ABSTRACT
(1) Background: T cells are important target cells, since they exert direct cytotoxic effects on infected/malignant cells, and affect the regulatory functions of other immune cells in a target antigen-specific manner. One of the current approaches for modifying the function of T cells is gene transfection by viral vectors. However, the insertion of the exogenous DNA molecules into the genome is attended by the risk of mutagenesis, especially when a transposon-based gene cassette is used. Based on this scenario, the transient expression of proteins by an in vitro-transcribed messenger RNA (IVT-mRNA) has become a subject of interest. The use of lipid nanoparticles (LNPs) for the transfection of IVT-mRNA is one of the more promising strategies for introducing exogenous genes. In this study, we report on the development of LNPs with transfection efficiencies that are comparable to that for electroporation in a T cell line (Jurkat cells). (2) Methods: Transfection efficiency was improved by optimizing the phospholipids and polyethylene glycol (PEG)-conjugated lipid components. (3) Results: Modification of the lipid composition resulted in the 221-fold increase in luciferase activity compared to a previously optimized formulation. Such a high transfection activity was due to the efficient uptake by clathrin/dynamin-dependent endocytosis and the relatively efficient escape into the cytoplasm at an early stage of endocytosis.
ABSTRACT
The world-first success of lipid nanoparticle (LNP)-based siRNA therapeutics (ONPATTRO®) promises to accelerate developments in siRNA therapeutics/gene therapy using LNP-type drug delivery systems (DDS). In this study, we explore the optimal composition of an LNP containing a self-degradable material (ssPalmO-Phe) for the delivery of oligonucleotides. siRNA or antisense oligonucleotides (ASO) were encapsulated in LNP with different lipid compositions. The hepatic knockdown efficiency of the target genes and liver toxicity were evaluated. The optimal compositions for the siRNA were different from those for ASO, and different from those for mRNA that were reported in a previous study. Extracellular stability, endosomal escape and cellular uptake appear to be the key processes for the successful delivery of mRNA, siRNA and ASO, respectively. Moreover, the compositions of the LNPs likely contribute to their toxicity. The lipid composition of the LNP needs to be optimized depending on the type of nucleic acids under consideration if the applications of LNPs are to be further expanded.
ABSTRACT
DNA vaccinations are promising strategies for treating diseases that require cellular immunity (i.e., cancer and protozoan infection). Here, we report on the use of a liposomal nanocarrier (lipid nanoparticles (LNPs)) composed of an SS-cleavable and pH-activated lipidlike material (ssPalm) as an in vivo DNA vaccine. After subcutaneous administration, the LNPs containing an ssPalmE, an ssPalm with vitamin E scaffolds, elicited a higher gene expression activity in comparison with the other LNPs composed of the ssPalms with different hydrophobic scaffolds. Immunization with the ssPalmE-LNPs encapsulating plasmid DNA that encodes ovalbumin (OVA, a model tumor antigen) or profilin (TgPF, a potent antigen of Toxoplasma gondii) induced substantial antitumor or antiprotozoan effects, respectively. Flow cytometry analysis of the cells that had taken up the LNPs in draining lymph nodes (dLNs) showed that the ssPalmE-LNPs were largely taken up by macrophages and a small number of dendritic cells. We found that the transient deletion of CD169+ macrophages, a subpopulation of macrophages that play a key role in cancer immunity, unexpectedly enhanced the activity of the DNA vaccine. These data suggest that the ssPalmE-LNPs are effective DNA vaccine carriers, and a strategy for avoiding their being trapped by CD169+ macrophages will be a promising approach for developing next-generation DNA vaccines.
Subject(s)
Lipids/chemistry , Nanoparticles/chemistry , Protozoan Infections/immunology , Vaccines, DNA/chemistry , Vaccines, DNA/immunology , Vitamin E/immunology , Animals , Cancer Vaccines/chemistry , Cancer Vaccines/immunology , DNA/immunology , Dendritic Cells/immunology , Female , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Immunity, Cellular/immunology , Immunization/methods , Liposomes/chemistry , Liposomes/immunology , Lymph Nodes/immunology , Macrophages/immunology , Mice, Inbred BALB C , Mice, Inbred C57BL , Neoplasms/immunology , Ovalbumin/immunology , Plasmids/immunology , Vitamin E/chemistryABSTRACT
The highly selective mono-C-allylation of oligoglycinates such as a diethylenetriaminepentaacetate, an iminodiacetate, and an ethylenediaminetetraacetate via insertion of a vacuum operation between the N-allylation and C-migration steps is reported. It is contrastive that one-pot N-allylation-C-allylation procedure gave a mixture including multiallylated products. In the reaction with N-ylides, gem-C-diallylation and α,α'-C-diallylation of oligoglycinates are strongly inhibited even with the use of an excess of allyl bromide and base. A mechanism to explain this control of the frequency of C-allylation on oligoglycinates via N-ylides is also proposed.
