Preparation of Block Copolymer-Stabilized Microspheres from Commercial Plastics and Their Use as Microplastic Proxies in Degradation Studies.

This study presents a novel one-pot procedure for preparing sub-10 µm poly(ethylene glycol) (MPEG)-stabilized glycol-modified poly(ethylene terephthalate), poly(ethylene terephthalate) (PET), poly(lactic acid) (PLA), polycarbonate, and polycaprolactone (PCL) particles from commercial plastics. The prepared particles can be dried and directly resuspended in water, making them easy to handle and relevant mimics of microplastics. In addition, the method was extended to the preparation of unstabilized PET particles and somewhat larger polyethylene (PE)-based particles. Selected stabilized microparticles were subjected to aerobic biodegradation studies and compared with nonstabilized PET particles. All of the particles exhibited some degradation. For PLA and PET particles, the degradation corresponded well to the amount of surface-stabilizing MPEG groups or known impurities, confirming that these polymers do not degrade under the applied conditions but that the stabilizing groups do. PCL particles degraded relatively rapidly, which is consistent with the literature data and their relatively small size. PE-based particles degraded more than expected if only degradation of the stabilizing groups was taken into account, indicating that the surface chemistry of these particles plays a role in bulk degradation.

Add-on testing: stability assessment of 63 biochemical analytes in centrifuged and capped samples stored at 16 °C.

OBJECTIVES: Integration of add-on testing in high-scale automated clinical laboratories constitute a valuable instrument not only for the clinicians and the general patient care, but also for the laboratory itself. Knowledge on sample quality and analytical stability upon storage is necessary to be able to offer add-on testing. The objectives of this study were to examine the analytical stability of 63 biochemical analytes in plasma and urine samples stored at 16 °C. METHODS: Samples were collected by professional laboratory technicians, analyzed at automated analyzers and stored in their primary, capped tube without separator for 10, 12, 16, 20 or 24 h at 16 °C. Stability was assessed by inspecting mean concentration of samples at baseline and examining if (A) mean concentration over time violated limits of bias, or if (B) individual sample concentrations violated limits of total error. RESULTS: The majority of the 63 analytes were stable for up to 24 h of storage. Few of the analytes were only suitable for add-on testing for 4, 6, 10, 12, 16 or 20 h of storage. One analyte, P-lactate dehydrogenase, was not found suitable for add-on testing when stored at 16 °C. CONCLUSIONS: Due to the increasing number of intelligent solutions for high-scale clinical laboratories, add-on testing has come to stay. Loss of stability could not be demonstrated for the majority of analytes after 10, 12, 16, 20 or 24 h of storage. This feature of analytical stability suggests that add-on testing is an acceptable tool for these analytes.

Motivational predictors of children's involvement in out-of-school activities: An application of a football program.

The "11 for Health in Denmark" in-school educational football program has shown to have numerous positive physiological and psychological effects in 10- to 12-year-old schoolchildren. A key part of the successful application of the program, however, has not yet been examined, namely the motivational processes underlying participation and behavioral changes. This study examined such motivational processes (i.e., autonomous motivation, beliefs, and intentions) using the trans-contextual model (TCM) and investigated if the 11 for Health in Denmark program increased intentions to participate in physical activity (PA) outside of school in 10- to 12-year-old schoolchildren. Using a web-based questionnaire, Danish-speaking schoolchildren (N = 276 [boys, 50.4%]; Mage  = 10.44, SD = 0.35) from three schools and seven classes completed TCM-based questionnaires at three time-points (weeks 0, 1, and 5) while participating in the 11 for Health in Denmark program. Single-indicator structural equation modeling was performed to examine goodness-of-fit and parameter estimates. A path analysis using maximum likelihood estimation was used to test the direct and indirect effects of the TCM model. The results partly supported a mediation sequence, as we found significant direct effects in eight of 13 motivational variables (ß = -0.25-0.83, p < 0.05), indirect effects in one of nine variables (ß = 0.15, p < 0.01), but no effects with regard to PA behavior. Findings provide evidence for a motivational link between Danish-speaking schoolchildren's autonomous and controlled motivation from in school to out of school, and may inform future interventions promoting motivation and participation in out of school PA.

Unbiased and efficient estimation of causal treatment effects in crossover trials.

We introduce causal inference reasoning to crossover trials, with a focus on thorough QT (TQT) studies. For such trials, we propose different sets of assumptions and consider their impact on the modeling strategy and estimation procedure. We show that unbiased estimates of a causal treatment effect are obtained by a g-computation approach in combination with weighted least squares predictions from a working regression model. Only a few natural requirements on the working regression and weighting matrix are needed for the result to hold. It follows that a large class of Gaussian linear mixed working models lead to unbiased estimates of a causal treatment effect, even if they do not capture the true data-generating mechanism. We compare a range of working regression models in a simulation study where data are simulated from a complex data-generating mechanism with input parameters estimated on a real TQT data set. In this setting, we find that for all practical purposes working models adjusting for baseline QTc measurements have comparable performance. Specifically, this is observed for working models that are by default too simplistic to capture the true data-generating mechanism. Crossover trials and particularly TQT studies can be analyzed efficiently using simple working regression models without biasing the estimates for the causal parameters of interest.

Highly Stretchable Silicone Elastomer Applied in Soft Actuators.

In this work, a highly stretchable silicone elastomer is incorporated into dielectric elastomer actuators (DEAs) in order to decrease operation voltages by applying high prestretches. Results show that the fabricated DEAs (5 mm diameter circle active region) can be actuated to a lateral strain of 30% at 4.3 kV for a 122 µm thick prestretched film, and to a lateral strain of 2.5% at only 250 V for a 6.9 µm thick prestretched film. Due to the significant viscous component of the silicone elastomer, the DEAs respond more slowly (2-14 s to reach 90% of full strain) and show greater strain changes over time compared to conventional silicone-based DEAs. While this inherent viscosity is not universally favorable, it can be advantageous in applications where actuator damping is desirable. The studied DEAs' mean lifetimes under DC actuation range significantly-from 0.9 h to more than 123.0 h-depending mainly on initial electrical fields (17.8-36.3 V µm-1 ). For instance, DEAs with a 150 µm initial thickness and a prestretch ratio of 3 show 1.4-2.6% lateral strains for the mean lifetime (123.0 h) at only 300 V. Given the strains achieved at low voltage, such DEAs show promise for applications that do not require fast response speeds.

Sampling in the case-time-control design among drug users when outcome prevents further treatment.

PURPOSE: The objective of this article is to advocate a new way of sampling controls in the case-time-control design in a cohort of drug users when the studied outcome prevents further treatment. METHODS: Mathematically we demonstrate how a standard sampling of controls, where controls are sampled among all subjects without an event at end-of-study, leads to a biased effect estimate. We propose to add the requirement that controls initiate treatment before the calendar time of event of their matched case to circumvent this. The standard and proposed sampling methods are compared in a simulation study and in an empirical data example examining the effect of nonsteroidal anti-inflammatory drug usage on the risk of upper gastrointestinal bleeding. RESULTS: When the controls are sampled the standard way, the case-time-control design confers a bias because cases and controls have a different time-trend of exposure. The bias has been upwards in all the scenarios we have investigated. The requirement we add to be a potential control ensures that cases and controls have the same time-trend of exposure when treatment and outcome are independent. The simulation study confirms that the proposed sampling method removes the bias between treatment and outcome. The proposed sampling method lowered the odds-ratio estimate from 3.72 to 3.26 in the data example. CONCLUSION: The proposed sampling method makes it possible to use the case-time-control design in a cohort of subjects with registered use of a drug when outcome prevents further treatment.

Higher free thyroxine associated with PFAS exposure in first trimester. The Odense Child Cohort.

BACKGROUND: Perfluoroalkyl substances (PFAS) are endocrine disrupting chemicals with elimination half-lives ranging from four to eight years. Experimental studies found PFAS able to interfere with thyroid hormone-binding proteins. During the first 20 weeks of gestation (GW), the fetus is reliant on placental transfer of maternal thyroid hormones, mainly free thyroxine (FT4). However, previous studies investigating associations between exposure to PFAS and thyroid hormone status mainly focused on blood samples from late pregnancy or umbilical cord with mixed findings. OBJECTIVES: To investigate associations between serum-PFAS concentrations and thyroid hormone status in early pregnancy as reflected by FT4 and thyroid-stimulating hormone (TSH). METHODS: In the Odense Child Cohort, a single-center study, we measured maternal pregnancy serum concentrations of five PFAS: perfluorohexane sulfonic acid (PFHxS), perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA); and FT4 and TSH in 1048 pregnant women at median GW 12 (25th, 75th percentile: 10, 15). Multivariate linear regression models were performed to estimate associations between PFAS exposure and thyroid hormone status. RESULTS: A doubling in PFOS, PFOA, and PFNA concentrations was associated with an increment in FT4 concentration by 1.85% (95% CI: 0.66%, 3.05%), 1.29% (95% CI: 0.21%, 2.39%), and 1.70% (95% CI: 0.48%, 2.94%), respectively, in adjusted analyses. A statistically significant dose-response relationship was observed across exposure quartiles for PFOS, PFOA, and PFNA in the association with FT4. No association was found between concentrations of PFAS and TSH in adjusted analyses. CONCLUSION: Exposure to PFOS, PFOA, and PFNA was associated with higher FT4 concentrations in women during early pregnancy. The potential clinical implications of these findings remain to be clarified.

Evaluation of the point-of-care devices KetoSureTM and StatStrip Express® blood ketone tests using ß-hydroxybutyrate spiked samples.

Measurement of ß-hydroxybutyrate (BHB) in blood is used clinically as a measure of ketosis when diabetic ketoacidosis is expected. With the introduction of point-of-care testing (POCT) for blood-BHB, nonproductive time is reduced to a minimum in a potential critical situation; however, studies have observed inferior quality of POCT-BHB. Recently, the POCT device KetoSure (Roche) has been introduced to the clinic. In this study, we evaluated the imprecision and linearity of KetoSure and compared this to the established StatStrip Express (Nova Biomedical) based on spiked full blood samples. We found comparable imprecision for KetoSure and StatStrip Express. However, linearity was only observed in the lower part of the measuring range for both devices. In a method comparison, higher values of BHB were measured by KetoSure than by an enzymatic endpoint spectrophotometric reference method (mean bias: 21% (95% confidence interval (CI): 4%-37%)). Conversely, StatStrip Express returned lower values of BHB (mean bias: -16% (95% CI: -38%-7%), while the widely applied POCT device FreeStyle Precision Neo (Abbott) returned values equivalent with the reference method (mean bias: 5% (95% CI: -14%-24%). In samples with concentrations of BHB above the measuring range, the POCT devices could be provoked to return falsely low results. In conclusion, the quality of KetoSure is in line with other established POCT devices; however, the KetoSure measures higher concentrations than other POCT devices. As, linearity only was observed in the lower part of the measuring range and as falsely low measures could be provoked, we advise users to interpret results with precaution.

Free thyroxine in early pregnancy is an independent negative predictor of 3rd trimester HbA1c. Odense child cohort.

BACKGROUND: Lower thyroid function outside pregnancy is associated with an increased risk of type 2 diabetes mellitus. The relationship between thyroid function in early pregnancy and glucose status in 3rd trimester has not been investigated. AIMS: To study the association between 1st trimester thyroid function and 3rd trimester glucose status. DESIGN: In the prospective study Odense Child Cohort (OCC), 1,041 women had 1st trimester blood samples analysed for thyroid-stimulating hormone (TSH), free T4 (FT4), thyroid peroxidase antibody and HbA1c. Third trimester (week 28) fasting blood samples included plasma glucose, insulin and HbA1c. Oral glucose tolerance test (OGTT, 75 g glucose) was performed in 509 women. First trimester FT4 was dichotomized >vs. ≤ the 25th percentile (25p = 12.9 pmol/L). Homeostatic model assessment-insulin resistance (HOMA)-IR and HOMA-ß were calculated. RESULTS: Women with FT4 ≤25p had significantly higher HbA1c in 1st and 3rd trimesters and higher 3rd trimester fasting glucose, insulin, HOMA-IR and HOMA-ß compared to women with FT4 >25p. In multiple regression analyses, FT4 was an independent negative predictor of 3rd trimester HbA1c. FT4 levels in 3rd and 4th quartiles (high-normal FT4 levels) showed closest inverse associations with HbA1c (p-trend <.001). TSH was not associated with 3rd trimester HbA1c. CONCLUSION: Women with lower levels of FT4 in early pregnancy had higher HbA1c in 3rd trimester and FT4 was an independent negative predictor of 3rd trimester HbA1c.

Daily monitoring of viral load measured as SARS-CoV-2 antigen and RNA in blood, IL-6, CRP and complement C3d predicts outcome in patients hospitalized with COVID-19.

OBJECTIVES: We hypothesized that the amount of antigen produced in the body during a COVID-19 infection might differ between patients, and that maximum concentrations would predict the degree of both inflammation and outcome for patients. METHODS: Eighty-four hospitalized and SARS-CoV-2 PCR swab-positive patients, were followed with blood sampling every day until discharge or death. A total of 444 serial EDTA plasma samples were analyzed for a range of biomarkers: SARS-CoV-2 nuclear antigen and RNA concentration, complement activation as well as several inflammatory markers, and KL-6 as a lung marker. The patients were divided into outcome groups depending on need of respiratory support and death/survival. RESULTS: Circulating SARS-CoV-2 nuclear antigen levels were above the detection limit in blood in 65 out of 84 COVID-19 PCR swab-positive patients on day one of hospitalization, as was viral RNA in plasma in 30 out of 84. In all patients, complete antigen clearance was observed within 24 days. There were definite statistically significant differences between the groups depending on their biomarkers, showing that the concentrations of virus RNA and antigen were correlated to the inflammatory biomarker levels, respiratory treatment and death. CONCLUSIONS: Viral antigen is cleared in parallel with the virus RNA levels. The levels of antigens and SARS-CoV-2 RNA in the blood correlates with the level of IL-6, inflammation, respiratory failure and death. We propose that the antigens levels together with RNA in blood can be used to predict the severity of disease, outcome, and the clearance of the virus from the body.

A Synthetic Overview of Preparation Protocols of Nonmetallic, Contact-Active Antimicrobial Quaternary Surfaces on Polymer Substrates.

Antibacterial surfaces have been researched for more than 30 years and remain highly desirable. In particular, there is an interest in providing antimicrobial properties to commodity plastics, because these, in their native state, are excellent substrates for pathogens to adhere and proliferate on. Therefore, efficient strategies for converting surfaces of commodity plastics into contact-active antimicrobial surfaces are of significant interest. Many systems have been prepared and tested for their efficacy. Here, the synthetic approaches to such active surfaces are reviewed, with the restriction to only include systems with tested antibacterial properties. The review focuses on the synthetic approach to surface functionalization of the most common materials used and tested for biomedical applications, which effectively has limited the study to quaternary materials. For future developments in the field, it is evident that there is a need for development of simple methods that permit scalable production of active surfaces. Furthermore, in terms of efficacy, there is an outstanding concern of a lack of universal antimicrobial action as well as rapid deactivation of the antibacterial effect through surface fouling.

Biochemical and structural analyses suggest that plasminogen activators coevolved with their cognate protein substrates and inhibitors.

Protein sequences of members of the plasminogen activation system are present throughout the entire vertebrate phylum. This important and well-described proteolytic cascade is governed by numerous protease-substrate and protease-inhibitor interactions whose conservation is crucial to maintaining unchanged protein function throughout evolution. The pressure to preserve protein-protein interactions may lead to either co-conservation or covariation of binding interfaces. Here, we combined covariation analysis and structure-based prediction to analyze the binding interfaces of urokinase (uPA):plasminogen activator inhibitor-1 (PAI-1) and uPA:plasminogen complexes. We detected correlated variation between the S3-pocket-lining residues of uPA and the P3 residue of both PAI-1 and plasminogen. These residues are known to form numerous polar interactions in the human uPA:PAI-1 Michaelis complex. To test the effect of mutations that correlate with each other and have occurred during mammalian diversification on protein-protein interactions, we produced uPA, PAI-1, and plasminogen from human and zebrafish to represent mammalian and nonmammalian orthologs. Using single amino acid point substitutions in these proteins, we found that the binding interfaces of uPA:plasminogen and uPA:PAI-1 may have coevolved to maintain tight interactions. Moreover, we conclude that although the interaction areas between protease-substrate and protease-inhibitor are shared, the two interactions are mechanistically different. Compared with a protease cleaving its natural substrate, the interaction between a protease and its inhibitor is more complex and involves a more fine-tuned mechanism. Understanding the effects of evolution on specific protein interactions may help further pharmacological interventions of the plasminogen activation system and other proteolytic systems.

Probing the local lipid environment of the Rhodobacter sphaeroides cytochrome bc1 and Synechocystis sp. PCC 6803 cytochrome b6f complexes with styrene maleic acid.

Intracytoplasmic vesicles (chromatophores) in the photosynthetic bacterium Rhodobacter sphaeroides represent a minimal structural and functional unit for absorbing photons and utilising their energy for the generation of ATP. The cytochrome bc1 complex (cytbc1) is one of the four major components of the chromatophore alongside the reaction centre-light harvesting 1-PufX core complex (RC-LH1-PufX), the light-harvesting 2 complex (LH2), and ATP synthase. Although the membrane organisation of these complexes is known, their local lipid environments have not been investigated. Here we utilise poly(styrene-alt-maleic acid) (SMA) co-polymers as a tool to simultaneously determine the local lipid environments of the RC-LH1-PufX, LH2 and cytbc1 complexes. SMA has previously been reported to effectively solubilise complexes in lipid-rich membrane regions whilst leaving lipid-poor ordered protein arrays intact. Here we show that SMA solubilises cytbc1 complexes with an efficiency of nearly 70%, whereas solubilisation of RC-LH1-PufX and LH2 was only 10% and 22% respectively. This high susceptibility of cytbc1 to SMA solubilisation is consistent with this complex residing in a locally lipid-rich region. SMA solubilised cytbc1 complexes retain their native dimeric structure and co-purify with 56±6 phospholipids from the chromatophore membrane. We extended this approach to the model cyanobacterium Synechocystis sp. PCC 6803, and show that the cytochrome b6f complex (cytb6f) and Photosystem II (PSII) complexes are susceptible to SMA solubilisation, suggesting they also reside in lipid-rich environments. Thus, lipid-rich membrane regions could be a general requirement for cytbc1/cytb6f complexes, providing a favourable local solvent to promote rapid quinol/quinone binding and release at the Q0 and Qi sites.

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin.

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process.

An Asymmetric Runaway Domain Swap Antithrombin Dimer as a Key Intermediate for Polymerization Revealed by Hydrogen/Deuterium-Exchange Mass Spectrometry.

Antithrombin deficiency is associated with increased risk of venous thrombosis. In certain families, this condition is caused by pathogenic polymerization of mutated antithrombin in the blood. To facilitate future development of pharmaceuticals against antithrombin polymerization, an improved understanding of the polymerogenic intermediates is crucial. However, X-ray crystallography of these intermediates is severely hampered by the difficulty in obtaining well-diffracting crystals of transient and heterogeneous noncovalent protein assemblies. Furthermore, their large size prohibits structural analysis by NMR spectroscopy. Here, we show how hydrogen/deuterium-exchange mass spectrometry (HDX-MS) provides detailed insight into the structural dynamics of each subunit in a polymerization-competent antithrombin dimer. Upon deuteration, this dimer surprisingly yields bimodal isotope distributions for the majority of peptides, demonstrating an asymmetric configuration of the two subunits. The data reveal that one subunit is very dynamic, potentially intrinsically disordered, whereas the other is considerably less dynamic. The local subunit-specific deuterium uptake of this polymerization-competent dimer strongly supports a ß4A-ß5A ß-hairpin runaway domain swap mechanism for antithrombin polymerization. HDX-MS thus holds exceptional promise as an enabling analytical technique in the efforts toward future pharmacological intervention with protein polymerization and associated diseases.

Removal of N-Linked Glycosylations at Acidic pH by PNGase A Facilitates Hydrogen/Deuterium Exchange Mass Spectrometry Analysis of N-Linked Glycoproteins.

Protein glycosylation is the most frequent post-translational modification and is present on more than 50% of eukaryotic proteins. Glycosylation covers a wide subset of modifications involving many types of complex oligosaccharide structures, making structural analysis of glycoproteins and their glycans challenging for most analytical techniques. Hydrogen/deuterium exchange monitored by mass spectrometry is a sensitive technique for investigation of protein conformational dynamics of complex heterogeneous proteins in solution. N-linked glycoproteins however pose a challenge for HDX-MS. HDX information can typically not be obtained from regions of the glycoprotein that contain the actual N-linked glycan as glycan heterogeneity combined with pepsin digestion yields a large diversity of peptic N-glycosylated peptides that can be difficult to detect. Here, we present a novel HDX-MS workflow for analysis of the conformational dynamics of N-linked glycoproteins that utilizes the enzyme PNGase A for deglycosylation of labeled peptic N-linked glycopeptides at HDX quench conditions, i.e., acidic pH and low temperature. PNGase A-based deglycosylation is thus performed after labeling (post-HDX) and the utility of this approach is demonstrated during analysis of the monoclonal antibody Trastuzumab for which it has been shown that the native conformational dynamics is dependent on the N-linked glycan. In summary, the HDX-MS workflow with integrated PNGase A deglycosylation enables analysis of the native HDX of protein regions containing N-linked glycan sites and should thus significantly improve our ability to study the conformational properties of glycoproteins.

Single-Molecule Encapsulation: A Straightforward Route to Highly Stable and Printable Enzymes.

A mild, fast, and sequence-independent method for controlled enzyme immobilization is presented. This novel approach involves the encapsulation of single-enzyme molecules and the covalent attachment of these nanobiocatalysts onto surfaces. Fast and mild immobilization conditions, combined with low nonspecific adsorption on hydrophobic substrates, enables well-defined surface patterns via microcontact printing. The biohybrid materials show enhanced activity in organic solvents.

Antimicrobial Graft Copolymer Gels.

In view of the growing worldwide rise in microbial resistance, there is considerable interest in designing new antimicrobial copolymers. The aim of the current study was to investigate the relationship between antimicrobial activity and copolymer composition/architecture to gain a better understanding of their mechanism of action. Specifically, the antibacterial activity of several copolymers based on 2-(methacryloyloxy)ethyl phosphorylcholine [MPC] and 2-hydroxypropyl methacrylate (HPMA) toward Staphylococcus aureus was examined. Both block and graft copolymers were synthesized using either atom transfer radical polymerization or reversible addition-fragmentation chain transfer polymerization and characterized via (1)H NMR, gel permeation chromatography, rheology, and surface tensiometry. Antimicrobial activity was assessed using a range of well-known assays, including direct contact, live/dead staining, and the release of lactate dehydrogenase (LDH), while transmission electron microscopy was used to study the morphology of the bacteria before and after the addition of various copolymers. As expected, PMPC homopolymer was biocompatible but possessed no discernible antimicrobial activity. PMPC-based graft copolymers comprising PHPMA side chains (i.e. PMPC-g-PHPMA) significantly reduced both bacterial growth and viability. In contrast, a PMPC-PHPMA diblock copolymer comprising a PMPC stabilizer block and a hydrophobic core-forming PHPMA block did not exhibit any antimicrobial activity, although it did form a biocompatible worm gel. Surface tensiometry studies and LDH release assays suggest that the PMPC-g-PHPMA graft copolymer exhibits surfactant-like activity. Thus, the observed antimicrobial activity is likely to be the result of the weakly hydrophobic PHPMA chains penetrating (and hence rupturing) the bacterial membrane.

Fine Adjustment of Interfacial Potential between pH-Responsive Hydrogels and Cell-Sized Particles.

We quantitatively determined interfacial potentials between cell-sized particles and stimulus-responsive hydrogels using a microinterferometer. The hydrogel is based on physically interconnected ABA triblock copolymer micelles comprising an inner biocompatible PMPC block and two outer pH-responsive PDPA blocks. The out-of-plane temporal fluctuation in the position of the cell-sized particles was calculated from changes in the interference pattern measured by Reflection Interference Contrast Microscopy (RICM), thus yielding the particle-substrate interaction potential V (Δh). Measurements in pH buffers ranging from 7.0 to 7.8 resulted in a systematic reduction in height of the potential minima ⟨Δh⟩ and a concomitant increase in the potential curvature V″ (Δh). The experimental data were analyzed by applying the modified Ross and Pincus model for polyelectrolytes, while accounting for gravitation, lubrication and van der Waals interactions. Elastic moduli calculated from V″ (Δh) were in good agreement with those measured by Atomic Force Microscopy. The ability to fine-tune both the gel elasticity and the interfacial potential at around physiological pH makes such triblock copolymer hydrogels a promising biocompatible substrate for dynamic switching of cell-material interactions.

Disulfide-Functionalized Diblock Copolymer Worm Gels.

Two strategies for introducing disulfide groups at the outer surface of RAFT-synthesized poly(glycerol monomethacrylate)-poly(2-hydroxypropyl methacrylate) (PGMA-PHPMA, or Gx-Hy for brevity) diblock copolymer worms are investigated. The first approach involved statistical copolymerization of GMA with a small amount of disulfide dimethacrylate (DSDMA, or D) comonomer to afford a G54-D0.50 macromolecular chain transfer agent (macro-CTA); this synthesis was conducted in relatively dilute solution in order to ensure mainly intramolecular cyclization and hence the formation of linear chains. Alternatively, a new disulfide-based bifunctional RAFT agent (DSDB) was used to prepare a G45-S-S-G45 (or (G45-S)2) macro-CTA. A binary mixture of a non-functionalized G55 macro-CTA was utilized with each of these two disulfide-based macro-CTAs in turn for the RAFT aqueous dispersion polymerization of 2-hydroxypropyl methacrylate (HPMA). By targeting a PHPMA DP of 130 and systematically varying the molar ratio of the two macro-CTAs, a series of disulfide-functionalized diblock copolymer worm gels were obtained. For both formulations, oscillatory rheology studies confirmed that higher disulfide contents led to stronger gels, presumably as a result of inter-worm covalent bond formation via disulfide/thiol exchange. Using the DSDB-based macro-CTA led to the strongest worm gels, and this formulation also proved to be more effective in suppressing the thermosensitive behavior that is observed for the nondisulfide-functionalized control worm gel. However, macroscopic precipitation occurred when the proportion of DSDB-based macro-CTA was increased to 50 mol %, whereas the DSDMA-based macro-CTA could be utilized at up to 80 mol %. Finally, the worm gel modulus could be reduced to that of a nondisulfide-containing worm gel by reductive cleavage of the inter-worm disulfide bonds using excess tris(2-carboxyethyl)phosphine (TCEP) to yield thiol groups. These new biomimetic worm gels are expected to exhibit enhanced muco-adhesion.