Drug delivery carrier material, drug delivery coating and preparation method thereof

Abstract

The invention discloses a drug delivery carrier material, a drug delivery coating and a preparation method thereof, and belongs to the technical field of biological materials. The preparation method of the drug delivery coating comprises the following steps: reacting polypeptide containing hydrophobic amino acid with acidic protein in a solution system in which the pH value is 3-7, the temperature is 20-50 DEG C and an oxidizing agent exists, and mixing the prepared coating with a drug solution to realize drug filling, and then mixing with a phosphate solution to induce the micro-nano pore structure in the coating to be closed. The isoelectric points of the polypeptide and the acidic protein are both less than 7, and the difference value of the isoelectric points is less than or equal to 2.5; the acidic protein is negatively charged under the condition that the pH is greater than or equal to 7. According to the method, the contradiction that high loading and low burst release of an existing carrier are difficult to achieve at the same time is effectively solved, the coating is mild in drug loading condition, effective loading and activity retention of various active biomolecules can be achieved, the coating can also adapt to various drug types through modular design, and a brand new mode is provided for constructing a next-generation intelligent drug delivery system.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a drug delivery carrier material and a drug delivery coating and a preparation method thereof. Background Art

[0002] In the field of biomedical engineering, the core challenge of drug delivery systems lies in balancing efficient loading and precisely controlled release while maintaining drug activity. While widely used, traditional carriers such as liposomes (e.g., Doxil® has a drug loading efficiency of only approximately 10%) and PLGA microspheres (burst release results in 30% drug release within 24 hours). However, their low loading efficiency (typically <30%), strong dependence on chemical modification (e.g., PEGylation increases the risk of biotoxicity), and uncontrollable release kinetics (e.g., difficulty achieving zero-order release) severely limit their application in targeted therapy and personalized medicine. Furthermore, traditional polymer carriers can lose up to 40% of drug activity due to residual crosslinkers (e.g., glutaraldehyde), while physical encapsulation methods can lead to rapid drug-carrier leakage due to insufficient drug-carrier affinity (e.g., paclitaxel in PLGA has a burst release rate of >50%).

[0003] In recent years, protein- and peptide-based self-assembling materials have attracted considerable attention due to their inherent biocompatibility (e.g., the nontoxicity of collagen degradation products) and molecular programmability (e.g., tunable hydrophobic / hydrophilic balance through sequence design). However, existing techniques, such as electrostatically driven assembly (e.g., heparin-lysozyme complexes), require strict pH control (e.g., stable structure formation requires a pH <4), resulting in complex processes and poor reproducibility. While chemical cross-linking methods (e.g., transglutaminase-mediated fibrin cross-linking) can enhance structural stability, they significantly reduce drug loading efficiency (drug loading decreases by approximately 30%). Furthermore, most studies have focused on single protein or peptide systems (e.g., β-lactoglobulin or RADA16-I peptide), which have low structural porosity (<20%) and limited surface area (<50 m² / g), making efficient loading of macromolecular drugs (e.g., antibodies or exosomes) difficult.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The object of the present invention is to provide a drug delivery carrier material and a drug delivery coating and a preparation method thereof, so as to solve or improve the above technical problems.

[0006] The present invention can be achieved like this: In a first aspect, the present invention provides a method for preparing a drug delivery coating, comprising the following steps: reacting a polypeptide containing a hydrophobic amino acid with an acidic protein in a solution system having a pH of 3 to 7, a temperature of 20° C. to 50° C., and the presence of an oxidant to prepare a coating having a micro-nanoporous structure; mixing the coating having the micro-nanoporous structure with a drug solution to achieve drug filling through the capillary effect of the micro-nanoporous structure; mixing the coating filled with the drug with a phosphate solution to induce closure of the micro-nanoporous structure in the coating, thereby obtaining a drug delivery coating; The isoelectric point of a polypeptide containing hydrophobic amino acids is pI1, and the isoelectric point of an acidic protein is pI2. pI1 < 7, pI2 < 7, ΔpI = |pI1-pI2| ≤ 2.5; and acidic proteins are negatively charged under conditions of pH ≥ 7.

[0007] In an optional embodiment, the molar ratio of the polypeptide containing hydrophobic amino acids to the acidic protein is 0.1:1 to 10:1, and the total concentration of the polypeptide containing hydrophobic amino acids and the acidic protein in the solution system is 0.01 mmol / mL to 10 mmol / L.

[0008] In an optional embodiment, the molar ratio of the polypeptide containing hydrophobic amino acids to the acidic protein is 0.1:1 to 5:1, and the total concentration of the polypeptide containing hydrophobic amino acids and the acidic protein in the solution system is 0.01 mmol / mL~5 mmol / L.

[0009] In an alternative embodiment, at least 10% of the amino acids in the amino acid sequence of the hydrophobic amino acid-containing polypeptide are hydrophobic amino acids.

[0010] In an alternative embodiment, the hydrophobic amino acid comprises at least one of alanine, valine, leucine, phenylalanine, tryptophan and isoleucine.

[0011] In an alternative embodiment, the acidic protein is a protein containing acidic amino acid residues.

[0012] In an alternative embodiment, the acidic amino acid residue comprises at least one of aspartic acid and glutamic acid.

[0013] In an optional embodiment, the polypeptide containing a hydrophobic amino acid reacts with the acidic protein in a solution system having a pH of 5-6.5, a temperature of 30° C.-40° C., and the presence of an oxidant.

[0014] In an optional embodiment, the mixing temperature of the coating having a micro-nano porous structure and the drug solution is 0°C to 40°C.

[0015] In an optional embodiment, the drug concentration in the drug solution is a saturation concentration.

[0016] In an alternative embodiment, the solvent of the drug solution does not contain phosphate ions.

[0017] In an alternative embodiment, the mixing of the coating layer having a micro-nanoporous structure and the drug solution is performed by immersing the coating layer in the drug solution.

[0018] In an optional embodiment, the immersion time of the coating having a micro-nano porous structure in the drug solution is not less than 1 min.

[0019] In an alternative embodiment, mixing the drug-filled coating with the phosphate solution is performed by immersing the coating in the phosphate solution.

[0020] In an optional embodiment, during the immersion process, the temperature is maintained at -10°C to 60°C, and the immersion time is at least 1 minute.

[0021] In an optional embodiment, during the immersion process, the immersion time is 5 minutes to 60 minutes.

[0022] In an optional embodiment, the concentration of the phosphate solution is 0.1 mmol / L to 10 mmol / L.

[0023] In an optional embodiment, the concentration of the phosphate solution is 3 mmol / L to 10 mmol / L.

[0024] In a second aspect, the present invention provides a drug delivery coating prepared by the preparation method of any one of the aforementioned embodiments.

[0025] In a third aspect, the present invention provides a drug delivery carrier material having the drug delivery coating according to the aforementioned embodiment.

[0026] The beneficial effects of the present invention include: The present invention creatively proposes a method for preparing a drug delivery coating, which involves first mixing a coating having a micro-nano porous structure with a drug solution, and then filling the drug through the capillary effect of the micro-nano porous structure; then mixing the coating filled with the drug with a phosphate solution, and inducing the closure of the micro-nano porous structure in the coating by phosphate, thereby effectively encapsulating the drug in the coating. Compared with mixing the coating having a micro-nano porous structure with a drug-phosphate solution at one time, the concentration of the drug in the solution can be better controlled, and the loading amount of the drug in the coating can be more accurately controlled, effectively solving the contradiction of "high load-low burst release" that is difficult to achieve in existing carriers. In addition, the coating has mild drug loading conditions, which can achieve effective loading and activity retention of various active biological molecules, and can also be adapted to various drug types through modular design, providing a new way to build the next generation of intelligent drug delivery systems. The resulting drug delivery coating has a high drug loading capacity, a low initial burst release rate, and a long sustained release period, which can meet the treatment needs of chronic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 This is an SEM image of the coating with micro-nano porous structure of Example 1 in Experimental Example 1; Figure 2 is a SEM image of the drug delivery coating of Example 1 in Experimental Example 1; Figure 3 Graph showing the thrombin time test results of the stainless steel sample with drug delivery coating and the soaking solution in Experimental Example 2; Figure 4 The fluorescence distribution diagram inside the coating of each sample in Experimental Example 3 was taken under a laser confocal microscope; Figure 5 This is the energy spectrum detection result diagram of the stainless steel sample with drug delivery coating obtained in Example 4 of Experimental Example 4; Figure 6 This is the energy spectrum detection result of the stainless steel sample with drug delivery coating obtained in Example 5 of Experimental Example 4; Figure 7 Graph showing the results of immersion experiments on stainless steel samples with drug delivery coatings obtained in each embodiment and comparative example ② in Experimental Example 5; Figure 8 Statistical graph of the porosity of the coating with micro-nano pore structure obtained in each embodiment and comparative example ① in Experimental Example 7; Figure 9 Statistical graph of the surface roughness of the coating with micro-nano porous structure obtained in each embodiment and comparative example ① in Experimental Example 7; Figure 10 The SEM images of the coatings with micro-nano porous structures obtained in each embodiment and comparative example ① in Experimental Example 8; Figure 11 Graph showing the results of immersion experiments on stainless steel samples with drug delivery coatings obtained in each embodiment and comparative example ② in Experimental Example 9; Figure 12 1 is a comparison chart of the drug loading of the stainless steel sample with the drug delivery coating obtained in Example 1 of Test Example 10 and in Comparative Example 19; Figure 13 Graph showing the thickness of the drug delivery layer in the stainless steel samples of Example 1 and Examples 21-23 in Test Example 11; Figure 14 The results of the active miRNA-22 content in the eluates corresponding to Example 1' and Examples 24 to 27 in Experimental Example 12 are shown in FIG. Figure 15 This is a graph showing the colchicine content in the eluate corresponding to the stainless steel samples with drug delivery coatings obtained in Example 1 and Examples 28 to 30 in Test Example 13. DETAILED DESCRIPTION

[0029] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0030] The drug delivery carrier material and drug delivery coating provided by the present invention and their preparation methods are described in detail below.

[0031] The present invention provides a method for preparing a drug delivery coating, comprising the following steps: reacting a polypeptide containing hydrophobic amino acids with an acidic protein in a solution system with a pH value of 3-7, a temperature of 20°C-50°C, and the presence of an oxidant to prepare a coating with a micro-nano porous structure; mixing the coating with the micro-nano porous structure with a drug solution to achieve drug filling through the capillary effect of the micro-nano porous structure; and mixing the coating filled with the drug with a phosphate solution to induce closure of the micro-nano porous structure in the coating to obtain the drug delivery coating.

[0032] The isoelectric point of a polypeptide containing hydrophobic amino acids is pI1, and the isoelectric point of an acidic protein is pI2. pI1 < 7, pI2 < 7, ΔpI = |pI1-pI2| ≤ 2.5; and acidic proteins are negatively charged under conditions of pH ≥ 7.

[0033] In some optional embodiments, pI1 can be 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1 or 0.5, or other values within the range of < 7. If pI1 is greater than 7, the formation of the coating may rely more on the electrostatic assembly of polypeptides and proteins, resulting in the structural collapse of the coating due to the influence of the ionic strength of the solution.

[0034] The pI2 can be 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1 or 0.5, or other values within the range of < 7. If the pI2 is greater than 7, the formation of the coating may rely more on the electrostatic assembly of polypeptides and proteins, resulting in the structural collapse of the coating due to the influence of the ionic strength of the solution.

[0035] ΔpI can be 2.5, 2, 1.5, 1, 0.5 or 0, or other values within the range of ≤2.5. If ΔpI>2.5, the formation of the coating may rely on the electrostatic assembly of the polypeptide and protein, thereby affecting the self-encapsulation performance of the coating.

[0036] The use of peptides containing hydrophobic amino acids and acidic proteins that meet the aforementioned isoelectric point conditions can suppress electrostatic interference at a pH of 3-7, preventing electrostatic assembly between the protein and peptide, thereby achieving a self-assembly process dominated by hydrophobic interactions. It is important to emphasize that traditional electrostatic assembly is highly sensitive to ionic strength, such as structural collapse at NaCl concentrations greater than 0.1 mmol / L. However, the self-assembly process in the present invention, which utilizes hydrophobic interactions, effectively avoids this structural collapse caused by ionic strength, resulting in a coating with a high porosity.

[0037] In some optional embodiments, at least 10% of the amino acids in the amino acid sequence of the hydrophobic amino acid-containing polypeptide are hydrophobic amino acids, wherein the hydrophobic amino acids may illustratively but not limitatively include at least one of alanine, valine, leucine, phenylalanine, tryptophan, and isoleucine.

[0038] Exemplarily, the polypeptide containing hydrophobic amino acids may include at least one of hormone polypeptides, antimicrobial polypeptides (AMPs), neurotransmitter polypeptides, immunomodulatory polypeptides, anti-tumor polypeptides, coagulation factor polypeptides, anti-inflammatory polypeptides, angiogenic polypeptides, growth factor polypeptides, antiviral polypeptides, receptor ligand polypeptides, functional structural peptides, cyclic polypeptides, synthetic and engineered polypeptides.

[0039] Among them, hormone polypeptides may include insulin, growth hormone, and thyroid-stimulating hormone. Antimicrobial polypeptides may include defensins, lysozymes, and bacterial membrane-permeating peptides. Neurotransmitter polypeptides may include enkephalins, neuropeptide Y, and endorphins. Immunomodulatory polypeptides may include interleukins, interferons, and tumor necrosis factors. Anti-tumor polypeptides may include tumor antigen peptides and tumor immunomodulatory peptides. Coagulation factor polypeptides may include coagulation factors and fibrinogen. Anti-inflammatory polypeptides may include anti-inflammatory cytokines and steroid hormone peptides. Angiogenic polypeptides may include VEGF and fibroblast growth factor. Growth factor polypeptides may include EGF and insulin-like growth factor. Antiviral polypeptides may include inhibitory peptides against HIV and influenza viruses. Receptor ligand polypeptides may include endorphins and erythropoietin. Functional structural peptides may include collagen peptides and antithrombotic peptides. Cyclic polypeptides may include cyclic peptide antibiotics and antimicrobial peptides. Synthetic and engineered polypeptides may include at least one of genetically engineered or chemically synthesized custom peptides.

[0040] In some optional embodiments, the acidic protein is a protein containing acidic amino acid residues. The acidic amino acid residues may, by way of example but not limitation, include at least one of aspartic acid and glutamic acid. For example, the acidic protein may include albumin, hemoglobin, and transferrin. Furthermore, the acidic protein may include structural proteins (such as collagen, fibrin, keratin, and silk) and other proteins (such as lysozyme and globulin).

[0041] In some optional embodiments, the oxidant may include at least one of an inorganic oxidant and an organic oxidant. The inorganic oxidant may illustratively include at least one of persulfate, chlorate, perchlorate, perchlorate, dichromate, periodate, permanganate, nitrate, and an inorganic peroxide. The inorganic peroxide may illustratively, but not limitatively, include at least one of Na2O2, K2O2, MgO2, CaO2, BaO2, and H2O2. The organic oxidant may illustratively include at least one of dichlorine peroxide and peracetic acid.

[0042] In some optional embodiments, the pH value of the solution system corresponding to the preparation of the coating having a micro-nanoporous structure can be 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7, or other values within the range of 3 to 7, to facilitate the binding of groups during the co-assembly process and avoid electrostatic repulsion. In some preferred embodiments, the pH value of the solution system corresponding to the preparation of the coating having a micro-nanoporous structure is 5 to 6.5.

[0043] If the reaction pH is less than 3 or greater than 7, the activity of polypeptides containing hydrophobic amino acids and acidic proteins may be affected, making it difficult to form a coating.

[0044] In some optional embodiments, the temperature of the solution system corresponding to the preparation of the coating having a micro-nano porous structure can be 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., or 50° C., or other values within the range of 20° C. to 50° C. In some preferred embodiments, the temperature of the solution system corresponding to the preparation of the coating having a micro-nano porous structure is 30° C. to 40° C.

[0045] If the reaction temperature is lower than 20°C, the coating may be too thin; if the reaction temperature is higher than 50°C, the coating may be too thick and may affect the activity of peptides and acidic proteins containing hydrophobic amino acids.

[0046] In some optional embodiments, the reaction time for preparing the coating having a micro-nano pore structure may be 2 hours or more.

[0047] In some optional embodiments, the molar ratio of the polypeptide containing hydrophobic amino acids to the acidic protein can be 0.1:1 to 10:1, such as 0.1:1, 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1 or 10:1, or other values within the range of 0.1:1 to 10:1. In some preferred embodiments, the molar ratio of the polypeptide containing hydrophobic amino acids to the acidic protein is 0.1:1 to 5:1.

[0048] If the amount of polypeptide containing hydrophobic amino acids is too small, or the amount of acidic protein is too large, it is not conducive to the oxidant to play the role of destroying protein disulfide bonds, affecting the assembly and formation of the coating; if the amount of polypeptide containing hydrophobic amino acids is too large, or the amount of acidic protein is too small, it is not conducive to the formation of the coating.

[0049] In some optional embodiments, the total concentration of the polypeptide containing hydrophobic amino acids and the acidic protein in the solution system can be 0.01 mmol / mL to 10 mmol / L, such as 0.01 mmol / mL, 0.05 mmol / mL, 1 mmol / mL, 1.5 mmol / mL, 2 mmol / mL, 2.5 mmol / mL, 3 mmol / mL, 3.5 mmol / mL, 4 mmol / mL, 4.5 mmol / mL, 5 mmol / mL, 5.5 mmol / mL, 6 mmol / mL, 6.5 mmol / mL, 7 mmol / mL, 7.5 mmol / mL, 8 mmol / mL, 8.5 mmol / mL, 9 mmol / mL, 9.5 mmol / mL or 10 mmol / mL, or other values within the range of 0.01 mmol / mL to 10 mmol / L. In some preferred embodiments, the total concentration of the polypeptide containing hydrophobic amino acids and the acidic protein in the solution system is 0.01 mmol / mL to 5 mmol / L.

[0050] In practice, a solution system can be obtained by mixing a hydrophobic amino acid-containing polypeptide solution, an acidic protein solution, and an oxidant solution. The solvent used for the hydrophobic amino acid-containing polypeptide solution, the acidic protein solution, and the oxidant solution can all be ultrapure water with a pH of approximately 6 (the pH value is adjusted with hydrochloric acid). The concentrations of the hydrophobic amino acid-containing polypeptide solution, the acidic protein solution, and the oxidant solution can each be between 0.01 mg / mL and 100 mg / mL, such as 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 50 mg / mL, 80 mg / mL, or 100 mg / mL, or other values within the range of 0.01 mg / mL to 100 mg / mL are also possible. By controlling the volume ratio of the hydrophobic amino acid-containing polypeptide solution, the acidic protein solution, and the oxidant solution, the molar ratio of the hydrophobic amino acid-containing polypeptide to the acidic protein is 0.1:1 to 10:1, and the total concentration of the hydrophobic amino acid-containing polypeptide and the acidic protein in the solution system is 0.01 mmol / mL to 10 mmol / L.

[0051] Continuing from the previous example, under the induction of oxygen free radicals from the oxidant, disulfide bonds in the acidic protein are broken and oxidized to form stable intermediate sulfides. During this process, the protein structure stretches, exposing numerous hydrophobic groups on its side chains. In an aqueous solution, the hydrophobic amino acids in the hydrophobic amino acid-containing polypeptide bind and assemble with the hydrophobic groups of the acidic protein, forming hydrophobic nucleation sites (acidic protein-hydrophobic amino acid-containing polypeptide particles). The resulting assembled acidic protein-hydrophobic amino acid-containing polypeptide particles are micro-nanosized and stacked on the substrate surface, resulting in a coating with a micro-nanoporous structure.

[0052] The above-mentioned coating with a micro-nano pore structure has a high porosity (e.g., up to 60% to 80%), and the pores are mainly micro-nano-sized gaps between acidic protein-polypeptide particles containing hydrophobic amino acids and acidic protein-polypeptide particles containing hydrophobic amino acids. These pore positions can provide more adsorption sites for drugs than conventional drug coatings, thereby increasing the drug loading capacity; and the above-mentioned coating with a micro-nano pore structure can undergo reconstructive self-encapsulation in phosphate solutions to achieve dynamic closure of the pores, which can significantly reduce the initial burst release rate of the drug and prolong the sustained-release period, which is conducive to meeting the needs of chronic disease treatment.

[0053] In some optional embodiments, after obtaining the coating with a micro-nano porous structure, the coating is cleaned and dried, and then mixed with the drug solution.

[0054] The cleaning liquid may be ultrapure water, and the drying temperature may be 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, or 45°C, or other values within the range of 10°C to 45°C.

[0055] In some optional embodiments, the drug solution may be dripped onto the coating having a micro-nanoporous structure, or the coating having a micro-nanoporous structure may be immersed in the drug solution to achieve mixing of the coating having a micro-nanoporous structure and the drug solution. Immersion is preferably used as the mixing method to ensure that the drug is fully and evenly applied to all locations of the coating.

[0056] In some optional embodiments, the concentration of the drug in the drug solution is not limited, but is preferably a saturating concentration. The above-mentioned drugs may, by way of example but not limitation, include traditional chemically synthesized drugs, biopharmaceutical drugs, gene therapy drugs, peptide and protein drugs, Chinese herbal medicines and plant extracts, and other drugs. Traditional chemically synthesized drugs may include anti-inflammatory drugs, statins, antibiotics, and anti-tumor drugs (such as rapamycin, paclitaxel, and targeted drugs). Biologic drugs may include monoclonal antibodies, vaccines, cytokines, and exosomes. Gene therapy drugs may include nucleic acid drugs (such as miRNA-22, RNAi, and siRNA for targeted gene expression) and gene editing technology drugs (such as CRISPR for gene repair). Peptide and protein drugs may include various antibodies. Chinese herbal medicines and plant extracts may include artemisinin, colchicine, oregano phenol, and allicin. Other drugs may include phenolic acids and metal ions. Furthermore, other drugs may be used as needed.

[0057] In some optional embodiments, the solvent of the drug solution does not contain phosphate ions, and may include at least one of an organic solvent and an inorganic solvent, such as ultrapure water, physiological saline, alcohol, anhydrous ethanol, phosphate buffered saline (PBS) and dimethyl sulfoxide (DMSO).

[0058] In some optional embodiments, the temperature of the drug solution may be 0°C to 40°C, such as 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, or 40°C, or other values within the range of 0°C to 40°C, depending on whether the drug activity is maintained. The pH value of the drug solution is not particularly limited and is also based on whether the drug activity is maintained.

[0059] In some optional embodiments, the coating having a micro-nano porous structure is immersed in the drug solution for no less than 1 minute to achieve sufficient filling of the drug.

[0060] Furthermore, the drug-filled coating is cleaned and dried, and then mixed with a phosphate solution to induce the closure of the micro-nano pore structure in the coating.

[0061] In some optional embodiments, the phosphate solution may be dripped onto the drug-filled coating, or the drug-filled coating may be immersed in the phosphate solution to achieve mixing of the drug-filled coating and the phosphate solution. Immersion is preferred for such mixing, as it closes the micro- and nano-pores present in various locations within the coating.

[0062] In some optional embodiments, during the immersion process, the temperature is maintained at -10°C to 60°C, such as -10°C, -5°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C, or other values within the range of -10°C to 60°C. The immersion time is at least 1 minute, such as 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, or 120 minutes, or other values within the range of 1 minute or more.

[0063] In some preferred embodiments, during the immersion process, the immersion time is 5 minutes to 60 minutes.

[0064] In some optional embodiments, the concentration of the phosphate solution can be 0.1 mmol / L to 10 mmol / L, such as 0.1 mmol / L, 0.5 mmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 5.5 mmol / L, 6 mmol / L, 6.5 mmol / L, 7 mmol / L, 7.5 mmol / L, 8 mmol / L, 8.5 mmol / L, 9 mmol / L, 9.5 mmol / L, or 10 mmol / L, or other values within the range of 0.1 mmol / L to 10 mmol / L. In some preferred embodiments, the concentration of the phosphate solution in the drug-phosphate solution can be 3 mmol / L to 10 mmol / L.

[0065] If the concentration of the phosphate solution is too low, it is easy to cause the pore structure of the coating to be loosely encapsulated, resulting in sudden release of the drug; if the concentration of the phosphate solution is too high, it is easy to cause the pore structure of the coating to rearrange too quickly, resulting in instability of the coating and thus falling off and dissociation.

[0066] The phosphate solution may include at least one of a phosphoric acid solution, a phosphate solution, a hydrogen phosphate solution and a dihydrogen phosphate solution, and preferably a phosphate solution is used.

[0067] It should be noted that the phosphate solution is highly hydrophilic and has a stronger ability to bind water than acidic white egg white. During the mixing process between the microporous coating and the phosphate solution, the phosphate solution can bind to water in the mixed system before the microporous coating, squeezing out the hydrophobic nucleation. During this squeezing process, due to the action of water and the thermodynamic trend toward minimum interfacial energy, the hydrophobic nucleation transitions to a state with the lowest interfacial energy, resulting in a smoother and denser coating. As the coating becomes smoother and denser, the drug molecules are effectively encapsulated within the porous structure.

[0068] Continuing from the above, the preparation method of the drug delivery coating proposed in the present invention is to first mix the coating with a micro-nano pore structure with a drug solution, and realize the filling of the drug through the capillary effect of the micro-nano pore structure; then mix the coating filled with the drug with a phosphate solution, and induce the closure of the micro-nano pore structure in the coating by phosphate, so as to effectively encapsulate the drug in the coating. Compared with mixing the coating with a micro-nano pore structure with the drug-phosphate solution at one time, it can better control the concentration of the drug in the solution and more accurately control the loading amount of the drug in the coating, effectively solving the contradiction of "high load-low burst release" that is difficult to achieve in existing carriers. In addition, the drug loading conditions of the coating are mild, which can achieve effective loading and activity retention of various active biological molecules, and can also be adapted to various types of drugs through modular design, providing a new way to build the next generation of intelligent drug delivery systems. The resulting drug delivery coating has a high drug loading capacity, a low initial burst release rate of the drug, and a long sustained release period, which can meet the treatment needs of chronic diseases.

[0069] Correspondingly, the present invention also provides a drug delivery coating, which is prepared by the above preparation method.

[0070] The drug delivery coating has a high drug loading capacity (for example, it can reach more than 40%), a low initial burst rate of drug release (for example, it can be reduced to less than 10%), and a long sustained release period (for example, it can reach more than 50 days), which can meet the treatment needs of chronic diseases.

[0071] The drug delivery coating has good biocompatibility, and the polypeptides and drug molecules containing hydrophobic amino acids can be released into tissues or blood environments to exert their effects as the acidic protein degrades in the human body.

[0072] In addition, the present invention also provides a drug delivery carrier material, which has the above-mentioned drug delivery coating.

[0073] The drug delivery carrier material may have a substrate, and the drug delivery coating is arranged on the surface of the substrate.

[0074] For example, the substrate may include at least one of a metal material, an inorganic material, a polymer material, a natural biological material, and an artificially synthesized polypeptide hydrogel material.

[0075] The metal material may illustratively include at least one of cobalt-based alloys, titanium and its alloys, nickel-titanium alloys, platinum and its alloys, magnesium and its alloys, iron and its alloys, and zinc and its alloys.

[0076] The inorganic material may illustratively include at least one of titanium oxide and its nanotubes, carbon materials, silicon, silicon dioxide, calcium phosphate, silicon nitride, silicon carbide, aluminosilicate, calcium aluminum system, bioglass, titanium nitride and biomedical micro-nanoparticles.

[0077] The biomedical micro-nanoparticles may illustratively include at least one of ferroferric oxide nanoparticles, silicon dioxide nanoparticles, titanium oxide nanoparticles, and zinc oxide nanoparticles.

[0078] The polymer material may illustratively include at least one of polyester, polyethylene, polyvinyl chloride, polytetrafluoroethylene, polyurethane, polystyrene, polyvinyl alcohol, polypropylene, polyoxymethylene, polycarbonate, carbon copolymer, polyglycolic acid, polymethyl methacrylate, polyvinyl acetate, polylactic acid, glycolide-lactide copolymer, polytrimethylene carbonate, polycaprolactone, polyhydroxyalkanoate, polybutylene succinate, polyamide, polydioxanone, epoxy resin, silicone rubber, silicone gel, polyacrylic acid and its derivatives, polyethylene glycol and its derivatives.

[0079] The natural biomaterial may illustratively include at least one of animal-derived decellularized tissues and organs, gelatin, collagen, fibrin, silk protein, keratin, and polysaccharides. The animal-derived decellularized tissues and organs may illustratively include at least one of blood vessels, valves, heart, bone, lungs, ligaments, bladder, mucosa, and cornea. The polysaccharide may illustratively include at least one of sodium hyaluronate, sodium alginate, agarose, plastic starch materials, cellulose, hemicellulose, lignin, chitin, and derivatives thereof.

[0080] The artificially synthesized polypeptide hydrogel material may illustratively include at least one of L-lysine and poly-L-glutamic acid.

[0081] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0082] Example 1 This embodiment provides a drug delivery carrier material, which can be obtained in the following manner: ① Add a pH 6 solution to a clean stainless steel surface, ensuring it fully covers the surface. The solution is then allowed to react in a 37°C incubator for 12 hours. After the reaction is complete, remove the sample and rinse with 25°C ultrapure water to remove loosely adhered material. Dry the sample (37°C for 12 hours) to obtain a coating with a micro-nanoporous structure.

[0083] The solution system is prepared by mixing a hydrophobic amino acid-containing peptide solution, an acidic protein solution, and an oxidant solution. The solvent for the hydrophobic amino acid-containing peptide solution, the acidic protein solution, and the oxidant solution is ultrapure water at a pH of 6 (pH adjusted with hydrochloric acid). The hydrophobic amino acid-containing peptide solution is bivalirudin with an isoelectric point (pI1) of 3.99, and the acidic protein is whey albumin with an isoelectric point (pI2) of 4.2 and a negative charge at pH ≥ 7, with a ΔpI = |pI1 - pI2| = 0.21. The oxidant is ammonium persulfate. The concentrations of the hydrophobic amino acid-containing peptide solution are 1 mg / mL, the acidic protein solution is 1 mg / mL, and the oxidant solution is 2 mg / mL. The volume ratio of the hydrophobic amino acid-containing peptide solution, the acidic protein solution, and the oxidant solution is 1:5:5. In other words, the molar ratio of the hydrophobic amino acid-containing polypeptide to the acidic protein is 0.2:1, and the total concentration of the hydrophobic amino acid-containing polypeptide and the acidic protein in the solution system is 0.11 mmol / L.

[0084] ② Immerse the stainless steel coated with the micro-nanoporous structure in a drug solution at 25°C for 10 minutes. After immersion, remove the stainless steel and rinse with ultrapure water. Dry the stainless steel in a 37°C oven for 12 minutes to obtain a drug-filled coating. The drug solution is a saturated colchicine aqueous solution.

[0085] ③. Immerse the drug-filled coating in a 3 mmol / L sodium hydrogen phosphate aqueous solution at 25°C for 10 minutes. Remove and rinse with ultrapure water, then dry in a constant temperature drying oven at 37°C for 12 minutes to obtain a drug delivery coating on the stainless steel surface.

[0086] Example 2 The difference between this embodiment and embodiment 1 is that the mixing temperature of the coating with micro-nanoporous structure and the drug solution is 25° C., colchicine is replaced by DAPI (4′,6-diamidino-2-phenylindole), and the rest is the same as embodiment 1.

[0087] Example 3 The difference between this embodiment and embodiment 1 is that the mixing temperature of the coating with a micro-nano porous structure and the drug solution is 25° C., and colchicine is replaced by rhodamine B. The rest is the same as embodiment 1.

[0088] Example 4 The difference between this embodiment and embodiment 1 is that the mixing temperature of the coating with a micro-nano porous structure and the drug solution is 25° C., colchicine is replaced by zinc oxide nanoparticles (ZnO, particle size of 20 nm to 40 nm), and the rest is the same as embodiment 1.

[0089] Example 5 The difference between this embodiment and embodiment 1 is that the mixing temperature of the coating with micro-nano porous structure and the drug solution is 25° C., colchicine is replaced by titanium dioxide nanoparticles (TiO 2 , 90 nm to 110 nm), and the rest is the same as embodiment 1.

[0090] Example 6 The difference between this embodiment and embodiment 1 is that the mixing temperature of the coating with micro-nanoporous structure and the drug solution is 25° C., colchicine is replaced by monodisperse fluorescent microspheres (MFM, with a diameter of about 1 μm), and the rest is the same as embodiment 1.

[0091] Example 7 The difference between this embodiment and embodiment 1 is that the reaction temperature of the polypeptide containing hydrophobic amino acids, the acidic protein and the oxidant is 30°C.

[0092] Example 8 The difference between this embodiment and embodiment 1 is that the reaction temperature of the polypeptide containing hydrophobic amino acids, the acidic protein and the oxidant is 40°C.

[0093] Example 9 The difference between this embodiment and embodiment 1 is that the reaction temperature of the polypeptide containing hydrophobic amino acids, the acidic protein and the oxidant is 50°C.

[0094] Example 10 The difference between this embodiment and embodiment 1 is that the reaction pH of the polypeptide containing hydrophobic amino acids, the acidic protein and the oxidant is 3.

[0095] Example 11 The difference between this embodiment and embodiment 1 is that the reaction pH of the polypeptide containing hydrophobic amino acids, the acidic protein and the oxidant is 5.

[0096] Example 12 The difference between this embodiment and embodiment 1 is that the reaction pH of the polypeptide containing hydrophobic amino acids, the acidic protein and the oxidant is 6.5.

[0097] Example 13 The difference between this embodiment and embodiment 1 is that the reaction pH of the polypeptide containing hydrophobic amino acids, the acidic protein and the oxidant is 7.

[0098] Example 14 The difference between this embodiment and embodiment 1 is that the polypeptide containing hydrophobic amino acids is an acidic antimicrobial peptide with an isoelectric point pI1=4.2, the acidic protein is whey protease with an isoelectric point pI2=4.2 and a negative charge under pH ≥7, ΔpI=|pI1-pI2|=0; and the oxidant is sodium peroxide.

[0099] Example 15 The difference between this embodiment and embodiment 1 is that the hydrophobic amino acid-containing polypeptide is gastrin with an isoelectric point pI1 = 5.7, the acidic protein is serum albumin with an isoelectric point pI2 = 4.7 and negatively charged at pH ≥ 7, ΔpI = |pI1-pI2| = 1; and the oxidant is sodium permanganate.

[0100] Example 16 The difference between this embodiment and embodiment 1 is that the polypeptide containing hydrophobic amino acids is a cell-penetrating peptide with an isoelectric point pI1=6, the acidic protein is pepsin with an isoelectric point pI2=3.5 and a negative charge at pH ≥7, ΔpI=|pI1-pI2|=2.5; and the oxidant is sodium peroxide.

[0101] Example 17 The difference between this embodiment and embodiment 1 is that the molar ratio of the polypeptide containing hydrophobic amino acids to the acidic protein is 0.1:1.

[0102] Example 18 The difference between this embodiment and embodiment 1 is that the molar ratio of the polypeptide containing hydrophobic amino acids to the acidic protein is 0.5:1.

[0103] Example 19 The difference between this embodiment and embodiment 1 is that the molar ratio of the polypeptide containing hydrophobic amino acids to the acidic protein is 5:1.

[0104] Example 20 The difference between this embodiment and embodiment 1 is that the molar ratio of the polypeptide containing hydrophobic amino acids to the acidic protein is 10:1.

[0105] Example 21 The difference between this embodiment and embodiment 1 is that the total concentration of the polypeptide containing hydrophobic amino acids and the acidic protein in the solution system is 0.01 mmol / L.

[0106] Example 22 The difference between this embodiment and embodiment 1 is that the total concentration of the polypeptide containing hydrophobic amino acids and the acidic protein in the solution system is 5 mmol / L.

[0107] Example 23 The difference between this embodiment and embodiment 1 is that the total concentration of the polypeptide containing hydrophobic amino acids and the acidic protein in the solution system is 10 mmol / L.

[0108] Example 24 The difference between this embodiment and embodiment 1 is that the drug selected is miRNA-22, and the mixing temperature of the coating filled with the drug and the phosphate solution is -10°C.

[0109] Example 25 The difference between this embodiment and embodiment 1 is that the drug selected is miRNA-22, and the mixing temperature of the coating filled with the drug and the phosphate solution is 4°C.

[0110] Example 26 The difference between this embodiment and embodiment 1 is that the drug is miRNA-22, and the mixing temperature of the coating filled with the drug and the phosphate solution is 37°C.

[0111] Example 27 The difference between this embodiment and embodiment 1 is that miRNA-22 is selected as the drug, and the mixing temperature of the coating filled with the drug and the phosphate solution is 50°C.

[0112] Example 28 The difference between this embodiment and embodiment 1 is that the mixing time of the coating filled with the drug and the phosphate solution is 1 minute.

[0113] Example 29 The difference between this embodiment and embodiment 1 is that the mixing time of the coating layer filled with the drug and the phosphate solution is 5 minutes.

[0114] Example 30 The difference between this embodiment and embodiment 1 is that the mixing time of the coating layer filled with the drug and the phosphate solution is 60 minutes.

[0115] Example 31 The difference between this embodiment and embodiment 1 is that the concentration of the phosphate solution is 0.1 mmol / L.

[0116] Example 32 The difference between this embodiment and embodiment 1 is that the concentration of the phosphate solution is 5 mmol / L.

[0117] Example 33 The difference between this embodiment and embodiment 1 is that the concentration of the phosphate solution is 10 mmol / L.

[0118] Example 34 The difference between this embodiment and embodiment 1 is that phosphoric acid aqueous solution is used as the phosphate solution, and the rest is the same as embodiment 1.

[0119] Example 35 The difference between this embodiment and embodiment 1 is that sodium dihydrogen phosphate aqueous solution is used as the phosphate solution, and the rest is the same as embodiment 1.

[0120] Comparative Example 1 The difference between this comparative example and Example 1 is that the polypeptide containing hydrophobic amino acids is melittin with an isoelectric point pI1>7.

[0121] Comparative Example 2 The difference between this comparative example and Example 1 is that the acidic protein is lysozyme with an isoelectric point pI2>7.

[0122] Comparative Example 3 The difference between this comparative example and Example 1 is that the polypeptide containing hydrophobic amino acids is bradykinin with an isoelectric point pI1 < 7, the acidic protein is calmodulin with an isoelectric point pI2 > 7, and ΔpI = |pI1-pI2| < 2.5.

[0123] Comparative Example 4 The difference between this comparative example and Example 1 is that the polypeptide containing hydrophobic amino acids is gastrin with an isoelectric point pI1 < 7, the acidic protein is lysozyme with an isoelectric point pI2 > 7, and ΔpI = |pI1-pI2| > 2.5.

[0124] Comparative Example 5 The difference between this comparative example and Example 1 is that the polypeptide containing hydrophobic amino acids is melittin with an isoelectric point pI1>7, the acidic protein is collagen with an isoelectric point pI2<7, and ΔpI=|pI1-pI2|<2.5.

[0125] Comparative Example 6 The difference between this comparative example and Example 1 is that the polypeptide containing hydrophobic amino acids is melittin with an isoelectric point pI1>7, the acidic protein is casein with an isoelectric point pI2<7, and ΔpI=|pI1-pI2|>2.5.

[0126] Comparative Example 7 The difference between this comparative example and Example 1 is that the polypeptide containing hydrophobic amino acids is gastrin with an isoelectric point pI1 <7, the acidic protein is collagen with an isoelectric point pI2 <7, and ΔpI = |pI1-pI2| > 2.5.

[0127] Comparative Example 8 The difference between this comparative example and Example 1 is that only 5% of the amino acids in the amino acid sequence of the polypeptide are hydrophobic amino acids.

[0128] Comparative Example 9 The difference between this comparative example and Example 1 is that the reaction temperature of the polypeptide containing hydrophobic amino acids, the acidic protein and the oxidant is 10°C.

[0129] Comparative Example 10 The difference between this comparative example and Example 1 is that the reaction temperature of the polypeptide containing hydrophobic amino acids, the acidic protein and the oxidant is 65°C.

[0130] Comparative Example 11 The difference between this comparative example and Example 1 is that the reaction pH of the polypeptide containing hydrophobic amino acids, the acidic protein and the oxidant is 2°C.

[0131] Comparative Example 12 The difference between this comparative example and Example 1 is that the reaction pH of the polypeptide containing hydrophobic amino acids, the acidic protein and the oxidant is 8°C.

[0132] Comparative Example 13 The difference between this comparative example and Example 1 is that the molar ratio of the polypeptide containing hydrophobic amino acids to the acidic protein is 0.05:1.

[0133] Comparative Example 14 The difference between this comparative example and Example 1 is that the molar ratio of the polypeptide containing hydrophobic amino acids to the acidic protein is 15:1.

[0134] Comparative Example 15 The difference between this comparative example and Example 1 is that the concentration of the phosphate solution is 0.05 mmol / L.

[0135] Comparative Example 16 The difference between this comparative example and Example 1 is that the concentration of the phosphate solution is 12 mmol / L.

[0136] Comparative Example 17 The difference between this comparative example and Example 1 is that the disodium hydrogen phosphate solution is replaced by ultrapure water.

[0137] Comparative Example 18 The difference between this comparative example and Example 1 is that the disodium hydrogen phosphate solution is replaced by a sodium sulfate aqueous solution.

[0138] Comparative Example 19 The difference between this comparative example and Example 1 is that the drug solution and the phosphate solution are first mixed to form a drug-phosphate solution, and then the stainless steel having the micro-nanoporous structure coating is immersed in the drug-phosphate solution. The remaining operations are the same as in Example 1.

[0139] Test Example 1 (1) The coating with micro-nano pore structure obtained in Example 1 was observed by scanning electron microscopy. The results are as follows: Figure 1shown. Figure 1 (a) is a surface SEM image of the coating having a micro-nano porous structure, (b) is a cross-sectional SEM image of the coating having a micro-nano porous structure, (c) is a local enlarged image of (a), and (d) is a local enlarged image of (b).

[0140] Depend on Figure 1 It can be seen that the coating is composed of stacked micro-nano particles and has a large number of pore structures.

[0141] (2) The drug delivery coating obtained in Example 1 (2) was observed by scanning electron microscopy. The results are as follows: Figure 2 shown. Figure 2 (a) is a surface SEM image of the drug delivery coating, (b) is a cross-sectional SEM image of the drug delivery coating, (c) is a partial enlarged image of (a), and (d) is a partial enlarged image of (b).

[0142] Depend on Figure 2 It can be seen that the drug delivery coating presents a dense and compact structure.

[0143] Test Example 2 The stainless steel sample with drug delivery coating prepared in Example 1 was subjected to an immersion experiment. The experimental method and conditions are as follows: a portion of the sample and immersion liquid were taken out at different immersion time points of the stainless steel sample with drug delivery coating prepared in Example 1. The control group was a bare substrate and simulated body fluid used for the immersion experiment. Equal amounts of platelet-poor plasma (PPP) were added to the sample surface and the immersion liquid, respectively. The samples were incubated in a 37°C incubator for 30 minutes. The PPP was taken out and mixed with an equal amount of thrombin reagent. The time when the mixed reaction liquid began to coagulate was observed and recorded. The thrombin time test results of the sample and the immersion liquid in the experiment were as follows: Figure 3 As shown in (a) and (b).

[0144] Depend on Figure 3 It can be seen that the drug delivery coating can achieve long-term sustained release of loaded drugs, and the sustained release time can be as long as more than 50 days.

[0145] Test Example 3 The stainless steel samples with drug delivery coatings obtained in Examples 2-3 and 6 were observed under a laser confocal microscope for the fluorescence distribution inside the coatings. The results are as follows: Figure 4 shown.

[0146] Depend on Figure 4It can be seen that there are a large number of fluorescent signals distributed inside the coating, indicating that the coating can achieve the loading of small molecules such as fluorescent signals and macromolecular drugs with a particle size of about 1 micron. The drug delivery coating can achieve efficient loading of the small molecules (DAPI and Rhodamine B) of Examples 2 to 3 and the macromolecular drug (MFM) of Example 6.

[0147] Test Example 4 The stainless steel samples with drug delivery coating obtained in Examples 4 and 5 were subjected to energy spectrum analysis, and the results were as follows: Figure 5 and Figure 6 shown. Figure 5 (a) is a cross-sectional SEM image of the drug delivery coating of Example 4, Figure 5 (b) to (g) correspond to the line scan results of C, N, O, Si, Pt, and Ti in the stainless steel sample of Example 4, respectively. Figure 6 (a) is a cross-sectional SEM image of the drug delivery coating of Example 5, Figure 6 (b) to (g) correspond to the line scan results of C, N, O, Si, Pt, and Zn in the stainless steel sample of Example 5, respectively.

[0148] Depend on Figure 5 and Figure 6 It can be seen that the energy spectrum line scan of the coating cross section shows that characteristic elements such as zinc and titanium are distributed throughout the coating, indicating that zinc oxide (20nm~40nm) and titanium oxide nanoparticles (90nm~110nm) are successfully loaded and evenly distributed inside the coating, that is, the drug delivery coating is able to achieve the loading of medium-molecular drugs (zinc oxide nanoparticles and titanium oxide nanoparticles).

[0149] Test Example 5 The stainless steel samples with drug delivery coatings obtained in Example 1, Examples 14 to 16, and Comparative Examples 1 to 8 were subjected to an immersion experiment. The experimental method and conditions were as follows: the absorbance of the immersion solution at each time period was measured at 350 nm, and the quantification was calculated based on the colchicine standard curve. The drug release amount of each stainless steel sample after immersion for 0 min, 10 min, 20 min, and 30 min was tested. The results are shown in FIG. Figure 7 shown.

[0150] Depend on Figure 7It can be seen that the isoelectric point or hydrophobicity of peptides and proteins will directly affect the drug loading or the initial burst rate of drug release. The reason may be that the isoelectric point or hydrophobicity of peptides and proteins will affect the structure of the coating obtained. If the isoelectric point of either the peptide or the protein is greater than 7, or the difference between the isoelectric points of the two is greater than 2.5, or the content of hydrophobic amino acids in the peptide is too low, it will result in the inability to obtain a coating with a micro-nanoporous structure, or the porosity of the coating will be low, which will lead to low drug loading or a high initial burst rate of drug release.

[0151] Test Example 6 The coatings with micro-nanoporous structures obtained in Example 1, Examples 7 to 13, and Comparative Examples 9 to 12 (1) were compared. The results showed that no coating could be formed in Comparative Examples 9 to 12. The reason is that the reaction temperature and pH value of the hydrophobic amino acid-containing polypeptide, acidic protein, and oxidant directly affect the structure of the coating obtained in (1). If the reaction temperature is too low, self-assembly cannot occur between the protein and polypeptide molecules, and then the coating cannot be formed; if the reaction temperature is too high, the protein and polypeptide structures will be severely damaged, and the coating cannot be formed; if the pH value is too low or too low, the protein and polypeptide structures will be severely damaged and precipitate immediately.

[0152] Test Example 7 The coatings with micro-nano pore structures obtained in Example 1, Examples 17-20, and Comparative Examples 13-14 were observed by scanning electron microscopy and the porosity of the coatings was quantified using Image J software. The results are as follows: Figure 8 As shown; the surface roughness of the above samples was measured using a 3D profilometer, and the results are as follows Figure 9 shown.

[0153] Depend on Figure 8 and Figure 9 It can be seen that the relationship between the amounts of the hydrophobic amino acid-containing polypeptide and the acidic protein will also directly affect the structure of the resulting coating. If there are too few hydrophobic amino acid-containing polypeptides, the pore structure of the coating will be small and the porosity will be low, which is not conducive to drug loading. If there are too many hydrophobic amino acid-containing polypeptides, the pore structure of the coating will be large but uneven, and the measured roughness of the coating will be large, which is not conducive to the complete self-encapsulation of the subsequent coating, thereby affecting drug loading.

[0154] Test Example 8 The coatings with micro-nanoporous structures obtained in Example 1, Examples 31-35, and Comparative Examples 15-18 were reacted with their respective corresponding drug-phosphate solutions at room temperature, and the SEM images of the drug delivery coatings corresponding to each sample were measured after 1 min, 5 min, 10 min, and 30 min of reaction. The results are shown in FIG. Figure 10 shown.

[0155] Depend on Figure 10It can be seen that using a phosphate solution significantly improves the density of the drug delivery coating compared to a non-phosphate solution. Furthermore, within a certain range, the higher the concentration of the phosphate solution, the better the density of the resulting drug delivery coating. However, excessively high concentrations of the phosphate solution can disrupt the uniformity of the coating, hindering drug loading and long-term release.

[0156] Test Example 9 The stainless steel samples with drug delivery coating obtained in Example 1, Examples 31-35, and Comparative Examples 15-18 were subjected to immersion experiments. The experimental methods and conditions are as follows: The specific method is shown in Experimental Example 5. The drug release amount of each stainless steel sample holder after immersion for 0 min, 10 min, 20 min, and 30 min was tested. The results are shown in Figure 5. Figure 11 shown.

[0157] Depend on Figure 11 It can be seen that Examples 31-32 have a small drug loading amount, a small burst release amount, and a small amount of drug released during the interval time, which may make it difficult to achieve an effective dose; in comparison, the stainless steel sample prepared in Example 1 has the best drug sustained release effect.

[0158] Test Example 10 Comparing Example 1 with Comparative Example 19, the mixed solution after drug loading and the solution of the washed sample were collected and the absorbance value was measured at 350 nm. The quantitative drug loading amount was converted by the colchicine standard curve. The results are as follows: Figure 12 shown.

[0159] Depend on Figure 12 It can be seen that immersing the stainless steel with a micro-nano porous structure coating in a drug-phosphate solution can significantly increase the drug loading capacity compared to immersing the stainless steel with a micro-nano porous structure coating in a drug solution first and then in a phosphate solution.

[0160] Test Example 11 The thickness of the drug delivery coating of the stainless steel samples of Example 1 and Examples 21 to 23 was measured by ellipsometry. The results are as follows: Figure 13 shown.

[0161] Depend on Figure 13 It can be seen that if the total concentration of the hydrophobic amino acid-containing peptide and the acidic protein in the solution system is too low, the drug delivery coating formed by the reaction will be too thin, which is not conducive to drug loading. On the other hand, if the total concentration of the hydrophobic amino acid-containing peptide and the acidic protein in the solution system is too high, the drug delivery coating will be thick, prone to cracking after substrate surface modification, and poor stability.

[0162] Test Example 12 The drug used in Example 1 was replaced with miRNA-22 and this example was defined as Example 1'. The coating of Example 1' and Examples 24 to 27 were completely eluted, and the active miRNA-22 in the eluate was quantified using a micro RNA quantification kit. The results are shown in FIG. Figure 14 shown.

[0163] Depend on Figure 14 It can be seen that the protein polypeptide coating can be loaded with drugs at low temperatures, maximally retaining the physiological activity of the active molecules.

[0164] Test Example 13 The stainless steel samples with drug delivery coatings obtained in Examples 1 and 28 to 30 were completely eluted, and the content of colchicine in the eluate was measured. The specific implementation steps are shown in Experimental Example 5. The results are shown in FIG. Figure 15 shown.

[0165] Depend on Figure 15 It can be seen that the drug loading amount and the phosphate treatment time are positively correlated within a certain range, which is speculated to be related to the degree of self-encapsulation of the drug delivery coating. When the drug delivery coating is incompletely self-encapsulated, the drug molecules leak out of the pores during the cleaning process and are easily eluted. When the drug delivery coating is completely self-encapsulated, the drug molecules are firmly sealed in the coating, not easily eluted, and can be released slowly over a long period of time.

[0166] In summary, the preparation method of the drug delivery coating provided by the present invention is to first mix the coating with a micro-nano pore structure with a drug solution, and realize the filling of the drug through the capillary effect of the micro-nano pore structure; then mix the coating filled with the drug with a phosphate solution, and induce the micro-nano pore structure in the coating to close by phosphate, thereby effectively encapsulating the drug in the coating. Compared with mixing the coating with a micro-nano pore structure with a drug-phosphate solution at one time, it can better control the concentration of the drug in the solution, and more accurately control the loading amount of the drug in the coating, effectively solving the contradiction that the existing carriers are difficult to achieve "high load-low burst release". In addition, the coating has mild drug loading conditions, can achieve effective loading and activity retention of various active biological molecules, and can also be adapted to various drug types through modular design, providing a new way to build the next generation of intelligent drug delivery systems. The resulting drug delivery coating has a high drug loading capacity, a low initial burst release rate of the drug, and a long sustained release period, which can meet the treatment needs of chronic diseases.

[0167] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing a drug delivery coating, characterized in that: The following steps are involved: A hydrophobic amino acid-containing polypeptide is reacted with an acidic protein in a solution system with a pH value of 3-7, a temperature of 20°C-50°C and an oxidant to prepare a coating with a micro-nano porous structure; The coating having the micro-nano pore structure is mixed with a drug solution to achieve drug filling through the capillary effect of the micro-nano pore structure; the coating filled with the drug is mixed with a phosphate solution to induce the micro-nano pore structure in the coating to close, thereby obtaining the drug delivery coating; The isoelectric point of the polypeptide containing hydrophobic amino acids is pI1, the isoelectric point of the acidic protein is pI2, pI1<7, pI2<7, ΔpI=|pI1-pI2|≤2.5; and the acidic protein is negatively charged under the condition of pH≥7.

2. The preparation method according to claim 1, characterized in that The molar ratio of the hydrophobic amino acid-containing polypeptide to the acidic protein is 0.1:1 to 10:1, and the total concentration of the hydrophobic amino acid-containing polypeptide and the acidic protein in the solution system is 0.01 mmol / mL to 10 mmol / L.

3. The preparation method according to claim 2, characterized in that The molar ratio of the hydrophobic amino acid-containing polypeptide to the acidic protein is 0.1:1 to 5:1, and the total concentration of the hydrophobic amino acid-containing polypeptide and the acidic protein in the solution system is 0.01 mmol / mL to 5 mmol / L.

4. The preparation method according to any one of claims 1 to 3, characterized in that At least 10% of the amino acids in the amino acid sequence of the hydrophobic amino acid-containing polypeptide are hydrophobic amino acids.

5. The preparation method according to claim 4, characterized in that The hydrophobic amino acid includes at least one of alanine, valine, leucine, phenylalanine, tryptophan and isoleucine.

6. The preparation method according to any one of claims 1 to 3, characterized in that The acidic protein is a protein containing acidic amino acid residues.

7. The preparation method according to claim 6, characterized in that The acidic amino acid residue includes at least one of aspartic acid and glutamic acid.

8. The preparation method according to any one of claims 1 to 3, characterized in that The polypeptide containing the hydrophobic amino acid reacts with the acidic protein in a solution system with a pH value of 5-6.5, a temperature of 30° C.-40° C. and the presence of an oxidant.

9. The preparation method according to any one of claims 1 to 3, characterized in that The mixing temperature of the coating with micro-nano pore structure and the drug solution is 0°C to 40°C.

10. The preparation method according to claim 9, characterized in that The drug concentration in the drug solution is a saturated concentration.

11. The preparation method according to claim 9, characterized in that The solvent of the drug solution does not contain phosphate ions.

12. The preparation method according to claim 9, characterized in that The coating with the micro-nano pore structure is mixed with the drug solution by immersing the coating in the drug solution.

13. The preparation method according to claim 12, characterized in that The coating is immersed in the drug solution for no less than 1 minute.

14. The preparation method according to any one of claims 1 to 3, characterized in that The coating filled with the drug is mixed with the phosphate solution by immersing the coating in the phosphate solution.

15. The preparation method according to claim 14, characterized in that During the immersion process, the temperature is maintained at -10℃~60℃ and the immersion time is at least 1 minute.

16. The preparation method according to claim 15, characterized in that During the immersion process, the immersion time is 5 minutes to 60 minutes.

17. The preparation method according to claim 1, characterized in that The concentration of the phosphate solution is 0.1 mmol / L to 10 mmol / L.

18. The preparation method according to claim 17, characterized in that: The concentration of the phosphate solution is 3 mmol / L to 10 mmol / L.

19. A drug delivery coating, characterized in that Prepared by the preparation method according to any one of claims 1 to 18.

20. A drug delivery carrier material, characterized in that The drug delivery carrier material has the drug delivery coating according to claim 19.

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