Providing insight into the mechanism of action of cationic lipidated oligomers using metabolomics.

The increasing resistance of clinically relevant microbes against current commercially available antimicrobials underpins the urgent need for alternative and novel treatment strategies. Cationic lipidated oligomers (CLOs) are innovative alternatives to antimicrobial peptides and have reported antimicrobial potential. An understanding of their antimicrobial mechanism of action is required to rationally design future treatment strategies for CLOs, either in monotherapy or synergistic combinations. In the present study, metabolomics was used to investigate the potential metabolic pathways involved in the mechanisms of antibacterial activity of one CLO, C12-o-(BG-D)-10, which we have previously shown to be effective against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. The metabolomes of MRSA ATCC 43300 at 1, 3, and 6 h following treatment with C12-o-(BG-D)-10 (48 µg/mL, i.e., 3× MIC) were compared to those of the untreated controls. Our findings reveal that the studied CLO, C12-o-(BG-D)-10, disorganized the bacterial membrane as the first step toward its antimicrobial effect, as evidenced by marked perturbations in the bacterial membrane lipids and peptidoglycan biosynthesis observed at early time points, i.e., 1 and 3 h. Central carbon metabolism and the biosynthesis of DNA, RNA, and arginine were also vigorously perturbed, mainly at early time points. Moreover, bacterial cells were under osmotic and oxidative stress across all time points, as evident by perturbations of trehalose biosynthesis and pentose phosphate shunt. Overall, this metabolomics study has, for the first time, revealed that the antimicrobial action of C12-o-(BG-D)-10 may potentially stem from the dysregulation of multiple metabolic pathways.IMPORTANCEAntimicrobial resistance poses a significant challenge to healthcare systems worldwide. Novel anti-infective therapeutics are urgently needed to combat drug-resistant microorganisms. Cationic lipidated oligomers (CLOs) show promise as new antibacterial agents against Gram-positive pathogens like methicillin-resistant Staphylococcus aureus (MRSA). Understanding their molecular mechanism(s) of antimicrobial action may help design synergistic CLO treatments along with monotherapy. Here, we describe the first metabolomics study to investigate the killing mechanism(s) of CLOs against MRSA. The results of our study indicate that the CLO, C12-o-(BG-D)-10, had a notable impact on the biosynthesis and organization of the bacterial cell envelope. C12-o-(BG-D)-10 also inhibits arginine, histidine, central carbon metabolism, and trehalose production, adding to its antibacterial characteristics. This work illuminates the unique mechanism of action of C12-o-(BG-D)-10 and opens an avenue to design innovative antibacterial oligomers/polymers for future clinical applications.

Lipid sulfoxide polymers as potential inhalable drug delivery platforms with differential albumin binding affinity.

Inhalable nanomedicines are increasingly being developed to optimise the pharmaceutical treatment of respiratory diseases. Large lipid-based nanosystems at the forefront of the inhalable nanomedicines development pipeline, though, have a number of limitations. The objective of this study was, therefore, to investigate the utility of novel small lipidated sulfoxide polymers based on poly(2-(methylsulfinyl)ethyl acrylate) (PMSEA) as inhalable drug delivery platforms with tuneable membrane permeability imparted by differential albumin binding kinetics. Linear PMSEA (5 kDa) was used as a hydrophilic polymer backbone with excellent anti-fouling and stealth properties compared to poly(ethylene glycol). Terminal lipids comprising single (1C2, 1C12) or double (2C12) chain diglycerides were installed to provide differing affinities for albumin and, by extension, albumin trafficking pathways in the lungs. Albumin binding kinetics, cytotoxicity, lung mucus penetration and cellular uptake and permeability through key cellular barriers in the lungs were examined in vitro. The polymers showed good mucus penetration and no cytotoxicity over 24 h at up to 1 mg ml-1. While 1C2-showed no interaction with albumin, 1C12-PMSEA and 2C12-PMSEA bound albumin with KD values of approximately 76 and 10 µM, respectively. Despite binding to albumin, 2C12-PMSEA showed reduced cell uptake and membrane permeability compared to the smaller polymers and the presence of albumin had little effect on cell uptake and membrane permeability. While PMSEA strongly shielded these lipids from albumin, the data suggest that there is scope to tune the lipid component of these systems to control membrane permeability and cellular interactions in the lungs to tailor drug disposition in the lungs.

Evaluation of a Clinical Decision Support Tool to Guide Adoption of the American Heart Association Telemetry Monitoring Practice Standards.

BACKGROUND: The objectives of this study were to (1) evaluate telemetry use pre- and postimplementation of clinical decision support tools to support American Heart Association practice standards for telemetry monitoring and (2) understand the factors that may contribute to variation of telemetry monitoring in practice. METHODS AND RESULTS: First, we captured overall variability in telemetry use pre- and postimplementation of the clinical decision support intervention. We then conducted semistructured interviews with telemetry-ordering providers to identify key barriers and facilitators to adoption. During the study period, 399 physicians met criteria for inclusion and were divided into excessive and nonexcessive orderers. Distribution of telemetry use was bimodal. Among nonexcessive users, 24.4% of patient days were with telemetry compared with 51.6% among excessive users. On average, both excessive (6.1% reduction) and nonexcessive users (2.8% reduction) decreased telemetry use postimplementation, and these reductions were sustained over a 16-month period. Sixteen interviews were conducted. Physicians believed that the tool was successful because it caused them to more closely consider if telemetry was indicated for each patient. Physicians also voiced frustration with interruptions to their workflow, and some noted that they commonly use telemetry outside of practice standards to monitor patients who were acutely but not critically ill. CONCLUSIONS: Embedding telemetry practice standards into the electronic health record in the form of clinical decision support is effective at reducing excess telemetry use. Although the intervention was well received, there are persistent barriers, such as preexisting views on telemetry and existing workflow habits, that may inhibit higher adoption of standards.

Impact of conjugation to different lipids on the lymphatic uptake and biodistribution of brush PEG polymers.

Delivery to peripheral lymphatics can be achieved following interstitial administration of nano-sized delivery systems (nanoparticles, liposomes, dendrimers etc) or molecules that hitchhike on endogenous nano-sized carriers (such as albumin). The published work concerning the hitchhiking approach has mostly focussed on the lymphatic uptake of vaccines conjugated directly to albumin binding moieties (ABMs such as lipids, Evans blue dye derivatives or peptides) and their subsequent trafficking into draining lymph nodes. The mechanisms underpinning access and transport of these constructs into lymph fluid, including potential interaction with other endogenous nanocarriers such as lipoproteins, have largely been ignored. Recently, we described a series of brush polyethylene glycol (PEG) polymers containing end terminal short-chain or medium-chain hydrocarbon tails (1C2 or 1C12, respectively), cholesterol moiety (Cho), or medium-chain or long-chain diacylglycerols (2C12 or 2C18, respectively). We evaluated the association of these materials with albumin and lipoprotein in rat plasma, and their intravenous (IV) and subcutaneous (SC) pharmacokinetic profiles. Here we fully detail the association of this suite of polymers with albumin and lipoproteins in rat lymph, which is expected to facilitate lymph transport of the materials from the SC injection site. Additionally, we characterise the thoracic lymph uptake, tissue and lymph node biodistribution of the lipidated brush PEG polymers following SC administration to thoracic lymph cannulated rats. All polymers had moderate lymphatic uptake in rats following SC dosing with the lymph uptake higher for 1C2-PEG, 2C12-PEG and 2C18-PEG (5.8%, 5.9% and 6.7% dose in lymph, respectively) compared with 1C12-PEG and Cho-PEG (both 1.5% dose in lymph). The enhanced lymph uptake of 1C2-PEG, 2C12-PEG and 2C18-PEG appeared related to their association profile with different lipoproteins. The five polymers displayed different biodistribution patterns in major organs and tissues in mice. All polymers reached immune cells deep within the inguinal lymph nodes of mice following SC dosing. The ability to access these immune cells suggests the potential of the polymers as platforms for the delivery of vaccines and immunotherapies. Future studies will focus on evaluating the lymphatic targeting and therapeutic potential of drug or vaccine-loaded polymers in pre-clinical disease models.

Lipidated brush-PEG polymers as low molecular weight pulmonary drug delivery platforms.

OBJECTIVES: Nanomedicines are being actively developed as inhalable drug delivery systems. However, there is a distinct utility in developing smaller polymeric systems that can bind albumin in the lungs. We therefore examined the pulmonary pharmacokinetic behavior of a series of lipidated brush-PEG (5 kDa) polymers conjugated to 1C2, 1C12 lipid or 2C12 lipids. METHODS: The pulmonary pharmacokinetics, patterns of lung clearance and safety of polymers were examined in rats. Permeability through monolayers of primary human alveolar epithelia, small airway epithelia and lung microvascular endothelium were also investigated, along with lung mucus penetration and cell uptake. RESULTS: Polymers showed similar pulmonary pharmacokinetic behavior and patterns of lung clearance, irrespective of lipid molecular weight and albumin binding capacity, with up to 30% of the dose absorbed from the lungs over 24 h. 1C12-PEG showed the greatest safety in the lungs. Based on its larger size, 2C12-PEG also showed the lowest mucus and cell membrane permeability of the three polymers. While albumin had no significant effect on membrane transport, the cell uptake of C12-conjugated PEGs were increased in alveolar epithelial cells. CONCLUSION: Lipidated brush-PEG polymers composed of 1C12 lipid may provide a useful and novel alternative to large nanomaterials as inhalable drug delivery systems.

Functionalisation of brush polyethylene glycol polymers with specific lipids extends their elimination half-life through association with natural lipid trafficking pathways.

Polymeric prodrugs have been applied to control the delivery of various types of therapeutics. Similarly, conjugation of peptide therapeutics to lipids has been used to prolong systemic exposure. Here, we extend on these two approaches by conjugating brush polyethylene glycol (PEG) polymers with different lipid components including short-chain (1C2) or medium-chain (1C12) monoalkyl hydrocarbon tails, cholesterol (Cho), and diacylglycerols composed of two medium-chain (2C12) or long-chain (2C18) fatty acids. We uniquely evaluate the integration of these lipid-polymers into endogenous lipid trafficking pathways (albumin and lipoproteins) and the impact of lipid conjugation on plasma pharmacokinetics after intravenous (IV) and subcutaneous (SC) dosing to cannulated rats. The IV and SC elimination half-lives of Cho-PEG (13 and 22 h, respectively), 2C12-PEG (11 and 17 h, respectively) and 2C18-PEG (12 h for both) were prolonged compared to 1C2-PEG (3 h for both) and 1C12-PEG (4 h for both). Interestingly, 1C2-PEG and 1C12-PEG had higher SC bioavailability (40 % and 52 %, respectively) compared to Cho-PEG, 2C12-PEG and 2C18-PEG (25 %, 24 % and 23 %, respectively). These differences in pharmacokinetics may be explained by the different association patterns of the polymers with rat serum albumin (RSA), bovine serum albumin (BSA) and lipoproteins. For example, in pooled plasma (from IV pharmacokinetic studies), 2C18-PEG had the highest recovery in the high-density lipoprotein (HDL) fraction. In conclusion, the pharmacokinetics of brush PEG polymers can be tuned via conjugation with different lipids, which can be utilised to tune the elimination half-life, biodistribution and effect of therapeutics for a range of medical applications. STATEMENT OF SIGNIFICANCE: Lipidation of therapeutics such as peptides has been employed to extend their plasma half-life by promoting binding to serum albumin, providing protection against rapid clearance. Here we design and evaluate innovative biomaterials consisting of brush polyethylene glycol polymers conjugated with different lipids. Importantly, we show for the first time that lipidated polymeric materials associate with endogenous lipoprotein trafficking pathways and this, in addition to albumin binding, controls their plasma pharmacokinetics. We find that conjugation to dialkyl lipids and cholesterol leads to higher association with lipid trafficking pathways, and more sustained plasma exposure, compared to conjugation to short and monoalkyl lipids. Our lipidated polymers can thus be utilised as delivery platforms to tune the plasma half-life of various pharmaceuticals.

Synthesis of Sulfur-35-Labeled Trisulfides and GYY-4137 as Donors of Radioactive Hydrogen Sulfide.

Hydrogen sulfide has emerged as a key gasotransmitter in humans and in plants, and the addition of exogenous hydrogen sulfide has many beneficial effects in vivo and in vitro. A challenge in investigating the effect of exogenous hydrogen sulfide is tracking the location of exogenous hydrogen sulfide on an organism and cellular level. In this article, we report the synthesis of three key chemicals (cysteine trisulfide, glutathione trisulfide, and GYY-4137) that release radiolabeled 35S as hydrogen sulfide. The synthesis started with the reduction of Na235SO4 mixed with Na2SO4 to generate hydrogen sulfide gas that was trapped with aq NaOH to yield radiolabeled Na2S. The Na2S was converted in one step to GYY-4137 at 65% yield. It was also converted to bis(tributyltin) sulfide that readily reacted with N-bromophthalimide to yield a monosulfur transfer reagent. Trisulfides were synthesized by reaction with the monosulfur transfer reagent and the corresponding thiols. The levels of radioactivity of the final products could be varied on a per gram basis to alter the radioactivity for applications that require different loadings of hydrogen sulfide donors.

Integrated analysis shows how the effects of extreme flooding events propagate through fish communities to impact amphibians.

Research Highlight: Davis, C. L., Walls, S. C., Barichivich, W. J., Brown, M. E., & Miller, D. A. (2022). Disentangling direct and indirect effects of extreme events on coastal wetland communities. Journal of Animal Ecology, https://doi.org/10.1111/1365-2656.13874. Catastrophic events such as floods, hurricanes, winter storms, droughts and wildfires increasingly touch our lives either directly or indirectly. These events draw our attention to the seriousness of changes in climate not only to human well-being but also to the integrity of ecological systems upon which we depend. Understanding the impacts of extreme events on ecological systems requires the ability to characterize the cascading effects of environmental changes on the environments in which organisms live and the altered biological interactions produced. This scientific ambition represents no small challenge for the study of animal communities, which are typically difficult to census as well as dynamic in time and space. Davis et al. (2022) in a recent study in the Journal of Animal Ecology examined the amphibian and fish communities found in depressional coastal wetlands to better understand how they respond to major rainfall and flooding events. Data from the U.S. Geological Survey's Amphibian Research and Monitoring Initiative provided an 8-year record of observations as well as environmental measurements. For this study, the authors integrated techniques for assessing the dynamics of animal populations with a Bayesian implementation of structural equation modelling. Using their integrated methodological approach permitted the authors to reveal the direct and indirect effects of extreme weather events on co-occurring amphibian and fish communities while accounting for observational uncertainty and temporal variation in population-level processes. Their findings indicate that the most prominent effects of flooding on the amphibian community were caused by changes in the fish community that led to increased predation and resource competition. In their conclusions, the authors emphasize the importance of understanding networks of abiotic and biotic effects if we are to predict and mitigate the influence of extreme weather events.

Discovery of pyrrolo[2,1-f][1,2,4]triazine-based inhibitors of adaptor protein 2-associated kinase 1 for the treatment of pain.

Adaptor protein 2-associated kinase 1 (AAK1) is a member of the Ark1/Prk1 family of serine/threonine kinases and plays a role in modulating receptor endocytosis. AAK1 was identified as a potential therapeutic target for the treatment of neuropathic pain when it was shown that AAK1 knock out (KO) mice had a normal response to the acute pain phase of the mouse formalin model, but a reduced response to the persistent pain phase. Herein we report our early work investigating a series of pyrrolo[2,1-f][1,2,4]triazines as part of our efforts to recapitulate this KO phenotype with a potent, small molecule inhibitor of AAK1. The synthesis, structure-activity relationships (SAR), and in vivo evaluation of these AAK1 inhibitors is described.

From bottom-up to top-down control of invertebrate herbivores in a retrogressive chronosequence.

In the long-term absence of disturbance, ecosystems often enter a decline or retrogressive phase which leads to reductions in primary productivity, plant biomass, nutrient cycling and foliar quality. However, the consequences of ecosystem retrogression for higher trophic levels such as herbivores and predators, are less clear. Using a post-fire forested island-chronosequence across which retrogression occurs, we provide evidence that nutrient availability strongly controls invertebrate herbivore biomass when predators are few, but that there is a switch from bottom-up to top-down control when predators are common. This trophic flip in herbivore control probably arises because invertebrate predators respond to alternative energy channels from the adjacent aquatic matrix, which were independent of terrestrial plant biomass. Our results suggest that effects of nutrient limitation resulting from ecosystem retrogression on trophic cascades are modified by nutrient-independent variation in predator abundance, and this calls for a more holistic approach to trophic ecology to better understand herbivore effects on plant communities.

Migration and transformation of coastal wetlands in response to rising seas.

Coastal wetlands are not only among the world's most valued ecosystems but also among the most threatened by high greenhouse gas emissions that lead to accelerated sea level rise. There is intense debate regarding the extent to which landward migration of wetlands might compensate for seaward wetland losses. By integrating data from 166 estuaries across the conterminous United States, we show that landward migration of coastal wetlands will transform coastlines but not counter seaward losses. Two-thirds of potential migration is expected to occur at the expense of coastal freshwater wetlands, while the remaining one-third is expected to occur at the expense of valuable uplands, including croplands, forests, pastures, and grasslands. Our analyses underscore the need to better prepare for coastal transformations and net wetland loss due to rising seas.

Sustained endosomal release of a neurokinin-1 receptor antagonist from nanostars provides long-lasting relief of chronic pain.

Soft polymer nanoparticles designed to disassemble and release an antagonist of the neurokinin 1 receptor (NK1R) in endosomes provide efficacious yet transient relief from chronic pain. These micellar nanoparticles are unstable and rapidly release cargo, which may limit the duration of analgesia. We examined the efficacy of stable star polymer nanostars containing the NK1R antagonist aprepitant-amine for the treatment of chronic pain in mice. Nanostars continually released cargo for 24 h, trafficked through the endosomal system, and disrupted NK1R endosomal signaling. After intrathecal injection, nanostars accumulated in endosomes of spinal neurons. Nanostar-aprepitant reversed mechanical, thermal and cold allodynia and normalized nociceptive behavior more efficaciously than free aprepitant in preclinical models of neuropathic and inflammatory pain. Analgesia was maintained for >10 h. The sustained endosomal delivery of antagonists from slow-release nanostars provides effective and long-lasting reversal of chronic pain.

Polymerization of Aniline Derivatives to Yield Poly[N,N-(phenylamino)disulfides] as Polymeric Auxochromes.

Polymerizations of phenylamines with a disulfide transfer reagent to yield poly[N,N-(phenylamino) disulfides] (poly-NADs) were investigated due to their unique repeat units that resulted in conjugation along the backbone that was perturbed by the aromatic rings and gave different colors for the polymers. These polymers were synthesized from 10 different anilines and sulfur monochloride in a step-growth polymerization. The polymers were characterized by nuclear magnetic resonance spectroscopy, size exclusion chromatography-multiangle light scattering, and UV-vis spectroscopy. These polymers possessed a polymeric backbone solely consisting of nitrogen and sulfur [-N(R)SS-], which was conjugated and yielded polymers of moderate molecular weight. Most notably, these polymers were an array of colors ranging from pale yellow to a deep purple depending on the substitution of the aromatic ring. The more electron-poor systems produced lighter yellow polymers, while the electron-rich systems gave orange, green, red, and even purple polymers.

Biodiversity effects on grape quality depend on variety and management intensity.

Interactions between plants can be beneficial, detrimental or neutral. In agricultural systems, competition between crop and spontaneous vegetation is a major concern. We evaluated the relative support for three non-exclusive ecological hypotheses about interactions between crop and spontaneous plants based on competition, complementarity or facilitation.The study was conducted in Swiss vineyards with different vegetation management intensities. In all, 33 vineyards planted with two different grape varieties were studied over 3 years to determine whether low-intensity vegetation management might provide benefits for grape quality parameters. Management intensity varied with the degree of control of spontaneous inter-row vegetation. Features of spontaneous vegetation measured included total cover, total species richness and abundance of nitrogen-fixing plants. Grape quality parameters of known importance to wine making (yeast assimilable nitrogen, sugars, tartaric acid and malic acid) were determined by Fourier-transform infrared spectroscopy (FTIR). Using structural equation modelling, we evaluated hypotheses about the multivariate responses of grape quality parameters as well as the direct and indirect (plant-mediated) effects of management.Observed effects of management differed between grape varieties. Management intensity and abundance of N-fixing plants significantly influenced grape quality parameters while total richness of spontaneous plants did not have detectable effects. Abundance of N-fixing plants was enhanced by low-intensity management resulting in increased N content in the red grape variety Pinot noir, potentially enhancing grape quality, while measured soil N content did not explain the increase.Synthesis and applications. Our study shows that crop quality can be enhanced by spontaneous plants, in this case by the abundance of a key functional group (N-fixers), most likely through plant-plant or plant-microbe facilitation. However, beneficial interactions may have a high specificity in terms of facilitation partners and may have contrasting effects at low taxonomic resolutions such as crop varieties. Generally, increasing plant biodiversity in agricultural systems may increase competition with crops. Thus, the identification of suitable interaction partners and a careful balance between crop variety and spontaneous plant species may be necessary to utilize beneficial interactions and to reduce the trade-off between agricultural production and biodiversity to achieve a sustainable ecological benefit in agricultural systems.

Synthesis and Hydrogen Sulfide Releasing Properties of Diaminodisulfides and Dialkoxydisulfides.

Heterosubstituted disulfides are an understudied class of molecules that have been used in biological studies, but they have not been investigated for their ability to release hydrogen sulfide (H2S). The synthesis of two sets of chemicals with the diaminodisulfide (NSSN) and dialkoxydisulfide (OSSO) functional groups was reported. These chemicals were synthesized from commercially available sulfur monochloride or a simple disulfur transfer reagent. Both the diaminodisulfide and dialkoxydisulfide functional groups were found to have rapid rates of H2S release in the presence of excess thiol. The release of H2S was complete with 10 min, and the only byproducts were conversion of the thiols into disulfides and the amines or alcohols originally used in the synthesis of the diaminodisulfide or dialkoxydisulfide functional groups. These results will allow the design of H2S releasing chemicals that also release natural, biocompatible alcohols or amines. Chemicals with the diaminodisulfide and dialkoxydisulfide functional groups may find applications in medicine where a controlled, burst release of H2S is needed.

The importance of natural versus human factors for ecological conditions of streams and rivers.

Streams are influenced by watershed-scale factors, such as climate, geology, topography, hydrology, and soils, which mostly vary naturally among sites, as well as human factors, agriculture and urban development. Thus, natural factors could complicate assessment of human disturbance. In the present study, we use structural equation modeling and data from the 2008-2009 United States National Rivers and Streams Assessment to quantify the relative importance of watershed-scale natural and human factors for in-stream conditions. We hypothesized that biological condition, represented using a diatom multimetric index (MMI), is directly affected by in-stream physicochemical environment, which in turn is regulated by natural and human factors. We evaluated this hypothesis at both national and ecoregion scales to understand how influences vary among regions. We found that direct influences of in-stream environment on diatom MMIs were greater than natural and human factors at the national scale and in all but one ecoregion. Meanwhile, in-stream environments were jointly explained by natural variations in precipitation, base flow index, hydrological stability, % volcanic rock, soil water table depth, and soil depth and by human factors measured as % crops, % other agriculture, and % urban land use. The explained variance of in-stream environment by natural and human factors ranged from 0.30 to 0.75, for which natural factors independently accounted for the largest proportion of explained variance at the national scale and in seven ecoregions. Covariation between natural and human factors accounted for a higher proportion of explained variance of in-stream environment than unique effects of human factors in most ecoregions. Ecoregions with relatively weak effects by human factors had relatively high levels of covariance, high levels of human disturbance, or small ranges in human disturbance. We conclude that accounting for effects of natural factors and their covariation with human factors will be important for accurate ecological assessments.

Scientist's guide to developing explanatory statistical models using causal analysis principles.

Recent discussions of model selection and multimodel inference highlight a general challenge for researchers: how to convey the explanatory content of a hypothesized model or set of competing models clearly. The advice from statisticians for scientists employing multimodel inference is to develop a well-thought-out set of candidate models for comparison, though precise instructions for how to do that are typically not given. A coherent body of knowledge, which falls under the general term causal analysis, now exists for examining the explanatory scientific content of candidate models. Much of the literature on causal analysis has been recently developed, and we suspect may not be familiar to many ecologists. This body of knowledge comprises a set of graphical tools and axiomatic principles to support scientists in their endeavors to create "well-formed hypotheses," as statisticians are asking them to do. Causal analysis is complementary to methods such as structural equation modeling, which provides the means for evaluation of proposed hypotheses against data. In this paper, we summarize and illustrate a set of principles that can guide scientists in their quest to develop explanatory hypotheses for evaluation. The principles presented in this paper have the capacity to close the communication gap between statisticians, who urge scientists to develop well-thought-out coherent models, and scientists, who would like some practical advice for exactly how to do that.

Thiol-Reactive Star Polymers Functionalized with Short Ethoxy-Containing Moieties Exhibit Enhanced Uptake in Acute Lymphoblastic Leukemia Cells.

PURPOSE: Directing nanoparticles to cancer cells without using antibodies is of great interest. Subtle changes to the surface chemistry of nanoparticles can significantly affect their biological fate, including their propensity to associate with different cell populations. For instance, nanoparticles functionalized with thiol-reactive groups can potentially enhance association with cells that over-express cell-surface thiol groups. The potential of such an approach for enhancing drug delivery for childhood acute lymphoblastic leukemia (ALL) cells has not been investigated. Herein, we investigate the impact of thiol-reactive star polymers on the cellular association and the mechanisms of uptake of the nanoparticles. METHODS: We prepared fluorescently labeled star polymers functionalized with an mPEG brush corona and pyridyl disulfide to examine how reactivity to exofacial thiols impacts cellular association with ALL cells. We also studied how variations to the mPEG brush composition could potentially be used as a secondary method for controlling the extent of cell association. Specifically, we examined how the inclusion of shorter diethylene glycol brush moieties into the nanoparticle corona could be used to further influence cell association. RESULTS: Star polymers incorporating both thiol-reactive and diethylene glycol brush moieties exhibited the highest cellular association, followed by those functionalized solely with thiol reactive groups compared to control nanoparticles in T and B pediatric ALL patient-derived xenografts harvested from the spleens and bone marrow of immunodeficient mice. Transfection of cells with an early endosomal marker and imaging with correlative light and electron microscopy confirmed cellular uptake. Endocytosis inhibitors revealed dynamin-dependent clathrin-mediated endocytosis as the main uptake pathway for all the star polymers. CONCLUSION: Thiol-reactive star polymers having an mPEG brush corona that includes a proportion of diethylene glycol brush moieties represent a potential strategy for improved leukemia cell delivery.

An optimised Cu(0)-RDRP approach for the synthesis of lipidated oligomeric vinyl azlactone: toward a versatile antimicrobial materials screening platform.

This report details the synthesis of lipidated 2-vinyl-4,4-dimethyl-5-oxazolone (VDM) oligomers via an optimised Cu(0)-mediated reversible-deactivation radical polymerisation approach, and the use of these oligomers as a versatile functional platform for the rapid generation of antimicrobial materials. The relative amounts of CuBr2 and Me6TREN were optimised to allow the fast and controlled polymerisation of VDM. These conditions were then used with the initiators ethyl 2-bromoisobutyrate, dodecyl 2-bromoisobutyrate, and (R)-3-((2-bromo-2-methylpropanoyl)oxy)propane-1,2-diyl didodecanoate to synthesise a library of oligo(VDM) (degree of polymerisation = 10) with ethyl, dodecyl or diglyceride end-groups. Subsequently, ring-opening of the pendant oxazolone group with various amines (i.e., 2-(2-aminoethyl)-1,3-di-Boc-guanidine, 1-(3-aminopropyl)imidazole, N-Boc-ethylenediamine, or N,N-dimethylethylenediamine) expanded the library to give 12 functional oligomers incorporating different cationic and lipid elements. The antimicrobial activities of these oligomers were assessed against a palette of bacteria and fungi: i.e. Staphylococcus aureus, Escherichia coli, Candida albicans, and Cryptococcus neoformans. The oligomers generally exhibited the greatest activity against the fungus, C. neoformans, with a minimum inhibitory concentration of 1 µg mL-1 (comparable to the clinically approved antifungal fluconazole). To assess haemocompatibility, the oligomers were assayed against erythrocytes, with the primary amine or guanidine containing C12 and 2C12 oligomers exhibiting greater lysis against the red blood cells (HC10 values between 7.1 and 43 µg mL-1) than their imidazole and tertiary amine counterparts (HC10 of >217 µg mL-1). Oligomers showed the greatest selectivity for C. neoformans, with the C12- and 2C12-tertiary amine and C12-imidazole oligomers possessing the greatest selectivity of >54-109. These results demonstrate the utility of reactive oligomers for rapidly assessing structure-property relationships for antibacterial and antifungal materials.