A freeze-dried, fast-dissolving composition containing fullerene and recombinant collagen and its preparation method

Abstract

This invention discloses a freeze-dried, rapidly soluble composition containing fullerene and recombinant collagen, and its preparation method, relating to the fields of biomedicine and cosmetics. The composition includes an active ingredient and a freeze-drying protectant. The active ingredient is a monocarboxylated fullerene-polyethylene glycol-recombinant collagen covalent complex, wherein the monocarboxylated fullerene and recombinant collagen are covalently linked by amide bonds through bifunctionalized polyethylene glycol. The freeze-drying protectant consists of trehalose, sucrose, and mannitol. The composition is a solid powder that dissolves in water to form a colloidal dispersion; it exhibits good antioxidant activity, skin repair efficacy, and anti-hair loss efficacy, and can be used to prepare skin repair products or anti-hair loss products.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and cosmetics, specifically to a freeze-dried, fast-dissolving composition containing fullerene and recombinant collagen and its preparation method. Background Technology

[0002] Fullerenes (primarily C60) are spherical molecules composed of 60 carbon atoms. They possess a unique nanostructure and extremely strong free radical scavenging ability, exhibiting antioxidant activity far exceeding that of traditional antioxidants such as vitamin C, earning them the title of "free radical sponges." However, the strong hydrophobicity of fullerenes (contact angle approximately 90°-100°) makes them difficult to apply directly to aqueous systems, limiting their widespread use in cosmetics and biopharmaceuticals. While water-soluble fullerene derivatives (such as hydroxylated fullerenes and pyrrolidine fullerenes) have been developed in existing technologies, these modifications often come at the cost of sacrificing the conjugated structure of the fullerene, resulting in a significant decrease in antioxidant activity.

[0003] Recombinant collagen is a type of collagen produced using genetic engineering technology. It possesses advantages such as high biocompatibility, low immunogenicity, and the ability to promote cell adhesion and proliferation. Recombinant type III collagen is mainly distributed in the dermis and has the effect of repairing the skin barrier and promoting wound healing. Recombinant type XVII collagen is an important transmembrane protein in the epidermal basement membrane zone, playing a crucial role in maintaining hair follicle stem cell homeostasis and regulating hair growth. However, as a large protein molecule, collagen is prone to degradation or conformational changes in aqueous solutions, leading to reduced activity.

[0004] Simply mixing fullerene with recombinant collagen physically makes it difficult to form a stable and uniform dispersion system. The main technical problems are as follows: (1) Fullerene is prone to agglomeration and loses its nano-effect; (2) Collagen cannot effectively encapsulate fullerene, and the complex is prone to precipitation; (3) Collagen is prone to inactivation in liquid formulations; (4) The two lack a molecular basis for synergistic effect.

[0005] Therefore, developing a freeze-dried formulation that can maintain the original structural activity of fullerene, achieve stable compounding of fullerene and collagen, and possess rapid dissolution properties has significant market value and application prospects. Summary of the Invention

[0006] To address the shortcomings of the prior art, this invention aims to provide a freeze-dried, fast-dissolving composition containing fullerene and recombinant collagen, and its preparation method.

[0007] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention provides a lyophilized and rapidly soluble composition containing fullerene and recombinant collagen, the composition comprising an active ingredient and a lyophilization protectant, the active ingredient being a monocarboxylated fullerene-polyethylene glycol-recombinant collagen covalent complex, wherein the monocarboxylated fullerene and recombinant collagen are covalently linked by amide bonds through bifunctionalized polyethylene glycol; the lyophilization protectant is composed of trehalose, sucrose and mannitol in a mass ratio of (1-2):(1-2):1; the mass ratio of the lyophilization protectant to the active ingredient is (5-10):1; the composition is a solid powder that dissolves in water to form a colloidal dispersion.

[0008] As a preferred embodiment of the present invention, the dual-functionalized polyethylene glycol is NH2-PEG-COOH, and its molecular weight is 1000-5000 Da.

[0009] As a preferred embodiment of the present invention, the monocarboxylated fullerene is obtained by introducing a monocarboxyl group into fullerene C60 or C70 through a Bingel cycloaddition reaction.

[0010] As a preferred embodiment of the present invention, the recombinant collagen is recombinant type III collagen or recombinant type XVII collagen.

[0011] Secondly, the present invention also provides a method for preparing the above-mentioned lyophilized and rapidly soluble composition containing fullerene and recombinant collagen, comprising the following steps: (1) A monocarboxyl group is introduced onto a fullerene via a Bingel cycloaddition reaction to obtain a monocarboxyl fullerene; (2) Dissolve monocarboxylated fullerene in DMF, add NH2-PEG-COOH, EDC and NHS, stir at room temperature under nitrogen protection; after the reaction is complete, add acetone to precipitate, collect the precipitate by centrifugation, wash with diethyl ether 3 times, and dry under vacuum to obtain monocarboxylated fullerene-PEG-COOH. (3) Disperse monocarboxylated fullerene-PEG-COOH in phosphate buffer at pH 7.4, add EDC and NHS, activate at room temperature, add recombinant collagen, stir at 4°C, and dialyze to obtain an aqueous dispersion of monocarboxylated fullerene-polyethylene glycol-recombinant collagen covalent complex. (4) Freeze-dry the aqueous dispersion of monocarboxylated fullerene-polyethylene glycol-recombinant collagen covalent complex obtained in step (3) to obtain monocarboxylated fullerene-polyethylene glycol-recombinant collagen covalent complex solid powder. (5) Mix the solid powder obtained in step (4) with the freeze-drying protectant until it is homogeneous.

[0012] Preferably, in step (2), the ratio of monocarboxylated fullerene, DMF, NH2-PEG-COOH, EDC, NHS and acetone is 100mg:10mL:300mg:50mg:30mg:50mL.

[0013] Preferably, in step (3), the ratio of monocarboxylated fullerene-PEG-COOH, phosphate buffer, EDC, NHS, and recombinant collagen is 2 mg: 20 mL: 10 mg: 6 mg: 500 mg. Preferably, in step (4), the freeze drying includes the steps of pre-freezing, primary drying, and secondary drying. Pre-freezing involves cooling to -45°C and holding for 3 hours; primary drying involves heating to -25°C, maintaining a vacuum of 15Pa for 20 hours; and secondary drying involves heating to 30°C, maintaining a vacuum of ≤5Pa for 6 hours.

[0014] Thirdly, the present invention also provides the use of the above-described composition for preparing skin repair products or hair loss prevention products.

[0015] Compared with the prior art, the beneficial technical effects of the present invention include: 1. This invention introduces a single carboxyl group onto fullerene via a Bingel cycloaddition reaction, modifying only one double bond of the fullerene without destroying the remaining conjugated structure, thus preserving the antioxidant activity of the fullerene. Furthermore, through EDC / NHS chemistry, a diamide bond is covalently formed between the collagen and NH2-PEG-COOH and recombinant collagen. The PEG bridging layer provides sufficient steric hindrance, keeping the collagen away from the fullerene surface and preventing conformational damage caused by hydrophobic interactions. The covalent connection ensures the long-term stability of the complex in aqueous solution; after lyophilization, it shows no significant degradation after storage at room temperature for more than 6 months.

[0016] 2. This invention uses monocarboxylated fullerene to scavenge free radicals, inhibit matrix metalloproteinase (MMP) activity, and reduce collagen degradation; recombinant collagen is responsible for replenishing the extracellular matrix, providing support and repair signals; the two achieve anti-aging and repair effects through a "scavenging + replenishment" mechanism; for type XVII collagen, it can also target and regulate hair follicle stem cells to promote hair growth.

[0017] 3. The composition of the present invention has rapid dissolution properties, dissolving quickly in water to form a uniform, clear to slightly opalescent colloidal dispersion, making it convenient to use.

[0018] 4. The process of this invention is highly operable, and all operation steps use conventional organic synthesis and freeze-drying equipment. The reaction conditions are mild and suitable for industrial production. Detailed Implementation

[0019] The technical solution of the present invention will be described in detail below with reference to the embodiments. Example

[0020] Preparation of monocarboxylated fullerenes: Under nitrogen protection, 1.0 g of fullerene C60 was dissolved in 500 mL of toluene, and 2.0 g of di-tert-butyl malonate, 1.5 g of carbon tetrabromide, and 1.5 mL of triethylamine were added. The mixture was stirred at room temperature for 24 hours. After the reaction was completed, the solvent was removed by vacuum distillation, and the product was separated by silica gel column chromatography (toluene / n-hexane = 1:1 elution). The product was collected to obtain the fullerene tert-butyl malonate intermediate. The intermediate was dissolved in trifluoroacetic acid and stirred at room temperature for 2 hours to remove the tert-butyl protection. The product was filtered, washed, and dried to obtain monocarboxylated fullerene C60-COOH.

[0021] Preparation of monocarboxylated fullerene-PEG-COOH: 100 mg of monocarboxylated fullerene C60-COOH was dissolved in 10 mL of anhydrous N,N-dimethylformamide (DMF), and 300 mg of NH2-PEG-COOH (MW=2000 Da), 50 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 30 mg of N-hydroxysuccinimide (NHS) were added. The mixture was stirred at room temperature for 24 hours under nitrogen protection. After the reaction was completed, 50 mL of acetone was added to precipitate the precipitate. The precipitate was collected by centrifugation, washed three times with diethyl ether, and dried under vacuum to obtain C60-PEG-COOH.

[0022] Preparation of monocarboxylated fullerene-polyethylene glycol-recombinant collagen covalent complex: 2 mg C60-PEG-COOH was dispersed in 20 mL of phosphate-buffered saline (PBS) at pH 7.4. 10 mg EDC and 6 mg NHS were added, and the mixture was activated at room temperature for 15 minutes. Then, 500 mg of recombinant type III collagen was added, and the mixture was stirred at 4 °C for 12 hours. After the reaction was completed, the mixture was dialyzed in PBS for 24 hours using a dialysis bag with a molecular weight cutoff of 100 kDa to remove unreacted collagen and small molecule byproducts, thus obtaining an aqueous dispersion of monocarboxylated fullerene-polyethylene glycol-recombinant collagen covalent complex. The above-mentioned aqueous dispersion of the complex was dispensed into freeze-drying trays and placed in a freeze dryer for freeze-drying. The freeze-drying procedure was as follows: pre-freezing and cooling to -45°C for 3 hours; first drying and heating to -25°C with a vacuum of 15 Pa for 20 hours; second drying and heating to 30°C with a vacuum of ≤5 Pa for 6 hours; after completion, the solid powder of the monocarboxylated fullerene-polyethylene glycol-recombinant collagen covalent complex was obtained.

[0023] Preparation of the freeze-dried and instantaneous composition: Weigh 0.5g of the above-mentioned monocarboxylated fullerene-polyethylene glycol-recombinant collagen covalent complex solid powder, mix it evenly with 1g of trehalose, 1g of sucrose and 0.5g of mannitol, fill it into a vial, and seal it with a cap to obtain the final product.

[0024] Performance testing: Take one bottle of the above-mentioned lyophilized instant soluble composition, add 10 mL of deionized water, shake gently to dissolve, and obtain a reconstituted solution. Perform the following tests, and the results are shown in Table 1.

[0025] Table 1 Performance test results of Example 1

[0026] Example 2 Preparation of monocarboxylated fullerenes: Same as in Example 1.

[0027] Preparation of monocarboxylated fullerene-PEG-COOH: Same as in Example 1.

[0028] Preparation of the monocarboxylated fullerene-polyethylene glycol-recombinant collagen covalent complex: 2 mg of C60-PEG-COOH was dispersed in 20 mL of phosphate-buffered saline (PBS) at pH 7.4. 10 mg of EDC and 6 mg of NHS were added, and the mixture was activated at room temperature for 15 minutes. Then, 500 mg of recombinant type XVII collagen was added, and the mixture was stirred at 4 °C for 12 hours. After the reaction, the mixture was dialyzed in PBS for 24 hours using a dialysis bag with a 100 kDa molecular weight cutoff to remove unreacted collagen and small molecule byproducts, yielding an aqueous dispersion of the monocarboxylated fullerene-polyethylene glycol-recombinant collagen covalent complex. The aqueous dispersion was dispensed into lyophilization trays and lyophilized in a lyophilizer. The lyophilization procedure was the same as in Example 1, yielding a solid powder of the monocarboxylated fullerene-polyethylene glycol-recombinant collagen covalent complex.

[0029] Preparation of the freeze-dried and instantaneous composition: Weigh 0.5g of the above-mentioned monocarboxylated fullerene-polyethylene glycol-recombinant collagen covalent complex solid powder, mix it evenly with 1g of trehalose, 1g of sucrose and 0.5g of mannitol, fill it into a vial, and seal it with a cap to obtain the final product.

[0030] Performance testing: Take one bottle of the above-mentioned lyophilized instant soluble composition, add 10 mL of deionized water, shake gently to dissolve, and obtain a reconstituted solution. Perform the following tests, and the results are shown in Table 2.

[0031] Table 2 Performance test results of Example 2

[0032] Comparative Example 1 Take 0.5 mg of monocarboxylated fullerene C60-COOH, 500 mg of recombinant type III collagen, and 300 mg of NH2-PEG-COOH (MW=2000 Da), and mix them directly with 1 g of trehalose, 1 g of sucrose, and 0.5 g of mannitol. Fill the mixture into vials and seal them with a pressure cap.

[0033] Upon testing, the comparative sample showed obvious precipitation after reconstitution. The particle size was >1μm as determined by dynamic light scattering method, and the DPPH scavenging rate (0.1mg / mL, 517nm colorimetric method) was 45.2%.

[0034] Comparative Example 2 The difference from Example 1 is that 2 mg of monocarboxylated fullerene C60-COOH was directly reacted with 500 mg of recombinant type III collagen in the presence of EDC / NHS, and the rest was the same as in Example 1.

[0035] The reconstituted sample of this comparative example had a particle size of 520 nm (dynamic light scattering method) and a PDI of 0.55. Circular dichroism spectroscopy determined the collagen activity retention rate to be 52% and the DPPH scavenging rate (0.1 mg / mL) to be 68.3%.

[0036] Comparative Example 3 The difference from Example 1 is that only 2g of trehalose and 2g of sucrose were used as freeze-drying protectants, without the addition of mannitol, and the rest is the same as in Example 1.

[0037] The reconstituted sample of this comparative example had a particle size of 380 nm (dynamic light scattering method) and a PDI of 0.55. The collagen activity retention rate was determined to be 78.5% by circular dichroism spectroscopy.

[0038] Comparative Example 4 The difference from Example 1 is that the amount of recombinant type III collagen used in this comparative example is reduced to 75 mg, while the rest is the same as in Example 1.

[0039] The comparative sample was found to have a reconstituted particle size (dynamic light scattering method) of 280 nm, PDI = 0.42, DPPH scavenging rate (0.1 mg / mL) of 72.6%, and collagen activity retention rate of 68.3% as determined by circular dichroism spectroscopy.

[0040] Experiment Example 1: Stability Test The lyophilized and rapidly soluble composition prepared in Example 1 was tested at 40°C / 75% RH for 6 months, and the results are shown in Table 3.

[0041] Table 3 Stability test results

[0042] Experiment Example 2: Batch-to-Batch Consistency Assessment Three batches of lyophilized instant soluble compositions were prepared consecutively according to the method in Example 1, and the results are shown in Table 4.

[0043] Table 4 Results of batch-to-batch consistency assessment

[0044] The freeze-dried, rapidly soluble composition of this invention can be widely used in the cosmetics field (such as freeze-dried powder masks, anti-aging essences, and hair growth essences), the medical device field (such as skin repair dressings and wound healing patches), and the biopharmaceutical field (such as adjuvant preparations for hair loss treatment). The preparation process is compatible with existing freeze-drying production lines, and the raw materials are all commercially available, demonstrating good prospects for industrialization.

[0045] The above description is a preferred embodiment of the present invention, used to explain the technical solution of the present invention, and is not intended to limit the present invention. Those skilled in the art can make conventional modifications, equivalent substitutions and improvements within the spirit and principles of the present invention, all of which are still included within the protection scope of the present invention.

Claims

1. A freeze-dried, fast-dissolving composition containing fullerene and recombinant collagen, characterized in that, The composition includes an active ingredient and a lyophilization protectant. The active ingredient is a monocarboxylated fullerene-polyethylene glycol-recombinant collagen covalent complex, wherein the monocarboxylated fullerene and recombinant collagen are covalently linked by amide bonds through bifunctionalized polyethylene glycol. The lyophilization protectant is composed of trehalose, sucrose, and mannitol in a mass ratio of (1-2):(1-2):

1. The mass ratio of the lyophilization protectant to the active ingredient is (5-10):

1. The composition is a solid powder that dissolves in water to form a colloidal dispersion.

2. The lyophilized, fast-dissolving composition containing fullerene and recombinant collagen according to claim 1, characterized in that: The dual-functionalized polyethylene glycol is NH2-PEG-COOH, with a molecular weight of 1000-5000 Da.

3. The freeze-dried, fast-dissolving composition containing fullerene and recombinant collagen according to claim 1, characterized in that: The monocarboxylated fullerene is obtained by introducing a monocarboxyl group into fullerene C60 or C70 via a Bingel cycloaddition reaction.

4. The freeze-dried, fast-dissolving composition containing fullerene and recombinant collagen according to claim 1, characterized in that: The recombinant collagen is recombinant type III collagen or recombinant type XVII collagen.

5. A method for preparing the freeze-dried, fast-dissolving composition containing fullerene and recombinant collagen as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) A monocarboxyl group is introduced onto a fullerene via a Bingel cycloaddition reaction to obtain a monocarboxyl fullerene; (2) Dissolve monocarboxylated fullerene in DMF, add NH2-PEG-COOH, EDC and NHS, stir at room temperature under nitrogen protection; after the reaction is complete, add acetone to precipitate, collect the precipitate by centrifugation, wash with diethyl ether 3 times, and dry under vacuum to obtain monocarboxylated fullerene-PEG-COOH. (3) Disperse monocarboxylated fullerene-PEG-COOH in phosphate buffer at pH 7.4, add EDC and NHS, activate at room temperature, add recombinant collagen, stir at 4°C, and dialyze to obtain an aqueous dispersion of monocarboxylated fullerene-polyethylene glycol-recombinant collagen covalent complex. (4) Freeze-dry the aqueous dispersion of monocarboxylated fullerene-polyethylene glycol-recombinant collagen covalent complex obtained in step (3) to obtain monocarboxylated fullerene-polyethylene glycol-recombinant collagen covalent complex solid powder. (5) Mix the solid powder obtained in step (4) with the freeze-drying protectant until it is homogeneous.

6. The preparation method according to claim 5, characterized in that: In step (2), the ratio of monocarboxylated fullerene, DMF, NH2-PEG-COOH, EDC, NHS and acetone is 100mg:10mL:300mg:50mg:30mg:50mL.

7. The preparation method according to claim 5, characterized in that: In step (3), the ratio of monocarboxylated fullerene-PEG-COOH, phosphate buffer, EDC, NHS, and recombinant collagen is 2mg:20mL:10mg:6mg:500mg.

8. The preparation method according to claim 5, characterized in that: In step (4), the freeze drying includes the steps of pre-freezing, primary drying, and secondary drying. Pre-freezing involves cooling to -45°C and holding for 3 hours; primary drying involves heating to -25°C, maintaining a vacuum of 15Pa for 20 hours; and secondary drying involves heating to 30°C, maintaining a vacuum of ≤5Pa for 6 hours.

9. Use of the composition according to any one of claims 1-4 or the composition prepared by the method according to any one of claims 5-8, characterized in that, Used to prepare skin repair products or hair loss prevention products.

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