Hollow fiber membrane modification with functional zwitterionic macromolecules for improved thromboresistance in artificial lungs.

Respiratory assist devices seek optimized performance in terms of gas transfer efficiency and thromboresistance to minimize device size and reduce complications associated with inadequate blood biocompatibility. The exchange of gas with blood occurs at the surface of the hollow fiber membranes (HFMs) used in these devices. In this study, three zwitterionic macromolecules were attached to HFM surfaces to putatively improve thromboresistance: (1) carboxyl-functionalized zwitterionic phosphorylcholine (PC) and (2) sulfobetaine (SB) macromolecules (mPC or mSB-COOH) prepared by a simple thiol-ene radical polymerization and (3) a low-molecular weight sulfobetaine (SB)-co-methacrylic acid (MA) block copolymer (SBMAb-COOH) prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. Each macromolecule type was covalently immobilized on an aminated commercial HFM (Celg-A) by a condensation reaction, and HFM surface composition changes were analyzed by X-ray photoelectron spectroscopy. Thrombotic deposition on the HFMs was investigated after contact with ovine blood in vitro. The removal of CO2 by the HFMs was also evaluated using a model respiratory assistance device. The HFMs conjugated with zwitterionic macromolecules (Celg-mPC, Celg-mSB, and Celg-SBMAb) showed expected increases in phosphorus or sulfur surface content. Celg-mPC and Celg-SBMAb experienced rates of platelet deposition significantly lower than those of unmodified (Celg-A, >95% reduction) and heparin-coated (>88% reduction) control HFMs. Smaller reductions were seen with Celg-mSB. The CO2 removal rate for Celg-SBMAb HFMs remained comparable to that of Celg-A. In contrast, the rate of removal of CO2 for heparin-coated HFMs was significantly reduced. The results demonstrate a promising approach to modifying HFMs using zwitterionic macromolecules for artificial lung devices with improved thromboresistance without degradation of gas transfer.

Surface modification of a biodegradable magnesium alloy with phosphorylcholine (PC) and sulfobetaine (SB) functional macromolecules for reduced thrombogenicity and acute corrosion resistance.

Siloxane functionalized phosphorylcholine (PC) or sulfobetaine (SB) macromolecules (PCSSi or SBSSi) were synthesized to act as surface modifying agents for degradable metallic surfaces to improve acute blood compatibility and slow initial corrosion rates. The macromolecules were synthesized using a thiol-ene radical photopolymerization technique and then utilized to modify magnesium (Mg) alloy (AZ31) surfaces via an anhydrous phase deposition of the silane functional groups. X-ray photoelectron spectroscopy surface analysis results indicated successful surface modification based on increased nitrogen and phosphorus or sulfur composition on the modified surfaces relative to unmodified AZ31. In vitro acute thrombogenicity assessment after ovine blood contact with the PCSSi and SBSSi modified surfaces showed a significant decrease in platelet deposition and bulk phase platelet activation compared with the control alloy surfaces. Potentiodynamic polarization and electrochemical impedance spectroscopy data obtained from electrochemical corrosion testing demonstrated increased corrosion resistance for PCSSi- and SBSSi-modified AZ31 versus unmodified surfaces. The developed coating technique using PCSSi or SBSSi showed promise in acutely reducing both the corrosion and thrombotic processes, which would be attractive for application to blood contacting devices, such as vascular stents, made from degradable Mg alloys.

Chemical patterning of ultrathin polymer films by direct-write multiphoton lithography.

We applied 2-photon laser ablation to write subdiffraction nanoscale chemical patterns into ultrathin polymer films under ambient conditions. Poly(ethylene glycol) methacrylate brush layers were prepared on quartz substrates via surface-initiated atom-transfer radical polymerization and ablated to expose the underlying substrate using the nonlinear 2-photon absorbance of a frequency-doubled Ti:sapphire femtosecond laser. Single-shot ablation thresholds of polymer films were ~1.5 times smaller than that of a quartz substrate, which allowed patterning of nanoscale features without damage to the underlying substrate. At a 1/e(2) laser spot diameter of 0.86 µm, the features of exposed substrate approached ~80 nm, well below the diffraction limit for 400 nm light. Ablated features were chemically distinct and amenable to chemical modification.

Surfactants influence polymer nanoparticle fate within the brain.

Drug delivery to the brain is limited by poor penetration of pharmaceutical agents across the blood-brain barrier (BBB), within the brain parenchyma, and into specific cells of interest. Nanotechnology can overcome these barriers, but its ability to do so is dependent on nanoparticle physicochemical properties including surface chemistry. Surface chemistry can be determined by a number of factors, including by the presence of stabilizing surfactant molecules introduced during the formulation process. Nanoparticles coated with poloxamer 188 (F68), poloxamer 407 (F127), and polysorbate 80 (P80) have demonstrated uptake in BBB endothelial cells and enhanced accumulation within the brain. However, the impact of surfactants on nanoparticle fate, and specifically on brain extracellular diffusion or intracellular targeting, must be better understood to design nanotherapeutics to efficiently overcome drug delivery barriers in the brain. Here, we evaluated the effect of the biocompatible and commonly used surfactants cholic acid (CHA), F68, F127, P80, and poly (vinyl alcohol) (PVA) on poly (lactic-co-glycolic acid)-poly (ethylene glycol) (PLGA-PEG) nanoparticle transport to and within the brain. The inclusion of these surfactant molecules decreases diffusive ability through brain tissue, reflecting the surfactant's role in encouraging cellular interaction at short length and time scales. After in vivo administration, PLGA-PEG/P80 nanoparticles demonstrated enhanced penetration across the BBB and subsequent internalization within neurons and microglia. Surfactants incorporated into the formulation of PLGA-PEG nanoparticles therefore represent an important design parameter for controlling nanoparticle fate within the brain.

Time-of-flight secondary ion mass spectrometry three-dimensional imaging of surface modifications in poly(caprolactone) scaffold pores.

Scaffolds composed of synthetic polymers such as poly(caprolactone) (PCL) are widely used for the support and repair of tissues in biomedicine. Pores are common features in scaffolds as they facilitate cell penetration. Various surface modifications can be performed to promote key biological responses to these scaffolds. However, verifying the chemistry of these materials post surface modification is problematic due to the combination of three-dimensional (3D) topography and surface sensitivity. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is commonly used to correlate surface chemistry with cell response. In this study, 3D imaging mass spectrometry analysis of surface modified synthetic polymer scaffolds is demonstrated using PCL porous scaffold, a pore filling polymer sample preparation, and 3D imaging ToF-SIMS. We apply a simple sample preparation procedure, filling the scaffold pores with a poly(vinyl alcohol)/glycerol mixture to remove topographic influence on image quality. This filling method allows the scaffold (PCL) and filler secondary ions to be reconstructed into a 3D chemical image of the pore. Furthermore, we show that surface modifications in the pores of synthetic polymer scaffolds can be mapped in 3D. Imaging of "dry" and "wet" surface modifications is demonstrated as well as a comparison of surface modifications with relatively strong ToF-SIMS peaks (fluorocarbon films [FC]) and to more biologically relevant surface modification of a protein (bovine serum albumin [BSA]). We demonstrate that surface modifications can be imaged in 3D showing that characteristic secondary ions associated with FC and BSA are associated with C3 F8 plasma treatment and BSA, respectively within the pore.

Development of zwitterionic sulfobetaine block copolymer conjugation strategies for reduced platelet deposition in respiratory assist devices.

Respiratory assist devices, that utilize ∼2 m2 of hollow fiber membranes (HFMs) to achieve desired gas transfer rates, have been limited in their adoption due to such blood biocompatibility limitations. This study reports two techniques for the functionalization and subsequent conjugation of zwitterionic sulfobetaine (SB) block copolymers to polymethylpentene (PMP) HFM surfaces with the intention of reducing thrombus formation in respiratory assist devices. Amine or hydroxyl functionalization of PMP HFMs (PMP-A or PMP-H) was accomplished using plasma-enhanced chemical vapor deposition. The generated functional groups were conjugated to low molecular weight SB block copolymers with N-hydroxysuccinimide ester or siloxane groups (SBNHS or SBNHSi) that were synthesized using reversible addition fragmentation chain transfer polymerization. The modified HFMs (PMP-A-SBNHS or PMP-H-SBNHSi) showed 80-95% reduction in platelet deposition from whole ovine blood, stability under the fluid shear of anticipated operating conditions, and uninhibited gas exchange performance relative to non-modified HFMs (PMP-C). Additionally, the functionalization and SBNHSi conjugation technique was shown to reduce platelet deposition on polycarbonate and poly(vinyl chloride), two other materials commonly found in extracorporeal circuits. The observed thromboresistance and stability of the SB modified surfaces, without degradation of HFM gas transfer performance, indicate that this approach is promising for longer term pre-clinical testing in respiratory assist devices and may ultimately allow for the reduction of anticoagulation levels in patients being supported for extended periods. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2681-2692, 2018.

Time of flight secondary ion mass spectrometry-A method to evaluate plasma-modified three-dimensional scaffold chemistry.

Biopolymers are used extensively in the manufacture of porous scaffolds for a variety of biological applications. The surfaces of these scaffolds are often modified to encourage specific interactions such as surface modification of scaffolds to prevent fouling or to promote a cell supportive environment for tissue engineering implants. However, few techniques can effectively characterize the uniformity of surface modifications in a porous scaffold. By filling the scaffold pores through polymer embedding, followed by analysis with imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS), the distribution and composition of surface chemical species though complex porous scaffolds can be characterized. This method is demonstrated on poly(caprolactone) scaffolds modified with a low-fouling plasma-deposited coating from octafluoropropane via plasma enhanced chemical vapor deposition. A gradient distribution of CF+/CF3+ is observed for scaffolds plasma treated for 5 min, whereas a 20 min treatment results in more uniform distribution of the surface modification throughout the entire scaffold. The authors expect this approach to be widely applicable for ToF-SIMS analysis of scaffolds modified by multiple plasma processing techniques as well as alternative surface modification approaches.

In vitro characterization of peptide-modified p(AAm-co-EG/AAc) IPN-coated titanium implants.

Interpenetrating polymer networks (IPNs) of poly(acrylamide-co-ethylene glycol/acrylic acid) [p(AAm-co-EG/AAc)] functionalized with an -Arg-Gly-Asp- containing peptide derived from rat bone sialoprotein [bsp-RGD(15)] were grafted to titanium implants in an effort to modulate osteoblast behavior in vitro. Surface characterization data were consistent with the presence of an IPN, and ligand density measurements established that the range of peptide density on the modified implants spanned three orders of magnitude (0.01-20 pmol/cm2). In vitro biological characterization of the modified implants employing the primary rat calvarial osteoblast (RCO) model resulted in the identification of a critical ligand density (0.01

Self-assembled antimicrobial and biocompatible copolymer films on titanium.

Copolymers of 4-vinyl-N-hexylpyridinium bromide and dimethyl(2-methacryloyloxyethyl) phosphonate self-assemble to form ultrathin layers on titanium surfaces that show antimicrobial activity, and biocompatibility. The copolymer layers are characterized by contact angle measurements, ellipsometry and XPS. Antibacterial activity is assessed by investigation of adherence of S. mutans. Biocompatibility is rated based on human gingival fibroblast adhesion and proliferation. By balancing the opposing effects of the chemical composition on biocompatibility and antimicrobial activity, copolymer coatings are fabricated that are able to inhibit the growth of S. mutans on the surface but still show attachment of gingival fibroblasts, and therefore might prevent biofilm formation on implants.

Kinetics of leucine-lysine peptide adsorption and desorption at -CH3 and -COOH terminated alkylthiolate monolayers.

The kinetics of adsorption and desorption of two highly asymmetrical model peptides were studied at methyl- and carboxylic acid-terminated alkylthiolate self-assembled monolayer (SAM) surfaces on gold. The model peptides were leucine-lysine (LK), α-helical (LKα14), and ß-strand (LKß15) peptides that have a well-defined secondary structure with the leucines localized on one side and the lysines on the other side. These secondary structures were previously shown to be maintained after adsorption and to control LK peptide orientation on these surfaces. The kinetics of peptide adsorption were analyzed by surface plasmon resonance as a function of peptide solution concentrations at pH 7.4. Peptide desorption was measured by rinsing with a buffer at various times along the adsorption curve. Both peptides had a saturation coverage of approximately 1 ML (monolayer) on the carboxyl SAM. Both peptides exhibited mostly irreversible binding on both surfaces, but the LKα14 peptide showed some limited reversible binding. Reversibly bound peptides could be in the second adlayer interacting only with peptides in the first layer or peptides interacting with a partially covered adsorption site and therefore not able to fully bind to the SAM surface. The near complete lack of reversible binding for LKß15 is possibly due to strong peptide-peptide hydrogen bonding in ß-sheet structures within the adsorbed layer. For a given dose of either peptide, much less peptide adsorbed on the methyl SAMs. The adsorption rate of irreversibly bound LKα14 on carboxylic acid SAMs was first-order with respect to solution concentration. Both peptides showed nucleation-like adsorption kinetics on the carboxylic acid SAM, indicating that peptide-peptide bonding is needed to stabilize the adsorbed layer. Adsorption on the methyl SAM was much lower in quantity for both peptides and seemed to require prior aggregation of the proteins in solution, at least for LKß15.

Simple surface modification of a titanium alloy with silanated zwitterionic phosphorylcholine or sulfobetaine modifiers to reduce thrombogenicity.

Thrombosis and thromboembolism remain problematic for a large number of blood contacting medical devices and limit broader application of some technologies due to this surface bioincompatibility. In this study we focused on the covalent attachment of zwitterionic phosphorylcholine (PC) or sulfobetaine (SB) moieties onto a TiAl(6)V(4) surface with a single step modification method to obtain a stable blood compatible interface. Silanated PC or SB modifiers (PCSi or SBSi) which contain an alkoxy silane group and either PC or SB groups were prepared respectively from trimethoxysilane and 2-methacryloyloxyethyl phosphorylcholine (MPC) or N-(3-sulfopropyl)-N-(methacryloxyethyl)-N,N-dimethylammonium betaine (SMDAB) monomers by a hydrosilylation reaction. A cleaned and oxidized TiAl(6)V(4) surface was then modified with the PCSi or SBSi modifiers by a simple surface silanization reaction. The surface was assessed with X-ray photoelectron spectroscopy (XPS), attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and contact angle goniometry. Platelet deposition and bulk phase activation were evaluated following contact with anticoagulated ovine blood. XPS results verified successful modification of the PCSi or SBSi modifiers onto TiAl(6)V(4) based on increases in surface phosphorous or sulfur respectively. Surface contact angles in water decreased with the addition of hydrophilic PC or SB moieties. Both the PCSi and SBSi modified TiAl(6)V(4) surfaces showed decreased platelet deposition and bulk phase platelet activation compared to unmodified TiAl(6)V(4) and control surfaces. This single step modification with PCSi or SBSi modifiers offers promise for improving the surface hemocompatibility of TiAl(6)V(4) and is attractive for its ease of application to geometrically complex metallic blood contacting devices.

Covalent surface modification of a titanium alloy with a phosphorylcholine-containing copolymer for reduced thrombogenicity in cardiovascular devices.

Our objective was to develop a surface modification strategy for a titanium alloy (TiAl6V4) to provide thromboresistance for surfaces in rigorous blood-contacting cardiovascular applications, such as that found in ventricular assist devices. We hypothesized that this could be accomplished by the covalent attachment of a phospholipid polymer, poly(2-methacryloyloxyethylphosphorylcholine (MPC)-co-methacryl acid) (PMA). TiAl6V4 was H2O plasma treated by radio frequency glow discharge, silanated with 3-aminopropyltriethoxysilane (APS), and ammonia plasma treated to increase surface reactivity. The TiAl6V4 surface was then modified with PMA via a condensation reaction between the amino groups on the TiAl6V4 surface and the carboxyl groups on PMA. The surface composition was verified by X-ray photoelectron spectroscopy, confirming successful modification of the TiAl6V4 surfaces with APS and PMA as evidenced by increased Si and P. Plasma treatments with H2O and ammonia were effective at further increasing the surface reactivity of TiAl6V4 as evidenced by increased surface PMA. The adsorption of ovine fibrinogen onto PMA-modified surfaces was reduced relative to unmodified surfaces, and in vitro ovine blood contact through a rocking test revealed marked reductions in platelet deposition and bulk phase platelet activation relative to unmodified TiAl6V4 and polystyrene controls. The results indicate that the PMA-modification scheme for TiAl6V4 surfaces offers a potential pathway to improve the thromboresistance of the blood-contacting surfaces of cardiovascular devices.

Surface modification of a titanium alloy with a phospholipid polymer prepared by a plasma-induced grafting technique to improve surface thromboresistance.

To improve the thromboresistance of a titanium alloy (TiAl(6)V(4)) surface which is currently utilized in several ventricular assist devices (VADs), a plasma-induced graft polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) was carried out and poly(MPC) (PMPC) chains were covalently attached onto a TiAl(6)V(4) surface by a plasma induced technique. Cleaned TiAl(6)V(4) surfaces were pretreated with H(2)O-vapor-plasma and silanated with 3-methacryloylpropyltrimethoxysilane (MPS). Next, a plasma-induced graft polymerization with MPC was performed after the surfaces were pretreated with Ar plasma. Surface compositions were verified by X-ray photoelectron spectroscopy (XPS). In vitro blood biocompatibility was evaluated by contacting the modified surfaces with ovine blood under continuous mixing. Bulk phase platelet activation was quantified by flow cytometric analysis, and surfaces were observed with scanning electron microscopy after blood contact. XPS data demonstrated successful modification of the TiAl(6)V(4) surfaces with PMPC as evidenced by increased N and P on modified surfaces. Platelet deposition was markedly reduced on the PMPC grafted surfaces and platelet activation in blood that contacted the PMPC-grafted samples was significantly reduced relative to the unmodified TiAl(6)V(4) and polystyrene control surfaces. Durability studies under continuously mixed water suggested no change in surface modification over a 1-month period. This modification strategy shows promise for further investigation as a means to reduce the thromboembolic risk associated with the metallic blood-contacting surfaces of VADs and other cardiovascular devices under development.

Surface characterization of biocidal polyurethane modifiers having poly(3,3-substituted)oxetane soft blocks with alkylammonium side chains.

This paper focuses on surface characterization of P[ AB] copolyoxetane soft block polyurethanes having either fluorous (3FOx, -CH2OCH 2CF3) or PEG-like (ME2Ox, -CH2(OCH2CH2) 2OCH3), A side chains and alkylammonium, B side chains. Physical surface characterization data were analyzed in light of the previously observed order of antimicrobial effectiveness for a set of four surface modifiers. Ample physical evidence for surface concentration of fluorous 2 wt % P[ AB]-polyurethane modifiers was obtained from XPS, contact angles, ATR-IR spectroscopy, and TM-AFM. In TM-AFM phase imaging, the most effective biocidal surface modifier, 2 wt % HMDI-BD(30)/P[(3FOx)(C12)-0.89:0.11]-PU, showed a nanoscale phase-separated structure consisting of 200 nm domains with background features about 10 times smaller. Despite similar surface characterization data, the 2 wt % fluorous C6 analog ranked third in contact biocidal effectiveness. Physical evidence for surface concentration of 2 wt % P[(ME2Ox)(C12)-0.86:0.14]-PU was modest, considering that antimicrobial effectiveness was second only to 2 wt % HMDI-BD(30)/P[(3FOx)(C12)-0.89:0.11]-PU. In this set of surface modifiers, nanoscale morphology is largely driven by the fluorous component, whereas antimicrobial effectiveness is more strongly influenced by alkylammonium chain length. The effect of alkylammonium side chain length on surface concentration and antimicrobial behavior is more pronounced for ME2Ox polyurethanes compared to the 3FOx analogs.

Microcontact printed antibodies on gold surfaces: function, uniformity, and silicone contamination.

The function of microcontact printed protein was investigated using surface plasmon resonance (SPR) imaging, X-ray photoelectron spectroscopy spectroscopy (XPS), and XPS imaging. We chose to analyze a model protein system, the binding of an antibody from solution to a microcontact printed protein antigen immobilized to a gold surface. SPR imaging experiments indicated that the microcontact printed protein antigen was less homogeneous, had increased nonspecific binding, and bound less antibody than substrates to which the protein antigen had been physically adsorbed. SPR images of substrates contacted with a poly(dimethylsiloxane) stamp inked with buffer alone (i.e., no protein) revealed that significant amounts of silicone oligomer were transferred to the surface. The transfer of the silicone oligomer was not homogeneous, and the oligomer nonspecifically bound protein (BSA and IgG) from solution. XPS spectroscopy and imaging were used to quantify the amount of silicon (due to the presence of silicone oligomer), as well as the amounts of other elements, transferred to the surface. The results suggest that the silicone oligomer introduced by the printing process reduces the overall binding capacity of the microcontact-printed protein compared to physically adsorbed protein.

Phosphonic acid monolayers for binding of bioactive molecules to titanium surfaces.

Two different phosphonic acid monolayer films for immobilization of bioactive molecules such as the protein BMP-2 on titanium surfaces have been prepared. Monolayers of (11-hydroxyundecyl)phosphonic acid and (12-carboxydodecyl)phosphonic acid molecules were produced by a simple dipping process (the T-BAG method). The terminal functional groups on these monolayers were activated (carbonyldiimidazole for hydroxyl groups and N-hydroxysuccinimide for carboxyl groups) to bind amine-containing molecules. The reactivity of the surfaces was investigated using trifluoroethylamine hydrochloride and BMP-2. Each step of the surface modification procedure was characterized by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry.

Synthesis and characterization of biocompatible polymer interlayers on titanium implant materials.

A bifunctional copolymer series of (4-vinylbenzyl)phosphonic acid diethylester and N-acryloxysuccinimide was developed as an interlayer with the aim of immobilizing proteins on titanium surfaces. Copolymers with varying compositions were synthesized, and an alternating copolymerization of the two monomers was found. The copolymers form ultrathin films of about 2-8 nm on titanium surfaces in a simple dipping process, as estimated from the attenuation of the titanium X-ray photoelectron spectroscopy (Ti-XPS) signal. The films were characterized by infrared spectroscopy, XPS, and time-of-flight secondary ion mass spectrometry. The results indicate that the immobilization is due to phosphonate groups, and thus the phosphonate content of the copolymers is decisive for the final film thickness. These polymer films were examined for their potential protein binding capacity by using trifluoroethylamine derivatization and subsequent XPS analysis as a reactivity assay.