Role of Lipid Nanoparticle (LNP) Characteristics on mRNA Delivery

What are mRNA-LNPs?

Messenger RNA (mRNA) is a nucleic acid molecule that carries genetic information and directs cells to synthesize proteins. mRNA vaccines use this mechanism to guide human cells to synthesize proteins that can trigger immune responses through artificially synthesized mRNA sequences, thereby achieving the purpose of preventing diseases. However, the main challenges associated with mRNA vaccines include their degradation tendency and low transfection efficiency. Therefore, delivery systems play a key role in the efficacy of mRNA vaccines. Currently, lipid nanoparticles (LNPs) are considered to be the most effective and widely used non-viral vectors.

LNPs are usually composed of four lipid components (ionizable lipids, phospholipids, cholesterol, and PEGylated lipids) mixed to form a uniform sphere with a diameter of 50 to 150 nm, which can encapsulate various RNA payloads. mRNA-LNPs have been successfully used in preventive vaccines against pathogens and have also been tested in oncology clinical trials to achieve intratumoral expression of immunostimulating cytokine combinations or as cancer vaccines. The encapsulation of mRNA within LNPs plays a protective role, significantly reduces the degradation of mRNA by RNase enzymes, and promotes mRNA penetration into target tissues. In addition, LNPs significantly enhanced transfection and protein expression in vivo and in vitro. It was reported that model mRNA encapsulated by LNPs was successfully delivered and translated into protein, and remained expressed for at least a week. In addition, repeated intramuscular injections of antigen mRNA through LNPs lead to massive production of neutralizing antibodies and antigen-specific T-cell immune responses. In addition, MRNA-containing LNPs have been used in chimeric antigen receptor (CAR)-T cell therapies. Previous research has shown that LNPs containing mRNA encoding fibroblast activating protein (FAP) antibodies can enable CAR-T therapy to treat heart injury in vivo, further emphasizing the importance of LNPs-mRNA therapy.

Generation of mRNA-LNPs

mRNA payloads are synthesized through in vitro transcription reactions, where mRNA is synthesized based on a DNA template encoding the required payload downstream of the phage promoter site (most common being T7 or SP6). The 5'and 3'untranslated regions (UTRs) of mRNA are encoded on the transcript, and the mRNA cap and poly(A) tail can be added either synergistically or post-transcriptional through an enzymatic process. These components contribute to the stabilization and enhanced expression of mRNA. In addition, mRNA payloads can bind modified nucleosides (such as N1 -methylpseudouridine), thereby increasing expression by inhibiting recognition by intrinsic immune sensors within cells.

mRNA-LNPs are prepared by mixing these payloads with various lipid components. Structurally, the ionizable lipid interacts hydrostatic with the nucleic acid payload to form inverted micelles around the nucleic acid. This allows helper lipids and sterols to spontaneously self-assemble into nuclei, producing solid nuclear LNPs. The particle size, charge and lipid composition of nanoparticles have been shown to significantly affect their transfection ability and biodistribution after intravenous administration.

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LNP characteristics influence mRNA transfection and in vivo distribution

Particle size of mRNA-LNPs

Some studies have shown that LNPs with a diameter of approximately 140nm show excellent transfection ability in vitro and in vivo, while smaller LNPs tend to accumulate in the liver after intramuscular injection. In contrast, a previous study showed that mRNA-LNPs vaccines of varying sizes elicited relatively strong immune responses in non-human primates. Research by Marianna YA et al. showed that smaller LNPs (e.g., 60 to 70 nanometers) are the best in terms of cellular uptake and protein expression. However, different studies have pointed out that the optimal particle size range for LNP may vary slightly, but it is generally believed that a range between 20 and 200 nanometers is appropriate. Research by Kong et al. found that smaller nanoparticles may penetrate cells more easily and enter the blood circulation faster. Proteins translated from mRNA are also present in the liver, a phenomenon that occurs independently of nanoparticle-mediated delivery after intramuscular injection. Proteins expressed at the injection site may be transported to the liver by other cells, such as dendritic cells or macrophages. The observed consistency in FRET ratios for different particle sizes may be due to the lack of synchronization between the disintegration of the nanoparticles and the expression of the encoded protein. This phenomenon can be explained by previous studies, which showed that the structural properties of larger LNPs are significantly different from those of smaller LNPs. This structural change may lead to the hypothesis that similar numbers of nanoparticles release different amounts of mRNA. Future surveys should be conducted to prove this assumption.

Surface charge of mRNA-LNPs

After intravenous injection, positively charged LNPs are preferentially localized in the lungs, while negatively charged LNPs are more likely to accumulate in the spleen. The main mechanism for the different in vivo distribution patterns of charged LNPs is attributed to changes in the corona of proteins adsorbed on the nanoparticles, which significantly affects uptake by cells. The impact of the surface charge of LNP on cellular uptake and toxicity is mainly reflected in the following aspects:

Cellular uptake: Positively charged lipids on the surface of LNP can bind to negatively charged mRNA through electrostatic interaction, thereby improving encapsulation efficiency and delivery effect. However, if the surface of LNP continues to be positively charged, non-specific binding and toxicity issues may arise.

Toxicity: Constant positively charged cationic lipids easily interact electrostatically with negatively charged cell membranes, causing cell membrane damage and causing cytotoxicity. In addition, positive charges may also activate TLR4-mediated signaling pathways in immune cells, causing systemic toxicity. Therefore, LNPs are usually designed with relatively neutral surface charges to reduce non-specific binding and toxicity.

A flow chart for the preparation and experiments of LNPs with
various charges. (Kong, W., 2024)

PEGylated lipids of LNPs

PEGylated lipids also play an important role in LNPs delivered by mRNA. PEGylated lipids promote LNP self-assembly by forming a hydrophilic spatial barrier on the LNP surface and prevent particle aggregation, thereby supporting particle stability. PEGylated lipids help escape capture by macrophages, thereby enhancing the stability of nanoparticles and extending their efficacy in vivo. The presence of PEGylated lipids in LNPs lengthens their circulation time, which is particularly beneficial for intravenous injection. After entering the circulation, PEGylated lipids promote the interaction of LNPs with apolipoprotein E(ApoE) in serum and promote subsequent binding to low-density lipoprotein receptors (LDLR), which are mainly located in the liver.

The content of PEGylated lipids in LNP usually does not exceed 2%. This is because excessive PEG may affect cell uptake and transfection, while appropriate amounts of PEG help achieve extrahepatic targeting. However, studies have shown that LNP containing 5% PEG-lipid is higher in tumor accumulation than 2.5% PEG-lipid LNP, suggesting that higher PEG levels may be beneficial in some cases.

The degree of PEGylation affects the in vivo distribution and cell interactions of LNP. Slightly PEGylated LNPs showed enhanced effects in activating and expanding tumor-resident antigen presenting cells (APCs), while highly PEGylated LNPs showed reduced effects. Therefore, when selecting the degree of PEGylation, factors such as treatment target, target organ or cell type, and route of administration need to be fully considered.

The type of PEGylated lipid affects the lysosomal escape of LNPs and depends mainly on the acyl chain length rather than the functional groups of the PEGylated lipid. Specifically, as the acyl chain length increased, a decrease in lysosomal escape efficiency was observed, a trend consistent with the trend of cellular uptake. For transfection of LNPs with various types of PEGylated lipids, lysosomal escape is as important as cellular uptake. Studies have shown that LNPs with DMG-PEG2k show higher transfection efficiency than similar LNPs.