Delivery of LNPs Part II: Active Targeting

Date: 01-June-2023

Previously, Part I of this two-article series reviewed passive targeting and local administration approaches for delivering LNPs to their site of action. In Part II, we will now look at strategies that involve incorporating ligands into LNPs for active targeting.

Targeted delivery of LNPs

Targeted delivery of LNP encapsulated gene therapies to their target tissues or cells by utilizing ligand-receptor binding, reducing the likelihood of off-target effects. (Figure created with

      1. Active targeting

Active targeting utilizes ligand-receptor binding to deliver the payload to the target tissues or cells. As the receptors are either overexpressed in or exclusive to a particular tissue or cell-type, the likelihood of off-target effects is greatly reduced. Most of the ligands used for targeting can be broadly classified as small molecules, peptides and antibodies, and oligonucleotides.

      1.1 Small molecules

Monosaccharides such as glucose, mannose and galactose have been commonly employed as ligands to target different cell-types for different applications. Mannosylated LNPs encapsulating self-amplifying RNA have been shown to have an increased uptake in to dendritic cells as well as higher vaccination rates compared to non-mannosylated LNPs after intradermal administration1. Mannose incorporation has also been used to target liver sinusoidal endothelial cells (LSECs)2. N-Acetylgalactosamine (GalNAc), an amino sugar derivative of galactose and a ligand for asialoglycoprotein receptor, has been effectively exploited to deliver oligonucleotides to the liver. Kasiewicz et al. developed GalNAc-LNPs that achieved a significantly higher gene editing efficacy livers in a non-human primate model of hypercholesterolemia compared to non-targeted LNPs3.

Another approach used for targeted LNPs is tethering small molecule ligands to lipidoids, lipid-like molecules, and incorporating the ligand-conjugated lipidoids into the LNPs. For example, Han et al. used anisamide, a ligand for sigma receptor expressed on proliferating active fibroblasts such hepatic stellate cells, for targeting of LNPs. They observed a two-fold increase in silencing by using siRNA LNPs with an anisamide-tethered lipidoid versus a non-targeted LNP4. A similar approach was used by Ma et al. to deliver LNPs across the blood brain barrier (BBB) by adding neurotransmitter-lipidoids to previously BBB-impermeable LNPs, enabling them to functionally deliver three types of payloads into the mouse brain5.

      1.2 Peptides and antibodies

The phage-display technique has been successfully used to screen and identify peptides and antibodies that target specific cell-types6–9. A common strategy used to decorate LNPs with these targeting moieties is to incorporate a functionalized PEG lipid in the LNPs followed by chemically grafting the targeting peptides to the PEG lipid. Targeting peptides identified using phage-display were conjugated to the PEG lipid, which guided the LNPs to the neural retina following intravitreal injection in rodents and non-human primates6. Using a similar approach, conjugation of a Fab-C4 antibody to the PEG lipid for caveolae targeting resulted in a 40-fold enhancement in protein expression in the lungs compared to the control LNPs9.

Several studies using antibodies as targeting ligands have successfully delivered LNPs to the desired organs or cell-types in pre-clinical studies. In one such study, Kedmi et al. developed a platform approach wherein LNPs were coated with antibodies using a lipoprotein. They observed that based on the type of antibody used, the LNPs showed affinity for different types of immune cells in vivo10. Another study used platelet endothelial cell adhesion molecule-1 (PECAM-1) antibodies conjugated on to the LNP surface to selectively deliver mRNA to the lung vasculature11. Some other examples include CD29 antibody-coated LNPs against mantle cell lymphoma cells10, CD4 antibody-coated LNPs for targeting CD4+ T cells and spleen12, EGFR antibody-LNPs against ovarian cancer cells13, and Ly6C antibody-LNPs targeting Ly6C+ leukocytes14.

      1.3 Oligonucleotides

Aptamers are single-stranded oligonucleotides with distinct tertiary structures that target specific cell-types. To achieve gene-silencing in osteoblasts, an aptamer targeting osteoblasts was first selected from a library using cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX). LNPs prepared with the aptamer CH6 showed significantly higher gene-silencing in osteoblasts compared to the unlabeled LNPs15. In an in vitro study, incorporation of a C-C chemokine receptor type 5 (CCR5)-selective RNA aptamer enabled transport of LNPs across a simplified BBB model and facilitated their uptake into CCR5-expressing cells7.

      2. Challenges and outlook

Some of the strategies described above seem rather promising, but translating these technologies into clinic will require rigorous development and testing. For active targeting strategies, the initial challenge would be to develop a scalable process to manufacture the LNPs decorated with the ligand. A review by Menon et al. describes different manufacturing approaches for production of active targeting LNPs16. Use of ligands will also potentially increase the cost of manufacturing, owing not only to the cost of the ligand itself, but also the extra processing steps required to incorporate the ligand into the LNPs.

Another key parameter to be evaluated and optimized would be the stability of the ligand as well as the LNPs during manufacturing and storage. Analysis of the ligand content and activity will likely entail thorough method development, in addition to other routine analytical methods for LNPs. This will be critical in assessing the efficiency of conjugation of ligands and conservation of their functional activity after manufacturing. Regulatory guidance would also need to be updated to keep up with the novel targeting strategies.

Some other targeting approaches not discussed here include incorporating cell membrane components in the LNPs, as well as stimuli responsive LNPs that would release their payloads when triggered with certain stimuli such as pH and temperature. Apart from targeting, it is also important to consider the transfection efficiency and intracellular release of the payload, both of which may be tissue or cell-type dependent.

A number of factors need to be considered when selecting an approach for targeting, such as the organ or tissue and presence of specific receptors, pathology, cost of manufacturing, stability of the product, feasibility of local administration, and off-target effects or toxicity. While active targeting is potentially more complex and expensive, it is a more precise approach compared to passive targeting.

Overall, with the several approaches being designed and assessed, it would not be long before technologies for precise delivery of LNPs to their site of action are developed and approved for clinical use, thus creating therapies and vaccines which are safer and more effective for patients.

Congratulations! Thank you for reading our first article series and as a token of appreciation, we would like to offer a discount code for your next mRNA purchase. Please use “LNPARTICLE20” for 20% off on your next mRNA order.


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