CN115350328A

CN115350328A - Long-acting particle III type collagen implant

Info

Publication number: CN115350328A
Application number: CN202211001395.8A
Authority: CN
Inventors: 韩淑萍; 丁劲松; 何伟; 陈文洁; 李佳悦
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: 2022-11-18

Abstract

The invention discloses a microparticle type III collagen implant with improved performance and a preparation method thereof. The collagen implant comprises: collagen type III microparticles crosslinked with glutamine transaminage, the microparticles having a particle size of between 0.5 μm and 25 μm. The collagen implant has the advantages of long acting, small effect period fluctuation, good needle penetration performance and the like.

Description

Long-acting micro-particle type III collagen implant

Technical Field

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

Technical Field

Type III collagen, the main collagen in human skin, fascia, and tendon, is in a ratio of 4 to 1 to type I collagen. Type III collagen, which is relatively fine and exists between the epidermis and dermis, is called "infant collagen". The collagen layer (cushinon Network) is mainly composed of type III collagen, is a tissue structure positioned between an epidermal layer and a dermal layer, is a key for supporting the epidermis, and is the first step of skin collapse.

Type iii collagen plays a minimal role in inflammation and antigenic responses and has been approved by food and drug administration in china, the united states, for many types of medical applications, including wound dressings and artificial skin.

However, type III collagen has a major disadvantage such as short residence time in vivo.

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 manufacturing method 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 effects are remarkable, there is a risk of chemical reagent remaining and harmful substances being formed during the modification, and further, there are 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 industrial 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 III collagen implant that has the advantages of long-lasting action, less fluctuation in the effective period, and good penetration.

Disclosure of Invention

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

The inventor finds that the common III type collagen is modified into the particle cross-linked collagen with basically uniform size by glutamine transaminase, so that the action time of the collagen 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 are solved, such as accurate subpackage dosage and better needle penetration during injection; 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, generate no by-products, improve the catalytic efficiency, improve the yield and the like.

Transglutaminase (transglutaminase, ec 2.3.2.13), abbreviated as TG enzyme, belongs to acyltransferase. Glutamine transaminase catalyzes an acyl transfer reaction between a gamma-hydroxylamine group of a glutamine residue in a protein and a primary amine compound (acyl acceptor) to generate an isopeptide bond, thereby covalently crosslinking the protein. During the reaction, one ammonia molecule is released per crosslink. If the amine-based substrate does not act as an acyl acceptor, TG enzymes can catalyze the deamination of glutamine residues using water as the acyl acceptor.

TG enzymes have a broad spectrum of acyl acceptor substrates.

The TG enzyme-mediated molecular crosslinking can improve the characteristics of the collagen such as thermal stability, water holding capacity and the like, is beneficial to forming a powerful gel and improving the performance of the collagen implant.

TG enzyme can also catalyze collagen to generate deamidation reaction, and improve foamability and emulsibility of collagen, thereby leading the collagen implant to be well micronized, degelatinize, reduce viscosity and greatly improve performance.

Based on this, the present invention has been completed.

The invention relates to a preparation method of a (performance-improved) long-acting microparticle type III collagen implant with small period fluctuation and good needle penetration, which 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, i.e. an aqueous phase, comprising type III collagen and transglutaminase, at a temperature not higher than 4 ℃;

(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 keeping the temperature at 4 ℃ to-4 ℃ for more than 24 hours, preferably within 48 hours, to substantially crosslink the 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 in the retained particles by a low-temperature freezing method after washing.

The invention relates to a (performance-improved) long-acting, little-fluctuation effective period and good-needle-penetration-performance micro-particle type III collagen implant, which comprises: collagen type III microparticles crosslinked with glutamine transaminage, the microparticles having a particle size of between 0.5 μm and 25 μm.

Detailed Description

Preferably, the mass concentration of the above aqueous solution, i.e., type III collagen in the aqueous phase, is 0.1% to 10%, more preferably 0.5% to 5%; preferably, the ratio of the amount of transglutaminase to the amount of type III collagen is 1 to 10U/g, preferably 4 to 8U/g, more preferably 5 to 6U/g.

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 esters; 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 transglutaminase should be added to the aqueous phase before the formation of the water-in-oil emulsion and should not be added to the oil phase before or after the formation of the water-in-oil emulsion, otherwise the type III collagen implant will degrade or disappear.

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, in the process of filtering the emulsion, filtering out particles with the particle size of more than 25 μm and less than 5 μm and keeping the particles with the particle size between 5 μm and 25 μm; most preferably, the reject size is greater than 25 μm and less than 10 μm or the reject size is greater than 15 μm and less than 5 μm, the retentate size is between 10 μm and 25 μm or the retentate size is between 5 μm and 15 μm.

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

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 ℃ containing 2% of surfactant: SPAN 80 ethyl acetate solution to obtain oil phase;

(2) Preparing an aqueous solution containing 5% of type III collagen and glutamine transaminase at 4 ℃, wherein the ratio of the dosage of the glutamine transaminase to the dosage of the type III collagen is 7U/g, and obtaining a water phase;

(3) Mixing the aqueous solution with the oil phase within 20 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 4 ℃ and 0 ℃ for 24 hours to ensure that the collagen is basically crosslinked, wherein the volume ratio of the oil phase to the water phase is 4.5;

(4) Filtering out the particles with the particle size of more than 25 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 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.

13 batches were made in the same way.

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 III collagen and glutamine transaminase at the temperature of 1 ℃, wherein the ratio of the dosage of the glutamine transaminase to the dosage of the type III collagen is 3U/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 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 3;

(4) Filtering out the particles with the particle size of more than 5 microns and less than 0.5 microns 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 microns and 5 microns;

13 batches were made in the same way.

Example 3

(1) Preparation temperature 0 ℃ of 10% surfactant: acetone solution of C12 acid (lauric acid) glyceride to obtain oil phase;

(2) Preparing an aqueous solution containing 8% of type III collagen and glutamine transaminase at the temperature of 1 ℃, wherein the ratio of the dosage of the glutamine transaminase to the dosage of the type III collagen is 10U/g, and obtaining a water phase;

(3) Mixing the aqueous solution with the oil phase within 1 to 5 minutes after the preparation of the aqueous solution is completed, and intensively stirring for 40 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 6;

(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;

13 batches were made in the same way.

Example 4

(1) Preparation temperature 0 ℃ 10% surfactant: propylene glycol stearate (C18) and 0.1% solution of TWEEN 80 in isoamyl isovalerate to form an oil phase;

(2) Preparing an aqueous solution containing 5% of type III collagen and transglutaminase at a temperature of 1 ℃, wherein the ratio of the amount of the transglutaminase to the amount of the type III collagen is 8U/g, thereby 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 8;

(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;

The same method is used for preparing 13 batches.

Example 5

(1) Preparation temperature 0 ℃ of 10% surfactant: a solution of SPAN20 in butyl acetate to obtain an oil phase;

(2) Preparing an aqueous solution containing 5% of type III collagen and glutamine transaminase at 1 ℃, wherein the ratio of the dosage of the glutamine transaminase to the dosage of the type III collagen is 4U/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 finished, and intensively stirring for 20 minutes to form water-in-oil type emulsion, keeping the temperature between 0 ℃ and-4 ℃ for 48 hours to basically crosslink the collagen, wherein the volume ratio of the oil phase to the water phase is 9;

(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;

The same method is used for preparing 13 batches.

Example 6

(1) Preparation temperature 0 ℃ of a surfactant containing 6%: sucrose palmitate (C16 acid) and 0.1% TWEEN 80 methyl acetate to obtain oil phase;

(2) Preparing an aqueous solution containing 2% of type III collagen and transglutaminase at a temperature of 1 ℃, wherein the ratio of the amount of the transglutaminase to the amount of the type III collagen is 5U/g, thereby 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 finished, and intensively stirring for 50 minutes to form water-in-oil type emulsion, keeping the temperature between 0 ℃ and-4 ℃ for 48 hours to basically crosslink the collagen, wherein the volume ratio of the oil phase to the water phase is 5;

The same method is used for preparing 13 batches.

Example 7

(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 preparing 13 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 corresponding examples, except that no particles having a particle size of more than 25 μm or 5 μm or 10 μm or 15 μm or 20 μm (which is the same as the respective corresponding examples) are filtered out.

Comparative examples 1 to 7 to 3

The same applies to the corresponding examples, except that no particles having a diameter of less than 0.5 μm or 5 μm or 10 μm or 15 μm (which is the same as the corresponding examples) are leached.

Comparative examples 1 to 7 to 4

The same applies to the respective examples, except that the transglutaminase is not added (directly) to the aqueous phase, but rather to the oil phase.

Comparative examples 1 to 7 to 5

The examples were identical with the respective corresponding examples, except that no surfactant was 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 the respective corresponding examples) and less than 0.5 μm or 5 μm or 10 μm or 15 μm (the same value as the respective corresponding examples) were filtered out.

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 weight (4 mg) of the collagen type III (4 mg, wei, hainan, shanxi, brocade) obtained in the above examples, comparative examples and commercial products was taken, the collagen solution was reconstituted (reconstituted) in the same manner (same shaking method) with the same amount (5 ml) of physiological saline for medical injection added in a vacuum freeze-dryer at-60- -80 ℃ for 48 hours to remove the solvent water, the same amount of the suspension was filled in the same syringe (the needle was not changed, and the syringe was cleaned and dried for repeated use), the syringe was injected with the same constant pressure to discharge the suspension, and the time required was measured. And finally, respectively calculating the ratio of the time measured by the embodiment and the comparison example to the time measured by a commercial product, and measuring the needle passing performance of the 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 amount (5 ml, i.e., 0.08%) of physiological saline for medical injection (pH 7.0) was added to the collagen solution obtained in the above examples, comparative examples and commercially available products (4 mg, wei-charming, shanxi brocade) of type III collagen type (4 mg) in the same weight and the same amount of the same amount was reconstituted into a suspension in the same manner and for the same time in a vacuum freeze-drying machine at-60- -80 ℃ for 48 hours to remove the solvent water. Adding collagen hydrolase (Bacillus cereus protease, purified and separated from Bacillus cereus) with the same amount into the suspension, wherein the weight ratio of the collagen hydrolase and the collagen protein is 4U/mg, placing in constant temperature and humidity environment with temperature of 37 deg.C and relative humidity of 70%, and sampling for 0hr, 1 hr, 3 hr, 5 hr, 7 hr, 9 hr, 11 hr, 13 hr, 15 hr, 17 hr, \8230, and \8230, until the absorbance value A is substantially stable (fluctuation range is less than 2%), and the absorbance value A is stableMean value 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 of the polypeptide C 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 commercially available products _0.99 Ratio (in average value) by which the relative in vivo retention performances of the above examples and comparative examples were measured, the larger the ratio, the stronger the in vivo retention (long-lasting) performance, and the ratioThe smaller the value, the weaker the in vivo retention (long-lasting) properties. The smaller the ratio fluctuation, the smaller the change in the persistence (long-lasting) properties of type III collagen in vivo; the greater the ratio fluctuation, the greater the change in the persistence (long-lasting) properties of type III collagen in the body.

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

0.24

0.42

0.49

0.22

-

Ratio of degradation time

4.3±1.0

4.8±3.0

6.7±4.1

3.6±2.2

1.3±0.2

8.5±5.2

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 passing

0.13

0.32

0.39

0.16

-

Ratio of time to degradation

2.1±0.4

3.2±1.8

4.5±2.1

1.8±0.6

1.2±0.1

6.9±4.4

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

5.6±1.5

6.8±4.1

8.3±6.0

4.2±2.6

1.5±0.4

10.2±7.3

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

3.2±0.8

5.3±3.1

6.5±4.1

2.6±1.3

1.4±0.2

9.7±5.8

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

5.4±1.3

7.3±4.2

9.2±6.3

4.5±2.4

1.6±0.3

11.8±8.7

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 degradation time

5.7±1.4

7.6±4.2

9.0±5.8

4.8±2.9

1.5±0.3

11.5±7.7

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

6.8±1.7

9.3±6.9

10.5±8.0

5.4±2.8

1.7±0.4

13.2±9.9

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 retention (long-lasting) properties than the control in which glutamine transaminase is not (directly) added to the aqueous phase but added to the oil phase, and the retention (long-lasting) properties in vivo are extremely weak;

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

5) The control example, in which only smaller particles are filtered out, has better in vivo persistence (long-lasting) performance and less fluctuation than the control example in which the in vivo persistence (long-lasting) performance fluctuates greatly;

6) The comparative example in which only larger particles were filtered out had better in vivo persistence (persistence) properties, longer in vivo persistence (persistence) properties and less fluctuation than the comparative example in which the in vivo persistence (persistence) properties were shorter and the fluctuation was larger;

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 the smaller particles are not filtered and only the larger particles are filtered.

Claims

1. A preparation method of a long-acting microparticle type III collagen implant with small period fluctuation and good needle penetration performance 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 ℃;

(3) Mixing the aqueous solution with the solution of the organic solvent within 30 minutes after the preparation of the aqueous solution is finished, forming the aqueous solution and the solution of the organic solvent into water-in-oil emulsion, and keeping the temperature between 4 ℃ below zero and 4 ℃ below zero for more than 24 hours to crosslink the 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 crosslinked, and keeping the particles with the particle size of between 0.5 microns and 25 microns;

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

2. The method according to claim 1, characterized in that the concentration by mass of the type III collagen in the aqueous solution is 0.1% to 10%.

3. The process according to claim 1, wherein the ratio of the amount of transglutaminase to the amount of type III collagen is 1 to 10U/g.

4. The method of claim 1, 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.

5. The process according to claim 1, characterized in that the surfactant is selected from lipophilic surfactants having an HLB value of from 3 to 8.

6. The production method according to claim 1, characterized in that the volume ratio of the oil phase to the aqueous phase is 2.5 to 10.

7. The process according to claim 1, characterized in that it comprises filtering off 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.

8. The process according to claim 1, characterized in that it comprises filtering the particles with a size greater than 25 μm and less than 10 μm and retaining the particles with a size between 10 μm and 25 μm.

9. The preparation method according to claim 1, characterized in that the low-temperature freezing method comprises a freeze-drying method or a spray-freeze-drying method.

10. A long-acting, small-period fluctuation, good needle penetration microparticle type III collagen implant, comprising: collagen type III microparticles crosslinked with glutamine transaminage, the microparticles having a particle size of between 0.5 μm and 25 μm.