Collagen fibers for hydrodermabrasion needles and their preparation and application

Abstract

The present invention provides a collagen fiber for a hydrodermoneedle, wherein the diameter of the collagen fiber is between 15 and 80 nm; the turbidity of fibrosis OD 313 nm = 0.85 - 1.3; the Tm value = 45.12 °C - 50.66 °C. The present invention also provides a preparation method and an application of the above-mentioned collagen fiber. The collagen fiber provided by the present invention has good fluidity and a degradation period that is more matched with the degradation period of hyaluronic acid. By adding sodium chloride in the preparation method, the present invention significantly improves the characteristics of the prepared collagen fiber.

Description

Technical Field

[0001] The present invention relates to collagen fibers for hydrodermabrasion needles and their preparation and application. Background Art

[0002] With the increase of age, the content of collagen in the body gradually decreases, and the water retention ability decreases, resulting in problems such as loose and dull skin and increased wrinkles. Hydrodermabrasion needle is a non-surgical beauty therapy for skin care, used for water retention and improving skin elasticity. In the prior art, the injection substance of the hydrodermabrasion needle is mainly sodium hyaluronate. Although the hydrodermabrasion needle with hyaluronic acid as the main component can retain water after injection, fine lines are still obvious. There are also literatures disclosing that a hydrodermabrasion needle is made with collagen molecules as the main component. The collagen molecules need a lower concentration to maintain the fluidity required for the hydrodermabrasion needle. After increasing the concentration of collagen molecules, it is easy to form a gel and the fluidity decreases (CN 114053166 B). In order to provide nutrition to the skin and improve fine lines while retaining water and maintaining skin elasticity, the prior art adds collagen molecules to the hydrodermabrasion needle. However, due to the reason of fluidity, the addition amount of collagen molecules is limited, and the degradation period is short (generally completely degraded in 15 days). After adding, the skin elasticity and gloss recovery effects are average, and the repair effect on skin fine lines is poor (CN 114053166B, CN 115957149A). Summary of the Invention

[0003] In order to increase the ingestible nutritional components, further improve the effect of removing fine lines, and further expand the selection space of hydrodermabrasion needle injections, the present invention provides a new hydrodermabrasion needle injection.

[0004] As one aspect of the present invention, it relates to a collagen fiber for a hydrodermabrasion needle, the diameter of the collagen fiber is between 15 - 80 nm, and more concentrated between 15 - 30 nm. The turbidity of fibrosis OD 313nm = 0.85 - 1.3, preferably 0.95 - 1.3, and the Tm value = 45.12 °C - 50.66 °C, preferably 48.99 - 50.66 °C.

[0005] As another aspect of the present invention, it relates to a hydrodermabrasion needle injection, and the injection contains the above-mentioned collagen fiber. The hydrodermabrasion needle injection may further contain sodium hyaluronate, vitamin C, vitamin E and / or niacinamide.

[0006] As yet another aspect of the present invention, it relates to a method for preparing the above-mentioned collagen fiber, including:

[0007] Removing immunogenicity and virus, purifying collagen molecules, and fibrillating them in an acidic environment, wherein the pH value of the acidic environment is 5.0 - 6.5. Further, NaCl is added during the fibrillation process of collagen molecules.

[0008] As can be seen from the embodiments of the present invention, the collagen fibers provided by the present invention have good fluidity and a degradation period that better matches the degradation period of hyaluronic acid, and are suitable for use in hydrodermabrasion needles. By adding sodium chloride to the preparation method, the present invention significantly improves the characteristics of the prepared collagen fibers. Detailed Embodiments

[0009] The invention of this application was jointly developed by Beijing Aibairui Biotechnology Co., Ltd. and Beijing Bairen Medical Technology Co., Ltd.

[0010] Referring to the prior art, the inventors prepared collagen fibers under conditions close to the pH value of the human body. However, the pushing force was too large and it was not suitable for use in hydrodermabrasion needles. After further experiments, the inventors surprisingly found that the pushing force of the collagen fibers prepared under acidic conditions was significantly reduced. After further treatment, it could be used for hydrodermabrasion needle operations.

[0011] The collagen fibers referred to in this application refer to the products of the fibrosis of collagen molecules under acidic conditions, and the main component is collagen fibers with a diameter of 15-30 nm.

[0012] The method for testing the injectability of the prepared collagen fibers in the present invention is as follows:

[0013] Mixing is carried out in a syringe connected by a two-way joint. One syringe contains 1 mL of the collagen fibers prepared in the embodiment of the present invention, and the other syringe contains 1 mL of normal saline. Physical mixing is carried out by reciprocating pushing to obtain a fluid that can be used in hydrodermabrasion needles.

[0014] Example 1:

[0015] (1) Pretreatment of animal-derived tissues: This step can be carried out with reference to the prior art with the goal of removing immunogenicity and viruses. After scraping the surface fat from fresh animal tissues, soak them in an acetone solution at room temperature for 16 h. Wash the treated materials 3-5 times with deionized water to remove excess acetone, and then put the materials into a 2.0 M NaOH solution and stir at room temperature for 16 h. After the treatment, soak the materials in deionized water and wash the materials until neutral to achieve defatting and virus inactivation.

[0016] (2) Extraction: This step can be carried out with reference to the prior art with the goal of extracting a crude extract of collagen molecules. Put the washed materials into 0.5 M acetic acid for crushing and homogenization. The homogenized tissue is subjected to collagen molecule extraction at a ratio of 20:1 (tissue weight / pepsin weight), control the temperature at 18 °C, and stir and extract for 72 hours. Centrifuge the extract to collect the supernatant, stir evenly and then carry out clarification filtration, control the turbidity to be less than 30 to obtain a crude extract of collagen molecules.

[0017] (3) Purification: This step can be carried out with reference to the prior art, aiming to obtain purified collagen molecules without immunogenicity. The crude extract of collagen molecules is ultrafiltered using a tangential flow system. After concentrating to 3 mg / ml, dialysis is started with a 20 mM acetic acid solution. After dialysis with 20 times the volume, collagen molecules are obtained. Electrophoresis detection shows no heteroprotein bands, and mass spectrometry detection shows that the helical region is intact and without immunogenicity.

[0018] (4) Fibrosis of collagen molecules: Take a certain volume of purified collagen molecules, add 1 / 10 volume of phosphate buffer (0.2 M Na2HPO4: 0.2 M NaH2PO4 = 7:3, v:v), mix well, and adjust the pH to 7.0. Place at 30 °C for 6 hours to obtain collagen fibers.

[0019] (5) Filling: Take a certain volume of collagen fibers, centrifuge at 8000 rpm for 30 min. The obtained precipitate is washed with normal saline and homogenized, and then the concentration is adjusted to 35 mg / ml. Then, the collagen fibers with the adjusted concentration are aseptically filled into 1 mL syringes and sealed.

[0020] The collagen fibers obtained in this example are detected and analyzed for the morphology of collagen molecules using a scanning electron microscope, an enzyme-labeled instrument, and a differential scanning calorimeter: 90% of the collagen fiber diameters are between 80 - 200 nm, the turbidity of fibrosis OD 313 nm = 1.4, and the Tm value = 53.41 °C. Further, its physical properties are detected by a push-pull testing machine: a 30G needle, tested at a speed of 1 mL / min, and the thrust is 22 N. Using a hydro-injection instrument, the injectability of the collagen fibers after being mixed with normal saline at a ratio of 1:1 in a three-way injection tube and passing through a conventional hydro-injection needle is tested. The test results show poor injectability. The aseptically filled collagen fibers are injected subcutaneously into both sides of the back of New Zealand white rabbits through a 30G needle and are completely degraded after 20 weeks.

[0021] Example 2

[0022] Steps (1)-(3) are the same as in Example 1.

[0023] (4) Fibrosis of collagen molecules: Take a certain volume of purified collagen molecules, add 1 / 10 volume of phosphate solution (0.2 M Na2HPO4: 0.2 M NaH2PO4 = 7:3, v:v), mix well and adjust the pH to 6.5. Place at 30 °C for 6 hours to obtain collagen fibers.

[0024] (5) Filling: Take a certain volume of collagen fibers, centrifuge at 8000 rpm for 30 min, add normal saline to the obtained precipitate for washing and homogenization, then adjust the concentration to 35 mg / ml. Next, aseptically fill the collagen fibers with adjusted concentration into syringes of 1 mL specification and seal them.

[0025] The collagen fibers obtained in this example were detected and analyzed for the molecular morphology of collagen by scanning electron microscope, enzyme-labeled instrument and differential scanning calorimeter: 80% of the collagen fiber diameters were 80 - 100 nm, the turbidity of fibrosis OD 313nm = 1.1, and the Tm value = 53.22 °C. Further, its physical properties were detected by a push-pull tester: with a 30G needle head, tested at a speed of 1 mL / min, the thrust was 18 N. Using a hydro-injection instrument, the injectability of the collagen fibers after being mixed with normal saline at a ratio of 1:1 in a three-way injection tube and passing through a conventional hydro-injection needle was tested, and the test results showed poor injectability. The aseptically filled collagen fibers were injected subcutaneously into both sides of the back of New Zealand white rabbits through a 30G needle head, and were completely degraded after 20 weeks.

[0026] Example 3

[0027] Steps (1)-(3) are the same as those in Example 1.

[0028] (4) Fibrosis of collagen molecules: Take a certain volume of purified collagen molecules, add 1 / 10 volume of phosphate solution (0.2M Na2HPO4:0.2M NaH2PO4 = 7:3, v:v), mix well and adjust the pH to 6.0. Place at 30 °C for 6 hours.

[0029] (5) Filling: Take a certain volume of collagen fibers, centrifuge at 8000 rpm for 30 min, add normal saline to the obtained precipitate for washing and homogenization, then adjust the concentration to 35 mg / ml. Next, aseptically fill the collagen fibers with adjusted concentration into syringes of 1 mL specification and seal them.

[0030] The collagen fibers obtained in this example were detected for the molecular morphology of collagen by scanning electron microscope, enzyme-labeled instrument and differential scanning calorimeter: 70% of the collagen fiber diameters were 50 - 80 nm, the turbidity of fibrosis OD 313nm = 0.90, and the Tm value = 51.9 °C. Further, its physical properties were detected by a push-pull tester: with a 30G needle head, tested at a speed of 1 mL / min, the thrust was 16 N. Using a hydro-injection instrument, the injectability of the collagen fibers after being mixed with normal saline at a ratio of 1:1 in a three-way injection tube and passing through a conventional hydro-injection needle was tested, and the test results showed poor injectability. The aseptically filled collagen fibers were injected subcutaneously into both sides of the back of New Zealand white rabbits through a 30G needle head, and were completely degraded after 18 weeks.

[0031] Example 4

[0032] Steps (1)-(3) are the same as those in Example 1.

[0033] (4) Collagen molecule fibrosis: Take a certain volume of purified collagen molecules, add 1 / 10 volume of phosphate solution (0.2M Na2HPO4: 0.2M NaH2PO4 = 7:3, v:v), mix well and adjust the pH to 5.0. Place at 30°C for 6 hours.

[0034] (5) Filling: Take a certain volume of collagen fibers, centrifuge at 8000 rpm for 30 min, add normal saline to wash the obtained precipitate, homogenize and adjust the concentration to 35 mg / ml, and then aseptically fill the collagen fibers with adjusted concentration into 1 mL syringes and seal.

[0035] The collagen fibers obtained in this example were used to detect the morphology of collagen molecules by scanning electron microscope, enzyme-linked immunosorbent assay and differential scanning calorimeter: 70% of the collagen fiber diameters were 15-30 nm, the turbidity of fibrosis OD 313 nm = 0.85, and the Tm value = 46.56°C. Further, the physical properties were detected by a push-pull tester: a 30G needle was used for testing at a speed of 1 mL / min, and the thrust was 7 N. A hydrodermabrasion injector was used to test the injectability of the collagen fibers after being mixed with normal saline at a ratio of 1:1 in a three-way injection tube through a conventional hydrodermabrasion injection needle. The test results showed good injectability. The aseptically filled collagen fibers were injected subcutaneously on both sides of the back of New Zealand white rabbits through a 30G needle and were completely degraded after 13 weeks.

[0036] Example 5

[0037] Steps (1)-(3) are the same as those in Example 1.

[0038] (4) Collagen molecule fibrosis: Take a certain volume of purified collagen molecules, add 1 / 10 volume of phosphate solution (0.2M Na2HPO4: 0.2M NaH2PO4 = 7:3, v:v), and then add 0.15M NaCl, mix well and adjust the pH to 5.0. Place at 30°C for 6 hours.

[0039] (5) Filling: Take a certain volume of collagen fibers, centrifuge at 8000 rpm for 30 min, add normal saline to wash the obtained precipitate, homogenize and adjust the concentration to 35 mg / ml, and then aseptically fill the collagen fibers with adjusted concentration into 1 mL syringes and seal.

[0040] The collagen fibers obtained in this example were detected for the molecular morphology of collagen using a scanning electron microscope, an enzyme-linked immunosorbent assay (ELISA) reader, and a differential scanning calorimeter: 90% of the collagen fiber diameters were 15 - 30 nm, the turbidity of fibrosis OD 313 nm = 1.3, and the Tm value = 50.66 °C. Further, its physical properties were detected using a push-pull tester: with a 30G needle, tested at a speed of 1 mL / min, the thrust force was 11 N. Using a hydro-injection device, the injectability of the collagen fibers after being mixed with normal saline at a ratio of 1:1 in a three-way injection tube and passing through a conventional hydro-injection needle was tested, and the test results showed good injectability. The sterile-filled collagen fibers were injected subcutaneously on both sides of the back of New Zealand white rabbits through a 30G needle and were completely degraded after 14 weeks.

[0041] Example 6

[0042] Steps (1)-(3) are the same as in Example 1.

[0043] (4) Fibrosis of collagen molecules: Take a certain volume of purified collagen molecules, add 1 / 10 volume of phosphate solution (0.2M Na2HPO4:0.2M NaH2PO4 = 7:3, v:v), then add 0.15M NaCl, mix well and adjust the pH to 5.0. Place at 35 °C for 4 hours.

[0044] (5) Filling: Take a certain volume of collagen fibers, centrifuge at 8000 rpm for 30 min, add the precipitate to normal saline for washing and homogenization, then adjust the concentration to 35 mg / ml, and then aseptically fill the collagen fibers with adjusted concentration into a 1 mL syringe and seal it.

[0045] The collagen fibers obtained in this example were detected for the molecular morphology of collagen using a scanning electron microscope, an ELISA reader, and a differential scanning calorimeter: 80% of the collagen fiber diameters were 15 - 30 nm, the turbidity of fibrosis OD 313 nm = 1.15, and the Tm value = 48.99 °C. Further, its physical properties were detected using a push-pull tester: with a 30G needle, tested at a speed of 1 mL / min, the thrust force was 10 N. Using a hydro-injection device, the injectability of the collagen fibers after being mixed with normal saline at a ratio of 1:1 in a three-way injection tube and passing through a conventional hydro-injection needle was tested, and the test results showed good injectability. The sterile-filled collagen fibers were injected subcutaneously on both sides of the back of New Zealand white rabbits through a 30G needle and were completely degraded after 15 weeks.

[0046] Example 7

[0047] Steps (1)-(3) are the same as in Example 1.

[0048] (4) Collagen molecule fibrosis: Take a certain volume of purified collagen molecules, add 1 / 10 volume of phosphate solution (0.2M Na2HPO4: 0.2M NaH2PO4 = 7:3, v:v), then add 0.15M NaCl, mix well and adjust the pH to 5.0. Leave it at 25 °C for 10 hours.

[0049] (5) Filling: Take a certain volume of collagen fibers, centrifuge at 8000 rpm for 30 min, add normal saline to wash the obtained precipitate, homogenize and adjust the concentration to 35 mg / ml. Then aseptically fill the collagen fibers with adjusted concentration into 1 mL syringes and seal.

[0050] The collagen fibers obtained in this example were used to detect the morphology of collagen molecules by scanning electron microscope, enzyme-labeled instrument and differential scanning calorimeter: 80% of the collagen fibers had a diameter of 15 - 30 nm, the turbidity of fibrosis OD 313 nm = 0.95, and the Tm value = 45.12 °C. Further, its physical properties were detected by a push-pull testing machine: with a 30G needle, tested at a speed of 1 mL / min, the thrust was 7.5 N. Using a hydro-injection instrument, the injectability of the collagen fibers after being mixed with normal saline at a ratio of 1:1 in a three-way injection tube through a conventional hydro-injection needle was tested, and the test results showed good injectability. The aseptically filled collagen fibers were injected subcutaneously on both sides of the back of New Zealand white rabbits through a 30G needle and were completely degraded after 13 weeks.

[0051] In the above examples, steps (1)-(3) are the same, mainly for degreasing and virus inactivation treatment of the materials and extracting and purifying collagen molecules. In Examples 1, 2, 3 and 4, fibrosis treatment was carried out using Na2HPO4 and NaH2PO4 solutions after steps (1)-(3). Examples 5, 6, 7 used Na2HPO4 and NaH2PO4 solutions and NaCl solution for fibrosis treatment.

[0052] The collagen fibers obtained in all examples were subjected to morphological measurement, turbidity measurement, melting point measurement, extrusion force test and degradation degree test. Among the collagen fibers obtained in Example 1, the fiber diameter was relatively large, the extrusion force was 20 N, and the fluidity of the collagen fibers was poor; the Tm value indicated strong intermolecular forces of the collagen molecules and a long in vivo degradation time. The collagen fiber diameters obtained in Examples 3-7 were significantly reduced, concentrated between 15 and 80 nm, and more concentrated between 15 and 30 nm. The collagen fiber diameters obtained in Examples 2-4 were significantly reduced and the melting point decreased, indicating that the formation of collagen fibers was strongly inhibited at low pH, the strength of the collagen fibers was relatively weak, the fluidity was better, but as the pH value decreased, the fiber turbidity (OD313nm) decreased and the number of collagen fibers was small. In Examples 5-7, NaCl was added to the fibrosis reaction solution: Na2HPO4 and NaH2PO4 solution, and better fibroblast formation effects (higher OD313nm) were obtained by adjusting the salt ions. Especially in Example 5, the collagen fibers obtained at pH 5.0 and 30 °C had the best effects. While maintaining the fiber diameter between 15 and 30 nm, the turbidity of the collagen fibers increased (OD 313nm = 1.3), the extrusion force was low (11 N) and the injectability was good. The melting point Tm = 50.66 °C, and the collagen fibers could be maintained in the body for 14 weeks, which was consistent with the therapeutic effect time of hyaluronic acid for 3 months. When used in combination with hyaluronic acid, as the collagen fibers slowly degraded, the effect of continuously providing nutritional components could be achieved.

[0053] The collagen fibers prepared by the present invention have diameters concentrated between 15 and 80 nm, and more concentrated between 15 and 30 nm, ensuring excellent fluid properties; the fibrosis turbidity OD 313nm = 0.85-1.3, preferably 0.95-1.3, reflecting a high yield of collagen fibers; the melting point Tm value = 45.12 °C-50.66 °C, preferably 48.99-50.66 °C, which can correspond to a longer degradation period. The comprehensive display of these properties enables the collagen fibers prepared by the present invention to not only be used alone as a hydroderm injection, but also be used in combination with hyaluronic acid, greatly improving the beauty effect of the hydroderm.

Claims

1. A collagen fiber for a hydrodermabrasion needle, characterized in that, The diameter of the collagen fiber is between 15 and 80 nm; the turbidity of fibrosis OD 313 nm = 0.85 - 1.3; the Tm value = 45.12 °C - 50.66 °C; the collagen fiber is obtained by fibrillating collagen molecules with immunogenicity removed and virus removed, and the fibrillation conditions are: 1 / 10 volume of phosphate solution, the concentration of NaCl is 0.15 M, and the acidic environment is pH 5.0; the phosphate solution is prepared by mixing 0.2 M Na2HPO4 and 0.2 M NaH2PO4 in a volume ratio of 7:

3.

2. The collagen fiber according to claim 1, characterized in that, The diameter of the collagen fiber is 15 - 30 nm; the turbidity of fibrosis OD 313 nm = 0.95 - 1.3; the Tm value = 48.99 - 50.66 °C.

3. The collagen fiber according to claim 2, wherein, 90% of the diameter of the collagen fiber is 15 - 30 nm, the turbidity of fibrosis OD 313 nm = 1.3, and the Tm value = 50.66 °C.

4. A water light needle injection, characterized in that: The injection contains the collagen fiber according to any one of claims 1 - 3.

5. The injection according to claim 4, wherein The injection also contains sodium hyaluronate.

6. The injection according to claim 5, characterized in that, The injection also contains vitamin C, vitamin E and / or niacinamide.