载天冬酰胺酶纳米囊的活性及体外稳定性评价

目的 制备载天冬酰胺酶（asparaginase，AN）透明质酸-聚乙二醇[hyaluronic acid-graft-poly（ethylene glycol），HA-g-PEG]/α-环糊精（α-cyclodextrin，α-CD）纳米囊（HA-g-PEG/α-CD hollow nanocapsules loaded with asparaginase，AHAPs），并对其体外活性及稳定性进行初步考察。方法 采用自组装法制备AHAPs，测定AHAPs的最适温度、最适pH、粒径、zeta电位和包封率，并通过热稳定性、酸碱稳定性、抗胰蛋白酶水解能力、抗金属离子和有机化合物能力、血浆稳定性和贮存稳定性实验对游离AN与AHAPs的体外稳定性差异进行考察。通过荧光实验对AN与空白HA-g-PEG/α-CD纳米囊的相互作用进行研究。结果 AHAPs的最适温度为50 ℃，最适pH值为7.0，测得平均粒径为（424.53±7.25）nm，zeta电位为（-48.77±0.99）mV。经计算，AHAPs的平均包封率为（64.40±1.82）%。稳定性实验结果显示，AHAPs中AN的体外稳定性及活性明显优于游离AN，且部分实验结果差异具有统计学意义（P<0.05）。荧光实验结果表明，AHAPs中AN生物活性的提高可能与AN和空白HA-g-PEG/α-CD纳米囊的相互作用引起蛋白质残基微环境和酶构象改变相关。结论 AHAPs不仅提高了AN的活性，而且明显增强了AN的体外稳定性。
Objective To prepare hyaluronic acid-graft-poly (ethylene glycol)/α-cyclodextrin hollow nanocapsules loaded with asparaginase (AHAPs) and to investigate their in vitro stability and activity. Methods We prepared the AHAPs by self-assembly method and detected the optimal temperature, optimal pH value, particle size, zeta potential and entrapment efficiency. Then the differences in in vitro stability between AHAPs and free asparaginase (AN) were investigated by measuring thermal stability, acid- and basic-stability, stability to trypsinase, stability to metal ions and organic compounds, plasma stability and storage stability. The interaction between AN and blank hyaluronic acid-graft-poly (ethylene glycol)/α-cyclodextrin hollow nanocapsules was studied through the fluorescence experiment. Results The optimal temperature for AHAPs was 50 ℃, the optimal pH value was 7.0, the mean particle size was (424.53±7.25) nm, and the mean zeta potential was (－48.77±0.99) mV. The entrapment efficiency of AHAPs was (64.40±1.82)%. The results of stability experiment showed that AHAPs had a significantly better in vitro stability than free AN, and some stability experimental results were statistically significant (P<0.05). Fluorescence experiment showed that the improvement of biological activity of AHAPs may be related to the change of protein residues microenvironment and enzyme conformation caused by the interaction between AN and blank hyaluronic acid-graft-poly (ethylene glycol)/α-cyclodextrin hollow nanocapsules. Conclusion It has been found that AHAPs can not only improve the activity but also greatly enhance the stability of AN in vitro