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Engineered targeting tLyp-1 exosomes as gene therapy vectors for efficient delivery of siRNA into lung cancer cells.

ABSTRACT: Natural exosomes can express specific proteins and carbohydrate molecules on the surface and hence have demonstrated the great potentials for gene therapy of cancer. However, the use of natural exosomes is restricted by their low transfection efficiency. Here, we report a novel targeting tLyp-1 exosome by gene recombinant engineering for delivery of siRNA to cancer and cancer stem cells. To reach such a purpose, the engineered tLyp-1-lamp2b plasmids were constructed and amplified in Escherichia coli. The tLyp-1-lamp2b plasmids were further used to transfect HEK293T tool cells and the targeting tLyp-1 exosomes were isolated from secretion of the transfected HEK293T cells. Afterwards, the artificially synthesized siRNA was encapsulated into targeting tLyp-1 exosomes by electroporation technology. Finally, the targeting siRNA tLyp-1 exosomes were used to transfect cancer or cancer stem cells. Results showed that the engineered targeting tLyp-1 exosomes had a nanosized structure (approximately 100?nm) and high transfection efficiency into lung cancer and cancer stem cells. The function verifications demonstrated that the targeting siRNA tLyp-1 exosomes were able to knock-down the target gene of cancer cells and to reduce the stemness of cancer stem cells. In conclusion, the targeting tLyp-1 exosomes are successfully engineered, and can be used for gene therapy with a high transfection efficiency. Therefore, the engineered targeting tLyp-1 exosomes offer a promising gene delivery platform for future cancer therapy.

SUBMITTER: Bai J

PROVIDER: S-EPMC7486479 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Engineered targeting tLyp-1 exosomes as gene therapy vectors for efficient delivery of siRNA into lung cancer cells.

Bai Jing J Duan Jialun J Liu Rui R Du Yafei Y Luo Qian Q Cui Yinuo Y Su Zhanbo Z Xu Jiarui J Xie Ying Y Lu Wanliang W

Asian journal of pharmaceutical sciences 20190506 4

Natural exosomes can express specific proteins and carbohydrate molecules on the surface and hence have demonstrated the great potentials for gene therapy of cancer. However, the use of natural exosomes is restricted by their low transfection efficiency. Here, we report a novel targeting tLyp-1 exosome by gene recombinant engineering for delivery of siRNA to cancer and cancer stem cells. To reach such a purpose, the engineered tLyp-1-lamp2b plasmids were constructed and amplified in Escherichia ...[more]

PMID: 32952669

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Project description:Rationale: Developing an effective nanoplatform to realize 'multi-in-one' is essential to broaden the therapeutic potential of combination therapy. Exosomes are ideal candidates since their intrinsic abilities of integrating multiple contents and functions. However, only limited efforts have been devoted to engineering exosomes to integrate the needed properties, also considering the safety and yield, for tumor-targeted and efficient gene/chemo combination therapy. Methods: Herein, by manipulating the exosome membrane, blood exosomes with high abundance and safety are engineered as a versatile combinatorial delivery system, where the doxorubicin (Dox) and cholesterol-modified miRNA21 inhibitor (miR-21i) are co-embedded into the lipid bilayer of exosomes, and the magnetic molecules and endosomolytic peptides L17E are bind to the exosome membrane through ligand-receptor coupling and electrostatic interactions, respectively. Results: It is proved that such engineering strategy not only preserves their intrinsic features, but also readily integrates multiple properties of tumor targeting, efficient transfection and gene/chemo combination therapy into blood exosomes. The lipid bilayer structure of exosomes allows them to co-load Dox and miR-21i with high-payloads. Moreover, profiting from the integration of magnetic molecules and L17E peptides, the engineered exosomes exhibit an enhanced tumor accumulation and an improved endosome escape ability, thereby specifically and efficiently delivering encapsulated cargos to tumor cells. As a result, a remarkable inhibition of tumor growth is observed in the tumor-bearing mice, and without noticeable side effects. Conclusions: This study demonstrates the potential of engineered blood exosomes as feasible co-delivery nanosystem for tumor-targeted and efficient combination therapy. Further development by replacing the drugs combined regimens can potentially make this engineered exosome become a general platform for the design of safe and effective combination therapy modality.

Project description:Stimuli-responsive polycations have been developed for improved nucleic acid transfection and enhanced therapeutic efficacy. The most reported mechanisms for controlled release of siRNA are based on polyelectrolyte exchange reactions in the cytoplasm and the degradation of polycations initiated by specific triggers. However, the degradation strategy has not always been sufficient due to unsatisfactory kinetics and binding of cationic fragments to siRNA, which limits the gene silencing effect. In this study, a new strategy that combines degradation and charge reversal is proposed. Methods: We prepared a polycation (CrossPPA) by crosslinking of phenylboronic acid (PBA)-grafted 1.8k PEI with alginate. It was compared with 25k PEI, 1.8k PEI and 1.8k PEI-PBA on siRNA encapsulation, ATP-responsive behavior and mechanism, cytotoxicity, cell uptake, siRNA transfection, in vivo biodistribution and in vivo anti-tumor efficacy. The in vitro and in vivo experiments were performed on 4T1 murine breast cancer cells and 4T1 tumor model separately. Results: The crosslinking strategy obviously improve the siRNA loading ability of 1.8k PEI. We validated that intracellular levels of ATP could trigger CrossPPA disassembly and charge reversal, which resulted in efficient and rapid siRNA release due to electrostatic repulsion. Besides, CrossPPA/siRNA showed strong cell uptake in 4T1 cells compared with 1.8k PEI/siRNA. Notably, the cytotoxicity of CrossPPA was pretty low, which was owing to its biodegradability. Furthermore, the crosslinked polyplexes significantly enhanced siRNA transfection and improved tumor accumulation. The high gene silencing ability of CrossPPA polyplex led to strong anti-tumor efficacy when using Bcl2-targeted siRNA. Conclusion: These results indicated that the ATP-triggered disassembly and charge reversal strategy provided a new way for developing stimuli-responsive siRNA carriers and showed potential for nucleic acid delivery in the treatment of cancer.

Dataset Information

Engineered targeting tLyp-1 exosomes as gene therapy vectors for efficient delivery of siRNA into lung cancer cells.

Publications

Engineered targeting tLyp-1 exosomes as gene therapy vectors for efficient delivery of siRNA into lung cancer cells.

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