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Physicochemical and biological characterization of chitosan-microRNA nanocomplexes for gene delivery to MCF-7 breast cancer cells.

Santos-Carballal B, Aaldering LJ, Ritzefeld M, Pereira S, Sewald N, Moerschbacher BM, Götte M, Goycoolea FM - Sci Rep (2015)

Bottom Line: Surface plasmon resonance spectroscopy shows that complexes formed by low degree of acetylation chitosans are highly stable, regardless of the molecular weight.We found no evidence that these complexes were cytotoxic towards MCF-7 cells.Furthermore, CS-miRNA nanocomplexes with degree of acetylation 12% and 29% were biologically active, showing successful downregulation of target mRNA expression in MCF-7 cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Biology and Biotechnology (IBBP), University of Münster, Schlossgarten 3, D-48149 Münster, Germany.

ABSTRACT
Cancer gene therapy requires the design of non-viral vectors that carry genetic material and selectively deliver it with minimal toxicity. Non-viral vectors based on cationic natural polymers can form electrostatic complexes with negatively-charged polynucleotides such as microRNAs (miRNAs). Here we investigated the physicochemical/biophysical properties of chitosan-hsa-miRNA-145 (CS-miRNA) nanocomplexes and the biological responses of MCF-7 breast cancer cells cultured in vitro. Self-assembled CS-miRNA nanocomplexes were produced with a range of (+/-) charge ratios (from 0.6 to 8) using chitosans with various degrees of acetylation and molecular weight. The Z-average particle diameter of the complexes was <200 nm. The surface charge increased with increasing amount of chitosan. We observed that chitosan induces the base-stacking of miRNA in a concentration dependent manner. Surface plasmon resonance spectroscopy shows that complexes formed by low degree of acetylation chitosans are highly stable, regardless of the molecular weight. We found no evidence that these complexes were cytotoxic towards MCF-7 cells. Furthermore, CS-miRNA nanocomplexes with degree of acetylation 12% and 29% were biologically active, showing successful downregulation of target mRNA expression in MCF-7 cells. Our data, therefore, shows that CS-miRNA complexes offer a promising non-viral platform for breast cancer gene therapy.

No MeSH data available.


Related in: MedlinePlus

Uptake of CS HDP-12–miRNA complexes into MCF-7 cells observed by confocal laser scanning microscopy.Horizontal axis: optical sections at increasing height (z-values) Vertical axis: incubation times: 0, 5, 24 and 48 h. Red fluorescent staining = CellMask Deep Red membrane staining, Green fluorescent staining = CS HDP-12–miRNA complexes labelled with 6-FAM-hsa-miR-5p.
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f7: Uptake of CS HDP-12–miRNA complexes into MCF-7 cells observed by confocal laser scanning microscopy.Horizontal axis: optical sections at increasing height (z-values) Vertical axis: incubation times: 0, 5, 24 and 48 h. Red fluorescent staining = CellMask Deep Red membrane staining, Green fluorescent staining = CS HDP-12–miRNA complexes labelled with 6-FAM-hsa-miR-5p.

Mentions: CLSM was used to investigate the uptake of CS HDP-12-miRNA complexes with a (+/−) charge ratio of 1.5 into MCF-7 breast cancer cells. The cell membranes were stained with CellMask Deep Red and the CS–miRNA complexes were formed with fluorescence-labelled miRNA-145 (Fig. 7).


Physicochemical and biological characterization of chitosan-microRNA nanocomplexes for gene delivery to MCF-7 breast cancer cells.

Santos-Carballal B, Aaldering LJ, Ritzefeld M, Pereira S, Sewald N, Moerschbacher BM, Götte M, Goycoolea FM - Sci Rep (2015)

Uptake of CS HDP-12–miRNA complexes into MCF-7 cells observed by confocal laser scanning microscopy.Horizontal axis: optical sections at increasing height (z-values) Vertical axis: incubation times: 0, 5, 24 and 48 h. Red fluorescent staining = CellMask Deep Red membrane staining, Green fluorescent staining = CS HDP-12–miRNA complexes labelled with 6-FAM-hsa-miR-5p.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555168&req=5

f7: Uptake of CS HDP-12–miRNA complexes into MCF-7 cells observed by confocal laser scanning microscopy.Horizontal axis: optical sections at increasing height (z-values) Vertical axis: incubation times: 0, 5, 24 and 48 h. Red fluorescent staining = CellMask Deep Red membrane staining, Green fluorescent staining = CS HDP-12–miRNA complexes labelled with 6-FAM-hsa-miR-5p.
Mentions: CLSM was used to investigate the uptake of CS HDP-12-miRNA complexes with a (+/−) charge ratio of 1.5 into MCF-7 breast cancer cells. The cell membranes were stained with CellMask Deep Red and the CS–miRNA complexes were formed with fluorescence-labelled miRNA-145 (Fig. 7).

Bottom Line: Surface plasmon resonance spectroscopy shows that complexes formed by low degree of acetylation chitosans are highly stable, regardless of the molecular weight.We found no evidence that these complexes were cytotoxic towards MCF-7 cells.Furthermore, CS-miRNA nanocomplexes with degree of acetylation 12% and 29% were biologically active, showing successful downregulation of target mRNA expression in MCF-7 cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Biology and Biotechnology (IBBP), University of Münster, Schlossgarten 3, D-48149 Münster, Germany.

ABSTRACT
Cancer gene therapy requires the design of non-viral vectors that carry genetic material and selectively deliver it with minimal toxicity. Non-viral vectors based on cationic natural polymers can form electrostatic complexes with negatively-charged polynucleotides such as microRNAs (miRNAs). Here we investigated the physicochemical/biophysical properties of chitosan-hsa-miRNA-145 (CS-miRNA) nanocomplexes and the biological responses of MCF-7 breast cancer cells cultured in vitro. Self-assembled CS-miRNA nanocomplexes were produced with a range of (+/-) charge ratios (from 0.6 to 8) using chitosans with various degrees of acetylation and molecular weight. The Z-average particle diameter of the complexes was <200 nm. The surface charge increased with increasing amount of chitosan. We observed that chitosan induces the base-stacking of miRNA in a concentration dependent manner. Surface plasmon resonance spectroscopy shows that complexes formed by low degree of acetylation chitosans are highly stable, regardless of the molecular weight. We found no evidence that these complexes were cytotoxic towards MCF-7 cells. Furthermore, CS-miRNA nanocomplexes with degree of acetylation 12% and 29% were biologically active, showing successful downregulation of target mRNA expression in MCF-7 cells. Our data, therefore, shows that CS-miRNA complexes offer a promising non-viral platform for breast cancer gene therapy.

No MeSH data available.


Related in: MedlinePlus