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Chitosan polyplex mediated delivery of miRNA-124 reduces activation of microglial cells in vitro and in rat models of spinal cord injury

Louw, Andrew M., Kolar, Mallappa K., Novikova, Liudmila N., Kingham, Paul J., Wiberg, Mikael, Kjems, Jørgen, Novikov, Lev N.·2016·Nanomedicine: Nanotechnology, Biology and Medicine
DOI 10.1016/j.nano.2015.10.011
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Figures

Figure 1 Chitosan particle characterization by photon correlation spectroscopy and laser Doppler velocimetry (LDV). Representative histograms of particle radius and dispersity ( A ). The size (diameter in nm), polydispersity index (PDI) and zeta potential of the particles ( B ).
Figure 1 Chitosan particle characterization by photon correlation spectroscopy and laser Doppler velocimetry (LDV). Representative histograms of particle radius and dispersity ( A ). The size (diameter in nm), polydispersity index (PDI) and zeta potential of the particles ( B ).CC-BY-NC-ND · From the PDF
Figure 2 Representative confocal scanning laser microscopy images of untreated primary rat microglia ( A -C ) labelled with wheat germ agglutinin Alexa Fluor® 488 conjugate (WGA, green) and DAPI (blue), and images showing intracellular uptake of chitosan particles containing 5 μ M ( D -F ), 25 μ M ( G -I ), 50 μ M ( J -L ) of siRNA-Cy3 (red) after 2 h treatment. Arrows indicate examples of external chitosan agglomerates. Scale bar, 20 μ m.
Figure 2 Representative confocal scanning laser microscopy images of untreated primary rat microglia ( A -C ) labelled with wheat germ agglutinin Alexa Fluor® 488 conjugate (WGA, green) and DAPI (blue), and images showing intracellular uptake of chitosan particles containing 5 μ M ( D -F ), 25 μ M ( G -I ), 50 μ M ( J -L ) of siRNA-Cy3 (red) after 2 h treatment. Arrows indicate examples of external chitosan agglomerates. Scale bar, 20 μ m.CC-BY-NC-ND · From the PDF
Image 3
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Image 4
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Figure 3 Isolated microglia were expanded with GM-CSF, activated using LPS and IFNγ , and treated with miR/chitosan particles. Cells were incubated with particles for 48 h with three different miRNA concentrations. Cytotoxicity of chitosan/miRNA particles measured by AlamarBlue® viability assay (A) . Percent viability is expressed relative to untreated controls ± SD (n=3). Activated microglia supernatant assays ( B, C ). Total supernatant was harvested each day for analysis by TNFα ELISA (B) and Greiss nitrite assay ( C ). Results indicate concentrations 48 h after particle treatment. ** and * P b 0.01 and 0.05 versus LPS and IFNγ alone (n=3).
Figure 3 Isolated microglia were expanded with GM-CSF, activated using LPS and IFNγ , and treated with miR/chitosan particles. Cells were incubated with particles for 48 h with three different miRNA concentrations. Cytotoxicity of chitosan/miRNA particles measured by AlamarBlue® viability assay (A) . Percent viability is expressed relative to untreated controls ± SD (n=3). Activated microglia supernatant assays ( B, C ). Total supernatant was harvested each day for analysis by TNFα ELISA (B) and Greiss nitrite assay ( C ). Results indicate concentrations 48 h after particle treatment. ** and * P b 0.01 and 0.05 versus LPS and IFNγ alone (n=3).CC-BY-NC-ND · From the PDF
Figure 4 Flow cytometry analysis of MHC-II (y-axis) and CD11b (x-axis) expression in non-activated (Control), activated (Vehicle), and activated microglia treated with chitosan/miR-124 or miR-CTRL particles. Upper quadrants represent MHC-II-positive cells.
Figure 4 Flow cytometry analysis of MHC-II (y-axis) and CD11b (x-axis) expression in non-activated (Control), activated (Vehicle), and activated microglia treated with chitosan/miR-124 or miR-CTRL particles. Upper quadrants represent MHC-II-positive cells.CC-BY-NC-ND · From the PDF
Figure 5 Horizontal spinal cord sections of C3 segments showing siRNA-Cy3/chitosan particles (Cy3) 3 days after injection into normal spinal cord ( A -I ) and after intraperitoneal injection in animals with spinal cord injury ( J -L ). Sections were immunostained for OX42 (CD11b, macrophages and microglia), GFAP (astrocytes), NeuN (neurons) and ED-1 (activated macrophages/microglia). Cell nuclei were stained using DAPI. Boxed area in ( A ) demonstrates details of injection site with small bright round particles and diffuse staining. Note association of Cy3 labelled particles with OX42 and ED-1 positive cells. Scale bar, 50 μ m ( A -F ), 100 μ m ( G -I ) and 20 μ m ( J -L ).
Figure 5 Horizontal spinal cord sections of C3 segments showing siRNA-Cy3/chitosan particles (Cy3) 3 days after injection into normal spinal cord ( A -I ) and after intraperitoneal injection in animals with spinal cord injury ( J -L ). Sections were immunostained for OX42 (CD11b, macrophages and microglia), GFAP (astrocytes), NeuN (neurons) and ED-1 (activated macrophages/microglia). Cell nuclei were stained using DAPI. Boxed area in ( A ) demonstrates details of injection site with small bright round particles and diffuse staining. Note association of Cy3 labelled particles with OX42 and ED-1 positive cells. Scale bar, 50 μ m ( A -F ), 100 μ m ( G -I ) and 20 μ m ( J -L ).CC-BY-NC-ND · From the PDF
Figure 6 Horizontal sections of C3 spinal segments with ED-1 positive macrophages and microglia 3 days after injection of miR-CTRL ( A ) and miR-124 ( B ) chitosan particles into normal spinal cord. Histogram showing the relative tissue area occupied by ED-1 positive macrophages and microglial cells ( C ). Error bars show S.E.M. P b 0.001 is indicated by ***. Scale bar, 50 μ m.
Figure 6 Horizontal sections of C3 spinal segments with ED-1 positive macrophages and microglia 3 days after injection of miR-CTRL ( A ) and miR-124 ( B ) chitosan particles into normal spinal cord. Histogram showing the relative tissue area occupied by ED-1 positive macrophages and microglial cells ( C ). Error bars show S.E.M. P b 0.001 is indicated by ***. Scale bar, 50 μ m.CC-BY-NC-ND · From the PDF
Figure 7 Horizontal spinal cord sections of C3-C4 segments ( A, B ) immunostained for ED-1 positive macrophages and microglial cells 3 days after spinal cord hemisection followed by intraspinal injection of miR-CTRL ( A ) and miR-124 ( B ) chitosan particles. Dotted lines indicate primary trauma zone. Boxed areas in ( A ) and ( B ) are enlarged in ( C ) and ( D ), respectively. Note significant reduction of ED-1 immunostaining in ( B ). Histogram showing the relative tissue area occupied by ED-1 positive macrophages and microglial cells ( E ). Error bars show S.E.M. P b 0.001 is indicated by ***. Scale bar, 500 μ m.
Figure 7 Horizontal spinal cord sections of C3-C4 segments ( A, B ) immunostained for ED-1 positive macrophages and microglial cells 3 days after spinal cord hemisection followed by intraspinal injection of miR-CTRL ( A ) and miR-124 ( B ) chitosan particles. Dotted lines indicate primary trauma zone. Boxed areas in ( A ) and ( B ) are enlarged in ( C ) and ( D ), respectively. Note significant reduction of ED-1 immunostaining in ( B ). Histogram showing the relative tissue area occupied by ED-1 positive macrophages and microglial cells ( E ). Error bars show S.E.M. P b 0.001 is indicated by ***. Scale bar, 500 μ m.CC-BY-NC-ND · From the PDF

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Citations

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Metadata

Authors
Louw, A. M., Kolar, M. K., Novikova, L. N., Kingham, P. J., Wiberg, M., Kjems, J., & Novikov, L. N.
Venue
Nanomedicine: Nanotechnology, Biology and Medicine
Publisher
Elsevier BV
Published
2016-04
Volume
12
Issue
3
Pages
643-653
ISSN
1549-9634
DOI
10.1016/j.nano.2015.10.011
Cited by
109
References
9
Language
en
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