CN115252898A

CN115252898A - Long-acting particle type I and type V collagen composite implant

Info

Publication number: CN115252898A
Application number: CN202211001402.4A
Authority: CN
Inventors: 韩淑萍; 何伟; 李贤明; 王雪; 陈文洁
Original assignee: Jiangsu Xihong Biomedical Co ltd
Current assignee: Jiangsu Xihong Biomedical Co ltd
Priority date: 2022-08-19
Filing date: 2022-08-19
Publication date: 2022-11-01
Anticipated expiration: 2042-08-19
Also published as: CN115252898B

Abstract

The invention discloses a particle type I and type V collagen composite implant with improved performance and a preparation method thereof. The method comprises the following steps: (1) Preparing a solution, namely an oil phase, which comprises a surfactant and a volatile organic solvent which is not completely dissolved in water and has the temperature of not higher than 4 ℃; (2) Preparing an aqueous solution (namely an aqueous phase) with the temperature not higher than 4 ℃, wherein the aqueous solution comprises type I and type V collagen, laccase and a protein activity protective agent; (3) Mixing said aqueous solution with said organic solvent solution within 30 minutes after said aqueous solution is prepared, and allowing them to form a water-in-oil emulsion, and maintaining the temperature at 4 ℃ to-4 ℃ for 24 hours or more, preferably within 48 hours, to substantially crosslink said collagen; (4) Filtering out particles with the particle size of more than 25 microns and less than 0.5 microns in the emulsion after the collagen is basically crosslinked, and keeping the particles with the particle size of between 0.5 microns and 25 microns; (5) And removing the solvent from the retained particles by low-temperature freezing. The implant has the advantages of long acting, small effect period fluctuation, good needle penetration performance and the like.

Description

Long-acting particle type I and type V collagen composite implant

Technical Field

The invention relates to an injection implant. In particular to a long-acting particle type I and type V collagen composite implant with excellent performance.

Technical Field

Type I collagen is the most abundant collagen in the human body. It forms large eosinophilic fibers, called collagen fibers. It is present in scar tissue, tissue healing in the final product through repair, and in the tendon, ligament, organic part of myofibril of the intima, in the dermis, dentin and organ capsules.

The extracellular peripheral collagen is usually collagen type V, and is abundantly present in connective tissue. Type v collagen is distributed around cells and type i collagen, and may act as a bridge between the basement membrane and connective tissue. After a wound or burn in the skin, some type v collagen is also formed and can be used to promote tissue repair.

The combination of type I and type V collagen can supplement and lack collagen in situ, the type I supports the depressed part, and the type V promotes tissue repair; after being filled, the collagen can be used as a cell growth attachment and a bracket, and can induce the generation of collagen tissues the same as those of a host, and the collagen regeneration and repair are stimulated.

However, the type I and type V collagen have serious deficiencies such as short in vivo residence time.

Although, some long-acting collagen technologies exist, such as:

CN 101648989 (long-acting collagen and its manufacturing method): the invention discloses a long-acting collagen and a preparation method thereof, which comprises the steps of scraping redundant tissues, removing grease, swelling, digesting, centrifugally separating, salting out, collecting lower-layer precipitates, freeze-drying to obtain collagen, mixing the collagen with gamma-polyglutamic acid (gamma-PGA), simultaneously adding a glutaraldehyde solution, uniformly stirring, carrying out first crosslinking, repeatedly adding the glutaraldehyde solution, uniformly stirring, and carrying out second crosslinking to obtain the long-acting collagen, wherein the defects that the conventional collagen has too short retention time, needs to be supplemented frequently, and high-concentration glutaraldehyde is remained, so that the biological toxicity can harm the health of a human body and the like can be overcome;

CN102924731A (a triple cross-linked collagen and its production and use): a method for producing triple cross-linked collagen, comprising: providing a soluble collagen sample; mixing the collagen sample with a first cross-linking agent to form a re-cross-linked collagen; mixing the double cross-linked collagen with a second cross-linking agent to form double cross-linked collagen; and mixing the double cross-linked collagen with a third cross-linking agent to form triple cross-linked collagen. Wherein the first cross-linking agent, the second cross-linking agent, and the third cross-linking agent are each selected from the group consisting of: aldehyde cross-linking agent, imine cross-linking agent and epoxide cross-linking agent, wherein the first cross-linking agent is different from the second cross-linking agent, and the third cross-linking agent is different from the first cross-linking agent and the second cross-linking agent;

however, although the above-mentioned techniques employ a chemical modification method, although the effect is remarkable, there is a risk of chemical reagent remaining and harmful substances being formed during the modification, and there are also disadvantages that the reaction conditions are not mild, by-products are generated, the specificity is poor, the catalytic efficiency and yield are low, and collagen is lost during the process.

More importantly, the collagen is crosslinked throughout the homogeneous phase, or is crosslinked into a large particle, not a very small particle, not a substantially uniform particle, but a smaller particle when used, and thus, the apparent therapeutic effect is not uniform, such as a large fluctuation in the effective period.

In addition, the preparation is basically gel, and the high viscosity of the water dispersion system causes inconvenience in production and use, such as inaccurate dosage split charging and poor needle penetration during injection.

Therefore, there is a need for a type I and type v collagen composite implant with the advantages of long-lasting effect, small fluctuation of effective period, good needle penetration, etc.

Disclosure of Invention

The invention aims to provide a particle type I and type V collagen composite implant with improved performance, long acting, small effect period fluctuation and good needle penetration and a preparation method thereof.

The inventor finds that the laccase modifies the combination of common type I collagen and type V collagen into particle cross-linked collagen with basically uniform size, so that the acting time of the laccase in vivo can be prolonged, the effective period fluctuation is small, the viscosity of a water dispersion system is reduced, and various problems caused in production and use, such as accurate subpackage dosage and better needle penetration during injection, are solved; in addition, the method can reduce the mixing of harmful substances, lead the reaction condition to be mild, reduce the loss of collagen in the process, improve the specificity, avoid generating byproducts, improve the catalytic efficiency, improve the yield and the like.

Laccase (laccase, EC 1.10.3.2) is a copper-containing polyphenol oxidase enzyme that catalyzes the oxidation of phenolic and aromatic compounds to the corresponding benzoquinones, with concomitant electron transfer to reduce molecular oxygen to water. Laccases have a broad substrate specificity, including monophenols, diphenols, polyphenols, thiophenols, aminophenols, anilines, polyamines, and lignin aromatic diamines [18]. Tyrosinase-catalyzed cross-linking is based on the formation of quinones, whereas laccase-catalyzed cross-linking is based on free radicals and their further reactions.

Based on this, the present invention has been completed.

The invention relates to a (performance-improved) long-acting microparticle type I and type V collagen composite implant with small effect period fluctuation and good needle penetration, which comprises: crosslinked type I and type V collagen composite particles having a particle size of 0.5 to 25 μm (using laccase).

The invention relates to a preparation method of a (performance-improved) long-acting microparticle type I and type V collagen composite implant with small effect period fluctuation and good needle penetration, which comprises the following steps:

(1) Preparing a solution, namely an oil phase, which comprises a surfactant and a volatile organic solvent which is not completely dissolved in water and has the temperature of not higher than 4 ℃;

(2) Preparing an aqueous solution (namely a water phase) which is at a temperature of not higher than 4 ℃ and comprises type I and type V collagen, laccase and a protein activity protective agent;

(3) Mixing said aqueous solution with said organic solvent solution within 30 minutes after said aqueous solution is prepared, and allowing them to form a water-in-oil emulsion, and maintaining the temperature at 4 ℃ to-4 ℃ for 24 hours or more, preferably within 48 hours, to substantially crosslink said collagen;

(4) Filtering out particles with the particle size of more than 25 microns and less than 0.5 microns in the emulsion after the collagen is basically crosslinked, and keeping the particles with the particle size of between 0.5 microns and 25 microns;

(5) And removing the solvent from the retained particles by a low-temperature freezing method.

The meaning of "composite" above is equivalent to "combination", both meaning being equivalent.

Detailed Description

Such protein activity protectors include, but are not limited to, sorbitol, mannitol, erythritol, maltitol, lactitol, xylitol, and combinations thereof. The protein activator is used for protecting the activity of type I and type V collagen and laccase.

Preferably, the total mass concentration of the above aqueous solution, i.e., the aqueous phase, of type I and type v collagen is 0.1% to 10%, more preferably 0.5% to 5%; preferably, the ratio of the amount of laccase to the total amount of type I and type V collagen is 1-10U/g, preferably 4-8U/g, more preferably 5-6U/g.

The dosage ratio of the type I collagen to the type V collagen is 8:1 to 1:8, preferably 5:1.

the organic solvent includes, but is not limited to, esters of C1-C6 acids and C1-C6 alcohols, such as ethyl acetate, methyl acetate, ethyl propionate, butyl acetate, amyl valerate, isoamyl isovalerate, etc.; ethers of C1-C6 alcohols with C1-C6 alcohols, such as diethyl ether, methylethyl ether; C3-C6 ketones, such as acetone.

Such surfactants include, but are not limited to, lipophilic surfactants, particularly those having an HLB value of 3 to 8, examples of which include span-type surfactants such as span20, 40, 60 or 80, sugar ester-type surfactants such as glucose esters of C12 to C18 acids; propylene glycol C12-C18 acid; glycerol (glycerin) C12-C18 acid; to co-combine them.

The lipophilic surfactant may be used in combination with 0.1-5 wt% of a hydrophilic surfactant, such as TWEEN 80.

The laccase should be added into the water phase before the water-in-oil emulsion is formed, and should not be added into the oil phase before or after the water-in-oil emulsion is formed, otherwise the type I and type V collagen implants are degraded or disappeared.

The volume ratio of the oil phase to the aqueous phase is 2.5 to 10, preferably 3 to 6.

After the preparation of the aqueous solution is completed, the aqueous solution and the oily solution are mixed within 30 minutes, preferably within 10 minutes, and most preferably within 5 minutes.

Preferably, during the filtration of said emulsion, the particles with a size greater than 25 μm and less than 5 μm are filtered out, and the particles with a size between 5 μm and 25 μm are retained; most preferably, particles having a size greater than 25 μm and less than 10 μm are leached or particles having a size greater than 15 μm and less than 5 μm are leached, with particles having a retained size between 10 μm and 25 μm or particles having a retained size between 5 μm and 15 μm.

The low-temperature freezing method includes freeze drying and spray freeze drying.

The process of the invention adopts an enzyme method to crosslink collagen, and has great advantages compared with a chemical crosslinking process and a physical crosslinking process:

mild reaction conditions, no by-product, high specificity, high catalytic efficiency and yield.

In addition, the mild reaction conditions of the enzyme reduce the loss of collagen during processing.

Examples

The following non-alternative example, a method of preparing an implant, further describes preferred embodiments within the scope of the present invention. Many variations of these embodiments are possible within the scope of the invention.

Example 1

(1) Preparation temperature 4 ℃ of 3.5% surfactant: ethyl acetate solution of SPAN 80 to obtain oil phase;

(2) Preparing an aqueous solution containing 4% of type I collagen, 1% of type V collagen, 6% of maltitol and laccase at the temperature of 4 ℃, wherein the ratio of the dosage of the laccase to the total dosage of the type I collagen and the type V collagen is 8U/g, and obtaining a water phase;

(3) Mixing the aqueous solution with the oil phase within 15 to 30 minutes after the preparation of the aqueous solution is finished, and intensively stirring for 30 minutes to form water-in-oil emulsion, keeping the temperature between 4 ℃ and 0 ℃ for 48 hours to ensure that the collagen is basically crosslinked, wherein the volume ratio of the oil phase to the water phase is 6;

(4) Filtering out particles with a diameter of more than 25 μm and less than 0.5 μm from the emulsion by using a filter membrane after the collagen is basically crosslinked, and keeping the particles with the diameter between 0.5 μm and 25 μm;

(5) Washing the retained particles with 10-30% ethanol water solution, and freeze drying in a vacuum freeze drier at-60- -80 deg.C for 48 hr to remove solvent and water.

15 batches were made in the same manner.

Example 2

(1) Preparation temperature 0 ℃ of 5% surfactant: the oleic acid glucose ester is dissolved in ethyl ether to obtain an oil phase;

(2) Preparing an aqueous solution containing 3% of type I collagen, 0.5% of type V collagen, 5% of sorbitol and laccase at the temperature of 1 ℃, wherein the ratio of the dosage of the laccase to the total dosage of the type I collagen and the type V collagen is 5U/g, and obtaining a water phase;

(3) Mixing the aqueous solution with the oil phase within 5 to 10 minutes after the preparation of the aqueous solution is finished, and intensively stirring for 60 minutes to form water-in-oil type emulsion, keeping the temperature between 0 ℃ and-4 ℃ for 24 hours to basically crosslink the collagen, wherein the volume ratio of the oil phase to the water phase is 5;

(4) Filtering out the particles with the particle size of more than 5 μm and less than 0.5 μm in the emulsion by using a filter membrane after the collagen is basically crosslinked, and keeping the particles with the particle size of between 0.5 μm and 5 μm;

The same method is used for manufacturing 15 batches.

Example 3

(1) Preparation temperature 0 ℃ of a surfactant containing 4%: solution of C14 acid (myristic acid) glyceride in acetone to obtain oil phase;

(2) Preparing an aqueous solution containing 10% of type I collagen, 2% of type V collagen, 6% of mannitol and laccase at the temperature of 1 ℃, wherein the ratio of the dosage of the laccase to the total dosage of the type I collagen and the type V collagen is 6U/g, and obtaining a water phase;

(3) Mixing the aqueous solution with the oil phase within 5 to 15 minutes after the preparation of the aqueous solution is completed, and intensively stirring for 30 minutes to form water-in-oil emulsion, keeping the temperature at 0 ℃ to-4 ℃ for 48 hours to basically crosslink the collagen, wherein the volume ratio of the oil phase to the water phase is 8;

(4) Filtering out the particles with the particle size of more than 25 μm and less than 5 μm in the emulsion by using a filter membrane after the collagen is basically crosslinked, and keeping the particles with the particle size between 5 μm and 25 μm;

15 batches were made in the same manner.

Example 4

(1) Preparation temperature 0 ℃ of 5% surfactant: propylene glycol oleate and 0.1% of solution of isoamyl isovalerate TWEEN 80 to obtain an oil phase;

(2) Preparing an aqueous solution containing 1% of type I collagen, 6% of type V collagen, 8% of erythritol and laccase at the temperature of 1 ℃, wherein the ratio of the amount of the laccase to the total amount of the type I collagen and the type V collagen is 6U/g, and obtaining a water phase;

(3) Mixing the aqueous solution with the oil phase within 5 to 25 minutes after the preparation of the aqueous solution is completed, and stirring strongly for 60 minutes to form water-in-oil emulsion, keeping the temperature at 0 to-4 ℃ for 48 hours to make collagen basically cross-linked, wherein the volume ratio of the oil phase to the water phase is 7;

(4) Filtering out the particles with the particle size of more than 15 μm and less than 5 μm in the emulsion by using a filter membrane after the collagen is basically crosslinked, and keeping the particles with the particle size between 5 μm and 15 μm;

15 batches were made in the same manner.

Example 5

(1) Preparation temperature 0 ℃ of 3% surfactant: SPAN20 butyl acetate solution to obtain oil phase;

(2) Preparing an aqueous solution containing 2% of type I collagen, 6% of type V collagen, 8% of lactitol and laccase at the temperature of 1 ℃, wherein the ratio of the dosage of the laccase to the total dosage of the type I collagen and the type V collagen is 5U/g, and obtaining a water phase;

(3) Mixing the aqueous solution with the oil phase within 15 to 30 minutes after the preparation of the aqueous solution is finished, stirring the aqueous solution and the oil phase strongly for 30 minutes to form water-in-oil emulsion, keeping the temperature between 0 ℃ and-4 ℃ for 48 hours to ensure that the collagen is basically crosslinked, wherein the volume ratio of the oil phase to the water phase is 9;

(4) Filtering out particles with a diameter of more than 25 μm and less than 5 μm from the emulsion by using a filter membrane after the collagen is basically crosslinked, and keeping the particles with the diameter between 5 μm and 25 μm;

15 batches were made in the same manner.

Example 6

(1) Preparation temperature 0 ℃ of a surfactant containing 4%: sucrose palmitate (C16 acid) and 0.1% TWEEN 80 methyl acetate to obtain oil phase;

(2) Preparing an aqueous solution containing 6% of type I collagen, 2% of type V collagen, 5% of xylitol and laccase at the temperature of 1 ℃, wherein the ratio of the dosage of the laccase to the total dosage of the type I collagen and the type V collagen is 10U/g, and obtaining a water phase;

(3) Mixing the aqueous solution with the oil phase within 5 to 20 minutes after the preparation of the aqueous solution is completed, and stirring strongly for 60 minutes to form water-in-oil emulsion, keeping the temperature at 0 ℃ to-4 ℃ for 48 hours to make collagen basically cross-linked, wherein the volume ratio of the oil phase to the water phase is 5;

The same method is used for manufacturing 15 batches.

Example 7

(1) Preparation temperature 0 ℃ of 5% surfactant: the solution of the oleic acid glucose ester in ether is used to obtain an oil phase;

(3) Mixing the aqueous solution with the oil phase within 5 to 10 minutes after the preparation of the aqueous solution is completed, and stirring strongly for 60 minutes to form water-in-oil emulsion, keeping the temperature at 0 to-4 ℃ for 24 hours to basically crosslink the collagen, wherein the volume ratio of the oil phase to the water phase is 5;

(4) Filtering out the particles with the particle size of more than 25 μm and less than 10 μm in the emulsion by using a filter membrane after the collagen is basically crosslinked, and keeping the particles with the particle size between 10 μm and 25 μm;

The same method is used for manufacturing 15 batches.

Comparative examples 1 to 7-1

The same applies to the respective corresponding examples, except that no particles having a particle size greater than 25 μm or 5 μm or 10 μm or 15 μm or 20 μm (which is the same as the respective corresponding examples) and less than 0.5 μm or 5 μm or 10 μm or 15 μm (which is the same as the respective corresponding examples) are filtered out.

Comparative examples 1 to 7-2

The same applies to the respective examples, except that no particles having a size greater than 25 μm or 5 μm or 10 μm or 15 μm or 20 μm (which is the same as the respective examples) are leached.

Comparative examples 1 to 7 to 3

The examples were identical with the corresponding examples except that fine particles having a particle diameter of less than 0.5 μm or 5 μm or 10 μm or 15 μm (which is the same as the corresponding examples) were not filtered out.

Comparative examples 1 to 7 to 4

The examples are identical with the respective corresponding examples, except that the laccase is not (directly) added to the aqueous phase, but rather to the oil phase.

Comparative examples 1 to 7 to 5

The same applies to the respective corresponding examples, except that no surfactant is added, no particles having a particle size of more than 25 μm or 5 μm or 10 μm or 15 μm or 20 μm (the same value as in the respective corresponding examples) and less than 0.5 μm or 5 μm or 10 μm or 15 μm (the same value as in the respective corresponding examples) are leached.

Test example 1 through needle Performance test

The principle is as follows:

the better the needle penetration performance, the less time it takes for the same amount of suspension to pass through the same needle under the same conditions.

The method comprises the following steps:

the same amount (5 ml) of physiological saline for medical injection was added to a lyophilizate prepared by removing the solvent water by lyophilization in a vacuum lyophilizer at-60 to-80 ℃ for 48 hours, which was used as a reference, in the same manner for the same time (e.g., a shaking method) to reconstitute the lyophilizate into a suspension, which was taken out from the above examples, comparative examples and commercially available collagen (35 mg, borteur: eel) in the same weight (35mg, total weight of type I and type V collagens), and the same amount of the suspension was taken out in the same syringe (the needle was not changed, washed and then dried and reused), and the suspension was discharged by the same constant pressure syringe, and the time required was measured. And finally, respectively calculating the ratio of the time measured in the above embodiment and the time measured in the comparison example to the time measured in a commercial product, and measuring the needle passing performance of the above embodiment and the comparison example by using the ratio, wherein the smaller the ratio is, the stronger the needle passing performance is, and the larger the ratio is, the weaker the needle passing performance is.

The test results are shown in tables 1 to 7.

Test example 2

In vitro degradation time test

The same weight of collagen (35mg, the total weight of type I and type V collagens) as in the above examples, the control example and the commercially available collagen (35 mg, trade name: borai: eel, and the method of the example: lyophilized preparation prepared by removing solvent water by lyophilization at-60 to-80 ℃ for 48 hours in a vacuum lyophilizer, which is used as a reference) was reconstituted into a suspension in the same manner and for the same time with the same amount (100 ml, concentration 0.035%) of physiological saline for medical injection (pH 7.0). Adding collagen hydrolase (Bacillus cereus protease, purified and separated from Bacillus cereus) in the same amount to the suspension, wherein the weight ratio of the collagen hydrolase to the collagen is 4U/mg, placing in a constant temperature and humidity environment with temperature of 37 deg.C and relative humidity of 70%, and sampling until absorbance value A is substantially stable (fluctuation range less than 2%), wherein the average value of the stable absorbance value A is represented as A ₀ 。

After sampling, inactivating enzyme at 90 ℃ for 10min, cooling to room temperature, and centrifuging at 4000 r/min for 10min to obtain clear hydrolysate. And (2) adding 5mL of 15% (W/W) trichloroacetic acid (TCA) aqueous solution into 5mL of the hydrolysate sample solution, uniformly mixing, standing for 10min, centrifuging for 10min at 4 000r/min, diluting the supernatant to a solution with the concentration of 1-10 mg/mL, adding 4mL of biuret reagent into 1mL of the diluent, uniformly mixing, and standing for 30min. The absorbance value A was measured at a wavelength of 540 nm. The concentration of polypeptide C (mg/mL) produced by degradation can be calculated according to a linear regression equation (see document 1: meat industry, 2011, (11), general No. 367, pp.21-24, liulili, yanglie, kinetic study of collagenase enzymolysis of bovine bone collagen).

The absorbance value A (or the concentration C of the polypeptide produced by degradation) reflects the amount of degraded collagen in the sample solution ₀ -A (or C) ₀ -C，C ₀ Represents the average value A after the absorbance value A is substantially stabilized ₀ The determined concentration of the polypeptide produced by degradation, i.e., the concentration of the polypeptide produced by complete degradation of collagen and the amount of collagen at the time of initiation of the reaction) reflects the amount of the collagen remaining in the sample solution, and it is known from the equation of mie that the protease degradation exhibits the first-order reaction characteristic at a lower concentration (see the above document 1), so ln (A) can be used ₀ -A) (or ln (C) ₀ -C)) is plotted against time T, and the degradation slope k is determined from said slope k and the formula T _0.99 Complete degradation of collagen (99% degradation or 1% residue), t = ln (C) was calculated for = ln100/k ₀ ＇/ C＇)/k, _t C＇/ C ₀ ' -0.01, wherein C ₀ "and C" represent the amount of original collagen and the amount of residual collagen, respectively)) _0.99 。

Finally, the time T for complete degradation of collagen measured in the above examples and comparative examples was calculated _0.99 Time T for complete degradation of collagen measured with commercial collagen _0.99 The ratio, as an average value thereof, by which the relative in vivo retention properties of the above examples and comparative examples are measured, is larger, the in vivo retention (depot) property is stronger, and smaller, the in vivo retention (depot) property is weaker. The smaller the ratio fluctuation is, the smaller the change of the retention (long-acting) performance of the collagen in the body is; the larger the ratio fluctuation, the larger the change in the retention (long-lasting) properties of collagen in vivo.

The test results are shown in tables 1 to 7.

TABLE 1

Example 1

Comparative example 1-1

Comparative examples 1 to 2

Comparative examples 1 to 3

Comparative examples 1 to 4

Comparative examples 1 to 5

Performance of needle passing

0.26

0.48

0.56

0.24

-

Ratio of time to degradation

4.5±1.1

6.5±3.9

8.1±5.3

3.7±2.4

1.3±0.3

10.8±7.2

TABLE 2

Example 2

Comparative example 2-1

Comparative example 2-2

Comparative examples 2 to 3

Comparative examples 2 to 4

Comparative examples 2 to 5

Performance of needle

0.13

0.32

0.39

0.16

-

Ratio of time to degradation

2.3±0.4

4.9±2.3

6.5±3.8

1.8±0.6

1.1±0.1

8.7±5.6

TABLE 3

Example 3

Comparative example 3-1

Comparative example 3-2

Comparative examples 3 to 3

Comparative examples 3 to 4

Comparative examples 3 to 5

Performance of needle passing

0.28

0.44

0.52

0.31

-

Ratio of degradation time

5.8±1.5

7.8±4.3

8.9±6.2

4.2±2.7

1.4±0.3

11.4±7.8

TABLE 4

Example 4

Comparative example 4-1

Comparative example 4-2

Comparative examples 4 to 3

Comparative examples 4 to 4

Comparative examples 4 to 5

Performance of needle passing

0.20

0.37

0.44

0.22

-

Ratio of time to degradation

3.6±0.8

5.8±3.1

6.9±4.8

3.1±2.0

1.3±0.2

10.5±7.3

TABLE 5

Example 5

Comparative example 5-1

Comparative example 5-2

Comparative examples 5 to 3

Comparative examples 5 to 4

Comparative examples 5 to 5

Performance of needle passing

0.30

0.47

0.56

0.28

-

Ratio of time to degradation

5.3±1.3

7.9±5.2

9.8±7.1

4.1±2.3

1.4±0.3

12.2±9.1

TABLE 6

Example 6

Comparative example 6-1

Comparative example 6-2

Comparative examples 6 to 3

Comparative examples 6 to 4

Comparative examples 6 to 5

Performance of needle passing

0.27

0.42

0.56

0.29

-

Ratio of time to degradation

5.6±1.4

7.9±4.8

9.3±5.7

4.3±2.8

1.5±0.3

12.5±8.9

TABLE 7

Example 7

Comparative example 7-1

Comparative example 7-2

Comparative examples 7 to 3

Comparative examples 7 to 4

Comparative examples 7 to 5

Performance of needle passing

0.32

0.57

0.69

0.35

-

Ratio of time to degradation

7.2±1.6

9.8±7.0

11.9±8.8

5.8±3.2

1.7±0.5

13.9±10.2

The results show that:

1) Examples have slower enzymatic hydrolysis rates, significantly longer in vivo retention (long-lasting) performance than commercial products;

2) The examples have better in vivo retention (long-acting) performance than the comparative example without adding the surfactant, and the fluctuation of the in vivo retention (long-acting) performance of the comparative example is extremely large;

3) The examples have better in vivo persistence (long-lasting) than the control example in which laccase is not (directly) added to the aqueous phase but to the oil phase, which is extremely weak in vivo persistence (long-lasting);

4) Examples the control, which did not filter out larger and smaller particles, had better in vivo retention (longevity) performance, less variability, and had a large fluctuation in vivo retention (longevity) performance;

5) Examples the control example in which only smaller particles were filtered out, but larger particles were filtered out, had better in vivo retention (retention) performance, less fluctuation, and the control example in which the in vivo retention (retention) performance fluctuated more;

6) The control example which only filters larger particles but filters smaller particles has better in-vivo persistence (long-acting) performance, longer in-vivo persistence (long-acting) performance and smaller volatility, and the control example has shorter in-vivo persistence (long-acting) performance and larger volatility;

7) The embodiment has better needle passing performance than the commercial products, the comparison example which does not filter out larger and smaller particles and the comparison example which only filters out smaller particles without filtering out larger particles; the examples have substantially the same stylet performance as the control example in which smaller particles are not filtered but only larger particles are filtered.

Claims

1. A long-acting, small-period-fluctuation, good-needle-penetration-performance microparticle type I and type III collagen composite implant comprises: the type I and type III collagen composite particles cross-linked by using sulfhydryl oxidase.

2. A preparation method of a microparticle type I and type III collagen composite implant with long-acting, small effect period fluctuation and good needle penetration performance comprises the following steps:

(1) Preparing a solution, namely an oil phase, which is not higher than 4 ℃ and comprises a surfactant and a volatile organic solvent which is not completely dissolved in water;

(2) Preparing an aqueous solution (namely an aqueous phase) with the temperature not higher than 4 ℃, wherein the aqueous solution comprises type I collagen, type III collagen, sulfydryl oxidase and a protein activity protective agent;

(3) Mixing the aqueous solution with the solution of the organic solvent within 30 minutes after the preparation of the aqueous solution is completed, and allowing them to form a water-in-oil emulsion, and maintaining the temperature at 4 ℃ to-4 ℃ for 24 hours or more to crosslink the collagen;

(4) Filtering the emulsion after the collagen is crosslinked to obtain microparticles, washing the microparticles by using an ethanol water solution, and removing the residues of the surfactant, the organic solvent, the protein activity protective agent, the enzyme and the like;

(5) And removing residual solvent in the particles by a low-temperature freezing method.

3. The method of claim 2, wherein the protein activity protector is selected from the group consisting of sorbitol, mannitol, erythritol, maltitol, lactitol, xylitol, and combinations thereof.

4. The production method according to claim 2, characterized in that the concentration of the ethanol aqueous solution is 10 to 40% by volume.

5. The method according to claim 2, characterized in that the total concentration by mass of the types I and III collagen in the aqueous solution is between 0.1% and 10%.

6. The method according to claim 2, wherein the ratio of the amount of thiol oxidase to the total amount of type I and type III collagen is 1 to 10U/g.

7. The method of claim 2, wherein the organic solvent comprises an ester of a C1-C6 acid with a C1-C6 alcohol, an ether of a C1-C6 alcohol with a C1-C6 alcohol, a C3-C6 ketone, and combinations thereof.

8. The process according to claim 2, characterized in that the surfactant is selected from lipophilic surfactants having an HLB value of from 3 to 8.

9. The production method according to claim 2, characterized in that the volume ratio of the oil phase to the aqueous phase is 2.5 to 10.

10. The process according to claim 2, characterized in that it comprises filtering out particles having a size greater than 25 μm and less than 0.5 μm and retaining particles having a size between 0.5 μm and 25 μm during the filtration of said emulsion to obtain particles.

11. The process according to claim 2, characterized by comprising filtering out particles having a size greater than 25 μm and less than 5 μm and retaining particles having a size between 5 μm and 25 μm during the filtration of said emulsion to obtain particles.

12. The process according to claim 2, characterized in that it comprises filtering out particles having a size greater than 25 μm and less than 10 μm or greater than 15 μm and less than 5 μm, retaining particles having a size between 10 μm and 25 μm or retaining particles having a size between 5 μm and 15 μm during the filtration of said emulsion to obtain particles.

13. The preparation method according to claim 2, characterized in that the low-temperature freezing method comprises a freeze-drying method or a spray-freeze-drying method.