Enhancing natural killer cells proliferation and cytotoxicity using imidazole-based lipid nanoparticles encapsulating interleukin-2 mRNA.

mRNA applications have undergone unprecedented applications-from vaccination to cell therapy. Natural killer (NK) cells are recognized to have a significant potential in immunotherapy. NK-based cell therapy has drawn attention as allogenic graft with a minimal graft-versus-host risk leading to easier off-the-shelf production. NK cells can be engineered with either viral vectors or electroporation, involving high costs, risks, and toxicity, emphasizing the need for alternative way as mRNA technology. We successfully developed, screened, and optimized novel lipid-based platforms based on imidazole lipids. Formulations are produced by microfluidic mixing and exhibit a size of approximately 100 nm with a polydispersity index of less than 0.2. They are able to transfect NK-92 cells, KHYG-1 cells, and primary NK cells with high efficiency without cytotoxicity, while Lipofectamine Messenger Max and D-Lin-MC3 lipid nanoparticle-based formulations do not. Moreover, the translation of non-modified mRNA was higher and more stable in time compared with a modified one. Remarkably, the delivery of therapeutically relevant interleukin 2 mRNA resulted in extended viability together with preserved activation markers and cytotoxic ability of both NK cell lines and primary NK cells. Altogether, our platforms feature all prerequisites needed for the successful deployment of NK-based therapeutic strategies.

Messenger RNA Lipid-Based Nanoparticles: Optimization of Formulations in the Lab.

Among the large variety of messenger RNA (mRNA) delivery systems, those developed with lipid-based formulations were the most widely used and efficient. In our lab, we produced different mRNA formulations made with liposomes, hybrid lipid polymer, and lipid nanoparticles. Our formulations were made with lipids bearing imidazole groups that trigger the endosomal escape of nanoparticles once protonated inside the mild acidic milieu of endosomes upon their cell uptake. Herein, we describe protocols that we used to produce, optimize, and characterize those formulations. The transfection efficiency is influenced by various factors including the physicochemical parameters of the nanoparticles, their efficiency to be internalized in cells, and their intracellular routing as well as their capacity to induce immune system sensors. We provide details on how to quantify the amount of mRNA nanoparticles uptake by cells and evaluate the acidity of the intracellular compartments where they are located, to investigate the endosomal escape, and to assess the activation of innate immune sensors as phosphorylation of PKR hampering mRNA translation.

Impact of net charge, targeting ligand amount and mRNA modification on the uptake, intracellular routing and the transfection efficiency of mRNA lipopolyplexes in dendritic cells.

Targeting mRNA formulations to achieve cell specificity is one of the challenges that must be tackled to mettle their therapeutic potential. Here, lipopolyplexes (LPR) bearing tri-mannose-lipid (TM) are used to target mannose receptor on dendritic cells. We investigated the impact of the net charge and percentage of TM units on the binding, uptake, transfection efficiency (TE) and RNA sensors activation. Binding and uptake capacities of naked and targeted LPR increase with the percent of cationic lipid, but the latter are 2-fold more up taken by the cells. Cationic LPR bearing 5 % and 10 % TM were localized in acidic compartments in contrast to naked LPR and 2.5 % TM-LPR. The drawback is the dramatic decrease of TE as the number of TM-units increases. Cationic LPR bearing 5 % and 10 % TM strongly induced NF-κB and PKR phosphorylation at 6 h. Conversely, mTOR is less activated in line with their low TE. Those side effects are overcome by using 5-methoxyuridine mRNA resulting in an improved TE due to non-phosphorylation of NF-κB and PKR and mTOR activation. Our results point out that targeting DC via mannose receptor triggers a higher uptake of cationic LPRs and fast routing to acidic compartments, and that efficient TE requires low number of TM units use or modified mRNA to escape RNA sensors activation to enhance the translation.

Structure-Activity Relationships in Nucleic-Acid-Templated Vectors Based on Peptidic Dynamic Covalent Polymers.

The use of nucleic acids as templates, which can trigger the self-assembly of their own vectors represent an emerging, simple and versatile, approach toward the self-fabrication of tailored nucleic acids delivery vectors. However, the structure-activity relationships governing this complex templated self-assembly process that accompanies the complexation of nucleic acids remains poorly understood. Herein, the class of arginine-rich dynamic covalent polymers (DCPs) composed of different monomers varying the number and position of arginines were studied. The combinations that lead to nucleic acid complexation, in saline buffer, using different templates, from short siRNA to long DNA, are described. Finally, a successful peptidic DCP featuring six-arginine repeating unit that promote the safe and effective delivery of siRNA in live cancer cells was identified.

mRNA, a Revolution in Biomedicine.

The perspective of using messenger RNA (mRNA) as a therapeutic molecule first faced some uncertainties due to concerns about its instability and the feasibility of large-scale production. Today, given technological advances and deeper biomolecular knowledge, these issues have started to be addressed and some strategies are being exploited to overcome the limitations. Thus, the potential of mRNA has become increasingly recognized for the development of new innovative therapeutics, envisioning its application in immunotherapy, regenerative medicine, vaccination, and gene editing. Nonetheless, to fully potentiate mRNA therapeutic application, its efficient production, stabilization and delivery into the target cells are required. In recent years, intensive research has been carried out in this field in order to bring new and effective solutions towards the stabilization and delivery of mRNA. Presently, the therapeutic potential of mRNA is undoubtedly recognized, which was greatly reinforced by the results achieved in the battle against the COVID-19 pandemic, but there are still some issues that need to be improved, which are critically discussed in this review.

Cell-Selective siRNA Delivery Using Glycosylated Dynamic Covalent Polymers Self-Assembled In†Situ by RNA Templating.

Dynamic covalent libraries enable exploring complex chemical systems from which bioactive assemblies can adaptively emerge through template effects. In this work, we studied dynamic covalent libraries made of complementary bifunctional cationic peptides, yielding a diversity of species from macrocycles to polymers. Although polymers are typically expressed only at high concentration, we found that siRNA acts as a template in the formation of dynamic covalent polymers at low concentration in a process guided by electrostatic binding. Using a glycosylated building block, we were able to show that this templated polymerization further translates into the multivalent presentation of carbohydrate ligands, which subsequently promotes cell uptake and even cell-selective siRNA delivery.

Hydrocarbon-Stapled Peptide Based-Nanoparticles for siRNA Delivery.

Small interfering RNAs (siRNAs) are promising molecules for developing new therapies based on gene silencing; however, their delivery into cells remains an issue. In this study, we took advantage of stapled peptide technology that has emerged as a valuable strategy to render natural peptides more structured, resistant to protease degradation and more bioavailable, to develop short carriers for siRNA delivery. From the pool of stapled peptides that we have designed and synthesized, we identified non-toxic vectors that were able to efficiently encapsulate siRNA, transport them into the cell and induce gene silencing. Remarkably, the most efficient stapled peptide (JMV6582), is composed of only eight amino-acids and contains only two cationic charges.

Reverse poly(ε-caprolactone)-g-dextran graft copolymers. Nano-carriers for intracellular uptake of anticancer drugs.

A new fully biodegradable "reverse" oligosaccharide-based amphiphilic graft copolymer structure with a hydrophobic backbone and hydrophilic side chains, poly(ε-caprolactone)-g-dextran (PCL-g-Dex) was synthetized. For this purpose, "clickable" propargylated PCL (PCL-yne) and azido-dextran (Dex-N3) were prepared to further synthesize PCL-g-Dex copolymer by a Huisgen's cycloaddition. This "reverse" copolymer architecture self-assembled in biodegradable nano-carriers, in the shape of dynamic polymeric micelles, and were loaded with doxorubicin (Dox) anti-cancer drug. Dox-loaded micelles showed different drug releases depending on the pH. Cytotoxicity tests showed that Dox-loaded micelles can selectively kill colon cancer cells (HCT-116) while they have no cytotoxic effect towards healthy cells (CCD-45SK). Fluorescent micelles based on FITC-labelled PCL-g-Dex copolymer were used for fluorescence imaging and flow cytometry assays. These experiments proved the effective and specific internalization of micelles by cancer cells, whereas healthy cells showed a very poor uptake. These results show that PCL-g-Dex micelles may be a promising Dox nano-carrier in cancer chemotherapy.

Combination of photodynamic therapy and gene silencing achieved through the hierarchical self-assembly of porphyrin-siRNA complexes.

In this work, we implemented a supramolecular approach in order to combine photodynamic therapy (PDT) with gene therapy. We made use of a simple cationic guanidylated porphyrin (H2­PG) with the hypothesis that porphyrin aggregates should be capable of complexing siRNA through multivalent interactions and thus contribute to its intracellular delivery, while remaining active photosensitizers for PDT. The PDT effect of H2­PG was shown by incubating human breast cancer cells (MDA-MB-231) with H2­PG followed by light-irradiation at 405 nm. On the other hand, while siRNA do not enter cells alone, we showed, by fluorescence confocal microscopy and flow cytometry, that H2­PG promotes the internalization of Atto-488 siRNA. Finally, studying the combined PDT and delivery of siRNA directed against inhibitory apoptotic protein (IAP) family, we found an additive effect of the two therapies, thereby demonstrating that H2­PG is capable of acting both as a photosensitizer and supramolecular siRNA vector.