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EpCAM aptamer mediated cancer cell specific delivery of EpCAM siRNA using polymeric nanocomplex.

Subramanian N, Kanwar JR, Athalya PK, Janakiraman N, Khetan V, Kanwar RK, Eluchuri S, Krishnakumar S - J. Biomed. Sci. (2015)

Bottom Line: Gel retardation assay confirmed the PEI-EpApt-SiEp nanoparticles formation.PEI-EpApt-SiEp downregulated EpCAM and inhibited selectively the cell proliferation of MCF-7 and WERI-Rb1 cells.The PEI nanocomplex fabricated with EpApt and siEp was able to target EpCAM tumor cells, deliver the siRNA and silence the target gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Nanobiotechnology, Vision Research Foundation, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, 18 College Road, Chennai, 600006, Tamil Nadu, India. nithyasnithya@gmail.com.

ABSTRACT

Background: Epithelial cell adhesion molecule (EpCAM) is overexpressed in solid tumors and regarded as a putative cancer stem cell marker. Here, we report that employing EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) dual approach, for the targeted delivery of siRNA to EpCAM positive cancer cells, efficiently inhibits cancer cell proliferation.

Results: Targeted delivery of siRNA using polyethyleneimine is one of the efficient methods for gene delivery, and thus, we developed a novel aptamer-PEI-siRNA nanocomplex for EpCAM targeting. PEI nanocomplex synthesized with EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) showed 198 nm diameter sized particles by dynamic light scattering, spherical shaped particles, of 151 ± 11 nm size by TEM. The surface charge of the nanoparticles was -30.0 mV using zeta potential measurements. Gel retardation assay confirmed the PEI-EpApt-SiEp nanoparticles formation. The difference in size observed by DLS and TEM could be due to coating of aptamer and siRNA on PEI nanocore. Flow cytometry analysis revealed that PEI-EpApt-SiEp has superior binding to cancer cells compared to EpApt or scramble aptamer (ScrApt) or PEI-ScrApt-SiEp. PEI-EpApt-SiEp downregulated EpCAM and inhibited selectively the cell proliferation of MCF-7 and WERI-Rb1 cells.

Conclusions: The PEI nanocomplex fabricated with EpApt and siEp was able to target EpCAM tumor cells, deliver the siRNA and silence the target gene. This nanocomplex exhibited decreased cell proliferation than the scrambled aptamer loaded nanocomplex in the EpCAM expressing cancer cells and may have potential for EpCAM targeting in vivo.

No MeSH data available.


Related in: MedlinePlus

Cell Uptake of the PEI nanocomplex by MCF-7 and WERI-Rb1 cells. The fabricated PEI complexes and free aptamer were added to MCF-7 cells (upper panel A-E), WERI-Rb1 cells (lower panel A-E) and incubated for their uptake at 37°C for 4 h followed DAPI counterstaining and microscopic evaluation. Images were taken at 40× using AxioObserver fluorescent microscope. Legend on the top of phase image represents the aptamer or nanocomplex added to the respective panels.
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Fig4: Cell Uptake of the PEI nanocomplex by MCF-7 and WERI-Rb1 cells. The fabricated PEI complexes and free aptamer were added to MCF-7 cells (upper panel A-E), WERI-Rb1 cells (lower panel A-E) and incubated for their uptake at 37°C for 4 h followed DAPI counterstaining and microscopic evaluation. Images were taken at 40× using AxioObserver fluorescent microscope. Legend on the top of phase image represents the aptamer or nanocomplex added to the respective panels.

Mentions: The cell binding and uptake of the PEI-EpApt-SiEp complex were studied in MCF-7 and WERI-Rb1 cell lines. Initially, we studied the expression of EpCAM in retinoblastoma and the data generated by us in WERI-Rb1 cell lines is represented in Additional file 2: Figure S1A, [8]. Similarly the expression of EpCAM in MCF-7 has also been studied earlier [2], and the binding of EpCAM aptamer to breast cancer cells, MCF-7 cell line is published [5]. The expression levels of EpCAM proteins in MCF-7 cells are higher compared to the WERI-Rb1 cells (Figure 3A). Similar to the expression levels of the protein, the aptamer binding was higher in MCF-7(Figure 3B). The uptake of aptamer and PEI-Apt-SiEp nanocomplexes was monitored using flow cytometry and the cells bound to PEI-EpApt-SiEp had increased fluorescent intensity compared to the cells bound with EpApt alone in both MCF-7 and WERI-Rb1 cell lines (Figure 3C and D). The ScrApt or PEI-ScrApt-SiEp did not show any binding onto the cell lines. The blocking of the cell surface EpCAM protein by the EpCAM antibody had decreased the binding of EpCAM aptamer alone or PEI-EpApt-SiEp (Figure 3E & F). The cellular uptake of the aptamer alone or the aptamer nanocomplex in MCF-7 and WERI-Rb1 cells was visualized using fluorescent microscopy. The PEI nanocomplex on MCF-7 and WERI-Rb1 cells showed intense membrane staining compared to the EpApt alone (Figure 4). The PEI-EpApt-siRNA nanocomplex exhibited greater binding than EpApt alone (Figure 4B and D). There was no binding when ScrApt or ScrApt-nanocomplex was used in both the cell lines (Figure 4C, E upper panel and 4C, E lower panel). Thus, the specificity of the EpCAM aptamer towards the target is in agreement with the above data.Figure 3


EpCAM aptamer mediated cancer cell specific delivery of EpCAM siRNA using polymeric nanocomplex.

Subramanian N, Kanwar JR, Athalya PK, Janakiraman N, Khetan V, Kanwar RK, Eluchuri S, Krishnakumar S - J. Biomed. Sci. (2015)

Cell Uptake of the PEI nanocomplex by MCF-7 and WERI-Rb1 cells. The fabricated PEI complexes and free aptamer were added to MCF-7 cells (upper panel A-E), WERI-Rb1 cells (lower panel A-E) and incubated for their uptake at 37°C for 4 h followed DAPI counterstaining and microscopic evaluation. Images were taken at 40× using AxioObserver fluorescent microscope. Legend on the top of phase image represents the aptamer or nanocomplex added to the respective panels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4307906&req=5

Fig4: Cell Uptake of the PEI nanocomplex by MCF-7 and WERI-Rb1 cells. The fabricated PEI complexes and free aptamer were added to MCF-7 cells (upper panel A-E), WERI-Rb1 cells (lower panel A-E) and incubated for their uptake at 37°C for 4 h followed DAPI counterstaining and microscopic evaluation. Images were taken at 40× using AxioObserver fluorescent microscope. Legend on the top of phase image represents the aptamer or nanocomplex added to the respective panels.
Mentions: The cell binding and uptake of the PEI-EpApt-SiEp complex were studied in MCF-7 and WERI-Rb1 cell lines. Initially, we studied the expression of EpCAM in retinoblastoma and the data generated by us in WERI-Rb1 cell lines is represented in Additional file 2: Figure S1A, [8]. Similarly the expression of EpCAM in MCF-7 has also been studied earlier [2], and the binding of EpCAM aptamer to breast cancer cells, MCF-7 cell line is published [5]. The expression levels of EpCAM proteins in MCF-7 cells are higher compared to the WERI-Rb1 cells (Figure 3A). Similar to the expression levels of the protein, the aptamer binding was higher in MCF-7(Figure 3B). The uptake of aptamer and PEI-Apt-SiEp nanocomplexes was monitored using flow cytometry and the cells bound to PEI-EpApt-SiEp had increased fluorescent intensity compared to the cells bound with EpApt alone in both MCF-7 and WERI-Rb1 cell lines (Figure 3C and D). The ScrApt or PEI-ScrApt-SiEp did not show any binding onto the cell lines. The blocking of the cell surface EpCAM protein by the EpCAM antibody had decreased the binding of EpCAM aptamer alone or PEI-EpApt-SiEp (Figure 3E & F). The cellular uptake of the aptamer alone or the aptamer nanocomplex in MCF-7 and WERI-Rb1 cells was visualized using fluorescent microscopy. The PEI nanocomplex on MCF-7 and WERI-Rb1 cells showed intense membrane staining compared to the EpApt alone (Figure 4). The PEI-EpApt-siRNA nanocomplex exhibited greater binding than EpApt alone (Figure 4B and D). There was no binding when ScrApt or ScrApt-nanocomplex was used in both the cell lines (Figure 4C, E upper panel and 4C, E lower panel). Thus, the specificity of the EpCAM aptamer towards the target is in agreement with the above data.Figure 3

Bottom Line: Gel retardation assay confirmed the PEI-EpApt-SiEp nanoparticles formation.PEI-EpApt-SiEp downregulated EpCAM and inhibited selectively the cell proliferation of MCF-7 and WERI-Rb1 cells.The PEI nanocomplex fabricated with EpApt and siEp was able to target EpCAM tumor cells, deliver the siRNA and silence the target gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Nanobiotechnology, Vision Research Foundation, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, 18 College Road, Chennai, 600006, Tamil Nadu, India. nithyasnithya@gmail.com.

ABSTRACT

Background: Epithelial cell adhesion molecule (EpCAM) is overexpressed in solid tumors and regarded as a putative cancer stem cell marker. Here, we report that employing EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) dual approach, for the targeted delivery of siRNA to EpCAM positive cancer cells, efficiently inhibits cancer cell proliferation.

Results: Targeted delivery of siRNA using polyethyleneimine is one of the efficient methods for gene delivery, and thus, we developed a novel aptamer-PEI-siRNA nanocomplex for EpCAM targeting. PEI nanocomplex synthesized with EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) showed 198 nm diameter sized particles by dynamic light scattering, spherical shaped particles, of 151 ± 11 nm size by TEM. The surface charge of the nanoparticles was -30.0 mV using zeta potential measurements. Gel retardation assay confirmed the PEI-EpApt-SiEp nanoparticles formation. The difference in size observed by DLS and TEM could be due to coating of aptamer and siRNA on PEI nanocore. Flow cytometry analysis revealed that PEI-EpApt-SiEp has superior binding to cancer cells compared to EpApt or scramble aptamer (ScrApt) or PEI-ScrApt-SiEp. PEI-EpApt-SiEp downregulated EpCAM and inhibited selectively the cell proliferation of MCF-7 and WERI-Rb1 cells.

Conclusions: The PEI nanocomplex fabricated with EpApt and siEp was able to target EpCAM tumor cells, deliver the siRNA and silence the target gene. This nanocomplex exhibited decreased cell proliferation than the scrambled aptamer loaded nanocomplex in the EpCAM expressing cancer cells and may have potential for EpCAM targeting in vivo.

No MeSH data available.


Related in: MedlinePlus