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Testing the <em>In Vitro</em> and <em>In Vivo</em> Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and ...

University of Pennsylvania

Here we describe two protocols for the construction of responsive and activable nanomedicines that regulate the tumor microenvironment (TME). The TME is composed of all non-cellular and cellular components surrounding a tumor, including the surrounding blood vessels, immune cells, fibroblasts, signaling molecules, and extracellular matrix and has a crucial role in tumor initiation, growth, and metastasis. Owing to the relatively stable properties of the TME compared to tumor cells, which exhibit frequent genetic mutations and epigenetic changes, therapeutic strategies targeting the TME using multifunctional nanomedicines hold great potential for anti-tumor therapy. By regulating tumor-associated platelets and pancreatic stellate cells (PSCs), the two major players in the TME, we can effectively manipulate the physiological barriers for enhanced drug delivery and significantly improve the tumor penetration and therapeutic efficacy of chemotherapeutics. The preparation and characterization of the multifunctional nanoparticles takes ~10 h for tumor-associated platelet regulation and 16 h for PSC regulation. These nanoformulations can be readily applied to regulate other components in the TME to realize synergistic or additive anti-tumor activity.

Responsive and activable nanomedicines for remodeling the tumor microenvironment.

Recent years have witnessed incredible growth in RNA therapeutics, which has benefited significantly from decades of research on lipid nanoparticles, specifically its key component—the ionizable lipid. This comment discusses the major ionizable lipid types, and provides perspectives for future development.

An ionizable lipid toolbox for RNA delivery.

Current nucleoside-modified RNA lipid nanoparticle (modmRNA-LNP) technology has successfully paved the way for the highest clinical efficacy data from next-generation vaccinations against SARS-CoV-2 during the COVID-19 pandemic. However, such modmRNA-LNP technology has not been characterized in common pre-existing inflammatory or immune-challenged conditions, raising the risk of adverse clinical effects when administering modmRNA-LNPs in such cases. Herein, we induce an acute-inflammation model in mice with lipopolysaccharide (LPS) intratracheally (IT), 1 mg kg, or intravenously (IV), 2 mg kg, and then IV administer modmRNA-LNP, 0.32 mg kg, after 4 h, and screen for inflammatory markers, such as pro-inflammatory cytokines. ModmRNA-LNP at this dose caused no significant elevation of cytokine levels in naive mice. In contrast, shortly after LPS immune stimulation, modmRNA-LNP enhanced inflammatory cytokine responses, Interleukin-6 (IL-6) in serum and Macrophage Inflammatory Protein 2 (MIP-2) in liver significantly. Our report identifies this phenomenon as inflammation exacerbation (IE), which was proven to be specific to the LNP, acting independent of mRNA cargo, and was demonstrated to be time- and dose-dependent. Macrophage depletion as well as TLR3 -/- and TLR4-/- knockout mouse studies revealed macrophages were the immune cells involved or responsible for IE. Finally, we show that pretreatment with anti-inflammatory drugs, such as corticosteroids, can partially alleviate IE response in mice. Our findings characterize the importance of LNP-mediated IE phenomena in gram negative bacterial inflammation, however, the generalizability of modmRNA-LNP in other forms of chronic or acute inflammatory and immune contexts needs to be addressed.

Added to pre-existing inflammation, mRNA-lipid nanoparticles induce inflammation exacerbation (IE).

Lipid nanoparticles (LNPs) play a crucial role in delivering messenger RNA (mRNA) therapeutics for clinical applications, including COVID-19 mRNA vaccines. While mRNA can be chemically modified to become immune-silent and increase protein expression, LNPs can still trigger innate immune responses and cause inflammation-related adverse effects. Inflammation can in turn suppress mRNA translation and reduce the therapeutic effect. Dexamethasone (Dex) is a widely used anti-inflammatory corticosteroid medication that is structurally similar to cholesterol, a key component of LNPs. Here, we developed LNP formulations with anti-inflammatory properties by partially substituting cholesterol with Dex as a means to reduce inflammation. We demonstrated that Dex-incorporated LNPs effectively abrogated the induction of tumor necrosis factor alpha (TNF-ɑ) in vitro and significantly reduced its expression in vivo. Reduction of inflammation using this strategy improved in vivo mRNA expression in mice by 1.5-fold. Thus, we envision that our Dex-incorporated LNPs could potentially be used to broadly to reduce the inflammatory responses of LNPs and enhance protein expression of a range of mRNA therapeutics.

Rational design of anti-inflammatory lipid nanoparticles for mRNA delivery.

Delivery of nucleic acids, such as mRNA, to immune cells has become a major focus in the past decade with ionizable lipid nanoparticles (LNPs) emerging as a clinically-validated delivery platform. LNPs-typically composed of ionizable lipids, cholesterol, phospholipids, and polyethylene glycol lipids -have been designed and optimized for a variety of applications including cancer therapies, vaccines, and gene editing. However, LNPs have only recently been investigated for delivery to T cells, which has various therapeutic applications including the engineering of T cell immunotherapies. While several LNP formulations have been evaluated for mRNA delivery, recent work has demonstrated that the utilization of cholesterol analogs may enhance mRNA delivery. Other studies have shown that cholesterols modified with hydroxyl groups can alter endocytic recycling mechanisms. Here, we engineered a library of LNPs incorporating hydroxycholesterols to evaluate their impact on mRNA delivery to T cells by leveraging endosomal trafficking mechanisms. Substitution of 25% and 50% 7α-hydroxycholesterol for cholesterol in LNPs enhanced mRNA delivery to primary human T cells ex vivo by 1.8-fold and 2.0-fold, respectively. Investigation of endosomal trafficking revealed that these modifications also increase late endosome production and reduce the presence of recycling endosomes. These results suggest that hydroxyl modification of cholesterol molecules incorporated into LNP formulations provides a mechanism for improving delivery of nucleic acid cargo to T cells for a range of immunotherapy applications.

Hydroxycholesterol substitution in ionizable lipid nanoparticles for mRNA delivery to T cells.

The development of lipid nanoparticle (LNP) formulations for targeting the bone microenvironment holds significant potential for nucleic acid therapeutic applications including bone regeneration, cancer, and hematopoietic stem cell therapies. However, therapeutic delivery to bone remains a significant challenge due to several biological barriers, such as low blood flow in bone, blood-bone marrow barriers, and low affinity between drugs and bone minerals, which leads to unfavorable therapeutic dosages in the bone microenvironment. Here, we construct a series of bisphosphonate (BP) lipid-like materials possessing a high affinity for bone minerals, as a means to overcome biological barriers to deliver mRNA therapeutics efficiently to the bone microenvironment . Following  screening of BP lipid-like materials formulated into LNPs, we identified a lead BP-LNP formulation, 490BP-C14, with enhanced mRNA expression and localization in the bone microenvironment of mice  compared to 490-C14 LNPs in the absence of BPs. Moreover, BP-LNPs enhanced mRNA delivery and secretion of therapeutic bone morphogenetic protein-2 from the bone microenvironment upon intravenous administration. These results demonstrate the potential of BP-LNPs for delivery to the bone microenvironment, which could potentially be utilized for a range of mRNA therapeutic applications including regenerative medicine, protein replacement, and gene editing therapies.

Rational Design of Bisphosphonate Lipid-like Materials for mRNA Delivery to the Bone Microenvironment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has already infected more than 500 million people globally (as of May 2022), creating the coronavirus disease 2019 (COVID-19) pandemic. Nanotechnology has played a pivotal role in the fight against SARS-CoV-2 in various aspects, with the successful development of the two highly effective nanotechnology-based messenger RNA vaccines being the most profound. Despite the remarkable efficacy of mRNA vaccines against the original SARS-CoV-2 strain, hopes for quickly ending this pandemic have been dampened by the emerging SARS-CoV-2 variants, which have brought several new pandemic waves. Thus, novel strategies should be proposed to tackle the crisis presented by existing and emerging SARS-CoV-2 variants. Here, we discuss the SARS-CoV-2 variants from biological and immunological perspectives, and the rational design and development of novel and potential nanotechnology-based strategies to combat existing and possible future SARS-CoV-2 variants. The lessons learnt and design strategies developed from this battle against SARS-CoV-2 variants could also inspire innovation in the development of nanotechnology-based strategies for tackling other global infectious diseases and their future variants.

Nanotechnology-based strategies against SARS-CoV-2 variants.

During long-term antiplatelet agents (APAs) administration, patients with thrombotic diseases take a fairly high risk of life-threatening bleeding, especially when in need of urgent surgery. Rapid functional reversal of APAs remains an issue yet to be efficiently resolved by far due to the lack of any specific reversal agent in the clinic, which greatly restricts the use of APAs. Although platelet transfusion has become a common practice to counteract the function of APAs, personalized dosage of platelet transfusion is difficult to be judged by assessing the trade-off between its effectiveness and additional thrombotic risk. Here we reported a platelet-mimicking perfluorocarbon-based nanosponge, which potently reversed the antiplatelet effect of APAs by competitively binding with APAs. Platelet-mimicking perfluorocarbon-based nanosponges showed high binding affinity comparable to fresh platelets in vitro with first-line APAs, ticagrelor and tirofiban, and efficiently reversed their function in both tail bleeding and postischemic-reperfusion models. Moreover, the deficiency of platelet intrinsic thrombotic activity diminished the risk of thrombogenesis. In summary, our results demonstrated the safety and effectiveness of the platelet-mimicking nanosponge in ameliorating the bleeding risk of different APAs, which offers a promising strategy for the management of bleeding complications induced by antiplatelet therapy.

Platelet-Mimicking Nanosponges for Functional Reversal of Antiplatelet Agents.

Lipid nanoparticle-mediated RNA delivery holds great potential to treat various liver diseases. However, targeted delivery of RNA therapeutics to activated liver-resident fibroblasts for liver fibrosis treatment remains challenging. Here, we develop a combinatorial library of anisamide ligand-tethered lipidoids (AA-lipidoids) using a one-pot, two-step modular synthetic method and adopt a two-round screening strategy to identify AA-lipidoids with both high potency and selectivity to deliver RNA payloads to activated fibroblasts. The lead AA-lipidoid AA-T3A-C12 mediates greater RNA delivery and transfection of activated fibroblasts than its analog without anisamide and the FDA-approved MC3 ionizable lipid. In a preclinical model of liver fibrosis, AA-T3A-C12 enables ~65% silencing of heat shock protein 47, a therapeutic target primarily expressed by activated fibroblasts, which is 2-fold more potent than MC3, leading to significantly reduced collagen deposition and liver fibrosis. These results demonstrate the potential of AA-lipidoids for targeted RNA delivery to activated fibroblasts. Furthermore, these synthetic methods and screening strategies open a new avenue to develop and discover potent lipidoids with targeting properties, which can potentially enable RNA delivery to a range of cell and tissue types that are challenging to access using traditional lipid nanoparticle formulations.

Xuexiang Han

Department of Bioengineering

University of Pennsylvania

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing