Research Article
Enhancement of dendritic cells transfection in vivo and of vaccination against B16F10 melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen messenger RNA

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Abstract

We report the preparation of mannosylated nanoparticles loaded with messenger RNA (mRNA) that enhance the transfection of dendritic cells (DCs) in vivo and the anti-B16F10 melanoma vaccination in mice. Mannosylated and histidylated lipopolyplexes (Man11-LPR100) were obtained by adding mannosylated and histidylated liposomes to mRNA-PEGylated histidylated polylysine polyplexes. Upon intravenous injection, ∼9% of the radioactivity of technetium 99 m–labeled lipopolyplexes measured in the liver, spleen, lungs, and kidneys was found in the spleen. We demonstrate that spleen from mice injected with enhanced green fluorescent protein (EGFP) mRNA-loaded Man11-LPR100 contained four times more DCs expressing EGFP than that from mice injected with sugar-free LPR100. This better transfection of DCs is correlated with a better inhibition of B16F10 melanoma growth and an increased survival time when mice were immunized with MART-1 mRNA-loaded Man11-LPR100. These results indicate that mannosylated and histidylated LPR is an efficient system for the delivery of tumor antigen mRNA in splenic DCs aiming to induce an anticancer immune response.

From the Clinical Editor

This paper discusses the preparation of mannosylated nanoparticles loaded with messenger RNA that enhance the transfection of dendritic cells (DCs) in vivo and the anti-B16F10 melanoma vaccination in mice. The authors describe an efficient system for the delivery of tumor antigen mRNA in splenic DCs aiming to induce an anticancer immune response.

Graphical Abstract

Mannosylated lipopolyplexes loaded with tumor antigen mRNA (Man-LPR) enhance the transfection of dendritic cells in vivo and inhibition of B16F10 melanoma growth.

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Section snippets

Methods

All reagents were purchased from Sigma-Aldrich (St. Quentin Fallavier, France) unless stated otherwise. PEGylated histidylated poly-l-lysine (PEG-HpK) was synthesized as described.16, 21 O,O-dioleyl-N-[3N-(N-methylimidazolium iodide)propylene] phosphoramidate (lipid 1) and O,O-dioleyl-N-histamine phosphoramidate (lipid 2) were synthesized as described (Supplementary Figure S1).18, 19 β-d-mannopyranosyl-N-dodecylhexadecanamide (Man-lipid) was synthesized as described.22

In vitro transfection of DCs with mRNA LPR100

mRNA and pDNA encoding EGFP were formulated as lipopolyplexes. Nucleic acids (5 μg) were first condensed with the PEG-HpK (15 μg) before adding 10 μg of either Lip100 or Man11-Lip100. The electrophoretic mobility shift assays showed that mRNA and pDNA did not migrate in the agarose gel, indicating that they were in complex with LPR100, LPD100, Man11-LPR100, and Man11-LPD100 at the ratio used (Supplementary Figs. S2 and S3, which can be found in the online version of this article). The nucleic

Discussion

In the present study we have incorporated mannose motifs into liposomes to increase the delivery and the transfection efficiency of mRNA lipopolyplexes into DCs. From an experimental point of view, EGFP or MART-1 mRNA (∼900 nt) was first condensed with a PEG-HpK. Next, a liposomal formulation comprising three lipids (a mannosylated lipid, an imidazole lipophosphoramidate, and an imidazolium lipophosphoramidate), was added. Lipophosphoramidates have a chemical structure that is bio-inspired from

Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.nano.2010.12.010.

The following is the supplementary material related to this article

. Lipid structures. 1: O,O-dioleyl-N-[3N-(N-methylimidazolium iodide)propylene] phosphoramidate; 2: O,O-dioleyl-N-histamine phosphoramidate; Man-lipid: β-D-mannopyranosyl-N-dodecylhexadecanamide.

. Electrophoretic mobility shift assay of LPR. LPR100 and Man11-LPR100 were formed with EGFP mRNA. LPR (1 μg mRNA) were

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    This work was supported by Ligue contre le Cancer (Région Centre) and Canceropole Grand Ouest. F.P. was a recipient of a fellowship from Ligue Nationale contre le Cancer.

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