Paul R. Van Tassel

History dependence of protein adsorption kinetics

The behavior of proteins at biological and synthetic interfaces is often characterized by a strong history dependence caused by long relaxation times or irreversible transitions. In this work, we introduce the rate of adsorption as a means to systematically quantify the extent, and identify the underlying causes, of history dependence. We use multistep kinetic experiments in which the i′th step is an exposure of a Si(Ti)O2 surface to a flowing fibronectin or cytochrome c solution of concentration ci for a time ti (ci = 0 corresponds to a rinse) and measure the protein adsorption by optical waveguide light mode spectroscopy. The rate of adsorption is sensitive to the structure of the adsorbed layer, and we observe it to greatly increase, for a given adsorbed density, when going from a first to a subsequent adsorption step. This increase is most pronounced when the duration of the initial adsorption step is long. We attribute these observations to the gradual and irreversible formation of protein clusters or locally ordered structures and use them to explain previous underestimates of kinetic data by mesoscopic model descriptions. A thorough understanding of these complex postadsorption events, and their impact on history dependence, is essential for many physiological and biotechnological processes. Optical waveguide lightmode spectroscopy is a promising technique for their macroscopic quantification.

Multilayer Nanofilms as Substrates for Hepatocellular Applications

Multilayer nanofilms, formed by the layer-by-layer (LbL) adsorption of positively and negatively charged polyelectrolytes, are promising substrates for tissue engineering. We investigate here the attachment and function of hepatic cells on multilayer films in terms of film composition, terminal layer, rigidity, charge, and presence of biofunctional species. Human hepatocellular carcinoma (HepG2) cells, adult rat hepatocytes (ARH), and human fetal hepatoblasts (HFHb) are studied on films composed of the polysaccharides chitosan (CHI) and alginate (ALG), the polypeptides poly(l-lysine) (PLL) and poly(l-glutamic acid) (PGA), and the synthetic polymers poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS). The influence of chemical cross-linking following LbL assembly is also investigated. We find HepG2 to reach confluence after 7 days of culture on only 2 of 18 candidate multilayer systems: (PAH-PSS)(n) (i.e. nPAH-PSS bilayers) and cross-linked (PLL-ALG)(n)-PLL. Cross-linked PLL-ALG and PLL-PGA films support attachment and function of ARH, independently of the terminal layer, provided collagen is adsorbed to the top of the film. (PAH-PSS)(n), cross-linked (PLL-ALG)(n), and cross-linked (PLL-PGA)(n)-PLL films all support attachment, layer confluence, and function of HFHb, with the latter film promoting the greatest level of function at 8 days. Overall, film composition, terminal layer, and rigidity are key variables in promoting attachment and function of hepatic cells, while film charge and biofunctionality are somewhat less important. These studies reveal optimal candidate multilayer biomaterials for human liver tissue engineering applications.

Antimicrobial biomaterials based on carbon nanotubes dispersed in poly(lactic-co-glycolic acid)

Biomaterials that inactivate microbes are needed to eliminate medical device infections. We investigate here the antimicrobial nature of single-walled carbon nanotubes (SWNTs) incorporated within the biomedical polymer poly(lactic-co-glycolic acid) (PLGA). We find Escherichia coli and Staphylococcus epidermidis viability and metabolic activity to be significantly diminished in the presence of SWNT-PLGA, and to correlate with SWNT length and concentration (<2% by weight). Up to 98% of bacteria die within one hour on SWNT-PLGA versus 15-20% on pure PLGA. Shorter SWNTs are more toxic, possibly due to increased density of open tube ends. This study demonstrates the potential usefulness of SWNT-PLGA as an antimicrobial biomaterial.

A kinetic model of partially reversible protein adsorption

We present a kinetic adsorption model for proteins that accounts for the experimentally observed properties of partial reversibility and surface induced conformational change. Particles (proteins) are modeled as disks that adsorb sequentially and without overlap at random positions onto a surface. Following adsorption, a particle can either desorb or spread symmetrically to a larger size. If the latter occurs, it remains adsorbed irreversibly. Both of these events obey first order kinetic rate laws. We derive analytical results in the asymptotic regime and report Monte Carlo results for shorter times. This model yields adsorbed phases that are more dense than those predicted by models of purely irreversible adsorption. We attribute this densification to a fluid structure that is quite liquidlike. We show that a number of experimentally observed kinetic behaviors can be reproduced with this model and that it is in good quantitative agreement with recent experiments.

Biosensing under an applied voltage using optical waveguide lightmode spectroscopy

An applied dc voltage offers a means of controlling immobilization during biosensor fabrication and detection during biosensing application. We present a method to directly and continuously measure the adsorption of biomacromolecules or other polyelectrolytes, under an applied potential difference, based on optical waveguide lightmode spectroscopy (OWLS). An indium tin oxide (ITO) film of thickness ca. 10 nm coated onto a silicon titanium oxide (STO) waveguiding film serves as the working (sensing) electrode. We observe the effective refractive index of the 0th transverse electric guided mode to increase significantly in the presence of an applied potential due to charging of the interfacial double layer and, possibly, modest electrochemical oxidation. Adsorption from solution onto the ITO electrode is detected by a further increase in the effective refractive index. We achieve accurate detection by employing an optical model in which the STO and ITO layers are combined into a single waveguiding film. No improvement is found using models treating the ITO as a separate layer, either dielectric or conducting. Using this method, we find the adsorption of human serum albumin and horse heart cytochrome c to be considerably enhanced in the presence of an applied potential exceeding 1 V. We attribute this behavior to adsorption at positions on the protein molecules of complementary charge.

Carbon nanotube-based antimicrobial biomaterials formed via layer-by-layer assembly with polypeptides.

Biomaterials capable of suppressing microbial infection are of clear importance in various health care applications, e.g. implantable devices. In this study, we investigate the antimicrobial activity of single-walled carbon nanotubes (SWNT) layer-by-layer (LbL) assembled with the polyelectrolytes poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA). SWNT dispersion in aqueous solution is achieved through the biocompatible nonionic surfactant polyoxyethylene(20) sorbitan monolaurate (Tween 20), and the amphiphilic polymer phospholipid-poly(ethylene glycol) (PL-PEG). Absorbance spectroscopy and transmission electron microscopy (TEM) show SWNT with either Tween 20 or PL-PEG in aqueous solution to be well dispersed, at about the level of SWNT in chloroform. Quartz crystal microgravimetry with dissipation (QCMD) measurements show both SWNT-Tween and SWNT-PL-PEG to LbL assemble with PLL and PGA into multilayer films, with the PL-PEG system yielding the greater final SWNT content. Escherichia coli and Staphylococcus epidermidis inactivation rates are significantly higher (up to 90%) upon 24h incubation with SWNT containing films, compared to control films (ca. 20%). This study demonstrates the potential usefulness of SWNT/PLL/PGA thin films as antimicrobial biomaterials.

Lattice model and simulation of dynamics of adsorbate motion in zeolites

Abstract Previous Monte Carlo simulations of the distribution of adsorbates in zeolites have suggested that adsorbates are confined to a lattice of sites in the micropore. In this work, we examine the effect of the structure and energetics of the adsorption site lattice on the mobility of small molecules in cage-like micropores using Monte Carlo lattice dynamics (MCLD) simulations. In MCLD, motion is modeled as a series of activated site-to-site hops over energetic and entropic barriers whose magnitudes can be functions of zeolite structure, sorbate chemistry, and loading. The topology of the lattice is such that adsorbate hops are of two types: (i) between sites in a given case, and (ii) between sites in neighboring cages. For comparison, an analytical model of adsorbate self-diffusivity is constructed by applying a random walk theory to this lattice. This model is exact at low loading and approximates the dynamics well even for a crowded lattice. We also compare MCLD to molecular dynamics (MD) simulations of methane adsorbed in zeolite A and observe qualitative agreement between the two approaches. However, the computational cost of MCLD is an order of magnitude lower than MD.

Nanofilm Biomaterials: Localized Cross-Linking To Optimize Mechanical Rigidity and Bioactivity

Nanofilm biomaterials, formed by the layer-by-layer assembly of charged macromolecules, are important systems for a variety of cell-contacting biomedical and biotechnological applications. Mechanical rigidity and bioactivity are two key film properties influencing the behavior of contacting cells. Increased rigidity tends to improve cells attachment, and films may be rendered bioactive through the incorporation of proteins, peptides, or drugs. A key challenge is to realize films that are simultaneously rigid and bioactive. Chemical cross-linking of the polymer framework--the standard means of increasing a films rigidity--can diminish bioactivity through deactivation or isolation of embedded biomolecules or inhibition of film biodegradation. We present here a strategy to decouple mechanical rigidity and bioactivity, potentially enabling nanofilm biomaterials that are both mechanically rigid and bioactive. Our idea is to selectively cross-link the outer region of the film, resulting in a rigid outer skin to promote cell attachment, while leaving the film interior (with any embedded bioactive species) unaffected. We propose an approach whereby an N-hydroxysulfosuccinimide (sulfo-NHS) activated poly(L-glutamic acid) is added as the terminal layer of a multilayer film and forms (covalent) amide bonds with amino groups of poly(L-lysine) placed previously within the film. We characterize film assembly and cross-linking extent via quartz crystal microbalance with dissipation monitoring (QCMD), Fourier transform infrared spectroscopy in attenuated total reflection mode (FTIR-ATR), and laser scanning confocal microscopy (LSCM) and measure the attachment and metabolic activity of preosteoblastic MC3T3-E1 cells. We show cross-linking to occur primarily at the film surface and the subsequent cell attachment and metabolic activity to be enhanced compared to native films. Our method appears promising as a means to realize films that are simultaneously mechanically rigid and bioactive.

Continuous polyelectrolyte adsorption under an applied electric potential

Interactions between charged macromolecules (e.g., proteins, nucleic acids, polyelectrolytes) and charged surfaces govern many natural and industrial processes. We investigate here the influence of an applied electric potential on the adsorption of charged polymers, and report the following significant result: the adsorption of certain amine side chain-containing polycations may become continuous, i.e., asymptotically linear (or nearly linear) in time over hours, upon the application of a modest anodic potential. Employing optical waveguide lightmode spectroscopy (OWLS) and an indium tin oxide (ITO) substrate, we show that asymptotic kinetics, and the adsorbed mass at the onset of the asymptotic regime, depend sensitively on polymer chemistry (in particular, side chain volume and charge location), increase with applied potential and ionic strength (conditions favoring a thicker initial layer), and are independent of bulk polymer concentration (suggesting postadsorption events to be rate limiting). X-ray photoelectron spectra reveal a suppressed polymer charge within layers formed via continuous adsorption, but no evidence of electrochemical reactions. We propose a mechanism based on polymer–polymer binding within the adsorbed layer, enabled by suppressed electrostatic repulsion and/or enhanced ionic correlations near the conducting surface, and stabilized by short-range attractive interactions. Continuous adsorption under an applied electric potential offers the possibility of nanoscale films of tailored polymer content realized in a single step.

Poly(lactide-co-glycolide) nanoparticle assembly for highly efficient delivery of potent therapeutic agents from medical devices.

Controlled delivery of therapeutic agents from medical devices can improve their safety and effectiveness in vivo, by ameliorating the surrounding tissue responses and thus maintaining the functional integrity of the devices. Previously, we presented a new method for providing simultaneous controlled delivery from medical devices, by surface assembly of biodegradable polymer nanoparticles (NPs) encapsulating fluorescent dyes. Here, we continue our investigation with NPs loaded with therapeutic agents, dexamethasone (DEX) or plasmid DNA, and evaluated the bioactivity of the released molecules with macrophage cells associated with inflammation. Over a period of one week, NPs encapsulating DEX released 24.9+/-0.8ng from the probe surface and was successful at suppressing macrophage cell growth by 40+/-10%. This percentage of suppression corresponded to approximately 100% drug delivery efficiency, in comparison with the unencapsulated drug. DNA NP coatings, in contrast, released approximately 1ng of plasmid DNA and were effective at transfecting macrophage cells to express the luciferase gene at 300+/-200 relative luminescence/mg total protein. This amount of luciferase activity corresponded to 100% gene delivery efficiency. Thus, NP coatings were capable of providing continuous release of bioactive agents in sufficient quantities to induce relevant biological effects in cell culture studies. These coatings also remained intact, even after 14 days of incubation with phosphate buffered saline. Although the maximum loading for NP coatings is inherently lower than the more established matrix coating, our study suggests that the NP coatings are a more versatile and efficient approach toward drug delivery or gene delivery from a medical device surface and are perhaps best suited for continuous release of highly potent therapeutic agents.