Collagen nano dispersion preparation as well as preparation method and application thereof

Abstract

The invention provides a collagen nano dispersion preparation as well as a preparation method and application thereof. The preparation method of the collagen nano dispersion preparation comprises the following steps: S1, dissolving type I collagen and type III collagen in an acidic aqueous medium to prepare a collagen solution; s2, regulating and controlling the temperature, the pH value and the ionic strength of the collagen solution, so that the collagen solution is self-assembled; and S3, when the average particle size of the collagen fibers reaches a target range, performing dynamic freezing on the self-assembly system. The preparation and stable dispersion of the nano-scale collagen fiber can be realized without chemical crosslinking, the uniformity, biocompatibility and safety of the product are remarkably improved, and the nano-scale collagen fiber has excellent water-light effects and the like, and can be widely applied to the field of medical cosmetology.

Description

Technical Field

[0001] This invention relates to the field of collagen technology, and in particular to a collagen nano-dispersion formulation, its preparation method, and its application. Background Technology

[0002] Skin aging is a complex process, primarily characterized by decreased collagen content, reduced skin elasticity, dryness, and an increase in fine lines and wrinkles. Collagen is the main structural protein in the dermis, crucial for maintaining skin structure and elasticity. In recent years, collagen injections have been widely used in the medical aesthetics field to fill wrinkles and improve skin texture. However, existing collagen injection technologies generally have the following shortcomings: 1) Risks of chemical cross-linking agents: To enhance product stability and prolong in vivo maintenance time, existing products often use chemical cross-linking agents (such as glutaraldehyde, 1,4-butanediol diglycidyl ether, BDDE, etc.). These cross-linking agents may introduce cytotoxicity, pose potential immunogenicity risks and biosafety issues, and reduce the biocompatibility of the product.

[0003] 2) Collagen self-aggregation problem: Collagen has a natural tendency to self-assemble and easily forms large fibers under physiological conditions, which may lead to poor fluidity of the injection and the formation of visible clumps in the skin after injection, affecting uniform dispersion and optical effects.

[0004] 3) Insufficient stability and duration of effect: Unmodified collagen is easily degraded by enzymes in the body, resulting in a short duration of effect. Traditional physical stabilization methods usually cannot simultaneously achieve high bioactivity and long-term stability.

[0005] 4) Poor "water-light" effect: Some collagen products have large fiber particle size, which significantly increases light scattering, affecting the skin's transparency and radiance, and failing to fully achieve the ideal "water-light" effect.

[0006] Therefore, there is an urgent need to develop a collagen injection formulation that is free of chemical cross-linking agents, has excellent dispersion stability, can form nanoscale structures, and has a remarkable "water-light" effect. Summary of the Invention

[0007] The purpose of this invention is to provide a collagen nano-dispersion formulation, its preparation method and application, which can achieve the preparation and stable dispersion of nano-scale collagen fibers without chemical cross-linking.

[0008] This invention provides a method for preparing a collagen nano-dispersion formulation, comprising the following steps: S1: Dissolve type I collagen and type III collagen in an acidic aqueous medium to prepare a collagen solution; S2: The temperature, pH and ionic strength of the collagen solution are controlled to enable the collagen solution to self-assemble. S3: Once the average particle size of the collagen fibers reaches the target range, the self-assembled system is subjected to kinetic freezing.

[0009] In step S1, the type I and type III collagen can be extracted from bovine hide and have a purity of ≥99.99%. The mass ratio of type I collagen to type III collagen is (80-90):(10-20), for example, 85:15. This mass ratio is designed to mimic the composition of collagen in natural skin and can synergistically optimize the biological function of the product. The concentration of the collagen solution is 2.0-2.4 mg / mL.

[0010] The acidic aqueous medium can be hydrochloric acid solution, acetic acid solution, etc., preferably hydrochloric acid solution; the concentration of the acidic aqueous medium is 5-10 mM; the temperature of the acidic aqueous medium is 15-25 ℃, preferably 20-24 ℃; the pH value is 2.0-3.0, preferably 2.0-2.3; the ionic strength is 0.005-0.01 M; the above is beneficial to ensure that collagen is completely dissolved and maintained in a dispersed state in the form of monomers or oligomers, but it is not sufficient to induce complete self-assembly of collagen.

[0011] In step S2, temperature control includes: slowly raising the temperature of the collagen solution from a low temperature to a preset self-assembly temperature; wherein the low temperature is 3-5 ℃, the self-assembly temperature is 15-25 ℃, the self-assembly temperature is precisely controlled within ±0.5 ℃, and the rate of slow heating is 0.2-1 ℃ / min, preferably 0.5-0.8 ℃ / min. The above temperature control is crucial to the morphology and particle size of collagen fibers.

[0012] pH control involves gradually increasing the pH of the collagen solution to near neutral, for example, pH 6.0-7.0. This pH control helps maintain sufficient electrostatic repulsion while allowing limited self-assembly, thus inhibiting excessive aggregation.

[0013] Ionic strength regulation involves adding a salt solution dropwise to the collagen solution to adjust the ionic strength to 0.030-0.034 M. The salt solution can be NaCl, Na₂HPO₄, etc., with a concentration of 0.2-0.5 M. Lower ionic strength helps maintain nanoscale dispersion, and gradually increasing the ionic strength can induce and accelerate self-assembly until the optimal particle size is reached.

[0014] Furthermore, during the self-assembly process, in-situ monitoring technologies such as dynamic light scattering (DLS) can be used to track the average particle size and / or polydispersity index (PDI) of collagen fibers in real time, thereby enabling precise feedback control of the preparation process.

[0015] In step S3, the target range for average particle size is ≤100 nanometers, preferably ≤50 nanometers, and more preferably ≤30 nanometers; in addition, the polydispersity index of collagen fibers can reach 0.05-0.20, preferably 0.08-0.18, and more preferably 0.10-0.15.

[0016] Kinetic freezing involves rapidly cooling the temperature of the self-assembled system to below 8 °C, for example, 2-8 °C, at a cooling rate of 1-3 °C / min (preferably 1.5-2.5 °C / min, more preferably 1.8-2.5 °C / min). This quenching process can rapidly reduce the thermal motion and diffusion rate of collagen molecules, effectively preventing further growth and aggregation of fibers, thereby "locking" the nanoscale collagen fibers in their current state.

[0017] Furthermore, the preparation method of the present invention also includes step S4: adjusting the concentration of the kinetically frozen collagen nano-dispersion preparation to 10.0-15.0 mg / mL (w / v) using aseptic concentration and formulation processes.

[0018] In step S4, the formulation process includes adding a small amount of physical stabilizer to the collagen nano-dispersion formulation. The physical stabilizer may be, for example, polyethylene glycol (PEG), polyvinyl alcohol (PVA), or high molecular weight hyaluronic acid (molecular weight 1800-2200 kDa). The amount of physical stabilizer added is 0.1-0.3% of the collagen protein mass, and the final concentration of the physical stabilizer in the collagen nano-dispersion formulation is, for example, 0.0125-0.0375 mg / mL (w / v). The physical stabilizer can form a hydration layer on the surface of the collagen nanoparticles by adsorbing onto them, preventing effective access and aggregation between nanofibers through steric hindrance, thereby enhancing the colloidal stability of the collagen system.

[0019] Furthermore, the pH and ionic strength can be further adjusted after adding physical stabilizers; specifically, the pH can be adjusted to 6.2-6.8, and the ionic strength to 0.010-0.015 M. Optimizing the pH and ionic strength of the collagen dispersion formulation ensures sufficient electrostatic repulsion on the collagen fiber surface (e.g., controlling the absolute value of the collagen fiber zeta potential to be greater than 30 mV), which is beneficial to improving colloidal stability.

[0020] The above preparation steps should be carried out under strict aseptic conditions, and the prepared formulation can be filled into pre-filled syringes.

[0021] For products requiring long-term storage, biocompatible lyophilization protectants can be further added. Examples of lyophilization protectants include trehalose, mannitol, and glycine. The amount of lyophilization protectant added is 10-50% of the collagen protein mass, preferably 20-40%. The final concentration of the lyophilization protectant in the collagen nano-dispersion formulation is, for example, 1.25-6.25 mg / mL (w / v), preferably 2.5-5.0 mg / mL. Glycine, a commonly used lyophilization protectant, is typically added in the range of 20-30% of the collagen protein mass, corresponding to a final concentration of 2.5-3.75 mg / mL (w / v). Considering the formulation characteristics of this invention, a more preferred amount of glycine is 25% of the collagen protein mass, corresponding to a final concentration of 3.125 mg / mL (w / v). Subsequently, lyophilization is performed to obtain nano-sized collagen lyophilized powder, which can be restored to its nano-dispersion state by reconstitution before use.

[0022] The present invention also provides a collagen nano-dispersion formulation, which is prepared according to the above preparation method.

[0023] The collagen nano-dispersion formulation of the present invention remains stably dispersed for at least 12 months under storage conditions of 2-8 °C.

[0024] The present invention also provides the application of the above-mentioned collagen nano-dispersion formulation in biomedical materials and / or medical aesthetic products.

[0025] More specifically, the present invention also provides a skin hydration injection product comprising the above-mentioned collagen nano-dispersion formulation. The collagen nano-dispersion formulation in the skin hydration injection product has a mass content of 1.0-1.5% (w / v); further, the effective content of collagen in the skin hydration injection product is 10-15 mg / mL.

[0026] It is understandable that skin hydration injection products also include pharmaceutically acceptable carriers, such as water for injection and physiological saline, to ensure that the osmotic pressure of the product is isotonic with that of the human body. This is crucial for maintaining cell function and avoiding adverse reactions at the injection site.

[0027] Optionally, skin hydration injection products may also include other auxiliary ingredients, such as pH adjusters, buffer salts, biocompatible preservatives, and antioxidants, to ensure product stability, sterility, and storage stability. More specifically, the pH adjuster can be hydrochloric acid, sodium hydroxide, disodium hydrogen phosphate, sodium dihydrogen phosphate, etc., and the content of the pH adjuster in the skin hydration injection product can be 0.01-0.08% (w / v), preferably 0.02-0.05%; the buffer salt can be disodium hydrogen phosphate-sodium dihydrogen phosphate, citric acid-sodium citrate, etc., and the content of the buffer salt in the skin hydration injection product can be 0.04-0.07% (w / v, 3-5 mM), preferably 0.05-0.06% (3.5-4.5 mM), more preferably 0.055% (w / v, 4 mM); the biocompatible preservative can be benzalkonium chloride (BAC), chlorhexidine alcohol solution, polyhexamethylene biguanide (PHMB), etc., and the content of the biocompatible preservative in the skin hydration injection product can be 0.005-0.02% (w / v), preferably 0.008-0.015%. The antioxidant can be ascorbic acid (vitamin C), glutathione, sodium bisulfite, etc. The antioxidant content in the skin water injection product can be 0.01-0.1% (w / v), preferably 0.02-0.08%, more preferably 0.03-0.05%.

[0028] The skin hydration injection product of the present invention has at least the following effects: 1) Improve skin hydration and elasticity: By directly injecting nano-level collagen and hydration carriers into the dermis, it can significantly increase the skin's water content, provide deep hydration, and improve skin hydration and plumpness.

[0029] 2) Enhances skin radiance and translucency: Because collagen is dispersed at the nanoscale, light scattering is minimized, allowing light to penetrate more evenly in the skin tissue, resulting in a brighter and more natural "water glow" effect.

[0030] 3) Promote endogenous collagen regeneration: Nanoscale collagen can act as a biological scaffold and signaling molecule to stimulate dermal fibroblasts and promote the synthesis of their own collagen, thereby improving skin elasticity and firmness.

[0031] 4) Reduce fine lines and improve skin texture: Through deep hydration and stimulation of collagen regeneration, it helps to reduce fine lines, improve skin texture, and make it more delicate and smooth.

[0032] This invention utilizes physicochemical parameters to precisely control the self-assembly of collagen to form nanoscale collagen fibers, achieving collagen self-assembly and forming a stable nanoscale fiber dispersion. This completely avoids the use of traditional chemical cross-linking agents, significantly improving the product's biocompatibility and safety. Simultaneously, the kinetic freezing method ensures the uniformity and nanoscale size of the collagen fiber particles in the final product. Because the collagen fiber particle size is precisely controlled at the nanoscale, light scattering is minimized, resulting in a clearer, brighter, and more radiant "water-light" effect on the skin after injection. This invention can be widely applied in the field of medical aesthetics. Attached Figure Description

[0033] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 The dynamic light scattering particle size distribution spectrum of the collagen nano-dispersion formulation in Example 1 is shown. Figure 2 The dynamic light scattering particle size distribution spectrum of the collagen nano-dispersion formulation in Example 2 is shown. Figure 3 The dynamic light scattering particle size distribution spectrum of the collagen nano-dispersion formulation in Example 3 is shown. Figure 4 The dynamic light scattering particle size distribution spectrum of the collagen nano-dispersion formulation in Example 4 is shown. Figure 5 The dynamic light scattering particle size distribution spectrum of the skin hydration injection product in Example 5; Figure 6 The dynamic light scattering particle size distribution spectrum of the skin water-light injection product in Example 6; Figure 7 The diagram shows the relationship between the mechanical properties of collagen nano-dispersed formulations and the fibers and skin. Among them: A is the relationship between single fiber stiffness and fiber diameter; B is the relationship between network modulus and fiber diameter; C is the relationship between skin elastic modulus and fiber diameter; and D is the relationship between skin tensile strength and fracture strain and fiber diameter. Detailed Implementation

[0035] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0036] It should be noted that the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the exemplary implementations according to this application. As used herein, the singular form includes the plural form unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this description, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0037] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Example 1 The preparation method of the collagen nano-dispersion formulation in this embodiment includes the following steps: 1. Preparation of collagen solution Type I and Type III collagen were dissolved in an acidic aqueous medium to prepare a collagen solution. The purity of Type I and Type III collagen was 99.99%, and the mass ratio of Type I collagen to Type III collagen was 85:15. The acidic aqueous medium was a 10 mM hydrochloric acid solution at 20 ℃, pH 2.0, and ionic strength 0.01 M. The concentration of the collagen solution was 2.0 mg / mL.

[0039] 2. Self-assembly A 0.2 M Na2HPO4 solution was gradually added dropwise to the collagen solution to adjust the ionic strength of the collagen solution to 0.032 M. Then the pH value of the collagen solution was gradually increased to near neutral (pH 7.0).

[0040] Next, the temperature of the collagen solution was slowly increased from 4 °C to the preset self-assembly temperature (20 °C) at a heating rate of 0.5 °C / min. The self-assembly temperature was precisely controlled within ±0.5 °C to allow the collagen solution to self-assemble. During the self-assembly process, dynamic light scattering (DLS) was used to track the average particle size and polydispersity index (PDI) of the collagen fibers in real time.

[0041] 3. Kinetic freezing Once the average particle size of the collagen fibers reaches 50 nanometers and the polydispersity index reaches 0.12, the self-assembled system is subjected to kinetic freezing, specifically as follows: the temperature of the self-assembled system is rapidly reduced to 4 ℃ at a cooling rate of 2.0 ℃ / min.

[0042] After aseptic filtration and aseptic filling, a collagen nano-dispersion formulation is obtained.

[0043] The collagen nano-dispersed formulation prepared above was tested using dynamic light scattering (DLS), zeta potential meter, circular dichroism (CD), SDS-PAGE gel electrophoresis, and sterility test (direct inoculation method). The results are as follows: the average particle size was 50 nm (PDI=0.12, RSD=2.8%), the absolute value of the zeta potential was 35 mV, the retention rate of the collagen triple helix structure was 99.3% (the characteristic peak intensity of 222 nm in circular dichroism was -29.8 mdeg, and the peak intensity of pure collagen standard was -30.0 mdeg; calculated according to the formula "retention rate = (sample peak intensity / standard peak intensity) × 100%), the purity was 99.99%, the sterility test was qualified, there were no aggregates ≥1 μm, and the osmotic pressure was 295 mOsm / kg.

[0044] The dynamic light scattering particle size distribution of collagen nano-dispersion formulation is shown in the figure. Figure 1 The spectrum shows that the particle size is symmetrically distributed with a single peak, the main peak is concentrated around 50 nm, the peak shape is narrow and without tailing, and there are no impurity peaks ≥1 μm. This characteristic is completely consistent with the above-mentioned detection of "average particle size 50 nm, PDI=0.12, no aggregates ≥1 μm", which directly proves the nanoscale uniform dispersion characteristics of the formulation.

[0045] Example 2 The preparation method of the collagen nano-dispersion formulation in this embodiment includes the following steps: 1. Preparation of collagen solution Type I and Type III collagen were dissolved in an acidic aqueous medium to prepare a collagen solution. The purity of both Type I and Type III collagen was 99.99%, and the mass ratio of Type I to Type III collagen was 85:15. The acidic aqueous medium was an 8 mM hydrochloric acid solution at 22 ℃, pH 2.1, and ionic strength of 0.008 M. The concentration of the collagen solution was 2.4 mg / mL.

[0046] 2. Self-assembly A 0.2 M Na2HPO4 solution was gradually added dropwise to the collagen solution to adjust the ionic strength of the collagen solution to 0.030 M. Then the pH value of the collagen solution was gradually increased to near neutral (pH 6.5).

[0047] Next, the temperature of the collagen solution was slowly increased from 3 °C to the preset self-assembly temperature (15 °C) at a heating rate of 0.5 °C / min. The self-assembly temperature was precisely controlled within ±0.5 °C to allow the collagen solution to self-assemble. During the self-assembly process, dynamic light scattering (DLS) was used to track the average particle size and polydispersity index (PDI) of the collagen fibers in real time.

[0048] 3. Kinetic freezing Once the average particle size of the collagen fibers reaches 30 nanometers and the polydispersity index reaches 0.10, the self-assembled system is subjected to kinetic freezing, specifically as follows: the temperature of the self-assembled system is rapidly reduced to 2 ℃ at a cooling rate of 2.5 ℃ / min.

[0049] After aseptic filtration and aseptic filling, a collagen nano-dispersion formulation is obtained.

[0050] The collagen nano-dispersed formulation was tested using the method described in Example 1. The results are as follows: the average particle size was 30 nm (PDI=0.10, RSD=2.3%), the absolute value of the Zeta potential was 32 mV, the retention rate of the collagen triple helix structure was 99.7% (the characteristic peak intensity at 222 nm in circular dichroism chromatogram was -29.9 mdeg, and the peak intensity of the pure collagen standard was -30.0 mdeg, calculated according to the formula "retention rate = (sample peak intensity / standard peak intensity) × 100%), the purity was ≥99.99%, the sterility test was qualified, there were no aggregates ≥1 μm, and the osmotic pressure was 293 mOsm / kg.

[0051] The dynamic light scattering particle size distribution of collagen nano-dispersion formulation is shown in the figure. Figure 2 The spectrum shows that the particle size is symmetrically distributed with a single peak, the main peak is concentrated around 30 nm, the peak shape is narrow and without tailing, and there are no impurity peaks ≥1 μm. This characteristic is completely consistent with the "average particle size of 30 nm, PDI=0.10, no aggregates ≥1 μm" detected in Example 2, which directly proves the nanoscale uniform dispersion characteristics of the formulation.

[0052] Example 3 The preparation method of the collagen nano-dispersion formulation in this embodiment includes the following steps: 1. Preparation of collagen solution Type I and Type III collagen were dissolved in an acidic aqueous medium to prepare a collagen solution. The purity of both Type I and Type III collagen was 99.99%, and the mass ratio of Type I to Type III collagen was 85:15. The acidic aqueous medium was a 5 mM hydrochloric acid solution at 24 ℃, pH 2.3, and ionic strength of 0.005 M. The concentration of the collagen solution was 2.3 mg / mL.

[0053] 2. Self-assembly A 0.5 M NaCl solution was gradually added dropwise to the collagen solution to adjust the ionic strength of the collagen solution to 0.034 M. Then the pH value of the collagen solution was gradually increased to near neutral (pH 6.0).

[0054] Next, the temperature of the collagen solution was slowly increased from 5 °C to the preset self-assembly temperature (25 °C) at a heating rate of 0.8 °C / min. The self-assembly temperature was precisely controlled within ±0.5 °C to allow the collagen solution to self-assemble. During the self-assembly process, dynamic light scattering (DLS) was used to track the average particle size and polydispersity index (PDI) of the collagen fibers in real time.

[0055] 3. Kinetic freezing Once the average particle size of the collagen fibers reaches 100 nanometers and the polydispersity index reaches 0.15, the self-assembled system is subjected to kinetic freezing, specifically as follows: the temperature of the self-assembled system is rapidly cooled to 8 ℃ at a cooling rate of 1.8 ℃ / min.

[0056] After aseptic filtration and aseptic filling, a collagen nano-dispersion formulation is obtained.

[0057] The collagen nano-dispersed formulation was tested using the method described in Example 1. The results are as follows: the average particle size was 100 nm (PDI=0.15, RSD=3.2%), the absolute value of the Zeta potential was 31 mV, the retention rate of the collagen triple helix structure was 99.0% (circular dichroism 222 nm characteristic peak intensity -29.7 mdeg, pure collagen standard peak intensity was -30.0 mdeg, calculated according to the formula "retention rate = (sample peak intensity / standard peak intensity) × 100%), purity ≥99.99%, sterility test qualified, no aggregates ≥1 μm, and osmotic pressure was 297 mOsm / kg.

[0058] The dynamic light scattering particle size distribution of collagen nano-dispersion formulation is shown in the figure. Figure 3 The spectrum shows that the particle size is symmetrically distributed with a single peak, the main peak is concentrated around 100 nm, the peak shape is narrow and without tailing, and there are no impurity peaks ≥1 μm. This characteristic is completely consistent with the above-mentioned detection of "average particle size of 100 nm, PDI=0.15, and no aggregates ≥1 μm", which directly proves the nanoscale uniform dispersion characteristics of the formulation.

[0059] Example 4 The preparation method of the collagen nano-dispersion formulation in this embodiment is the same as that in Example 1, except that steps 4 and 5 are included before aseptic filtration and aseptic filling, as detailed below: 4. Add physical stabilizers In the formulation process after kinetic freezing and aseptic concentration, high molecular weight hyaluronic acid with a molecular weight of 1800-2200 kDa is added to the collagen dispersion as a physical stabilizer. The amount of physical stabilizer added is 0.2% of the collagen protein mass.

[0060] 5. Adjust pH and ionic strength The final pH of the collagen dispersion was 6.5, the ionic strength was 0.013 M, and the absolute value of the zeta potential of the collagen fibers was 35 mV.

[0061] The collagen nano-dispersion formulation was tested using the method described in Example 1. The results are as follows: average particle size was 50 nm (PDI=0.11, RSD=2.5%), absolute zeta potential was 35 mV, and the retention rate of the collagen triple helix structure was 99.8% (circular dichroism chromatogram 222 nm characteristic peak intensity was -29.95 mdeg, pure collagen standard peak intensity was -30.0 mdeg, calculated using the formula "retention rate = (sample peak intensity / standard peak intensity) × 100%), purity ≥ 99.99%, sterility was satisfactory, no aggregates ≥ 1 μm were found, and osmotic pressure was 298 mOsm / kg. After storage at 4℃ for 18 months, the average particle size remained at 51 nm (PDI=0.12, RSD=2.7%), absolute zeta potential was ≥ 33 mV, the retention rate of the collagen triple helix structure was ≥ 99.5%, and the ionic strength remained between 0.012 and 0.014. Between M and 6.6, the pH remained stable at 6.4-6.6, with no stratification, precipitation or abnormal particle size growth, and the stability was significantly improved compared to Example 1 (stored at 4°C for 12 months).

[0062] The dynamic light scattering particle size distribution of collagen nano-dispersion formulation is shown in the figure. Figure 4 The spectrum shows that the particle size is symmetrically distributed with a single peak, the main peak is concentrated around 50 nm, the peak shape is narrow and without tailing, and there are no impurity peaks ≥1 μm. This characteristic is completely consistent with the above-mentioned detection of "average particle size of 50 nm, PDI=0.11, and no aggregates ≥1 μm", which directly proves the nanoscale uniform dispersion characteristics of the formulation (it still maintains excellent dispersion uniformity after the addition of physical stabilizers).

[0063] Example 5 The skin hydration injection product of this embodiment includes the collagen nano-dispersion formulation of Example 1 and a pharmaceutically acceptable carrier; wherein, the collagen nano-dispersion formulation in the skin hydration injection product has a mass content of 1.25% (w / v) (corresponding to an effective collagen content of 12.5 mg / mL), and the pharmaceutically acceptable carrier is water for injection.

[0064] The efficacy of skin hydration injection products was evaluated using the following methods: in vitro skin moisturizing rate assay (weighing method), skin brightness L-value assay (spectrophotometer method), dermal thickness ultrasound assay, skin irritation test (single / multiple skin irritation tests), in vitro fibroblast proliferation assay, accelerated stability assay, sterility and safety assays (direct inoculation method, heavy metal inductively coupled plasma mass spectrometry, allergen ELISA assay), dynamic light scattering (DLS), Zeta potential meter, circular dichroism spectroscopy (CD), and SDS-PAGE gel electrophoresis. The results are as follows: 1. Core physicochemical indicators: The average particle size was 50 nm (PDI=0.12, RSD=2.8%), the absolute value of the Zeta potential was 34 mV, the retention rate of the collagen triple helix structure was 99.2% (the characteristic peak intensity at 222 nm in circular dichroism chromatogram was -29.76 mdeg, and the peak intensity of pure collagen standard was -30.0 mdeg, calculated according to the formula "retention rate = (sample peak intensity / standard peak intensity) × 100%), purity ≥ 99.99%, sterility test qualified, no aggregates ≥ 1 μm, and osmotic pressure was 298 mOsm / kg.

[0065] See the dynamic light scattering particle size distribution map of the skin hydration injection product. Figure 5 The spectrum shows that the particle size is symmetrically distributed with a single peak, the main peak is concentrated around 50 nm, the peak shape is narrow and without tailing, and there are no impurity peaks ≥1 μm. This characteristic is completely consistent with the above-mentioned detection of "average particle size 50 nm, PDI=0.12, no aggregates ≥1 μm", which directly proves the nanoscale uniform dispersion characteristics of the product.

[0066] 2. Moisturizing effect: The in vitro skin stratum corneum moisturization rate was increased by 55% compared with the blank control group. The skin moisture content (Corneometer test) remained above 50 AU 28 days after injection (32 AU in the blank group), and the moisturizing duration was ≥3.5 months.

[0067] 3. Brightening effect: Fourteen days after injection, the skin luminance L value increased by 15% compared to the baseline (detected by spectrophotometer), skin transparency was significantly improved, there was no whitening or mask-like appearance, and the degree of light scattering was reduced by 40% compared to conventional collagen injections.

[0068] 4. Filling and repair effects: Ultrasound examination showed that 4 weeks after injection, the dermal layer thickness increased by 0.25 mm compared with the baseline, the fibroblast proliferation rate increased by 28% (in vitro cell experiment), and the improvement rate of fine lines (such as dry lines and shallow lines) was ≥55%.

[0069] 5. Biocompatibility: The single skin irritation test score is ≤0.5 (non-irritating), and there is no redness, swelling, bruising, or foreign body reaction after multiple injections. The incidence of skin irritation is <0.03%, which meets the biocompatibility requirements of GB / T 16886.10-2017 Biological Evaluation of Medical Devices Part 10: Irritation and Skin Sensitization Tests.

[0070] 6. Stability: After 12 months of storage at 4 ℃ and 6 months of accelerated storage at 25 ℃, the average particle size of the product remains 50 nm, PDI≤0.14, absolute value of Zeta potential≥32 mV, and there are no aggregates ≥1 μm.

[0071] 7. Sterility and safety: The sterility test (direct inoculation method) met the requirements, the content of heavy metals (lead, mercury, arsenic) was <10 μg / g, the allergen was not detected by ELISA, and there were no reports of allergic reactions in clinical use.

[0072] Example 6 The skin hydration injection product of this embodiment includes the collagen nano-dispersion formulation of Example 1, a pharmaceutically acceptable carrier, a pH adjuster, a buffer salt, a biocompatible preservative, and an antioxidant. The collagen nano-dispersion formulation in the skin hydration injection product has a mass content of 1.25% (w / v) (corresponding to an effective collagen content of 12.5 mg / mL). The pharmaceutically acceptable carrier is water for injection. The pH adjuster is disodium hydrogen phosphate with a content of 0.02% (w / v). The buffer salt is a disodium hydrogen phosphate-sodium dihydrogen phosphate mixture (mass ratio 3:2) with a content of 0.055% (w / v, 4 mM). The biocompatible preservative is benzalkonium chloride (BAC) with a content of 0.01% (w / v). The antioxidant is ascorbic acid (vitamin C) with a content of 0.04% (w / v). The final osmotic pressure of the product is 295 mOsm / kg.

[0073] The efficacy of the skin hydration injection product was tested using the method described in Example 5; the results are as follows: 1. Core physicochemical indicators: The average particle size was 50 nm (PDI=0.13, RSD=2.7%), the absolute value of the Zeta potential was 34 mV, the retention rate of the collagen triple helix structure was 99.5% (the characteristic peak intensity at 222 nm in circular dichroism chromatogram was -29.85 mdeg, and the peak intensity of the pure collagen standard was -30.0 mdeg, calculated according to the formula "retention rate = (sample peak intensity / standard peak intensity) × 100%), the purity was ≥99.99%, the sterility test was qualified, there were no aggregates ≥1 μm, and the osmotic pressure was 295 mOsm / kg.

[0074] See the dynamic light scattering particle size distribution map of the skin hydration injection product. Figure 6 The spectrum shows that the particle size is symmetrically distributed with a single peak, the main peak is concentrated around 50 nm, the peak shape is narrow and without tailing, and there are no impurity peaks ≥1 μm. This characteristic is completely consistent with the above-mentioned detection of "average particle size 50 nm, PDI=0.13, no aggregates ≥1 μm", which directly proves the nanoscale uniform dispersion characteristics of the product (it still maintains excellent dispersion uniformity after adding pH adjuster, buffer salt, preservative and antioxidant).

[0075] 2. Core Functions: The in vitro skin stratum corneum moisturization rate was increased by 68% compared with the blank control group. The skin moisture content (Corneometer test) remained above 56AU 28 days after injection (32AU in the blank group), and the moisturizing duration was extended to ≥4.5 months.

[0076] 3. Brightening effect: Fourteen days after injection, the skin brightness L value increased by 20% compared to the baseline, and the translucency score increased by 28%; the light scattering degree decreased by 15% compared to Example 5, with no whitening or mask-like appearance, and the "water light" visual effect was more natural.

[0077] 4. Filling and repair effects: Ultrasound examination showed that 4 weeks after injection, the dermal layer thickness increased by 0.30 mm compared to the baseline (0.05 mm compared to Example 5), the in vitro fibroblast proliferation rate increased by 35% (due to the synergistic effect of the auxiliary components in promoting collagen regeneration), and the improvement rate of fine lines (such as dry lines and fine lines) was ≥65%.

[0078] 5. Biocompatibility: The single skin irritation test score was ≤0.3 (better than Example 5), the irritation rate after multiple injections was <0.01%, and there were no adverse reactions such as redness, swelling, itching, or bruising, which meets the requirements of GB / T 16886.10-2017 Biological evaluation of medical devices - Part 10: Irritation and skin sensitization test.

[0079] 6. Stability: After 18 months of storage at 4 ℃ and 9 months of accelerated storage at 25 ℃, the average particle size of the product remains 50 nm (PDI=0.13, RSD≤2.9%), the absolute value of the Zeta potential is ≥33 mV, the retention rate of the collagen triple helix structure is ≥99.0%, and after 72 hours of storage at 25 ℃, there is no stratification, precipitation or abnormal particle size change.

[0080] 7. Safety: The sterility test (direct inoculation method) results were satisfactory, the benzalkonium chloride residue was <0.008% (w / v), the ascorbic acid oxidation rate was <5%, and the content of heavy metals (lead, mercury, arsenic) was <10 μg / g (superior to Example 5). The allergen was not detected by ELISA, and there were no reports of allergies or serious adverse reactions in clinical practice.

[0081] The correlation diagram between the fiber and skin mechanical properties of collagen nano-dispersion formulations is shown below. Figure 7 Among them, the "Relationship between single fiber stiffness and fiber diameter" shows the positive correlation between the diameter (50 nm, 100 nm, etc.) of collagen nanofibers and single fiber stiffness; the "Relationship between network modulus and fiber diameter" shows the trend of the network structure modulus formed by nano-dispersed formulations changing with fiber diameter (50 nm, etc.); the "Relationship between skin elastic modulus and fiber diameter" quantitatively reflects the effect of collagen fibers of different diameters (30 nm, 50 nm, etc.) in injectable products on improving the skin elastic modulus; and the "Relationship between skin tensile strength and fracture strain and fiber diameter" proves that nano-sized collagen fibers (50 nm, etc.) can significantly improve the skin's tensile strength and fracture strain.

[0082] Compare with Example 1 Except for the different self-assembly steps, the rest is the same as in Example 1.

[0083] The self-assembly steps for this comparative example are as follows: Add Na3PO4 buffer to the collagen solution prepared in Example 1 to adjust the ionic strength of the collagen solution to 0.2 M, and then gradually increase the pH of the collagen solution to near neutral (pH 7.0).

[0084] Next, the temperature of the collagen solution was increased from the initial temperature of 25 ℃ to 35 ℃ at a heating rate of 0.2 ℃ / s, held at 35 ℃ for 50 s, and then cooled down to 25 ℃ at a cooling rate of 0.2 ℃ / s to complete the self-assembly.

[0085] The method described in Example 1 was used for detection; the results are as follows: the average particle size was 620 nm (PDI=0.38, RSD=8.5%), the absolute value of the Zeta potential was 18 mV, the retention rate of the collagen triple helix structure was 71.0% (the characteristic peak intensity at 222 nm in circular dichroism spectroscopy was -21.3 mdeg, and the peak intensity of the pure collagen standard was -30.0 mdeg; calculated according to the formula "retention rate = (sample peak intensity / standard peak intensity) × 100%), the purity was still 99.99%, the sterility test was qualified, it contained a large number of aggregates ≥1 μm (accounting for 23%), and the osmotic pressure was 385 mOsm / kg (exceeding the isotonic range of 280-320 mOsm / kg for the human body); after storage at 4 ℃ for 1 month, the average particle size increased to 1250 nm (PDI=0.45, RSD=11.2%), the retention rate of the collagen triple helix structure decreased to 65.0%, and the proportion of aggregates increased to 42%). %, showing obvious stratification and sedimentation, with no practical application value.

[0086] Compare with Example 2 Except for replacing the kinetic freezing of Example 1 with conventional freezing, the rest is the same as Example 1.

[0087] The standard freezing procedure for this comparative example is as follows: the temperature of the self-assembled system was cooled to 4 °C at a cooling rate of 0.3 °C / min.

[0088] The method described in Example 1 was used for detection; the results are as follows: the average particle size was 170 nm (PDI=0.27, RSD=6.5%), the absolute value of the Zeta potential was 24 mV, the retention rate of the collagen triple helix structure was 92.0% (the characteristic peak intensity at 222 nm in circular dichroism chromatogram was -27.6 mdeg, and the peak intensity of pure collagen standard was -30.0 mdeg; calculated according to the formula "retention rate = (sample peak intensity / standard peak intensity) × 100%), the purity was ≥99.99%, the sterility test was qualified, it contained aggregates ≥1 μm (accounting for 7.8%), and the osmotic pressure was 296 mOsm / kg (within the isotonic range of human body); after storage at 4 ℃ for 3 months, the average particle size increased to 320 nm (PDI=0.35, RSD=9.8%), the retention rate of the collagen triple helix structure decreased to 88.0%, the absolute value of the Zeta potential decreased to 20 mV, and the proportion of aggregates increased to 15.2%. %, slight stratification occurred, and the dispersion stability could not meet the requirements for long-term storage (medical aesthetic injectables are generally required to be stable for at least 12 months).

[0089] Compare with Example 3 Except for replacing the acidic aqueous medium in Example 1 with a 10 mM acetic acid solution (pH 3.5, ionic strength 0.003M), the rest was the same as in Example 1.

[0090] The method described in Example 1 was used for detection; the results are as follows: the average particle size was 220 nm (PDI=0.32, RSD=7.8%), the absolute value of the Zeta potential was 23 mV, the retention rate of the collagen triple helix structure was 91.0% (the characteristic peak intensity at 222 nm in circular dichroism spectroscopy was -27.3 mdeg, and the peak intensity of the pure collagen standard was -30.0 mdeg; calculated according to the formula "retention rate = (sample peak intensity / standard peak intensity) × 100%), the purity was ≥99.99%, the sterility test was qualified, it contained aggregates ≥1 μm (accounting for 11.2%), and the osmotic pressure was 294 mOsm / kg (within the isotonic range of the human body); after storage at 4℃ for 2 months, the average particle size increased to 450 nm (PDI=0.41, RSD=10.5%), the retention rate of the collagen triple helix structure decreased to 87.0%, and the Zeta potential decreased. The absolute value of the potential dropped to 20mV, the proportion of aggregates increased to 20.5%, and obvious stratification and precipitation appeared, which could not meet the core requirement of "stable storage for at least 12 months".

[0091] Compare with Example 4 Except for the different self-assembly steps, the rest is the same as in Example 1.

[0092] The self-assembly steps of this comparative example are as follows: 0.2 M PBS buffer (pH 11.2) at room temperature was added directly to the collagen solution prepared in Example 1, and the pH value of the system was adjusted from 2.0 to 7.0 and the ionic strength was adjusted from 0.01 M to 0.032 M in one step. Then, self-assembly was carried out according to the temperature conditions of Example 1 (4 ℃→20 ℃, 0.5 ℃ / min).

[0093] The method described in Example 1 was used for detection; the results are as follows: the average particle size was 320 nm (PDI=0.38, RSD=8.2%), the absolute value of the Zeta potential was 22 mV, the retention rate of the collagen triple helix structure was 82.0% (the characteristic peak intensity at 222 nm in circular dichroism spectroscopy was -24.6 mdeg, and the peak intensity of pure collagen standard was -30.0 mdeg; calculated according to the formula "retention rate = (sample peak intensity / standard peak intensity) × 100%), the purity was ≥99.99%, the sterility test was qualified, it contained aggregates ≥1 μm (accounting for 18.6%), and the osmotic pressure was 296 mOsm / kg (within the isotonic range of human body); after storage at 4 ℃ for 1.5 months, the average particle size increased to 650 nm (PDI=0.48, RSD=12.5%), the retention rate of the collagen triple helix structure decreased to 75.0%, the absolute value of the Zeta potential decreased to 19 mV, and the proportion of aggregates increased to 29.8%). %, with a large amount of sedimentation and stratification, poor fluidity, and uneven dispersion.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a collagen nano-dispersion formulation, characterized in that, Includes the following steps: S1: Dissolve type I collagen and type III collagen in an acidic aqueous medium to prepare a collagen solution; S2: The temperature, pH and ionic strength of the collagen solution are controlled to enable the collagen solution to self-assemble. S3: Once the average particle size of the collagen fibers reaches the target range, the self-assembled system is subjected to kinetic freezing.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of type I collagen to type III collagen is (80-90):(10-20); the concentration of the collagen solution is 2.0-2.4 mg / mL.

3. The preparation method according to claim 1, characterized in that, In step S1, the concentration of the acidic aqueous medium is 5-10 mM, the pH value is 2.0-3.0, and the ionic strength is 0.005-0.01 M.

4. The preparation method according to claim 1, characterized in that, In step S2, temperature control includes: slowly raising the temperature of the collagen solution from a low temperature to a preset self-assembly temperature; wherein the low temperature is 3-5 ℃, the self-assembly temperature is 15-25 ℃, and the rate of slow temperature increase is 0.2-1 ℃ / min.

5. The preparation method according to claim 1, characterized in that, In step S2, pH adjustment includes gradually increasing the pH of the collagen solution to 6.0-7.

0.

6. The preparation method according to claim 1, characterized in that, In step S2, the ionic strength adjustment includes: adding a salt solution dropwise to the collagen solution to adjust the ionic strength of the collagen solution to 0.030-0.034 M; wherein the concentration of the salt solution is 0.2-0.5 M.

7. The preparation method according to claim 1, characterized in that, In step S3, the target range for average particle size is ≤100 nanometers; the polydispersity index of collagen fibers is 0.05-0.

20.

8. The preparation method according to claim 1, characterized in that, In step S3, kinetic freezing includes rapidly cooling the temperature of the self-assembled system to below 8 °C at a cooling rate of 1.5-2.5 °C / min.

9. A collagen nano-dispersion formulation, characterized in that, Prepared according to the preparation method according to any one of claims 1-8.

10. The application of the collagen nano-dispersion formulation according to claim 9 in biomedical materials and / or medical aesthetic products.

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