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Improving Nucleic Acid Delivery for Immunotherapy Applications


Scientists have recently assessed that substituting hydroxycholesterol candidates in ionizable nanoparticles could elevate the possibility of messenger RNA (mRNA) delivery to T cells. This finding suggests that the efficacy of immunotherapies against various diseases could be significantly improved. This study is available in the Journal of Controlled Release.


​​​​​​​Study: Hydroxycholesterol substitution in ionizable lipid nanoparticles for mRNA delivery to T cells. Image Credit: isocortex/

Efficacy of Immunotherapy against Various Diseases

Several studies have revealed the importance of immunotherapy in treating various diseases, including blood cancer, solid cancers, and autoimmune diseases. Some of the common immunotherapies used are antibody-based inhibitors, mRNA-based immunotherapies, and genetically-engineered immune cells.

Researchers stated that in mRNA-based immunotherapies, mRNA is translated in the cytoplasm, which plays an important role in minimizing various risks related to genomic integration, e.g., insertional mutagenesis.

Scientists have standardized the manufacturing parameters and techniques for the optimal production of highly potent mRNA. For this reason, mRNA platforms are effectively used in the development of vaccines for coronavirus disease 2019 (COVID-19), Zika virus, and influenza. mRNA-based immunotherapy is also used for cancer treatment, for example, tumor-infiltrating T cell therapy and chimeric antigen receptor (CAR) T cell therapy. 

mRNA Delivery to Immune Cells

The delivery of mRNA to immune cells is promoted by the electroporation technique. This technique is only viable for ex vivo applications. Two of the major limitations of electroporation ex vivo are that it is a highly expensive process and is extremely toxic to targeted cells, and might modify its genomic expression. Therefore, scientists have expressed the need for an effective platform for mRNA delivery to immune cells with low cytotoxicity.

Ionizable lipid nanoparticles (LNPs) are used as an alternative means to deliver mRNA to immune cells. Typically, LNPs are composed of cholesterol, ionizable lipids, phospholipids, lipid-anchored polyethylene glycol, along with their nucleic acid cargo. Previous studies have shown that compared to electroporated methods, the LNP technique is more effective in delivering mRNA to immune cells (T cells) and induces lower toxicity in cells. Recently, LNP platforms have been used to develop COVID-19 mRNA vaccines by Moderna and Pfizer/BioNTech.

Scientists have highlighted some of the challenges related to the LNP platform that includes an accumulation in the liver, which inhibits proper biodistribution, and endosomal recycling. To overcome this limitation, researchers have adopted various strategies, for example, the use of modified LNP formulation ratios, novel LPN components, etc.

Evaluation of Hydroxycholesterols Substitution LNP-mediated mRNA Delivery to T cells – A New Study

In a new study, researchers evaluated the impact of hydroxycholesterols (cholesterol analogs), on LNP-based mRNA delivery to immune cells, i.e., T cells. In this study, researchers created a library of twenty-four LNPs, with various substitution percentages of unmodified cholesterol (12.5%, 25%, 50%, or 100%), assessed for improved mRNA delivery to T cells. 

Researchers screened the LNP library candidates via in vitro and ex vivo studies, with the best candidates selected to determine their dose-response behavior and endosomal trafficking. They observed that substituting 25% and 50% 7αhydroxycholesterol for cholesterol in LNPs significantly increased mRNA delivery to T cells. Scientists estimated these increases to be 1.8-fold (25%) and 2.0-fold (50%), respectively.

In this study, each of the hydroxycholesterol substitutes was referred to as A1, A2, A3, B1, B2, and B3, where A stands for analogs containing hydroxyl group along with ring structure of the cholesterol molecule, and B stands for analogs containing hydroxyl group along with hydrophobic pole or tail of the cholesterol molecule. 

Scientists evaluated hydroxycholesterol substitution into LNP formulations to explore its effect on particle stability. Stability was estimated using the measurement of z-average diameter, mRNA concentration polydispersity index (PDI), and encapsulation efficiency over a period of twenty-eight days.

This study reported that LNPs remained stable for the entire assessment period. However, compared to the A series, the B series was more unstable due to partial lipid aggregation. In the screening of LNPs, A1–25 and A1–50 were found to be top performers. More specifically, A1–25 exhibited higher colocalization with the endosome, reduced endosomal recycling, and enhancement in the generation of late endosomes.

To examine endosomal trafficking, researchers characterized the progression of LNP candidates through different stages of the endosome. Previous studies have reported that endosomal trafficking through cells can be evaluated via tracking the Ras-associated binding (Rab) family of proteins (e.g., Rab5, Rab7, and Rab11). 

In the current study, scientists hypothesized that hydroxycholesterol substitution into LNP formulations can effectively decrease the recycling of endocytosed LNPs out of the cell. The team observed that hydroxyl modification of cholesterol molecules not only enhanced late endosome production but also lowered the presence of recycling endosomes.


The authors stated that the results of the current study indicate that hydroxyl modification of cholesterol molecules, which are inserted into LNP formulations, offers an improved mechanism for the delivery of nucleic acid cargo to T cells. Thereby, this strategy could be applied to a wide range of immunotherapies.


Patel, K.S. et al. (2022) Hydroxycholesterol substitution in ionizable lipid nanoparticles for mRNA delivery to T cells. Journal of Controlled Release, 347, pp. 521–532.

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