A predictive microfluidic model of human glioblastoma to assess trafficking of blood-brain barrier-penetrant nanoparticles.

The blood­brain barrier represents a significant challenge for the treatment of high-grade gliomas, and our understanding of drug transport across this critical biointerface remains limited. To advance preclinical therapeutic development for gliomas, there is an urgent need for predictive in vitro models with realistic blood­brain-barrier vasculature. Here, we report a vascularized human glioblastoma multiforme (GBM) model in a microfluidic device that accurately recapitulates brain tumor vasculature with self-assembled endothelial cells, astrocytes, and pericytes to investigate the transport of targeted nanotherapeutics across the blood­brain barrier and into GBM cells. Using modular layer-by-layer assembly, we functionalized the surface of nanoparticles with GBM-targeting motifs to improve trafficking to tumors. We directly compared nanoparticle transport in our in vitro platform with transport across mouse brain capillaries using intravital imaging, validating the ability of the platform to model in vivo blood­brain-barrier transport. We investigated the therapeutic potential of functionalized nanoparticles by encapsulating cisplatin and showed improved efficacy of these GBM-targeted nanoparticles both in vitro and in an in vivo orthotopic xenograft model. Our vascularized GBM model represents a significant biomaterials advance, enabling in-depth investigation of brain tumor vasculature and accelerating the development of targeted nanotherapeutics.

In Vivo mRNA CAR T Cell Engineering via Targeted Ionizable Lipid Nanoparticles with Extrahepatic Tropism.

With six therapies approved by the Food and Drug Association, chimeric antigen receptor (CAR) T cells have reshaped cancer immunotherapy. However, these therapies rely on ex vivo viral transduction to induce permanent CAR expression in T cells, which contributes to high production costs and long-term side effects. Thus, this work aims to develop an in vivo CAR T cell engineering platform to streamline production while using mRNA to induce transient, tunable CAR expression. Specifically, an ionizable lipid nanoparticle (LNP) is utilized as these platforms have demonstrated clinical success in nucleic acid delivery. Though LNPs often accumulate in the liver, the LNP platform used here achieves extrahepatic transfection with enhanced delivery to the spleen, and it is further modified via antibody conjugation (Ab-LNPs) to target pan-T cell markers. The in vivo evaluation of these Ab-LNPs confirms that targeting is necessary for potent T cell transfection. When using these Ab-LNPs for the delivery of CAR mRNA, antibody and dose-dependent CAR expression and cytokine release are observed along with B cell depletion of up to 90%. In all, this work conjugates antibodies to LNPs with extrahepatic tropism, evaluates pan-T cell markers, and develops Ab-LNPs capable of generating functional CAR T cells in vivo.

Ionizable Lipid Nanoparticles for In Vivo mRNA Delivery to the Placenta during Pregnancy.

Ionizable lipid nanoparticles (LNPs) are the most clinically advanced nonviral platform for mRNA delivery. While they have been explored for applications including vaccines and gene editing, LNPs have not been investigated for placental insufficiency during pregnancy. Placental insufficiency is caused by inadequate blood flow in the placenta, which results in increased maternal blood pressure and restricted fetal growth. Therefore, improving vasodilation in the placenta can benefit both maternal and fetal health. Here, we engineered ionizable LNPs for mRNA delivery to the placenta with applications in mediating placental vasodilation. We designed a library of ionizable lipids to formulate LNPs for mRNA delivery to placental cells and identified a lead LNP that enables in vivo mRNA delivery to trophoblasts, endothelial cells, and immune cells in the placenta. Delivery of this top LNP formulation encapsulated with VEGF-A mRNA engendered placental vasodilation, demonstrating the potential of mRNA LNPs for protein replacement therapy during pregnancy to treat placental disorders.

Rational Design of Nanomedicine for Placental Disorders: Birthing a New Era in Women's Reproductive Health.

The placenta is a transient organ that forms during pregnancy and acts as a biological barrier, mediating exchange between maternal and fetal circulation. Placental disorders, such as preeclampsia, fetal growth restriction, placenta accreta spectrum, and gestational trophoblastic disease, originate in dysfunctional placental development during pregnancy and can lead to severe complications for both the mother and fetus. Unfortunately, treatment options for these disorders are severely lacking. Challenges in designing therapeutics for use during pregnancy involve selectively delivering payloads to the placenta while protecting the fetus from potential toxic side effects. Nanomedicine holds great promise in overcoming these barriers; the versatile and modular nature of nanocarriers, including prolonged circulation times, intracellular delivery, and organ-specific targeting, can control how therapeutics interact with the placenta. In this review, nanomedicine strategies are discussed to treat and diagnose placental disorders with an emphasis on understanding the unique pathophysiology behind each of these diseases. Finally, prior study of the pathophysiologic mechanisms underlying these placental disorders has revealed novel disease targets. These targets are highlighted here to motivate the rational design of precision nanocarriers to improve therapeutic options for placental disorders.

Orthogonal Design of Experiments for Engineering of Lipid Nanoparticles for mRNA Delivery to the Placenta.

During healthy pregnancy, the placenta develops to allow for exchange of nutrients and oxygen between the mother and the fetus. However, placental dysregulation can lead to several pregnancy disorders, such as preeclampsia and fetal growth restriction. Recently, lipid nanoparticle (LNP)-mediated delivery of messenger RNA (mRNA) has been explored as a promising approach to treat these disorders. Here, iterative libraries of LNPs with varied excipient molar ratios are screened in vitro for enhanced mRNA delivery to placental cells with minimal cytotoxicity when compared to an LNP formulation with a standard excipient molar ratio. LNP C5, the top formulation identified by these screens, demonstrates a fourfold increase in mRNA delivery in vitro compared to the standard formulation. Intravenous administration of LNP C5 to pregnant mice achieves improved in vivo placental mRNA delivery compared to the standard formulation and mediates mRNA delivery to placental trophoblasts, endothelial cells, and immune cells. These results identify LNP C5 as a promising optimized LNP formulation for placental mRNA delivery and further validates the design of experiments strategy for LNP excipient optimization to enhance mRNA delivery to cell types and organs of interest.

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.

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.

Bile acid-containing lipid nanoparticles enhance extrahepatic mRNA delivery.

Lipid nanoparticles (LNPs) have emerged as a viable, clinically-validated platform for the delivery of mRNA therapeutics. LNPs have been utilized as mRNA delivery systems for applications including vaccines, gene therapy, and cancer immunotherapy. However, LNPs, which are typically composed of ionizable lipids, cholesterol, helper lipids, and lipid-anchored polyethylene glycol, often traffic to the liver which limits the therapeutic potential of the platform. Several approaches have been proposed to resolve this tropism such as post-synthesis surface modification or the addition of synthetic cationic lipids. Methods: Here, we present a strategy for achieving extrahepatic delivery of mRNA involving the incorporation of bile acids, a naturally-occurring class of cholesterol analogs, during LNP synthesis. We synthesized a series of bile acid-containing C14-4 LNPs by replacing cholesterol with bile acids (cholic acid, chenodeoxycholic acid, deoxycholic acid, or lithocholic acid) at various ratios. Results: Bile acid-containing LNPs (BA-LNPs) were able to reduce delivery to liver cells in vitro and improve delivery in a variety of other cell types, including T cells, B cells, and epithelial cells. Our subsequent in vivo screening of selected LNP candidates injected intraperitoneally or intravenously identified a highly spleen tropic BA-LNP: CA-100, a four-component LNP containing cholic acid and no cholesterol. These screens also identified BA-LNP candidates demonstrating promise for other mRNA therapeutic applications such as for gastrointestinal or immune cell delivery. We further found that the substitution of cholic acid for cholesterol in an LNP formulation utilizing a different ionizable lipid, C12-200, also shifted mRNA delivery from the liver to the spleen, suggesting that this cholic acid replacement strategy may be generalizable. Conclusion: These results demonstrate the potential of a four-component BA-LNP formulation, CA-100, for extrahepatic mRNA delivery that could potentially be utilized for a range of therapeutic and vaccine applications.

Massively parallel pooled screening reveals genomic determinants of nanoparticle delivery.

To accelerate the translation of cancer nanomedicine, we used an integrated genomic approach to improve our understanding of the cellular processes that govern nanoparticle trafficking. We developed a massively parallel screen that leverages barcoded, pooled cancer cell lines annotated with multiomic data to investigate cell association patterns across a nanoparticle library spanning a range of formulations with clinical potential. We identified both materials properties and cell-intrinsic features that mediate nanoparticle-cell association. Using machine learning algorithms, we constructed genomic nanoparticle trafficking networks and identified nanoparticle-specific biomarkers. We validated one such biomarker: gene expression of SLC46A3, which inversely predicts lipid-based nanoparticle uptake in vitro and in vivo. Our work establishes the power of integrated screens for nanoparticle delivery and enables the identification and utilization of biomarkers to rationally design nanoformulations.