What Really Makes LNP Work? From Lipids to Enabling Genetic Medicines

Imagine you’ve engineered the perfect mRNA sequence. Now what? mRNA is fragile, easily degraded, and does not easily enter cells. It needs an escort: something stealthy, biocompatible, and capable of releasing it inside the cell.

That’s where lipid nanoparticles (LNPs) come in. But they’re more than a simple shell. They’re engineered vehicles, finely tuned at the nanoscale to navigate biological chaos. LNPs not only act as a delivery system, but also contribute to vaccine immunogenicity.

The story of LNPs didn’t start with COVID-19, it began with liposomes in the 1960s. Over six decades, breakthroughs such as the ethanol dilution method, PEGylated lipids, and ionizable lipid chemistry transformed simple bilayer vesicles into highly engineered RNA carriers.

But here’s the key point: each advancement in lipid formulation has brought us closer to scalable, tissue-specific RNA therapeutics. Rationally designing next-generation LNP technologies with improved potency & tolerability are key toward realizing extrahepatic nucleic acid delivery.

Figure 1 – Ten key highlights in the 56-year evolution of LNPs (1964-2020)

In the next four articles we will unpack the science (and the art) behind LNP technologies: key components & formulation methods, rigorous characterization & morphology, pharmacokinetics & biodistribution, and delivery beyond the liver. Let’s start with the basics.

The Core Four: Lipids That Build an LNP

Formulation design plays a critical role and must account for key factors such as administration route, stability, release profile, targeting, biocompatibility, manufacturability, scalability, and regulatory strategy. Explore our previous article to learn more.

All clinically approved LNPs from Onpattro® (Alnylam), Comirnaty® (BNT162b2; BioNTech), Spikevax® (mRNA-1273; Moderna), or Kostaive® (ARCT-154, Arcturus) employ a core mix of four lipid types:

Table 1 - Lipids That Build an LNP

Figure 2 – LNP composition

Ionizable Lipids: The Heart of RNA Delivery

Ionizable cationic lipids are the workhorse of RNA delivery. They possess neutral charge at physiological pH (reducing systemic toxicity) and become positively charged in acidic environments, such as endosomes, triggering membrane fusion and cargo release.

Types of Ionizable Lipids (ILs)

A 2021 review grouped ionizable lipids into five broad categories:

Unsaturated ionizable lipids - contain unsaturated lipid chains (like DLin-MC3-DMA used in Onpattro®) and enhance membrane fusion through bilayer destabilization, supporting liver-targeted siRNA delivery.

Multi-tail ionizable lipids - contain more than two lipophilic tails, offer a cone-shaped geometry that boosts endosomal disruption and potency, although their low biodegradability raises safety considerations.

Ionizable polymer-lipids - contain polymer or dendrimer bonds, enable endothelial or tumor-selective delivery through hydrophobic aggregation, but face challenges due to backbone toxicity and complexity.

Biodegradable ionizable lipids - include cleavable bonds (esters, disulfides) that allow for safer repeated dosing by breaking down into non-toxic byproducts, ideal for chronic therapies.

Branched-tail ionizable lipids - contain branched tails, like ALC-0315 in Comirnaty® and SM-102 in Spikevax®, enhance endosomal escape and mRNA expression by forming cone-shaped structures and slowing degradation for extended intracellular presence.

Each class contributes uniquely to balancing efficacy, safety, and tissue targeting in RNA-based therapeutics. Recent work by NanoVation has proposed a heuristic design framework for ionizable lipids (published soon), identifying structural motifs such as branched alkyl tails and biodegradable linkers that promote endosomal escape while minimizing systemic toxicity.

Each tweak impacts:

• RNA binding strength
• Lipid packing and morphology
• Immunogenicity and tolerability

For a deeper dive into the role of other lipids, take a look at this review article and our previous article here.

It’s Not Just Lipids: The Role of Buffers and Salts

LNPs are sensitive to their environment - pH, salt concentration, and excipients all matter.

Recent study showed:

• pH 4 in the mRNA aqueous phase yields the highest encapsulation efficiency and improves luciferase expression 2.9-fold compared to pH 6.
• Sucrose at 300 mM acts as a cryoprotectant, preserving structure during freeze–thaw cycles.
• Buffer choice matters: Tris preserved transfection efficiency post-lyophilization better than PBS.

These aren’t trivial details. They influence final biodistribution, mRNA integrity, and therapeutic performance.

Why pKa Matters

Ionizable lipids are key to how LNPs deliver nucleic acids. Generally, ionizable lipids  contain amino groups that can gain or lose a proton depending on the pH (how acidic the environment is). The pKa is the specific pH where half of these ionizable lipids are charged (protonated) and half are not. When incorporated into LNPs, ionizable lipids show a lower pKa than when they're free in solution - meaning the surrounding lipids and mRNA affect their behavior. The pKa value of ionizable lipids within an LNP is called the apparent pKa. This value is crucial, because at a pH below their pKa, ionizable lipids become positively charged, allowing for more efficient encapsulation of negatively charged mRNA, as well as improving delivery efficiency. Examples of ionizable lipids with optimized pKa values include MC3, SM-102, ALC-0315, and ATX-126.

The apparent pKa of ionizable lipids is critical. Data suggests that a sweet spot between 6.2 and 6.5 enables efficient endosomal escape while maintaining a reasonable safety profile.

Different RNA payloads may require fine-tuning:

• MC3 is sufficiently potent for siRNA
• SM-102 or ALC-0315 are better suited for mRNA

pKa too low? The lipid won’t protonate in endosomes. Too high? It might stay charged in blood, increasing off-target effects and toxicity.

Endosomal Escape: The Key Mechanism

LNPs deliver mRNA by first entering cells through a process called endocytosis, a natural mechanism where the cell membrane folds inward to "swallow" external particles. Once inside, LNPs become trapped in small compartments called endosomes.

As these endosomes mature, their internal environment becomes increasingly acidic. This drop in pH is crucial: it triggers the ionizable lipids within the LNPs to gain a positive charge (they become protonated). These positively charged lipids interact with the negatively charged lipids in the endosomal membrane. This interaction disrupts the endosomal membrane by rearranging lipids into a cone-like shape (called the hexagonal HII phase) driven by an ion pair of the lipid headgroups, which promotes membrane fusion. This allows the mRNA to escape into the cytosol, where it can be used by the cell’s machinery to produce proteins.

However, this step - called endosomal escape - is often limited. Some studies have shown that less than 2% of LNPs successfully release their mRNA into the cytosol. Most of the cargo remains trapped and eventually degrades.

Improving endosomal escape is a major focus in mRNA-LNP research, as it plays a critical role in ensuring that the therapeutic mRNA reaches its target and produces the desired effect. Recent research emphasizes rational lipid structure engineering to improve efficacy while avoiding toxic positive charge accumulation - a critical factor for clinical translation.

Why LNPs are primarily taken up by the liver

LNPs are mostly taken up by the liver after injection due to their interaction with proteins in the bloodstream - especially apolipoproteins such as ApoE.

Here’s how it works:

• ApoE binds to LNPs, making them look like chylomicron remnants (natural fat particles the body clears via the liver).
• This "disguise" allows LNPs to be recognized by LDL receptors (LDLR) on hepatocytes, the main cells in the liver.
• As a result, LNPs are efficiently taken up by the liver through ApoE-LDLR interactions.

The surface charge of LNPs (which depends on their pKa) plays a key role in how much ApoE binds. At blood pH (~7.4), ionizable lipids with a pKa of ~6.4 are ~10% positively charged, enough to affect which proteins stick to the LNP surface (also called the protein corona – this will be explored more in depth in one of our next articles).

Finally, particle size matters: smaller or larger LNPs can be tuned to target specific liver cell types like hepatocytes, Kupffer cells, or liver endothelial cells. For example, Onpattro®, the first FDA-approved siRNA-LNP drug, was designed to exploit ApoE binding for liver delivery.

Going Beyond the Liver

LNPs naturally accumulate in the liver due to apoE-mediated uptake by hepatocytes, as described above. But delivering RNA elsewhere is the next frontier. New strategies for delivery to tissues beyond the liver following systemic administration include:

• Bilayer-rich liposomal LNPs (phosholipids / cholesterol) with extended systemic circulation and uptake in tissues beyond the liver
• SORT (Selective Organ Targeting) systems with additional lipids to tweak surface charge and direct LNPs to tissues like lung and spleen
• Ligand-modified LNPs (e.g. CD4 for T cells, mannose for dendritic cells) to enable active targeting

An alternative to IV delivery beyond the liver, is local injection into specific tissues. Insights into extrahepatic delivery and how to rationally design LNPs will be covered in one of our upcoming articles.

LNP Formation: What’s Really Going On?

To develop effective LNP-based therapies, it’s essential to first understand how lipid nanoparticles form and why some still form without cargo.

Although the exact formation mechanism was unclear for years, scientists discovered a fusion-dependent mechanism of formation. Li et al. proposed another key insight: during rapid mixing, solvent quality changes too fast, leading to the formation of empty LNPs before RNA and lipids even interact. This is especially problematic for large molecules like mRNA, which require precise timing to encapsulate efficiently.

That’s why mastering LNP preparation techniques is critical - each method offers a different balance of control, efficiency, and safety:

• Solvent-Based Rapid Mixing involves dissolving lipids in ethanol before mixing into an aqueous phase containing the nucleic acid. Various  Mixing systems have been developed to precisely regulate flow conditions for creating uniform particles with high encapsulation efficiency (EE).

While rapid mixing helps minimize solvent gradients and reduce the number of empty particles, some degree of inefficiency remains - especially with large RNA cargos like mRNA.

We’re still learning how to optimize this critical step to boost RNA loading and minimize waste, in addition to developing robust methods to address this key challenge.

CMC and Manufacturing: More Than Just a Recipe

Developing RNA-LNP therapeutics isn’t just about good science. It’s about controlling variability.

Table 2 - The Three-Legged Framework

Without solid links between these, scalability and regulatory approval become serious hurdles.

Formulation design plays a critical role and must account for key factors such as administration route, stability, release profile, targeting, biocompatibility, manufacturability, scalability, and regulatory strategy. Explore our previous article to learn more.

The physical properties of the particles, including size and size distribution, significantly influence their efficacy. These properties affect which cells take up the particles and how efficiently this uptake occurs. To achieve consistent and reproducible results, it's crucial to promote optimized conditions during the formation process to ensure a uniform product with homogeneous size distribution. This consistency is vital for the quality and effectiveness of LNPs in various applications, both in vivo and in vitro.

Why does this matter? Because inconsistency at the particle level can lead to unpredictable drug performance - some LNPs might deliver the mRNA well, while others might fail. This has big implications for companies developing RNA vaccines or therapeutics: product quality and clinical outcomes can be affected by subtle differences in LNP composition.

The takeaway is clear: next-gen delivery platforms must go beyond just average size and encapsulation efficiency. There’s growing value in rigorously characterizing LNPs which can be produced at scales required for therapeutic potential, which can lead to more reliable products and faster regulatory approval.

Freeze-Thaw: The Cold Chain Dilemma

Freeze-drying (lyophilization) solves a major challenge in mRNA-LNP stability. Hydrated LNPs are highly sensitive to ice crystal damage during freezing, which disrupts their structure and reduces mRNA encapsulation efficiency.

Key solutions:

• Use of Tris or phosphate buffers (recent study showed that Tris preserved transfection efficiency post-lyophilization better than PBS.)

• Sucrose as a cryoprotectant (recent study suggests Sucrose at 300 mM)
• Continuous spin-freeze drying improves throughput and quality control

Results:

• Lyophilized LNPs remained stable for 12+ weeks at 4°C, 22°C, and 37°C
• Transfection efficiency was preserved in vitro and in vivo (mouse luciferase model)
• Cryo-EM imaging revealed buffer-dependent behavior during drying

This is the first study to show that freeze-dried mRNA LNPs can be stored safely at room and body temperature without losing function - making mRNA vaccines more accessible globally.

IVIVC: The In Vitro–In Vivo Correlation Gap

The performance of mRNA-LNPs is governed by their ability to encapsulate, protect, and deliver mRNA efficiently. Once inside the body, many factors, including biodistribution, immune response, and endosomal escape, influence efficacy. Researchers tested four widely used LNPs (a standard lipid composition containing SM-102, ALC-0315, MC3, and C12-200) with similar physicochemical properties in both in vitro (cell lines) and in vivo (mice) settings.

Key findings:

• In vitro transfection efficiency did not correlate with in vivo protein expression or immune responses.
• LNP uptake mechanisms vary: ApoE-dependent for some (like SM-102), and macropinocytosis for others (like C12-200).
• Endosomal escape efficiency, influenced by lipid structure and pKa, plays a major role in successful mRNA translation.
• The immune-stimulating properties of LNPs (e.g., through TLR4/CD1d interaction) act as adjuvants, rendering antibody levels independent of protein expression.

Current in vitro assays using immortalized cell lines and even primary cell lines are not reliable predictors of in vivo performance or vaccine efficacy. Bridging this gap requires advanced models that capture tissue complexity, circulation effects, and immune system behavior. Ionizable lipids also influence immune responses, making LNPs active participants (not just carriers) in vaccine formulations.

Final Thoughts: Where Are We Headed?

We believe translating nanomedicines requires both scientific depth and business clarity. What you’ve just read is the foundation.

Therapeutic LNP development is not plug-and-play. Rational design of fit-for-purpose LNP formulations is key to improving therapeutic index and clinical translatability. It’s a balance of:

• The right lipid mixture with appropriate safety and efficacy
• Scalable manufacturing techniques and components
• Appropriate preclinical characterization

The design choices outlined in this article, i.e. ionizable lipids, pKa tuning, helper lipid selection, and formulation buffers, are all levers for improving LNP performance. LNP experts are crucial to optimize these parameters for engineering the delivery systems that are not just liver-optimized but customized for a variety of tissues and tuned for diverse therapeutic applications.

Ready to dive deeper? If you haven’t yet begun with our foundational overview of nanomedicine basics, start here with our first article, then check all RNA modalities here, and finally join us next as we unpack LNP CQAs and morphology - from blebs to bilayers, and what they mean for delivery success.

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Written by

Dr. Dominik Witzigmann 

Dominik is an entrepreneurial scientist with deep expertise in nanomedicines and nucleic acid delivery. In 2024, he was named Highly Cited Researcher, recognizing him among the world’s most influential researchers in the field. Dominik obtained his Ph.D. in Pharmaceutical Technology from the University of Basel in Switzerland, and held research positions at leading institutions including University College London (safety/tox), German Cancer Research Center (RNAi & cancer), University of Basel (targeted nanomedicines & DNA delivery) and the University of Zurich (mRNA-based genome editing). To focus on extrahepatic RNA delivery, he later joined the team of Prof. Pieter Cullis at the University of British Columbia. Dominik has held leadership roles in Canada’s NanoMedicines Innovation Network (NMIN) and served on the Board of the CRS Gene Delivery and Genome Editing Focus Group. To translate next-generation LNP technologies into the clinic, Dominik co-founded and leads the LNP-nucleic acid company NanoVation Therapeutics. 

Dr. Jayesh Kulkarni

Dr. Kulkarni obtained his PhD from the University of British Columbia and has over 15 years experience in the nanoparticle drug delivery field. He has published over 40 peer-reviewed articles and is a co-inventor on numerous patents. Dr. Kulkarni’s research has focused on the role of the various lipid components in LNP and the biophysics that governs particle formation. His work has contributed to clinical translation, including scale-up and manufacturing of LNP systems in accordance with GLP and GMP regulations. Dr. Kulkarni is a leader in the design and development of lipid nanoparticle (LNP) formulations of small molecule and nucleic acid therapeutics. He currently serves as the Chief Scientific Officer of NanoVation Therapeutics, an LNP-RNA formulation developer.

Daniel Kurek, MSc

Daniel has over 10 years experience in organic synthesis, formulation development, and scale-up of LNPs. He started his career in lipid nanoparticles at Evonik Canada in formulation and process development. In his current role as Associate Principal Scientist, Formulation – Team Lead at NanoVation Therapeutics™, he oversees the development of novel long-circulating LNPs for targeting extrahepatic tissue, improving the potency and characteristics of LNPs encapsulating various nucleic acid payloads, and is a co-inventor on several of NanoVation’s key patents. Daniel holds an MSc in organic chemistry from the University of British Columbia.

Dr. Marija Petrovic 

Marija is a pharmacist with a PhD in Biopharmacy from the University of Geneva, and a cancer research (ISREC)–trained professional through EPFL, with over seven years of experience in nanomedicine. During her PhD, she worked on miRNA and STING ligand nanocomplexes for cancer immunotherapy, gaining deep expertise in nanoparticle characterization and translational workflows. Certified by the EU-NCL in nanobiotechnology and awarded by Innosuisse (Swiss Innovation Agency) with two prizes (jury and public) for the best life science project, she also earned support from FONGIT, Geneva’s leading deep-tech incubator. As the founder of NanoSphere and an active contributor to the Controlled Release Society (Communication Chair for the Gene Delivery and Editing Group (GDGE), and Industry representative at Nanomedicine and Nanoscale Delivery (NND)), Marija focuses on making next-gen medicine scientific advances more visible, understandable, and useful to the communities that can turn them into impact.