Branched-Tail Lipid Nanoparticles for Intravenous mRNA Delivery to Lung Immune, Endothelial, and Alveolar Cells in Mice.

Lipid nanoparticles (LNPs) are proven as safe and effective delivery systems on a global scale. However, their efficacy has been limited primarily to liver and immune cells targets. To extend the potential applicability of mRNA drugs, we have synthesized and examined a library of 580 ionizable lipidoids for delivery to extrahepatocellular targets following intravenous administration. Of these lipidoids, over 40 enabled protein expression in mice, with the majority transfecting the liver. Beyond the liver, several LNPs containing new, branched-tail ionizable lipidoids potently delivered mRNA to the lungs, with cell-level specificity depending on helper lipid chemistry. Incorporation of the neutral helper lipid DOPE at 16 mol% enabled highly specific delivery to natural killer and dendritic cells within the lung. Although inclusion of the cationic helper lipid, DOTAP, improved lung tropism, it did so at the expense of cell specificity, resulting in equal transfection of endothelial and lymphoid cells. DOTAP formulations were also less favorable than DOPE formulations in that they elevated liver enzyme level and the overall cytokine response. Together, these data identify a new branched-tailed LNP formulation with a unique ability to selectively transfect lung immune cell populations without the use of toxicity-prone cationic helper lipids. This novel vehicle may unlock RNA therapies for lung diseases associated with immune cell dysregulation, including cancer, viral infections, and autoimmune disorders. This article is protected by copyright. All rights reserved.

Lipid Nanoparticle-Associated Inflammation is Triggered by Sensing of Endosomal Damage: Engineering Endosomal Escape Without Side Effects.

Lipid nanoparticles (LNPs) have emerged as the dominant platform for RNA delivery, based on their success in the COVID-19 vaccines and late-stage clinical studies in other indications. However, we and others have shown that LNPs induce severe inflammation, and massively aggravate pre-existing inflammation. Here, using structure-function screening of lipids and analyses of signaling pathways, we elucidate the mechanisms of LNP-associated inflammation and demonstrate solutions. We show that LNPs' hallmark feature, endosomal escape, which is necessary for RNA expression, also directly triggers inflammation by causing endosomal membrane damage. Large, irreparable, endosomal holes are recognized by cytosolic proteins called galectins, which bind to sugars on the inner endosomal membrane and then regulate downstream inflammation. We find that inhibition of galectins abrogates LNP-associated inflammation, both in vitro and in vivo . We show that rapidly biodegradable ionizable lipids can preferentially create endosomal holes that are smaller in size and reparable by the endosomal sorting complex required for transport (ESCRT) pathway. Ionizable lipids producing such ESCRT-recruiting endosomal holes can produce high expression from cargo mRNA with minimal inflammation. Finally, we show that both routes to non-inflammatory LNPs, either galectin inhibition or ESCRT-recruiting ionizable lipids, are compatible with therapeutic mRNAs that ameliorate inflammation in disease models. LNPs without galectin inhibition or biodegradable ionizable lipids lead to severe exacerbation of inflammation in these models. In summary, endosomal escape induces endosomal membrane damage that can lead to inflammation. However, the inflammation can be controlled by inhibiting galectins (large hole detectors) or by using biodegradable lipids, which create smaller holes that are reparable by the ESCRT pathway. These strategies should lead to generally safer LNPs that can be used to treat inflammatory diseases.

Lipid nanoparticle structure and delivery route during pregnancy dictate mRNA potency, immunogenicity, and maternal and fetal outcomes.

Treating pregnancy-related disorders is exceptionally challenging because the threat of maternal and/or fetal toxicity discourages the use of existing medications and hinders new drug development. One potential solution is the use of lipid nanoparticle (LNP) RNA therapies, given their proven efficacy, tolerability, and lack of fetal accumulation. Here, we describe LNPs for efficacious mRNA delivery to maternal organs in pregnant mice via several routes of administration. In the placenta, our lead LNP transfected trophoblasts, endothelial cells, and immune cells, with efficacy being structurally dependent on the ionizable lipid polyamine headgroup. Next, we show that LNP-induced maternal inflammatory responses affect mRNA expression in the maternal compartment and hinder neonatal development. Specifically, pro-inflammatory LNP structures and routes of administration curtailed efficacy in maternal lymphoid organs in an IL-1ß-dependent manner. Further, immunogenic LNPs provoked the infiltration of adaptive immune cells into the placenta and restricted pup growth after birth. Together, our results provide mechanism-based structural guidance on the design of potent LNPs for safe use during pregnancy.

The mixing method used to formulate lipid nanoparticles affects mRNA delivery efficacy and organ tropism.

mRNA is a versatile drug molecule with therapeutic applications ranging from protein replacement therapies to in vivo gene engineering. mRNA delivery is often accomplished using lipid nanoparticles, which are formulated via mixing of aqueous and organic solutions. Although this has historically been accomplished by manual mixing for bench scale science, microfluidic mixing is required for scalable continuous manufacturing and batch to batch control. Currently, there is limited understanding on how the mixing process affects mRNA delivery efficacy, particularly in regard to tropism. To address this knowledge gap, we examined the influence of the type of mixing and microfluidic mixing parameters on the performance of lipid nanoparticles in mice. This was accomplished with a Design of Experiment approach using four nanoparticle formulations with varied ionizable lipid chemistry. We found that each formulation required unique optimization of mixing parameters, with the total delivery efficacy of each lipid nanoparticle generated with microfluidics ranging from 100-fold less to 4-fold more than manually mixed LNPs. Further, mixing parameters influenced organ tropism, with the most efficacious formulations disproportionately increasing liver delivery compared to other organs. These data suggest that mixing parameters for lipid nanoparticle production may require optimization for each unique chemical formulation, complicating translational efforts. Further, microfluidic parameters must be chosen carefully to balance overall mRNA delivery efficacy with application-specific tropism requirements.

Placental drug transport and fetal exposure during pregnancy is determined by drug molecular size, chemistry, and conformation.

Pregnant people are unable to take many prescription and over-the-counter medications because of suspected or known risk to the fetus. This undermedication contributes to the high maternal mortality rate in the United States and detracts from the quality of life of pregnant people. As such, there is an urgent need to develop safe pharmaceutical formulations for use during pregnancy. Most drugs are small molecules that easily cross the placenta, which is the biological barrier that separates the maternal and fetal bloodstreams. One potential approach to preventing fetal drug accumulation is to design drug compounds that are excluded by the placenta; however, there is little understanding of how macromolecular drug properties affect transplacental transport. To address this knowledge gap, we examined the transport behavior of fluorescently-labeled polymers with varying size, conformation, and chemistry. We compared these polymers to unconjugated fluorescein, a small molecule model drug that readily crosses biological barriers. We found that molecular size affected transplacental transport in an in vitro model, BeWo b30 monolayers, as well as in pregnant mice, with larger polymers having lower permeability. In addition to size, polymer chemistry altered behavior, with polyethylene glycol (PEG) molecules permeating the placental barrier to a greater extent than dextrans of equivalent molecular weight. PEG molecules were also more readily taken up into placental cells in vivo. These findings will inform the future development of drug conjugates or other macromolecular medicines that can safely be used during pregnancy.

Ionizable lipid nanoparticles deliver mRNA to pancreatic ß cells via macrophage-mediated gene transfer.

Systemic messenger RNA (mRNA) delivery to organs outside the liver, spleen, and lungs remains challenging. To overcome this issue, we hypothesized that altering nanoparticle chemistry and administration routes may enable mRNA-induced protein expression outside of the reticuloendothelial system. Here, we describe a strategy for delivering mRNA potently and specifically to the pancreas using lipid nanoparticles. Our results show that delivering lipid nanoparticles containing cationic helper lipids by intraperitoneal administration produces robust and specific protein expression in the pancreas. Most resultant protein expression occurred within insulin-producing ß cells. Last, we found that pancreatic mRNA delivery was dependent on horizontal gene transfer by peritoneal macrophage exosome secretion, an underappreciated mechanism that influences the delivery of mRNA lipid nanoparticles. We anticipate that this strategy will enable gene therapies for intractable pancreatic diseases such as diabetes and cancer.

The replacement of helper lipids with charged alternatives in lipid nanoparticles facilitates targeted mRNA delivery to the spleen and lungs.

The broad clinical application of mRNA therapeutics has been hampered by a lack of delivery vehicles that induce protein expression in extrahepatic organs and tissues. Recently, it was shown that mRNA delivery to the spleen or lungs is possible upon the addition of a charged lipid to a standard four-component lipid nanoparticle formulation. This approach, while effective, further complicates an already complex drug formulation and has the potential to slow regulatory approval and adversely impact manufacturing processes. We were thus motivated to maintain a four-component nanoparticle system while achieving shifts in tropism. To that end, we replaced the standard helper lipid in lipidoid nanoparticles, DOPE, with one of eight alternatives. These lipids included the neutral lipids, DOPC, sphingomyelin, and ceramide; the anionic lipids, phosphatidylserine (PS), phosphatidylglycerol, and phosphatidic acid; and the cationic lipids, DOTAP and ethyl phosphatidylcholine. While neutral helper lipids maintained protein expression in the liver, anionic and cationic lipids shifted protein expression to the spleen and lungs, respectively. For example, replacing DOPE with DOTAP increased positive LNP surface charge at pH 7 by 5-fold and altered the ratio of liver to lung protein expression from 36:1 to 1:56. Similarly, replacing DOPE with PS reduced positive charge by half and altered the ratio of liver to spleen protein expression from 8:1 to 1:3. Effects were consistent across ionizable lipidoid chemistries. Regarding mechanism, nanoparticles formulated with neutral and anionic helper lipids best transfected epithelial and immune cells, respectively. Further, the lung-tropic effect of DOTAP was linked to reduced immune cell infiltration of the lungs compared to neutral or anionic lipids. Together, these data show that intravenous non-hepatocellular mRNA delivery is readily achievable while maintaining a four-component formulation with modified helper lipid chemistry.

Lipid nanoparticle chemistry determines how nucleoside base modifications alter mRNA delivery.

Therapeutic mRNA has the potential to revolutionize the treatment of myriad diseases and, in 2020, facilitated the most rapid vaccine development in history. Among the substantial advances in mRNA technology made in recent years, the incorporation of base modifications into therapeutic mRNA sequences can reduce immunogenicity and increase translation. However, experiments from our lab and others have shown that the incorporation of base modifications does not always yield superior protein expression. We hypothesized that the variable benefit of base modifications may relate to lipid nanoparticle chemistry, formulation, and accumulation within specific organs. To test this theory, we compared IV-injected lipid nanoparticles formulated with reporter mRNA incorporating five base modifications (ψ, m1ψ, m5U, m5C/ψ, and m5C/s2U) and four ionizable lipids (C12-200, cKK-E12, ZA3-Ep10, and 200Oi10) with tropism for different organs. In general, the m1ψ base modification best enhanced translation, producing up to 15-fold improvements in total protein expression compared to unmodified mRNA. Expression improved most dramatically in the spleen (up to 50-fold) and was attributed to enhanced protein expression in monocytic lineage splenocytes. The extent to which these effects were observed varied with delivery vehicle and correlated with differences in innate immunogenicity. Through comparison of firefly luciferase and erythropoietin mRNA constructs, we also found that mRNA modification-induced enhancements in protein expression are limited outside of the spleen, irrespective of delivery vehicle. These results highlight the complexity of mRNA-loaded lipid nanoparticle drug design and show that the effectiveness of mRNA base modifications depend on the delivery vehicle, the target cells, and the site of endogenous protein expression.

Elucidating the Electrochemical Mechanism of NG-Hydroxy-L-arginine.

NG-Hydroxy-L-arginine (NOHA) is a stable intermediate product in the urea cycle that can be used to monitor the consumption of L-arginine by nitrous oxide synthase (NOS) to produce nitric oxide (NO) and L-citrulline. Research has implicated the urea cycle in many diseases and NO has cultivated interest as a potential biomarker for neural health. Electrochemical detection is an established, cost-effective method that can successfully detect low levels of analyte concentrations. As one of the few electrochemically active species in the urea cycle, NOHA shows promise as a biomarker for monitoring disruptions in this biochemical process. In this study, we show that NOHA has an oxidation peak at +355 mV vs Ag/AgCl at a glassy carbon electrode. In addition, cyclic voltammetry studies with structural analogs - alanine and N-hydroxyguanidine - allowed us to approximate the oxidation wave at +355 mV vs Ag/AgCl to be a one electron process. Diffusivity of NOHA was found using linear scan voltammetry with a rotating disk electrode and approximated at 5.50×10-5 cm2/s. Ample work is still needed to make a robust biosensor, but the results here characterize the electrochemical activity and represent principle steps in making a NOHA biosensor.

Electrochemical Detection of NG-Hydroxy-L-arginine.

NG-hydroxy-L-arginine (NOHA) is a stable intermediate product in the consumption of L-arginine in the urea cycle by nitric oxide synthase (NOS) to produce nitric oxide (NO) and L-citrulline. Research has shown that the urea cycle is disrupted in various diseases. As one of the few electrochemically active species in the urea cycle, NOHA shows promise as a marker for detection of various diseases. Electrochemical detection is an established, cost-effective method that is able to successfully detect low levels of analyte concentrations. NOHA, to the best of our knowledge, has not been electrochemically detected previously. Using cyclic voltammetry with a glassy carbon electrode, we have found that NOHA has an oxidation peak at 355 mV with a sensitivity of 5.4 nA/µM. We also investigated detecting NOHA with differential pulse voltammetry, which shows similar sensitivity and oxidation peaks. While there is significant work ahead to understand the kinetics of NOHA detection, the results here represent the first steps in making a NOHA biosensor.