The Problem That Most mRNA Gets Wrong
The appeal of mRNA therapeutics is straightforward: deliver the genetic instructions for a beneficial protein directly into cells, let the cell’s own machinery produce the protein, and achieve a therapeutic effect without permanently altering the genome. The challenge is equally clear: mRNA is a large, negatively charged, inherently unstable molecule that cannot cross cell membranes unaided, is degraded rapidly by extracellular RNases, and triggers innate immune responses that can abort expression before it begins.
Lipid nanoparticles have become the delivery system of choice for mRNA therapeutics precisely because they address these barriers. But not all LNPs deliver mRNA with equal efficiency. Understanding the intracellular journey from LNP uptake to protein expression, and the points at which that journey can fail, is essential for designing formulations that maximise therapeutic protein output.
The Intracellular Delivery Pathway
Step 1 — Cellular Uptake
LNPs enter cells primarily through endocytosis, a process in which the cell membrane engulfs the particle and internalises it within an endosome. The efficiency of uptake depends on the particle size, surface properties, and the extent to which the LNP has adsorbed apolipoprotein E (ApoE) from plasma, which facilitates receptor-mediated uptake in hepatocytes via the LDL receptor pathway. For non-hepatic targets, surface modification with targeting ligands can enhance uptake in specific cell types.
Step 2 — Endosomal Escape
This is the critical bottleneck in LNP-mediated mRNA delivery. After cellular uptake, the LNP is contained within an endosome, and the endosome matures progressively from early endosome through late endosome to lysosome, with decreasing pH at each stage. If the mRNA does not escape the endosome before lysosomal degradation occurs, it is destroyed and no protein expression results.
Ionisable lipids are the key functional component that drives endosomal escape. At physiological pH they are largely neutral, which reduces non-specific toxicity in circulation. At the acidic pH of the late endosome, they become protonated and positively charged, disrupting the endosomal membrane and enabling mRNA release into the cytoplasm. The efficiency of this process, often described as endosomal escape efficiency, is typically low: published estimates suggest that fewer than 2% of endocytosed LNPs successfully deliver their mRNA cargo to the cytoplasm.
Step 3 — Translation
Once mRNA reaches the cytoplasm, it is translated by ribosomes into the encoded protein. The efficiency of translation depends on the quality and integrity of the mRNA, including its 5-prime cap structure, the design of the untranslated regions (UTRs), and the codon optimisation of the coding sequence. mRNA degradation by cytoplasmic RNases competes with translation throughout the expression period.
Formulation Variables That Drive Delivery Efficiency
| Formulation Variable | Effect on Delivery | Optimisation Approach |
| Ionisable lipid selection | Primary driver of endosomal escape efficiency; pKa affects pH-response profile | Screen ionisable lipids across a range of apparent pKa values; target pKa 6.2-6.8 for hepatic delivery |
| Lipid molar ratios | Balance between encapsulation, stability, and endosomal escape activity | Design of experiments to map formulation space; optimise N:P ratio for each mRNA |
| PEG-lipid content and PEG chain length | Modulates particle size, colloidal stability, and cellular uptake | Higher PEG content reduces uptake; shed-able PEG chains can improve delivery |
| mRNA : lipid ratio (N:P ratio) | Determines encapsulation efficiency and particle charge | Typically optimised to achieve greater than 85% encapsulation with near-neutral zeta potential |
| Microfluidics mixing parameters | Affects particle size and homogeneity during formulation | Optimise flow rate ratio and total flow rate for target particle size |
Measuring Delivery Efficiency In Vitro
The standard cell-based assay for measuring LNP transfection efficiency uses a reporter system, typically luciferase or green fluorescent protein (GFP), where the mRNA encodes a readily detectable protein. Cells are incubated with the LNP at defined concentrations for a defined period, and protein expression is measured by luminescence or fluorescence. Comparing luminescence or fluorescence across formulations with identical mRNA doses gives a direct readout of relative delivery efficiency.
In vitro delivery efficiency is a useful screening tool for ranking formulations, but it does not always predict in vivo performance. Cell lines used for screening may have different uptake and endosomal biology to the target cell type in vivo, and the absence of serum proteins including ApoE in some in vitro assays can significantly underestimate hepatic delivery efficiency.
Ardena’s mRNA LNP Formulation Expertise at Oss
Ardena’s formulation scientists in Oss have expertise in ionisable lipid-based LNP formulation for mRNA and nucleic acid payloads. The site operates microfluidics and nanoparticle extrusion platforms for LNP manufacture at both screening and GMP scale, with integrated analytical capability for particle size, PDI, encapsulation efficiency, and zeta potential measurement.
Formulation development programmes at Oss are structured to move efficiently from initial lipid screening through to a robust formulation with defined critical quality attributes, supported by in vitro characterisation and stability data suitable for an IND or IMPD filing.
