Expand mRNA-LNP nanomedicine potential by optimizing lipids

Nanomedicine offers a promising new avenue for therapeutic solutions, like cell and gene therapies, to treat autoimmune diseases, cancer, rare inherited diseases, and more. Some new applications to explore include early disease detection or molecularly tailored treatments for patients who have complex diseases and are not responding to existing therapies.

Three areas of medicines ― gene editing, gene therapy, and mRNA ― are currently evolving at an especially rapid pace, backed by a range of new technology platforms supporting novel diagnostic and therapeutic solutions. Non-viral gene delivery approaches, such as lipid nanoparticles (LNP), have the potential to deliver a range of RNA-based therapeutics and vaccines, through optimized LNP processes that yield precise formulations.

Lipid nanoparticles are helping to make nanomedicine a reality

Finding an effective method of delivery remains a key challenge in developing cell and gene therapies. DNA, RNA, and other nucleic acid-based formulations face several obstacles in the journey to their target cells. Even if a therapeutic reaches a target cell, rapid degradation in the cells' biological fluids and the inability to accumulate or penetrate threaten efficacy. Innovative, effective delivery systems are therefore necessary for targeted, safe, and efficient intracellular nucleic acid delivery.

LNP are non-viral delivery systems that can carry large payloads with design flexibility, easy manufacture, and multi-dosing capabilities. They can be made with lipid excipients modified to deliver a variety of nucleic acid-based active pharmaceutical ingredients (API) to different targets in the body.

Each lipid nanoparticle consists of the following four components

• Ionizable lipids bind nucleic acid and hold a neutral charge under physiological pH to minimize toxicity and allow for the charge to shift with pH changes, limiting cytotoxic effects.  
• Helper lipids aid in LNP stability, intracellular uptake, and endosomal escape. 
• Cholesterol binds apolipoprotein E (ApoE)​ and mediates endocytosis via the low-density lipoprotein (LDL) receptor. 
• Hydrophilic PEGylated lipids create a barrier of water to protect LNP from aggregating during assembly and increase bloodstream circulation lifetime. 

Even a slight alteration in the chemical structure and the ratio of the four components can alter an LNP properties and delivery efficiency. This adaptability allows for selective organ targeting (SORT), where therapeutic developers modify particle design to overcome the challenges presented by extrahepatic mRNA delivery and gene editing systems.

A study found that augmenting conventional four-component LNP for mRNA delivery to the liver with a fifth component, termed a SORT molecule, enabled diverse cargoes (nucleic acids and proteins) to achieve gene expression and CRISPR/Cas-based gene editing in therapeutically relevant cell types, such as epithelial cells, endothelial cells, B cells, T cells, and hepatocytes (1). Tuning the nanoparticle’s molecular composition aided in its binding to specific proteins in the serum to allow for delivery to the target site and facilitated mRNA biodistribution to the target organ. These properties make it possible to engineer a wide array of nanomaterials for extrahepatic delivery.   

Ionizable lipids are the main component of LNPs that enable efficient cellular delivery and overcome payload limitations. In another study, a team of researchers used ionizable cationic lipids from a novel ionizable amino lipid library to co-encapsulate larger Cas9 mRNA and single-guide RNA (sgRNA) (2). The data showed that single or double administration of CRISPR-LNP (cLNP) formulations targeting the PLK1 gene, a kinase required for mitosis to avoid cell cycle arrest and cell death in dividing cells, could inhibit tumor growth and improve survival in two aggressive cancer models.

These findings demonstrate the potential for targeted lipid nanoparticles with customized ionizable cationic lipids to be used in targeted gene editing and the delivery of novel therapeutics. Testing and altering lipids in LNPs therefore offer a vast array of opportunities to enable the broader translation of RNA therapeutics and vaccines.

Categorizing lipids to find the best option for LNP

Ionizable lipids are a strategic component of lipid nanoparticles that play a significant role in protecting nucleic acids and facilitating their cytosolic transport. They have a unique pH sensitivity that allows them to shift charge with pH changes. In acidic pH, they are positively charged to condense nucleic acids into LNP but, at physiological pH, they’re neutral to minimize toxicity. Despite the FDA approval of ionizable lipids for RNA delivery, there are several challenges to overcome when using them in clinical applications.

There are five types of ionizable lipids typically used for RNA delivery:

• Unsaturated ionizable lipids:
  • enhance membrane disruption and payload release
  • don’t always correspond with potent in vivo RNA delivery, so both rational design and screening are necessary
• Multi-tail ionizable lipids:
  • offer greater endosome-disrupting ability
  • often have stable backbones and low degradability, so toxicity and immunogenicity pose limitations
• Ionizable-polymer lipids:
  • support particle formation through hydrophobic aggregation
  • can be re-optimized to achieve potent gene silencing
  • comprise a mixture of different substitution compounds even after purification, which increases their complexity
  • toxic polycation core and non-degradable backbone pose extra hurdles for clinical translation 
• Biodegradable ionizable lipids:
  • stable at physiological pH but can enzymatically hydrolyze within tissues and cells.
  • position and steric effect of the ester groups can significantly affect ionizable lipid clearance and potency
  • synthesis difficulties and the risk of premature release can limit their applications
• Branch-tailed ionizable lipids:
  • enhances endosomal escape with the potential for integrating different therapeutic modalities, such as gene silencing, expression, and editing
  • increased tail branching requires a thorough investigation

Ionizable lipids that can deliver nanoparticles to cells in culture do not necessarily translate to successful animal studies. Clinical ionizable lipids are synthesized in multiple steps, posing scalability challenges. Furthermore, the labor-intensive synthesis process makes it difficult to prepare rationally designed ionizable lipid candidates.

In addition to focusing on safety and potency, drug developers also face the challenge of optimizing ionizable lipids for structural properties and additional functionalities such as targeting and immunomodulation. Decades of research on lipid nanoparticles suggest a need to profile and characterize ionizable lipids to ensure the product quality of vaccines and other mRNA-based therapies. To address this issue, a proprietary lipid library that systematically categorizes these lipids based on their structures can be accessed to develop custom formulations. 

Optimizing and analyzing lipid formulations

According to Dr. Anna Blakney, Assistant Professor in the Michael Smith Laboratories and School of Biomedical Engineering at UBC, LNP are prominent leaders for RNA delivery ― following mRNA COVID-19 vaccines and the LNP-based drug Patisiran ― and optimizing them is essential to success. She highlights two primary challenges while working with LNP: the formulation and the assembly process, and believes the most intricate work comes in finding a suitable LNP formulation, which is both an art and a science.

With ionizable lipids offering an exciting path forward for nanomedicine, it’s important to focus on testing and optimizing promising formulations according to their physicochemical attributes ― including size, size distribution, and encapsulation efficiency ― cellular uptake, and in vitro potency. Changing the solvent combination while completing analytical tests for physical parameters, such as encapsulation or particle size, and assays for composition, identity, and purity is a key step during formulation design.

In a case study, Optimizing LNP formulations for plasmid expression in induced pluripotent stem cells (iPSCs), it was found that formulations had very different in vitro performance despite similar physical properties (3). Microliter formulations containing microgram quantities of mRNA were rapidly produced using the NanoAssemblr™ Spark™ system to systematically screen compositions for properties and activity.

A panel of formulations containing PNI-Ila (a proprietary ionizable cationic lipid) with different helper lipids was created at different amine-to-phosphate (N/P) ratios. This ratio of positively chargeable polymer amine groups to negatively charged nucleic acid phosphate groups can influence many properties of polymer-based gene delivery vehicles, such as net surface charge, size, and stability. The N/P ratio is vital to formulation efficacy for plasmid delivery to human iPSC-derived cortical neurons.

Analytical testing to identify candidate formulations with optimal performance in this therapeutically relevant cell type revealed that a higher N/P ratio formulation had better encapsulation efficiency. However, in vitro, there was a measured decrease in neurite length, showing a negative impact on cell health. This study shows that performing empirical testing is critical in determining the ideal formulation.

Exploring the possibilities for nanomedicine

Further optimization, including functionalization of LNP for developing drugs to target tissues, is likely to improve formulation efficiency and avoid off-target effects. Although optimizing LNP formulations is complex, at Cytiva, we provide a wide range of cGMP-manufactured lipids to enable researchers and developers to tailor drug delivery systems to suit their unique needs. The Genvoy-ILM™ delivery platform comprises off-the-shelf research-use-only reagents, such as the GenVoy-ILM™ lipid mixture, and a library of proprietary lipids available for custom formulations on your path to the clinic.

Our BioPharma Services technical team can help in developing the right analytical assays for both viral and non-viral nanomedicines, including:

• Drug product identity confirmation
• Physical characterization
• Acceptance testing
• Stability studies
• GMP release testing
• Toxicology testing
• Raw material testing

Optimization with delivery platforms enables seamless scaling of drug products from preclinical to clinical development, reducing costly raw material requirements and, in turn, saving time and money. Our team not only helps scientists and drug developers select appropriate formulations, but we also offer LNP manufacturing instruments with NxGen™ technology that are scalable to advanced preclinical and clinical production.

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