Development of Human Hepatocellular Carcinoma Cell-Targeted Protein Cages

Abstract

We described herein a human hepatocellular carcinoma (HCC) cell-targeted protein cage for which the HCC-binding peptide termed SP94 was modified at the surface of a naturally occurred heat shock protein (Hsp) cage. Six types of HCC-targeted Hsp cages were chemically synthesized using two types of heterobifunctional linker (SM(PEG)(n)) with different lengths and two types of SP94 peptide, which contained a unique Cys residue at the N- or C-terminus of the peptide. These Hsp cages were characterized using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) analyses, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, and dynamic light scattering (DLS) measurement. Fluorescence microscopic observations revealed that all the engineered protein cages bind selectively to HCC cells but not to the other cell lines tested (including normal liver cell). Moreover, the number of SP94 peptides on Hsp cages, conjugation site of SP94 peptide, and linker length between a Hsp cage and a SP94 peptide had important effects upon the binding of engineered Hsp cages to HCC cells. An engineered Hsp cage conjugated to the N-terminus of SP94 peptide via a longer linker molecule and containing high SP94 peptide levels showed greater binding toward HCC cells. Surprisingly, through optimization of these three factors, up to 10-fold greater affinity toward HCC cells was achieved. These results are critically important not only for the development of HCC cell-targeting devices using SP94 peptide, but also to create other cell-targeting materials that utilize other peptide ligands.

Biocompatible and biodegradable fibrinogen microspheres for tumor-targeted doxorubicin delivery

“…Similarly, the published result of Toita et al explained that more flexible longer linkers could make the binding of drug carriers to cells easier. 30 Therefore, the feasibility of the DOX-linker-Fbg microsphere-based drug carrier system was confirmed by the cell experiments.…”

Ligand-based targeted therapy: a novel strategy for hepatocellular carcinoma

“…Studies that they implemented on mice bearing HCC xenografts confirmed the targeting efficiency of SP94 and demonstrated the superior targeting ability of SP94 over the phage clone PC94 and the enhanced therapeutic effect of SP94-LD compared with control peptide conjugated LD (CP-LD) and LD. Toita et al 188 formulated HspG41C-SM(PEG)n-SP94 peptide conjugates by modifying a naturally occurred heat shock protein cage (HspG41C cage) with SP94 through the heterobifunctional linker (SM(PEG)n). The cellular binding investigation demonstrated the binding selectivity of this conjugate to human HCC-derived cell lines, while suggesting that the binding efficiency could be influenced by the amount of SP94 peptides on Hsp cages, conjugation site of SP94 peptide, and linker length between a Hsp cage and a SP94 peptide.…”

Biological evaluation of protein nanocapsules containing doxorubicin

“…Also, for in vivo application, specific drug delivery to target cells was very important in order to avoid any undesirable side effects. An advantage of Hsp cages is that the peptide ligand can be modified by the chemical/genetic method, 22,23 enabling the delivery of cytotoxic reagents to specific cells. It was previously reported that Hsp cages could specifically target human hepatoma cells after binding human hepatoma binding peptides (SP94 peptides) to the surface of the Hsp cages using poly(ethylene glycol) linkers.…”

“…The resulting HspSP94 cage was specifically taken up by human hepatoma cells but not by normal liver cells. 22 Accordingly, an attempt will be made to modify the peptide to Hsp cages and changing the linker between Hsp and DOX to a more acid-responsive linker, which will enhance the usefulness of Hsp cages in a drug delivery system.…”

“…Hsp cages have also been used for biomedical applications due to their advantageous biocompatibility, biodegradability, and easy fabrication through chemical/genetic strategies. [22][23][24][25] Although Hsp cages have several favorable characteristics making them suitable as drug carriers, to the best of the authors' knowledge, only a few reports have examined this possibility. 11 In this study, an Hsp cage-based prodrug was developed.…”