Patrick Vermette

Interactions of phospholipid- and poly(ethylene glycol)-modified surfaces with biological systems: relation to physico-chemical properties and mechanisms

Abstract This paper critically reviews reports on the interactions of phospholipids and poly(ethylene glycol) (PEG)—also called poly(ethylene oxide) (PEO)—with biological systems (model polypeptides, proteins, macrophages, etc). The review arises from the need to rationalize the widely varying findings reported in the literature. The paper discusses the interaction of simple polypeptides with phospholipids, followed by the interaction of proteins with supported phospholipid layers and liposomes. In addition, the interactions of liposomes with more complex biological systems, in vitro and in vivo, are discussed. Since many proposed liposome formulations contain grafted PEG molecules, a discussion of the interactions of PEG molecules and PEG surface layers with proteins is also provided as well as a substantial section dealing with the mechanistic aspects of these interactions. As well as providing an introduction to the multidisciplinary and complex literature available, we hope that this review article will provide the necessary information for more rational design of systems and devices for in vivo application. In particular, the article stresses the need for much better characterization of the systems and materials used.

Oxidized-LDL induce morphological changes and increase stiffness of endothelial cells

There is increasing evidence suggesting that oxidized low-density lipoproteins (ox-LDL) play a critical role in endothelial injury contributing to the age-related physio-pathological process of atherosclerosis. In this study, the effects of native LDL and ox-LDL on the mechanical properties of living human umbilical vein endothelial cells (HUVEC) were investigated by atomic force microscopy (AFM) force measurements. The contribution of filamentous actin (F-actin) and vimentin on cytoskeletal network organization were also examined by fluorescence microscopy. Our results revealed that ox-LDL had an impact on the HUVEC shape by interfering with F-actin and vimentin while native LDL showed no effect. AFM colloidal force measurements on living individual HUVEC were successfully used to measure stiffness of cells exposed to native and ox-LDL. AFM results demonstrated that the cell body became significantly stiffer when cells were exposed for 24 h to ox-LDL while cells exposed for 24 h to native LDL displayed similar rigidity to that of the control cells. Youngs moduli of LDL-exposed HUVEC were calculated using two models. This study thus provides quantitative evidence on biomechanical mechanisms related to endothelial cell dysfunction and may give new insight on strategies aiming to protect endothelial function in atherosclerosis.

Physico-chemical properties and cytotoxicity assessment of PEG-modified liposomes containing human hemoglobin.

PEGylated liposomes encapsulating human hemoglobin as oxygen carriers were prepared from purified carbonylhemoglobin (HbCO) solution and a lipid mixture composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 2000] (DMPE-PEG(2000)) and palmitic acid. Hemoglobin was extracted and purified from human blood samples. SDS-PAGE was used to assess its purity. Diameter of liposomes containing hemoglobin was controlled to approximately 200 nm using extrusion as measured by dynamic light scattering and transmission electron microscopy. Liposome size distributions were shown to remain unimodal over 14 days, even at different storage temperatures. Zeta potential measurements revealed that liposome containing hemoglobin have a net surface charge of -7.16+/-0.33 mV. Also, hemoglobin encapsulated in liposomes was able to perform several cycles of oxygen loading and unloading using oxygen (O(2)) and carbon monoxide (CO). The hemoglobin vesicle dispersion showed some toxicity as revealed by three in vitro assays in which endothelial cell (HUVECs) monolayers were exposed to these dispersions. Cytotoxicity was function of the liposome concentration in the culture medium.

Bridging the gap between physicochemistry and interpretation prevalent in cell-surface interactions.

Bridging the Gap Between Physicochemistry and Interpretation Prevalent in Cell-Surface Interactions Evan A. Dubiel, Yves Martin, and Patrick Vermette* Laboratoire de bio-ing enierie et de biophysique de l’Universit e de Sherbrooke, Department of Chemical and Biotechnological Engineering, Universit e de Sherbrooke, 2500 Boulevard de l’Universit e, Sherbrooke, Qu ebec, Canada J1K 2R1 Research Centre on Aging, Institut universitaire de g eriatrie de Sherbrooke, 1036 rue Belv ed ere Sud, Sherbrooke, Qu ebec, Canada J1H 4C4

Immobilized liposome layers for drug delivery applications: inhibition of angiogenesis.

Liposomes were immobilized onto the surface of perfluorinated polymer tape samples and tissue culture polystyrene well-plates using a multilayer immobilization strategy. In the first step, a thin interfacial bonding layer with surface aldehyde groups was deposited from a glow discharge struck in acetaldehyde vapour. Polyethylenimine was then covalently bound onto the aldehyde groups by reductive amination, followed by covalent binding of NHS-PEG-biotin molecules onto the surface amine groups by carbodiimide chemistry. Next, NeutrAvidin protein molecules were bound onto the PEG-biotin layer. Finally, liposomes containing PEG-biotinylated lipids were docked onto the remaining binding sites of the surface-immobilized NeutrAvidin molecules. AFM was used to image surface-bound liposomes and revealed a density well below close packing. The release characteristics of the surface-bound liposomes were measured by the fluorescence intensity changes of carboxyfluorescein upon release. Liposomes filled with sodium orthovanadate were surface immobilized and used in two in vitro angiogenesis assays. Marked differences compared to various control samples were observed, demonstrating the utility of drug-filled, surface-bound liposomes for evoking localized, controlled biological host responses proximal to an implanted biomedical device.

Albumin and fibrinogen adsorption onto phosphatidylcholine monolayers investigated by Fourier transform infrared spectroscopy

Abstract Dipalmitoylphosphatidylcholine (DPPC) monolayers were deposited onto a germanium attenuated total reflectance (ATR) crystal using the Langmuir–Blodgett technique. The DPPC-coated crystal was then exposed to human serum albumin or human fibrinogen solutions while measuring the protein adsorption by recording FTIR spectra. The effect of the zwitterionic nature of the DPPC polar headgroup towards protein adsorption has been ascertained by exposing either the phospholipid headgroup or the acyl chains to the protein solution; this was possible by the use of a silanized or a bare germanium crystal. Calibration curves have been made to measure the protein surface concentrations. After 3 h, the albumin surface concentration on DPPC monolayers was about three times higher when the proteins were exposed to the lipid acyl chains instead of the polar headgroups (e.g. 3 vs. 1 μg cm −2 ). As for fibrinogen (FGN) adsorption, when the lipid polar headgroups were exposed to the protein solution, the FGN adsorption was low reaching a maximum value of 0.5 μg cm −2 . When interacting with the lipid acyl chains, the FGN adsorption reached a plateau at a value of 2.1 μg cm −2 after 3 h. Clearly, both albumin and FGN showed a low tendency to adsorb on surfaces where the lipid polar headgroups are exposed toward the protein solution.

The effects of co-culture with fibroblasts and angiogenic growth factors on microvascular maturation and multi-cellular lumen formation in HUVEC-oriented polymer fibre constructs

In the present study, polymer monofilaments were embedded in fibrin seeded with human umbilical vein endothelial cells (HUVEC) to guide HUVEC attachment and migration in order to form oriented vessel-like structures between adjacent monofilaments. Histology and fluorescent fibrin experiments confirmed that microvessel-like structures, which were developing between polymer monofilaments embedded in fibrin, contained a lumen. The effect of human fibroblasts and growth factors (VEGF and bFGF) over the microvessel formation process was tested. The effects of VEGF and bFGF were dose-dependent. The effect of VEGF was optimum at the lower concentration tested (2 ng/mL), while that of bFGF was optimum at the higher tested concentration (20 ng/mL). Furthermore, the use of fibroblasts significantly improved the maturation of the microvessels compared to control and to samples cultured with VEGF and bFGF.

Enhanced smooth muscle cell adhesion and proliferation on protein‐modified polycaprolactone‐based copolymers

Smooth muscle cells (SMC) were cultured for up to 6 days on copolymer films fabricated from a PCL-PEG-PCL block copolymer or P(epsilon-CL-co-D,L-LA)-PEG-P(epsilon-CL-co-D,L-LA), named P(100/0) and P(70/30), respectively. The films were modified by aminolysis using 1,6-hexanediamine, and fibronectin, fibrinogen, or fibrin layers were subsequently immobilized by physisorption or by covalent coupling using imidoester chemistry. Immobilization of all the tested proteins resulted in significantly enhanced cell adhesion on these polymers. Moreover, we found that covalently immobilized proteins supported significantly greater cell proliferation than physisorbed proteins over 6 days. SMC cultured on P(100/0) films modified by covalently attached fibronectin or fibrin layers proliferated at a rate comparable to that observed on control tissue culture polystyrene. The proposed surface modification schemes were much less efficient in improving cell attachment and proliferation on P(70/30) films. However, prewetting P(70/30) with a phosphate buffer prior to aminolysis significantly improved cell numbers following immobilization of fibronectin. Immunostaining of smooth muscle-specific alpha-actin of SMC grown on protein-modified P(100/0) 8 h and 48 h after cell seeding, confirmed preserved SMC phenotype on all modified surfaces.

Young's Moduli of Surface-Bound Liposomes by Atomic Force Microscopy Force Measurements

Mechanical properties of layers of intact liposomes attached by specific interactions on solid surfaces were studied by atomic force microscopy (AFM) force measurements. Force-distance measurements using colloidal probe tips were obtained over liposome layers and used to calculate Youngs moduli by using the Hertz contact theory. A classical Hertz model and a modified Hertz one have been used to extract Youngs moduli from AFM force curves. The modified model, proposed by Dimitriadis, is correcting for the finite sample thickness since Hertzs classical model is assuming that the sample is infinitely thick. Values for Youngs moduli of 40 and 8 kPa have been obtained using the Hertz model for one and three layers of intact liposomes, respectively. Youngs moduli of approximately 3 kPa have been obtained using the corrected Hertz model for both one and three layers of surface-bound liposomes. Compression work performed by the colloidal probe to compress these liposome layers has also been calculated.

Biofouling of dextran-derivative layers investigated by quartz crystal microbalance

This study reports the fouling of carboxymethyl dextran (CMD) layers in cell culture medium, fibronectin, and serum solutions. CMD layers were covalently immobilized onto amine groups available either on an n-heptylamine plasma polymer (HApp) layer or onto a polyethylenimine (PEI) coating grafted to an acetaldehyde plasma polymer (AApp) layer. The successful immobilization of the graft layers was demonstrated by X-ray photoelectron spectroscopy (XPS). Primary amines on HApp and AApp+PEI surfaces were quantified using a colorimetric assay. Quartz crystal microbalance (QCM) was used to investigate in real-time the fouling of the graft layers upon incubation in cell culture medium (RPMI), fibronectin, and foetal bovine serum (FBS) solutions. HApp, AApp and AApp+PEI layers exhibited large fouling in fibronectin and FBS solutions, while fouling in RPMI cell culture medium was not significant. Protein repellent properties of CMD layers in FBS and fibronectin have been demonstrated compared to the other tested surfaces. QCM has shown that both CMD layers were fouled to a small extent in RPMI medium.