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Lipid nanoparticles (LNPs) are the most advanced delivery system currently available for RNA therapeutics. Their development has accelerated since the success of Patisiran, the first siRNA-LNP therapeutic, and the mRNA vaccines that emerged during the COVID-19 pandemic. Designing LNPs with specific targeting, high potency, and minimal side effects is crucial for their successful clinical use. However, our understanding of how the composition and mixing method influences the structural, biophysical, and biological properties of the resulting LNPs remains limited, hindering the development of LNPs. Our lack of structural understanding extends from the physical and compositional polydispersity of LNPs, which render traditional characterization methods, such as dynamic light scattering (DLS), unable to accurately quantitate the physicochemical characteristics of LNPs. In this study, we address the challenge of structurally characterizing polydisperse LNP formulations using emerging solution-based biophysical methods that have higher resolution and provide biophysical data beyond size and polydispersity. These techniques include sedimentation velocity analytical ultracentrifugation (SV-AUC), field-flow fractionation followed by multi-angle light scattering (FFF-MALS), and size-exclusion chromatography in-line with synchrotron small-angle X-ray scattering (SEC-SAXS). Here, we show that the LNPs have intrinsic polydispersity in size, RNA loading, and shape, and that these parameters are dependent on both the formulation technique and lipid composition. Lastly, we demonstrate that these biophysical methods can be employed to predict transfection in human primary T cells, intravenous administration, and intramuscular administration by examining the relationship between mRNA translation and physicochemical characteristics. We envision that employing solution-based biophysical methods will be essential for determining LNP structure-function relationships, facilitating the creation of new design rules for LNPs., Competing Interests: Competing Interests The authors declare that they have no conflicts of interest with the contents of this article.

Lipid nanoparticles (LNPs) are the preeminent non-viral drug delivery vehicle for mRNA-based therapies. Immense effort has been placed on optimizing the ionizable lipid (IL) structure, which contains an amine core conjugated to lipid tails, as small molecular adjustments can result in substantial changes in the overall efficacy of the resulting LNPs. However, despite some advancements, a major barrier for LNP delivery is endosomal escape. Here, we develop a platform for synthesizing a class of branched ILs that improve endosomal escape. These compounds incorporate terminally branched groups that increase hepatic mRNA and ribonucleoprotein complex delivery and gene editing efficiency as well as T cell transfection compared to non-branched lipids. Through an array of complementary experiments, we determine that our lipid architecture induces greater endosomal penetration and disruption. This work provides a scheme to generate a class of ILs for both mRNA and protein delivery., Competing Interests: Competing interests: M.S.P. and M.J.M. have filed a patent application related to the structure of the BEND lipids and biological applications (U.S. Provisional Patent Appl. No. 63/373,793, filed August 29, 2022). J.M.W. is a paid advisor to and holds equity in iECURE, Passage Bio, and the Center for Breakthrough Medicines (CBM). He also holds equity in the former G2 Bio asset companies and Ceva Santé Animale. He has sponsored research agreements with Alexion Pharmaceuticals, Amicus Therapeutics, CBM, Ceva Santé Animale, Elaaj Bio, FA212, Foundation for Angelman Syndrome Therapeutics, former G2 Bio asset companies, iECURE, and Passage Bio, which are licensees of Penn technology. JMW is an inventor on patents that have been licensed to various biopharmaceutical companies and for which he may receive payments. The remaining authors declare no competing interests, (© 2025. The Author(s).)

Systemic delivery of large nucleic acids, such as mRNA, to the brain remains challenging in part due to the blood-brain barrier (BBB) and the tendency of delivery vehicles to accumulate in the liver. Here, we design a peptide-functionalized lipid nanoparticle (LNP) platform for targeted mRNA delivery to the brain. We utilize click chemistry to functionalize LNPs with peptides that target receptors overexpressed on brain endothelial cells and neurons, namely the RVG29, T7, AP2, and mApoE peptides. We evaluate the effect of LNP targeting on brain endothelial and neuronal cell transfection in vitro , investigating factors such as serum protein adsorption, intracellular trafficking, endothelial transcytosis, and exosome secretion. Finally, we show that LNP peptide functionalization enhances mRNA transfection in the mouse brain and reduces hepatic delivery after systemic administration. Specifically, RVG29 LNPs improved neuronal transfection in vivo , establishing its potential as a nonviral platform for delivering mRNA to the brain.

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., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

Background and Aim: Colorectal cancer (CRC) is the fourth leading cause of cancer death globally. CRC surveillance is a common indication for colonoscopy, representing a considerable burden for endoscopy services. Accurate identification of high-risk patients who would benefit from more intensive surveillance, as well as low-risk patients suitable for less frequent follow-up, could improve the effectiveness of surveillance protocols and resource use. Our aim was to identify and critically appraise published risk models for the occurrence of metachronous advanced colorectal neoplasia (ACN), defined here as CRC or advanced adenomas detected during surveillance colonoscopy., Methods: We searched PubMed and EMBASE for primary research studies reporting the development and/or validation of multivariable models that predict metachronous ACN risk. Screening of studies for inclusion, data extraction, and risk of bias assessment were conducted by two researchers independently., Results: We identified nine studies describing nine risk models. Six models were internally validated and two were externally validated. No model underwent both internal and external validation. Good model discrimination (concordance index > 0.7) was reported for two models during internal validation and for one model during external validation. Calibration was acceptable when assessed (n = 4). Methodological limitations and a high risk of bias were observed for all studies., Conclusions: Several published models predicting metachronous ACN risk showed some promise. However, adherence to methodological standards was limited, and only two models were externally validated. Head-to-head comparisons of existing models using populations independent from model development cohorts should be prioritized to identify models suitable for use in clinical practice., (© 2024 The Author(s). Journal of Gastroenterology and Hepatology published by Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

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., (© 2024 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Chimeric antigen receptor (CAR) monocyte and macrophage therapies are promising solid tumor immunotherapies that can overcome the challenges facing conventional CAR T cell therapy. mRNA lipid nanoparticles (mRNA-LNPs) offer a viable platform for in situ engineering of CAR monocytes with transient and tunable CAR expression to reduce off-tumor toxicity and streamline cell manufacturing. However, identifying LNPs with monocyte tropism and intracellular delivery potency is difficult using traditional screening techniques. Here, ionizable lipid design and high-throughput in vivo screening are utilized to identify a new class of oxidized LNPs with innate tropism and mRNA delivery to monocytes. A library of oxidized (oLNPs) and unoxidized LNPs (uLNPs) is synthesized to evaluate mRNA delivery to immune cells. oLNPs demonstrate notable differences in morphology, ionization energy, and p Ka , therefore enhancing delivery to human macrophages, but not T cells. Subsequently, in vivo library screening with DNA barcodes identifies an oLNP formulation, C14-O2, with innate tropism to monocytes. In a proof-of-concept study, the C14-O2 LNP is used to engineer functional CD19-CAR monocytes in situ for robust B cell aplasia (45%) in healthy mice. This work highlights the utility of oxidized LNPs as a promising platform for engineering CAR macrophages/monocytes for solid tumor CAR monocyte therapy.

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 ∼twofold increase in mRNA delivery in murine placentas and a ∼twofold 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., Competing Interests: Declaration of competing interest H.C.G., K.L.S., H.C.S., and M.J.M. have filed a patent application related to this study. The remaining authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable clinical success in the treatment of hematological malignancies. However, producing these bespoke cancer-killing cells is a complicated ex vivo process involving leukapheresis, artificial T cell activation, and CAR construct introduction. The activation step requires the engagement of CD3/TCR and CD28 and is vital for T cell transfection and differentiation. Though antigen-presenting cells (APCs) facilitate activation in vivo, ex vivo activation relies on antibodies against CD3 and CD28 conjugated to magnetic beads. While effective, this artificial activation adds to the complexity of CAR T cell production as the beads must be removed prior to clinical implementation. To overcome this challenge, this work develops activating lipid nanoparticles (aLNPs) that mimic APCs to combine the activation of magnetic beads and the transfection capabilities of LNPs. It is shown that aLNPs enable one-step activation and transfection of primary human T cells with the resulting mRNA CAR T cells reducing tumor burden in a murine xenograft model, validating aLNPs as a promising platform for the rapid production of mRNA CAR T cells., (© 2024 The Authors. Advanced Materials published by Wiley‐VCH GmbH.)

In this study, an oil-in-water (o/w) nanoemulsion is used to deliver siRNA targeting Twist1, a protein that contributes to tumor metastasis in a variety of cancers. The FDA-approved oil, medium chain triglycerides (MCT), is used as the hydrophobic phase for the nanoemulsion. The siRNA is paired with dioleoyl-3-trimethylammonium-propane (DOTAP) to form a hydrophobic salt that is soluble at high concentrations in MCT. The resulting MCT/siRNA-DOTAP solution is formulated into a nanoemulsion with an average particle size of 140 nm. The nanoemulsion displays long term stability over the course of 195 days. In an in vivo murine tumor model, the nanoemulsion facilitates a 46% decrease in Twist1 mRNA after 48 h., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Lipid nanoparticle (LNP)-mediated nucleic acid therapies, including mRNA protein replacement and gene editing therapies, hold great potential in treating neurological disorders including neurodegeneration, brain cancer, and stroke. However, delivering LNPs across the blood-brain barrier (BBB) after systemic administration remains underexplored. In this work, we engineered a high-throughput screening transwell platform for the BBB (HTS-BBB), specifically optimized for screening mRNA LNPs. Unlike most transwell assays, which only assess transport across an endothelial monolayer, HTS-BBB simultaneously measures LNP transport and mRNA transfection of the endothelial cells themselves. We then use HTS-BBB to screen a library of 14 LNPs made with structurally diverse ionizable lipids and demonstrate it is predictive of in vivo performance by validating lead candidates for mRNA delivery to the mouse brain after intravenous injection. Going forward, this platform could be used to screen large libraries of brain-targeted LNPs for a range of protein replacement and gene editing applications.

Lipid nanoparticles (LNPs) are a potent delivery technology that have made it possible for the recent clinical breakthroughs in mRNA therapeutics and vaccines. A key challenge to the broader implementation of mRNA therapeutics and vaccines is the development of technology to produce precisely defined LNP formulations, with throughput that can scale from discovery to commercial manufacturing and meet the stringent manufacturing standards of the pharmaceutical industry. To address these challenges, we have developed a microfluidic chip that incorporates 1×, 10×, or 256× LNP-generating units that achieve scalable production rates of up to 17 L/h of precisely defined LNPs. Using these chips, we demonstrate that LNP physical properties and potency in vivo are unchanged as throughput is scaled. Our chips are fabricated out of silicon and glass substrates, which have excellent solvent compatibility, compatibility with pharmaceutical manufacturing, and can be fully reset and reused. SARS-CoV-2 mRNA-LNP vaccines formulated by our chips triggered potent antibody responses in a preclinical study. These results demonstrate the feasibility of directly translating microfluidic-generated LNPs to the scale necessary for commercial production.

Malaria is the most lethal parasitic disease worldwide; the severity of symptoms and mortality are higher in men than in women, exhibiting an evident sexual dimorphism in the immune response; therefore, the contribution of 17β-estradiol and testosterone to this phenomenon has been studied. Both hormones differentially affect several aspects of innate and adaptive immunity. Dehydroepiandrosterone (DHEA) is the precursor of both hormones and is the sexual steroid in higher concentrations in humans, with immunomodulatory properties in different parasitic diseases; however, the involvement of DHEA in this sexual dimorphism has not been studied. In the case of malaria, the only information is that higher levels of DHEA are associated with reduced Plasmodium falciparum parasitemia. Therefore, this work aims to analyze the DHEA contribution to the sexual dimorphism of the immune response in malaria. We assessed the effect of modifying the concentration of DHEA on parasitemia, the number of immune cells in the spleen, cytokines, and antibody levels in plasma of CBA/Ca mice infected with Plasmodium berghei ANKA ( P. berghei ANKA). DHEA differentially affected the immune response in males and females: it decreased IFN-γ, IL-2 and IL-4 concentrations only in females, whereas in gonadectomized males, it increased IgG2a and IgG3 antibodies. The results presented here show that DHEA modulates the immune response against Plasmodium differently in each sex, which helps to explain the sexual dimorphism present in malaria.

Mutated RAS proteins are potent oncogenic drivers and have long been considered "undruggable". While RAS-targeting therapies have recently shown promise, there remains a clinical need for RAS inhibitors with more diverse targets. Small proteins represent a potential new therapeutic option, including K27, a designed ankyrin repeat protein (DARPin) engineered to inhibit RAS. However, K27 functions intracellularly and is incapable of entering the cytosol on its own, currently limiting its utility. To overcome this barrier, we have engineered a lipid nanoparticle (LNP) platform for potent delivery of functional K27-D30─a charge-modified version of the protein─intracellularly in vitro and in vivo . This system efficiently encapsulates charge-modified proteins, facilitates delivery in up to 90% of cells in vitro , and maintains potency after at least 45 days of storage. In vivo , these LNPs deliver K27-D30 to the cytosol of cancerous cells in the liver, inhibiting RAS-driven growth and ultimately reducing tumor load in an HTVI-induced mouse model of hepatocellular carcinoma. This work shows that K27 holds promise as a new cancer therapeutic when delivered using this LNP platform. Furthermore, this technology has the potential to broaden the use of LNPs to include new cargo types─beyond RNA─for diverse therapeutic applications.

Nanoparticle (NP)-based therapeutics have ushered in a new era in translational medicine. However, despite the clinical success of NP technology, it is not well-understood how NPs fundamentally change in biological environments. When introduced into physiological fluids, NPs are coated by proteins, forming a protein corona (PC). The PC has the potential to endow NPs with a new identity and alter their bioactivity, stability, and destination. Additionally, the conformation of proteins is sensitive to their physical and chemical surroundings. Therefore, biological factors and protein-NP-interactions can induce changes in the conformation and orientation of proteins in vivo . Since the function of a protein is closely connected to its folded structure, slight differences in the surrounding environment as well as the surface characteristics of the NP materials may cause proteins to lose or gain a function. As a result, this can alter the downstream functionality of the NPs. This review introduces the main biological factors affecting the conformation of proteins associated with the PC. Then, four types of NPs with extensive utility in biomedical applications are described in greater detail, focusing on the conformation and orientation of adsorbed proteins. This is followed by a discussion on the instances in which the conformation of adsorbed proteins can be leveraged for therapeutic purposes, such as controlling protein conformation in assembled matrices in tissue, as well as controlling the PC conformation for modulating immune responses. The review concludes with a perspective on the remaining challenges and unexplored areas at the interface of PC and NP research.

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.

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and Pfizer/BioNTech. These vaccines have demonstrated the efficacy of mRNA-LNP therapeutics and opened the door for future clinical applications. In mRNA-LNP systems, the LNPs serve as delivery platforms that protect the mRNA cargo from degradation by nucleases and mediate their intracellular delivery. The LNPs are typically composed of four components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene glycol (PEG) conjugate (lipid-PEG). Here, LNPs encapsulating mRNA encoding firefly luciferase are formulated by microfluidic mixing of the organic phase containing LNP lipid components and the aqueous phase containing mRNA. These mRNA-LNPs are then tested in vitro to evaluate their transfection efficiency in HepG2 cells using a bioluminescent plate-based assay. Additionally, mRNA-LNPs are evaluated in vivo in C57BL/6 mice following an intravenous injection via the lateral tail vein. Whole-body bioluminescence imaging is performed by using an in vivo imaging system. Representative results are shown for the mRNA-LNP characteristics, their transfection efficiency in HepG2 cells, and the total luminescent flux in C57BL/6 mice.

Post-traumatic stress disorder (PTSD) is a debilitating mental health condition for which current treatments have long-term efficacy in 50% of patients. There is a clear need for better understanding of the mechanisms underlying PTSD and the development of new treatment approaches. Analog trauma procedures in animals, such as the stress-enhanced fear learning (SEFL) procedure, can be used to produce behavioral and neurobiological changes that have validity in modeling PTSD. However, by necessity, the modeling of PTSD in animals requires them to potentially experience pain and suffering. Consistent with the '3Rs' (reduction, refinement and replacement) of animal research, this study aimed to determine whether the SEFL procedure can be refined to reduce potential animal pain and suffering while retaining the same behavioral and neurobiological changes. Here we showed that PTSD-relevant changes could be produced in both behavior and the brain of rats that were group- rather than single-housed and that received lower-magnitude electric shocks in the 'trauma analog' session. We also varied the number of shock exposures in the trauma analog session, finding SEFL-susceptible and SEFL-resilient populations at all levels of shock exposure, but with greater levels of shock increasing the proportion of rats showing the SEFL-susceptible phenotype. These data demonstrate that the SEFL procedure can be used as an animal analog of PTSD with reduced potential pain and suffering to the animals and that variations in the procedure could be used to generate specific proportions of SEFL-susceptible and SEFL-resilient animals in future studies., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Malaria is the most lethal parasitic disease worldwide; men exhibit higher mortality and more severe symptomatology than women; however, in most studies of immune response in malaria, sex is not considered a variable. Sex hormones 17β-oestradiol and testosterone are responsible for the main physiological differences between sexes. When interacting with their receptors on different immune cells, they modify the expression of genes that modulate cell proliferation, differentiation, and synthesis of cytokines. The immunosuppressive activity of testosterone is well accepted; however, its participation in the sexual dimorphism of the immune response to malaria has not been studied. In this work, we analysed whether altering the concentration of testosterone, through increasing the concentration of this hormone for exogenous administration for three weeks, or gonadectomy before infection with Plasmodium berghei ANKA affects different cells of the immune response necessary for parasite clearance. We also assessed the concentration of pro-and anti-inflammatory cytokines in male and female CBA/Ca mice infected or not with the parasite. Our results show that testosterone changes affect females more than males, resulting in sex-associated patterns. Testosterone administration increased parasitaemia in intact males while reducing it in intact females leading to a dimorphic pattern. In addition, gonadectomy increased parasitaemia in both sexes. Moreover, testosterone administration prevented both weight loss caused by the infection in females and hypothermia in gonadectomized mice of both sexes. Boosting testosterone concentration increased CD3 + and CD8 + populations but decreased the B220 + cells exclusively in females. Additionally, testosterone reduced IFN-γ concentration and increased IL-6 levels only in females, while in males, testosterone increased the number of NK cells. Finally, gonadectomy decreased TNF-α concentration in both sexes. Our results demonstrate that testosterone induces different patterns depending on sex and testosterone concentration. The results of this work contribute to understanding the impact of modifying testosterone concentration on the immune response specific against Plasmodium and the participation of this hormone in sexual dimorphism in malaria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Aguilar-Castro, Cervantes-Candelas, Buendía-González, Fernández-Rivera, Nolasco-Pérez, López-Padilla, Chavira-Ramírez, Cervantes-Sandoval and Legorreta-Herrera.)

The unfolded protein response has been increasingly implicated as an important pathological pathway and target for therapeutic intervention in neurodegeneration. The licensed antidepressant trazodone is one drug which has been proposed to act on this pathway and may therefore be a potential therapy. Previous examination of existing data for patients with dementia prescribed trazodone did not find a signal suggesting a disease modifying effect. Here we add to that literature by examining the electronic patient record of patients with dementia in Cambridgeshire UK. We found that trazodone is rarely prescribed and where it is used it is at a dose less than half that predicted to be disease modifying. We also found that patients prescribed trazodone had higher levels of neuropsychiatric symptoms and were relatively late in the disease course, likely beyond the optimal point for therapeutic intervention. We suggest it is therefore premature to discard potential therapies based on observational data alone, particularly when experimental medicine approaches to examine the effects of trazodone are feasible., (© 2022 The Authors. International Journal of Geriatric Psychiatry published by John Wiley & Sons Ltd.)

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 in vivo . Following in vitro 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 in vivo 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.

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., (© 2022 Wiley Periodicals LLC.)

Malaria is the leading cause of parasitic infection-related death globally. Additionally, malaria-associated mortality is higher in men than in women, and this sexual dimorphism reflects differences in innate and adaptive immune responses that are influenced by sex hormones. Normally, females develop more robust immune responses against parasites than males. However, most clinical and laboratory studies related to the immune response to malaria do not consider sex as a variable, and relatively few studies have compared the sex-dependent role of 17β-estradiol in this process. In this study, we decreased in vivo the levels of 17β-estradiol by gonadectomy or administered 17β-estradiol to intact or gonadectomized male and female CBA/Ca mice infected with Plasmodium berghei ANKA. Subsequently, we assessed the effects of 17β-estradiol on parasite load; the percentages of different immune cells in the spleen; the plasma levels of antibodies and pro- and anti-inflammatory cytokines; and the mRNA expression levels of cytokine-encoding genes in the brain. The results showed that the administration of 17β-estradiol increased parasitemia and decreased body weight in intact female mice. Moreover, intact females exhibited higher levels of CD8 + T cells and lower levels of NK1.1 + cells than their male counterparts under the same condition. Gonadectomy increased IFN-γ and decreased TNF-α concentrations only in intact female mice. Additionally, IL-10 levels were higher in intact females than in their male counterparts. Finally, the mRNA expression levels of cytokines coding genes in the brain showed a dimorphic pattern, i.e., gonadectomy upregulated Tnf , Il1b , and Il10 expression in males but not in females. Our findings explain the sexual dimorphism in the immune response to malaria, at least in part, and suggest potential sex-dependent implications for the efficacy of vaccines or drugs targeting malaria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial of financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cervantes-Candelas, Aguilar-Castro, Buendía-González, Fernández-Rivera, Nolasco-Pérez, López-Padilla, Chavira-Ramírez and Legorreta-Herrera.)

Malaria is the parasitic disease with the highest mortality worldwide; males exhibit higher mortality and more severe symptomatology than females, suggesting the participation of sexual hormones in protection and pathology. We have documented that gonadectomy modifies oxidative stress in Plasmodium berghei ANKA-infected mice in a dimorphic manner. However, gonadectomy decreases all sexual steroids levels, making it difficult to determine the contribution of each hormone to the results. This study aimed to explore the participation of 17β-oestradiol (E2) in oxidative stress in the blood, spleen, liver and brain of P. berghei-infected female and male mice. E2 was administered to intact or gonadectomized (GX) male and female mice to assess their effects on parasitaemia, body weight loss and hypothermia. We also measured the effect of E2 on the specific activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and on malondialdehyde (MDA) levels in the blood, spleen, liver and brain of CBA/Ca male and female mice infected with P. berghei ANKA. We detected the effects of E2 and sexual dimorphism on all tissues and variables analysed. Administration of E2 increased parasitaemia in intact mice. However, reconstitution of GX female mice with E2 decreased parasitaemia. E2 decreased body weight and differentially modulated oxidative stress depending on the sex, infection and tissue analysed. Low antioxidant activity was detected in the brain, suggesting additional protective antioxidant mechanisms in the brain independent of antioxidant enzymes. Our results explained, at least in part, the sexual dimorphism in this experimental model of malaria., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Importance: Metformin hydrochloride is emerging as a repurposed anticancer drug. Preclinical and retrospective studies have shown that it improves outcomes across a wide variety of neoplasms, including lung cancer. Particularly, evidence is accumulating regarding the synergistic association between metformin and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs)., Objective: To assess the progression-free survival (PFS) in patients with advanced lung adenocarcinoma who received treatment with EGFR-TKIs plus metformin compared with those who received EGFR-TKIs alone., Design, Setting, and Participants: Open-label, randomized, phase 2 trial conducted at the Instituto Nacional de Cancerología (INCan), Mexico City, Mexico. Eligible patients were 18 years or older, had histologically confirmed stage IIIB-IV lung adenocarcinoma with an activating EGFR mutation., Interventions: Patients were randomly allocated to receive EGFR-TKIs (erlotinib hydrochloride, afatinib dimaleate, or gefitinib at standard dosage) plus metformin hydrochloride (500 mg twice a day) or EGFR-TKIs alone. Treatment was continued until occurrence of intolerable toxic effects or withdrawal of consent., Main Outcomes and Measures: The primary outcome was PFS in the intent-to-treat population. Secondary outcomes included objective response rate, disease control rate, overall survival (OS), and safety., Results: Between March 31, 2016, and December 31, 2017, a total of 139 patients (mean [SD] age, 59.4 [12.0] years; 65.5% female) were randomly assigned to receive EGFR-TKIs (n = 70) or EGFR-TKIs plus metformin (n = 69). The median PFS was significantly longer in the EGFR-TKIs plus metformin group (13.1; 95% CI, 9.8-16.3 months) compared with the EGFR-TKIs group (9.9; 95% CI, 7.5-12.2 months) (hazard ratio, 0.60; 95% CI, 0.40-0.94; P = .03). The median OS was also significantly longer for patients receiving the combination therapy (31.7; 95% CI, 20.5-42.8 vs 17.5; 95% CI, 11.4-23.7 months; P = .02)., Conclusions and Relevance: To our knowledge, this is the first study to prospectively show that the addition of metformin to standard EGFR-TKIs therapy in patients with advanced lung adenocarcinoma significantly improves PFS. These results justify the design of a phase 3, placebo-controlled study., Trial Registration: ClinicalTrials.gov identifier: NCT03071705.

Objective: Conduct a cost-effectiveness analysis of the Fuel Your Life (FYL) program dissemination., Methods: Employees were recruited from three workplaces randomly assigned to one of the conditions: telephone coaching, small group coaching, and self-study. Costs were collected prospectively during the efficacy trial. The main outcome measures of interest were weight loss and quality-adjusted life years (QALYs)., Results: The phone condition was most costly ($601 to $589/employee) and the self-study condition was least costly ($145 to $143/employee). For weight loss, delivering FYL through the small group condition was no more effective, yet more expensive, than the self-study delivery. For QALYs, the group delivery of FYL was in an acceptable cost-effectiveness range ($22,400/QALY) relative to self-study (95% confidence interval [CI]: $10,600/QALY-dominated)., Conclusions: Prevention programs require adaptation at the local level and significantly affect the cost, effectiveness, and cost-effectiveness of the program.

Objective: A survey assessed use of and attitudes toward online mental health services among community college students to inform how such services may contribute to reducing unmet treatment need., Methods: A total of 6,034 students completed a Web-based survey on mental health and use of and attitudes toward mental health services. Logistic regression assessed the relationship between prior mental health treatment and attitudes among students with current serious psychological distress., Results: Among students with psychological distress (N=1,557), 28% reported prior in-person service use and 3% reported online mental health services use; most (60%) reported willingness to use online services. Students with no prior in-person treatment were less likely than those with history of in-person treatment to endorse preferences for in-person services (adjusted odds ratio=.54)., Conclusions: Students reported being open to using online mental health services, but utilization was low. Targeted outreach efforts may be required if these services are to reduce unmet treatment need.

The prevalence of 1,3-dipolar cycloadditions of azides and alkynes within both biology and chemistry highlights the utility of these reactions. However, the use of a copper catalyst can be prohibitive to some applications. Consequently, we have optimized a copper-free microwave-assisted reaction to alleviate the necessity for the copper catalyst. A small array of triazoles was prepared to examine the scope of this approach, and the methodology was translated to a protein context through the use of unnatural amino acids to demonstrate one of the first microwave-mediated bioconjugations involving a full length protein., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: Worksite health promotion interventions have the potential to reach half of Americans nationally, but low participation rates hinder optimal intervention effectiveness. This study examines factors associated with employee interest in worksite health-related discussions/events., Method: We analyzed cross-sectional survey data from a representative sample of employed adults in California with one or more chronic conditions. An ordinal regression model was developed., Results: Employees who reported more interest in worksite health-related discussions/events had higher coworkers support, perceived greater value from learning health-related knowledge and getting practical tips from others, and reported higher interest in health discussions/events held in community settings., Conclusion: Efforts are needed to enhance the culture of worksite health and encourage communication and support among workers. Practitioners should consider connecting different settings to enhance reach and accessibility, and applying multiple delivery strategies to increase employee interest and engagement.

Objective: The aim of this study was to evaluate the effectiveness of the Fuel Your Life program, an adaptation of the Diabetes Prevention Program (DPP), utilizing implementation strategies commonly used in worksite programs-telephone coaching, small group coaching, and self-study., Methods: The primary outcomes of body mass index and weight were examined in a randomized control trial conducted with city/county employees., Results: Although the majority of participants in all three groups lost some weight, the phone group lost significantly more weight (4.9 lb), followed by the small groups (3.4 lb) and the self-study (2.7 lb). Of the total participants, 28.3% of the phone group, 20.6% of the small group, and 15.7% of the self-study group lost 5% or more of their body weight., Conclusions: Fuel Your Life (DPP) can be effectively disseminated using different implementation strategies that are tailored to the workplace.

Reversing a bioconjugation in a spatial and temporal fashion has widespread applications, especially toward targeted drug delivery. We report the synthesis and incorporation of an unnatural amino acid with an alkyne modified dimethoxy- ortho -nitrobenzyl caging group. This unnatural amino acid can be utilized in a Glaser-Hay conjugation to generate a bioconjugate, but also is able to disrupt the bioconjugate when irradiated with light. These combined features allow for the preparation of bioconjugates with a high degree of site-specificity and allow for the separation of the two components if necessary.

The incorporation of unnatural amino acids represents a unique mechanism for the modulation of protein function. This approach has been utilized to generate photoswitchable GFP mutants, capable of demonstrating modulated fluorescence upon exposure to UV irradiation. Overall these photosensitive GFP mutants can be employed in various biosensing and diagnostic techniques to better understand protein function and processing., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Polyynes exhibit both unique photophysical properties and biological activities, necessitating efficient syntheses towards these core structures. A novel methodology for the construction of highly conjugated asymmetrical polyynes has been developed in a chemoselective fashion utilizing a solid-support. The synthesis has been applied to prepare a small library of polyynes in good to moderate yield. Moreover, their interesting fluorescence properties have been investigated, demonstrating the ability to tune fluorescence through selection of appropriate synthetic building blocks.