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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

Effect of citrate on the nanocomplex size, charge and characterization of the nanocomplex. Graph showing the hydrodynamic sizes (A) and surface charge (B) of PEI: citrate nanocomplexes formed using different ratio of PEI to citrate measured using zetasizer. C. Titration of different concentration of aptamer and siRNA was carried out and loaded onto 2% agarose gel with ethidium bromide and checked for the retention of the PEI complex on the wells. Lane 3 shows 200nM of aptamer and 200nM of siRNA is required to saturate 0.3μgs of PEI and the next highest concentration of 300nM of aptamer & siRNA respectively had some amount of free siRNA and aptamer (lane 4). Lane 5 & 6 indicates free aptamer and siRNA indicated with red and black arrow respectively. On the right, histogram plot showing Particle size distribution of the PEI-Apt-siRNA nanocomplex. D. Histogram overlay plot showing the percent number distribution of the PEI nanocore alone and PEI-nanocomplex with aptamer and siRNA (hydrodynamic diameter in nm) measured using zetasizer. E. Graph showing the total counts of representative zeta-potential (mV) of the PEI nanocore and the PEI-Apt-siEp nanocomplex. F. TEM images of the PEI nanocomplex left panel showing the uniformity of particle distribution and histogram showing distinct particles with a spherical shape (G). H. Graph showing the percentage cell proliferation upon treating with different concentration from 0.1 to 3 μg/mL of PEI on MCF-7 and WERI-Rb1 cell line till 48 h. Inhibitory effect of PEI on the cell proliferation and mitrochondrial activity was assessed by MTT assay.
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Fig2: Effect of citrate on the nanocomplex size, charge and characterization of the nanocomplex. Graph showing the hydrodynamic sizes (A) and surface charge (B) of PEI: citrate nanocomplexes formed using different ratio of PEI to citrate measured using zetasizer. C. Titration of different concentration of aptamer and siRNA was carried out and loaded onto 2% agarose gel with ethidium bromide and checked for the retention of the PEI complex on the wells. Lane 3 shows 200nM of aptamer and 200nM of siRNA is required to saturate 0.3μgs of PEI and the next highest concentration of 300nM of aptamer & siRNA respectively had some amount of free siRNA and aptamer (lane 4). Lane 5 & 6 indicates free aptamer and siRNA indicated with red and black arrow respectively. On the right, histogram plot showing Particle size distribution of the PEI-Apt-siRNA nanocomplex. D. Histogram overlay plot showing the percent number distribution of the PEI nanocore alone and PEI-nanocomplex with aptamer and siRNA (hydrodynamic diameter in nm) measured using zetasizer. E. Graph showing the total counts of representative zeta-potential (mV) of the PEI nanocore and the PEI-Apt-siEp nanocomplex. F. TEM images of the PEI nanocomplex left panel showing the uniformity of particle distribution and histogram showing distinct particles with a spherical shape (G). H. Graph showing the percentage cell proliferation upon treating with different concentration from 0.1 to 3 μg/mL of PEI on MCF-7 and WERI-Rb1 cell line till 48 h. Inhibitory effect of PEI on the cell proliferation and mitrochondrial activity was assessed by MTT assay.

Mentions: We have synthesized PEI nanocore and PEI-Apt-SiEp nanocomplex as shown in schematic representation (Figure 1). The illustration describes the process of PEI nanocomplex synthesis followed by siRNA and aptamer addition. We tested the hypothesis that the nanocomplex when added to cells, would specifically bind to the EpCAM receptor on the membrane, and would release the aptamers, and siRNA into the cytoplasm, leading to the silencing of EpCAM mRNA. PEI nanocore was prepared by stabilizing its charge using sodium citrate to form an optimal core in aqueous solution. The size of the PEI nanocore depended on the ratios of citrate to PEI i.e., carboxyl group charge/amine group charge ratio (R). The PEI: citrate nanocore synthesized with different R ratios showed particles sizes ranging from 75–250 nm and zeta-potential ranging from 34 mV to 48 mV (positively charged). The ratio 1:1.5 of PEI: citrate resulted in optimum size of 156 ± 6.8 nm and zeta potential of 34.6 mV (Figure 2A & B). The nanocomplex formation was mediated by stabilization of the positively-charged PEI by negatively charged sodium citrate, and further by the electrostatic interaction between the nanocore, the siRNA and the aptamer. The PEI-Apt-siRNA nanocomplexes were synthesized in aqueous media with varying amounts of the aptamer and siRNA. To the PEI nanocore, siRNA was added first followed by aptamer. The aptamer was added finally to form the complex as it enables the complex to recognize the EpCAM on the cell surface. The addition of the aptamer and the siRNA together would lead to a lesser occupancy of aptamer on the particle surface thereby leading to lower binding efficiency of the particles; hence the stepwise complex formation was maintained. The synthesized complexes exhibited retardation on an agarose gel (Figure 2C). We observed that 200 nM of aptamer and siRNA, were able to saturate the PEI: citrate nanocore beyond which free aptamer and siRNA were present, in addition to that the complex retained in the well (Figure 2C, lane 4). Therefore, 200 nM of EpApt and 200 nM SiEp complexed with PEI-citrate (PEI-EpApt-SiEp) nanocomplex was used for further studies. This nanocomplex exhibited a hydrodynamic diameter of 198 ± 14.2 nm and zeta potential of −30.0 mV. The percent number distribution of the sizes of PEI nanocore alone and nanocomplex are shown in Figure 2D. Figure 2E shows the zeta potential of the PEI nanocore alone and nanocomplex, respectively. There was a complete shift in the surface charge(34.6mV) due to the aptamer and siRNA addition leading to negative surface charge(−30.0). The TEM analysis exhibited a particle size of 151 ± 11 nm. The TEM analysis of both PEI and PEI-Apt-siRNA nanocomplexes showed spherical particles (Figure 2F). The frequency of sizes of the particles as observed by TEM is shown as histogram (Figure 2G).Figure 1


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)

Effect of citrate on the nanocomplex size, charge and characterization of the nanocomplex. Graph showing the hydrodynamic sizes (A) and surface charge (B) of PEI: citrate nanocomplexes formed using different ratio of PEI to citrate measured using zetasizer. C. Titration of different concentration of aptamer and siRNA was carried out and loaded onto 2% agarose gel with ethidium bromide and checked for the retention of the PEI complex on the wells. Lane 3 shows 200nM of aptamer and 200nM of siRNA is required to saturate 0.3μgs of PEI and the next highest concentration of 300nM of aptamer & siRNA respectively had some amount of free siRNA and aptamer (lane 4). Lane 5 & 6 indicates free aptamer and siRNA indicated with red and black arrow respectively. On the right, histogram plot showing Particle size distribution of the PEI-Apt-siRNA nanocomplex. D. Histogram overlay plot showing the percent number distribution of the PEI nanocore alone and PEI-nanocomplex with aptamer and siRNA (hydrodynamic diameter in nm) measured using zetasizer. E. Graph showing the total counts of representative zeta-potential (mV) of the PEI nanocore and the PEI-Apt-siEp nanocomplex. F. TEM images of the PEI nanocomplex left panel showing the uniformity of particle distribution and histogram showing distinct particles with a spherical shape (G). H. Graph showing the percentage cell proliferation upon treating with different concentration from 0.1 to 3 μg/mL of PEI on MCF-7 and WERI-Rb1 cell line till 48 h. Inhibitory effect of PEI on the cell proliferation and mitrochondrial activity was assessed by MTT assay.
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Related In: Results  -  Collection

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Fig2: Effect of citrate on the nanocomplex size, charge and characterization of the nanocomplex. Graph showing the hydrodynamic sizes (A) and surface charge (B) of PEI: citrate nanocomplexes formed using different ratio of PEI to citrate measured using zetasizer. C. Titration of different concentration of aptamer and siRNA was carried out and loaded onto 2% agarose gel with ethidium bromide and checked for the retention of the PEI complex on the wells. Lane 3 shows 200nM of aptamer and 200nM of siRNA is required to saturate 0.3μgs of PEI and the next highest concentration of 300nM of aptamer & siRNA respectively had some amount of free siRNA and aptamer (lane 4). Lane 5 & 6 indicates free aptamer and siRNA indicated with red and black arrow respectively. On the right, histogram plot showing Particle size distribution of the PEI-Apt-siRNA nanocomplex. D. Histogram overlay plot showing the percent number distribution of the PEI nanocore alone and PEI-nanocomplex with aptamer and siRNA (hydrodynamic diameter in nm) measured using zetasizer. E. Graph showing the total counts of representative zeta-potential (mV) of the PEI nanocore and the PEI-Apt-siEp nanocomplex. F. TEM images of the PEI nanocomplex left panel showing the uniformity of particle distribution and histogram showing distinct particles with a spherical shape (G). H. Graph showing the percentage cell proliferation upon treating with different concentration from 0.1 to 3 μg/mL of PEI on MCF-7 and WERI-Rb1 cell line till 48 h. Inhibitory effect of PEI on the cell proliferation and mitrochondrial activity was assessed by MTT assay.
Mentions: We have synthesized PEI nanocore and PEI-Apt-SiEp nanocomplex as shown in schematic representation (Figure 1). The illustration describes the process of PEI nanocomplex synthesis followed by siRNA and aptamer addition. We tested the hypothesis that the nanocomplex when added to cells, would specifically bind to the EpCAM receptor on the membrane, and would release the aptamers, and siRNA into the cytoplasm, leading to the silencing of EpCAM mRNA. PEI nanocore was prepared by stabilizing its charge using sodium citrate to form an optimal core in aqueous solution. The size of the PEI nanocore depended on the ratios of citrate to PEI i.e., carboxyl group charge/amine group charge ratio (R). The PEI: citrate nanocore synthesized with different R ratios showed particles sizes ranging from 75–250 nm and zeta-potential ranging from 34 mV to 48 mV (positively charged). The ratio 1:1.5 of PEI: citrate resulted in optimum size of 156 ± 6.8 nm and zeta potential of 34.6 mV (Figure 2A & B). The nanocomplex formation was mediated by stabilization of the positively-charged PEI by negatively charged sodium citrate, and further by the electrostatic interaction between the nanocore, the siRNA and the aptamer. The PEI-Apt-siRNA nanocomplexes were synthesized in aqueous media with varying amounts of the aptamer and siRNA. To the PEI nanocore, siRNA was added first followed by aptamer. The aptamer was added finally to form the complex as it enables the complex to recognize the EpCAM on the cell surface. The addition of the aptamer and the siRNA together would lead to a lesser occupancy of aptamer on the particle surface thereby leading to lower binding efficiency of the particles; hence the stepwise complex formation was maintained. The synthesized complexes exhibited retardation on an agarose gel (Figure 2C). We observed that 200 nM of aptamer and siRNA, were able to saturate the PEI: citrate nanocore beyond which free aptamer and siRNA were present, in addition to that the complex retained in the well (Figure 2C, lane 4). Therefore, 200 nM of EpApt and 200 nM SiEp complexed with PEI-citrate (PEI-EpApt-SiEp) nanocomplex was used for further studies. This nanocomplex exhibited a hydrodynamic diameter of 198 ± 14.2 nm and zeta potential of −30.0 mV. The percent number distribution of the sizes of PEI nanocore alone and nanocomplex are shown in Figure 2D. Figure 2E shows the zeta potential of the PEI nanocore alone and nanocomplex, respectively. There was a complete shift in the surface charge(34.6mV) due to the aptamer and siRNA addition leading to negative surface charge(−30.0). The TEM analysis exhibited a particle size of 151 ± 11 nm. The TEM analysis of both PEI and PEI-Apt-siRNA nanocomplexes showed spherical particles (Figure 2F). The frequency of sizes of the particles as observed by TEM is shown as histogram (Figure 2G).Figure 1

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