DNA-silk fibroin composite hydrogel microsphere and preparation method therefor

Abstract

The present invention relates to the field of biomedical materials. Particularly provided is a DNA-silk fibroin composite hydrogel microsphere. The DNA-silk fibroin composite hydrogel microsphere is prepared from DNA and silk fibroin on the basis of the micro-fluidic technology by means of combination of self-assembly physical crosslinking and photo-crosslinking. The introduction of a silk fibroin network into the composite hydrogel microsphere can provide mechanical performance for a DNA hydrogel network and reduce the production cost. RGD can remarkably improve the cell loading capability of the microsphere. The hydrogel microsphere can simulate a microenvironment of cartilage tissue by means of three-dimensional loading of high-density cells, so as to enhance substance exchange and signaling between cells, and more effectively promote the proliferation and adhesion of bone marrow mesenchymal stem cells and regulating the chondrogenic differentiation, thus promoting cartilage regeneration and repair, and providing an available material for preparing medicaments or medical instruments for cartilage defect repair.

Description

DNA-silk fibroin composite hydrogel microspheres and preparation method thereof Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to a DNA-silk fibroin composite hydrogel microsphere and a preparation method and application thereof. Background Art

[0002] Osteoarthritis is a common degenerative joint disease characterized by degeneration of articular cartilage and is one of the leading causes of disability in adults worldwide. According to statistics, over 500 million people worldwide suffer from osteoarthritis, placing a significant social and economic burden on patients and society. Currently, commonly used cartilage repair strategies, including microfracture, cartilage autografting, cartilage allografting, and implantation of processed allogeneic cartilage, are widely used in clinical practice, but each has limitations and shortcomings. For example, microfracture can lead to poor defect filling and cartilage fibrosis; cartilage autografting is limited by donor area and can cause donor-to-graft incompatibility; and cartilage allografting carries the risk of immune rejection and infection, as well as difficulties in matching allogeneic tissue with autologous cartilage, which can lead to biomechanical load imbalance and decreased joint bearing capacity. Therefore, existing methods are unable to effectively address cartilage degeneration in the long term. In recent years, tissue engineering techniques for cartilage regeneration, such as autologous cartilage cell transplantation and matrix-induced autologous chondrocyte implantation, have provided new strategies for cartilage repair and demonstrated significant success. However, because existing chondrocyte expansion strategies that rely on two-dimensional systems may lead to cell dedifferentiation, in vitro three-dimensional cell culture is needed to better maintain cell morphology, function, and the biomechanical integrity of the graft. Therefore, new technologies and methods are urgently needed to solve the problem of cartilage repair.

[0003] DNA hydrogel is a material with DNA as its main component, forming a hydrophilic three-dimensional network structure. It retains the good biocompatibility, biodegradability and programmability of DNA, and perfectly integrates the mechanical properties of its skeleton, making it one of the ideal materials for constructing cartilage organoids. Compared with pure DNA hydrogels, composite DNA hydrogels exhibit better mechanical properties. The DNA hydrogels synthesized in current research are generally measured in microliters, and the synthesis cost is high. The development of hybrid hydrogels can alleviate the high cost problem brought by pure DNA hydrogels to a certain extent. And compared with the generally weak mechanical properties of pure DNA hydrogels, composite DNA hydrogels have stronger mechanical properties. However, the use of traditional block hydrogels to construct complex functional tissues and structures remains challenging due to the low solute diffusion efficiency and the limitation of submicron applications.

[0004] DNA hydrogels synthesized in current research are generally measured in microliters, resulting in high synthesis costs. The development of hybrid hydrogels could, to some extent, alleviate the high cost associated with pure DNA hydrogels. Therefore, the introduction of a silk fibroin network could provide mechanical properties to the DNA hydrogel network and reduce costs. However, the lack of cell binding sites on the smooth microsphere surface makes it difficult to load cells, necessitating surface modification to promote cell adhesion. Therefore, we chose to use RGD-modified hydrogel microspheres to achieve efficient loading of bone marrow mesenchymal stem cells. Summary of the Invention

[0005] To address the challenges of the existing technology, the present invention provides DNA-silk fibroin composite hydrogel microspheres. These hydrogel microspheres can simulate the microenvironment of cartilage tissue by loading high-density cells in three dimensions, thereby enhancing intercellular material exchange and signal transduction. This more effectively promotes the proliferation and adhesion of bone marrow mesenchymal stem cells and regulates their differentiation into cartilage, thereby promoting cartilage regeneration and repair. The DNA-silk fibroin composite hydrogel microspheres are prepared using microfluidic technology by combining self-assembly, physical crosslinking, and photocrosslinking of DNA and silk fibroin.

[0006] The purpose of the present invention can be specifically achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a DNA-silk fibroin composite hydrogel microsphere. The hydrogel can be prepared by combining DNA and silk fibroin through self-assembly physical crosslinking and photocrosslinking using microfluidic technology.

[0008] Furthermore, the DNA is a DNA supramolecular network composed of Y-shaped DNA nanounits and L-shaped DNA nanounits.

[0009] Furthermore, the Y-shaped DNA nanounit comprises three DNA single strands (Y1, Y2 and Y3) of 39 nucleotides, each of which has three sticky ends of 13 nucleotides.

[0010] Furthermore, the L-shaped DNA nanounit comprises two DNA single strands (L1 and L2) of 44 nucleotides, each of which has a sticky end of 13 nucleotides at both ends, which can be completely complementary to the sticky end of the Y-shaped DNA nanounit.

[0011] Furthermore, the nucleotide sequences of the Y-shaped and L-shaped DNA single strands are as follows:

[0012] In a second aspect, the present invention provides a method for preparing the DNA-silk fibroin composite hydrogel microspheres of the present invention, the specific steps comprising:

[0013] Step 1, construction of DNA supramolecular network: Y-shaped and L-shaped DNA single strands are mixed in phosphate buffered saline solution, and the first network structure of DNA supramolecular is formed by cross-linking the sticky ends;

[0014] Step 2, preparation of DNA-silk fibroin composite hydrogel microspheres:

[0015] Preparation of a premix: Mixing methacryloylated silk fibroin and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate with the DNA solution in step 1 to obtain a DNA-silk fibroin premix;

[0016] Preparation of microspheres: Using coaxial needles combined with microfluidics technology, microspheres are formed under ultraviolet light irradiation;

[0017] Step 3, surface cleaning of the microspheres: washing the microspheres with a phosphate buffered saline solution containing 30% ethanol to remove the paraffin on the surface, and then washing with a phosphate buffered saline solution multiple times to remove the residual ethanol;

[0018] Step 4, surface modification of microspheres: RGD solution was added dropwise to the microspheres, and irradiated under ultraviolet light for 30 seconds for surface RGD modification to obtain the final DNA-silk fibroin composite hydrogel microspheres.

[0019] Furthermore, the concentration of the single-stranded DNA in step 1 is 500 nmol / mL.

[0020] Furthermore, the concentration of the silk fibroin dissolved in the DNA solution in step 2 is 10 wt %, and the concentration of 2,4,6-trimethylbenzoyl lithium phosphate is 0.25 wt %.

[0021] Furthermore, in step 2, a coaxial needle having an inner diameter of 340 μm and an outer diameter of 1110 μm and an outer needle having an inner diameter of 640 μm and an outer diameter of 1490 μm was used.

[0022] Furthermore, the water phase flow rate of the microfluidic system described in step 2 was 5 μL / min, the oil phase flow rate was 50 μL / min, and the UV light was set to 365 nm wavelength and 8.0 mW cm -2 Light intensity.

[0023] Furthermore, the RGD solution in step 4 is prepared by dissolving 2.5 wt % of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate and 5 wt % of acryloyl RGD peptide in phosphate buffered saline.

[0024] Furthermore, the ultraviolet light in step 4 is 365 nm in wavelength and 8.0 mW cm -2 strength.

[0025] Furthermore, the preparation method of the DNA-silk fibroin composite hydrogel microspheres specifically comprises the following steps:

[0026] Step 1, formation of a DNA supramolecular network: Y-shaped and L-shaped DNA single strands at a concentration of 500 nmol / mL were thoroughly mixed in a phosphate buffered saline solution at pH 7.4 at 37°C to rapidly form a first network structure through cross-linking of the sticky ends;

[0027] Step 2, preparation of DNA-silk fibroin composite hydrogel microspheres:

[0028] Preparation of DNA-silk fibroin premix: Add 10 wt% methacrylylated silk fibroin and 0.25 wt% lithium phenyl (2,4,6-trimethylbenzoyl) phosphate to the DNA solution in step 1 and mix well to obtain a premix;

[0029] Preparation of microspheres: An inner needle with an inner diameter of 340 μm and an outer diameter of 1110 μm and an outer needle with an inner diameter of 640 μm and an outer diameter of 1490 μm were used. The premixed solution and liquid paraffin containing 5 wt% Span 80 were injected into the inner and outer inlets of the coaxial needle, respectively. The flow rates of the aqueous phase and the oil phase were adjusted to 5 μL / min and 50 μL / min, respectively. The wavelength was 365 nm and the power was 8.0 mW cm -2 Cross-linking was performed under ultraviolet light of high intensity, and the microspheres were collected using a 15 ml centrifuge tube;

[0030] Step 3, cleaning of the microspheres: using a phosphate buffered saline solution containing 30% ethanol to clean the paraffin on the surface of the microspheres, and then washing with a phosphate buffered saline solution multiple times to remove the residual ethanol;

[0031] Step 4: RGD modification of the microsphere surface: 2.5 wt% of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and 5 wt% of acryloyl RGD peptide were dissolved in phosphate buffered saline solution, added dropwise to the microspheres, and then irradiated at a wavelength of 365 nm and 8.0 mW cm -2 The film was irradiated with ultraviolet light for 30 seconds to achieve cross-linking.

[0032] In a third aspect, the present invention provides a use of the DNA-silk fibroin composite hydrogel microspheres of the present invention in the preparation of a medicament or medical device for repairing cartilage defects.

[0033] Compared with the prior art, the present invention has the following effects:

[0034] First, the introduction of the silk fibroin network in the present invention can provide mechanical properties for the DNA hydrogel network and reduce costs;

[0035] Secondly, the RGD introduced in the present invention can significantly enhance the ability of microspheres to load cells;

[0036] Third, the present invention can simulate the microenvironment of cartilage tissue by three-dimensionally loading high-density cells, thereby enhancing material exchange and signal conduction between cells, more effectively promoting the proliferation, adhesion and regulation of cartilage differentiation of bone marrow mesenchymal stem cells, and thus promoting cartilage regeneration and repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a flow chart of the preparation of DNA-silk fibroin composite hydrogel microspheres obtained in Example 1 of the present invention.

[0038] Figure 2. Actual images of DNA-silk fibroin composite hydrogel microspheres without RGD modification and DNA-silk fibroin composite hydrogel microspheres with RGD modification prepared in Example 1 of the present invention.

[0039] FIG3 is a scanning electron micrograph of DNA-silk fibroin composite hydrogel microspheres without RGD modification and DNA-silk fibroin composite hydrogel microspheres with RGD modification prepared in Example 2 of the present invention.

[0040] FIG4 is a graph showing the swelling curves of the DNA-silk fibroin composite hydrogel microspheres without RGD modification and the DNA-silk fibroin composite hydrogel microspheres with RGD modification prepared in Example 2 of the present invention in phosphate buffer solution.

[0041] FIG5 is a diagram showing the results of a cytoskeleton staining experiment of bone marrow mesenchymal stem cells loaded with DNA-silk fibroin composite hydrogel microspheres prepared in Example 3 of the present invention.

[0042] FIG6 is a graph showing the results of Alcian blue staining experiments of DNA-silk fibroin composite hydrogel microspheres without RGD modification and DNA-silk fibroin composite hydrogel microspheres with RGD modification prepared in Example 3 of the present invention.

[0043] Figure 7. Graph showing the results of immunostaining experiments on cartilage organoid precursors constructed using DNA-silk fibroin composite hydrogel microspheres in Example 4 of the present invention.

[0044] Figure 8. Macroscopic observation of cartilage repair using DNA-silk fibroin composite hydrogel microspheres and cartilage organoid precursors in Example 5 of the present invention.

[0045] Figure 9 is a graph showing the results of a small animal fluorescence imaging experiment on the in vivo degradation of DNA-silk fibroin composite hydrogel microspheres in Example 6 of the present invention. DETAILED DESCRIPTION

[0046] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several variations and improvements can be made without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0047] The present invention has not been completed. Unless otherwise specified, the experimental reagents, materials or instruments used in the examples were purchased from commercial products.

[0048] Example 1 Preparation of DNA-silk fibroin composite hydrogel microspheres

[0049] Step 1, construction of DNA supramolecular network: Mixing DNA single strands: 500 nmol / mL of Y-shaped and L-shaped DNA single strands were mixed in a pH 7.4 phosphate buffer solution at 37°C, and the first network structure of the DNA supramolecular was formed by cross-linking the sticky ends.

[0050] Step 2, preparation of DNA-silk fibroin composite hydrogel microspheres

[0051] Preparation of premix: 10 wt % of methacryloylated silk fibroin and 0.25 wt % of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate were mixed with the DNA solution in step 1 to obtain a DNA-silk fibroin premix.

[0052] Preparation of microspheres: Using coaxial needles combined with microfluidics, an inner needle with an inner diameter of 340 μm and an outer diameter of 1110 μm, and an outer needle with an inner diameter of 640 μm and an outer diameter of 1490 μm, water (DNA-silk fibroin premix) and oil (liquid paraffin containing 5 wt% Span 80) were injected into the inner and outer channels, respectively. The water phase flow rate was set at 5 μl / min and the oil phase flow rate was 50 μl / min. The formed droplets were irradiated at a wavelength of 365 nm and a current of 8.0 mW cm -2 The microspheres were cross-linked under ultraviolet light of high intensity and collected in 15 ml centrifuge tubes.

[0053] Step 3, surface cleaning of the microspheres: the microspheres were cleaned with a phosphate buffered saline solution containing 30% ethanol to remove the paraffin on the surface, and then washed with a phosphate buffered saline solution several times to remove the residual ethanol, thereby obtaining unmodified DNA-silk fibroin composite hydrogel microspheres.

[0054] Step 4, surface modification of microspheres: 2.5 wt% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and 5 wt% acryloyl RGD peptide were dissolved in phosphate buffered saline, added dropwise to the microspheres, and then irradiated with a wavelength of 365 nm and 8.0 mW cm -2The surface was irradiated with ultraviolet light of high intensity for 30 seconds for RGD modification to obtain the final DNA-silk fibroin composite hydrogel microspheres.

[0055] The process flow chart of the DNA-silk fibroin composite hydrogel microspheres prepared in this example is shown in Figure 1, and the actual picture is shown in Figure 2. It can be seen from the figure that the DNA-silk fibroin composite hydrogel microspheres (SD-MS) without RGD modification and the DNA-silk fibroin composite hydrogel microspheres (RSD-MS) modified with RGD both exhibit good dispersibility, size consistency and complete shape.

[0056] Example 2

[0057] This example mainly examines whether RGD modification has an effect on hydrogel microspheres. Figure 3 is a scanning electron micrograph of DNA-silk fibroin composite hydrogel microspheres prepared in Example 2 that have not been modified with RGD and DNA-silk fibroin composite hydrogel microspheres that have been modified with RGD. From the figure, it can be seen that both groups of hydrogel microspheres have porous structures, which are conducive to the transport of cellular nutrients and the discharge of metabolic waste, as well as cell adhesion. Figure 4 is the swelling curve of the two groups of hydrogel microspheres. From the figure, it can be seen that both groups of hydrogel microspheres have similar and good swelling properties. This shows that RGD modification has little effect on the surface of DNA-silk fibroin composite hydrogel microspheres.

[0058] Example 3

[0059] In order to explore the effect of DNA-silk fibroin composite hydrogel microspheres on the behavior of bone marrow mesenchymal stem cells, bone marrow mesenchymal stem cells were cultured on the surface of hydrogel microspheres for nucleus staining and skeleton staining. The experimental results are shown in Figure 5. It can be seen from the figure that DNA-silk fibroin composite hydrogel microspheres can promote the proliferation and adhesion behavior of bone marrow mesenchymal stem cells; bone marrow mesenchymal stem cells were co-cultured with DNA-silk fibroin composite hydrogel microspheres, and Alcian blue staining experiments were performed after chondrogenic induction culture. The experimental results are shown in Figure 6. The experimental results show that compared with the control group and the DNA-silk fibroin composite hydrogel microsphere group without RGD modification, the staining positive signal of the DNA-silk fibroin composite hydrogel microsphere group modified with RGD is stronger, indicating that the effect of DNA-silk fibroin composite hydrogel microspheres in promoting the chondrogenic differentiation of bone marrow mesenchymal stem cells is more significant.

[0060] Example 4

[0061] Bone marrow mesenchymal stem cells were cultured on the surface of RGD-modified DNA-silk fibroin composite hydrogel microspheres and subjected to 14 days of chondrogenic induction to construct cartilage organoid precursors (COPs). The cartilage organoid precursors were paraffin-embedded and sectioned, and immunofluorescence staining for ACAN, SOX9, and CVOL II was performed. The experimental results are shown in Figure 7. The experimental results show that the cartilage organoid precursors express chondrogenic differentiation proteins such as ACAN, SOX9, and CVOL II, indicating that DNA-silk fibroin composite hydrogel microspheres can support the long-term chondrogenic differentiation of bone marrow mesenchymal stem cells.

[0062] Example 5

[0063] (1) Construction of rat cartilage defect model: SD male rats aged 7-8 weeks (weighing about 250 g) were taken and, after general anesthesia, the joint capsule was cut to expose the femoral trochlea, and a cartilage defect with a diameter of 2 mm and a depth of 1.5 mm was constructed in the middle of the femoral trochlea using a punch.

[0064] (2) Repair of cartilage defects using DNA-silk fibroin composite hydrogel microspheres and cartilage organoid precursors: The mixed solution prepared above was injected in situ into the cartilage defect. After gelation, the rats were sutured layer by layer. Rats were maintained normally after surgery and sacrificed 10 weeks later for analysis of the repair effect.

[0065] This example mainly examines the repair effects of DNA-silk fibroin composite hydrogel microspheres and cartilage organoid precursors on cartilage defects. Figure 8 shows the macroscopic observation of the femurs of each animal group at the 10th week of the experiment. Compared with the control group and the DNA-silk fibroin composite hydrogel microsphere group, almost no cartilage defects were visible in the cartilage organoid precursor group, which was closest to the sham operation group. The results show that cartilage organoid precursors can promote cartilage regeneration and repair.

[0066] Example 6

[0067] This example primarily investigates the in vivo degradation properties of DNA-silk fibroin composite hydrogel microspheres. DNA-silk fibroin composite hydrogel microspheres were immersed in a sulfonated Cy5.5-NHS solution for 1 hour to label them with Cy5.5. Subsequently, the microspheres were washed three times to remove excess fluorescent markers, thereby constructing Cy5.5-labeled microspheres. These Cy5.5-labeled DNA-silk fibroin composite hydrogel microspheres were then injected into the knee joint cavity of SD rats. An in vivo imaging system was used to assess the in vivo retention of the microspheres. Figure 9 shows that the DNA-silk fibroin composite hydrogel microspheres gradually degraded over time, demonstrating their good biodegradability. Specifically, at week 3, the fluorescence signal of the DNA-silk fibroin composite hydrogel microspheres was extremely weak, and by week 4, the fluorescence signal had almost disappeared. The appropriate degradation rate not only promoted cartilage regeneration but also demonstrated that the DNA-silk fibroin composite hydrogel microspheres had strong in vivo stability.

Claims

1. A DNA-silk fibroin composite hydrogel microsphere, characterized in that: The hydrogel microspheres are prepared by combining self-assembly physical cross-linking and photo-cross-linking of DNA and silk fibroin through microfluidic technology.

2. The composite hydrogel microsphere according to claim 1, characterized in that The DNA is a DNA supramolecular network, which is composed of Y-shaped DNA nanounits and L-shaped DNA nanounits.

3. The composite hydrogel microsphere according to claim 2, characterized in that The Y-shaped DNA nanounit comprises three DNA single strands (Y1, Y2 and Y3) of 39 nucleotides, each of which has three sticky ends of 13 nucleotides.

4. The composite hydrogel microsphere according to claim 2, characterized in that The L-shaped DNA nanounit comprises two DNA single strands (L1 and L2) of 44 nucleotides, each of which has a sticky end of 13 nucleotides at both ends, which can be completely complementary to the sticky end of the Y-shaped DNA nanounit.

5. A method for preparing the DNA-silk fibroin composite hydrogel microspheres according to any one of claims 1 to 4, characterized in that: The specific steps include: Step 1, construction of DNA supramolecular network: Y-shaped and L-shaped DNA single strands are mixed in phosphate buffered saline solution, and the first network structure of DNA supramolecular is formed by cross-linking the sticky ends; Step 2, preparation of DNA-silk fibroin composite hydrogel microspheres: Preparation of a premix: Mixing methacryloylated silk fibroin and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate with the DNA solution in step 1 to obtain a DNA-silk fibroin premix; Preparation of microspheres: Using coaxial needles combined with microfluidics technology, microspheres are formed under ultraviolet light irradiation; Step 3, surface cleaning of the microspheres: washing the microspheres with a phosphate buffered saline solution containing 30% ethanol to remove the paraffin on the surface, and then washing with a phosphate buffered saline solution multiple times to remove the residual ethanol; Step 4, surface modification of microspheres: RGD solution was added dropwise to the microspheres and irradiated under ultraviolet light for 30 seconds for surface RGD modification to obtain the final DNA-silk fibroin composite hydrogel microspheres.

6. The preparation method according to claim 5, characterized in that The concentration of the single-stranded DNA in step 1 is 500 nmol / mL.

7. The preparation method according to claim 5, characterized in that The concentration of the silk fibroin dissolved in the DNA solution in step 2 is 10 wt %, and the concentration of 2,4,6-trimethylbenzoyl lithium phosphate is 0.25 wt %.

8. The preparation method according to claim 5, characterized in that In step 2, an inner needle with an inner diameter of 340 μm and an outer diameter of 1110 μm and an outer needle with an inner diameter of 640 μm and an outer diameter of 1490 μm were used as coaxial needles. The water phase flow rate of the microfluidic system described in step 2 was 5 μl / min, the oil phase flow rate was 50 μl / min, and the UV light was set to 365 nm wavelength and 8.0 mW cm -2 Light intensity.

9. The preparation method according to claim 5, characterized in that The RGD solution in step 4 is prepared by dissolving 2.5 wt% of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and 5 wt% of acryloyl RGD peptide in phosphate buffered saline; the ultraviolet light in step 4 is 365 nm in wavelength and 8.0 mW cm -2 strength.

10. Use of the DNA-silk fibroin composite hydrogel microspheres according to any one of claims 1 to 4 in the preparation of a medicine or medical device for repairing cartilage defects.

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