Development of a Recombinant Multifunctional Biomacromolecule for Targeted Gene Transfer to Prostate Cancer Cells

Abstract

The objective of this study was to genetically engineer a fully functional single chain fusion peptide composed of motifs from diverse biological and synthetic origins that can perform multiple tasks including DNA condensation, cell targeting, cell transfection, particle shielding from immune system and effective gene transfer to prostate tumors. To achieve the objective, a single chain biomacromolecule (vector) consisted of four repeatative units of histone H2A peptide, fusogenic peptide GALA, short elastin-like peptide, and PC-3 cell targeting peptide was designed. To examine the functionality of each motif in the vector sequence, it was characterized in terms of size and zeta potential by Zetasizer, PC-3 cell targeting and transfection by flowcytometry, IgG induction by immunogenicity assay, and PC-3 tumor transfection by quantitative live animal imaging. Overall, the results of this study showed the possibility of using genetic engineering techniques to program various functionalities into one single chain vector and create a multifunctional nonimmunogenic biomacromolecule for targeted gene transfer to prostate cancer cells. This proof-of-concept study is a significant step forward toward creating a library of vectors for targeted gene transfer to any cancer cell type at both in vitro and in vivo levels.

Figure 1

SDS–PAGE analysis of the purified fusion peptides. The theoretical molecular weights of H4G, TpH4G, TpE_SH4G and TpE_EH4G are 19,752 Da, 23,109 Da, 24,352 Da and 24,562 Da, respectively.

Figure 2

The analysis of particle size and charge for TpH4G (A), TpE_EH4G (B), TpE_SH4G (C) and H4G (D) in complex with pEGFP at various N:P ratios.

Figure 3

Qualitative (fluorescent microscopy) and quantitative (flow cytometry) demonstration of the targeted gene transfer to PC-3 cells with TpH4G/pEGFP complexes (N:P 10) and non-targeted gene transfer by H4G/pEGFP (N:P 10).

Figure 4

Evaluation of the transfection efficiency of TpH4G, TpE_EH4G and TpE_SH4G vectors in complexation with pEGFP at N:P ratios of 8, 10, and 12. A) Flow cytometry histograms displaying the percentage of GFP positive cells. Each display is an overlay of three independent histograms. B) Fluorescent microscopy images of the transfected PC3-3 cells. C) A bar chart that summarizes the percent transfection and total green fluorescent protein expression in each group. The results are presented as mean ± s.d (n = 3).

Figure 5

WST-1 cell toxicity assay. A) Percentage of viable PC-3 cells after transfection with TpH4G, TpE_EH4G and TpE_SH4G vectors in complexation with 1μg pEGFP. B) Evaluation of cell killing efficiency of TpE_SH4G vector in complexation with plasmid encoding yCD:UPRT and in combination with various concentrations of 5-FC. * indicates statistical significance.

Figure 6

Evaluation of IgG response against the vector/pDNA nanocomplexes. A) The dosing schedule that was used to immunize mice and induce IgG response. B) Measurement of fold increase in IgG response against vector/pCpGfree nanocomplexes and control groups. Statistical significance is shown by * (p<0.05).

Figure 7

Measurement of luciferase expression in transfected PC-3 tumors. A-C) Bioluminescence intensity of PC-3 tumors transfected with H4G/pLuc, TpH4G/pLuc and TpE_SH4G/pLuc nanoparticles. D) Quantitative analysis of gene expression in transfected tumors. The gene expression in tumors transfected with H4G was not quantified as they did not show any detectable bioluminescence. The results are shown as mean ± s.d. (n=6). * indicates statistical significance.