Tackling Challenges In RNA Stability And Organ Targeting

By Jyotsna Jajula, Research Assistant, Wayne State University

RNA-based vaccines and medicines have rapidly evolved from experimental tools to frontline therapies^1,2,3. But as the field expands, it faces two persistent barriers to broader clinical adoption: the instability of RNA in biological systems and the challenge of delivering RNA to organs beyond the liver^4,5. These issues are increasingly critical as developers target diseases of the lungs, heart, brain, and immune system — areas in which current delivery platforms fall short^7,8. The need for tissue-specific targeting is becoming more urgent, as rapid degradation by nucleases and activation of innate immune pathways continue to undermine potency and safety^4,9.

RNA Instability In Biological Systems

Despite their promise, RNA molecules remain chemically and biologically fragile. Once introduced into the body, they encounter an environment designed to break them down. Ubiquitous ribonucleases (RNases) rapidly degrade single-stranded RNA, while hydrolysis in aqueous environments shortens half-life. These effects are worsened by temperature, pH, and mechanical stress during manufacturing and formulation^4,13,15.

Beyond structural decay, unmodified RNA can also trigger innate immune responses. Toll-like receptors (TLR7 and TLR8), expressed in immune cells, recognize foreign RNA as a danger signal leading to inflammation, cytokine release, and reduced efficacy^9,16. This immune activation also complicates repeat dosing, critical for chronic conditions.

To mitigate these risks, researchers use nucleoside modifications, structural optimization, and encapsulation in nanocarriers:

• Nucleoside modifications like pseudouridine and N1-methylpseudouridine improve enzymatic stability and reduce immune recognition^4,9.
• Structural optimization (e.g., 5’ capping, optimized UTRs, poly(A) tails) enhances intracellular stability and translation^4,15.
• Encapsulation, particularly in lipid-based systems, shields RNA from RNases to promote cellular uptake^3,5,13.

Despite advancements in RNA therapeutics, even the most robust RNA must reach the right tissue to be effective, bringing us to the next challenge: delivery.

Lipid Nanoparticles For RNA Stabilization And Limitations In Organ-Specific Targeting

To counter RNA’s inherent instability, researchers have turned to encapsulation technologies. Among various platforms, lipid nanoparticles (LNPs) have become the most clinically validated and effective system for stabilizing and delivering RNA in vivo^3,5,13.

LNPs form a protective shell around RNA, shielding it from nucleases during circulation. Their nanoscale size enables cellular uptake via endocytosis, while ionizable lipids drive pH-triggered endosomal escape, allowing the RNA payload to reach the cytoplasm for translation^3,13.

Each component of the formulation plays a distinct role:

• Ionizable lipids are neutral at physiological pH but positively charged in acidic endosomes, enabling membrane fusion and RNA release^3,15.
• Cholesterol enhances membrane fluidity and structural stability³.
• Phospholipids and PEG-lipids support bilayer formation, reduce aggregation, and extend circulation time^3,4.

Despite their proven success in stabilizing RNA, conventional LNPs show a strong preference for the liver due to their size, charge, and composition^5,7,13. Once administered, nanoparticles 50 nm to 150 nm in diameter are readily filtered by the liver’s fenestrated endothelium. The reticuloendothelial system (RES) also captures them, concentrating delivery in hepatic tissue^5,13.

While advantageous for liver-targeted therapies such as those treating hemophilia or metabolic diseases, this bias limits therapeutic use for non-hepatic organs, including:

• Lungs: sites for cystic fibrosis and pulmonary fibrosis^7,9
• Heart: targets for myocarditis and cardiomyopathies¹⁶
• Spleen and lymph nodes: crucial for vaccine and immune modulation^7,16
• Brain (central nervous system [CNS]): inaccessible due to the blood–brain barrier but vital in neurodegenerative diseases like ALS or Alzheimer’s⁹

This delivery challenge constrains even the most optimized RNA formulations. Stability alone is not enough; tissue access defines efficacy⁷. Overcoming this hepatic bias is therefore not just a formulation challenge but a strategic requirement for the next generation of RNA medicines.^5,7,16

Tissue-Targeted LNP Design

To overcome liver-dominant delivery, researchers are applying modular design principles to tune LNP properties for tissue-specific targeting^5,7,13. By adjusting lipid composition and nanoparticle characteristics, it’s now possible to influence which organs accumulate RNA payloads⁷.

Particle size is a key lever: smaller LNPs may penetrate deeper into tissues, while specific vascular beds retain larger ones^5,7. Zeta potential, a measure of surface charge, affects circulation time and interactions with cell membranes. Anionic or neutral particles circulate longer, while cationic particles may enhance uptake by specific cells or be sequestered by immune mechanisms^5,7,13.

The inclusion of helper lipids such as DOTAP (cationic), DODAP (ionizable), or 18PA (anionic) enables further tuning of membrane fusion, pKa behavior, and biodistribution^5,13,16. These lipids influence endosomal escape, particle stability, and tissue selectivity under physiological conditions^5,7.

To evaluate organ targeting, preclinical teams often use in vivo imaging systems (IVIS) to track fluorescently labeled LNPs in real time⁷. Several studies show that tweaking charge or lipid ratios leads to preferential accumulation in lungs, spleen, or lymph nodes — a promising signal for non-hepatic delivery^7,9.

Notably, charge-based targeting has emerged as a stand-alone strategy in RNA delivery⁷. By modulating the surface charge of LNPs to align with natural biodistribution patterns, researchers are steering LNPs to specific tissues without relying on ligands or highly complex designs^7,9,16. This approach offers a simpler yet effective way to target specific tissues, expanding the reach of RNA medicines beyond the liver^5,7.

These advances reflect a shift from passive biodistribution toward active control over therapeutic localization^7,15.

Innovations In RNA Delivery Systems

While lipid nanoparticles (LNPs) remain the dominant platform for RNA delivery, two major innovation areas include ligand-directed targeting and stimuli-responsive carriers.

Ligand-Directed LNPs

One strategy involves functionalizing the LNP surface with targeting ligands such as peptides, antibodies, aptamers, or small molecules that bind to receptors overexpressed on specific cell types^15,16. For example:

• Peptide-modified LNPs can guide RNA to endothelial or immune cells¹⁶.
• Antibody-conjugated LNPs are under study for selective targeting in cancer and autoimmune diseases¹⁶.
• GalNAc-LNPs, while optimized for liver delivery, demonstrate the potential of receptor-mediated uptake^5,15.

These ligands exploit cell-specific endocytosis, increasing delivery precision while reducing off-target exposure^15,16.

Stimuli-Responsive Systems

Another approach uses delivery vehicles that respond to local physiological cues, remaining stable in circulation but releasing RNA when triggered by:

• pH gradients (e.g., tumor microenvironments, endosomes)⁹
• enzymes (e.g., MMPs in inflamed or fibrotic tissue)⁹
• redox conditions (e.g., elevated intracellular glutathione)⁹.

These “smart” carriers enable localized RNA release, improving safety and allowing access to challenging organs like the CNS or pancreas^9,15.

Translational And Regulatory Challenges

As RNA delivery systems grow more advanced, so do the hurdles in translating them into safe, scalable, and regulatory-compliant therapies^12,15. While modular LNPs and next-generation carriers show strong promise in preclinical studies, their path to clinical deployment remains complex^12,15.

Scale-Up and Manufacturing Complexity

Innovative delivery platforms, especially those incorporating ligands or stimuli-responsive elements, pose significant manufacturing challenges¹². Achieving consistency across batches for critical parameters — such as particle size and polydispersity, encapsulation efficiency, release kinetics, and stability/shelf life — requires highly controlled nanoscale production and sophisticated analytics¹². This becomes a bottleneck during tech transfer and scale-up, particularly for academic or early-stage biotech teams lacking prior cGMP experience¹².

Unlike small molecules or monoclonal antibodies, RNA-loaded nanoparticles require novel CMC (chemistry, manufacturing, and controls) strategies. Regulatory expectations for these platforms are still evolving, making process documentation and validation more challenging.

Immunogenicity and Off-Target Distribution

Despite advances in nucleoside modification and formulation, immune activation remains a key concern, particularly with chronic or repeated dosing. Novel lipid chemistry and synthetic polymers may trigger unexpected innate or adaptive responses, limit tolerability, and complicate long-term use^9,16.

Off-target accumulation in non-target tissues such as kidneys, reproductive organs, or CNS raises toxicity risks, especially in systemically delivered therapies^9,15. Preclinical toxicology and biodistribution studies are critical yet often challenged by limited historical precedent and a lack of standardized evaluation frameworks for these emerging delivery materials¹².

Lack of Delivery Efficiency Standards

Despite remarkable advances in RNA formulation and delivery, benchmarking efficiency remains a persistent challenge¹⁵. Tools like IVIS, biodistribution assays, and protein expression analyses yield valuable insights, but cross-platform comparisons are often limited by differences in models, methodologies, and endpoints^9,15.

The lack of standardized performance metrics hinders the objective evaluation of emerging systems. Without consistent scoring frameworks, regulators, investors, and collaborators struggle to determine whether a new platform is truly superior or merely different. As the field matures, there is a growing consensus around the need for harmonized benchmarks to evaluate delivery performance across tissues, indications, and routes of administration.

Regulatory Uncertainty

RNA delivery systems often straddle regulatory definitions — neither purely drug, device, nor biologic. When developers introduce novel materials or modular designs, approval pathways become less defined. Agencies like the FDA and EMA are actively evolving guidance on nanoparticle-based therapeutics, yet ambiguity persists, particularly for IND submissions¹².

To navigate this gray area, early and proactive engagement with regulators is essential. Clear, well-documented CMC strategies, backed by robust characterization data, are critical. Developers must demonstrate not only efficacy but also scalability, reproducibility, and safety to advance next-generation delivery systems through clinical and regulatory milestones¹².

Conclusion

RNA therapeutics have already reshaped the biomedical landscape, but their long-term potential depends on overcoming two foundational barriers: molecular stability and precise, tissue-specific delivery. While significant progress has been made, primarily through chemical modifications and LNPs, most delivery systems remain liver-biased, limiting their utility in diseases of the lungs, heart, CNS, and immune system.

Recent innovations in modular LNP design, such as ligand-guided targeting and stimuli-responsive carriers, signal a more flexible and intelligent future — one in which RNA drugs are guided by molecular addressability, not just passive pharmacokinetics. But realizing this future will require more than scientific creativity.

It will demand:

• scalable, reproducible manufacturing processes
• clear, standardized benchmarks for delivery performance
• regulatory frameworks that evolve with technological complexity.

To unlock the next generation of RNA medicines, delivery must become a design priority, not a downstream concern. As the field moves from pandemic-scale vaccines to personalized and systemic RNA therapies, the question is no longer whether delivery innovation matters — it’s how quickly the scientific community can make it happen.

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About The Expert:

Jyotsna Jajula holds a master’s degree in Pharmaceutical Sciences with a research focus on lipid nanoparticles, RNA stability, and targeted delivery systems. Her research focuses on optimizing biodistribution and immunocompatibility of RNA therapeutics across oncology, immunology, and gene-therapy applications.