Controlled swelling of biomaterial devices for improved antifouling polymer coatings.

Nonspecific interactions between cells and implantable elastomers often leads to failure modes for devices such as catheters, cosmetic and reconstructive implants, and sensors. To reduce these interactions, device surfaces can be coated with hydrophilic polymers, where greater polymer density enhances antifouling properties. Although graft-from coating techniques result in higher density polymer films and lower fouling in controlled settings, simpler graft-to methods show similar results on complex implanted devices, despite limited density. To address the need for improved graft-to methods, we developed Graft then shrink (GtS) where elastomeric materials are temporarily swollen during polymer grafting. Herein, we demonstrate a graft-to based method for poly(oligo(ethylene glycol) methyl ether methacrylate) (pOEGMA) on swollen silicone, GtS, that enhances grafted polymer content and fouling resistance. Total grafted polymer content of pOEGMA on toluene swollen silicone increased over ~ 13 × compared to non-swollen controls, dependent on the degree of silicone swelling. Increases in total grafted polymer within the top 200 µm of the material led to bacterial and mammalian cell adhesion reductions of 75% and 91% respectively, compared to Shrink then Graft (StG) antifouling polymer coated controls. GtS allows for the simple 3D coating of swellable elastomers (e.g., silicone medical devices) with improved antifouling pOEGMA coatings.

Graft-Then-Shrink: Simultaneous Generation of Antifouling Polymeric Interfaces and Localized Surface Plasmon Resonance Biosensors.

Antifouling polymer coatings that are simple to manufacture are crucial for the performance of medical devices such as biosensors. "Grafting-to", a simple technique where presynthesized polymers are immobilized onto surfaces, is commonly employed but suffers from nonideal polymer packing leading to increased biofouling. Herein, we present a material prepared via the grafting-to method with improved antifouling surface properties and intrinsic localized surface plasmon resonance (LSPR) sensor capabilities. A new substrate shrinking fabrication method, Graft-then-Shrink, improved the antifouling properties of polymer-coated Au surfaces by altering graft-to polymer packing while simultaneously generating wrinkled Au structures for LSPR biosensing. Thiol-terminated, antifouling, hydrophilic polymers were grafted to Au-coated prestressed polystyrene (PS) followed by shrinking upon heating above the PS glass transition temperature. Interestingly, the polymer molecular weight and hydration influenced Au wrinkling patterns. Compared to Shrink-then-Graft controls, where polymers are immobilized post shrinking, Graft-then-Shrink increased the polymer content by 76% in defined footprints and improved the antifouling properties as demonstrated by 84 and 72% reduction in macrophage adhesion and protein adsorption, respectively. Wrinkled Au LSPR sensors had sensitivities of ∼200-1000 Δλ/ΔRIU, comparing favorably to commercial LSPR sensors, and detected biotin-avidin and desthiobiotin-avidin complexation in a concentration-dependent manner using a standard plate reader and a 96-well format.

Soil invertebrate toxicity and bioaccumulation of nano copper oxide and copper sulphate in soils, with and without biosolids amendment.

The fate, toxicity and bioaccumulation of copper oxide nanoparticles (nCuO) was investigated in soil, with and without biosolids amendment, through chronic exposures using the earthworm, Eisenia andrei, and the collembolan, Folsomia candida. The effects of copper sulphate (CuSO4) were included so as to compare the behavior of nCuO to a readily soluble counterpart. The fate of nCuO was evaluated through characterization of dissolved and nano-particulate fractions (via single particle ICP-MS) as well as extractable Cu2+ throughout the duration of select tests. Neither Cu form was particularly toxic to F. candida, but effects on E. andrei reproduction were significant in all treatments (IC50 range: 98 - 149 mg Cu kg-1 dry soil). There were no significant differences in toxicity between the Cu forms, nor in extractable Cu2+ activities, indicative that particle dissolution within the soil and, subsequent activity of Cu2+ was likely the primary mode of toxicity in the nCuO exposures. The presence of biosolids did not significantly alter toxicity of nCuO, but did affect Cu2+ activity over time. Bioaccumulation of total Cu in E. andrei when exposed to nCuO (kinetic bioaccumulation factor (BAFk): 0.80 with biosolids and 0.81 without) was lower than exposure to CuSO4 (BAFk: 2.31 with biosolids and 1.12 without). Enhanced dark-field hyperspectral imaging showed accumulation of nCuO along the epidermis and gut of E. andrei, with trace amounts observed in muscle and chloragogenous tissue, providing evidence of nCuO translocation within the organism. The present study demonstrates that the current risk assessment approach for trace metals in the environment, based on substance solubility and bioavailability of the dissolved free ion, are applicable for nCuO exposure to soil invertebrates, but that the rate of particle dissolution in different soil environments is an important factor for consideration.

Controlling Experimental Parameters to Improve Characterization of Biomaterial Fouling.

Uncontrolled protein adsorption and cell binding to biomaterial surfaces may lead to degradation, implant failure, infection, and deleterious inflammatory and immune responses. The accurate characterization of biofouling is therefore crucial for the optimization of biomaterials and devices that interface with complex biological environments composed of macromolecules, fluids, and cells. Currently, a diverse array of experimental conditions and characterization techniques are utilized, making it difficult to compare reported fouling values between similar or different biomaterials. This review aims to help scientists and engineers appreciate current limitations and conduct fouling experiments to facilitate the comparison of reported values and expedite the development of low-fouling materials. Recent advancements in the understanding of protein-interface interactions and fouling variability due to experiment conditions will be highlighted to discuss protein adsorption and cell adhesion and activation on biomaterial surfaces.

Fabrication of low-fouling, high-loading polymeric surfaces through pH-controlled RAFT.

Low-fouling and high-loading surfaces are increasingly important for biosensing and blood purification technologies. Selective and efficient target binding from complex media can be achieved with poly(carboxybetaine) (pCB) surfaces that consist of a dense brush layer to resist non-specific protein adsorption and a sparse "mushroom" upper layer for high-density capture agent immobilization (i.e. high-loading). We developed pH-controlled surface-reversible addition-fragmentation chain-transfer (S-RAFT) polymerization to simplify fabrication of multi-modal, low-fouling and high-loading pCB surfaces without the need for quenching or re-initiation steps, toxic transition metals or light irradiation. Multi-modal polymer layers were produced through partial polymer termination by temporarily raising the pH to aminolyse a fraction of dormant chain transfer agents (CTAs); remaining polymer chains with intact CTAs continued uninterrupted extension to create the "mushroom" upper layer. The multi-modal pCB surfaces were low-fouling towards proteins (<6.7 ng cm-2), and macrophages. Compared to mono-modal brush surfaces, multi-modal pCB surfaces were high-loading with 5-fold greater capture agent immobilization (e.g. antibody) and 4-fold greater target binding (e.g. biotin-fluorescein).

Improved Efficacy of Antibody Cancer Immunotherapeutics through Local and Sustained Delivery.

Antibodies are a growing class of cancer immunotherapeutics that facilitate immune-cell-mediated killing of tumors. However, the efficacy and safety of immunotherapeutics are limited by transport barriers and poor tumor uptake, which lead to high systemic concentrations and potentially fatal side effects. To increase tumor antibody immunotherapeutic concentrations while decreasing systemic concentrations, local delivery vehicles for sustained antibody release are being developed. The focus of this review is to define the material properties required for implantable controlled antibody delivery and highlight the controlled-release strategies that are applicable to antibody immunotherapeutics.

Controlled degradation of low-fouling poly(oligo(ethylene glycol)methyl ether methacrylate) hydrogels.

Degradable low-fouling hydrogels are ideal vehicles for drug and cell delivery. For each application, hydrogel degradation rate must be re-optimized for maximum therapeutic benefit. We developed a method to rapidly and predictably tune degradation rates of low-fouling poly(oligo(ethylene glycol)methyl ether methacrylate) (P(EG) x MA) hydrogels by modifying two interdependent variables: (1) base-catalysed crosslink degradation kinetics, dependent on crosslinker electronics (electron withdrawing groups (EWGs)); and, (2) polymer hydration, dependent on the molecular weight (M W) of poly(ethylene glycol) (PEG) pendant groups. By controlling PEG M W and EWG strength, P(EG) x MA hydrogels were tuned to degrade over 6 to 52 d. A 6-member P(EG) x MA copolymer library yielded slow and fast degrading low-fouling hydrogels suitable for short- and long-term delivery applications. The degradation mechanism was also applied to RGD-functionalized poly(carboxybetaine methacrylamide) (PCBMAA) hydrogels to achieve slow (∼50 d) and fast (∼13 d) degrading low-fouling, bioactive hydrogels.

Influence of Hydrophobic Cross-Linkers on Carboxybetaine Copolymer Stimuli Response and Hydrogel Biological Properties.

Poly(carboxybetaine) (pCB) hydrogels do not elicit a foreign body response due to their low-fouling properties, making them ideal implantable materials for in vivo drug and cell delivery. Current reported pCB hydrogels are cross-linked using cytotoxic UV-initiated radical polymerization limiting clinical and in vivo translation. For clinical translation, we require in situ and biorthogonal cross-linking of pCB hydrogels that are both low-fouling and low-swelling to limit nonspecific interactions and minimize tissue damage, respectively. To this end, we synthesized carboxybetaine (CB) random copolymers (molecular weight (MW): ∼7-33 kDa; D: 1.1-1.36) containing azide (pCB-azide) or strained alkyne (Dibenzocyclooctyne (DBCO); pCB-DBCO) that rapidly cross-link upon mixing. Unlike CB homopolymers and other CB copolymers studied, high DBCO content pCB-DBCO30 (30% DBCO mole fraction) is thermoresponsive with a upper critical solution temperature (UCST; cloud point of ∼20 °C at 50 g/L) in water due to electrostatic associations. Due to the antipolyelectrolyte effect, pCB-DBCO30 is salt-responsive and is soluble even at low temperatures in 5 M NaCl, which prevents zwitterion electrostatic associations. pCB-azide and pCB-DBCO with 0.05 to 0.16 cross-linker mole fractions rapidly formed 10 wt % hydrogels upon mixing that were low-swelling (increase of ∼10% in wet weight) while remaining low-fouling to proteins (∼10-20 µg cm-2) and cells, making them suitable for in vivo applications. pCB-X31 hydrogels composed of pCB-azide32 and pCB-DBCO30 formed opaque gels in water and physiological conditions that shrunk to ∼70% of their original wet weight due to pCB-DBCO30's greater hydrophobicity and interchain electrostatic interactions, which promotes nonspecific protein adsorption (∼35 µg cm-2) and cell binding. Once formed, the electrostatic interactions in pCB-X31 hydrogels are not fully reversible with heat or salt. Although, pCB-X31 hydrogels are transparent when initially prepared in 5 M NaCl. This is the first demonstration of a thermo- and salt-responsive CB copolymer that can tune hydrogel protein and cell fouling properties.

The toxicity of silver to soil organisms exposed to silver nanoparticles and silver nitrate in biosolids-amended field soil.

The use of engineered silver nanoparticles (AgNPs) is widespread, with expected release to the terrestrial environment through the application of biosolids onto agricultural lands. The toxicity of AgNPs and silver nitrate (AgNO3 ; as ionic Ag+ ) to plant (Elymus lanceolatus and Trifolium pratense) and soil invertebrate (Eisenia andrei and Folsomia candida) species was assessed using Ag-amended biosolids applied to a natural sandy loam soil. Bioavailable Ag+ in soil samples was estimated using an ion-exchange technique applied to KNO3 soil extracts, whereas exposure to dispersible AgNPs was verified by single-particle inductively coupled plasma-mass spectrometry and transmission electron microscopy-energy dispersive X-ray spectroscopy analysis. Greater toxicity to plant growth and earthworm reproduction was observed in AgNP exposures relative to those of AgNO3 , whereas no difference in toxicity was observed for F. candida reproduction. Transformation products in the AgNP-biosolids exposures resulted in larger pools of extractable Ag+ than those from AgNO3 -biosolids exposures, at similar total Ag soil concentrations. The results of the present study reveal intrinsic differences in the behavior and bioavailability of the 2 different forms of Ag within the biosolids-soils pathway. The present study demonstrates how analytical methods that target biologically relevant fractions can be used to advance the understanding of AgNP behavior and toxicity in terrestrial environments. Environ Toxicol Chem 2017;36:2756-2765. © 2017 Crown in the Right of Canada. Published Wiley Periodicals Inc., on behalf of SETAC.

Extracting Metallic Nanoparticles from Soils for Quantitative Analysis: Method Development Using Engineered Silver Nanoparticles and SP-ICP-MS.

The lack of an efficient and standardized method to disperse soil particles and quantitatively subsample the nanoparticulate fraction for characterization analyses is hindering progress in assessing the fate and toxicity of metallic engineered nanomaterials in the soil environment. This study investigates various soil extraction and extract preparation techniques for their ability to remove nanoparticulate Ag from a field soil amended with biosolids contaminated with engineered silver nanoparticles (AgNPs), while presenting a suitable suspension for quantitative single-particle inductively coupled plasma mass spectroscopy (SP-ICP-MS) analysis. Extraction parameters investigated included reagent type (water, NaNO3, KNO3, tetrasodium pyrophosphate (TSPP), tetramethylammonium hydroxide (TMAH)), soil-to-reagent ratio, homogenization techniques as well as procedures commonly used to separate nanoparticles from larger colloids prior to analysis (filtration, centrifugation, and sedimentation). We assessed the efficacy of the extraction procedure by testing for the occurrence of potential procedural artifacts (dissolution, agglomeration) using a dissolved/particulate Ag mass ratio and by monitoring the amount of Ag mass in discrete particles. The optimal method employed 2.5 mM TSPP used in a 1:100 (m/v) soil-to-reagent ratio, with ultrasonication to enhance particle dispersion and sedimentation to settle out the micrometer-sized particles. A spiked-sample recovery analysis shows that 96% ± 2% of the total Ag mass added as engineered AgNP is recovered, which includes the recovery of 84.1% of the particles added, while particle recovery in a spiked method blank is ∼100%, indicating that both the extraction and settling procedure have a minimal effect on driving transformation processes. A soil dilution experiment showed that the method extracted a consistent proportion of nanoparticulate Ag (9.2% ± 1.4% of the total Ag) in samples containing 100%, 50%, 25%, and 10% portions of the AgNP-contaminated test soil. The nanoparticulate Ag extracted by this method represents the upper limit of the potentially dispersible nanoparticulate fraction, thus providing a benchmark with which to make quantitative comparisons, while presenting a suspension suitable for a myriad of other characterization analyses.

Single Particle-Inductively Coupled Plasma Mass Spectroscopy Analysis of Metallic Nanoparticles in Environmental Samples with Large Dissolved Analyte Fractions.

There is an increasing interest to use single particle-inductively coupled plasma mass spectroscopy (SP-ICPMS) to help quantify exposure to engineered nanoparticles, and their transformation products, released into the environment. Hindering the use of this analytical technique for environmental samples is the presence of high levels of dissolved analyte which impedes resolution of the particle signal from the dissolved. While sample dilution is often necessary to achieve the low analyte concentrations necessary for SP-ICPMS analysis, and to reduce the occurrence of matrix effects on the analyte signal, it is used here to also reduce the dissolved signal relative to the particulate, while maintaining a matrix chemistry that promotes particle stability. We propose a simple, systematic dilution series approach where by the first dilution is used to quantify the dissolved analyte, the second is used to optimize the particle signal, and the third is used as an analytical quality control. Using simple suspensions of well characterized Au and Ag nanoparticles spiked with the dissolved analyte form, as well as suspensions of complex environmental media (i.e., extracts from soils previously contaminated with engineered silver nanoparticles), we show how this dilution series technique improves resolution of the particle signal which in turn improves the accuracy of particle counts, quantification of particulate mass and determination of particle size. The technique proposed here is meant to offer a systematic and reproducible approach to the SP-ICPMS analysis of environmental samples and improve the quality and consistency of data generated from this relatively new analytical tool.