Performance of a polyurethane vascular prosthesis carrying a dipyridamole (Persantin) coating on its lumenal surface

A porous polyurethane vascular prosthesis with an internal diameter of 5 mm was studied. The graft carries a coating of immobilized dipyridamole (Persantin(R)) on the surface of its lumen. Dipyridamole is a potent nontoxic inhibitor of platelet activation/aggregation, and also a strong inhibitor of vascular smooth muscle cell proliferation. The polyurethane material is also known as Chronoflex(R), and already finds use as a vascular access graft. The coated vascular graft was studied in vitro (hemocompatibility, interaction with blood platelets and cultured endothelial cells), as well as in two established in vivo models. In the first in vivo study, coated grafts were implanted in goats, as a bypass of the carotid artery (four animals, eight grafts, length of the graft was approximately 12 cm). Four uncoated grafts were used as controls in otherwise identical experiments. In the second in vivo experiment, eight sheep were used. Each animal received one coated and one uncoated prosthesis as an interposition graft in the carotid artery (length of the graft was 4 cm). The in vitro experiments revealed that the dipyridamole coating has three beneficial effects: reduced thrombogenicity, reduced adherence of blood platelets, and accommodation of a confluent monolayer of endothelial cells. The goat experiments showed patency of the coated grafts in three of the eight cases. The sheep experiments were not useful for the evaluation of the dipyridamole coating because deterioration of the polyurethane material was observed. The in vivo results indicate that the dipyridamole coating may positively influence the patency rate, probably because the coating promotes the growth of an endothelial cell lining. The sheep data show, however, that the limited stability of the Chronoflex(R) material precludes its issue for the construction of permanent small-bore vascular grafts.     

A common pathway for the uptake of surfactant lipids by alveolar cells

The uptake of different surfactant lipids-dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol (PG), or phosphatidylinositol (PI)-and liposomes with a surfactant-like composition by alveolar type II cells (alveolar type II cells) and macrophages (alveolar macrophages) was studied in vitro. Fluorescent-labeled liposomes containing either 86% of the studied lipid, i.e., DPPC, PG, PI, and 6% labeled phosphatidylethanolamine (PE) and 8% cholesterol or a lipid mixture similar to surfactant (DPPC, PG, PI, phosphatidylcholine, PE, and cholesterol in a weight ratio of 55:8:2:21:8:6) were incubated with alveolar macrophages and alveolar type II cells. The cell-associated fluorescence assessed by flow cytometry demonstrated a higher uptake of PG and PI by both alveolar macrophages and alveolar type II cells, and a lower uptake of DPPC by alveolar macrophages. In addition, fewer alveolar type II cells take up DPPC, whereas there are no differences for the alveolar macrophages in the number of cells involved in the uptake. Competition experiments with Texas Red-labeled liposomes and either DPPC liposomes or PI liposomes labeled with Bodipy indicated that all these liposomes are internalized via the same pathway by alveolar cells. Thus, lipid composition directly influences the (re)uptake of surfactant.     

Encapsulation and release of a hydrophobic drug from hydroxyapatite coated liposomes

Hydroxyapatite (HA) coated liposomes (HACL) have been successfully manufactured and filled with a model hydrophobic (lipophilic) drug, indomethacin (IMC). These HACL particles have been characterized in terms of particle size and zeta-potential. The liposomes are formed from 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Altering their relative proportions caused the zeta-potential to change from -38.8 to -67.0 mV, with a concomitant change in phase transition temperature from 36.4 to 53.3 degrees C. These changes also affect the drug loading efficiency. The release profiles of IMC have been measured. HA coating of the liposome reduces the release rate of IMC over uncoated liposomes. Under the present experimental conditions 70% of the drug is released after approximately 5h from the liposome, but coating with HA changes this time to over 20 h. Perhaps most importantly, it has been observed that for uncoated liposomes, IMC is released at a greater rate at pH=7.4 than at pH=4. However, coating with HA reduced the rate at pH=7.4 compared to pH=4. This behaviour arises because IMC is more soluble under basic conditions, but HA is more soluble under acidic conditions. This behaviour shows that it is now possible to have environmental control over the release of drugs from HA-coated liposomes.     

Improved endothelialization and reduced thrombosis by coating a synthetic vascular graft with fibronectin and stem cell homing factor SDF-1α

Failure of synthetic small-diameter vascular grafts is determined mainly by the lack of endothelial cells, as these cells inhibit thrombosis and intimal hyperplasia. Coating of graft material with homing factors for circulating stem cells has the potential to improve endogenous endothelialization of these grafts and to reduce graft failure. Synthetic knitted polyester grafts (6mm diameter) were coated with FN and SDF-1α before surgical interposition in the carotid artery of sheep. Similar uncoated vascular grafts were implanted in the contralateral side as internal controls. To study the early attraction of stem cells, grafts were implanted in a first series of nine sheep and explanted after 1 or 3 days. In coated grafts, four times higher fractions of CD34(+) and three to four times higher fractions of CD117(+) cells adhering to the vessel walls were found than in control grafts (P<0.05). When such coated and non-coated grafts were implanted in 12 other sheep and explanted after 3 months, all coated grafts were patent, while one control graft was occluded. EcNOS staining revealed that FN-SDF-1α coating significantly increased coverage with endothelial cells from 27 ± 4% of the graft to 48 ± 4% compared with the controls (P=0.001). This was associated with a significant reduction of intimal hyperplasia (average thickness 1.03 ± 0.09 mm in controls vs. 0.69 ± 0.04 mm in coated grafts; P=0.009) and significantly less adhesion of thrombotic material in the middle part of the graft (P=0.029). FN-SDF-1α coating of synthetic small-caliber vascular grafts stimulated the attraction of stem cells and was associated with improved endothelialization and reduced intimal hyperplasia and thrombosis.     

Glycocalyx-mimetic dextran-modified poly(vinyl amine) surfactant coating reduces platelet adhesion on medical-grade polycarbonate surface

A dextran-modified poly(vinyl amine) comb-like surfactant polymer, poly(N-vinyl dextran aldonamide-co-N-vinyl hexanamide), that can surface-adsorb on hydrophobic polymeric substrates, was designed to improve the interfacial blood-compatibility of polymeric biomaterials. Medical-grade polycarbonate was selected as a model substrate because of its extensive use in blood-contacting biomedical devices like hemodialyzers, blood pumps and oxygenators. The surfactant polymer was physisorbed from aqueous solution onto the polycarbonate substrate. The surfactant coating was stable under dynamic shear conditions in whole blood, as confirmed by fluorescence microscopy and total internal reflection fluorescence (TIRF) experiments with fluorescein-labeled surfactant polymer. The coated disks and uncoated control disks were exposed to platelet-rich plasma (PRP) and whole human blood in a rotating disk system (RDS) to study platelet-adhesion under dynamic shear stress environments. Adhered platelets were stained with fluorescein isothiocyante (FITC)-tagged anti-CD41a monoclonal antibody and imaged by epifluorescence microscopy. Complimentary images were obtained by phase-contrast microscopy. Platelet adhesion on the surfactant-coated disks was reduced by approximately 90%, compared with uncoated disks. The images also showed a concomitant reduction in platelet-derived microparticles on surfactant-coated disks, compared with uncoated disks. The results suggest potential application of carbohydrate-modified surfactant polymers as a glycocalyx-mimetic non-thrombogenic interfacial coating for blood-contacting biomaterials.     

Overcoming drug-resistant lung cancer by paclitaxel loaded dual-functional liposomes with mitochondria targeting and pH-response

Mitochondrion-orientated transportation of smart liposomes has been developed as a promising strategy to deliver anticancer drugs directly to tumor sites, and these have a tremendous potential for killing cancer cells, especially those with multidrug resistance (MDR). Herein we report a novel dual-functional liposome system possessing both extracellular pH response and mitochondrial targeting properties to enhance drug accumulation in mitochondria and trigger apoptosis of drug-resistant cancer cells. Briefly, peptide _D[KLAKLAK]₂ (KLA) was modified with 2, 3-dimethylmaleic anhydride (DMA) and combined with 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) to yield a DSPE-KLA-DMA (DKD) lipid. This dual-functional DKD was then mixed with other commercially available lipids to fabricate liposomes. In vitro anticancer efficacy of this liposome system was evaluated in human lung cancer A549 cells and drug-resistant lung cancer A549/Taxol cells. At tumor extracellular pH (∼6.8), liposomes could reverse their surface charge (negative to positive), facilitating liposome internalization. After cellular uptake, KLA peptide directed delivery-enabled selective accumulation of these liposomes into mitochondria and favored release of their cargo paclitaxel (PTX) into desired sites. Specifically, enhanced apoptosis of MDR cancer cells through mitochondrial signaling pathways was evidenced by release of cytochrome c and increased activity of caspase-9 and -3. These dual-functional liposomes had the greatest efficacy for treating A549 cells and A549/Taxol cells in vitro, and in treating drug-resistant lung cancer A549/Taxol cells xenografted onto nude mice (tumor growth inhibition 86.7%). In conclusion, dual-functional liposomes provide a novel and versatile approach for overcoming MDR in cancer treatment.     

Heparin and non-heparin-like dextrans differentially modulate endothelial cell proliferation: in vitro evaluation with soluble and crosslinked polysaccharide matrices

Proliferation of endothelial cells (ECs) is a cellular step of particular importance for implanted cardiovascular biomaterials. Heparin and some synthetic water-soluble non-anticoagulant polysaccharides derived from dextran and bearing anionic carboxymethyl and hydrophobic benzylamine groups were first investigated for their effects on EC proliferation in vitro. The results assessed by cell counting, 3H-thymidine uptake, and flow cytometry analysis, showed that the derivatized dextran-bearing hydrophobic groups stimulated the EC growth in the presence of serum, whereas native dextran or dextran-bearing anionic carboxymethyl groups were inactive and heparin was slightly inhibitory. Then, we showed that the derivatized dextran enhanced EC proliferation by potentiation of the mitogenic activities of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2), two potent EC growth factors. In the presence of 2 nM of derivatized dextran, a 3-fold and 13-fold increase of 3H-thymidine uptake was obtained with VEGF and FGF-2, respectively. Finally, proliferation of ECs was investigated on crosslinked gels made of polysaccharides. It is of interest that EC proliferation was higher on gels containing the derivatized dextran than on plain hydrogels, and heparinized gels inhibited cell proliferation. From the obtained results, we propose that the synthetic non-heparin-like dextran may be of interest as a coating for the endothelialization of cardiovascular biomaterials.     

Effect of liposomes on lymphocytes: induction of proliferation of B lymphocytes and potentiation of the cytotoxic response of T lymphocytes to alloantigens

Liposomes of certain lipid composition prepared by the detergent removal method (Brunner, J. et al., Biochim. Biophys. Acta 1976. 455: 322) induced the proliferation of spleen cells from different mouse strains. Spleen cell populations enriched in B lymphocytes and those obtained from nude mice were induced to proliferate, whereas spleen cell fractions enriched in T lymphocytes and thymocytes were not. The mitogenic effect of liposomes resembled that of lipopolysaccharide (LPS) and it depended upon their lipid composition. Liposomes prepared from dimyristoyl lecithin (DML), 2:1 dimyristoyl lecithin:cholesterol (DML:C), 2:1 dioleoyl lecithin: cholesterol (DOL:C), and 2:1 egg yolk lecithin:cholesterol (EYL:C) were mitogenic, whereas liposomes prepared from egg yolk lecithin (EYL) alone were not mitogenic for spleen cells. The mitogenic effects of these liposome preparations were in the decreasing order DML greater than DOL:C greater than or equal to EYL:C greater than DML:C greater than EYL. The results suggest a correlation between the membrane fluidity of liposomes and their mitogenic effect. Although no proliferative response was induced on T lymphocytes, two of these liposomes, DML and EYL:C, had the ability to potentiate the cytotoxic response of T lymphocytes to alloantigens in mixed leukocyte culture. In responder-stimulator combinations which differed for the H-2K, H-2D or the entire H-2 region, these liposomes potentiated the cytotoxic response significantly. The results suggest that liposomes have an ability to modulate T lymphocyte response.     

Improved neo-endothelialization of small diameter ePTFE grafts with titanium coating

BACKGROUND: Patency of small synthetic bypass grafts is inferior compared to autologous grafts for revascularization procedures. Titanium coating of foreign surfaces has shown to decrease thrombogenicity, enhance biocompatibility and promote adhesion of endothelial cells. The aim of this study was to test the effect of titanium coating of small diameter ePTFE grafts on short term patency, neo-endothelialization and neointimal proliferation. METHODS: Bilateral carotid graft interposition was performed in 5 pigs with uncoated (n=5) and titanium-coated (n=5) ePTFE grafts (internal diameter=4 mm, length=5 cm), thus each pig served as its own control. At the end of the study (30 +/- 3 days), patency and stenosis severity was assessed by carotid angiography. Animals were sacrificed and grafts were excised for histology and scanning electron microscopy. Morphometry of histologic sections was carried out to determine neointimal proliferation and percentage of neo-endothelial coverage. RESULTS: Patency rate was 80% for uncoated and titanium-coated grafts. Quantitative angiography did not show any significant difference in lumen size between two groups. Morphometry revealed a significantly higher cellular coverage with CD31 positive endothelial cells for titanium-coated (84 +/- 19%) than uncoated grafts (48 +/- 26%, p<0.001). There was a non significant trend (p=0.112) towards increased neointimal proliferation in titanium-coated (94 +/- 61 micron2/micron) compared to uncoated grafts (60 +/- 57 micron2/micron). CONCLUSIONS: Patency rate in uncoated and titanium-coated ePTFE grafts is similar at one month. However, titanium coated grafts show a significant improvement in neo-endothelialization compared to uncoated grafts.     

Cell response to dextran-derivatised iron oxide nanoparticles post internalisation

Magnetic nanoparticles have been used for many years as magnetic resonance imaging contrast agents. Despite the fact that there are currently several dextran-coated iron oxide nanoparticles are in preclinical and clinical use, there is very little information available concerning the influence such particles have on cells in culture. The prerequisite for particles employed as contrast agents is capture and subsequent uptake by cells. This study involved the use of magnetic nanoparticles synthesised and derivatised with dextran, compared to similar underivatised plain particles. The influence in vitro was assessed using human dermal fibroblasts and various techniques to observe cell-particles interaction, including light and fluorescence microscopy, scanning and transmission electron microscopy. The results indicate that although both the uncoated and the dextran-derivatised particles are uptaken into the cell, the derivatised particles induce alterations in cell behaviour and morphology distinct from the plain particles, suggesting that cell response is dependent on the particles coating.     

An increased adjuvanticity of liposomes by the inclusion of phosphatidylserine in immunization with surface-coupled liposomal antigen

BACKGROUND: Exposure of phosphatidylserine (PS) on apoptotic cells is known to result in the enhanced recognition of apoptotic cells by phagocytes. By the inclusion of PS in the lipid component of liposomes, increased liposome immune adjuvant activity was expected. METHODS: In the present study, two different liposome preparations containing either PS, i.e. PS-liposome, or phosphatidylcholine (PC), i.e. PC-liposome, were made, and macrophage recognition, processing, and antigen presentation of surface-coupled liposomal antigen were compared. RESULTS: When ovalbumin-liposome conjugates were added to a culture of macrophages, enhanced recognition and processing of ovalbumin by the macrophages were observed by the inclusion of PS in the liposomes. The results correlated well with those regarding macrophage antigen presentation of liposome-coupled ovalbumin. Furthermore, in vivo immunization in mice with ovalbumin-liposome conjugates made with PS-liposomes induced a significantly higher level of anti-ovalbumin IgG antibody production than was induced by ovalbumin-liposome conjugates made with PC-liposomes. IgE-selective unresponsiveness was induced by ovalbumin-liposome conjugates regardless of the lipid components of liposomes. CONCLUSIONS: These results suggest that the inclusion of PS in liposomes enhances recognition and processing of surface-coupled liposomal antigen by macrophages, and increases liposome immune adjuvant activity.     

Construction of silica-enhanced S-layer protein cages

The work presented here shows for the first time that it is possible to silicify S-layer coated liposomes and to obtain stable functionalized hollow nano-containers. For this purpose, the S-layer protein of Geobacillus stearothermophilus PV72/p2 was recombinantly expressed and used for coating positively charged liposomes composed of dipalmitoylphosphatidylcholine, cholesterol and hexadecylamine in a molar ratio of 10:5:4. Subsequently, plain (uncoated) liposomes and S-layer coated liposomes were silicified. Determination of the charge of the constructs during silicification allowed the deposition process to be followed. After the particles had been silicified, lipids were dissolved by treatment with Triton X-100 with the release of previously entrapped fluorescent dyes being determined by fluorimetry. Both, ζ-potential and release experiments showed differences between silicified plain liposomes and silicified S-layer coated liposomes. The results of the individual preparation steps were examined by embedding the respective assemblies in resin, ultrathin sectioning and inspection by bright-field transmission electron microscopy (TEM). Energy filtered TEM confirmed the successful construction of S-layer based silica cages. It is anticipated that this approach will provide a key to enabling technology for the fabrication of nanoporous protein cages for applications ranging from nano medicine to materials science.     

Isolation of vascular endothelial cells for investigations on hybrid prostheses

Hybrid vascular prostheses are coated with vascular endothelial cells (EC) in an attempt to reduce thrombogenicity through the metabolic activities of living cells. The present studies were planned to develop a standardized method of isolating bovine aortic EC with high yield for studies of endothelial coating of vascular prostheses. The best results were achieved using a combination of incubation with collagenase and mechanically scraping the mobilised cells from the donor vessel. Isolated adult male bovine endothelial cells were identified by the typical "cobble stone" morphology in culture and characterized by factor VIII related antigen immunofluorescence microscopy. The cells were seeded successfully on PTFE vascular prostheses.     

Reduced thrombogenicity of artificial materials by coating with ADPase.

A novel coating solution for the improvement of biocompatibility of polyurethane-based vascular prostheses was tested in rabbits and rats in vivo. Segments of coated and uncoated vascular prostheses were implanted into the peritoneal cavity of rats, followed by induction of experimental haemorrhage; otherwise whole vascular prostheses were implanted in the carotid artery of rabbits using microsurgical procedures. While in both rats and rabbits, the uncoated material showed abundant formation of fibrinoid thrombi, clear reduction of thrombus formation was seen in all ADPase coated materials following implantation in vivo.     

负载多柔比星的EGFR靶向光敏脂质体对胃癌细胞的杀伤作用研究

目的:构建靶向EGFR的光敏脂质体,提高化疗药物对胃癌细胞系的杀伤效果,并降低相关毒副反应.方法:以联乙炔基甘油磷脂酰胆碱(Diacetylenic glycerophosphatidylcholine,PC)(以下简称PC)为原料,采用薄膜分散法制备光反应性脂质体,表面修饰表皮生长因子受体(Epidermal gro...     

A Novel Approach to Increase the Stability of Liposomal Containers via In Prep Coating by Poly[N‐(2‐Hydroxypropyl)Methacrylamide] with Covalently Attached Cholesterol Groups

Highly stable liposomes are developed by coating phosphatidylcholine liposomes with amphiphilic N‐(2‐hydroxypropyl)methacrylamide copolymer. Two approaches in the preparation of coated liposomes are employed: the copolymer is added during (“in prep”) or after (“ex post”) the liposome formation. The influence of polymer concentration and coating method is evaluated using the cryogenic transmission electron microscopy, dynamic light scattering, and small‐angle X‐ray scattering techniques. The in prep modification significantly increases, up to four weeks, the stability of liposomes against aggregation and makes the liposomal membrane nonpermeable toward an inorganic salt. Such enhanced longevity is attributed to the different structure of in prep coated liposomal membranes.     

The in vitro kinetics of the interactions between PEG-ylated magnetic-fluid-loaded liposomes and macrophages

Binding and uptake kinetics of magnetic-fluid-loaded liposomes (MFL) by endocytotic cells were investigated in vitro on the model cell-line J774. MFL consisted of unilamellar phosphatidylcholine vesicles (mean hydrodynamic diameter close to 200nm) encapsulating 8-nm nanocrystals of maghemite (gamma-Fe(2)O(3)) and sterically stabilized by introducing 5mol% of distearylphosphatidylcholine poly(ethylene glycol)(2,000) (DSPE-PEG(2,000)) in the vesicle bilayer. The association processes with living macrophages were followed at two levels. On one hand, the lipid vesicles were imaged by confocal fluorescence microscopy. For this purpose 1mol% of rhodamine-marked phosphatidylethanolamine was added to the liposome composition. On the other hand, the iron oxide particles associated with cells were independently quantified by magnetophoresis. All the experiments were similarly performed with PEG-ylated or conventional MFL to point out the role of polymer coating. The results showed cell association with both types of liposomes resulting from binding followed by endocytosis. Steric stabilization by PEG chains reduced binding efficiency limiting the amount of MFL internalized by the macrophages. In contrast, PEG coating did not change the kinetics of endocytosis which exhibited the same first-order rate constant for both conventional and PEG-ylated liposomes. Moreover, lipids and iron oxide particle uptakes were perfectly correlated, indicating that MFL vesicle structure and encapsulation rate were preserved upon cell penetration.     

Mineral coatings modulate β-TCP stability and enable growth factor binding and release

β-Tricalcium phosphate (β-TCP) is an attractive ceramic for bone tissue repair because of its similar composition to bone mineral and its osteoconductivity. However, compared with other ceramics β-TCP has a rapid and uncontrolled rate of degradation. In the current study β-TCP granules were mineral coated with the aim of influencing the dissolution rate of β-TCP, and also to use the coating as a carrier for controlled release of the growth factors recombinant human vascular endothelial growth factor (rhVEGF), modular VEGF peptide (mVEGF), and modular bone morphogenetic protein 2 peptide (mBMP2). The biomineral coatings were formed by heterogeneous nucleation in aqueous solution using simulated body fluid solutions with varying concentrations of bicarbonate (HCO(3)). Our results demonstrate that we could coat β-TCP granules with mineral layers possessing different dissolution properties. The presence of a biomineral coating delays the dissolution rate of the β-TCP granules. As the carbonate (CO(3)(2-)) content in the coating was increased the dissolution rate of the coated β-TCP also increased, but remained slower than the dissolution of uncoated β-TCP. In addition, we showed sustained release of multiple growth factors, with release kinetics that were controllable by varying the identity of the growth factor or the CO(3)(2-) content in the mineral coating. Released rhVEGF induced human umbilical vein endothelial cell (HUVEC) proliferation, and mVEGF enhanced migration of mouse embryonic endothelial cells in a scratch wound healing assay, indicating that each released growth factor was biologically active.     

Quantitative and ultrastructural studies on the uptake of drug loaded liposomes by mononuclear phagocytes infected with Leishmania donovani

This study compared splenic and hepatic uptake of free and liposome-entrapped sodium antimony gluconate after i.v. administration to mice infected with Leishmania donovani. It was demonstrated that entrapment within liposomes greatly altered the kinetics of uptake of the drug. We were also able to show that liposomes composed of sphingomyelin, stearylamine and cholesterol were marginally better than any other preparation in delivering entrapped drug to liver and spleen. X-ray microanalytical studies on the uptake of liposomes by Kupffer cells infected with L. donovani have indicated that internalised liposomes probably fuse with parasitophorous vacuoles, transferring their contents into the immediate locality of the leishmanial parasites. It is proposed that this is the way in which liposome entrapped antileishmanial agents have an enhanced therapeutic effect over free drug therapy.     

Experimental evaluation of a gelatin-coated polyester graft used as an arterial substitute

Protein coating and endothelial cell preseeding have been proposed and studied as improvements to arterial prostheses. In this paper, an impervious polyester vascular graft which had been coated with cross-linked gelatin was compared to a porous one over a period of up to 8 months in dogs. This evaluation involved in vivo methods using radio tracers to study patency and thrombogenicity and in vitro controls of the healing processes. The main advantages offered by coated grafts over uncoated include the absence of preclotting and better biointegration.