A METHOD OF PREPARING POLYMERIC MICROPARTICLES, POLYMERIC MICROPARTICLES, MEDICAL COMPOSITION, COSMETIC COMPOSITION, MEDICAL ARTICLES AND COSMETIC ARTICLES USING THE SAME

Abstract

According to the present disclosure, a method of preparing polymeric microparticles comprising further crosslinking with an organic crosslinking agent after crosslinking with metal ions, polymeric microparticles, medical compositions, cosmetic compositions, medical articles and cosmetic articles comprising the same can be provided.

Claims

1. A method of preparing polymeric microparticles comprising the steps of: subjecting a mixture containing a biocompatible polymer and a metal ion to a crosslinking reaction to form crosslinked polymer particles; and further crosslinking the crosslinked polymer particles in a polar solvent phase including an organic crosslinking agent containing at least one reactive functional group.

2. The method of claim 1, wherein: the step of subjecting a mixture containing a biocompatible polymer and a metal ion to a crosslinking reaction to form crosslinked polymer particles is performed in a polar solvent.

3. The method of claim 1, wherein: the step of subjecting a mixture containing a biocompatible polymer and a metal ion to a crosslinking reaction to form crosslinked polymer particles comprises forming an aqueous solution in which the biocompatible polymer is dissolved; adding a compound containing the metal ion to a polar solvent to form a solution containing the metal ion; and mixing droplets of the aqueous solution in which the biocompatible polymer is dissolved and the solution containing the metal ion to form a mixed solution.

4. The method of claim 3, wherein: the aqueous solution in which the biocompatible polymer is dissolved contains the biocompatible polymer in an amount of 0.01% by weight or more and 10% by weight or less with respect to the total weight of the aqueous solution in which the biocompatible polymer is dissolved.

5. The method of claim 3, wherein: the compound containing the metal ion is contained in an amount of 200 parts by weight or more and 1000 parts by weight or less with respect to 100 parts by weight of the biocompatible polymer.

6. The method of claim 1, wherein: in the step of further crosslinking the crosslinked polymer particles in a polar solvent phase including an organic crosslinking agent containing at least one reactive functional group, the organic crosslinking agent containing at least one reactive functional group is contained in an amount of 150 parts by weight or more and 1000 parts by weight or less with respect to 100 parts by weight of the biocompatible polymer.

7. (canceled)

8. The method of claim 1, wherein: the organic crosslinking agent containing at least one reactive functional group comprises at least one formyl group or epoxy group.

9. (canceled)

10. The method of claim 1, wherein: the biocompatible polymer comprises a mixture of hyaluronic acid and gelatin.

11. The method of claim 10, wherein: the mixture of hyaluronic acid and gelatin contains the gelatin in an amount of 50 parts by weight or more and 500 parts by weight or less with respect to 100 parts by weight of the hyaluronic acid.

12. Polymeric microparticles having a core-shell structure, comprising: a core including a first biocompatible polymer, a metal ion, and an organic crosslinking agent containing at least one reactive functional group; and a shell surrounding all or part of the core and including a second biocompatible polymer, a metal ion, and an organic crosslinking agent containing at least one reactive functional group.

13. The polymeric microparticles of claim 12, wherein: the core comprises a polymer matrix in which the first biocompatible polymer is crosslinked through the metal ion and the organic crosslinking agent containing at least one reactive functional group, and the shell comprises a polymer matrix in which the second biocompatible polymer is crosslinked through the metal ion and the organic crosslinking agent containing at least one reactive functional group.

14. The polymeric microparticles of claim 12, wherein: the first biocompatible polymer comprises hyaluronic acid, and the second biocompatible polymer comprises gelatin.

15. The polymeric microparticles of claim 13, wherein: the core contains a polymer matrix in which hyaluronic acid is crosslinked through the metal ion and the organic crosslinking agent containing at least one reactive functional group, in an amount of more than 50% by volume of with respect to the total volume of the polymer matrix contained in the core.

16. The polymeric microparticles of claim 13, wherein: the shell contains a polymer matrix in which gelatin is crosslinked through the metal ion and the organic crosslinking agent containing at least one reactive functional group, in an amount of more than 50% by volume with respect to the total volume of the polymer matrix contained in the shell.

17. (canceled)

18. The polymeric microparticles of claim 12, wherein: a thickness of the shell is 95% or less of a longest diameter of the polymeric microparticles, based on a cross section having the longest diameter of the polymeric microparticles.

19. The polymeric microparticles of claim 12, wherein: the organic crosslinking agent containing at least one reactive functional group comprises a crosslinking agent having 1 to 30 carbon atoms and containing at least one reactive functional group.

20. (canceled)

21. (canceled)

22. A medical composition comprising the polymeric microparticles of claim 12 and a pharmaceutically effective substance contained in the polymeric microparticles.

23. A cosmetic composition comprising the polymeric microparticles of claim 12 and a cosmetically effective substance contained in the polymeric microparticles.

24. A medical article comprising the medical composition of claim 22.

25. A cosmetic article comprising the cosmetic composition of claim 23.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0127] FIG. 1 is an optical microscope (OM) photograph of the polymeric microparticles of Example 1;

[0128] FIG. 2 is an IR photograph of the gelatin characteristic peak (1650 cm.sup.−1) of the polymeric microparticles of Example 1; and

[0129] FIG. 3 is an IR photograph of the hyaluronic acid characteristic peak (1080 cm.sup.−1) relative to the gelatin characteristic peak (1650 cm.sup.−1) of the polymeric microparticles of Example 1.

[0130] Hereinafter, the present disclosure will be described in more detail by way of examples. However, these examples are for illustrative purposes only, and the scope of the present disclosure is not limited thereby.

EXAMPLE 1

Preparation of Polymeric Microparticles

[0131] 200 mg of hyaluronic acid salt (weight average molecular weight: 500 kDa, maker: SK Bioland) was dissolved in 0.1N NaOH aqueous solution at 2 wt. %, and 250 mg of gelatin (gel strength: 300 g Bloom, maker: Sigma-Aldrich, product name G2500) was dissolved in distilled water at 2.5 wt. %. 10 mL of the respective solutions thus obtained were mixed to produce 20 mL of a mixed solution. To 80 mL of an ethanol solution added with 4 g of FeCl.sub.3 as a compound containing iron ions (Fe.sup.3+), the droplet of the mixed solution formed using an encapsulator (BUCHI, B-390) was added, subjected to a crosslinking reaction at 4° C. for 2 hours, and then washed with ethanol to prepare crosslinked particles.

[0132] 2.2 g of 1,4-butandiol diglycidyl ether (BDDE) was mixed with 80% ethanol solution containing 20% 0.1N NaOH aqueous solution, to which the crosslinked particles were then added and subjected to a crosslinking reaction at room temperature for 3 days to prepare polymeric microparticles. The prepared particles were washed in the order of ethanol and distilled water, and then the crosslinked particles were recovered using a sieve with a mesh size of 45 μm. The recovered crosslinked particles were filtered through a sieve with a mesh size of 500 μm, and the remaining crosslinked particles were analyzed.

[0133] An optical microscope (OM) photograph of the prepared polymeric microparticles is shown in FIG. 1.

[0134] An IR photograph of the gelatin characteristic peak (1650 cm.sup.−1) of the prepared polymeric microparticles is shown in FIG. 2. It was confirmed that the portion containing gelatin was brightly displayed due to the difference in intensity of the characteristic peak (1650 cm.sup.−1) of gelatin, and gelatin was distributed in the shell of the prepared polymeric microparticles.

[0135] An IR photograph of the hyaluronic acid characteristic peak (1080 cm.sup.−1) relative to the gelatin characteristic peak (1650 cm.sup.−1) of the prepared polymeric microparticles is shown in FIG. 3. It was confirmed that the portion containing hyaluronic acid in a high relative amount with respect to gelatin was displayed brightly, and hyaluronic acid was distributed in a relatively high amount compared to gelatin in the core of the prepared polymeric microparticles.

EXAMPLE 2

Preparation of Polymeric Microparticles

[0136] Polymeric microparticles were prepared in the same manner as in Example 1, except that an ethanol solution added with 4 g of AlCl.sub.3 as a compound containing aluminum ions (Al.sup.3+) was used instead of an ethanol solution added with 4 g of FeCl.sub.3 as a compound containing iron ions (Fe.sup.3+).

EXAMPLE 3

Preparation of Polymeric Microparticles

[0137] Polymeric microparticles were prepared in the same manner as in Example 1, except that 2.2 g of 50% glutaraldehyde was used instead of 2.2 g of 1,4-butandiol diglycidyl ether (BDDE).

Comparative Example 1

Preparation of Polymeric Microparticles

[0138] Hyaluronic acid salt (weight average molecular weight: 500 kDa, maker: SK Bioland) and gelatin (gel strength: 300 g Bloom, maker: Sigma-Aldrich, product name: G2500) were respectively dissolved in distilled water at 2 wt. % and 20 wt. % to prepare 5 ml each, and a mixed solution of these two solutions was mixed with a liquid paraffin solution to prepare a mixed solution containing a microemulsion. Then, 2.2 g of 1,4-butandiol diglycidyl ether (BDDE) as a crosslinking agent was added to the mixed solution, and subjected to a crosslinking reaction at room temperature for 5 days to prepare polymeric microparticles. The prepared particles were washed in the order of acetone, dichloromethane and distilled water, and then the crosslinked particles were recovered using a sieve with a mesh size of 45 μm. The recovered crosslinked particles were filtered through a sieve with a mesh size of 500 μm, and the remaining crosslinked particles were analyzed.

Comparative Example 2

Preparation of Polymeric Microparticles

[0139] Hyaluronic acid salt (weight average molecular weight: 500 kDa, maker: SK Bioland) was dissolved in 0.1N NaOH aqueous solution at 2 wt. %, and gelatin (gel strength: 300 g Bloom, maker: Sigma-Aldrich, product name G2500) was dissolved in distilled water at 2.5 wt. %. 10 mL of the respective solutions thus obtained were mixed to produce 20 mL. To 80 mL of an ethanol solution added with 4 g of FeCl.sub.3 as a compound containing iron ions (Fe.sup.3+), the droplet formed using an encapsulator (BUCHI, B-390) was added, subjected to a crosslinking reaction at 4° C. for 2 hours, and then washed with ethanol and distilled water to prepare crosslinked particles. The crosslinked particles were recovered using a sieve with a mesh size of 45 μm. The recovered crosslinked particles were filtered through a mesh sieve with a sieve size of 500 μm, and the remaining crosslinked particles were analyzed.

Comparative Example 3

Preparation of Polymeric Microparticles

[0140] 200 mg of hyaluronic acid salt (weight average molecular weight: 500 kDa, maker: SK Bioland) was dissolved in 0.1 N NaOH aqueous solution at a concentration of 2 wt. %, and 250 mg of gelatin (gel strength: 300 g Bloom, maker: Sigma-Aldrich, product name G2500) was dissolved in distilled water at a concentration of 2.5 wt. % to produce 10 mL each. Then, a mixed solution of these two solutions was mixed with a liquid paraffin solution to prepare a mixed solution containing a microemulsion. Then, 2.2 g of 1,4-butandiol diglycidyl ether (BDDE) as a crosslinking agent was added to the mixed solution, and subjected to a crosslinking reaction at room temperature for 5 days. The prepared particles were washed in the order of acetone, dichloromethane, and distilled water, and then the crosslinked particles were recovered using a sieve with a mesh size of 45 μm. The recovered crosslinked particles were filtered through a sieve with a mesh size of 500 μm, and the remaining crosslinked particles were analyzed.

Comparative Example 4

Preparation of Polymeric Microparticles

[0141] Hyaluronic acid salt (weight average molecular weight: 500 kDa, maker: SK Bioland) was dissolved in 0.1 N NaOH aqueous solution at a concentration of 2 wt. %, and gelatin (gel strength: 300 g Bloom, maker: Sigma-Aldrich, product name G2500) was dissolved in distilled water at a concentration of 2.5 wt. % to prepare 5 ml each. Then, a mixed solution of these two solutions was mixed with a liquid paraffin solution to prepare a mixed solution containing a microemulsion. Then, 2.2 g of 1,4-butandiol diglycidyl ether (BDDE) as a crosslinking agent was added to the mixed solution, and subjected to a crosslinking reaction at room temperature for 5 days. The prepared particles were washed in the order of acetone, dichloromethane, and distilled water, and then the crosslinked particles were recovered using a sieve with a mesh size of 45 μm. The recovered crosslinked particles were filtered through a mesh sieve with a sieve size of 500 μm, and the remaining crosslinked particles were analyzed.

Comparative Example 5

Preparation of Polymeric Microparticles

[0142] Hyaluronic acid salt (weight average molecular weight: 500 kDa, maker: SK Bioland) and gelatin (gel strength: 300 g Bloom, maker: Sigma-Aldrich, product name: G2500) were respectively dissolved in distilled water at 2 wt. % and 2.5 wt. % to produce 5 mL each. Then, a mixed solution of these two solutions was mixed with a liquid paraffin solution to prepare a mixed solution containing a microemulsion. Then, to the mixed solution, 2.2 g of 1,4-butandiol diglycidyl ether (BDDE) as a crosslinking agent was added to the mixed solution, and subjected to a crosslinking reaction at room temperature for 5 days. The prepared particles were washed in the order of acetone, dichloromethane, and distilled water, and then the crosslinked particles were recovered using a sieve with a mesh size of 45 μm. The recovered crosslinked particles were filtered through a sieve with a mesh size of 500 μm, and the remaining crosslinked particles were analyzed.

Comparative Example 6

Preparation of Polymeric Microparticles

[0143] Alginate (maker: Sigma-Aldrich, product name: 180947) and cellulose (maker: Sigma-Aldrich, product name: C5678) were respectively dissolved in distilled water at 2.5 wt. %, and 10 mL of the respective solutions thus obtained were mixed to produce 20 mL. To 80 mL of an ethanol solution added with 4 g of CaC.sub.2 as a compound containing calcium ion (Ca.sup.2+), the droplet formed using an encapsulator (BUCHI, B-390) was added, subjected to a crosslinking reaction at room temperature for 2 hours, and then washed with ethanol to prepare crosslinked particles.

[0144] 2.2 g of 1,4-butandiol diglycidyl ether (BDDE) was mixed with 80% ethanol solution containing 20% 0.1N NaOH aqueous solution, to which the crosslinked particles were then added and subjected to a crosslinking reaction at room temperature for 3 days to prepare polymeric microparticles. The prepared particles were washed in the order of ethanol and distilled water, and then the crosslinked particles were recovered using a sieve with a mesh size of 45 μm. The recovered crosslinked particles were filtered through a sieve with a mesh size of 500 μm, and the remaining crosslinked particles were analyzed.

Comparative Example 7

Preparation of Polymeric Microparticles

[0145] 200 mg of hyaluronic acid salt (weight average molecular weight: 500 kDa, maker: SK Bioland) was dissolved in 0.1 N NaOH aqueous solution at a concentration of 2 wt. %, and 250 mg of gelatin (gel strength: 300 g Bloom, maker: Sigma-Aldrich, product name G2500) was dissolved in distilled water at a concentration of 2.5 wt. %. 10 mL of the respective solutions thus obtained were mixed to produce 20 mL. To 80 mL of an ethanol solution added with 4 g of FeC13 as a compound containing iron ion (Fe.sup.3+), the droplet formed using an encapsulator (BUCHI, B-390) was added, and the mixture was subjected to a crosslinking reaction at 4° C. for 2 hours, and then washed with ethanol to prepare crosslinked particles.

[0146] The crosslinked particles were added to 80 mL of an ethanol solution added with 4 g of CaCl.sub.2 as a compound containing calcium ion (Ca.sup.2+), and subjected to a crosslinking reaction at 4° C. for 2 hours to prepare polymeric microparticles. The prepared particles were washed in the order of ethanol and distilled water, and then the crosslinked particles were recovered using a sieve with a mesh size of 45 μm. The recovered crosslinked particles were filtered through a sieve with a mesh size of 500 μm, and the remaining crosslinked particles were analyzed.

Experimental Example

Measurement of Physical Properties of Polymeric Microparticles

[0147] For the polymeric microparticles prepared in the above Examples and Comparative Examples, the average diameter, sphericity degree, strength, cell culture suitability, and stability of the polymeric microparticles were evaluated by the following methods.

[0148] 1. Average Diameter

[0149] The average diameter of the polymeric microparticles of Examples and Comparative Examples in distilled water was measured using a laser particle size analyzer (Horiba, Partica LA-960).

[0150] 2. Sphericity Degree

[0151] Optical (Olympus, BX53) photographs of the polymeric microparticles of Examples and Comparative Examples were taken, and the sphericity degree was calculated therefrom.

[0152] The sphericity degree according to the present disclosure was calculated as the average value of the ratio of the longest diameter to the shortest diameter (major axis/minor axis ratio) of 30 arbitrary particles in the optical photograph.

[0153] At this time, the closer the sphericity value 1 refers to the closer to the spherical shape.

[0154] 3. Strength

[0155] For the polymeric microparticles of Examples and Comparative Examples, the strength of the microparticles was measured using a texture analyzer equipment. 30 microparticles swollen with distilled water for 24 hours were placed as a single layer on the region under the flat cylindrical probe of the equipment equipped with a 5 N load cell. The initial trigger force was set to 1 mN, and the particles were compressed at a rate of 1 mm/s. The force when deformed to a level of 25% of the average particle diameter was defined as the compressive force.

[0156] The average compressive strength was calculated by dividing the compressive force by 30, which is the number of microparticles to be measured.

[0157] 4. Cell Culture Suitability

[0158] A cell culture medium was filled in a 6-well plate, polymeric microparticles and cells were added and the cells were cultured by plate-rock method. At this time, the temperature of the culture medium was maintained at 37° C., and the cells were cultured for 3 days, and the number of cells cultured with the polymeric microparticles was confirmed.

[0159] At this time, the cell culture suitability was evaluated based on the following criteria.

[0160] Suitable: when the number of cultured cells relative to the number of injected cells is 100% or more

[0161] Unsuitable: when the number of cultured cells relative to the number of injected cells is less than 100%, or when microparticles are decomposed during culture

[0162] 5. Stability

[0163] The stability of the polymeric microparticles placed in phosphate-buffered saline solution during the sterilization process using a high-temperature and high-pressure sterilizer (Autoclave) and the particle stability after long-term culture were evaluated based on the following criteria:

[0164] Suitable: when the weight reduction rate of the dried polymeric microparticles before and after use of autoclave is 20% or less

[0165] Unsuitable: when the weight reduction rate of the dried polymeric microparticles before and after use of autoclave exceeds 20%

TABLE-US-00001 TABLE 1 Average Average compressive diameter Sphericity strength Cell culture Category (μm) degree (mN) suitability Stability Example 1 317 ± 21 0.95 ± 0.08 2.10 Suitable Suitable Example 2 346 ± 26 0.96 ± 0.13 1.37 Suitable Suitable Example 3 319 ± 32 0.95 ± 0.05 0.37 Suitable Suitable Comparative Example 1 385 ± 13 0.98 ± 0.09 0.23 Suitable Unsuitable Comparative 193 ± 21 0.96 ± 0.03 0.10 Unsuitable Unsuitable Example 2 Comparative Particle preparation impossible Example 3 Comparative Particle preparation impossible Example 4 Comparative Particle preparation impossible Example 5 Comparative 301 ± 48 0.94 ± 0.11 0.29 Unsuitable Suitable Example 6 Comparative Example 7 194 ± 17 0.95 ± 0.04 0.12 Unsuitable Unsuitable

[0166] As shown in Table 1, it could be confirmed that in the polymeric microparticles of Examples, not only the number of cultured cells is 100% or more compared to the number of cells injected first, which is thus suitable for cell culture, but also the weight reduction rate of the dried polymeric microparticles before and after autoclave treatment appears to be 20% or less, which is thus suitable for sterilization treatment and long-term culture.

[0167] In addition, it could be confirmed that in the polymeric microparticles of Examples, the average compressive strength appears to be 0.37 mN or more, and thus excellent mechanical properties are realized and, at the same time, the proportion of particles exhibiting a high crosslinking density is high.

[0168] That is, the polymeric microparticles of Examples are suitable for cell culture, sterilization treatment, and long-term culture, and also can realize excellent crosslinking density and mechanical properties.

[0169] On the other hand, it could be confirmed that in the polymeric microparticles of Comparative Example 1, the weight reduction rate of the dried polymeric microparticles before and after autoclave treatment appears to be more than 20%, which is not suitable for sterilization and long-term culture, but also the average compressive strength appears to be 0.23 mN, indicating inferior mechanical properties, and the proportion of particles showing a low crosslinking density is high.

[0170] Moreover, it was observed that in the polymeric microparticles of Comparative Example 2, the polymer that was not crosslinked is dissolved in the distilled water washing step, so that the particles are contracted. Further, it could be confirmed that not only the microparticles are decomposed during cell culture, and the number of cultured cells compared to the number of injected cells is less than 100%, which is not suitable for cell culture, but also the weight reduction rate of the dried polymeric microparticles before and after autoclave treatment appears to be more than 20%, which is not suitable for sterilization treatment and long-term culture. Further, it could be confirmed that the average compressive strength appears to be 0.1 mN, showing inferior mechanical properties.

[0171] Further, it could be confirmed that in the polymeric microparticles of Comparative Examples 3 to 5, unlike Comparative Example 1, the concentration of the aqueous solution in which the biocompatible polymer was dissolved is adjusted to the same level as in Example 1, whereby the polymeric microparticles are not formed, and thus in the case of Examples, the polymeric microparticles can be formed even at a low biocompatible polymer concentration.

[0172] It could be confirmed that the polymeric microparticles of Comparative Example 6 exhibited an average compressive strength of 0.29 mN in distilled water state due to chemical crosslinking by the crosslinking agent, but they are not suitable for cell culture because alginate and cellulose, having no cell adhesion, are used as biocompatible polymers. In addition, by performing the crosslinking using calcium ions (Ca.sup.2+) as a metal ion, it can react reversibly with the calcium ion present in the cell culture medium, and thus, the particle strength may be lowered during cell culture.

[0173] It could be confirmed that as the polymeric microparticles of Comparative Example 7 were prepared only through ionic crosslinking, some microparticles are degraded during sterilization and cell culture, which are not suitable for cell culture.