Antibacterial and Biocompatible Coating for Cardiovascular Grafts

In chapter 1: Polyethylene terephthalate (PET) is considered as the gold standard cardiovascular graft to restore the function of damaged vessels and heart valves. However, the post implantation complications essentially distract the long-term patency of PET grafts resulting in prolonged hospitalization, graft failure, and patient death. Most of the prominent shortcomings of PET are the substantial thrombogenic property and the associated infections as well as the biocompatibility issues. Therefore, in this thesis, the improvement of the biocompatibility and the infection-resistance properties of PET grafts were our foremost perspective. We fundamentally minimized the bacterial adhesion and enhanced the biocompatibility of woven and knitted forms of crimped PET cardiovascular grafts. Our results proved an effective strategy for graft surface modification in terms of biocompatible and infection-resistant. In chapter 2: the initial bacterial adhesion was minimized by a multifunctional network-structured film coat using a newly synthesized amphiphilic SD-PHA-b-MPEO diblock copolymer. A versatile coating technique was described based on the repulsion forces between the surface and the used polymer to preserve the flexibility and tensile ability of crimped PET grafts. The surface modified graft was confirmed by Fourier transform infrared spectroscopy (FTIR) and by scanning electron microscope (SEM). The employed polymer manifested suitable biocompatibility to host cell as established using mouse L929 fibroblast cell line. Importantly, the negative charge and the hydrophobic properties of the polymer augmented the bactericidal effect of the sulfadimethoxine moiety as reported by the significant bacterial anti-adhesion efficiency for Gram-positive S. aureus and Gram-negative E. coli bacteria, and for the previously vein isolated Gram-positive S. epidermidis. In chapter 3: Unlike previous studies, the bacterial adhesion was enzymatically inhibited using a bacterial lytic enzyme, lysozyme. Accordingly, graft with broad-spectrum bacteria-resistant was developed. The lysozyme enzyme was covalently immobilized on PET graft by end-point method and proved by FTIR and X-ray photoelectron spectroscopy (XPS). The activity of immobilized enzyme against M. lysodeikticus cells displayed a significant reduction as compared to the free enzyme. However, the remaining activity remarkably decreased the adhesion of Gram-positive S. epidermidis and S. aureus bacteria and to less extent of Gram-negative E.coli. The anti-adhesion efficiency showed bacterial cells specificity while, showed no significant effect on L929 cells adhesion and growth. This indicated the utility of the employed strategy to modulate the initial bacteria adhesion to inhibit the graft-associated infection. In chapter 4: FITC-dextran loaded Poly lactic-glycolic acid (PLGA) nanoparticles were covalently immobilized onto two different cardiovascular prostheses namely; woven crimped PET and expanded polytetrafluoroethylene (ePTFE, Teflon®). The grafts surface was modified by introduction of amino groups on the surface. The surface modified graft was characterized by electro kinetic analyzer, and FTIR before the covalent coupling to the carboxyl group of PLGA Nanoparticles was performed. The prepared model manifested homogenous monolayer of nanoparticles on grafts surface and displayed a satisfactory stability under appropriate human-mimic continuous flow conditions for 24h. Additionally, the established biocompatibility of nano-coated grafts highlighted the utility of the immobilized nanoparticles on the graft’s surface as an attractive strategy for local drug delivery to treat the common complications after graft’s implantation, and hence increasing the grafts long-term patency. In chapter 5: A thrombus-resistant graft was developed by covalent immobilization of heparin. Additionally, the host cell compatibility of PET grafts was enhanced by co-immobilization of collagen. Heparin and collagen were immobilized by end-point method into previously functionalized PET grafts and characterized using FTIR and XPS. The modified grafts manifested a significant biological activity in-vitro under human-mimic conditions mainly, substantial resistance of the graft to clot and fibrin formation. Importantly, the co-immobilization of heparin and collagen supported the host cell adhesion and growth, and showed synergistic inhibition effect of platelets deposition after continuous flow for 30 minutes to simulate the massive blood flow conditions. Consequently, this approach minimized the inherent thrombogenicity of the PET grafts and the corresponding host response, hence ensuring a rapid coating of grafts with host cells required for the grafts biocompatibility

Determination of Hypoglycemic, Hypolipidemic and Nephroprotective Effects of <i>Berberis Calliobotrys</i> in Alloxan-Induced Diabetic Rats

Many plants of the Berberis genus have been reported pharmacologically to possess anti-diabetic potential, and Berberis calliobotrys has been found to be an inhibitor of α-glucosidase, α-amylase and tyrosinase. Thus, this study investigated the hypoglycemic effects of Berberis calliobotrys methanol extract/fractions using in vitro and In vivo methods. Bovine serum albumin (BSA), BSA–methylglyoxal and BSA–glucose methods were used to assess anti-glycation activity in vitro, while in vivo hypoglycemic effects were determined by oral glucose tolerance test (OGTT). Moreover, the hypolipidemic and nephroprotective effects were studied and phenolics were detected using high performance liquid chromatography (HPLC). In vitro anti-glycation showed a significant reduction in glycated end-products formation at 1, 0.25 and 0.5 mg/mL. In vivo hypoglycemic effects were tested at 200, 400 and 600 mg/kg by measuring blood glucose, insulin, hemoglobin (Hb) and HbA1c. The synergistic effect of extract/fractions (600 mg/kg) with insulin exhibited a pronounced glucose reduction in alloxan diabetic rats. The oral glucose tolerance test (OGTT) demonstrated a decline in glucose concentration. Moreover, extract/fractions (600 mg/kg) exhibited an improved lipid profile, increased Hb, HbA1c levels and body weight for 30 days. Furthermore, diabetic animals significantly exhibited an upsurge in total protein, albumin and globulin levels, along with a significant improvement in urea and creatinine after extract/fractions administration for 42 days. Phytochemistry revealed alkaloids, tannins, glycosides, flavonoids, phenols, terpenoids and saponins. HPLC showed the presence of phenolics in ethyl acetate fraction that could be accountable for pharmacological actions. Therefore, it can be concluded that Berberis calliobotrys possesses strong hypoglycemic, hypolipidemic and nephroprotective effects, and could be a potential therapeutic agent for diabetes treatment

Antibacterial and Biocompatible Coating for Cardiovascular Grafts

In chapter 1: Polyethylene terephthalate (PET) is considered as the gold standard cardiovascular graft to restore the function of damaged vessels and heart valves. However, the post implantation complications essentially distract the long-term patency of PET grafts resulting in prolonged hospitalization, graft failure, and patient death. Most of the prominent shortcomings of PET are the substantial thrombogenic property and the associated infections as well as the biocompatibility issues. Therefore, in this thesis, the improvement of the biocompatibility and the infection-resistance properties of PET grafts were our foremost perspective. We fundamentally minimized the bacterial adhesion and enhanced the biocompatibility of woven and knitted forms of crimped PET cardiovascular grafts. Our results proved an effective strategy for graft surface modification in terms of biocompatible and infection-resistant. In chapter 2: the initial bacterial adhesion was minimized by a multifunctional network-structured film coat using a newly synthesized amphiphilic SD-PHA-b-MPEO diblock copolymer. A versatile coating technique was described based on the repulsion forces between the surface and the used polymer to preserve the flexibility and tensile ability of crimped PET grafts. The surface modified graft was confirmed by Fourier transform infrared spectroscopy (FTIR) and by scanning electron microscope (SEM). The employed polymer manifested suitable biocompatibility to host cell as established using mouse L929 fibroblast cell line. Importantly, the negative charge and the hydrophobic properties of the polymer augmented the bactericidal effect of the sulfadimethoxine moiety as reported by the significant bacterial anti-adhesion efficiency for Gram-positive S. aureus and Gram-negative E. coli bacteria, and for the previously vein isolated Gram-positive S. epidermidis. In chapter 3: Unlike previous studies, the bacterial adhesion was enzymatically inhibited using a bacterial lytic enzyme, lysozyme. Accordingly, graft with broad-spectrum bacteria-resistant was developed. The lysozyme enzyme was covalently immobilized on PET graft by end-point method and proved by FTIR and X-ray photoelectron spectroscopy (XPS). The activity of immobilized enzyme against M. lysodeikticus cells displayed a significant reduction as compared to the free enzyme. However, the remaining activity remarkably decreased the adhesion of Gram-positive S. epidermidis and S. aureus bacteria and to less extent of Gram-negative E.coli. The anti-adhesion efficiency showed bacterial cells specificity while, showed no significant effect on L929 cells adhesion and growth. This indicated the utility of the employed strategy to modulate the initial bacteria adhesion to inhibit the graft-associated infection. In chapter 4: FITC-dextran loaded Poly lactic-glycolic acid (PLGA) nanoparticles were covalently immobilized onto two different cardiovascular prostheses namely; woven crimped PET and expanded polytetrafluoroethylene (ePTFE, Teflon®). The grafts surface was modified by introduction of amino groups on the surface. The surface modified graft was characterized by electro kinetic analyzer, and FTIR before the covalent coupling to the carboxyl group of PLGA Nanoparticles was performed. The prepared model manifested homogenous monolayer of nanoparticles on grafts surface and displayed a satisfactory stability under appropriate human-mimic continuous flow conditions for 24h. Additionally, the established biocompatibility of nano-coated grafts highlighted the utility of the immobilized nanoparticles on the graft’s surface as an attractive strategy for local drug delivery to treat the common complications after graft’s implantation, and hence increasing the grafts long-term patency. In chapter 5: A thrombus-resistant graft was developed by covalent immobilization of heparin. Additionally, the host cell compatibility of PET grafts was enhanced by co-immobilization of collagen. Heparin and collagen were immobilized by end-point method into previously functionalized PET grafts and characterized using FTIR and XPS. The modified grafts manifested a significant biological activity in-vitro under human-mimic conditions mainly, substantial resistance of the graft to clot and fibrin formation. Importantly, the co-immobilization of heparin and collagen supported the host cell adhesion and growth, and showed synergistic inhibition effect of platelets deposition after continuous flow for 30 minutes to simulate the massive blood flow conditions. Consequently, this approach minimized the inherent thrombogenicity of the PET grafts and the corresponding host response, hence ensuring a rapid coating of grafts with host cells required for the grafts biocompatibility