Polyphenolic Nanoparticle Platforms (PARCELs) for In Vitro and In Vivo mRNA Delivery.

Despite their successful implementation in the COVID-19 vaccines, lipid nanoparticles (LNPs) still face a central limitation in the delivery of mRNA payloads: endosomal trapping. Improving upon this inefficiency could afford improved drug delivery systems, paving the way toward safer and more effective mRNA-based medicines. Here, we present polyphenolic nanoparticle platforms (PARCELs) as effective mRNA delivery systems. In brief, our investigation begins with a computationally guided structural analysis of 1825 discrete polyphenolic structural data points across 73 diverse small molecule polyphenols and 25 molecular parameters. We then generate structurally diverse PARCELs, evaluating their in vitro mechanism and activity, ultimately highlighting the superior endosomal escape properties of PARCELs relative to analogous LNPs. Finally, we examine the in vivo biodistribution, protein expression, and therapeutic efficacy of PARCELs in mice. In undertaking this approach, the goal of this study is to establish PARCELs as viable delivery platforms for safe and effective mRNA delivery.

A Lung-Expressing mRNA Delivery Platform with Tunable Activity in Hypoxic Environments.

Messenger RNA (mRNA) delivery platforms often facilitate protein expression in the liver following intravenous injection and have been optimized for use in normally oxygenated cells (21% O2 atmosphere). However, there is a growing need for mRNA therapy in diseases affecting non-liver organs, such as the lungs. Additionally, many diseases are characterized by hypoxia (<21% O2 atmosphere), a state of abnormally low oxygenation in cells and tissues that can reduce the efficacy of mRNA therapies by upwards of 80%. Here, we report a Tunable Lung-Expressing Nanoparticle Platform (TULEP) for mRNA delivery, whose properties can be readily tuned for optimal expression in hypoxic environments. Briefly, our study begins with the synthesis and characterization of a novel amino acrylate polymer that can be effectively complexed with mRNA payloads into TULEPs. We study the efficacy and mechanism of mRNA delivery using TULEP, including analysis of the cellular association, endocytosis mechanisms, endosomal escape, and protein expression in a lung cell line. We then evaluate TULEP under hypoxic conditions and address hypoxia-related deficits in efficacy by making our system tunable with adenosine triphosphate (ATP). Finally, we conclude our study with an in vivo analysis of mRNA expression, biodistribution, and tolerability of the TULEP platform in mice. In presenting these data, we hope that our work highlights the utility of TULEPs for tunable and effective mRNA delivery while more broadly highlighting the utility of considering oxygen levels when developing mRNA delivery platforms.

Regioselective Palladium-Catalyzed Chain-Growth Allylic Amination Polymerization of Vinyl Aziridines.

Organometallic-mediated chain growth polymerization of readily accessible chemical building blocks is responsible for important commercial and technological advances in polymer science, but the incorporation of heteroatoms into the polymer backbone through these mechanisms remains a challenge. Transition metal π-allyl complexes are well-developed organometallic intermediates for carbon-heteroatom bond formation in small-molecule catalysis yet remain underexplored in polymer science. Here, we developed a regioselective palladium-phosphoramidite-catalyzed chain-growth allylic amination polymerization of vinyl aziridines for the synthesis of novel nitrogen-rich polymers via ambiphilic π-allyl complexes. The polymerization accessed a linear microstructure with four carbons between each nitrogen, which is challenging to achieve through other chain-growth polymerization approaches. The highly regioselective allylic amination polymerization demonstrated the characteristics of a controlled polymerization and was able to achieve molar masses exceeding 20 kg mol-1 with low dispersities (D̵ < 1.3). The identification of the polymer structure and well-defined chain ends were supported by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and chain extension experiments demonstrate opportunities for building more complex materials from this method. A Hammett study was performed to understand the role of the catalyst and monomer structure on regioselectivity, and the data supported a mechanism wherein regioselectivity was primarily controlled by the ligand-metal complex. Postpolymerization desulfonylation provided access to a novel polyamine that demonstrated broad anticancer activity in vitro, which highlights the benefits of unlocking novel polyamine microstructures through regioselective chain-growth allylic amination polymerization.

Enhancing the Functionality of Immunoisolated Human SC-ßeta Cell Clusters through Prior Resizing.

The transplantation of immunoisolated stem cell derived beta cell clusters (SC-ß) has the potential to restore physiological glycemic control in patients with type I diabetes. This strategy is attractive as it uses a renewable ß-cell source without the need for systemic immune suppression. SC-ß cells have been shown to reverse diabetes in immune compromised mice when transplanted as ≈300 µm diameter clusters into sites where they can become revascularized. However, immunoisolated SC-ß clusters are not directly revascularized and rely on slower diffusion of nutrients through a membrane. It is hypothesized that smaller SC-ß cell clusters (≈150 µm diameter), more similar to islets, will perform better within immunoisolation devices due to enhanced mass transport. To test this, SC-ß cells are resized into small clusters, encapsulated in alginate spheres, and coated with a biocompatible A10 polycation coating that resists fibrosis. After transplantation into diabetic immune competent C57BL/6 mice, the "resized" SC-ß cells plus the A10 biocompatible polycation coating induced long-term euglycemia in the mice (6 months). After retrieval, the resized A10 SC-ß cells exhibited the least amount of fibrosis and enhanced markers of ß-cell maturation. The utilization of small SC-ß cell clusters within immunoprotection devices may improve clinical translation in the future.

Messenger RNA Therapy for Female Reproductive Health.

Female reproductive health has traditionally been an underrepresented area of research in the drug delivery sciences. This disparity is also seen in the emerging field of mRNA therapeutics, a class of medicines that promises to treat and prevent disease by upregulating protein expression in the body. Here, we review advances in mRNA therapies through the lens of improving female reproductive health. Specifically, we begin our review by discussing the fundamental structure and biochemical modifications associated with mRNA-based drugs. Then, we discuss various packaging technologies, including lipid nanoparticles, that can be utilized to protect and transport mRNA drugs to target cells in the body. Last, we conclude our review by discussing the usage of mRNA therapy for addressing pregnancy-related health and vaccination against sexually transmitted diseases in women. Of note, we also highlight relevant clinical trials using mRNA for female reproductive health while also providing their corresponding National Clinical Trial identifiers. In undertaking this review, our aim is to provide a fundamental background understanding of mRNA therapy and its usage to specifically address female health issues with an overarching goal of providing information toward addressing gender disparity in certain aspects of health research.

A Unified Strategy to Improve Lipid Nanoparticle Mediated mRNA Delivery Using Adenosine Triphosphate.

A central goal of chemical and drug delivery sciences is to maximize the therapeutic efficacy of a given drug at the lowest possible dose. Here, we report a generalizable strategy that can be utilized to improve the delivery of mRNA drugs using lipid nanoparticles (LNPs), the clinically approved chemistry platforms utilized in the Moderna and Pfizer/BioNTech COVID-19 vaccines. In brief, our strategy updates the chemistry of LNPs to incorporate adenosine triphosphate (ATP) alongside mRNA, a modification that results in upward of a 79-fold increase in LNP-delivered mRNA-encoded protein expression in vitro and a 24-fold increase in vivo when compared to parent mRNA LNP formulations that do not contain ATP. Notably, we find that our ATP co-delivery strategy increases LNP-delivered mRNA-encoded protein expression across eight different LNP chemistries and three different cell lines, under normoxia and hypoxia, and in a well-tolerated fashion. Notably, our strategy also improves the expression of mRNA encoding for intracellular and secreted proteins both in vitro and in vivo, highlighting the utility of leveraging ATP co-delivery within mRNA LNPs as a means to increase protein expression. In developing this strategy, we hope that we have provided a simple yet powerful approach to improving mRNA LNPs that may one day be useful in developing therapies for human disease.

An Efficacy and Mechanism Driven Study on the Impact of Hypoxia on Lipid Nanoparticle Mediated mRNA Delivery.

Hypoxia is a common hallmark of human disease that is characterized by abnormally low oxygen levels in the body. While the effects of hypoxia on many small molecule-based drugs are known, its effects on several classes of next-generation medications including messenger RNA therapies warrant further study. Here, we provide an efficacy- and mechanism-driven study that details how hypoxia impacts the cellular response to mRNA therapies delivered using 4 different chemistries of lipid nanoparticles (LNPs, the frontrunner class of drug delivery vehicles for translational mRNA therapy utilized in the Moderna and Pfizer/BioNTech COVID-19 vaccines). Specifically, our work provides a comparative analysis as to how various states of oxygenation impact LNP-delivered mRNA expression, cellular association, endosomal escape, and intracellular ATP concentrations following treatment with 4 different LNPs across 3 different cell lines. In brief, we first identify that hypoxic cells express less LNP-delivered mRNA into protein than normoxic cells. Next, we identify generalizable cellular reoxygenation protocols that can reverse the negative effects that hypoxia imparts on LNP-delivered mRNA expression. Finally, mechanistic studies that utilize fluorescence-activated cell sorting, confocal microscopy, and enzyme inhibition reveal that decreases in mRNA expression correlate with decreases in intracellular ATP (rather than with differences in mRNA LNP uptake pathways). In presenting this data, we hope that our work provides a comprehensive efficacy and mechanism-driven study that explores the impact of differential oxygenation on LNP-delivered mRNA expression while simultaneously establishing fundamental criteria that may one day be useful for the development of mRNA drugs to treat hypoxia-associated disease.

Ongoing Clinical Trials of Nonviral siRNA Therapeutics.

Short interfering RNAs (siRNA) are a powerful class of genetic medicines whose clinical translation can be hindered by their suboptimal delivery properties in vivo. Here, we provide a clinically focused overview that summarizes ongoing siRNA clinical trials from the perspective of innovations in nonviral delivery strategies. More specifically, our review begins by highlighting the delivery barriers and physiochemical properties of siRNA that make it challenging to deliver it in vivo. We then provide commentary on specific delivery strategies, including sequence modification, siRNA ligand conjugation, and nanoparticle and exosomal packaging, each of which can be used to control the delivery of siRNA therapies in living systems. Last, we provide a summary table of ongoing siRNA clinical trials which also highlights the indication of use, target, and National Clinical Trial (NCT) number associated with each entry. In writing this review, our work aims to highlight the key challenges and strategies for effective nonviral siRNA delivery in vivo, while simultaneously summarizing information on ongoing clinical trials for siRNA therapy in humans.

Effects of dairy processing sludge and derived biochar on greenhouse gas emissions from Danish and Irish soils.

Globally, to ensure food security bio-based fertilizers must replace a percentage of chemical fertilizers. Such replacement must be deemed sustainable from agronomic and greenhouse gas (GHG) emission perspectives. For agronomic performance several controlled protocols are in place but not for testing GHG emissions. Herein, a pre-screening tool is presented to examine GHG emissions from bio-waste as fertilizers. The various treatments examined are as follows: soil with added mineral nitrogen (N, 140 kg N ha-1) fertilizer (MF), the same amount of MF combined with dairy processing sludge (DS), sludge-derived biochar produced at 450 °C (BC450) and 700 °C (BC700) and untreated control (CK). These treatments were combined with Danish (sandy loam) or Irish (clay loam) soils, with carbon dioxide (CO2), methane (CH4) and nitrous oxide (N2O) emissions and soil inorganic-N contents measured on selected days. During the incubation, biochar mitigated N2O emissions by regulating denitrification. BC450 reduced N2O emissions from Danish soil by 95.5% and BC700 by 97.7% compared to emissions with the sludge application, and for Irish soil, the N2O reductions were 93.6% and 32.3%, respectively. For both soils, biochar reduced CO2 emissions by 50% as compared to the sludge. The lower N2O reduction potential of BC700 for Irish soil could be due to the high soil organic carbon and clay content and pyrolysis temperature. For the same reasons emissions of N2O and CO2 from Irish soil were significantly higher than from Danish soil. The temporal variation in N2O emissions was correlated with soil inorganic-N contents. The CH4 emissions across treatments were not significantly different. This study developed a simple and cost-effective pre-screening method to evaluate the GHG emission potential of new bio-waste before its field application and guide the development of national emission inventories, towards achieving the goals of circular economy and the European Green Deal.

A comparative study of thermally and chemically treated dairy waste: Impacts on soil phosphorus turnover and availability using 33P isotope dilution.

Dairy processing sludge (DPS) and DPS-derived secondary products such as struvite, biochar, hydrochar and ash (collectively known as SRUBIAS) are emerging as alternatives to fertilizers produced from mined rock phosphate. However, little is known about how these products affect soil P availability and daily P turnover rates.. A lack of such information prevents precision nutrient management planning using these products out on farms. This study used a novel isotope dilution technique (IPD) with 33P as a tracer to compare P turnover in soils amended with chemically (alum-treated DPS and struvite) and thermally (biochar, hydrochar, ash) treated DPS. Results showed that thermally treated products exhibited poor agronomic performance as P fertilizers, potentially inhibiting P availability when applied to soils. For example, a P deficient soil amended with hydrochar treatment at the highest application rates did not record a build-up of available P to agronomic target values. In ash and biochar treated P deficient soils, available P increased but only with very high application rates of 150 and 80 mg P kg -1. The application of these products as fertilizers could have negative implications for both environmental and agronomic goals. Conversely, chemically treated fertilisers demonstrated better agronomic performance. The same agronomic target value was reached with application rates of only 20 mg P kg -1 soil for DPS and 50 mg P kg -1 soil for struvite. However, the techniques deployed revealed that these products exhibited slower rates of available and exchangeable P build-up when compared with chemical fertilisers. This suggests that these bio-based alternatives require higher application rates or earlier application times compared to conventional chemical fertilizers. Regulations providing advice on P use in agricultural soils need to account for slower P turnover in soils receiving recycled fertilizers. The IPD technique is transferrable to all wastes to examine their performance as fertilizers.

Investigation of differential levels of phosphorus fixation in dolomite and calcium carbonate amended red soil.

BACKGROUND: The pH adjustment of acidic red soils with lime materials is beneficial for the reduction of phosphorus (P) fixation. However, the reasons for varying levels of P activation after adding different lime materials have not been fully investigated. Therefore, this study examined changes in soil labile P and P forms after phosphate application to calcium carbonate (CaCO3 ) and dolomite amended red soil during a 120-day incubation period. Also change of P sorption properties in the amended soil samples from day 120 were examined through a sorption-desorption experiment. RESULTS: The increase of soil H2 O-P and NaHCO3 -P in the CaCO3 and dolomite amended soil treatments was mainly ascribed to the decline of the NaOH-P. However, when compared with the control treatment after 120 days, soil Olsen-P significantly increased by 34% and 66% in the CaCO3 and dolomite treatments. The Hedley P fractionation results demonstrated that the CaCO3 application caused a notable increase of HCl-P (stable Ca-P), which was 88.4% higher than that in the dolomite treatment. However, the formation of stable P was strongly suppressed in the dolomite treatment due to the presence of magnesium (Mg), which was identified by the negative relationship between M3-Mg and HCl-P. In line with these findings, P sorption-desorption work showed weaker P binding energy in the dolomite treatment relative to the CaCO3 treatment. CONCLUSION: In terms of increasing P availability in red soil, this study suggests that dolomite should be used to substitute CaCO3 in order to reduce the soil P fixation. © 2021 Society of Chemical Industry.

Nucleic acid delivery and nanoparticle design for COVID vaccines.

ABSTRACT: Nucleic acid therapeutics offer a new paradigm to rapidly respond to global health problems. The versatility of nucleic acids, especially in RNA therapies, provides the ability to tune levels of specific protein expression, achieving downregulation through short interfering RNA (siRNA) or upregulation by messenger RNA (mRNA) administration. Recent advances in the development of delivery vehicles, including nonviral nanoparticles are crucial to overcome the innate barriers to nucleic acid delivery. Toward this end, current clinical approaches have utilized mRNA and lipid nanoparticles (LNPs) to address the COVID-19 pandemic through novel vaccine strategies, producing efficacious vaccines within one year of sequencing the SARS-CoV-2 genome. Here, we review fundamental concepts required to achieve successful nucleic acid delivery, including the design of LNP systems optimized for mRNA vaccine applications.

A Nanoprimer To Improve the Systemic Delivery of siRNA and mRNA.

Despite tremendous interest in gene therapies, the systemic delivery of nucleic acids still faces substantial challenges. To successfully administer nucleic acids, one approach is to encapsulate them in lipid nanoparticles (LNPs). However, LNPs administered intravenously substantially accumulate in the liver where they are taken up by the reticuloendothelial system (RES). Here, we administer prior to the LNPs a liposome designed to transiently occupy liver cells, the Nanoprimer. This study demonstrates that the pretreatment of mice with the Nanoprimer decreases the LNPs' uptake by the RES. By accumulating rapidly in the liver cells, the Nanoprimer improves the bioavailability of the LNPs encapsulating human erythropoietin (hEPO) mRNA or factor VII (FVII) siRNA, leading respectively to more hEPO production (by 32%) or FVII silencing (by 49%). The use of the Nanoprimer offers a new strategy to improve the systemic delivery of RNA-based therapeutics.

Quantitative microbial human exposure model for faecal indicator bacteria and risk assessment of pathogenic Escherichia coli in surface runoff following application of dairy cattle slurry and co-digestate to grassland.

Animal waste contains high numbers of microorganisms and therefore can present a potential biological threat to human health. During episodic rainfall events resulting in runoff, microorganisms in the waste and soil may migrate into surface runoff, contaminating surface water resources. A probabilistic human exposure (HE) model was created to determine exposure to faecal indicator bacteria (FIB): total coliforms (TC), E. coli and enterococci following application of bio-based fertiliser (dairy cattle slurry, digestate) to grassland; using a combination of experimental field results and literature-based data. This step was followed by a quantitative microbial risk assessment (QMRA) model for pathogenic E. coli based on a literature-based dose-response model. The results showed that the maximum daily HE (HEdaily) is associated with E. coli for unprocessed slurry (treatment T1) on day 1, the worst-case scenario where the simulated mean HEdaily was calculated as 2.84 CFU day -1. The results indicate that the overall annual probability of risk (Pannual) of illness from E. coli is very low or low based on the WHO safe-limit of Pannual as 10 -6. In the worst-case scenario, a moderate risk was estimated with simulated mean Pannual as 1.0 × 10 -5. Unpasteurised digestate application showed low risk on day 1 and 2 (1.651 × 10 -6, 1.167 × 10 -6, respectively). Pasteurised digestate showed very low risk in all scenarios. These results support the restriction imposed on applying bio-based fertiliser if there is any rain forecast within 48 h from the application time. This study proposes a future extension of the probabilistic model to include time, intensity, discharge, and distance-dependant dilution factor. The information generated from this model can help policymakers ensure the safety of surface water sources through the quality monitoring of FIB levels in bio-based fertiliser.

Alum split applications strengthened phosphorus fixation and phosphate sorption in high legacy phosphorus calcareous soil.

High phosphorus (P) saturation arising from historic P inputs to protected vegetable fields (PVFs) drives high P mobilisation to waterbodies. Amendment of soils with alum has shown potential in terms of fixing labile P and protecting water quality. The present 15 month pot experiment investigated P stabilisation across single alum application (Alum-1 treatment, 20 g alum/kg soil incorporated into soil before the maize was sown), alum split applications (Alum-4 treatment, 5 g alum/kg soil incorporated into soil before each crop was sown i.e. 4 × 5 g/kg) and soil only treatment (Control). Results showed that the Alum-1 treatment caused the strongest stabilisation of soil labile P after maize plant removal, whereas the P stabilisation effect was gradually weakened due to the transformation of soil non-labile P to labile P and the reduced active Al3+ in soil solution. For the Alum-4 treatment, soil labile P decreased gradually with each crop planting and was lower than the Alum-1 treatment at the end of the final crop removal, without any impairment on plant growth. The better P stabilisation at the end of Alum-4 treatment was closely correlated with a progressive supply of Al3+ and a gradual decrease of pH, which resulted in higher contents of poorly-crystalline Al, Fe and exchangeable Ca. These aspects were conducive to increasing the soil P stabilisation and phosphate sorption. In terms of management, growers in continuous cropping systems could utilise split alum applications as a strategy to alleviate P losses in high-P enriched calcareous soil.

Polymers for extended-release administration.

Developing strategies to deliver the required dose of therapeutics into target tissues and cell populations within the body is a principal aim of controlled release and drug delivery. Specifically, there is an interest in developing formulations that can achieve drug concentrations within the therapeutic window, for extended periods of time, with tunable release profiles, and with minimal complication and distress for the patient. To date, drug delivery systems have been developed to serve as depots, triggers, and carriers for therapeutics including small molecules, biologics, and cell-based therapies. Notably, the efficacy of these systems is intricately tied to the manner in which they are administered. For example, systemic and oral routes of administration are common, but both can result in rapid clearance from the organism. Towards this end, what formulation and administration route strategies are available to prolong the bioavailability of therapeutics? Here, we discuss historical and modern drug delivery systems, with the intention of exploring how properties including formulation, administration route and chemical structure influence the ability to achieve extended-release drug release profiles within the body.

Injectable Polymer-Nanoparticle Hydrogels for Local Immune Cell Recruitment.

The ability to engineer immune function has transformed modern medicine, highlighted by the success of vaccinations and recent efforts in cancer immunotherapy. Further directions in programming the immune system focus on the design of immunomodulatory biomaterials that can recruit, engage with, and program immune cells locally in vivo. Here, we synthesized shear-thinning and self-healing polymer-nanoparticle (PNP) hydrogels as a tunable and injectable biomaterial platform for local dendritic cell (DC) recruitment. PNP gels were formed from two populations of poly(ethylene glycol)-block-polylactide (PEG-b-PLA) NPs with the same diameter but different PEG brush length (2 or 5 kDa). PEG-b-PLA NPs with the longer PEG brush exhibited improved gel formation following self-assembly and faster recovery after shear-thinning. In all cases, model protein therapeutics were released via Fickian diffusion in vitro, and minor differences in the release rate between the gel formulations were observed. PNP hydrogels were loaded with the DC cytokine CCL21 and injected subcutaneously in a murine model. CCL21-loaded PNP hydrogels recruited DCs preferentially to the site of injection in vivo relative to non-CCL21-loaded hydrogels. Thus, PNP hydrogels comprise a simple and tunable platform biomaterial for in vivo immunomodulation following minimally invasive subcutaneous injection.

Optimization of a Degradable Polymer-Lipid Nanoparticle for Potent Systemic Delivery of mRNA to the Lung Endothelium and Immune Cells.

mRNA therapeutics hold great potential for treating a variety of diseases through protein-replacement, immunomodulation, and gene editing. However, much like siRNA therapy the majority of progress in mRNA delivery has been confined to the liver. Previously, we demonstrated that poly(ß-amino esters), a class of degradable polymers, are capable of systemic mRNA delivery to the lungs in mice when formulated into nanoparticles with poly(ethylene glycol)-lipid conjugates. Using experimental design, a statistical approach to optimization that reduces experimental burden, we demonstrate herein that these degradable polymer-lipid nanoparticles can be optimized in terms of polymer synthesis and nanoparticle formulation to achieve a multiple order-of-magnitude increase in potency. Furthermore, using genetically engineered Cre reporter mice, we demonstrate that mRNA is functionally delivered to both the lung endothelium and pulmonary immune cells, expanding the potential utility of these nanoparticles.

Lipid Nanoparticle Assisted mRNA Delivery for Potent Cancer Immunotherapy.

The induction of a strong cytotoxic T cell response is an important prerequisite for successful immunotherapy against many viral diseases and tumors. Nucleotide vaccines, including mRNA vaccines with their intracellular antigen synthesis, have been shown to be potent activators of a cytotoxic immune response. The intracellular delivery of mRNA vaccines to the cytosol of antigen presenting immune cells is still not sufficiently well understood. Here, we report on the development of a lipid nanoparticle formulation for the delivery of mRNA vaccines to induce a cytotoxic CD 8 T cell response. We show transfection of dendritic cells, macrophages, and neutrophils. The efficacy of the vaccine was tested in an aggressive B16F10 melanoma model. We found a strong CD 8 T cell activation after a single immunization. Treatment of B16F10 melanoma tumors with lipid nanoparticles containing mRNA coding for the tumor-associated antigens gp100 and TRP2 resulted in tumor shrinkage and extended the overall survival of the treated mice. The immune response can be further increased by the incorporation of the adjuvant LPS. In conclusion, the lipid nanoparticle formulation presented here is a promising vector for mRNA vaccine delivery, one that is capable of inducing a strong cytotoxic T cell response. Further optimization, including the incorporation of different adjuvants, will likely enhance the potency of the vaccine.