Nucleic acid and gene therapies stand to revolutionize the treatment of cancers, genetic diseases, neurological disorders, and more. This Comment discusses critical biological phenomena across scales that should be considered when designing lipid nanoparticles for targeted delivery of nucleic acids, as well as computational and high-throughput technologies for studying these phenomena.

Interest continues to grow in the use of mRNA vaccines and therapeutics. While effective for immunization against infectious diseases, lipid nanoparticle (LNP) formulations used for other mRNA delivery applications suffer from off-target accumulation, poor immune transfection, and reactogenicity, limiting their application to immunoengineering. Development of new mRNA LNPs is severely bottlenecked by the LNP discovery process, which is historically low-throughput due to reliance on low-plex measurements. Here, we develop a high-throughput in vivo mRNA LNP screening platform based on barcoded mRNA (b-mRNA). Using this b-mRNA screening platform to simultaneously evaluate 122 LNPs, we identify novel LNP formulations capable of potent hepatic and extrahepatic transfection. We evaluate a lead LNP candidate for in situ immune modulation in a syngeneic mouse model of melanoma and demonstrate a significant reduction in tumor burden and extended survival compared to mice treated with a gold standard mRNA LNP formulation. We employ novel biochemical characterization techniques to analyze nanoparticle protein corona formation with single-particle resolution and gain insight into the influence of protein adsorption on hepatic and splenic transfection. Together, our results demonstrate the value of advanced LNP screening and characterization techniques for the development of next-generation mRNA LNPs for immunoengineering.

The therapeutic potential of chimeric antigen receptor (CAR) T cells has long been limited by the static nature of the engineered receptor; once manufactured, the cell is committed to a single antigen, unable to respond to the immune evasion and antigen heterogeneity that define solid tumors. Although solutions such as bispecific constructs and logic-gated circuits have been proposed, each encodes additional complexity into the cell product at the time of manufacture, with no capacity for post-infusion reprogramming. In this issue, Kuo and colleagues report a fundamentally different strategy-the meditope-enabled CAR-in which a small, structurally orthogonal peptide-docking interface is embedded into the extracellular domain of the CAR, converting a fixed cellular product into a programmable platform that can be redirected, selectively expanded, or precisely tracked by administering a modular adapter molecule. See related article by Kuo et al., p. XX.

RNA therapeutics are rapidly redefining the landscape of modern medicine, offering programmable solutions to target diseases at the genetic level. Their success, exemplified by FDA-approved siRNA drugs and mRNA vaccines in clinical use, is primarily enabled by lipid nanoparticles (LNPs), which protect RNA, facilitate its intracellular delivery, and enhance endosomal escape. However, LNPs exhibit limited organ selectivity, often accumulating in the liver, which restricts broader clinical translation. This review presents a materials-centered framework for engineering targeted LNPs that improve therapeutic efficacy in target organs while minimizing off-target effects. We first examine surface functionalization strategies using active targeting ligands such as antibodies, peptides, aptamers, carbohydrates, and small molecules. We then highlight approaches to modulate organ tropism through intrinsic lipid component design, such as rational design of ionizable lipids, use of lipid additives, and tuning of lipid composition. Key analytical methods for evaluating targeting efficiency, including in vitro and in vivo assays, are also discussed. Finally, we examine emerging applications of targeted LNPs across diverse disease areas, including cancer, women’s health disorders and neurological diseases, with an outline on future directions. Overall, this review aims to guide the rational design of next-generation targeted LNPs by presenting a toolbox of material strategies to facilitate the safe and effective application of RNA therapeutics.

Since the first market authorization of RNA therapies, just eight years ago, the field has witnessed an extraordinary expansion, ranging from hepatic delivery for rare genetic diseases to global-scale vaccination during the COVID-19 pandemic, and now to cutting-edge cancer vaccines and gene editing strategies entering late-stage clinical trials. In parallel, the RNA therapeutics landscape has evolved rapidly, progressing from small interfering RNAs to next-generation and combinatorial RNA modalities. None of these breakthroughs would have been possible without the development of sophisticated RNA delivery technologies capable of navigating complex biological environments, enabling precise cellular targeting, and facilitating efficient intracellular trafficking. In this Editorial Note, we take a step back to reflect on key lessons learned throughout the RNA delivery journey. Featuring insights from leading and experienced voices in the field, this manuscript highlights critical milestones, persistent challenges, and the roles of lipid nanoparticles (LNPs) and polymer nanoparticles (PNPs) as RNA delivery platforms. These experts reflect on the features that have positioned LNPs as the current RNA delivery gold standard, while also exploring the untapped potential and distinctive advantages of polymer-based nanosystems. Collectively, these perspectives underscore a striking truth: we are only beginning to unlock the full therapeutic potential of RNA, and nanomedicine will certainly continue to shape the future clinical translation of RNA-based therapies.

Solid tumors remain difficult to treat via conventional and novel therapeutic strategies. Immunotherapies such as chimeric antigen receptor T (CAR-T) cell therapy have been remarkably effective in treating hematological cancers, but their efficacy is limited in solid tumors. Recently, CAR macrophages (CAR-Ms) have emerged as a promising solid tumor immunotherapy, primarily for their intrinsic tumor infiltration and effector functions. However, CAR-Ms are engineered using viral transduction, which is associated with aberrant immunogenicity and toxicity. To overcome these challenges, we developed a bioinspired oxidized lipid nanoparticle (LNP) platform for mRNA-based engineering of human CAR-Ms. A library of 24 ionizable lipids was synthesized, formulated into LNPs, and screened for delivery to human macrophages. The top LNP was subsequently optimized using an orthogonal design of experiments and the physicochemical properties, such as size and mRNA encapsulation, were tuned via optimization of microfluidic mixing parameters, yielding an LNP formulation that significantly outperformed a gold standard C12-200 LNP. Utilizing small molecule and antibody inhibitors, we demonstrate that uptake of optimized LNPs into macrophages is driven by apolipoprotein E independent macropinocytosis, which is further supported by potent extrahepatic spleen tropism upon intravenous administration to mice. Lastly, we demonstrate the translatability of this LNP platform and utilize it to engineer functional primary human HER2-CAR-Ms ex vivo with potent antigen-specific tumor cell killing, validated in an ex vivo co-culture with ovarian cancer cells. This bioinspired oxidized LNP platform demonstrates potential for engineering a range of human CAR-M immunotherapies to treat various types of solid tumors.

Oral squamous cell carcinoma (OSCC) represents 90% of all head and neck cancers. Despite decades of research, the 5-year survival rate is 50%. Strikingly, the overall incidence rate is projected to increase by 30% in the next 10 years, which may result in a sharp increase in mortality. Two fundamental aspects of OSCC are that it progresses via the inactivation and mutation of tumor suppressor genes (TSGs) and has a "cold" tumor microenvironment (TME). A major barrier in the treatment of OSCC is the lack of novel therapies clinicians have at their disposal that are designed to disrupt tumor progression by reshaping the cold tumors into inflammatory "hot" tumors. To overcome these obstacles, we employed a lipid nanoparticle (LNP) that co-encapsulates p53 mRNA and the small molecule ciclopirox (CPX). We demonstrate that both drugs have innate chemotherapeutic properties by facilitating caspase activation. Moreover, these therapies can create a less immunosuppressive TME in part by repolarizing tumor-associated macrophages (TAMs) to M1-like phenotypes. When formulated together, our platform provides an all-in-one approach for OSCC, effective in both p53-therapy-susceptible and p53-therapy-resistant models. Additionally, this work offers a template for a delivery platform capable of tackling multiple mechanisms of OSCC progression and survival.

Regulating T cell phenotypes between activation and exhaustion remains a significant challenge for messenger RNA-based cancer immunotherapy. A potential approach to improve anti-cancer T cell activity is to co-deliver interleukin-12 (IL-12), to stimulate effector T cells, and indoleamine 2,3-dioxygenase (IDO) inhibitor, to suppress T cell exhaustion. Here we design prodrug ionizable lipid nanoparticles (pLNPs), via a library of prodrug ionizable lipids (pILs), incorporating an intracellularly cleavable IDO inhibitor within the pIL structure and encapsulating IL-12 messenger RNA. The lead pIL shows enhanced mRNA transfection over a clinically utilized ionizable lipid, as well as strong immunomodulatory effects via release of the IDO inhibitor. In a subcutaneous colon cancer mouse model, pLNP drives complete regression of primary tumours by eliciting effector T cell infiltration while reducing exhaustion, induces a memory T cell response and stimulates a systemic immune response that allows for regression of distal tumours in this study. These results highlight the promise of pLNPs for small-molecule drug and mRNA combination cancer immunotherapy.

Nanodiamonds (NDs) have emerged as versatile and promising nanocarriers in pharmacological applications, attributed to their distinctive physicochemical characteristics such as tunable size, customizable surface chemistry, and outstanding biocompatibility. These properties have allowed NDs to be explored extensively across various nanomedicine and biomedical research domains. Despite the significant promise of NDs, a comprehensive understanding of their biodistribution, pharmacokinetics (PK), and pharmacodynamics (PD) remains limited. This review systematically summarizes the critical factors influencing the PK and PD profiles of NDs, highlighting recent advances in their design and functionalization. Particular attention is given to the development of stimuli-responsive NDs that can actively respond to endogenous triggers such as pH, ionic concentrations, hypoxia, and enzymatic activity, as well as exogenous triggers including temperature and light. The integration of dual stimuli-responsive strategies further expands the potential applications of NDs for controlled and targeted drug delivery. Collectively, these advancements underscore the evolving role of NDs as next-generation therapeutic platforms with significant potential to enhance precision and efficacy in drug delivery systems.

Chimeric antigen receptor (CAR) T cell therapy has achieved clinical success in hematological malignancies, but its reliance on viral vectors and complex ex vivo manufacturing poses challenges related to safety, cost, and scalability. Next-generation strategies, including universal ("off-the-shelf") and in vivo CAR-T cell therapies, have emerged to address these limitations. The latter strategy employs targeted delivery systems to directly program patients' T cells in situ, bypassing ex vivo manipulation and offering a more streamlined, scalable, and safer therapeutic paradigm. The success of in vivo CAR-T cell therapy relies on targeted delivery systems. While engineered lentiviruses enable stable integration, non-viral vectors for transient CAR expression offer superior pharmacological control. This approach, exemplified by lipid nanoparticles in combination with mRNA, avoids risks of insertional mutagenesis and enables titratable, short-lived CAR expression, thereby enhancing safety management and suitability for applications beyond oncology. In this chapter, we first delineate the pharmacological imperative for transient CAR expression. Next, various delivery strategies are systematically reviewed, including ex vivo electroporation, in vivo non-viral systems, and engineered virus-like particles. Afterwards, we summarize the ongoing clinical trials of transient CAR-T cell therapy for oncology and non-oncology indications. Finally, we provide perspectives on the development of next-generation transient CAR-T cell therapies.

Lipid nanoparticles (LNPs) have been immensely successful in facilitating the delivery of nucleic acids to tissues of interest and continue to be optimized for mRNA delivery. These vehicles are particularly advantageous for vaccination due to their ease of production, highly tunable composition, and ability to induce robust immune responses without an adjuvant, as exemplified most recently by the clinical success of the Pfizer/BioNTech and Moderna COVID-19 vaccines. However, LNPs are known to exhibit off-target liver tropism. To address this limitation, we developed a library of aromatic bioreducible ionizable lipids that potently transfect secondary lymphoid tissues with liver detargeting capabilities compared with an industry standard ionizable lipid used in the COVID-19 vaccine. The library consists of three modular components: amine core structure, lipid tail length, and regiochemistry. These aromatic ionizable lipids employ benzene rings both as a scaffold for regiochemical differences and as a moiety to improve transfection. Bioreducible disulfide bonds in the lipids additionally serve to increase their biodegradability. When these aromatic ionizable lipids are formulated as LNPs, top-performing aromatic LNPs (aroLNPs) accumulate in and transfect lymph nodes while minimizing off-target liver tropism. Top-performing aroLNPs also induce strong antigen-specific immune responses, increased effector memory T cell generation, and decreased terminal effector T cell generation in mice when utilized in a preclinical SARS-CoV-2 vaccine study. Additionally, aroLNPs are strongly retained in the injection site and induce low levels of systemic inflammatory cytokines. Together, these results establish aroLNPs as a promising platform for vaccine delivery and potentially other immune-focused therapeutic applications.

Lipid nanoparticles are a burgeoning technology which has vast potential to improve chimeric antigen receptor (CAR) T cell immunotherapy. This focused review provides an overview of CAR T cell therapy - highlighting its promises, limitations, and challenges - and describes ways in which lipid nanoparticles (LNPs) can be rationally designed to circumvent some of the challenges. Of particular note are antigen presenting cell-mimetic LNPs, which have the potential to streamline the CAR T cell production process by activating T cells and delivering the CAR transgene in a single step. Although the current clinical standard is ex vivo CAR T cell production, in vivo CAR T cell production represents a potentially transformative alternative. Recent innovations in each production method are described, with a particular emphasis on ways in which LNPs may enable in vivo CAR T cell production. The review concludes with a discussion of safety, immunogenicity, scalability, manufacturing, and regulatory factors which will be essential as LNP-based CAR T cell immunotherapies move toward clinical translation.

Targeted lipid nanoparticles (tLNPs) enable cell-specific nucleic acid delivery through covalent attachment of targeting ligands that drive receptor-mediated LNP uptake. tLNPs are potentially promising for pregnancy‑associated applications where precise delivery is required to minimize maternal toxicity and protect fetal health. Yet, rational tLNP design is limited by an incomplete understanding of how physicochemical properties influence biological performance. Traditional analytical methods report only ensemble-averaged properties, leaving the nanoscale heterogeneity of tLNPs unresolved. Here, we utilize asymmetric flow field-flow fractionation integrated with in-line UV spectral analysis, light scattering, and synchrotron small-angle X-ray scattering (AF4-UV-DLS-MALS-SAXS) to resolve ligand-dependent tLNP subpopulations that differ in size, shape, composition, and relative abundance. Protein conjugation preserves the internal lipid-RNA nanostructure of base LNPs but substantially increases particle heterogeneity, particularly for larger and multivalent ligands. Despite increased heterogeneity, tLNPs functionalized with higher-avidity ligands achieve more effective targeted placental RNA delivery in mice. Chemometric SAXS analyses reveal that only SAXS-resolved tLNP subpopulations, not ensemble-averaged parameters, correlate with targeted placental transfection in vivo, whereas bulk physicochemical metrics more strongly associate with nonspecific hepatic delivery. Together, this work harnesses a separation-coupled biophysical platform to resolve previously inaccessible tLNP subpopulations and provides insights to inform rational engineering of next-generation targeted RNA therapeutics.

Achieving systemic nonviral delivery of large nucleic acids such as mRNA to the brain is challenging due to high off-target delivery and the blood-brain barrier (BBB), a cellular barrier which prevents most nucleic acids in circulation from entering the brain. Ionizable lipid nanoparticles (LNPs) are a promising class of nanocarriers to facilitate the delivery of mRNA, as their highly modular nature enables fine-tuning of the LNP formulation for targeted delivery applications. In this work, we explore the role of ionizable lipid chemical structure and lipid molar ratios within the LNP formulation on mRNA delivery to and transfection of the brain. We utilize a high-throughput in vivo screening approach based on mRNA barcoding to study a large library of LNPs made with systematically varied ionizable lipid structures, amounts of ionizable lipid, and amounts of lipid-polyethylene glycol (PEG). We find that ionizable lipids with longer tail structures and linear amine cores can facilitate mRNA delivery to the mouse brain, and ultimately identify a specific ionizable lipid, C14-306, that facilitates brain transfection coupled with reduced liver transfection compared to an FDA-approved benchmark formulation. Furthermore, the lead LNP formulated with C14-306 is able to increase neuronal transfection and facilitate Cre-mediated recombination in the brain. Finally, safety analyses demonstrate that the lead LNP does not induce BBB leakage, increases in serum inflammatory cytokine levels, or increases in serum liver enzyme levels. Overall, our work highlights the utility of molecular barcoding for high-throughput screening of LNPs for delivery to the brain and suggests several design principles to guide the engineering of next-generation brain-tropic LNPs.

Children are a heterogeneous population, and their physiology differs from adults in terms of physical size, body composition, organ development and metabolism; all of which impact the pharmacokinetics and pharmacodynamics of administered medications. However, less than 50% of therapeutics are evaluated in paediatric patients, and more than 40% of medications prescribed to children are used off-label. Therefore, there is a critical need to design medicines intentionally for children. Precision therapies for children require constituent biomaterial-based drug delivery systems that are engineered to target distinct microenvironments of paediatric patients. In this Review, we examine the specific anatomy, physiology and immune profiles of paediatric populations. By exploring the biological barriers and opportunities for drug delivery across three developmental stages — fetal (before birth), infant (birth to 23 months) child (2 years to adolescence) — we highlight considerations for the design of biomaterial-based drug delivery systems aimed at advancing translational paediatric medicines. Designing therapeutics for children requires careful consideration of age-dependent anatomy, physiology and immunology. This Review discusses the engineering and optimization of drug delivery systems for paediatric microenvironments to prevent, treat and cure childhood diseases.

Control of cell identity and number is central to tissue function, yet principles governing the organization of malignant cells remain poorly understood. Using genetically engineered mouse models and orthotopic allografts with dual WNT reporter systems, we discover that pancreatic ductal adenocarcinoma (PDAC) organizes in a stereotypical pattern, whereby PDAC cells responding to WNT signals (WNT-R) neighbor WNT-secreting cancer cells (WNT-S). Lineage tracing reveals that the WNT-R state is transient and gives rise to a stable WNT-S state. A subset of WNT-S cells expressing DLL1 forms a functional niche for WNT-R cells. The genetic inactivation of WNT secretion or Notch pathway components, or the cytoablation of WNT-S cells, disrupts PDAC tissue organization, suppressing tumor growth and metastasis. Analysis of human PDAC tissues confirms conservation of these populations. PDAC growth depends on an intricately controlled equilibrium of functionally distinct cancer cell states, revealing the fundamental principles governing solid tumor organization and therapeutic opportunities.