CN115317668B

CN115317668B - Long-acting microparticle type I collagen implant

Info

Publication number: CN115317668B
Application number: CN202211000723.2A
Authority: CN
Inventors: 付劼; 周文虎; 韩淑萍; 陈文洁; 李贤明
Original assignee: Beijing Xihong Runmei Pharmaceutical Technology Co ltd
Current assignee: Beijing Xihong Runmei Pharmaceutical Technology Co ltd
Priority date: 2022-08-19
Filing date: 2022-08-19
Publication date: 2023-12-29
Anticipated expiration: 2042-08-19
Also published as: CN115317668A

Abstract

The invention discloses a microparticle type I collagen implant with improved performance and a preparation method thereof. The collagen implant comprises: tyrosinase cross-linked type i collagen microparticles were used. The implant has the advantages of long-acting, consistent and long-acting period (small fluctuation) and good needle penetrating property.

Description

Long-acting microparticle type I collagen implant

Technical Field

The present invention relates to an injection implant. In particular to a long-acting microparticle type I collagen implant with excellent performance.

Technical Field

Type I collagen is the most abundant collagen in humans. It forms large eosinophilic fibers, called collagen fibers. It is present in scar tissue and tissue healing at the end product is through repair, as well as tendons, ligaments, organic parts of the bones of the myofibrils of the intima, in the dermis, dentin and organ capsules.

Type I collagen has minimal effects in inflammation and antigenic response and has been approved by the food and drug administration in china, the united states for many types of medical applications, including wound dressings and artificial skin.

However, type I collagen has significant drawbacks such as short residence time in vivo.

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

CN 101648989 (long-acting collagen and method for producing the same): the invention discloses a long-acting collagen and a preparation method thereof, which are characterized in that a pigskin is scraped, grease is removed, swelling, digestion, centrifugal separation, salting out, lower layer sediment collection and freeze drying are carried out to obtain a collagen, then the collagen is mixed with gamma-polyglutamic acid (gamma-PGA), a glutaraldehyde solution is added and uniformly stirred, the first crosslinking is carried out, the glutaraldehyde solution is repeatedly added and uniformly stirred, and the second crosslinking is carried out, thus obtaining the long-acting collagen, which can solve the defects that the residence time of the conventional collagen is too short, frequent supplement and beating are needed, high concentration glutaraldehyde can be remained, the biotoxicity can harm the health of human body, and the like;

CN102924731a (a triple cross-linked collagen and method of manufacture and use): a method for producing triple-crosslinked 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 heavy cross-linked collagen with a second cross-linking agent to form double cross-linked collagen; and mixing the double-crosslinked collagen with a third crosslinking agent to form triple-crosslinked collagen. Wherein the first crosslinking agent, the second crosslinking agent, and the third crosslinking agent are each selected from the group consisting of: the cross-linking agent comprises 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, the above-mentioned techniques use chemical modification methods, which have remarkable effects, but involve the risk of formation of harmful substances during the modification process and the disadvantages of low reaction conditions, production of byproducts, poor specificity, low catalytic efficiency and yield, and loss of collagen during the process.

More importantly, the collagen is cross-linked in a homogeneous phase or cross-linked into a large particle, not a very small particle, and not a substantially uniform particle, but a smaller particle when used, and thus, the apparent therapeutic effect is inconsistent, such as a long duration of therapeutic effect.

In addition, the preparation is basically gel, and the high viscosity of the aqueous dispersion system causes a plurality of inconveniences in production and use, such as inaccurate dosage and split charging and poor needle penetrating property during injection.

Therefore, there is a real need for a type I collagen implant that has the advantages of long efficacy, consistent long duration (small fluctuation), good needle penetration, and the like.

Disclosure of Invention

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

The inventor discovers that the tyrosinase is used for modifying the common type I collagen into the microparticle crosslinked collagen with basically uniform size, so that the action time of the microparticle crosslinked collagen in the body can be prolonged, the effect period is consistent and long (the fluctuation is small), the viscosity of a water dispersion system of the microparticle crosslinked collagen is reduced, various problems caused in the production and use of the microparticle crosslinked collagen are solved, such as accurate split charging of the dosage, and the needle penetrating property is good during injection; in addition, the method can reduce the mixing of harmful substances, make the reaction condition mild, reduce the loss of collagen in the process, improve the specificity, avoid the generation of byproducts, improve the catalytic efficiency, improve the yield and the like.

Tyrosinase has a unique dual catalytic function that catalyzes the oxidation of monophenols to ortho dihydric phenols and further oxidizes dihydric phenols to o-benzoquinone. In the whole catalytic process, oxygen molecules act as electron acceptors, are reduced to water instead of hydrogen peroxide, and 1 mol/L oxygen molecules are required for oxidizing 1 mol/L monohydric phenol, while 0.5 mol/L oxygen molecules are required for oxidizing 1 mol/L dihydric phenol. The final released products of these 2 catalytic processes are all quinones, which are precursors to melanin formation. Thus, tyrosinase can oxidize tyrosine in protein side chains to quinone, thereby further crosslinking with lysine, tyrosine, and cysteine residues present in the protein.

Based on this, the present invention has been completed.

The invention relates to a particulate type I collagen implant with improved performance, long-acting and consistent duration (small fluctuation) and good needle penetration, which comprises the following components: tyrosinase cross-linked type i collagen microparticles were used.

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

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

(2) Preparing an aqueous solution, i.e. an aqueous phase, comprising type I collagen and tyrosinase and a sugar alcohol at a temperature of not more than 4 ℃;

(3) Mixing the aqueous solution with the solution of the organic solvent within 30 minutes after the preparation of the aqueous solution, and allowing the aqueous solution and the solution to form a water-in-oil emulsion, and maintaining the temperature at 4 ℃ to-4 ℃ for more than 24 hours, preferably for less than 48 hours, so as to substantially crosslink the collagen;

(4) Filtering the emulsion to obtain fine particles after the collagen is substantially crosslinked, and washing the fine particles with a 10-40% ethanol aqueous solution to remove residues such as the surfactant, the organic solvent, the sugar alcohol, and the enzyme;

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

Detailed Description

The particle size of the microparticles in the above-mentioned microparticle 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.

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

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

The above organic solvents include, but are not limited to, esters of C1-C6 acids with C1-C6 alcohols, such as ethyl acetate, methyl acetate, ethyl propionate, butyl acetate, pentyl valerate, isopentyl isovalerate, and the like; 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 are span-type surfactants such as span20, 40, 60 or 80, sugar ester surfactants such as glucose esters of C12 to C18 acids; propylene glycol esters of C12-C18 acids; C12-C18 glycerol (glycerol) esters; to co-operate in combination.

The above 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 prior to formation of the water-in-oil emulsion, and should not be added to the oil phase prior to or after formation of the water-in-oil emulsion, otherwise the type I collagen implant would have decreased or disappeared.

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

The aqueous solution should be mixed with the oily solution within 30 minutes, preferably within 10 minutes, and most preferably within 5 minutes after the completion of the preparation of the aqueous solution.

Preferably, in the process of filtering the emulsion to obtain particles, the particles with the particle diameters of more than 25 μm and less than 0.5 μm are filtered, and the particles with the particle diameters of between 0.5 μm and 25 μm are reserved; more preferably, particles having a particle size greater than 25 μm and less than 5 μm are filtered off, leaving particles having a particle size between 5 μm and 25 μm; most preferably, particles having a particle size of greater than 25 μm and less than 10 μm or particles having a particle size of greater than 15 μm and less than 5 μm are filtered off, particles having a particle size of between 10 μm and 25 μm are retained or particles having a particle size of between 5 μm and 15 μm are retained.

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

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

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

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

Examples

The following non-alternative examples, namely methods of making implants, further describe 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) 3% of surfactant at a preparation temperature of 4 ℃): ethyl acetate solution of SPAN 80 to obtain oil phase;

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

(3) Mixing said aqueous solution with said oil phase within 10 to 30 minutes after completion of said preparation of said aqueous solution, and vigorously stirring for 30 minutes to form a water-in-oil emulsion, maintaining the temperature at not more than 4 ℃ to 0 ℃ for 48 hours to substantially crosslink collagen, said oil phase and said aqueous phase having a volume ratio of 5;

(4) Filtering the emulsion after the collagen is substantially crosslinked to obtain microparticles: filtering out particles having a particle size of more than 25 μm and less than 0.5 μm with a filter membrane, retaining particles having a particle size of between 0.5 μm and 25 μm, washing the particles with a 15% aqueous ethanol solution, and removing residues such as the surfactant, the organic solvent, the sugar alcohol, and the enzyme;

(5) Freeze drying at-60- -80deg.C in vacuum freeze dryer for 48 hr to remove residual solvent.

15 batches were prepared in the same manner.

Example 2

(1) 5% of surfactant at preparation temperature 0 ℃): the solution of the oleic acid glucose ester in diethyl ether is used for obtaining an oil phase;

(2) Preparing an aqueous solution containing 3% of type I collagen, 8% of mannitol and tyrosinase at a temperature of 1 ℃, wherein the ratio of the amount of tyrosinase to the type I collagen is 7U/g, and obtaining a water phase;

(3) Mixing said aqueous solution with said oil phase within 5 to 10 minutes after completion of said preparation of said aqueous solution, and vigorously stirring for 60 minutes to form a water-in-oil emulsion, maintaining the temperature at 0 ℃ to-4 ℃ for 24 hours to substantially crosslink collagen, said oil phase and said water phase having a volume ratio of 3;

(4) Filtering the emulsion after the collagen is substantially crosslinked to obtain microparticles: filtering out particles having a particle size of more than 5 μm and less than 0.5 μm with a filter membrane, retaining particles having a particle size of between 0.5 μm and 5 μm, washing the particles with a 30% aqueous ethanol solution, and removing residues such as the surfactant, the organic solvent, the sugar alcohol, and the enzyme;

15 batches were prepared in the same manner.

Example 3

(1) Preparation temperature 0 ℃ containing 6% of surfactant: the acetone solution of the glyceride of C12 acid (lauric acid) is used for obtaining an oil phase;

(2) Preparing an aqueous solution containing 10% of type I collagen, 3% of erythritol and tyrosinase at a temperature of 1 ℃, wherein the ratio of the dosage of tyrosinase to the type I collagen is 10U/g, and obtaining a water phase;

(3) Mixing said aqueous solution with said oil phase within 1 to 5 minutes after completion of said preparation of said aqueous solution, and vigorously stirring for 40 minutes to form a water-in-oil emulsion, maintaining the temperature at 0 ℃ to-4 ℃ for 48 hours to substantially crosslink collagen, said oil phase and said water phase having a volume ratio of 8;

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

15 batches were prepared in the same manner.

Example 4

(1) 5% of surfactant at preparation temperature 0 ℃): a solution of propylene glycol stearyl (C18) acid and 0.1% of isoamyl isovalerate of TWEEN 80 to obtain an oil phase;

(2) Preparing an aqueous solution containing 2% of type I collagen, 6% of maltitol and tyrosinase at a temperature of 1 ℃, wherein the ratio of the amount of tyrosinase to the type I collagen is 5U/g, and obtaining a water phase;

(3) Mixing said aqueous solution with said oil phase within 5 to 25 minutes after completion of said preparation of said aqueous solution, and vigorously stirring for 60 minutes to form a water-in-oil emulsion, maintaining the temperature at 0 ℃ to-4 ℃ for 48 hours to substantially crosslink collagen, said oil phase and said water phase having a volume ratio of 6;

(4) Filtering the emulsion after the collagen is substantially crosslinked to obtain microparticles: filtering out particles with a particle size of more than 15 μm and less than 5 μm with a filter membrane, retaining particles with a particle size of between 5 μm and 15 μm, washing the particles with 15% ethanol aqueous solution, and removing residues such as the surfactant, the organic solvent, the sugar alcohol, and the enzyme;

15 batches were prepared in the same manner.

Example 5

(1) 3% of surfactant at preparation temperature of 0 ℃): butyl acetate solution of SPAN20 is used for obtaining an oil phase;

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

(3) Mixing said aqueous solution with said oil phase within 5 to 25 minutes after completion of said preparation of said aqueous solution, and vigorously stirring for 20 minutes to form a water-in-oil emulsion, maintaining the temperature at 0 ℃ to-4 ℃ for 48 hours to substantially crosslink collagen, said oil phase and said water phase having a volume ratio of 7.5;

(4) Filtering the emulsion after the collagen is substantially crosslinked to obtain microparticles: filtering out particles having a particle size of more than 25 μm and less than 5 μm with a filter membrane, retaining particles having a particle size of between 5 μm and 25 μm, washing the particles with a 25% aqueous ethanol solution, and removing residues such as the surfactant, the organic solvent, the sugar alcohol, and the enzyme;

15 batches were prepared in the same manner.

Example 6

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

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

(3) Mixing said aqueous solution with said oil phase within 5 to 25 minutes after completion of said preparation of said aqueous solution, and vigorously stirring for 50 minutes to form a water-in-oil emulsion, maintaining the temperature at 0 ℃ to-4 ℃ for 48 hours to substantially crosslink collagen, said oil phase and said water phase having a volume ratio of 8;

(4) Filtering the emulsion after the collagen is substantially crosslinked to obtain microparticles: filtering out particles with a particle size of more than 25 μm and less than 5 μm with a filter membrane, retaining particles with a particle size of between 5 μm and 25 μm, washing the particles with 35% ethanol aqueous solution, and removing residues such as the surfactant, the organic solvent, the sugar alcohol, and the enzyme;

15 batches were prepared in the same manner.

Example 7

(3) Mixing said aqueous solution with said oil phase within 5 to 10 minutes after completion of said preparation of said aqueous solution, and vigorously stirring for 60 minutes to form a water-in-oil emulsion, maintaining the temperature at 0 ℃ to-4 ℃ for 48 hours to substantially crosslink collagen, said oil phase and said water phase having a volume ratio of 3;

(4) Filtering the emulsion after the collagen is substantially crosslinked to obtain microparticles: filtering out particles having a particle size of more than 25 μm and less than 10 μm with a filter membrane, retaining particles having a particle size of between 10 μm and 25 μm, washing the particles with a 30% aqueous ethanol solution, and removing residues such as the surfactant, the organic solvent, the sugar alcohol, and the enzyme;

15 batches were prepared in the same manner.

Comparative examples 1 to 7-1

The same applies to the respective examples, except that particles having a particle size of greater than 25 μm or 5 μm or 10 μm or 15 μm or 20 μm (which is the same as the respective examples) and less than 0.5 μm or 5 μm or 10 μm or 15 μm (which is the same as the respective examples) are not filtered out.

Comparative examples 1 to 7-2

The same applies to the respective examples, except that particles having a particle diameter of greater than 25 μm or 5 μm or 10 μm or 15 μm or 20 μm (which are the same as those of the respective examples) are not filtered out.

Comparative examples 1 to 7-3

The same applies to the respective examples, except that 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 that of the respective examples) are not filtered out.

Comparative examples 1 to 7-4

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

Comparative examples 1 to 7-5

The examples were the same as the examples, except that no surfactant was added and no particles having a particle size of greater than 25 μm or 5 μm or 10 μm or 15 μm or 20 μm (which was the same as the examples) and less than 0.5 μm or 5 μm or 10 μm or 15 μm (which was the same as the examples) were filtered.

Test example 1 through needle performance test

Principle of: the better the needle performance, the less time is required 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 (8 mg) of the above-mentioned examples, control and commercial products type I collagen (8 mg, lyophilized preparation, shanxi Bo biomedical Co., ltd.) was taken, and the same amount (5 ml) of physiological saline for medical injection was added to prepare a suspension by reconstitution (reconstitution) in the same manner for the same time (as in the shaking method), and the same amount of the suspension was taken and put into the same syringe (needle was not changed, dried and reused after washing), and the syringe was pushed with the same constant pressure to complete the discharge of the suspension, and the required time was measured. Finally, the ratio of the time measured by the above examples and the control example to the time measured by the commercial product is calculated, and the needle passing performance of the above examples and the control example is measured by the ratio, 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 the above example, control and commercial type I collagen (8 mg, lyophilized preparation, shanxi Bo biomedical Co., ltd.) was taken, and the same amount (10 ml, concentration 0.08%) of physiological saline for medical injection (pH 7.0) was added to reconstitute the suspension in the same manner over the same time. Adding the same amount of collagenase (Bacillus cereus protease, purified and separated from Bacillus cereus) into the suspension, mixing the above collagen protease and collagen at a weight ratio of 4U/mg, placing into constant temperature and humidity environment with a temperature of 37deg.C and a relative humidity of 70%, and maintaining at a temperature of 0hr, 1 hr, 3 hr, 5 hr, 7 hr, 9 hr, 11 hr,13 hr, 15 hr, 17 hr, … … until the absorbance value A described below is substantially stable (fluctuation range less than 2%), the average value of the stable absorbance value A being expressed as A ₀ 。

After sampling, enzyme is immediately deactivated for 10min at 90 ℃, the temperature is reduced to room temperature, and the centrifugation is carried out for 10min at 4000 r/min, so that clear hydrolysate is obtained. Taking 5mL of the hydrolysate sample solution, adding 5mL of 15% (W/W) trichloroacetic acid (TCA) aqueous 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, taking 1mL of the diluent, adding 4mL of biuret reagent, uniformly mixing, and standing for 30min. Absorbance value a was measured at wavelength 540 nm. The concentration C (mg/mL) of polypeptide produced by degradation can be calculated according to a linear regression equation (see document 1: meat industry. 2011 (11), general phase 367, pages 21-24, liu Lili, yang Xieli, kinetics study of collagenase enzymatic hydrolysis of bovine bone collagen).

The absorbance value A (or the concentration C of the polypeptide produced by the degradation) reflects the amount of collagen that has been degraded in the sample solution ₀ -A (or C) ₀ -C，C ₀ Represents the average value A after the absorbance value A is basically stable ₀ The concentration of the polypeptide produced by the degradation, i.e., the concentration of the polypeptide produced by complete degradation of collagen, the amount of collagen remaining in the sample solution is reflected in the amount of collagen remaining in the sample solution, and since the degradation of the protease is characterized by the first order reaction at a lower concentration as known from the Emi equation (see document 1 above), ln (A) ₀ -A) (or ln (C) ₀ -C)) is plotted against time T, and the degradation slope k is determined by the slope k and the formula T _0.99 Complete degradation of collagen (99% degradation or residual 1%, t=ln (C) ₀ ＇/ C＇)/k, _t C＇/ C ₀ ' 0.01, wherein C ₀ 'and C' respectively represent the amount of initial collagen and the amount of residual collagen) time T _0.99 。

Finally, the time T for complete degradation of the collagen measured in the above examples and comparative examples is calculated _0.99 Time T of complete degradation of collagen measured with commercially available products _0.99 Ratio (in terms of its average value)The relative in vivo retention performance of the above examples and comparative examples was measured as the ratio, the greater the in vivo retention (long-acting) performance, and the smaller the ratio, the weaker the in vivo retention (long-acting) performance. The smaller the fluctuation of the ratio, the smaller the change of the in-vivo persistence (long-acting) performance of the collagen; the greater the fluctuation of this ratio, the greater the change in the in vivo persistence (long-acting) properties of collagen.

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

Needle passing performance

0.26

0.49

0.56

0.25

-

Degradation time ratio

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

Needle passing performance

0.11

0.33

0.40

0.14

-

Degradation time ratio

2.3±22%

3.5±47%

4.9±56%

1.7±36%

1.0±18%

7.2±68%

TABLE 3 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

Needle passing performance

0.31

0.46

0.58

0.34

-

Degradation timeRatio of

5.2±32%

6.9±52%

8.2±70%

4.1±43%

1.3±28%

10.8±75%

TABLE 4 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

Needle passing performance

0.22

0.41

0.48

0.24

-

Degradation time ratio

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

Needle passing performance

0.33

0.51

0.63

0.31

-

Degradation time ratio

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

Needle passing performance

0.29

0.47

0.59

0.27

-

Degradation time ratio

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

Needle passing performance

0.35

0.59

0.72

0.37

-

Degradation time ratio

6.9±25%

9.1±56%

10.7±72%

4.4±47%

1.4±23%

13.4±89%

The results show that:

1) The examples have a slower rate of enzymatic hydrolysis than the commercial products, significantly longer in vivo retention (long-lasting) performance;

2) The examples have better in vivo retention (long-acting) performance than the control examples without surfactant, which have extremely high in vivo retention (long-acting) performance variability;

3) The examples have better in vivo retention (long-acting) properties than the control examples in which tyrosinase is not added (directly) to the aqueous phase, but rather to the oil phase, which are very weak in vivo retention (long-acting);

4) The control examples, which had less filtering of larger and smaller particles, had better in vivo retention (long-term) performance and less volatility, which had very much fluctuation in vivo retention (long-term) performance;

5) The control examples, which were less prone to larger particle removal and which were only prone to smaller particle removal, had better in vivo retention (long-term) performance and less volatility;

6) The comparative example, which is less filtered of smaller particles and only filtered of larger particles, has better in vivo retention (long-acting) performance, longer in vivo retention (long-acting) performance, less volatility, and the comparative example has shorter in vivo retention (long-acting) performance and larger variability;

7) The examples have better needle penetration performance than commercial products, comparative examples that do not filter larger and smaller particles, and comparative examples that do not filter larger particles but only smaller particles; the examples have substantially the same needle passing performance as the control with only larger particles without smaller particles.

Claims

1. A method for preparing a microparticle type-I collagen implant with long-acting and small fluctuation of the effective period and good needle penetration, which comprises the following steps:

(4) Filtering the emulsion after the collagen is substantially crosslinked to obtain microparticles: filtering out particles having a particle size of more than 25 μm and less than 10 μm with a filter membrane, retaining particles having a particle size of between 10 μm and 25 μm, washing the particles with a 30% aqueous ethanol solution, and removing the surfactant, the organic solvent, the sugar alcohol, and the enzyme residue;