CN115317668A

CN115317668A - Long-acting particle type I collagen implant

Info

Publication number: CN115317668A
Application number: CN202211000723.2A
Authority: CN
Inventors: 付劼; 周文虎; 韩淑萍; 陈文洁; 李贤明
Original assignee: Jiangsu Dongfang Yanmei Biotechnology Development Co ltd
Current assignee: Beijing Xihong Runmei Pharmaceutical Technology Co ltd
Priority date: 2022-08-19
Filing date: 2022-08-19
Publication date: 2022-11-11
Anticipated expiration: 2042-08-19
Also published as: CN115317668B

Abstract

The invention discloses a microparticle type I collagen implant with improved performance and a preparation method thereof. The collagen implant comprises: collagen type i microparticles crosslinked using tyrosinase. The implant has the advantages of long acting, long effective period (small fluctuation) and good needle penetration.

Description

Long-acting particle type I collagen implant

Technical Field

The invention relates to an injection implant. In particular to a long-acting particle type I collagen 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, the healing of the tissue in the final product by repair, and the organic part of the bone of the tendons, ligaments, myofibrils of the intima, the dermis, dentin and organ capsules.

Type I collagen plays the least 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, the major disadvantages of the type I collagen include 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 method of manufacture 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 consistent, e.g., the duration of the therapeutic effect is not long enough.

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 collagen implant that has the advantages of long duration, consistent duration of action (less fluctuation), and good penetration.

Disclosure of Invention

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

The inventor finds that the tyrosinase modifies the common type I collagen into the particle cross-linked collagen with basically uniform size, so that the action time of the collagen in the body can be prolonged, the effective period is consistent and long (the fluctuation is small), the viscosity of a water dispersion system is reduced, and a plurality of 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, avoid generating byproducts, improve the catalytic efficiency, improve the yield and the like.

Tyrosinase has a unique dual catalytic function, and can catalyze the oxidation of monophenol to ortho-diphenol and further oxidize diphenol to ortho-benzoquinone. Throughout the catalytic process, oxygen molecules act as electron acceptors, are reduced to water rather than forming hydrogen peroxide, and 1 mol/L of oxygen molecules are required to oxidize 1 mol/L of monohydric phenol, while 0.5 mol/L of oxygen molecules are required to oxidize 1 mol/L of dihydric phenol. The final products released in these 2 catalytic processes are all quinones, which are precursors for melanin. Thus, tyrosinase can oxidize tyrosine in the side chains of proteins to quinones, thereby further crosslinking with lysine, tyrosine and cysteine residues present in proteins.

Based on this, the present invention has been completed.

The invention relates to a (performance-improved) long-acting, long-lasting (fluctuation is small), fine-particle type I collagen implant with good needle penetration, which comprises: collagen type i microparticles crosslinked using tyrosinase.

The invention relates to a preparation method of a microparticle type I collagen implant with long effect (improved performance), consistent and long effective period (small 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 containing type I collagen, tyrosinase and sugar alcohol at a temperature of not higher than 4 ℃, namely an aqueous phase;

(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 the emulsion after the collagen is basically crosslinked to obtain microparticles, washing the microparticles by using 10-40% ethanol water solution, and removing the residues of the surfactant, the organic solvent, the sugar alcohol, the enzyme and the like;

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

Detailed Description

The particle size of the particles in the above particulate collagen implant is preferably between 5 μm and 25 μm, more preferably between 10 μm and 25 μm or between 5 μm and 15 μm.

Such sugar alcohols include, but are not limited to, sorbitol, mannitol, erythritol, maltitol, lactitol, xylitol, and combinations thereof. The sugar alcohol is used for protecting the activity of type I collagen and tyrosinase.

Preferably, the mass concentration of the above aqueous solution, i.e. type I collagen in the aqueous phase, is 0.1% to 10%, more preferably 0.5% to 5%; preferably, the ratio of the amount of tyrosinase to the amount of collagen type I is 1-10U/g, preferably 4-8U/g, more preferably 5-6U/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 formed by C1-C6 alcohols and C1-C6 alcohols, such as diethyl ether, methyl ethyl 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, such as span surfactants, e.g., span20, 40, 60 or 80, sugar ester surfactants, e.g., 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 hydrophilic surfactant, such as TWEEN 80.

Tyrosinase should be added to the aqueous phase before the water-in-oil emulsion is formed, and should not be added to the oil phase before or after the water-in-oil emulsion is formed, otherwise the type I collagen implant performance is degraded or disappears.

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, 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 to obtain particles, filtering out particles with the particle size of more than 25 μm and less than 0.5 μm, and keeping the particles with the particle size of between 0.5 μm and 25 μm; more preferably, filtering out particles with a size greater than 25 μm and less than 5 μm, and retaining particles with a size between 5 μm and 25 μm; 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, and particles having a size between 10 μm and 25 μm are retained or particles having a size between 5 μm and 15 μm are retained.

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 ℃ containing 3% of surfactant: SPAN 80 ethyl acetate solution to obtain oil phase;

(2) Preparing an aqueous solution containing 5% of type I collagen, 5% of sorbitol and tyrosinase at the temperature of 4 ℃, wherein the ratio of the amount of the tyrosinase to the type I collagen is 5U/g, and obtaining a water phase;

(3) Mixing the aqueous solution with the oil phase within 10 to 30 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 not higher than 4 ℃ to 0 ℃ for 48 hours to basically crosslink the collagen, wherein the volume ratio of the oil phase to the water phase is 5;

(4) Filtering the emulsion after the collagen is basically crosslinked to obtain particles: filtering with filter membrane to remove particles with particle diameter of more than 25 μm and less than 0.5 μm, retaining particles with particle diameter of 0.5 μm and 25 μm, washing the particles with 15% ethanol water solution, and removing residues of the surfactant, the organic solvent, the sugar alcohol, and the enzyme;

(5) And freeze-drying in a vacuum freeze-drying machine at-60- -80 deg.C for 48 hr to remove residual solvent from the particles.

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, 8% of mannitol and tyrosinase at the temperature of 1 ℃, wherein the ratio of the amount of the tyrosinase to the type I collagen is 7U/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 24 hours to basically crosslink the collagen, wherein the volume ratio of the oil phase to the water phase is 3;

(4) Filtering the emulsion after the collagen is basically crosslinked to obtain particles: filtering with filter membrane to remove particles with particle diameter of more than 5 μm and less than 0.5 μm, retaining particles with particle diameter of 0.5 μm and 5 μm, washing the particles with 30% ethanol water solution, and removing residues of the surfactant, the organic solvent, the sugar alcohol, and the enzyme;

15 batches were made in the same manner.

Example 3

(1) Preparation temperature 0 ℃ of a surfactant containing 6%: acetone solution of C12 acid (lauric acid) glyceride to obtain oil phase;

(2) Preparing an aqueous solution containing 10% of type I collagen, 3% of erythritol and tyrosinase at the temperature of 1 ℃, wherein the ratio of the amount of the tyrosinase to the type I 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 8;

(4) Filtering the emulsion to obtain particles: filtering with filter membrane to remove particles with particle size of more than 25 μm and less than 5 μm, retaining particles with particle size between 5 μm and 25 μm, washing the particles with 20% ethanol water solution, and removing residues of the surfactant, the organic solvent, the sugar alcohol, the enzyme, etc.;

The same method is used for manufacturing 15 batches.

Example 4

(1) Preparation temperature 0 ℃ of 5% 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 2% of type I collagen, 6% of maltitol and tyrosinase at the temperature of 1 ℃, wherein the ratio of the amount of the tyrosinase to the type I collagen is 5U/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 6;

(4) Filtering the emulsion after the collagen is basically crosslinked to obtain particles: filtering with filter membrane to remove particles with particle size of more than 15 μm and less than 5 μm, retaining particles with particle size between 5 μm and 15 μm, washing the particles with 15% ethanol water solution, and removing residues of the surfactant, the organic solvent, the sugar alcohol, the enzyme, etc.;

(5) And freeze-drying in a vacuum freeze-drying machine at-60- -80 deg.C for 48 hr to remove residual solvent in the microparticles.

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 5% of type I collagen, 10% of xylitol and tyrosinase at the temperature of 1 ℃, wherein the ratio of the amount of the tyrosinase to the type I collagen is 6U/g, and obtaining a water phase;

(3) Mixing the aqueous solution and the oil phase within 5 to 25 minutes after the preparation of the aqueous solution is finished, forcibly stirring the aqueous solution and the oil phase for 20 minutes to form water-in-oil 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 7.5;

(4) Filtering the emulsion after the collagen is basically crosslinked to obtain particles: filtering with filter membrane to remove particles with particle size of more than 25 μm and less than 5 μm, retaining particles with particle size between 5 μm and 25 μm, washing the particles with 25% ethanol water solution, and removing residues of the surfactant, the organic solvent, the sugar alcohol, the enzyme, etc.;

15 batches were made in the same manner.

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 6% of type I collagen, 5% of lactitol and tyrosinase at the temperature of 1 ℃, wherein the ratio of the amount of the tyrosinase to the type I collagen is 10U/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 intensively stirring for 50 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 the emulsion after the collagen is basically crosslinked to obtain particles: filtering with filter membrane to remove particles with particle size of more than 25 μm and less than 5 μm, retaining particles with particle size between 5 μm and 25 μm, washing the particles with 35% ethanol water solution, and removing residues of the surfactant, the organic solvent, the sugar alcohol, the enzyme, etc.;

15 batches were made in the same manner.

Example 7

(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 the emulsion after the collagen is basically crosslinked to obtain particles: filtering with filter membrane to remove particles with particle size of more than 25 μm and less than 10 μm, retaining particles with particle size between 10 μm and 25 μm, washing the particles with 30% ethanol water solution, and removing residues of the surfactant, the organic solvent, the sugar alcohol, the enzyme, etc.;

15 batches were made in the same manner.

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 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 same applies to the respective corresponding examples, except that tyrosinase was not added (directly) 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 (8 mg) of collagen type I (8 mg, lyophilized preparation, hizibo Bio-pharmaceuticals Co., ltd.) as in the above examples, comparative examples and commercial products was added to the same amount (5 ml) of physiological saline for medical injection and reconstituted (reconstituted) in the same manner and for the same time (by the same shaking method), the same amount of suspension was filled in the same syringe (the needle was also changed, and the syringe was dried and reused after cleaning), 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 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 above examples, comparative examples and commercial products of type I collagen (8 mg) were reconstituted in the same manner and for the same time into suspensions by adding the same amount (10 ml, concentration 0.08%) of medical injectable physiological saline (pH 7.0) to the same amount (8 mg, lyophilized preparation, shanxi brocade biomedical Co., ltd.) of type I collagen (8 mg). In the above suspensionAdding the same amount of collagenase (Bacillus cereus protease, purified and separated from Bacillus cereus) at a weight ratio of 4U/mg to the collagen, and sampling in a constant temperature and humidity environment at 37 deg.C and 70% relative humidity for 0hr, 1 hr, 3 hr, 5 hr, 7 hr, 9 hr, 11 hr, 13 hr, 15 hr, 17 hr, … … until the absorbance value A is substantially stable (fluctuation range is less than 2%), wherein the average value of the stability value A is expressed 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 the 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, liu Lili, yang Xieli, 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 Respectively representing original collagenAmount of protein and amount of residual collagen) of the collagen composition _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 The ratio, which is a measure of the relative in vivo retention properties of the above examples and controls, is larger, the greater the in vivo retention (longevity) property, and smaller, the weaker the in vivo retention (longevity) property. 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

0.26

0.49

0.56

0.25

-

Ratio of time to degradation

4.6±37%

5.8±62%

7.1±75%

3.4±51%

1.2±26%

9.5±83%

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

0.11

0.33

0.40

0.14

-

Ratio of degradation time

2.3±22%

3.5±47%

4.9±56%

1.7±36%

1.0±18%

7.2±68%

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

0.31

0.46

0.58

0.34

-

Ratio of time to degradation

5.2±32%

6.9±52%

8.2±70%

4.1±43%

1.3±28%

10.8±75%

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

0.22

0.41

0.48

0.24

-

Ratio of degradation time

3.0±23%

5.5±51%

6.8±68%

2.1±41%

1.2±26%

9.3±79%

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

0.33

0.51

0.63

0.31

-

Ratio of time to degradation

5.6±31%

7.6±55%

9.4±71%

4.1±47%

1.4±27%

11.6±84%

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

0.29

0.47

0.59

0.27

-

Ratio of time to degradation

5.4±32%

7.3±53%

9.2±68%

3.9±43%

1.3±24%

11.8±85%

TABLE 7

Example 7

Comparative example 7-1

Comparative example 7-2

Comparative example 7-3

Comparative examples 7 to 4

Comparative examples 7 to 5

Performance of needle passing

0.35

0.59

0.72

0.37

-

Ratio of time to degradation

6.9±25%

9.1±56%

10.7±72%

4.4±47%

1.4±23%

13.4±89%

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-lasting) performance than the control example without the addition of the surfactant, and the fluctuation of the in vivo retention (long-lasting) performance of the control example is extremely large;

3) The examples have better in vivo persistence (long-acting) properties than the control example in which tyrosinase was not (directly) added to the aqueous phase but to the oil phase, and the in vivo persistence (long-acting) properties of the control example were 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 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 product, 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 preparation method of a long-acting microparticle type I collagen implant with small effect period fluctuation and good needle penetration performance comprises the following steps:

(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 the emulsion after the collagen crosslinking to obtain microparticles, filtering to remove microparticles with a particle size of more than 25 μm and less than 0.5 μm, retaining microparticles with a particle size of between 0.5 μm and 25 μm, washing the microparticles with 10-40% aqueous ethanol solution, and removing the residues of the surfactant, the organic solvent, the sugar alcohol, the enzyme, etc.;

2. Preparation process according to claim 1, characterized in that the sugar alcohol is selected from the group consisting of sorbitol, mannitol, erythritol, maltitol, lactitol, xylitol, and combinations thereof.

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

4. The method according to claim 1, characterized in that the ratio of the amount of tyrosinase to the amount of collagen type i is 1-10U/g.

5. 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.

6. 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.

7. The method according to claim 1, wherein the volume ratio of the oil phase to the aqueous phase is 2.5 to 10.

8. 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.

9. 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.

10. 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.

11. A long-acting, low-fluctuation effective period, good needle penetration microparticle type I collagen implant, comprising: tyrosinase crosslinked type I collagen microparticles having a particle size of between 0.5 μm and 25 μm are used.