효소를 이용한 탈아세틸화 히할루론산의 제조와 특성분석

Abstract

히알루론산은 글리코스아미노글리칸의 일종으로, N-아세틸-D-글루코사민과 D-글루쿠론산으로 이루어진 반복 단위가 선형으로 연결되어 있는 천연고분자이다. 히알루론산은 생체 적합성, 생분해성 및 높은 함수성 등의 특성을 가지고 있기 때문에 생체재료로서 광범위하게 사용될 수 있다. 그러나 히알루론산은 생체 내에서 기계적 물성이 약하며, 분해속도가 빠르다는 단점이 있어 생의학적 응용에 한계가 있다. 그러므로 히알루론산을 생체 재료로 이용하기 위해서는 생체 내에서의 분해 속도 문제를 극복하고 분자량 저하를 최소화하여 히알루론산의 구조를 유지할 수 있는 형태의 유도체의 개발이 필요하다. 그렇기 때문에 본 연구에서는 분자량 감소가 작은 효소적 탈아세틸화 히알루론산(Enz-DeHyA)을 제조하고 물리화학적 특성 및 생체 적합성을 평가하였다. 효소적 탈아세틸화 히알루론산을 제조하기 위하여 히알루론산 탈아세틸화 효소를 Scopulariopsis brevicaulis (S.brevicaulis)균에서 추출 정제한 후, 최적반응조건을 선정하였다. 이를 바탕으로 효소적 탈아세틸화 히알루론산(Enz-DeHyA)을 제조 한 후 물리화학적 특성을 측정한 결과 효소적 탈아세틸화 히알루론산의 분자량과 점도 모두 화학적 탈아세틸화 히알루론산 보다 높게 측정되었다. 또한 세포생존율과 산화질소 생성 억제 능력을 각각 측정한 결과, 효소적 탈아세틸화 히알루론산은 hMSC의 세포생존율에 큰 영향을 미치지 않으며, LPS에 의해 유도된 산화질소의 생성을 억제 시킬 수 있는 능력이 화학적 탈아세틸화 히알루론산 보다 뛰어남을 확인하였다. 이러한 결과들을 토대로 효소적 탈아세틸화 히알루론산은 조직공학 응용 분야에서 사용 가능성이 있다는 것을 알 수 있다. |Hyaluronic acid (HyA) is consist of β-1,4-D-glucuronic acid and β-1,3-N-acetyl-D-glucosamine. Because of its biocompatibility, biodegradability, and high moisture retention capacity, HyA has wide-range applicability in biomaterials. However, drawback of HyA, rapid biodegradation rate in vivo, limit biomedical application. Therefore, HyA needs to be modified and developed into a derivative that retains its unique properties and do not undergo the rapid degradation in vivo. In this study, enzymatically deacetylated hyaluronic acid (Enz-DeHyA) having higher molecular weight than chemically deacetylated hyaluronic acid (Chem-DeHyA) was prepared by hyaluronic acid deacetylase, isolated from Scopulariopsis brevicaulis (S.brevicaulis). Enz-DeHyA was characterized by fourier transformed infrared spectroscopy (FT-IR), 1H nuclear magnetic resonance (1H NMR) and circular dichroism (CD) spectroscopy. The molecular weight and viscosity of HyA and its deacetylated HyA were measured by gel-permeation chromatography and rheometer, respectively. The Enz-DeHyA and Chem-DeHyA had a molecular weight of 605kDa and 56-146kDa, respectively. Viscosity of Enz-DeHyA (1.56 log cP) was higher than that of Chem-DeHyA (0.58-1.09 log cP). The cell viability of human mesenchymal stem cells (hMSC) cultured on the HyA and its deacetylated HyA was evaluated by cell counting kit-8 assay. It was show that Enz-DeHyA has no significantly effect2 on hMSC cell viability. Also, nitric oxide (NO) production on raw264.7 cells cultured on the HyA and its deacetylated HyA was evaluated by NO assay. In these results, Enz-DeHyA will be a good biomaterial of candidates for tissue engineering applications.; Hyaluronic acid (HyA) is consist of β-1,4-D-glucuronic acid and β-1,3-N-acetyl-D-glucosamine. Because of its biocompatibility, biodegradability, and high moisture retention capacity, HyA has wide-range applicability in biomaterials. However, drawback of HyA, rapid biodegradation rate in vivo, limit biomedical application. Therefore, HyA needs to be modified and developed into a derivative that retains its unique properties and do not undergo the rapid degradation in vivo. In this study, enzymatically deacetylated hyaluronic acid (Enz-DeHyA) having higher molecular weight than chemically deacetylated hyaluronic acid (Chem-DeHyA) was prepared by hyaluronic acid deacetylase, isolated from Scopulariopsis brevicaulis (S.brevicaulis). Enz-DeHyA was characterized by fourier transformed infrared spectroscopy (FT-IR), 1H nuclear magnetic resonance (1H NMR) and circular dichroism (CD) spectroscopy. The molecular weight and viscosity of HyA and its deacetylated HyA were measured by gel-permeation chromatography and rheometer, respectively. The Enz-DeHyA and Chem-DeHyA had a molecular weight of 605kDa and 56-146kDa, respectively. Viscosity of Enz-DeHyA (1.56 log cP) was higher than that of Chem-DeHyA (0.58-1.09 log cP). The cell viability of human mesenchymal stem cells (hMSC) cultured on the HyA and its deacetylated HyA was evaluated by cell counting kit-8 assay. It was show that Enz-DeHyA has no significantly effect2 on hMSC cell viability. Also, nitric oxide (NO) production on raw264.7 cells cultured on the HyA and its deacetylated HyA was evaluated by NO assay. In these results, Enz-DeHyA will be a good biomaterial of candidates for tissue engineering applications.