High-bioactivity recombinant human collagen microsphere filling material and preparation method thereof

Abstract

The invention discloses a high-biological-activity recombinant human collagen microsphere filling material and a preparation method thereof, the filling material is composed of components with good biological compatibility, no irritant component is added in the preparation process, and fibroblast proliferation is supported. The invention further provides application of the high-biological-activity recombinant human collagen microsphere filling material in the field of medical beauty filling materials. The collagen micro-filling material prepared by the invention has the characteristics of antibiosis, antioxidation and anti-inflammation, and has high biological activity. Meanwhile, the stability is good, the stability of the material in a human body is improved, and the filling effect is maintained; the mechanical property is excellent, the elastic characteristic is more remarkable than the viscous characteristic, and the good filling effect is guaranteed.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical aesthetic materials, and in particular relates to a highly bioactive recombinant human collagen microsphere filling material and a preparation method thereof. Background Art

[0002] Collagen is a very important protein, which is widely distributed in human skin, bones, muscles and other tissues. Collagen has excellent moisturizing properties, which can help the skin maintain moisture and promote the regeneration and repair of skin cells. It is widely used in the fields of beauty and medical beauty. Collagen filling material is a popular beauty product, which is natural, safe and effective. This filling material is mainly derived from collagen in the body and has excellent biocompatibility. Collagen filling material can effectively improve signs of aging such as skin sagging and wrinkles, and restore the skin to firmness, smoothness and youthful vitality. In addition, it can also be used to fill facial contours and create more three-dimensional and natural facial lines. Chitosan is a natural polysaccharide derived from the shells of crustaceans. The extracted chitosan is a safe and environmentally friendly beauty ingredient. Chitosan has good biocompatibility and excellent antibacterial properties, which can effectively prevent infection at the filling site.

[0003] The existing technology provides collagen chitosan composite scaffolds, which have the characteristics of rigidity and toughness, but the biological activity and antioxidant properties of the materials need to be improved, and it cannot be guaranteed that the filling effect can be retained for a long time. Summary of the invention

[0004] In order to solve the deficiencies of the prior art, the present invention proposes a highly bioactive recombinant human collagen microsphere filling material and a preparation method thereof, wherein the filling material has antibacterial, antioxidant, and anti-inflammatory characteristics and has high bioactivity. In order to achieve this effect, the present invention provides the following technical solutions:

[0005] In one aspect, the present invention provides a method for preparing a highly bioactive recombinant human collagen microsphere filling material, comprising:

[0006] Step 1, preparing recombinant human collagen;

[0007] In the embodiment of the present invention, the amino acid sequence of the recombinant human collagen is as shown in SEQ ID NO.1, and the sequence is obtained by splicing the active sequence in type III collagen;

[0008] The pet28a-rCol plasmid carrying the target amino acid sequence was transformed into E. coli BL21 competent cells to prepare the BL21 / pet28a-rCol strain seed solution, which was inoculated in a fermenter, induced, and fermented to obtain the fermentation solution; the bacteria were collected, crushed by high-pressure homogenization, and centrifuged to obtain the bacterial supernatant; and the target protein was purified by nickel ion affinity chromatography.

[0009] Step 2, synthesizing dopamine-modified chitosan;

[0010] In the embodiment of the present invention, it includes:

[0011] S2.1, dissolving chitosan powder in acetic acid solution to prepare chitosan solution;

[0012] S2.2, adding dopamine hydrochloride to the chitosan solution to obtain a chitosan dopamine mixed solution;

[0013] S2.3, dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in 2-(N-morpholinyl)ethanesulfonic acid MES buffer solution, and then adding them to the prepared chitosan-dopamine mixed solution to react at room temperature;

[0014] S2.4, the solution after the reaction is placed in a dialysis bag with a molecular weight cutoff of 8KD-14KD, dialyzed with deionized water, and freeze-dried.

[0015] Step 3, expression and purification of American cockroach thymosin THY;

[0016] In the embodiment of the present invention, (a) the amino acid sequence of thymosin THY from American cockroach is shown in SEQ ID NO.2.

[0017] In step 3, the American cockroach thymosin THY is recombinantly expressed using E. coli BL21 (DE3), and the obtained protein is purified by gravity column; the eluate is placed in a dialysis bag with a molecular weight cutoff of 8KD-14KD and dialyzed in deionized water.

[0018] Step 4, preparing chitosan composite recombinant human collagen microsphere filling material:

[0019] In an embodiment of the present invention, recombinant human collagen, dopamine-modified chitosan and American cockroach thymosin THY are dissolved in a pH 3.8 sodium acetate buffer to obtain a mixed solution; the mixed solution is added dropwise to an N-hydroxysuccinimide EDC / NHS solution by electrospinning, and the chitosan composite collagen microsphere filling material is obtained by cross-linking; the EDC / NHS solution is prepared by dissolving 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in a 2-(N-morpholino)ethanesulfonic acid MES buffer solution.

[0020] In step 4, in the mixed solution, the concentrations of recombinant human collagen, dopamine-modified chitosan and American cockroach thymosin THY dissolved in pH 3.8 sodium acetate buffer are 10-20 mg / mL, 2-5 mg / mL and 1-3 mg / mL respectively.

[0021] In step 4, during the cross-linking process, the electrospinning voltage is 10-18 kV, the volume ratio of the mixed solution to the EDC / NHS solution is 1:10, the inner diameter of the needle used in the electrospinning process is 0.5 mm, the receiving distance of the electrospinning is 20 cm, the push speed of the electrospinning is 0.8 mL / min, and the chemical cross-linking time is 12 hours. The cross-linked microspheres are washed and freeze-dried to obtain the product.

[0022] The average particle size is 285±26μm and the spheroidization degree is 98±15%.

[0023] The invention also provides a high-bioactive recombinant human collagen microsphere filling material prepared by the preparation method.

[0024] As a third aspect of the present invention, it is to provide the application of the highly bioactive recombinant human collagen microsphere filling material in the field of medical beauty filling materials.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention provides a method for preparing a filling material composed of recombinant human collagen and dopamine-modified chitosan. The collagen microspheres prepared by the present invention have good stability, which helps to improve the stability of the material in the human body and maintain the filling effect; at the same time, it has a good antibacterial effect.

[0027] 2. The collagen microsphere material provided by the present invention has excellent mechanical properties, and its elastic characteristics are more significant than its viscous characteristics, ensuring a good filling effect.

[0028] 3. The collagen microsphere filling material provided by the present invention is composed of ingredients with good biocompatibility, and no irritating ingredients are added during the preparation process. In a 7-day cell activity experiment, the cell activity of the filling material treatment group did not decrease significantly compared with the DMEM cell culture medium, indicating that the filling material did not have an adverse effect on fibroblast proliferation. Among them, recombinant human collagen can avoid problems such as immunity and virality compared to animal-extracted collagen.

[0029] 4. The present invention encapsulates a thymosin THY of American cockroach in the dressing. The prepared material can be integrated with human tissue in 7 days with a fast integration speed and can significantly reduce the phenomenon of inflammatory scars and the like.

[0030] 5. The present invention uses collagen as the main material, and compounds it with chitosan, which is antibacterial, dopamine, which is antioxidant and anti-inflammatory, and thymosin from American cockroaches, which is anti-inflammatory, promotes angiogenesis, and promotes cell migration. This gives the collagen filler richer biological activity and can promote the migration of fibroblasts in a cell scratch experiment lasting 48 hours. At the same time, the composite chitosan can also improve the stability of the collagen filler in the in vivo environment. Its good biocompatibility can support the material to stay in the body for a longer time, so that the filling effect can be better retained, and a satisfactory use effect can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0032] Figure 1 : The stability of collagen microspheres in PBS at different chitosan contents in Experimental Example 1.1.

[0033] Figure 2 : Antibacterial efficiency of collagen microspheres at different chitosan contents in Experimental Example 1.2.

[0034] Figure 3 : Rheological characterization of 2 mg / mL chitosan collagen microspheres in Experimental Example 1.3.

[0035] Figure 4 : In the 2 mg / mL chitosan collagen microsphere cell compatibility experiment in Experimental Example 1.4, the cells used were L929 cells, wherein the control group was cultured in DMEM medium for 7 days, and the experimental group was cultured in DMEM medium containing 500 μL 2 mg / mL chitosan microsphere extract for 7 days.

[0036] Figure 5 : HE staining results of normal tissue and microsphere injection site tissue in Experimental Example 1.5.

[0037] Figure 6 : Scratch migration experiment of L929 cells treated with the control group and microsphere extract in Experimental Example 1.6. DETAILED DESCRIPTION

[0038] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0039] Example 1: Preparation method of recombinant human collagen

[0040] (I) Obtaining the amino acid sequence of recombinant human collagen, the sequence is obtained by splicing the active sequence in type III collagen, the amino acid sequence is SEQ ID NO.1:

[0041]

[0042] (ii) The pet28a-rCol plasmid carrying the target amino acid sequence was transformed into E. coli BL21 competent cells to prepare the BL21 / pet28a-rCol strain seed solution, which was inoculated in a fermenter, induced, and fermented to obtain the fermentation broth. The cells were collected, crushed by high-pressure homogenization, and centrifuged to obtain the cell supernatant. The target protein was purified by nickel ion affinity chromatography.

[0043] Example 2: Synthesis of dopamine-modified chitosan

[0044] (i) 1.2 g of chitosan powder (95% deacetylated, MW = 340,000 g / mol) was dissolved in 40 ml of 1.5% (v / v) acetic acid solution to prepare a 3% (w / v) chitosan solution. Dopamine hydrochloride was added to the solution at 0.1 g / mL, 0.2 g / mL or 0.4 g / mL and stirred for 30 minutes. EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide, 0.4 mmol) and NHS (N-hydroxysuccinimide, 0.2 mmol) were dissolved in 5 mL of MES (2-(N-morpholino)ethanesulfonic acid, 0.01 M, pH = 6) buffer solution and then added to the prepared chitosan-dopamine mixed solution and reacted at room temperature for 12 hours.

[0045] (ii) After the reaction, the solution was placed in a dialysis bag with a molecular weight cutoff of 8KD-14KD and dialyzed with deionized water for 3 times. The solution was frozen at -80°C and freeze-dried.

[0046] Example 3: Expression and purification of American cockroach thymosin THY

[0047] (I) The amino acid sequence of thymosin of American cockroach is

[0048] MAKTKGSSHHHHHHHHGSSGSSSSGSSGMSAPVSPQLKDLPKVNDLLKSELEGFKTVNMKKAETHEKNV

[0049] LPTAEDVKQERQHSELIQGVESFKPERLKRTNTQEKIVLPNAQDVATEKTQKALLQGVEAFDTGKLKHT

[0050] ETQEKNPLPDKDVVKQEKVHQNLLEGVEHFDKTTMKPTQTQEKNPLPDPEAIEQERGKQNLIAGIENFDPRKLKHTETQEKNPLPTKEAIDEEKKA*. (SEQ ID NO.2)

[0051] (ii) Use E.coli BL21 (DE3) for recombinant expression, and purify the protein by gravity column: first wash 4 times with 5mL deionized water, and then wash 4 times with 10mM imidazole buffer. Hang the filtered supernatant on the column 3-5 times, and take a sample as the flow-through. Wash the column with a buffer containing 20mM imidazole, and take a sample as the wash liquid. Wash the column with a buffer containing 400mM imidazole, take a sample and collect the eluate. Wash the column twice with a buffer containing 500mM imidazole, wash once with deionized water and 20% ethanol respectively, store in 20% ethanol, and place at 4°C. The eluate is placed in a dialysis bag with a molecular weight cutoff of 8KD-14KD, and dialyzed in deionized water until the buffer is completely replaced.

[0052] Example 4: Preparation of chitosan composite recombinant human collagen microsphere filling material

[0053] (i) 10 mg / mL recombinant human collagen, 2 mg / mL dopamine-modified chitosan, and 1 mg / mL THY were dissolved in pH 3.8 sodium acetate buffer.

[0054] (ii) The above solution was added dropwise to a MES solution containing a molar concentration of 400mM EDC / 100mM NHS by electrospinning, and chitosan composite collagen microsphere filling material was prepared by cross-linking. The electrospinning voltage was 10-18kV, the volume ratio of the solution in step (i) to the EDC / NHS solution was 1:10, the inner diameter of the needle used in the electrospinning process was 0.5mm, the receiving distance of the electrospinning was 20cm, the push speed of the electrospinning was 0.8mL / min, and the chemical cross-linking time was 12 hours. The cross-linked microspheres were washed and freeze-dried to obtain a product with an average particle size of 285±26μm and a spheroidization degree of 98±15%.

[0055] Test Example 1,

[0056] Collagen microspheres were prepared in the manner described in Examples 1 to 4, and in step 4, the concentrations of dopamine-modified chitosan were 0 mg / mL, 0.2 mg / mL, and 1 mg / mL. The other preparation methods remained unchanged.

[0057] 1.1. Stability test of collagen microspheres in PBS with different chitosan contents

[0058] The microspheres were placed in PBS buffer and the chitosan content retained at different times (days) was measured.

[0059] like Figure 1 As shown, adding chitosan to collagen and increasing the chitosan content can prolong the degradation time of the material in PBS. It can be seen that it helps to improve the stability of the material in the human body and maintain the filling effect.

[0060] 1.2. Antibacterial efficiency of collagen microspheres at different chitosan contents

[0061] The experiment used Escherichia coli, and the bacterial solution (LB-broth, 10 6 CFU / mL) was mixed with the microsphere suspension and incubated at 37 degrees in a humidified environment for 16 hours. The bacterial solution that was not in contact with the microspheres was grown in the same environment for 16 hours. After 16 hours, 100 μL was taken and diluted with 10 5 The 100-fold bacterial solution was smeared on the agar plate, and the plate was incubated in a humid environment at 37 degrees for 16 hours. After 16 hours, the number of colonies was counted.

[0062] The results are as follows Figure 2 As shown, the filling material has good antibacterial effect when chitosan is added, and the antibacterial effect is enhanced with the increase of chitosan content.

[0063] 2.3, Rheological characterization of 2 mg / mL chitosan collagen microspheres

[0064] Since the filling material with 2 mg / mL chitosan has the highest stability ( Figure 1 ) and antibacterial effect ( Figure 2 ), this example further tests the rheological characteristics of the material at a chitosan concentration of 2 mg / mL. Figure 3 As shown, at a chitosan concentration of 2 mg / mL, the storage modulus (G') of the filler in the rheological frequency sweep test is higher than the loss modulus, indicating that the elastic characteristics of the material are more significant than the viscous characteristics, and its mechanical properties ensure a good filling effect.

[0065] 2.4, 2mg / mL chitosan collagen microsphere cell compatibility experiment

[0066] L929 cells were used. The control group was cultured in DMEM medium for 7 days, and the experimental group was cultured in DMEM medium containing 500 μL 2 mg / mL chitosan microsphere extract for 7 days.

[0067] like Figure 4 It can be seen that in the 7-day cell activity experiment, the cell activity of the filler material treatment group did not decrease significantly compared with the DMEM cell culture medium, indicating that the filler material did not have an adverse effect on the proliferation of fibroblasts.

[0068] 2.5. HE staining

[0069] The collagen microspheres were injected subcutaneously into the back of mice, and the control group was normal mouse skin tissue. Samples were taken and HE staining was performed on the samples.

[0070] First, remove the tissue and fix it in pre-prepared 4% paraformaldehyde for more than 24 hours. After successful fixation, wash off the excess fixative with water, then put it in 70% ethanol solution for 30 minutes, 80% ethanol solution for 30 minutes, 90% ethanol solution for 30 minutes, 95% ethanol solution for 30 minutes, anhydrous ethanol for 60 minutes, anhydrous ethanol for 60 minutes, xylene for 60 minutes, and paraffin for 60 minutes. Repeat twice. Dehydrate and make the tissue transparent. Pour the melted paraffin into the dehydrated and transparent tissue, put it in a wax melting box and keep it warm for a period of time. When the paraffin completely penetrates into the tissue block, embed it with paraffin liquid, cool and solidify it into a block. Fix the embedded wax block on the slicer, cut it into 5-8μm thick slices, stick it on a slide and put it in a 45-degree constant temperature box to dry. The sections were stained with hematoxylin for 4 minutes, rinsed with tap water for 10 minutes, differentiated with hydrochloric acid and ethanol for 3 seconds, rinsed with tap water for 10 minutes to return to blue, and stained with eosin for 25 seconds. The stained sections were dehydrated with pure alcohol and then made transparent with xylene.

[0071] The results are as follows Figure 5 As shown, the prepared material can be quickly integrated with human tissue within 7 days, with a fast integration speed, and can significantly reduce phenomena such as inflammatory scars.

[0072] 2.6. Detection of cell migration ability using scratch assay

[0073] The results of the scratch migration experiment of L929 cells treated with the microsphere extract and the control group are shown in Figure 6 The figure shows the morphology of cells in the scratch test at different time points. It can be seen that the protein microspheres provided by the present invention can promote the migration of fibroblasts in a cell scratch test lasting 48 hours.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a highly bioactive recombinant human collagen microsphere filling material, comprising: Step 1, preparing recombinant human collagen; Step 2, synthesizing dopamine-modified chitosan; Step 3, expression and purification of American cockroach thymosin THY; Step 4, preparing chitosan composite recombinant human collagen microsphere filling material.

2. The preparation method according to claim 1, characterized in that: In step 1, the amino acid sequence of the recombinant human collagen is as shown in SEQ ID NO.1, and the sequence is obtained by splicing the active sequence in type III collagen; The pet28a-rCol plasmid carrying the target amino acid sequence was transformed into E. coli BL21 competent cells to prepare the BL21 / pet28a-rCol strain seed solution, which was inoculated in a fermenter, induced, and fermented to obtain the fermentation solution; the bacteria were collected, crushed by high-pressure homogenization, and centrifuged to obtain the bacterial supernatant; and the target protein was purified by nickel ion affinity chromatography.

3. The preparation method according to claim 1, characterized in that: Step 2 includes: S2.1, dissolving chitosan powder in acetic acid solution to prepare chitosan solution; S2.2, adding dopamine hydrochloride to the chitosan solution to obtain a chitosan dopamine mixed solution; S2.3, dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in 2-(N-morpholinyl)ethanesulfonic acid MES buffer solution, and then adding them to the prepared chitosan-dopamine mixed solution to react at room temperature; S2.4, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 8KD-14KD, dialyzed with deionized water, and freeze-dried.

4. The preparation method according to claim 1, characterized in that: In step 3, the amino acid sequence of American cockroach thymosin THY is shown in SEQ ID NO.

2.

5. The preparation method according to claim 1, characterized in that: In step 3, the American cockroach thymosin THY is recombinantly expressed using E. coli BL21 (DE3), and the obtained protein is purified by gravity column; the eluate is placed in a dialysis bag with a molecular weight cutoff of 8KD-14KD and dialyzed in deionized water.

6. The preparation method according to claim 1, characterized in that: In step 4, recombinant human collagen, dopamine-modified chitosan and American cockroach thymosin THY are dissolved in a pH 3.8 sodium acetate buffer to obtain a mixed solution; The above mixed solution was added dropwise into the EDC / NHS solution by electrospinning, and the chitosan composite collagen microsphere filling material was obtained by cross-linking; the EDC / NHS solution was prepared by dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in 2-(N-morpholino)ethanesulfonic acid MES buffer solution.

7. The preparation method according to claim 6, characterized in that: In step 4, in the mixed solution, the concentrations of recombinant human collagen, dopamine-modified chitosan and American cockroach thymosin THY dissolved in pH 3.8 sodium acetate buffer are 10-20 mg / mL, 2-5 mg / mL and 1-3 mg / mL respectively.

8. The preparation method according to claim 7, characterized in that: In step 4, during the cross-linking process, the electrospinning voltage is 10-18 kV, the volume ratio of the mixed solution to the EDC / NHS solution is 1:10, the inner diameter of the needle used in the electrospinning process is 0.5 mm, the receiving distance of the electrospinning is 20 cm, the pushing speed of the electrospinning is 0.8 mL / min, and the chemical cross-linking time is 12 hours; the cross-linked microspheres are washed and freeze-dried to obtain the product.

9. A recombinant human collagen microsphere filling material with high biological activity obtained according to the preparation method according to any one of claims 1 to 8.

10. Use of the highly bioactive recombinant human collagen microsphere filling material according to claim 9 in the field of medical aesthetic filling materials.

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