KR102645182B1 - Preparing method for gelatin crosslinked particle - Google Patents
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- 239000002245 particle Substances 0.000 title claims abstract description 114
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000004132 cross linking Methods 0.000 claims abstract description 18
- 238000010257 thawing Methods 0.000 claims abstract description 12
- 238000007710 freezing Methods 0.000 claims description 22
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- 238000004519 manufacturing process Methods 0.000 claims description 20
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 12
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 5
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- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 6
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- 238000004807 desolvation Methods 0.000 description 4
- 238000013178 mathematical model Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
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- JUJBNYBVVQSIOU-UHFFFAOYSA-M sodium;4-[2-(4-iodophenyl)-3-(4-nitrophenyl)tetrazol-2-ium-5-yl]benzene-1,3-disulfonate Chemical compound [Na+].C1=CC([N+](=O)[O-])=CC=C1N1[N+](C=2C=CC(I)=CC=2)=NC(C=2C(=CC(=CC=2)S([O-])(=O)=O)S([O-])(=O)=O)=N1 JUJBNYBVVQSIOU-UHFFFAOYSA-M 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
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- 102000008186 Collagen Human genes 0.000 description 1
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- AZKVWQKMDGGDSV-BCMRRPTOSA-N Genipin Chemical compound COC(=O)C1=CO[C@@H](O)[C@@H]2C(CO)=CC[C@H]12 AZKVWQKMDGGDSV-BCMRRPTOSA-N 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 229940117975 chromium trioxide Drugs 0.000 description 1
- WGLPBDUCMAPZCE-UHFFFAOYSA-N chromium trioxide Inorganic materials O=[Cr](=O)=O WGLPBDUCMAPZCE-UHFFFAOYSA-N 0.000 description 1
- GAMDZJFZMJECOS-UHFFFAOYSA-N chromium(6+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[Cr+6] GAMDZJFZMJECOS-UHFFFAOYSA-N 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000013270 controlled release Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
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- 125000000524 functional group Chemical group 0.000 description 1
- AZKVWQKMDGGDSV-UHFFFAOYSA-N genipin Natural products COC(=O)C1=COC(O)C2C(CO)=CCC12 AZKVWQKMDGGDSV-UHFFFAOYSA-N 0.000 description 1
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- 238000002844 melting Methods 0.000 description 1
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- 239000002953 phosphate buffered saline Substances 0.000 description 1
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical compound OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 description 1
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- 231100000331 toxic Toxicity 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5169—Proteins, e.g. albumin, gelatin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5192—Processes
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- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Nanotechnology (AREA)
- Optics & Photonics (AREA)
- Physics & Mathematics (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
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Abstract
본 발명은 냉동된 젤라틴 입자를 해동시키면서 가교하는 단계를 포함함으로써, 작교 균질한 젤라틴 가교입자를 제조하는 방법에 관한 것이다.The present invention relates to a method of producing homogeneous cross-linked gelatin particles by including the step of cross-linking frozen gelatin particles while thawing them.
Description
본 발명은 젤라틴 가교입자의 제조 방법에 관한 것이다.The present invention relates to a method for producing crosslinked gelatin particles.
최근 수십 년 동안 나노입자는 작은 크기, 안정성 및 생체적합성으로 인해 약물 전달 시스템 및 조직 공학을 비롯한 다양한 생물 의학 분야에서 주목을 받아왔다. 특히, 고분자 기반 나노입자는 다른 나노입자 시스템에 비해 캡슐화 공정이 더 발달되어 질병 표적화에 더 높은 효율을 보였다. In recent decades, nanoparticles have received attention in various biomedical fields, including drug delivery systems and tissue engineering, due to their small size, stability, and biocompatibility. In particular, polymer-based nanoparticles have a more advanced encapsulation process compared to other nanoparticle systems, showing higher efficiency in targeting diseases.
이러한 나노입자의 표적화 효율은 주로 입자 크기, 표면 전하 및 표면의 변형에 의해 영향을 받으며, 특히, 나노입자의 크기는 세포막과의 상호작용과 생물학적 장벽의 침투를 결정하는 가장 중요한 요인 중 하나로 알려져 있다. 예를 들어, 100 nm 미만의 크기인 나노입자는 뇌-혈액 장벽과 같은 다양한 생물학적 장벽을 관통할 수 있다.The targeting efficiency of these nanoparticles is mainly influenced by particle size, surface charge, and surface modification. In particular, the size of nanoparticles is known to be one of the most important factors determining interaction with cell membranes and penetration of biological barriers. . For example, nanoparticles less than 100 nm in size can penetrate various biological barriers, such as the brain-blood barrier.
천연고분자인 젤라틴은 나노입자 제조 재료로서 친수성, 생분해성, 무독성, 생체적합성, 낮은 가격 등 많은 장점을 가지고 있다. 이러한 다양한 장점 때문에 많은 연구자들이 다양한 생의학 응용 분야에 젤라틴 기반 나노입자의 사용을 연구해왔다. 그러나, 이전 연구에서 일반적인 탈용매화 방법을 사용해 제조된 젤라틴 입자는 입자가 불안정하고 응집되기 쉬운 경향이 있었다. 이러한 한계를 극복하기 위해 응집되지 않고 안정적인 작은 젤라틴 입자를 제조할 수 있는 2단계(침전-가교) 탈용매화방법이 보고되었으나, 기존의 2단계 탈용매화 방법으로 제조된 젤라틴 입자의 평균 크기는 150~300 nm로 사용상의 제약이 있었다. 예를 들어, 100 nm 보다 큰 나노입자는 간과 비장에 대량으로 축적되는 문제가 있다.Gelatin, a natural polymer, is a material for manufacturing nanoparticles and has many advantages such as hydrophilicity, biodegradability, non-toxicity, biocompatibility, and low price. Because of these various advantages, many researchers have studied the use of gelatin-based nanoparticles in various biomedical applications. However, in previous studies, gelatin particles prepared using common desolvation methods tended to make the particles unstable and prone to agglomeration. To overcome these limitations, a two-step (precipitation-crosslinking) desolvation method that can produce stable small gelatin particles without agglomeration has been reported. However, the average size of gelatin particles prepared by the existing two-step desolvation method is 150~150. There were limitations in use at 300 nm. For example, nanoparticles larger than 100 nm have the problem of accumulating in large quantities in the liver and spleen.
한국공개특허 제2010-0081291호는 침전반응을 통한 젤라틴 나노입자의 제조방법을 제시하고 있으나, 상기의 문제점을 해결하지는 못하고 있다.Korean Patent Publication No. 2010-0081291 proposes a method for producing gelatin nanoparticles through precipitation reaction, but does not solve the above problems.
이에, 작고 균질한 젤라틴 입자를 제조하는 방법에 대한 연구가 필요하다.Accordingly, research on methods for producing small and homogeneous gelatin particles is needed.
본 발명은 작고 균질한 젤라틴 가교입자의 제조 방법을 제공하는 것을 목적으로 한다.The purpose of the present invention is to provide a method for producing small and homogeneous crosslinked gelatin particles.
1. 냉동된 젤라틴 입자를 해동시키면서 가교하는 단계를 포함하는 젤라틴 가교입자의 제조 방법.1. A method for producing crosslinked gelatin particles comprising the step of crosslinking frozen gelatin particles while thawing them.
2. 위 1에 있어서, 상기 해동은 냉장 하에 수행되는 젤라틴 가교입자의 제조 방법.2. The method of producing cross-linked gelatin particles according to 1 above, wherein the thawing is performed under refrigeration.
3. 위 1에 있어서, 상기 가교는 글루타르알데하이드와의 반응으로 수행되는 젤라틴 가교입자의 제조 방법.3. The method for producing crosslinked gelatin particles according to 1 above, wherein the crosslinking is performed by reaction with glutaraldehyde.
4. 위 1에 있어서, 젤라틴 용액으로부터 젤라틴 침전액을 얻고, 이를 냉동하여 상기 냉동된 젤라틴 입자를 얻는 단계를 더 포함하는, 젤라틴 가교입자의 제조 방법.4. The method of producing crosslinked gelatin particles according to 1 above, further comprising obtaining a gelatin precipitate from a gelatin solution and freezing it to obtain the frozen gelatin particles.
5. 위 4에 있어서, 상기 용액의 용매는 물이며, 상기 침전은 아세톤을 첨가하여 수행되는 젤라틴 가교입자의 제조 방법.5. In item 4 above, the solvent of the solution is water, and the precipitation is performed by adding acetone.
6. 위 1에 있어서, 젤라틴 용액에서 젤라틴을 침전시키고, 그 침전물을 재용해시키고 산 첨가 후에 재침전시켜 젤라틴 침전액을 얻고, 이를 냉동하여 상기 냉동된 젤라틴 입자를 얻는 단계를 더 포함하는 젤라틴 가교입자의 제조 방법.6. Gelatin cross-linking according to 1 above, further comprising the step of precipitating gelatin from a gelatin solution, re-dissolving the precipitate, re-precipitating after adding acid to obtain a gelatin precipitate, and freezing the gelatin solution to obtain the frozen gelatin particles. Method for producing particles.
7. 위 1에 있어서, 가교된 젤라틴 입자에 유기 용매를 첨가하고 원심분리하여 젤라틴 가교입자를 정제하는 단계를 더 포함하는 젤라틴 가교입자의 제조 방법.7. The method of producing cross-linked gelatin particles according to 1 above, further comprising the step of purifying the cross-linked gelatin particles by adding an organic solvent to the cross-linked gelatin particles and centrifuging.
본 발명의 실시예에 따른 젤라틴 가교입자의 제조 방법은 작고 균질한 젤라틴 가교입자를 제조할 수 있다.The method for producing cross-linked gelatin particles according to an embodiment of the present invention can produce small and homogeneous cross-linked gelatin particles.
본 발명의 실시예에 따른 젤라틴 가교입자는 세포독성이 없다.Gelatin cross-linked particles according to an embodiment of the present invention are not cytotoxic.
본 발명의 실시예에 따른 젤라틴 가교입자는 약물 전달 시스템 및 조직 공학 등 생체의학 분야에 적용될 수 있다.Gelatin cross-linked particles according to embodiments of the present invention can be applied to biomedical fields such as drug delivery systems and tissue engineering.
도 1은 기존 젤라틴 입자 제조 방법과 실시예의 제조 방법을 도식화하여 비교한다.
도 2는 기존 젤라틴 입자 제조 방법으로 제조된 젤라틴 입자와 실시예의 제조 방법으로 제조된 젤라틴 입자의 SEM 관찰 결과(2a, 2b) 및 크기 분포를 비교한다(2c, 2d).
도 3은 젤라틴과 실시예의 제조 방법으로 제조된 젤라틴 입자의 FT-IR 분석 결과를 나타낸다.
도 4는 실시예의 제조 방법으로 제조된 젤라틴 입자의 농도에 따른 조골세포 및 섬유아세포의 세포생존률을 나타낸다.
도 5는 실시예의 제조 방법으로 제조된 젤라틴 입자의 트리판블루 농도에 따른 약물방출 프로파일을 나타낸다.
도 6은 실시예의 제조 방법으로 제조된 젤라틴 입자의 실제 약물방출 프로파일과 수학적 모델에 따른 약물방출 프로파일을 비교한다. Figure 1 schematically compares the existing gelatin particle manufacturing method with the manufacturing method of the example.
Figure 2 compares the SEM observation results (2a, 2b) and size distribution (2c, 2d) of gelatin particles manufactured by the existing gelatin particle manufacturing method and gelatin particles manufactured by the manufacturing method of the example.
Figure 3 shows the results of FT-IR analysis of gelatin and gelatin particles prepared by the manufacturing method of the example.
Figure 4 shows the cell survival rate of osteoblasts and fibroblasts according to the concentration of gelatin particles prepared by the manufacturing method of the example.
Figure 5 shows the drug release profile according to trypan blue concentration of gelatin particles prepared by the preparation method of the example.
Figure 6 compares the drug release profile according to the mathematical model with the actual drug release profile of gelatin particles produced by the manufacturing method of the example.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 냉동된 젤라틴 입자를 해동시키면서 가교하는 단계를 포함하는 젤라틴 가교입자의 제조 방법에 관한 것이다.The present invention relates to a method for producing crosslinked gelatin particles, comprising the step of crosslinking frozen gelatin particles while thawing them.
상기 젤라틴은 동물의 가죽·힘줄·연골 등을 구성하는 천연 단백질인 콜라겐에서 얻어지는 유도 단백질의 일종을 의미한다. 젤라틴은 제조공정에서 원료를 산 처리 하여 얻어지는 A타입과, 염기 처리하여 얻어지는 B타입으로 구별되며, 본 발명의 젤라틴은 A타입 또는 B타입의 젤라틴일 수 있다.The gelatin refers to a type of derived protein obtained from collagen, a natural protein that constitutes animal skin, tendons, and cartilage. Gelatin is divided into type A, which is obtained by treating the raw material with acid during the manufacturing process, and type B, which is obtained by treating the raw material with base. The gelatin of the present invention may be type A or type B gelatin.
상기 젤라틴 입자는 젤라틴이 알갱이 형태로 있는 것을 의미하며, 예를 들면 젤라틴을 용해시킨 후 침전 반응을 통해 얻을 수 있다. The gelatin particles refer to gelatin in the form of granules, and can be obtained, for example, through precipitation reaction after dissolving gelatin.
상기 냉동은 젤라틴 입자의 온도를 상온 보다 낮은 온도로 내리는 것을 의미한다. 상기 냉동된 젤라틴 입자는 젤라틴 입자의 부피가 온도 감소로 인하여 상온에서의 부피보다 줄어든 상태이면 특별히 제한되지 않으며, 냉동되는 온도는 극저온도를 포함한다. 냉동은 급속 냉동일 수 있으며, 급속 냉동은 물질의 온도를 급속하게 떨어트려 보존하는 방법을 의미한다. 급속 냉동은 예를 들면 급속 냉동 기능이 구비된 냉동고를 통해 수행할 수 있고, 급속 냉동의 방법은 예를 들면 찬 공기를 이용하는 송풍 냉동, 선반에 냉매를 순환시키는 접촉 냉동, 또는 액체 질소를 쓰는 극저온 냉동법일 수 있으나, 이에 제한되는 것은 아니다.The freezing means lowering the temperature of the gelatin particles to a temperature lower than room temperature. The frozen gelatin particles are not particularly limited as long as the volume of the gelatin particles is reduced from the volume at room temperature due to a decrease in temperature, and the freezing temperature includes extremely low temperatures. Freezing can be quick freezing, and quick freezing refers to a method of preserving a material by rapidly dropping its temperature. Quick freezing can be performed, for example, through a freezer equipped with a quick freezing function, and methods of quick freezing include, for example, blast freezing using cold air, contact freezing by circulating refrigerant on a shelf, or cryogenic freezing using liquid nitrogen. It may be a freezing method, but is not limited thereto.
냉동된 젤라틴 입자는 예를 들면 용매에 분산된 젤라틴 입자를 냉동시킨 것일 수 있다. 그 경우, 용매에 분산된 젤라틴 입자를 용매의 녹는점 이하의 온도로 냉동시킨 것일 수 있다. 용매는 예를 들면 물일 수 있다.Frozen gelatin particles may be, for example, frozen gelatin particles dispersed in a solvent. In that case, the gelatin particles dispersed in the solvent may be frozen at a temperature below the melting point of the solvent. The solvent may be water, for example.
상기 해동은 젤라틴 입자의 온도를 상기 냉동된 온도와 상온 사이의 온도로 올리는 것을 의미하며, 따라서 상기 해동시키면서 가교하는 단계는 젤라틴 입자가 냉동 전 상태로 완전히 돌아가지 않은 상태에서 가교가 이루어짐을 의미한다. 해동은 예를 들면 냉장 하에 수행될 수 있으며, 냉장 온도는 예를 들면 0 ℃ 내지 10 ℃일 수 있다. The thawing means raising the temperature of the gelatin particles to a temperature between the frozen temperature and room temperature. Therefore, the step of crosslinking while thawing means that the crosslinking is performed in a state where the gelatin particles have not completely returned to their pre-frozen state. . Thawing may be performed, for example, under refrigeration, and the refrigeration temperature may be, for example, 0° C. to 10° C.
상기 가교는 젤라틴 입자를 이루는 사슬모양의 젤라틴 분자가 화학결합에 의해 서로 연결되는 것을 의미한다. 상기 젤라틴을 가교하는 단계는 당업자가 통상의 지식에 의해 적절히 선택할 수 있는 가교결합 방법을 통해 수행될 수 있으며 특별히 제한되지 않는다. 예를 들면, 젤라틴의 가교결합은 글루타르알데하이드 또는 제니핀과의 반응으로 수행될 수 있다.The crosslinking means that the chain-shaped gelatin molecules that make up the gelatin particles are connected to each other by chemical bonds. The step of crosslinking the gelatin may be performed through a crosslinking method that can be appropriately selected by a person skilled in the art based on his or her common knowledge, and is not particularly limited. For example, crosslinking of gelatin can be accomplished by reaction with glutaraldehyde or genipin.
냉동된 젤라틴 입자가 용매에 분산된 젤라틴 입자를 냉동시킨 것이라면, 냉동 상태로 가교제와 반응되기는 어려우므로, 이를 해동시키면서 용매에서 가교제와 젤라틴 입자가 접촉할 수 있도록 하여 가교시킬 수 있다. 젤라틴 입자가 완전히 해동되고 온도가 상승한다면 부피가 다시 팽창될 것이므로, 가교는 완전히 해동되기 전에 수행되는 것이고, 냉동된 젤라틴 입자가 용매에 분산된 젤라틴 입자를 냉동시킨 것이라면, 가교는 완전히 해동되기 전, 예를 들면 살얼음이 끼어 있는 상태에서 수행될 수 있다.If the frozen gelatin particles are frozen gelatin particles dispersed in a solvent, it is difficult to react with the cross-linking agent in a frozen state, so cross-linking can be achieved by thawing the gelatin particles and allowing the cross-linking agent to come into contact with the gelatin particles in the solvent. If the gelatin particles are completely thawed and the temperature rises, the volume will expand again, so cross-linking is performed before complete thawing. If the frozen gelatin particles are frozen gelatin particles dispersed in a solvent, cross-linking is performed before completely thawing. For example, it can be performed in the presence of thin ice.
본 발명은 젤라틴 용액으로부터 젤라틴 침전액을 얻고, 이를 냉동하여 상기 냉동된 젤라틴 입자를 얻는 단계를 포함할 수 있다.The present invention may include the step of obtaining a gelatin precipitate from a gelatin solution and freezing it to obtain the frozen gelatin particles.
상기 침전액은 젤라틴 용액에서 젤라틴이 포화에 도달하여 용액 속에 나온 상태를 의미하며, 젤라틴 용액으로부터 젤라틴 침전액을 얻는 방법은 당업자가 통상의 지식에 의해 적절히 선택할 수 있는 침전 반응을 통해 수행될 수 있으며 특별히 제한되지 않는다. 예를 들면 젤라틴 침전 반응은 젤라틴 용액에 침전 시약을 첨가하여 일어날 수 있다. 젤라틴 용액은 예를 들면 젤라틴을 40 ℃ 내지 60 ℃의 용매(ex. 물)에 녹이는 방법으로 얻을 수 있으며, 침전 시약은 예를 들면 젤라틴 수용액일 때, 아세톤, 에탄올, 피크린산용액 및 삼산화크롬용액 중에서 선택된 것일 수 있으나 이에 제한되는 것은 아니다. 상기 침전 반응에 사용되는 용매 및 시약의 양, pH 및 온도 등은 실시 목적에 따라 당업자에 의해 적절히 선택될 수 있다.The precipitate refers to a state in which gelatin reaches saturation in a gelatin solution and comes out of the solution. The method of obtaining a gelatin precipitate from a gelatin solution can be performed through a precipitation reaction that can be appropriately selected by a person skilled in the art based on his or her ordinary knowledge. There are no particular restrictions. For example, gelatin precipitation reaction can occur by adding a precipitation reagent to a gelatin solution. A gelatin solution can be obtained, for example, by dissolving gelatin in a solvent (e.g. water) at 40°C to 60°C, and the precipitation reagent may be, for example, an aqueous gelatin solution, acetone, ethanol, picric acid solution, or chromium trioxide solution. It may be selected, but is not limited thereto. The amount of solvent and reagent used in the precipitation reaction, pH, temperature, etc. may be appropriately selected by a person skilled in the art depending on the purpose of implementation.
상기 냉동은 전술한 냉동과 동일한 의미를 가진다.The freezing has the same meaning as the above-mentioned freezing.
본 발명은 젤라틴 용액에서 젤라틴을 침전시키고, 그 침전물을 재용해시키고 산 첨가 후에 재침전시켜 젤라틴 침전액을 얻고, 이를 냉동하여 상기 냉동된 젤라틴 입자를 얻는 단계를 포함할 수 있다.The present invention may include the steps of precipitating gelatin from a gelatin solution, re-dissolving the precipitate and re-precipitating it after adding acid to obtain a gelatin precipitate, and freezing the gelatin solution to obtain the frozen gelatin particles.
상기 젤라틴의 침전 및 냉동은 전술한 젤라틴의 침전 및 냉동과 동일한 의미를 가진다.The precipitation and freezing of gelatin have the same meaning as the precipitation and freezing of gelatin described above.
침전 반응이 수행되기 전의 젤라틴 용액의 pH는 침전을 통해 생성된 젤라틴 입자의 형태 및 크기에 영향을 미친다. 상기 산 첨가는 젤라틴 용액의 pH를 낮춤으로써 용액 속 젤라틴 분자의 표면 전하를 높이며, 이로 인한 젤라틴 분자 사이의 반발력이 침전 반응 시 생성되는 젤라틴 입자의 형태를 균일하고 작게 할 수 있다. 상기 산 첨가를 통해 얻어지는 젤라틴 용액의 pH는 예를 들면 2 내지 4일 수 있으며, 구체적으로 3일 수 있다. 상기 산은 HCl일 수 있으나 이에 제한되는 것은 아니다.The pH of the gelatin solution before the precipitation reaction is performed affects the shape and size of gelatin particles produced through precipitation. The addition of the acid lowers the pH of the gelatin solution, thereby increasing the surface charge of the gelatin molecules in the solution, and the resulting repulsive force between gelatin molecules can make the shape of the gelatin particles generated during the precipitation reaction uniform and small. The pH of the gelatin solution obtained through the addition of the acid may be, for example, 2 to 4, and specifically 3. The acid may be HCl, but is not limited thereto.
본 발명은 이후 가교된 젤라틴 입자에 유기 용매를 첨가하고, 원심분리하여 젤라틴 가교입자를 정제하는 단계를 포함할 수 있다.The present invention may then include the step of adding an organic solvent to the cross-linked gelatin particles and purifying the cross-linked gelatin particles by centrifugation.
상기 유기 용매는 아세톤, 아세토니트릴, 클로로포름, 메탄올, 에탄올 중에서 선택된 것일 수 있으나 이에 제한되는 것은 아니다.The organic solvent may be selected from acetone, acetonitrile, chloroform, methanol, and ethanol, but is not limited thereto.
상기 정제는 냉장 하에 수행될 수 있다. 상기 냉장 온도는 예를 들면 0 ℃ 내지 10 ℃일 수 있다.The purification can be performed under refrigeration. The refrigeration temperature may be, for example, 0°C to 10°C.
본 발명에서 후술하는 약물방출 프로파일은 젤라틴 입자에 약물이 로드될 경우 약물이 방출되는 속도와 경향성을 분석한 것으로서, 본 발명의 젤라틴 입자를 약물 전달 시스템에 사용할 경우 이를 바탕으로 입자의 적정량 사용과 방출 조절이 가능하도록 돕기 위하여 기재되었다.The drug release profile described later in the present invention analyzes the rate and tendency of drug release when the drug is loaded into gelatin particles. When the gelatin particles of the present invention are used in a drug delivery system, the appropriate amount of particles is used and released based on this. It was written to help make control possible.
이하, 본 발명의 이해를 돕기 위하여 구체적인 실시예를 제시하나, 이는 본 발명을 예시하는 것일 뿐 첨부된 특허청구범위를 제한하는 것이 아니며, 본 발명의 범주 및 기술사상 범위 내에서 실시예에 대한 다양한 변경 및 수정이 가능함은 당업자에게 있어서 명백한 것이며, 이러한 변형 및 수정이 첨부된 특허청구범위에 속하는 것도 당연한 것이다. Hereinafter, specific examples are presented to aid understanding of the present invention, but these are only illustrative of the present invention and do not limit the scope of the appended claims, and various embodiments are included within the scope and technical idea of the present invention. It is obvious to those skilled in the art that changes and modifications are possible, and it is natural that such changes and modifications fall within the scope of the appended patent claims.
실험Experiment
1. 냉동-해동 방법을 통한 젤라틴 가교입자의 제조1. Production of gelatin crosslinked particles through freeze-thaw method
젤라틴 A타입 (Sigma, 1.25g)을 50 ℃에서 25 mL의 물에 용해하였다. 젤라틴이 충분히 용해된 후, 25 mL의 아세톤을 첨가한 다음, 혼합액을 빠르게 저어 젤라틴의 침전을 빠르게 하였다. 그 후 상층액을 제거하여 높은 몰 질량의 젤라틴 침전물만을 남겼다. 침전물을 다시 50 ℃에서 25 mL의 물에 용해한 후, HCl 첨가를 통해 혼합액의 pH를 3으로 조정하였다. 그 후 75 mL의 아세톤(Sigma)을 첨가하고 40 ℃에서 600 rpm으로 회전시킨 후, 젤라틴을 압축하기 위해, 젤라틴 용액을 초저온냉동고에서 빠르게 냉동시켰다. 1시간 후, 냉동된 젤라틴 용액을 녹이고 4 ℃의 저온냉장쳄버에서 저어주었다. 그 후 젤라틴 입자의 가교 결합 형성을 위해 280 μL 의 글루타르알데하이드 (25%, v/v)를 첨가하였다. 그 후 젤라틴 입자를 75%의 아세톤 용액에 용해한 후, 4 ℃에서 6500 rpm으로 3회 원심분리하여 젤라틴 가교입자를 정제하였다. 이후 3일 동안 젤라틴 가교입자를 동결 건조하였다.Gelatin type A (Sigma, 1.25 g) was dissolved in 25 mL of water at 50°C. After the gelatin was sufficiently dissolved, 25 mL of acetone was added, and the mixture was quickly stirred to accelerate precipitation of the gelatin. The supernatant was then removed, leaving only a high molar mass gelatin precipitate. The precipitate was again dissolved in 25 mL of water at 50°C, and the pH of the mixture was adjusted to 3 by adding HCl. Afterwards, 75 mL of acetone (Sigma) was added and the solution was rotated at 600 rpm at 40°C, and then the gelatin solution was quickly frozen in an ultra-low temperature freezer to compress the gelatin. After 1 hour, the frozen gelatin solution was thawed and stirred in a low-temperature refrigerated chamber at 4°C. Afterwards, 280 μL of glutaraldehyde (25%, v/v) was added to form cross-links of gelatin particles. Afterwards, the gelatin particles were dissolved in a 75% acetone solution and centrifuged three times at 6500 rpm at 4°C to purify the gelatin crosslinked particles. Afterwards, the gelatin cross-linked particles were freeze-dried for 3 days.
2. 젤라틴 가교입자의 SEM 분석2. SEM analysis of gelatin cross-linked particles
젤라틴 가교입자의 크기를 ImageJ 소프트웨어를 사용한 주사전자현미경(SEM) (JSM-7500F) 으로 관찰하였다. PerkinElmer Spectrum 400 기구를 사용하여 푸리에변환적외선분광법(FT-IR)을 수행하였다.The size of gelatin cross-linked particles was observed using a scanning electron microscope (SEM) (JSM-7500F) using ImageJ software. Fourier transform infrared spectroscopy (FT-IR) was performed using a PerkinElmer Spectrum 400 instrument.
3. 젤라틴 가교입자의 세포독성 분석3. Cytotoxicity analysis of gelatin cross-linked particles
MG-63 조골세포 및 건세포(tenocyte)에 대한 젤라틴 가교입자의 독성을 분석하기 위해, WST-1 assay를 수행하였다. MG-63 조골세포 및 건세포 각각을 1 × 104 cells/well의 밀도로 96-well plate에 분주한 후, 배양 배지(Cellgro)에서 24시간 동안 배양하였다. 24시간 후, 배양 배지를 젤라틴 가교입자가 각 0.05%, 0.1%, 0.5%, 1% 및 2%(w/v) 포함된 배양 배지로 교체한 후, 다시 24시간 동안 배양하였다. 그 후 WST-1 assay를 통해 생존한 세포의 수를 측정하였다.To analyze the toxicity of gelatin cross-linked particles to MG-63 osteoblasts and tenocytes, WST-1 assay was performed. MG-63 osteoblasts and tenocytes were each distributed in a 96-well plate at a density of 1 × 104 cells/well and cultured in culture medium (Cellgro) for 24 hours. After 24 hours, the culture medium was replaced with a culture medium containing 0.05%, 0.1%, 0.5%, 1%, and 2% (w/v) of gelatin cross-linked particles, respectively, and then cultured for another 24 hours. Afterwards, the number of surviving cells was measured through WST-1 assay.
4. 생체외 젤라틴 가교입자의 약물방출 분석4. Drug release analysis of in vitro gelatin cross-linked particles
젤라틴 가교입자의 약물방출 프로파일을 위해 트리판블루를 모델 약물로 사용하였다. 젤라틴 가교입자를 트리판블루 용액(용액의 농도는 각 0.05%, 0.1% 및 0.2% (w/v))에 5 μL/mg의 비율로 함침시킨 후 상온에서 2시간 동안 보존하였다. 그 후, 젤라틴 가교입자를 인산염완충식염수(phosphate-buffered saline)에 1.5 mL/5 mg의 비율로 담근 후 인큐베이터에서 37 ℃에서 20 rpm으로 인버팅(inverting)한다. 이후 분광기에서 595 nm의 파장을 측정하여 상층액의 트리판블루 농도를 측정하였다.Trypan blue was used as a model drug for the drug release profile of gelatin cross-linked particles. Gelatin cross-linked particles were impregnated with trypan blue solution (solution concentrations of 0.05%, 0.1%, and 0.2% (w/v), respectively) at a rate of 5 μL/mg and stored at room temperature for 2 hours. Afterwards, the gelatin cross-linked particles were immersed in phosphate-buffered saline at a ratio of 1.5 mL/5 mg and then inverted at 20 rpm at 37°C in an incubator. Afterwards, the trypan blue concentration of the supernatant was measured by measuring the wavelength of 595 nm on a spectrometer.
5. 약물방출의 수학적 모델 제안5. Proposal of a mathematical model of drug release
Fick’s second law를 바탕으로 간단한 1차원 수학적 모델을 제안하였다. 하기 식은 일반 확산 방정식이다.A simple one-dimensional mathematical model was proposed based on Fick’s second law. The equation below is the general diffusion equation.
(1) (One)
상기 식 중, CA는 시간에 따른 약물 농도이며, A는 입자의 내부 위치, B는 입자의 외부 위치, t는 시간, DAB는 젤라틴 입자의 처음 3일 간의 약물방출 결과를 모델링하여 얻어진 확산 계수를 의미한다.In the above formula, C A is the drug concentration over time, A is the internal position of the particle, B is the external position of the particle, t is time, and D AB is the diffusion obtained by modeling the drug release results of the first 3 days of gelatin particles. It means coefficient.
결과result
본 발명은 작은 크기의 젤라틴 가교입자를 제조하기 위하여, 냉동-해동 단계를 바탕으로 한, 두 단계의 탈용매화 방법을 사용하였다. 이 방법은 온도 감소에 따라 물질의 분자의 압축이 증가하고 부피가 줄어드는 자연현상을 바탕으로 한 것이다. The present invention used a two-step desolvation method based on a freeze-thaw step to produce small-sized gelatin crosslinked particles. This method is based on the natural phenomenon that the compression of the molecules of a substance increases and the volume decreases as the temperature decreases.
도 1에서 상기 방법을 도식화하였다. 기존 방법에서는 젤라틴을 먼저 침전시키고 입자가 형성되면 글루타르알데하이드를 추가하는 것과 대조적으로, 본 발명에서는 젤라틴의 참전 후, 급속 냉동에 의해 젤라틴 분자 사이의 압축이 촉진되어 평균 100 nm 이하의 젤라틴 나노입자가 제조되었다. 도 2a은 기존 방법으로 제조된 젤라틴 입자의 SEM 이미지를 나타낸 것이며, 더 응집된 형태의 젤라틴 입자가 관찰된다. 도 2b는 냉동-해동 방법을 통해 제조된 젤라틴 가교입자의 SEM 이미지를 나타낸 것이며, 기존 방법으로 제조된 젤라틴 입자와 비교할 때 응집되지 않고 구형의 작은 크기의 입자가 제조되었음을 확인할 수 있다. 기존 방법으로 제조된 젤라틴 입자의 평균적인 크기는 도 2c에서와 같이 150 nm 이상이며, 냉동-해동 방법으로 제조된 젤라틴 가교입자는 평균적으로 최소 40~60 nm에서 최대 100 nm의 크기를 갖는다. The method is schematized in Figure 1. In contrast to the existing method in which gelatin is first precipitated and glutaraldehyde is added once the particles are formed, in the present invention, compression between gelatin molecules is promoted by quick freezing after the gelatin is added to form gelatin nanoparticles with an average size of 100 nm or less. was manufactured. Figure 2a shows an SEM image of gelatin particles prepared by a conventional method, and gelatin particles in a more aggregated form are observed. Figure 2b shows an SEM image of gelatin crosslinked particles prepared through the freeze-thaw method, and it can be seen that compared to gelatin particles prepared by the conventional method, small spherical particles were produced without agglomeration. The average size of gelatin particles produced by the conventional method is 150 nm or more as shown in Figure 2c, and the cross-linked gelatin particles produced by the freeze-thaw method have an average size of at least 40 to 60 nm and up to 100 nm.
젤라틴과 본 발명의 젤라틴 가교입자의 화학적 구조를 비교하기 위하여, 젤라틴 및 젤라틴 가교입자의 작용기를 FT-IR로 분석한 결과를, 도 3에서 나타내었다. 3466 cm-1 에 분포하는 N-H stretching으로 인한 amide-A 피크는 가교결합의 정도와 관련되어 있으며, amide I 및 N-H denaturation에 대한 1630 cm-1의 C=0 stretching, amide II에 대한 1565 cm-1 의 C-N stretching이 있다.In order to compare the chemical structures of gelatin and the gelatin crosslinked particles of the present invention, the results of analyzing the functional groups of gelatin and gelatin crosslinked particles by FT-IR are shown in Figure 3. The amide-A peak due to NH stretching distributed at 3466 cm -1 is related to the degree of crosslinking, C=0 stretching at 1630 cm -1 for amide I and NH denaturation, and 1565 cm -1 for amide II. There is CN stretching of .
입자의 크기는 입자의 세포독성을 결정하는 중요한 요소로 보고되어 왔다. 일반적으로, 100 nm 이하의 입자가 가장 독성이 적으며, 반면에 매우 작은 크기(1 nm 이하)의 입자는 세포독성이 매우 높은 것으로 알려져 있다. 젤라틴 가교입자의 세포독성을 조사하기 위하여, MG-63 조골세포 및 건세포를 각각 조골세포 및 섬유아세포의 모델로 사용하여 WST-1 assay를 수행하였다. 두 세포그룹 모두, 젤라틴 가교입자가 0%인 대조군과 비교하여 더 높은 세포생존력을 나타냈다. 흥미롭게도, 조골세포의 경우는 젤라틴 가교입자 농도가 증가함에 따라 세포생존력이 증가하였다. 상기 결과를 통해 젤라틴 가교입자가 세포독성이 없음을 알 수 있다.Particle size has been reported to be an important factor in determining the cytotoxicity of particles. In general, particles of 100 nm or less are the least toxic, while particles of very small size (1 nm or less) are known to have very high cytotoxicity. To investigate the cytotoxicity of gelatin cross-linked particles, WST-1 assay was performed using MG-63 osteoblasts and tenocytes as models of osteoblasts and fibroblasts, respectively. Both cell groups showed higher cell viability compared to the control group with 0% gelatin cross-linked particles. Interestingly, in the case of osteoblasts, cell viability increased as the concentration of gelatin cross-linked particles increased. The above results show that the gelatin cross-linked particles are not cytotoxic.
입자의 크기는 일반적으로 생체내외에서 약물방출속도를 결정하는 중요 요소 중 하나로 알려져 있다. 젤라틴 가교입자의 양물방출속도 프로파일을 확인하기 위하여, 생체외에서 약물방출속도 실험을 수행하였다. 도 5에서, 모델 약물(트리판블루)의 농도가 각 0.05%, 0.1%, 및 0.2% 일 때의 약물방출 프로파일을 나타내었다. 실험결과, 상기 세 가지 농도에서, 각 40%, 30% 및 20%로 로드된(loaded) 약물에 대하여, 처음 3일 동안 초기 약물급속방출이 관찰되었다. 이는 트리판블루가 젤라틴 가교입자의 외부 표면에 흡수되었던 것으로 해석될 수 있다. 더욱이, 젤라틴의 약한 기계적 성질로 인해 초기 방출이 높았던 것으로 해석된다. 위 실험 결과는 초기의 약물급속방출을 보여줄 뿐만 아니라, 세 가지 농도에서 약물방출 곡선이 유사하게 나타남을 보여준다. 그러나, 약물방출속도는 로드된 약물의 양이 많을수록 높았다. 젤라틴 가교입자의 약물방출 메커니즘은, solid matrices의 약물방출에 대한 Higuchi 방정식에 따라, 확산에 의해 제어되는 것으로 알려져 있다. Particle size is generally known to be one of the important factors that determine the drug release rate in vitro and in vivo. To confirm the drug release rate profile of the gelatin cross-linked particles, an in vitro drug release rate experiment was performed. In Figure 5, the drug release profile is shown when the concentrations of the model drug (trypan blue) are 0.05%, 0.1%, and 0.2%, respectively. As a result of the experiment, initial rapid drug release was observed for the first 3 days for the drugs loaded at 40%, 30%, and 20%, respectively, at the above three concentrations. This can be interpreted as trypan blue being absorbed into the outer surface of the gelatin cross-linked particles. Moreover, it is interpreted that the initial release was high due to the weak mechanical properties of gelatin. The above experimental results not only show the initial rapid release of the drug, but also show that the drug release curves appear similar at three concentrations. However, the drug release rate was higher as the amount of loaded drug increased. The drug release mechanism from gelatin cross-linked particles is known to be controlled by diffusion according to the Higuchi equation for drug release from solid matrices.
생체의학에서의 응용에서, 신규한 입자의 약물방출 프로파일에 대한 수학적 모델링은 매우 중요한 요소이다. 수학적 모델링을 통한 입자의 약물방출 프로파일 및 방출 경향에 대한 이해는 입자에 대하여 적정량의 사용과 방출 조절을 가능하도록 해준다. 따라서, 기존 확산 방정식을 사용하여 간단한 수학적 모델을 제시하였다. 전술한 방정식 (1)은, 처음 3일 동안의 약물방출 프로파일에서 얻어진 확산 계수를 사용하였다. 추가적으로, 처음 3일 동안의 급속방출을 고려하여 결과를 보정하였다. 도 6에서 세 가지 농도에서의 방출 모델 결과와 실험 결과를 비교하여 나타내었다. In biomedical applications, mathematical modeling of the drug release profile of novel particles is a very important factor. Understanding the drug release profile and release tendency of particles through mathematical modeling allows the use of appropriate amounts and controlled release of particles. Therefore, a simple mathematical model was presented using the existing diffusion equation. The above-mentioned equation (1) used the diffusion coefficient obtained from the drug release profile for the first 3 days. Additionally, the results were corrected to take into account rapid release during the first 3 days. In Figure 6, the release model results and experimental results at three concentrations are compared.
Claims (7)
상기 젤라틴 입자를 냉동하여 냉동된 젤라틴 입자를 얻는 단계; 및
상기 냉동된 젤라틴 입자를 해동시키면서 가교하는 단계를 포함하는 젤라틴 가교입자의 제조 방법.
Obtaining gelatin particles from a gelatin solution through a precipitation reaction;
Freezing the gelatin particles to obtain frozen gelatin particles; and
A method for producing crosslinked gelatin particles comprising the step of crosslinking the frozen gelatin particles while thawing them.
The method of claim 1, wherein the thawing is performed under refrigeration.
The method of claim 1, wherein the crosslinking is performed by reaction with glutaraldehyde.
The method of claim 1, wherein the solvent of the solution is water, and the precipitation is performed by adding acetone.
젤라틴 용액에서 젤라틴을 침전시키고, 그 침전물을 재용해시키고 산 첨가 후에 재침전시켜 젤라틴 입자의 침전액을 얻는 단계인 젤라틴 가교입자의 제조 방법.
The method according to claim 1, wherein the step of obtaining gelatin particles from the gelatin solution through a precipitation reaction,
A method for producing crosslinked gelatin particles, which includes precipitating gelatin from a gelatin solution, redissolving the precipitate, adding acid, and then reprecipitating to obtain a precipitate solution of gelatin particles.
The method of claim 1, further comprising adding an organic solvent to the cross-linked gelatin particles and purifying the cross-linked gelatin particles by centrifugation.
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