WO2020258883A1

WO2020258883A1 - Biological scaffold and preparation method therefor and use thereof

Info

Publication number: WO2020258883A1
Application number: PCT/CN2020/074195
Authority: WO
Inventors: 马少华; 赵浩然; 蒋盛威
Original assignee: 清华-伯克利深圳学院筹备办公室
Priority date: 2019-06-27
Filing date: 2020-02-03
Publication date: 2020-12-30
Also published as: CN110180026A; WO2020258883A9; CN110180026B

Abstract

A biological scaffold and a preparation method therefor and use thereof. The biological scaffold comprises a biological matrix and long chain DNA entangled in the biological matrix; the biological scaffold has a pore structure therein. The biological scaffold employs the biological matrix as the main scaffold material, long chain DNA is entangled in the biological matrix as a mechanical lock to lock a biological matrix fiber, the sol containing a large amount of monodisperse droplets is quickly gelated under the action of shear force, shortening the gelation time of the natural biological matrix, and increasing the gelation speed by 10 to 100 times, the pore structure promotes the exchange of substances in the biological matrix, providing oxygen and nutrients for the cells, discharging metabolic wastes, and improving the survival of the cell transplantation.

Description

Biological scaffold and preparation method and application thereof

Technical field

This application belongs to the field of biomedical technology, and relates to a biological scaffold and its preparation method and application.

Background technique

In recent years, cell transplantation therapy has made great progress and has been widely used in the treatment of trauma, endometrial injury, heart failure, liver failure, spinal cord injury and type I diabetes. In regenerative medicine, the biological scaffold that supports cell growth plays a vital role in simulating the growth and development of human organs. Under the dual regulation of physical factors (such as viscoelasticity, porosity, surface morphology) and biological factors (such as biocompatibility, biodegradability, antigenicity/immunogenicity), the new scaffold material can be reshaped by cells , Promote morphogenesis, bind to cell surface receptors and release growth factors.

At present, the most common biomatrix gel in clinical practice is hyaluronic acid (HA), which is mainly used to prevent tissue adhesion after operation, such as filling the uterine cavity with cross-linked sodium hyaluronate gel to prevent postoperative adhesion. . Biomatrices such as collagen are mostly used in skin care products and health care products (oral), rarely used in clinical tissue repair related treatment methods, and rarely used directly as cell scaffolds for tissue repair. However, collagen accounts for about 1/3 of the total protein of the organism and is an important structure of the connective tissue. HA also shows excellent performance in various clinical indicators, and plays a very important role in tissue repair and regenerative medicine.

There have been studies using natural extracellular matrix (ECM) such as collagen, fibronectin, and elastin as a scaffold material to simulate the cellular microenvironment. Among them, type I collagen is the most abundant protein in the human body. It mainly exists in the form of fibers in the skin, tendons, vascular tissue, internal organs and bones. It has the functions of cell remodeling and transduction of biological and mechanical signals to cells. However, natural ECM materials are very soft (Young's modulus E<200Pa), gel is slow, and cannot maintain structural integrity under the action of shearing force. Therefore, it is necessary to provide a new method to enhance the engineering properties of the natural ECM scaffold while retaining its cellular remodeling.

Munoz-pinto, etc.(Munoz-pinto, DJ, Jimenez-vergara, AC, Gharat, TP&Hahn, MS(2015) Characterization of sequential collagen-poly(ethylene glycol)diacrylate interpenetrating networks and initial asset rating issue of their potential engineering for vascular. Biomaterials 40,32-42.) Adding polymers with engineering properties to collagen, such as alginate, gelatin, and polyethylene glycol, forms an interpenetrating network with collagen, which overcomes the limitations of collagen applications. However, due to the inherent biodegradability, porosity, viscoelasticity and immunogenicity of natural collagen, the interpenetrating network formed has gradually weakened the promotion of cell remodeling and morphogenesis. DNA molecules have highly programmable properties and have been considered as a promising bio-scaffold material in the fields of smart hydrogels, controlled release and tissue engineering. However, the use of DNA gel as a scaffold material for cell culture is limited due to its high cost, easy degradation by nucleases, lack of mechanical transduction properties, and lack of the viscoelasticity, porosity and surface morphology of natural ECM. Widely used in the field of regenerative medicine.

Cells that are 200μm deep in the matrix or far away from the capillaries, due to the lack of oxygen and nutrients, and the inability of metabolic waste to be discharged through the porous matrix, cannot perform normal functions and have a low survival rate. At present, the cell microenvironment engineering limits the particle size of the microgel to be less than 400μm to ensure sufficient material transportation and overcome the problem of forming functional blood vessels in large-volume tissues. Due to its small size and only a limited number of cells, microgels cannot be used in the field of regenerative medicine.

This problem can be solved by implanting a microgel containing cells. The cells in the microgel reshape the scaffold to form a complete scaffold structure. Compared with large-scale tissue transplantation technology, microgel transplantation is injectable, minimally invasive and low immunogenic. In addition, the fluid interstitium in the microgel is conducive to providing sufficient nutrients and oxygen to the cells during transplantation and vascularization, and expelling metabolic waste.

The preparation methods of microgels include flow lithography, PRINT (particle replication in the non-wetting template) and step and flash imprint lithography (step and flash imprint lithography). However, the above method requires the use of high-end modalities and is only suitable for rigid and fast gel materials. Microfluidic technology based on PDMS or coaxial glass capillaries can be used to prepare monodisperse microgels. This method uses gel precursor droplets as a template for the preparation of microgels, but requires the addition of cytocompatible surfactants. To stabilize the water-oil interface, repeated washing and centrifugation are required after the droplets are gelled to induce oil-water phase transfer.

Therefore, a new type of biological scaffold material is provided, which not only has excellent mechanical properties, but also has good biocompatibility and biodegradability, low antigenicity and immunogenicity, simple preparation process, low cost, and promotes natural The functionalization of ECM materials has important significance and broad application prospects in the field of regenerative medicine.

Summary of the invention

In view of the shortcomings of the prior art, this application provides a biological scaffold and a preparation method and application thereof. The biological scaffold uses a biological matrix as the main material and uses long-strand DNA as an interpenetrating scaffold. Under the premise of plasticity, it provides proper engineering performance, improves the gel speed, strengthens the structural stability of the gel, and provides a new way for functionalized natural ECM materials.

To achieve this goal, this application adopts the following technical solutions:

In the first aspect, the present application provides a biological scaffold, which includes a biological matrix and long-chain DNA entangled in the biological matrix; and

The biological scaffold has a pore structure.

In this application, the biological scaffold uses biological matrix glue as the main scaffold material. Long-strand DNA is entangled in the biological matrix to act as a mechanical lock to lock the biological matrix fibers. The sol containing a large number of monodisperse droplets quickly coagulates under the action of shearing force. Glue, shorten the gelation time of natural biological matrix.

Preferably, the biological matrix includes any one or a combination of at least two of collagen, fibronectin and elastin, preferably collagen.

Preferably, the length of the long-chain DNA is not less than 10000nt, for example, it can be 10000nt, 20000nt, 50000nt, 100000nt or 200000nt.

In this application, long-chain DNA with a length of not less than 10,000 nt is added to the biological matrix, so that the long-chain DNA can quickly form a tangled structure at a lower weight fraction, which significantly increases the viscosity of the sol; After the junction structure reaches a certain density, the sol gels.

Preferably, the biological scaffold also includes a complementary strand of long-strand DNA;

The long-strand DNA hybridizes with the complementary strand to form an interlocking structure.

In this application, the complementary strand of long-strand DNA is added, and the biological matrix has a certain steric hindrance to the combination of long-strand DNA and complementary strand. The long-strand DNA and complementary strand partially hybridize under the action of dynamic driving force. A mechanical interlocking structure is formed in the matrix, which realizes the tight interlocking of the chain structure of the biological matrix.

In this application, due to the steric hindrance of the biological matrix, the long-strand DNA and complementary strands basically do not hybridize under static conditions, and the sol maintains fluidity in a low-temperature environment.

Preferably, the biological matrix is loaded with cells.

Preferably, the long-chain DNA and/or the complementary strand of the long-chain DNA include functional sequences, preferably including any one or at least two of drug loading sites, RNA complementary sites and nucleic acid aptamer sites It is further preferred that the combination includes a nucleic acid aptamer site.

Preferably, the nucleic acid aptamer targets a functional protein, preferably a target growth factor, and more preferably a target vascular endothelial growth factor.

This application introduces a nucleic acid aptamer with specificity and high affinity for growth factors into the long-stranded DNA by special design of the DNA sequence. When the DNA is degraded by nuclease, the growth factor can be released from the biological scaffold in a controlled manner to promote Cell remodeling and morphogenesis of the scaffold are improved. In the specific embodiment of the present application, when vascular endothelial growth factor (VEGF) is connected to a nucleic acid aptamer and slowly released into the endothelial microenvironment, the formation of blood vessels is promoted.

Preferably, the long-chain DNA amplification template includes the nucleic acid molecule described in SEQ ID NO:1;

The nucleotide sequence shown in SEQ ID NO:1 is:

Preferably, the complementary strand amplification template of the long-strand DNA includes a nucleic acid molecule as shown in SEQ ID NO: 2;

The nucleotide sequence shown in SEQ ID NO: 2 is:

Preferably, the pore size of the pore structure is 10-100 μm, for example, it may be 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm or 100 μm.

In the present application, the pore structure formed between the separated biological substrates promotes the exchange of substances in the biological substrates, provides oxygen and nutrients for the cells, discharges metabolic wastes, improves cell viability, and helps improve the survival rate of cell transplantation.

In the second aspect, the present application provides a method for preparing the biological scaffold as described in the first aspect, and the method includes the following steps:

(1) Design the DNA sequence and carry out rolling circle amplification to obtain long DNA and complementary strands with repetitive DNA sequences;

(2) Mix and incubate the biological matrix and the long-chain DNA in proportion to obtain the biological matrix-long-chain DNA complex; and

Mixing and incubating the biological matrix and the complementary strands of long-strand DNA in proportion to obtain a biological matrix-long-strand DNA complementary strand complex; and

(3) After mixing the biological matrix-long-chain DNA complex and the biological matrix-long-chain DNA complementary strand complex obtained in step (2), they are added to the microfluidic pipeline and reacted to obtain the biological scaffold.

Preferably, the weight ratio of the biological substrate and the long-chain DNA in step (2) is (0.2-100):1, for example, it can be 0.2:1, 1:1, 5:1, 10:1, 20: 1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1 or 100:1, preferably (0.2-50):1.

Preferably, the weight ratio of the biological matrix and the complementary strand of the long-strand DNA in step (2) is (0.2-100):1, for example, it can be 0.2:1, 1:1, 5:1, 10:1 , 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1 or 100:1, preferably (0.2-50):1.

Preferably, the pH of the incubation in step (2) is 2.0 to 6.5, for example, it can be 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 or 6.5.

In this application, under the condition of pH 2.0-6.5, the long-stranded DNA or complementary strand is negatively charged, the biological matrix is positively charged, and the DNA and the biological matrix are combined by electrostatic force.

Preferably, the incubation temperature in step (2) is 0-40°C, for example, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10℃, 11℃, 12℃, 13℃, 14℃, 15℃, 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃ , 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃ or 40℃.

Preferably, the incubation time in step (2) is 10min-24h, for example, it can be 10min, 20min, 30min, 40min, 50min, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h , 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h or 24h.

Preferably, the pH of the incubation in step (3) is 6.0 to 8.0, for example 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

Preferably, the incubation temperature in step (3) is 0-40°C, for example, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C 、10℃、11℃、12℃、13℃、14℃、15℃、16℃、17℃、18℃、19℃、20℃、21℃、22℃、23℃、24℃、25℃、26 ℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃ or 40℃.

Preferably, the incubation time in step (3) is 15s-60min, for example, it can be 15s, 20s, 30s, 40s, 50s, 1min, 10min, 20min, 30min, 40min, 50min or 60min.

Preferably, the reaction temperature in step (3) is 0-40°C, for example, it can be 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C 、10℃、11℃、12℃、13℃、14℃、15℃、16℃、17℃、18℃、19℃、20℃、21℃、22℃、23℃、24℃、25℃、26 ℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃ or 40℃.

Preferably, the reaction time in step (3) is 15s to 1.5h, for example, it can be 15s, 20s, 30s, 40s, 50s, 1min, 10min, 20min, 30min, 40min, 50min, 1h or 1.5h.

In the present application, the formation of the DNA interlocking structure promotes the occurrence of gelation and significantly shortens the gelation time. Compared with a single biological matrix, the gel speed of the DNA-biological matrix complex is increased by 10 to 100 times.

Preferably, before step (2), a step of adding functional molecules to the long-chain DNA and/or the complementary strand of the long-chain DNA is further included.

Preferably, the functional molecule includes any one or a combination of at least two of drugs, RNA and growth factors, preferably growth factors.

Preferably, the step of adding cells to the biological matrix is further included before step (2).

Preferably, step (3) further includes the step of adding the oil phase to the microfluidic pipeline.

In this application, the addition of the oil phase while adding the DNA-biomatrix complex to the microfluidic pipeline facilitates the formation of gel microspheres.

In this application, under the joint adjustment of shear force and capillary force, the DNA-biomatrix emulsifies to form monodisperse gel precursor droplets. The droplets in the pipeline are separated by the oil phase and occupy the entire cross-section of the pipeline. , To ensure that there is no collision with each other under the condition of no surfactant, which is beneficial to maintain the stability of the liquid phase.

As a preferred technical solution, this application provides a method for preparing the biological scaffold as described in the first aspect, and the method includes the following steps:

(1) Design the DNA sequence and perform rolling circle amplification to obtain long DNA and complementary strands with repetitive DNA sequences, and add drugs, RNA or growth factors to the long DNA and/or complementary strands of the long DNA;

(2) The weight ratio of the biological matrix carrying the cells to the long-strand DNA is (0.2-50):1, mixed and incubated at a pH of 2.0-6.5 and a temperature of 0-40°C for 10min-24h to obtain a biological matrix-length Strand DNA complex;

The cell-loaded biological matrix and the complementary strands of long-strand DNA are mixed and incubated at a pH of 2.0 to 6.5 and a temperature of 0 to 40°C for 10 minutes to 24 hours in a weight ratio of (0.2-50): 1 to obtain a biological matrix-long Complementary strand complex of strand DNA;

(3) After mixing the biological matrix-long-chain DNA complex and the biological matrix-long-strand DNA complementary strand complex obtained in step (2), incubating at a pH of 6.0～8.0 and a temperature of 0～40℃ for 15s～60min , Adding the oil phase into the microfluidic pipeline, at the same time adding the oil phase to the microfluidic pipeline, and reacting at 0-40°C for 15s-1.5h to obtain the biological scaffold.

In the third aspect, the present application provides a pharmaceutical composition, the pharmaceutical composition comprising the biological scaffold as described in the first aspect.

Preferably, the pharmaceutical composition further includes any one or a combination of at least two of pharmaceutically acceptable carriers, excipients and diluents.

In a fourth aspect, this application provides an application of the biological scaffold described in the first aspect and/or the pharmaceutical composition described in the third aspect in the preparation of disease treatment drugs, medical materials or medical devices.

Preferably, the disease includes any one or a combination of at least two of trauma, endometrial injury, heart failure, liver failure, spinal cord injury and type I diabetes.

Compared with the prior art, this application has the following beneficial effects:

(1) The biological scaffold of the present application uses biological matrix glue as the main scaffold material. Long-chain DNA is entangled in the biological matrix to act as a mechanical lock to lock the biological matrix fibers. The sol containing a large number of monodisperse droplets is under the action of shearing force. Quick gel, shorten the gelation time of natural biological matrix, gel speed is 10-100 times faster than natural collagen;

(2) The pore structure in the biological scaffold of the present application promotes the material exchange in the biological matrix, provides oxygen and nutrients for the cells, discharges metabolic waste, and improves the survival rate of cell transplantation;

(3) The long-chain DNA contains functional nucleic acid aptamers that target growth factors. When the DNA is degraded by nuclease, the growth factors are released from the biological scaffold in a controlled manner, which promotes the cell remodeling and morphogenesis of the scaffold. The biological scaffold of VEGF nucleic acid aptamer promotes the formation of blood vessels in the implanted tissue;

(4) The preparation method of the present application is simple, low in cost, fast in gel formation, no surfactant or chemical modification is added, and the gel morphology is accurate and controllable, and it has broad application prospects.

Description of the drawings

Figure 1 is a schematic diagram of the preparation of a biological scaffold;

Figure 2 is a bright field picture of the pellet obtained by shearing glue;

Figure 3(A) shows the survival rate of cells cultured in vitro for 1 day, Figure 3(B) shows the survival rate of cells cultured in vitro for 3 days, and Figure 3(C) shows the survival rate of cells cultured in vitro for 7 days, where PI is the death rate. Cell dye, Calcein-AM is a living cell dye;

Figure 4 is an H&E staining diagram of transplant angiogenesis in Example 9 of the application;

Figure 5 is an immunofluorescence imaging image of transplanted angiogenesis in Example 9 of the application.

Detailed ways

In order to further illustrate the technical means adopted by this application and its effects, the application will be further described below in conjunction with embodiments and drawings. It can be understood that the specific implementations described here are only used to explain the application, but not to limit the application.

For those who do not indicate specific technologies or conditions in the examples, follow the technologies or conditions described in the literature in the field or follow the product instructions. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased through formal channels.

Example 1 DNA rolling circle amplification and purification

Design a nucleic acid aptamer targeting VEGF and perform rolling circle amplification to obtain long-chain DNA with a length of more than 10,000 nt. The specific steps are as follows:

(1) Annealing: The reaction system is 150.8μL of H ₂ O, 5.2μL of 10×T4 ligase buffer, 12μL of 5μM complementary strand template, 12μL of 10μM aptamer template, and after mixing, incubate at 90℃ for 10min at 300r speed , Slowly cool to room temperature;

(2) Connection: The reaction system is 180μL of annealing product, 14.8μL of 10×T4 ligase buffer, and 5.2μL of 10U/μL T4 ligase. After mixing, incubate at 16℃ and 300r for 16h, and then at 65℃ and 300r Incubate at speed for 10 minutes and slowly cool to room temperature;

(3) Rolling circle amplification (RCA): The reaction system is H ₂ O 300μL, 10×phi29 polymerase buffer 50μL, 100mM dNTP 10μL/species (four bases), ligation product 100μL, 10U/μL phi29 polymerase 10μL, after mixing, incubate at 30°C for 36h at a rotation speed of 300r, heat to 90°C for 10min, and slowly cool to room temperature;

(4) Alcohol purification: add 0.5M EDTA to the RCA product until the RCA product becomes clear, add the same volume of phenol-chloroform-isoamyl alcohol mixture as the RCA product, shake and mix, centrifuge at 3000r for one minute, and take the supernatant. Add 1200μL ethanol, put it in the refrigerator overnight at -20℃, centrifuge at 8000r at 4℃ for 15min, remove the supernatant, put the precipitate in a vacuum drying oven and take it out, add an appropriate amount of water, heat at 90℃ for 300r until the DNA is completely dissolved In 1×PBS buffer, slowly cooled to room temperature to obtain purified RCA products SEQ ID NO: 1 and SEQ ID NO: 2.

Example 2 Preparation of biological scaffold containing VEGF

The schematic diagram of the preparation of the biological scaffold is shown in Figure 1. The long-chain DNA of the rolling circle amplification product containing functional sites is entangled in the biological matrix after binding to functional molecules, which acts as a mechanical lock to lock the biological matrix fibers, and the complementary chain Partial hybridization occurs, forming a mechanical interlocking structure in the biological matrix, and the sol containing a large number of monodisperse droplets quickly gels under the action of shearing force to form a biological scaffold.

The steps of the preparation method of the biological scaffold containing VEGF are as follows:

(1) Mix 5 μL of 0.01 mg/mL VEGF165 with 10 μL of 2 mg/mL RCA product SEQ ID NO:1, and incubate at 30°C for 30 minutes at a speed of 300r;

(2) Take 100μL of type I collagen (containing 1×PBS buffer) at a concentration of 2mg/mL, and mix with 2mg/mL of 10μL RCA product SEQ ID NO:1, 20μL RCA under the condition of pH 6.0 The complementary chain (c-RCA) SEQ ID NO: 2 is mixed and placed in a metal bath shaker at 4°C and incubated at 1000r for 30min;

(3) After adjusting the pH of the system to 7.0 with 1.0M NaOH, place it in a metal bath shaker at 4°C and incubate at a speed of 1000r for 30 minutes to obtain RCA-collagen complex (RCA-Col) and RCA complementary chain-collagen complex ( c-RCA-Col);

(4) Shake and mix RCA-Col and c-RCA-Col at 37°C for about 1 hour to obtain DNA-collagen complex (DNA-Col), which is stored at 4°C until use.

The results are shown in Figure 2. The DNA-Col was shaken and mixed at 37°C for 1 hour. The sol containing a large number of monodisperse droplets quickly gelled under the action of shearing force, while the transaction time of the natural biological matrix was about 10-100 hours. The preparation method of this embodiment significantly shortens the gelation time of the natural biological matrix, and the gelation speed is increased by 10 to 100 times.

Example 3 Preparation of biological scaffold containing VEGF

(2) Take 500μL of type I collagen (containing 1×PBS buffer) at a concentration of 2mg/mL, and mix with 2mg/mL of 10μL RCA product SEQ ID NO:1, 20μL RCA under the condition of pH 6.5 The complementary chain (c-RCA) SEQ ID NO: 2 is mixed and placed in a metal bath shaker at 0°C and incubated at 1000r for 24h;

(3) After adjusting the pH of the system to 8.0 with 1.0M NaOH, place it in a metal bath shaker at 0°C and incubate at 1000r for 60 minutes to obtain RCA-collagen complex (RCA-Col) and RCA complementary chain-collagen complex ( c-RCA-Col);

(4) Shake and mix RCA-Col and c-RCA-Col at 0°C for about 1.5 hours to obtain DNA-collagen complex (DNA-Col), which is stored at 4°C until use.

Example 4 Preparation of biological scaffold containing VEGF

(2) Take 2μL of type I collagen (containing 1×PBS buffer) at a concentration of 2mg/mL, and mix with 2mg/mL of 10μL RCA product SEQ ID NO:1, 20μL RCA under the condition of pH 2.0 The complementary chain (c-RCA) SEQ ID NO: 2 is mixed and placed in a metal bath shaker at 40°C and incubated at 1000r for 10 min;

(3) After adjusting the pH of the system to 6.0 with 1.0M NaOH, place it in a metal bath shaker at 40°C and incubate for 15s at a speed of 1000r to obtain RCA-collagen complex (RCA-Col) and RCA complementary chain-collagen complex ( c-RCA-Col);

(4) Shake and mix RCA-Col and c-RCA-Col at 40°C for about 15s to obtain DNA-collagen complex (DNA-Col), which is stored at 4°C until use.

Example 5 Preparation of bio-scaffold containing functional drugs

(1) Mix 5 μL of 0.01 mg/mL doxorubicin (DOX) with 10 μL of 2 mg/mL RCA product containing drug-carrying sites, and incubate for 30 minutes at 30°C and 300 rpm;

(2) Take 1000μL of fibronectin (containing 1×PBS buffer) at a concentration of 2mg/mL, and mix with 2mg/mL of 10μL RCA product and 20μL RCA complementary chain (c- RCA) Mix well, put it in a metal bath shaker at 4°C and incubate at 1000r speed for 30min;

(3) After adjusting the pH of the system to 7.0 with 1.0M NaOH, place it in a metal bath shaker at 4°C and incubate at a speed of 1000r for 30 minutes to obtain RCA-fibronectin complex and RCA complementary chain-fibronectin complex;

(4) The two complexes obtained in step (3) were shaken and mixed at 37°C for about 1.5 hours to obtain the DNA-fibronectin complex, which was stored at 4°C for later use.

Example 6 Preparation of biological scaffold containing functional RNA

(1) Mix 5μL of 0.01mg/mL functional RNACOX_2 primer sequence with 10μL of 2mg/mL RCA product carrying RNA complementary sequence, and incubate for 30min at 30℃ and 300r;

(2) Take 100μL of elastin (containing 1×PBS buffer) at a concentration of 2mg/mL, and mix with 2mg/mL of 10μL RCA product, 20μL RCA complementary chain (c-RCA) under the condition of pH 6.0. ) Mix well and incubate in a metal bath shaker at 4°C for 30 minutes at a speed of 1000r;

(3) After adjusting the pH of the system to 7.0 with 1.0M NaOH, place it in a metal bath shaker at 4°C and incubate at 1000r for 30min to obtain RCA-elastin complex and RCA complementary chain-elastin complex;

(4) The two complexes obtained in step (3) were shaken and mixed at 37°C for about 1.5 hours to obtain the DNA-elastin complex, which was stored at 4°C for later use.

Example 7 Preparation of Gel Microspheres

(1) Mix RCA-Col and c-RCA-Col and add it to the syringe as the water phase. Due to steric hindrance, the DNA will not complement each other and form a gel temporarily;

(2) Add fluorine oil to another syringe as the oil phase;

(3) Inject two syringe pumps into the three-way structure at a speed of 1:2 oil phase: water phase;

(4) The oil phase divides the water phase into small balls, and provides shearing force for the water phase by flowing in the microtubes, breaking the steric hindrance effect, making the DNA complementary, and the small balls become gelled to form gel microspheres. The diameter of the microspheres is about 800 μm.

Example 8 The pore structure enhances the material exchange in the biological scaffold

According to the above method, microtubes of different diameters were used to prepare cell-containing microsphere DNA-Col with particle diameters of about 300, 800, and 1000, and cultured in vitro.

The structure is shown in Figure 3(A), Figure 3(B) and Figure 3(C). It can be seen that over time, the cell metabolism in the microsphere DNA-Col is vigorous, even if the cells in the 1000μm microsphere After being cultured in vitro for a week, it still has a high survival rate, indicating that the pore structure of the microspheres is conducive to effective material exchange among internal cells.

The prepared DNA-fibronectin complex and DNA-elastin complex also have the effect of promoting material exchange in internal cells.

Example 9 VEGF promotes angiogenesis in tissue implanted with blood vessels

The DNA-Col pellets mixed with human venous endothelial cells (HUVEC) were subcutaneously injected into balb/c mice subcutaneously. After two weeks, they were taken out for H&E staining and immunofluorescence labeling. The results are shown in Figure 4 and Figure 5. Complete vascular structure generation, where the left column of Figure 5 is the vascular structure formed by cells marked by the targeting marker of human vascular vein endothelial cells, the middle column is DAPI-stained cell nuclei (for cell positioning), and the right column is the superimposed field .

In summary, the biological scaffold of the present application uses biological matrix glue as the main scaffold material. Long-chain DNA is entangled in the biological matrix as a mechanical lock to lock the biological matrix fibers. The sol containing a large number of monodisperse droplets is affected by the shearing force. Under the action of rapid gelation, the gelation time of the natural biological matrix is shortened; the pore structure in the biological scaffold promotes the material exchange in the biological matrix, provides oxygen and nutrients for the cells, discharges metabolic waste, and improves the cell The long-chain DNA contains functional aptamers that target growth factors. When the DNA is degraded by nuclease, the growth factors are released from the biological scaffold in a controlled manner, which promotes the cell remodeling and Morphogenesis; the preparation method of the present application is simple, the gel forming speed is fast, no chemical modification is added, and it has broad application prospects.

The applicant declares that this application uses the above-mentioned embodiments to illustrate the detailed methods of this application, but this application is not limited to the above-mentioned detailed methods, which does not mean that this application must rely on the above-mentioned detailed methods to be implemented. Those skilled in the art should understand that any improvement to this application, the equivalent replacement of each raw material of the product of this application, the addition of auxiliary components, the selection of specific methods, etc., fall within the scope of protection and disclosure of this application.

Claims

A biological scaffold includes a biological matrix and long-chain DNA entangled in the biological matrix; and the biological scaffold has a pore structure.
The biological scaffold according to claim 1, wherein the biological matrix comprises any one or a combination of at least two of collagen, fibronectin and elastin, preferably collagen.
The biological scaffold according to claim 1 or 2, wherein the length of the long-stranded DNA is not less than 10000 nt.
The biological scaffold according to any one of claims 1 to 3, wherein the biological scaffold further comprises a complementary strand of long DNA,

Wherein, the long-strand DNA hybridizes with the complementary strand to form an interlocking structure.
The biological scaffold according to any one of claims 1 to 4, wherein the biological matrix is loaded with cells.
The biological scaffold according to any one of claims 1-5, wherein the long-strand DNA and/or the complementary strand of the long-strand DNA includes functional sequences, preferably including drug loading sites and RNA complementary sites And any one or a combination of at least two of the nucleic acid aptamer sites, and further preferably include a nucleic acid aptamer site;

Preferably, the nucleic acid aptamer targets a functional protein, preferably a target growth factor, and more preferably a target vascular endothelial growth factor.
The biological scaffold according to any one of claims 1-6, wherein the long-strand DNA comprises the nucleic acid molecule set forth in SEQ ID NO:1;

Preferably, the complementary strand of the long-strand DNA includes a nucleic acid molecule as shown in SEQ ID NO: 2.
The biological scaffold according to any one of claims 1-7, wherein the pore size of the pore structure is 10-100 μm.
A method for preparing a biological scaffold according to any one of claims 1-8, which comprises the following steps:

(1) Design the DNA sequence and carry out rolling circle amplification to obtain long DNA and complementary strands with repetitive DNA sequences;

(2) Mix and incubate the biological matrix and the long-chain DNA in proportion to obtain the biological matrix-long-chain DNA complex; and

Mix and incubate the biological matrix and the complementary strands of the long-chain DNA in proportion to obtain the biological matrix-long-chain DNA complementary strand complex; and

(3) After mixing the biological matrix-long-chain DNA complex and the biological matrix-long-chain DNA complementary strand complex obtained in step (2), they are added to the microfluidic pipeline and reacted to obtain the biological scaffold.
The preparation method according to claim 9, wherein the weight ratio of the biological substrate to the long-chain DNA in step (2) is (0.2-100):1, preferably (0.2-50):1;

Preferably, the weight ratio of the biological matrix and the complementary strand of the long-strand DNA in step (2) is (0.2-100):1, preferably (0.2-50):1.
The preparation method according to claim 10, wherein the pH of the incubation in step (2) is 2.0-6.5;

Preferably, the incubation temperature in step (2) is 0-40°C;

Preferably, the incubation time in step (2) is 10min-24h;

Preferably, the pH of the incubation in step (3) is 6.0-8.0;

Preferably, the incubation temperature in step (3) is 0-40°C;

Preferably, the incubation time in step (3) is 15s-60min;

Preferably, the reaction temperature in step (3) is 0-40°C;

Preferably, the reaction time in step (3) is 15s to 1.5h.
The preparation method according to any one of claims 9-11, wherein before step (2), it further comprises a step of adding functional molecules to the long-strand DNA and/or the complementary strand of the long-strand DNA;

Preferably, the functional molecule includes any one or a combination of at least two of drugs, RNA and growth factors, preferably growth factors;

Preferably, before step (2), it further includes the step of adding cells to the biological matrix;

Preferably, step (3) further includes the step of adding the oil phase to the microfluidic pipeline.
The preparation method according to any one of claims 9-12, wherein the method comprises the following steps:

(1) Design the DNA sequence and perform rolling circle amplification to obtain long DNA and complementary strands with repetitive DNA sequences, and add drugs, RNA or growth factors to the long DNA and/or complementary strands of the long DNA;

(2) The weight ratio of the biological matrix carrying the cells to the long-strand DNA is (0.2-50):1, mixed and incubated at a pH of 2.0-6.5 and a temperature of 0-40°C for 10min-24h to obtain a biological matrix-length Strand DNA complex;

The cell-loaded biological matrix and the complementary strands of long-strand DNA are mixed and incubated at a pH of 2.0 to 6.5 and a temperature of 0 to 40°C for 10 minutes to 24 hours in a weight ratio of (0.2-50): 1 to obtain a biological matrix-long Complementary strand complex of strand DNA;

(3) After mixing the biological matrix-long-chain DNA complex and the biological matrix-long-strand DNA complementary strand complex obtained in step (2), incubating at a pH of 6.0～8.0 and a temperature of 0～40℃ for 15s～60min , Adding the oil phase into the microfluidic pipeline, at the same time adding the oil phase to the microfluidic pipeline, and reacting at 0-40°C for 15s-1.5h to obtain the biological scaffold.
A pharmaceutical composition, wherein the pharmaceutical composition comprises the biological scaffold according to any one of claims 1-7;

Preferably, the pharmaceutical composition further includes any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient and diluent.
An application of the biological scaffold according to any one of claims 1-7 and/or the pharmaceutical composition according to claim 9 in the preparation of disease treatment drugs, medical materials or medical devices;

Preferably, the disease includes any one or a combination of at least two of trauma, endometrial injury, heart failure, liver failure, spinal cord injury and type I diabetes.