用于眼部给药的槲皮素与microRNA-150共载阳离子固体脂质纳米粒制备及初步评价

Preparation and preliminary evaluation of quercetin and micro RNA-150 co-loaded cationic solid lipid nanoparticles for ocular administration

目的制备槲皮素与microRNA-150(m R150)共载阳离子固体脂质纳米粒(Que/mR150 SLNs),考察其制备工艺,并评价其体外释放、细胞摄取能力以及眼部给药安全性。方法采用薄膜分散法制备包载槲皮素的阳离子固体脂质纳米粒(Que-SLNs),以平均粒径、多分散指数(PDI)、包封率为指标,优化其制备工艺;采用静电吸附法将m R150共载于纳米粒中,制备Que/mR150 SLNs,通过琼脂糖凝胶电泳实验考察纳米粒对miRNA的吸附效率;并考察Que/mR150 SLNs中槲皮素的体外释药性能;采用MTT法测定Que/mR150 SLNs对人脐静脉血管内皮细胞HUVEC增殖的影响,并对其进行荧光标记,观察其在HUVEC细胞中的摄取情况;并通过兔眼病理组织切片考察Que/mR150 SLNs对兔眼的刺激性。结果经过工艺优化,制得的阳离子纳米Que-SLNs载药性、粒径分布、稳定性均较好,其外观呈类球形,放置2个月能保持稳定,槲皮素包封率为(85.25±1.29)%,载药量(1.67±0.02)%,平均粒径(110.00±2.10)nm,Zeta电位(53.20±5.12)m V;体外药物释放结果表明,槲皮素在纳米粒中释放较缓慢,48 h内累积释放量约(80.69±1.29)%;在不同阳离子材料双十八烷基二甲基溴化铵与m R150的质量比(DDAB/RNA)为6∶1时,阳离子固体脂质纳米粒可基本将m R150包载完全,且对其粒径、电位影响较小;MTT实验表明,50~150 mg/L的空白纳米质量浓度对HUVEC细胞无明显增殖毒性;细胞摄取实验表明,Cy5与香豆素6(coumarin-6,C6)双荧光标记共载纳米能有效进入HUVEC细胞;兔眼病理组织切片显示Que/mR150SLNs多次给药对眼部角膜组织无明显损伤。结论Que/mR150SLNs固体脂质纳米粒制备工艺稳定可靠、重复性好、贮藏稳定性、生物安全性好,有利于高效递送槲皮素与m R150进入HUVEC细胞,为年龄相关性黄斑变性等血管增生相关疾病的治疗提供思路。

Objective To prepare the cationic solid lipid nanoparticles(Que/mR150 SLNs) co-loaded with quercetin(Que) and microRNA-150(mR150) and investigate the preparation process, then assess its in vitro release, cell uptake capacity and safety of ocular administration. Method First, thin-film dispersion method was used to prepare quercetin-encapsulated cationic solid lipid nanoparticles(Que-SLNs), and the preparation process was optimized based on the particle size, PDI and encapsulation rate;Using electrostatic adsorption method to co-load mR150 in nanoparticles(Que/mR150 SLNs), and the adsorption efficiency of the miRNA by the nanoparticles was examined by agarose gel electrophoresis experiment;The in vitro release performance of quercetin in Que/mR150 SLNs was investigated;The effect of Que/mR150 SLNs on the proliferation of HUVEC of human umbilical vein endothelial cells was measured by MTT method, and fluorescence labeling was used to observe their uptake in HUVEC;And the irritancy of Que/mR150 SLNs to rabbit eyes was examined by pathological tissue sections of rabbit eyes. Result After process optimization, the cationic nano Que-SLNs had good drug-loading, particle size distribution and stability. The appearance of the cationic nano-Que-SLNs was spherical, and it could be kept stable for two months. The quercetin encapsulation rate was(85.25 ± 1.29)%, the drug load was(1.67 ± 0.02)%, the average particle size was(110.00 ± 2.10) nm, and the Zeta potential is(53.2 ± 5.12) m V;The in vitro drug release results showed that the release of quercetin in the nanoparticles was slow, and the cumulative release amount within 48 h was about(80.69 ± 1.29)%;When the mass ratio of dioctadecyl dimethyl ammonium bromide to m R150(DDAB/RNA) of different cationic materials was 6∶1, the cationic solid lipid nanoparticles basically encapsulated m R150 completely with little effect on its particle size and potential. MTT experiments showed that blank nanometer mass concentration of 50—150 mg/L had no significant proliferation toxicity on HUVEC cells;Cell uptake experiments showed that Cy5 and coumarin-6 dual fluorescently labeled and co-loaded nanometers could effectively enter HUVEC cells;Pathological tissues of rabbit eyes showed that Que/mR150 SLNs had no obvious damage to the eyes. Conclusion The preparation process of Que/mR150 SLNs solid lipid nanoparticles is stable and reliable, with good reproducibility, storage stability and good biological safety, which is conducive to the efficient delivery of quercetin and m R150 into HUVEC cells, which provides the ideas for the treatment of diseases related to angiogenesis.