Single-cell membrane drug delivery using porous pen nanodeposition.

Delivering molecules onto the plasma membrane of single cells is still a challenging task in profiling cell signaling pathways with single cell resolution. We demonstrated that a large quantity of molecules could be targeted and released onto the membrane of individual cells to trigger signaling responses. This is achieved by a porous pen nanodeposition (PPN) method, in which a multilayer porous structure, serving as a reservoir for a large amount of molecules, is formed on an atomic force microscope (AFM) tip using layer-by-layer assembly and post processing. To demonstrate its capability for single cell membrane drug delivery, PPN was employed to induce a calcium flux triggered by the binding of released antibodies to membrane antigens in an autoimmune skin disease model. This calcium signal propagates from the target cell to its neighbors in a matter of seconds, proving the theory of intercellular communication through cell-cell junctions. Collectively, these results demonstrated the effectiveness of PPN in membrane drug delivery for single cells; to the best of our knowledge, this is the first technique that can perform the targeted transport and delivery in single cell resolution, paving the way for probing complex signaling interactions in multicellular settings.

Synchronous Generation of Nano- and Microscaled Hierarchical Porous Polyelectrolyte Multilayers for Superwettable Surfaces.

We created both a superhydrophilic polymer surface and a superhydrophobic surface by using the poly(acrylic acid) (PAA)/poly(allylamine hydrochloride) (PAH) multilayers with the synchronously generated hierarchical porous surface structures. The formation of surface and pore structures induced at acidic pH values is subject to the composition, distribution, and molecular weights of polyelectrolytes in the layer-by-layer (LbL) assembled film, leading to a variety of unique surface topographies and porous structures located on different scales. During the porous induction at pH 2.0, both nano- and microscaled features synchronously developed on the surface as a result of the unique combination of high-molecular-weight PAH (900K g/mol) and low molecular weight PAA (15K g/mol), along with a much reduced deposition time of 1 min. Although thermally cross-linked, the porous surface with hierarchical structure could achieve superhydrophilicity due to the remaining free amine and carboxylate groups on the porous structures. A complete switch from the superhydrophilic to the superhydrophobic surface was achieved via a simple chemical vapor deposition of trichloro(1H,1H,2H,2H-perfluoro-octyl)silane. In this work, the effects of molecular weight of polyelectrolytes (15K-900K g/mol), deposition time (10-900 s) during the LbL assembly, and pH (1.8 to 2.4) for the porous induction on the surface topography, pore structures, and wetting behavior were investigated in detail. A variety of unique porous surface structures on different length scales were systematically studied by controlling the above parameters.

Development of Layered Multiscale Porous Thin Films by Tuning Deposition Time and Molecular Weight of Polyelectrolytes.

This work focuses on the design of porous polymeric films with nano- and micro-sized pores existing in distinct zones. The porous thin films are fabricated by the post-treatment of layer-by-layer assembled poly(allylamine hydrochloride) (PAH)/poly(acrylic acid) (PAA) multilayers. In order to improve the processing efficiency, the deposition time is shortened to ≈ 10 s. It is found that fine porous structures can be created even by significantly reducing the processing time. The effect of using polyelectrolytes with widely different molecular weights is also studied. The pore size is increased by using high molecular weight PAH, while high molecular weight PAA minimizes the pore size to nanometer scale. Having gained a precise control over the pore size, layered multiscale porous thin films are further built up with either a microsized porous zone on top of a nanosized porous zone or vice versa.

Recent advances in the fabrication of nanostructured barrier films.

The fabrication of barrier packaging films has gained significant momentum in recent years. Besides its dominance in the food industry as a means to extend the shelf-life of perishable goods and facilitate ease of handling and transportation, the use of barrier films to protect semiconductor and flexible electronics from dust, oxidation and moisture has generated considerable interest in recent years. This has ushered in new challenges for researchers to design and develop novel thin film barrier coatings that could be made available at a fraction of the cost. The emergence of the multidisciplinary field of nanotechnology has provided innovative solutions in the fields of medicine, catalysis and energy. In this review, we will be examining the integration of nanoscience driven techniques with barrier film technology with applications in both food and electronics industry. Details regarding permeation theory, some key parameters governing gas/moisture barrier properties and the market potential of nanostructured barrier films have been included. This review also explores several past and current examples of successful inclusion of functional nanostructured or colloidal materials to fabricate tailor-made barrier films. Finally a brief discussion regarding novel emerging trends for this industry has been included.

Recent progress in the applications of layer-by-layer assembly to the preparation of nanostructured ion-rejecting water purification membranes.

Reverse osmosis (RO) and nanofiltration (NF) are the two dominant membrane separation processes responsible for ion rejection. While RO is highly efficient in removal of ions it needs a high operating pressure and offers very low selectivity between ions. Nanofiltration on the other hand has a comparatively low operating pressure and most commercial membranes offer selectivity in terms of ion rejection. However in many nanofiltration operations rejection of monovalent ions is not appreciable. Therefore a high flux high rejection membrane is needed that can be applied to water purification systems. One such alternative is the usage of polyelectrolyte multilayer membranes that are prepared by the deposition of alternately charged polyelectrolytes via layer-by-layer (LbL) assembly method. LbL is one of the most common self-assembly techniques and finds application in various areas. It has a number of tunable parameters like deposition conditions, number of bilayers deposited etc. which can be manipulated as per the type of application. This technique can be applied to make a nanothin membrane skin which gives high rejection and at the same time allow a high water flux across it. Several research groups have applied this highly versatile technique to prepare membranes that can be employed for water purification. Some of these membranes have shown better performance than the commercial nanofiltration and reverse osmosis membranes. These membranes have the potential to be applied to various different aspects of water treatment like water softening, desalination and recovery of certain ions. Besides the conventional method of LbL technique other alternative methods have also been suggested that can make the technique fast, more efficient and thereby make it more commercially acceptable.

Dynamic encapsulation of hydrophilic nisin in hydrophobic poly (lactic acid) particles with controlled morphology by a single emulsion process.

Hydrophilic nisin-loaded hydrophobic poly (lactic acid) (PLA) particles with controlled size and shape were successfully produced utilizing a one-step single emulsification method. Preliminary shear stress and temperature tests showed that there was no significant loss in the nisin inhibition activity during this process. PLA/nisin composite particles were prepared into solid nanocomposite spheres (50-200 nm) or hollow microcomposite spheres (1-5 µm) under the operative conditions developed in our previous study, in which the hydrophilic nisin in the aqueous phase solution could be entrapped in the hydrophobic polymer in the emulsification process generating either single or multiple emulsions. The incorporation of nisin in PLA had little effect on key processing conditions, which allows the dynamic control of the morphology and property of resulting particles. Microscopic and surface analyses suggested that nisin was dispersed uniformly inside the polymer matrix and adsorbed on the particle surface. The encapsulation amount and efficiency were enhanced with the increase in nisin loading in the aqueous solution. Unique reversible control of particle size and shape by this process was successfully applied in the nisin encapsulation. Alternating temperature in the repeating emulsification steps improved the encapsulation efficiency while generated particles in desired size and shape.

Conductive oxygen barrier films using supramolecular assembly of graphene embedded polyelectrolyte multilayers.

The supramolecular self-assembly of polyelectrolyte multilayers (PEMs) provides robust bottom-up strategies to assemble a broad spectrum of nanostructures on the host substrates. In this study, we discuss the formation of graphene nanoplatelet (GNP) embedded polyelectrolyte films to enhance the oxygen barrier properties of poly(ethylene terephthalate) (PET) films. Despite cheaper costs and high mechanical strength, the diffusion of small gas molecules such as oxygen through PET films remains a matter of great concern. The simple yet robust supramolecular deposition of GNP/polyelectrolyte on PET substrates significantly increases the tortuous path the oxygen molecule has to travel, making it harder to diffuse through the PET film. With permeability coefficients in the range of 10-18 cc cm/cm(2) s Pa, the coatings developed in this study show three orders of magnitude reduction as compared to the permeability coefficient of the bare PET film, significantly lower than that of ethylene vinyl alcohol (EVOH) and comparable to silicon oxide thin films used in commercial gas barrier foils. The use of GNPs in the multilayered films also helped reduce the electrical sheet resistance to about 1MΩ which is five orders of magnitude lower than the original PET substrate opening up promising opportunities for future use in semiconductor and electronics industry. Making suitable modifications in the deposition process, three configurations of GNP embedded PEM multilayers namely hydrogen bonded, electrostatic, and composite films were developed and their effect on oxygen barrier property and sheet resistance was monitored. Oxygen permeability of films was tested in accordance with ASTM D-3985 using a MOCON 2/21 ML instrument, whereas electrical sheet resistance was quantified using a Gamry Femtostat Electrochemical Impedance station.

Enzyme production by the mixed fungal culture with nano-shear pretreated biomass and lignocellulose hydrolysis.

Cellulase, xylanase, and ß-glucosidase production was studied on novel nano-shear pretreated corn stover by the mixed fungi culture. The high shear force from a modified Tayor-Couette nano-shear mixing reactor efficiently disintegrated corn stover, resulting in a homogeneous watery mash with particles in much reduced size. Scanning electron microscope study showed visible mini-pores on the fiber cell wall surface, which could improve the accessibility of the pretreated corn stover to microorganisms. Mixed fungal culture of Trichoderma reesei RUT-C30 and Aspergillus niger produced enzymes with higher cellulolytic and xylanolytic activities on corn stover pretreated with nano-shear mixing reactor, in comparison with other pretreatment methods, including acid and ammonia fiber explosion (AFEX) pretreatment. The hydrolytic potential of the whole fermentation broth from the mixed fungi was studied, and the possibility of applying the whole cell saccharification concept was also investigated to further reduce the cost of lignocellulose hydrolysis.

Impact of cationic polyelectrolyte on the nanoshear hybrid alkaline pretreatment of corn stover: morphology and saccharification study.

Cationic polyelectrolyte was first used as the additive in the nanoshear hybrid alkaline pretreatment of corn stover. The novel nanoshear hybrid pretreatment process was recently developed at MSU. The chemical compositions and morphologies were investigated by SEM, TEM, confocal CLSM, and XPS to elucidate the degradation mechanism of cellular structures. At room temperature and fast processing conditions (~2 min), lignin was found to redistribute on the inner and outer surfaces of the cell wall as lignin aggregate droplets instead of being extracted. Free microfibrils in the residues were also observed. The yields of enzymatic hydrolysis were enhanced for the pretreated corn stover with the aid of polyelectrolyte as an additive. We speculate that lignin was effectively modified which opened up the cell wall structure during the short pretreatment process and prevented non-productive binding of enzymes in the enzyme hydrolysis reaction.

Molecular self-assembly: smart design of surface and interface via secondary molecular interactions.

The molecular self-assembly of macromolecular species such as polymers, colloids, nano/microparticles, proteins, and cells when they interface with a solid/substrate surface has been studied for many years, especially in terms of molecular interactions, adsorption, and adhesion. Such fundamental knowledge is practically important in designing smart micro- and nanodevices and sensors, including biologically implantable ones. This review gives a brief sketch of molecular self-assembly and nanostructured multifunctional thin films that utilize secondary molecular interactions at surfaces and interfaces.

Carbon nanotubes tuned foam structures as novel nanostructured biocarriers for lignocellulose hydrolysis.

The use of immobilized enzymes during saccharification of lignocelluloses enables the continuous process of enzymatic hydrolysis and repeatable use of enzyme, resulting in reduced operational cost. Novel nano-biocarriers were developed by layer-by-layer deposition of carbon nanotube (CNT) on the foam structures, and their efficiency for enzyme immobilization was demonstrated with cellulase and ß-glucosidase. A three-fold enhancement was achieved in the activity of cellulase immobilized on CNT coated polyurethane foam. In addition, both cellulase and ß-glucosidase immobilized on the CNT-foam showed much better storage stability and operational stability than the ones immobilized on the commercial biocarrier (Celite), which is critical for a continuous operation. CNT coated monolith was also developed as a biocarrier, offering high surface area and geometric stability. These nano-biocarriers are promising candidates for the efficient saccharification of biomass and to reduce carbon footprint and cost of the equipment.

Time controlled release of arabinofuranosylcytosine (Ara-C) from agarose hydrogels using layer-by-layer assembly: an in vitro study.

Experimentally induced axonal regeneration is compromised by glial scar formation arising from leptomeningeal fibroblasts cells in and around the hydrogel scaffold implanted for nerve repair. Strategies are needed to prevent such fibroblastic reactive cell layer formation for enhanced axonal regeneration. Here, we implement the technique of layer-by-layer assembled degradable, hydrogen bonded multilayers on agarose hydrogels to incorporate an anti-mitotic drug (1-ß-D-arabinofuranosylcytosine (Ara-C)) within the agarose hydrogels. We show controlled release of Ara-C under physiological conditions over a period of days. The concentrations of Ara-C released from agarose at the different time points were sufficient to inhibit fibroblast growth in vitro, while not adversely affecting the viability of the neuronal cells.

POLYELECTROLYTE MULTILAYER STAMPING IN AQUEOUS PHASE AND NON-CONTACT MODE.

Polyelectrolyte multilayer (PEM) transfer printing has been previously achieved by stamping under dry conditions. Here, we show for the first time, that PEM can be transferred from a stamp to the base substrate under aqueous conditions whereby the two surfaces are in a non-contact mode. Degradable multilayers of (PAA/PEG)(10.5) followed by non-degradable multilayers of (PDAC/SPS)(80.5) were fabricated under acidic pH conditions on either PDMS or glass (stamp), and subsequently transferred over top of another multilayer prepared on a different substrate (base substrate), with a spacing of ~ 200 µm between the stamping surface and the base substrate. This multilayer transfer was performed under physiological pH conditions. This process is referred to herein as non-contact, aqueous-phase multilayer (NAM) transfer. NAM transfer can be useful for applications such as fabricating three-dimensional (3-D) cellular scaffolds. We attempted to create a 3-D cellular scaffold using NAM transfer, and characterized the scaffolds with conventional and fluorescence microscopy.

Identification of a novel benzimidazole that inhibits bacterial biofilm formation in a broad-spectrum manner.

Bacterial biofilm formation causes significant industrial economic loss and high morbidity and mortality in medical settings. Biofilms are defined as multicellular communities of bacteria encased in a matrix of protective extracellular polymers. Because biofilms have a high tolerance for treatment with antimicrobials, protect bacteria from immune defense, and resist clearance with standard sanitation protocols, it is critical to develop new approaches to prevent biofilm formation. Here, a novel benzimidazole molecule, named antibiofilm compound 1 (ABC-1), identified in a small-molecule screen, was found to prevent bacterial biofilm formation in multiple Gram-negative and Gram-positive bacterial pathogens, including Pseudomonas aeruginosa and Staphylococcus aureus, on a variety of different surface types. Importantly, ABC-1 itself does not inhibit the growth of bacteria, and it is effective at nanomolar concentrations. Also, coating a polystyrene surface with ABC-1 reduces biofilm formation. These data suggest ABC-1 is a new chemical scaffold for the development of antibiofilm compounds.

Cell adhesive behavior on thin polyelectrolyte multilayers: cells attempt to achieve homeostasis of its adhesion energy.

Linearly growing ultrathin polyelectrolyte multilayer (PEM) films of strong polyelectrolytes, poly(diallyldimethylammonium chloride) (PDAC), and sulfonated polystyrene, sodium salt (SPS) exhibit a gradual shift from cytophilic to cytophobic behavior, with increasing thickness for films of less than 100 nm. Previous explanations based on film hydration, swelling, and changes in the elastic modulus cannot account for the cytophobicity observed with these thin films as the number of bilayers increases. We implemented a finite element analysis to help elucidate the observed trends in cell spreading. The simulation results suggest that cells maintain a constant level of energy consumption (energy homeostasis) during active probing and thus respond to changes in the film stiffness as the film thickness increases by adjusting their morphology and the number of focal adhesions recruited and thereby their attachment to a substrate.

Time Controlled Protein Release from Layer-by-Layer Assembled Multilayer Functionalized Agarose Hydrogels.

Axons of the adult central nervous system exhibit an extremely limited ability to regenerate after spinal cord injury. Experimentally generated patterns of axon growth are typically disorganized and randomly oriented. Support of linear axonal growth into spinal cord lesion sites has been demonstrated using arrays of uniaxial channels, templated with agarose hydrogel, and containing genetically engineered cells that secrete brain-derived neurotrophic factor (BDNF). However, immobilizing neurotrophic factors secreting cells within a scaffold is relatively cumbersome, and alternative strategies are needed to provide sustained release of BDNF from templated agarose scaffolds. Existing methods of loading the drug or protein into hydrogels cannot provide sustained release from templated agarose hydrogels. Alternatively, here it is shown that pH-responsive H-bonded poly(ethylene glycol)(PEG)/poly(acrylic acid)(PAA)/protein hybrid layer-by-layer (LbL) thin films, when prepared over agarose, provided sustained release of protein under physiological conditions for more than four weeks. Lysozyme, a protein similar in size and isoelectric point to BDNF, is released from the multilayers on the agarose and is biologically active during the earlier time points, with decreasing activity at later time points. This is the first demonstration of month-long sustained protein release from an agarose hydrogel, whereby the drug/protein is loaded separately from the agarose hydrogel fabrication process.

Multilayer mediated forward and patterned siRNA transfection using linear-PEI at extended N/P ratios.

Gene delivery from a substrate depends, in part, on the vector-nucleic acid complex that is bound to the surface and the cell adhesive properties of the surface. Here, we present a method to deliver patterns of small interfering RNA (siRNA) that capitalize on a forward transfection method (transfection by introducing siRNA transfection reagent complexes onto plated cells); herein denoted as multilayer mediated forward transfection (MFT). This method separates the substrate-mediated delivery from the cell adhesive properties of the surface. pH responsive layer-by-layer (LbL) assembled multilayers were used as the delivery platform and microcontact printing technique (microCP) was used to pattern nanoparticles of transfection reagent-siRNA complexes onto degradable multilayers. Efficient MFT depend on optimal formulation of the nanoparticles. 25 kDa linear polyethylenimine (LPEI) was optimized as the siRNA transfection reagent for normal forward transfection (NFT) of the nanoparticles. A broad range of LPEI-siRNA nitrogen/phosphate (N/P) ratios (ranging from 5 to 90) was evaluated for the relative amounts of siRNA incorporated into the nanoparticles, nanoparticle size and NFT efficiencies. All the siRNA was incorporated into the nanoparticles at N/P ratio near 90. Increasing the amount of siRNA incorporated into the nanoparticles, with increasing N/P ratio correlated with a linear blue shift in the ultraviolet/visible (UV/vis) absorbance spectrum of the LPEI-siRNA nanoparticles. NFT efficiency greater than 80% was achieved with minimal cytotoxicity at N/P ratio of 30 and siRNA concentration of 200 nM. Similarly, MFT efficiency 60% was achieved for LPEI-siRNA nanoparticles at N/P ratios greater than 30.

Versatile bioelectronic interfaces on flexible non-conductive substrates.

Bioelectronic interfaces that establish electrical communication between redox enzymes and electrodes have potential applications as biosensors, biocatalytic reactors, and biological fuel cells. These interfaces are commonly formed on gold films deposited using physical vapor deposition (PVD) or chemical vapor deposition (CVD). PVD and CVD require deposition of a primer layer, such as titanium or chromium, and require the use of expensive equipment and cannot be used on a wide range of substrates. This paper describes a versatile new bench-top method to form bioelectronic interfaces containing a gold film, electron mediator, cofactor, and dehydrogenase enzyme (secondary alcohol dehydrogenase, and sorbitol dehydrogenase) on nonconductive substrates such as polystyrene and glass. The method combines layer-by-layer deposition of polyelectrolytes, electroless metal deposition, and directed molecular self-assembly. Cyclic voltammetry, chronoamperometry, field emission X-ray dispersive spectroscopy, scanning electron microscopy, and atomic force microscopy were used to characterize the bioelectronic interfaces. Interfaces formed on flexible polystyrene slides were shown to retain their activity after bending to a radius of curvature of 18mm, confirming that the approach can be applied on cheap and flexible substrates for applications where traditional wafer-scale electronics is not suitable, such as personal or structural health monitors and rolled microtube biosensors.

Nanometal-decorated exfoliated graphite nanoplatelet based glucose biosensors with high sensitivity and fast response.

We report the novel fabrication of a highly sensitive, selective, fast responding, and affordable amperometric glucose biosensor using exfoliated graphite nanoplatelets (xGnPs) decorated with Pt and Pd nanoparticles. Nafion was used to solubilize metal-decorated graphite nanoplatelets, and a simple cast method with high content organic solvent (85 wt %) was used to prepare the biosensors. The addition of precious metal nanoparticles such as platinum (Pt) and palladium (Pd) to xGnP increased the electroactive area of the electrode and substantially decreased the overpotential in the detection of hydrogen peroxide. The Pt-xGnP glucose biosensor had a sensitivity of 61.5+/-0.6 microA/(mM x cm(2)) and gave a linear response up to 20 mM. The response time and detection limit (S/N=3) were determined to be 2 s and 1 microM, respectively. Therefore, this novel glucose biosensor based on the Pt nanoparticle coated xGnP is among the best reported to date in both sensing performance and production cost. In addition, the effects of metal nanoparticle loading and the particle size on the biosensor performance were systematically investigated.

Primary Neuron/Astrocyte Co-Culture on Polyelectrolyte Multilayer Films: A Template for Studying Astrocyte-Mediated Oxidative Stress in Neurons.

We engineered patterned co-cultures of primary neurons and astrocytes on polyelectrolyte multilayer (PEM) films without the aid of adhesive proteins/ligands to study the oxidative stress mediated by astrocytes on neuronal cells. A number of studies have explored engineering co-culture of neurons and astrocytes predominantly using cell lines rather than primary cells owing to the difficulties involved in attaching primary cells onto synthetic surfaces. To our knowledge this is the first demonstration of patterned co-culture of primary neurons and astrocytes for studying neuronal metabolism. In our study, we used synthetic polymers, namely poly(diallyldimethylammoniumchloride) (PDAC) and sulfonated poly(styrene) (SPS) as the polycation and polyanion, respectively, to build the multilayers. Primary neurons attached and spread preferentially on SPS surfaces, while primary astrocytes attached to both SPS and PDAC surfaces. SPS patterns were formed on PEM surfaces, either by microcontact printing SPS onto PDAC surfaces or vice-versa, to obtain patterns of primary neurons and patterned co-cultures of primary neurons and astrocytes. We further used the patterned co-culture system to study the neuronal response to elevated levels of free fatty acids as compared to the response in separated monoculture by measuring the level of reactive oxygen species (ROS; a widely accepted marker of oxidative stress). The elevation in the ROS levels was observed to occur earlier in the patterned co-culture system than in the separated monoculture system. The results suggest that this technique may provide a useful tool for engineering neuronal co-culture systems, that may more accurately capture neuronal function and metabolism, and thus could be used to obtain valuable insights into neuronal cell function and perhaps even the pathogenesis of neurodegenerative diseases.