Background: The combination of the unique properties of cancer cells makes it possible to find specific ligands that interact directly with the tumor, and to conduct targeted tumor therapy. Phage display is one of the most common methods for searching for specific ligands. Bacteriophages display peptides, and the peptides themselves can be used as targeting molecules for the delivery of diagnostic and therapeutic agents. Phage display can be performed both in vitro and in vivo. Moreover, it is possible to carry out the phage display on cells pre-enriched for a certain tumor marker, for example, CD44 and CD133. Methods: For this work we used several methods, such as phage display, sequencing, cell sorting, immunocytochemistry, phage titration. Results: We performed phage display using different screening systems (in vitro and in vivo), different phage libraries (Ph.D-7, Ph.D-12, Ph.D-C7C) on CD44+/CD133+ and without enrichment U-87 MG cells. The binding efficiency of bacteriophages displayed tumor-targeting peptides on U-87 MG cells was compared in vitro. We also conducted a comparative analysis in vivo of the specificity of the accumulation of selected bacteriophages in the tumor and in the control organs (liver, brain, kidney and lungs). Conclusions: The screening in vivo of linear phage peptide libraries for glioblastoma was the most effective strategy for obtaining tumor-targeting peptides providing targeted delivery of diagnostic and therapeutic agents to glioblastoma.
The binding efficiency of tumor-targeting phage particles, which exhibited tumor-addressing peptides and were obtained by phage display, to the human glioblastma cell line U-87 MG and health brain cells DKM- 5 has been evaluated by flow cytometry and enzyme-linked immunosorbent assay (ELISA). The specific binding of the selected bacteriophages to U-87 MG cells was shown by fluorescence microscopy. Based on the obtained data, tumor-targeting phage particles were selected that provide the most efficient binding to the U-87 MG cells in vitro for further studies in order to create targeted antitumor compounds.
phage display, tumor-addressing peptides, glioblastoma, flow cytometry, ELISA
The authors are grateful to S. I. Baiborodin, Head of the Microscopic Analysis of Biological Objects Core Facilities Center, SB RAS, for assistance in conducting confocal microscopy. This study was supported by the Russian Science Fund (grant no. 19-44-02006).
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