Delivery of LNPs (Part I): Passive targeting and localized administration

Date: 23-May-2023

The two lipid nanoparticle (LNP)-based vaccines at the forefront of the Covid-19 pandemic have validated the potential of LNP technology for nucleic acid delivery1. However, reports of rare side-effects from these vaccines have also necessitated the development of more precisely targeted LNPs, that will minimize off-target effects and reduce toxicity 2. In addition to reducing side effects, precise delivery also facilitates a reduction in the dose required to elicit a response. The significance of dose reduction is amplified when the payload is expensive or difficult to manufacture.

Delivery of LNP encapsulated gene therapies to their intended site of action decreases off-target effects while improving efficacy of therapies (Figure created with

Current FDA-approved LNP vaccines and therapeutics do not include any targeting moieties or strategies to deliver to a specific organ, tissue or cell type. Inherently, LNPs localize in the liver after systemic administration3, and this property has been exploited by several groups to develop therapeutics against hepatic pathologies4. However, to harness the full potential of LNPs and to advance them as vaccines or therapies encompassing a wide range of indications, it is necessary to develop strategies to deliver LNPs more precisely to their site of action.

Strategies for restricting LNPs to their site of action include passive targeting, localized administration, and active targeting. Part I of this two-article series will focus on passive targeting and local administration approaches for LNPs.

        1. Passive targeting strategies

Passive targeting involves modifications of physicochemical attributes such as the particle size or surface charge of LNPs to enhance their distribution to a particular organ, tissue or cell-type without the need for the incorporation of targeting ligands. This strategy relies on the anatomy and physiology of the tissue or organ to preferentially internalize particles of a specific charge or size. A well-known example of passive targeting is therapies that exploit the leaky vasculature of solid tumors, which allows higher permeation of therapeutics compared to normal tissues with intact blood vessels. This phenomenon is also known as the enhanced permeation and retention (EPR) effect5.

Size, surface charge and chemistry are important parameters that can be optimized to obtain desired biodistribution. Findings from some studies that investigate size, charge and lipid compositions for passive targeting are cited below. It should be noted that some of these parameters affect multiple physicochemical attributes (e.g., the concentration of PEG lipid affects surface properties as well as particle size of the LNPs).

        1.1 Size

Particle size of LNPs is typically measured using dynamic light scattering (DLS) and the mean hydrodynamic diameter is reported as the particle size. Many have studied the impact of LNP size on the efficacy of therapy and it has been highlighted in various observations that the optimal size largely depends on the route of administration and the intended indication. Chen et al. demonstrated that optimal size of LNPs for hepatocyte gene silencing was around 80 nm. Particles smaller than 80 nm are likely to be less fusogenic whereas larger particles would not be able to pass through the endothelial fenestrations6. Similar observations were noted by Sato et al.; LNPs larger than the fenestrae diameters showed a significantly reduced gene silencing activity in hepatocytes. On the other hand, this reduction in activity was not observed for LNPs up to 200 nm in liver sinusoidal endothelial cells (LSECs)7.

In studies comparing the effect of size on distribution of LNPs to lymph nodes following subcutaneous injection in mice, the authors observed that particles with an average diameter of 30 nm were more efficiently translocated into the lymph nodes and taken up by the CD8+ cells compared to larger LNPs with 100 nm or 200 nm diameters8. In contrast, when delivered via intravenous injection, smaller particles showed poor serum stability and hence lower activity9.

When comparing particle size of LNPs for delivery to the eye, larger particles ~150 nm containing smaller amounts of PEG lipid (0.5%) performed better compared to their smaller counterparts ~ 50 nm (containing 5% PEG lipid) and were able to transfect different regions of the eye based on the route of administration10.

        1.2 Charge and lipid composition

Altering the surface charge of LNPs has been employed in the selective organ targeting (SORT) approach to selectively deliver payloads to the spleen, liver or lung. For example, incorporating a cationic lipid such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) enhanced delivery to the lung, while anionic lipid such as 1,2-dioleoyl-sn-glycero-3-phosphate (18PA) enhanced delivery to the spleen11,12. A study comparing the charge of LNPs also found that negatively charged LNPs distributed into the lymph nodes more efficiently than neutral or positively charged LNPs8. It has also been shown that base LNPs without charged lipids preferentially accumulated in the liver11,12.

In a study comparing helper lipids, LNPs containing 1,2-dioleoyl-sn-glycero-3-PE (DOPE) accumulated in the liver, whereas those with distearoylphosphatidylcholine (DSPC) target the spleen. This was observed for siRNA and mRNA containing LNPs, with a larger fold change in distribution for mRNA LNPs13. Choice of ionizable cationic lipids or lipidoids have also been shown to impact distribution of LNPs. For example, LNPs prepared with the ionizable lipid DLin-KC2-DMA showed more transfection of the spleen compared to those prepared with DLin-MC3-DMA14. Guimaraes et al. observed that changing the ratios of lipids or the lipid to RNA ratios had an effect on the biodistribution profiles of LNPs15. In a recent study, Naidu et al. observed that distribution of LNPs was greatly influenced by the linkers and hydrophobic tail chains of the ionizable amino lipids. From the screen, the authors identified one lipid specifically delivered LNPs to the liver and another targeting a specific macrophage population16.

        1.3 Impact of the “biomolecular corona” on biodistribution

Upon administration into the body, LNPs first interact with physiological environment containing a myriad of biomolecules such as proteins, lipids, carbohydrates and electrolytes, that adsorb on to the LNP surface and form the “biomolecular corona”17. Physicochemical characteristics of the LNPs influence the composition of this biomolecular corona, which in turn determines the biodistribution and cellular uptake of the LNPs. This effect is exemplified by Onpattro’s liver tropism. Upon administration, one of the molecules that adsorbs on the Onpattro LNP surface is apolipoprotein E (ApoE), which is suggested to enable their delivery to hepatocytes18.

        2. Local administration

This approach is a simple solution to limit distribution of LNPs within organs or tissues that are accessible for direct administration. Some examples are discussed below.

Ocular delivery can be achieved by several routes including topical, subretinal, intravitreal and suprachoroidal. When LNPs were administered subretinally, transfection was observed primarily in the retinal pigment epithelium (RPE), whereas upon intravitreal delivery, the expression was seen in the Muller glia, optic nerve head (ONH) and the trabecular meshwork (TM)19.

Lungs are another organ accessible for local delivery via nebulization. Chronic respiratory diseases such as cystic fibrosis, asthma, chronic obstructive pulmonary disease (COPD), and pulmonary infections are some conditions against which inhaled LNPs are being investigated in several pre-clinical studies20. ReCode Therapeutics has developed RCT1100, an LNP formulation for direct administration to the airways as an aerosol. Phase 1 trials of RCT1100 for treatment of primary ciliary dyskinesia are expected this year21.

LNPs containing human frataxin (hFXN) mRNA were injected intrathecally to deliver them to the dorsal root ganglia (DRG) in mice as a therapy against Friedreich’s ataxia. Their uptake in the spinal cord or cerebellum was not detected. When the same formulation was injected intravenously, LNPs were primarily distributed in the liver, and not in the cardiac tissue or central nervous system, which are the organs affected in Friedreich’s ataxia22.

        3. Challenges and outlook

Passive targeting of LNPs does not involve incorporation of ligands and the manufacturing process is usually similar to that of non-targeted LNPs. Hence, there is relatively less complexity in developing LNPs that are delivered to the site of action via passive targeting. However, as LNPs with different particle sizes or charges have different biodistribution profiles, comprehensive safety and toxicity evaluations will be essential. In addition, specificity achieved by passive targeting is variable as it is to a large extent impacted by the physiological environment in which they are introduced. The physiological environment is in turn influenced by several factors such as age, body weight, muscle mass and disease state. These factors should therefore be considered during testing and development of passively targeted vaccines or therapies.

Local administration can, to a certain extent, improve the specificity of deliver of LNPs to their site of action. However, the main drawback of local administration is the limited organs or sites available for direct administration. Furthermore, local administration for certain routes may be challenging and may result in lower patient compliance or an increase in cost due to specific skills and expertise required for administration. It would also be critical to assess whether and to what extent does the product escape into systemic circulation, and its safety profile. Although local administration confines the distribution to a particular organ or tissue, it would be necessary to assess the uptake into different cell-types within the site of administration. To limit distribution to a particular cell-type within a tissue, strategies that combine active or passive targeting approaches along with local delivery are being studied10,20,21.

Unlike passive targeting and local administration, active targeting strategies use ligand-receptor binding to guide LNPs to their site of action. Some examples of active targeting approaches will be discussed in Part II of this articles series. Stay tuned!

Congratulations! Thank you for reading our first article 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.


  1. Milane L, Amiji M. Clinical approval of nanotechnology-based SARS-CoV-2 mRNA vaccines: impact on translational nanomedicine. Drug Deliv Transl Res. 2021;11.
  2. Hosseini R, Askari N. A review of neurological side effects of COVID-19 vaccination. Vol. 28, Eur J Med Res. 2023.
  3. Shi B, Keough E, Matter A, Leander K, Young S, Carlini E, et al. Biodistribution of small interfering RNA at the organ and cellular levels after lipid nanoparticle-mediated delivery. J Histochem Cytochem. 2011;59.
  4. Witzigmann D, Kulkarni JA, Leung J, Chen S, Cullis PR, van der Meel R. Lipid nanoparticle technology for therapeutic gene regulation in the liver. Vol. 159, Adv Drug Deliv Rev. 2020.
  5. Wu J. The enhanced permeability and retention (EPR) effect: The significance of the concept and methods to enhance its application. J Pers Med. 2021;11.
  6. Chen S, Tam YYC, Lin PJC, Sung MMH, Tam YK, Cullis PR. Influence of particle size on the in vivo potency of lipid nanoparticle formulations of siRNA. J Control Release. 2016;235.
  7. Sato Y, Note Y, Maeki M, Kaji N, Baba Y, Tokeshi M, et al. Elucidation of the physicochemical properties and potency of siRNA-loaded small-sized lipid nanoparticles for siRNA delivery. J Control Release. 2016;229.
  8. Nakamura T, Kawai M, Sato Y, Maeki M, Tokeshi M, Harashima H. The effect of size and charge of lipid nanoparticles prepared by microfluidic mixing on their lymph node transitivity and distribution. Mol Pharm. 2020;17.
  9. Sato Y, Hatakeyama H, Hyodo M, Harashima H. Relationship between the physicochemical properties of lipid nanoparticles and the quality of siRNA delivery to liver cells. Mol Ther. 2016;24.
  10. Ryals RC, Patel S, Acosta C, McKinney M, Pennesi ME, Sahay G. The effects of PEGylation on LNP based mRNA delivery to the eye. PLoS One. 2020;15.
  11. Dilliard SA, Cheng Q, Siegwart DJ. On the mechanism of tissue-specific mRNA delivery by selective organ targeting nanoparticles. Proc Natl Acad Sci USA. 2021;118.
  12. Cheng Q, Wei T, Farbiak L, Johnson LT, Dilliard SA, Siegwart DJ. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nat Nanotechnol. 2020;20:701–10.
  13. Zhang R, El-Mayta R, Murdoch TJ, Warzecha CC, Billingsley MM, Shepherd SJ, et al. Helper lipid structure influences protein adsorption and delivery of lipid nanoparticles to spleen and liver. Biomater Sci. 2021;9.
  14. Algarni A, Pilkington EH, Suys EJA, Al-Wassiti H, Pouton CW, Truong NP. In vivo delivery of plasmid DNA by lipid nanoparticles: the influence of ionizable cationic lipids on organ-selective gene expression. Biomater Sci. 2022;
  15. Guimaraes PPG, Zhang R, Spektor R, Tan M, Chung A, Billingsley MM, et al. Ionizable lipid nanoparticles encapsulating barcoded mRNA for accelerated in vivo delivery screening. J Control Release. 2019;316.
  16. Gonna Somu Naidu SBYSRRRPSAEMGIHHSCAADP. A Combinatorial Library of Lipid Nanoparticles for Cell Type-Specific mRNA Delivery. Adv Sci. 2023;2301929.
  17. Francia V, Schiffelers RM, Cullis PR, Witzigmann D. The Biomolecular Corona of Lipid Nanoparticles for Gene Therapy. Bioconjug Chem. 2020;31.
  18. Akinc A, Querbes W, De S, Qin J, Frank-Kamenetsky M, Jayaprakash KN, et al. Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol Ther. 2010;18.
  19. Patel S, Ryals RC, Weller KK, Pennesi ME, Sahay G. Lipid nanoparticles for delivery of messenger RNA to the back of the eye. J Control Release. 2019;303.
  20. Lokugamage MP, Vanover D, Beyersdorf J, Hatit MZC, Rotolo L, Echeverri ES, et al. Optimization of lipid nanoparticles for the delivery of nebulized therapeutic mRNA to the lungs. Nat Biomed Eng. 2021;5.
  21. Study Evaluating the Safety and Tolerability of RCT1100 in Healthy Subjects. 2023.
  22. Nabhan JF, Wood KM, Rao VP, Morin J, Bhamidipaty S, Labranche TP, et al. Intrathecal delivery of frataxin mRNA encapsulated in lipid nanoparticles to dorsal root ganglia as a potential therapeutic for Friedreich’s ataxia. Sci Rep. 2016;6.