Jadidi-Niaragh Farhad, Atyabi Fatemeh, Rastegari Ali, Mollarazi Esmail, Kiani Melika, Razavi Alireza, Yousefi Mehdi, Kheshtchin Nasim, Hassannia Hadi, Hadjati Jamshid, Shokri Fazel
Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Tumour Biol. 2016 Jun;37(6):8403-12. doi: 10.1007/s13277-015-4732-0. Epub 2016 Jan 5.
The immunosuppressive factors in tumor microenvironment enhance tumor growth and suppress anti-tumor immune responses. Adenosine is an important immunosuppressive factor which can be secreted by both tumor and immune cells trough action of two cell surface ecto-nucleotidase molecules CD39 and CD73. Blocking the adenosine generating molecules has emerged as an effective immunotherapeutic approach for treatment of cancer. In this study, CD73-siRNA encapsulated into chitosan-lactate (ChLa) nanoparticles (NPs) was employed to suppress the expression of CD73 molecule on 4T1 breast tumor cells, in vitro. ChLa NPs were generated through ionic gelation of ChLa by tripolyphosphate (TPP). Small interfering RNA (SiRNA)-loaded NPs had about 100 nm size with a polydispersive index below 0.3 and a zeta potential about 13. Our results showed that ChLa NPs with Ch 50 kDa exhibit the best physicochemical features with the high siRNA encapsulation capacity. Synthesized NPs were able to fully bind with siRNA, protect them against serum and heparin degradation, and promote the transfection process. While the NPs exhibited low toxicity during 72 h cell culture, the transfection of Ch-plasmid expressing green fluorescent protein (pEGFP) NPs was efficient in 4T1 cells with a transfection rate of 53.6 % as detected by flow cytometry. In addition, CD73-siRNA-loaded ChLa NPs could efficiently suppress the expression of CD73 as assayed by real-time polymerase chain reaction and flow cytometry. As a conclusion, CD73-siRNA-loaded ChLa NPs may be considered as a promising therapeutic tool for cancer therapy; however, further in vivo investigations are necessary.
肿瘤微环境中的免疫抑制因子可促进肿瘤生长并抑制抗肿瘤免疫反应。腺苷是一种重要的免疫抑制因子，肿瘤细胞和免疫细胞均可通过两种细胞表面外切核苷酸酶分子CD39和CD73的作用分泌该因子。阻断腺苷生成分子已成为一种有效的癌症免疫治疗方法。在本研究中，将包裹于壳聚糖乳酸盐（ChLa）纳米颗粒（NPs）中的CD73小干扰RNA（siRNA）用于体外抑制4T1乳腺肿瘤细胞上CD73分子的表达。ChLa NPs通过三聚磷酸钠（TPP）对ChLa进行离子凝胶化反应生成。负载小干扰RNA（SiRNA）的NPs粒径约为100 nm，多分散指数低于0.3，zeta电位约为13。我们的结果表明，具有50 kDa壳聚糖的ChLa NPs具有最佳的物理化学特性和高siRNA包封率。合成的NPs能够与siRNA充分结合，保护其免受血清和肝素的降解，并促进转染过程。虽然NPs在72小时细胞培养过程中表现出低毒性，但通过流式细胞术检测，表达绿色荧光蛋白（pEGFP）的Ch-质粒NPs在4T1细胞中的转染效率很高，转染率为53.6%。此外，通过实时聚合酶链反应和流式细胞术检测，负载CD73-siRNA的ChLa NPs能够有效抑制CD73的表达。综上所述，负载CD73-siRNA的ChLa NPs可被视为一种有前景的癌症治疗工具；然而，还需要进一步的体内研究。
