Long-term adherence of human brain cells in vitro is enhanced by charged amine-based plasma polymer coatings.

Advances in cellular reprogramming have radically increased the use of patient-derived cells for neurological research in vitro. However, adherence of human neurons on tissue cultureware is unreliable over the extended periods required for electrophysiological maturation. Adherence issues are particularly prominent for transferable glass coverslips, hindering imaging and electrophysiological assays. Here, we assessed thin-film plasma polymer treatments, polymeric factors, and extracellular matrix coatings for extending the adherence of human neuronal cultures on glass. We find that positive-charged, amine-based plasma polymers improve the adherence of a range of human brain cells. Diaminopropane (DAP) treatment with laminin-based coating optimally supports long-term maturation of fundamental ion channel properties and synaptic activity of human neurons. As proof of concept, we demonstrated that DAP-treated glass is ideal for live imaging, patch-clamping, and optogenetics. A DAP-treated glass surface reduces the technical variability of human neuronal models and enhances electrophysiological maturation, allowing more reliable discoveries of treatments for neurological and psychiatric disorders.

To be a radical or not to be one? The fate of the stable nitroxide radical TEMPO [(2,2,6,6-Tetramethylpiperidin-1-yl)oxyl] undergoing plasma polymerization into thin-film coatings.

The stable nitroxide radical TEMPO [(2,2,6,6-Tetramethylpiperidin-1-yl)oxyl] has a multitude of applications in fields ranging from energy storage to biomedical applications and many more. However, to date, the processes of incorporating nitroxide radicals into thin-film coatings are laborious and not cost-effective, which hinders their wider use in many applications. In contrast, the authors have recently demonstrated the facile method of plasma polymerization of TEMPO into thin-film coatings that retain the stable nitroxide radicals. In this work, we are using three types of mass spectroscopic methods (plasma-mass spectrometry, time of flight secondary ion mass spectrometry, and high-performance liquid chromatography-mass spectrometry) and electron spin resonance to track the fate of the TEMPO molecule from monomer flask through the plasma and inside the resulting coatings. The results of this study demonstrate that TEMPO is a versatile monomer that can be used across different plasma reactors and reliably retain the stable nitroxide radical in the resulting thin-film coatings if certain process conditions are observed, namely, higher process pressures and lower powers.

S-Nitrosothiol Plasma-Modified Surfaces for the Prevention of Bacterial Biofilm Formation.

The development of novel strategies for the prevention of bacterial infections is of utmost importance because of the exponential growth in the number of patient morbidity related to nosocomial and chronic infections. Nitric oxide (NO) is known to be a potent inhibitor of bacterial growth and adhesion to surfaces. Here, we develop an antibiofilm coating that possesses S-nitrosothiol NO donors via plasma polymerization (PP) for biofilm prevention applications. Cell culture dishes of four different film thicknesses ranging from 125 to 1000 nm were coated via PP using a thiol monomer. The thiol functionality on the substrates was converted to S-nitrosothiol NO precursors using tert-butyl nitrite. The successful conjugation of thiol and subsequent formation of S-nitrosothiol functionalities on the substrates were confirmed using X-ray photoelectron spectroscopy and UV-vis analysis. These coatings are capable of releasing NO over 2 days, and the NO loading is tunable by the polymer film thickness. The antibiofilm activity of the surfaces was assessed using Gram-negative bacteria, Pseudomonas aeruginosa. Higher film thickness (and hence, higher NO loading) demonstrate better antibiofilm activity, and the best performing coating shows 81 and 60% inhibition of bacterial attachment to the surface after exposure to bacterial culture solution for 24 and 36 h, respectively. Overall, the NO-releasing plasma-modified surfaces present a potential viable strategy to inhibit bacterial biofilm formation.

"Thunderstruck": Plasma-Polymer-Coated Porous Silicon Microparticles As a Controlled Drug Delivery System.

Controlling the release kinetics from a drug carrier is crucial to maintain a drug's therapeutic window. We report the use of biodegradable porous silicon microparticles (pSi MPs) loaded with the anticancer drug camphothecin, followed by a plasma polymer overcoating using a loudspeaker plasma reactor. Homogenous "Teflon-like" coatings were achieved by tumbling the particles by playing AC/DC's song "Thunderstruck". The overcoating resulted in a markedly slower release of the cytotoxic drug, and this effect correlated positively with the plasma polymer coating times, ranging from 2-fold up to more than 100-fold. Ultimately, upon characterizing and verifying pSi MP production, loading, and coating with analytical methods such as time-of-flight secondary ion mass spectrometry, scanning electron microscopy, thermal gravimetry, water contact angle measurements, and fluorescence microscopy, human neuroblastoma cells were challenged with pSi MPs in an in vitro assay, revealing a significant time delay in cell death onset.

Fabrication and Characterization of a Porous Silicon Drug Delivery System with an Initiated Chemical Vapor Deposition Temperature-Responsive Coating.

This paper reports on the fabrication of a pSi-based drug delivery system, functionalized with an initiated chemical vapor deposition (iCVD) polymer film, for the sustainable and temperature-dependent delivery of drugs. The devices were prepared by loading biodegradable porous silicon (pSi) with a fluorescent anticancer drug camptothecin (CPT) and coating the surface with temperature-responsive poly(N-isopropylacrylamide-co-diethylene glycol divinyl ether) (pNIPAM-co-DEGDVE) or non-stimulus-responsive poly(aminostyrene) (pAS) via iCVD. CPT released from the uncoated oxidized pSi control with a burst release fashion (∼21 nmol/(cm(2) h)), and this was almost identical at temperatures both above (37 °C) and below (25 °C) the lower critical solution temperature (LCST) of the switchable polymer used, pNIPAM-co-DEGDVE (28.5 °C). In comparison, the burst release rate from the pSi-pNIPAM-co-DEGDVE sample was substantially slower at 6.12 and 9.19 nmol/(cm(2) h) at 25 and 37 °C, respectively. The final amount of CPT released over 16 h was 10% higher at 37 °C compared to 25 °C for pSi coated with pNIPAM-co-DEGDVE (46.29% vs 35.67%), indicating that this material can be used to deliver drugs on-demand at elevated temperatures. pSi coated with pAS also displayed sustainable drug delivery profiles, but these were independent of the release temperature. These data show that sustainable and temperature-responsive delivery systems can be produced by functionalization of pSi with iCVD polymer films. Benefits of the iCVD approach include the application of the iCVD coating after drug loading without causing degradation of the drug commonly caused by exposure to factors such as solvents or high temperatures. Importantly, the iCVD process is applicable to a wide array of surfaces as the process is independent of the surface chemistry and pore size of the nanoporous matrix being coated.

Attachment of Poly(l-lactide) Nanoparticles to Plasma-Treated Non-Woven Polymer Fabrics Using Inkjet Printing.

Active dressings that based on fabric materials are an area of interest for the treatment of wounds. Poly(l-lactide) nanoparticles containing the antimicrobial agent octenidine can be controllably lysed by toxins released by pathogenic bacteria thus releasing antimicrobial material in response to the presence of the bacterial toxins and so counteracting the infection. We developed an integrated engineering solution that allows for the stable immobilisation of nanoparticles on non-woven fabrics. The process involves coating nanoparticles on non-woven polymer surfaces by using an inkjet printing process. In order to improve the adhesion and retention of the nanoparticles on the fabric, surface pretreatment of the non-woven fabric using plasma jet treatment can be applied to increase its surface energy.

Surface protein gradients generated in sealed microchannels using spatially varying helium microplasma.

Spatially varied surface treatment of a fluorescently labeled Bovine Serum Albumin (BSA) protein, on the walls of a closed (sealed) microchannel is achieved via a well-defined gradient in plasma intensity. The microchips comprised a microchannel positioned in-between two microelectrodes (embedded in the chip) with a variable electrode separation along the length of the channel. The channel and electrodes were 50 µm and 100 µm wide, respectively, 50 µm deep, and adjacent to the channel for a length of 18 mm. The electrode separation distance was varied linearly from 50 µm at one end of the channel to a maximum distance of 150, 300, 500, or 1000 µm to generate a gradient in helium plasma intensity. Plasma ignition was achieved at a helium flow rate of 2.5 ml/min, 8.5 kVpk-pk, and 10 kHz. It is shown that the plasma intensity decreases with increasing electrode separation and is directly related to the residual amount of BSA left after the treatment. The plasma intensity and surface protein gradient, for the different electrode gradients studied, collapse onto master curves when plotted against electrode separation. This precise spatial control is expected to enable the surface protein gradient to be tuned for a range of applications, including high-throughput screening and cell-biomolecule-biomaterial interactions.

Surface engineering of porous silicon to optimise therapeutic antibody loading and release.

The proinflammatory cytokine, tumor necrosis factor-α (TNF-α), is elevated in several diseases such as uveitis, rheumatoid arthritis and non-healing chronic wounds. Adding Infliximab, a chimeric IgG1 monoclonal antibody raised against TNF-α, to chronic wound fluid can neutralise human TNF-α, thereby providing a potential therapeutic option for chronic wound healing. However, to avoid the need for repeated application in a clinical setting, and to protect the therapeutic antibody from the hostile environment of the wound, suitable delivery vehicles are required. Porous silicon (pSi) is a biodegradable high surface area material commonly employed for drug delivery applications. In this study, the use of pSi microparticles (pSi MPs) for the controlled release of Infliximab to disease environments, such as chronic wounds, is demonstrated. Surface chemistry and pore parameters for Infliximab loading are first optimised in pSi films and loading conditions are transferred to pSi MPs. Loading regimens exceeding 60 µg of Infliximab per mg of pSi are achieved. Infliximab is released with zero-order release kinetics over the course of 8 days. Critically, the released antibody remains functional and is able to sequester TNF-α over a weeklong timeframe; suitable for a clinical application in chronic wound therapy.

ToF-SIMS analysis of poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) ultrathin adlayers.

Understanding of the interfacial chemistry of ultrathin polymeric adlayers is fundamentally important in the context of establishing quantitative design rules for the fabrication of nonfouling surfaces in various applications such as biomaterials and medical devices. In this study, seven poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-PMOXA) copolymers with grafting density (number of PMOXA chains per lysine residue) 0.09, 0.14, 0.19, 0.33, 0.43, 0.56, and 0.77, respectively, were synthesized and characterized by means of nuclear magnetic resonance spectroscopy (NMR). The copolymers were then adsorbed on Nb2O5 surfaces. Optical waveguide lightmode spectroscopy method was used to monitor the surface adsorption in situ of these copolymers and provide information on adlayer masses that were then converted into PLL and PMOXA surface densities. To investigate the relationship between copolymer bulk architecture (as shown by NMR data) and surface coverage as well as surface architecture, time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis was performed. Furthermore, ToF-SIMS method combined with principal component analysis (PCA) was used to verify the protein resistant properties of PLL-PMOXA adlayers, by thorough characterization before and after adlayer exposure to human serum. ToF-SIMS analysis revealed that the chemical composition as well as the architecture of the different PLL-PMOXA adlayers indeed reflects the copolymer bulk composition. ToF-SIMS results also indicated a heterogeneous surface coverage of PLL-PMOXA adlayers with high grafting densities higher than 0.33. In the case of protein resistant surface, PCA results showed clear differences between protein resistant and nonprotein-resistant surfaces. Therefore, ToF-SIMS results combined with PCA confirmed that the PLL-PMOXA adlayer with brush architecture resists protein adsorption. However, low increases of some amino acid signals in ToF-SIMS spectra were detected after the adlayer has been exposed to human serum.

On the effect of monomer chemistry on growth mechanisms of nonfouling PEG-like plasma polymers.

It has been shown that both ions and neutral species may contribute to plasma polymer growth. However, the relative contribution from these mechanisms remains unclear. We present data elucidating the importance of considering monomer structure with respect to which the growth mechanism dominates for nonfouling PEG-like plasma polymers. The deposition rate for saturated monomers is directly linked with ion flux to the substrate. For unsaturated monomers, the neutral flux also plays a role, particularly at low power. Increased fragmentation of the monomer at high power reduces the ability of unsaturated monomers to grow via neutral grafting. Chemical characterization by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) confirm the role that plasma phase fragmentation plays in determining the deposition rate and surface chemistry of the deposited film. The simple experimental method used here may also be used to determine which mechanisms dominate plasma deposition for other monomers. This knowledge may enable significant improvement in future reactor design and process control.

Combination of iCVD and porous silicon for the development of a controlled drug delivery system.

We describe a pH responsive drug delivery system which was fabricated using a novel approach to functionalize biodegradeable porous silicon (pSi) by initiated chemical vapor deposition (iCVD). The assembly involved first loading a model drug (camptothecin, CPT) into the pores of the pSi matrix followed by capping the pores with a thin pH responsive copolymer film of poly(methacrylic acid-co-ethylene dimethacrylate) (p(MAA-co-EDMA)) via iCVD. Release of CPT from uncoated pSi was identical in two buffers at pH 1.8 and pH 7.4. In contrast, the linear release rate of CPT from the pSi matrix with the p(MAA-co-EDMA) coating was dependent on the pH; release of CPT was more than four times faster at pH 7.4 (13.1 nmol/(cm(2) h)) than at pH 1.8 (3.0 nmol/(cm(2) h)). The key advantage of this drug delivery approach over existing ones based on pSi is that the iCVD coating can be applied to the pSi matrix after drug loading without degradation of the drug because the process does not expose the drug to harmful solvents or high temperatures and is independent of the surface chemistry and pore size of the nanoporous matrix.

Microplasma patterning of bonded microchannels using high-precision "injected" electrodes.

A rapid, high-precision method for localised plasma-treatment of bonded PDMS microchannels is demonstrated. Patterned electrodes were prepared by injection of molten gallium into preformed microchannel guides. The electrode guides were prepared without any additional fabrication steps compared to conventional microchannel fabrication. Alignment of the "injected" electrodes is precisely controlled by the photomask design, rather than positioning accuracy of alignment tools. Surface modification is detected using a fluorescent dye (Rhodamine B), revealing a well-defined micropattern with regions less than 100 µm along the length of the microchannel.

Covalent immobilization of antibacterial furanones via photochemical activation of perfluorophenylazide.

N-(3-trimethoxysilylpropyl)-4-azido-2,3,5,6-tetrafluorobenzamide (PFPA-silane) was used as a photoactive cross-linker to immobilize antibacterial furanone molecules on silicon oxide surfaces. This immobilization strategy is useful, especially for substrates and molecules that lack reactive functional groups. To this end, cleaned wafers were initially incubated in solutions of different concentrations of PFPA-silane to form a monolayer presenting azido groups on the surface. The functionalized surfaces were then treated with a furanone solution followed by illumination with UV light and extensive rinsing with ethanol to remove noncovalently adhered molecules. In the presented study, we demonstrate the ability to control the surface density of the immobilized furanone molecules by adjusting the concentration of PFPA-silane solution used for surface functionalization using complementary surface analytical techniques. The fluorine in PFPA-silane and the bromine in furanone molecules were convenient markers for the XPS study. The ellipsometric layer thickness of the immobilized furanone molecules on the surface decreased with decreasing PFPA-silane concentration, which correlated with a decline of water contact angle as a sign of film collapse. The intensity of characteristic azide vibration in the MTR IR spectra was monitored as a function of PFPA-silane concentration, and the peak disappeared completely after furanone application followed by UV irradiation. As a complementary technique to XPS, TOF-SIMS provided valuable information on the chemical and molecular structure of the modified surfaces and spatial distribution of the immobilized furanone molecules. Finally, this report presents a convenient, reproducible, and robust strategy to design antibacterial coating based on furanone compounds for applications in human health care.

Furanone at subinhibitory concentrations enhances staphylococcal biofilm formation by luxS repression.

Brominated furanones from marine algae inhibit multicellular behaviors of gram-negative bacteria such as biofilm formation and quorum sensing (QS) without affecting their growth. The interaction of furanone with QS in gram-positive bacteria is unknown. Staphylococci have two QS systems, agr and luxS, which lower biofilm formation by two different pathways, RNAIII upregulation and bacterial detachment, and polysaccharide intercellular adhesin (PIA) reduction, respectively. We synthesized natural furanone compound 2 [(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone] from Delisea pulchra and three analogues to investigate their effect on biofilm formation in gram-positive bacteria. Compound 2, but not the analogues, enhanced the biofilms of Staphylococcus epidermidis 1457 and 047 and of S. aureus Newman at concentrations between 1.25 and 20 microM. We show the growth inhibition of S. epidermidis and S. aureus by free furanone and demonstrate bactericidal activity. An induction of biofilm occurred at concentrations of 10 to 20% of the MIC and correlated with an increase in PIA. The biofilm effect was agr independent. It was due to interference with luxS, as shown by reduced luxS expression in the presence of compound 2 and independence of the strong biofilm formation in a luxS mutant upon furanone addition. Poly(l-lysine)-grafted/poly(ethylene glycol)-grafted furanone was ineffective on biofilm and not bactericidal, indicating the necessity for free furanone. Free furanone was similarly toxic for murine fibroblasts as for staphylococci, excluding a therapeutic application of this compound. In summary, we observed a biofilm enhancement by furanone in staphylococci at subinhibitory concentrations, which was manifested by an increase in PIA and dependent on luxS.

TOF-SIMS and principal component analysis characterization of the multilayer surface grafting of small molecules: antibacterial furanones.

Furanone compounds (fimbrolides) have attracted interest as antibacterial compounds for use in human health care, for instance, as an antibacterial coating for medical devices to combat device-centered infections. To ensure effectiveness for extended periods of time, they must be immobilized covalently onto a device surface; in this study, this was done via azide/nitrene chemistry and photochemical coupling. However, the detection and quantification of surface-immobilized small molecules such as furanones presents a considerable analytical challenge, yet is necessary for optimization of coatings and reliable interpretation of biological responses. We have utilized the surface sensitivity and chemical specificity of time-of-flight secondary ion mass spectrometry (TOF-SIMS) to characterize each step of the grafting sequence. On account of the complexity of the data, principal component analysis (PCA) was used to interpret and compare spectra. The results demonstrate the utility of TOF-SIMS with PCA for the detection of the surface-grafted small molecules azidoaniline and a brominated furanone; imaging of the bromine ion peaks also enabled assessment of grafting uniformity. Thus, successful multilayer coating and furanone grafting was observed, and substantial and uniform coverage of furanone molecules on the surface. Even multiple grafting steps involving, in the present case, two low molecular weight compounds can readily be disentangled by PCA. The utility of TOF-SIMS analysis with PCA is particularly well illustrated in the present case by the grafting of the furanone molecules, which did not yield a singular unique peak in the positive ion mass spectra, whereas the collective spectral changes elucidated by PCA provided unambiguous verification of successful grafting of this low molecular weight compound.