Mofed Dina, Wahba Mohamed A, Salem Tamer Z
Molecular Biology and Virology Lab, Biomedical Sciences Program, UST, Zewail City of Science and Technology, October Gardens, 6th of October City, Giza 12578, Egypt.
Department of Microbial Genetics, Agricultural Genetic Engineering Research Institute (AGERl), ARC, Giza 12619, Egypt.
Curr Issues Mol Biol. 2022 Aug 20;44(8):3746-3756. doi: 10.3390/cimb44080256.
Targeted cancer therapy is a challenging area that includes multiple chemical and biological vehicles. Virus-like particles (VLPs) combine safety and efficacy in their roles as potential vaccines and drug delivery vehicles. In this study, we propose a novel drug delivery system based on HCV-LPs engineered with SP94 and RGD peptides mediated by a specific molecular chaperone (Grp78) associated with cancer drug resistance. The PCR primers were designed for engineering two constructs, SP94-EGFP-CORE-HIS and RGD-EGFP-CORE-HIS, by sequential PCR reactions. The two fragments were cloned into pFastBac Dual under the polyhedrin promoter and then used to produce two recombinant baculoviruses (AcSP94 and AcRGD). The VLP's expression was optimized by recombinant virus infection with different MOIs, ranging from 1 to 20 MOI. Recombinant VLP2 were purified by Ni-NTA and their sizes and shapes were confirmed with TEM. They were incubated with different types of cells prior to examination using the fluorescence microscope to test the binding specificity. The effect of the overexpression of the Grp78 on the binding affinity of the engineered VLPs was tested in HepG2 and HeLa cells. The protocol optimization revealed that MOI 10 produced the highest fluorescence intensities after 72 h for the two recombinant proteins (SP94-core and RGD-core). Moreover, the binding assay tested on different types of mammalian cells (HeLa, HEK-293T, and HepG2 cells) showed green fluorescence on the periphery of all tested cell lines when using the RGD-core protein; while, the SP94-core protein showed green fluorescence only with the liver cancer cells, HepG2 and HuH7. Overexpression of Grp78 in HepG2 and HeLa cells enhanced the binding efficiency of the engineered VLPs. We confirmed that the SP94 peptide can be specifically used to target liver cancer cells, while the RGD peptide is sufficiently functional for most types of cancer cells. The overexpression of the Grp78 improved the binding capacity of both SP94 and RGD peptides. It is worth noting that the SP94 peptide can function properly as a recombinant peptide, and not only as a chemically conjugated peptide, as heretofore commonly used.
靶向癌症治疗是一个具有挑战性的领域，其中包括多种化学和生物载体。病毒样颗粒（VLP）作为潜在的疫苗和药物递送载体，兼具安全性和有效性。在本研究中，我们提出了一种基于丙型肝炎病毒样颗粒（HCV-LP）的新型药物递送系统，该颗粒经与癌症耐药相关的特定分子伴侣（Grp78）介导，用SP94和RGD肽进行工程改造。通过连续PCR反应设计PCR引物，用于构建两种构建体，即SP94-EGFP-CORE-HIS和RGD-EGFP-CORE-HIS。将这两个片段克隆到多角体蛋白启动子下的pFastBac Dual中，然后用于产生两种重组杆状病毒（AcSP94和AcRGD）。通过用1至20 MOI范围内的不同感染复数（MOI）进行重组病毒感染来优化VLP的表达。重组VLP2通过镍-亚氨基三乙酸（Ni-NTA）进行纯化，并用透射电子显微镜（TEM）确认其大小和形状。在使用荧光显微镜检查之前，将它们与不同类型的细胞孵育，以测试结合特异性。在肝癌细胞系HepG2和人宫颈癌细胞系HeLa中测试Grp78过表达对工程化VLP结合亲和力的影响。方案优化显示，对于两种重组蛋白（SP94核心和RGD核心），感染复数为10时在72小时后产生最高的荧光强度。此外，在不同类型的哺乳动物细胞（HeLa、人胚肾细胞系HEK-293T和HepG2细胞）上进行的结合试验表明，当使用RGD核心蛋白时，所有测试细胞系的周边均显示绿色荧光；而SP94核心蛋白仅在肝癌细胞HepG2和HuH7中显示绿色荧光。在HepG2和HeLa细胞中Grp78的过表达提高了工程化VLP的结合效率。我们证实，SP94肽可特异性用于靶向肝癌细胞，而RGD肽对大多数类型的癌细胞具有充分的功能。Grp78的过表达提高了SP94和RGD肽的结合能力。值得注意的是，SP94肽可以作为重组肽正常发挥作用，而不仅像迄今为止常用的那样作为化学偶联肽发挥作用。
