Yuri Rochev

Interaction of soft condensed materials with living cells: Phenotype/transcriptome correlations for the hydrophobic effect

The assessment of biomaterial compatibility relies heavily on the analysis of macroscopic cellular responses to material interaction. However, new technologies have become available that permit a more profound understanding of the molecular basis of cell–biomaterial interaction. Here, both conventional phenotypic and contemporary transcriptomic (DNA microarray-based) analysis techniques were combined to examine the interaction of cells with a homologous series of copolymer films that subtly vary in terms of surface hydrophobicity. More specifically, we used differing combinations of N-isopropylacrylamide, which is presently used as an adaptive cell culture substrate, and the more hydrophobic, yet structurally similar, monomer N-tert-butylacrylamide. We show here that even discrete modifications with respect to the physiochemistry of soft amorphous materials can lead to significant impacts on the phenotype of interacting cells. Furthermore, we have elucidated putative links between phenotypic responses to cell–biomaterial interaction and global gene expression profile alterations. This case study indicates that high-throughput analysis of gene expression not only can greatly refine our knowledge of cell–biomaterial interaction, but also can yield novel biomarkers for potential use in biocompatibility assessment.

Poly(N-isopropylacrylamide) co-polymer films as potential vehicles for delivery of an antimitotic agent to vascular smooth muscle cells

INTRODUCTION Local delivery of antimitotic agents is a potential therapeutic strategy for protection of injured coronary vasculature against intimal hyperplasia and restenosis. This study sought to establish the principle that thermoresponsive poly(N-isopropylacrylamide) co-polymer films can be used to deliver, in a controlled manner, an antimitotic agent to vascular smooth muscle cells (VSMC). METHODS A series of co-polymer films was prepared, using varying ratios (w/w) of N-isopropylacrylamide (NiPAAm) monomer to N-tert-butylacrylamide (NtBAAm) and loaded with the antimitotic agent colchicine (100 nmol/film) at room temperature. RESULTS The extent of colchicine release at 37 degrees C was inversely proportional to the amount of NtBAAm in co-polymer films: release after 48 h from 85:15, 65:35 and 50:50 (NiPAAm:NtBAAm) films was 26, 17 and 0.5 nmol, respectively. In cytotoxicity studies, when medium incubated with co-polymers for 24 h (in the absence of colchicine) was further incubated with target bovine aortic smooth muscle cells (BASMC), no loss of cell viability occurred. Colchicine released from all three co-polymer films significantly inhibited proliferation and random migration of BASMC: 100 nM colchicine (released from 65:35 NiPAAm:NtBAAm) reduced cell proliferation to 25.7+/-1.7% of levels seen in the absence of colchicine (control) and random cell migration to 37.7+/-5.7% of control (mean+/-S.E.M., n = 3, P < .01 and P < .05, respectively). The magnitudes of these effects were comparable to those seen in separate experiments with native colchicine and were observed in samples of released colchicine which had been stored at -20 degrees C for up to 6 months. CONCLUSIONS This study has shown that the release of the antimitotic agent colchicine, from NiPAAm/NtBAAm co-polymer films can be manipulated by changes in co-polymer composition. Furthermore, such drug released at 37 degrees C retains comparable bioactivity to that of native colchicine.

Intact endothelial cell sheet harvesting from thermoresponsive surfaces coated with cell adhesion promoters

Recently, with the development of smart polymers, research has looked to using thermoresponsive polymers as cell culture substrates. These novel surfaces allow the cultivation of cells without enzymes using the thermoresponsive phase transition property of poly(N-isopropylacrylamide) (PNIPAAm). However, this requires expensive techniques to generate a sufficiently thin film that allows cell adhesion. In this study, we looked at simple solvent cast films which normally show poor cell adhesion, but here the films are coated with cell adhesion promoters (CAPs) to improve cell growth without altering the copolymer thermoresponsive behaviour. A copolymer of PNIPAAm and N-tert-butylacrylamide (NtBAm) with a ratio of 85 : 15, respectively, was synthesized and solvent cast. The copolymer films were coated with CAPs, such as collagen, fibronectin and laminin, to increase their cell adhesion and growth properties. Cell activity measured by the alamarBlue assay showed similar results for coated copolymer films and standard tissue culture plastic controls. Deposition of CAPs on to the copolymer films was characterized by scanning electron microscopy and atomic force microscopy. Cell detachment from the copolymer films is not affected by the surface coatings of CAPs, and endothelial cells are recovered as an intact sheet, which has great potential for uses in tissue engineering applications. The results demonstrate a versatile method for the cultivation of cells while eliminating the need for the use of digestive enzymes such as trypsin. This study shows that cultivation on physically bonded PNIPAAm copolymers is viable and achievable by relatively simple methods.

Human coronary artery smooth muscle cell response to a novel PLA textile/fibrin gel composite scaffold.

Previous studies have demonstrated the potential of fibrin as a cell carrier for cardiovascular tissue engineering applications. Unfortunately, fibrin exhibits poor mechanical properties. One method of addressing this issue is to incorporate a textile in fibrin to provide structural support. However, it is first necessary to develop a deeper understanding of the effect of the textile on cell response. In this study, the cytotoxicity of a polylactic acid (PLA) warp-knit textile was assessed with human coronary artery smooth muscle cells (HCASMC). Subsequently, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was employed to examine the gene expression of HCASMC embedded in fibrin with and without the textile. Five genes were examined over a 3-week period: smooth muscle alpha-actin (SMalphaA), myosin heavy chain 11 smooth muscle (SM1/SM2), calponin, myosin heavy chain 10 non-muscle (SMemb) and collagen. Additionally, a microarray analysis was performed to examine a wider range of genes. The knitting process did not adversely affect the cell response; there was no dramatic change in cell number or metabolic rate compared to the negative control. After 3 weeks, there was no significant difference in gene expression, except for a slight decrease of 10% in SMemb in the fibrin with textile. After 3 weeks, there were no obvious cytotoxic effects observed as a result of the knitting process and the gene expression profile did not appear to be altered in the presence of the mesh in the fibrin gel.

Polarity Assessment of Thermoresponsive Poly(NIPAM-co-NtBA) Copolymer Films Using Fluorescence Methods

The in-situ, non-contact, and non-destructive measurement of the physicochemical properties such as the polarity of thin, hydrophilic polymer films is desirable in many areas of polymer science. Polarity is a complex factor and encompasses a range of non-covalent interactions including dipolarity/polarizability and hydrogen bonding. A polarity measurement method based on fluorescence would be ideal, but the key challenge is to identify suitable probes which can accurately measure specific polarity related parameters. In this manuscript we assess a variety of fluorophores for measuring the polarity of a series of relatively hydrophilic, thermoresponsive N-isopropylacrylamide/N-tert-butylacrylamide (NIPAM/NtBA) copolymers. The emission properties of both pyrene and 3-Hydroxyflavone (3-HF) based fluorophores were measured in dry polymer films. In the case of pyrene, a relatively weak, linear relationship between polymer composition and the ratio of the first to the third vibronic band of the emission spectrum (I1/I3) is observed, but pyrene emission is very sensitive to temperature and thus not suitable for robust polarity measurements. The 3-HF fluorophores which can undergo an excited-state intramolecular proton transfer (ESIPT) reaction have a dual band fluorescence emission that exhibits strong solvatochromism. Here we used 4′-diethylamino-3-hydroxyflavone (FE), 5,6-benzo-4′-diethylamino-3-hydroxyflavone (BFE), and 4´-diethylamino-3-hydroxy-7-methoxyflavone (MFE). The log ratio of the dual band fluorescence emission (log (IN*/IT*)) of 3-HF doped, dry, NIPAM-NtBA copolymer films were found to depend linearly on copolymer composition, with increasing hydrophobicity (greater NtBA fraction) leading to a decrease in the value of log (IN*/IT*). However, the ESIPT process in the polymer matrix was found to be irreversible, non-equilibrated and occurs over a much longer timescale in comparison to the results previously reported for liquid solvents.

Measuring the Micro-Polarity and Hydrogen-Bond Donor/Acceptor Ability of Thermoresponsive N -Isopropylacrylamide/ N -tert-Butylacrylamide Copolymer Films Using Solvatochromic Indicators

Thin polymer films are important in many areas of biomaterials research, biomedical devices, and biological sensors. The accurate in situ measurement of multiple physicochemical properties of thin polymer films is critical in understanding biocompatibility, polymer function, and performance. In this work we demonstrate a facile spectroscopic methodology for accurately measuring the micro-polarity and hydrogen-bond donor/acceptor ability for a series of relatively hydrophilic thermoresponsive copolymers. The micro-polarity of the N-isopropylacrylamide (NIPAM) and N-tert-butylacrylamide (NtBA) co-polymers was evaluated by means of the ET(30), α, β, and π* empirical solvatochromic polarity parameters. The data shows that increasing the NtBA fraction in the dry copolymer film reduces polarity and hydrogen-bonding ability. Within the Kamlet–Taft polarity framework, the NIPAM/NtBA copolymer films are strong hydrogen-bond acceptors, strongly dipolar/polarizable, and rather moderate hydrogen-bond donors. This characterization provides a more comprehensive physicochemical description of polymers, which aids the interpretation of film performance. Comparison of the measured ET(30) values with literature data for other water-soluble polymers show that dry NIPAM/NtBA copolymers are slightly more polar than poly(ethylene oxide), less polar than polyvinyl-alcohol, and approximately the same polarity as poly(N-vinyl-2-pyrrolidone). These findings indicate that this spectroscopic method is a facile, rapid, and nondestructive methodology for measuring polymer properties in situ, suitable for most biomaterials research laboratories.

Film Thickness Determines Cell Growth and Cell Sheet Detachment from Spin-Coated Poly(N-Isopropylacrylamide) Substrates

Poly(N-isopropylacrylamide) (pNIPAm) is widely used to fabricate thermoresponsive surfaces for cell sheet detachment. Many complex and expensive techniques have been employed to produce pNIPAm substrates for cell culture. The spin-coating technique allows rapid fabrication of pNIPAm substrates with high reproducibility and uniformity. In this study, the dynamics of cell attachment, proliferation, and function on non-cross-linked spin-coated pNIPAm films of different thicknesses were investigated. The measurements of advancing contact angle revealed increasing contact angles with increasing film thickness. Results suggest that more hydrophilic 50 and 80 nm thin pNIPAm films are more preferable for cell sheet fabrication, whereas more hydrophobic 300 and 900 nm thick spin-coated pNIPAm films impede cell attachment. These changes in cell behavior were correlated with changes in thickness and hydration of pNIPAm films. The control of pNIPAm film thickness using the spin-coating technique offers an effective tool for cell sheet-based tissue engineering.

The effects of vinblastine on smooth muscle cells in vitro: evaluation of a therapeutic window for the treatment of restenosis.

The development of drug-eluting stents (DES) to combat the problem of in-stent restenosis has revolutionized interventional cardiology. However, concerns have emerged about the risk of late angiographic stent thromboses associated with DES. The evaluation and width of the therapeutic window of a particular DES system is of huge importance to its safety and efficacy. In this study, the effects of vinblastine, an antimitotic drug, on smooth muscle cells in vitro is analyzed. The change in levels of proliferation, activity, migration, and viability in human coronary artery smooth muscle cells was measured at a range of concentrations and over a number of time points. These findings were then compared with those of a previous study on the effects of vinblastine on endothelial cells, and an optimum working concentration range was evaluated. This study suggests that the concentration of vinblastine most appropriate in restenosis treatment would be between 0.1 and 1 nM. At this concentration, vinblastine exerts a distinct effect on smooth muscle cell proliferation without detrimental effects on endothelial cell viability. It was also found that vinblastine affects certain cellular activities such as migration in a threshold-independent manner, suggesting that very low doses could be active against the processes of restenosis.