Hydrophobic needle injection as well as preparation and application thereof

Abstract

The invention provides a water-light needle injection which comprises collagen fibers, and the diameter of the collagen fibers ranges from 15 nm to 80 nm. The fibrosis turbidity OD (optical density) 313nm is equal to 0.85 to 1.3; and the Tm value is equal to 45.12 DEG C to 50.66 DEG C. The invention also provides a preparation method and application of the hydro-optical needle injection. In the hydro-optical needle injection provided by the invention, the collagen fiber has relatively good fluidity and a degradation period which is more matched with a degradation period of hyaluronic acid. The sodium chloride is added in the preparation method, so that the characteristics of the prepared collagen fiber are obviously improved.

Description

[0001] This application is a divisional application of the Chinese invention patent application with an application date of February 5, 2024, an invention name of collagen fibers for hyaluronic acid injections, their preparation and application, and application number of 202410165029.9. This application retains the application date of invention patent application No. 202410165029.9. Technical Field

[0002] The present invention relates to a water light needle injection and its preparation and application. Background Art

[0003] As we age, the collagen content in the body gradually decreases, and the ability to retain water decreases, which can lead to problems such as sagging, dull skin, and increased wrinkles. Hydrating injection is a non-surgical cosmetic treatment for skin care that is used to retain water and improve skin elasticity. In the prior art, the injection substance of hyaluronic acid injection is mainly sodium hyaluronate. Although hyaluronic acid-based hyaluronic acid injection can retain water after injection, fine lines are still obvious. There are also documents that disclose that hyaluronic acid injection is made with collagen molecules as the main component. Collagen molecules need a lower concentration to maintain the fluidity required by hyaluronic acid injection. After increasing the concentration of collagen molecules, gel is easily formed, and the fluidity is reduced (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 hyaluronic acid injection. However, due to fluidity, the amount of collagen molecules added is limited, and the degradation cycle is short (generally 15 days for complete degradation). After addition, the effect of restoring skin elasticity and gloss is average, and the effect of repairing fine lines on the skin is poor (CN 114053166B, CN 115957149A). Summary of the Invention

[0004] To increase the amount of nutrients available, further improve the effectiveness of fine line removal, and further expand the selection of hyaluronic acid injections, the present invention provides a new hyaluronic acid injection. As one aspect of the present invention, the invention relates to a hyaluronic acid injection comprising collagen fibers with a diameter between 15 and 80 nm, a fibrosis turbidity OD 313 nm of 0.85-1.3, and a Tm value of 45.12°C-50.66°C.

[0005] Preferably, the collagen fiber diameter is 15-30 nm; the fibrosis turbidity OD 313 nm = 0.95-1.3; and the Tm value = 48.99-50.66°C.

[0006] As a further preference, 90% of the collagen fibers have a diameter of 15-30 nm, a fibrosis turbidity OD 313 nm = 1.3, and a Tm value = 50.66°C.

[0007] In a specific embodiment, the hyaluronic acid injection has collagen fibers as the main functional component.

[0008] In a specific embodiment, the injection contains the above-mentioned collagen fibers. The hyaluronic acid injection may also contain sodium hyaluronate, vitamin C, vitamin E and / or niacinamide.

[0009] As another aspect of the present invention, it relates to a method for preparing the above-mentioned water light needle injection, comprising preparing the collagen fiber by the following method:

[0010] The purified collagen molecules that have been free of immunogenicity and viruses are fibroticized in an acidic environment, wherein the pH value of the acidic environment is 5.0-6.5. Furthermore, NaCl is added during the fibroticization of the collagen molecules.

[0011] As can be seen from the examples of the present invention, the collagen fibers provided by the present invention have good fluidity and a degradation cycle that is more compatible with the degradation cycle of hyaluronic acid, making them suitable for use in hyaluronic acid injections. The present invention significantly improves the properties of the prepared collagen fibers by adding sodium chloride during the preparation process. DETAILED DESCRIPTION

[0012] The invention of this application was jointly developed by Beijing Aibairui Biotechnology Co., Ltd. and Beijing Bairen Medical Technology Co., Ltd.

[0013] The inventors, referring to existing technologies, prepared collagen fibers at a pH close to that of the human body, but the pushing force was too high, making them unsuitable for hyaluronic acid injections. Further experiments surprisingly revealed that collagen fibers prepared under slightly acidic conditions exhibited significantly lower pushing force, and after further processing, could be used for hyaluronic acid injections.

[0014] The collagen fibers referred to in this application refer to: the products of collagen molecules fibrosing under acidic conditions, wherein collagen fibers with a diameter of 15-30 nm are the main components.

[0015] The method for testing the injectability of the prepared collagen fibers is as follows:

[0016] Mixing was performed in syringes connected by two-way connectors, one of which was filled with 1 mL of collagen fibers prepared according to an embodiment of the present invention, and the other was filled with 1 mL of normal saline, and the mixture was physically mixed by pushing back and forth to obtain a fluid that could be used for hyaluronic acid injection.

[0017] Example 1:

[0018] (1) Animal-derived tissue pretreatment: This step can be performed with reference to existing technologies, with the goal of removing immunogenicity and viruses. Fresh animal tissue is scraped to remove surface fat, then soaked in an acetone solution at room temperature for 16 hours. The treated material is then rinsed 3-5 times with deionized water to remove excess acetone. The material is then placed in a 2.0 M NaOH solution and stirred at room temperature for 16 hours. After treatment, the material is rinsed with deionized water until neutral to achieve fat removal and viral inactivation.

[0019] (2) Extraction: This step can be carried out with reference to existing technologies, with the goal of extracting a crude collagen molecule extract. The cleaned material is placed in 0.5M 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), the temperature is controlled at 18°C, and the extraction is stirred for 72 hours. The extract is centrifuged to collect the supernatant, and after stirring evenly, it is clarified and filtered to control the turbidity to less than 30 to obtain a crude collagen molecule extract.

[0020] (3) Purification: This step can be performed with reference to existing technologies, with the goal of obtaining purified collagen molecules that are non-immunogenic. The crude collagen extract is ultrafiltered using a tangential flow system and concentrated to 3 mg / ml. Dialysis is then initiated against a 20 mM acetic acid solution. After 20 times the volume of the dialysis solution, the collagen molecules are obtained. Electrophoresis detection shows no impurity protein bands, and mass spectrometry detection shows that the helical region is intact and non-immunogenic.

[0021] (4) Collagen molecule fibrosis: Take a certain volume of purified collagen molecules, add 1 / 10 volume of phosphate buffer (0.2M Na2HPO4:0.2M NaH2PO4=7:3, v:v), mix well, and adjust the pH to 7.0. Place at 30℃ for 6 hours to obtain collagen fibers.

[0022] (5) Filling: Take a certain volume of collagen fiber and centrifuge it at 8000 rpm for 30 min. Add physiological saline to the obtained precipitate for washing and homogenization, and adjust the concentration to 35 mg / ml. Then, aseptically fill the collagen fiber with the adjusted concentration into a 1 mL syringe and seal it.

[0023] The collagen fibers obtained in this example were detected and analyzed using a scanning electron microscope, an enzyme marker and a differential scanning calorimeter to detect the molecular morphology of collagen: 90% of the collagen fibers had a diameter between 80-200nm, the fibrosis turbidity OD 313nm = 1.4, and the Tm value = 53.41°C. The physical properties were further tested using a push-pull tester: a 30G needle, tested at a speed of 1mL / min, with a thrust of 22N. A water-light injection instrument was used to test the injectability of the collagen fibers after they were mixed with physiological saline in a 1:1 ratio in a three-way injection tube and then passed through a conventional water-light injection needle. The test results showed that the injectability was poor. The collagen fibers obtained by sterile filling were injected into the subcutaneous tissue on both sides of the back of New Zealand white rabbits through a 30G needle and were completely degraded after 20 weeks.

[0024] Example 2

[0025] Steps (1)-(3) are the same as in Example 1.

[0026] (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 6.5. Place at 30℃ for 6 hours to obtain collagen fibers.

[0027] (5) Filling: Take a certain volume of collagen fiber and centrifuge it at 8000 rpm for 30 min. Add physiological saline to the obtained precipitate for washing and homogenization, and adjust the concentration to 35 mg / ml. Then, aseptically fill the collagen fiber with the adjusted concentration into a 1 mL syringe and seal it.

[0028] The collagen fibers obtained in this example were detected and analyzed using a scanning electron microscope, an enzyme marker and a differential scanning calorimeter to detect the molecular morphology of collagen: 80% of the collagen fibers had a diameter of 80-100nm, the fibrosis turbidity OD 313nm = 1.1, and the Tm value = 53.22°C. The physical properties were further tested using a push-pull tester: a 30G needle, tested at a speed of 1mL / min, with a thrust of 18N. A water-light injection instrument was used to test the injectability of the collagen fibers after they were mixed with physiological saline in a 1:1 ratio in a three-way injection tube and then passed through a conventional water-light injection needle. The test results showed that the injectability was poor. The collagen fibers obtained by sterile filling were injected into the subcutaneous tissue on both sides of the back of New Zealand white rabbits through a 30G needle and were completely degraded after 20 weeks.

[0029] Example 3

[0030] Steps (1)-(3) are the same as in Example 1.

[0031] (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 6.0. Place at 30℃ for 6 hours.

[0032] (5) Filling: Take a certain volume of collagen fiber and centrifuge it at 8000 rpm for 30 min. Add physiological saline to the obtained precipitate for washing and homogenization, and adjust the concentration to 35 mg / ml. Then, aseptically fill the collagen fiber with the adjusted concentration into a 1 mL syringe and seal it.

[0033] The collagen fibers obtained in this example were tested for their molecular morphology using a scanning electron microscope, an enzyme marker, and a differential scanning calorimeter: 70% of the collagen fibers had a diameter of 50-80 nm, a fibrosis turbidity OD 313 nm = 0.90, and a Tm value = 51.9°C. The physical properties were further tested using a push-pull tester: a 30G needle was used for testing at a speed of 1 mL / min, with a thrust of 16 N. A water-light injection instrument was used to test the injectability of the collagen fibers after they were mixed with physiological saline in a 1:1 ratio in a three-way injection tube and then passed through a conventional water-light injection needle. The test results showed that the injectability was poor. The collagen fibers obtained by sterile filling were injected into the subcutaneous tissue on both sides of the back of New Zealand white rabbits through a 30G needle and were completely degraded after 18 weeks.

[0034] Example 4

[0035] Steps (1)-(3) are the same as in Example 1.

[0036] (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℃ for 6 hours.

[0037] (5) Filling: Take a certain volume of collagen fiber and centrifuge it at 8000 rpm for 30 min. Add physiological saline to the obtained precipitate for washing and homogenization, and adjust the concentration to 35 mg / ml. Then, aseptically fill the collagen fiber with the adjusted concentration into a 1 mL syringe and seal it.

[0038] The collagen fibers obtained in this example were tested for their molecular morphology using a scanning electron microscope, an enzyme marker, and a differential scanning calorimeter: 70% of the collagen fibers had a diameter of 15-30 nm, the fibrosis turbidity OD 313 nm = 0.85, and the Tm value = 46.56°C. The physical properties were further tested using a push-pull tester: a 30G needle, tested at a speed of 1 mL / min, with a thrust of 7 N. A water-light injection instrument was used to test the injectability of the collagen fibers after they were mixed with physiological saline in a 1:1 ratio in a three-way injection tube and then passed through a conventional water-light injection needle. The test results showed that the injectability was good. The collagen fibers obtained by sterile filling were injected into the subcutaneous tissue on both sides of the back of New Zealand white rabbits through a 30G needle and were completely degraded after 13 weeks.

[0039] Example 5

[0040] Steps (1)-(3) are the same as in Example 1.

[0041] (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. Place at 30℃ for 6 hours.

[0042] (5) Filling: Take a certain volume of collagen fiber and centrifuge it at 8000 rpm for 30 min. Add physiological saline to the obtained precipitate for washing and homogenization, and adjust the concentration to 35 mg / ml. Then, aseptically fill the collagen fiber with the adjusted concentration into a 1 mL syringe and seal it.

[0043] The collagen fibers obtained in this example were tested for their molecular morphology using a scanning electron microscope, an enzyme marker, and a differential scanning calorimeter: 90% of the collagen fibers had a diameter of 15-30 nm, the fibrosis turbidity OD 313 nm = 1.3, and the Tm value = 50.66°C. The physical properties were further tested using a push-pull tester: a 30G needle, tested at a speed of 1 mL / min, with a thrust of 11 N. A water-light injection instrument was used to test the injectability of the collagen fibers after they were mixed with physiological saline in a 1:1 ratio in a three-way injection tube and then passed through a conventional water-light injection needle. The test results showed that the injectability was good. The collagen fibers obtained by sterile filling were injected into the subcutaneous tissue on both sides of the back of New Zealand white rabbits through a 30G needle and were completely degraded after 14 weeks.

[0044] Example 6

[0045] Steps (1)-(3) are the same as in Example 1.

[0046] (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. Place at 35℃ for 4 hours.

[0047] (5) Filling: Take a certain volume of collagen fiber and centrifuge it at 8000 rpm for 30 min. Add physiological saline to the precipitate for washing and homogenization, and adjust the concentration to 35 mg / ml. Then, aseptically fill the collagen fiber with the adjusted concentration into a 1 mL syringe and seal it.

[0048] The collagen fibers obtained in this example were tested for their molecular morphology using a scanning electron microscope, an enzyme marker, and a differential scanning calorimeter: 80% of the collagen fibers had a diameter of 15-30 nm, a fibrosis turbidity OD 313 nm = 1.15, and a Tm value = 48.99°C. The physical properties were further tested using a push-pull tester: a 30G needle was used, tested at a speed of 1 mL / min, and the thrust was 10 N. A water-light injection instrument was used to test the injectability of the collagen fibers after they were mixed with physiological saline in a 1:1 ratio in a three-way injection tube and then passed through a conventional water-light injection needle. The test results showed that the injectability was good. The collagen fibers obtained by sterile filling were injected into the subcutaneous tissue on both sides of the back of New Zealand white rabbits through a 30G needle and were completely degraded after 15 weeks.

[0049] Example 7

[0050] Steps (1)-(3) are the same as in Example 1.

[0051] (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. Place at 25℃ for 10 hours.

[0052] (5) Filling: Take a certain volume of collagen fiber and centrifuge it at 8000 rpm for 30 min. Add physiological saline to the obtained precipitate for washing and homogenization, and adjust the concentration to 35 mg / ml. Then, aseptically fill the collagen fiber with the adjusted concentration into a 1 mL syringe and seal it.

[0053] The collagen fibers obtained in this example were tested for their molecular morphology using a scanning electron microscope, an enzyme marker, and a differential scanning calorimeter: 80% of the collagen fibers had a diameter of 15-30 nm, a fibrosis turbidity OD 313 nm = 0.95, and a Tm value = 45.12°C. The physical properties were further tested using a push-pull tester: a 30G needle was used for testing at a speed of 1 mL / min, with a thrust of 7.5 N. A water-light injection instrument was used to test the injectability of the collagen fibers after they were mixed with physiological saline in a 1:1 ratio in a three-way injection tube and then passed through a conventional water-light injection needle. The test results showed that the injectability was good. The collagen fibers obtained by sterile filling were injected into the subcutaneous tissue on both sides of the back of New Zealand white rabbits through a 30G needle, and were completely degraded after 13 weeks.

[0054] In the above examples, steps (1) to (3) are the same, mainly involving degreasing and sterilizing the material and extracting and purifying the collagen molecules. Examples 1, 2, 3, and 4 all used Na2HPO4 and NaH2PO4 solutions for fibrosis treatment after steps (1) to (3). Examples 5, 6, and 7 used Na2HPO4 and NaH2PO4 solutions and NaCl solutions for fibrosis treatment.

[0055] The collagen fibers obtained in all examples were subjected to morphological measurements, turbidity measurements, melting point measurements, pushing force tests and degradation tests. Among the collagen fibers obtained in Example 1, the fiber diameter was relatively large, the pushing force was 20N, and the collagen fibers had poor fluidity; the Tm value indicated that the intermolecular forces of collagen were strong and the degradation time in vivo was long. The diameter of the collagen fibers obtained in Examples 3-7 was significantly reduced, concentrated between 15-80nm, and more concentrated between 15-30nm. The diameter of the collagen fibers obtained in Examples 2-4 was significantly reduced, and the melting point was lowered, indicating that at low pH, the formation of collagen fibers was strongly inhibited, the strength of the collagen fibers was relatively weak, and the fluidity was good, but as the pH value decreased, the fiber turbidity (OD313nm) decreased, and the number of collagen fibers was small. Examples 5-7 added NaCl to the fibrosis reaction solutions: Na2HPO4 and NaH2PO4 solutions. By adjusting the salt ions, better fiber-forming effects (higher OD313nm) were achieved. In particular, Example 5 achieved the best collagen fiber effects at pH 5.0 and 30°C. While maintaining a fiber diameter of 15-30nm, the collagen fiber turbidity increased (OD313nm=1.3), the pushing force was low (11N), and the injectability was good. The melting point Tm=50.66°C, the collagen fibers can be maintained in the body for 14 weeks, consistent with the 3-month efficacy of hyaluronic acid. When used in combination with hyaluronic acid, the collagen fibers can be continuously provided with nutrients as they slowly degrade.

[0056] The collagen fibers prepared by the present invention have diameters ranging from 15 to 80 nm, with a higher concentration of 15 to 30 nm, ensuring excellent fluid properties. Their fibrosis turbidity (OD) at 313 nm is 0.85 to 1.3, preferably 0.95 to 1.3, reflecting a high collagen fiber yield. Their melting point (Tm) is 45.12°C to 50.66°C, preferably 48.99 to 50.66°C, corresponding to a longer degradation cycle. These combined properties make the collagen fibers prepared by the present invention suitable not only for use as a standalone injection for hyaluronic acid injections, but also for use in combination with hyaluronic acid, significantly enhancing the cosmetic effects of hyaluronic acid injections.

Claims

1. A water light needle injection, characterized in that: The injection contains collagen fibers, the diameter of the collagen fibers is between 15-80 nm, the fibrosis turbidity OD 313 nm is 0.85-1.3, and the Tm value is 45.12° C.-50.66° C.

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

3. The water light needle injection according to claim 2, characterized in that: 90% of the collagen fibers have a diameter of 15-30 nm, a fibrosis turbidity OD 313 nm = 1.3, and a Tm value = 50.66°C.

4. The water light needle injection according to claim 3, characterized in that: The hyaluronic acid injection has collagen fiber as the main functional component.

5. The water light needle injection according to claim 4, characterized in that: The injection further contains sodium hyaluronate.

6. The water light needle injection according to claim 5, characterized in that: The injection further contains vitamin C, vitamin E and / or nicotinamide.

7. A method for preparing the water light needle injection according to any one of claims 1 to 6, characterized in that: The method comprises the following steps of preparing the collagen fiber: removing immunogenicity and virus from the purified collagen molecules and subjecting them to fibrosis under an acidic environment.

8. The method according to claim 7, characterized in that The pH value of the acidic environment is 5.0-6.

5.

9. The method according to claim 7, characterized in that NaCl is added into the acidic environment.

10. The method according to claim 7, characterized in that The acidic environment is a phosphate buffer solution, and the collagen molecules are animal-derived collagen molecules.