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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

Number of viable cells in samples treated with the various APs / AP-based reagents alone (grey bars: AP-7, AP-8, AP-11, AP-15, AP-15/DOPE, AP-15/DOPE/DMEM) and AP:pGFP / AP-15 and DOPE-containing complexes (patterned bars: AP:pGFP: AP-7, AP-8, AP-11 r = 1.7, AP-15 r = 2.0; AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP 2.5 μg of lipids/μg of pDNA), analysed by FACS.Transfections were performed with the use of 4 μg of pDNA. *P<0.05.
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pone.0153633.g006: Number of viable cells in samples treated with the various APs / AP-based reagents alone (grey bars: AP-7, AP-8, AP-11, AP-15, AP-15/DOPE, AP-15/DOPE/DMEM) and AP:pGFP / AP-15 and DOPE-containing complexes (patterned bars: AP:pGFP: AP-7, AP-8, AP-11 r = 1.7, AP-15 r = 2.0; AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP 2.5 μg of lipids/μg of pDNA), analysed by FACS.Transfections were performed with the use of 4 μg of pDNA. *P<0.05.

Mentions: 7-AAD-staining of non-viable cells allowed their subsequent discrimination with use of flow cytometry from viable cells in the sample. Study with 7-AAD showed a high viability of cells treated with AP:pGFP complexes (71–91%, Fig 4a). However, in the preliminary studies, in the case of cells transfected with AP-7 and AP-8 the viability and the number of cells studied under the microscope was very low. Therefore, in contrast to low-toxic AP-11 and AP-15, during the transfection procedure with AP-7 and AP-8 the media containing the latter APs had to be replaced by fresh standard (APs-free) media. The viability and number of cells transfected with AP-8 complexes containing 2 or 3 μg pGFP was higher than that observed for complexes with 4 μg of pDNA, whereas the transfection efficiency was similar (data not shown). Interestingly, the number of the cells in the samples subjected to transfection with APs, shown as percentage of non-transfected cells, was decreased for cells transfected by complexes with AP-8 and-7 (total cell count 28% and 23% of the control, respectively) (Fig 4b). Such an effect on cell number was not observed when complexes containing AP-15 or AP-11 were used (total cell count was approximately 59% and 46% of the control, respectively; Fig 4b). Effects of cell treatment with free APs alone, apart from AP-7, resulted in smaller decreases of cell viability (decrease in viable cell count by 96%, 58%, 17% and 25% for AP-7, -8, -11 and -15, respectively) than that observed for AP:pGFP complexes (Fig 6). It should be emphasized that AP-15:pGFP complex triggered lower decrease in cell number than Atractene, PEI and Lipofectamine (total cell count 56%, 12% and 24% of the control, respectively) (Fig 4b).


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)

Number of viable cells in samples treated with the various APs / AP-based reagents alone (grey bars: AP-7, AP-8, AP-11, AP-15, AP-15/DOPE, AP-15/DOPE/DMEM) and AP:pGFP / AP-15 and DOPE-containing complexes (patterned bars: AP:pGFP: AP-7, AP-8, AP-11 r = 1.7, AP-15 r = 2.0; AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP 2.5 μg of lipids/μg of pDNA), analysed by FACS.Transfections were performed with the use of 4 μg of pDNA. *P<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153633.g006: Number of viable cells in samples treated with the various APs / AP-based reagents alone (grey bars: AP-7, AP-8, AP-11, AP-15, AP-15/DOPE, AP-15/DOPE/DMEM) and AP:pGFP / AP-15 and DOPE-containing complexes (patterned bars: AP:pGFP: AP-7, AP-8, AP-11 r = 1.7, AP-15 r = 2.0; AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP 2.5 μg of lipids/μg of pDNA), analysed by FACS.Transfections were performed with the use of 4 μg of pDNA. *P<0.05.
Mentions: 7-AAD-staining of non-viable cells allowed their subsequent discrimination with use of flow cytometry from viable cells in the sample. Study with 7-AAD showed a high viability of cells treated with AP:pGFP complexes (71–91%, Fig 4a). However, in the preliminary studies, in the case of cells transfected with AP-7 and AP-8 the viability and the number of cells studied under the microscope was very low. Therefore, in contrast to low-toxic AP-11 and AP-15, during the transfection procedure with AP-7 and AP-8 the media containing the latter APs had to be replaced by fresh standard (APs-free) media. The viability and number of cells transfected with AP-8 complexes containing 2 or 3 μg pGFP was higher than that observed for complexes with 4 μg of pDNA, whereas the transfection efficiency was similar (data not shown). Interestingly, the number of the cells in the samples subjected to transfection with APs, shown as percentage of non-transfected cells, was decreased for cells transfected by complexes with AP-8 and-7 (total cell count 28% and 23% of the control, respectively) (Fig 4b). Such an effect on cell number was not observed when complexes containing AP-15 or AP-11 were used (total cell count was approximately 59% and 46% of the control, respectively; Fig 4b). Effects of cell treatment with free APs alone, apart from AP-7, resulted in smaller decreases of cell viability (decrease in viable cell count by 96%, 58%, 17% and 25% for AP-7, -8, -11 and -15, respectively) than that observed for AP:pGFP complexes (Fig 6). It should be emphasized that AP-15:pGFP complex triggered lower decrease in cell number than Atractene, PEI and Lipofectamine (total cell count 56%, 12% and 24% of the control, respectively) (Fig 4b).

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