Nucleic acid-modified silk fibroin biomaterial and preparation method thereof

Abstract

The application provides a nucleic acid modified silk fibroin biomaterial and a preparation method. The preparation method comprises the following steps: providing a silk fibroin solution, wherein the silk fibroin molecules in the silk fibroin solution have carboxyl and amino groups; adding an amino protecting agent into the silk fibroin solution to selectively protect the amino groups in the silk fibroin molecules, thereby obtaining amino-protected silk fibroin; adding a nucleic acid material containing amino groups and a carboxyl activating agent into the solution containing the amino-protected silk fibroin, so that the silk fibroin molecules after carboxyl activation and the nucleic acid material are subjected to amidation reaction, the nucleic acid material is covalently grafted on the silk fibroin molecules through an amide bond, and a nucleic acid modified silk fibroin biomaterial is obtained. The nucleic acid material and the silk fibroin molecules are covalently connected through an amide bond, which significantly enhances the delivery stability, slow-release effect and bioavailability of the nucleic acid material.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomaterials, and particularly relates to a nucleic acid modified silk fibroin biomaterial and a preparation method thereof. BACKGROUND

[0002] Nucleic acid materials, such as polydeoxyribonucleotide PDRN, oligonucleotide, DNA aptamer, etc., have been widely used in multiple biomedical fields such as tissue repair, anti-inflammatory regulation, angiogenesis promotion and gene expression regulation due to their good biological activity, target recognition ability and high programmability. However, nucleic acid materials face problems such as rapid enzymatic degradation in vivo, thermodynamic instability, high clearance rate in blood, etc., which lead to low bioavailability and short functional duration, seriously restricting their effective delivery and biological function in clinical treatment.

[0003] To overcome the above problems, various nucleic acid delivery systems have been developed in the prior art, including using cationic polymers, liposomes, polysaccharides or inorganic nanomaterials as delivery carriers to realize the delivery of nucleic acids through electrostatic adsorption, encapsulation or physical packaging. However, such systems have the following defects: on the one hand, the electrostatic adsorption method has limited binding stability in physiological environment and is easy to dissociate due to changes in ionic strength, leading to nucleic acid leakage and loss of activity; on the other hand, some cationic polymers and inorganic nanomaterials have potential biological toxicity or immunogenicity, limiting their long-term use in vivo. In addition, some delivery systems have complex structures and difficult to scale up in processing technology, and their ability to regulate the release behavior of nucleic acids is poor, which is difficult to meet the demand for functional controllability of nucleic acid delivery systems in application scenarios such as tissue engineering and precision therapy. SUMMARY

[0004] In order to solve the above problems, according to the first aspect of the present application, a preparation method of a nucleic acid modified silk fibroin biomaterial is provided, comprising:

[0005] Step S100, providing a silk fibroin solution, wherein the silk fibroin molecules in the silk fibroin solution have carboxyl and amino groups;

[0006] Step S200, adding an amino protecting agent to the silk fibroin solution to selectively protect the amino groups in the silk fibroin molecules, to obtain amino-protected silk fibroin;

[0007] Step S300, adding nucleic acid materials containing amino groups and a carboxyl activating agent to the solution containing the amino-protected silk fibroin, so that the silk fibroin molecules after carboxyl activation and the nucleic acid materials undergo amidation reaction, so that the nucleic acid materials are covalently grafted on the silk fibroin molecules through amide bond, to obtain a nucleic acid modified silk fibroin biomaterial.

[0008] Optionally, the step S300 comprises:

[0009] In step S310, an acid buffer is added to the solution containing the amino-protected silk fibroin to adjust the surface charge of the silk fibroin molecules.

[0010] In step S320, the nucleic acid material is added to the amino-protected silk fibroin solution after charge adjustment, so that the two form a nucleic acid-silk fibroin complex intermediate solution through electrostatic interaction.

[0011] In step S330, a carboxyl activator is added to the nucleic acid-silk fibroin complex intermediate solution, and the carboxyl-activated silk fibroin molecules are amidated with the nucleic acid material to obtain a nucleic acid-modified silk fibroin biomaterial.

[0012] Optionally, the mass ratio of the amino-protected silk fibroin solution and the nucleic acid material is 1:20-100:1.

[0013] Optionally, the acid buffer is a combination of one or more of 2-(N-morpholino) ethanesulfonic acid buffer, phosphate buffer, citric acid buffer, acetic acid buffer, tartaric acid buffer, propionic acid buffer, and succinic acid buffer.

[0014] Optionally, in step S310, an anti-aggregation agent is also added to the solution containing the amino-protected silk fibroin.

[0015] Optionally, the step of adding a carboxyl activator to the nucleic acid-silk fibroin complex intermediate solution, and amidating the carboxyl-activated silk fibroin molecules with the nucleic acid material to obtain a nucleic acid-modified silk fibroin biomaterial comprises the following steps:

[0016] The carboxyl activator is added to the nucleic acid-silk fibroin complex intermediate solution, and the temperature is raised from a first preset temperature to a second preset temperature, and maintained at a pH value of a first preset value for a first preset time.

[0017] The pH value is adjusted to a second preset value, and the mixture is mixed at the second preset temperature for a second preset time, and the second preset value is greater than the first preset value.

[0018] Small molecules or byproducts in the complex solution after reaction are separated to obtain the nucleic acid-modified silk fibroin biomaterial.

[0019] Optionally, the first preset temperature is 0-10°C, and the second preset temperature is 25-37°C.

[0020] The first preset value is 3-5, and the second preset value is 6-8.

[0021] The first preset time is 2h-4h, and the second preset time is 4h-8h.

[0022] Optionally, the step S200 comprises the following steps:

[0023] A hydrogen bond inhibitor is added into the silk fibroin solution, and the mass ratio of the silk fibroin molecules in the silk fibroin solution to the hydrogen bond inhibitor is any value in the range of 1:3-1:500;

[0024] An amino protecting agent is further added to obtain amino-protected silk fibroin, and the mass ratio of the silk fibroin molecules in the silk fibroin solution to the amino protecting agent is any value in the range of 50:1-5:1.

[0025] Optionally, the silk fibroin molecules in the silk fibroin solution are silk fibroin molecules subjected to enzymatic treatment.

[0026] According to a second aspect of the present application, a nucleic acid modified silk fibroin biomaterial is provided, in which nucleic acid materials are covalently grafted on the silk fibroin molecules through amide bonds.

[0027] According to the scheme of the present application, the introduction of the amino protecting agent effectively shields the in-situ amino active sites in the silk fibroin molecules, preventing non-specific amidation with the activated carboxyl groups in itself or other components, thereby significantly reducing the probability of background reaction and the generation of cross-linking byproducts, and improving the reaction purity and controllability of the system. Moreover, through selective activation of the carboxyl groups in the silk fibroin by the carboxyl activating agent, the activated carboxyl groups are only reactive to the exposed amino groups of the nucleic acid materials, and under the premise of amino protection, the activated carboxyl groups can be precisely directed to the amino groups of the nucleic acid materials, thereby forming specific amide bonds. In addition, the nucleic acid materials and the silk fibroin molecules are covalently connected through amide bonds, avoiding the problems of weak binding force and easy shedding caused by physical adsorption or electrostatic combination in traditional delivery systems, thereby significantly enhancing the delivery stability, slow-release effect and bioavailability of the nucleic acid materials.

[0028] The above description is only a summary of the technical scheme of the present application. In order to more clearly understand the technical means of the present application, and to implement the content of the description, the following will describe the preferred embodiments of the present application in detail. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 A schematic flow chart of a preparation method of a nucleic acid modified silk fibroin biomaterial according to an embodiment of the present application is shown;

[0030] Figure 2Pictures of the silk fibroin precipitate and the PDRN-silk fibroin composite biomaterial after freeze-drying according to the embodiment one of the present application are shown, the left sample is the silk fibroin precipitate, and the right sample is the PDRN-silk fibroin composite biomaterial;

[0031] Figure 3 Fourier transform infrared spectra of the silk fibroin precipitate after enzyme cutting according to the embodiment one of the present application and the PDRN-silk fibroin composite biomaterial prepared in the embodiment one are shown;

[0032] Figure 4 A release rate comparison chart according to the embodiment one and the comparative example one of the present application is shown.

[0033] Figure 5 Zeta potential chart of the system in step eight according to the embodiment one of the present application. DETAILED DESCRIPTION

[0034] The technical solutions of the present application are further described below in combination with specific embodiments.

[0035] Figure 1 A schematic flow chart of the preparation method of the nucleic acid modified silk fibroin biomaterial according to an embodiment of the present application is shown. As shown in the figure, Figure 1 the preparation method includes:

[0036] Step S100, providing a silk fibroin solution, the silk fibroin molecules in the silk fibroin solution have carboxyl and amino groups;

[0037] Step S200, adding an amino protecting agent to the silk fibroin solution to selectively protect the amino groups in the silk fibroin molecules, to obtain amino-protected silk fibroin;

[0038] Step S300, adding a nucleic acid material containing amino groups and a carboxyl activating agent to the solution containing the amino-protected silk fibroin, to make the silk fibroin molecules after carboxyl activation and the nucleic acid material undergo amide reaction, so that the nucleic acid material is covalently grafted on the silk fibroin molecules through amide bond, to obtain a nucleic acid modified silk fibroin biomaterial.

[0039] According to the scheme of the embodiment of the present application, the introduction of the amino protecting agent effectively shields the in-situ amino active site in the silk fibroin molecule, preventing non-specific amidation with the activated carboxyl in itself or other components, thereby significantly reducing the probability of background reaction and the generation of cross-linking byproducts, and improving the reaction purity and controllability of the system. Moreover, by selectively activating the carboxyl in the silk fibroin with a carboxyl activating agent, the carboxyl is only reactive to the amino group of the exposed nucleic acid material, and under the premise of amino protection, the activated carboxyl can be precisely directed to the amino group of the nucleic acid material, thereby forming a specific amide bond. In addition, the nucleic acid material and the silk fibroin molecule are covalently connected through an amide bond, avoiding the problems of weak binding force and easy shedding caused by physical adsorption or electrostatic binding in traditional delivery systems, thereby significantly enhancing the delivery stability, slow-release effect and bioavailability of the nucleic acid material.

[0040] It should be noted that before the present application was proposed, the skilled person in the art generally used physical methods to combine nucleic acid materials with protein carriers, such as solution blending, electrostatic adsorption, microsphere loading, hydrogel encapsulation, etc. to construct a delivery system. These methods are simple to operate and can achieve nucleic acid loading to some extent, but due to the dependence on non-covalent interaction, they have limitations such as unstable binding, easy burst release, and low drug loading in physiological environment.

[0041] In addition, since nucleic acid molecules such as PDRN, siRNA, etc. have a typical phosphate backbone structure, the whole molecule is negatively charged, highly hydrophilic, and has a long chain length and poor chemical stability, and is prone to degradation or structural denaturation under the action of temperature, pH and activating agents. Therefore, in the long-term research and practice in the field, the skilled person generally believes that nucleic acids are not suitable for direct participation in covalent reactions, especially to avoid reactions under the condition of a water-soluble coupling system containing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) and other commonly used carboxyl activating agents, in order to prevent damage to nucleic acids. Thus, a technical bias against covalent grafting strategies for nucleic acids has been formed.

[0042] The application first proposes a technical route of covalently grafting nucleic acid materials on the silk fibroin backbone by means of amino protection and carboxyl activation, which breaks through the inherent understanding of the skilled in the art that nucleic acid cannot be covalently grafted. The experimental results show that the method realizes the synergistic enhancement effect of long-period slow release, excellent hydrophilicity and promotion of cell proliferation.

[0043] In step S100, the preparation method of the silk fibroin solution comprises:

[0044] In step S110, a silk fibroin raw material is provided and dissolved in a buffer solution containing a hydrogen bond inhibitor;

[0045] In step S120, a protease is added for incubation reaction at a preset temperature, and the protease is inactivated after the incubation is completed;

[0046] In step S130, the mixed solution after reaction is subjected to centrifugal treatment to obtain a precipitate phase;

[0047] In step S140, the precipitate phase is dispersed in a pre-dispersed medium without reactive components to obtain a silk fibroin solution.

[0048] In step S110, the silk fibroin raw material is a natural silk fibroin raw material, such as silk protein. The silk fibroin raw material is dissolved in a buffer solution containing a hydrogen bond inhibitor to destroy the extensive intermolecular and intramolecular hydrogen bond structure in its natural conformation, forming a molecular state solution that can participate in subsequent reactions. The hydrogen bond inhibitor can be one or more of lithium bromide, urea, 1,6-hexanediol, and ethanolamine. The pH value of the buffer solution is preferably 6-8.5, for example, it can be 6, 7 or 8.5.

[0049] In step S120, a protease is added for incubation reaction to selectively cleave the flexible segments in the silk fibroin molecules, exposing the carboxyl functional groups shielded by the native silk fibroin structure. The preset temperature is preferably 30-45°C, for example, it can be 30°C, 40°C or 45°C. The incubation reaction time is 1-3h, for example, it can be 1h, 2h or 3h. The protease can be one or more of the biological enzymes such as trypsin, chymotrypsin, proteinase K, etc. The mass ratio of protease to silk fibroin is 1:20-1:200, more preferably 1:50-1:100. Within this range, selective hydrolysis of the flexible segments can be achieved without excessive degradation of the backbone structure. After the incubation is completed, the protease is inactivated by heating or adding metal chelating agents, etc. to terminate the hydrolysis reaction and avoid subsequent structural interference. By controlling the conditions such as the mass ratio of protease to silk fibroin, temperature and reaction time, the molecular weight of the product is tested by the method for testing molecular weight in patent publication No. CN119365865A, mainly distributed in the range of 10-50kDa, and then in subsequent step S130, the precipitate phase is separated by centrifugation and selected, further removing small molecular fragments below 10kDa and enriching segments with higher crystallinity, so as to stabilize the relative molecular mass of the precipitate phase to 10-50kDa.

[0050] In step S130, the precipitate phase of the obtained silk fibroin has a dual-phase structure characteristic. First, the β-sheet crystalline structure containing 30%-50% is retained. In some preferred embodiments, the β-sheet crystalline structure accounts for 35%-45%, and the β-sheet content is quantitatively calculated according to the method in patent publication No. CN119741964A. In addition, it is all "amorphous region", which refers to the whole of all conformations other than β-sheet crystal structure, such as random coil, α-helix, etc. The β-sheet crystalline structure can provide a stable three-dimensional skeleton framework, which not only endows the material with higher structural stability and mechanical strength, but also exposes a large number of reactive functional groups such as carboxyl and amino groups in space, thereby providing stable and intensive reaction site support for subsequent covalent grafting reaction. The amorphous region is embedded between the crystalline regions, which retains a certain proportion of flexible segments, and this part of the segment has relatively good swelling and solubility, so that the precipitate phase silk fibroin still has certain reaction accessibility and molecular flexibility while maintaining overall stability. It is the synergistic existence of the crystalline region and the amorphous region that makes the precipitate phase silk fibroin not only serve as a stable structural scaffold, but also provide sufficient segment flexibility at the molecular level.

[0051] In addition, the molecular weight of the fibroin in the precipitated phase is 10-50 kDa. Compared with the low molecular weight fibroin peptide fragments below 10 kDa commonly used in the prior art, the fibroin segment in the molecular weight range of the embodiments of the present application exhibits a significantly slowed enzymatic degradation rate and a longer half-life during in vivo degradation. Specifically, low molecular weight fragments are extremely prone to rapid degradation in a protease or hydrolysis environment due to their short chain length and insufficient crystallinity, resulting in the mechanical support of the material disappearing too quickly, and the release process of grafted functional molecules such as nucleic acids being too burst, making it difficult to maintain long-term stable effects, and due to the lack of stable spatial conformation, resulting in insufficient number and spatial distribution of reactive sites available for covalent coupling. In contrast, the precipitated phase fibroin with a molecular weight of 10-50 kDa selected by the embodiments of the present application has moderate chain length stability and partial beta-sheet crystalline regions, which can resist rapid enzymatic degradation on a certain time scale, slow down the overall degradation rate of the material, and provide sufficient reactive sites and flexible segments through amorphous regions to ensure the accessibility and uniformity of covalent grafting reactions. However, when the molecular weight of fibroin exceeds 50 kDa, the molecular chain segment is too long, and the proportion of crystalline regions increases significantly, resulting in increased overall rigidity and decreased dispersibility, which causes severe aggregation and accumulation between molecules, thereby weakening the spatial exposure of carboxyl, amino and other reactive functional groups, reducing the reaction accessibility between them and the added nucleic acid materials. At the same time, high molecular weight fibroin has significantly reduced solubility in an aqueous environment, resulting in increased system viscosity, which not only increases the difficulty of process operation, but also easily causes uneven coupling efficiency. In addition, fibroin with a molecular weight exceeding 50 kDa exhibits a too slow degradation kinetics in vivo, which can result in a too long material residue time and pose a biological compatibility risk. In contrast, by controlling the molecular weight in the range of 10-50 kDa, 30%-50% of the beta-sheet crystalline skeleton is retained to provide a stable three-dimensional framework, and 50%-70% of the amorphous segment is maintained to maintain solubility and flexibility, thereby balancing reaction accessibility, coupling efficiency and in vivo degradability.

[0052] It needs to be explained that after the enzyme cutting treatment, the silk fibroin is divided into supernatant and precipitate phase after centrifugal treatment. Among them, the supernatant part is mostly amorphous, has good hydrophilicity and processability, therefore, the person skilled in the art usually preferentially selects the supernatant as the raw material for subsequent research or composite material when further applying, and pays less attention to the precipitate phase part. The present application does not follow the above-mentioned conventional idea, but selects to use the precipitate phase after enzyme cutting as the main raw material for grafting reaction, which breaks through the technical route of relying on supernatant in the prior art. Although the precipitate phase has the disadvantages of difficult dispersion and easy agglomeration under the conventional understanding, the embodiments of the present application realize the controllable activation and efficient grafting of the precipitate phase through subsequent pH control, carboxyl activation and amino protection strategies. The present application not only overcomes the inherent cognition of the difficulty of processing the precipitate phase, but also realizes the covalent anchoring of nucleic acid molecules, thereby obtaining a binding efficiency and release stability far exceeding the prior art.

[0053] In step S140, the pre-dispersion medium is a buffer solution or aqueous solution containing no reactive functional groups, which is used to realize uniform resuspension of the silk fibroin precipitate without introducing reaction interference components. The pre-dispersion medium can be selected from one or more combinations of Tris-HCl buffer, phosphate buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, deionized water, and Milli-Q pure water. The pH value of the pre-dispersion medium is preferably 6.5-8.5, for example, it can be 6.5, 7.5 or 8.5.

[0054] The distribution of reactive sites such as carboxyl and amino groups in natural silk fibroin is complex, and random crosslinking or side reactions are prone to occur, which lacks controllability. In the embodiment of the present application, the hydrogen bond inhibitor in step S110 partially depolymerizes the silk fibroin segment, improves the spatial accessibility of the amorphous region and the potential reactive site (containing Asp / Glu side chain), and creates conditions for subsequent selective enzymatic hydrolysis. Through step S120, selective cleavage occurs preferentially in the amorphous region under controlled temperature, time and enzyme amount, so that part of the originally shielded Asp / Glu side chain and end group carboxyl are exposed, while 30%-50% of the β-sheet crystal structure skeleton is retained to enhance the controllability and structural stability of the reaction site, and 50%-70% of the amorphous region is retained as an embedded flexible segment to provide necessary swelling and molecular mobility. Through phase separation in step S130, part of the β-sheet rich and still containing some amorphous segments is enriched in the precipitated phase. Further through step S140, the structural state and site exposure state obtained in step S130 are maintained and stabilized in a medium without reactive components, avoiding further rearrangement or side reactions. Thus, a stable and controllable functional group basis is provided for subsequent carboxyl activation and directional covalent grafting of nucleic acids and / or multi-site, thereby improving the specificity and modification efficiency of grafting.

[0055] In one embodiment, the step S200 comprises:

[0056] Step S210, adding a hydrogen bond inhibitor to the silk fibroin solution;

[0057] Step S220, adding an amino protecting agent to the system in step S210 to selectively protect the reactive amino groups in the silk fibroin molecules to generate protected amino derivatives;

[0058] Step S230, after incubation for a period of time, dialysis treatment is performed to remove unreacted amino protecting agent, hydrogen bond inhibitor and by-products, and obtain amino-protected silk fibroin.

[0059] The silk fibroin solution in step S210 is the silk fibroin solution obtained in step S140. The hydrogen bond inhibitor is added to the silk fibroin solution to interfere with the hydrogen bonding between the silk fibroin molecule chains and between the silk fibroin molecule chains and the solvent molecules, promote the unfolding of the protein segments, enhance the solubility, and significantly improve the exposure degree of internal reactive sites such as amino functional groups. The hydrogen bond inhibitor can be selected from one or more of lithium bromide, urea, 1,6-hexanediol, and ethanolamine.

[0060] The dispersion concentration of the precipitated phase of silk fibroin in the hydrogen bond inhibitor solution is 2 mg / mL-100 mg / mL, more preferably 5 mg / mL-50 mg / mL, for example, it can be 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL or 50 mg / mL. The setting of this concentration parameter helps to obtain a good dispersion state and reaction activity, providing a controllable and efficient reaction basis for the subsequent amino protection reaction. The concentration of the hydrogen bond inhibitor is 0.01 mol / L-10 mol / L, more preferably 0.5 mol / L-5 mol / L, for example, it can be 0.5 mol / L, 1 mol / L, 3 mol / L or 5 mol / L, in order to ensure sufficient interference with the intermolecular hydrogen bond network of silk fibroin, thereby promoting the complete depolymerization of the protein segment and the effective exposure of the reaction site. The mass ratio of the hydrogen bond inhibitor to the precipitated phase of silk fibroin is 3:1-500:1, for example, it can be 3:1, 50:1, 100:1, 300:1, 400:1 or 500:1. Selecting the mass ratio of the hydrogen bond inhibitor to the precipitated phase of silk fibroin within the above mass ratio range can meet the balance between the destruction of intermolecular forces and the stability of protein conformation. In this step S220, the target group of the amino selective protection reaction is the primary amino functional group in the silk fibroin molecule. This primary amino group is usually located at the a-carbon position of the amino acid residue and mainly includes the N-terminal amino group and the free primary amino group in the lysine side chain. Compared with other types of amino groups, the primary amino group has higher nucleophilic activity and can preferentially participate in the formation reaction of amide bonds or carbamate bonds. There are almost no free amino groups in the form of secondary or tertiary amino groups in natural proteins, even if they exist, they are mostly introduced by subsequent chemical modification, and their reaction activity is significantly lower than that of primary amino groups.

[0061] Based on this, the amino protection agent is preferably a compound that can selectively react with the side chain amino group and form a stable blocked derivative. The amino protection agent can be selected from one or more combinations of acetic anhydride, maleic anhydride, tert-butyloxycarbonyl or its derivatives. Through the above amino selective protection treatment, the highly reactive amino site in silk fibroin can be effectively shielded to avoid non-specific crosslinking with the activated carboxyl group in the subsequent amidation coupling reaction, thereby improving the selectivity of the subsequent grafting reaction and the structural uniformity of the target product.

[0062] The mass ratio of the silk fibroin solution to the amino-protecting agent is in the range of 50:1-5:1, more preferably 20:1-30:1, for example, 20:1, 25:1 or 30:1. The ratio is set to control the amount of the amino-protecting agent to be added at a moderate level, so that it can fully react with the primary amino functional groups in the silk fibroin molecules and avoid unnecessary chemical modification of the protein backbone structure due to the excess of the amino-protecting agent. The amino-protection reaction in the above ratio range can effectively shield the target amino sites and reduce the possibility of non-specific crosslinking in the subsequent carboxyl activation process, while maximizing the stability of the spatial conformation of the silk fibroin and the reactivity as the grafting backbone.

[0063] The selective protection reaction is carried out under mild reaction conditions to avoid irreversible changes in the conformation of the silk fibroin molecules and ensure the structural stability and biocompatibility of the reaction system. Specifically, the pH value of the protection reaction system is preferably controlled in the range of 7.5-8.5, more preferably 7.8 or 8. The reaction temperature is preferably controlled in the range of 20-30°C, for example, room temperature. Under the above conditions, the amino-protecting agent can selectively react with the primary amino functional groups in the silk fibroin molecules to form stable protective groups, without causing the unfolding or aggregation and precipitation of the protein backbone. The mild conditions not only help to improve the amino-protection efficiency, but also effectively inhibit the occurrence of side reactions, thereby improving the consistency and purity of the product. In addition, the moderate pH environment can maintain the activity of the amino-protecting agent, while avoiding the alkaline degradation or conformational changes of the protein, thereby providing a high-quality precursor basis for the subsequent carboxyl activation and nucleic acid material grafting reaction.

[0064] In this step S230, the dialysis treatment process usually uses a dialysis membrane with a molecular cut-off range of 3000-10000 Da, which is selected according to the molecular weight of the target product and the reaction conditions. The dialysis treatment process can be carried out in a non-reactive solution such as deionized water, Tris-HCl buffer and / or PBS buffer at 0-4°C, and maintained for 12-48 h under the condition of continuous replacement of the dialysis solution, so as to realize the sufficient diffusion and removal of small molecular impurities. After dialysis, the obtained solution can be subjected to pretreatment operations such as low-speed centrifugation and filtration to further improve the purity and stability of the product.

[0065] After the above steps, the amino-protected silk fibroin obtained has an intact backbone structure, while the primary amino active sites are selectively shielded, which has good adaptability for subsequent reactions and can be directly used in the carboxyl activation coupling reaction system for covalent grafting of target functional molecules.

[0066] In one embodiment, the step S300 comprises:

[0067] Step S310, adding an acidic buffer to the solution containing the amino-protected silk fibroin to adjust the surface charge of the silk fibroin molecules;

[0068] Step S320, adding nucleic acid materials to the amino-protected silk fibroin solution after adjusting the charge, so that the two form a nucleic acid-silk fibroin complex intermediate solution through electrostatic interaction;

[0069] Step S330, adding a carboxyl activator to the nucleic acid-silk fibroin complex intermediate solution, so that the carboxyl-activated silk fibroin molecules and the nucleic acid materials undergo amidation reaction to obtain nucleic acid-modified silk fibroin biomaterials.

[0070] In step S310, an acidic buffer is added to the solution containing the amino-protected silk fibroin to adjust the pH of the solution system, thereby regulating the surface charge state of the silk fibroin molecules. That is, by adjusting the ionic environment, changing the net charge distribution of the silk fibroin molecules in the dispersion system, enhancing the electrostatic adsorption between them and the nucleic acid materials with opposite charges, laying the foundation for the subsequent formation of stable nucleic acid-silk fibroin complex intermediates.

[0071] The pH value of the acidic buffer is preferably 3-7, more preferably 4.5-6. The acidic buffer can be one or more of 2-(N-morpholino)ethanesulfonic acid (MES), phosphate buffer, citric acid buffer, acetic acid buffer, tartaric acid buffer, propionic acid buffer, and succinic acid buffer. The amount of acidic buffer added can be adjusted according to the target pH value and the initial pH of the system to achieve a controllable change in the surface potential of the silk fibroin from neutral or negative to weakly positive.

[0072] The surface potential of the adjusted silk fibroin molecules is any value between +5mV and +8mV, which helps to enhance the electrostatic affinity with nucleic acid molecules, thereby improving the efficiency of nucleic acid enrichment and the stability of the complex structure. By adjusting the charge of the silk fibroin through this step, not only the targeting enrichment ability of the nucleic acid molecules is improved, but also the spatial positioning and reaction pairing efficiency of the molecules is promoted, which helps to improve the selectivity and uniformity of the subsequent covalent grafting reaction.

[0073] In step S310, during the adjustment of the surface charge of the silk fibroin molecules, in order to avoid non-specific aggregation of the silk fibroin molecules due to conformational changes or charge shielding under acidic conditions, an anti-aggregation agent is further added to the amino-protected silk fibroin solution to improve the stability of the protein dispersion and maintain its reactivity.

[0074] The anti-aggregation agent can be selected from polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer (Pluronic F-127), polyethylene glycol (PEG), Tween non-ionic surfactant (such as Tween 20, Tween 80), dimethyl sulfoxide (DMSO) or a combination thereof. Among them, Pluronic F-127, PEG and Tween surfactant mainly prevent the hydrophobic interaction between protein molecules from inducing aggregation by providing steric hindrance effect and interfacial stability. DMSO belongs to a polar aprotic small molecule solvent, which can improve the solubility and thermodynamic stability of the system by interfering with the intermolecular hydrogen bond interaction and hydrophobic aggregation tendency.

[0075] In some preferred embodiments, the addition concentration of DMSO is any value between 0.01% and 1% of the volume of the system, and more preferably any value between 0.05% and 0.3%. The specific selection and amount of the above anti-aggregation agent can be optimized according to the concentration of silk fibroin in the system, the type and pH value of the acid buffer used, the charge density and stability of the nucleic acid material, and other factors, so as to ensure the electrostatic complexing efficiency of silk fibroin and nucleic acid material while inhibiting the spontaneous precipitation or gelation of silk fibroin, and ensure that the system has good homogeneity and reaction controllability.

[0076] In step S320, based on the surface charge regulation treatment of the amino-protected silk fibroin solution in step S310, the amino-protected silk fibroin molecules are in a positively charged state. Nucleic acid materials naturally have multiple anionic groups in their phosphate backbone and exhibit significant negative electric properties in physiological or weakly acidic environments. After contacting with silk fibroin molecules with a positively charged surface, such molecules can spontaneously perform electrostatic adsorption in the solution system, thereby forming a uniformly distributed nucleic acid adsorption layer on the surface of the silk fibroin molecule chain. This electrostatic adsorption process promotes the local enrichment of nucleic acid material molecules on the surface of the silk fibroin backbone, effectively increasing the site concentration and reaction efficiency of the subsequent amidation coupling reaction. Moreover, this electrostatic adsorption process is carried out in a mild aqueous system without the need to introduce organic solvents or use harsh physical means, which helps to maintain the structural integrity and biological functional activity of the nucleic acid material, avoiding problems such as degradation, denaturation or loss of function that may be caused by conventional chemical reaction conditions. At the same time, the formed complex intermediate structure has a certain stability and can maintain good colloidal uniformity and dispersibility under conventional temperature and buffer system, facilitating the smooth progress of the subsequent coupling reaction.

[0077] The nucleic acid material may, for example, be a natural material without additional chemical modification, which does not have additional amino groups or other active groups introduced on the molecule, and can generally be purchased through commercial channels. To further improve the performance of the nucleic acid modified silk fibroin biomaterial, the embodiments of the present application preferably use nucleic acid materials with multiple reactive amino groups to achieve the covalent anchoring of the nucleic acid material on the silk fibroin backbone. The nucleic acid material has active amino groups distributed at multiple positions on the molecular chain, which can react with the carboxyl groups of the silk fibroin molecule through amide reaction, and is not limited to a single end group position. Preferred molecular structure features include: 1) introducing one primary amino group at the 5' end and the 3' end, which can be achieved by using amino-modified primer adapters during solid-phase synthesis; 2) introducing amino-modified nucleotides, such as amino-modified dT and amino-modified dC, into the interior of the oligonucleotide chain, and controlling the nucleotide spacing between adjacent internal modified sites to be 8-15 nucleotides, to balance steric hindrance and reaction accessibility; 3) connecting the internal amino groups to the nucleotide backbone through C6 alkyl chains or PEG4-PEG12 spacers to reduce conformational constraints and increase the probability of contact with the carboxyl sites of the silk fibroin; 4) using nucleic acid derivatives with high structural stability under aqueous carboxyl activation conditions, such as locked nucleic acids (LNA), peptide nucleic acids (PNA), or partially modified oligodeoxynucleotides (ODN), to prevent degradation during coupling.

[0078] In some embodiments, the nucleic acid material with multiple reactive amino groups is a nucleic acid derivative containing reactive amino functional groups in its molecular structure, which can be an oligodeoxynucleotide (ODN), polydeoxyribonucleotide (PDRN), small interfering RNA (siRNA), or other artificially synthesized nucleic acid mimics such as peptide nucleic acid PNA, locked nucleic acid LNA, etc. with primary amino groups introduced at one or more ends through chemical modification. The amino groups of such nucleic acid derivatives are usually located at the 5' end, 3' end, or sugar-phosphate backbone side chain, and can be introduced through pre-modification or solid-phase synthesis. The amino functional groups react with the carboxyl groups of the silk fibroin molecule after carboxyl activation in the subsequent step to form a stable covalent bond, thereby achieving the directional grafting of the nucleic acid material on the silk fibroin backbone.

[0079] The multi-site amino-modified nucleic acid satisfying the above structural features can be obtained by a conventional solid-phase synthesis method. For example, on a DNA / RNA solid-phase synthesis column, a 5' end amino-modified phosphoramidite compound (such as Amino-Modifier C6 dT) is introduced according to a preset nucleotide sequence, an amino-modified nucleotide monomer with a C6 or PEG spacer arm is introduced at a specified internal position, and a primary amino group is introduced at the 3' end through an amino-modified linker. After deprotection, cleavage and high-performance liquid chromatography purification, a nucleotide material with multi-site amino modification is obtained.

[0080] The multi-site anchoring method has many technical advantages. In the early stage of release, due to the steric hindrance and multiple chemical bonds generated by multi-site covalent bonding, the nucleic acid molecule is difficult to dissociate quickly, thereby significantly reducing the burst release amount. The uniform distribution of multi-sites in the silk fibroin skeleton makes the release rate of the nucleic acid molecule closer to constant, and the release curve presents a near zero-order kinetic characteristic, significantly prolonging the effective release time. Under the simulated body fluid conditions containing serum nucleases, the multi-site anchoring structure provides enhanced spatial protection for the nucleic acid molecule, significantly prolongs the half-life of the nucleic acid molecule, and maintains the structural integrity and biological activity for a longer time.

[0081] The mass ratio of the amino-protected silk fibroin solution to the nucleic acid material is 1:20-100:1, for example, it can be 1:20, 1:10, 30:1, 60:1 or 100:1. In a preferred embodiment, the mass ratio of the amino-protected silk fibroin solution to the nucleic acid material is 1:1-50:1, for example, it can be 1:1, 10:1, 20:1, 40:1 or 50:1. The mass ratio range is obtained based on the electrostatic complexing efficiency between the nucleic acid material and the silk fibroin, the subsequent amide bond coupling efficiency and the system stability.

[0082] The step S330 includes:

[0083] In step S331, a carboxyl activating agent is added to the nucleic acid-silk fibroin complex intermediate solution, the temperature is increased from the first preset temperature to the second preset temperature, and the pH value is maintained at the first preset value for the first preset time;

[0084] In step S332, the pH value is adjusted to the second preset value, and the mixture is maintained at the second preset temperature for the second preset time, and the second preset value is greater than the first preset value.

[0085] In step S333, small molecules or by-products in the complex solution after reaction are separated to obtain a nucleic acid-modified silk fibroin biomaterial.

[0086] In step S331, the first preset temperature is 0-10°C, for example, 0°C, 3°C, 5°C, 7°C, 9°C or 10°C. Under this temperature condition, the nucleic acid-silk fibroin complex intermediate solution is placed in a low-temperature reaction environment, and the carboxyl activator is introduced in a constant or slow dropwise manner to cause selective activation reaction of the carboxyl functional groups exposed in the silk fibroin molecules after enzymatic treatment. The setting of the low-temperature environment not only can reduce the risk of chemical degradation, chain breakage or base modification of the nucleic acid material caused by the carboxyl activator, but also can effectively control the rate of carboxyl activation, inhibit non-specific crosslinking and by-product generation, thereby ensuring the specificity of the coupling reaction and the structural integrity of the product. Subsequently, the reaction system is warmed to a second preset temperature, which is 25-37°C, for example, 25°C, 30°C, 33°C or 37°C. The warming process helps to improve the kinetic rate and reaction completeness of the subsequent amidation coupling reaction, realizes efficient covalent grafting effect under the premise of ensuring the stability of the molecular structure and biological activity of the nucleic acid material.

[0087] The first preset value is 3-5, for example, 3, 4 or 5. The pH range can improve the selective reaction efficiency of the carboxyl activator on the carboxyl functional groups under acidic conditions, while inhibiting the premature removal of the amino protecting group, ensuring the controllability and specificity of the activation process. The first preset time is 2-4h, for example, 2h, 3h or 4h. This time range can effectively avoid side reactions such as carboxyl self-condensation, crosslinking by-product generation or nucleic acid molecular structure degradation caused by excessive activation time while ensuring sufficient carboxyl activation.

[0088] The carboxyl activating agent may, for example, include one or more of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) in combination, preferably using a coupling system that can exert high activation efficiency in an aqueous phase, such as a combination of EDC and NHS. Nucleic acid materials such as PDRN, siRNA, etc. have long-chain phosphate backbones, strong overall negative charge and high hydrophilicity, and are prone to base modification, chain breakage or spatial conformation destruction under the action of carboxyl activating agents. The present application inhibits the destructive effect of carboxyl activating agents on nucleic acid materials through a combination process of amino protection, acid activation, buffer system control, etc., thereby breaking through the inherent cognition in the art that nucleic acids are not suitable for carboxyl activation covalent coupling in an aqueous phase.

[0089] In step S332, the second preset value is 6-8, for example, it can be 6, 7 or 8. The pH range is close to neutral to weak alkaline environment, which is conducive to promoting the nucleophilic attack of the amino functional group in the nucleic acid material molecule under the premise that the carboxyl group has been activated, thereby efficiently generating stable amide bond connection. At the same time, the pH condition can reduce the risk of hydrolysis of the phosphate backbone of the nucleic acid under acidic conditions, and avoid the alkali-catalyzed degradation reaction initiated under strong alkaline conditions, ensuring the structural integrity and biological activity of the nucleic acid molecule.

[0090] The second preset time is 4h-8h, for example, it can be 4h, 6h or 8h. The time range ensures that the amidation coupling reaction proceeds sufficiently while effectively preventing side reactions such as crosslinking between protein segments, accumulation of by-products or non-specific modification of nucleic acid molecules caused by long-term exposure of the reaction system to the carboxyl activating agent residue environment, thereby maintaining the purity and functional stability of the product while ensuring high grafting efficiency.

[0091] In step S333, a separation reaction process is used to remove residual small molecule reactants, unreacted nucleic acid materials, carboxyl activator byproducts, and other low molecular weight impurities in the reaction system, thereby obtaining a purified nucleic acid modified silk fibroin biomaterial. The separation can be achieved by a single method or a combination of multiple methods, including one or more of dialysis, membrane separation technology for ultrafiltration concentration, molecular sieve or ion exchange chromatography, PEG multistage precipitation, and salting-out.

[0092] Steps S331 to S333 significantly improve the selectivity and specificity of the coupling reaction by implementing staged temperature and pH control throughout the carboxyl activation and amidation coupling reaction, combined with precise definition of the time window and post-treatment purification, while minimizing the risk of chemical degradation, chain breakage, and base modification of nucleic acid materials when exposed to carboxyl activators in aqueous phase. Specifically, the low-temperature acidic conditions of step S331 inhibit side reactions and achieve efficient and directional activation of carboxyl groups, the neutral to weakly basic conditions of step S332 accelerate the nucleophilic condensation of amino groups with activated carboxyl groups, improving coupling efficiency and maintaining nucleic acid structural stability, and the purification treatment of step S333 effectively removes residual small molecule reactants, byproducts, and unreacted components, ensuring the production of nucleic acid-silk fibroin composite materials with high purity and structural integrity. Under the synergistic effect of the above multi-stage control, not only stable and controllable covalent grafting is achieved, but also the grafting efficiency of nucleic acid molecules on the silk fibroin backbone is significantly improved, and the uniformity of the grafting site in spatial distribution is improved, reducing batch-to-batch differences and enhancing functional performance consistency.

[0093] In summary, the scheme of the embodiments of the present application achieves limited grafting effect in the improved process even under ordinary nucleic acid conditions (such as commercially available PDRN) by using strategies such as amino protection, carboxyl activation, temperature control, and charge adjustment, thereby breaking through the technical prejudice in the field that nucleic acid materials cannot be covalently grafted. At the same time, by optimizing the multi-site aminated nucleic acid, the grafting efficiency is further significantly improved, and the release period is prolonged.

[0094] In particular, the present application also provides a nucleic acid modified silk fibroin biomaterial, in which the nucleic acid material is covalently grafted on the silk fibroin molecule through an amide bond. In one embodiment, the nucleic acid modified silk fibroin biomaterial is prepared using the aforementioned preparation method.

[0095] The following will be described in detail with specific embodiments.

[0096] Example One:

[0097] The embodiment provides a preparation method of a nucleic acid modified silk fibroin biomaterial, comprising:

[0098] Step one, take the degummed natural silk protein raw material 1g, add to 100ml containing 8mol / L lithium bromide in pH 5.6 phosphate buffer solution, the final silk fibroin mass concentration is 10mg / mL, in the magnetic stirring conditions in 60℃ stirring 1h;

[0099] Step two, under the solution temperature stability in 37℃, to the above silk fibroin solution is added to the mass concentration of 8mg / mL trypsin solution, the mass ratio of trypsin and silk fibroin is 1:100, maintain stirring and incubation 2h, after the incubation is completed, the system is heated to 90℃ for 20min, to inactivate the protease, terminate the enzyme cutting reaction;

[0100] Step three, the above reaction after mixing solution in 8000rpm speed centrifugal 5min, discard the supernatant, collect the precipitate phase, the precipitate phase freeze-dried powder as Figure 2 The left sample in the middle;

[0101] Step four, the precipitate phase is dispersed in PBS buffer solution with pH 5.6, after stirring, the silk fibroin solution is obtained;

[0102] Step five, take the silk fibroin solution 50ml containing silk fibroin 1g, under the magnetic stirring at room temperature, to the above silk fibroin solution is slowly added lithium bromide to the final concentration of 4mol / L, maintain stirring 30min, at this time the dispersion concentration of silk fibroin is 20mg / mL, the mass ratio of lithium bromide and silk fibroin is about 12:1, to ensure that the protein chain segment is fully depolymerized and the amino site is exposed;

[0103] Step six, adjust the pH of the system to 8.5, add acetic anhydride at 25℃, control the mass ratio of silk fibroin and acetic anhydride to be 20:1, after 15min of dropwise addition, continue to stir for 2h, during which 0.5mol / L NaHCO3 is used to slowly adjust the pH to 7.8-8, to ensure the selective acylation of the primary amino group and avoid side reactions;

[0104] Step seven, transfer the reaction solution into a dialysis bag with a molecular weight cut-off of 3.5kDa, dialyze in deionized water at 4℃ for 24h, replace the dialysate every 4h, then use PBS buffer solution with pH 5.6, dialyze for 12h, replace 2-3 times, to remove unreacted amino protecting agent, hydrogen bond inhibitor and byproducts, after dialysis, remove the insoluble material by low speed centrifugation, and filter with 0.22μm filter membrane, to obtain the amino protected silk fibroin solution with a concentration of 20mg / mL;

[0105] Step eight, take 20 mL of amino-protected silk fibroin solution, add MES buffer to make its final concentration 0.1 mol / L, adjust the pH value of the system to 4, under the condition of magnetic stirring, add Pluronic F-127 with a mass ratio of 0.3% of the total reaction system and DMSO with a volume ratio of 0.1% of the total reaction system to the solution in turn, so that the Zeta potential of the system is stable at +5 mV to +8 mV, as shown in Figure 5 ;

[0106] Step nine, at 4℃, add nucleic acid material PDRN to the above amino-protected silk fibroin solution treated by charge adjustment, so that the final concentration of PDRN is 5 mg / mL. Under the condition of low-speed oscillation at 60 rpm-80 rpm, react for 30 min, so that the PDRN molecules combine with the positively charged regions on the surface of the silk fibroin molecules under the action of electrostatic attraction, forming a PDRN-silk fibroin complex intermediate solution. The nucleic acid material PDRN is purchased from sigma, product number is 100403-24-5.

[0107] Step ten, place the PDRN-silk fibroin complex intermediate solution in a low-temperature condition of 4℃, slowly add 10 mM of DMTMM solution, gradually increase the temperature from 4℃ to 25℃, and react for 3 h at pH 4.

[0108] Step eleven, adjust the pH value of the reaction system to 7.5, and continue to oscillate at 25℃ for 6 h. Under this condition, the amino functional groups present in the PDRN molecules can perform nucleophilic attack on the activated carboxyl groups to form stable amide bond connections.

[0109] Step twelve, after the reaction is completed, the reaction solution is loaded into a dialysis bag with a molecular cut-off range of 3.5 kDa, and dialyzed against deionized water at 4℃ for 72 h, and the dialysis solution is replaced every 6 h-8 h to remove small molecule reactants, unreacted nucleic acid materials and DMTMM byproducts. After dialysis is completed, the dialysis product is transferred to a centrifuge tube, PEG 8000 precipitant is added to make its final concentration 8%, and it is placed at 4℃ for 1 h to promote the precipitation of the grafted product. The supernatant is discarded and the PEG fractionation precipitation step is repeated 2-3 times. The collected precipitate is the PDRN-silk fibroin composite biomaterial. The powder of the PDRN-silk fibroin composite biomaterial after lyophilization is shown in the right sample in Figure 2 .

[0110] The product obtained in step four is tested for molecular weight using the method of patent publication number CN119365865A, and the test gives a molecular weight of 35 kDa. The amount of β-sheet in the product obtained in step four is quantitatively calculated using the method of patent publication number CN119741964A, and the amount of β-sheet is 41%.

[0111] To verify the effectiveness of nucleic acid grafting, the silk fibroin precipitate obtained after enzyme digestion in step four of this embodiment of the invention (… Figure 3 The upper middle figure) and the PDRN-silk fibroin composite biomaterial prepared in the embodiments of the present invention ( Figure 3 The structures in the lower and middle figures were characterized using Fourier transform infrared spectroscopy (FTIR). Figure 3 The test results show that at 3200cm -1 -3400cm -1 At 1640 cm⁻¹, the absorption peak of the OH stretching vibration of the composite material is significantly broadened, indicating that the hydrogen bonding interaction within the system is enhanced after the introduction of PDRN. -1 -1680cm -1 Amide I band and 1550cm -1 The increased absorption intensity of the amide II band indicates that an amide bond grafting reaction occurred between silk fibroin and PDRN. At 1238 cm⁻¹ -1 At 1091 cm⁻¹, the enhanced absorption peak intensity of the composite biomaterial can be attributed to the introduction of the P=O group in the PDRN molecule. -1 A new characteristic absorption peak appeared, which is the characteristic absorption of the POC bond, further confirming that the nucleic acid backbone structure has been successfully covalently grafted onto the silk fibroin molecule. In summary, the changes in characteristic peaks in the infrared spectrum confirm that PDRN has been successfully grafted into the silk fibroin backbone through an amidation reaction, thereby forming a stable nucleic acid-silk fibroin composite biomaterial.

[0112] To verify the amino protection effect, the amino content of silk fibroin samples after enzymatic digestion and acetic anhydride protection was determined using the 2,4,6-trinitrobenzenesulfonic acid (TNBS) colorimetric method. This detection method differs from the preparation process conditions of this invention and is only used as a quantitative analysis of the amino protection effect; it does not constitute a preparation step in this invention. This detection method includes sample preparation and reaction steps.

[0113] In the sample preparation step, two sets of samples were prepared, namely Sample A and Sample B. Sample A was the silk fibroin solution obtained according to steps one through four, with a mass concentration of 2 mg / mL, dissolved in 0.1 mol / L MES buffer at pH 4, containing 0.3% Pluronic F-127 by mass. Sample B was the amino-protected silk fibroin solution obtained according to steps one through seven, with the same concentration, buffer system, and solubilizing components as Sample A. Both sets of samples were diluted with equal volumes or equal amounts of protein to ensure consistent protein mass. The final detection concentration was adjusted to 2 mg / mL to ensure the sample concentration was within the optimal detection range of the TNBS colorimetric method, thereby ensuring the accuracy and comparability of the detection results.

[0114] The reaction step comprises: uniformly mixing 400 μL of the sample, 400 μL of NaHCO3 and 200 μL of TNBS, incubating at 60°C in a constant temperature water bath for 2 h in the dark, to obtain a reaction solution; adding 1 mL of SDS, 0.5 mL of HCl with a concentration of 6 mol / L in 1 mL of the reaction solution, terminating the reaction and stabilizing the color development, uniformly mixing and then standing at room temperature for 10 min to 15 min; and taking the supernatant for determination after centrifugation.

[0115] The detection sample obtained by the above detection method is matched with a blank to zero on a spectrophotometer, the absorbance A of each sample is determined at 420 nm, a standard amino acid solution is colored in the same way, and an A 420 - Concentration standard curve, correct batch difference. The protection rate (%) is calculated according to the following formula:

[0116] ;

[0117] A 保护后 of sample B, A 420 of sample A. 保护前 420

[0118] According to the above detection method, the amino acid protection rate is determined to be 85%, which indicates that the above method can effectively shield the amino sites with reactivity in the silk fibroin molecules after enzyme cutting.

[0119] To verify the influence of PDRN grafting on the hydrophilicity of the material surface, the PDRN-silk fibroin composite biomaterial obtained in Example One of the application is subjected to contact angle testing. The testing method is different from the preparation process conditions of the application, and is only used as a comparative analysis means for the surface properties of the material, and does not constitute the preparation steps of the application. The detection method comprises a sample preparation step and a testing step.

[0120] In the preparation step, two groups of samples are prepared, which are sample C and sample D. Sample C is a silk fibroin precipitate prepared according to steps one to three, which is freeze-dried to obtain a powder, and is pressed into a circular sheet sample with a diameter of about 10 mm and a thickness of about 1 mm. Sample D is a PDRN-silk fibroin composite biomaterial prepared according to steps one to eleven, which is subjected to the same freeze-drying and tabletting treatment to obtain a circular sheet sample. Both groups of samples are tested after being equilibrated in an environment with a temperature of 25°C and a relative humidity of 40% for 24 h.

[0121] ​​In the testing step, the contact angle measurement instrument of Dataphysics OCA series was used to test at room temperature. 5 μL of ultrapure water was dropped on the surface of each sample, the static morphology of the droplet was recorded by high-speed camera, and the contact angle value was calculated by automatically fitting the droplet profile using the test software. Five different positions of each sample were tested, and the average value was taken as the final test result.

[0122] The test results show that the contact angle of sample C is about 85°, and the contact angle of sample D is about 52°, which indicates that the contact angle of the PDRN-silk fibroin composite biomaterial of the embodiment of the present application is significantly reduced, indicating that the hydrophilicity of the material surface is obviously enhanced. This change is due to the introduction of a large number of polar groups such as phosphate groups and hydroxyl groups in the PDRN molecule, which increases the surface energy and hydrophilicity of the material, thereby improving the wettability and dispersibility of the material in the aqueous environment. This improvement in hydrophilicity provides wider applicability for subsequent biomedical applications such as drug loading, tissue repair and cell compatibility.

[0123] To verify the proliferation-promoting ability of the PDRN-silk fibroin composite biomaterial of the embodiment of the present application on fibroblasts, the CCK-8 cell viability kit was used to determine the proliferation rate of the composite biomaterial of Example 1 and different control groups for 72 h and 7 days. This detection method is different from the preparation process conditions of the present application, and is only used as a means of biological performance evaluation, and does not constitute the preparation steps of the present application. The detection method includes a sample preparation step and a testing step.

[0124] In the preparation step, sample group and control group samples were prepared, the sample group sample was sample E, and the control group samples were sample F, sample G and sample H. Sample E was the PDRN-silk fibroin composite biomaterial of the embodiment of the present application, which was freeze-dried and crushed, then an appropriate amount of powder was weighed in a clean bench, dissolved and prepared into a sterile solution with a final mass concentration of 1 mg / mL using sterile PBS solution, filtered and sterilized through a 0.22 μm sterile filter membrane, and stored at 4°C for standby. Sample F was a silk fibroin solution prepared according to steps one to four, which was also freeze-dried and crushed, then an appropriate amount of powder was weighed in a clean bench, dissolved and prepared into a sterile solution with a final mass concentration of 1 mg / mL using sterile PBS, filtered and sterilized through a 0.22 μm sterile filter membrane, and stored at 4°C for standby. Sample G was sterile freeze-dried PDRN powder, which was weighed in a clean bench, dissolved and prepared into a solution with a final mass concentration of 1 mg / mL using sterile PBS solution, filtered and sterilized through a 0.22 μm sterile filter membrane, and stored at 4°C for standby. Sample H was sterile PBS buffer with pH 7.4, which was directly taken in a clean bench without adding any active ingredients, filtered and sterilized through a 0.22 μm sterile filter membrane, and stored at 4°C for standby.

[0125] In the testing procedure, human skin fibroblasts (HDFs) were selected and cultured to the logarithmic growth phase in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C in a humidified incubator with 5% CO2. The cells were then sputtered at a rate of 5 × 10⁶ cells / year. 3 Seeds were planted at a density of samples per well in 96-well plates and incubated for 24 hours to facilitate adhesion. Equal volumes of sample E, sample F, sample G, and sample H solutions were added to each well, and the plates were incubated for 72 hours and 7 days, respectively, with the culture medium containing the corresponding sample replaced every 2 days. For assay, 10 μL of CCK-8 working solution was added to each well, and incubation continued for 2 hours. The absorbance (OD) was measured at 450 nm using a microplate reader. 450 The OD of sample D at 72h or 7 days was used as the metric. 450 Based on the value, the relative cell proliferation rate of each group was calculated using the following formula:

[0126] ;

[0127] The results showed that the 72-hour measurement result for sample E was 160%, and compared to sample H as a baseline, the 7-day measurement result was 2.8 times higher. The 72-hour measurement result for sample F was 130%, and compared to sample H as a baseline, the 7-day measurement result was 1.5 times higher. The 72-hour measurement result for sample G was 135%, and compared to sample G as a baseline, the 7-day measurement result was 1.9 times higher. The 72-hour measurement result for sample H was 100%, and compared to sample H as a baseline, the 7-day measurement result was 1 times higher.

[0128] The test results showed that sample E exhibited significantly better proliferation-promoting effects than single components in both the 72-hour and 7-day fibroblast proliferation experiments. Compared with PDRN alone (sample G) or silk fibroin alone (sample F), the cell proliferation rate of the experimental groups increased by approximately 47% and 87%, respectively, based on 7-day data. These results demonstrate that the grafted material of this invention possesses the bioactivity of both nucleic acids and silk fibroin, and through the structural support of silk fibroin and the synergistic activation of cell proliferation by the nucleic acid signaling stimulation of PDRN, it significantly improves cell growth rate and metabolic activity.

[0129] Example 2:

[0130] The difference between this embodiment and embodiment one lies only in step ten. In this embodiment two, step ten in embodiment one is replaced with the following steps:

[0131] The PDRN-silk fibroin complex intermediate solution was placed at a low temperature of 4℃, and 10mM DMTMM solution was slowly added dropwise. The reaction was carried out at pH 4 and temperature of 4℃ for 1 hour, then the temperature was raised to 37℃ and the reaction was carried out for another 2 hours.

[0132] Example 3:

[0133] The embodiment three is only different from the embodiment one in step ten, and the step ten in the embodiment one is replaced by the following steps:

[0134] The PDRN-silk fibroin complex intermediate solution is placed in a low-temperature condition of 4°C, and a 15 mM DMTMM solution is slowly added dropwise, and the reaction is carried out at a pH of 4 and a temperature of 4°C for 1 h, and then the temperature is increased to 37°C, and the reaction is carried out for another 2 h.

[0135] To verify the grafting efficiency of the embodiments one to three, the free amino content of the amino-protected non-grafted silk fibroin sample and the nucleic acid-silk fibroin complex sample in the above embodiments one to three is detected by using the TNBS colorimetric method. The detection method is different from the preparation process condition of the embodiments, and is only used as a quantitative analysis means of the grafting effect, and does not constitute the preparation step of the present application. The detection method comprises a sample preparation step and a reaction step.

[0136] In the sample preparation step, two groups of samples are prepared, which are sample I and sample J. The sample I is the PDRN-silk fibroin complex intermediate solution obtained from steps one to nine in the embodiment one. The sample J is the PDRN-silk fibroin complex biomaterial obtained from the embodiments one to three. The above two groups of samples are diluted by equal volume or equal protein mass to ensure the consistency of the protein mass in the detection, and the final detection concentration is adjusted to 2 mg / mL to ensure that it is in the best detection range of the TNBS colorimetric method, so as to ensure the accuracy and comparability of the detection results.

[0137] The reaction step is consistent with the TNBS colorimetric method used to verify the amino protection effect, and will not be described here. By using the above detection method, the absorbance A of each sample is measured at 420 nm on a spectrophotometer with a matched blank zero, and the standard amino acid solution is colored in the same way to draw the A 420 concentration standard curve to correct the batch difference. The grafting efficiency (%) is calculated according to the following formula:

[0138] ;

[0139] wherein, A 已接枝 is the A 420 of the sample J, and A 未接枝 is the A 420 of the sample I.

[0140] The results show that the grafting efficiency of the embodiment one is 56%, the grafting efficiency of the embodiment two is 64.5%, and the grafting efficiency of the embodiment three is 74%. It can be known that under the conditions of increasing the activation temperature and increasing the concentration of the carboxyl activator, the grafting efficiency can be significantly improved, thereby verifying the controllability and high efficiency of the process of the embodiments.

[0141] To verify the PDRN loadings of Example One to Example Three, the PDRN content of the PDRN-silk fibroin composite biomaterials of Example One to Example Three was determined by ultraviolet spectrophotometry. The detection method is different from the preparation process conditions of the embodiments of the present application, and is only used as a quantitative analysis means for the PDRN loading effect, and does not constitute the preparation steps of the present application.

[0142] The PDRN-silk fibroin composite biomaterials obtained in Example One to Example Three were used as test samples, and the absorbance A of the test samples at a wavelength of 260 nm was determined on an ultraviolet spectrophotometer 260 . At the same time, a series of PDRN standard solutions with known concentrations were prepared, and their A 260 values were determined under the same conditions, an A 260 -concentration standard curve was drawn, and the concentration of unreacted PDRN in the test sample was calculated by a linear regression equation. According to the total amount of PDRN added before the reaction and the amount of unreacted PDRN detected, the PDRN loading (%) was calculated according to the following formula:

[0143] ;

[0144] wherein C 未反应 is the detected ungrafted PDRN concentration, and C 初始 is the PDRN concentration before the reaction.

[0145] The detection results show that PDRN can be successfully grafted onto the silk fibroin molecular skeleton through the carboxyl activation coupling reaction provided in the embodiments of the present application. When the concentration of the carboxyl activator in the reaction conditions is increased from 10 mM to 15 mM, or the activation process temperature is increased from 25℃ to 37℃, the PDRN loading is significantly increased, indicating that increasing the activation efficiency can effectively improve the final grafting effect and nucleic acid loading.

[0146] Example Four:

[0147] The difference between this Example Four and Example One is only that this Example Four does not include Step Eight, and in Step Nine, instead of the amino-protected silk fibroin solution treated by charge adjustment, it is the amino-protected silk fibroin solution that has not been treated by charge adjustment.

[0148] Example Five:

[0149] The difference between this Example Five and Example One is only that the PDRN in this Example Five is a PDRN prepared by a solid-phase synthesis method and having multiple amino reaction sites. The preparation method of the PDRN is:

[0150] The CPG resin loaded with initial nucleotide dT was loaded into the synthesis column, washed with acetonitrile, and then treated with a 3% (mass / volume) solution of trichloroacetic acid in dichloromethane to remove the 5'-DMT protecting group at a flow rate of 1 mL / min for 2 min, followed by washing with acetonitrile until neutral;

[0151] The 0.1 M phosphoramidite nucleotide monomer was mixed with 0.3 M tetrazole activator in acetonitrile and injected into the synthesis column, and the new nucleotide was coupled with the free 5'-OH at room temperature for 5 min. The phosphite bond was oxidized to form a stable phosphodiester bond with a 0.1 M iodine solution, and the reaction time was 1 min.

[0152] The unreacted 5'-OH was then blocked with acetic anhydride / N-methylimidazole solution to avoid side reactions. A 0.2 M solution of Fmoc-aminoalkane carboxylic acid and 0.2 M HBTU condensing agent in DMF was prepared and injected into the synthesis column. The amino reagent was coupled with the PDRN terminal carboxyl or hydroxyl group for 30 min, and the reaction was checked by ninhydrin detection. When the resin was colorless, the coupling was confirmed to be complete. The Fmoc group was removed with 20% piperidine / DMF solution for 10 min to expose the free amino group for reaction.

[0153] The PDRN was cleaved from the CPG resin and the base protecting group was removed by treating with concentrated ammonia at 55°C for 12 h to obtain the multi-reactive site PDRN raw material.

[0154] After obtaining the raw material, the nitrogen content of the PDRN and the multi-reactive site PDRN raw material was calculated using the Kjeldahl method. The results showed that the nitrogen content of the multi-reactive site PDRN raw material was increased by 4.2% compared to the original PDRN material.

[0155] The raw material was used to prepare the PDRN-silk fibroin product according to the process conditions of Example 1.

[0156] Comparative Example 1

[0157] The difference between this comparative example and Example 1 is that in Comparative Example 1, the silk fibroin solution obtained from Steps 1 to 4 was directly physically mixed with a PDRN solution with a concentration of 5 mg / mL, followed by the addition of a sodium alginate solution and uniform mixing. The PDRN was only physically embedded and encapsulated, and did not undergo chemical grafting.

[0158] To verify the advantage of the grafted silk fibroin composite material of the embodiment of the present application in the drug release performance, the PDRN release behavior thereof is evaluated by using the ultraviolet spectrophotometry, and is compared with the physical encapsulation type silk fibroin gel in the comparative example. The detection method is different from the preparation process condition of the embodiment of the present application, and is only used as a verification means of the release performance effect, and does not constitute the preparation step of the present application.

[0159] The samples of the embodiment one and the comparative example one are respectively placed in dialysis bags containing PBS buffer solution with a pH value of 7.4, and the molecular weight cut-off range of the dialysis bag is 3.5kDa-8kDa, so as to ensure that the PDRN can freely diffuse. The dialysis bag is hung in a constant temperature oscillation water bath, and the external dialysate supernatant is taken at a set time interval (0h, 2h, 6h, 12h, 24h, 48h, 72h, 7d, 14d, etc.), and an equal volume of fresh PBS is supplemented to keep the solution volume constant. The absorbance of the supernatant at each time point is measured by using an ultraviolet spectrophotometer at 260nm, and the corresponding PDRN release rate is calculated according to the PDRN standard curve drawn in advance, and the calculation formula of the PDRN release rate is as follows:

[0160]

[0161] Figure 4 The release rate comparison diagram of the embodiment one and the comparative example one according to the present application is shown. As shown in Figure 4 The silk fibroin-sodium alginate gel of the comparative example one shows a faster release in the initial stage, the release curve shows a logarithmic characteristic, and the release period is shorter. The PDRN release process of the embodiment one of the present application is more gentle, the curve is close to the zero-order equation release mode, the total release period is significantly prolonged, and the early burst effect is obviously reduced, which verifies the structural advantage and functional stability of the PDRN-silk fibroin composite biomaterial of the embodiment of the present application in the drug release field.

[0162] Comparative example two:

[0163] The difference between the comparative example two and the embodiment one is only that the order of step eight and step nine in the process flow of the comparative example two is adjusted, that is, the nucleic acid material PDRN is directly added to the system before the Zeta potential adjustment of the amino-protected silk fibroin solution, and then the system charge adjustment treatment is performed.

[0164] Comparative example three:

[0165] The difference between the comparative example three and the embodiment one is only that steps five to seven of the embodiment one are omitted in the process flow of the comparative example three, that is, the amino protection treatment is not performed on the silk fibroin. In step eight, the silk fibroin solution obtained in step four of the embodiment one is directly taken as the raw material for the subsequent charge adjustment and grafting reaction.​

[0166] Comparative Example Four:

[0167] The difference between Comparative Example Four and Example One is that the PDRN used in Step Nine of Example One is replaced by an oligonucleotide with a length of 17 bases in the process flow of Comparative Example Four. The short-chain oligonucleotide is a commercially available product from the company Macron Reagent, with product number 1258984-36-9.

[0168] Comparative Example Five:

[0169] The difference between Comparative Example Five and Example One is that Steps Five to Nine in Example 1 are not performed, i.e., the silk fibroin is not subjected to amino protection treatment and surface charge adjustment, and the remaining steps remain the same. However, Step Ten is replaced by the following operation: add nucleic acid material PDRN to the silk fibroin solution obtained in Step Four, so that the final concentration of PDRN is 5 mg / mL, and place the obtained mixed solution at 4°C. Slowly add 10 mM DMTMM solution, gradually increase the temperature from 4°C to 25°C, and react at pH 4 for 3 h.

[0170] Comparative Example Six:

[0171] The difference between Comparative Example Six and Example One is only in the treatment method of Step Three: in Comparative Example Six, Step Three is to collect the supernatant after centrifugation, rather than collecting the precipitate phase. Accordingly, in Step Four, the supernatant obtained in Step Three is dispersed in PBS buffer at pH 7.4 and stirred uniformly to obtain a silk fibroin solution.

[0172] Comparative Example Seven:

[0173] The difference between Comparative Example Seven and Example One is that Comparative Example Seven omits the silk fibroin molecular chain segment depolymerization and carboxyl exposure pretreatment process involved in Steps One to Four of Example One, i.e., without lithium bromide-buffer dissolution and enzyme treatment to increase the degree of carboxyl functional group exposure, but directly adding the untreated silk fibroin raw material to Step Five for subsequent treatment.

[0174] Table 1 lists the results of various performance tests of each embodiment and comparative example of the present application, including amino protection rate, grafting efficiency, PDRN loading capacity, slow-release period and release curve characteristics, contact angle, fibroblast 72h and 7-day proliferation rate, etc. The test data is shown in Table 1.

[0175]

[0176] The amino protection rates of Examples 1 to 3 are all 85%, indicating that the pretreatment and acetic anhydride protection process can stably achieve efficient amino shielding and reduce non-specific reactions. The protection rate of Example 4 is significantly decreased, indicating that omitting the charge adjustment step can significantly reduce the effective protection degree of primary amino groups and adversely affect the selectivity of the subsequent grafting reaction. Although Comparative Example 2 has a certain protection rate, the protection effect fails to effectively cooperate with the grafting reaction due to the adjustment of the process sequence, resulting in poor grafting efficiency. In terms of grafting efficiency, Examples 1 to 3 are significantly higher than the comparative examples, verifying the synergistic effect of the staged activation, temperature control, and protection strategy. The grafting rate of Example 5 is significantly better than other examples and comparative examples, proving that nucleic acid materials can react with the carboxyl groups of the carboxyl-activated carboxyl sites of silk fibroin molecules at multiple positions in the molecular chain. The PDRN loading of Comparative Example 4 is significantly lower, indicating that short-chain oligonucleotides are inferior to long-chain PDRN in terms of coupling efficiency and loading capacity. In terms of sustained release period, each example is significantly longer than the comparative examples. Although Example 4 is a grafting type structure, the sustained release period is reduced to 19 days, indicating that charge adjustment plays an important role in forming a dense and stable covalent network. Example 5 has the highest grafting efficiency and the longest release time, indicating that nucleic acids with multiple amino reaction sites can better bind to silk fibroin, and the binding is more stable. This fully proves that the steric hindrance and multiple chemical bonds produced by multiple covalent connections make it difficult for nucleic acid molecules to quickly dissociate, thereby significantly reducing the burst release amount. Comparative Example 1 releases the fastest and has a significant burst effect, verifying that the physical encapsulation mode is inferior to the chemical grafting mode in terms of sustained release performance.

[0177] Based on the above data analysis, in Comparative Example 2, the initial binding efficiency between PDRN molecules and silk fibroin molecules is significantly reduced because the system has not established a positive charge environment conducive to electrostatic adsorption when the PDRN molecules are added. A large amount of PDRN is dispersed in the solution in a free state, resulting in insufficient PDRN available for the subsequent carboxyl activation and coupling reaction, and a decrease in grafting efficiency and the final PDRN loading. At the same time, during the subsequent Zeta potential adjustment, the combined PDRN molecules partially dissociate due to the change in the charge environment, further reducing the binding stability.

[0178] In Comparative Example 3, due to the lack of a selective amino protection step with acetic anhydride, the system is prone to self-crosslinking reactions between silk fibroin molecules after the carboxyl groups are activated, resulting in an increase in solution viscosity, a decrease in dispersibility, and the formation of insoluble aggregates, which reduces the reaction uniformity and the structural stability of the final product. In addition, the unprotected primary amino groups are prone to side reactions with non-target sites during the coupling reaction, further affecting the grafting specificity and biological function performance of the product.

[0179] In the Comparative Example Four, the molecular weight of the short-chain oligonucleotide is significantly lower than that of the PDRN, the spatial conformation is relatively compact, the number of polar groups provided by the molecular skeleton is limited, and the electrostatic adsorption force between the short-chain oligonucleotide and the charged region of the silk fibroin surface is weak. In the carboxyl activation coupling reaction, the short-chain oligonucleotide is limited by the number of amino and carboxyl sites available for reaction and the conformation matching degree, and the grafting efficiency and unit mass loading are significantly lower than those of the PDRN. At the same time, due to the faster diffusion rate of the short-chain oligonucleotide in the aqueous phase system, its fixation ability in the composite material is insufficient, and it is easy to fall off during subsequent washing or dialysis, resulting in a decrease in the effective load retention rate. In addition, the biological action persistence of the short-chain oligonucleotide is weak, and it is difficult to achieve the long-acting release characteristics exhibited by the PDRN, thereby significantly degrading the overall effect in the application scenarios such as cell proliferation promotion and tissue repair.

[0180] In the Comparative Example Five, the amino protection step and the charge adjustment step in the Example One are omitted, and the PDRN molecules are directly contacted with the silk fibroin obtained in Step Four under the DMTMM activation condition. Under this condition, a large number of exposed primary amino groups in the silk fibroin molecules will compete with the activated carboxyl groups, resulting in a significant reduction in the effective coupling sites between the PDRN and the silk fibroin. At the same time, the silk fibroin surface is negatively charged without charge adjustment, and there is a significant electrostatic repulsion between the negatively charged PDRN skeleton, making it difficult for the two to approach and combine. More importantly, in the presence of acidic and DMTMM activators, nucleic acid molecules are prone to structural degradation such as phosphodiester bond rupture, base depurination, or oxidation, thereby destroying their integrity and biological activity. Therefore, this comparative example will exhibit low grafting efficiency and PDRN loading, and the structural integrity of the PDRN will be significantly reduced, verifying the viewpoint in the prior art that "nucleic acids are not suitable for direct covalent coupling under EDC / NHS, DMTMM, etc.".

[0181] In the Comparative Example Six, the supernatant is used instead of the precipitate phase, and the supernatant mainly contains silk fibroin peptide fragments with a low molecular weight (generally less than 10 kDa). Such fragments are mainly in amorphous structure and lack a β-sheet crystalline skeleton. Due to the short length of the small-molecule silk fibroin peptide chains in the supernatant, the number of exposed carboxyl, amino, and other reaction functional groups is limited, making it difficult to form stable and high-density covalent grafting, thereby reducing the grafting efficiency. Moreover, the amorphous silk fibroin peptide lacks a stable three-dimensional crystalline skeleton and cannot effectively fix nucleic acid molecules, resulting in poor binding stability and difficulty in maintaining long-term stability.

[0182] In Comparative Example 7, a large number of Asp / Glu side chains and end group carboxyl groups in the natural silk fibroin macromolecule are embedded in the highly crystalline region, which is difficult to be activated by DMTMM and the like. The natural silk fibroin structure is highly ordered and dense, and most of the amino sites are hindered or embedded, so that the protection agent cannot be uniformly contacted, resulting in a decrease in the amino protection rate. The number of activated carboxyl sites is insufficient, and the spatial distribution is not conducive, so that the PDRN amino group is difficult to effectively attack nucleophilically. In addition, without chain segment depolymerization, the protein chain is rigid and lacks flexible segments to cooperate, making it more difficult to provide a uniform grafting environment.

[0183] The above-described examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the scope of protection of the present application patent should be subject to the appended claims.

Claims

1. A method for preparing nucleic acid-modified silk fibroin biomaterials, characterized in that, include: Step S100: Provide a silk fibroin solution, wherein the silk fibroin molecules in the silk fibroin solution have carboxyl and amino groups; Step S200: An amino protectant is added to the silk fibroin solution to selectively protect the amino groups in the silk fibroin molecules, thereby obtaining amino-protected silk fibroin. Step S300: Add an amino-containing nucleic acid material and a carboxyl activator to a solution containing the amino-protected silk fibroin, so that the carboxyl-activated silk fibroin molecules undergo an amidation reaction with the nucleic acid material, so that the nucleic acid material is covalently grafted onto the silk fibroin molecules through amide bonds, thereby obtaining nucleic acid-modified silk fibroin biomaterials. Step S300 includes: Step S310: Add acidic buffer solution to the solution containing the amino-protected silk fibroin to adjust the surface charge of the silk fibroin molecules to a positive state. Step S320: Add the nucleic acid material to the charge-adjusted amino-protected silk fibroin solution, so that the two form a nucleic acid-silk fibroin complex intermediate solution through electrostatic interaction; Step S330: Add a carboxyl activator to the nucleic acid-silk fibroin complex intermediate solution to cause the carboxyl-activated silk fibroin molecules to undergo an amidation reaction with the nucleic acid material to obtain nucleic acid-modified silk fibroin biomaterial. The method for preparing the silk fibroin solution includes: Step S110: Provide silk fibroin raw material and dissolve it in a buffer solution containing hydrogen bond inhibitors; Step S120: Add a protease at a preset temperature for incubation, and inactivate the protease after incubation. The protease is one or more of trypsin, chymotrypsin, and proteinase K. Step S130: The mixed solution after the reaction is centrifuged to obtain a precipitate phase, wherein the precipitate phase contains 30%-50% β-sheet crystal structure and 50%-70% amorphous region; Step S140: The precipitate phase is dispersed in a pre-dispersion medium free of reactive components to obtain a silk fibroin solution.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the amino-protected silk fibroin solution to the nucleic acid material is 1:20-100:

1.

3. The preparation method according to claim 2, characterized in that, The acidic buffer solution is one or more of the following: 2-(N-morpholine)ethanesulfonic acid buffer, phosphate buffer, citrate buffer, acetate buffer, tartrate buffer, propionic acid buffer, and succinate buffer.

4. The preparation method according to claim 1, characterized in that, Step S310 further includes adding an anti-aggregation agent to the solution containing the amino-protected silk fibroin.

5. The preparation method according to any one of claims 1-4, characterized in that, Step S330 includes the following steps: A carboxyl activator is added to the nucleic acid-silk fibroin complex intermediate solution, the temperature is increased from a first preset temperature to a second preset temperature, and the solution is maintained at a first preset pH value for a first preset time. Adjust the pH value to a second preset value, mix at a second preset temperature for a second preset time, where the second preset value is greater than the first preset value; Small molecules or byproducts in the composite solution after the separation reaction are obtained to obtain the nucleic acid-modified silk fibroin biomaterial.

6. The preparation method according to claim 5, characterized in that, The first preset temperature is 0℃-10℃, and the second preset temperature is 25℃-37℃; The first preset value is 3-5, and the second preset value is 6-8; The first preset time is 2h-4h, and the second preset time is 4h-8h.

7. The preparation method according to any one of claims 1-4 and 6, characterized in that, Step S200 includes the following steps: A hydrogen bond inhibitor is added to the silk fibroin solution, wherein the mass ratio of silk fibroin molecules to the hydrogen bond inhibitor in the silk fibroin solution is any value between 1:3 and 1:

500. An amino protectant is then added to obtain amino-protected silk fibroin, wherein the mass ratio of silk fibroin molecules to the amino protectant in the silk fibroin solution is any value between 50:1 and 5:

1.

8. The preparation method according to any one of claims 1-4 and 6, characterized in that, The silk fibroin molecules in the silk fibroin solution are silk fibroin molecules that have undergone enzymatic digestion.

9. A nucleic acid-modified silk fibroin biomaterial, characterized in that, The nucleic acid-modified silk fibroin biomaterial is prepared by any one of the preparation methods described in claims 1-8, wherein the nucleic acid material is covalently grafted onto the silk fibroin molecule via amide bonds.

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