Advancing RNA Delivery Beyond Vaccines

cytiva.com Lipid nanoparticles beyond vaccines New frontiers in targeted and personalized therapeutics2 cytiva.com Contents Foreword ..................................................................................................................................3 Chapter 1: Designing LNPs for repeat dosing: advancing safety and tolerability beyond vaccines.....................................................................................................................4 Chapter 2: LNP stability and lyophilization: enabling real-world RNA therapeutics..............10 Chapter 3: Lipid nanoparticles in personalized cancer vaccines ...............................................15 Chapter 4: Beyond the liver: advancing LNP delivery to the lung ..............................................21 Chapter 5: Expanding the horizon: targeted LNPs for extrahepatic delivery ..........................263 cytiva.com Foreword We are entering a new era of medicine. RNA therapeutics are transforming how we approach disease, not simply by managing symptoms, but by addressing their underlying biological mechanisms. By harnessing the body’s own cellular machinery, these therapies are opening new possibilities for treating cancer, rare diseases, infectious diseases, and many other conditions. As our scientific understanding deepens and technology continues to advance, RNA-based medicines are becoming an increasingly versatile platform for developing more precise, adaptable, and personalized therapies. Realizing their potential, however, depends on solving one of the field’s greatest challenges: delivery. RNA molecules are inherently fragile. Without an effective delivery system, they are rapidly degraded before reaching their intended target. Lipid nanoparticles (LNPs) have emerged as the leading non-viral delivery platform, protecting RNA, enabling intracellular delivery, and accelerating the translation of RNA therapeutics from research to the clinic. Yet delivery is no longer just about getting RNA into cells. As the field expands beyond prophylactic vaccines toward protein replacement therapies, gene editing, personalized cancer vaccines, and chronic diseases requiring repeat administration, the demands placed on delivery systems are changing. Future LNP platforms must not only deliver efficiently, but also demonstrate long-term safety, repeat-dose tolerability, enhanced stability, scalable manufacturing, and the ability to precisely target tissues beyond the liver. These challenges represent more than technical hurdles. They define the next phase of innovation in RNA therapeutics. Across academia and industry, researchers are rethinking every aspect of LNP design: engineering formulations for repeat dosing, developing alternative stabilizers, improving storage through lyophilization, and developing targeting strategies that extend delivery beyond traditional organs. Together, these advances are expanding the therapeutic reach of RNA medicines while bringing them closer to real-world clinical application. This eBook explores several of the scientific advances shaping the future of RNA delivery. Drawing on insights from researchers working across LNP development, it examines how innovation in formulation, stability, repeat dosing, and organ-specific targeting is helping address some of the field’s most important challenges. While significant challenges remain, the momentum across the field is unmistakable, moving us closer to a future where RNA therapeutics can reach more diseases, more patients, and more healthcare settings than ever before.4 cytiva.com Chapter 1: Designing LNPs for repeat dosing: advancing safety and tolerability beyond vaccines Lipid nanoparticles (LNPs) have transformed nucleic acid delivery, most visibly through the success of mRNA vaccines. Yet, as therapeutic ambitions extend beyond prophylaxis toward chronic and complex indications, the expectations placed on delivery systems are changing. Previously, vaccine design assumptions focused on single or limited dosing, primarily as prophylactic measures, relying on a singular prime dose or a few booster doses. In that context, LNPs were designed for stability, efficient delivery, and acute tolerability. Today, the landscape is evolving beyond acute, one-time interventions and into applications that require sustained or repeated delivery, including protein replacement, immune modulation, and emerging gene editing approaches. When repeated dosing becomes the foundation of therapeutic efficacy, a fundamentally different safety paradigm is introduced. Subtle tolerability liabilities can compound over time, amplifying concerns around systemic inflammation, immunogenicity, and pharmacokinetic variability. As Nikita Jain, Senior Scientist at Cytiva, explains, “The strategy must shift from potency-driven to tolerance-driven. If a therapy is administered repeatedly or even life-long, tolerability, clearance, and safety become the primary design criteria.” For developers, this raises a critical question: Can LNP platforms maintain safety, efficacy, and predictable immune compatibility? Repeated dosing introduces new biological challenges and potential risks, including immune activation, organ toxicity, and anti-polyethylene glycol (PEG) antibody (APA) formation. Each of these can reduce therapeutic efficacy, alter biodistribution, or trigger adverse reactions. Among these risks associated with repeat exposure, immune recognition remains one of the most closely scrutinized. In particular, PEG, used pervasively in creams, cosmetics,1 food,2 and medicine,3 is also used in LNP formulations (typically 1%–2% by mole) to enhance stability and prolong circularization. Its exposure has been associated with the induction of APAs in both preclinical and clinical settings,3 raising challenges for dose optimization, regulatory confidence, and long-term patient management.5 cytiva.com The PEG challenge: understanding the immunological landscape While PEG has long been the stabilizer of choice for extending LNP circulation time and preventing aggregation, recent data highlights a growing hurdle regarding its immunogenicity.4 Recent data shows: • Pre-existing APAs have also been detected in individuals with no prior exposure to PEGylated drugs, raising additional clinical concerns.5 • Repeated LNP administration can boost these antibodies, trigger accelerated blood clearance (ABC), which causes the rapid clearance and decreased efficacy of therapeutic agents, and reduce therapeutic payload delivery.6 • Elevated APAs have been linked to enhanced reactogenicity and altered biodistribution, which can compromise clinical outcomes, and it is important to validate this potential association with larger clinical mRNA health conference studies.7 These findings underscore a critical reality: for therapies requiring repeated or chronic dosing, PEG immunogenicity is a practical barrier to effective translation. Environmental exposure to PEG Anti-PEG antibodies develop Pre-existing antibodies can reduce effectiveness of PEGylated drugs 1 2 3 Figure 1. Overview of environmental PEG exposure, anti-PEG antibodies, and therapeutic outcomes6 cytiva.com Designing LNPs for safer, repeated dosing Researchers at Cytiva are using a unique approach by combining a deep understanding of lipid chemistry with rigorous preclinical evaluation. In one of their recent studies on repeat-dose safety, Nikita and her team evaluated multiple LNP formulations containing proprietary ionizable lipids in a nine-dose intramuscular study in mice, with injections administered every 21 days. The threeweek interval was selected based on precedent from early clinical-stage cancer vaccine program studies, providing a practical starting point for evaluating repeated exposure. The study was presented at the 2025 mRNA Health Conference in Berlin, Germany. This extended regimen enabled evaluation of both acute and cumulative effects, capturing endpoints that short-term studies may overlook. Clinical observations were monitored throughout the study, alongside comprehensive immunological assessments. Key readouts included: • Clinical observations • Liver enzyme functions • Inflammatory markers • Anti-PEG antibody responses/ immune responses Across all dosing cycles, the results were encouraging: • No differences in clinical observations were seen compared with phosphate-buffered saline (PBS) controls. • Measures of liver enzyme, often considered sensitive indicators of systemic and hepatic stress, remained stable. This indicated an absence of hepatotoxicity and suggested preserved metabolic homeostasis despite repeated exposure. • No sustained elevation in inflammatory markers was detected. Chronic inflammation represents a central concern for repeat administration strategies, as even low-grade inflammatory signaling can contribute to cumulative tissue stress, immune activation, and patient discomfort. The absence of sustained inflammatory responses under extended dosing supports the favorable tolerability profile of these formulations. • Critically, no long-term APAs were observed. Addressing anti-PEG immunity – a critical translational barrier Beyond general tolerability, immune recognition of nanoparticle components remains a defining challenge for chronic delivery platforms. The absence of detectable APAs following nine-dose LNP administration represents a meaningful advance. Immune profiling revealed no long-term adaptive response against PEG components, with antibody levels remaining comparable to PBS-treated controls throughout the study duration. These findings demonstrate that careful lipid formulation and dosing strategy alignment can mitigate immune risk, supporting repeated LNP administration without compromising safety or7 cytiva.com tolerability. While continued clinical investigation remains essential, the ability to maintain immune tolerance under repeat dosing provides an important scientific credibility and clinical relevance for expanding LNP-based therapies into chronic and high-frequency treatment paradigms. A crucial insight gained from this study is that a universal formulation simply doesn’t exist. Even when using the same lipid components, small changes in composition can significantly alter how LNPs behave in the body. As Nikita describes, “We may use the same components, but by tuning the composition, we can direct where the LNP goes. If the goal is liver targeting, the formulation will look very different from one designed for spleen delivery or localized expression.” The formulation tuning is critical because different applications require different delivery profiles: • Protein replacement therapies often require liver targeting. • Gene editing applications depend on organspecific delivery. • Vaccines benefit from localized expression and controlled immune activation. For vaccines in particular, excessive systemic exposure can increase the risk of adverse immune responses. As a result, LNPs are often designed to remain localized after intramuscular injections, with controlled drainage to lymph nodes. This level of precision reinforces a key principle: LNP design must start with the therapeutic goal, not the formulation. Interpreting the implications of repeated dosing for RNA therapeutics Repeated dosing capability opens the door to chronic, long-term, and multi-dose therapies for mRNA therapeutics targeting: • Chronic protein replacement, where consistent expression is needed over months. • Gene editing, which may require multiple dosing to achieve sufficient editing efficiency. • Immunotherapies, where dosing schedules may be patient specific. Moreover, these results reinforce a critical insight: safety and tolerability must be evaluated longitudinally under clinically relevant conditions. Demonstrating preserved tolerability across repeated administrations, therefore, extends beyond risk mitigation—it actively expands the therapeutic design space. Developers gain flexibility to explore higher cumulative doses, extended treatment windows, and adaptive dosing schedules tailored to individual patients’ needs. At the platform level, these findings reinforce the importance of integrating lipid chemistry, immunological insight, and formulation engineering early in development. As Nikita explains, “If a therapy needs to be administered over years, the LNP is no longer just a delivery system; it effectively becomes part of the drug.” By addressing tolerability and immunogenicity at the design stage, formulation strategies can evolve8 cytiva.com in parallel with therapeutic ambition rather than becoming downstream constraints (Table 1). Short-term focus Long-term focus High potency High tolerability Strong endosomal escape Controlled release Transient cytotoxicity acceptable Near-zero cytotoxicity Rapid effect Sustained, predictable response Table 1. Formulation design priorities This approach allows developers to anticipate challenges early and reduce risks during clinical translation. Preparing for the next evolution of LNP design While the favorable safety profile observed in the study supports continued use of PEG-containing formulations under optimized conditions, it also sharpens focus on long-term platform sustainability. Growing awareness of PEG immunogenicity, which limits the efficacy of both commercially available medications and future therapeutics, is stimulating the development of alternative stabilization strategies.8 This recognition motivated the development of Cytiva’s non-PEG stabilizers, a complementary innovation pathway aimed at expanding formulation options, enhancing operational flexibility, and meeting evolving regulatory expectations. Cytiva offers a library of ionizable lipids and custom formulation services. By building accessibility and optionality into LNP chemistry, developers can proactively adapt to shifting clinical, regulatory, and commercial landscapes. In this context, repeat-dosing safety is not an endpoint. Instead, it becomes a foundation for continued platform evolution, enabling next-generation LNP systems capable of supporting increasingly sophisticated therapeutic designs. Enabling robust genetic medicines The transition from single-dose vaccines to chronic genetic therapies marks a defining inflection point for LNP technology. As this unfolds, safety under repeated exposure emerges as a central determinant of clinical viability. Through rigorous preclinical evaluation, Cytiva’s LNP formulations demonstrate sustained tolerability, preserved liver function, and the absence of detectable anti-PEG immune responses under extended dosing conditions. These results establish a robust safety foundation while informing ongoing innovation in non-PEG stabilization strategies. Together, this integrated approach, combining lipid chemistry, formulation engineering, and immunological insight, positions LNP platforms to support not only today’s mRNA vaccines but the next generation of genetic medicines. As therapeutic ambition accelerates, delivery systems designed for durability, flexibility, and long-term tolerability will define the pace of clinical translation.9 cytiva.com References 1. Jang HJ, Shin CY, Kim KB. Safety Evaluation of polyethylene glycol (PEG) compounds for cosmetic use. Toxicol Res. 2015;31(2):105–136. doi: 10.5487/tr.2015.31.2.105 2. Younes M, Aggett P, Aguilar F, et al. Refined exposure assessment of polyethylene glycol (E 1521) from its use as a food additive. EFSA J. 2018;16(6):5293. doi: 10.2903/j.efsa.2018.5293 3. Chen BM, Cheng TL, Roffler SR. Polyethylene glycol immunogenicity: theoretical, clinical, and practical aspects of anti-polyethylene glycol antibodies. ACS Nano. 2021;15(9):14022–14048. doi: 10.1021/acsnano.1c05922 4. Fu J, Wu E, Li G, Wang B, Zhan C. Anti-PEG antibodies: Current situation and countermeasures. Nano Today. 2024;55:102163. doi: 10.1016/j.nantod.2024.102163 5. Fu S, Zhu X, Huang F, Chen X. Anti-PEG antibodies and their biological impact on PEGylated drugs: Challenges and strategies for optimization. Pharmaceutics. 2025;17(8):1074. doi: 10.3390/pharmaceutics17081074 6. Gao P. PEGylated lipids in lipid nanoparticle delivery dynamics and therapeutic innovation. Beilstein J Nanotechnol. 2025;16:1914–1930. doi: 10.3762/bjnano.16.133 7. Ju Y, Carreño JM, Simon V, Dawson K, Krammer F, Kent SJ. Impact of anti-PEG antibodies induced by SARS-CoV-2 mRNA vaccines. Nat Rev Immunol. 2023;23(3):135–136. doi: 10.1038/s41577-022-00825-x 8. Thi TTH, Pilkington EH, Nguyen DH, Lee JS, Park KD, Truong NP. The Importance of Poly(ethylene glycol) alternatives for overcoming PEG immunogenicity in drug delivery and bioconjugation. Polymers. 2020;12(2):298. doi: 10.3390/polym12020298 Expert Perspective While stabilizers account for just a few mole percent of a formulation, their impact can be significant. To improve efficacy and overcome traditional PEG related toxicity, my team is developing a portfolio of non-PEG stabilizers with diverse functionalities. For example, some include end groups that can enable click-chemistry conjugation of targeting molecules, opening new possibilities for antibody-targeted applications. Nikita Jain Senior Manager I, Delivery, Cytiva PhD, Organic Chemistry, The University of British Columbia10 cytiva.com Chapter 2: LNP stability and lyophilization: enabling real-world RNA therapeutics The rapid rise of mRNA–lipid nanoparticle (mRNA– LNP) technologies has transformed what is possible in modern science. Yet beyond efficacy and delivery, a quieter constraint continues to shape how far these therapies can reach: stability.1,2 LNPs are dynamic assemblies that, while enabling flexibility and modularity, also raise concerns about their stability. Additionally, both the lipids and the encapsulated RNA molecules themselves are highly sensitive to environmental stress, particularly temperature fluctuations during storage and handling.1 As a result, many current formulations rely on ultra-low temperature storage, often at -80⁰C.2 However, this requirement introduces a fundamental challenge: it limits scalability, complicates distribution, and restricts access, particularly in settings where cold-chain infrastructure is not readily available.2 Why LNPs are so sensitive to storage conditions The instability of the LNP system is not driven by a single factor, but by a combination of physical and chemical processes that occur over time and are amplified during freeze–thaw cycles. At the molecular level, degradation pathways affect both components of the system: • Lipids can undergo hydrolysis and oxidation.1 • RNA payloads are susceptible to hydrolytic degradation and structural disruption.1 These changes directly impact critical quality attributes such as: • Particle size • Polydispersity index (PDI) • Encapsulation efficiency • RNA integrity However, one of the most overlooked challenges arises during freezing and thawing. When LNPs are frozen, ice crystal formation and phase separation introduce mechanical stress. Upon thawing, rapid structural reorganization can further destabilize this system.311 cytiva.com Moving beyond ultra-cold storage To address these limitations, researchers are increasingly turning to lyophilization (freeze-drying) to stabilize LNP formulations. Rather than storing LNPs as frozen liquids, lyophilization converts them into a dry, solid form that can be stored at refrigerated temperatures. However, successful lyophilization is not simply a matter of removing water. Without proper formulation design, the process itself can introduce damage through ice crystal formation, internal stress during drying, pH changes, and structural collapse.4 This makes formulation, particularly excipient selection, central to success. Protecting LNP structure through formulation design Lyophilization is a critical strategy for improving the stability, shelf life, and manufacturability of RNA–LNP therapeutics, but successful freeze-dried formulations depend heavily on the rational selection of excipients. Formulation additives play a crucial role in maintaining nanoparticle structure, ensuring RNA integrity, and enhancing performance during freezing, drying, and storage processes. The role of excipients in stabilizing LNPs Excipients play a central, multifunctional role in lyophilized RNA–LNP formulations, acting as structural and stabilizing enablers rather than passive ingredients. Disaccharides such as trehalose and sucrose form an amorphous glass that replaces water–lipid interactions during freezing, protecting membranes from ice stress and maintaining particle integrity after reconstitution. The polymeric excipients (e.g., hydroxypropyl β-cyclodextrin, PVP, dextrans) raise the glass transition temperature to reduce molecular mobility and improve solid-state stability under less stringent storage conditions. Moreover, crystallizing bulking agents like mannitol provide mechanical strength to the lyophilized cake while buffers such as histidine or Tris preserve pH and RNA/lipid integrity during freezing and drying. Additionally, low levels of nonionic surfactants (e.g., poloxamer 188) mitigate aggregation during processing.3 Together, these excipient systems define the performance envelope of lyophilized RNA therapeutics, transforming lyophilization into a platform strategy that enables robust manufacturing, distribution, and patient access.3 In a recent lyophilization study, researchers at Cytiva evaluated how lyophilization strategies impact LNP stability, specifically whether optimized buffer systems and excipient combinations can preserve particle integrity and functional performance after drying and rehydration. Researchers identified lyophilization-suitable buffers that retain the critical quality attributes and potency of both mRNA and saRNA LNPs following 10 months of storage at 4°C. Across key metrics, lyophilized formulations demonstrated: • Minimal changes in size and PDI • Stable encapsulation efficiency • Comparable in vivo efficacy12 cytiva.com A key observation from this work was that LNP composition itself remained constant, while stability outcomes were driven by differences in the lyophilization buffer system. “The LNPs perform very similarly to fresh formulations. Even after months, we didn’t observe significant changes in efficacy,” notes Hossein Yazdani Ahmadabadi, Associate Senior Scientist at Cytiva. From centralized manufacturing to global access The implications of improved stability extend far beyond the lab. During the COVID-19 pandemic, cold-chain requirements created major logistical challenges, particularly in regions with limited infrastructure. Lyophilized LNPs offer a path to overcome these barriers. “If you want to ship therapeutics globally without ultra-cold storage, lyophilization makes a big difference by enabling distribution using standard refrigerated conditions,” Hossein adds. This shift would enable: • Broader global distribution of LNP medicines • Improved pandemic preparedness • Reduced cost and complexity of logistics Lyophilization is not just a formulation improvement—it reshapes how RNA therapies can be deployed. Key advantages: • Eliminates reliance on ultra-cold storage • Enables refrigerated (2°C to 8°C) distribution • Preserves efficacy over extended periods • Simplifies handling in clinical and field settings Why lyophilization changes the game COVID era mRNA cold chain Manufacturing Ultra-cold storage - specialized freezers Dry ice temperature controlled transport Limited distribution Limited stability Patient Future lyophilization supply chain Manufacturing Lyophilization Standard Patient refrigerated or room temp shipping More healthcare settings Reconstitute before use13 cytiva.com Enabling new therapeutic models Beyond large-scale distribution, stability plays a critical role in enabling emerging therapeutic paradigms. Lyophilization supports on-demand manufacturing workflows, decentralized production models, and faster turnaround for patient-specific therapies. These capabilities are particularly relevant for personalized cancer vaccines, rare disease treatments, and small-batch clinical applications. Remaining challenges and path to lyophilization adoption While promising, several challenges remain before widespread clinical adoption. The Cytiva study data demonstrate that lyophilization can preserve LNP integrity and in vivo performance after storage, but translating this into routine practice requires addressing several practical and scientific challenges. Stability is formulation specific, not universal In the study, the LNP composition remains unchanged across samples. But LNPs are diverse, and formulations are adapted to meet the needs of different indications. What drives stability during lyophilization is how well the buffer is matched to the formulation. Hence, there is no single excipient system that will translate across all payloads and LNP chemistries. Importantly, though, there are numerous levers available to developers to adapt their lyophilization buffers accordingly. In practice, this means teams need to treat stability as a design variable, grounded in a clear understanding of the target product profile (TPP) and the real-world circumstances of treatment it defines, from storage and transport to handling at the point of care. Until definitive evidence demonstrates that lyophilization can be developed independently from formulation as a downstream fix, it is prudent to align formulation, process, and stability requirements early, designing with the end use in mind. This means: 1. Defining stability requirements directly from the TPP (e.g., temperature excursions, shelf life, reconstitution constraints) 2. Screening formulations through the entire workflow, including freeze-drying, testing excipients and buffer systems for compatibility 3. Assessing particle integrity and potency across freeze–thaw and drying stresses 4. Considering process implications such as fill volume, container closure, and cycle scalability While adding lyophilization introduces additional steps, timelines, and equipment, this integrated approach can reduce the risk of late-stage surprises, especially for programs moving quickly from preclinical to clinical phases. Scaling the process introduces new variables Lyophilization performs well at a small scale, but consistency becomes more difficult as batch sizes increase. Key factors include: • Freezing rate and uniformity across larger volumes14 cytiva.com • Control of cake structure and residual moisture • Reproducibility during reconstitution A small shift in these parameters can affect particle size, PDI, and ultimately performance. Analytical and regulatory expectations remain high Transitioning from liquid to lyophilized formats requires clear evidence of compatibility and effectiveness. Developers need to demonstrate consistent particle size, distribution, maintained encapsulation efficiency, and preserved biological activity. The findings from the Cytiva study are encouraging, but building a robust dataset will be essential to support regulatory submissions. Application across modalities is still evolving The study data provide a strong foundation, but broader validation is still needed to answer the open questions: • How different payloads respond to the same buffer strategies • Whether similar outcomes can be achieved across delivery routes • How complex or sensitive constructs behave during drying and rehydration Looking ahead Lyophilization changes how LNPs can be stored, handled, and deployed. However, its full impact depends on how early it is integrated into the development strategy. The opportunity is clear. The next step is bringing formulation, process, and application requirements together to make stability a built-in feature. References 1. Schoenmaker L, Witzigmann D, Kulkarni JA, et al. mRNA-lipid nanoparticle COVID-19 vaccines: structure and stability. Int J Pharm. 2021;601:120586. doi: 10.1016/j.ijpharm.2021.120586 2. Crommelin DJA, Anchordoquy TJ, Volkin DB, Jiskoot W, Mastrobattista E. Addressing the cold reality of mRNA vaccine stability. J Pharm Sci. 2021;110(3):997–1001. doi: 10.1016/j.xphs.2020.12.006 3. Khan MDFH, Baudin F, Perumal AS, Kamen AA. Freezedrying of mRNA-LNPs vaccines: A review. Vaccines. 2025;13(8):853. doi: 10.3390/vaccines13080853 4. Ruppl A, Hutanu A, Köll-Weber M, Allmendinger A. Freezing-induced stress in mRNA-lipid nanoparticles during lyophilization: mechanistic insights from process and formulation studies. Pharm Res. 2026;43:877–889. doi: 10.1007/s11095-026-04039-x Expert Perspective LNP lyophilization is particularly challenging because water plays a critical role in LNP structure, and no two formulations are exactly alike. Through this work, we’ve gained valuable process insights, and a strong understanding of the key variables that our services team can apply to help customers develop lyophilization strategies tailored to their formulations. Hossein Yazdani Formulation Scientist II, Cytiva PhD, Chemistry, The University of British Columbia15 cytiva.com Chapter 3: Lipid nanoparticles in personalized cancer vaccines Cancer is not a single disease but rather a dynamic, evolving system of mutations. Even tumors originating in the same tissue can vary dramatically between patients and, at times, within the same tumor. This heterogeneity has driven growing interest in personalized cancer vaccines (PCVs), an emerging immunotherapy approach designed to train the immune system to recognize the unique molecular fingerprints of an individual patient’s tumor.1 Rather than targeting a universal tumor antigen, personalized vaccines rely on neoantigens, which are mutated proteins that arise from tumor-specific genetic changes. Advances in genome sequencing and computational prediction allow researchers to identify these neoantigens by comparing tumor DNA and RNA with healthy tissue from the same patient.2 Once identified, these neoantigens can be encoded with mRNA sequences and delivered as a vaccine capable of stimulating anti-tumor immune responses. However, the success of this strategy depends on a critical enabling technology: efficient delivery of complex RNA constructs into immune cells. Why delivery matters in cancer vaccines mRNA molecules are inherently fragile and cannot easily enter cells on their own. Without protection, they degrade rapidly in biological environments. Lipid nanoparticles (LNPs) solve this challenge by encapsulating mRNA and facilitating cellular uptake. Once inside the target cells, the RNA can be translated into antigen proteins that are presented to the immune system, triggering immune activation. This delivery capability has become central to the rapid progress of mRNA-based therapeutics and vaccines. Moreover, LNP delivery technologies have matured significantly in recent years, with advances in formulation design improving not only delivery but also safety, tolerability, and stability. These improvements have enabled the transition from simple RNA payloads to more complex therapeutic constructs. As a result, researchers can now explore applications such as multi-antigen personalized cancer vaccines, where both delivery performance and formulation robustness are critical to success.16 cytiva.com Multi-antigen vaccine for complex tumors Tumors rarely rely on a single mutation to drive progression. Instead, they often harbor numerous genetic alterations that drive tumor growth and immune evasion. For this reason, many personalized vaccine strategies encode multiple tumor antigens within a single mRNA construct.3 In a recent study at Cytiva, researchers evaluated multi-epitope mRNA constructs encoding combinations of melanoma-associated antigens designed to stimulate broad anti-tumor immune responses in a melanoma model. Why LNPs uniquely enable multi-antigen vaccines Personalized cancer vaccines often encode multiple neoantigens simultaneously. Delivering such complex payloads would be extremely challenging with many traditional vaccine platforms. Lipid nanoparticles enable this strategy by: • Protecting fragile RNA molecules from degradation • Facilitating efficient intracellular delivery • Enabling flexible mRNA design, where multiple antigens can be encoded and quickly reconfigured without re-engineering the delivery platform Tumor biopsy Mutation identification Neoantigen prediction Nucleic acids Multiple epitopes Delivery 5’ neo1 neo2 neo9 neo1017 cytiva.com These vaccines aimed to activate both: • CD8+ cytotoxic T cells, which directly kill tumor cells • CD4+ helper T cells, which support and sustain immune responses4 Activating multiple immune pathways simultaneously may help overcome tumor heterogeneity and reduce the risk of immune escape. Formulation choices shape delivery performance While mRNA sequence design is critical, the LNP formulation itself plays an equally important role in determining therapeutic performance. Key parameters that influence LNP behavior include: • The choice of ionizable lipid • The ratio of lipid components • Particle size and structural organization • Encapsulation efficiency and stability According to Jay Paquette, a scientist at Cytiva who worked on the melanoma model, formulation differences can significantly influence biological outcomes. “Picking the right ionizable lipid matters a great deal,” Jay explains. “Some ionizable lipids work extremely well in certain models, while others don’t work at all. Even the ratios of those components can change how the nanoparticle performs. That’s why it’s important to test them across different systems before drawing conclusions.” This sensitivity means that formulation development often requires extensive screening and empirical testing. Applying a validated LNP platform in a melanoma model Rather than designing a completely new nanoparticle formulation for the melanoma study, the research team selected a lead LNP platform that had previously demonstrated strong performance across multiple in vivo models. This approach allowed the investigators to focus on evaluating the multi-antigen mRNA constructs themselves while relying on a delivery system that had already been characterized. “We had already screened several LNP formulations in different applications,” Jay explains. “This particular formulation had shown strong performance across models, so we used it as the starting point for this melanoma study.” The mRNA construct encoding melanoma antigens were encapsulated within lipid nanoparticles and characterized using standard metrics, including: • particle size • polydispersity index (PDI) • encapsulation efficiency The resulting formulations were then evaluated in a tumor-bearing mouse model of melanoma.18 cytiva.com Early signals of therapeutic potential The vaccine candidates demonstrated promising activity in vivo. Several multi-antigen constructs produced measurable anti-tumor responses, resulting in slower tumor progression and improved survival compared with untreated controls. Although the work represents an early-stage proof of concept, it illustrated how LNP-mediated delivery can support complex mRNA vaccine strategies targeting multiple tumor antigens simultaneously. Jay emphasizes that these findings represent an important but preliminary step. “It’s important to remember that this is still a mouse melanoma model,” he says. “What we’re demonstrating is that this approach can generate an immune response and influence tumor growth. Translating that into therapies will require a lot more work.” Translational challenges remain Despite encouraging progress, several scientific and logistical challenges must be addressed before personalized cancer vaccines can become routine clinical therapies. Neoantigen identification: the real bottleneck in personalization Identifying the most immunogenic tumor mutations that will generate a meaningful immune response remains a complex task. Modern sequencing approaches can reveal dozens to hundreds of candidate neoantigens from a single tumor sample. However, only a small subset of these is likely to be presented effectively by antigen-presenting cells and recognized by T cells.5 “Finding the right epitopes is one of the hardest parts,” Jay explains. “You may identify dozens of mutations, but figuring out which ones will actually trigger a strong immune response is far from straightforward.” One common strategy is to prioritize neoantigens based on factors such as expression level, binding affinity to major histocompatibility complex (MHC) molecules, and predicted immunogenicity. However, these parameters do not always translate directly into clinical response. “Just because an epitope is highly expressed doesn’t mean it will generate a strong immune response,” Jay notes. “If it were that simple, the immune system would have already cleared the tumor.” This uncertainty introduces an element of empirical decision making into vaccine design. In some cases, researchers select a diverse set of epitopes, including highly expressed and moderately expressed candidates, to increase the likelihood of eliciting a productive immune response. At the same time, there is a need to carefully balance specificity and safety. “You also have to consider how similar these epitopes are to normal proteins,” Jay explains. “If you select something too close to what’s already present in healthy tissue, you risk triggering an unintended autoimmune response.”19 cytiva.com As a result, neoantigen selection represents a multidimensional challenge requiring integration of genomic data, immunological insight, and predictive modeling. Increasingly, computational approaches and machine learning tools are being explored to improve the accuracy and speed of this process, particularly as personalized therapies move toward clinical implementation. Manufacturing speed Personalized vaccines require a rapid turnaround between tumor sequencing and therapeutic administration. “You’re essentially developing a new drug for a single patient,” Jay says. “That means identifying the epitopes, producing the mRNA sequence, formulating it into nanoparticles, and delivering it quickly enough to make a clinical difference.” Automation and scalable RNA manufacturing platforms will be essential to support this workflow.6 Combination immunotherapy strategies Many researchers expect personalized cancer vaccines to work best when combined with other immunotherapies. Immune checkpoint inhibitors, which prevent tumors from suppressing immune responses, have already demonstrated a transformative impact in oncology.7 Combining these therapies with personalized vaccines may produce stronger, more durable antitumor responses. “A cancer vaccine may alone not be the full answer,” Jay explains. “But when combined with checkpoint inhibitors or other immunotherapies, there’s a real potential to create a much stronger therapeutic effect.” Towards individualized cancer treatment As genomic sequencing, RNA synthesis, and delivery technologies continue to evolve, personalized cancer vaccines are emerging as a promising addition to the oncology treatment landscape. These approaches are often framed in terms of individualization, tailoring therapies to the unique mutation profile of each patient’s tumor. However, their potential impact extends beyond personalization alone. One of the most important advantages of immuno-oncology approaches lies in their ability to address residual disease, recurrence, and metastasis—challenges that remain difficult to manage with conventional therapies. Surgeries and radiation can effectively remove or reduce primary tumors, but they do not always eliminate micrometastatic disease, where a small population of persisting cancer cells can eventually relapse and spread from the primary tumor to another part of the body. Similarly, systemic therapies may reduce tumor burden but often lack the specificity or durability needed for long-term control.20 cytiva.com Personalized cancer vaccines offer a different mechanism. By training the immune system to recognize tumor-specific antigens, they have the potential to establish ongoing immune surveillance, enabling the body to detect and eliminate cancer cells that re-emerge over time. “Personalized cancer vaccines could eventually become part of follow-up treatment,” Jay says. “After removing the primary tumor, a vaccine could help the immune system eliminate remaining cancer cells and prevent metastasis.” In this context, individualized vaccine design is not the endpoint; it is the enabler of a broader therapeutic goal: durable, system-wide protection against recurrence. Realizing this vision will require advances across multiple domains, including neoantigen selection, manufacturing speed, and clinical integration. Yet the convergence of these capabilities suggests a future in which cancer treatment extends beyond tumor removal toward long-term disease management driven by the patient’s own immune system. References 1. Lu L, Lu X, Luo W. Personalized Cancer vaccines: Current advances and emerging horizons. Vaccines. 2025;13(12):1231. doi: 10.3390/vaccines13121231 2. Sahin U, Derhovanessian E, Miller M, et al. Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer. Nature. 2017;547:222–226. doi: 10.1038/nature23003 3. Fennemann FL, de Vries IJM, Figdor CG, Verdoes M. Attacking tumors from all sides: Personalized multiplex vaccines to tackle intratumor heterogeneity. Front Immunol. 2019;10:824. doi: 10.3389/fimmu.2019.00824 4. Ostroumov D, Fekete-Drimusz N, Saborowski M, Kühnel F, Woller N. CD4 and CD8 T lymphocyte interplay in controlling tumor growth. Cell Mol Life Sci. 2018;75:689– 713. doi: 10.1007/s00018-017-2686-7 5. Zhang Y, Chen TT, Li X, et al. Advances and challenges in neoantigen prediction for cancer immunotherapy. Front Immunol. 2025;16:1617654. doi: 10.3389/ fimmu.2025.1617654 6. Magoola M, Niazi SK. Current progress and future perspectives of RNA-Based cancer vaccines: A 2025 update. Cancers. 2025;17(11):1882 doi: 10.3390/ cancers17111882 7. Ribas A, Wolchok JD. Cancer immunotherapy using checkpoint blockade. Science. 2018;359(6382):1350– 1355. doi: 10.1126/science.aar4060 Expert Perspective Manufacturing personalized cancer vaccines at small scale remains a significant challenge. The NxGen™ scale ready cartridge provides a gamma-irradiated, animal origin-free, fully traceable solution that helps researchers transition from process development to small-scale clinical studies using the NanoAssemblr Ignite™ system. Jay Paquette Senior Manager, Nucleic Acids and Nanomedicine, Cytiva PhD, Psychology: Brain, Behaviour and Cognitive Science, Queen’s University21 cytiva.com Chapter 4: Beyond the liver: advancing LNP delivery to the lung In previous chapters, we explored how advances in lipid nanoparticle (LNP) formulation are enhancing efficiency, manufacturability, and their application in personalized cancer vaccines. Yet, formulation optimization alone cannot unlock the full potential of RNA therapeutics. The next major challenge lies in directing those systems beyond their natural destination—the liver.1 Today, most lipid nanoparticle systems used to deliver RNA accumulate primarily in the liver following systemic administration. This biological bias has enabled important therapeutic advances, but it also limits the range of diseases that RNA medicines can target. Researchers across academia and industry are now working to overcome this constraint. Achieving extrahepatic delivery to other organs has become one of the most important frontiers in RNA medicine. Among the emerging targets, the lung represents both a compelling opportunity and a formidable scientific challenge. Why lung delivery presents a unique challenge Pulmonary diseases represent a major unmet medical need. Conditions such as cystic fibrosis, chronic obstructive pulmonary disease (COPD), and rare genetic lung disorders could potentially benefit from RNA therapies capable of restoring or modifying protein expression directly within airway cells. However, the lung is not an easy target for nanoparticle delivery. Unlike hepatocytes, pulmonary cells are protected by multiple biological barriers, including mucus layers, airway branching structures, and rapid clearance mechanisms. Inhaled particles must navigate these barriers before reaching epithelial cells capable of expressing the therapeutic RNA.2 “Pulmonary cells are completely different from other cells in the body”, explains Cytiva scientist Noorjahan Aibani, whose work focuses on developing LNP systems for pulmonary RNA delivery. “That means the design rules we use for liver delivery cannot simply be applied to lung tissue.” Traditional intravenous administration also performs poorly for lung targeting. As a result, researchers are exploring inhalation-based delivery methods, including nebulization, that can deposit nanoparticles directly into the respiratory tract. However, this strategy faces formulation challenges. Only a small fraction of inhaled particles ultimately deposit in the lower respiratory tract, meaning the delivery system must achieve strong transfection efficiency even at relatively low effective doses.22 cytiva.com Key barriers to pulmonary RNA delivery Pulmonary tissue presents several biological and engineering challenges for RNA delivery systems: • Airway mucus layers that trap nanoparticles • Rapid mucociliary clearance mechanism • Complex branching airway architecture • Low deposition efficiency during inhalation delivery • Mechanical stress during aerosolization • Distinct pulmonary cell biology compared with hepatocytes Overcoming these barriers requires nanoparticle composition specifically optimized for pulmonary tissues. Engineering LNPs for pulmonary delivery LNPs typically contain four core components: • An ionizable lipid that facilitates RNA encapsulation and endosomal escape • Cholesterol that stabilizes the lipid layer • Helper phospholipids that support particle structure • Surface modifying lipids that regulate particle size, colloidal stability, and biological interaction Adjusting the relative proportions of these components can significantly influence biodistribution, cellular uptake, and intracellular RNA release.3 To better understand how nanoparticle composition influences pulmonary delivery, researchers recently evaluated a panel of LNP formulations designed to deliver mRNA to the lung through nebulization. In the study, multiple formulations were screened to identify combinations capable of producing measurable expression in lung tissue following inhalation delivery. One observation was particularly notable: several formulations containing lower relative amounts of structural helper lipids produced higher reporter gene expression in lung tissue, a result that initially appeared counterintuitive. Conventional lipid nanoparticle designs usually rely on helper lipids to stabilize their structure. However, the specific mechanisms involved in the pulmonary environment are still under investigation. Several hypotheses can explain this behavior. One possibility is that a reduced quantity of structural lipids may increase the flexibility of the nanoparticle membranes, enhancing their interaction with pulmonary cell membranes and aiding in endosomal escape after cellular uptake. These findings reinforce a broader theme emerging across RNA delivery research: each organ requires its own formulation design strategy.23 cytiva.com Ensuring nanoparticle stability during nebulization Pulmonary delivery also introduces a mechanical challenge that does not occur with conventional intravenous administration: aerosolization. During nebulization, nanoparticles experience shear stress and repeated air–liquid interface exposure. These forces can destabilize lipid structure or reduce RNA encapsulation efficiency. For inhaled RNA therapeutics to be viable, LNPs must retain their physiological integrity during this process. To address this concern, researchers evaluated nanoparticle size, encapsulation efficiency, and other critical quality attributes before and after nebulization. Encouragingly, the tested LNPs maintained structural integrity under aerosolization conditions. “Our work showed that the particles remained stable after nebulization,” Noorjahan notes. “That is essential if pulmonary RNA delivery is going to work in practice.” Maintaining stability during aerosolization is a key prerequisite for translating inhaled RNA therapies into clinical applications. Building a platform for pulmonary RNA therapeutics Although pulmonary delivery research is often motivated by specific diseases such as cystic fibrosis, the implications extend well beyond a single indication. Pulmonary delivery Inhalation can access lung tissue in ways intravenous administration cannot. However, during nebulization, nanoparticles experience shear stress and repeated air–liquid interface exposure. These forces can destabilize lipid structure or reduce RNA encapsulation efficiency. What if LNPs could be engineered to overcome this challenge?24 cytiva.com “What we are developing is not limited to one disease,” Noorjahan explains. “This type of delivery platform could support long-term expression studies, gene editing approaches, and other advanced RNA therapies in the lung.” Efficient pulmonary delivery could enable: • Gene replacement strategies for inherited lung disease • Localized immunotherapies for respiratory cancers • CRISPR-based gene editing targeting airway epithelial cells Given the limited success of previous therapeutic approaches for some pulmonary genetic disorders, improved RNA delivery systems could represent a significant breakthrough. Designing RNA therapies for real-world accessibility Scientific performance is only one aspect of successful RNA therapeutics. Practical considerations such as storage stability, manufacturing scalability, and patient accessibility also play critical roles. Many current LNP therapeutics require ultra-cold storage conditions, sometimes as low as -80⁰C. While manageable for controlled vaccine distribution, such requirements could complicate long-term treatment strategies for chronic respiratory diseases. Researchers are therefore exploring alternative formats such as lyophilized dry-powder inhalation systems. These systems could potentially enable RNA medicines to be delivered using inhaler devices similar to those already used for asthma treatments. “Accessibility is an important consideration,” Noorjahan says. “It is not only about scaling up manufacturing. In some cases, it is also about producing smaller batches that can reach individual patients.” This perspective reflects a broader shift towards flexible manufacturing models that can support both large-scale production and personalized medicine applications. A rapidly evolving field beyond vaccines Momentum in RNA therapeutics continues to grow across multiple areas of medicine. Research programs that began with vaccine development are now expanding into cancer immunotherapy, gene editing technologies, and treatments for rare genetic diseases. As these applications emerge, delivery remains the central challenge. The ability to direct RNA therapeutics to specific tissues will ultimately determine which diseases these technologies can address. Pulmonary delivery research provides a clear example of how the field is beginning to tackle this challenge. By combining formulation engineering with inhalation-based administration strategies, researchers are starting to move RNA medicines beyond their natural liver tropism into previously difficult-to-reach organs.25 cytiva.com Expanding the reach of RNA medicine The ability to deliver RNA beyond the liver represents one of the most important milestones in the evolution of RNA therapeutics. Advances in nanoparticle formulation, aerosol delivery technologies, and translational research are beginning to expand the reach of RNA medicines. What once appeared to be a fundamental limitation of the lipid nanoparticle system is now becoming an active area of innovation. Pulmonary-targeted LNPs illustrate how this progress is unfolding. By adapting nanoparticle design to the unique biology of airway tissues and ensuring that formulations remain stable during inhalation delivery, researchers are beginning to unlock new possibilities for treating respiratory diseases. While significant scientific and translational challenges remain, continued advances in delivery technology may soon allow RNA therapeutics to reach organs that were once considered inaccessible. For pulmonary diseases such as cystic fibrosis and COPD, these innovations could ultimately open the door to entirely new therapeutic approaches. References 1. Cullis PR, Hope MJ. Lipid nanoparticle systems for enabling gene therapies. Mol Ther. 2017;25(7):1467–1475. doi: 10.1016/j.ymthe.2017.03.013 2. Paranjpe M, Müller-Goymann CC. Nanoparticle-mediated pulmonary drug delivery: A review. Int J Mol Sci. 2014;15(4):5852–5873. doi: 10.3390/ijms15045852 3. Hou X, Zaks T, Langer R, Dong Y. Lipid nanoparticles for mRNA delivery. Nat Rev Mater. 2021;6:1078–1094. doi: 10.1038/s41578-021-00358-0 Expert Perspective Because this study involved screening dozens of formulations, consistency was critical. The NanoAssemblr Ignite™ system allowed us to produce formulations quickly and reproducibly, making it easier to distinguish chemistry-driven effects from formulation variability. The resulting volumes also aligned well with the needs of our in vivo studies. Noorjahan Aibani Scientist II, Delivery, Cytiva PhD, Pharmaceutical Sciences, Ulster University26 cytiva.com Chapter 5: Expanding the horizon: targeted LNPs for extrahepatic delivery As discussed in the previous chapter, advancing extrahepatic RNA delivery using lipid nanoparticles (LNPs) marks a pivotal step in overcoming the liver’s intrinsic role as a biological “sink”. While the lung presents a distinct and accessible target via inhalation, the true inflection point lies beyond localized delivery. The field is now converging on the more complex challenge of systemic administration, where precise modulation of LNP biodistribution is essential to unlock efficient targeted delivery to distal organs. Achieving this level of control will define the next era of RNA therapeutics, requiring deeper integration of particle engineering, biological insight, and translational strategy. The spleen as a strategic target for next-generation therapies In vivo CAR T therapy is emerging as a defining application of LNP technology, enabling direct genetic modification of T cells to recognize and eliminate cancer. Conventional CAR T approaches rely on complex ex vivo workflows where patient cells are harvested, modified in labs, and reinfused weeks later, introducing significant time, cost, and manufacturing constraints.1 LNPs carrying messenger RNA (mRNA) offer a streamlined alternative by genetically modifying T cells directly within the patient’s body. This allows for temporary expression of chimeric antigen receptors (CARs), enabling CAR T therapy to be administered as an “off-the-shelf” injection. This approach has the potential to reduce logistical and manufacturing complexity, treatment time, and cost.1 However, making in vivo CAR-T therapy clinically viable depends on overcoming a major biological challenge: directing LNPs away from the liver and toward immune-relevant organs such as the spleen. As a primary lymphoid organ enriched with T cells and antigen-presenting cells, the spleen offers a uniquely permissive environment for immune-cell targeting and activation. Achieving this level of organ-specific targeting is an important step towards expanding therapeutic applications of RNA–LNP technology. Achieving cell-specific targeting Even if a delivery system successfully reaches the intended organ, such as the spleen, an additional challenge remains: targeting specific cell populations within a heterogeneous tissue environment.27 cytiva.com Off-target delivery at the cellular level introduces significant risk, including unintended gene disruption, aberrant cell activation, or immune-mediated toxicity, causing immune attacks on healthy cells.2 As a result, precision at the cellular scale is not simply optimization, it is also a prerequisite for clinical viability. To improve cellular specificity, delivery systems often incorporate ligands such as antibodies or peptides. However, these approaches introduce additional design complexity requiring careful optimization.3 Ligand density, for example, must be finely tuned, as insufficient density can reduce binding efficiency while excessive functionalization may cause aggregation or rapid immune clearance. Ultimately, for in vivo cell therapy to move beyond experimental trials and into routine medicine, researchers must prove that they can successfully deliver payloads to the correct cell, in the correct organ, with high precision and reproducibility. Enabling spleen tropism with advanced LNP formulations To overcome the inherent “liver sink” that often limits systemic RNA therapies, Cytiva is developing an advanced LNP-based delivery platform designed to enable more selective, extrahepatic distribution. To evaluate its potential for extrahepatic delivery, proprietary LNP–mRNA compositions were tested in mouse models.4 The results showed high levels of mRNA translation across multiple organs, such as the lungs, kidneys, and heart, confirming efficient mRNA delivery and expression. This ability to bypass the hepatic “sink” represents a pivotal step in expanding the reach of RNA–LNP technology. The most notable observation, however, was the platform’s distinct spleen tropism: The lead LNP formulation achieved a 34-fold increase in trafficking to the spleen relative to the liver (Figure 1). By leveraging the spleen’s role as a primary hub for immune cell activity, this approach unlocks new opportunities for immune-focused in vivo delivery strategies. 40 30 20 10 0 PBS LNP 1 LNP 8 LNP 9 Spleen/liver ratio (from avg. total flux) Figure 1. Cytiva’s LNPs enabled 34-fold higher spleen expression compared to the liver. Refining specificity: from organ to cell Reaching the correct organ is only half the battle; the payload must then reach the specific cells capable of mounting an immune response. Analysis of spleens from mice injected with LNPs revealed targeting across a range of cell types.4 Specifically, macrophages and natural killer cells showed higher transfection rates than T cells, B cells, and dendritic cells. This represents a promising avenue for chimeric antigen receptor macrophage (CAR-M) therapy and next-generation vaccines.28 cytiva.com To enhance cell-specific activity, CD8+ targeting moieties were conjugated to LNPs. When tested in a humanized mouse model, these targeted LNPs (tLNPs) successfully directed mRNA to the target T-cell populations, as evidenced by significantly higher transgene expression in CD8+ T-cell populations than in CD4+ T-cell populations (Figure 2). Not to be overlooked, advanced analytics developed by Cytiva’s BioPharma Services team were crucial in measuring the state of conjugation of targeting moieties, providing a readout to fine-tune the conjugation reaction. This combination of Cytiva’s advanced LNP formulations, ligand targeting, and analytical confirmation has enabled immune-cell programming in vivo—an important capability for translating targeted RNA therapies into scalable and clinically practical treatment platforms. Demonstrating therapeutic efficacy A key test of the platform was its ability to generate functional in vivo CAR-T cells capable of eliminating cancer. Mice with humanized tumors received tLNPs containing CAR mRNA, and the growth or regression of their cancer cells was tracked in real time. The results demonstrated: • Regression of cancer cells in treated mice • Dose-dependent effects • No observed adverse effects, suggesting the formulations were well tolerated Figure 2. Key insights into Cytiva’s innovation in LNP development Conventional LNP Cytiva LNP Intravenous administration Intravenous administration Natural accumulation in liver 34x trafficking to spleen over liver Uptake in liver cells Ligand conjugation enabling cell-specific targeting29 cytiva.com The selective and dose-dependent killing of tumor cells illustrates the potential of the tLNP platform for transient in vivo CAR-T cell therapy. A comprehensive platform for extrahepatic delivery As LNP formulations incorporate targeting moieties, advanced analytical methods are required to: • Characterize surface conjugation • Detect unconjugated particles or residual targeting moieties • Assess targeting specificity and activity Cytiva’s BioPharma Services bring together formulation and analytics into an end-to-end unified workflow, supported by NanoAssemblr™ systems, to enable the design, production, and characterization of complex LNP constructs. Central to this platform is an extensive library of ionizable lipids, including those explored above, distinguished by unique linker structures that offer enhanced biodegradability while maintaining flexibility in design and application. This portfolio is further supported by specialized non-PEG stabilizers, providing a complete toolkit for researchers looking to optimize delivery, stability, and targeting performance for in vivo cell therapy. Broadening the scope of RNA therapeutics While the potential of RNA therapeutics is vast, clinical progress has long been constrained by challenges of delivery beyond the liver. The demonstration of successful targeting of the spleen via LNPs represents a substantial leap toward piercing that horizon, demonstrating that the liver “sink” is no longer an insurmountable barrier. By ensuring the payload reaches the “right postal code” through the LNP formulation and the “right address” through targeting moieties, this platform paves the way for therapies that avoid the costs and complexities of traditional ex vivo processes. Ultimately, this core technology provides a blueprint to potentially enable RNA delivery to a wide array of non-liver targets. As active targeting strategies continue to mature, the goal of making the next generation of genomic medicine accessible to every patient is moving steadily within reach. References 1. Pinto E, Lione L, Compagnone M, et al. From ex vivo to in vivo chimeric antigen T cells manufacturing: new horizons for CAR T-cell based therapy. J Transl Med. 2025;23:10. doi: 10.1186/s12967-024-06052-3 2. Kalter N, Fuster-García C, Silva A, et al. Off-target effects in CRISPR-Cas genome editing for human therapeutics: Progress and challenges. Mol Ther Nucleic Acids. 2025;36(3):102636. doi: 10.1016/j.omtn.2025.102636 3. Lin X, Xiang H, Wu J, et al. The evolution of lipid nanoparticles: Paving the way for next-generation nucleic acid medicines. Asian J Pharm Sci. 2026;21(1):101121. doi: 10.1016/j.ajps.2026.101121 4. Golla K, Sams S, Yazdani H. Extrahepatic delivery of mRNA LNP to spleen: Towards in vivo CAR therapeutics. Cytotherapy. 2026;28(5):102591. doi: 10.1016/j.jcyt.2026.10259130 cytiva.com Expert Perspective Targeted LNPs have the potential to transform the future of RNA medicines by enabling precise delivery to relevant tissues and cell types. Realizing this potential involves navigating significant scientific and technical complexity, along with the establishment of appropriate CMC frameworks. Compared with conventional LNPs, targeted LNPs require more sophisticated analytical strategies to comprehensively characterize their attributes and understand how those attributes influence biological performance. By leveraging our extensive portfolio of ionizable lipids and stabilizers, robust analytical capabilities, and close collaboration across our chemistry, formulation, bioassay, process development, and analytical teams, and through strong partnerships with our collaborators, we generate the insights needed to accelerate the development and success of our partner programs. We ultimately believe this close collaboration is key to pushing the boundaries of targeted delivery and advancing programs toward clinical readiness. Anitha Thomas Director, R&D, Cytiva PhD, Chemistry, Interdisciplinary Chemistry & Biology, Indian Institute of Science (IISc)31 cytiva.com Featured resources Accelerating RNA therapeutics from discovery to clinic Explore Cytiva’s NanoAssemblr™ systems powered by NxGen™ technology, trusted by thousands of global users for early research to clinical and commercial-stage programs, enabling reproducible LNP formulation and scalable manufacturing. Explore formulation systems Enabling lipid design and RNA delivery Access Cytiva’s LNP portfolio, combining ionizable lipids, pre-optimized formulations, and specialist services, including liver de-targeting and antibody conjugation to accelerate RNA therapeutic development and delivery optimization. Explore LNP portfolioCytiva and the Drop logo are trademarks of Life Sciences IP Holdings Corporation or an affiliate doing business as Cytiva. NanoAssemblr and NxGen are trademarks of Global Life Sciences Solutions USA LLC or an affiliate doing business as Cytiva. Any other trademarks are the property of their respective owners. © 2026 Cytiva For local office contact information, visit cytiva.com/contact CY61101-05Aug26-EB cytiva.com About This eBook This eBook was developed through interviews and contributions from Cytiva subject matter experts and written by the editorial team at Cytiva. Written by Reshma Kapatrala Marketing Writer, Cytiva Subject Matter Experts Nikita Jain, PhD Senior Manager I, Delivery, Cytiva Hossein Yazdani Ahmadabadi, PhD Formulation Scientist II, Cytiva Jay Paquette, PhD Senior Manager, Nucleic Acids and Nanomedicine, Cytiva Noorjahan Aibani, PhD Scientist II, Delivery, Cytiva Anitha Thomas Director, R&D, Cytiva Shell Ip, PhD Client Learning and Scientific Content, Nanomedicine, Cytiva