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. 2013;8(1):e54182.
doi: 10.1371/journal.pone.0054182. Epub 2013 Jan 23.

Nanoparticle mediated P-glycoprotein silencing for improved drug delivery across the blood-brain barrier: a siRNA-chitosan approach

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Nanoparticle mediated P-glycoprotein silencing for improved drug delivery across the blood-brain barrier: a siRNA-chitosan approach

Jostein Malmo et al. PLoS One. 2013.

Abstract

The blood-brain barrier (BBB), composed of tightly organized endothelial cells, limits the availability of drugs to therapeutic targets in the central nervous system. The barrier is maintained by membrane bound efflux pumps efficiently transporting specific xenobiotics back into the blood. The efflux pump P-glycoprotein (P-gp), expressed at high levels in brain endothelial cells, has several drug substrates. Consequently, siRNA mediated silencing of the P-gp gene is one possible strategy how to improve the delivery of drugs to the brain. Herein, we investigated the potential of siRNA-chitosan nanoparticles in silencing P-gp in a BBB model. We show that the transfection of rat brain endothelial cells mediated effective knockdown of P-gp with subsequent decrease in P-gp substrate efflux. This resulted in increased cellular delivery and efficacy of the model drug doxorubicin.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Chitosan-mediated siRNA uptake in RBE4 cells.
A) Levels of internalized Alexa-647 conjugated siRNA at different nanoparticle N/P ratios and siRNA concentrations expressed as the median fluorescence intensities (FI) of the analyzed cells. Data represents mean values ± s.d., n = 3. B) Representative histograms of siRNA fluorescence from flow cytometry analysis of untreated cells, cells with added naked siRNA or transfected with nanoparticles having N/P 10, 30 or 60 and a siRNA concentration of 100 nM. C) Representative CLSM images of a) untreated cells, b) cells with added naked siRNA or nanoparticles having N/P c) 10, d) 30 or e) 60 and a siRNA concentration of 100 nM. The cellular plasma membrane was stained with CellMask Orange (blue) and the fluorescent siRNA is indicated with the red color. The bar size is 20 µm.
Figure 2
Figure 2. Particle concentrations measured by nanoparticle tracking analysis.
The samples consisted of complexes in formulations having N/P 10, 30 or 60 and a siRNA concentration of 500 nM. Data represents mean values ± s.d., n = 3.
Figure 3
Figure 3. Knockdown of GAPDH measured by levels of mRNA and protein activity.
The nanoparticles had a N/P ratio of 30 and a concentration of 50 nM GAPDH targeting (T) or non-targeting (NT) siRNA. Cells were also treated with naked siRNA (siRNA). Data represents mean values ± s.d., n = 3.
Figure 4
Figure 4. Knockdown of P-gp measured at mRNA level by qRT-PCR.
The cells were transfected with only chitosan (mock, M) or nanoparticles having N/P 30 and P-gp targeting (T) or non-targeting (NT) siRNA concentrations of 100 nM. Cells were also treated with naked siRNA (siRNA). Data represents mean values ± s.d., n = 3.
Figure 5
Figure 5. The effect of P-gp knockdown on R123 efflux.
Intracellular levels of R123 as a function of A) siRNA concentration and B) days post-transfection. The relative levels of R123 are expressed as the median FI of the cells. The cells were transfected with nanoparticles having N/P 30 and P-gp targeting (T) or non-targeting (NT) siRNA concentrations of 100 nM. Data represents mean values ± s.d., n = 3. C) Representative histograms of R123 fluorescence from flow cytometry analysis of untreated cells or cells transfected with nanoparticles having N/P 30 and a siRNA concentration of 100 nM at one to five days post-transfection. D) Representative CLSM images after incubation with R123 post-transfection with a) T or b) NT siRNA or c) untreated cells. The cells were transfected with nanoparticles having N/P ratios of 30 and a siRNA concentration of 100 nM. The cellular plasma membranes were stained with CellMask Deep Red (blue) and R123 fluorescence is indicated with the green color. The bar size is 20 µm.
Figure 6
Figure 6. The effect of P-gp knockdown on doxorubicin efficacy and delivery.
A) Metabolic activity of cells after two days of incubation with doxorubicin. The cells were transfected with nanoparticles having N/P 30 and P-gp targeting (T) or non-targeting (NT) siRNA concentrations of 50 or 100 nM. Data represents mean values ± s.d., n = 4. B) Intracellular uptake and accumulation of doxorubicin expressed as the median FI of the cells. The cells were transfected with nanoparticles having N/P 30 and T or NT siRNA concentrations of 100 nM. Data represents mean values ± s.d., n = 3. C) Representative histograms of doxorubicin fluorescence from flow cytometry analysis of untreated or transfected cells. D) Representative CLSM images after transfection with a) T or b) NT siRNA or c) untreated cells. E) Enlarged images of RBE4 nuclei. The cells were transfected with nanoparticles having N/P 30 and a siRNA concentration of 100 nM. Doxorubicin fluorescence is indicated with the green color. The bar size is 20 µm.

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This work was supported by a Ph.D. grant from NTNU (Norges Teknisk-Naturvitenskapelige Universitet) (Norwegian University of Science and Technology) to JM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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