Delivery and Cytotoxicity of Type I Shiga Toxin A Subunit by pH-Sensitive Polymer Micelles
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摘要: 探究了pH敏感型聚合物胶束递送和释放I型志贺毒素A亚基至肿瘤细胞Hela的效率和功能。首先将在大肠杆菌中分别重组表达I型志贺毒素A亚基Stx1A和减毒突变A亚基Mu-Stx1A,然后将对pH敏感的聚合物胶束PEG8-PDPA100-PEG8(其中PEG为聚乙二醇,PDPA为聚异丙基甲基烯酸酯)分别运载至宫颈癌细胞Hela中。体外活性实验证明重组蛋白Stx1A具有明显的抑制蛋白合成作用,而减毒变异型Mu-Stx1A不具备这种作用。用聚合物胶束将Stx1A及Mu-Stx1A转运至Hela细胞中,发现随着蛋白浓度的增加细胞转染效率增大。包载了Stx1A的胶束进入细胞后,释放活性Stx1A导致细胞病变和凋亡,该现象随着Stx1A浓度的增加表现更显著。实验证明聚合物胶束可以成功包载、运输和稳定释放具有活性的Stx1A分子至肿瘤细胞内,发挥毒性功能诱导细胞程序性死亡。该聚合物胶束可以在蛋白类药物运输中发挥有效作用,为后续I型志贺毒素A亚基在肿瘤治疗中的应用性研究提供重要的理论基础。
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关键词:
- I型志贺毒素 /
- pH敏感型聚合物胶束 /
- 蛋白类药物 /
- 胞内递送 /
- 肿瘤治疗
Abstract: This paper aims to explore the efficiency and function of pH sensitive polymer micelles for targeted delivery and release of Shiga toxin 1 subunit A to HeLa cells. Recombinantly expressed the Shiga toxin 1 A subunit (Stx1A) and the attenuated variant A subunit (Mu-Stx1A) in Escherichia coli, and used pH-sensitive polymer micelles formed by self-assembly of PEG8-PDPA100-PEG8 to deliver them to the cervical cancer cells Hela. In vitro activity test showed that the recombinant protein Stx1A could significantly inhibit protein synthesis, but the attenuated mutant Mu-Stx1A could not. Stx1A and Mu-Stx1A were successfully transported into HeLa cells by the polymeric micelles, and the transfection efficiency increased with the protein concentration. After Stx1A micelles entered the cells, it successfully released the active subunit Stx1A, leading to cytopathy and apoptosis, which became more evident with the concentration of Stx1A. Experiments have proved that the polymer micelles can successfully encapsulate, transport and stably release Stx1A molecules into tumor cells, exerting toxic functions to induce programmed cell death. This work indicates that polymer micelles can play an effective role in protein transport, which provides a theoretical basis for the subsequent research and application of Shiga toxin 1 subunit A in tumor therapy.-
Key words:
- Shiga toxin type I /
- pH sensitive polymer micelles /
- peptide drugs /
- targeted delivery /
- tumor therapy
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表 1 胶束及蛋白的性质和参数
Table 1. Properties and parameters of micelle and free protein
Samples Diameter/nm PDI ζ/mV Unloaded micelle 173.4±1.8 0.04±0.011 −8.8±1.2 Free-Stx1A 44.47±8.1 0.558±0.051 −11.0±1.8 Free-Mu-Stx1A 65.92±0.6 0.608±0.060 −12.4±2.0 Stx1A micelle 189.0±2.0 0.172±0.013 −12.2±0.7 Mu-Stx1A micelle 208.5±2.2 0.223±0.012 −14.9±2.4 -
[1] WEERAKKODY L R, WITHARANA C. The role of bacterial toxins and spores in cancer therapy[J]. Life Sciences, 2019, 235: 116839. doi: 10.1016/j.lfs.2019.116839 [2] MELLAERT V L, BARBE S, ANNE J. Clostridium spores as anti-tumour agents[J]. Trends in Microbiology, 2006, 14(4): 190-196. doi: 10.1016/j.tim.2006.02.002 [3] BAINDARA P, MANDAL S M. Bacteria and bacterial anticancer agents as a promising alternative for cancer therapeutics[J]. Biochimie, 2020, 177(20): 164-189. [4] RYAN R M, GREEN J, LEWIS C E. Use of bacteria in anti-cancer therapies[J]. Bioessays, 2006, 28(1): 84-94. doi: 10.1002/bies.20336 [5] JOHANNES L, ROMER W. Shiga toxins-from cell biology to biomedical applications[J]. Nature Reviews Microbiology, 2010, 8(2): 105-116. doi: 10.1038/nrmicro2279 [6] MELTON-CELSA A R. Shiga toxin (Stx) classification, structure, and function[J]. Microbiology Spectrum, 2014, 2(2): 24-45. [7] NG Y S, CHEN T B. Shiga toxins: From structure and mechanism to applications[J]. Apply Microbiology Biotechnology, 2016, 100(3): 1597-1610. [8] CHERLA R P, LEE S Y, TESH V L. Shiga toxins and apoptosis[J]. FEMS Microbiology Letters, 2003, 228(2): 159-166. doi: 10.1016/S0378-1097(03)00761-4 [9] WILLYSSON A, STAHL A L, GILLET D, et al. Shiga toxin uptake and sequestration in extracellular vesicles is mediated by its B-subunit[J]. Toxins (Basel), 2020, 12(7): 449-464. doi: 10.3390/toxins12070449 [10] VERNON L, JENNIFER A B, JENNIE W O, et al. Comparison of the relative toxicities of Shiga-like toxins type I and type II for mice[J]. Infection and Immunity, 1993, 61(8): 3392-3402. doi: 10.1128/iai.61.8.3392-3402.1993 [11] RUSSO L M, MELTON-CELSA A R, SMITH M J, et al. Comparisons of native Shiga toxins (Stxs) type 1 and 2 with chimeric toxins indicate that the source of the binding subunit dictates degree of toxicity[J]. Plos One, 2014, 9(3): e93463. doi: 10.1371/journal.pone.0093463 [12] HUGHES A K, STRICKLETT P K, KOHAN D E. Cytotoxic effect of Shiga toxin-1 on human proximal tubule cells[J]. Kidney International, 1998, 54(2): 426-437. doi: 10.1046/j.1523-1755.1998.00015.x [13] LIU X, WU F, JI Y, et al. Recent advances in anti-cancer protein/peptide delivery[J]. Bioconjug Chemistry, 2019, 30(2): 305-324. doi: 10.1021/acs.bioconjchem.8b00750 [14] LEE Y W, LUTHER D C, KRETZMANN J A, et al. Protein delivery into the cell cytosol using non-viral nanocarriers[J]. Theranostics, 2019, 9(11): 3280-3292. doi: 10.7150/thno.34412 [15] FANG J, NAKAMURA H, MAEDA H. The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect[J]. Advanced Drug Delivery Reviews, 2011, 63(3): 136-151. doi: 10.1016/j.addr.2010.04.009 [16] LIU Y, WANG W, YANG J, et al. pH-Sensitive polymeric micelles triggered drug release for extracellular and intracellular drug targeting delivery[J]. Asian Journal of Pharmaceutical Sciences, 2013, 8(3): 159-167. doi: 10.1016/j.ajps.2013.07.021 [17] KAUR S, PRASAD C, BALARISHNAN B, et al. Trigger responsive polymeric nanocarriers for cancer therapy[J]. Biomaterials Science, 2015, 3(7): 955-987. doi: 10.1039/C5BM00002E [18] SHEN Y, ZHANG J, HAO W, et al. Copolymer micelles function as pH-responsive nanocarriers to enhance the cytotoxicity of a HER2 aptamer in HER2-positive breast cancer cells[J]. International Journal of Nanomedicine, 2018, 13: 537-553. doi: 10.2147/IJN.S149942 [19] HAO W J, LIU D Y, SHANG Y Z, et al. pH-Triggered copolymer micelles as drug nanocarriers for intracellular delivery[J]. Royal Society of Chemistry Advances, 2016, 6(35): 29149-29158. doi: 10.1039/C6RA00673F [20] CAROLYN J, HOVDE S B C, JOHN J, et al. Evidence that glutamic acid 167 is an active-site residue of Shiga toxin[J]. Proceedings of the National Academy of Sciences, 1988, 85: 2568-2572. doi: 10.1073/pnas.85.8.2568 [21] EISENHABER F, ARGOS P. Hydrophobic regions on protein surfaces: Definition based on hydration shell structure and a quick method for their computation[J]. Protein Engine, 1996, 9(12): 1121-1133. doi: 10.1093/protein/9.12.1121 [22] RAY M, LEE Y W, SCALETTI F, et al. Intracellular delivery of proteins by nanocarriers[J]. Nanomedicine (Lond), 2017, 12(8): 941-952. doi: 10.2217/nnm-2016-0393 [23] VARKOUHI A K, SCHOLTE M, STORM G, et al. Endosomal escape pathways for delivery of biologicals[J]. Journal of Controlled Release, 2011, 151(3): 220-228. doi: 10.1016/j.jconrel.2010.11.004 -