Equilibrium state model for surfactants in oils: Colloidal assembly and adsorption.

An equilibrium state model addressing the aggregation and adsorption of colloidal assemblies in apolar solvents (oils) via monomer exchange is presented. The model is based on the previously reported step-wise aggregation response of fatty acids and monoglycerides in bio-oils, and captures surface crowding via scaled particle theory (SPT). The sensitivity of key observables - mean aggregation number, adsorbed surfactant amount, and free monomer concentration - to model parameters is demonstrated. Fits to molecular modelling based aggregation and adsorption data of oleic acid and monoolein reveal that the model accurately reproduces chemically specific aggregate exponential distributions in both bulk and surface phases, even outside of its parameterization conditions. A biased state model, where the initial bulk aggregation step (dimer formation) differs from other steps results in a notable improvement in accuracy. Fits to various phospholipid adsorption isotherms demonstrate the applicability of the model to isotherm type experimental data. The fits reveal either monolayer or aggregate like adsorption structures, depending on surfactant head group charge. The presented model provides an easily accessible, computationally feasible means to estimate colloidal assembly and adsorption in oil environments, and enables assessment of surfactant aggregation propensity and adsorption energetics.

Polyelectrolyte Influence on Beta-Hairpin Peptide Stability: A Simulation Study.

Assemblies of proteins and charged macromolecules (polyelectrolytes) find important applications as pharmaceutical formulations, biocatalysts, and cell-contacting substrates. A key question is how the polymer component influences the structure and function of the protein. The present paper addresses the influence of charged polymers on the thermal stability of two model beta-hairpin-forming peptides through an all-atom, replica exchange molecular dynamics simulation. The (negatively charged) peptides consist of the terminal 16 amino acids of the B1 domain of Protein G (GB1) and a variant with three of the GB1 residues substituted with tryptophan (Tryptophan Zipper 4, or TZ4). A (cationic) lysine polymer is seen to thermally stabilize TZ4 and destabilize GB1, while a (also cationic) chitosan polymer slightly stabilizes GB1 but has essentially no effect on TZ4. Free energy profiles reveal folded and unfolded conformations to be separated by kinetic barriers generally acting in the direction of the thermodynamically favored state. Through application of an Ising-like statistical mechanical model, a mechanism is proposed based on competition between (indirect) entropic stabilization of folded versus unfolded states and (direct) competition for hydrogen-bonding and hydrophobic interactions. These findings have important implications to the design of polyelectrolyte-based materials for biomedical and biotechnological applications.

Weak polyelectrolyte-based multilayers via layer-by-layer assembly: Approaches, properties, and applications.

Layer-by-layer (LbL) assembly is a nanoscale technique with great versatility, simplicity and molecular-level processing of various nanoscopic materials. Weak polyelectrolytes have been used as major building blocks for LbL assembly providing a fundamental and versatile tool to study the underlying mechanisms and practical applications of LbL assembly due to its pH-responsive charge density and molecular conformation. Because of high-density uncompensated charges and high-chain mobility, weak polyelectrolyte exponential multilayer growth is considered one of the fastest developing areas for organized molecular films. In this article, we systematically review the current status and developments of weak polyelectrolyte-based multilayers including all-weak-polyelectrolyte multilayers, weak polyelectrolytes/other components (e.g. strong polyelectrolytes, neutral polymers, and nanoparticles) multilayers, and exponentially grown weak polyelectrolyte multilayers. Several key aspects of weak polyelectrolytes are highlighted including the pH-controllable properties, the responsiveness to environmental pH, and synergetic functions obtained from weak polyelectrolyte/other component multilayers. Throughout this review, useful applications of weak polyelectrolyte-based multilayers in drug delivery, tunable biointerfaces, nanoreactors for synthesis of nanostructures, solid state electrolytes, membrane separation, and sensors are highlighted, and promising future directions in the area of weak polyelectrolyte-based multilayer assembly such as fabrication of multi-responsive materials, adoption of unique building blocks, investigation of internal molecular-level structure and mechanism of exponentially grown multilayers, and exploration of novel biomedical and energy applications are proposed.

Repulsion between oppositely charged rod-shaped macromolecules: Role of overcharging and ionic confinement.

The interaction between two oppositely charged rod-shaped macro-ions in a micro-ion solution is investigated via Monte Carlo simulations of the primitive model. The focus is on the asymmetry in rod and/or ion charge, i.e., conditions where oppositely charged objects can repel one another. For equally and oppositely charged rods with asymmetric z:1 micro-ions, repulsion may be induced by overcharging one of the rods with the z valent ions. For asymmetrically charged rods in a symmetric z:z micro-ion solution, a repulsive interaction-at separation of the order of one ion diameter-can arise via an unbalanced osmotic pressure contribution from the ionic atmosphere in the inter-rod space, and an attractive interaction-at a smaller separation-may occur due to a "squeezing out" of the micro-ions from the space between the rods (with a consequent gain in entropy). The thermodynamics of each mechanism is investigated in terms of rod charge and size and micro-ion valence, size, and concentration. Our findings contribute to the understanding of the complex role of charge asymmetry on the interaction of, for example, oppositely charged polyelectrolytes, functionalized nanotubes, and rod-like biomolecules, e.g., viruses.

Fibronectin-based multilayer thin films.

Thin films mimicking the structure and composition of the extra-cellular matrix (ECM) are potentially attractive as biomaterials for cell contacting applications. Layer-by-layer (LbL) assembly of a biological polycation, poly(l-lysine) (PLL), and a common ECM protein, fibronectin (Fn), was employed here to construct nanoscale, ECM mimicking films. Incremental film thickness and interfacial charge magnitude are observed to diminish with layer number, resulting in sub-linear film growth scaling and saturation after about 10 layers. Infrared spectroscopy and electron microscopy together reveal the formation of Fn containing aggregates, whose presence correlates with diminished charge reversal and suppressed LbL assembly. PLL-Fn films induce a significantly greater murine MC3T3-E1 pre-osteoblastic cell proliferation, while maintaining a much higher proportion of Fn in the molecular (as opposed to fibrillar) state, compared to a Fn monolayer, suggesting the enhanced Fn content of these ECM-mimicking films to significantly, and positively, affect cell behavior.

Interaction modes between asymmetrically and oppositely charged rods.

The interaction of oppositely and asymmetrically charged rods in salt-a simple model of (bio)macromolecular assembly-is observed via simulation to exhibit two free energy minima, separated by a repulsive barrier. In contrast to similar minima in the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, the governing mechanism includes electrostatic attraction at large separation, osmotic repulsion at close range, and depletion attraction near contact. A model accounting for ion condensation and excluded volume is shown to be superior to a mean-field treatment in predicting the effect of charge asymmetry on the free-energy profile.

Nanotemplated polyelectrolyte films as porous biomolecular delivery systems. Application to the growth factor BMP-2.

Biomaterials capable of delivering controlled quantities of bioactive agents, while maintaining mechanical integrity, are needed for a variety of cell contacting applications. We describe here a nanotemplating strategy toward porous, polyelectrolyte-based thin films capable of controlled biomolecular loading and release. Films are formed via the layer-by-layer assembly of charged polymers and nanoparticles (NP), then chemically cross-linked to increase mechanical rigidity and stability, and finally exposed to tetrahydrofuran to dissolve the NP and create an intra-film porous network. We report here on the loading and release of the growth factor bone morphogenetic protein 2 (BMP-2), and the influence of BMP-2 loaded films on contacting murine C2C12 myoblasts. We observe nanotemplating to enable stable BMP-2 loading throughout the thickness of the film, and find the nanotemplated film to exhibit comparable cell adhesion, and enhanced cell differentiation, compared with a non-porous cross-linked film (where BMP-2 loading is mainly confined to the film surface).

Layer-by-layer films as biomaterials: bioactivity and mechanics.

Layer-by-layer (LbL) assembly offers a facile approach toward biomaterials of nanoscale thickness. Bioactivity may be realized through incorporation of biomolecular species within the film architecture, and film mechanics may be controlled through post-formation chemical cross-linking steps. This short review focuses on recent efforts toward the simultaneous control of LbL film bioactive and mechanical properties, with a particular focus on approaches yielding cellular outcomes dependent on both of these properties. Challenges and opportunities toward the independent control of film rigidity and bioactivity are highlighted.

Nanotechnology in medicine: nanofilm biomaterials.

By interrogating nature at the length scale of important biological molecules (proteins, DNA), nanotechnology offers great promise to biomedicine. We review here our recent work on nanofilm biomaterials: "nanoscopically" thin, functional, polymer-based films serving as biocompatible interfaces. In one thrust, films containing carbon nanotubes are shown to be highly antimicrobial and, thus, to be promising as biomedical device materials inherently resistive to microbial infection. In another thrust, strategies are developed toward films of independently controllable bioactivity and mechanical rigidity - two key variables governing typical biological responses.

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.

Molecular recognition effects in atomistic models of imprinted polymers.

In this article we present a model for molecularly imprinted polymers, which considers both complexation processes in the pre-polymerization mixture and adsorption in the imprinted structures within a single consistent framework. As a case study we investigate MAA/EGDMA polymers imprinted with pyrazine and pyrimidine. A polymer imprinted with pyrazine shows substantial selectivity towards pyrazine over pyrimidine, thus exhibiting molecular recognition, whereas the pyrimidine imprinted structure shows no preferential adsorption of the template. Binding sites responsible for the molecular recognition of pyrazine involve one MAA molecule and one EGDMA molecule, forming associations with the two functional groups of the pyrazine molecule. Presence of these specific sites in the pyrazine imprinted system and lack of the analogous sites in the pyrimidine imprinted system is directly linked to the complexation processes in the pre-polymerization solution. These processes are quite different for pyrazine and pyrimidine as a result of both enthalpic and entropic effects.

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 film's 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.

Pre-osteoblasts on poly(L-lactic acid) and silicon oxide: Influence of fibronectin and albumin adsorption.

Cell adhesion and subsequent viability are critical initial steps in biomaterial-tissue integration and are strongly dependent on the material properties and the presence of matrix proteins. In the present study MC3T3-E1 osteoblast-like cell behavior on silicon oxide (SO) and poly(L-lactic acid) (PLLA) substrates has been examined, with a focus on the influence of the adhesive protein fibronectin and the non-adhesive protein albumin adsorbed on the substrates. Quartz crystal microgravimetry showed adsorption of fibronectin and albumin to be nearly identical on SO and PLLA. Subsequent exposure a previously adsorbed fibronectin layer to albumin decreased the rigidity of the adsorbed layer without any measurable increase in adsorbed mass. Cell adhesion and spreading were significantly enhanced on both SO and PLLA substrates coated with fibronectin or with fibronectin and albumin, compared with uncoated or albumin-coated substrates. The only statistically significant difference between the two substrates in these assays was increased spreading on PLLA compared with SO in the presence of fibronectin and albumin. Cell proliferation was significantly higher on SO compared with PLLA after 7 days culture, but depended on the presence of fibronectin only in the PLLA system. In contrast, mitochondrial activity was higher on PLLA than on SO, and was enhanced by fibronectin on both substrates. PLLA substrates coated with fibronectin and subsequently exposed to albumin exhibited the highest level of cell differentiation, as assayed via alkaline phosphatase activity. These results demonstrate the importance of adsorbed proteins on osteoblast-like cell-surface interactions.

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.

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.

Simultaneous release of multiple molecules from poly(lactide-co-glycolide) nanoparticles assembled onto medical devices.

Cell and tissue responses to implanted biomaterials often limit their effectiveness and lifetime. This is particularly true for materials implanted into the brain. We present here a new approach for the modification of materials to enable release of multiple agents, which might be useful in modulating tissue responses, without changing the properties of the underlying material, in this case, a silicon probe. Poly(lactide-co-glycolide) nanoparticles (NPs) were assembled onto silicon probe surfaces by electrostatic interactions. Charged NPs were fabricated by altering the properties of the surfactant. NPs formed with poly(ethylene-alt-maleic anhydride) (PEMA) were strongly negatively charged; these NPs assembled onto probes best when suspended at nearly physiological conditions (surface density approximately 83,600+/-3000 particles/mm(2)). The percentage of surface area coverage by the NPs was estimated to be approximately 13% and was maintained over two weeks during constant exposure to PBS. Multiple fluorescent NP populations were attached to the same probe to allow visualization of simultaneous delivery of multiple agents by fluorescence microscopy. Release from NP coatings was reproducible and controllable. The distinct release profiles of each agent from the coatings were preserved upon attachment to the surfaces. The unique feature of this new system is that NPs encapsulating various molecules (i.e. drugs, proteins, or DNA) can be fabricated separately, in advance, and simply mixed prior to attachment. The versatility of this delivery system, therefore, makes it suitable for many applications.

Influence of charged nanoparticles on colloidal forces: a molecular simulation study.

We employ the grand canonical Monte Carlo simulation technique to investigate the influence of charged nanoparticles (macro-ions) on the force between colloidal objects. Specifically, the structure and osmotic pressure of a system of screened Coulomb (Yukawa) particles confined between charged planar walls are simulated. We observe osmotic pressure to oscillate with wall separation and these oscillations to correspond to changes in the number of nanoparticle layers present in the slit pore. Using the Derjaguin approximation, we estimate the overall force between a colloidal sphere and a flat surface and compare our predictions to recent atomic force microscopy (AFM) results (Tulpar, A.; Van Tassel, P. R.; Walz, J. Y. Langmuir 2006, 22, 2876-2883). In excellent agreement with experiment, we find the wavelength of the force versus distance oscillations to scale as c(nu), with c being the bulk nanoparticle concentration and nu = -0.31 +/- 0.01; that is, slightly lower in magnitude from the expected value -1/3 based on average molecule spacing. By considering an order parameter measuring the extent to which neighboring particles form hexagonal symmetry, we show structural order within confined nanoparticle systems to be significantly enhanced as compared to that of bulk systems, despite being quite insensitive to wall separation. Wavelength scaling and order parameter analysis together suggest the confined macro-ion systems to be somewhat glasslike.

Polyelectrolyte adsorption kinetics under an applied electric potential: Strongly versus weakly charged polymers.

Previous work has demonstrated adsorption of weakly basic polycations to a conducting substrate to be continuous, i.e. asymptotically linear in time over hours, under an applied anodic potential [A.P. Ngankam, P.R. Van Tassel, Proc. Natl. Acad. Sci. USA 104 (2007) 1140]. Adsorption without apparent saturation requires an interfacial charge regulation, which is possible for weakly charged polymers via segment deprotonation. We investigate here whether deprotonation is a necessary condition for continuous adsorption by comparing the behavior of a weakly and a strongly charged polyelectrolyte, the latter containing permanently charged segments incapable of deprotonation. We employ optical waveguide lightmode spectroscopy (OWLS) to measure adsorption of poly(N-vinyl imidazole) (PVI), a weakly basic polycation, and quaternized poly(N-vinyl imidazole) (QPVI), a structurally similar polymer with ca. 20% of its monomers containing a permanent positive charge, onto indium tin oxide (ITO). Under open circuit conditions, we observe both PVI and QPVI adsorption to reach a rapid saturation and be essentially irreversible. In contrast, at an ITO potential of 1.5 V (versus hydrogen) in a 0.1 M NaCl solution, we observe adsorption of both PVI and QPVI to be continuous and reversible. In salt free solution, we observe PVI but not QPVI to exhibit continuous adsorption at 1.5 V, and for both polymers to be essentially irreversibly attached. We propose interfacial charge regulation to occur via a deprotonation mechanism for PVI, and via a counterion condensation mechanism for QPVI. Continuous adsorption is therefore possible for a strongly charged polyelectrolyte, via a counterion condensation mechanism; this finding opens the door to nanofilms of controlled polymer content containing permanent charges.

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.