Immobilisation of sulphated hyaluronan for improved biocompatibility

Hyaluronan (Hyal) was modified by the insertion of sulphate to hydroxyl groups. A series of heparin-like compounds with controllable properties was obtained. The physicochemical and biological behaviours of all these sulphated hyaluronan acids (HyalSx) and their complexes with heavy metal ions (Cu2+ and Zn2+) were investigated. HyalS, derivatives showed a good anticoagulant activity and low platelet aggregation which increased with increasing degree of sulphation. Moreover HyalSx and their Cu2+ complexes were demonstrated to favour the growth of human endothelial cells. However, the utilisation of HyalSx as a material is hindered by its high solubility in physiological solution. Our approach to improve its stability was directed to the synthesis of new HyalSx-based hydrogels and on the preparation of new biocompatible polymeric surfaces obtained through covalent photoimmobilisation of HyalSx. The reaction of primary ovine chondrocytes and B10D2 endothelial cells was studied on both matrices in terms of cell number, F-actin and CD44 receptor immunostaining. Analysis of cell movement showed that the cells respond to HyalSx showing good adhesion and spreading. These results suggest that HyalSx containing materials could be used as biomaterials to aid cartilage repair and vessel endothelisation.     

Chemical modification of the surfaces of bacterial cell walls.

The surfaces of the isolated cell walls of four bacterial species were studied by microelectrophoresis following chemical treatments intended to remove specific charged groups. Acid-base titrations of the walls were used to assess specificity and extent of the modifications. Carboxyl groups were specifically and completely modified by activation with a water-soluble carbodiimide and subsequent reaction with a nucleophile, such as glycinamide, to give an uncharged pH-stable product. Aqueous media and mild reaction conditions make the method suitable for modifying carboxyl groups on cell surfaces too labile to withstand the harsh conditions required for conventional esterification reactions. Use of the carbodiimide-mediated reaction for discharging carboxyl groups, along with fluorodinitrobenzene for discharging amino groups and extraction procedures for removing constituents carrying phosphoester groups (teichoic acids), made it possible to obtain information about the spatial arrangement of charged groups on the wall surfaces. Removal of the exterior negative charge dominating wall surfaces allowed underlying amino groups to become electrokinetically effective and, in the case of E. coli, also revealed a lipophilic region with an affinity for a cationic surfactant.     

纳米羟基磷灰石表面修饰及其转染细胞的研究

目的:探讨羟基磷灰石-聚乙烯亚胺(nHA-PEI 10KD)纳米颗粒的癌细胞基因转染效率.方法:通过透射电子显微镜(TEM)观察HA-PEI(10KD)纳米颗粒的形态及粒径,Zeta电位仪测定nHA-PEI和HA在酸、碱、中性环境中的电位,用琼脂糖凝胶电泳检测nHAP-PEI(10KD)与DNA结合的能力,MTT比色法检测nHAP-PEI(10KD)对nepG2细胞的毒性,选用增强型绿色荧光蛋白质粒pEGFP1与nHA-PEI结合后,分别转染真核细胞HepG2、Hela、SW620,计算其转染率.结果:nHA-PEI(10KD)分散程度好,粒径60-80nm,在PH7.2时,Zeta电位42.87mV,能转染实验中的细胞,转然效果最好的是HepG2细胞,其次Hela、SW620,转染率高于PEI(10KD)、nHA,但低于脂质体.结论:通过阳离子PEI修饰HA,可有效将增强型绿色荧光蛋白质粒转入HepG2细胞,HA-PEI(10KD)纳米颗粒复合物有望成为基因传递的有效栽体.     

Submicron patterning of DNA oligonucleotides on silicon

The covalent attachment of DNA oligonucleotides onto crystalline silicon (100) surfaces, in patterns with submicron features, in a straightforward, two-step process is presented. UV light exposure of a hydrogen-terminated silicon (100) surface coated with alkenes functionalized with N-hydroxysuccinimide ester groups resulted in the covalent attachment of the alkene as a monolayer on the surface. Submicron-scale patterning of surfaces was achieved by illumination with an interference pattern obtained by the transmission of 248 nm excimer laser light through a phase mask. The N-hydroxysuccinimide ester surface acted as a template for the subsequent covalent attachment of aminohexyl-modified DNA oligonucleotides. Oligonucleotide patterns, with feature sizes of 500 nm, were reliably produced over large areas. The patterned surfaces were characterized with atomic force microscopy, scanning electron microscopy, epifluorescence microscopy and ellipsometry. Complementary oligonucleotides were hybridized to the surface-attached oligonucleotides with a density of 7 × 10¹² DNA oligonucleotides per square centimetre. The method will offer much potential for the creation of nano- and micro-scale DNA biosensor devices in silicon.     

Collagen and fibronectin surface modification of nanoporous anodic alumina and macroporous silicon for endothelial cell cultures

Background

The ability to direct the cellular response by means of biomaterial surface topography is important for biomedical applications. Substrate surface topography has been shown to be an effective cue for the regulation of cellular response. Here, the response of human aortic endothelial cells to nanoporous anodic alumina and macroporous silicon with collagen and fibronectin functionalization has been studied.

Methods

Confocal microscopy and scanning electron microscopy were employed to analyse the effects of the material and the porosity on the adhesion, morphology, and proliferation of the cells. Cell spreading and filopodia formation on macro- and nanoporous material was characterized by atomic force microscopy. We have also studied the influence of the protein on the adhesion.

Results

It was obtained the best results when the material is functionalized with fibronectin, regarding cells adhesion, morphology, and proliferation.

Conclusion

These results permit to obtain chemical modified 3D structures for several biotechnology applications such as tissue engineering, organ-on-chip or regenerative medicine.

     

Surface modification for DNA and protein microarrays

Microarrays of biomolecules are emerging as powerful tools for genomics, proteomics, and clinical assays, since they make it possible to screen biologically important binding events in a parallel and high throughput fashion. Because the microarrays are fabricated on a solid support, coating of the surface and immobilization strategy of the biomolecules are major issues for successful microarray fabrication. This review deals with both DNA microarrays and protein microarrays, and focuses on the various modification approaches for the two-dimensional surface materials and three-dimensional ones. In addition, the immobilization strategies including adsorption, covalent attachment, physical entrapment, and affinity attachment of the biomolecules are summarized, and advantage and limitation of representative efforts are discussed.     

Surface modification of silicone intraocular implants to inhibit cell proliferation

Photo-cross-linkable polymers bearing cinnamic, sulfonate, and carboxylate groups were synthesized by radical polymerization leading to randomly distributed copolymers. These polymers were used to coat silicone intraocular lenses in order to reduce posterior capsule opacification, also named "secondary cataract". We previously demonstrated that polymers containing both carboxylate and sulfonate groups inhibit cell proliferation, and formulations with the ratio R = COO-/(COO- + SO3-) equal to 0.64 provided the highest inhibitory effect. Ionic polymers with this formulation were synthesized to contain a monomer with pendant siloxane groups in order to get compatibility with the silicone matrix of the intraocular lenses. Anchorage of the ionic polymer at the surface of the silicone implant was achieved by a cycloaddition reaction of the photosensitive groups according to two options. These modified silicone surfaces grafted onto intraocular lenses were shown to inhibit cell proliferation to 60%.     

Surface characterization and biocompatibility of restorative resin containing nanoparticles

Composite resins that are used to restore hard tissues have several drawbacks including the accumulation of biofilm on teeth and restorations. Recently, quaternary ammonium poly(ethylene imine) (QA-PEI) nanoparticles were developed for additional antibacterial activity of restorative composite resins. QA-PEI nanoparticles were synthesized from cross-linked poly(ethylene imine) that was N-alkylated with octyl halide, followed by quaternary methylation with methyl iodide. QA-PEI particles that were embedded in restorative composite resin at 1% w/w resulted in the complete growth inhibition of Streptococcus mutans. Moreover, the antibacterial activity was retained for at least 3 months. The active substances on the surface of the restorative composite resin that were incorporated with QA-PEI particles were detected by X-ray photoelectron spectroscopy (XPS) and confocal microscopy measurements. The in vitro cytotoxicity tests showed a similar effect on the viability of the cell line that was tested with composites including modified and unmodified dental composite resins. In vivo toxicity studies, which were assessed on Wistar rats by the implantation of modified composite specimens, revealed no inflammation response 1 week after the implantation of restorative composite resin that was embedded with up to 2% w/w QA-PEI.     

Immobilization of neuraminidase and its potential application in the modification of cell surfaces.

Vibrio cholerae neuraminidase was immobilized on the inside of 1.0 mm inner diameter nylon tubing with retention of enzyme activity, when assayed at 37 degrees C and pH 5.5 with mucin as substrate. The stabilities of the immobilized and soluble enzymes were similar for up to 3 hr at 37 degrees C. Preliminary data indicated that immobilized neuraminidase will release sialic acid from the surface of leukemic AKR mouse thymus and spleen lymphocytes; however, the level of immobilized enzyme activity needs to be increased for practical applications. With this improvement immobilized neuraminidase could become a novel preparation for carrying out cell surface modifications with minimal enzyme contamination of the cell.     

Whole genome expression profiling using DNA microarray for determining biocompatibility of polymeric surfaces

There is an ever increasing need to find surfaces that are biocompatible for applications like medical implants and microfluidics-based cell culture systems. The biocompatibility of five different surfaces with different hydrophobicity was determined using gene expression profiling as well as more conventional methods to determine biocompatibility such as cellular growth rate, morphology and the hydrophobicity of the surfaces. HeLa cells grown on polymethylmethacrylate (PMMA) or a SU-8 surface treated with HNO3-ceric ammonium nitrate (HNO3-CAN) and ethanolamine showed no differences in growth rate, morphology or gene expression profiles as compared to HeLa cells grown in cell culture flasks. Cells grown on SU-8 treated with only HNO3-CAN showed almost the same growth rate (36 +/- 1 h) and similar morphology as cells grown in cell culture flasks (32 +/- 1 h), indicating good biocompatibility. However, more than 200 genes showed different expression levels in cells grown on SU-8 treated with HNO3-CAN compared to cells grown in cell culture flasks. This shows that gene expression profiling is a simple and precise method for determining differences in cells grown on different surfaces that are otherwise difficult to find using conventional methods. It is particularly noteworthy that no correlation was found between surface hydrophobicity and biocompatibility.     

Development of motorized plasma lithography for cell patterning

The micropatterning of cells, which restricts the adhesive regions on the substrate and thus controls cell geometry, is used to study mechanobiology-related cell functions. Plasma lithography is a means of providing such patterns and uses a spatially-selective plasma treatment. Conventional plasma lithography employs a positionally-fixed mask with which the geometry of the patterns is determined and thus is not suited for producing on-demand geometries of patterns. To overcome this, we have manufactured a new device with a motorized mask mounted in a vacuum chamber of a plasma generator, which we designate motorized plasma lithography. Our pilot tests indicate that various pattern geometries can be obtained with the control of a shielding mask during plasma treatment. Our approach can thus omit the laborious process of preparing photolithographically microfabricated masks required for the conventional plasma lithography.     

Cold-plasma modification of oxide surfaces for covalent biomolecule attachment

While many processes have been developed to modify the surface of glass and other oxides for biomolecule attachment, they rely primarily upon wet chemistry and are costly and time-consuming. We describe a process that uses a cold plasma and a subsequent in vacuo vapor-phase reaction to terminate a variety of oxide surfaces with epoxide chemical groups. These epoxide groups can react with amine-containing biomolecules, such as proteins and modified oligonucleotides, to form strong covalent linkages between the biomolecules and the treated surface. The use of a plasma activation step followed by an in vacuo vapor-phase reaction allows for the precise control of surface functional groups, rather than the mixture of functionalities normally produced. By maintaining the samples under vacuum throughout the process, adsorption of contaminants is effectively eliminated. This process modifies a range of different oxide surfaces, is fast, consumes a minimal amount of reagents, and produces attachment densities for bound biomolecules that are comparable to or better than commercially available substrates.     

Functional patterning of DNA origami by parallel enzymatic modification

We demonstrate here a rapid and cost-effective technique for nanoscale patterning of functional molecules on the surface of a DNA origami. The pattern is created enzymatically by transferring a functionalized dideoxynucleotide to the 3'-end of an arbitrary selected set of synthetic DNA oligonucleotides positioned approximately 6 nm apart in a 70 × 100 nm(2) rectangular DNA origami. The modifications, which are performed in a single-tube reaction, provide an origami surface modified with a variety of functional groups including chemical handles, fluorescent dyes, or ligands for subsequent binding of proteins. Efficient labeling and patterning was demonstrated by gel electrophoresis shift assays, reverse-phase HPLC, mass spectrometry, atomic force microscopy (AFM) analysis, and fluorescence measurements. The results show a very high yield of oligonucleotide labeling and incorporation in the DNA origami. This method expands the toolbox for constructing several different modified DNA origami from the same set of staple strands.     

Genetic modification of lignin biosynthesis for improved biofuel production

The energy in cellulosic biomass largely resides in plant cell walls. Cellulosic biomass is more difficult than starch to break down into sugars because of the presence of lignin and the complex structure of cell walls. Transgenic down-regulation of major lignin genes led to reduced lignin content, increased dry matter degradability, and improved accessibility of cellulases for cellulose degradation. This review provides background information on lignin biosynthesis and focuses on genetic manipulation of lignin genes in important monocot species as well as the dicot potential biofuel crop alfalfa. Reduction of lignin in biofuel crops by genetic engineering is likely one of the most effective ways of reducing costs associated with pretreatment and hydrolysis of cellulosic feedstocks, although some potential fitness issues should also be addressed.     

Method for patterning stretched DNA molecules on mica surfaces by soft lithography

Lambda DNA was stretched and patterned on mica surface using soft lithography. A highly diluted solution of amino propyl trimethoxy silane in hexane was deposited on a line patterned polydimethylsiloxane (PDMS) stamp. The functionalized stamp was then used to pick up DNA by molecular combing while the line patterns are parallel to the liquid surface. The stamp was then microcontact printed on freshly cleaved mica. We successfully obtained stretched DNA pattern on mica surface. DNA was found to be stretched in patterns perpendicular to those carved on the stamp. The stretched DNA population was large enough to be used for molecular biology mapping studies. Furthermore, the possibility of locating stretched DNA molecules in the desired position by stamping makes this method a good candidate for assembling non-semiconductor molecular devices.     

In vitro assessment of the biocompatibility of chemically modified GaAs surfaces

GaAs has excellent optical, electrical, and mechanical properties and shows promise to be used in the fabrication of novel devices. However, the unprotected GaAs surface can release heavy metal compounds such as AsOx, which are toxic to living cells. A promising approach to reduce or eliminate this release relies on the passivation of the GaAs surface using different chemical approaches. In this work, we compared three different passivation methods aimed at enhancing the viability of cells on GaAs. Protective layers composed of self-assembled alkyl thiols, polypeptides, thick polymer layers, and shells of polyelectrolytes were tested. We confirmed that the GaAs surface can be made biocompatible for several days based on in vitro tests with HeLa and KB cells. In addition, we compared the cell spreading behavior on the GaAs substrates modified by different chemical approaches. Our results suggest that when the toxicity of the GaAs surface is reduced or eliminated, the cells’viability and spreading depend on the chemical and topographical nature of the surface.     

Methods for the topographical patterning and patterned surface modification of hydrogels based on hydroxyethyl methacrylate

Hydrogels have gained broad acceptance as a class of biocompatible materials. In this paper, we report the topographic patterning and regiospecific functionalization of hydrogel surfaces. Both photolithography and soft lithography are combined in a hybrid process to form these topographic features. By functionalization of a base layer surface followed by lithographic patterning steps, it is possible to introduce chemical functions to specific regions of the patterned surface. The model systems investigated were based on 2-hydroxyethyl methacrylate (HEMA), which is well-known for its low toxicity and widespread use in biomedical applications. Tests of Ni-NTA modified hydrogel surfaces showed successful binding of fluorescently labeled proteins to selected regions of the patterned hydrogel surface. These processes can be expanded to a wide range of monomer systems.     

Region-specific requirement for cholesterol modification of sonic hedgehog in patterning the telencephalon and spinal cord

Sonic hedgehog (Shh) secreted from the axial signaling centers of the notochord and prechordal plate functions as a morphogen in dorsoventral patterning of the neural tube. Active Shh is uniquely cholesterol-modified and the hydrophobic nature of cholesterol suggests that it might regulate Shh spreading in the neural tube. Here, we examined the capacity of Shh lacking the cholesterol moiety (ShhN) to pattern different cell types in the telencephalon and spinal cord. In mice expressing ShhN, we detected low-level ShhN in the prechordal plate and notochord, consistent with the notion that ShhN can rapidly spread from its site of synthesis. Surprisingly, we found that low-level ShhN can elicit the generation of a full spectrum of ventral cell types in the spinal cord, whereas ventral neuronal specification and ganglionic eminence development in the Shh(N/-) telencephalon were severely impaired, suggesting that telencephalic patterning is more sensitive to alterations in local Shh concentration and spreading. In agreement, we observed induction of Shh pathway activity and expression of ventral markers at ectopic sites in the dorsal telencephalon indicative of long-range ShhN activity. Our findings indicate an essential role for the cholesterol moiety in restricting Shh dilution and deregulated spread for patterning the telencephalon. We propose that the differential effect of ShhN in patterning the spinal cord versus telencephalon may be attributed to regional differences in the maintenance of Shh expression in the ventral neuroepithelium and differences in dorsal tissue responsiveness to deregulated Shh spreading behavior.