Collagen nano polymer particle and preparation method thereof

Abstract

The invention discloses a preparation method of collagen nano polymer particles, which comprises the following steps: carrying out surface hydrolysis on organized collagen to prepare a microsphere base material; the preparation method comprises the following steps: uniformly mixing a microsphere base material, a target load, a nonionic surfactant and polyol ester in proportion to prepare a microsphere modified base material; and uniformly mixing the microsphere modified base material, an ether compound and a chemical auxiliary agent according to a ratio to prepare collagen microspheres, namely the collagen nano polymer particles. The preparation method comprises the following steps: firstly, reducing the volume of collagen through enzyme hydrolysis, reducing biological stimulation possibly caused by drug loading, and strengthening the drug loading property of the collagen nano polymer; and secondly, a polymer crystal region appears in the carrier protein in an aqueous solution by utilizing subsequent surface modification, so that the drug carrier can control the release progress of the loaded drug, and the drug carrier can permeate into deep tissues through the surface layer of the skin.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomass capsules, and in particular relates to collagen nano-polymer particles and a preparation method thereof. Background Art

[0002] Collagen, a protein ubiquitous in human tissue, exhibits significant potential as a drug carrier due to its exceptional biocompatibility and biodegradability. As a drug carrier, collagen, through precise manipulation of its structure and properties, can achieve controlled drug release, thereby prolonging drug action and enhancing drug stability and bioavailability. Collagen's porous structure enables it to adsorb and activate platelets, release growth factors and cell adhesion proteins, and promote angiogenesis and tissue regeneration, making it essential for its function as a drug carrier. Furthermore, collagen can serve as a carrier for gene therapy, effectively delivering therapeutic genes to target cells and achieving efficient gene expression. In practical applications, collagen has been widely used for the topical delivery of low-molecular-weight drugs, including antibiotics. In the treatment of severe burns and various types of trauma, collagen sponges can absorb large amounts of tissue permeate, preventing wounds and promoting rapid drying. Furthermore, collagen can be used as a carrier for ocular drug delivery, enabling gradual drug release within the conjunctival sac, ensuring that intraocular drug concentrations reach high levels quickly and are maintained for extended periods, effectively reducing systemic drug toxicity.

[0003] However, collagen also faces challenges in cosmetics and medical applications, including poor stability and transdermal absorption. Its hydrogen bonds and cross-linked structures are susceptible to environmental factors such as temperature, pH, and air during cosmetic preparation and use, causing them to lose their activity. Furthermore, collagen's large molecular weight makes it difficult to penetrate the skin's surface into deeper tissues, which to some extent limits its application in these areas. Summary of the Invention

[0004] In order to overcome the shortcomings of the existing technology, the present invention provides a collagen nanoparticle and a preparation method thereof, aiming at the technical difficulties in traditional technology such as the inactivation of transdermal drug delivery materials due to external influences and weak biological permeability.

[0005] The technical solution adopted by the present invention to solve its technical problem is:

[0006] A method for preparing collagen nanoparticles comprises the following steps:

[0007] The surface of the organized collagen is hydrolyzed to prepare a microsphere substrate;

[0008] The microsphere substrate, the target load, the nonionic surfactant and the polyol ester are mixed uniformly in proportion to prepare a microsphere modified substrate;

[0009] The microsphere modified substrate, ether compound and chemical additives are mixed evenly in proportion to prepare collagen microspheres, namely collagen nano-polymer particles.

[0010] As a preference,

[0011] The organic collagen is prefabricated collagen, and its preparation method includes the following steps: uniformly mixing collagen, methacrylic anhydride and phosphate buffer solution in a mass ratio of 1: (0.20-0.30): (9.5-11.5), continuously stirring for 2-6 hours in an environment at a temperature of 50-60°C, pouring the solution into a dialysis bag with a molecular weight cutoff of 500D after stirring, dialyzing in deionized water for 24 hours, cooling and drying to obtain the organic collagen.

[0012] As a preference,

[0013] The surface hydrolysis step is to use protease to perform enzymatic hydrolysis, and the amount of the protease used is 1 to 2 wt% of the organized collagen.

[0014] As a preference,

[0015] The protease is 1398 neutral protease.

[0016] As a preference,

[0017] Distilled water or deionized water is added in the surface hydrolysis step, and then reacted for 2 to 6 hours in an environment with a temperature of 35 to 45° C. and a pH of 6.5 to 7.5 to complete the preparation of the microsphere substrate, wherein the amount of distilled water or deionized water used is 2.5 to 3.5 times the volume of the organized collagen.

[0018] As a preference,

[0019] The target load is an imidazole compound; the nonionic surfactant is glyceryl monostearate; the polyol ester is polysorbate; the microsphere substrate, the target load, the nonionic surfactant and the polyol ester are evenly mixed in a mass ratio of 1:(3-4):(0.4-0.8):0.3.

[0020] As a preference,

[0021] The step of preparing the microsphere modified substrate is to react in an environment with a temperature of 40 to 50° C. for 2 to 4 hours.

[0022] As a preference,

[0023] The ether compound is anhydrous ether; the chemical additive is liquid paraffin, which contains 0.3-0.7 wt% of Span 80; the microsphere modified substrate, the ether compound and the chemical additive are evenly mixed in a mass ratio of 6: (0.90-1.00): (18-22).

[0024] As a preference,

[0025] The step of preparing collagen microspheres is to stir the microsphere modified substrate, ether compound and chemical additive for 20 to 40 minutes under the conditions of protective gas atmosphere, temperature of 30 to 40° C. and ultrasonic power of 160 to 200W.

[0026] A collagen nanoparticle,

[0027] Prepared by the preparation method according to any one of claims 1 to 9.

[0028] The core of the technology of the present invention is to change the affinity of collagen by hydrolyzing and surface modifying collagen. Functional modification can enhance the drug-loading capacity of collagen nanopolymers, improve the biosafety of collagen nanopolymers, and reduce irritation. At the same time, the modified collagen molecular structure can penetrate through the surface of the skin into the deep tissue.

[0029] Among the existing technical solutions, collagen has demonstrated its unique potential as a drug carrier. By connecting drugs to collagen, targeted drug delivery can be achieved, thereby significantly reducing the potential side effects of drugs during translocation; in addition, collagen is also widely used in the preparation of biomaterial substrates due to its excellent biocompatibility and bioactivity, such as artificial skin, bones, and blood vessels. However, although collagen has many advantages as a drug carrier, there are still some limitations that cannot be ignored. First, the extraction and purification process of collagen is relatively complex, which may have a certain impact on its stability and bioactivity; secondly, the immunogenicity of collagen may limit its permeability when delivering cell membranes; in addition, the degradation rate and method of collagen may also have a significant impact on the release rate and final effect of the drug.

[0030] In this technical solution, collagen is modified to adjust its molecular structure and properties to achieve precise control of drug release, thereby extending the effective duration of the drug in the body and significantly enhancing the stability and bioavailability of the drug. Through surface modification technology, collagen exhibits a unique porous structure at the macro level. These structures can effectively adsorb and activate platelets, promote the release of growth factors and the activation of cell adhesion proteins, thereby accelerating the process of angiogenesis and cell tissue regeneration. In addition, the modified collagen also has the ability to accurately identify intracellular targets and achieve efficient regulation of gene expression. At the same time, as a drug carrier, collagen can stably and slowly release drugs in an in vitro environment, ensuring that the drug continues to act at the target location, thereby reducing nonspecific effects on surrounding cell structures.

[0031] In the present invention, an enzymatic method is used to decompose the long-chain structure of collagen, thereby obtaining smaller protein fragments and monomers. Under specific acidic conditions, the amide group undergoes a hydrolysis reaction, during which the active groups amino and carboxyl groups at the ends of the polyamide fiber are exposed. The enzymatic hydrolysis process has a significant effect on the physical and chemical properties of the protein, which is specifically reflected in the solubility, viscosity and molecular size of the modified collagen. The collagen fragments that have been hydrolyzed show higher potential and are easy to form nanoscale polymers. This type of nanoscale collagen has significant advantages as a transport carrier for drug delivery. It can effectively penetrate tissues and blood vessels and prolong the circulation time of drugs inside tissues.

[0032] In the present invention, an innovative method is adopted, namely, chemical groups and molecular structures are precisely introduced on the surface of collagen nanopolymers to achieve precise control of their affinity. By introducing these specific functional groups, the binding strength between nanoparticles and drug molecules is successfully enhanced, thereby significantly improving the drug loading of nanopolymers and achieving fine control of drug release rate. In addition, through surface modification, the present invention also significantly reduces the immunogenicity and cytotoxicity of the material, significantly improves its compatibility with biological tissues, reduces immune recognition and macrophage uptake, thereby further improving its biological application potential. And by connecting specific ligands, such as antibodies, multi-peptides and other targeting molecules to the surface of nanoparticles, the loaded drug delivery system is successfully given targeting, ensuring that the drug can be accurately delivered to the tissue that needs repair. This clever modification of collagen also significantly improves the ability of nanoparticles to pass through the skin barrier, allowing the loaded drug to reach deep tissues.

[0033] The modification technology of the present invention not only achieves slow drug release but also, by introducing modifications that are sensitive to external stimuli, enables precise regulation of drug release by the intracellular environment. Under initial conditions, the nanoparticles are in a thermodynamically stable state. However, as the temperature rises, the nanoparticles begin to expand, gradually increasing in size. When the diffusion front reaches half the volume of the swelling medium, the spatial distance between the molecules gradually increases, and the non-covalent forces that maintain the stability of the nanoparticles gradually weaken, ultimately leading to continued swelling of the polymer backbone and gradual release of the drug.

[0034] It is worth noting that although continued heating will lead to the dissociation of the nano drug carrier, due to the difference in particle size and hydrophilicity between the modified drug particle size and the drug carrier, the interaction between the drug and the polymer is still strong under the initial conditions. In addition, although the expansion state of the core inside the nano drug carrier leads to an increase in particle size, the surface-modified cross-linked collagen shell can effectively limit the continued expansion of its core, avoiding the expansion and dissociation phenomenon of conventional self-assembled nano drug carriers. Therefore, the nano drug carrier of the present invention exhibits good stability at different temperatures, providing reliable technical support for the precise delivery and release of drugs.

[0035] Further research and analysis, based on conformational analysis of the binding of the nanomaterials provided by the present invention to receptors, revealed that the three-dimensional structure of the nano-drug carriers prepared by the present invention primarily utilizes passive targeting, utilizing the composition, particle size, and material exchange of the drug carrier, as well as capillary retention and the high permeability of capillaries in damaged tissues, to retain the drug at the target site. After the drug carrier enters the target cell, a diffusion front appears on the carrier surface, allowing for slow release. The drug in the core is released to exert its therapeutic effect, thus preventing drug release in other cellular and tissue locations. The quaternary stability of the protein structure of the collagen nano-drug carriers described in the present invention primarily relies on hydrophobic interactions between amino acid residues between subunits. The surface-modified collagen nano-carriers readily bind to receptors on cells, forming pharmacologically active prodrugs. In addition to the polar metabolic processes of the loaded drug itself, the nano-polymer particles also exhibit dynamic changes in pharmacological activity. It should be noted that the nano-polymer particles themselves do not possess significant pharmacological activity; rather, their pharmacological activity is exhibited after entering the cellular environment and undergoing demethylation catalyzed by drug-metabolizing enzymes.

[0036] In response to the various problems that exist in the prior art when converting collagen into prodrugs, such as the impact of partial structures on cell permeability, the toxic side effects that may be caused by irregular modified structures, insufficient water solubility, a short half-life in cells, and poor performance of carrier stabilizers, the present invention aims to optimize the pharmacokinetic properties of prodrugs by chemically modifying the drug structure with known pharmacological effects, thereby overcoming its biological defects and enabling the modified drugs to more fully exert their efficacy.

[0037] The present invention relates to a nanoparticle carrier technology. The resulting collagen microsphere emulsion exhibits minimal biological side effects during drug loading. This emulsion, composed of nanospheres, exhibits excellent stability and is resistant to changes in external pH, salt concentration, temperature, and organic phase composition. The nanosphere emulsion not only serves as a drug carrier but also allows the viscosity of the emulsion to be adjusted by controlling rheological properties, thereby enabling precise control of the nucleation density of collagen nanoparticles. This process allows nanoparticles to nucleate and grow at a single location, providing a controllable factor for controlling the size of the collagen nanoparticle aggregates. The present invention enables precise control of drug delivery, initially forming multiple nucleation sites within the crystal region. Subsequently, uniformly oriented crystal domains gradually grow and chain together to form a stable lattice structure. This structure renders the polymer as a whole amphiphilic in aqueous solution, effectively slowing the relaxation process between the collagen peptide chains and the loaded drug. As the peptide chains continue to swell, the loaded drug is slowly released.

[0038] In the technical solution of the present invention, the surface after hydrolysis modification has collagen nanoparticles reinforced with tunnel nanotubes, and this reinforcement significantly improves the connection stability between the drug and the peptide chain. At the same time, the modified surface chemical bonds have a further restrictive effect on the release of the loaded drug. Under the impetus of the internal environment of the release environment system (including concentration gradient, chemical potential gradient and stress gradient), the drug particles achieve directional migration due to thermal motion, thereby presenting a directional transport phenomenon of the substance on a macro scale. The affinity between the loaded drug and the drug carrier is affected by the functionalized modified polyamide terminal active groups (amino and carboxyl groups), resulting in a significant enhancement of the affinity. In addition, a polymer crystal region is formed inside the collagen nanoparticles provided by the present invention. This crystal region is an impenetrable barrier for most drug molecules. Therefore, the drug molecules must bypass the crystal region during the release process, thereby affecting their movement path and achieving a sustained release effect. During the molecular sustained release process, structural changes will occur between the drug carrier and the loaded drug, forming a diffusion interface, a diffusion front, a swelling interface and a swelling medium in the center from the outside to the inside. When the drug-loaded glassy polymer comes into contact with the aqueous solution in the cellular environment, the aqueous solution will penetrate into the diffusion interface and the swelling interface, and at the same time will be affected by the crystalline region to slow down the release rate of the loaded drug.

[0039] In this technical solution, the loading strength of collagen carrier and imidazole compound shows significant advantages. Specifically, the collagen carrier prepared by the present invention, after chemical modification, can be efficiently combined with imidazole compound to form a hydrogel that is easy for cells to absorb. This hydrogel is not only a key component of the extracellular matrix, but also provides a stable scaffold structure for cell adhesion and growth. It also significantly promotes the distribution and stable maintenance of imidazole compounds in cells. Further, imidazole compounds must be combined with specific target receptors to give full play to their pharmacological effects, thereby producing a synergistic or antagonistic effect. After being combined with the collagen carrier prepared by the present invention, since collagen has been pharmacologically activated, it can specifically bind to the imidazole group. This characteristic greatly improves the delivery efficiency of imidazole compounds, ensuring that they can reach the target location accurately and quickly. Finally, the collagen carrier prepared by the present invention also shows excellent biocompatibility. This characteristic makes it possible to minimize the adverse effects on the organism during the delivery of the loaded imidazole compound, ensuring that the entire delivery process has a high degree of biosafety.

[0040] The beneficial effects of the present invention are: 1) reducing the volume of collagen through enzymatic hydrolysis, reducing the biological stimulation that may be caused by drug loading, and enhancing the drug loading capacity of collagen nanopolymers; 2) utilizing subsequent surface modification to make the carrier protein appear polymer crystal regions in the aqueous solution, so that the drug carrier can control the release progress of the loaded drug and enable it to penetrate through the skin surface to the deep tissue; 3) achieving precise control of drug release, thereby extending the effective action time of the drug in the body and significantly enhancing the stability and bioavailability of the drug; 4) by hydrolyzing and surface modifying collagen, changing the affinity of collagen, and performing functional modification, the drug loading capacity of collagen nanopolymers can be enhanced, the biosafety of collagen nanopolymers can be improved, and irritation can be reduced. At the same time, the modified collagen molecular structure can penetrate through the skin surface to the deep tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The porous structure of the collagen of the present invention is displayed through surface modification technology. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the solutions of the present invention, the following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0043] Unless otherwise specified, the raw materials used in the examples of the present invention are all commercially available or available to those skilled in the art; unless otherwise specified, the methods used in the examples of the present invention are all methods known to those skilled in the art.

[0044] Example 1

[0045] A method for preparing collagen nanoparticles comprises the following steps:

[0046] (1) Collagen, methacrylic anhydride, and phosphate buffer solution were mixed uniformly in a mass ratio of 1:0.20:9.5, and stirred continuously at 50°C for 6 h. After stirring, the solution was poured into a dialysis bag with a molecular weight cutoff of 500 D, dialyzed in deionized water for 24 h, and cooled and dried to obtain organic collagen;

[0047] The phosphate buffer is a phosphate buffer with a concentration of 0.2 M and a pH of 7.5;

[0048] (2) hydrolyzing the surface of the organized collagen with 1398 neutral protease at a concentration of 1 wt% of the organized collagen, and reacting for 6 h at a temperature of 35° C. and a pH of 6.5 to prepare a microsphere substrate. During the process, distilled water or deionized water was added at a concentration of 2.5 times the volume of the organized collagen.

[0049] The pH adjustment was performed using a phosphate buffer solution with a concentration of 1 / 15 M and a pH of 8.2;

[0050] (3) The microsphere substrate, cimetidine, glyceryl monostearate, and polysorbate were mixed uniformly in a mass ratio of 1:3:0.4:0.3, and reacted at a temperature of 40°C for 4 hours to prepare a microsphere modified substrate;

[0051] (4) The microsphere modified substrate, anhydrous ether and liquid paraffin containing 0.3 wt% Span 80 were mixed uniformly in a mass ratio of 6:0.90:18, and stirred for 40 minutes under a nitrogen atmosphere at a temperature of 30°C and an ultrasonic power of 160 W to prepare a collagen microsphere emulsion, i.e., collagen nanopolymer particles.

[0052] The collagen microsphere emulsion prepared in step 4) was separated by ultrafiltration to obtain collagen nanoparticles, and the nanoparticles were characterized, which showed the following Figure 1 In addition, the collagen nanoparticles obtained in the example were subjected to biological phase detection, and the specific characterization results are as follows.

[0053] Polymer particle transdermal penetration test: The skin of the back area of ​​16-week-old Bama miniature pigs was depilated and rinsed clean, and the collagen microsphere emulsion prepared in the example (the collagen nano-polymer particles obtained by ultrafiltration separation were dispersed in pure water to prepare an emulsion of 10 μg / mL) was applied to the depilated area. A 20 mm × 20 mm × 1 mm test sample was cut from the coated area, and a conventional diffusion cell and a receiving cell were built at room temperature and pressure. Raman spectroscopy was used to detect and calculate the transmittance of the collagen polymer particles. The characterization data are as follows.

[0054] Time, min 0 30 60 90 120 150 180 210 240 Transmittance of test group, % 0 63 82 90 95 98 99 ＞99 ＞99

[0055] In vitro skin depth test: Fresh abdominal skin of 16-week-old Bama miniature pigs was used. After hair removal, the fat layer and part of the dermis were scraped off with a scraper, and the skin was cut into pieces of about 3.14 cm 2 A circular-shaped microsphere was fixed to the permeation device with the stratum corneum facing upwards. A collagen microsphere emulsion was applied to the stratum corneum. FITC-HA fluorescent material was used as the coating drug for the collagen polymer particles. The FITC-HA fluorescent material was diluted to 50 μg / mL using pH 7.4 PBS buffer. 0.5 mL of this dilution was added to the permeation device and incubated in the dark for 30 minutes. After removal, the skin was removed and repeatedly rinsed with clean water until the surface fluorescence showed no discoloration. The surface moisture was dried with absorbent paper and incubated in the dark for 10 minutes to dry slightly. The skin was then sectioned and observed under a fluorescence microscope. The penetration depth of the fluorescence in the skin was 576 ± 20 μm.

[0056] In vitro release testing: An RYJ-12B transdermal drug diffusion tester was used to test the drug. An artificial semipermeable membrane was placed in the transdermal instrument to simulate the in vitro release platform. Every hour, 1 mL of the receptor solution was aspirated, followed by the addition of the corresponding volume of isotonic solution. After each time point, the solution was placed in a UV spectrophotometer to measure the absorbance, and the in vitro release rate was calculated. Characterization data are shown below. The receptor solution consisted of a collagen microsphere emulsion containing 2 mg / mL of collagen nanoparticles diluted with propylene glycol.

[0057] Time, h 0 2 4 8 12 18 24 28 32 Drug release rate, % 0 11 26 43 55 71 88 93 96

[0058] In addition to the above utility tests, the collagen nanoparticles prepared in the examples were also subjected to performance tests, and the specific characterization results are as follows.

[0059] Antioxidant activity assay: Prepare a 0.05 mol / L Tris-HCl solution with a pH of 8.2 using 0.1 mol / L HCl. Add 4.5 mL of Tris-HCl solution to 4.2 mL of collagen microsphere emulsion, mix thoroughly, and react in a 25°C water bath for 20 minutes. Preheat 3 mmol / L pyrogallol in a 25°C water bath. Add 0.3 mL of pyrogallol to the heated reaction solution and immediately measure its absorbance at 300 nm. Measure the absorbance every 30 seconds. The slope of the absorbance over a 4-minute period is used to calculate the superoxide anion radical scavenging rate, as shown below.

[0060]

[0061] Where:

[0062] ——Anion free radical scavenging rate;

[0063] K0——Distilled water comparison table;

[0064] K——test sample.

[0065] Reducibility test: Add 2.5 mL of phosphate buffer and 2.5 mL of 1% potassium ferricyanide to 1 mL of collagen microsphere emulsion, mix well, and react in a 50°C constant temperature water bath for 20 minutes. Finally, add 2.5 mL of 10% trichloroacetic acid to terminate the reaction. Centrifuge at 3000 rpm for 30 minutes, collect the supernatant, mix the supernatant with 2.5 mL of distilled water and 0.5 mL of ferric chloride, and let it stand for 10 minutes. Measure the absorbance at a wavelength of 300 nm. The absorbance value of the test group is the reducing property of the collagen nanoparticles.

[0066] Antioxidant activity, % Reducibility 57.26 0.47

[0067] Based on the above data and analysis results, the collagen nanoparticles prepared in this example have excellent sustained-release properties, achieving extremely efficient penetration within 4 hours and a stable and sustained release process within 32 hours. This characteristic of the present invention makes it an ideal choice for long-lasting release of ingredients in vitro. Furthermore, the collagen microsphere emulsion in the present invention also exhibits extremely high transdermal release rate and deep release effect.

[0068] A collagen nano-polymer particle is prepared by the above preparation method.

[0069] Example 2

[0070] A method for preparing collagen nanoparticles comprises the following steps:

[0071] (1) Collagen, methacrylic anhydride, and phosphate buffer solution were mixed uniformly in a mass ratio of 1:0.25:10.5, and stirred continuously at 55°C for 4 h. After stirring, the solution was poured into a dialysis bag with a molecular weight cutoff of 500 D, dialyzed in deionized water for 24 h, and cooled and dried to obtain organic collagen;

[0072] The phosphate buffer is a phosphate buffer with a concentration of 0.2 M and a pH of 7.5;

[0073] (2) The surface of the organized collagen was hydrolyzed using 1398 neutral protease, with the amount of 1398 neutral protease used being 1.5 wt% of the organized collagen, and the reaction was carried out at a temperature of 40° C. and a pH of 7.0 for 4 h to prepare a microsphere substrate. During the process, distilled water or deionized water was added in an amount of 3 times the volume of the organized collagen;

[0074] The pH adjustment was performed using a phosphate buffer solution with a concentration of 1 / 15 M and a pH of 8.2;

[0075] (3) The microsphere substrate, cimetidine, glyceryl monostearate, and polysorbate were mixed uniformly in a mass ratio of 1:3.5:0.6:0.3, and reacted at a temperature of 45°C for 3 hours to prepare a microsphere modified substrate;

[0076] (4) The microsphere modified substrate, anhydrous ether and liquid paraffin containing 0.5 wt% Span 80 were mixed uniformly in a mass ratio of 6:0.95:20, and stirred for 30 minutes under a nitrogen atmosphere at a temperature of 35°C and an ultrasonic power of 180 W to prepare a collagen microsphere emulsion, i.e., collagen nanopolymer particles.

[0077] A collagen nano-polymer particle is prepared by the above preparation method.

[0078] Referring to the collagen nano-polymer particle detection method of Example 1, the same performance test was performed on the example, and the specific characterization results are as follows.

[0079] Polymeric particle transdermal penetration testing:

[0080] Time, min 0 30 60 90 120 150 180 210 240 Transmittance of test group, % 0 64 84 90 94 98 99 ＞99 ＞99

[0081] In vitro release assay:

[0082]

[0083] Based on the above data and analysis results, the collagen nanoparticles prepared in this example have excellent sustained-release properties, achieving extremely efficient penetration within 4 hours and a stable and sustained release process within 32 hours. This characteristic of the present invention makes it an ideal choice for long-lasting release of ingredients in vitro. Furthermore, the collagen microsphere emulsion in the present invention also exhibits extremely high transdermal release rate and deep release effect.

[0084] Example 3

[0085] A method for preparing collagen nanoparticles comprises the following steps:

[0086] (1) Collagen, methacrylic anhydride, and phosphate buffer solution were mixed uniformly in a mass ratio of 1:0.30:11.5, and stirred continuously at 60°C for 2 h. After stirring, the solution was poured into a dialysis bag with a molecular weight cutoff of 500 D, dialyzed in deionized water for 24 h, and cooled and dried to obtain organic collagen;

[0087] The phosphate buffer is a phosphate buffer with a concentration of 0.2 M and a pH of 7.5;

[0088] (2) The surface of the organized collagen was hydrolyzed using 1398 neutral protease, with the amount of 1398 neutral protease used being 2 wt% of the organized collagen, and the reaction was carried out at a temperature of 45° C. and a pH of 7.5 for 2 h to prepare a microsphere substrate. During the process, distilled water or deionized water was added in an amount of 3.5 times the volume of the organized collagen;

[0089] The pH adjustment was performed using a phosphate buffer solution with a concentration of 1 / 15 M and a pH of 8.2;

[0090] (3) The microsphere substrate, cimetidine, glyceryl monostearate, and polysorbate were mixed uniformly in a mass ratio of 1:4:0.8:0.3, and reacted at a temperature of 50°C for 2 h to prepare a microsphere modified substrate;

[0091] (4) The microsphere modified substrate, anhydrous ether and liquid paraffin containing 0.7 wt% Span 80 were mixed uniformly in a mass ratio of 6:1:22, and stirred for 20 minutes under a nitrogen atmosphere at a temperature of 40°C and an ultrasonic power of 200 W to prepare a collagen microsphere emulsion, i.e., collagen nanopolymer particles.

[0092] A collagen nano-polymer particle is prepared by the above preparation method.

[0093] Referring to the collagen nano-polymer particle detection method of Example 1, the same performance test was performed on the example, and the specific characterization results are as follows.

[0094] Polymeric particle transdermal penetration testing:

[0095] Time, min 0 30 60 90 120 150 180 210 240 Transmittance of test group, % 0 64 81 89 94 98 99 ＞99 ＞99

[0096] In vitro release assay:

[0097]

[0098] Based on the above data and analysis results, the collagen nanoparticles prepared in this example have excellent sustained-release properties, achieving extremely efficient penetration within 4 hours and a stable and sustained release process within 32 hours. This characteristic of the present invention makes it an ideal choice for long-lasting release of ingredients in vitro. Furthermore, the collagen microsphere emulsion in the present invention also exhibits extremely high transdermal release rate and deep release effect.

[0099] In addition, the collagen carriers prepared in Example 1, Example 2, and Example 3 were tested for temperature resistance, and the specific characterization results are as follows.

[0100] Temperature resistance test: Prepare a sodium chloride solution with a concentration of 1.0%, dilute the volume of the collagen nanoparticles prepared in the embodiment of the present invention and the prepared 1.0% sodium chloride solution to 1 mL, let it stand at room temperature for 48 hours, and then detect the particle size of the collagen nanoparticles. Set a gradient temperature of 20 to 60°C, keep it warm and stand for 24 hours, then detect the particle size of the collagen nanoparticles, and calculate the particle size volume change rate. The specific calculation formula is as follows.

[0101]

[0102] Where:

[0103] α——particle size volume change rate, %;

[0104] V0——particle size of procollagen nanopolymer particles, nm;

[0105] V t ——Particle size of collagen nanopolymer particles after temperature change, nm.

[0106] 20℃ 30℃ 40℃ 50℃ 60℃ Blank 11.7％ 25.2％ 32.6％ 26.7％ 23.5％ Example 1 8.3％ 9.6％ 9.8％ 9.9％ 10.1％ Example 2 8.4％ 9.5％ 9.7％ 9.9％ 10.0％ Example 3 8.4％ 9.5％ 9.8％ 9.9％ 10.1％

[0107] The blank sample is commercially available collagen particles with a particle size of no more than 100 nm.

[0108] Analyzing the above characterization results and comparing them with the blank sample test results, the overall trend of collagen particle size change with increasing temperature is that the particle size first increases and then decreases. Since the drug carrier is in a relatively thermodynamically stable state, the overall energy increases as the temperature rises, causing the protein segments to tend to move to a state to reduce the overall energy, resulting in a decrease in the interaction between the molecular segments, structural expansion, and an increase in particle size. When the expansion reaches a certain level, the non-covalent forces are insufficient to maintain the three-dimensional structure of the protein particles, causing the protein particles to gradually dissociate and the particle size to gradually decrease.

[0109] The collagen polymer particle size prepared in the embodiment of the present invention does not change significantly with temperature, and only a small increase in particle size occurs. This phenomenon is accompanied by an increase in temperature, and the core inside the drug carrier expands, resulting in an increase in particle size. However, since the polymer crystal region formed inside the collagen nanopolymer particles will limit the continued expansion of the nanodrug core, expansion dissociation will not occur.

[0110] Comparative Example 1

[0111] A method for preparing collagen nanoparticles is provided, wherein the preparation method of microspheres is changed to prepare collagen nanoparticles. The method comprises the following steps:

[0112] (1) Collagen, methacrylic anhydride, and phosphate buffer solution were mixed uniformly in a mass ratio of 1:0.20:9.5, and stirred continuously at 50°C for 6 h. After stirring, the solution was poured into a dialysis bag with a molecular weight cutoff of 500 D, dialyzed in deionized water for 24 h, and cooled and dried to obtain organic collagen;

[0113] The phosphate buffer is a phosphate buffer with a concentration of 0.2 M and a pH of 7.5;

[0114] (2) hydrolyzing the surface of the organized collagen with 1398 neutral protease at a concentration of 1 wt% of the organized collagen, and reacting for 6 h at a temperature of 35° C. and a pH of 6.5 to prepare a microsphere substrate. During the process, distilled water or deionized water was added at a concentration of 2.5 times the volume of the organized collagen.

[0115] The pH adjustment was performed using a phosphate buffer solution with a concentration of 1 / 15 M and a pH of 8.2;

[0116] (3) The microsphere substrate, cimetidine, glyceryl monostearate, and polysorbate were mixed uniformly in a mass ratio of 1:3:0.4:0.3, and reacted at a temperature of 40°C for 4 hours to prepare a microsphere modified substrate;

[0117] (4) The microsphere modified substrate and dichloromethane were mixed evenly in a mass ratio of 1:8. After the microsphere modified substrate was completely dissolved, the mixed solution was transferred to the dispersed phase container of the SPG membrane emulsifier. In a nitrogen atmosphere and a pressure of 1.2 MPa, a continuous phase container was used. The washing water pump was turned on to circulate the continuous phase. The operation was stopped when the pressure dropped to 0 KPa. The continuous phase suspension was stirred at a low speed for 24 hours to evaporate the solvent, centrifuged and washed with water three times, and the sediment was freeze-dried to prepare a collagen microsphere emulsion, i.e., collagen nanopolymer particles.

[0118] The collagen nanoparticles prepared in the comparative example were subjected to partial performance testing in the same manner as in Example 1, and the characterization results are as follows.

[0119] Polymeric particle transdermal penetration testing:

[0120] Time, min 0 30 60 90 120 150 180 210 240 Transmittance of test group, % 0 46 51 56 70 72 74 75 76

[0121] In vitro release assay:

[0122]

[0123] Temperature resistance test:

[0124] 20℃ 30℃ 40℃ 50℃ 60℃ 14.5％ 15.8％ 18.4％ 22.1％ 26.7％

[0125] Comparative Example 2

[0126] A method for preparing collagen nanoparticles is provided, wherein the preparation method of microspheres is changed to prepare collagen nanoparticles. The method comprises the following steps:

[0127] (1) Collagen, methacrylic anhydride, and phosphate buffer solution were mixed uniformly in a mass ratio of 1:0.20:9.5, and stirred continuously at 50°C for 6 h. After stirring, the solution was poured into a dialysis bag with a molecular weight cutoff of 500 D, dialyzed in deionized water for 24 h, and cooled and dried to obtain organic collagen;

[0128] The phosphate buffer is a phosphate buffer with a concentration of 0.2 M and a pH of 7.5;

[0129] (2) hydrolyzing the surface of the organized collagen with 1398 neutral protease at a concentration of 1 wt% of the organized collagen, and reacting for 6 h at a temperature of 35° C. and a pH of 6.5 to prepare a microsphere substrate. During the process, distilled water or deionized water was added at a concentration of 2.5 times the volume of the organized collagen.

[0130] The pH adjustment was performed using a phosphate buffer solution with a concentration of 1 / 15 M and a pH of 8.2;

[0131] (3) The microsphere substrate, cimetidine, glyceryl monostearate, and polysorbate were mixed uniformly in a mass ratio of 1:3:0.4:0.3, and reacted at a temperature of 40°C for 4 hours to prepare a microsphere modified substrate;

[0132] (4) The microsphere modified substrate and the sodium alginate solution were mixed evenly in a mass ratio of 1:6. Under normal temperature and pressure and a flow rate of 2 μL / min, the microsphere modified substrate was allowed to generate droplets inside the chip. The droplets generated inside the channel flowed through a silicon tube into a collection dish containing 0.1 mol / L 0.25% calcium chloride solution for cross-linking reaction to prepare a collagen microsphere emulsion, i.e., collagen nanopolymer particles.

[0133] The throat structure of the microfluidic chip in the above microfluidic system is a gradually widening throat structure, with a narrow throat width of 150 μm and a width of 300 μm for the remaining parts;

[0134] The above microfluidic system requires continuous oil phase emulsification. The oil phase additive is mineral oil with 0.1% Span 80. The injection flow rate is 20 μL / min, and the injection volume is 5 times the volume of the mixed solution of microsphere modified substrate and sodium alginate.

[0135] The collagen nanoparticles prepared in the comparative example were subjected to partial performance testing in the same manner as in Example 1, and the characterization results are as follows.

[0136] Polymeric particle transdermal penetration testing:

[0137] Time, min 0 30 60 90 120 150 180 210 240 Transmittance of test group, % 0 17 27 42 63 68 71 75 78

[0138] In vitro release assay:

[0139]

[0140] Temperature resistance test:

[0141]

[0142]

[0143] Comprehensive analysis of the characterization results from Comparative Examples 1 and 2 reveals that the collagen nanoparticles prepared in this comparative example exhibit a significant decrease and fluctuation in in vitro release rate compared to the examples, failing to ensure a stable and effective in vitro release process. Furthermore, the transdermal release rate and in vitro skin penetration depth also exhibited a significant downward trend, directly leading to a significant reduction in their practical application effectiveness.

[0144] After an in-depth comparison and analysis of the data of Comparative Example 1 and Comparative Example 2, the nano drug carrier of the present invention exhibits a unique operating mechanism. The core of this is that, through the efficient combination of passive targeting strategy and receptor protein, the interception effect of capillaries and the enhanced permeability of damaged tissue are cleverly utilized to achieve the precise retention of drug carriers in the target area. Once inside the cell, the carrier can accurately release the drug at the target site in a slow and strictly controlled mode, thereby significantly reducing the amount of drug released in non-target cell areas. In addition, the collagen microsphere emulsion used in the present invention performs well in terms of stability. It is not only qualified as an efficient drug carrier, but also has the unique ability to regulate the viscosity of the emulsion. This characteristic can accurately control the nucleation density of nano-polymer particles, realize the single nucleation and orderly growth of nanoparticles at a specific position, and then construct a stable lattice structure, significantly extending the release cycle of the drug.

[0145] Comparative Example 3

[0146] A method for preparing collagen nanoparticles is described. The specific preparation method is the same as that in Example 1. In the comparative example, the enzymatic hydrolysis of the present invention is omitted, i.e., 1398 neutral protease is not used for biological surface hydrolysis. Instead, mixed acid hydrolysis is used. The specific steps are as follows:

[0147] (1) Collagen, methacrylic anhydride, and phosphate buffer solution were mixed uniformly in a mass ratio of 1:0.20:9.5, and stirred continuously at 50°C for 6 h. After stirring, the solution was poured into a dialysis bag with a molecular weight cutoff of 500 D, dialyzed in deionized water for 24 h, and cooled and dried to obtain organic collagen;

[0148] The phosphate buffer is a phosphate buffer with a concentration of 0.2 M and a pH of 7.5;

[0149] (2) hydrolyzing the surface of the organized collagen using a mixed acid at a concentration of 3 wt % of the organized collagen in an environment at a temperature of 35° C. and a pH of 6.5 for 6 h to prepare a microsphere substrate;

[0150] The mixed acid component is a mixed acid of sulfuric acid and formic acid, the content of sulfuric acid is 4wt% and the balance is formic acid, wherein the sulfuric acid is 15mol / L concentrated sulfuric acid and the concentration of formic acid is 5mol / L;

[0151] (3) The microsphere substrate, cimetidine, glyceryl monostearate, and polysorbate were mixed uniformly in a mass ratio of 1:3:0.4:0.3, and reacted at a temperature of 40°C for 4 hours to prepare a microsphere modified substrate;

[0152] (4) The microsphere modified substrate, anhydrous ether and liquid paraffin containing 0.3 wt% Span 80 were mixed uniformly in a mass ratio of 6:0.90:18, and stirred for 40 minutes under a nitrogen atmosphere at a temperature of 30°C and an ultrasonic power of 160 W to prepare a collagen microsphere emulsion, i.e., collagen nanopolymer particles.

[0153] The collagen nanoparticles prepared in the comparative example were subjected to partial performance testing in the same manner as in Example 1, and the characterization results are as follows.

[0154] Polymeric particle transdermal penetration testing:

[0155] Time, min 0 30 60 90 120 150 180 210 240 Transmittance of test group, % 0 14 27 41 52 64 66 67 68

[0156] In vitro release assay:

[0157]

[0158] Temperature resistance test:

[0159] 20℃ 30℃ 40℃ 50℃ 60℃ 12.7％ 15.9％ 22.5％ 26.1％ 32.4％

[0160] Analyzing the above characterization results, the collagen nanoparticles prepared in this comparative example are compared with the examples, and it can be clearly seen that the in vitro release of the comparative example has the characteristics of ultra-high efficiency release between 4 and 12 hours. However, its early release rate and late release rate process are very slow, and it does not actually have the characteristics of long-term stable sustained release. After evaluation, it was found that the inclusion effect of the nanopolymer was not ideal, showing poor inclusion capacity. At the same time, the transdermal release rate of the comparative example remained low, and its penetration depth was far lower than the data of the examples. In addition, observing the temperature resistance test of the comparative example, the force between the molecular segments prepared by the comparative example was reduced, and the structural expansion phenomenon occurred, resulting in a decrease in the heat resistance of the collagen nanoparticles loaded with drugs.

[0161] Comparative Example 4

[0162] A method for preparing collagen nanoparticles is disclosed. The specific preparation method is the same as that in Example 1. The comparative example only does not change the functional modification process unique to the present invention, that is, the microsphere substrate is not modified using cimetidine, glyceryl stearate, and polysorbate, and the collagen nanoparticles are prepared. The collagen nanoparticles prepared in the comparative example are partially tested for performance in the same manner as in Example 1, and the characterization results are as follows.

[0163] Polymeric particle transdermal penetration testing:

[0164] Time, min 0 30 60 90 120 150 180 210 240 Transmittance of test group, % 0 8 14 20 28 36 47 54 59

[0165] In vitro release assay:

[0166]

[0167] Temperature resistance test:

[0168] 20℃ 30℃ 40℃ 50℃ 60℃ 12.1％ 24.7％ 30.3％ 25.6％ 22.1％

[0169] Analyzing the above characterization results, the collagen nano-polymer particles prepared in this comparative example are compared with the examples, and it can be clearly seen that the in vitro release and transdermal release processes of the comparative example are relatively slow, and do not have the characteristics of long-term stable sustained release. The nano drug carrier provided by the present invention has targeted delivery technology, which realizes targeted delivery of drugs by precisely connecting specific ligands. By adopting modification technology, the ability of nanoparticles to cross the skin barrier is significantly improved, so that they can penetrate deep into the tissue. In addition, the carrier has excellent thermodynamic stability and can expand and effectively release drugs when the temperature rises. The cross-linked collagen shell ensures its stability and supports precise delivery and release processes. Further analysis of the temperature resistance test data shows that the overall trend of the particle size increases and then decreases as the temperature rises. After the temperature rises, the overall energy increases, resulting in a decrease in the force between the binding segments, causing the internal structure to collapse and mutate, resulting in unpredictable changes in properties.

[0170] The above specific embodiments are used to illustrate the present invention rather than to limit the present invention. Any modifications and changes made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A method for preparing collagen nanoparticles, characterized in that: The following steps are involved: The surface of the organized collagen is hydrolyzed to prepare a microsphere substrate; The microsphere substrate, the target load, the nonionic surfactant and the polyol ester are mixed uniformly in proportion to prepare a microsphere modified substrate; The microsphere modified substrate, ether compound and chemical additives are mixed evenly in proportion to prepare collagen microspheres, namely collagen nano-polymer particles.

2. The method for preparing collagen nanoparticles according to claim 1, wherein: The organized collagen is prefabricated collagen, and its preparation method comprises the following steps: collagen, methacrylic anhydride and phosphate buffer solution are mixed according to 1: The mixture was uniformly mixed in a mass ratio of (0.20-0.30): (9.5-11.5), and stirred continuously for 2-6 hours in an environment with a temperature of 50-60°C. After stirring, the solution was poured into a dialysis bag with a molecular weight cutoff of 500D, dialyzed in deionized water for 24 hours, cooled and dried to obtain the organic collagen.

3. The method for preparing collagen nanoparticles according to claim 1 or 2, wherein: The surface hydrolysis step is to use protease to perform enzymatic hydrolysis, and the amount of the protease used is 1 to 2 wt% of the organized collagen.

4. The method for preparing collagen nanoparticles according to claim 3, wherein: The protease is 1398 neutral protease.

5. The method for preparing collagen nanoparticles according to claim 1, wherein: Distilled water or deionized water is added in the surface hydrolysis step, and then reacted for 2 to 6 hours in an environment with a temperature of 35 to 45° C. and a pH of 6.5 to 7.5 to complete the preparation of the microsphere substrate, wherein the amount of distilled water or deionized water used is 2.5 to 3.5 times the volume of the organized collagen.

6. The method for preparing collagen nanoparticles according to claim 1, wherein: The target load is an imidazole compound; the nonionic surfactant is glyceryl monostearate; the polyol ester is polysorbate; the microsphere substrate, the target load, the nonionic surfactant and the polyol ester are evenly mixed in a mass ratio of 1:(3-4):(0.4-0.8):0.

3.

7. The method for preparing collagen nanoparticles according to claim 1 or 6, characterized in that: The step of preparing the microsphere modified substrate is to react in an environment with a temperature of 40 to 50° C. for 2 to 4 hours.

8. The method for preparing collagen nanoparticles according to claim 1, wherein: The ether compound is anhydrous ether; the chemical additive is liquid paraffin, which contains 0.3-0.7 wt% of Span 80; the microsphere modified substrate, the ether compound and the chemical additive are evenly mixed in a mass ratio of 6: (0.90-1.00): (18-22).

9. The method for preparing collagen nanoparticles according to claim 1, wherein: The step of preparing collagen microspheres is to stir the microsphere modified substrate, ether compound and chemical additive for 20 to 40 minutes under the conditions of protective gas atmosphere, temperature of 30 to 40° C. and ultrasonic power of 160 to 200W.

10. A collagen nanoparticle, characterized by: Prepared by the preparation method according to any one of claims 1 to 9.

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