Polyelectrolyte Coating of Ferumoxytol Differentially Impacts the Labeling of Inflammatory and Steady-State Dendritic Cell Subtypes.

Engineered magnetic nanoparticles (MNPs) are emerging as advanced tools for medical applications. The coating of MNPs using polyelectrolytes (PEs) is a versatile means to tailor MNP properties and is used to optimize MNP functionality. Dendritic cells (DCs) are critical regulators of adaptive immune responses. Functionally distinct DC subsets exist, either under steady-state or inflammatory conditions, which are explored for the specific treatment of various diseases, such as cancer, autoimmunity, and transplant rejection. Here, the impact of the PE coating of ferumoxytol for uptake into both inflammatory and steady-state DCs and the cellular responses to MNP labeling is addressed. Labeling efficiency by uncoated and PE-coated ferumoxytol is highly variable in different DC subsets, and PE coating significantly improves the labeling of steady-state DCs. Uncoated ferumoxytol results in increased cytotoxicity of steady-state DCs after labeling, which is abolished by the PE coating, while no increased cell death is observed in inflammatory DCs. Furthermore, uncoated and PE-coated ferumoxytol appear immunologically inert in inflammatory DCs, but they induce activation of steady-state DCs. These results show that the PE coating of MNPs can be applied to endow particles with desired properties for enhanced uptake and cell type-specific responses in distinct target DC populations.

Singapore's health-care system: key features, challenges, and shifts.

Since Singapore became an independent nation in 1965, the development of its health-care system has been underpinned by an emphasis on personal responsibility for health, and active government intervention to ensure access and affordability through targeted subsidies and to reduce unnecessary costs. Singapore is achieving good health outcomes, with a total health expenditure of 4·47% of gross domestic product in 2016. However, the health-care system is contending with increased stress, as reflected in so-called pain points that have led to public concern, including shortages in acute hospital beds and intermediate and long-term care (ILTC) services, and high out-of-pocket payments. The main drivers of these challenges are the rising prevalence of non-communicable diseases and rapid population ageing, limitations in the delivery and organisation of primary care and ILTC, and financial incentives that might inadvertently impede care integration. To address these challenges, Singapore's Ministry of Health implemented a comprehensive set of reforms in 2012 under its Healthcare 2020 Masterplan. These reforms substantially increased the capacity of public hospital beds and ILTC services in the community, expanded subsidies for primary care and long-term care, and introduced a series of financing health-care reforms to strengthen financial protection and coverage. However, it became clear that these measures alone would not address the underlying drivers of system stress in the long term. Instead, the system requires, and is making, much more fundamental changes to its approach. In 2016, the Ministry of Health encapsulated the required shifts in terms of the so-called Three Beyonds-namely, beyond health care to health, beyond hospital to community, and beyond quality to value.

Design and Multicenter Clinical Validation of a 3-Dimensionally Printed Nasopharyngeal Swab for SARS-CoV-2 Testing.

Importance: Three-dimensionally printed nasopharyngeal swabs (3DP swabs) have been used to mitigate swab shortages during the coronavirus disease 2019 (COVID-19) pandemic. Clinical validation for diagnostic accuracy and consistency, as well as patient acceptability, is crucial to evaluate the swab's performance. Objective: To determine the accuracy and acceptability of the 3DP swab for identifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Design, Setting, and Participants: A diagnostic study was conducted from May to July 2020 at 2 tertiary care centers in Singapore with different reference swabs (FLOQSwab [COPAN Diagnostics] or Dacron swab [Deltalab]) and swab processing techniques (wet or dry) to evaluate the performance of the 3DP swab compared with traditional, standard-of-care nasopharyngeal swabs used in health care institutions. The participants were patients with COVID-19 in the first 2 weeks of illness and controls with acute respiratory illness with negative test results for SARS-CoV-2. Paired nasopharyngeal swabs were obtained from the same nostril and tested for SARS-CoV-2 by reverse-transcriptase polymerase chain reaction. The sequence of swabs was randomized based on odd and even participant numbers. Main Outcomes and Measures: Primary outcome measures were overall agreement (OA), positive percentage agreement (PPA), and negative percentage agreement of the 3DP swab compared with reference swabs. Secondary outcome measures were the correlation of cycle threshold (Ct) values of both swabs. Results: The mean (SD) age of participants was 45.4 (13.1) years, and most participants were men (87 of 89 [97.8%]), in keeping with the epidemiology of the COVID-19 pandemic in Singapore. A total of 79 patients with COVID-19 and 10 controls were recruited. Among the patients with COVID-19, the overall agreement and PPA of the 3DP swab was 91.1% and 93.5%, respectively, compared with reference swabs. The PPA was 100% for patients with COVID-19 who were tested within the first week of illness. All controls tested negative. The reverse-transcriptase polymerase chain reaction Ct values for the ORF1ab and E-gene targets showed a strong correlation (intraclass correlations coefficient, 0.869-0.920) between the 3DP and reference swab on independent testing at each institution despite differences in sample processing. Discordant results for both gene targets were observed only at high Ct values. Conclusions and Relevance: In this diagnostic study of 79 patients with COVID-19 and 10 controls, the 3DP swab performed accurately and consistently across health care institutions and could help mitigate strained resources in the escalating COVID-19 pandemic.

Clinical Diagnostic Study of a Novel Injection Molded Swab for SARS-Cov-2 Testing.

INTRODUCTION: The gold standard for COVID-19 diagnosis is currently a real-time reverse transcriptase polymerase chain reaction (RT-PCR) to detect SARS-CoV-2. This is most commonly performed on respiratory secretions obtained via a nasopharyngeal swab. Due to supply chain limitations and high demand worldwide because of the COVID-19 pandemic, access to commercial nasopharyngeal swabs has not been assured. 3D printing methods have been used to meet the shortfall. For longer-term considerations, 3D printing may not compare well with injection molding as a production method due to the challenging scalability and greater production costs of 3D printing. METHODS: To secure sufficient nasopharyngeal swab availability for our national healthcare system, we designed a novel injection molded nasopharyngeal swab (the IM2 swab). We performed a clinical diagnostic study comparing the IM2 swab to the Copan FLOQSwab. Forty patients with a known diagnosis of COVID-19 and 10 healthy controls were recruited. Paired nasopharyngeal swabs were obtained from the same nostril of each participant and tested for SARS-CoV-2 by RT-PCR. RESULTS: When compared to the Copan FLOQswab, results from the IM2 swab displayed excellent overall agreement and positive percent agreement of 96.0% and 94.9%, respectively. There was no significant difference in mean RT-PCR cycle threshold values for the ORF1ab (28.05 vs. 28.03, p = 0.97) and E-gene (29.72 vs. 29.37, p = 0.64) targets, respectively. We did not observe any significant adverse events and there was no significant difference in patient-reported pain. CONCLUSION: In summary, the IM2 nasopharyngeal swab is a clinically safe, highly accurate option to commercial nasopharyngeal swabs.

Electrostatic expansion of polyelectrolyte microgels: Effect of solvent quality and added salt.

We report dynamic light scattering data for a PNIPAM based microgel copolymerised with 2% ionic groups. The hydrodynamic radius is measured as a function of temperature (which varies the solvent quality) and added salt concentration (cS) from salt-free conditions up to cS = 0.1 M. Incorporation of the ionic co-monomer leads to an increase of the volume phase transition temperature with respect to a non-ionic microgel from T≃306 K to T≃317 K in salt-free water. The hydrodynamic radius varies as RH∝cS-0.05 in good solvent (T≃278-298 K) and RH∝cS-0.15 in poor solvent (T≃313-319 K). Deep into the poor solvent regime the microgel is collapsed for all salt concentrations and RH∝cS0. Data are fitted to the Flory-Rehner model modified to account for the osmotic pressure of the counterions through the Donnan model. We find that the Flory-Rehner-Donnan (FRD) model does not fully account for the experimental observations, particularly at low ionic strengths, possibly due to the influence of electrostatic excluded volume. The disagreements between theory and experiments are greatly reduced if a cross-linking density three times lower than that expected from synthesis is assumed. The scaling theory exponents for the variation of gel size with temperature and added salt are not in agreement with the experimental results presented.

Global implementation of genomic medicine: We are not alone.

Around the world, innovative genomic-medicine programs capitalize on singular capabilities arising from local health care systems, cultural or political milieus, and unusual selected risk alleles or disease burdens. Such individual efforts might benefit from the sharing of approaches and lessons learned in other locales. The U.S. National Human Genome Research Institute and the National Academy of Medicine recently brought together 25 of these groups to compare projects, to examine the current state of implementation and desired near-term capabilities, and to identify opportunities for collaboration that promote the responsible practice of genomic medicine. Efforts to coalesce these groups around concrete but compelling signature projects should accelerate the responsible implementation of genomic medicine in efforts to improve clinical care worldwide.

Temperature-modulated water filtration using microgel-functionalized hollow-fiber membranes.

In the present work, we investigate the potential of aqueous polymer microgels in membrane technology, especially for filtration applications. The poly(N-vinylcaprolactam)-based microgels exhibit thermoresponsive behavior and were employed to coat hollow-fiber membranes used for micro- and ultrafiltration. We discuss the preparation of microgel-modified membranes (by "inside-out" as well as "outside-in" filtration in dead-end mode). The clean-water permeability and stability of these membranes was studied not only as a function of time, but also of temperature. The microgel-modified membranes exhibit a reversible thermoresponsive behavior whereby both the resistance and the retention increased with decreasing temperature.

Catalytic polyelectrolyte multilayers at the bipolar membrane interface.

Bipolar membranes are laminated anion and cation exchange membranes that split water at their interface very efficiently upon application of an electric field. This paper investigates the layer-by-layer (LbL) deposition of polyelectrolyte multilayers, as a tool to introduce molecularly thin catalyst groups at this interface of bipolar membranes. The bipolar membranes were prepared by first modifying an anion exchange membrane by consecutive dipping LbL assembly, then casting a thin highly charged intermediate layer followed by casting a cation exchange layer. The results reveal that polyelectrolytes of higher charge density coated on the anion exchange layer yield better performance. Several parameters of the LbL interface deposition were varied. Out of the investigated LbL assembly parameters, ionic strength and number of layers have shown the largest influence on catalytic activity as well as ionic selectivity. The membrane with two bilayers of poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS) and poly(ethyleneimine) (PEI), where the PEI was prepared in 0.5 M NaCl, gave rise to the best performance. Surprisingly, detailed data analysis at low electrical potential suggests that the interface layers of a bipolar membrane play a major role in its permselectivity. Previously, only the bulk thickness of the anion and cation exchange membrane was assumed to influence the bipolar membrane selectivity.

Magnetically triggered clustering of biotinylated iron oxide nanoparticles in the presence of streptavidinylated enzymes.

This work deals with the production and characterization of water-compatible, iron oxide based nanoparticles covered with functional poly(ethylene glycol) (PEG)-biotin surface groups (SPIO-PEG-biotin). Synthesis of the functionalized colloids occurred by incubating the oleate coated particles used as precursor magnetic fluid with anionic liposomes containing 14 mol% of a phospholipid-PEG-biotin conjugate. The latter was prepared by coupling dimyristoylphosphatidylethanolamine (DC(14:0)PE) to activated α-biotinylamido-ω -N-hydroxy-succinimidcarbonyl-PEG (NHS-PEG-biotin). Physical characterization of the oleate and PEG-biotin iron oxide nanocolloids revealed that they appear as colloidal stable clusters with a hydrodynamic diameter of 160 nm and zeta potentials of - 39 mV (oleate coated particles) and - 14 mV (PEG-biotin covered particles), respectively, as measured by light scattering techniques. Superconducting quantum interference device (SQUID) measurements revealed specific saturation magnetizations of 62-73 emu g(-1) Fe(3)O(4) and no hysteresis was observed at 300 K. MR relaxometry at 3 T revealed very high r(2) relaxivities and moderately high r(1) values. Thus, both nanocolloids can be classified as small, superparamagnetic, negative MR contrast agents. The capacity to functionalize the particles was illustrated by binding streptavidin alkaline phosphatase (SAP). It was found, however, that these complexes become highly aggregated after capturing them on the magnetic filter device during high-gradient magnetophoresis, thereby reducing the accessibility of the SAP.

Fluorescent magnetoliposomes as a platform technology for functional and molecular MR and optical imaging.

Here, we present a detailed characterisation of rhodamine B-containing magnetoliposomes (FLU-ML), emphasising the dependence of their fluorescence properties on the presence of iron oxide cores, and the molar fraction of the fluorophore. The magnetoliposome types used exist as colloidally stable, negatively charged clusters with an average hydrodynamic diameter of 95 nm. The molar rhodamine B fractions were 0.67 % and 1.97 %. Rhodamine B normalised fluorescence, quantum yields and fluorescence lifetimes were substantially reduced by inner filter effects as the magnetoliposome concentration is increased, by increasing molar rhodamine B fraction, and by quenching originating from the iron oxide cores. MR relaxometry at 3 T revealed extremely high r2 relaxivities (440 to 554 s-1mM-1) and moderately high r1 values (2.06 to 3.59 s-1mM-1). Upon incubating human prostate carcinoma (PC-3) cells with FLU-ML, a dose-dependent particle internalisation was found by MR relaxometry. In addition, the internalised FLU-ML were clearly visible by fluorescence microscopy. At the FLU-ML concentrations used (up to 3 × 10³ M Fe) cell viability was not substantially impaired. These results provide valuable insights on the fluorescence properties of bimodal magnetoliposomes and open promising perspectives for the use of these materials as a platform technology for advanced functional and molecular MR and optical imaging applications.

Polyelectrolyte coating of iron oxide nanoparticles for MRI-based cell tracking.

Iron oxide-based magnetic nanoparticles (MNPs) offer unique properties for cell tracking by magnetic resonance imaging (MRI) in cellular immunotherapy. In this study, we investigated the uptake of chemically engineered NPs into antigen-presenting dendritic cells (DCs). DCs are expected to perceive MNPs as foreign antigens, thus exhibiting the capability to immunologically sense MNP surface chemistry. To systematically evaluate cellular uptake and T2/T2(⁎) MR imaging properties of MNPs, we synthesized polymer-based MNPs by employing layer-by-layer (LbL) technology. Thereby, we achieved modification of particle shell parameters, such as size, surface charge, and chemistry. We found that subcellular packaging of MNPs rather than MNP content in DCs influences MR imaging quality. Increased local intracellular electron density as inferred from transmission electron microscopy (TEM) strongly correlated with enhanced contrast in MRI. Thus, LbL-tailoring of MNP shells using polyelectrolytes that impact on uptake and subcellular localization can be used for modulating MR imaging properties. FROM THE CLINICAL EDITOR: In this study, layer-by-layer tailoring of magnetic NP shells was performed using polyelectrolytes to improve uptake by dendritic cells for cell-specific MR imaging. The authors conclude that polyelectrolyte modified NP-s can be used for modulating improving MR imaging quality by increasing subcellular localization.

Breast cancer in a multi-ethnic Asian setting: results from the Singapore-Malaysia hospital-based breast cancer registry.

Two hospital-based breast cancer databases (University Malaya Medical Center, Malaysia [n = 1513] and National University Hospital, Singapore [n = 2545]) were merged into a regional registry of breast cancer patients diagnosed between 1990 and 2007. A review of the data found 51% of patients diagnosed before the age of 50 years. and 72% percent of the women were Chinese followed by Malays (16%), Indians (8%), and other races (4%). Median tumor size at presentation was 26 mm and about 25% of patients presented with TNM stage III or IV disease. Most tumors were of ductal histology (87%). Fifty-seven percent of tumors were estrogen receptor positive and 40% were poorly differentiated. Of those patients who had surgery, 70% had mastectomy while 30% had breast conserving surgery. Overall, chemotherapy was administered to 56% of patients and hormonal treatment to 60%. Five-year overall survival was 82.5% in patients with TNM stage 0 to stage II cancer, and 30.2% in those with later stages.

Effect of layer-by-layer confinement of polypeptides and polysaccharides onto thermoresponsive microgels: a comparative study.

The confinement of polyelectrolyte multilayers of poly-L-lysine (PLL)/poly-L-glutamic acid (PGA) and chitosan (CHIT)/dextran sulfate (DEX) onto soft and porous thermoresponsive poly(N-isopropylacrylamide-co-methacrylic acid) (P(NiPAM-co-MAA)) microgel was studied by dynamic light scattering (DLS) and electrophoretic measurements. DLS demonstrates an "odd-even" effect in the hydrodynamic radius depending on the type of polyelectrolyte in the outermost layer and that coated microgels retain their native thermoresponsive property. Strong hysteresis is found between the swelling and deswelling processes of microgels coated with polypeptides, whilst for those coated with polysaccharides are nearly reversible. Electrophoretic mobility results indicate charge reversal after each layer deposition, which magnitude decreases with increasing number of layers. Microgels coated with polysaccharides exhibit higher mobility values. Stability studies reveal spatial and temporal reorganization of the polymer chains over several weeks. Upon increasing time, the hydrodynamic radius of polypeptide-terminated microgels decreases, whereas that of polysaccharide-terminated increases to almost the original size of the uncoated microgel. Both systems exhibit an exponential growth of the bilayer thickness with the number of deposition steps, more pronounced for microgels coated with polypeptides. Our results demonstrate the feasibility of the layer-by-layer assembly of these biopolymers onto microgels, which could have potential application for storage and release of biomolecules.

Study of layer-by-layer films on thermoresponsive nanogels using temperature-controlled dual-focus fluorescence correlation spectroscopy.

While a few studies have reported on the layer-by-layer (LbL) assembly of polyelectrolytes on soft and porous templates, none have really demonstrated direct proof that the layers are actually on the template. Thermoresponsive nanogels present challenges that render a quantitative proof of successful polyelectrolyte deposition extremely difficult. Additionally, the fate of the polyelectrolyte has never been investigated during the phase transition of the coated nanogel. Here, the auto- and cross-correlation functions of a labeled polyelectrolyte assembled via the LbL technique onto soft and porous thermoresponsive labeled nanogels using dual-focus fluorescence correlation spectroscopy (2f-FCS) are presented. Performing 2f-FCS as a function of temperature, hydrodynamic radii of nanogels coated with various numbers of layers are determined, which are found to be in excellent agreement with values obtained from dynamic light scattering. This study presents irrefutable quantitative evidence of successful LbL assembly on thermoresponsive nanogels and demonstrates that the layers are not stripped off during the phase transition of the nanogels. Forster Resonance Energy Transfer (FRET) detection also supports our findings.