氧化还原响应性二氧化硅载三氧化二砷纳米递药系统的制备及体外评价

Preparation and in vitro evaluation of redox-responsive silica-loaded arsenic trioxide nano-drug delivery system

目的以介孔二氧化硅(mesoporous silica nanoparticles,MSN)为载体材料,通过具有氧化还原敏感的二硫键修饰,酰化反应连接无毒无免疫原性的聚乙二醇(polyethylene glycol,PEG),静电吸附作用负载三氧化二砷(arsenic trioxide,As2O3),构建一种载As2O3的氧化还原响应性二氧化硅(MSN-SS-PEG@As2O3)纳米递药系统,并进行体外评价。方法采用共沉淀法合成二氧化硅,以二氧化硅、(3-巯基丙基)三甲氧基硅烷、2-(2-吡啶基二硫基)乙胺盐酸盐以及甲氧基封端的PEG为基础,合成氧化还原响应性载体(MSN-SS-PEG)。通过马尔文粒度测定仪测定其粒径、Zeta电位;红外光谱法验证载体结构;采用透射电镜、小角粉末衍射仪等方法考察载体的形态及理化性质;电感耦合等离子发射光谱法(inductively coupled plasma emission spectrum,ICP)考察了载As2O3的二硫键修饰二氧化硅(MSN-SS-NH2@As2O3)和MSN-SS-PEG@As2O3的载药量,利用热重分析仪进一步验证其载药量。透析袋法考察了不同p H条件下递药系统的体外释药特性。MTT法考察载体以及递药系统对人正常肝细胞(L02)或人肝癌Hep G2细胞的毒性。结果 MSN、MSN-SS-NH2、MSN-SS-PEG的电位分别为(-13.40±0.87)、(31.63±0.90)、(27.70±5.60)m V,经修饰后的载体最终电位为正。MSN-SS-PEG的粒径为(159.60±3.10)nm。透射电镜结果表明MSN、MSN-SS-NH2、MSN-SS-PEG均呈圆形或类圆形;通过ICP测定MSN-SS-PEG@As2O3的载药量4.38%;体外释放实验结果表明MSN-SS-PEG@As2O3具有氧化还原的敏感响应。相比于L02细胞,Hep G2细胞对载体的毒性更敏感,且随着载体质量浓度的增加MSN-SS-PEG组的细胞存活率大于MSN-SS-NH2组,表明PEG修饰可进一步降低载体的细胞毒性,提高载体的生物相容性。另外MTT结果表明MSN-SS-PEG@As2O3组抑制Hep G2细胞生长的效果明显高于其他组。结论载体MSN-SS-PEG外观圆整,带正电,粒径均一,经修饰后的二氧化硅系统能在肿瘤特殊微环境下响应下释放药物,增加药物在肿瘤部位的积累。该纳米递药系统作为肿瘤微环境响应性载体在肿瘤治疗方面具有较好的应用前景。

Objective The redox-responsive drug delivery system of MSN-SS-PEG@As2O3 was constructed based on mesoporous silica nanoparticle(MSN), which was modified by both redox-sensitive disulfide bonds and non-toxic, non-immunogenic polyglycols(PEG), and loaded the arsenic trioxide(As2O3) by electrostatic adsorption and evaluated in vitro. Methods Silica was synthesized by coprecipitation method. The redox-responsive carrier(MSN-SS-PEG) was synthesized on the basis of silica,(3-mercaptopropyl) trimethoxysilane, 2-(2-pyridyldithio) ethylamine hydrochloride and methoxy terminated PEG. The particle size and Zeta potential of MSN-SS-PEG were measured by Malvern particle size analyzer;The structure of the carrier was verified by infrared spectroscopy;The morphology and physical and chemical properties of the carrier were investigated by transmission electron microscopy(TEM) and small angle powder diffraction;The drug loading efficiency of MSN-SS-NH2@As2O3 and MSN-SS-PEG@As2O3 were investigated by inductively coupled plasma emission spectrum(ICP). The drug loading was further verified by thermogravimetry(TGA). In vitro release characteristics of the drug delivery system under different pH conditions were investigated by dialysis bag method. MTT assay was used to investigate the toxicity of carrier and delivery system to human normal hepatocytes(L02) or human hepatocarcinoma(HepG2) cells. Results The potential of MSN, MSN-SS-NH2, MSN-SS-PEG was(-13.40 ± 0.87),(31.63 ± 0.90),(27.70 ± 5.60) mV, respectively. The final potential of modified carrier was positive. The particle size of MSN-SS-PEG was(159.60 ± 3.10) nm. The results of TEM showed that MSN, MSN-SS-NH2 and MSN-SS-PEG were all round or quasi round;The drug loading of MSN-SS-PEG@As2O3 was 4.38%, which measured by ICP;The release in vitro showed that MSN-SS-PEG@As2O3 was redox sensitive response. Compared with L02 cells, HepG2 cells were more sensitive to the toxicity of the carrier, and with the increase of the carrier concentration, the cell survival rate of MSN-SS-PEG was higher than that of MSN-SS-NH2, suggesting that PEG modification can further reduce the cytotoxicity of the carrier and improve the biocompatibility of the carrier. In addition, MTT results showed that the inhibitory effect of MSN-SS-PEG@As2O3 on HepG2 cell was significantly higher than that of other groups. Conclusion The carrier prepared in this paper had a round and uniform particle size. The modified silica can release under the special microenvironment of the tumor and increase the accumulation of the drug in the tumor site. The delivery system has a good application in tumor therapy as a tumor micro-environment responsive carrier.