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Prenyl Ammonium Salts--New Carriers for Gene Delivery: A B16-F10 Mouse Melanoma Model.

Grecka E, Statkiewicz M, Gorska A, Biernacka M, Grygorowicz MA, Masnyk M, Chmielewski M, Gawarecka K, Chojnacki T, Swiezewska E, Malecki M - PLoS ONE (2016)

Bottom Line: AP-15/DOPE complexes were also efficient to introduce pDNA to cells, without much effect on cell viability.Furthermore, complexes containing AP-15 and therapeutic plasmid, encoding the TIMP metallopeptidase inhibitor 2 (TIMP2), introduced the TIMP2 gene with high efficiency to B16-F10 melanoma cells but not to B16-F10 melanoma tumors in C57BL/6 mice, as confirmed by TIMP2 protein level determination.Obtained results indicate that APs have a potential as non-viral vectors for cell transfection.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Translational Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland.

ABSTRACT

Purpose: Prenyl ammonium iodides (Amino-Prenols, APs), semi-synthetic polyprenol derivatives were studied as prospective novel gene transfer agents.

Methods: AP-7, -8, -11 and -15 (aminoprenols composed of 7, 8, 11 or 15 isoprene units, respectively) were examined for their capacity to form complexes with pDNA, for cytotoxicity and ability to transfect genes to cells.

Results: All the carriers were able to complex DNA. The highest, comparable to commercial reagents, transfection efficiency was observed for AP-15. Simultaneously, AP-15 exhibited the lowest negative impact on cell viability and proliferation--considerably lower than that of commercial agents. AP-15/DOPE complexes were also efficient to introduce pDNA to cells, without much effect on cell viability. Transfection with AP-15/DOPE complexes influenced the expression of a very few among 44 tested genes involved in cellular lipid metabolism. Furthermore, complexes containing AP-15 and therapeutic plasmid, encoding the TIMP metallopeptidase inhibitor 2 (TIMP2), introduced the TIMP2 gene with high efficiency to B16-F10 melanoma cells but not to B16-F10 melanoma tumors in C57BL/6 mice, as confirmed by TIMP2 protein level determination.

Conclusion: Obtained results indicate that APs have a potential as non-viral vectors for cell transfection.

No MeSH data available.


Related in: MedlinePlus

Evaluation of transfection efficiency of B16-F10 cells transfected with use of carrier:pDNA complexes.(a) Percentage of GFP-positive cells transfected with complexes: AP:pGFP at different N/P ratios (r = 1.7 for AP-7, AP-8, AP-11, r = 2.0 for AP-15) and AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP lipoplexes containing 2.5 μg of lipids/μg of pGFP, analysed by FACS; Ap- significant difference from AP-15 treatment, At- significant difference from Attractene treatment, L- significant difference from Lipofectamine treatment, P- significant difference from PEI treatment, (b) Activity of β-galactosidase in cells transfected with complexes: AP-11:pLacZ, AP-15:pLacZ, PEI:pLacZ, at r = 2.0–2.5 N/P ratio, studied by β-Gal test; *P<0.05, **P<0.005. Transfections were performed using 4 μg of respective pDNA.
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pone.0153633.g003: Evaluation of transfection efficiency of B16-F10 cells transfected with use of carrier:pDNA complexes.(a) Percentage of GFP-positive cells transfected with complexes: AP:pGFP at different N/P ratios (r = 1.7 for AP-7, AP-8, AP-11, r = 2.0 for AP-15) and AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP lipoplexes containing 2.5 μg of lipids/μg of pGFP, analysed by FACS; Ap- significant difference from AP-15 treatment, At- significant difference from Attractene treatment, L- significant difference from Lipofectamine treatment, P- significant difference from PEI treatment, (b) Activity of β-galactosidase in cells transfected with complexes: AP-11:pLacZ, AP-15:pLacZ, PEI:pLacZ, at r = 2.0–2.5 N/P ratio, studied by β-Gal test; *P<0.05, **P<0.005. Transfections were performed using 4 μg of respective pDNA.

Mentions: The effect of AP on efficacy of transfection was assessed using a pDNA bearing an GFP encoding gene (Fig 3a), in this experiment 4 μg of pGFP was used per each transfection. Before subjecting to flow cytometry analysis, the cells transfected with the formulations containing optimal amounts of pDNA and respective APs in the most appropriate N/P ratio (what resulted in introducing pDNA to the cells with the highest efficiency) were selected using microscopic observations. Flow cytometry analysis of transfected B16-F10 cells confirmed high transfection efficiency for complexes containing AP-8 and AP-15. The highest numbers of GFP-positive cells—58% and 61% of viable GFP expressing cells were noticed for complexes with AP-8 (r = 1.7) and AP-15 (r = 2.0), respectively. These numbers were higher than those recorded for commercial agents—Attractene (52% of viable GFP-positive cells), PEI (55% of viable GFP-positive cells), however lower than estimated for Lipofectamine3000 (86% of viable GFP-positive cells). The complexes containing AP-7 (r = 1.7) and AP-11 (r = 1.7) transfected the cells with lower efficiency (approx. 13% and 30% of viable GFP-positive cells, respectively) (Fig 3a).


Prenyl Ammonium Salts--New Carriers for Gene Delivery: A B16-F10 Mouse Melanoma Model.

Grecka E, Statkiewicz M, Gorska A, Biernacka M, Grygorowicz MA, Masnyk M, Chmielewski M, Gawarecka K, Chojnacki T, Swiezewska E, Malecki M - PLoS ONE (2016)

Evaluation of transfection efficiency of B16-F10 cells transfected with use of carrier:pDNA complexes.(a) Percentage of GFP-positive cells transfected with complexes: AP:pGFP at different N/P ratios (r = 1.7 for AP-7, AP-8, AP-11, r = 2.0 for AP-15) and AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP lipoplexes containing 2.5 μg of lipids/μg of pGFP, analysed by FACS; Ap- significant difference from AP-15 treatment, At- significant difference from Attractene treatment, L- significant difference from Lipofectamine treatment, P- significant difference from PEI treatment, (b) Activity of β-galactosidase in cells transfected with complexes: AP-11:pLacZ, AP-15:pLacZ, PEI:pLacZ, at r = 2.0–2.5 N/P ratio, studied by β-Gal test; *P<0.05, **P<0.005. Transfections were performed using 4 μg of respective pDNA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835110&req=5

pone.0153633.g003: Evaluation of transfection efficiency of B16-F10 cells transfected with use of carrier:pDNA complexes.(a) Percentage of GFP-positive cells transfected with complexes: AP:pGFP at different N/P ratios (r = 1.7 for AP-7, AP-8, AP-11, r = 2.0 for AP-15) and AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP lipoplexes containing 2.5 μg of lipids/μg of pGFP, analysed by FACS; Ap- significant difference from AP-15 treatment, At- significant difference from Attractene treatment, L- significant difference from Lipofectamine treatment, P- significant difference from PEI treatment, (b) Activity of β-galactosidase in cells transfected with complexes: AP-11:pLacZ, AP-15:pLacZ, PEI:pLacZ, at r = 2.0–2.5 N/P ratio, studied by β-Gal test; *P<0.05, **P<0.005. Transfections were performed using 4 μg of respective pDNA.
Mentions: The effect of AP on efficacy of transfection was assessed using a pDNA bearing an GFP encoding gene (Fig 3a), in this experiment 4 μg of pGFP was used per each transfection. Before subjecting to flow cytometry analysis, the cells transfected with the formulations containing optimal amounts of pDNA and respective APs in the most appropriate N/P ratio (what resulted in introducing pDNA to the cells with the highest efficiency) were selected using microscopic observations. Flow cytometry analysis of transfected B16-F10 cells confirmed high transfection efficiency for complexes containing AP-8 and AP-15. The highest numbers of GFP-positive cells—58% and 61% of viable GFP expressing cells were noticed for complexes with AP-8 (r = 1.7) and AP-15 (r = 2.0), respectively. These numbers were higher than those recorded for commercial agents—Attractene (52% of viable GFP-positive cells), PEI (55% of viable GFP-positive cells), however lower than estimated for Lipofectamine3000 (86% of viable GFP-positive cells). The complexes containing AP-7 (r = 1.7) and AP-11 (r = 1.7) transfected the cells with lower efficiency (approx. 13% and 30% of viable GFP-positive cells, respectively) (Fig 3a).

Bottom Line: AP-15/DOPE complexes were also efficient to introduce pDNA to cells, without much effect on cell viability.Furthermore, complexes containing AP-15 and therapeutic plasmid, encoding the TIMP metallopeptidase inhibitor 2 (TIMP2), introduced the TIMP2 gene with high efficiency to B16-F10 melanoma cells but not to B16-F10 melanoma tumors in C57BL/6 mice, as confirmed by TIMP2 protein level determination.Obtained results indicate that APs have a potential as non-viral vectors for cell transfection.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Translational Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland.

ABSTRACT

Purpose: Prenyl ammonium iodides (Amino-Prenols, APs), semi-synthetic polyprenol derivatives were studied as prospective novel gene transfer agents.

Methods: AP-7, -8, -11 and -15 (aminoprenols composed of 7, 8, 11 or 15 isoprene units, respectively) were examined for their capacity to form complexes with pDNA, for cytotoxicity and ability to transfect genes to cells.

Results: All the carriers were able to complex DNA. The highest, comparable to commercial reagents, transfection efficiency was observed for AP-15. Simultaneously, AP-15 exhibited the lowest negative impact on cell viability and proliferation--considerably lower than that of commercial agents. AP-15/DOPE complexes were also efficient to introduce pDNA to cells, without much effect on cell viability. Transfection with AP-15/DOPE complexes influenced the expression of a very few among 44 tested genes involved in cellular lipid metabolism. Furthermore, complexes containing AP-15 and therapeutic plasmid, encoding the TIMP metallopeptidase inhibitor 2 (TIMP2), introduced the TIMP2 gene with high efficiency to B16-F10 melanoma cells but not to B16-F10 melanoma tumors in C57BL/6 mice, as confirmed by TIMP2 protein level determination.

Conclusion: Obtained results indicate that APs have a potential as non-viral vectors for cell transfection.

No MeSH data available.


Related in: MedlinePlus