KR20220113856A - Hyaluronic acid hydrogel based on supramolecular chemistry and covalent bond - Google Patents
Hyaluronic acid hydrogel based on supramolecular chemistry and covalent bond Download PDFInfo
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- KR20220113856A KR20220113856A KR1020210016643A KR20210016643A KR20220113856A KR 20220113856 A KR20220113856 A KR 20220113856A KR 1020210016643 A KR1020210016643 A KR 1020210016643A KR 20210016643 A KR20210016643 A KR 20210016643A KR 20220113856 A KR20220113856 A KR 20220113856A
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- peg
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- 239000000017 hydrogel Substances 0.000 title claims abstract description 134
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 95
- 229920002674 hyaluronan Polymers 0.000 title claims abstract description 90
- 229960003160 hyaluronic acid Drugs 0.000 title claims abstract description 90
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 claims abstract description 72
- 239000000203 mixture Substances 0.000 claims abstract description 66
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- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 claims abstract description 18
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- 238000012377 drug delivery Methods 0.000 claims abstract description 10
- ADEBPBSSDYVVLD-UHFFFAOYSA-N donepezil Chemical compound O=C1C=2C=C(OC)C(OC)=CC=2CC1CC(CC1)CCN1CC1=CC=CC=C1 ADEBPBSSDYVVLD-UHFFFAOYSA-N 0.000 claims description 62
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- CGIGDMFJXJATDK-UHFFFAOYSA-N indomethacin Chemical compound CC1=C(CC(O)=O)C2=CC(OC)=CC=C2N1C(=O)C1=CC=C(Cl)C=C1 CGIGDMFJXJATDK-UHFFFAOYSA-N 0.000 claims description 6
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims description 6
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- VHVPQPYKVGDNFY-DFMJLFEVSA-N 2-[(2r)-butan-2-yl]-4-[4-[4-[4-[[(2r,4s)-2-(2,4-dichlorophenyl)-2-(1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazin-1-yl]phenyl]-1,2,4-triazol-3-one Chemical compound O=C1N([C@H](C)CC)N=CN1C1=CC=C(N2CCN(CC2)C=2C=CC(OC[C@@H]3O[C@](CN4N=CN=C4)(OC3)C=3C(=CC(Cl)=CC=3)Cl)=CC=2)C=C1 VHVPQPYKVGDNFY-DFMJLFEVSA-N 0.000 claims description 3
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 claims description 3
- VOVIALXJUBGFJZ-KWVAZRHASA-N Budesonide Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@@H]2[C@@H]1[C@@H]1C[C@H]3OC(CCC)O[C@@]3(C(=O)CO)[C@@]1(C)C[C@@H]2O VOVIALXJUBGFJZ-KWVAZRHASA-N 0.000 claims description 3
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- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 claims description 3
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- QJJXYPPXXYFBGM-LFZNUXCKSA-N Tacrolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1\C=C(/C)[C@@H]1[C@H](C)[C@@H](O)CC(=O)[C@H](CC=C)/C=C(C)/C[C@H](C)C[C@H](OC)[C@H]([C@H](C[C@H]2C)OC)O[C@@]2(O)C(=O)C(=O)N2CCCC[C@H]2C(=O)O1 QJJXYPPXXYFBGM-LFZNUXCKSA-N 0.000 claims description 3
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
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Abstract
Description
본 발명은 초분자화학 및 공유결합에 기반한 히알루론산 하이드로겔에 관한 것으로, 더욱 상세하게는 히알루론산(HA)에 도파민과 PEG가 결합된 HA-도파민-PEG 접합체; 사이클로덱스트린; 및 약학적으로 사용가능한 염의 양이온을 포함하는 하이드로겔 조성물, 및 이를 포함하는 약물 전달체에 관한 것이다.The present invention relates to a hyaluronic acid hydrogel based on supramolecular chemistry and covalent bonding, and more particularly, a HA-dopamine-PEG conjugate in which dopamine and PEG are bonded to hyaluronic acid (HA); cyclodextrins; And it relates to a hydrogel composition comprising a cation of a pharmaceutically usable salt, and a drug delivery system comprising the same.
하이드로겔은 물을 흡수할 수 있는 중합체로 구성된 3 차원 (3D) 네트워크로 정의되며, 하이드로겔을 이용하여 다양한 유형의 제형 (예: 필름, 마이크로 입자 및 나노 입자)을 제조할 수 있다. 하이드로겔은 높은 수분 함량으로 인해 친수성 약물의 탑재를 가능하게 한다. 가교된 고분자 네트워크는 하이드로겔의 유동학적 및 기계적 특성을 조절할 수 있으므로 다양한 분자량 범위를 갖는 약물 (즉, 소분자 화합물, 핵산, 펩타이드, 단백질 및 세포)의 제어 방출에 기여할 수 있다. 하이드로겔 중의 친수성 중합체 사슬의 빠른 분해를 방지하고 하이드로겔로부터의 즉각적인 약물 방출을 제어하기 위하여 몇몇 물리적 및 화학적 가교 전략이 도입되고 있다. 화학적 가교 방법 (즉, 부가 반응, 축합 반응, 효소 반응 및 라디칼 중합)과 물리적 가교 방법 (즉, 수소 결합, 이온 상호작용 및 단백질 상호작용)은 각각 고유한 장점과 단점이 있는 것으로 알려져 있다.Hydrogels are defined as three-dimensional (3D) networks of polymers capable of absorbing water, which can be used to prepare various types of formulations (e.g. films, microparticles and nanoparticles). Hydrogels enable loading of hydrophilic drugs due to their high water content. The cross-linked polymer network can control the rheological and mechanical properties of the hydrogel, and thus can contribute to the controlled release of drugs (ie, small molecule compounds, nucleic acids, peptides, proteins and cells) with various molecular weight ranges. Several physical and chemical crosslinking strategies are being introduced to prevent rapid degradation of the hydrophilic polymer chains in the hydrogel and to control the immediate drug release from the hydrogel. It is known that chemical crosslinking methods (ie, addition reactions, condensation reactions, enzymatic reactions and radical polymerization) and physical crosslinking methods (ie hydrogen bonding, ionic interactions and protein interactions) each have their own advantages and disadvantages.
하이드로겔 매트릭스로서 사용되는 다양한 물질 중에서 히알루론산 (hyaluronic acid, HA) (대부분 D-글루쿠론산[D-glucuronic acid]과 N-아세틸-D-글루코사민[N-acetyl-D-glucosamine]으로 구성됨)은 하이드로겔 시스템의 생체 적합성으로 인해 생분해성 매트릭스로서 널리 사용되고 있다. HA는 상피 조직 및 결합 조직의 구성 요소 중 하나이며 세포 외 기질 (extracellular matrix, ECM)의 주요 구성 요소이다. HA는 히알루론산 분해효소에 의해 분해될 수 있으며 체내에서는 간 및 림프관에서 분해될 수 있다. 약물 전달 시스템에 HA이 광범위하게 적용되는 것 외에도, 생체 적합성 및 생분해성으로 인해 상업적으로 피부 필러 및 상처 드레싱제로 사용되고 있다.Among the various substances used as hydrogel matrix, hyaluronic acid (HA) (mostly composed of D-glucuronic acid and N -acetyl-D-glucosamine [ N -acetyl-D-glucosamine]) Silver is widely used as a biodegradable matrix due to the biocompatibility of the hydrogel system. HA is one of the components of epithelial and connective tissue and is a major component of the extracellular matrix (ECM). HA can be degraded by hyaluronic acid degrading enzymes and can be degraded in the liver and lymphatic vessels in the body. In addition to the widespread application of HA in drug delivery systems, it is also used commercially as dermal fillers and wound dressings due to its biocompatibility and biodegradability.
본 발명이 해결하고자 하는 과제는 다양한 분자량 범위를 갖는 약물의 제어 방출에 있어서, 하이드로겔 중의 친수성 중합체 사슬의 빠른 분해를 방지하고 하이드로겔로부터의 즉각적인 약물 방출을 보다 효율적으로 제어할 수 있는 하이드로겔 시스템을 제공하는 것이다.The problem to be solved by the present invention is a hydrogel system that can prevent the rapid decomposition of the hydrophilic polymer chain in the hydrogel and control the immediate drug release from the hydrogel more efficiently in the controlled release of drugs having various molecular weight ranges is to provide
본 발명이 해결하고자 하는 과제는 이상에서 언급한 해결 과제로 제한되지 않으며, 언급되지 않은 또 다른 기술적 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.The problem to be solved by the present invention is not limited to the above-mentioned problems, and other technical problems not mentioned can be clearly understood by those of ordinary skill in the art to which the present invention belongs from the description below. There will be.
상기 해결 과제를 달성하기 위하여, 본 발명의 일 측면에 따라, 히알루론산(HA)에 도파민과 PEG가 결합된 HA-도파민-PEG 접합체; 사이클로덱스트린; 및 약학적으로 사용 가능한 염의 양이온을 포함하고, pH 7 ~ 11인 하이드로겔 조성물이 제공된다.In order to achieve the above object, according to an aspect of the present invention, HA-dopamine-PEG conjugate in which dopamine and PEG are coupled to hyaluronic acid (HA); cyclodextrins; and a cation of a pharmaceutically usable salt, and a hydrogel composition having a pH of 7 to 11 is provided.
일 구현예에서, 상기 히알루론산은 평균 분자량 10 ~ 3000 kDa일 수 있다.In one embodiment, the hyaluronic acid may have an average molecular weight of 10 to 3000 kDa.
일 구현예에서, 상기 PEG는 분자량 100 ~ 100000 Da일 수 있다.In one embodiment, the PEG may have a molecular weight of 100 to 100000 Da.
일 구현예에서, 상기 약학적으로 사용가능한 염의 양이온은 나트륨 이온, 칼륨 이온, 칼슘 이온, 마그네슘 이온, 바륨 이온, 알루미늄 이온 및 이들의 혼합물로 이루어진 군에서 선택되는 1종의 양이온일 수 있으며, 바람직하게는 수산화나트륨(NaOH), 제1인산칼륨(KH2PO4), 제2인산칼륨(K2HPO4), 염화칼륨(KCl), 제1인산나트륨(NaH2PO4), 제2인산나트륨(Na2HPO4), 염화나트륨(NaCl) 및 이들의 혼합물로 이루어진 군에서 선택되는 1종의 염의 양이온일 수 있다.In one embodiment, the cation of the pharmaceutically usable salt may be one cation selected from the group consisting of sodium ion, potassium ion, calcium ion, magnesium ion, barium ion, aluminum ion, and mixtures thereof, preferably sodium hydroxide (NaOH), potassium phosphate monobasic (KH 2 PO 4 ), potassium phosphate dibasic (K 2 HPO 4 ), potassium chloride (KCl), sodium phosphate monobasic (NaH 2 PO 4 ), sodium phosphate dibasic (Na 2 HPO 4 ), sodium chloride (NaCl), and may be a cation of one salt selected from the group consisting of mixtures thereof.
일 구현예에서, 상기 하이드로겔 조성물은 난용성 약물을 추가로 포함할 수 있으며, 상기 난용성 약물은 도네페질, 암포테리신 B, 커큐민, 플로레틴, 파클리탁셀, 도세탁셀, 덱사메타손, 부데소나이드, 시클로스포린, 타크로리무스, 라파마이신, 페노피브레이트, 프레드니솔론, 이트라코나졸, 인슐린(중성 pH), 인도메타신 및 이들의 혼합물로 이루어진 군에서 선택되는 1종일 수 있으며, 상기 난용성 약물은 마이크로스피어(microsphere)에 캡슐화된 것일 수 있다.In one embodiment, the hydrogel composition may further include a poorly soluble drug, wherein the poorly soluble drug is donepezil, amphotericin B, curcumin, phloretin, paclitaxel, docetaxel, dexamethasone, budesonide, cyclo It may be one selected from the group consisting of sporin, tacrolimus, rapamycin, fenofibrate, prednisolone, itraconazole, insulin (neutral pH), indomethacin, and mixtures thereof, and the poorly soluble drug is encapsulated in microspheres. it could be
본 발명의 다른 측면에 따라, 상기 하이드로겔 조성물을 포함하는 약물 전달체가 제공된다.According to another aspect of the present invention, a drug delivery system comprising the hydrogel composition is provided.
본 발명의 또 다른 측면에 따라, 상기 하이드로겔 조성물을 포함하는 피하주사용 주사제가 제공된다.According to another aspect of the present invention, there is provided an injection for subcutaneous injection comprising the hydrogel composition.
본 발명의 또 다른 측면에 따라, 상기 하이드로겔 조성물을 포함하는 필러가 제공된다.According to another aspect of the present invention, a filler comprising the hydrogel composition is provided.
본 발명의 또 다른 측면에 따라, 상기 하이드로겔 조성물을 포함하는 드레싱제가 제공된다.According to another aspect of the present invention, there is provided a dressing comprising the hydrogel composition.
본 발명의 또 다른 측면에 따라, 히알루론산(HA)에 도파민과 PEG를 결합시켜 HA-도파민-PEG 접합체를 얻는 단계; 및 상기 HA-도파민-PEG 접합체 및 사이클로덱스트린을 혼합한 후 약학적으로 사용 가능한 염을 혼합하여 pH 7 ~ 11로 조절하는 단계를 포함하는 하이드로겔 조성물의 제조 방법이 제공된다.According to another aspect of the present invention, the method comprising: conjugating dopamine and PEG to hyaluronic acid (HA) to obtain a HA-dopamine-PEG conjugate; And after mixing the HA-dopamine-PEG conjugate and cyclodextrin, a method for preparing a hydrogel composition comprising the step of adjusting the pH to 7 to 11 by mixing a pharmaceutically usable salt is provided.
일 구현예에서, 상기 HA-도파민-PEG 접합체는 히알루론산의 카르복시산 기와 도파민의 아민 기; 및 히알루론산의 카르복시산 기와 PEG의 아민 기 간에 형성되는 아미드 결합에 의해 제조될 수 있다.In one embodiment, the HA-dopamine-PEG conjugate comprises a carboxylic acid group of hyaluronic acid and an amine group of dopamine; and an amide bond formed between the carboxylic acid group of hyaluronic acid and the amine group of PEG.
일 구현예에서, 상기 하이드로겔 조성물의 제조 방법은 상기 HA-도파민-PEG 접합체 및 사이클로덱스트린을 혼합한 후 난용성 약물을 혼합하는 단계를 추가로 포함할 수 있다.In one embodiment, the method for preparing the hydrogel composition may further include mixing the poorly soluble drug after mixing the HA-dopamine-PEG conjugate and the cyclodextrin.
본 발명에 의해, 히알루론산(HA)에 도파민과 PEG가 결합된 HA-도파민-PEG 접합체; 사이클로덱스트린; 및 약학적으로 사용가능한 염의 양이온을 포함하는 하이드로겔 조성물은 즉각적인 겔화, 단일 주사기를 통한 주사가능성, 자가치유능, 전단 박화 거동, 약물의 서방출 특성 및 피하주사 후의 느린 생분해 특성을 나타내므로, 하이드로겔로부터의 즉각적인 약물 방출을 보다 효율적으로 제어할 수 있는 약물 서방출 제제(피하주사 제형 등)로 개발될 수 있다는 것이 밝혀졌다.According to the present invention, HA-dopamine-PEG conjugate in which dopamine and PEG are bonded to hyaluronic acid (HA); cyclodextrins; And a hydrogel composition comprising a cation of a pharmaceutically usable salt exhibits immediate gelation, injectability through a single syringe, self-healing ability, shear thinning behavior, sustained release properties of the drug and slow biodegradation properties after subcutaneous injection. It has been found that it can be developed into a sustained-release drug formulation (such as a subcutaneous injection formulation) that can more efficiently control the immediate drug release from the gel.
따라서, 본 발명의 히알루론산 하이드로겔 조성물, 및 이를 포함하는 피하주사용 주사제 등의 약물 전달체는 의약, 제약, 피부 시술 분야에서 최소 침습성으로 피하조직에 주사될 수 있으며, 약물 서방출에 의한 투여 빈도의 감소로 환자 순응도를 향상시킬 수 있는 제형으로서 유용하게 사용될 수 있다.Therefore, the hyaluronic acid hydrogel composition of the present invention, and a drug delivery system such as a subcutaneous injection containing the same can be injected into the subcutaneous tissue with minimal invasiveness in the fields of medicine, pharmaceuticals, and skin procedures, and the frequency of administration by sustained release of the drug It can be usefully used as a formulation that can improve patient compliance by reducing
본 발명의 효과는 상기한 효과로 한정되는 것은 아니며, 본 발명의 설명 또는 특허청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.The effects of the present invention are not limited to the above-described effects, and it should be understood to include all effects that can be inferred from the configuration of the invention described in the description or claims of the present invention.
도 1은 HDP[HA-도파민-PEG]의 합성 및 특성에 관한 결과로서, (A) HA로부터 HDP를 합성한 과정의 개략도이다. (B) HDP의 1H-NMR 스펙트럼으로서, HA의 N-아세틸 기 (시프트 1), 도파민 (시프트 2-4) 및 PEG의 메틸렌 기 (시프트 5 및 6)에 대한 양성자 NMR 화학적 이동이 스펙트럼에 표시되어 있다. (C) 중량비 (mPEG2K-NH2/HA)와 적분비 (3.6-3.7/1.8-2.0 ppm) 사이의 선형 회귀선이다. (D) HD 및 HDP의 13C-NMR 스펙트럼으로서, HA의 N-아세틸 기 (시프트 1), 도파민 (시프트 2-4) 및 PEG의 메틸렌 기 (시프트 5 및 6)에 대한 탄소 NMR 화학적 이동이 나타나 있다. (E) HD, mPEG2K-NH2 및 HDP의 FT-IR 스펙트럼이다.
도 2는 PDM의 입자 특성으로서, (A) FE-SEM 이미지 및 (B) PDM의 입자 크기 분포이다.
도 3은 겔 가교 메커니즘을 설명하는 결과들로서, (A) aCD 및 HDP/aCD의 1H-NMR 스펙트럼이다. (B) HDP/aCD의 2D NOESY (NMR nuclear Overhauser effect spectroscopy) 결과이다. (C) HDP 및 HDP 8.5 군의 UV-Vis 흡광도 결과이다. (D) aCD, HDP 및 HDP/aCD 검체의 XRD 프로파일이다.
도 4는 하이드로겔 시스템의 유동학적 특성으로서, (A) HA/aCD, HD/aCD, HP/aCD, HP/aCD/PDM, HDP/aCD, HDP/aCD 8.5, HDP/aCD/PDM 8.5 및 HDP/PDM 8.5 군의 반전 시험 결과로서, 0 시간 (정상 위치) 및 6 시간 (반전 위치)에 겔화 거동을 시험하였다. (B) HDP/aCD/PDM 8.5 하이드로겔의 인큐베이션 시간에 따른 겔화 거동으로서, 겔화 시간을 측정하기 위하여 180 분 동안 반전 시험을 수행하였다. (C) 주사기 바늘을 통한 HDP/aCD/PDM 8.5 하이드로겔 (0.2 mg/mL 메틸렌블루 함유)의 주사가능성 시험 결과이다. (D) HP/aCD/PDM, HDP/aCD 8.5, HDP/PDM 8.5 및 HDP/aCD/PDM 8.5 검체의 유동학 결과로서, 변형률 변화 시험(strain sweep), 진동수 변화 시험(frequency sweep), 단계적 시간-점탄성 및 전단 속도-전단 응력 결과를 측정하였다.
도 5는 가교된 하이드로겔 시스템의 물리화학적 특성으로서, (A) HDP/aCD 8.5 및 HDP/aCD/PDM 8.5 군의 FE-SEM 이미지로서, 동결건조된 샘플의 단면 이미지를 관찰하였고, 스케일 바 (흰색)의 길이는 20 μm이다. (B) 도네페질 용액 및 HDP/aCD/PDM 8.5 하이드로겔 군에서의 약물의 방출 프로파일로서, 각 측정값은 평균±SD (n=3)로 표시되어 있다. (C) HAase의 존재 또는 부재 하에서 HDP/aCD/PDM 8.5 하이드로겔의 시험관내 분해 결과로서, 각 측정값은 평균±표준편차 (n=4)로 표시되어 있다.
도 6은 설계된 하이드로겔 시스템의 마우스에서의 생체 내(in vivo) 분해 특성으로서, (A) 대조군, PDM, HDP/aCD 8.5, HDP/PDM 8.5, HDP/aCD/PDM 및 HDP/aCD/PDM 8.5 군에서 겔 주입 부위를 절개한 사진이다. (B) HDP/aCD 8.5, HDP/aCD/PDM 및 HDP/aCD/PDM 8.5 군에서 절제한 겔 사진이다. (C) HDP/aCD 8.5, HDP/aCD/PDM 및 HDP/aCD/PDM 8.5 군의 잔여 겔 중량이고, 각 측정값은 평균±표준편차 (n=5)로 표시되어 있다. 1 is a schematic view of the synthesis and properties of HDP [HA-dopamine-PEG], (A) the process of synthesizing HDP from HA. (B) 1 H-NMR spectrum of HDP, where the proton NMR chemical shifts for the N -acetyl group of HA (shift 1), dopamine (shift 2-4) and methylene group of PEG (
2 is a particle characteristic of PDM, (A) FE-SEM image and (B) particle size distribution of PDM.
Figure 3 is the results explaining the gel crosslinking mechanism, (A) 1 H-NMR spectrum of aCD and HDP/aCD. (B) 2D NOESY (NMR nuclear Overhauser effect spectroscopy) results of HDP/aCD. (C) UV-Vis absorbance results of HDP and HDP 8.5 groups. (D) XRD profiles of aCD, HDP and HDP/aCD samples.
4 shows the rheological properties of the hydrogel system, (A) HA/aCD, HD/aCD, HP/aCD, HP/aCD/PDM, HDP/aCD, HDP/aCD 8.5, HDP/aCD/PDM 8.5 and HDP. As a result of the reversal test of the /PDM 8.5 group, the gelation behavior was tested at 0 hours (normal position) and 6 hours (inversion position). (B) Gelation behavior according to incubation time of HDP/aCD/PDM 8.5 hydrogel. In order to measure the gelation time, an inversion test was performed for 180 minutes. (C) Result of injectability test of HDP/aCD/PDM 8.5 hydrogel (containing 0.2 mg/mL methylene blue) through a syringe needle. (D) Rheology results of HP/aCD/PDM, HDP/aCD 8.5, HDP/PDM 8.5 and HDP/aCD/PDM 8.5 specimens, strain sweep, frequency sweep, step-by-step time -Viscoelasticity and shear rate-shear stress results were measured.
Figure 5 is the physicochemical properties of the cross-linked hydrogel system, (A) FE-SEM images of HDP / aCD 8.5 and HDP / aCD / PDM 8.5 groups. Cross-sectional images of the lyophilized samples were observed, and scale bars ( The length of white) is 20 μm. (B) Drug release profiles in donepezil solution and HDP/aCD/PDM 8.5 hydrogel groups, with each measurement expressed as mean±SD (n=3). (C) In vitro degradation results of HDP/aCD/PDM 8.5 hydrogels in the presence or absence of HAase, with each measurement expressed as mean±standard deviation (n=4).
6 is an in vivo degradation characteristic in mice of the designed hydrogel system, (A) control, PDM, HDP / aCD 8.5, HDP / PDM 8.5, HDP / aCD / PDM and HDP / aCD / PDM 8.5 It is a photograph of the gel injection site incised in the group. (B) A photograph of a gel excised from HDP/aCD 8.5, HDP/aCD/PDM and HDP/aCD/PDM 8.5 groups. (C) Residual gel weights of HDP/aCD 8.5, HDP/aCD/PDM and HDP/aCD/PDM 8.5 groups, and each measured value is expressed as mean±standard deviation (n=5).
본 발명은 히알루론산(HA)에 도파민과 PEG가 결합된 HA-도파민-PEG 접합체; 사이클로덱스트린; 및 약학적으로 사용가능한 염의 양이온을 포함하고, pH 7 ~ 11인 하이드로겔 조성물을 제공한다.The present invention is a HA-dopamine-PEG conjugate in which dopamine and PEG are bonded to hyaluronic acid (HA); cyclodextrins; and a cation of a pharmaceutically usable salt, and provides a hydrogel composition having a pH of 7 to 11.
본 발명에서는 HA 기반 하이드로겔의 유동학적 및 기계적 특성을 최적화하기 위하여 다양한 화학적 및 물리적 가교 접근법이 시도되었다. 화학 반응 (즉, 클릭 화학[click chemistry], 이황화 결합, 효소 가교 및 에스테르화) 및 물리적 상호작용 (즉, 호스트-게스트 상호작용 및 소수성 상호작용)이 하이드로겔의 물리화학적 및 기계적 특징을 조정하는데 사용되고 있다. 이전 연구 (Lee et al., 2020; Seo et al., 2020; Yang et al., 2020)에서는 HA-도파민 접합체와 미네랄 또는 완충 염 (예: FeSO4, KH2PO4 및 Na2SeO3) 간의 결합(coordination)과 카테콜 중합이 HA 하이드로겔 시스템의 주요한 가교 메커니즘으로 도입되었다. 이러한 연구의 공통된 목적은 피하 또는 종양 내 주사를 위한 겔화의 조절, 단일 주사기를 통한 주사가능성, 낮은 생분해율, 자가치유능 및 지속적인 약물 방출이다. 본 발명에서는 주사제 사용에 있어서의 안전성을 확보하기 위하여 높은 독성을 나타내는 가교제의 사용은 제외되었다. 대신, 하이드로겔 시스템을 가교시키기 위한 HA 유도체의 반응물로서 생리학적으로 필수적인 미네랄과 완충 염이 추가되었다.In the present invention, various chemical and physical crosslinking approaches were attempted to optimize the rheological and mechanical properties of HA-based hydrogels. Chemical reactions (i.e., click chemistry, disulfide bonds, enzymatic crosslinking and esterification) and physical interactions (i.e., host-guest interactions and hydrophobic interactions) are used to modulate the physicochemical and mechanical properties of hydrogels. is being used Previous studies (Lee et al., 2020; Seo et al., 2020; Yang et al., 2020) have shown that HA-dopamine conjugates and mineral or buffered salts (eg, FeSO 4 , KH 2 PO 4 and Na 2 SeO 3 ) Hepatic coordination and catechol polymerization were introduced as major crosslinking mechanisms of the HA hydrogel system. The common objectives of these studies are control of gelation for subcutaneous or intratumoral injection, injectability through a single syringe, low biodegradability, self-healing capacity, and sustained drug release. In the present invention, the use of a crosslinking agent exhibiting high toxicity is excluded to ensure safety in the use of injections. Instead, physiologically essential minerals and buffer salts were added as reactants of HA derivatives to crosslink the hydrogel system.
또한, 본 발명에서는 HA 기반 하이드로겔의 유동학적 특성을 조절하기 위하여 초분자화학 (물리적 상호작용)을 이용하였다. 초분자화학 영역에서 사이클로덱스트린 (CD)은 분자 인식 특성으로 인해 호스트 부분으로 광범위하게 사용되고 있으며, 다른 게스트 분자가 결합되어 포접 복합체가 형성되도록 한다. 또한, CD의 내부 공동에 작은 소수성 분자를 포함하는 것 대신에, CD 분자내 공동을 관통하는 선형 중합체 사슬을 기반으로 한 "분자 목걸이" 개념(예: 알파-CD (aCD)-폴리에틸렌 글리콜 (PEG))을 개발하여 폴리로탁산 구조를 형성하기도 한다. 또 다른 연구에서는, 말단 선형 사슬이 대형 마개 분자로 제한될 수 있는 폴리로탁산 시스템과 함께, 특정 조건에서 쉽게 결합이 해제될 수 있는 폴리슈도로탁산 구조도 하이드로겔 가교를 위하여 고안되었다. 게스트 분자로 선형 중합체를 사용하는 것 외에도, 폴리슈도로탁산 하이드로겔 구조를 위한 aCD의 대응물로 분지형 또는 그래프트된(grafted) 중합체가 도입되기도 한다. 특히, 호스트-게스트 포접 복합체 조립 구조로서 CD 분자내 공동을 관통하는 PEG 사슬이 연결된(grafted) 알지네이트, 키토산, 덱스트란 및 헤파린 등이 제작되기도 하였다.In addition, in the present invention, supramolecular chemistry (physical interaction) was used to control the rheological properties of the HA-based hydrogel. In the field of supramolecular chemistry, cyclodextrin (CD) is widely used as a host moiety due to its molecular recognition properties, allowing other guest molecules to bind to form inclusion complexes. Furthermore, instead of including small hydrophobic molecules in the inner cavity of the CD, a “molecular necklace” concept based on linear polymer chains penetrating the CD intramolecular cavity (e.g., alpha-CD (aCD)-polyethylene glycol (PEG) )) to form a polyrotaxane structure. In another study, a polypseudotaxane structure, which can be easily dissociated under certain conditions, along with a polyrotaxane system in which the terminal linear chain can be restricted to large stopper molecules, was also designed for hydrogel crosslinking. In addition to using linear polymers as guest molecules, branched or grafted polymers have also been introduced as counterparts of aCDs for polypseudotaxane hydrogel structures. In particular, as a host-guest inclusion complex assembly structure, alginate, chitosan, dextran, and heparin, in which a PEG chain penetrating a CD intramolecular cavity is grafted, has also been prepared.
본 발명에서는 PEG (HA-도파민-PEG (HDP) 중합체에 연결된 형태) 및 도파민 (HDP 접합체에 부착된 형태)이 각각 폴리슈도로탁산 및 카테콜 중합 형성에 참여하여 HA 하이드로겔 가교를 형성하는 구조를 제공한다. 이전에 HA-PEG 및 aCD를 기반으로 한 하이드로겔 구조는 보고된 적이 있지만, aCD-PEG 상호작용과 함께 HDP 하이드로겔의 화학적 가교를 위하여 폴리도파민 연결을 도입한 것은 본 발명에서 최초로 제공하는 것이다. 이러한 겔 가교 전략에 의해, 설계된 HDP 기반 하이드로겔의 점탄성을 조절하여, 이후 설명할 즉각적인 겔화, 단일 주사기를 통한 주사가능성, 자가치유능, 전단 박화 거동 및 피하주사 후의 느린 생분해를 달성하였다. 또한, 도네페질 캡슐화된 마이크로스피어를 가교된 HA 하이드로겔에 함입시켜 투여 빈도를 감소시킬 수 있도록 하였다.In the present invention, PEG (form linked to HA-dopamine-PEG (HDP) polymer) and dopamine (form attached to HDP conjugate) participate in the formation of polypseudotaxane and catechol polymerization, respectively, to form HA hydrogel cross-links provides Although hydrogel structures based on HA-PEG and aCD have been previously reported, the introduction of polydopamine linkages for chemical crosslinking of HDP hydrogels with aCD-PEG interaction is provided for the first time in the present invention. By this gel crosslinking strategy, the viscoelasticity of the designed HDP-based hydrogel was controlled to achieve immediate gelation, injectability through a single syringe, self-healing ability, shear thinning behavior and slow biodegradation after subcutaneous injection, which will be described later. In addition, donepezil encapsulated microspheres were incorporated into the cross-linked HA hydrogel to reduce the dosing frequency.
본 발명의 하이드로겔 조성물에 포함되는 히알루론산(HA)-도파민-PEG 접합체는 히알루론산(HA)에 도파민과 PEG가 결합되어 형성된 것으로서, 여기에 사용되는 히알루론산은 본 발명이 속하는 기술 분야에서 통상적으로 사용되는 히알루론산을 사용할 수 있으며, 바람직하게는 평균 분자량이 10 ~ 3000 kDa, 더욱 바람직하게는 저분자량 등급인 100 ~ 1000 kDa인 히알루론산일 수 있다.The hyaluronic acid (HA)-dopamine-PEG conjugate included in the hydrogel composition of the present invention is formed by combining dopamine and PEG to hyaluronic acid (HA). Hyaluronic acid may be used as a hyaluronic acid, preferably hyaluronic acid having an average molecular weight of 10 to 3000 kDa, more preferably from 100 to 1000 kDa, which is a low molecular weight grade.
본 발명에서 사용되는 도파민(dopamine)은 하기 화학식 1의 화학구조를 가지며, 화학구조 중 아민기가 히알루론산의 카르복시산 기와 아미드 결합하여 접합체를 형성한다.Dopamine used in the present invention has a chemical structure of
<화학식 1><
본 발명에서 사용되는 폴리에틸렌글리콜(polyethylene glycol)은 에틸렌글리콜의 중축합으로 생성되는 폴리에테르(polyether) 화합물로서 통상적으로 H-[O-CH2-CH2]n-OH로 표시되며, PEG, 폴리에틸엔옥사이드(polyethylene oxide, PEO), 폴리옥시에틸렌(polyoxyethylene, POE) 등으로 지칭되기도 한다. 본 발명에서 지칭하는 폴리에틸렌글리콜(PEG)은 폴리에틸렌글리콜 및 그 양 말단이 일부 변형된 폴리에틸렌글리콜 유도체를 포함하며, 예를 들어, 메톡시 폴리에틸렌 글리콜 아민(methoxy polyethylene glycol amine, mPEG2K-NH2) 등을 포함할 수 있다. 상기 PEG는 분자량 100 ~ 100000 Da일 수 있으며, 바람직하게는 500 ~ 20000 Da, 더욱 바람직하게는 1000 ~ 10000 Da일 수 있다. 상기 PEG는 히알루론산(HA)-도파민-PEG 접합체 총량에 대하여 10 ~ 70 중량%로 포함될 수 있으며, 바람직하게는 20 ~ 60 중량%로 포함될 수 있다.Polyethylene glycol used in the present invention is a polyether compound produced by polycondensation of ethylene glycol and is usually represented by H-[O-CH 2 -CH 2 ] n -OH, PEG, poly It is also referred to as ethylen oxide (polyethylene oxide, PEO), polyoxyethylene (POE), and the like. Polyethylene glycol (PEG) referred to in the present invention includes polyethylene glycol and polyethylene glycol derivatives having both ends partially modified, for example, methoxy polyethylene glycol amine (mPEG2K-NH 2 ) and the like. may include The PEG may have a molecular weight of 100 to 100000 Da, preferably 500 to 20000 Da, more preferably 1000 to 10000 Da. The PEG may be included in an amount of 10 to 70% by weight, preferably 20 to 60% by weight, based on the total amount of the hyaluronic acid (HA)-dopamine-PEG conjugate.
본 발명에서 사용되는 사이클로덱스트린은 전분 분해시에 사이클릭 덱스트린으로 형성되는 사이클로아밀로오스 또는 사이클로글루칸으로서, 6, 7 또는 8개가 α-1,4 결합된 글루코오스 단위의 하나의 사이클러스(cyclus)를 형성하기 때문에, α-, β- 또는 γ-사이클로덱스트린으로 지칭된다. 이것은 하나의 결정 격자 내에 결합되어 관통하는 내부 분자 채널을 형성하며 상기 채널 내에 소수성 게스트 분자, 예를 들면, 알코올 또는 탄화수소가 가변량으로 포화 상태까지 포함될 수 있도록 쌓여질 수 있다. 상기 사이클로덱스트린은 하이드로겔 조성물에서 히알루론산(HA)-도파민-PEG 접합체 중량 대비 0.5 ~ 50배의 비율로 포함될 수 있으며, 바람직하게는 1 ~ 10배의 비율로 포함될 수 있다.The cyclodextrin used in the present invention is cycloamylose or cycloglucan formed from cyclic dextrin upon starch degradation, and 6, 7 or 8 α-1,4-linked glucose units form one cyclus. Therefore, they are referred to as α-, β- or γ-cyclodextrins. It binds within one crystal lattice to form a penetrating internal molecular channel, which can be stacked to contain variable amounts of hydrophobic guest molecules, such as alcohols or hydrocarbons, to saturation. The cyclodextrin may be included in a ratio of 0.5 to 50 times the weight of the hyaluronic acid (HA)-dopamine-PEG conjugate in the hydrogel composition, preferably in a ratio of 1 to 10 times.
본 명세서에서, "약학적으로 사용가능한 염"이라 함은 본 발명이 속하는 기술분야에서 통상적으로 의약품에 사용가능한 염으로서, 바람직하게는 피하주사 등의 인체에 적용되는 제형에 사용가능한 염이다. 예를 들어, 나트륨염, 칼륨염 등과 같은 알칼리 금속염; 칼슘염, 마그네슘염, 바륨염 등과 같은 알칼리 토금속염; 알루미늄염 등이 포함될 수 있으나, 이에 제한되는 것은 아니다. 상기 약학적으로 사용가능한 염의 구체적인 예로서는 수산화나트륨(NaOH), 제1인산칼륨(KH2PO4), 제2인산칼륨(K2HPO4), 염화칼륨(KCl), 제1인산나트륨(NaH2PO4), 제2인산나트륨(Na2HPO4), 염화나트륨(NaCl) 및 이들의 혼합물로 이루어진 군에서 선택되는 1종의 염이 포함될 수 있으나, 이에 제한되는 것은 아니다. 상기 약학적으로 사용가능한 염의 양이온은 나트륨 이온, 칼륨 이온, 칼슘 이온, 마그네슘 이온, 바륨 이온, 알루미늄 이온 및 이들의 혼합물로 이루어진 군에서 선택되는 1종의 양이온이다.In the present specification, the term "pharmaceutically usable salt" refers to a salt that can be used in pharmaceuticals in the art to which the present invention belongs, preferably a salt that can be used in formulations applied to the human body such as subcutaneous injection. alkali metal salts such as, for example, sodium salts, potassium salts and the like; alkaline earth metal salts such as calcium salts, magnesium salts and barium salts; Aluminum salts and the like may be included, but are not limited thereto. Specific examples of the pharmaceutically usable salt include sodium hydroxide (NaOH), potassium phosphate monobasic (KH 2 PO 4 ), potassium phosphate dibasic (K 2 HPO 4 ), potassium chloride (KCl), sodium phosphate monobasic (NaH 2 PO). 4 ), sodium phosphate dibasic (Na 2 HPO 4 ), sodium chloride (NaCl), and one salt selected from the group consisting of mixtures thereof may be included, but is not limited thereto. The cation of the pharmaceutically usable salt is one kind of cation selected from the group consisting of sodium ion, potassium ion, calcium ion, magnesium ion, barium ion, aluminum ion, and mixtures thereof.
상기와 같이 약학적으로 사용가능한 염을 하이드로겔 조성물 중에서 졸-겔 전이를 나타내는 농도로 사용하는 경우에 하이드로겔 조성물은 pH 7 ~ 11인 특성을 나타내며, 바람직하게는 pH 7.5 ~ 10, 더욱 바람직하게는 pH 8 ~ 9인 특성을 나타낸다.As described above, when the pharmaceutically usable salt is used at a concentration showing sol-gel transition in the hydrogel composition, the hydrogel composition exhibits a characteristic of
일 구현예에서, 상기 하이드로겔 조성물은 난용성 약물을 추가로 포함할 수 있으며, 상기 난용성 약물은 수용해도가 비교적 낮은 약물을 의미한다. 예를 들어, 상기 난용성 약물은 도네페질, 암포테리신 B, 커큐민, 플로레틴, 파클리탁셀, 도세탁셀, 덱사메타손, 부데소나이드, 시클로스포린, 타크로리무스, 라파마이신, 페노피브레이트, 프레드니솔론, 이트라코나졸, 인슐린(중성 pH), 인도메타신 및 이들의 혼합물로 이루어진 군에서 선택되는 1종일 수 있으나, 이에 제한되지 않는다.In one embodiment, the hydrogel composition may further include a poorly soluble drug, and the poorly soluble drug means a drug having relatively low water solubility. For example, the poorly soluble drug is donepezil, amphotericin B, curcumin, floretin, paclitaxel, docetaxel, dexamethasone, budesonide, cyclosporine, tacrolimus, rapamycin, fenofibrate, prednisolone, itraconazole, insulin (neutral pH ), may be one selected from the group consisting of indomethacin and mixtures thereof, but is not limited thereto.
일 구현예에서, 상기 난용성 약물은 마이크로스피어(microsphere)에 캡슐화될 수 있다. 상기 마이크로스피어는 통상적으로 사용되는 유화 및 용매 증발 방법을 이용하여 제조될 수 있으며, 요약하면 하기와 같다. 폴리(락틱-코-글리콜산)[poly(lactic-co-glycolic acid), PLGA]과 난용성 약물을 유기용매에 용해시킨 후 폴리비닐알코올 수용액에 첨가하여 균질화시킴으로써 에멀젼을 얻는다. 이 때 사용되는 PLGA는 본 발명이 속하는 기술 분야에서 통상적으로 사용되는 PLGA일 수 있으며, 바람직하게는 락트산:글리콜산(10:90 ~ 90:10), 더욱 바람직하게는 락트산:글리콜산(40:60 ~ 60:40)으로 구성된 PLGA일 수 있으며, 평균 분자량 10 ~ 100 kDa일 수 있으나, 이에 제한되지 않는다. 얻어진 에멀젼을 교반하여 유기상을 증발시킨 다음, 적절한 분리 방법(예를 들어, 원심분리 등)을 이용하여 마이크로스피어를 분리한다.In one embodiment, the poorly soluble drug may be encapsulated in microspheres. The microspheres can be prepared using commonly used emulsification and solvent evaporation methods, which are summarized as follows. After dissolving poly(lactic-co-glycolic acid) [poly( lactic-co-glycolic acid), PLGA] and a poorly soluble drug in an organic solvent, it is added to an aqueous polyvinyl alcohol solution and homogenized to obtain an emulsion. The PLGA used at this time may be PLGA commonly used in the art to which the present invention belongs, preferably lactic acid: glycolic acid (10:90 to 90:10), more preferably lactic acid: glycolic acid (40: 60 to 60:40), and may have an average molecular weight of 10 to 100 kDa, but is not limited thereto. The resulting emulsion is stirred to evaporate the organic phase, and then the microspheres are separated using an appropriate separation method (eg, centrifugation, etc.).
상기와 같은 방법으로 제조된 마이크로스피어는 평균 부피 밀도-연관 직경이 1 ~ 100 μm, 바람직하게는 5 ~ 50 μm일 수 있으나, 이에 제한되지 않는다.The microspheres prepared by the above method may have an average bulk density-associated diameter of 1 to 100 μm, preferably 5 to 50 μm, but is not limited thereto.
본 발명은 또한, 상기 하이드로겔 조성물을 포함하는 약물 전달체를 제공한다. 본 발명의 하이드로겔 조성물은 상기 설명한 바와 같은 겔 가교 전략에 의해 HDP 기반 하이드로겔의 점탄성을 조절함으로써 즉각적인 겔화, 단일 주사기를 통한 주사가능성, 자가치유능, 전단 박화 거동 및 피하주사 후의 느린 생분해와 같은 특성을 달성할 수 있다. 따라서, 약물 성질에 따라 마이크로스피어에 캡슐화된 약물-마이크로스피어가 HA-도파민-PEG (HDP) 중합체에 함입되도록 적절히 설계될 수 있으므로, 이와 같은 특성이 조절된 약물 전달체 및 피하주사용 주사제로 사용될 수 있다.The present invention also provides a drug delivery system comprising the hydrogel composition. The hydrogel composition of the present invention controls the viscoelasticity of the HDP-based hydrogel by the gel crosslinking strategy as described above, such as immediate gelation, injectability through a single syringe, self-healing ability, shear thinning behavior, and slow biodegradation after subcutaneous injection. characteristics can be achieved. Therefore, depending on the drug properties, the drug-microspheres encapsulated in the microspheres can be appropriately designed to be incorporated into the HA-dopamine-PEG (HDP) polymer. have.
또한, 본 발명의 하이드로겔 조성물은 상기와 같은 점탄성 조절 특성을 이용하여 설계될 수 있으므로 하이드로겔 조성물을 포함하는 필러 또는 드레싱제로서 사용될 수 있다.In addition, since the hydrogel composition of the present invention can be designed using the viscoelasticity control properties as described above, it can be used as a filler or a dressing including the hydrogel composition.
본 발명은 또한, 히알루론산(HA)에 도파민과 PEG를 결합시켜 HA-도파민-PEG 접합체를 얻는 단계; 및 상기 HA-도파민-PEG 접합체 및 사이클로덱스트린을 혼합한 후 약학적으로 사용가능한 염을 혼합하여 pH 7 ~ 11로 조절하는 단계를 포함하는 하이드로겔 조성물의 제조 방법를 제공한다.The present invention also comprises the steps of conjugating dopamine and PEG to hyaluronic acid (HA) to obtain a HA-dopamine-PEG conjugate; And after mixing the HA-dopamine-PEG conjugate and cyclodextrin, it provides a method for preparing a hydrogel composition comprising the step of adjusting the pH to 7 to 11 by mixing a pharmaceutically usable salt.
본 발명의 하이드로겔 조성물의 제조 방법에는 히알루론산(HA)에 도파민과 PEG를 결합시켜 HA-도파민-PEG 접합체를 얻는 단계가 포함된다. 본 단계에서는 히알루론산의 카르복시산 기와 도파민의 아민 기; 및 히알루론산의 카르복시산 기와 PEG의 아민 기 간에 아미드 결합을 형성시켜 HA-도파민-PEG 접합체를 제조할 수 있다. 히알루론산-도파민 간 아미드 결합시에는 EDC-NHS 커플링을 이용하여 아미드 결합 반응시킬 수 있으며, 제조 과정을 요약하면, 히알루론산을 물에 분산시킨 후, EDC 및 NHS의 유기용매 용액과 상기 히알루론산 분산액을 혼합하여 히알루론산의 카르복실산 기를 활성화시킨다. 상기 혼합액의 pH를 산성화한 다음, 도파민 용액을 첨가하여 반응시킴으로써 히알루론산-도파민 접합체를 얻을 수 있다. 히알루론산-PEG 간 아미드 결합시에는 히알루론산-도파민 접합체를 물에 분산시킨 후, EDC 및 NHS의 유기용매 용액과 상기 히알루론산-도파민 접합체 분산액을 혼합하여 히알루론산의 카르복실산 기를 활성화시킨다. 상기 혼합액의 pH를 산성화한 다음, 아민 기 포함 구조의 PEG 용액을 첨가하여 반응시킴으로써 HA-도파민-PEG 접합체를 얻을 수 있다.The method for preparing the hydrogel composition of the present invention includes the step of obtaining a HA-dopamine-PEG conjugate by binding dopamine and PEG to hyaluronic acid (HA). In this step, the carboxylic acid group of hyaluronic acid and the amine group of dopamine; and forming an amide bond between the carboxylic acid group of hyaluronic acid and the amine group of PEG to prepare a HA-dopamine-PEG conjugate. In the case of amide bond between hyaluronic acid and dopamine, the amide bond reaction can be performed using EDC-NHS coupling. The dispersion is mixed to activate the carboxylic acid groups of the hyaluronic acid. A hyaluronic acid-dopamine conjugate can be obtained by acidifying the pH of the mixed solution and then reacting by adding a dopamine solution. When amide bonding between hyaluronic acid and PEG, the hyaluronic acid-dopamine conjugate is dispersed in water, and then the organic solvent solution of EDC and NHS and the hyaluronic acid-dopamine conjugate dispersion are mixed to activate the carboxylic acid group of hyaluronic acid. The HA-dopamine-PEG conjugate can be obtained by acidifying the pH of the mixed solution and then reacting by adding a PEG solution having an amine group-containing structure.
본 발명의 하이드로겔 조성물의 제조 방법에는 상기 HA-도파민-PEG 접합체 및 사이클로덱스트린을 혼합한 후 약학적으로 사용가능한 염을 혼합하여 pH 7 ~ 11로 조절하는 단계가 포함된다. 본 단계에서는 HA-도파민-PEG 접합체 및 사이클로덱스트린을 물에 분산시켜 분산액을 제조한 후, 약학적으로 사용가능한 염의 용액을 상기 분산액에 첨가하여 제조할 수 있다. 이 때, HA-도파민-PEG 접합체 분산액에 난용성 약물을 추가로 첨가하여 난용성 약물이 도입된 하이드로겔 조성물을 제조할 수 있다. 상기 난용성 약물은 마이크로스피어에 캡슐화된 형태로 첨가될 수 있다. 난용성 약물 및 마이크로스피어는 상기에서 설명한 바와 같다. 또한, 상기 약학적으로 사용가능한 염은 상기에서 설명한 바와 같다.The method for preparing the hydrogel composition of the present invention includes mixing the HA-dopamine-PEG conjugate and the cyclodextrin and then adjusting the pH to 7 to 11 by mixing a pharmaceutically usable salt. In this step, after preparing a dispersion by dispersing the HA-dopamine-PEG conjugate and cyclodextrin in water, it can be prepared by adding a solution of a pharmaceutically usable salt to the dispersion. In this case, a poorly soluble drug may be further added to the HA-dopamine-PEG conjugate dispersion to prepare a hydrogel composition into which the poorly soluble drug is introduced. The poorly soluble drug may be added in encapsulated form in microspheres. Poorly soluble drugs and microspheres are as described above. In addition, the pharmaceutically usable salt is as described above.
이하, 본 발명을 실시예를 통하여 더욱 상세히 설명한다. 그러나, 하기 실시예는 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이에 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.
<실시예><Example>
1. 물질 및 실험방법1. Substances and test methods
1.1. 물질1.1. matter
히알루론산(hyaluronic acid [HA], 150-600 kDa; 겔 투과 크로마토그래피로 측정한 평균 Mw: 667 kDa)는 (주)바이오랜드사 (대한민국 천안)에서 구입하였다. 3-히드록시티라민 히드로클로라이드(3-Hydroxytyramine hydrochloride, 도파민)은 Tokyo Chemical Industry Co., Ltd. (일본 도쿄)에서 입수하였다. 메톡시 폴리에틸렌 글리콜 아민 (Methoxy polyethylene glycol amine, 분자량: 2K) (mPEG2K-NH2)은 Creative PEGWorks (Chapel Hill, NC, USA)에서 구입하였다. 폴리(락틱-코-글리콜산)[poly(lactic-co-glycolic acid), PLGA] (젖산:글리콜산=50:50, Mn: 45-55 kDa)은 PolySciTech (Akina, Inc., West Lafayette, IN, USA)에서 구입하였다. 도네페질 염기는 종근당 제약사 (서울, 한국)에서 입수하였다. 물-d2 (D2O), 히알루론산 분해효소 (hyaluronidase, HAase; 소 고환 유래, 400∼1000 units/mg 고체), N-하이드록시숙신이미드 (N-hydroxysuccinimide, NHS), 폴리비닐 알코올 (polyvinyl alcohol, PVA; MW: 67 kDa), N-(3-디메틸아미노프로필)-N'-에틸카르보디이미드 히드로클로라이드(N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, EDC) 및 트윈 80은 시그마-알드리치사 (Saint Louis, MO, USA)에서 구입하였다.Hyaluronic acid (hyaluronic acid [HA], 150-600 kDa; average Mw measured by gel permeation chromatography: 667 kDa) was purchased from Bioland Co., Ltd. (Cheonan, Korea). 3-Hydroxytyramine hydrochloride (dopamine) is manufactured by Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Methoxy polyethylene glycol amine (Methoxy polyethylene glycol amine, molecular weight: 2K) (mPEG2K-NH 2 ) was purchased from Creative PEGWorks (Chapel Hill, NC, USA). Poly(lactic-co-glycolic acid), PLGA (lactic acid:glycolic acid=50:50, M n: 45-55 kDa) is a polySciTech (Akina, Inc., West Lafayette) , IN, USA). Donepezil base was obtained from Chong Kun Dang Pharmaceutical (Seoul, Korea). Water-d 2 (D 2 O), hyaluronic acid degrading enzyme (hyaluronidase, HAase ; from bovine testis, 400-1000 units/mg solid), N -hydroxysuccinimide (NHS), polyvinyl alcohol (polyvinyl alcohol, PVA; MW: 67 kDa), N -(3-dimethylaminopropyl)- N ' -ethylcarbodiimide hydrochloride ( N -(3-dimethylaminopropyl)- N' -ethylcarbodiimide hydrochloride, EDC) and
1.2. HDP 접합체의 합성 및 확인1.2. Synthesis and Identification of HDP Conjugates
HA-도파민 (HD) 중합체 유도체는 이전 연구에 보고된 바(Lee et al., 2020)에 따라 EDC 및 NHS 매개 아미드 결합 형성에 의해 합성하였다. HA (100 mg)를 자석 교반하면서 증류수 (DW, 10 mL)에 분산시켰다. EDC (57.5 mg) 및 NHS (34.5 mg)가 용해되어 있는 디메틸설폭사이드 (DMSO, 10 mL)를 상기 HA 분산액과 혼합하고 1N HCl을 첨가하여 최종 pH 5로 조절하였다. 그 다음 DMSO (5 mL)에 용해된 도파민 (37.9 mg)을 HA, EDC 및 NHS의 혼합물에 첨가하고 하루 동안 교반하였다. 결과물을 튜브형 투석막 (분자량 컷오프 (MWCO): 7 kDa)으로 옮기고 DW로 3 일 동안 투석하였다. 저장을 위하여 투석된 물질을 동결건조하였다.HA-dopamine (HD) polymer derivatives were synthesized by EDC and NHS mediated amide bond formation as reported in a previous study (Lee et al., 2020). HA (100 mg) was dispersed in distilled water (DW, 10 mL) with magnetic stirring. Dimethylsulfoxide (DMSO, 10 mL) in which EDC (57.5 mg) and NHS (34.5 mg) were dissolved was mixed with the HA dispersion and 1N HCl was added to adjust the final pH to 5. Then dopamine (37.9 mg) dissolved in DMSO (5 mL) was added to the mixture of HA, EDC and NHS and stirred for one day. The resultant was transferred to a tubular dialysis membrane (molecular weight cutoff (MWCO): 7 kDa) and dialyzed against DW for 3 days. The dialyzed material was lyophilized for storage.
HDP를 제조하기 위하여, HA(HD 중)의 카르복시산 기 및 mPEG2K-NH2의 아민 기 사이의 아미드 결합을 이용해 하기와 같이 HD에 mPEG2K-NH2를 결합시켰다 (Cho et al., 2012). 먼저, DW (10 mL)에 분산된 HD (96 mg)의 분산액과, DMSO (10 mL) 중의 EDC (38.3 mg) 및 NHS (22.3 mg)의 혼합물을 섞고, 얻어진 최종 혼합물을 1N HCl로 pH 5로 조절하였다. DMSO (5 mL) 중 mPEG2K-NH2 (200 mg)를 HD, EDC 및 NHS의 반응 혼합물에 첨가하였다. 이를 실온에서 15 시간 동안 교반하고 튜브형 투석막 (분자량 컷오프 (MWCO): 7 kDa)에 포함된 DW에서 36 시간 동안 투석하였다. 투석된 결과물을 동결건조한 후 아세톤으로 세척하여 미 반응 mPEG2K-NH2를 제거하였다. 최종 제품은 추가 보관을 위하여 동결건조하였다.To prepare HDP, mPEG2K-NH 2 was coupled to HD using an amide bond between the carboxylic acid group of HA (in HD) and the amine group of mPEG2K-NH 2 as follows (Cho et al., 2012). First, a dispersion of HD (96 mg) dispersed in DW (10 mL) and a mixture of EDC (38.3 mg) and NHS (22.3 mg) in DMSO (10 mL) were mixed, and the resulting final mixture was
HD 중합체 유도체가 올바르게 합성되었는지 확인하기 위하여 HDP의 양성자 핵자기 공명 (1H-NMR) (600MHz, JNM-ECA-600, JEOL Ltd., Akishima, Japan) 스펙트럼을 측정하였다. HDP 접합체에서 mPEG2K-NH2의 중량 백분율을 계산하기 위하여, 상이한 중량비 (mPEG2K-NH2/HA)의 표준 샘플로부터 선형 회귀선을 얻었다. 추가로 HDP 중합체의 합성을 확인하기 위하여 HD 및 HDP의 탄소-13 핵자기 공명 (13C-NMR) (800MHz, Bruker AVANCE III HD, Bruker, Billerica, MA, USA) 스펙트럼을 측정하였다.To confirm that the HD polymer derivative was correctly synthesized, the proton nuclear magnetic resonance ( 1 H-NMR) (600 MHz, JNM-ECA-600, JEOL Ltd., Akishima, Japan) spectrum of HDP was measured. To calculate the weight percentage of mPEG2K-NH 2 in the HDP conjugate, a linear regression line was obtained from standard samples of different weight ratios (mPEG2K-NH 2 /HA). In addition, to confirm the synthesis of the HDP polymer, carbon-13 nuclear magnetic resonance ( 13 C-NMR) (800 MHz, Bruker AVANCE III HD, Bruker, Billerica, MA, USA) spectra of HD and HDP were measured.
HDP의 합성은 푸리에-변환 적외선 (Fourier-transform infrared, FT-IR) 분광기 (Frontier ™FT-IR spectrometer, PerkinElmer Inc., Buckinghamshire, UK)로도 검증하였다. HD, mPEG2K-NH2 및 HDP의 스펙트럼은 400-4000 cm-1 파수 범위에서 ATR (attenuated total reflectance) 모드로 스캔하였다.The synthesis of HDP was also verified with a Fourier-transform infrared (FT-IR) spectrometer (Frontier ™ FT-IR spectrometer, PerkinElmer Inc., Buckinghamshire, UK). Spectra of HD, mPEG2K-NH 2 and HDP were scanned in the attenuated total reflectance (ATR) mode in the 400-4000 cm -1 wavenumber range.
1.3. 하이드로겔 구조의 제조 및 검증1.3. Preparation and validation of hydrogel structures
PLGA/도네페질 마이크로스피어 (PLGA/donepezil microsphere, PDM)는 이전 연구에 보고된 바(Lee et al., 2020; Seo et al., 2020)에 따라 수중유 (oil-in-water, o/w) 유화 및 용매증발법을 이용하여 제조하였다. 도네페질 염기 (5 mg) 및 PLGA (50 mg)를 디클로로메탄 (1.5 mL)에 용해시키고 DW (15 mL) 중 PVA (0.5 %, w/v)와 혼합하였다. 그 다음, 해당 o/w 에멀젼을 고속 균질기를 사용하여 9,500 rpm으로 블렌딩하였다. 실온에서 30 분 동안 교반하여 유기상을 제거하고, 입자 현탁액을 16,100 g에서 5 분 동안 원심분리하여 입자 펠렛을 분리하였다. 상층액을 제거한 후 입자 펠렛을 DW로 재분산하고 동결건조하였다.As reported in previous studies (Lee et al., 2020; Seo et al., 2020), PLGA/donepezil microspheres (PLGA/donepezil microspheres, PDM) are oil-in-water (o/w) ) was prepared using emulsification and solvent evaporation. Donepezil base (5 mg) and PLGA (50 mg) were dissolved in dichloromethane (1.5 mL) and mixed with PVA (0.5 %, w/v) in DW (15 mL). Then, the o/w emulsion was blended at 9,500 rpm using a high-speed homogenizer. The organic phase was removed by stirring at room temperature for 30 minutes, and the particle suspension was centrifuged at 16,100 g for 5 minutes to separate the particle pellet. After removing the supernatant, the particle pellet was redispersed in DW and lyophilized.
전계-방출 주사 전자 현미경(FE-SEM; SUPRA 55VP, Carl Zeiss, Oberkochen, Germany)을 사용하여 동결건조된 PDM의 확대 형태 사진을 촬영하였다. PDM의 부피 밀도 프로파일은 입자 크기 분석기 (Mastersizer 3000, Malvern Instruments Ltd., Malvern, UK)로 측정하였다.Magnified morphological pictures of the lyophilized PDM were taken using a field-emission scanning electron microscope (FE-SEM; SUPRA 55VP, Carl Zeiss, Oberkochen, Germany). The bulk density profile of PDM was measured with a particle size analyzer (
PDM에서 도네페질의 캡슐화율 수치는 자동 샘플러(1260 Vialsampler), 펌프(1260 Quat Pump VL) 및 UV-Vis 검출기(1260 VWD)가 장착된 고성능 액체 크로마토그래피(HPLC) 시스템(1260 Infinity II, Agilent Technologies, Santa Clara, CA, USA)으로 분석하였다. 이동상은 제1인산칼륨 완충액(potassium phosphate monobasic, 10 mM) 및 아세토니트릴 혼합액(65:35, v/v)으로 제조하였고, 유속은 1mL/분으로 유지하였다. 샘플의 일정량 (20 μL)을 역상 C18 컬럼 (Kinetex, 250 mm×4.6 mm, 5 μm; Phenomenex, Torrance, CA, USA)에 주입하고 흡광도는 268 nm에서 검출하였다.Encapsulation rate figures of donepezil in PDM were obtained using a high performance liquid chromatography (HPLC) system (1260 Infinity II, Agilent Technologies) equipped with an autosampler (1260 Vialsampler), pump (1260 Quat Pump VL) and UV-Vis detector (1260 VWD). , Santa Clara, CA, USA). The mobile phase was prepared with potassium phosphate monobasic (10 mM) and acetonitrile mixed solution (65:35, v/v), and the flow rate was maintained at 1 mL/min. An aliquot (20 μL) of the sample was injected into a reversed-phase C18 column (Kinetex, 250 mm×4.6 mm, 5 μm; Phenomenex, Torrance, CA, USA) and absorbance was detected at 268 nm.
이후, 블렌딩 및 pH 조절 방법으로 설계된 하이드로겔 시스템을 제조하였다. HDP/aCD/PDM 8.5 샘플 1 mL을 제조하기 위하여, HDP (20 mg)를 aCD (100 mg)를 포함하는 DW (0.79 mL)에 균일하게 분산시켰고, DW (0.1 mL) 중 PDM 분산액 (도네페질 1 mg에 해당함)과 혼합하였다. 1N NaOH로 얻어진 혼합물의 pH 값을 8.5로 조절하였고 DW 첨가하여 최종 부피 1 mL로 하였다.Thereafter, a hydrogel system designed by blending and pH control methods was prepared. To prepare 1 mL of HDP/aCD/PDM 8.5 sample, HDP (20 mg) was uniformly dispersed in DW (0.79 mL) containing aCD (100 mg), and PDM dispersion (Donepezil) in DW (0.1 mL) corresponding to 1 mg). The pH value of the mixture obtained with 1N NaOH was adjusted to 8.5, and DW was added to make a final volume of 1 mL.
HDP와 aCD 간 폴리슈도로탁산 구조 형성은 1H-NMR 분석 (600MHz; JNM-ECA-600, JEOL Ltd.)에 의해 확인하였다. aCD (25 mg/mL) 또는 HDP/aCD (5:25, mg/mL)는 1H-NMR 분석을 위하여 D2O에 용해시켰다.Polypseudotaxane structure formation between HDP and aCD was confirmed by 1 H-NMR analysis (600 MHz; JNM-ECA-600, JEOL Ltd.). aCD (25 mg/mL) or HDP/aCD (5:25, mg/mL) was dissolved in D 2 O for 1 H-NMR analysis.
HDP와 aCD 간의 호스트-게스트 상호작용은 2 차원 (2D) NOESY (NMR nuclear Overhauser effect spectroscopy) (850MHz, Bruker AVANCE III HD, Bruker, Billerica, MA, USA) 실험으로 확인하였다. HDP (5mg/mL)와 aCD (25 mg/mL)의 혼합물도 D2O에 용해시켜 제조하였다.The host-guest interaction between HDP and aCD was confirmed by two-dimensional (2D) NOESY (NMR nuclear Overhauser effect spectroscopy) (850 MHz, Bruker AVANCE III HD, Bruker, Billerica, MA, USA) experiments. A mixture of HDP (5 mg/mL) and aCD (25 mg/mL) was also prepared by dissolving in D 2 O.
HDP 접합체에서 도파민 분자의 중합은 UV-Vis 분광계 (Libra S80 이중 빔 분광 광도계, Biochrom Ltd., Cambridge, UK)로 확인하였다. 일반 HDP (DW 중 20 mg/mL) 및 가교 HDP (pH 8.5; DW 중 20 mg/mL) 샘플을 제조하고 6 시간 동안 인큐베이션하였다. 두 샘플의 흡광도 프로파일은 250 ~ 900 nm 범위에서 모니터링하였다.Polymerization of dopamine molecules in the HDP conjugate was confirmed with a UV-Vis spectrophotometer (Libra S80 dual beam spectrophotometer, Biochrom Ltd., Cambridge, UK). Samples of normal HDP (20 mg/mL in DW) and cross-linked HDP (pH 8.5; 20 mg/mL in DW) were prepared and incubated for 6 hours. The absorbance profiles of both samples were monitored in the range of 250 to 900 nm.
HDP의 PEG 사슬이 aCD의 내부 공동을 관통하는 메커니즘은 X-선 회절분석기(X-ray diffractometry, XRD) (Philips X'Pert PRO MPD diffractometer; PANalytical Corp., Almero, Netherlands)를 사용하여 추가로 조사하였다. aCD, HDP 및 HDP/aCD (동결건조된 형태)의 강도 값은 10 ~ 60 ° 각도 범위에서 스캔하였다. 발전기 전압 및 튜브 전류는 각각 40 kV 및 30 mA로 설정하고 시험하였다.The mechanism by which the PEG chains of HDP penetrate the inner cavity of aCD were further investigated using X-ray diffractometry (XRD) (Philips X'Pert PRO MPD diffractometer; PANalytical Corp., Almero, Netherlands). did. Intensity values of aCD, HDP, and HDP/aCD (lyophilized form) were scanned at angles ranging from 10 to 60°. Generator voltage and tube current were set and tested at 40 kV and 30 mA, respectively.
1.4. 제조된 하이드로겔 구조의 유동학적 특성1.4. Rheological properties of the prepared hydrogel structure
설계된 하이드로겔 시스템의 겔화 특성은 반전 시험으로 평가하였다. HA/aCD (20:100, mg/mL), HD/aCD (20:100, mg/mL), HP/aCD (20:100, mg/mL), HP/aCD/PDM (20:100:1 (도네페질 농도에서), mg/mL), HDP/aCD (20:100, mg/mL), HDP/aCD 8.5 (20:100, mg/mL; pH 8.5), HDP/aCD/PDM 8.5 (20:100:1 (도네페질 농도에서), mg/mL; pH 8.5) 및 HDP/PDM 8.5 (20:1 (도네페질 농도에서), mg/mL; pH 8.5) 검체를 실온에서 6 시간 동안 인큐베이션한 후에 반전시켜 역 위치에서의 겔화 거동을 관찰하였다.The gelling properties of the designed hydrogel system were evaluated by an inversion test. HA/aCD (20:100, mg/mL), HD/aCD (20:100, mg/mL), HP/aCD (20:100, mg/mL), HP/aCD/PDM (20:100:1 (at donepezil concentration), mg/mL), HDP/aCD (20:100, mg/mL), HDP/aCD 8.5 (20:100, mg/mL; pH 8.5), HDP/aCD/PDM 8.5 (20 :100:1 (at donepezil concentration), mg/mL; pH 8.5) and HDP/PDM 8.5 (20:1 (at donepezil concentration), mg/mL; pH 8.5) samples were incubated at room temperature for 6 hours. After inversion, the gelation behavior in the reverse position was observed.
HDP/aCD/PDM 8.5 (20:100:1 (도네페질 농도에서), mg/mL; pH 8.5)의 졸-겔 전이에 필요한 시간은 역 위치에서 1, 3, 5, 10, 30, 60, 120, 및 180 분에 거동을 관찰하여 측정하였다.The time required for the sol-gel transition of HDP/aCD/PDM 8.5 (20:100:1 (at donepezil concentration), mg/mL; pH 8.5) was 1, 3, 5, 10, 30, 60, The behavior was measured by observing at 120, and 180 minutes.
HDP/aCD/PDM 8.5 하이드로겔 (0.2 mg/mL 메틸렌 블루 포함)의 주사가능성을 단일 주사기로 시험하였다. 주사가능성을 평가하기 위하여 주사기 바늘을 통하여 하이드로겔 시스템을 압출시켜 그 양상을 관찰하였다.The injectability of HDP/aCD/PDM 8.5 hydrogel with 0.2 mg/mL methylene blue was tested with a single syringe. To evaluate injectability, the hydrogel system was extruded through a syringe needle and the behavior was observed.
HP/aCD/PDM, HDP/aCD 8.5, HDP/PDM 8.5 및 HDP/aCD/PDM 8.5 군의 유동학적 특성은 레오미터 (rheometer, MCR 302, Anton Paar GmbH, Graz, Austria)를 사용하여 실온에서 1일간 인큐베이션하여 평가하였다. 10 rad/s 고정 진동수 및 37 ℃ 온도에서 변형률 변화 시험(strain sweep test)으로 변형 변화 범위 (1-400 %)에 따른 저장 계수 (G') 및 손실 계수 (G") 값을 측정하였다. 진동수 (1-100 rad/s)-의존 G' 및 G" 값은 10 % 변형 및 37 ℃ 온도에서 측정하였다. 시간에 따른 G' 및 G" 값을 100 % 변형 (0-1, 2-3, 및 4-5 분) 및 0 % 변형 (1-2, 3-4, 및 5-6 분)의 3주기에서 측정하였다. 0.01-100 s-1 범위에서 전단 속도에 따른 전단 응력(shear stress) 및 점도의 상관관계(plot)를 얻었다.The rheological properties of the HP/aCD/PDM, HDP/aCD 8.5, HDP/PDM 8.5 and HDP/aCD/PDM 8.5 groups were measured at room temperature using a rheometer (MCR 302, Anton Paar GmbH, Graz, Austria). It was evaluated by incubation for a day. The storage coefficient (G') and loss coefficient (G") values were measured according to the strain change range (1-400 %) by a strain sweep test at a fixed frequency of 10 rad/s and a temperature of 37°C. (1-100 rad/s)-dependent G' and G" values were measured at 10% strain and 37°C temperature. 3 cycles of 100% strain (0-1, 2-3, and 4-5 min) and 0% strain (1-2, 3-4, and 5-6 min) of G' and G" values over time A correlation (plot) between shear stress and viscosity according to the shear rate was obtained in the range of 0.01-100 s -1 .
1.5. 시험관 내(1.5. in vitro ( in vitroin vitro ) 약물 방출 및 분해성 평가) drug release and degradation evaluation
전계-방출 주사 전자 현미경 (FE-SEM; SUPRA 55VP, Carl Zeiss, Oberkochen, Germany)을 사용하여 동결건조된 HDP/aCD 8.5 및 HDP/aCD/PDM 8.5 겔의 단면 사진을 얻었다.Cross-sectional pictures of lyophilized HDP/aCD 8.5 and HDP/aCD/PDM 8.5 gels were obtained using a field-emission scanning electron microscope (FE-SEM; SUPRA 55VP, Carl Zeiss, Oberkochen, Germany).
시험관 내(in vitro)에서 HDP/aCD/PDM 8.5 시스템으로부터 DPZ 방출 프로파일을 pH 7.4에서 측정하였다. 도네페질 염기 용액 (50 % DMSO 중 1 mg/mL) 및 HDP/aCD/PDM 8.5 (1 mg/mL 도네페질 포함)를 제조하고 각 검체의 일정량 (0.6 mL)을 투석 튜브(Midi-GeBAflex tube, MWCO:3.5 kDa; Gene Bio-Application Ltd., Yavne, Israel)로 옮겼다. 상기 투석 튜브를 0.3 % 트윈 80 (Tween 80, 30 mL) 포함 PBS (pH 7.4)에 담그고 37 ℃ 및 50 rpm의 수조에서 인큐베이션하였다. 도네페질 용액 군의 경우 6 시간 및 24 시간에, HDP/aCD/PDM 8.5 군의 경우 6, 24, 48, 72, 168, 192, 216, 336, 504 및 672 시간에 각각 방출액 일정량 (0.2 mL)을 취하였다. 상기 설명한 HPLC 방법으로 취한 방출액의 도네페질 농도를 분석하였다.The DPZ release profile was measured at pH 7.4 from the HDP/aCD/PDM 8.5 system in vitro . Prepare donepezil base solution (1 mg/mL in 50% DMSO) and HDP/aCD/PDM 8.5 (with 1 mg/mL donepezil) and transfer an aliquot (0.6 mL) of each sample into a dialysis tube (Midi-GeBAflex tube, MWCO: 3.5 kDa; Gene Bio-Application Ltd., Yavne, Israel). The dialysis tube was immersed in PBS (pH 7.4) containing 0.3% Tween 80 (
HDP/aCD/PDM 8.5 시스템의 시험관내 분해 특성을 HAase의 부재 또는 존재 하에서 평가하였다. HDP/aCD/PDM 8.5 (0.5 mL)를 HAase (1 mg/mL)를 포함하는 DW (0.5 mL) 또는 포함하지 않은 DW (0.5 mL)와 혼합하고 마이크로 원심분리 튜브에 넣었다. 각 샘플링 시점 (24, 48, 72, 168, 240, 336 및 504 시간)에서 상층액을 제거하고 남은 겔 질량을 측정하였다. 그 후, 동일한 부피의 새로운 매질(medium)을 보충하고 37 ℃에서 인큐베이션하였다. 남아 있는 겔 부분의 상대적 백분율은 초기 값 (0 시간)과 비교하여 계산하였다.The in vitro degradation properties of the HDP/aCD/PDM 8.5 system were evaluated in the absence or presence of HAase. HDP/aCD/PDM 8.5 (0.5 mL) was mixed with DW (0.5 mL) or without HAase (1 mg/mL) (0.5 mL) and placed in a microcentrifuge tube. At each sampling time point (24, 48, 72, 168, 240, 336 and 504 hours), the supernatant was removed and the remaining gel mass was measured. Thereafter, the same volume of fresh medium was replenished and incubated at 37°C. The relative percentage of the remaining gel fraction was calculated compared to the initial value (0 h).
1.6. 생분해 시험1.6. biodegradation test
개발된 하이드로겔 시스템의 생분해 특성은 마우스 모델에서 부피 감소 프로파일을 측정하여 평가하였다. 설계된 하이드로겔 시스템을 ICR 마우스 (Institute of Cancer Research mouse, 수컷, 5 주령; 오리엔트 바이오, 성남, 한국)에 피하주사하였다. 모든 검체는 실온에서 하루 동안 인큐베이션한 후, PDM, HDP/aCD 8.5, HDP/PDM 8.5, HDP/aCD/PDM 및 HDP/aCD/PDM 8.5 검체의 일정량 (20 g 체중에 대해 0.1 mL)을 마우스의 등쪽에 피하주사하였다. 주사 후 14 일에 마우스를 흡입 마취 하에 희생시키고 잔류 겔을 절제하여 중량을 측정하였다.The biodegradation properties of the developed hydrogel system were evaluated by measuring the volume reduction profile in a mouse model. The designed hydrogel system was subcutaneously injected into ICR mice (Institute of Cancer Research mouse, male, 5 weeks old; Orient Bio, Seongnam, Korea). All samples were incubated for one day at room temperature, and then an aliquot of PDM, HDP/aCD 8.5, HDP/PDM 8.5, HDP/aCD/PDM and HDP/aCD/PDM 8.5 samples (0.1 mL for 20 g body weight) was administered to the mouse. It was injected subcutaneously in the back. On
1.7. 데이터 분석1.7. data analysis
얻어진 실험 데이터의 통계 분석을 위하여 양측 t-검정 및 일원 분산 분석 (ANOVA) 후 사후검정시험(post hoc test)을 수행하였다. 각 실험은 3 회 이상 반복하였으며, 얻은 데이터는 평균±표준편차 (SD)로 표시하였다.For statistical analysis of the obtained experimental data, a two-sided t -test and one-way analysis of variance (ANOVA) followed by a post hoc test were performed. Each experiment was repeated three or more times, and the obtained data were expressed as mean±standard deviation (SD).
2. 결과 및 논의2. Results and Discussion
2.1. 하이드로겔 시스템 설계 및 HDP 중합체 합성2.1. Hydrogel system design and HDP polymer synthesis
물리적으로 상호 연결되고 화학적으로 가교된 하이드로겔 시스템을 제작하기 위하여, 본 발명에서는 PEG (HDP 접합체에 연결된 형태)와 aCD 간에 형성되는 폴리슈도로탁산 구조, 및 도파민 기 (HDP 접합체에 부착된 형태)의 폴리도파민 결합을 도입하였다. aCD는 PEG 사슬(HDP에 연결된 형태)로 연결(내부 관통)되어 HDP 구조를 묶을 수 있다. 인접한 aCD 분자들 간의 수소 결합과 이들의 응집으로 결정질 복합체가 형성될 수 있다. 또한, 알칼리성 pH에서의 도파민 중합은 HDP 네트워크의 화학적 가교를 제공할 수 있다. 이러한 결합 메커니즘에 의해 최적화된 피하주사 제형을 개발하기 위한 하이드로겔의 유동학적 특성 및 기계적 특성을 조절할 수 있다. 또한, 약물의 지속적인 방출을 위하여 설계된 하이드로겔 시스템에 도네페질-캡슐화된 PLGA 마이크로스피어 (PDM)를 함입시켰다.In order to fabricate a physically interconnected and chemically crosslinked hydrogel system, in the present invention, a polypseudotaxane structure formed between PEG (in the form linked to the HDP conjugate) and aCD, and a dopamine group (in the form attached to the HDP conjugate) of polydopamine binding was introduced. aCD can be linked (penetrated inside) with a PEG chain (form linked to HDP) to bind the HDP structure. Hydrogen bonds between adjacent aCD molecules and their aggregation can form crystalline complexes. In addition, dopamine polymerization at alkaline pH can provide chemical crosslinking of the HDP network. By this binding mechanism, the rheological and mechanical properties of the hydrogel can be controlled to develop an optimized subcutaneous injection formulation. In addition, donepezil-encapsulated PLGA microspheres (PDM) were incorporated in a hydrogel system designed for sustained release of the drug.
주된 겔 매트릭스로서, HA 골격에 도파민 기 및 PEG 기를 공유결합시켜 HDP 접합체를 최종적으로 합성하였다 (도 1A). 도파민은 EDC 및 NHS에 의해 촉매되는 아미드 결합을 통해 HA 골격에 연결시켰다. 그 다음, 합성된 HD 중합체에 mPEG2K-NH2의 아민 기와 HA (HD 중의)의 카르복시산 기 간의 아미드 결합을 기반으로 PEG를 추가로 연결하였다.As the main gel matrix, a dopamine group and a PEG group were covalently linked to the HA backbone to finally synthesize an HDP conjugate (Fig. 1A). Dopamine was linked to the HA backbone via an amide bond catalyzed by EDC and NHS. Then, PEG was further linked to the synthesized HD polymer based on the amide bond between the amine group of mPEG2K-NH 2 and the carboxylic acid group of HA (in HD).
HDP의 합성은 NMR 분석으로 확인하였다 (도 1B-1D). HDP의 1H-NMR 스펙트럼에서 HA의 N-아세틸 기, 도파민의 페닐 고리, PEG의 메틸렌 기에 대한 화학적 이동은 각각 1.8 ~ 2.0 ppm (피크 1), 6.6 ~ 6.8 ppm (피크 2 ~ 4) 및 3.6 ~ 3.7 ppm (피크 5 및 6)에서 나타났다 (도 1B). HD 합성은 이전 연구 (Lee et al., 2020)에서 분광학 분석을 통해 이미 확인되었으므로 관련 분석 데이터는 본 명세서에 포함시키지 않았다. HDP 중합체의 PEG 함량은 표준 샘플 (mPEG2K-NH2 및 HD의 혼합물)로 회귀선을 작성하여 결정하였다. 회귀선 (도 1C)으로부터 계산된, 본 발명에서의 HDP 접합체의 평균 PEG 함량은 50.7 %였다. HD와 HDP의 합성은 13C-NMR 분석에 의해 확인하였다 (도 1D). HD의 13C-NMR 스펙트럼에서 HA의 N-아세틸 기에 대한 탄소 이동 (22.4 ppm; 피크 1) 및 도파민의 페닐 고리 기에 대한 탄소 이동 (116.5 및 121.2 ppm; 피크 2-4)이 나타났다 (Ling et al., 2015; Zhang et al., 2016). HA의 N-아세틸 기 (22.5 ppm; 피크 1) 및 도파민의 페닐 고리 기 (116.6 및 121.2 ppm; 피크 2-4)와 함께, HDP 스펙트럼에서 PEG의 메틸렌 탄소에 대한 탄소 이동은 69.6 및 71.0 ppm (피크 5 및 6)에서 나타났다 (Mahou and Wandrey, 2012). 이러한 13C-NMR 데이터 또한 HDP이 적절히 합성되었음을 뒷받침한다.The synthesis of HDP was confirmed by NMR analysis ( FIGS. 1B-1D ). In the 1 H-NMR spectrum of HDP, the chemical shifts for the N -acetyl group of HA, the phenyl ring of dopamine, and the methylene group of PEG were 1.8 to 2.0 ppm (peak 1), 6.6 to 6.8 ppm (
HDP의 합성은 FT-IR 분석으로도 입증되었다 (도 1E). O-H 신축 (3286 cm-1), 피라노즈(pyranose) 고리에서 C-H 신축 (2917 cm-1), 및 NH-Ac에서 C=O 신축 (1643 cm-1)에 대한 피크가 HD 접합체의 스펙트럼에 나타났다. HDP 스펙트럼에서 O-H 신축 (3361 cm-1), 피라노즈 고리에서 C-H 신축 (2875 cm-1), 및 NH-Ac에서 C=O 신축 (1649 cm-1)에 대한 피크도 관찰되었고, 1466 cm-1의 피크는 HA와 mPEG2K-NH2 간의 아미드 결합을 의미한다 (Chatterjee et al., 2019). 이러한 모든 FT-IR 데이터로부터 HA 골격에 대하여 도파민 및 PEG 모이어티가 적절히 결합되었음을 확인하였다.The synthesis of HDP was also verified by FT-IR analysis ( FIG. 1E ). Peaks for OH stretching (3286 cm -1 ), CH stretching in the pyranose ring (2917 cm -1 ), and C=O stretching in NH-Ac (1643 cm -1 ) appeared in the spectrum of the HD conjugate. . In the HDP spectrum, peaks for OH stretching (3361 cm -1 ), CH stretching in the pyranose ring (2875 cm -1 ), and C=O stretching in NH-Ac (1649 cm -1 ) were also observed, and 1466 cm -1 The peak of 1 means the amide bond between HA and mPEG2K-NH 2 (Chatterjee et al., 2019). From all these FT-IR data, it was confirmed that the dopamine and PEG moieties were properly bound to the HA backbone.
2.2. 하이드로겔 구조의 제조 및 하이드로겔 가교 메커니즘의 규명2.2. Preparation of hydrogel structure and identification of hydrogel crosslinking mechanism
초분자 구조 및 카테콜 기능화된 화학-연관 하이드로겔 시스템을 블렌딩 방법으로 간편하게 제조하였다 (Lee et al., 2020; Seo et al., 2020). 도네페질 염기-캡슐화된 마이크로스피어 (PDM)를 제조하고 겔 가교 공정에서 하이드로겔의 전구체와 혼합하였다. 피하주사 후 도네페질의 지속적인 방출을 목표로 PDM을 하이드로겔 네트워크에 함입시켰다.Chemical-associated hydrogel systems functionalized with supramolecular structures and catechols were conveniently prepared by a blending method (Lee et al., 2020; Seo et al., 2020). Donepezil base-encapsulated microspheres (PDM) were prepared and mixed with the precursor of the hydrogel in a gel crosslinking process. PDM was incorporated into the hydrogel network with the goal of sustained release of donepezil after subcutaneous injection.
PDM은 이전 연구에 보고된 바에 따라 유화/증발 방법을 사용하여 제조하였다 (Lee et al., 2020; Seo et al., 2020). 수용성이 낮은 도네페질 염기는 지속 방출을 위하여 PLGA 마이크로스피어에 도입하였다. 도 2A에 나타난 바와 같이, FE-SEM 사진에서 표면이 매끄러운 구형으로 관찰되었다. 크기에 따른 부피 밀도 (Dv) 빈도를 측정하였으며, PDM의 Dv (10), Dv (50), Dv (90) 값은 각각 10, 19, 34 μm였다 (도 2B). 이러한 입자 크기 분포 프로파일은 유사한 실험 프로토콜로 수행된 이전에 보고된 데이터와 비슷하였다 (Seo et al., 2020). PDM에서 도네페질의 평균 캡슐화 효율은 68 %였으며, 이는 마이크로스피어에서 적절한 약물 캡슐화를 의미한다.PDM was prepared using an emulsification/evaporation method as reported in previous studies (Lee et al., 2020; Seo et al., 2020). Donepezil base with low water solubility was introduced into PLGA microspheres for sustained release. As shown in FIG. 2A, a smooth spherical surface was observed in the FE-SEM photograph. The bulk density (Dv) frequency according to size was measured, and the Dv (10), Dv (50), and Dv (90) values of PDM were 10, 19, and 34 μm, respectively ( FIG. 2B ). This particle size distribution profile was comparable to previously reported data performed with similar experimental protocols (Seo et al., 2020). The average encapsulation efficiency of donepezil in PDM was 68%, indicating adequate drug encapsulation in microspheres.
초분자화학과 카테콜 중합에 기반한 하이드로겔 가교 메커니즘은 NMR, UV-Vis 및 XRD 분석으로 규명하였다 (도 3). aCD와 PEG 사이의 호스트-게스트 상호작용은 1H-NMR 분석으로 조사하였다 (도 3A). 양성자 이동 (H3-H6)은 aCD 스펙트럼의 3.5 ~ 3.9 ppm에서 관찰되었으며 이러한 이동은 HDP/aCD의 1H-NMR 스펙트럼에서도 나타났다. HDP/aCD의 스펙트럼에서 PEG의 메틸렌 기에서의 양성자 이동도 약 3.6 ppm에서 관찰되었다. 이러한 데이터는 HDP 및 aCD가 동결건조된 HDP/aCD 하이드로겔 시스템에 포함되어 있음을 나타낸다. PEG 사슬 (HDP 접합체에 연결된 형태로)이 aCD의 내부 공동을 관통하는 것은 2D NOESY NMR 분석에 의해 추가로 입증되었다 (도 3B). HDP/aCD 폴리슈도로탁산 구조의 2D NOESY NMR 스펙트럼에서는 PEG 메틸렌 기의 수소 원자와 aCD 단위의 수소 원자 사이의 상호작용에 기반한 크로스피크(crosspeak)가 나타났다. 이 2D NMR 결과는 설계된 HDP/aCD 구조의 로탁산화를 의미한다.The hydrogel crosslinking mechanism based on supramolecular chemistry and catechol polymerization was identified by NMR, UV-Vis and XRD analysis (FIG. 3). The host-guest interaction between aCD and PEG was investigated by 1 H-NMR analysis ( FIG. 3A ). A proton shift (H3-H6) was observed at 3.5 to 3.9 ppm of the aCD spectrum, and this shift was also shown in the 1 H-NMR spectrum of HDP/aCD. In the spectrum of HDP/aCD, a proton shift in the methylene group of PEG was also observed at about 3.6 ppm. These data indicate that HDP and aCD are included in the lyophilized HDP/aCD hydrogel system. The penetration of PEG chains (in the form linked to the HDP conjugate) through the inner cavity of aCD was further demonstrated by 2D NOESY NMR analysis (Fig. 3B). The 2D NOESY NMR spectrum of the HDP/aCD polypseudotaxane structure showed a crosspeak based on the interaction between the hydrogen atom of the PEG methylene group and the hydrogen atom of the aCD unit. This 2D NMR result indicates rotaxylation of the designed HDP/aCD structure.
카테콜 중합으로 지칭되는 다른 하이드로겔 가교 메커니즘은 UV-Vis 분광법 분석으로 연구하였다 (도 3C). HDP 스펙트럼에서 280 nm 부근의 높은 흡광도 값은 도파민 분자의 페닐 고리의 기여를 의미한다. HDP 8.5의 스펙트럼에서는 400 nm 부근의 흡광도 값이 HDP 자체에서보다 높은 것으로 측정되었다. pH 제어에 의한 폴리도파민 형성이 흡광도 프로파일에 영향을 미쳤으므로 이러한 데이터로부터 카테콜 (HDP 접합체에 연결된 형태로) 중합을 통한 하이드로겔 가교임을 알 수 있다.Another hydrogel crosslinking mechanism, termed catechol polymerization, was studied by UV-Vis spectroscopy analysis ( FIG. 3C ). The high absorbance value near 280 nm in the HDP spectrum indicates the contribution of the phenyl ring of the dopamine molecule. In the spectrum of HDP 8.5, it was measured that the absorbance value near 400 nm was higher than that of HDP itself. Since polydopamine formation by pH control affected the absorbance profile, it can be seen from these data that it is hydrogel crosslinking through polymerization of catechol (in the form linked to the HDP conjugate).
aCD와 PEG 간의 호스트-게스트 화학과 aCD 응집체에 기반한 결정질 복합체는 XRD 분석으로 추가로 평가하였다 (도 3D). 보고된 바와 같이(Feng et al., 2016), aCD의 스펙트럼에서 결정질 속성이 관찰되었다. PEG 사슬은 aCD의 터널 구조를 관통할 수 있으며, PEG/aCD의 포접 복합체는 aCD 분자들 간의 강한 수소결합력으로 인해 물에 불용성이 되는 것으로 알려져 있다. HDP/aCD의 XRD 패턴에서는 19.8˚에서 선명한 피크가 나타났고, 이는 채널형 결정 구조를 의미한다. 관찰된 XRD 패턴은 본 연구에서 PEG/aCD에 기반한 초분자 구조가 형성되었음을 뒷받침한다.Host-guest chemistry between aCD and PEG and crystalline complexes based on aCD aggregates were further evaluated by XRD analysis ( FIG. 3D ). As reported (Feng et al., 2016), crystalline properties were observed in the spectrum of aCD. It is known that the PEG chain can penetrate the tunnel structure of aCD, and the inclusion complex of PEG/aCD becomes insoluble in water due to the strong hydrogen bonding force between aCD molecules. In the XRD pattern of HDP/aCD, a clear peak appeared at 19.8˚, indicating a channel-type crystal structure. The observed XRD patterns support the formation of supramolecular structures based on PEG/aCD in this study.
2.3. 하이드로겔의 유동학적 특성2.3. Rheological properties of hydrogels
설계된 하이드로겔 구조의 겔화성, 주사가능성 및 점탄성 특성을 하기와 같이 평가하였다 (도 4). HA/aCD, HD/aCD, HP/aCD, HP/aCD/PDM, HDP/aCD, HDP/aCD 8.5, HDP/aCD/PDM 8.5 및 HDP/PDM 8.5 군의 겔화 거동을 반전 시험으로 평가하였다 (도 4A). HA/aCD 및 HD/aCD 군에서는 제안된 가교 메커니즘 (즉, 초분자화학 및 폴리도파민 연결)이 적용되지 않아서 점탄성 측면이 증가되는 것으로는 나타나지 않았다. HP/aCD 및 HP/aCD/PDM 군은 이론적으로는 PEG/aCD 기반 물리적 상호작용 전략이 적용되었지만, 이러한 겔 검체에서는 불충분한 점탄성 거동으로 인해 반전 시험에서 역 위치에 그대로 머물러 있지 못하였다. HDP/aCD 군은 역 상태에서 부분적으로 유지되었고 HDP/aCD 8.5 군은 완전한 겔 경화 거동을 나타냈다. HDP/aCD 8.5 군에서는 HDP/aCD 군과 비교하여 폴리도파민 형성으로 인한 색상 변화가 나타났다. HDP/aCD/PDM 8.5 군에서 나타난 것처럼 HDP/aCD 8.5 네트워크에 PDM을 통합하는 것이 겔화 현상을 방해하지는 않았다. 또한, HDP/PDM 8.5 군도 초분자화학 (PEG와 aCD 간)없이 카테콜 중합으로 인해 점탄성 특성이 상승하는 것으로 나타냈다.The gelling properties, injectability and viscoelastic properties of the designed hydrogel structure were evaluated as follows ( FIG. 4 ). The gelation behavior of HA/aCD, HD/aCD, HP/aCD, HP/aCD/PDM, HDP/aCD, HDP/aCD 8.5, HDP/aCD/PDM 8.5 and HDP/PDM 8.5 groups was evaluated by inversion test (Fig. 4A). In the HA/aCD and HD/aCD groups, the proposed cross-linking mechanism (ie, supramolecular chemistry and polydopamine linkage) was not applied, so the viscoelastic aspect did not appear to be increased. The HP/aCD and HP/aCD/PDM groups were theoretically applied with a PEG/aCD-based physical interaction strategy, but in these gel samples, they could not remain in the inverse position in the inversion test due to insufficient viscoelastic behavior. The HDP/aCD group partially remained in the inverted state and the HDP/aCD 8.5 group showed complete gel curing behavior. The HDP/aCD 8.5 group showed color change due to polydopamine formation compared to the HDP/aCD group. Incorporation of PDM into the HDP/aCD 8.5 network did not interfere with the gelation phenomenon as shown in the HDP/aCD/PDM 8.5 group. In addition, HDP/PDM 8.5 group also showed an increase in viscoelastic properties due to catechol polymerization without supramolecular chemistry (between PEG and aCD).
겔화 시간은 도 4B와 같이 반전 시험으로 측정하였다. HDP/aCD/PDM 8.5 검체는 이미 1 분에도 역 위치에서 겔이 유지됨을 보였고 겔 경화 특성은 360 분 동안 유지되었다. HDP/aCD/PDM 8.5 군의 주사가능성은 주사기 바늘을 통한 압출로 시험하였다 (도 4C). HDP/aCD/PDM 8.5 군에 있어서 주입 가능성이 관찰된 것은 편리한 피하주사의 가능성을 암시한다.The gelation time was measured by an inversion test as shown in FIG. 4B. The HDP/aCD/PDM 8.5 sample already showed that the gel was retained in the inverted position even after 1 min and the gel hardening properties were maintained for 360 min. The injectability of the HDP/aCD/PDM 8.5 group was tested by extrusion through a syringe needle ( FIG. 4C ). The observed infusion potential in the HDP/aCD/PDM 8.5 group suggests the possibility of convenient subcutaneous injection.
HP/aCD/PDM, HDP/aCD 8.5, HDP/PDM 8.5 및 HDP/aCD/PDM 8.5 샘플의 점탄성 특성을 레오미터로 평가하였다 (도 4D). HDP/PDM 8.5 군은 역 위치에서 그대로 유지되었지만 (도 4A), 변형률 변화(strain sweep) 및 진동수 변화(frequency sweep) 시험에서는 반전 및 유동학 시험 사이의 시험 방법 및 샘플 인큐베이션 시간의 차이로 추정되는 요인에 의하여, 해당 군의 저장 계수 (G') 및 손실 계수 (G") 값은 다른 초분자 하이드로겔 구조 (주로 aCD 및 PEG 상호작용에 기반함) 보다 상당히 낮았다. 반전 시험 결과에서 나타난 바와 같이 (도 4A), 진동수 변화 시험 및 점도 시험에서 HP/aCD/PDM 군의 G' 및 G" 값은 HDP/aCD 8.5 및 HDP/aCD/PDM 8.5 군에서보다 낮았다. HDP/aCD 8.5 및 HDP/aCD/PDM 8.5 군 모두 변형율 변화 및 진동수 변화 시험에서 HP/aCD/PDM 및 HDP/PDM 8.5 군보다 높은 G' 및 G" 값을 나타냈다. 이는 초분자화학 및 카테콜 중합의 복합 전략의 도입은 하이드로겔의 점탄성 특성을 크게 향상시킬 수 있다는 것을 의미한다. HDP/aCD 8.5 및 HDP/aCD/PDM 8.5 군은 모두 G"(점성 거동을 나타냄) 값 보다 높은 G'(탄성 거동을 나타냄) 값을 나타내었고, 이는 진동수 변화 데이터에서 주로 고체 상태 특성임을 시사한다. 높은 전단 하에서 하이드로겔 구조의 파괴와 그에 따른 회복을 평가하기 위하여 시간 의존적 단계적 시험을 수행하였다. HDP/aCD 8.5 및 HDP/aCD/PDM 8.5 하이드로겔 모두에서 첫 번째 단계 (0 % 변형, 1-2 분)에 나타난 것과 같은, 두 번째 및 세 번째의 휴지 동안 (0 % 변형, 3-4 및 5-6 분)의 고체 유사 특성 (G'> G")의 복원은 자가치유 거동을 시사한다. 이러한 HDP/aCD 8.5 및 HDP/aCD/PDM 8.5 하이드로겔은 전단 속도가 증가함에 따라 점도 데이터선의 기울어짐을 나타냈다. HDP/aCD/PDM 8.5 하이드로겔의 이러한 전단-박화 거동(shear-thinning behavior)은 피하조직에서 주사기 바늘을 통한 주입을 용이하게 할 수 있다. 관찰된 모든 데이터는 설계된 HDP/aCD/PDM 8.5 하이드로겔의 즉각적인 겔화, 단일 주사기를 통한 주사가능성, 자가치유능, 및 전단 박화 거동을 의미하였고, 이러한 특성은 피하주사에 적합할 것임을 시사하는 것이다.The viscoelastic properties of the HP/aCD/PDM, HDP/aCD 8.5, HDP/PDM 8.5 and HDP/aCD/PDM 8.5 samples were evaluated with a rheometer ( FIG. 4D ). The HDP/PDM 8.5 group remained in the inverted position (Fig. 4A), but in the strain sweep and frequency sweep tests, it was estimated that the difference in test method and sample incubation time between the inversion and rheology tests was By factor, the storage coefficient (G') and loss coefficient (G") values of this group were significantly lower than that of other supramolecular hydrogel structures (mainly based on aCD and PEG interactions). As shown in the inversion test results ( 4A), the G' and G" values of the HP/aCD/PDM group in the frequency change test and the viscosity test were lower than in the HDP/aCD 8.5 and HDP/aCD/PDM 8.5 groups. Both HDP/aCD 8.5 and HDP/aCD/PDM 8.5 groups showed higher G' and G" values than HP/aCD/PDM and HDP/PDM 8.5 groups in strain change and frequency change tests. The introduction of the composite strategy means that the viscoelastic properties of the hydrogel can be greatly improved. Both the HDP/aCD 8.5 and HDP/aCD/PDM 8.5 groups have higher G' (elastic behavior) than the G" (indicating viscous behavior) values. ) values, suggesting that it is mainly a solid state characteristic in the frequency change data. A time-dependent stepwise test was performed to evaluate the destruction and subsequent recovery of the hydrogel structure under high shear. During the second and third resting periods (0% strain, 3-4 and Restoration of the solid-like properties (G'>G") of 5-6 min) suggests a self-healing behavior. These HDP/aCD 8.5 and HDP/aCD/PDM 8.5 hydrogels exhibited an increase in the viscosity data line with increasing shear rate. This shear-thinning behavior of the HDP/aCD/PDM 8.5 hydrogel can facilitate injection through the syringe needle in the subcutaneous tissue.All observed data are the designed HDP/aCD/ Immediate gelation of PDM 8.5 hydrogel, injectability through a single syringe, self-healing ability, and shear thinning behavior were implied, suggesting that these properties would be suitable for subcutaneous injection.
2.4. 하이드로겔의 물리화학적 특징2.4. Physicochemical properties of hydrogels
동결건조된 HDP/aCD 8.5 및 HDP/aCD/PDM 8.5 군의 내부 구조를 FE-SEM 이미징으로 관찰하였다 (도 5A). HDP/aCD 8.5 및 HDP/aCD/PDM 8.5 군 모두에서 벌집(honeycomb)과 같은 구조가 나타났으며, 이는 폴리슈도로탁산 구조 및 폴리도파민 연결에 기반한 이중 가교 전략 때문일 수 있다. 특히, HDP/aCD/PDM 8.5 군의 사진에서는 PDM이 HDP/aCD 8.5 하이드로겔 시스템의 내부 표면에 존재하는 것으로 확인되었다.The internal structures of the lyophilized HDP/aCD 8.5 and HDP/aCD/PDM 8.5 groups were observed by FE-SEM imaging (FIG. 5A). A honeycomb-like structure appeared in both the HDP/aCD 8.5 and HDP/aCD/PDM 8.5 groups, which may be due to the polypseudotaxane structure and the double crosslinking strategy based on polydopamine linkages. In particular, in the picture of the HDP/aCD/PDM 8.5 group, it was confirmed that PDM was present on the inner surface of the HDP/aCD 8.5 hydrogel system.
HDP/aCD/PDM 8.5 군의 약물 방출 패턴을 도네페질 용액 군(pH 7.4)과 비교하였다 (도 5B). 약물 방출 시험에서 도네페질 용액은 중합체 막을 통해 즉시 확산되었다. 로딩 약물 양의 약 94.4 %가 24 시간 이내에 투석 튜브에서 방출액으로 확산되었다. 이와는 반대로, HDP/aCD/PDM 8.5 하이드로겔에서 방출된 도네페질의 양은 24 시간에 6.0 %에 불과하였다. 7일 및 14일에 HDP/aCD/PDM 8.5 하이드로겔로부터 방출된 도네페질의 양은 각각 39.9 % 및 56.7 %였다. HDP/aCD/PDM 8.5 하이드로겔 군은 본 연구에서 pH 7.4에서 4주 동안 지속적인 약물 방출을 나타냈다. 마이크로스피어-함입된 가교 하이드로겔 시스템에서 관찰된 약물 서방출은 투여 빈도를 감소시켜 환자 순응도를 높일 수 있다.The drug release pattern of the HDP/aCD/PDM 8.5 group was compared with the donepezil solution group (pH 7.4) ( FIG. 5B ). In the drug release test, the donepezil solution immediately diffused through the polymer membrane. Approximately 94.4% of the loading drug amount diffused from the dialysis tube into the release solution within 24 h. In contrast, the amount of donepezil released from the HDP/aCD/PDM 8.5 hydrogel was only 6.0% at 24 h. The amount of donepezil released from the HDP/aCD/PDM 8.5 hydrogel on
설계된 HDP/aCD/PDM 8.5 하이드로겔의 시험관 내(in vitro) 분해 프로파일은 HAase의 존재 또는 부재 하에서 평가하였다 (도 5C). HAase 부재 하에서는 하이드로겔의 상대 중량은 72 시간 동안 초기 값의 1.64 배로 증가하였으며, 이는 HA 골격의 팽창 특성에 의해 유발된 것일 수 있다. 그 후 21일째에 겔의 상대 중량은 초기 중량의 118.7%로 감소하였다. HAase가 포함된 군의 경우 48 시간에 하이드로겔의 상대 중량은 119.9%로 측정되었다. 그 후, 감소하는 패턴이 관찰되었고, 21 일에 최종적으로 초기 값의 61.8 %에 도달하였다. HAase 부재 군에 비해 HAase 존재 군에서 하이드로겔의 상대 중량이 더 낮다는 것은 HDP/aCD/PDM 8.5 하이드로겔에서 HA 중합체의 효소 분해가 진행되었음을 의미할 수 있다. HAase 존재 하에서 21 일째에 잔류된 하이드로겔 중량 (61.8 %)은 HDP 네트워크에 테더링된 PEG (HA 골격에 연결됨)와 aCD 사이의 분해 불가능한 포접 복합체 부분을 의미할 수 있다. 관찰된 시험관 내(in vitro) 분해 데이터로부터, 설계된 하이드로겔 시스템은 HAase를 포함한 생물학적 조직에서 천천히 분해될 수 있음을 알 수 있다. 가교 결합된 하이드로겔로부터의 지속적인 약물 방출 및 HAase의 존재 하에서도 낮은 하이드로겔의 분해 속도는 투여 빈도를 감소시키고 장기간 지속되는 약물 전달 시스템을 만드는 데 기여할 수 있다.The in vitro degradation profile of the designed HDP/aCD/PDM 8.5 hydrogel was evaluated in the presence or absence of HAase ( FIG. 5C ). In the absence of HAase, the relative weight of the hydrogel increased to 1.64 times its initial value for 72 hours, which may be caused by the expansive nature of the HA backbone. Thereafter, on day 21, the relative weight of the gel decreased to 118.7% of the initial weight. In the case of the HAase-containing group, the relative weight of the hydrogel at 48 hours was measured to be 119.9%. After that, a decreasing pattern was observed and finally reached 61.8% of the initial value on day 21. The lower relative weight of the hydrogel in the group with HAase compared to the group without HAase may mean that enzymatic degradation of the HA polymer proceeded in the HDP/aCD/PDM 8.5 hydrogel. The residual hydrogel weight (61.8%) at day 21 in the presence of HAase may represent the portion of the non-cleavable inclusion complex between PEG (linked to the HA backbone) and aCD tethered to the HDP network. From the observed in vitro degradation data, it can be seen that the designed hydrogel system can be degraded slowly in biological tissues including HAase. The sustained drug release from the cross-linked hydrogel and the low degradation rate of the hydrogel even in the presence of HAase may contribute to reducing the dosing frequency and creating a long-lasting drug delivery system.
2.5. 생분해 특성2.5. Biodegradation properties
설계된 하이드로겔 시스템의 생분해 패턴을 마우스에서 피하주사한 후 시험하였다 (도 6). PDM, HDP/aCD 8.5, HDP/PDM 8.5, HDP/aCD/PDM 및 HDP/aCD/PDM 8.5 검체를 마우스의 등쪽에 피하주사하고 생체내(in vivo) 머무름 특성을 모니터링하였다. PDM 및 HDP/PDM 8.5 군에서는 남아 있는 샘플 조각을 얻을 수 없었는데, 이는 PDM 및 HDP/PDM 8.5 하이드로겔이 피하조직으로 즉각적으로 확산하여 분해되었기 때문일 것이다. HDP/PDM 8.5 군에서는 가교 방법으로 카테콜 중합이 사용되었지만 시료 분산물을 주입한 후 14 일에 피하조직에서 남아있지 않았다. 유동학 데이터 (도 4D)에서 볼 수 있듯이, 변형 및 진동수 변화 시험에서 HDP/PDM 8.5 군의 최대 G' 값은 각각 20.6 및 18.6 Pa에 불과하였고, 이러한 낮은 점탄성 특성은 피하조직에서의 빠른 생분해와 관련이 있는 것으로 보인다. HDP/aCD 8.5, HDP/aCD/PDM 및 HDP/aCD/PDM 8.5 군에서 남아 있는 겔 부분을 수집하여 측정한 겔 중량의 순서는 HDP/aCD/PDM 8.5> HDP/aCD 8.5> HDP/aCD/PDM이었다. HDP/aCD/PDM 8.5 군의 잔류 겔 중량은 HDP/aCD 8.5 군보다 유의적으로 높았다 (p <0.05). 상기 두 군 간의 차이는 동일한 주입량에 포함된 PDM의 중량보다 컸으며, 이는 하이드로겔 네트워크에 분산된 PDM이 주입된 부위에서 하이드로겔 구조를 유지하고 생분해 속도를 지연시키는 데에 기여하였음을 의미한다. HDP/aCD/PDM 8.5 군의 남아 있는 겔 중량은 HDP/aCD/PDM 군의 수치보다 5.8 배 더 높았다 (p <0.05). PEG/aCD 기반 초분자 구조와 함께 도파민 중합과 HDP 접합체의 연결을 기반으로 한 화학적 가교 전략은 낮은 생분해 속도에 크게 기여하는 것으로 보인다. 제시된 데이터는 HDP/aCD/PDM 8.5 하이드로겔이 생분해성 장기 지속성 약물전달 시스템으로 사용될 수 있음을 시사한다.The biodegradation pattern of the designed hydrogel system was tested after subcutaneous injection in mice ( FIG. 6 ). PDM, HDP/aCD 8.5, HDP/PDM 8.5, HDP/aCD/PDM and HDP/aCD/PDM 8.5 specimens were injected subcutaneously into the dorsal side of mice, and retention properties were monitored in vivo . The remaining sample fragments could not be obtained from the PDM and HDP/PDM 8.5 groups, probably because the PDM and HDP/PDM 8.5 hydrogels immediately diffused into the subcutaneous tissue and degraded. In the HDP/PDM 8.5 group, catechol polymerization was used as a crosslinking method, but it did not remain in the subcutaneous tissue at 14 days after injection of the sample dispersion. As can be seen from the rheological data (FIG. 4D), the maximum G' values of the HDP/PDM 8.5 group in the strain and frequency change tests were only 20.6 and 18.6 Pa, respectively, and this low viscoelastic property is due to the rapid biodegradation and rapid biodegradation in the subcutaneous tissue. seems to be related. The order of gel weight measured by collecting the remaining gel fractions from HDP/aCD 8.5, HDP/aCD/PDM and HDP/aCD/PDM 8.5 groups is: HDP/aCD/PDM 8.5> HDP/aCD 8.5> HDP/aCD/PDM It was. The residual gel weight of the HDP/aCD/PDM 8.5 group was significantly higher than that of the HDP/aCD 8.5 group (p <0.05). The difference between the two groups was larger than the weight of the PDM included in the same injection amount, which means that the PDM dispersed in the hydrogel network contributed to maintaining the hydrogel structure at the injection site and delaying the biodegradation rate. The residual gel weight of the HDP/aCD/PDM 8.5 group was 5.8 times higher than that of the HDP/aCD/PDM group (p <0.05). Chemical crosslinking strategies based on dopamine polymerization and ligation of HDP conjugates with PEG/aCD-based supramolecular structures appear to contribute significantly to the low biodegradation rate. The presented data suggest that the HDP/aCD/PDM 8.5 hydrogel can be used as a biodegradable long-acting drug delivery system.
3. 결론3. Conclusion
도파민과 PEG 결합 HA 중합체 접합체 (HDP)를 합성하고 aCD와 PEG와 폴리도파민 결합 사이에 포접 복합체를 형성하여 가교 결합된 하이드로겔을 제조하였다. 초분자 시스템 (aCD와 PEG 사이) 및 카테콜 중합 (HA 중합체에 연결된(grafted) 도파민 분자 사이)은 이중 물리적 및 화학적 하이드로겔 가교 전략으로서 도입되었으며 이에 의해 점탄성 특성이 크게 향상되었다. 또한, 설계된 하이드로겔 시스템에 대하여 신속한 졸(sol)-대-겔(gel) 변환, 단일 주사기를 통한 주사가능성, 자가치유능 및 전단 박화 거동을 검증하였다. 지속 방출을 위하여 가교된 하이드로겔 네트워크에 PDM을 함입시켰고 HDP/aCD/PDM 8.5 하이드로겔은 마우스에 피하주사 후에 낮은 생분해율 및 무시할 수 있는 정도의 독성을 나타냈다. 이러한 모든 결과로부터 설계된 HDP/aCD/PDM 8.5 하이드로겔은 피하주사를 통하여 도네페질을 장기간 전달에 안전하게 사용할 수 있음을 알 수 있다.A cross-linked hydrogel was prepared by synthesizing a dopamine and PEG-linked HA polymer conjugate (HDP) and forming an inclusion complex between aCD and PEG and polydopamine bonds. The supramolecular system (between aCD and PEG) and catechol polymerization (between dopamine molecules grafted to the HA polymer) were introduced as dual physical and chemical hydrogel crosslinking strategies, whereby the viscoelastic properties were greatly improved. In addition, for the designed hydrogel system, rapid sol-to-gel conversion, injectability through a single syringe, self-healing ability, and shear thinning behavior were verified. For sustained release, PDM was incorporated into the cross-linked hydrogel network and the HDP/aCD/PDM 8.5 hydrogel showed low biodegradation rate and negligible toxicity after subcutaneous injection into mice. From all these results, it can be seen that the designed HDP/aCD/PDM 8.5 hydrogel can be safely used for long-term delivery of donepezil through subcutaneous injection.
4. 요약4. Summary
본 발명에서는 약물(도네페질)을 피하주사 후 지속적으로 전달하기 위하여 폴리슈도로탁산 구조와 폴리도파민 결합에 기반한 가교 히알루론산(HA) 하이드로겔을 개발하였다. 도파민 및 폴리에틸렌 글리콜(PEG) 분자를 각각 카테콜 중합을 위한 아미드 결합 및 사이클로덱스트린(CD)과의 포접 복합체화(inclusion complexation)를 위하여 HA 중합체에 공유결합시켰다. HA-도파민-PEG (HDP) 접합체의 PEG 사슬에 알파-사이클로덱스트린 (aCD)이 결합하여 폴리슈도로탁산 구조를 만들고, pH를 알칼리성 조건 (pH 8.5)으로 조정하여 중합된(polymerized) 도파민을 형성하였다 (HD 접합체에 부착된 형태로). 지속적인 방출을 위하여 폴리(락틱-코-글리콜산)[poly(lactic-co-glycolic acid), PLGA]/도네페질(donepezil) 마이크로스피어(마이크로스피어, MS) (PDM)를 HDP 고분자 네트워크에 함입시켰다. 설계된 HDP/aCD/PDM 8.5 하이드로겔 시스템은 즉각적인 겔화 패턴, 단일 주사기를 통한 주사가능성, 자가치유능 및 전단 박화 거동(shear-thinning behavior)을 나타냈다. 도네페질은 HDP/aCD/PDM 8.5 하이드로겔에서 지속적인 패턴으로 방출되었고, HDP/aCD/PDM 8.5 하이드로겔은 히알루론산 분해효소에 의해 분해되는 특성을 나타냈다. 피하주사 후, HDP/aCD/PDM 8.5 하이드로겔은 14 일이 지난 후에도 다른 군보다 더 많은 양이 남아 있었다. 관찰된 결과에 의해 개발된 HDP/aCD/PDM 8.5 하이드로겔은 피하주사 제제로서 높은 임상 타당성을 시사한다.In the present invention, a cross-linked hyaluronic acid (HA) hydrogel based on a polypseudotaxane structure and polydopamine bond was developed to continuously deliver the drug (donepezil) after subcutaneous injection. Dopamine and polyethylene glycol (PEG) molecules were covalently linked to the HA polymer for amide linkage for catechol polymerization and inclusion complexation with cyclodextrin (CD), respectively. Alpha-cyclodextrin (aCD) is bound to the PEG chain of the HA-dopamine-PEG (HDP) conjugate to form a polypseudotaxane structure, and the pH is adjusted to alkaline conditions (pH 8.5) to form polymerized dopamine (in the form attached to the HD conjugate). Poly(lactic-co-glycolic acid) [poly(lactic-co-glycolic acid), PLGA]/donepezil microspheres (microspheres, MS) (PDM) were incorporated into the HDP polymer network for sustained release. . The designed HDP/aCD/PDM 8.5 hydrogel system exhibited an immediate gelation pattern, injectability through a single syringe, self-healing ability and shear-thinning behavior. Donepezil was released in a continuous pattern from the HDP/aCD/PDM 8.5 hydrogel, and the HDP/aCD/PDM 8.5 hydrogel exhibited the property of being degraded by hyaluronic acid degrading enzyme. After subcutaneous injection, the amount of HDP/aCD/PDM 8.5 hydrogel remained larger than that of the other groups even after 14 days. Based on the observed results, the developed HDP/aCD/PDM 8.5 hydrogel suggests high clinical feasibility as a subcutaneous injection formulation.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 예를 들어, 단일형으로 설명되어 있는 각 구성 요소는 분산되어 실시될 수도 있으며, 마찬가지로 분산된 것으로 설명되어 있는 구성 요소들도 결합된 형태로 실시될 수 있다.The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and likewise components described as distributed may also be implemented in a combined form.
본 발명의 범위는 후술하는 청구범위에 의하여 나타내어지며, 청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.
Claims (15)
상기 HA-도파민-PEG 접합체 및 사이클로덱스트린을 혼합한 후 약학적으로 사용가능한 염을 혼합하여 pH 7 ~ 11로 조절하는 단계
를 포함하는 하이드로겔 조성물의 제조 방법.conjugating dopamine and PEG to hyaluronic acid (HA) to obtain a HA-dopamine-PEG conjugate; and
After mixing the HA-dopamine-PEG conjugate and cyclodextrin, adjusting the pH to 7-11 by mixing a pharmaceutically usable salt
A method for producing a hydrogel composition comprising a.
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CN115779158A (en) * | 2022-11-28 | 2023-03-14 | 上海赢生医疗科技有限公司 | Self-repairing coating for medical apparatus and instruments and preparation method thereof |
CN115779158B (en) * | 2022-11-28 | 2024-02-09 | 上海赢生医疗科技有限公司 | Self-repairing coating for medical instrument and preparation method thereof |
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