Targeted lipid nanoparticle delivery of calcitriol to human monocyte-derived macrophages in vitro and in vivo: investigation of the anti-inflammatory effects of calcitriol

Abstract

Background

Vitamin D₃ possesses anti-inflammatory and modulatory properties in addition to its role in calcium and phosphate homeostasis. Upon activation, macrophages (M) can initiate and sustain pro-inflammatory cytokine production in inflammatory disorders and play a pathogenic role in certain cancers.

Purpose

The main purpose of this study was to encapsulate and specifically target calcitriol to macrophages and investigate the anti-inflammatory properties of calcitriol in vitro and in vivo.

Methods

In this study we have designed and developed near-infrared calcitriol PEGylated nanoparticles (PEG-LNP(Cal)) using a microfluidic mixing technique and modified lipid nanoparticles (LNPs) to target the M specific endocytic receptor CD163. We have investigated LNP cellular uptake and anti-inflammatory effect in LPS-induced M in vitro by flow cytometry, confocal microscopy and gene expression analyses. LNP pharmacodynamics, bio-distribution and organ specific LNP accumulation was also investigated in mice in vivo.

Results

In vitro, we observed the specific uptake of PEG-LNP(Cal)-hCD163 in human M, which was significantly higher than the non-specific uptake of control PEG-LNP(Cal)-IgG(h) in M. Pretreatment with encapsulated calcitriol was able to attenuate intracellular TNF-expression, and M surface marker HLA-DR expression more efficiently than free calcitriol in LPS-induced M in vitro. Encapsulated calcitriol diminished mRNA gene levels of TNF-, NF-B, MCP-1 and IL-6, while upregulating IL-10. TNF- and IL-6 protein secretion also decreased. In mice, an in vivo pharmacodynamic study of PEG-LNP(Cal) showed a rapid clearance of IgG and CD163 modified LNPs compared to PEG-LNP(Cal). Antibody modified PEG-LNP(Cal) accumulated in the liver, spleen and kidney, whereas unmodified PEG-LNP(Cal) accumulation was only observed in the liver.

Conclusion

Our results show that calcitriol can be effectively targeted to M. Our data confirms the anti-inflammatory properties of calcitriol and this may be a potential way to deliver high dose bioactive calcitriol to M during inflammation in vivo.

Keywords:

• macrophages
• 1.25(OH)₂D₃
• calcitriol
• lipid nanoparticles
• pro-inflammatory cytokines
• CD163 targeted drug delivery
• gene expression analyses
• in vivo pharmacodynamics

Supplementary materials

Figure S1 Flow cytometry gating strategy and uptake of nIR IgG- and CD163-targeted PEG-LNPs by Mφ and CHO-CD163 transfected cells visualized by confocal microscopy. (A) Gating strategy: Mφ were stained with Live/Dead nIR and live cells (cell viability >75%) were gated. Mφ were gated using a forward/side scatter plot and [CD163+DID⁻], [CD163-DID⁺], and [CD163+DID⁺] gates were placed according to unstimulated (Live/Dead stained) Mφ blank controls. (B) Mφ (Ia–Ic), human CD163-transfected CHO cells (IIa–IIc), and control CHO-MOCK(h) (IIIa–IIIc) were stimulated for 24 hours with either control medium, 25 nM DID PEG-LNP(c)-IgG, or 25 nM DID PEG-LNP(c)-αCD163 (DID: red). Cell membranes were stained with wheat germ agglutinin Alexa Flour 488 (green), and cell nuclei were counterstained with DAPI (blue). White arrows indicate the uptake of DID PEG-LNP(c)-IgG and DID-PEG-LNP(c)-αCD163. (C) Murine CD163-transfected CHO cells and control CHO-MOCK(m) cells were treated with IgG- and CD163-targeted PEG-LNP(Cal) (Ia–Ic, IIa–IIc) or IgG- and CD163-targeted PEG-LNP(c) (IIIa–IIIc, IVa–IVc). Murine CD163-transfected CHO cells and control CHO-MOCK(m) cells were stained as described for human CHO cells.

Abbreviations: Mφ, macrophages; nIR, near-infrared; LNPs, lipid nanoparticles; LNP(c), conventional empty LNPs; PEG, polyethylene glycol; PEG-LNP(Cal), calcitriol PEGylated lipid nanoparticles.

Figure S2 mRNA gene expression analyses of target genes (TNF-α, MCP-1, NF-κB, IL-6, IL-10, and CD163). mRNA target gene expressions of untreated Mφ, Mφ treated with LPS (1 μg/mL for 4 hours), and LPS-induced Mφ treated with dexamethasone (10 μM for 24 hours). Target gene expression levels were normalized to the stable housekeeping gene GAPDH, and the resulting mRNA ratios were normalized to untreated control Mφ and given the value 1. Controls for (A) TNF-α, (B) MCP-1, (C) NF-κB, (D) IL-6, (E) IL-10, and (F) CD163.

Abbreviations: Mφ, macrophages; TNF-α, tumor necrosis factor-alpha; MCP-1, monocyte chemoattractant protein; NF, nuclear factor; IL, interleukin; LPS, lipopolysaccharide; DEXA, dexamethasone; CTRL, control.

Acknowledgments

We acknowledge laboratory technicians Christina Strande Søndeskov and Helle Hausser Ryom, and animal caretaker Kristoffer Augustensen for their excellent technical assistance. Danish Council for Strategic Research (TRAIN 10-092797), Vilhelm Bangs Fund, Lily Benthine Lunds Fund of 1.6.1978, and Laegefonden supported the studies.

Author contributions

All authors contributed toward data analysis, drafting and revising the paper, gave final approval of the version to be published and agree to be accountable for all aspects of the work.

Disclosure

Dr Jonas H Graversen reports assistance from Affinicon during the conduct of the study. The authors report no other conflicts of interest in this work.