LNPs as drug delivery platforms: Overcoming bioanalytical hurdles

By Jane Byrne contact

- Last updated on GMT

© GettyImages/Love Employee
© GettyImages/Love Employee

Related tags: lipid, Nanoparticles, Rna, Oligonucleotide, LNPs

Lipid nanoparticle systems (LNPs) as a delivery mechanism for novel drug modalities are proving effective, but there are bioanalytical challenges associated with encapsulation, says an expert.

There is a need to further understand the exposure and distribution of LNPs as a means of developing effective nucleic acid therapies and LNP toxicity arising from localized accumulation, says Iain Love, director, chromatographic bioanalysis and residues at Charles River.

We ran a Q&A with the specialist to hear about the modalities that LNPs support and why ​bioanalysis of LNP components is important.

BioPharma-Reporter: What opportunities are offered by LNPs as a delivery system for novel drug modalities?

Iain Love: ​Lipid nanoparticles (LNPs) are complex, non-viral drug delivery systems that can confer stability, selectivity and delivery efficiency of highly labile therapeutics. Primarily, the encapsulating nanoparticle helps to protect the active payload from systemic degradation. In addition, relative to the naked therapy, customization of LNPs with surfaced attached ligands can help direct the particle to target tissues and promote internalization of the active therapeutic agent into cells.

BPR: What modalities do they support?

IL:​ The first approval of a liposoma drug formulation was Doxil in the mid-1990s. Doxil is a liposomal formulation of the small molecule oncology drug, doxorubicin. Where LNPs really come into their own, however, is in the delivery of nucleic acid therapies. Nucleic acid therapies such as oligonucleotide and RNA-based drugs are a fascinating class of molecules with long-lasting pharmacological effects through gene editing, inhibition, addition or replacement. However, naked oligonucleotide and RNA molecules are rapidly cleared by the body. Encapsulation in LNPs can improve the effectiveness of drugs through promoting stability, improving affinity for their cellular targets and increasing transfection efficiency. 

BPR:Why is bioanalysis of LNP components important?

IL: ​As the structure and morphology of LNPs largely determines their function, analysis of LNP components can provide useful mechanistic information on exposure and the fate of the encapsulated product. The lipid constituents of an LNP may also demonstrate a mild pharmacological or toxic effect and LNP bioanalysis may inform on exposure:effect relationships. Moreover, as more is understood about the behaviors of these delivery platforms, regulators are increasingly interested to see exposure data included in filings.

BPR: Why do LNPs present new challenges for bioanalysts?

IL: ​The bioanalytical challenges presented by lipid encapsulation are two-fold: the first is related to measurement of the active material, the second to measurement of the lipid particle itself.

For a conventional formulation, the active agent is typically bioavailable and the associated, conventional bioanalytics are well understood. For lipid-based delivery, the molecule is retained within a lipid shell and it is common for the bioanalyst to be challenged with the need to determine free, bound and/or total concentrations of the active material. In practice, this is challenging owing to generally limited ex-vivo stability of lipid-based particles or degradation of the LNP through sample handling as part of a bioanalytical sample clean up.

As LNPs are large, heterogenous packets of ionizable lipids, a quantitative measure of intact LNP is not possible by conventional bioanalytical approaches. To determine exposure to LNP particles it is, therefore, more common to measure concentrations of individual LNP components as a surrogate measure for LNP exposure.

In identifying the need to assess LNP exposure, it is most effective to identify a specific lipid within the LNP to act as an appropriate surrogate. Polyethylene glycols, a helper lipid, often incorporated into LNP structure to promote systemic stability, are not amenable to analysis by conventional liquid chromatography-mass spectrometry. Indeed, this class of molecules is well known to have a deleterious effect on the performance of mass spectrometry-based assays. Other helper lipids, such as cholesterol and other phytosterols, which are incorporated to enable structure rigidity and promote cellular fusion, are credible surrogate candidates. These lipids typically present a challenge because they are often endogenous and/or introduced through diet. They are less amenable to development of sensitive bioanalytical methods compared to cationic lipids due to fewer charge carrying moieties. To this end, non-natural cationic lipids, present as a major component in the LNP macro structure, are generally employed as surrogate analytes for the determination of LNP exposure.

BPR: What elements need to be considered in building a robust surrogate lipid?

IL: ​Selection of an appropriate surrogate lipid helps to bring the challenges of LNP exposure in line with those that can commonly be encountered in small molecule bioanalysis.

Development of a selective assay for a surrogate lipid is commonly a challenge due to the significant overlap in structure and mass of lipids. 

Chromatographic bioanalysts will be familiar with potentially interfering lipids in the form of phospholipids. Phospholipids are a significant component in cell membranes and can significantly suppress or enhance mass spectrometer signals. There are specialist sample preparation techniques available that can selectively remove phospholipids, but these typically exploit lipophilicity as a way of separating small molecules from lipids. When the target analyte itself is a lipid, these technologies are often redundant and natural lipids will remain in the prepared analytical sample. Consequently, it becomes critical to develop a set of chromatographic conditions to adequately separate the target lipid from any lipidic interference.

Chromatographic separation is often afforded by the incorporation of a small amount of alcohol in reverse phase mobile phase systems. Alcohol helps to promote gaussian reverse phase peak shapes and a strong mass spectral response. When utilized in conjunction with high-quality, efficiently end-capped, reverse phase chromatography columns, it is possible to minimize unwanted secondary interactions between the cationic lipid head and any residual silanols on reverse phase column media.

A further challenge in the development of robust surrogate lipid assays is that of sample handling and preparation.

Lipids, particularly unsaturated lipids, are famously oxidatively labile. In addition to this, it is commonly required to account for liabilities related to plasma esterases by using modifiers or environmental controls to promote incurred sample stability. Lipids are also known to exhibit high potential for a specific binding to surfaces. For example, long aliphatic tales can readily bind to the surfaces of commercial plasticware while positively charged cationic lipids can readily bind to the surfaces of glassware. To this end, the bioanalyst should exercise care when selecting operating pH and include alcohol in the preparation of lipid solutions to promote solubility. 

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