目的 通過(guò)引入殼聚糖增加丙交酯乙交酯共聚物（PLGA）載體中小干擾RNA（siRNA）的包封率，對(duì)制備的攜siRNA-殼聚糖（siRNA-CS）的PLGA納米載體（siRNA-CPG）進(jìn)行表征。方法 選用傳統(tǒng)的乳化-溶劑揮發(fā)法制備siRNACPG，以粒徑、Zeta電位及包封率為指標(biāo)，對(duì)處方（siRNA與殼聚糖質(zhì)量比，PLGA質(zhì)量濃度，內(nèi)水相、二氯甲烷、外水相的體積比）進(jìn)行單因素考察及星點(diǎn)設(shè)計(jì)-響應(yīng)面法優(yōu)化。對(duì)優(yōu)化后制備的siRNA-CPG進(jìn)行粒徑、Zeta電位、包封率質(zhì)量評(píng)價(jià)；根據(jù)粒徑及包封率考察siRNA-CPG在生理鹽水及血清中的穩(wěn)定性；制備熒光標(biāo)記的siRNA-CPG，與MCF-7細(xì)胞孵育4 h，觀察細(xì)胞攝取情況。結(jié)果 殼聚糖的引入可將siRNA包封率從3.14%提高到90%以上。優(yōu)化后的處方中殼聚糖、siRNA、PLGA質(zhì)量濃度分別為1、0.5、10 mg·mL^-1，內(nèi)水相、二氯甲烷、外水相的體積比為1∶10∶100。制備3批siRNA-CPG凍干粉，復(fù)溶后室溫放置48 h及血清中24 h內(nèi)粒徑和包封率均無(wú)明顯變化；siRNA-CPG可被細(xì)胞攝取并從溶酶體中逃逸出來(lái)。結(jié)論 制備的siRNA-CPG包封率高、穩(wěn)定性好，可以實(shí)現(xiàn)siRNA的細(xì)胞質(zhì)有效遞送。

Objective To increase the encapsulation efficiency of siRNA in PLGA nano particles by incorprating chitosan, to prepare and characterise the PLGA nanoparticles carrying siRNA (siRNA-CPG). Methods The traditional emulsification solvent volatilization method was used to prepare siRNA-CPG. The formulation (mass ratio of siRNA to chitosan, mass concentration of PLGA, volume ratio of internal water phase, dichloromethane and external water phase) was optimized with the particle size, Zeta potential, entrapment efficiency as the index by single factor experiment and central composite design-response surface method. The particle size, Zeta potential and encapsulation rate of the optimized siRNA-CPG were evaluated. The stability of siRNA-CPG in normal saline and serum was investigated according to particle size and encapsulation rate. Fluorescent-labeled siRNA-CPG was prepared and incubated with MCF-7 cells for 4 h to observe cell uptake. Results The addition of chitosan increased the entrapment efficiency of siRNA from 3.14% to more than 90% in the PLGA nanoparticles. The optimized formula was as follows:the concentrations of chitosan, siRNA and PLGA were 1, 0.5 and 10 mg·mL^-1 respectively. Three batches of siRNA-CPG had little change in particle size and entrapment efficiency after redissolution by normal saline at room temperature for 48 h, and it could be uptaken by cells and then escape from lysosomes. Conclusion The siRNA-CPG prepared in this study has high encapsulation efficiency and good stability, and can realize cytoplasmic delivery of siRNA.

R943

中國(guó)博士后基金（2020M682926）