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Biomimetic engineering of non-adhesive glycocalyx-like surfaces using oligosaccharide surfactant polymers.

The external region of a cell membrane, known as the glycocalyx, is dominated by glycosylated molecules, which direct specific interactions such as cell-cell recognition and contribute to the steric repulsion that prevents undesirable non-specific adhesion of other molecules and cells. Mimicking the non-adhesive properties of a glycocalyx provides a potential solution to the clinical problems, such as thrombosis, that are associated with implantable devices owing to non-specific adsorption of plasma proteins. Here we describe a biomimetic surface modification of graphite using oligosaccharide surfactant polymers, which, like a glycocalyx, provides a dense and confluent layer of oligosaccharides. The surfactant polymers consist of a flexible poly(vinyl amine) with dextran and alkanoyl side chains. We show that alkanoyl side chains assemble on graphite through hydrophobic interaction and epitaxial adsorption. This constrains the polymer backbone to lie parallel to the substrate, with solvated dextran side chains protruding into the aqueous phase, creating a glycocalyx-like coating. The resulting biomimetic surface is effective in suppressing protein adsorption from human plasma protein solution.

Adhesiveness↗

Immunoselection of a human melanoma resistant to specific lysis by autologous tumor-infiltrating lymphocytes. Possible mechanisms for immunotherapeutic failures.

Intratumoral heterogeneity has been proposed as a possible basis for immunotherapeutic failure when tumor-specific agents such as tumor infiltrating lymphocytes (TIL) are employed for cancer therapy. To examine this issue, highly specific oligoclonal MHC class I-restricted cytolytic TIL grown in bulk culture from patient 397 were used to immunoselect a TIL-resistant variant tumor from the autologous cultured melanoma line 397-mel. Four cycles of immunoselection produced tumor 397-R4, a variant completely resistant to 397 TIL but not to allogeneic LAK cell lysis in 4-h 51Cr release assays. By flow microfluorometry analysis, this tumor variant had not lost MHC molecules, adhesion molecules, or a variety of tumor-associated Ag expressed by the parent tumor but showed decreased expression of many Ag examined. Failure of 397-R4 to cold target inhibit TIL lysis of 397-mel suggested that cell-surface modification was at least one mechanism causing TIL resistance. The inherent lysability of 397-R4 was equal to 397-mel, as confirmed by lectin-dependent cellular cytotoxicity, lysis by non-MHC restricted allogeneic TIL, and lysis by a second line of 397 TIL grown independently from tumor 397. Treatment of 397-R4 with IFN-alpha or IFN-gamma, +/- TNF-alpha for 72 h before cytolytic assays enhanced TIL lysis of this target slightly, and enhanced surface expression of MHC class I and II molecules and the adhesion molecule ICAM-1. The resistant phenotype of 397-R4 was evident in all clones of 397-R4 examined and has been maintained in serial culture for over 13 mo and through passage in nude mice, suggesting that such stable tumor variants may provide an in vivo escape mechanism from specific immune reagents such as TIL. Evolving patterns of TIL culture clonality over time, as well as the spontaneous emergence of different clones in two long term TIL cultures grown under identical conditions from the same source of cryopreserved tumor, were documented by analyzing TCR gene rearrangements and suggest that TIL from different culture passages or lines may be used to overcome resistant tumor subpopulations.

Animals↗

The influence of surfactant on PLGA microsphere glass transition and water sorption: remodeling the surface morphology to attenuate the burst release.

PURPOSE: The stability of protein unloaded and loaded poly(lactic-co-glycolic acid) (PLGA) microspheres fabricated with surfactant was challenged through exposure to environmental conditions of different relative humidity. METHODS: Polyvinyl alcohol (PVA) or Triton X-100 was added to the primary emulsion of the double-emulsion solvent evaporation technique. After storage at ambient humidity and 75% relative humidity, the mechanical stability of the polymer was tested to reveal PLGA chain mobility using differential scanning calorimetry. Subsequent surface modifications were examined by atomic force microscopy (AFM), and protein release profiles were collected. RESULTS: Residual amounts of PVA and particularly Triton X-100 raised the hydrophilicity of the microspheres. When exposed to ambient humidity or 75% relative humidity, PVA and Triton X-100 had, respectively, an antiplasticizing and a plasticizing effect upon PLGA, and both led to physical aging. The high-resolution AFM imaging of microspheres containing model protein and Triton X-100 showed that the depth of the surface pores was reduced when exposed to 75% relative humidity, and the initial burst release subsequently decreased. CONCLUSION: These studies suggested that the mechanical stability of PLGA was influenced by the addition of surfactants, which, depending on the formulation, led to surface pore remodeling under high humidity, reducing the initial burst release while maintaining the spherical integrity of the microsphere.

Delayed-Action Preparations↗

Effect of heparin-surface-modified poly(methyl methacrylate) intraocular lenses on the postoperative inflammation in an Asian population.

PURPOSE: To compare the efficacy of heparin-surface-modified (HSM), poly(methyl methacrylate) (PMMA) posterior chamber intraocular lenses (IOLs) with that of unmodified PMMA IOLs in reducing postoperative complications caused by inflammatory reactions after extracapsular cataract extraction in an Asian population. SETTING: Departments of Ophthalmology, University of Malaya, Kuala Lumpur, Malaysia, and Tan Tock Seng Hospital, Singapore. METHODS: In a randomized, double-blind study performed at two centers, 51 patients received an HSM PMMA lens and 48, an unmodified PMMA IOL. Cell and pigment deposits were evaluated by slitlamp at 1 to 6 days, 2 to 3 weeks, and 3 to 6 months postoperatively. RESULTS: Significantly more eyes with unmodified IOLs had inflammatory cell deposits than those with HSM IOLs at 3 to 6 months (P < .001) and 12 to 14 months (P = .018) postoperatively. The HSM group also had significantly fewer cell deposits per patient at these two follow-ups. Significantly more eyes in the non-HSM group had pigment deposits 3 to 6 months after surgery (P = .049). One year postoperatively, about 85% of patients in both groups had a best corrected visual acuity of 0.5 or better. CONCLUSION: Heparin surface modification significantly reduced the inflammatory response to PMMA IOLs in an Asian population for at least 12 to 14 months.

Adult↗

A direct route toward assembly of nanoparticle-carbon nanotube composite materials.

The preparation of nanocomposite materials from carbon nanotubes (CNTs) and metal or metal oxide nanoparticles has important implications to the development of advanced catalytic and sensory materials. This paper reports findings of an investigation of the preparation of nanoparticle-coated carbon nanotube composite materials. Our approach involves molecularly mediated assembly of monolayer-capped nanoparticles on multiwalled CNTs via a combination of hydrophobic and hydrogen-bonding interactions between the capping/mediating shell and the CNT surface. The advantage of this route is that it does not require tedious surface modification of CNTs. We have demonstrated its simplicity and effectiveness for assembling alkanethiolate-capped gold nanoparticles of 2-5 nm core sizes onto CNTs with controllable coverage and spatially isolated character. The loading and distribution of the nanoparticles on CNTs depend on the relative concentrations of gold nanoparticles, CNTs, and mediating or linking agents. The composite nanomaterials can be dispersed in organic solvent, and the capping/linking shells can be removed by thermal treatment to produce controllable nanocrystals on the CNT surfaces. The nanocomposite materials are characterized using transmission electron microscopy and Fourier transform infrared spectroscopy techniques. The results will be discussed in terms of developing advanced catalytic and sensory nanomaterials.

Gold↗

Preparation of bioactive Ti and its alloys via simple chemical surface treatment.

A simple chemical method was established for inducing bioactivity of Ti and its alloys. When pure Ti, Ti-6A1-4V, Ti-6A1-2Nb-Ta, and Ti-15Mo-5Zr-3A1 substrates were treated with 10M NaOH aqueous solution and subsequently heat-treated at 600 degrees C, a thin sodium titanate layer was formed on their surfaces. Thus, treated substrates formed a dense and uniform bonelike apatite layer on their surfaces in simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma. This indicates that the alkali- and heat-treated metals bond to living bone through the bonelike apatite layer formed on their surfaces in the body. The apatite formation on the surfaces of Ti and its alloys was assumed to be induced by a hydrated titania which was formed by an ion exchange of the alkali ion in the alkali titanate layer and the hydronium ion in SBF. The resultant surface structure changed gradually from the outermost apatite layer to the inner Ti and its alloys through a hydrated titania and titanium oxide layers. This provides not only the strong bonding of the apatite layer to the substrates but also a uniform gradient of stress transfer from bone to the implants. The present chemical surface modification is therefore expected to allow the use the bioactive Ti and its alloys as artificial bones even under load-bearing conditions.

Alkalies↗

Modification of kaolinite surfaces through mechanochemical treatment--a mid-IR and near-IR spectroscopic study.

The modification of kaolinite surfaces through mechanochemical treatment has been studied using a combination of mid-IR and near-IR spectroscopy. Kaolinite hydroxyls were lost after 10 h of grinding as evidenced by the decrease in intensity of the OH stretching vibrations at 3695 and 3619 cm(-1) and the deformation modes at 937 and 915 cm(-1). Concomitantly an increase in the hydroxyl-stretching vibrations of water is observed. The mechanochemical activation (dry grinding) causes destruction in the crystal structure of kaolinite by the rupture of the O-H, Al-OH, Al-O-Si and Si-O bonds. Evidence of this destruction may be followed using near-IR spectroscopy. Two intense bands are observed in the spectral region of the first overtone of the hydroxyl-stretching vibration at 7065 and 7163 cm(-1). These two bands decrease in intensity with mechanochemical treatment and two new bands are observed at 6842 and 6978 cm(-1) assigned to the first overtone of the hydroxyl-stretching band of water. Concomitantly the water combination bands observed at 5238 and 5161 cm(-1) increase in intensity with mechanochemical treatment. The destruction of the kaolinite surface may be also followed by the loss of intensity of the two hydroxyl combination bands at 4526 and 4623 cm(-1). Infrared spectroscopy shows that the kaolinite surface has been modified by the removal of the kaolinite hydroxyls and their replacement with water adsorbed on the kaolinite surface. NIR spectroscopy enables the determination of the optimum time for grinding of the kaolinite. Further NIR allows the possibility of continual on-line analysis of the mechanochemical treatment of kaolinite.

Hydroxides↗

Nanoscale glassification of gold substrates for surface plasmon resonance analysis of protein toxins with supported lipid membranes.

Surface plasmon resonance (SPR) spectroscopy, a powerful tool for biosensing and protein interaction analysis, is currently confined to gold substrates and the relevant surface chemistries involving dextran and functional thiols. Drawbacks of using self-assembled monolayers (SAMs) for SPR-related surface modification include limited stability, pinhole defects, bioincompatibility, and nonspecific protein adsorption. Here we report the development of stable nanometer-scale glass (silicate) layers on gold substrates for SPR analysis of protein toxins. The nanoscale silicate layers were built up with layer-by-layer deposition of poly(allylamine hydrochloride) and sodium silicate, followed by calcination at high temperature. The resulting silicate films have a thickness ranging from 2 to 15 nm and demonstrate outstanding stability in flow cell conditions. The use of these surfaces as a platform to construct supported bilayer membranes (SBMs) is demonstrated, and improved performance against protein adsorption on SBM-coated surfaces is quantified by SPR measurements. SBMs can be formed reproducibly on the silicate surface via vesicle fusion and quantitatively removed using injection of 5% Triton X-100 solution, generating a fresh surface for each test. Membrane properties such as lateral diffusion of the SBMs on the silicate films are characterized with photobleaching methods. Studies of protein binding with biotin/avidin and ganglioside/cholera toxin systems show detection limits lower than 1 microg/mL (i.e., nanomolar range), and the response reproducibility is better than 7% RSD. The method reported here allows many assay techniques developed for glass surfaces to be transferred to label-free SPR analysis without the need for adaptation of protocols and time-consuming synthetic development of thiol-based materials and opens new avenues for developing novel bioanalytical technologies for protein analysis.

Avidin↗

Bonding strength and durability of alkaline-treated titanium to veneering resin.

The shear bonding strengths of a veneering resin to polished, sandblasted, and retention bead-cast commercially pure titanium (cpTi) plates with and without alkaline treatment were measured before and after thermal cycling. The bonding strengths to polished cpTi with and without alkaline treatment decreased remarkably with thermal cycling (p<0.01). The bonding strength to sandblasted cpTi with alkaline treatment at 5,000 thermal cycles showed no significant differences from those before thermal cycling (p>0.05), and those at 20,000 thermal cycles showed values which were quite small (p<0.01). On the other hand, there were no significant differences in the bonding strengths of veneering resin to retention bead-cast cpTi in all conditions (p>0.05). These results suggested that although alkaline treatment is a simple and effective surface modification technique for titanium improving adhesion to resin due to formation of tight-fine rutile particles, it does not provide sufficient bonding durability for long-period restorations.

Composite Resins↗

Surface-modified LPD nanoparticles for tumor targeting.

We have developed a tumor-targeted LPD formulation (liposome-polycation-DNA complex) for siRNA. With surface modification, the targeted, PEGylated LPD increased the delivery efficiency by four-fold and the gene-silencing effect by two- to three-fold. Downregulation of survivin in human lung cancer cells by targeted LPD induced 90% of apoptosis and sensitized the cells to cisplatin by four-fold. PEGylated LPD formulation also significantly improved the tumor localization of siRNA in the NCI-H460 human lung cancer xenograft model. The tumor appeared to be the major uptake organ for siRNA formulated in surface-modified LPD. Our encouraging results indicate that surface-modified LPD may be a potent carrier for RNAi-based tumor therapy.

Animals↗

Ultrastructural histochemical alteration of the plasma membrane in chronic myelocytic leukemia.

Ultrastructural histochemical evaluation of the surface of normal human blood and bone marrow cells exposed to the pyroantimonate-osmium (PAO) reaction indicated the selective binding of pyroantimonate to certain cations (calcium, magnesium, and possibly sodium) associated with the plasma membrane of neutrophilic leukocytes and their developmental forms. Other leukocytes and their precursors did not exhibit plasma membrane PAO reactivity. The extent of surface binding was related to cell maturity, with maximal labeling evident in the mid and late promyelocytes; decreased binding occurred with subsequent maturation while myeloblasts were nonreactive. This study was initiated to ascertain if histochemical surface modifications of neutrophilic cells occur in certain myeloproliferative disorders. In this regard, we have been able to demonstrate a distinctive defect in the plasma membrane PAO binding characteristics of the leukemic cells in chronic myelocytic leukemia (CML). Limited binding of pyroantimonate to the plasma membrane of the leukemic cell series in four patients with CML contrasted with that of the normal granulocytic cell series and the neutrophilic cells seen in myelomonocytic leukemia (two patients), myelofibrosis (one patient), and acute myelocytic leukemia (three patients). Comparison of surface PAO reactivity of neutrophilic cells in all stages of maturation in two patients with CML in blast crisis revealed that, in the patient with 30% circulating blast cells, PAO reactivity was identical to that noted in CML, while in the patient with 80% circulating blast forms, the PAO reactivity of the maturing neutrophilic cells more nearly resembled that observed in neutrophilic cells from normal individuals. Many neutrophilic cells from patients with myelofibrosis and myelomonocytic leukemia and from one patient in severe blast crisis had large surface deposits of pyroantimonate considered to reflect increased membrane-associated reactive cation.

Antimony↗

Effect of alternative crosslinking techniques on the enzymatic degradation of bovine pericardia and their calcification.

The in vitro calcification and enzymatic degradation of bovine pericardia (BP) after a series of surface treatments were studied as a function of exposure time. The degradation of these treated surfaces was monitored by scanning electron micrography and tensile strength measurements. Polyethylene glycol-(PEG) grafted BP and glutaraldehyde-(GA) treated BPs retained maximum stability in collagenase digestion compared with SDS-treated BP. The ability of alpha chymotrypsin, bromelain, esterase, trypsin, and collagenase to modulate the degradation of SDS-, GA-, PEG-, Carbodiimide-, and glycidylether-treated BPs also was investigated. Incubation of various enzymes to these crosslinked pericardia variably reduced the tensile strength of these tissues. It is conceivable that chemical treatments of pericardial tissues might have altered their physical and chemical configuration and the subsequent degradation properties. In vitro calcification studies showed a substantial reduction in the calcification profile of PEG-grafted bovine pericardia compared to other treated tissues. Furthermore, the biocompatibility aspects of pericardial tissues were established by platelet adhesion and octane contact angle. In conclusion, it seems that the surface modification of bovine pericardia via GA-PEG grafting may provide new ways of controlling biodegradation and calcification.

Animals↗

Bacterial adhesion on PEG modified polyurethane surfaces.

Polyurethane surface was modified with poly(ethylene glycol) (mol. wt. 1000, PEG1k) carrying terminal hydroxyl, amino and sulfonate groups, poly(ethylene glucol) (mol. wt. 3350, PEG3.4k) and PEG3.4k-Heparin, respectively. These surfaces were investigated for bacterial adhesion using S. epidermidis and E. coli in tryptic soya broth (TSB), brain heart infusion (BHI), and human plasma. All PEG modified surfaces reduced bacterial adhesion significantly and the adhesion level differs depending on surfaces as well as media. In the case of PEG1k surfaces, no reduction of S. epidermidis adhesion was demonstrated in TSB media, regardless of terminal functional groups of PEG1k. However, adhesion in plasma was reduced to the different degree, depending on terminal groups of PEG1k (least adhesion on sulfonated PEG surface). Relatively longer PEG surface (PEG3.4k) and PEG3.4k-heparin surface minimized bacterial adhesion in both media. In the case of E. coli adhesion, significant reduction in adherent bacteria was observed on all PEG1k, PEG3.4k, and PEG-heparin surfaces in both media compared to controls. In contrast, no reduction in bacterial adhesion was demonstrated on poly(propylene glycol) (PPG1k) grafted PU surface as compared to control PU. These results suggest that surface modification with PEG1k-SO3, PEG3.4k and PEG3.4k-heparin seems to be effective for prevention of bacterial adhesion and subsequent infection.

Animals↗

A glycosyltransferase involved in biosynthesis of triglycosylated glycopeptidolipids in Mycobacterium smegmatis: impact on surface properties.

The cell envelope of mycobacteria is a complex structure that plays an important role in the interactions of the cell with its environment and in the protection against the antimicrobial activity of the immune system. Glycopeptidolipids (GPLs) are species- or type species-specific glycolipids that are present at the surface of a number of mycobacteria and that are characterized by a high variability in glycosylation patterns. These GPLs possess various biological activities that depend mostly on the sugars capping the core molecule. In Mycobacterium smegmatis, the GPL core can be substituted by either two or three deoxyhexoses. In this study, we show that Gtf3 is a glycosyltransferase responsible for the synthesis of the triglycosylated GPLs. Biochemical analysis of these molecules, with a combination of mass spectrometry and chemical degradation methods, has shown that they contain three deoxyhexose moieties. The presence of the triglycosylated GPLs is associated with cell surface modifications that lead to a decrease in sliding motility as well as a modification in cellular aggregation and colony appearance on Congo red. Phylogenetic analysis indicated that Gtf3 is a member of a yet-uncharacterized glycosyltransferase family conserved among the mycobacteria.

Bacterial Proteins↗

Quantifying surface coverage of colloidal silica by a cationic peptide using a combined centrifugation/time-resolved fluorescence anisotropy approach.

Recent experimental studies have shown that time-resolved fluorescence anisotropy (TRFA) is a promising methodology for in situ characterization of the surface modification of aqueous silica nanocolloids. Here we provide a more fundamental insight into the principle of this approach and discuss how the adsorption parameters for a cationic peptide, Lys-Trp-Lys (denoted using the standard shortform KWK), onto Ludox nanoparticles (NPs) are linked to the rotational dynamics of rhodamine 6G (R6G) dispersed in the KWK/Ludox mixture. First, the adsorption isotherm of KWK on hydrophilic controlled pore glass (CPG-3000) was obtained using the traditional centrifugation method, which provides the total molar amount of KWK per unit surface area of the silica. Assuming that both CPG and Ludox particles possess identical surface properties when suspended in the same aqueous buffer, both materials should also have identical adsorption properties. Thus, the adsorbed amount of KWK per unit area at a given total KWK concentration, as determined by the centrifugation method, can be plotted against the fractions of R6G anisotropy decay components at the same KWK concentration to relate the anisotropy components to the absolute surface coverage. Using this approach, it was determined that the concentration of KWK at which the CPG surface was saturated corresponded to the condition g = 0 in the R6G decay, where g is the fraction of the nondecaying anisotropy component. This condition means that there is no R6G bound to the fraction of Ludox NPs with a radius R > 2.5 nm at maximum KWK coverage, consistent with the adsorbed peptide forming a continuous layer on the Ludox surface. Hence, the g value obtained from TRFA analysis can be used to assess the absolute surface coverage of monolayer coatings on colloidal nanoparticles.

Centrifugation↗

Novel O-palmitoylscleroglucan-coated liposomes as drug carriers: development, characterization and interaction with leuprolide.

Polysaccharide-coated liposomes have been studied for their potential use for peptide drug delivery by the oral route because they are able to minimize the disruptive influences on peptide drugs of gastrointestinal fluids. The aim of this work was to synthesize and characterize a modified polysaccharide, O-palmitoylscleroglucan (PSCG), and to coat unilamellar liposomes for oral delivery of peptide drugs. To better evaluate the coating efficiency of PSCG, also scleroglucan (SCG)-coated liposomes were prepared. We studied the surface modification of liposomes and the SCG- and PSCG-coated liposomes were characterized in terms of size, shape, zeta potential, influence of polymer coating on bilayer fluidity, stability in serum, in simulated gastric and intestinal fluids and against sodium cholate and pancreatin. Leuprolide, a synthetic superpotent agonist of luteinizing hormone releasing hormone (LHRH) receptor, was chosen as a model peptide drug. After polymer coating the vesicle dimensions increased and the zeta potential shifted to less negative values. These results indicate that both SCG- and PSCG-coated liposomes surface and DSC results showed that PSCG was anchored on the liposomal surface. The stability of coated-liposomes in SGF, sodium cholate solution and pancreatin solution was increased. From this preliminary in vitro studies, it seems that PSCG-coated liposomes could be considered as a potential carrier for oral administration.

Animals↗

Poly(L-lysine)-GRGDS as a biomimetic surface modifier for poly(lactic acid).

The immobilization of adhesion peptide sequences (such as RGD) at the surfaces of poly(alpha-hydroxyacid)s, including poly(lactic acid) (PLA), is complicated by an absence of functional groups to support covalent attachment. We demonstrate a method to overcome this problem, by attaching the peptide to poly(L-lysine) (PLL), which immobilizes the sequence through adsorption at the poly(alpha-hydroxyacid) surface. When coated using a 0.01% w/v solution of PLL-GRGDS, bovine aortic endothelial cells seeded upon the modified PLA showed a marked increase in spreading over unmodified PLA. However, inhibition of the cell-spreading effect occurred when using higher concentrations of PLL-GRGDS, which we attribute to the PLL component. This inhibitory effect can be challenged by increasing the amount of GRGDS attached to each PLL molecule. Potentially, this is a flexible method of surface modification that can engineer many different types of tissue engineering scaffolds with a variety of biomolecules, thus allowing initial cell adhesion to be controlled.

Animals↗

Endothelial cell--interactions with polyelectrolyte multilayer films.

The seeding of endothelial cells (ECs) on biomaterial surfaces became a major challenge, allowing to improve the non-thrombogenic properties of these surfaces. Recently, the use of polyelectrolyte films has been suggested as a new versatile technique of surface modification aimed at tissue engineering. In this study, we evaluate the adhesion properties of ECs on two types of polyelectrolyte films ending either by poly(D-lysine) (PDL), or poly(allylamine hydrochloride) (PAH), and compared them to data obtained on PDL or PAH monolayers, glass and fibronectin (Fn)-coated glass. ECs seeded on polyelectrolyte films showed a good morphology, allowing ECs to resist physiological shear stress better compared to ECs seeded on glass or Fn. The expression of beta1 integrins was slightly lower on polyelectrolyte films than on control surfaces. However, the phosphorylation of focal adhesion kinase, involved in the transduction of adhesion signal, was not modified on PAH ending films compared to control surfaces; whereas it became lower on PDL ending films. Finally, PAH ending films improve strongly ECs adhesion without disturbing the adhesion mechanism, necessary for the development of a new endothelium. These types of films or similar build-ups could thus be used in the future as a way to modify surfaces for vascular tissue engineering.

Actins↗