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Molybdenum dioxide (MoO2), considering its near-metallic conductivity and surface plasmonic properties, is a great material for electronics, energy storage devices and biosensing. Yet to this day, room-temperature synthesis of large area MoO2, which allows deposition on arbitrary substrates, has remained a challenge. Due to their reactive interfaces and specific solubility conditions, gallium-based liquid metal alloys offer unique opportunities for synthesizing materials that can meet these challenges. Herein, a substrate-independent liquid metal-based method for the room temperature deposition and patterning of MoO2 is presented. By introducing a molybdate precursor to the surrounding of a eutectic gallium-indium alloy droplet, a uniform layer of hydrated molybdenum oxide (H2MoO3) is formed at the interface. This layer is then exfoliated and transferred onto a desired substrate. Utilizing the transferred H2MoO3 layer, a laser-writing technique is developed which selectively transforms this H2MoO3 into crystalline MoO2 and produces electrically conductive MoO2 patterns at room temperature. The electrical conductivity and plasmonic properties of the MoO2 are analyzed and demonstrated. The presented metal oxide room-temperature deposition and patterning method can find many applications in optoelectronics, sensing, and energy industries.

The stripe phase formed by long-chain alkane derivatives on the graphite lattice provides a unique opportunity for the study of molecular adsorption, aggregation, and reaction on patterns. Fatty acids, such as arachidic acid (AA), self-assemble on graphite into a sheet of parallel stripes with a periodicity of twice its molecular chain length. The molecular pattern is thus defined precisely by the size and functionality of the headgroup and tailgroup of the amphiphile. Complexation of metal ions to AA fixes the number and location of the ion, which can serve as a precursor to semiconductor nanocrystal arrays. In order to understand the effect of the ion complexation, we carry out atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) investigations of AA self-assembly in the presence of various metal ions. While the stripe orientation is dictated by the graphite lattice and the stripe periodicity is determined by the AA chain length, the size, shape, and degree of order of the stripe crystalline domain are influenced by the metal ion bond strength to the carboxylic ligand. The change of morphology in the self-assembled pattern shows a trend along the Irving-Williams series.

The current study was aimed to develop a targeted dendrimer formulation of 3, 4-difluorobenzylidene curcumin (CDF) and evaluate its potential in CD44 targeted therapy for pancreatic cancer. Using amine terminated fourth generation poly(amidoamine) (PAMAM) dendrimer nanocarrier and hyaluronic acid (HA) as a targeting ligand, we engineered a CD44-targeted PAMAM dendrimer (HA-PAMAM) formulation of CDF. The resulting dendrimer nanosystem (HA-PAMAM-CDF) had a particle size and surface charge of 9.3 ± 1.5 nm and -7.02 ± 9.53 mV, respectively. When CD44 receptor overexpressing MiaPaCa-2 and AsPC-1 human pancreatic cancer cells were treated with HA-PAMAM-CDF, a dose-dependent cytotoxicity was observed. Furthermore, blocking the CD44 receptors present on the MiaPaCa-2 cells using free excess soluble HA prior to treatment with HA-PAMAM-CDF nano-formulation resulted in 1.71 fold increase in the IC50 value compared to non-targeted formulation (PAMAM-CDF), confirming target specificity of HA-PAMAM-CDF. Additionally, HA-PAMAM-CDF formulation when compared to PAMAM-CDF, displayed higher cellular uptake in MiaPaCa-2 cancer cell lines as shown by fluorescence studies. In summary, the novel CD44 targeted dendrimer based nanocarriers appear to be proficient in mediating site-specific delivery of CDF via CD44 receptors, with an improved therapeutic margin and safety.

Lingxiao Xie, Xiong Ding, Rachel Budry, Guangzhao Mao Department of Chemical Engineering and Materials Science, Wayne State University, Detroit, MI, USA Background: The layer-by-layer (LbL) assembly method offers a molecular level control of the amount and spatial distribution of bioactive molecules. However, successful clinical translation of LbL film technology will most certainly require a better understanding and control of not only the film assembly process, but also film disassembly kinetics in physiologic conditions. Purpose: This work focuses on the understanding and control of degradation properties of LbL films for localized gene delivery. Methods: Bioreducible poly(amido amine)s (PAAs) containing cystaminebisacrylamide (CBA), methylenebisacrylamide, and 5-amino-1-pentanol (APOL) were synthesized by Michael addition polymerization for the construction of bioreducible LbL films capable of sequential gene delivery. Results: The synthesized PAAs were screened for desirable buffering capacity, cell transfection, and cytotoxicity characteristics together with 25 kDa branched polyethylenimine (PEI) and cross-linked 800 Da PEI. By screening the various polycations we were able to identify a copolymer of CBA and APOL for the subsequent construction of the LbL films. By incorporating a highly transfecting polycation and a nondiffusing polycation we were able to improve the overall transfection of HEK293 and MC3T3 cells from the bioreducible LbL films. We also demonstrated the dual-stage release and transfection of two different DNAs from the LbL films. Conclusion: The results indicate that LbL films consisting of bioreducible PAAs and non-diffusing polyelectrolytes have excellent degradation properties for the development of LbL coating technology for localized gene delivery applications. Keywords: biodegradable polymers, cell transfection, interlayer diffusion, localized gene delivery, polyelectrolyte multilayers, sequential release

This paper describes electrochemical synthesis of nanorods of tetrathiafulvalene (TTF) charge-transfer salt on gold nanoparticle (GNP) seeds. The seed-mediated process was monitored by cyclic voltammetry, AFM, and field-emission SEM. The electrodeposition of GNPs (nucleation seeds) on highly oriented pyrolytic graphite (HOPG) electrodes was studied as a function of the electrolytic conditions. The GNP size increases with increasing HAuCl4 concentration and decreasing applied overpotential. A morphological transition from quasi-spherical particles to dendritic aggregates occurs when the HAuCl4 concentration increases from 0.5 to 1 mM. The electrocrystallization of (TTF)Br0.76 on the GNP-decorated HOPG was investigated as a function of TTF concentration and GNP morphology. We observed a preferential nucleation of (TTF)Br0.76 on the GNP seed. The seed-mediated (TTF)Br0.76 crystals display a confined crystal morphology in comparison to those nucleated on bare HOPG. (TTF)Br0.76 nanorods as small as 7 nm in hei...

Nanoparticles (NPs) are 1-100 nm constructs explored for their application in cancer diagnosis and treatment. Exposure of cells to ionizing irradiation leads to DNA damage, with DNA double strand breaks (DSBs) being the most toxic that can lead to apoptosis. The presence of gold in NPs enhances radiation damage and DSBs, the latter measured by the levels of phosphorylated DNA histone protein H2AX (γH2AX). The goal of this study was to decipher biological mechanisms of NP radio-sensitization. We analyzed the effects of two different sizes of gold nanoparticles (AuNP) on DSBs in MCF-7 breast cancer cells by assessing H2AX phosphorylation at three photon energies. Cells were incubated with either 4 nm or 14 nm AuNP and irradiated with 2, 4 or 8 Gy using 2.5 FFF MV (60 MU/min), 6 MV (600 MU/min) or 10 FFF MV (2400 MU/min) X-rays. Percent of cells positive for γH2AX was determined by flow cytometry. Live cells (100000) were gated using forward (FSC; cell size) versus side scatter (SSC; complexity) characteristics (gate P1). Cells positive for γH2AX were further gated to identify subpopulations exhibiting brighter (gate P2) or dimmer (gate P3) fluorescence intensity. Treatment with 4 nm AuNP resulted in significantly more γH2AX positive cells after irradiation at all three energies, compared to their respective controls. When treated at 6 MV energy, increases in the percentage of cells positive for H2AX phosphorylation was detected at all three doses (2, 4 and 8 Gy). Increased phosphorylation was accompanied by an increase of cells in the P2 gate accompanied by an increase in their FCS and SSC characteristics, consistent with activation. In cells treated with 10 MV the effect was most pronounced at 4Gy dose, while 2 and 8 Gy resulted in a slight increase in the percentage of γH2AX positive cells. Nevertheless, these cells still exhibited prominent increases within the P2 gate and FCS vs SSC characteristics, compared to their irradiated controls. In cells treated with 2.5 MV energy similar results were observed using 4 and 8 Gy, while no effect was detected with 2 Gy. Data from the experiments using 14 nm size AuNP were less clear and require further investigation. The difference may be due to the localization of the smaller NPs within the nucleus and therefore cause more DNA damage thereby greater H2AX phosphorylation. We hypothesize that the NP radio-sensitization mechanism involves directing cells towards apoptosis by enhancing DNA damage and interfering with DNA repair. Hence, γH2AX positive cells exhibiting bright fluorescence (P2) and an increase in FCS and SSC identified even in the conditions without significant increase in the percent of total γH2AX positive cells, may reflect cells primed for apoptosis. Future studies are planned to elucidate the exact intracellular mechanisms of NP biological radio-sensitization effect. Citation Format: Branislava Janic, Fangchao Liu, Kevin Bobbitt, Stephen Brown, Guangzhao Mao, Indrin J. Chetty, Benjamin Movsas, Ning Winston Wen. Effect of gold nanoparticle on radiation induced DNA damage in MCF7 breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1376.

Dendrimers, with their well-defined globular shape and a high density of functional groups, are ideal nanoscale materials for templating sensor surfaces. This work exploits dendrimers as a versatile platform for capturing biomarkers with improved sensitivity and specificity. Synthesis, characterization, fabrication, and functional validation of the dendrimer-based assay platform are described. Bifunctional hydroxyl/thiol functionalized G4-polyamidoamine (PAMAM) dendrimer is synthesized and immobilized on to the polyethylene-glycol (PEG)-functionalized assay plate by coupling PEG-maleimide and dendrimer thiol groups. Simultaneously, part of the dendrimer thiol groups are converted to hydrazide functionalities. The resulting dendrimer-modified surface is coupled to the capture antibody in the Fc region of the oxidized antibody. This preserves the orientation flexibility of the antigen binding region (Fv) of the antibody. To validate the approach, the fabricated plates are further used as a solid phase for developing a sandwich type ELISA to detect IL-6 and IL-1β, important biomarkers for early stages of chorioamnionitis. The dendrimer-modified plate provides assays with significantly enhanced sensitivity, lower nonspecific adsorption, and a detection limit of 0.13 pg ml-1 for IL-6 luminol detection and 1.15 pg ml-1 for IL-1β TMB detection, which are significantly better than those for the traditional ELISA. The assays were validated in human serum samples from normal (non-pregnant) woman and pregnant women with pyelonephritis. The specificity and the improved sensitivity of the dendrimer-based capture strategy could have significant implications for the detection of a wide range of cytokines and biomarkers since the capture strategy could be applied to multiplex microbead assays, conductometric immunosensors and field effect biosensors.

The molecular disassembly and DNA release dynamics of bioreducible poly(amido amine) polyplexes were studied in real time by atomic force microscopy (AFM). Molecular disassembly of polyplexes constitutes a critical step along the nonviral gene delivery pathway. DNA release is triggered by the depolymerization of high-molecular-weight polycations into low-molecular-weight oligocations utilizing the thiol and disulfide exchange reaction mechanism. The research is motivated by low cytotoxicity and higher transfection of the poly(amido amine)s containing the disulfide bond than those without the disulfide linkage. AFM images were captured in simulated physiological reducing environment by employing dithiothreitol (DTT). Distinctive stages of polyplex disassembly common to the polyplexes with different disulfide content, molecular weight, and polymer architecture are revealed though the DNA release rate is dependent on the disulfide content. In stage one, upon the depolymerization, polyplexes evolve from metastable structures into the thermodynamically favorable toroid structure. In the second stage, toroids aggregate and sometimes fuse into larger ones. In the last stage DNA gradually unravels from the polyplex resulting in highly decondensed wormlike chains and loops that are held by a central compact core. The results affirm the promising aspect of bioreducible poly(amido amine)s as controlled DNA delivery vectors. The study offers new physical insights into the DNA release pathway including intermediate structures with a high degree of structural heterogeneity and disassembly-induced particle growth. The study identifies disassembly-induced colloidal and morphological instability as an important issue to be addressed in the development of successful polyelectrolyte-based gene delivery systems.

Drug resistance limits the success of many anticancer drugs. Reduced accumulation of the drug at its intracellular site of action because of overexpression of efflux transporters such as P-glycoprotein (P-gp) is a major mechanism of drug resistance. In this study, we investigated whether photodynamic therapy (PDT) using methylene blue, also a P-gp inhibitor, can be used to enhance doxorubicin-induced cytotoxicity in drug-resistant tumor cells. Aerosol OT (AOT)-alginate nanoparticles were used as a carrier for the simultaneous cellular delivery of doxorubicin and methylene blue. Methylene blue was photoactivated using light of 665 nm wavelength. Induction of apoptosis and necrosis following treatment with combination chemotherapy and PDT was investigated in drug-resistant NCI/ADR-RES cells using flow cytometry and fluorescence microscopy. Effect of encapsulation in nanoparticles on the intracellular accumulation of doxorubicin and methylene blue was investigated qualitatively using fluorescence microscopy and was quantitated using HPLC. Encapsulation in AOT-alginate nanoparticles significantly enhanced the cytotoxicity of combination therapy in resistant tumor cells. Nanoparticle-mediated combination therapy resulted in a significant induction of both apoptosis and necrosis. Improvement in cytotoxicity could be correlated with enhanced intracellular and nuclear delivery of the two drugs. Further, nanoparticle-mediated combination therapy resulted in significantly elevated reactive oxygen species (ROS) production compared to single drug treatment. In conclusion, nanoparticle-mediated combination chemotherapy and PDT using doxorubicin and methylene blue was able to overcome resistance mechanisms and resulted in improved cytotoxicity in drug-resistant tumor cells.

Layer-by-layer (LbL) assemblies of DNA and polycations on the surface of colloidal templates can be used for gene delivery. Plasmid DNA encoding for secreted alkaline phosphatase (SEAP) was used to deposit LbL films with poly(ethylenimine) (PEI) on the surface of polystyrene and poly(lactide-co-glycolide) microparticles. The formation of LBL films was confirmed by zeta potential analysis and fluorescence and atomic force microscopy techniques. The LbL particles were rapidly internalized in a dose-dependent manner by J774.1 murine macrophages. Transfection activity of the LbL particles was evaluated in J774.1 cells using three different doses (5, 10, 25 particle per cell). The levels of SEAP expression increased with increasing dose but were lower than transfection levels mediated by control PEI/DNA polyplexes at corresponding DNA doses. The LbL particles reported here present a promising platform for delivery of DNA to phagocytic cells.

Controlled intracellular disassembly of polyelectrolyte complexes of polycations and DNA (polyplexes) is a crucial step for the success of nonviral gene delivery. Motivated by our previous observation of different gene delivery performances among multiblock reducible copolypeptide vectors (Manickam, D. S., and Oupický, D. Bioconjugate Chem. 2006, 17, 1395−1403), atomic force microscopy is used to visualize plasmid DNA in various decondensed states from reducible polypeptide polyplexes under simulated physiological reducing conditions. DNA decondensation is triggered by reductive degradation of disulfide-containing cationic polypeptides. Striking differences in DNA release dynamics between polyplexes based on polypeptides of histidine-rich peptide (HRP, CKHHHKHHHKC) and nuclear localization signal (NLS, CGAGPKKKRKVC) peptide are presented. The HRP and NLS polyplexes are similar to each other in their initial morphology with a majority of them containing only one DNA plasmid. Upon reductive degradation by d...

Photodynamic therapy (PDT) is a promising treatment modality for cancer. PDT is based on the concept that photosensitizers, when exposed to light of specific wavelength, generate cytotoxic reactive oxygen species (ROS) capable of killing tumor cells. The effectiveness of PDT has been limited in part by the lack of photosensitizers that accumulate sufficiently in tumor cells and poor yield of ROS from existing photosensitizers. In this report, we investigated whether aerosol OT-alginate nanoparticles can be used as a carrier to enhance the therapeutic efficacy of a model photosensitizer, methylene blue. Methylene blue loaded nanoparticles were evaluated for PDT effectiveness in two cancer cell lines, MCF-7 and 4T1. Encapsulation of methylene blue in nanoparticles significantly enhanced intracellular ROS production, and the overall cytotoxicity following PDT. It also resulted in higher incidence of necrosis. Greater effectiveness of nanoparticles could be correlated with higher yield of ROS with nanoparticle-encapsulated methylene blue. Further, treatment of tumor cells with nanoparticle-encapsulated methylene blue resulted in significant nuclear localization of methylene blue while free drug treatment resulted in its accumulation mainly in the endolysosomal vesicles. In conclusion, encapsulation of methylene blue in aerosol OT-alginate nanoparticles enhanced its anticancer photodynamic efficacy in vitro. Increased ROS production and favorable alteration in the subcellular distribution contribute to the enhanced PDT efficacy of nanoparticle-encapsulated photosensitizer.

Poor drug encapsulation efficiency and rapid release of the encapsulated drug limit the use of nanoparticles in biomedical applications involving water-soluble drugs. We have developed a novel polymer-surfactant nanoparticle formulation, using the anionic surfactant Aerosol OT (AOT) and polysaccharide polymer alginate, for sustained release of water-soluble drugs. Particle size of nanoparticles, as determined by atomic force microscopy and transmission electron microscopy, was in the range of 40-70 nm. Weakly basic molecules like methylene blue, doxorubicin, rhodamine, verapamil, and clonidine could be encapsulated efficiently in AOT-alginate nanoparticles. In vitro release studies with basic drug molecules indicate that nanoparticles released 60-70% of the encapsulated drug over 4 weeks, with near zero-order release during the first 15 days. Studies with anionic drug molecules demonstrate poorer drug encapsulation efficiency and more rapid drug release than those observed with basic drugs. Further studies investigating the effect of sodium concentration in the release medium and the charge of the drug suggest that calcium-sodium exchange between nanoparticle matrix and release medium and electrostatic interaction between drug and nanoparticle matrix are important determinants of drug release. In conclusion, we have formulated a novel surfactant-polymer drug delivery carrier demonstrating sustained release of water-soluble drugs.

We report synthesis of temperature- and redox-responsive multiblock copolymers by reversible addition-fragmentation chain transfer (RAFT) polymerization. Well-defined α,ω-bis(dithioester)-functionalized poly(N-isopropylacrylamide) (PNIPAM) and poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA) were prepared using 1,4-bis(thiobenzoylthiomethyl)benzene and 1,4-bis(2-(thiobenzoylthio)prop-2-yl)benzene as RAFT agents, respectively. Dually responsive multiblock copolymers were synthesized in a single aminolysis/oxidation step from the α,ω-bis(dithioester)-terminated PNIPAM and PDMAEMA. The copolymers and their stimulus-responsive behavior were characterized by size exclusion chromatography, NMR, light scattering and atomic force microscopy. Due to the presence of redox-sensitive disulfide bonds between the blocks, the copolymers were readily reduced to the starting polymer blocks. The presence of temperature-responsive PNIPAM blocks provided the copolymers with the ability to assemble into core-shell nanostructures with hydrophobic PNIPAM as a core and cationic PDMAEMA as stabilizing shell when above the phase transition temperatures of PNIPAM. The temperature-induced assembly of the copolymers also showed substantial pH sensitivity. The phase transition temperature increased with decreasing pH, while molecular weight of the assemblies decreased.

This paper reports the disassembly of layer-by-layer (LbL) films of plasmid DNA and a reducible cationic polypeptide. To utilize a reducing microenvironment of cellular plasma membrane as a potential trigger, LbL films are assembled to contain both DNA and the TAT-based polypeptide (PTAT) with reducible disulfide bonds in the backbone. The assembly and disassembly processes are monitored by goniometry, ellipsometry, and atomic force microscopy (AFM). The structure of the PTAT films is compared with that of non-reducible poly( l -lysine) (PLL) films. Both PTAT and PLL films exhibit exponential growth but with the contact angle alternating between characteristic values. Ellipsometry and AFM show a gradual and complete disassembly of the PTAT but not the PLL films in a 24 h period in the reducing environment in vitro. This study suggests a potential of using reducible LbL films for controlled DNA delivery.

Increasingly, organosilane self-assembled monolayers (SAMs) are used to modify the surfaces of silicon-based sensors and atomic force microscope (AFM) probes. Organosilane SAMs are preferred due to their fast and easy preparation, stability, and applicability to a wide range of substrates. The traditional dip coating method from solution often yields ill-defined particulate aggregates on the two-dimensional SAM. The presence of such three-dimensional aggregates seriously reduces the performance of miniaturized biosensor devices and AFM probes. It is difficult to control the amount of water in solution-based deposition. This paper describes a chemical vapor deposition (CVD) method to deposit octadecyltrichlorosilane (OTS) monolayers on silicon wafers and AFM probes under vacuum condition. OTS coated surfaces with static water contact angle ranging from 20° to 107° can be obtained by controlling the deposition conditions. The silicon substrates and AFM probes after CVD are characterized by AFM, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and contact angle goniometry. The OTS monolayer is in a uniform low-density state below 65%. Above 65%, densely packed crystalline-like domains start to form. It takes 24 h to reach the adsorption saturation. The time span in the CVD deposition is much longer than the solution case and thus allowing precise variation of the substrate hydrophobicity for biosensor applications.

The reactivity of aminosilane and vinylsilane with a dihydroxy (Bisphenol AF) crosslinkable fluoroelastomer containing only vinylidene fluoride (VF2) and hexafluoropropylene (HFP) was monitored using Differential Scanning Calorimetry (DSC) and X-ray Photoelectron Spectroscopy (XPS). The DSC results showed that the introduction of an aminosilane, or a combination of aminosilane and vinylsilane solutions, to the cure system of the fluoroelastomer resulted in a peak shift. This shift indicates that crosslinking in fluoroelastomer-silane system occurs at a lower temperature when aminosilane is present. Results also confirm that the organofunctional group on the silane reacts with the elastomer. XPS analysis of the silane-coated fluoroelastomer heated at 120 °C, 160 °C and 200 °C reveals that dehydrofluorination of the polymer is one of the steps in the reaction. It is our hypothesis that the amine group on the aminosilane is a contributor to the dehydrofluorination of the fluoroelastomer.

Layer-by-layer (LbL) films containing cationic polyelectrolytes and anionic bioactive molecules such as DNA are promising biomaterials for controlled and localized gene delivery for a number of biomedical applications including cancer DNA vaccine delivery. Bioreducible LbL films made of disulfide-containing poly(amido amine)s (PAAs) and plasmid DNA can be degraded by redox-active membrane proteins through the thiol-disulfide exchange reaction to release DNA exclusively into the extracellular microenvironment adjacent to the film. In order to better understand the film degradation mechanism and nature of the released species, the bioreducible film degradation is studied by atomic force microscopy, fluorescence, and dynamic light scattering in solutions containing a reducing agent. The PAA/DNA LbL film undergoes fast bulk degradation with micrometer-sized pieces breaking off from the substrate. This bulk degradation behavior is arrested by periodic insertions of a nonbioreducible poly(ethylenimine) (PEI) layer. The LbL films containing PAA/DNA and PEI/DNA bilayers display sequential film disassembly and are capable of continuously releasing DNA nanoparticles over a prolonged time. Insertion of the PEI layer enables the bioreducible LbL films to transfect human embryonic kidney 293 cells. The data conclude that the PEI layer is effective as a barrier layer against interlayer diffusion during LbL film assembly and more importantly during film disassembly. Without the barrier layer, the high mobility of cleaved PAA fragments is responsible for bulk degradation of bioreducible LbL films, which may prevent their ultimate gene-delivery applications. This work establishes a direct link among film internal structure, disassembly mechanism, and transfection efficiency. It provides a simple method to design bioreducible LbL films for sequential and long-time DNA release.

This paper reports the synthesis and characterization of polystyrene nanorods in hemicylindrical hemimicelles of a nonionic polyoxyethylene surfactant, C 12 E 5, on graphite. The surface structure is characterized by atomic force microscopy (AFM), Fourier transform infrared spectroscopy, and contact angle goniometry. Uniformly aligned polystyrene nanorods are captured by AFM. The nanorod dimensions are studied as a function of the reaction time and styrene monomer concentration. The template synthesis using self-assembled surfactant surface aggregates promises to create functional and stable nanostructures for optoelectronics and surface engineering.

The influence of solvents (alcohols and binary mixtures of alcohol and water) on the structure and permeability of self-assembled layer-by-layer polyelectrolyte multilayers (PEMs) has been investigated by in situ atomic force microscopy (AFM), cyclic voltammetry (CV), and confocal laser scanning microscopy (CLSM). A decrease in the dielectric constant of the solvent medium increases the strength of electrostatic interactions between the polyelectrolyte chains because the Coulombic force is inversely proportional to the dielectric constant. The stronger attractions as a result of increased alcohol volume percentage in water drive the polyelectrolyte chains to contract, collapse, and coagulate. Consequently, subtle changes in the PEM film structure are observed. AFM images show that the originally smooth surface of the PEM film becomes rougher with aggregates and holes developing with increasing alcohol amount. The diffusion of Fe(CN)63-/4- through PEM films is promoted upon solvent treatment. The entrapmen...

Temperature- and pH-controlled association of terpolymers of N -isopropylacrylamide (NIPA) with 1-vinylimidazole (VI) and polyethylene glycol (PEG) has been investigated by light scattering and atomic force microscopy (AFM) in situ. The polymers contained 0–15 mol% VI and 0–2 mol% PEG. The phase transition temperatures ( T p ) have been in the range of 32–45 °C and exhibited significant dependence on the pH of solution in the pH range between 5 and 8. The T p of the polymers increased with increasing VI content and with decreasing pH, confirming major effect of VI ionization status on T p . The presence of PEG grafts in the polymer structure had augmenting effect on the magnitude of pH-responsiveness and on the pH-independent colloidal stability of the polymer particles formed above T p . Incorporation of VI into the polymer structure had similar, but pH-dependent effect on colloidal stabilization of the polymer particles. The size of the particles formed after the phase transition is driven by the association of the collapsed NIPA segments in the globule conformation and it decreased with decreasing pH. The phase transition temperature of the polymers could be adjusted to increase from temperatures below, to temperatures above body temperature upon decreasing pH from 7 to 6, suggesting that such polymers could provide a material platform for a variety of biomedical applications. AFM analysis in situ showed a fully reversible formation of particles in the solutions of the polymers above their T p .

Use of bioactive cationic peptides as gene carriers is limited by instability of their DNA complexes in vivo and by the loss of their biological activity due to undesired interactions of their bioactive parts with the DNA. To overcome the two major limitations, biodegradable high-molecular-weight form of TAT peptide (POLYTAT) sensitive to cellular redox-potential gradients was synthesized in this study by oxidative polycondensation. Physicochemical and transfection properties of DNA polyplexes based on POLYTAT were investigated and compared with polyplexes based on TAT polymer prepared by in situ template-assisted polymerization. Physicochemical properties of TAT-based polyplexes were affected by the molecular weight and method of polymerization of the TAT peptide. All TAT-based DNA polyplexes exhibited reduced cytotoxicity when compared with control polyethylenimine (PEI) polyplexes. Polyplexes based on both high-molecular-weight TAT polypeptides exhibited increased transfection efficiency compared to control TAT peptide but lower than that of PEI polyplexes. The evidence shows that transfection activity of TAT-based polyplexes is strongly dependent on the presence of chloroquine and therefore suggests that TAT polyplexes are internalized by an endocytosis. Overall, high-molecular-weight reducible polycations based on bioactive peptides has the potential as versatile carriers of nucleic acids that display low cytotoxicity and can prove to be especially beneficial in cases that require surface presentation of membrane-active or cell-specific targeting peptides.

The Layer-by-layer deposition of positively and negatively charged macromolecular species is an ideal method for constructing thin films incorporating biological molecules. We investigate the adsorption of fibronectin onto polyelectrolyte multilayer (PEM) films using optical waveguide lightmode spectroscopy (OWLS) and atomic force microscopy (AFM). PEM films are formed by adsorption onto Si(Ti)O2 from alternately introduced flowing solutions of anionic poly(sodium 4-styrenesulfonate) (PSS) and cationic poly(allylamine hydrochloride) (PAH). Using OWLS, we find the initial rate and overall extent offibronectin adsorption to be greatest on PEM films terminated with a PAH layer. The polarizability density of the adsorbed protein layer, as measured by its refractive index, is virtually identical on both PAH- and PSS-terminated films; the higher adsorbed density on the PAH-terminated film is due to an adsorbed layer of roughly twice the thickness. The binding of monoclonal antibodies specific to the protein's cell binding site is considerably enhanced to fibronectin adsorbed to the PSS layer, indicating a more accessible adsorbed layer. With increased salt concentration, we find thicker PEM films but considerably thinner adsorbed fibronectin layers, owing to increased electrostatic screening. Using AFM, we find adsorbed fibronectin layers to contain clusters; these are more numerous and symmetric on the PSS-terminated film. By considering the electrostatic binding of a segmental model fibronectin molecule, we propose a picture of fibronectin adsorbed primarily in an end-on-oriented monolayer on a PAH-terminated film and as clusters plus side-on-oriented isolated molecules onto a PSS-terminated film.

Sonicated small unilamellar egg yolk phosphatidylcholine (EggPC) vesicles were investigated using atomic force microscopy (AFM) imaging and force measurements. Three different topographies (convex, planar, and concave shape) of the EggPC vesicles on the mica surface were observed by tapping mode in fluid, respectively. It was found that the topography change of the vesicles could be attributed to the interaction force between the AFM tip and vesicles. Force curves between an AFM tip and an unruptured vesicle were obtained in contact mode. During approach, two breaks corresponding to the abrupt penetration of upper and lower bilayer of vesicle were exhibited in the force curve. Both breaks spanned a distance of around 4 nm close to the EggPC bilayer thickness. Based on Hertz analysis of AFM approach force curves, the Young’s modulus (E) and the bending modulus (kc) for pure EggPC vesicles were measured to be (1.97 ± 0.75) × 10 6 Pa and (0.21 ± 0.08) × 10 −19 J, respectively. The results show that the AFM can be used to obtain good images of intact and deformed vesicles by tapping mode, as well as to probe the integrity and bilayer structure of the vesicles. AFM force curve compare favorably with other methods to measure mechanical properties of soft samples with higher spatial resolution. © 2003 Elsevier B.V. All rights reserved.

We investigate the making of semiconductor nanorods using well-defined molecular templates. The films are characterized by AFM and XPS. The spin-coated monolayer of arachidic acid assembles on graphite as 1D nanostripes with bilayer periodicity. The copper arachidate film shows curved stripes, which become straight after reacting with hydrogen sulfide. The new template scheme may facilitate the fabrication of nanorod arrays of a variety of inorganic and semiconducting materials.

icone polyether (SPE) surfactants. The SPE surfactants exhibit several unique properties (4): (1) they can lower the air/water interfacial tension to about 20 mN/m; (2) the siloxane chain remains flexible and in the liquid state up to high molecular weight; (3) they are surface active in both aqueous and nonaqueous media; and (4) they are suitable for cosmetic applications because they cause little skin irritation. The base of the SPE surfactants is polydimethylsiloxane (PDMS). PDMS with sufficient molecular weight, e.g., about 1000, is well known for its hydrophobicity as well as oleophobicity. In particular, the oleophobicity distinguishes the silicone-based surfactants from their hydrocarbon counterparts. The molecular structure of SPEs is either comblike, with the polyoxyalkylene chain side-grafted to a PDMS backbone, or linear diblock (AB) and triblock (ABA) types, with A representing the polyoxyalkylene hydrophile and B the PDMS hydrophobe. SPEs incorporating copolymers of polyoxyethylene (PEO) and polyoxypropylene (PPO) are widely used in polyurethane foam manufacture and personal care prod

The interaction forces provided by silicone polyether (SPE) surfactants, adsorbed at the interface of octadecyltrichlorosilan monolayer and aqueous surfactant solution, were measured by atomic force microscopy. Changes in the forces in the presence of ethanol were examined for a series of comb-type SPE surfactants. It was found that the steric repulsion provided by the SPE molecules persisted up to an 80% or higher ethanol level. Conversely, conventional hydrocarbon surfactants such as dodecyl polyoxyethylene ether lose their interfacial activity at much lower ethanol levels. The force measurement results were compared favorably with the findings from emulsion-stability and interfacial-tension measurements. The difference between the conventional hydrocarbon surfactants and the SPE surfactants is attributed to the polysiloxane moiety being hydrophobic but not oleophilic. The polyoxypropylene units in the polyether chain were found to add unique characteristics to the surface behavior of the SPE surfactants by modifying the hydrophile's solubility in the alcoholic media. The surface activity and colloidal stability can be fine-tuned by the ratio of polyoxypropylene to polyoxyethylene. In general, emulsion and colloidal products and processes that contain alcohol or other nonaqueous media will benefit from the unique properties of this special class of SPE surfactants.

A mixed film of microscopic heterogeneity was formed by stepwise deposition of a long-chain organosilane n -octadecyltrichlorosilane (OTS) and a short-chain organosilane aminobutyldimethylmethoxysilane (ABS). The amount of silane deposited on an oxidized silicon substrate was controlled kinetically by varying the dip time in each silane solution. The binary film was characterized by the atomic force microscopy (AFM), infrared attenuated total internal reflection (IR-ATR), and contact angle goniometry. OTS forms a monolayer structure whose fractional surface coverage and surface energy can be precisely controlled. ABS agglomerates slowly to form island structure and is ineffective in surface energy or composition variation. The contact angles on sub-monolayers of OTS follows the Cassie equation, while the contact angle of the binary OTS and ABS film deviates positively from the equation.

Langmuir–Blodgett (LB) multilayers of arachidic acid (AA) and octadecylamine (ODA) were used as templates in order to produce cadmium sulfide (CdS) nanoparticle arrays. Both atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) were used to characterize the film. In the absence of ODA, the cadmium arachidate trilayers were highly crystalline with a lattice constant of 0.42 nm. Upon reaction with hydrogen sulfide (H2S), an ordered array of CdS nanoparticles with a particle separation of 4.2 nm was imaged. The existence of CdS was also confirmed by XPS. No CdS nanoparticles were formed when ODA was used as the sole component. In the presence of ODA, the crystalline structure of the AA film was disturbed, resulted in randomly distributed particles. The average particle height was reduced from 4 to 1 nm when the amount of ODA was increased. It is concluded that there is a direct correlation between the ordering of the organic template and that of the nanoparticle array. There is also a correlation between the particle size and the ratio of AA and ODA. Mixed LB films are promising in synthesizing semiconducting nanoparticle arrays of limited particle sizes.

A recently developed method of spherulitic crystallization was used for the preparation of highly ordered 80 nm thin films of the dye 1.7-bis(dimethylamino)heptamethinium perchlorate (BDH+ClO4-). Depending on the crystallization temperature, the film color and surface topography varied widely, while the crystal structure and film thickness remained the same. At low undercooling, two homogeneous regions were obtained. One showed in-plane symmetrical and the other asymmetrical growth behaviors. At high undercooling, a banded spherulitic structure with rainbow-like colors developed, whose formation is attributed to the out-of-plane asymmetrical growth. The spherulitic growth kinetics, microstructure, and optical properties were investigated by optical microscopy, atomic force, and scanning tunneling microscopy in static and real time mode. A molecular mechanism is proposed which originates from different directions of macroscopic growth and microscopic molecular attachment dictated by the three-dimensional c...

Synthetic vector-based gene delivery continues to gain strength as viable alternatives to viral vectors due to safety and other concerns. DNA release dynamics is key to the understanding and control of gene delivery from nanosystems. Here we describe atomic force microscope (AFM) application to the understanding of DNA release dynamics from bioreducible polycation-based nanosystems. The two nanosystems are polyplex nanoparticles and layer-by-layer (LbL) films.

DNA release dynamics from layer-by-layer (LbL) films is an important aspect to consider with regards to localized gene delivery systems. The rate of DNA release and the condensation state of DNA during release are of particular interest in the field of gene delivery. A hyperbranched poly(amido amine) (RHB) containing bioreducible disulfide bonds is used to form interpolyelectrolyte complexes with DNA during LbL film assembly. During film disassembly, DNA is released in physiologic conditions due to the reducing nature of the RHB. Uncondensed DNA deposited on the surface was compared to DNA condensed by RHB in polyplex form by using two types of LbL films, RHB/DNA/RHB and polyplex terminated films, RHB/DNA/polyplex. LbL films with up to three layers are used in order to facilitate high-resolution atomic force microscopy (AFM) imaging. X-ray reflectivity, ellipsometry, and Fourier transform infrared spectroscopy are also used. The film disassembly, rearrangement, and release of molecules from the surface due to thiol-disulfide exchange is conducted in reducing dithiothreitol (DTT) solutions. Salt is found to accelerate the overall rate of film disassembly. Additionally, it was found that the polyplex layer disassembles faster than the DNA layer. The predominant intermediate structure is the toroid structure for the polyplex layer and the fiber bundle structure for the DNA layer during film disassembly. This study offers a simple means to modulate DNA release from LbL films by utilizing both condensed and uncondensed DNA in different layers. The study highlights nanostructures, toroids, and bundles as dominant intermediate DNA structures during DNA release from LbL films.

Layer-by-layer (LbL) films were assembled on flexible stainless steel substrate using plasmid DNA and reducible hyperbranched poly(amido amine) (RHB) polycation. The films were characterized by XPS and their disassembly in reducing conditions confirmed by ellipsometry. Fibroblast and smooth muscle cell attachment and proliferation on DNA/RHB films were indistinguishable from those on control DNA/poly(ethylenimine) (PEI) films. In vitro transfection activity was evaluated using reporter plasmids encoding for secreted alkaline phosphatase (SEAP) and green fluorescent protein (GFP). DNA/RHB films showed higher and longer lasting transfection activity than control DNA/PEI films using SEAP plasmid. It was revealed through the use of GFP plasmid that DNA/RHB films transfected almost the entire cell population growing on the films. In vivo transfection activity was evaluated by subcutaneously implanting a stainless steel substrate coated with the DNA/RHB films containing SEAP plasmid DNA and measuring the levels of SEAP secreted into the blood circulation of rats. It was found that the plasma levels of SEAP peaked at approximately 160 ng SEAP/mL five days post-implantation.

Aspirin and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) are deposited from their alcoholic mixed solution onto highly oriented pyrolytic graphite (HOPG) by spin coating. The film structure and morphology are characterized by atomic force microscopy (AFM). The barely soluble DMPE forms a highly oriented stripe phase as a result of its one-dimensional epitaxy with the HOPG lattice. The bilayer stripe pattern exposes the cross section of the lipid bilayer lamellae and enables the direct visualization of the molecular interactions of drug or biological molecules with either the hydrophobic or the hydrophilic part of the phospholipid bilayer. The bilayer pattern affects the aspirin molecular deposition and aggregation. AFM shows that the aspirin molecules prefer to deposit and aggregate along the aliphatic interior part of the bilayer pattern, giving rise to parallel dimer rods in registry with the underlying pattern. The nonpolar interactions between aspirin and the phospholipid bilayer are consistent with the lipophilic nature of aspirin. The bilayer pattern not only stabilizes the rodlike aggregate structure of aspirin at low aspirin concentration but also inhibits crystallization of aspirin at high aspirin concentration. Molecular models show that the width of the DMPE aliphatic chain interior can accommodate no more than two aspirin dimers. The bilayer confinement may prevent aspirin from reaching its critical nucleus size. This study illustrates a general method to induce a metastable or amorphous form of an active pharmaceutical ingredient (API) by chemical confinement under high undercooling conditions. Metastable and amorphous solids often display better solubility and bioavailability than the stable crystalline form of the API.

The morphology and stability of small unilamellar egg yolk phosphatidylcholine (EggPC) liposomes modified with the Pluronic® copolymer (poly (oxyethylene)–poly (oxypropylene)–poly (oxyethylene) (PEO–PPO–PEO)) with different compositions on mica surface have been investigated using atomic force microscopy. Morphology studies reveal significant morphological changes of liposomes upon incorporating the Pluronic® copolymer. Bilayers are observed for Pluronic® with small hydrophilic (PEO) chain lengths such as L81 [(PEO)2(PPO)40(PEO)2] and L121 [(PEO)4(PPO)60(PEO)4]; bilayer and vesicle coexistence is observed for P85 [(PEO)26(PPO)39.5(PEO)26] and F87 [(PEO)61.1(PPO)39.7(PEO)61.1]; and stable vesicles are observed for F88 [(PEO)103.5(PPO)39.2(PEO)103.5], F127 [(PEO)100(PPO)65(PEO)100], and F108 [(PEO)132.6(PPO)50.3(PEO)132.6]. The micromechanical properties of Pluronic®-modified EggPC vesicles were studied by analyzing AFM approaching force curve. The bending modulus ( k c ) of the Pluronic®-modified EggPC vesicles increased several-fold compared with that of the pure EggPC vesicles. The significant difference is due to the enhanced rigidity of the EggPC vesicles as a result of the incorporation of PPO molecules and PEO chains. Based on the analysis of onset point by AFM and diameters of vesicles by light scattering, it was concluded that the favorable model to describe the polymer–bilayer interaction is the membrane-spanning model.

The micromechanical properties of pure and cholesterol modified egg yolk phosphatidylcholine (EggPC) vesicles prepared by sonication were studied by atomic force microscopy (AFM) on mica surface. The force curves between an AFM tip and an unruptured vesicle were obtained by contact mode. During approach, two repulsion regions with two breaks were observed. The slopes of the two repulsive force regimes for the pure EggPC vesicles are determined to be several times lower than that of EggPC/cholesterol vesicles. The elastic properties from force plot analysis based on the Hertzian model showed that Young's modulus (E) and the bending modulus (kc) of cholesterol-modified vesicles increased several-fold compared with pure EggPC vesicles. The significant difference is attributed to the enhanced rigidity of the EggPC vesicles as a result of the incorporation of cholesterol molecules. The behavior of cholesterol-modified vesicles upon adsorption is different from that in solution as revealed by mechanical properties. The results indicate that AFM can provide a direct method to measure the mechanical properties of adsorbed small liposomes and to detect the stability change of liposomes.

This communication describes a spin-coating method to nucleate organic molecular rods of uniform size from an inorganic nanoparticle at a solid surface. The particle-rod hybrid structure spontaneously forms when a film is spin coated from a mixed 2-propanol solution of arachidic acid (AA) and nanoparticles of cadmium selenide capped by mercaptoundecanoic acid (MUA-CdSe) on graphite. AFM images show that MUA-CdSe nanoparticles nucleate single crystalline rods of AA with a cross section of a single unit cell of the C-form. The solution-based process potentially allows the precise tuning of the wetting profile of the solution on the surface-attached nanoparticle, which provides the reservoir for the growth of the single crystalline rods. The results suggest that nanoparticles can be regarded as nanoseeds for the nucleation of guest crystals. It should be possible to further functionalize the AA rods by electrostatic complexation with metal or organic ions.

The failure of prosthesis after total joint replacement is due to the lack of early implant osseointegration. In this study polyvinyl alcohol-collagen-hydroxyapatite (PVA-Col-HA) electrospun nanofibrous meshes were fabricated as a biomimetic bone-like extracellular matrix for the modification of orthopedic prosthetic surfaces. In order to reinforce the PVA nanofibers, HA nanorods and Type I collagen were incorporated into the nanofibers. We investigated the morphology, biodegradability, mechanical properties and biocompatibility of the prepared nanofibers. Our results showed these inorganic-organic blended nanofibers to be degradable in vitro. The encapsulated nano-HA and collagen interacted with the PVA content, reinforcing the hydrolytic resistance and mechanical properties of nanofibers that provided longer lasting stability. The encapsulated nano-HA and collagen also enhanced the adhesion and proliferation of murine bone cells (MC3T3) in vitro. We propose the PVA-Col-HA nanofibers might be promising modifying materials on implant surfaces for orthopedic applications.

We have created nanospirals by self-assembly during droplet evaporation. The nanospirals, 60–70 nm in diameter, formed when solvent mixtures of methanol and m-cresol were used. In contrast, spin coating using only methanol as the solvent produced epitaxial films of stripe nanopatterns and using only m-cresol disordered structure. Due to the disparity in vapor pressure between the two solvents, droplets of m-cresol solution remaining on the substrate serve as templates for the self-assembly of carboxylic acid molecules, which in turn allows the visualization of solution droplet evaporation one molecule at a time.