Polycondensed Peptide Carriers Modified with Cyclic RGD Ligand for Targeted Suicide Gene Delivery to Uterine Fibroid Cells

Abstract

Suicide gene therapy was suggested as a possible strategy for the treatment of uterine fibroids (UFs), which are the most common benign tumors inwomen of reproductive age. For successful suicide gene therapy, DNAtherapeutics should be specifically delivered to UF cells. Peptide carriers are promising non-viral gene delivery systems that can be easily modified with ligands and other biomolecules to overcome DNA transfer barriers. Here we designed polycondensed peptide carriers modified with a cyclic RGD moiety for targeted DNA delivery to UF cells. Molecular weights of the resultant polymers were determined, and inclusion of the ligand was confirmed by MALDI-TOF. The physicochemical properties of the polyplexes, as well as cellular DNA transport, toxicity, and transfection efficiency were studied, and the specificity of αvβ3 integrin-expressing cell transfection was proved. The modification with the ligand resulted in a three-fold increase of transfection efficiency. Modeling of the suicide gene therapy by transferring the HSV-TK suicide gene to primary cells obtained from myomatous nodes of uterine leiomyoma patients was carried out. We observed up to a 2.3-fold decrease in proliferative activity after ganciclovir treatment of the transfected cells. Pro- and anti-apoptotic gene expression analysis confirmed our findings that the developed polyplexes stimulate UF cell death in a suicide-specific manner.

Keywords: DNA delivery; gene therapy; integrins; peptide-based carriers; polycondensation; thymidine kinase; uterine fibroids.

Figure 1

EtBr displacement assay of DNA complexes with R6p and R6p-cRGD carriers. Values are the mean ± SD of the mean of triplicates.

Figure 2

DNase I protection assay of DNAcomplexes formed with R6p (a) and R6p-cRGD (b) carriers. Charge ratio (CR) in bold indicates full DNA protection. C– = ‘naked’ plasmid DNA treated with DNase I; C+ = untreated plasmid DNA.

Figure 3

DNA release after DTT treatment of DNAcomplexes with R6p and R6p-cRGD carriers formed at anN/P ratio 8/1. Values are the mean ± SEM of the mean of triplicates. ** p < 0.01 compared to untreated complexes.

Figure 4

Relaxation of DNAcomplexes with R6p and R6p-cRGD carriers (N/P ratio 8/1) after 24 h of DS treatment in three-fold charge excess. Values are the mean ± SEM of the mean of triplicates. ** p < 0.01 compared to untreated complexes.

Figure 5

Cytotoxicity after transfection of DNA complexes formed with RGD1-R6, RGD0-R6, and R6 carriers at charge ratios 4/1, 8/1, and 12/1. Values are the mean ± SD of the mean of triplicates. *** p < 0.001 compared to intact cells.

Figure 6

Normalized fluorescence intensity of PANC-1 and 293T cells after uptake of R6p/DNA and R6p-cRGD/DNA complexes at 8/1 charge ratios labeled with YOYO-1. ** p < 0.01, *** p < 0.001, when compared with R6p/DNA polyplexes.

Figure 7

Transfection efficacy evaluation in the PANC-1 cells: (a) the cells were transfected with R6p/DNA and R6p-cRGD/DNA complexes at charge ratios of 4/1, 8/1, and 12/1, ‘naked’ plasmid pCMV-lacZ alone, and PEI/DNA complexes; (b) the cells were transfected with complexes of the pEXPR-IBA5-eGFP plasmid with R6p and R6p-cRGD carriers at a charge ratio of 8/1 withPEI/DNA polyplexes as a control; (c) the cells were transfected in the presence of the free cyclo(RGDfK) ligand with R6p/DNA and R6p-cRGD/DNA complexes at a charge ratio of 8/1, with thepCMV-lacZ plasmid alone and PEI/DNA polyplexes used as controls. lacZ gene expression was calculated as milliunits (mU) of beta-galactosidase per milligram of total cell extract protein. GFP gene expression was presented as a percentage of GFP-positive cells determined by flow cytometry assay. Values are the mean ± SD of the mean of triplicates. ** p < 0.01, *** p < 0.001, when compared with R6p/DNA polyplexes (a,b) and after free cyclo(RGDfK) ligand addition in transfection medium (c).

Figure 8

UF cell viability (a) and the number of living UF cells (b) after HSV thymidine kinase expression and GCV treatment as determined by Alamar Blue assay (a) and Trypan Blue exclusion assay (b). Values are the mean ± SEM of the mean of triplicates. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to pCMV-LacZpolyplexes.

Figure 9

Representative microphotographs in bright field made 96 h after GCV treatment. The UF cells were transfected with R6p-cRGD/pCMV-lacZ polyplexes at N/Pratios of 8/1 (a,b) and 12/1 (c) with 0.7 µg (a) and 0.35 µg (b,c) of DNA per well; with R6p-cRGD/pPTK1 complexes at 8/1 (d,e) and 12/1 (f) charge ratios with 0.7 µg (d) and 0.35 µg (e,f) of DNA per well. Control wells contained GCV-treated intact cells (g) and untreated intact ones (h).

Figure 9

Representative microphotographs in bright field made 96 h after GCV treatment. The UF cells were transfected with R6p-cRGD/pCMV-lacZ polyplexes at N/Pratios of 8/1 (a,b) and 12/1 (c) with 0.7 µg (a) and 0.35 µg (b,c) of DNA per well; with R6p-cRGD/pPTK1 complexes at 8/1 (d,e) and 12/1 (f) charge ratios with 0.7 µg (d) and 0.35 µg (e,f) of DNA per well. Control wells contained GCV-treated intact cells (g) and untreated intact ones (h).

Figure 10

Relative amount of apoptotic (a) and necrotic (b) UF cells induced by GCV treatment after cell transfection with R6p-cRGD/DNA polyplexes formed with pPTK and pCMV-lacZ plasmids. Values are the mean ± SEM of the mean of four independent experiments. * p < 0.05, ** p < 0.01, compared to pCMV-lacZ-complexes.

Figure 10

Relative amount of apoptotic (a) and necrotic (b) UF cells induced by GCV treatment after cell transfection with R6p-cRGD/DNA polyplexes formed with pPTK and pCMV-lacZ plasmids. Values are the mean ± SEM of the mean of four independent experiments. * p < 0.05, ** p < 0.01, compared to pCMV-lacZ-complexes.

Figure 11

Relative gene expression levels of pro-apoptotic (p53, DAXX, bax) and anti-apoptotic factors in UF cells induced by GCV treatment after cell transfection with R6p-cRGD/DNA polyplexes formed with the pPTK plasmid. Values are the mean ± SEM of the mean of three independent experiments. * p < 0.05, compared to intact cells.