核黄素修饰的脂质复合纳米粒共递送CXCR4 siRNA和阿霉素治疗高转移性癌症

doi:10.5246/jcps.2021.03.015

中国药学(英文版) ›› 2021, Vol. 30 ›› Issue (3): 189-205.DOI: 10.5246/jcps.2021.03.015

• 【研究论文】 • 下一篇

核黄素修饰的脂质复合纳米粒共递送CXCR4 siRNA和阿霉素治疗高转移性癌症

李锴森¹^,², 王如东¹^,², 彭祎玮¹^,², 董达文³, 齐宪荣¹^,²^,^*()

1. 北京大学医学部药学院药剂学系, 北京 100191
2. 北京大学医学部分子药剂学与新释药系统北京市重点实验室, 北京 100191
3. 扬子江药业集团药物制剂新技术国家重点实验室, 江苏 225300

收稿日期:2020-09-05 修回日期:2020-10-11 接受日期:2020-11-16 出版日期:2021-03-29 发布日期:2021-03-29
通讯作者: 齐宪荣
作者简介:
+ Tel.: +86-10-82801584, E-mail: qixr@bjmu.edu.cn
基金资助:
The National Nature Science Foundation of China (Grant No. 81973258 and 81673365) and State Key Laboratory of Advanced Pharmaceutical Formulation with High Technology in Yangtze River Pharmaceutical Group.

Riboflavin-modified lipo-polyplexes co-delivering CXCR4 siRNA and doxorubicin for treatment of highly metastatic cancer

Kaisen Li¹^,², Rudong Wang¹^,², Yiwei Peng¹^,², Dawen Dong³, Xianrong Qi¹^,²^,^*()

1 Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
2 Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery System, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
3 State Key Laboratory of Advanced Pharmaceutical Formulation with High Technology, Yangtze River Pharmaceutical Group, Jiangsu 225300, China

Received:2020-09-05 Revised:2020-10-11 Accepted:2020-11-16 Online:2021-03-29 Published:2021-03-29
Contact: Xianrong Qi

摘要/Abstract

摘要：

多项研究揭示SDF-1/CXCR4是与癌症转移最相关的趋化因子通路之一, 因此靶向CXCR4的siRNA能够改善高转移性癌症治疗效果, 尤其将其与化疗联合使用时潜力巨大。本研究中, 我们构建了素修饰的脂质复合纳米粒, 实现了对CXCR4 siRNA和阿霉素的共递送, 用于癌症治疗。以酸性环境中可断裂的腙键, 将阿霉素共价连接至聚乙烯亚胺(PEI), 所得酸敏感性偶联物与siRNA紧密压缩形成聚合物纳米粒, 进一步以尾端含核黄素的脂质薄膜对其进行包覆, 制备了核黄素修饰的脂质复合纳米粒。利用肿瘤细胞高表达核黄素受体的事实, 核黄素修饰有效增强了肿瘤细胞对脂质复合纳米粒的摄取。共包载CXCR4 siRNA和阿霉素的核黄素修饰脂质复合纳米粒在体外有效降低了肿瘤细胞的生存率和侵袭能力; 在体内, 抑制了原位肿瘤的生长, 同时抑制了肿瘤转移。

关键词: 核黄素, 脂质复合纳米粒, 共递送, 小干扰RNA, 阿霉素

Abstract:

SDF-1/CXCR4 has been recognized as one of the most relevant chemokine signaling pathways to cancer metastasis, and siRNA targeting CXCR4 may provide potential improvements to treat those highly metastatic cancers, especially when combined with chemotherapy. In the present study, we constructed riboflavin-modified lipo-polyplexes to co-deliver CXCR4 siRNA and doxorubicin for cancer therapy. Doxorubicin was covalently conjugated to polyethyleneimine (PEI) with acid-cleavable hydrazine bond, and the obtained acid-sensitive conjugate was efficiently condensed with siRNA to form polyplexes, which were further coated with riboflavin-tailed lipid-membrane to prepare the lipo-polyplexes conveniently. Utilizing the fact that tumor cells overexpress riboflavin receptors, the riboflavin modification effectively enhanced uptake of lipo-polyplexes by tumor cells in a receptor-mediated manner. The riboflavin-modified lipo-polyplexes co-delivering CXCR4 siRNA and doxorubicin effectively decreased viability and invasiveness of tumor cells in vitro, and inhibited primary tumor growth and tumor metastasis in vivo.

Key words: Riboflavin, Lipo-polyplexes, Co-delivery, SiRNA, Doxorubicin

Supporting:

李锴森, 王如东, 彭祎玮, 董达文, 齐宪荣. 核黄素修饰的脂质复合纳米粒共递送CXCR4 siRNA和阿霉素治疗高转移性癌症[J]. 中国药学(英文版), 2021, 30(3): 189-205.

Kaisen Li, Rudong Wang, Yiwei Peng, Dawen Dong, Xianrong Qi. Riboflavin-modified lipo-polyplexes co-delivering CXCR4 siRNA and doxorubicin for treatment of highly metastatic cancer[J]. Journal of Chinese Pharmaceutical Sciences, 2021, 30(3): 189-205.

图/表 8

Figure 1. (A) Synthetic scheme of DSPE-PEG-RF. (B) The 1H NMR spectra of DSPE-PEG-NH2 and DSPE-PEG-RF. (C, D) MAIDI-TOF mass spectra of DSPE-PEG-NH2 (C) and DSPE-PEG-RF (D).

Figure 2. (A) Synthetic scheme of PhD. (B) The 1H NMR spectra of PhD. (C) HPLC spectra of DOX, PhD and PhD incubated within HCl solution for 30 min.

Figure 3. (A) Preparation procedure of PhD/siRNA/lip-RF by the lipid film hydration method. (B) siRNA binding ability of PhD/siRNA and PhD/siRNA/lip-RF analyzed by agarose gel electrophoresis. (C, D) The particle size and zeta potential of PhD/siRNA (C) and PhD/siRNA/lip-RF (D) at an N/P ratio of 15 by DLS. (E, F) Transmission electron micrograph photos of PhD/siRNA (E) and PhD/siRNA/lip (F) at an N/P ratio of 15.

Table 1. Size and zeta potential of the lipo-polyplexes.

Figure 4. (A) Confocal microscope images of intracellular distribution of the polyplexes and lipo-polyplexes co-loaded with Cy5-siRNA (red) and DOX (green) in MDA-MB-231 cells after incubation for 2 h. The concentrations of Cy5-siRNA and DOX were 200 nM and 1.5 μM, respectively. Cell nuclei were stained with Hoechst 33258 (blue). Scale bar: 75 μm. (B, C) Fluorescence intensity of DOX (B) and fluorescence of FAM-siRNA (C) with different formulations in MDA-MB-231 cells after incubation for 4 h measured by flow cytometry. In the competition experiment, cells were pre-incubated with a 20-fold excess of FMN or RF-modified liposomes for 40 min, followed by incubation with PhD/FAM-siRNA/lip-RF for 4 h. The data are presented as the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5. Cell viability of MDA-MB-231 cells cultured with various formulations after 48 h (A) and 72 h (B), respectively. The concentrations of siRNA and DOX were 200 nM and 3.2 μM, respectively. The data are presented as the mean ± SD (n = 6).

Figure 6. (A) CXCR4 mRNA in MDA-MB-231 cells determined by qRT-PCR after incubating with various formulations. (B) Cellular membrane CXCR4 protein expression in MDA-MB-231 cells after incubating with various formulations. The concentrations of siRNA were 100 nM. The data are presented as the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. (C, D) Matrigel invasion assays in normoxia (C) and hypoxia conditions (D) of MDA-MB-231 cells pre-treated with different formulations, respectively. Magnification: 20×.

Figure 7. Antitumor effect and safety evaluation of the lipo-polyplexes in 4T1 tumor-bearing mice. The doses of siRNA and DOX were 1 mg/kg and 0.52 mg/kg, respectively. (A) Tumor growth curve. The black arrow denotes the time of drug administration. (B) The body weight variation in BALB/c nude mice after intravenous treatment. The data are presented as the mean ± SD (n = 5~7). (C) Representative histopathologic images of lung, liver, and heart.

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核黄素修饰的脂质复合纳米粒共递送CXCR4 siRNA和阿霉素治疗高转移性癌症

Riboflavin-modified lipo-polyplexes co-delivering CXCR4 siRNA and doxorubicin for treatment of highly metastatic cancer

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