Brightfield and fluorescence in-channel staining of thin blood smears generated in a pumpless microfluidic.

Effective staining of peripheral blood smears by increasing contrast of intracellular components and biomarkers is essential for the accurate characterization, diagnosis, and monitoring of various diseases such as malaria. To assess the potential for automation of stained whole human blood smears at the point-of-care (POC), brightfield and fluorescence staining protocols were adapted for smears generated in channels of pumpless microchannels and compared to a standard glass smear. A 3× concentration Giemsa brightfield staining solutions (10, 33, and 50% dilution), and Acridine Orange fluorescence staining solutions (12 µg mL-1) were evaluated with human blood smears containing malaria parasites within a microfluidic channel. Giemsa staining at 33% dilution showed an optimal combination of contrast and preservation of cellular morphology, while 50% dilutions showed significant cellular crenation and 10% dilutions did not show desired contrast in brightfield imaging. Fluorescence staining at 12 µg mL-1 using Acridine Orange showed clear separability between the fluorescent intensities of the malaria parasites and that of the red blood cells (RBCs) and background. However, compared to glass smears, these exhibited reduced signal intensity as well as inverted contrast of RBCs and background. These results demonstrate that peripheral thin blood smears generated in pumpless microfluidic can be successfully stained in-channel with a simple, one-step procedure to permit brightfield and fluorescence imaging.

Amphiphilic, thixotropic additives for extrusion-based 3D printing of silica-reinforced silicone.

The ability to utilize extrusion-based, direct ink write (DIW) 3D printing to create silica-reinforced silicones with complex structures could expand their utility in industrial and biomedical applications. Sylgard 184, a common Pt-cure silicone, lacks the thixotropic behavior necessary for effective printing and its hydrophobicity renders cured structures susceptible to biofouling. Herein, we evaluated the efficacy of various PEO-silane amphiphiles (PEO-SAs) as thixotropic and surface modifying additives in Sylgard 184. Eight amphiphilic PEO-SAs of varying architecture (e.g. linear, star, and graft), crosslinkability, and PEO content were evaluated. Modified formulations were also prepared with additional amounts of silica filler, both hexamethyldisilazane (HMDS)-treated and dimethyldichlorosilane (DiMeDi)-treated types. Numerous PEO-SA modified silicone formulations demonstrated effective water-driven surface hydrophilicity that was generally diminished with the addition of HMDS-treated silica filler. While increased yield stress was observed for PEO-SA modified silicones with added HMDS-treated filler, none achieved the initial target for 3D printing (>1000 Pa). Only the formulations containing the DiMeDi-treated filler (17.3 wt%) were able to surpass this value. These formulations were then tested for their thixotropic properties and all surpassed the targets for recovered storage modulus (G') (>1000 Pa) and loss factor (<0.8). In particular, the triblock linear PEO-SA produced exceptionally high recovered G', low loss factor, and substantial water-driven restructuring to form a hydrophilic surface. Combined, these results demonstrate the potential of silicones modified with PEO-SA surface-modifying additives (SMAs) for extrusion-based, DIW 3D printing applications.

Amphiphilic silicones to reduce the absorption of small hydrophobic molecules.

Silicones (i.e. crosslinked poly(dimethylsiloxane), PDMS) are commonly used material for microfluidic device fabrication. Nonetheless, due to the uncontrollable absorption of small hydrophobic molecules (<1 kDa) into the bulk, its applicability to cell-based drug assays and sensing applications has been limited. Here, we demonstrate the use of substrates made of silicones bulk modified with a poly(ethylene oxide) silane amphiphile (PEO-SA) to reduce hydrophobic small molecule sequestration for cell-based assays. Modified silicone substrates were generated with concentrations of 2 wt.%, 9 wt.% and, 14 wt.% PEO-SA. Incorporation of PEO-SA into the silicone bulk was assessed by FTIR analysis in addition to water contact angle analysis to evaluate surface hydrophobicity. Cell toxicity, absorption of small hydrophobic drugs, and cell response to hydrophobic molecules were also evaluated. Results showed that the incorporation of the PEO-SA into the silicone led to a reduction in water contact angle from 114° to as low as 16° that was stable for at least three months. The modified silicones showed viability values above 85% for NIH-3T3, MCF7, MDA-MB-468, and MDA-MB-231 cell lines. A drug response assay using tamoxifen and the MCF7 cell line showed full recovery of cell toxicity response when exposed to PDMS modified with 9 wt.% or 14 wt.% PEO-SA compared to tissue culture plastic. Therefore, our study supports the use of PEO-SA at concentrations of 9 wt.% or higher for enhanced surface wettability and reduced absorption of small hydrophobic molecules in PDMS-based platforms.

Thromboresistance of Polyurethanes Modified with PEO-Silane Amphiphiles.

Surface-induced thrombosis is problematic in blood-contacting devices composed of silicones or polyurethanes (PUs). Poly(ethylene oxide)-silane amphiphiles (PEO-SA) are previously shown effective as surface modifying additives (SMAs) in silicones for enhanced thromboresistance. This study investigates PEO-SAs as SMAs in a PU at various concentrations: 5, 10, 25, 50, and 100 µmol g-1 PU. PEO-SA modified PUs are evaluated for their mechanical properties, water-driven surface restructuring, and adhesion resistance against a human fibrinogen (HF) solution as well as whole human blood. Stability is assessed by monitoring hydrophilicity, water uptake, and mass loss following air- or aqueous-conditioning. PEO-SA modified PUs do not demonstrate plasticization, as evidenced by minimal changes in glass transition temperature, modulus, tensile strength, and percent strain at break. These also show a concentration-dependent increase in hydrophilicity that is sustained following air- and aqueous-conditioning for concentrations ≥25 µmol g-1 . Additionally, water uptake and mass loss are minimal at all concentrations. Although protein resistance is not enhanced versus an HF solution, PEO-SA modified PUs have significantly reduced protein adsorption and platelet adhesion from human blood at concentrations ≥10 µmol g-1 . Overall, this study demonstrates the versatility of PEO-SAs as SMAs in PU, which leads to enhanced and sustained hydrophilicity as well as thromboresistance.

Thromboresistance of Silicones Modified with PEO-Silane Amphiphiles.

The antifouling properties of poly(ethylene oxide) (PEO)-silane amphiphiles as surface-modifying additives (SMAs) in a condensation cure silicone have been previously demonstrated against simple protein solutions. Comprising an oligo(dimethylsiloxane) tether (m = 13 or 30) and PEO segment (n = 8), sustained protein resistance was achieved even in the absence of a cross-linkable triethoxysilane group, particularly when comprising the longer tether. To probe their potential for thromboresistance, PEO-silane amphiphile SMAs were used to bulk-modify silicones and evaluated for adhesion resistance against whole human blood under both static and dynamic conditions. Both a cross-linkable (XL diblock, m = 13) and a non-cross-linkable (Diblock, m = 30) SMA were evaluated at various concentrations (5-50 µmol SMA/g silicone) in a condensation cure silicone. Under static conditions, silicones modified with either SMA at concentrations of 10 µmol/g or greater were effective in reducing adhesion of human fibrinogen and platelets. Dynamic testing further showed that modified silicones were able to reduce protein adsorption and thrombus formation. This occurred at 5 and 10 µmol/g for silicones modified with XL diblock, m = 13 and Diblock, m = 30 SMAs, respectively. Combined, these results indicate the effectiveness of PEO-silane amphiphiles as SMAs in silicone for improved thromboresistance.

Anti-protein and anti-bacterial behavior of amphiphilic silicones.

Silicones with improved water-driven surface hydrophilicity and anti-biofouling behavior were achieved when bulk-modified with poly(ethylene oxide) (PEO) -silane amphiphiles of varying siloxane tether length: α-(EtO)3Si-(CH2)2-oligodimethylsiloxane m -block-poly(ethylene oxide)8-OCH3 (m = 0, 4, 13, 17, 24, and 30). A PEO8-silane [α-(EtO)3Si-(CH2)3-PEO8-OCH3] served as a conventional PEO-silane control. To examine anti-biofouling behavior in the absence versus presence of water-driven surface restructuring, the amphiphiles and control were surface-grafted onto silicon wafers and used to bulk-modify a medical-grade silicone, respectively. While the surface-grafted PEO-control exhibited superior protein resistance, it failed to appreciably restructure to the surface-water interface of bulk-modified silicone and thus led to poor protein resistance. In contrast, the PEO-silane amphiphiles, while less protein-resistant when surface-grafted onto silicon wafers, rapidly and substantially restructured in bulk-modified silicone, exhibiting superior hydrophilicity and protein resistance. A reduction of biofilm for several strains of bacteria and a fungus was observed for silicones modified with PEO-silane amphiphiles. Longer siloxane tethers maintained surface restructuring and protein resistance while displaying the added benefit of increased transparency.

Protein Resistant Polymeric Biomaterials.

Toward improving implantable medical devices as well as diagnostic performance, the development of polymeric biomaterials having resistance to proteins remains a priority. Herein, we highlight key strategies reported in the recent literature that have relied upon improvement of surface hydrophilicity via direct surface modification methods or with bulk modification using surface modifying additives (SMAs). These approaches have utilized a variety of techniques to incorporate the surface hydrophilization agent, including physisorption, hydrogel network formation, surface grafting, layer-by-layer (LbL) assembly and blending base polymers with SMAs. While poly(ethylene glycol) (PEG) remains the gold standard, new alternatives have emerged such as polyglycidols, poly(2-oxazoline)s (POx), polyzwitterions, and amphiphilic block copolymers. While these new strategies provide encouraging results, the need for improved correlation between in vitro and in vivo protein resistance is critical. This may be achieved by employing complex protein solutions as well as strides to enhance the sensitivity of protein adsorption measurements.

Antifouling silicones based on surface-modifying additive amphiphiles.

Surface modifying additives (SMAs), which may be readily blended into silicones to improve anti-fouling behavior, must have excellent surface migration potential and must not leach into the aqueous environment. In this work, we evaluated the efficacy of a series of poly(ethylene oxide) (PEO)-based SMA amphiphiles which varied in terms of crosslinkability, siloxane tether length (m) and diblock versus triblock architectures. Specifically, crosslinkable, diblock PEO-silane amphiphiles with two oligodimethylsiloxane (ODMS) tether lengths [(EtO)3Si-(CH2)3-ODMS m -PEO8, m = 13 and 30] were compared to analogous non-crosslinkable, diblock (H-Si-ODMS m -PEO8) and triblock (PEO8-ODMS m -PEO8) SMAs. Prior to water conditioning, while all modified silicone coatings exhibited a high degree of water-driven surface restructuring, that prepared with the non-crosslinkable diblock SMA (m = 13) was the most hydrophilic. After conditioning, all modified silicone coatings were similarly hydrophilic and remained highly protein resistant, with the exception of PEO8-ODMS 30 -PEO8. Notably, despite twice the PEO content, triblock SMAs were not superior to diblock SMAs. For diblock SMAs, it was shown that water uptake and leaching were also similar whether or not the SMA was crosslinkable.