Development of novel membranes for blood purification therapies based on copolymers of N-vinylpyrrolidone and n-butylmethacrylate.

Developments in membrane based blood purification therapies often come with longer treatment times and therefore longer blood-material contact, which requires long-term membrane biocompatibility. In this study, we develop for the first time membranes for blood purification using the material SlipSkin™, which is a copolymer, made from N-vinylpyrrolidone (NVP) and butylmethacrylate (BMA). Specific attention is focused on understanding the mechanism of pore formation and the tailoring of the membrane mechanical and transport properties to obtain the optimal membrane for blood purification therapies. Polymer composition, solvent type and solvent evaporation time influence membrane morphology and membranes with sieving properties of cascade filters in plasma fractionation applications are developed. The new membranes have very good blood compatibility properties; in fact compared to benchmark flat membranes currently used in the clinic, they have lower platelet adhesion while all other properties (contact activation, thrombogenicity, leukocyte adhesion, hemolysis and complement activation) are also very good and comparable to the benchmarks.

A sensitive HPLC method for the assessment of metabolic conversion of estrogens.

Disorders of estrogen-responsive tissues are frequently associated with aberrations in steroid metabolism due to altered expression of synthesizing and metabolizing enzymes. For instance, overexposure to unopposed 17beta-estradiol has been associated with the pathogenesis of endometrial proliferative disorders, such as endometriosis. Investigations into the metabolic conversion in tissues and cells have been rather limited. This is mostly due to fact that such studies have to make use of radioactive steroid hormones and expensive equipment to obtain sufficient sensitivity. We adapted a sensitive non-radioactive HPLC method to study estrogen metabolism in more detail. This HPLC method is based on the solid phase extraction of estrogens and the derivatization of the steroids with 2-(4-carboxy-phenyl)-5,6-dimethylbenzimidazole. The technique is sensitive, robust and is useful for the detection of aromatase, 17beta-HSD types 1 and 2 and sulfatase activities in lysates of placenta and endometrium.

Platelet adhesion studies on dipyridamole coated polyurethane surfaces.

Surface modification of polyurethanes (PUs) by covalent attachment of dipyridamole (Persantin) is known to reduce adherence of blood platelets upon exposure to human platelet rich plasma (PRP). This effect was investigated in further detail. First platelet adhesion under static conditions was studied with four different biomaterial surfaces: untreated PU, PU immobilised with conjugate molecule 1, PU immobilised with conjugate molecule 2, and PU immobilised with conjugate molecule 3. In PU immobilised with 1 dipyridamole is directly linked to the surface, in PU immobilised with 2 there is a short hydrophilic spacer chain in between the surface and the dipyridamole, while conjugate molecule 3 is merely the spacer chain. Scanning electron microscopy (SEM) was used to characterise platelet adhesion from human PRP under static conditions, and fluorescence imaging microscopy was used to study platelet adhesion from whole blood under flow. SEM experiments encompassed both density measurements and analysis of the morphology of adherent platelets. In the static experiments the surface immobilised with 2 showed the lowest platelet adherence. No difference between the three modified surfaces emerged from the flow experiments. The surfaces were also incubated with washed blood platelets and labeled with Oregon-Green Annexin V. No capture of Oregon-Green Annexin V was seen, implying that the adhered platelets did not expose any phosphatidyl serine at their exterior surface.

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.

Sustained local drug delivery from a radiopaque implanted reservoir.

A new polymeric biomaterial that contains covalently bound iodine, and is therefore radiopaque, was used to construct a sustained local drug-delivery device. A polymeric wall was designed to be porous (i.e., passage of low-molecular-weight molecules across the wall is possible), self-healing, and biocompatible. Once implanted, the sphere cavity can be filled and refilled with a concentrated solution of a (cytostatic) drug, which is subsequently released by slow diffusion into the tissue region surrounding the sphere. This principle of sustained local drug delivery is shown by a series of in vitro experiments on the release of 5-fluorouracil, and in vivo animal experiments, using x-ray fluoroscopic and scintigraphic techniques.

Synthesis of a new photoreactive derivative of dipyridamole and its use in the manufacture of artificial surfaces with low thrombogenicity.

Photoimmobilization of dipyridamole (Persantin) was accomplished through the use of a new synthetic conjugate molecule, 1. Persantin is a powerful inhibitor of platelet activation and aggregation and is widely used as a vasodilator. Conjugate 1 consists of triply protected dipyridamole [three of the four hydroxyl groups carry a tert-butyldimethylsilyl (TBDMS) protective group) and the photoreactive 4-azidobenzoyl group. A short hydrophilic spacer chain, derived from triethylene glycol, separates the protected dipyridamole system and the photoreactive group. Compound 1 was immobilized on polyurethane sheets (Pellethane D-55) through irradiation with ultraviolet (UV) light, and the protective groups were removed afterward. The resulting modified polyurethane surfaces were characterized by different physicochemical techniques: UV extinction, contact angle measurements (captive bubble technique), and X-ray photoelectron spectroscopy (XPS). The UV extinction measurements showed the presence of 13 +/- 1 nmol of immobilized dipyridamole/cm2. The contact angle measurements revealed that the modified surface was markedly more hydrophilic than the control (i.e. unmodified polyurethane). XPS measurements clearly established the presence of immobilized dipyridamole in the outermost layers of the modified surface. This was especially clear from the XPS spectra recorded at a low take-off angle (approximately 6 degrees). Furthermore, the XPS spectra showed that the TBDMS protective groups had been quantitatively removed during the deprotection/washing treatment. The in vitro blood compatibility of the modified surface was studied with the thrombin generation assay as developed in our group, as well as with scanning electron microscopy. The thrombin generation test produced a lag time of 1275 s for the modified surface, as opposed to 569 s for the control. Scanning electron microscopy showed that far fewer platelets adhere to the modified surface (approximately 7 x 10(3)/mm2) as compared to the control (approximately 6 x 10(2)/mm2). Taken together, the experimental data reveal that the modified surface has excellent blood compatibility in vitro. It is discussed that the use of conjugate 1 leads to simultaneous exposure of dipyridamole at the modified surface and to a marked increase of the surface hydrophilicity, which is likely to hamper adsorption of plasma proteins. The combination of these effects is uniquely related to the molecular buildup of 1. Conjugate 1 will be used in future work that is aimed at preparing small-caliber polyurethane vascular grafts with a blood compatible lumenal surface.

Photo-immobilization of dipyridamole (Persantin) at the surface of polyurethane biomaterials: reduction of in-vitro thrombogenicity.

Dipyridamole is a well-known vasodilator and a powerful inhibitor of activation and aggregation of blood platelets. Moreover, dipyridamole is essentially non-toxic. The drug is used extensively in clinical anti-coagulation regimes, for example pre- and post-coronary angioplasty procedures. Recently, we have found that photochemical, covalent coupling of dipyridamole to polyurethane surfaces leads to improved thromboresistance in vitro. This phenomenon is now studied in more detail. Both qualitative and more quantitative biochemical experiments were performed in order to characterize the in vitro blood compatibility of a set of polyurethane surfaces onto which dipyridamole was immobilized. First, scanning electron microscopy was used to examine the morphology of platelets which adhered during incubation with platelet-rich plasma. These experiments showed that immobilization of dipyridamole leads to a clearly decreased number of adherent platelets and to a largely diminished propensity of the surface to activate adherent platelets. Secondly, an in vitro thrombogenicity assay was run. These experiments showed that the thromboresistance increased with increasing surface density of immobilized dipyridamole. A short spacer chain separating dipyridamole from the polymer surface, was found to improve the thromboresistance further. Such a spacer chain apparently increases the efficacy of the immobilized drug. Collectively, the present results further substantiate the idea that dipyridamole retains its inhibitory activity with respect to activation and aggregation of blood platelets, when the compound is covalently attached to a polymer surface. The possible utility of these findings with respect to the development of an artificial blood vessel prosthesis is discussed briefly.

Preparation, characterization and radiolabeling of a new amphiphilic derivative of DTPA.

A new derivative (1) of diethylenetriamine pentaacetic acid (DTPA) is described. Compound 1 contains a hydrophobic unit (a triphenyl methyl group) and a hydrophilic unit (a DTPA ester), and therefore behaves as an amphiphile in aqueous solution. Compound 1 appears to form a stable inclusion complex with 99mTc. A rabbit model was used in a scintigraphic study of the biodistribution of the complex 99mTc-1, using 99mTc-DTPA as a control. The resulting images revealed marked differences: 99mTc-1 showed rapid uptake in the liver followed by excretion in the gallbladder and intestines within 1 h, whereas 99mTc-DTPA appears, as expected, in the renal-pelvico system and in the bladder. These findings are significant as they provide more insight into the complex relationship between structural and physicochemical properties of radiopharmaceuticals and their biodistribution. Knowledge of such relationships is absolutely mandatory with respect to the development of new radiopharmaceuticals with increased efficacy and/or specificity.

Studies on a new strategy for surface modification of polymeric biomaterials.

A new methodology to improve the hemocompatibility of polyurethane (medical grade Pellethane D-55) surfaces is reported. The approach is essentially based on a photochemical immobilization reaction. Two new conjugate molecules, compounds 2 and 3, were prepared. They consist of (i) dipyridamole, a well-known inhibitor of platelet activation, and a vasodilating drug with clinical application, for instance before and during pecutaneous transluminar coronary angioplasty (Dottering); and (ii) an aryl azide, a moiety that exhibits marked photoreactivity. In 2, the dipyridamole unit is directly linked to the aryl azide (via an ester bond), while a short spacer chain separates both units in 3. Upon irradiation of 2 or 3, adsorbed onto the polyurethane foil, the aryl azide is converted into a highly reactive species which reacts with a nucleophilic group on the polymer surface. In this way, the dipyridamole is covalently linked to the polymer. The underlying principle is also used in photoaffinity labeling, a well-known technique in biochemical studies on enzyme structure and function. From UV extinction experiments it could be deduced that the surface-density of immobilized 2 is 4.9 nmol/cm2. The surface density for 3 was 14.6 nmol/cm2. The surfaces were subjected to an in vitro thrombin generation assay. This assay gives a valuable impression about the hemocompatibility of artificial surfaces. These experiments revealed that the clotting times were substantially prolonged as a result of the photoimmobilization of dipyridamole. This was especially the case for immobilized 3. This effect cannot be readily explained. Possibly, the enhanced activity of immobilized 3 is due to the spacer chain. An alternative explanation is that the surface density is larger for 3 than for 2. In addition, the photomodified surfaces were incubated with platelet-rich blood plasma (37 degrees C, 30 min) and subsequently examined by scanning electron microscopy. The morphology of the blood platelets adhered to the surface also showed that hemocompatibility increased in the order untreated polyurethane < polyurethane with immobilized 2 < polyurethane with immobilized 3. Future work will concentrate on evaluation of the role of the spacer (length, hydrophilicity, etc.), as well as on the possible use of this approach with respect to the construction of biomaterials with improved in vivo biocompatibility, in particular hemocompatibility.