peptides for 3 h, graft at a density of 1.5~1.8 μmol / cm², wash with PBS and freeze dry.

[0181] S6, Preparation of biomimetic cell infiltration layer 12

[0182] Prepare a 4% (w / v) PBS solution by mixing hyaluronic acid and sodium alginate in a 5:5 mass ratio, and add genipin (final concentration 0.4%).

[0183] Electrostatic spray parameters: voltage 7kV, flow rate 0.15mL / h, receiving distance 9cm;

[0184] Pre-freeze at -20℃ for 2 hours, freeze-dry at -80℃ for 12 hours to form a hydrogel layer with a porosity of 65~75% and a thickness of 70~100μm.

[0185] S8, Self-adhesive module assembly

[0186] A 5% (w / v) solution of gelatin and dopamine in a mass ratio of 9:1 was prepared by dissolving the gelatin in PBS.

[0187] Apply the coating evenly to the edge of patch 1 (circumferential width 1.5~2mm) with a coating pen, and dry at room temperature for 12 hours to form an adhesive layer with a thickness of 30~40μm. The adhesion strength in a humid environment is ≥0.6N / cm².

[0188] S9. Sterilization and Packaging

[0189] Sterilize with 22 kGy gamma rays, package in sterile aluminum foil bags, and store at 4°C.

[0190] An application of a biomimetic tissue equivalent patch 1 based on nano-CRISPR assistance, specifically for use in the above-mentioned biomimetic tissue equivalent patch 1, can be applied in the preparation of implantable repair devices for defects of intervertebral disc annulus fibrosus, articular cartilage or tendon.

[0191] Implantable repair devices are implanted into the body through minimally invasive surgery. Their biomimetic structure is equivalent to the mechanical properties and pore distribution of the target tissue. Nano-CRISPR enables precise dual-target regulation to inhibit abnormal proliferation and matrix degradation and promote tissue function regeneration.

[0192] It can also be used in the preparation of surface / minimally invasive repair instruments for skin wound defects;

[0193] Skin wounds include chronic ulcers, acute traumatic wounds, and burn healing wounds. Repair instruments are applied to the wound site through self-adhesive fixation or minimally invasive bonding. Bionic structures mimic the natural layered structure of the skin, and nano-CRISPR is used to promote epithelialization of the wound and reduce scar formation and recurrence.

[0194] Example 7: Application of minimally invasive implantation repair for knee cartilage defects

[0195] Background: The patient was a 45-year-old male who presented with a medial femoral condyle cartilage defect in his right knee due to a sports injury. The defect measured 1.8cm × 1.2cm and was 3mm deep. Preoperatively, he presented with weight-bearing pain in the knee (VAS score 6.5) and limited flexion and extension range (105°). MRI revealed a full-thickness cartilage defect with exposed subchondral bone, accompanied by local synovitis and mild cartilage matrix degradation. The patient had experienced recurrence after traditional arthroscopic microfracture surgery. Therefore, precise repair of the cartilage structure and inhibition of abnormal proliferation were required. A biomimetic tissue equivalent patch specifically designed for articular cartilage was selected.

[0196] Bionic mechanical adaptation layer 10: PCL-gelatin composite nanofiber membrane (mass ratio 6:4), porosity 38%, thickness 160μm, elastic modulus 6.2MPa (12% deviation from normal articular cartilage).

[0197] Nano-CRISPR smart release layer 11: gelatin-chitosan composite nanofiber membrane (mass ratio 7:3), porosity 45%, thickness 110μm, grafted with SEQ ID NO.1 MMP sensitive peptide chain (grafting density 1.6μmol / cm²).

[0198] Dual-target CRISPR composite nanoparticles: Combination B (VEGF gene knockout plasmid + ADAMTS-5 gene knockout plasmid), particle size 120 nm, loading 80 μg / cm².

[0199] 4. Bionic cell infiltration layer 12: Hyaluronic acid-sodium alginate composite hydrogel (mass ratio 5:5), porosity 70%, thickness 85μm;

[0200] Fixing module: self-adhesive module (gelatin-dopamine adhesive layer, thickness 35μm, wet adhesion strength 0.7N / cm²).

[0201] Preoperative preparation: Remove the patch from the refrigerator at 14℃, allow it to warm to room temperature for 30 minutes, and rinse with saline to remove any remaining protective agent.

[0202] Minimally invasive surgical procedure: Using a knee arthroscopy minimally invasive approach, degenerated cartilage and inflammatory tissue at the edge of the defect area are cleaned, and the subchondral bone is ground down until slight bleeding occurs (to create a repair microenvironment).

[0203] Patch 1 implantation: The biomimetic cell infiltration layer 12 of patch 1 is placed towards the bone surface of the defect area, precisely covering the defect area. Press the edge for 30 seconds to achieve self-adhesion fixation (the circular adhesive layer adheres to the surrounding normal cartilage).

[0204] Postoperative management: Intra-articular injection of antibiotics to prevent infection, closure of the incision, and application of an elastic bandage for pressure dressing.

[0205] Postoperative follow-up: Short-term (1 month): VAS pain score dropped to 2.0, knee flexion and extension range of motion recovered to 135°, no obvious swelling or signs of infection, MRI showed that patch 1 and the surrounding cartilage had blurred boundaries and began to fuse;

[0206] Mid-term (6 months): Clinical symptoms completely disappeared, and the patient could walk normally and engage in light exercise. Arthroscopic re-examination revealed that the defect area was covered with hyaline cartilage-like tissue. Histological examination showed that the chondrocytes were arranged in an orderly manner and type II collagen expression was positive.

[0207] Long-term (12 months): MRI showed that the repaired tissue matched the mechanical properties of normal cartilage (elastic modulus 5.8 MPa), and nano-CRISPR continuously inhibited VEGF (reducing abnormal angiogenesis by 82%) and ADAMTS-5 (reducing cartilage matrix degradation rate by 75%), with no scar hyperplasia or degeneration of the repaired tissue.

[0208] Example 8: Application in the repair of chronic diabetic foot ulcers on the body surface

[0209] Background: The patient is a 62-year-old female with a 15-year history of type 2 diabetes. She has a chronic ulcer on the dorsum of her left foot, with a wound area of ​​3.5cm × 2.8cm and a depth of 0.8cm, and a course of 8 months. The wound shows slow granulation tissue growth, arrested epithelialization at the edges, and mild infection (Staphylococcus aureus was found in the secretion culture). Previous treatments (iodine disinfection + Vaseline gauze) have been ineffective, and there is a risk of scar recurrence. A biomimetic tissue equivalent patch for skin wounds was selected.

[0210] Bionic mechanical adaptation layer 10: PLGA-hyaluronic acid composite nanofiber membrane (mass ratio 5:5), porosity 38%, thickness 110μm, elastic modulus 2.2MPa (simulating the mechanical properties of the dermis layer of skin).

[0211] Nano-CRISPR smart release layer 11: gelatin-chitosan composite nanofiber membrane (mass ratio 6:4), porosity 50%, thickness 90μm, grafted with SEQ ID NO.3 MMP-9 sensitive peptide chain (grafting density 1.3μmol / cm²).

[0212] Dual-target CRISPR composite nanoparticles: Combination D (BFGF gene knockout plasmid + MMP-2 gene knockout plasmid), particle size 100 nm, loading 60 μg / cm².

[0213] Bionic cell infiltration layer 12: Hyaluronic acid-sodium alginate composite hydrogel (mass ratio 6:4), porosity 78%, thickness 65μm;

[0214] Fixing module: self-adhesive module (gelatin-dopamine adhesive layer, annular width 2mm, thickness 30μm).

[0215] Wound pretreatment: Thoroughly clean the wound (remove necrotic tissue and purulent secretions), rinse with 3% hydrogen peroxide solution, clean with physiological saline, disinfect the skin around the wound (within a 5cm diameter range) with povidone-iodine, and allow it to dry;

[0216] Application of patch 1: With the biomimetic cell infiltration layer 12 of patch 1 facing the granulation tissue of the wound, completely cover the wound and 0.5cm of normal skin at the edge, press the edge for 1 minute to achieve self-adhesion and fixation (avoid air bubble residue).

[0217] Postoperative care: Cover the wound with sterile gauze (to keep it moist), change the outer gauze daily, and change patch 1 every 3 days (adjust according to the amount of secretions from the wound). Monitor blood glucose and control it between 4.8 and 7.2 mmol / L.

[0218] Postoperative follow-up: Short-term (2 weeks): Wound secretions were significantly reduced, granulation tissue was bright red, epithelial cells migrated from the edge of the wound to the center, and the wound area shrank to 2.0cm×1.5cm (healing rate 68%).

[0219] Intermediate stage (4 weeks): The wound is completely epithelialized, covered with a thin layer of new skin, with no obvious scar protrusion, good skin elasticity, and no pain when the patient walks;

[0220] Long-term (6 months): The new skin has the same color and texture as the surrounding normal skin, the scar area is <0.3cm² (scar formation rate reduced by 89%), there is no ulcer recurrence during the follow-up period, and nano-CRISPR effectively inhibits BFGF (scar hyperplasia-related factor expression downregulated by 76%) and MMP-2 (excessive wound degradation reduced by 69%), achieving functional healing.

[0221] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These are all equivalent modifications and improvements made to the above embodiments based on the essential technology of the present invention, and all of these fall within the protection scope of the present invention.

Claims

1. A biomimetic tissue equivalent patch based on nano-CRISPR assistance, characterized in that, include: From the outside to the inside, the biomimetic mechanical adaptation layer, the nano CRISPR smart release layer, the biomimetic cell infiltration layer, and the self-fixing / self-adhesion module distributed in a ring at the edge of the patch are connected in a layered composite manner. The nano-CRISPR smart release layer is loaded with composite nanoparticles containing a dual-target CRISPR system, and the surface is grafted with matrix metalloproteinase-sensitive peptide chains. The structure of each layer of the biomimetic mechanical adaptation layer, the nano-CRISPR smart release layer, and the biomimetic cell infiltration layer is equivalent to the mechanical properties and pore distribution of the natural structure of the target tissue. The biomimetic mechanical adapter layer is selected from any of the following options: Option 1: PCL-collagen composite nanofiber membrane, mass ratio 6:4~7:3, porosity 25~35%, thickness 150~250μm, elastic modulus deviation from the natural structure of the target tissue ≤15%; Option 2: PCL-gelatin composite nanofiber membrane, mass ratio 5:5~7:3, porosity 30~40%, thickness 120~200μm, elastic modulus deviation from the natural structure of the target tissue ≤15%; Option 3: PLGA-hyaluronic acid composite nanofiber membrane, with a mass ratio of 4:6 to 6:4, a porosity of 35 to 40%, a thickness of 100 to 150 μm, and an elastic modulus that deviates from the natural structure of the target tissue by ≤15%; The nano-CRISPR smart release layer is selected from any of the following: Option 1: PCL-gelatin composite nanofiber membrane, mass ratio 7:3~9:1, porosity 40~60%, thickness 80~180μm, elastic modulus deviation from the natural structure of the target tissue ≤20%, porosity deviation from the natural structure of the target tissue ≤20%; Option 2: Gelatin-chitosan composite nanofiber membrane, mass ratio 6:4~8:2, porosity 40~60%, thickness 80~180μm, elastic modulus deviation from the natural structure of the target tissue ≤20%, porosity deviation from the natural structure of the target tissue ≤20%; The biomimetic cell infiltration layer is selected from collagen-hyaluronic acid composite hydrogel or hyaluronic acid-sodium alginate composite hydrogel. If a collagen-hyaluronic acid composite hydrogel is selected, the mass ratio should be 5:5~7:3, the porosity should be 60~80%, the thickness should be 50~120μm, the elastic modulus should deviate from the natural structure of the target tissue by ≤20%, and the porosity should deviate from the natural structure of the target tissue by ≤20%. If a hyaluronic acid-sodium alginate composite hydrogel is selected, the mass ratio is 4:6~6:4, the porosity is 60~80%, the thickness is 50~120μm, the elastic modulus deviates from the natural structure of the target tissue by ≤20%, and the porosity deviates from the natural structure of the target tissue by ≤20%. The collagen-hyaluronic acid composite hydrogel or hyaluronic acid-sodium alginate composite hydrogel is used to simulate the microenvironment for the growth of inner cells in the target tissue. The self-fixing / self-adhesive module is either a self-fixing module or a self-adhesive module. The self-fixing module is a PLGA micro-spiky array with an array height of 200~350μm and a diameter of 50~80μm. The self-adhesive module is a gelatin-dopamine adhesive layer with a thickness of 20~50μm; Both the self-fixing module and the self-adhesion module are adapted to the surface characteristics of the target tissue.

2. The biomimetic tissue equivalent patch based on nano-CRISPR assistance according to claim 1, characterized in that: The dual-target CRISPR system composite nanoparticles comprise a target plasmid and a nanocarrier, with a particle size of 80–200 nm; the target plasmid is an expression plasmid encoding the Cas protein and the corresponding target gRNA, and is selected from one of the following combinations: Combination A.VEGF gene knockout plasmid + MMP-13 gene knockout plasmid; Combination of B.VEGF gene knockout plasmid + ADAMTS-5 gene knockout plasmid; Combination of C.PDGF gene knockout plasmid + MMP-9 gene knockout plasmid; Combination of D.BFGF gene knockout plasmid + MMP-2 gene knockout plasmid.

3. The biomimetic tissue equivalent patch based on nano-CRISPR assistance according to claim 1, characterized in that: The amino acid sequence of the matrix metalloproteinase-sensitive peptide chain is selected from SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3, and the grafting density is 1.0~2.5 μmol / cm².

4. A method for preparing a biomimetic tissue equivalent patch based on nano-CRISPR assistance, characterized in that: The preparation of the biomimetic tissue equivalent patch according to any one of claims 1-3 includes the following steps: raw material pretreatment, spinning solution preparation, layered electrospinning / spraying, nano-CRISPR loading, peptide linking and branching, freeze drying, module assembly, sterilization and packaging. The electrospinning environment is controlled at 30-50% humidity and 20-26°C, and the biomimetic cell infiltration layer achieves a pore distribution equivalent to the target tissue through freeze-drying.

5. An application of a biomimetic tissue equivalent patch based on nano-CRISPR assistance, characterized in that: Used for the biomimetic tissue equivalent patch as described in any one of claims 1-4, and applied in the preparation of implantable repair devices for defects in the annulus fibrosus of the intervertebral disc, articular cartilage, or tendon. The implantable repair device is implanted into the body through minimally invasive surgery. Its biomimetic structure is equivalent to the mechanical properties and pore distribution of the target tissue. The nano-CRISPR enables precise dual-target regulation to inhibit abnormal proliferation and matrix degradation and promote tissue function regeneration.

6. The application of a biomimetic tissue equivalent patch based on nano-CRISPR assistance according to claim 5, characterized in that: Application in the preparation of surface / minimally invasive repair instruments for skin wound defects; The skin wounds include chronic ulcer wounds, acute traumatic wounds, and burn healing wounds. The repair device is applied to the wound site through self-adhesive fixation or minimally invasive bonding. The biomimetic structure simulates the natural layered structure of the skin, and nano-CRISPR is used to promote epithelialization of the wound and reduce scar formation and recurrence.

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