KR102641841B1 - Porous microspheres containing magnetic nanoparticles for delivery of a cell and a drug and manufacturing method thereof - Google Patents
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- 239000003814 drug Substances 0.000 title claims abstract description 29
- 239000002122 magnetic nanoparticle Substances 0.000 title claims abstract description 14
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 14
- 239000004005 microsphere Substances 0.000 title claims abstract description 8
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- 239000003937 drug carrier Substances 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 22
- WTFXARWRTYJXII-UHFFFAOYSA-N iron(2+);iron(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[O-2].[Fe+2].[Fe+3].[Fe+3] WTFXARWRTYJXII-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229940031182 nanoparticles iron oxide Drugs 0.000 claims abstract description 16
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- 238000012377 drug delivery Methods 0.000 claims abstract description 13
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- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 12
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- 239000004626 polylactic acid Substances 0.000 claims description 10
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- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 claims description 2
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- CVOFKRWYWCSDMA-UHFFFAOYSA-N 2-chloro-n-(2,6-diethylphenyl)-n-(methoxymethyl)acetamide;2,6-dinitro-n,n-dipropyl-4-(trifluoromethyl)aniline Chemical compound CCC1=CC=CC(CC)=C1N(COC)C(=O)CCl.CCCN(CCC)C1=C([N+]([O-])=O)C=C(C(F)(F)F)C=C1[N+]([O-])=O CVOFKRWYWCSDMA-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1611—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
- A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/40—Cyclodextrins; Derivatives thereof
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- A61K9/00—Medicinal preparations characterised by special physical form
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- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
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- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
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Abstract
본 발명은 세포-약물을 전달하기 위한 자성 나노입자를 함유하는 다공성 마이크로 입자(microspheres) 및 이의 제조방법에 관한 것으로서, 생분해성 고분자를 이용하여 초상자성 산화철 나노입자(superparamagnetic iron oxide nanoparticles)를 함유한 다공성 마이크로 입자를 제조하여 다공성 부위에 세포를 부착할 수 있을 뿐만 아니라, 다공성 마이크로 입자의 표면에 약물 담지체를 결합하여 단순한 혼합만으로 약물을 로딩할 수 있고, 외부 자기 구동 장치에 의해 세포-약물을 동시에 함유한 다공성 마이크로 입자를 병소 부위에 정확하게 전달함으로써 현재보다 그 치료효과를 크게 높일 수 있고, 세포와 약물이 비침습적으로 국소적 병소 부위로 축적 가능하므로, 이를 효과적으로 관절염을 포함하는 다양한 국소적 염증 질환의 치료 및 조직 재생 유도에 이용할 수 있다.The present invention relates to porous microspheres containing magnetic nanoparticles for cell-drug delivery and a method for manufacturing the same. The present invention relates to porous microspheres containing superparamagnetic iron oxide nanoparticles using biodegradable polymers. By manufacturing porous micro-particles, not only can cells be attached to porous areas, but also the drug carrier can be loaded on the surface of the porous micro-particles by simple mixing, and the cell-drug combination can be achieved by an external magnetic driving device. At the same time, by accurately delivering the contained porous micro-particles to the lesion site, the therapeutic effect can be greatly increased compared to the present, and since cells and drugs can accumulate in the local lesion site non-invasively, they can effectively treat various local inflammations, including arthritis. It can be used to treat diseases and induce tissue regeneration.
Description
본 발명은 세포-약물을 동시에 전달하기 위한 자성 나노입자를 함유하는 다공성 마이크로 입자(microspheres) 및 이의 제조방법에 관한 것으로서, 더욱 상세하게는 생분해성 고분자에 초상자성 산화철 나노입자(superparamagnetic iron oxide nanoparticles)를 혼합하여 다공성 마이크로 입자를 제조하고 그 표면에 약물 담지체를 결합시킴으로써, 외부 자기장에 의해 병소 부위로 정밀하게 이송할 수 있을 뿐 아니라 기공에 세포를 부착하여 배양할 수 있고 동시에 약물을 전달할 수 있으므로, 일관된 치료 효과를 보장할 수 있는 세포-약물 전달체에 관한 것이다.The present invention relates to porous microspheres containing magnetic nanoparticles for simultaneous cell-drug delivery and a method of manufacturing the same. More specifically, it relates to superparamagnetic iron oxide nanoparticles in biodegradable polymers. By mixing porous micro-particles and binding a drug carrier to their surface, not only can they be precisely transported to the lesion site by an external magnetic field, but cells can also be attached to the pores and cultured, and drugs can be delivered at the same time. , relates to a cell-drug delivery system that can ensure consistent therapeutic effects.
국내 퇴행성 관절염 환자 수는 2020년 기준 약 700만 명을 넘어설 것으로 전망되고 있다. 건강보험심사평가원의 보고에 의하면, 2019년 연령별 환자 비율은 20대가 0.9%, 30대는 1.6%, 40대는 5.6%, 50대는 21.0%, 60대는 32.9%, 70대가 26.8%, 80세 이상은 11.0%로 나타났다.The number of degenerative arthritis patients in Korea is expected to exceed 7 million by 2020. According to a report by the Health Insurance Review and Assessment Service, the proportion of patients by age in 2019 was 0.9% in their 20s, 1.6% in their 30s, 5.6% in their 40s, 21.0% in their 50s, 32.9% in their 60s, 26.8% in their 70s, and 11.0% in their 80s and older. It was expressed as %.
현재까지 관절염 치료는 통증의 완화를 유도할 뿐, 기능 개선 및 구조적 질병 진행 억제를 통한 근본적인 골관절염 치료제로 인정받은 제품은 없는 실정이다. 최근 들어, 수술 없이 줄기세포 주사만으로 말기 관절염을 치료하는 방법도 개발되는 등 퇴행성 관절염 치료의 패턴이 바뀌고 있다.To date, arthritis treatment only induces pain relief, and there are no products recognized as a fundamental treatment for osteoarthritis by improving function and suppressing structural disease progression. Recently, the pattern of treatment for degenerative arthritis is changing, with the development of methods to treat late-stage arthritis using only stem cell injections without surgery.
이러한 치료법도 몇 가지 한계점을 가지고 있다. 예를 들어, 자가연골세포 이식 수술의 경우 세포만을 단독적으로 사용하는 치료는 세포의 이식 후에 해당 세포의 생착률 문제와 병변 부위에 고르지 않게 분포되는 등의 단점을 극복하기 위한 접근법이 필요하다. 또한 세포뿐 아니라 약물을 관절염 질환 병소 부위로 정확하고 균일하게 전달함으로써 치료의 효과를 높이는 새로운 패러다임의 치료법이 필요하다.These treatments also have some limitations. For example, in the case of autologous chondrocyte transplantation, treatment using cells alone requires an approach to overcome disadvantages such as problems with the engraftment rate of the cells and uneven distribution in the lesion area after cell transplantation. In addition, a new paradigm of treatment that increases the effectiveness of treatment by accurately and uniformly delivering not only cells but also drugs to the arthritic lesion site is needed.
한편, 외부 자기장 줄기세포 전달 장치(Stem cell navigator®)를 이용할 경우, 자기미세입자의 이동을 조절할 수 있고 병소 부위로 전달할 수 있다. 따라서, 지금까지의 관절염 치료의 문제점을 해결하고 관절염 치료의 효과를 높이기 위해 관절염 부위에 외부 자기장 줄기세포 전달 장치를 통해 정확하게 세포와 약물을 전달하기 위한 다공성 마이크로 입자를 개발할 필요가 있다.Meanwhile, when using an external magnetic field stem cell delivery device (Stem cell navigator ® ), the movement of magnetic microparticles can be controlled and delivered to the lesion site. Therefore, in order to solve the problems of arthritis treatment so far and increase the effectiveness of arthritis treatment, there is a need to develop porous microparticles to accurately deliver cells and drugs to the arthritis site through an external magnetic field stem cell delivery device.
이에 본 발명자들은 초상자성 산화철 나노입자(superparamagnetic iron oxide nanoparticles)를 포함하는 다공성 마이크로 입자(microspheres) 표면에 약물 담지체를 결합함으로써, 시술 전에 세포를 부착한 다공성 마이크로 입자 용액과 약물 용액을 단순히 혼합하는 과정을 통해 다공성 마이크로 입자에 약물을 담지할 수 있으며, 이를 환자에게 시술할 경우 세포와 함께 약물을 담지한 다공성 마이크로 입자를 병소로 전달함으로써 그 치료 효과를 높일 수 있음을 확인하였다.Accordingly, the present inventors combined a drug carrier on the surface of porous microparticles containing superparamagnetic iron oxide nanoparticles, thereby simply mixing the drug solution with the porous microparticle solution to which cells were attached before the procedure. Through this process, it was confirmed that drugs can be loaded into porous microparticles, and that when this procedure is performed on a patient, the therapeutic effect can be increased by delivering the drug-loaded porous microparticles along with cells to the lesion.
이에, 본 발명의 목적은 자성 나노입자를 포함하는 생분해성 고분자 기반의 세포-약물을 동시에 전달할 수 있는 다공성 마이크로 입자를 제공하는 것이다.Accordingly, the purpose of the present invention is to provide porous microparticles that can simultaneously deliver cell-drugs based on biodegradable polymers containing magnetic nanoparticles.
본 발명의 다른 목적은 다음 단계를 포함하는 세포-약물 전달용 다공성 마이크로 입자의 제조방법을 제공하는 것이다:Another object of the present invention is to provide a method for producing porous microparticles for cell-drug delivery comprising the following steps:
생분해성 고분자 및 자성 나노입자의 혼합물로 다공성 마이크로 입자를 제조하는 다공성 구조 형성 단계; 및A porous structure forming step of manufacturing porous micro-particles from a mixture of biodegradable polymers and magnetic nanoparticles; and
다공성 마이크로 입자의 표면에 약물 담지체를 도입하는 약물 담지체 도입 단계.A drug carrier introduction step in which the drug carrier is introduced onto the surface of the porous microparticles.
본 발명의 또 다른 목적은 자성 나노입자를 포함하는 생분해성 고분자 기반의 다공성 마이크로 입자를 이용한 세포-약물 전달 용도에 관한 것이다.Another object of the present invention relates to the use of cell-drug delivery using porous microparticles based on biodegradable polymers containing magnetic nanoparticles.
본 발명은 자성 나노입자를 함유하는 세포-약물 전달용 다공성 마이크로 입자(microspheres) 및 이의 제조방법에 관한 것으로, 본 발명에 따른 마이크로 입자는 자기장을 이용하여 정밀하게 이송할 수 있고 세포 및 약물을 동시에 병소에 전달할 수 있다.The present invention relates to porous microspheres for cell-drug delivery containing magnetic nanoparticles and a method for manufacturing the same. The microparticles according to the present invention can precisely transport cells and drugs using a magnetic field and deliver cells and drugs at the same time. It can be delivered to the lesion.
본 발명자들은 상기의 목적을 달성하기 위하여 a) 외부 자기장에 의해 이동이 가능하도록 초상자성 산화철 나노입자(superparamagnetic iron oxide nanoparticles)를 함유하고, b) 세포를 부착할 수 있으며, c) 약물을 단순한 혼합만으로 담지할 수 있는 마이크로 입자를 구현하였다.In order to achieve the above object, the present inventors a) contain superparamagnetic iron oxide nanoparticles that can be moved by an external magnetic field, b) can attach cells, and c) simply mix drugs. We have implemented micro particles that can be supported by just one surface.
이하 본 발명을 더욱 자세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 양태는 자성 나노입자를 포함하는 생분해성 고분자 기반의 세포-약물 전달용 다공성 마이크로 입자이다.One aspect of the present invention is a porous micro-particle for cell-drug delivery based on a biodegradable polymer containing magnetic nanoparticles.
본 발명에 있어서 생분해성 고분자는 폴리락틱엑시드(polylactic acid), 폴리락틱글리콜엑시드(poly(lactic-co-glycolic acid)), 폴리카프로락톤(poly(ε-carprolactone)), 폴리디옥산논(polydioxanone), 폴리하이드록시부틸레이트(poly(β-hydroxybutyrate)) 및 폴리메틸시아노아크릴레이트(poly(methyl 2-cyanoacylate))로 이루어진 군으로부터 선택되는 1종 이상인 것일 수 있다.In the present invention, biodegradable polymers include polylactic acid, poly(lactic-co-glycolic acid), poly(ε-carprolactone), and polydioxanone. ), poly(β-hydroxybutyrate), and polymethyl cyanoacrylate (poly(methyl 2-cyanoacylate)).
본 명세서상의 용어 “폴리락틱엑시드”는 옥수수에서 녹말을 분리하여 포도 당 발효를 통해 젖산을 생산하고 중합하여 제조되는 생분해성 고분자이다. 폴리락틱 엑시드는 생분해성이 우수하며 미생물에 의해 완전히 분해되어 이산화탄소와 물을 형성한다. 가공성이 우수하고 대부분의 합성 플라스틱 용도에 적합하고 의료용 소재로도 널리 활용된다.The term “polylactic acid” in this specification is a biodegradable polymer manufactured by separating starch from corn, producing lactic acid through glucose fermentation, and polymerizing it. Polylactic acid has excellent biodegradability and is completely decomposed by microorganisms to form carbon dioxide and water. It has excellent processability, is suitable for most synthetic plastic applications, and is also widely used as a medical material.
본 발명에 있어서 자성 나노입자는 초상자성, 반자성, 상자성 또는 강자성 물질인 것일 수 있고, 예를 들어, 초상자성 산화철 나노입자인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the magnetic nanoparticles may be superparamagnetic, diamagnetic, paramagnetic, or ferromagnetic materials, for example, superparamagnetic iron oxide nanoparticles, but are not limited thereto.
본 발명에 있어서 다공성 마이크로 입자는 표면에 약물 담지체를 추가적으로 포함하는 것일 수 있다.In the present invention, the porous microparticles may additionally include a drug carrier on the surface.
상기 약물 담지체는 아미노-베타-사이클로덱스트린(amino-β-cyclodextrin) 또는 사이클로덱스트린 유도체인 것일 수 있다.The drug carrier may be amino-β-cyclodextrin or a cyclodextrin derivative.
상기 약물 담지체는 정전기적 인력에 의하여 다공성 마이크로 입자 표면에 도입되는 것일 수 있다.The drug carrier may be introduced to the surface of porous micro particles by electrostatic attraction.
본 발명의 다른 양태는 다음 단계를 포함하는 세포-약물 전달용 다공성 마이크로 입자 제조방법이다:Another aspect of the present invention is a method for preparing porous microparticles for cell-drug delivery comprising the following steps:
생분해성 고분자 및 자성 나노입자의 혼합물로 다공성 마이크로 입자를 제조하는 다공성 구조 형성 단계; 및A porous structure forming step of manufacturing porous micro-particles from a mixture of biodegradable polymers and magnetic nanoparticles; and
다공성 마이크로 입자의 표면에 약물 담지체를 도입하는 약물 담지체 도입 단계.A drug carrier introduction step of introducing a drug carrier onto the surface of porous microparticles.
본 발명에 있어서 생분해성 고분자는 폴리락틱엑시드, 폴리락틱글리콜엑시드, 폴리카프로락톤, 폴리디옥산논, 폴리하이드록시부틸레이트 및 폴리메틸시아노아크릴레이트로 이루어진 군으로부터 선택되는 1종 이상인 것일 수 있다.In the present invention, the biodegradable polymer may be one or more selected from the group consisting of polylactic acid, polylactic glycol acid, polycaprolactone, polydioxanone, polyhydroxybutyrate, and polymethylcyanoacrylate. .
본 발명의 일 구현예에서, 상기 방법에 따라 제조된 다공성 마이크로 입자는 구조적으로 자성 나노입자가 분포되어 내재된 형태로 구현되었다.In one embodiment of the present invention, the porous micro-particles prepared according to the above method were implemented in a form in which magnetic nanoparticles were structurally distributed.
본 발명에 있어서 다공성 구조 형성 단계는 기포발포법의 사용 또는 친수성 고분자의 첨가를 통해 다공성 마이크로 입자에 기공을 형성하여 수행되는 것일 수 있다.In the present invention, the step of forming a porous structure may be performed by forming pores in porous micro particles through the use of a bubble foaming method or the addition of a hydrophilic polymer.
상기 기포발포법은 생분해성 고분자 및 자성 나노입자의 혼합물에 대한 소듐바이카보네이트(sodium bicarbonate)의 첨가를 통해 수행되는 것일 수 있다.The bubble foaming method may be performed through the addition of sodium bicarbonate to a mixture of biodegradable polymer and magnetic nanoparticles.
상기 친수성 고분자는 젤라틴(geltain), 히알우로산(hyaluronic acid), 알부민(albumin), 폴리에틸렌글리콜(polyethylene glycol), 플루란(pullulan), 알지네이트(alginate) 및 키토산(chitosan)으로 이루어진 군으로부터 선택되는 1종 이상인 것일 수 있으나, 이에 한정되는 것은 아니다.The hydrophilic polymer is selected from the group consisting of gelatin, hyaluronic acid, albumin, polyethylene glycol, pullulan, alginate, and chitosan. There may be one or more types, but it is not limited thereto.
본 발명에 있어서 세포-약물 전달용 다공성 마이크로 입자 제조방법은 다공성 마이크로 입자에 형성된 기공에 세포를 배양하는 세포 배양 단계를 추가적으로 포함하는 것일 수 있고, 예를 들어, 상기 세포는 줄기세포인 것일 수 있다.In the present invention, the method for producing porous microparticles for cell-drug delivery may additionally include a cell culture step of culturing cells in pores formed in the porous microparticles. For example, the cells may be stem cells. .
상기 약물 담지체는 아미노-베타-사이클로덱스트린 또는 사이클로덱스트린 유도체인 것일 수 있다.The drug carrier may be amino-beta-cyclodextrin or a cyclodextrin derivative.
상기 약물 담지체는 정전기적 인력에 의하여 다공성 마이크로 입자 표면에 도입되는 것일 수 있다.The drug carrier may be introduced to the surface of porous micro particles by electrostatic attraction.
본 발명에 있어서 다공성 마이크로 입자와 목표 약물을 단순히 혼합하는 공정만으로도 다공성 마이크로 입자 표면에 도입된 약물 담지체에 약물을 로딩할 수 있다.In the present invention, the drug can be loaded onto the drug carrier introduced on the surface of the porous microparticles simply by mixing the porous microparticles and the target drug.
본 발명의 일 구현예에서, 약물 담지체로 아미노-베타-사이클로덱스트린이 도입된 다공성 마이크로 입자는 분산액 내에서 목표 약물인 메소트렉세이트(methotrexate)를 첨가하여 혼합하는 간단한 방법을 통하여 관절염 약물을 담지하여 의료기기 조성물로 사용 가능한 다공성 마이크로 입자로서 준비되었다.In one embodiment of the present invention, porous microparticles into which amino-beta-cyclodextrin is introduced as a drug carrier carry an arthritis drug through a simple method of adding and mixing the target drug, methotrexate, in the dispersion. It was prepared as a porous micro particle that can be used as a medical device composition.
본 발명의 세포-약물 전달용 다공성 마이크로 입자를 질환의 치료 및 국소 조직 재생 용도로 사용하게 되는 경우, 예를 들어, 목표 약물은 관절염을 포함하는 다양한 국소적 질환에 적용할 수 있고 비침습적인 치료 방법에 사용됨에 있어서 외부 자기 구동 장치를 이용해 병소 부위로 세포와 약물을 정확하게 전달할 수 있다. 특히 다공성 마이크로 입자에는 약물을 담지할 수 있을 뿐만 아니라 세포를 부착 및 배양할 수 있어 관절염의 치료효과를 극대화시킬 수 있다.When the porous microparticles for cell-drug delivery of the present invention are used for disease treatment and local tissue regeneration, for example, the target drug can be applied to various local diseases, including arthritis, and non-invasive treatment When used in the method, cells and drugs can be accurately delivered to the lesion site using an external magnetic drive device. In particular, porous micro particles can not only carry drugs but also attach and culture cells, maximizing the therapeutic effect of arthritis.
본 발명은 세포-약물을 전달하기 위한 자성 나노입자를 함유하는 다공성 마이크로 입자(microspheres) 및 이의 제조방법에 관한 것으로서, 생분해성 고분자를 이용하여 초상자성 산화철 나노입자(superparamagnetic iron oxide nanoparticles)를 함유한 다공성 마이크로 입자를 제조하여 다공성 부위에 세포를 부착할 수 있을 뿐만 아니라, 다공성 마이크로 입자의 표면에 약물 담지체를 결합하여 단순한 혼합만으로 약물을 로딩할 수 있고, 외부 자기 구동 장치에 의해 세포-약물을 동시에 함유한 다공성 마이크로 입자를 병소 부위에 정확하게 전달함으로써 현재보다 그 치료효과를 크게 높일 수 있고, 세포와 약물이 비침습적으로 국소적 병소 부위로 축적 가능하므로, 이를 효과적으로 관절염을 포함하는 다양한 국소적 염증 질환의 치료 및 조직 재생 유도에 이용할 수 있다.The present invention relates to porous microspheres containing magnetic nanoparticles for cell-drug delivery and a method for manufacturing the same. The present invention relates to porous microspheres containing superparamagnetic iron oxide nanoparticles using biodegradable polymers. By manufacturing porous micro-particles, not only can cells be attached to porous areas, but also the drug carrier can be loaded on the surface of the porous micro-particles by simple mixing, and the cell-drug combination can be achieved by an external magnetic driving device. At the same time, by accurately delivering the contained porous micro-particles to the lesion site, the therapeutic effect can be greatly increased compared to the present, and since cells and drugs can accumulate in the local lesion site non-invasively, they can effectively treat various local inflammations, including arthritis. It can be used to treat diseases and induce tissue regeneration.
도 1은 본 발명의 일 실시예에 따라 제조된 초상자성 산화철 나노입자(superparamagnetic iron oxide nanoparticles)를 함유한 폴리락틱엑시드(polylactic acid) 다공성 마이크로 입자(microspheres)의 전계 방사형 주사전자현미경(Field emission scanning electromicroscope; FE-SEM) 사진이다.
도 2는 본 발명의 일 실시예에 따라 제조된 초상자성 산화철 나노입자를 함유한 다공성 마이크로 입자 용액(좌, OFF)과 영구자석에 끌려온 일부(우, ON)를 나타낸 사진이다.
도 3은 본 발명의 일 실시예에 따라 제조된 초상자성 산화철 나노입자를 함유한 폴리락틱엑시드 다공성 마이크로 입자의 표면에서 약물 담지체의 결합 전후 제타 포텐셜(zeta potential)을 나타낸 그래프이다.
도 4는 본 발명의 일 실시예에 따라 제조된 세포-약물 동시 전달을 위한 다공성 마이크로 입자의 모식도이다.
도 5는 본 발명의 일 실시예에 따라 제조된 세포-약물 동시 전달을 위한 다공성 마이크로 입자로부터 약물인 메소트렉세이트의 방출 결과를 나타낸 그래프이다.1 is a field emission scanning electron microscope view of polylactic acid porous microparticles containing superparamagnetic iron oxide nanoparticles prepared according to an embodiment of the present invention. This is an electromicroscope; FE-SEM) photo.
Figure 2 is a photograph showing a porous micro-particle solution containing superparamagnetic iron oxide nanoparticles prepared according to an embodiment of the present invention (left, OFF) and a portion attracted to a permanent magnet (right, ON).
Figure 3 is a graph showing the zeta potential before and after binding of a drug carrier on the surface of polylactic acid porous microparticles containing superparamagnetic iron oxide nanoparticles prepared according to an embodiment of the present invention.
Figure 4 is a schematic diagram of porous microparticles for simultaneous cell-drug delivery prepared according to an embodiment of the present invention.
Figure 5 is a graph showing the release results of the drug methotrexate from porous microparticles for cell-drug simultaneous delivery manufactured according to an embodiment of the present invention.
이하, 본 발명을 하기의 실시예에 의하여 더욱 상세히 설명한다. 그러나 이들 실시예는 본 발명을 예시하기 위한 것일 뿐이며, 본 발명의 범위가 이들 실시예에 의하여 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
본 명세서 전체에 걸쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 "%"는 별도의 언급이 없는 경우, 고체/고체는 (중량/중량)%, 고체/액체는 (중량/부피)%, 그리고 액체/액체는 (부피/부피)%이다.Throughout this specification, “%” used to indicate the concentration of a specific substance means (weight/volume)% for solid/solid, (weight/volume)% for solid/liquid, and Liquid/liquid is (volume/volume)%.
실시예 1: 초상자성 산화철 나노입자를 함유한 다공성 마이크로 입자의 제조Example 1: Preparation of porous microparticles containing superparamagnetic iron oxide nanoparticles
폴리락틱엑시드(polylactic acid, Sigma-aldrich Co., USA)를 디클로로메탄(dichloromethane)에 6.25%(w/v)으로 용해한 용액 4 mL에 80 mg의 초상자성 산화철 나노입자(superparamagnetic iron oxide nanoparticles, Sigma-aldrich Co., USA)를 첨가한 후 볼텍스(vortex) 믹서로 충분하게 혼합하였다. 소듐바이카보네이트(sodium bicarbonate, Sigma-aldrich Co., USA)를 물에 용해하여 위의 용액에 5%(v/v)가 되도록 첨가한 후 호모게나이저(homogenizer)로 11,000 rpm으로 3분간 혼합하였다. 제조한 용액을 0.1%(w/v) 폴리비닐알코올(poly vinylalcohol) 용액에 떨어뜨리고 4시간 동안 교반하였다. 마지막으로 다공성 구조를 형성하기 위해 0.2 M 소듐하이드록사이드(sodium hydroxide, Sigma-aldrich Co., USA)에 30분 동안 담근 후 물로 세척하고 동결건조하여 샘플을 얻었다.80 mg of superparamagnetic iron oxide nanoparticles (Sigma) were added to 4 mL of a 6.25% (w/v) solution of polylactic acid (Sigma-aldrich Co., USA) in dichloromethane. -aldrich Co., USA) was added and thoroughly mixed with a vortex mixer. Sodium bicarbonate (Sigma-aldrich Co., USA) was dissolved in water and added to the above solution to make it 5% (v/v), then mixed with a homogenizer at 11,000 rpm for 3 minutes. . The prepared solution was dropped into a 0.1% (w/v) polyvinyl alcohol solution and stirred for 4 hours. Finally, to form a porous structure, the sample was obtained by soaking it in 0.2 M sodium hydroxide (Sigma-aldrich Co., USA) for 30 minutes, washing it with water, and lyophilizing it.
실시예 2: 다공성 마이크로 입자의 형태 분석Example 2: Morphological analysis of porous micro particles
제조된 초상자성 산화철 나노입자를 함유한 다공성 마이크로 입자의 형태를 관찰하기 위해 전계 방사형 주사전자현미경(Field emission scanning electromicroscope; FE-SEM)을 이용하여 분석하였다.To observe the shape of the prepared porous microparticles containing superparamagnetic iron oxide nanoparticles, they were analyzed using a field emission scanning electron microscope (FE-SEM).
도 1에서 확인할 수 있듯이, 다공성 마이크로 입자는 약 250~300 μm이고, 다공성 구조의 형태를 보이며, 다공성 마이크로 입자 내의 소듐 바이카보네이트가 잘 제거되어 다공성 구조가 형성되었음을 알 수 있었다. 제조된 다공성 마이크로 입자의 크기 및 형태는 폴리비닐알콜과 폴리락틱엑시드의 비율을 조정하여 수십 나노미터 내지 수십 마이크로미터 크기 범위까지 다양하게 조절할 수 있다. 다공성 구조를 이루는 기공의 크기는 소듐 바이카보네이트의 양과 소듐하이드록사이드 용액에 담그는 시간에 따라 조절될 수 있다. 따라서, 본 발명의 다공성 마이크로 입자를 이용하여, 다공성 영역에 약물을 결합시키고 줄기세포를 함유시킬 수 있는 가능성을 입증하였다.As can be seen in Figure 1, the porous microparticles were about 250-300 μm in size and showed a porous structure, and it was found that the sodium bicarbonate in the porous microparticles was well removed to form a porous structure. The size and shape of the prepared porous micro particles can be varied from tens of nanometers to tens of micrometers by adjusting the ratio of polyvinyl alcohol and polylactic acid. The size of the pores forming the porous structure can be adjusted depending on the amount of sodium bicarbonate and the time of immersion in the sodium hydroxide solution. Therefore, using the porous microparticles of the present invention, the possibility of binding a drug to the porous region and containing stem cells was demonstrated.
실시예 3: 다공성 마이크로 입자의 자기 특성 평가Example 3: Evaluation of magnetic properties of porous micro particles
실시예의 다공성 마이크로 입자의 자성 특성을 확인하기 위해, 다공성 마이크로 입자를 PBS(phosphate buffer saline) 용액 내에 분산시킨 후, 외부의 영구자석에 대한 반응성을 육안으로 확인하였다.In order to confirm the magnetic properties of the porous microparticles of the example, the porous microparticles were dispersed in a phosphate buffer saline (PBS) solution and then their reactivity to an external permanent magnet was visually confirmed.
도 2에서 확인할 수 있듯이, 자기장을 가하기 전(좌, OFF)에는 다공성 마이크로 입자가 PBS 용액 내에서 분산된 상태로 유지되어 있지만, 반대로 자기장이 가해지면(우, ON) 자기장이 있는 부분을 따라 반응하여 영구 자석 쪽으로 이동하는 것을 확인하였다.As can be seen in Figure 2, before applying a magnetic field (left, OFF), the porous microparticles remain dispersed in the PBS solution, but on the contrary, when the magnetic field is applied (right, ON), they react along the area where the magnetic field is applied. It was confirmed that it was moving toward the permanent magnet.
상기와 같은 결과는 초상자성 산화철 나노입자를 함유한 다공성 마이크로 입자가 외부의 자기장에 반응할 수 있음을 뒷받침하는 것으로, 비침습적으로 상기 다공성 마이크로 입자의 이동을 조절할 수 있고 또한 병소 부위로 선택적으로 전달할 수 있으므로 치료 효과를 높일 수 있음을 예상할 수 있다.The above results support the fact that porous microparticles containing superparamagnetic iron oxide nanoparticles can respond to an external magnetic field, and can non-invasively control the movement of the porous microparticles and selectively deliver them to the lesion site. Therefore, it can be expected that the treatment effect can be increased.
실시예 4: 다공성 마이크로 입자에 도입된 약물 담지체의 특성 평가Example 4: Evaluation of properties of drug carrier introduced into porous microparticles
초상자성 산화철 나노입자를 함유한 다공성 마이크로 입자에 아미노-베타-사이클로덱스트린(amino-β-cyclodextrin)을 정전기적 인력에 의해 표면에 결합시킨 후, 제타 포텐셜(zeta potential) 값의 변화를 측정하였다.After amino-β-cyclodextrin was bound to the surface of porous microparticles containing superparamagnetic iron oxide nanoparticles by electrostatic attraction, the change in zeta potential value was measured.
도 3에서 확인할 수 있듯이, 다공성 마이크로 입자는 -30.9 mV로 음의 전하(negative charge)를 띄었고, 아미노-베타-사이클로덱스트린(@β-CD)을 함유한 다공성 마이크로 입자는 +6.9 mV로 측정되었다. 상대적으로 양의 전하(positive charge) 값을 나타내는 상기와 같은 결과는 다공성 마이크로 입자 표면에 아미노-베타-사이클로덱스트린이 정전기적 인력으로 상호작용함을 의미한다.As can be seen in Figure 3, the porous microparticles had a negative charge of -30.9 mV, and the porous microparticles containing amino-beta-cyclodextrin (@β-CD) were measured at +6.9 mV. . The above results showing a relatively positive charge value mean that amino-beta-cyclodextrin interacts with the surface of the porous micro particles through electrostatic attraction.
제조한 다공성 마이크로 입자를 PBS 용액에 분산시킨 후, 아미노-베타-사이클로덱스트린을 분산액에 첨가하고, 정전기적 인력을 이용하여 다공성 마이크로 입자 표면에 결합시켰다. 그 후, 원심분리 및 세척과정을 거쳐 모델 약물인 메소트렉세이트(methotrexate, Sigma-aldrich Co., USA) 40 μg/200 μL를 상기 분산액에 단순 혼합하여 관절염 약물 세포 동시 치료용 마이크로 입자 의료기기 조성물을 도 4와 같이 제조하였다.After the prepared porous microparticles were dispersed in a PBS solution, amino-beta-cyclodextrin was added to the dispersion and bound to the surface of the porous microparticles using electrostatic attraction. Afterwards, through centrifugation and washing processes, 40 μg/200 μL of methotrexate (Sigma-aldrich Co., USA), a model drug, was simply mixed into the dispersion to produce a microparticle medical device composition for simultaneous treatment of arthritis drug cells. was prepared as shown in Figure 4.
실시예 5: 다공성 마이크로 입자에 도입된 약물 담지체의 특성 평가Example 5: Evaluation of properties of drug carrier introduced into porous microparticles
메소트렉세이트 2 mg을 초상자성 산화철 나노입자를 함유한 아미노-베타사이클로덱스트린-다공성 마이크로 입자 용액에 첨가한 후 2시간 동안 교반하여 로딩하였다. 이후 결합되지 않은 메소트렉세이트를 제거하기 위해 원심분리하고 세척하였다. 메소트렉세이트가 로딩된 다공성 마이크로 입자를 pH 5.5, 6.5 및 7.4의 PBS 용액에 분산하고 37℃에서 12시간 동안 방출을 시행하였고, 방출된 메소트렉세이트의 방출량은 345 nm에서 UV 분광광도계로 측정하여 계산하였다.2 mg of methotrexate was added to the amino-betacyclodextrin-porous microparticle solution containing superparamagnetic iron oxide nanoparticles and stirred for 2 hours before loading. Afterwards, it was centrifuged and washed to remove unbound methotrexate. Methotrexate-loaded porous microparticles were dispersed in PBS solutions of pH 5.5, 6.5, and 7.4 and released at 37°C for 12 hours. The amount of released methotrexate was measured with a UV spectrophotometer at 345 nm. Calculated.
도 5에서 확인할 수 있듯이, pH 값이 산성화될수록 약물의 방출속도가 증가하는 경향을 보였다. 따라서, 정상조직에서의 약물의 방출량보다 pH가 약산성인 암 병소에서의 약물의 방출량이 많을 것으로 예상된다. 폴리락틱엑시드는 산성조건에서 가수분해 속도가 빨라지기 때문에 약물의 방출 속도가 증가한다.As can be seen in Figure 5, the drug release rate tended to increase as the pH value became more acidic. Therefore, it is expected that the amount of drug released from cancerous lesions with a slightly acidic pH will be greater than that from normal tissues. Because polylactic acid undergoes faster hydrolysis in acidic conditions, the release rate of the drug increases.
Claims (15)
폴리락틱엑시드(polylactic acid)를 디클로로메탄에 6.25%(w/v)로 용해한 용액 및 상기 용액 1 mL 당 20 mg의 초상자성 산화철 나노입자의 혼합물을 제조하고, 소듐바이카보네이트(sodium bicarbonate)를 물에 용해하여 상기 혼합물에 5%(w/v)가 되도록 첨가한 용액을 제조하고, 이를 0.1%(w/v) 폴리비닐알코올(poly vinylalcohol) 용액에 떨어뜨려 4시간 동안 교반함으로써 마이크로 입자를 형성하고, 상기 마이크로 입자를 다시 0.2 M 소듐하이드록사이드(sodium hydroxide) 용액에 30분 동안 담근 후 물로 세척하고 동결건조함으로써 다공성 마이크로 입자를 제조하는 다공성 구조 형성 단계;
상기 다공성 마이크로 입자의 표면에 약물 담지체로 아미노-베타-사이클로덱스트린(amino-β-cyclodextrin)을 도입하는 약물 담지체 도입 단계; 및
상기 다공성 마이크로 입자의 표면에 도입된 약물 담지체에 메소트렉세이트(methotrexate)를 담지시키는 약물 담지 단계.Method for manufacturing porous microparticles (microspheres) for cell-drug delivery comprising the following steps:
A mixture of polylactic acid dissolved at 6.25% (w/v) in dichloromethane and 20 mg of superparamagnetic iron oxide nanoparticles per 1 mL of the solution was prepared, and sodium bicarbonate was dissolved in water. A solution was prepared by dissolving and adding 5% (w/v) to the mixture, dropping it into a 0.1% (w/v) polyvinyl alcohol solution and stirring for 4 hours to form micro particles. A porous structure forming step of preparing porous micro particles by immersing the micro particles in a 0.2 M sodium hydroxide solution for 30 minutes, washing them with water, and freeze-drying them;
A drug carrier introduction step of introducing amino-β-cyclodextrin as a drug carrier onto the surface of the porous microparticles; and
A drug loading step of loading methotrexate on the drug carrier introduced to the surface of the porous microparticles.
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| KR102042210B1 (en) | 2017-07-21 | 2019-11-27 | 서울대학교산학협력단 | Platform for target delivery of stem cell using magnet |
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| KR102315368B1 (en) | 2018-08-13 | 2021-10-20 | 재단법인대구경북과학기술원 | Magnetic actuated drug delivery microstructure and manufacturing method thereof |
| KR102366334B1 (en) * | 2019-03-29 | 2022-02-22 | 전남대학교산학협력단 | Porous Microscaffold for tissue regeneration having increased Magnetic driving force and Preparation Method thereof |
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2021
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