Journal: International Journal of Nanomedicine
Article Title: Targeted lipid nanoparticle delivery of calcitriol to human monocyte-derived macrophages in vitro and in vivo: investigation of the anti-inflammatory effects of calcitriol
doi: 10.2147/IJN.S192113
Figure Lengend Snippet: mRNA gene expression analyses of selected target genes, protein analyses of IL-6 and TNF-α, and analysis of surface marker expression of HLA-DR and CD80 to investigate the effects of non-targeted and targeted PEG-LNP(Cal) in LPS-induced human Mφ. ( A – H ) Human Mφ (1×10 6 cells/well) from buffy coats (n=3) were pretreated with different doses (0.5, 10, and 100 nM) of either PEG-LNP(Cal), PEG-LNP(Cal)-IgG(h), or PEG-LNP(Cal)-αhCD163 for 24 hours following LPS challenge (μL/mL) for 4 hours. The second derivative max method was used to calculate the relative mRNA concentration of each sample. Target gene expression levels (NF-κB, TNF-α, MCP-1, IL-6, IL-10, and CD163) were normalized to the stable housekeeping gene GAPDH expression level, and the resulting mRNA ratios were normalized to untreated control Mφ and given the value 1 (dotted horizontal line). Two-way ANOVA along with Tukey’s multiple comparisons test was performed on all targets to investigate the difference between LNP types per dose and the specific dose-dependent response per LNP type. * P ≤0.05, ** P ≤0.01, *** P ≤0.001, and **** P ≤0.0001. Asterisks over the horizontal line indicate dose-dependent significance. ( A ) # 10 nM PEG-LNP(Cal) vs 10 nM PEG-LNP(Cal)-αhCD163, * and **PEG-LNP(Cal)-IgG(h) vs PEG-LNP(Cal)-αhCD163. ( B ) ## PEG-LNP(Cal) vs PEG-LNP(Cal)-αhCD163. ( C ) **The horizontal line above shows dose-dependent significance for non-targeted and targeted PEG-LNP(Cal). ( D ) *The horizontal line above illustrates significant dose-dependent response for PEG-LNP(Cal) and PEG-LNP(Cal)-IgG(h). *For both 0.5 and 10 nM PEG-LNP(Cal)-IgG(h) vs 0.5 and 10 nM PEG-LNP(Cal)-αhCD163. ( E ) # 0.5 nM PEG-LNP(Cal) vs 0.5 nM PEG-LNP(Cal)-αhCD163, *0.5 and 100 nM PEG-LNP(Cal)-IgG(h) vs 0.5 and 100 nM PEG-LNP(Cal)-αhCD163. ( G , H ) Cell culture supernatant was collected from Mφ (n=3). Protein concentration of ( G ) IL-6 *(outlier removed from IL-6 ELISA) and ( H ) TNF-α protein level (pg/mL) was measured using a commercial human IL-6 and TNF-α kit. Dotted horizontal line indicates the protein level detected in control Mφ. Two-way ANOVA with Tukey’s multiple comparisons test was performed. ***Statistically significant difference for all 100 nM LNP types. **For all 10 nM LNP types. @@ For 0.5 nM PEG-LNP(Cal) and 0.5 nM PEG-LNP(Cal)-IgG(h). # 0.5 nM PEG-LNP(Cal) vs 0.5 nM PEG-LNP(Cal)-αhCD163. *0.5 nM PEG-LNP(Cal)-IgG(h) vs 0.5 nM PEG-LNP(Cal)-αhCD163. ( I , J ) MFI of surface markers HLA-DR and CD80 on DID + LPS-induced Mφ pretreated with 10 and 100 nM PEG-LNP(Cal)-αhCD163. Overall MFI of surface markers on live LPS-induced Mφ population; mean MFI HLA-DR: 1978 and mean MFI CD80: 446. Abbreviations: Mφ, macrophages; LNPs, lipid nanoparticles; PEG, polyethylene glycol; PEG-LNP(Cal), calcitriol PEGylated lipid nanoparticles; LPS, lipopolysaccharide; MFI, median fluorescence intensity; TNF-α, tumor necrosis factor-alpha; MCP-1, monocyte chemoattractant protein; NF, nuclear factor; IL, interleukin.
Article Snippet: Specific antibodies for flow cytometry and confocal microscopy included the following: human CD80 HRZN V450 (clone: L307.4) was obtained from Becton Dickinson (Franklin Lakes, NJ, USA); FITC antihuman HLA-DR (clone L243) and PE-antihuman CD163 (clone: GHI) were obtained from Nordic BioSite (Taby, Sweden); wheat germ agglutinin Alexa Flour 488 (cat no W11261), Live/Dead (near-infrared stain), and antihuman TNF-α MAB11 EF450 were obtained from Thermo Fisher Scientific.
Techniques: Expressing, Marker, Concentration Assay, Cell Culture, Protein Concentration, Enzyme-linked Immunosorbent Assay, Fluorescence
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