Combinatorial design of siloxane-incorporated lipid nanoparticles augments intracellular processing for tissue-specific mRNA therapeutic delivery.

Systemic delivery of messenger RNA (mRNA) for tissue-specific targeting using lipid nanoparticles (LNPs) holds great therapeutic potential. Nevertheless, how the structural characteristics of ionizable lipids (lipidoids) impact their capability to target cells and organs remains unclear. Here we engineered a class of siloxane-based ionizable lipids with varying structures and formulated siloxane-incorporated LNPs (SiLNPs) to control in vivo mRNA delivery to the liver, lung and spleen in mice. The siloxane moieties enhance cellular internalization of mRNA-LNPs and improve their endosomal escape capacity, augmenting their mRNA delivery efficacy. Using organ-specific SiLNPs to deliver gene editing machinery, we achieve robust gene knockout in the liver of wild-type mice and in the lungs of both transgenic GFP and Lewis lung carcinoma (LLC) tumour-bearing mice. Moreover, we showed effective recovery from viral infection-induced lung damage by delivering angiogenic factors with lung-targeted Si5-N14 LNPs. We envision that our SiLNPs will aid in the clinical translation of mRNA therapeutics for next-generation tissue-specific protein replacement therapies, regenerative medicine and gene editing.

Breaking the final barrier: Evolution of cationic and ionizable lipid structure in lipid nanoparticles to escape the endosome.

In the past decade, nucleic acid therapies have seen a boon in development and clinical translation largely due to advances in nanotechnology that have enabled their safe and targeted delivery. Nanoparticles can protect nucleic acids from degradation by serum enzymes and can facilitate entry into cells. Still, achieving endosomal escape to allow nucleic acids to enter the cytoplasm has remained a significant barrier, where less than 5% of nanoparticles within the endo-lysosomal pathway are able to transfer their cargo to the cytosol. Lipid-based drug delivery vehicles, particularly lipid nanoparticles (LNPs), have been optimized to achieve potent endosomal escape, and thus have been the vector of choice in the clinic as demonstrated by their utilization in the COVID-19 mRNA vaccines. The success of LNPs is in large part due to the rational design of lipids that can specifically overcome endosomal barriers. In this review, we chart the evolution of lipid structure from cationic lipids to ionizable lipids, focusing on structure-function relationships, with a focus on how they relate to endosomal escape. Additionally, we examine recent advancements in ionizable lipid structure as well as discuss the future of lipid design.

Lipid nanoparticle-mediated RNA delivery for immune cell modulation.

Lipid nanoparticles (LNPs) have emerged as the preeminent nonviral drug delivery vehicles for nucleic acid therapeutics, as exemplified by their usage in the mRNA COVID-19 vaccines. As a safe and highly modular delivery platform, LNPs are attractive for a wide range of applications. In addition to vaccines, LNPs are being utilized as platforms for other immunoengineering efforts, especially as cancer immunotherapies by modulating immune cells and their functionality via nucleic acid delivery. In this review, we focus on the methods and applications of LNP-based immunotherapy in five cell types: T cells, NK cells, macrophages, stem cells, and dendritic cells. Each of these cell types has wide-reaching applications in immunotherapy but comes with unique challenges and delivery barriers. By combining knowledge of immunology and nanotechnology, LNPs can be developed for improved immune cell targeting and transfection, ultimately working toward novel clinical therapeutics.

Tumour-derived small extracellular vesicles act as a barrier to therapeutic nanoparticle delivery.

Nanoparticles are promising for drug delivery applications, with several clinically approved products. However, attaining high nanoparticle accumulation in solid tumours remains challenging. Here we show that tumour cell-derived small extracellular vesicles (sEVs) block nanoparticle delivery to tumours, unveiling another barrier to nanoparticle-based tumour therapy. Tumour cells secrete large amounts of sEVs in the tumour microenvironment, which then bind to nanoparticles entering tumour tissue and traffic them to liver Kupffer cells for degradation. Knockdown of Rab27a, a gene that controls sEV secretion, decreases sEV levels and improves nanoparticle accumulation in tumour tissue. The therapeutic efficacy of messenger RNAs encoding tumour suppressing and proinflammatory proteins is greatly improved when co-encapsulated with Rab27a small interfering RNA in lipid nanoparticles. Together, our results demonstrate that tumour cell-derived sEVs act as a defence system against nanoparticle tumour delivery and that this system may be a potential target for improving nanoparticle-based tumour therapies.

Flow cytometric analysis of the murine placenta to evaluate nanoparticle platforms during pregnancy.

Clinically approved therapeutics for obstetric conditions are extremely limited, with over 80% of drugs lacking appropriate labeling information for pregnant individuals. The pathology for many of these obstetric conditions can be linked to the placenta, necessitating the development of therapeutic platforms for selective drug delivery to the placenta. When evaluating therapeutics for placental delivery, literature has focused on ex vivo delivery to human placental cells and tissue, which can be difficult to source for non-clinical researchers. Evaluating in vivo drug delivery to the placenta using small animal models can be more accessible than using human tissue, but robust, quantitative methods to characterize delivery remain poorly established. Here, we report a flow cytometric method to evaluate in vivo drug delivery to the murine placenta. Specifically, we describe techniques to identify key cell types in the murine placenta - trophoblasts, endothelial cells, and immune cells - via flow cytometric analysis. While we have employed this method to detect lipid nanoparticle-mediated nucleic acid delivery, this approach can extend to a variety of drug carriers (e.g., liposomes, exosomes, polymeric and metallic nanoparticles) and payloads (e.g., small molecules, proteins, other nucleic acids). Similarly, we describe the application of this method toward immunophenotypic analysis to assess changes in the placental immune environment during disease or in response to a therapeutic. Together, the techniques reported herein aim to broaden the accessibility of placental research in an effort to encourage collaboration between physician-scientists, engineers, placental biologists, and clinicians for developing novel therapeutics to treat placental conditions during pregnancy.

OLAH connects fatty acid metabolism to the severity of respiratory viral disease.

Respiratory virus infections may cause profound respiratory illness, yet the factors that underlie disease severity are not well understood. In this issue of Cell, Jia, Crawford, et al.1 identify the role of oleoyl-ACP-hydrolase (OLAH) in mediating life-threatening inflammation associated with viral respiratory disease severity.

Enhancing in situ cancer vaccines using delivery technologies.

In situ cancer vaccination refers to any approach that exploits tumour antigens available at a tumour site to induce tumour-specific adaptive immune responses. These approaches hold great promise for the treatment of many solid tumours, with numerous candidate drugs under preclinical or clinical evaluation and several products already approved. However, there are challenges in the development of effective in situ cancer vaccines. For example, inadequate release of tumour antigens from tumour cells limits antigen uptake by immune cells; insufficient antigen processing by antigen-presenting cells restricts the generation of antigen-specific T cell responses; and the suppressive immune microenvironment of the tumour leads to exhaustion and death of effector cells. Rationally designed delivery technologies such as lipid nanoparticles, hydrogels, scaffolds and polymeric nanoparticles are uniquely suited to overcome these challenges through the targeted delivery of therapeutics to tumour cells, immune cells or the extracellular matrix. Here, we discuss delivery technologies that have the potential to reduce various clinical barriers for in situ cancer vaccines. We also provide our perspective on this emerging field that lies at the interface of cancer vaccine biology and delivery technologies.

Optimized microfluidic formulation and organic excipients for improved lipid nanoparticle mediated genome editing.

mRNA-based gene editing platforms have tremendous promise in the treatment of genetic diseases. However, for this potential to be realized in vivo, these nucleic acid cargos must be delivered safely and effectively to cells of interest. Ionizable lipid nanoparticles (LNPs), the most clinically advanced non-viral RNA delivery system, have been well-studied for the delivery of mRNA but have not been systematically optimized for the delivery of mRNA-based CRISPR-Cas9 platforms. In this study, we investigated the effect of microfluidic and lipid excipient parameters on LNP gene editing efficacy. Through in vitro screening in liver cells, we discovered distinct trends in delivery based on phospholipid, cholesterol, and lipid-PEG structure in LNP formulations. Combination of top-performing lipid excipients produced an LNP formulation that resulted in 3-fold greater gene editing in vitro and facilitated 3-fold greater reduction of a therapeutically-relevant protein in vivo relative to the unoptimized LNP formulation. Thus, systematic optimization of LNP formulation parameters revealed a novel LNP formulation that has strong potential for delivery of gene editors to the liver to treat metabolic disease.

Fast and facile synthesis of amidine-incorporated degradable lipids for versatile mRNA delivery in vivo.

Lipid nanoparticles (LNPs) are widely used for mRNA delivery, with cationic lipids greatly affecting biodistribution, cellular uptake, endosomal escape and transfection efficiency. However, the laborious synthesis of cationic lipids limits the discovery of efficacious candidates and slows down scale-up manufacturing. Here we develop a one-pot, tandem multi-component reaction based on the rationally designed amine-thiol-acrylate conjugation, which enables fast (1 h) and facile room-temperature synthesis of amidine-incorporated degradable (AID) lipids. Structure-activity relationship analysis of a combinatorial library of 100 chemically diverse AID-lipids leads to the identification of a tail-like amine-ring-alkyl aniline that generally affords efficacious lipids. Experimental and theoretical studies show that the embedded bulky benzene ring can enhance endosomal escape and mRNA delivery by enabling the lipid to adopt a more conical shape. The lead AID-lipid can not only mediate local delivery of mRNA vaccines and systemic delivery of mRNA therapeutics, but can also alter the tropism of liver-tropic LNPs to selectively deliver gene editors to the lung and mRNA vaccines to the spleen.

Lipid-mediated intracellular delivery of recombinant bioPROTACs for the rapid degradation of undruggable proteins.

Recently, targeted degradation has emerged as a powerful therapeutic modality. Relying on "event-driven" pharmacology, proteolysis targeting chimeras (PROTACs) can degrade targets and are superior to conventional inhibitors against undruggable proteins. Unfortunately, PROTAC discovery is limited by warhead scarcity and laborious optimization campaigns. To address these shortcomings, analogous protein-based heterobifunctional degraders, known as bioPROTACs, have been developed. Compared to small-molecule PROTACs, bioPROTACs have higher success rates and are subject to fewer design constraints. However, the membrane impermeability of proteins severely restricts bioPROTAC deployment as a generalized therapeutic modality. Here, we present an engineered bioPROTAC template able to complex with cationic and ionizable lipids via electrostatic interactions for cytosolic delivery. When delivered by biocompatible lipid nanoparticles, these modified bioPROTACs can rapidly degrade intracellular proteins, exhibiting near-complete elimination (up to 95% clearance) of targets within hours of treatment. Our bioPROTAC format can degrade proteins localized to various subcellular compartments including the mitochondria, nucleus, cytosol, and membrane. Moreover, substrate specificity can be easily reprogrammed, allowing modular design and targeting of clinically-relevant proteins such as Ras, Jnk, and Erk. In summary, this work introduces an inexpensive, flexible, and scalable platform for efficient intracellular degradation of proteins that may elude chemical inhibition.

In utero delivery of targeted ionizable lipid nanoparticles facilitates in vivo gene editing of hematopoietic stem cells.

Monogenic blood diseases are among the most common genetic disorders worldwide. These diseases result in significant pediatric and adult morbidity, and some can result in death prior to birth. Novel ex vivo hematopoietic stem cell (HSC) gene editing therapies hold tremendous promise to alter the therapeutic landscape but are not without potential limitations. In vivo gene editing therapies offer a potentially safer and more accessible treatment for these diseases but are hindered by a lack of delivery vectors targeting HSCs, which reside in the difficult-to-access bone marrow niche. Here, we propose that this biological barrier can be overcome by taking advantage of HSC residence in the easily accessible liver during fetal development. To facilitate the delivery of gene editing cargo to fetal HSCs, we developed an ionizable lipid nanoparticle (LNP) platform targeting the CD45 receptor on the surface of HSCs. After validating that targeted LNPs improved messenger ribonucleic acid (mRNA) delivery to hematopoietic lineage cells via a CD45-specific mechanism in vitro, we demonstrated that this platform mediated safe, potent, and long-term gene modulation of HSCs in vivo in multiple mouse models. We further optimized this LNP platform in vitro to encapsulate and deliver CRISPR-based nucleic acid cargos. Finally, we showed that optimized and targeted LNPs enhanced gene editing at a proof-of-concept locus in fetal HSCs after a single in utero intravenous injection. By targeting HSCs in vivo during fetal development, our Systematically optimized Targeted Editing Machinery (STEM) LNPs may provide a translatable strategy to treat monogenic blood diseases before birth.

High-Throughput In Vivo Screening Identifies Differential Influences on mRNA Lipid Nanoparticle Immune Cell Delivery by Administration Route.

Immune modulation through the intracellular delivery of nucleoside-modified mRNA to immune cells is an attractive approach for in vivo immunoengineering, with applications in infectious disease, cancer immunotherapy, and beyond. Lipid nanoparticles (LNPs) have come to the fore as a promising nucleic acid delivery platform, but LNP design criteria remain poorly defined, making the rate-limiting step for LNP discovery the screening process. In this study, we employed high-throughput in vivo LNP screening based on molecular barcoding to investigate the influence of LNP composition on immune tropism with applications in vaccines and systemic immunotherapies. Screening a large LNP library under both intramuscular (i.m.) and intravenous (i.v.) injection, we observed differential influences on LNP uptake by immune populations across the two administration routes, gleaning insight into LNP design criteria for in vivo immunoengineering. In validation studies, the lead LNP formulation for i.m. administration demonstrated substantial mRNA translation in the spleen and draining lymph nodes with a more favorable biodistribution profile than LNPs formulated with the clinical standard ionizable lipid DLin-MC3-DMA (MC3). The lead LNP formulations for i.v. administration displayed potent immune transfection in the spleen and peripheral blood, with one lead LNP demonstrating substantial transfection of splenic dendritic cells and another inducing substantial transfection of circulating monocytes. Altogether, the immunotropic LNPs identified by high-throughput in vivo screening demonstrated significant promise for both locally- and systemically-delivered mRNA and confirmed the value of the LNP design criteria gleaned from our screening process, which could potentially inform future endeavors in mRNA vaccine and immunotherapy applications.

EGFR-targeted ionizable lipid nanoparticles enhance in vivo mRNA delivery to the placenta.

The full potential of ionizable lipid nanoparticles (LNPs) as an in vivo nucleic acid delivery platform has not yet been realized given that LNPs primarily accumulate in the liver following systemic administration, limiting their success to liver-centric conditions. The engineering of LNPs with antibody targeting moieties can enable extrahepatic tropism by facilitating site-specific LNP tethering and driving preferential LNP uptake into receptor-expressing cell types via receptor-mediated endocytosis. Obstetric conditions stemming from placental dysfunction, such as preeclampsia, are characterized by overexpression of cellular receptors, including the epidermal growth factor receptor (EGFR), making targeted LNP platforms an exciting potential treatment strategy for placental dysfunction during pregnancy. Herein, an EGFR antibody-conjugated LNP (aEGFR-LNP) platform was developed by engineering LNPs with increasing densities of antibody functionalization. aEGFR-LNPs were screened in vitro in immortalized placental trophoblasts and in vivo in non-pregnant and pregnant mice and compared to non-targeted formulations for extrahepatic, antibody-targeted mRNA LNP delivery to the placenta. Our top performing LNP with an intermediate density of antibody functionalization (1:5 aEGFR-LNP) mediated a âˆ¼twofold increase in mRNA delivery in murine placentas and a âˆ¼twofold increase in LNP uptake in EGFR-expressing trophoblasts compared to non-targeted counterparts. These results demonstrate the potential of antibody-conjugated LNPs for achieving extrahepatic tropism, and the ability of aEGFR-LNPs in promoting mRNA delivery to EGFR-expressing cell types in the placenta.

Mechanisms and Barriers in Nanomedicine: Progress in the Field and Future Directions.

In recent years, steady progress has been made in synthesizing and characterizing engineered nanoparticles, resulting in several approved drugs and multiple promising candidates in clinical trials. Regulatory agencies such as the Food and Drug Administration and the European Medicines Agency released important guidance documents facilitating nanoparticle-based drug product development, particularly in the context of liposomes and lipid-based carriers. Even with the progress achieved, it is clear that many barriers must still be overcome to accelerate translation into the clinic. At the recent conference workshop "Mechanisms and Barriers in Nanomedicine" in May 2023 in Colorado, U.S.A., leading experts discussed the formulation, physiological, immunological, regulatory, clinical, and educational barriers. This position paper invites open, unrestricted, nonproprietary discussion among senior faculty, young investigators, and students to trigger ideas and concepts to move the field forward.

Ionizable lipid nanoparticles for RAS protease delivery to inhibit cancer cell proliferation.

Mutations in RAS, a family of proteins found in all human cells, drive a third of cancers, including many pancreatic, colorectal, and lung cancers. However, there is a lack of clinical therapies that can effectively prevent RAS from causing tumor growth. Recently, a protease was engineered that specifically degrades active RAS, offering a promising new tool for treating these cancers. However, like many other intracellularly acting protein-based therapies, this protease requires a delivery vector to reach its site of action within the cell. In this study, we explored the incorporation of cationic lipids into ionizable lipid nanoparticles (LNPs) to develop a RAS protease delivery platform capable of inhibiting cancer cell proliferation in vitro and in vivo. A library of 13 LNPs encapsulating RAS protease was designed, and each formulation was evaluated for in vitro delivery efficiency and toxicity. A subset of four top-performing LNP formulations was identified and further evaluated for their impact on cancer cell proliferation in human colorectal cancer cells with mutated KRAS in vitro and in vivo, as well as their in vivo biodistribution and toxicity. In vivo, both the concentration of cationic lipid and type of cargo influenced LNP and cargo distribution. All lead candidate LNPs showed RAS protease functionality in vitro, and the top-performing formulation achieved effective intracellular RAS protease delivery in vivo, decreasing cancer cell proliferation in an in vivo xenograft model and significantly reducing tumor growth and size. Overall, this work demonstrates the use of LNPs as an effective delivery platform for RAS proteases, which could potentially be utilized for cancer therapies.

Vascular endothelial-derived SPARCL1 exacerbates viral pneumonia through pro-inflammatory macrophage activation.

Inflammation induced by lung infection is a double-edged sword, moderating both anti-viral and immune pathogenesis effects; the mechanism of the latter is not fully understood. Previous studies suggest the vasculature is involved in tissue injury. Here, we report that expression of Sparcl1, a secreted matricellular protein, is upregulated in pulmonary capillary endothelial cells (EC) during influenza-induced lung injury. Endothelial overexpression of SPARCL1 promotes detrimental lung inflammation, with SPARCL1 inducing 'M1-like' macrophages and related pro-inflammatory cytokines, while SPARCL1 deletion alleviates these effects. Mechanistically, SPARCL1 functions through TLR4 on macrophages in vitro, while TLR4 inhibition in vivo ameliorates excessive inflammation caused by endothelial Sparcl1 overexpression. Finally, SPARCL1 expression is increased in lung ECs from COVID-19 patients when compared with healthy donors, while fatal COVID-19 correlates with higher circulating SPARCL1 protein levels in the plasma. Our results thus implicate SPARCL1 as a potential prognosis biomarker for deadly COVID-19 pneumonia and as a therapeutic target for taming hyperinflammation in pneumonia.

Influence of ionizable lipid tail length on lipid nanoparticle delivery of mRNA of varying length.

RNA-based therapeutics have gained traction for the prevention and treatment of a variety of diseases. However, their fragility and immunogenicity necessitate a drug carrier. Lipid nanoparticles (LNPs) have emerged as the predominant delivery vehicle for RNA therapeutics. An important component of LNPs is the ionizable lipid (IL), which is protonated in the acidic environment of the endosome, prompting cargo release into the cytosol. Currently, there is growing evidence that the structure of IL lipid tails significantly impacts the efficacy of LNP-mediated mRNA translation. Here, we optimized IL tail length for LNP-mediated delivery of three different mRNA cargos. Using C12-200, a gold standard IL, as a model, we designed a library of ILs with varying tail lengths and evaluated their potency in vivo. We demonstrated that small changes in lipophilicity can drastically increase or decrease mRNA translation. We identified that LNPs formulated with firefly luciferase mRNA (1929 base pairs) and C10-200, an IL with shorter tail lengths than C12-200, enhance liver transfection by over 10-fold. Furthermore, different IL tail lengths were found to be ideal for transfection of LNPs encapsulating mRNA cargos of varying sizes. LNPs formulated with erythropoietin (EPO), responsible for stimulating red blood cell production, mRNA (858 base pairs), and the C13-200 IL led to EPO translation at levels similar to the C12-200 LNP. The LNPs formulated with Cas9 mRNA (4521 base pairs) and the C9-200 IL induced over three times the quantity of indels compared with the C12-200 LNP. Our findings suggest that shorter IL tails may lead to higher transfection of LNPs encapsulating larger mRNAs, and that longer IL tails may be more efficacious for delivering smaller mRNA cargos. We envision that the results of this project can be utilized as future design criteria for the next generation of LNP delivery systems for RNA therapeutics.

Ionizable Lipid Nanoparticle-Mediated TRAIL mRNA Delivery in the Tumor Microenvironment to Inhibit Colon Cancer Progression.

Introduction: Immunotherapy has revolutionized cancer treatment by harnessing the immune system to enhance antitumor responses while minimizing off-target effects. Among the promising cancer-specific therapies, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted significant attention. Methods: Here, we developed an ionizable lipid nanoparticle (LNP) platform to deliver TRAIL mRNA (LNP-TRAIL) directly to the tumor microenvironment (TME) to induce tumor cell death. Our LNP-TRAIL was formulated via microfluidic mixing and the induction of tumor cell death was assessed in vitro. Next, we investigated the ability of LNP-TRAIL to inhibit colon cancer progression in vivo in combination with a TME normalization approach using Losartan (Los) or angiotensin 1-7 (Ang(1-7)) to reduce vascular compression and deposition of extracellular matrix in mice. Results: Our results demonstrated that LNP-TRAIL induced tumor cell death in vitro and effectively inhibited colon cancer progression in vivo, particularly when combined with TME normalization induced by treatment Los or Ang(1-7). In addition, potent tumor cell death as well as enhanced apoptosis and necrosis was found in the tumor tissue of a group treated with LNP-TRAIL combined with TME normalization. Discussion: Together, our data demonstrate the potential of the LNP to deliver TRAIL mRNA to the TME and to induce tumor cell death, especially when combined with TME normalization. Therefore, these findings provide important insights for the development of novel therapeutic strategies for the immunotherapy of solid tumors.