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. 2022 Mar 8;9(3):112.
doi: 10.3390/bioengineering9030112.

Magnetic Nanoparticles as a Component of Peptide-Based DNA Delivery System for Suicide Gene Therapy of Uterine Leiomyoma

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Magnetic Nanoparticles as a Component of Peptide-Based DNA Delivery System for Suicide Gene Therapy of Uterine Leiomyoma

Sofia Shtykalova et al. Bioengineering (Basel). .

Abstract

Suicidegene therapy is considered a promising approach for the treatment of uterine leiomyoma (UL), a benign tumor in women characterized by precise localization. In this study, we investigate the efficiency of αvβ3 integrin-targeted arginine-rich peptide carrier R6p-cRGD electrostatically bound to magnetic nanoparticles (MNPs) for targeted DNA delivery into the UL cells. The physico-chemical and cytotoxic properties, transfection efficiency, and specificity of R6p-cRGD/DNA/MNPs polyplexes were evaluated. The addition of MNPs resulted in a decrease in the time needed for successful transfection with simultaneous increase in efficiency. We revealed a therapeutic effect on primary UL cells after delivery of plasmid encoding the herpes simplex virus type 1 (HSV-1) thymidine kinase gene. Treatment with ganciclovir resulted in 20% efficiency of suicide gene therapy in UL cells transfected with the pPTK-1 plasmid. Based on these results, we conclude that the use of cationic peptide carriers with MNPs can be promising for the development of modular non-viral carriers for suicide gene delivery to UL cells.

Keywords: DNA delivery; gene therapy; integrins; magnetic nanoparticles; peptide-based carriers; thymidine kinase; uterine leiomyoma.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Ethidium bromide (EtBr) exclusion from R6p-cRGD/DNA polyplexes with an increasein MNPs content. Values are the mean ± S.D. of triplicate experiments.
Figure 2
Figure 2
Efficiency of DNA protection from nuclease degradation within R6p-cRGD/DNA/MNPs polyplexes after DNase I enzyme digestion. The polyplexes containing 0.5 µg of DNA were treated with 0.2 units of DNase I for 30 min at 37 °C. C− is DNA treated with DNase I; C+ is intact DNA. (a) R6p-cRGD/DNA N/P ratio is of 8/1; (b) R6p-cRGD/DNA N/P ratio is of 12/1.
Figure 3
Figure 3
DNA release after (a)DTT treatment of R6p-cRGD/DNA/MNPs polyplexes; (b) relaxation of R6p-cRGD/DNA/MNPs polyplexes after 24h of DS treatment in three-fold charge excess. Values are the mean ± SD of the mean of triplicates.
Figure 4
Figure 4
Typical micrographs of the R6p-cRGD/DNA/MNPs polyplexes obtained by transmission electron microscopy: (a,b) N/P ratio of R6p-cRGD/DNA is of 8/1; (c,d) N/P ratio is of 12/1.
Figure 5
Figure 5
Survival rate of PANC-1 cells (%) after transfection with R6p-cRGD/DNA/MNPs polyplexes. DNA:MNP quantity ratio is 1:0.05. The threshold indicates the acceptable level of cell survival (80%).
Figure 6
Figure 6
Activity of β-galactosidase in PANC-1 cells after transfection with pCMV-lacZ polyplexes with MNPs. The results are presented as mean and S.E.M. Cells transfected with R6p-cRGD/DNA/MNPs were compared to unpacked DNA; statistical significance was assessed by ordinary one-way ANOVA with Sidak’s multiple comparisons test (n = 9; ** 0.01, **** 0.0001).
Figure 7
Figure 7
Activity of β-galactosidase after transfection of PANC-1 cells with nucleopeptide polyplexes with MNPs (a) in the presence and absence of a magnet; (b) with and without the addition of a free ligand c(RGDfK). Statistical significance was assessed by the Kruskal–Wallis test with Dunn’s multiple comparisons test (n = 6; * 0.05, ** 0.01).
Figure 8
Figure 8
Relative number of UL cells (%) (a) accessed by Alamar Blue test and amount of living UL cells (b)accessed by Trypan blue staining after suicidal gene therapy by transfection with pPTK-1-bearing polyplexes with MNPs and subsequent incubation with ganciclovir. Statistical significance was determined using One-way ANOVA with Sidak’s multiple comparisons test (n = 9, * 0.05, **** 0.0001).
Figure 9
Figure 9
Typical micrographs in bright field conducted 96 h after GCV treatment. The UL cells were transfected with R6p-cRGD/pCMV-lacZ/MNPs polyplexes at N/P ratios of (a) 8/1, (c) 12/1; and with pPTK1/R6p-cRGD/MNPs polyplexes at (b) 8/1, (d) 12/1 charge ratio. Control wells contained (e) GCV-treated cells.

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