Cytochrome P450-3A4/copper-poly(propylene imine)- polypyrrole star co-polymer Nanobiosensor system for delavirdine – a non-nucleoside reverse transcriptase inhibitor HIV drug

Abstract

HIV and AIDS are among the world's pandemics that pose serious concern to almost every individual in the world. With the current level of availability of anti-retroviral (ARV) drugs and the ease of accessibility of treatment in many countries such as South Africa, the disease can be controlled by suppressing the viral load of an infected individual. These anti HIV drugs such as delavirdine are metabolised by enzymes which are found in the liver microsomes, particularly those of the cytochrome P450 family. Due to the fact that the metabolic rate of a patient determines the effect of the drug, the drug could either have a beneficial or an adverse effect once it is administered. It is therefore imperative that the metabolic profile of a patient is determined at point-of-care is necessary for proper dosing of the ARV drugs. In this project a nanobiosensor system was devised and used for the determination of the metabolism of delavirdine, a non-nucleoside reverse transcriptase inhibitor (NNRTI) ARV drug. The nanobiosensor was prepared by the entrapment of the isoenzyme CYP3A4 into a pre-formed electro active carrier matrice consisting of a dendrimeric copper generation-2 poly (propylene imine)-co-polypyrrole star copolymer (Cu(G2PPI)-co-PPy). The metallo-dendrimer was used as a host for the enzyme and provided thenecessary bio-compatible environment that allowed the direct transfer of electrons between the enzyme's active centres and platinum electrode surface. Copper was the choice of metal used in the study due to its properties. Copper is a malleable, ductile and a good conductor of both heat and electricity. It is a better conductor than most metals. Silver which also belongs to group 1b in the periodic table is a better electrical conductor than copper but copper has better corrosion resistance and is a more abundant and hence it is a cheaper material to use. Cu(G2PPI)-co-PPy was prepared by the incorporation of the copper metal into the G2PPI and the electropolymerization of pyrrole onto the Cu(G2PPI). The incorporation of Cu into G2PPI was determined by FTIR which did not show the presence of the Cu but showed an increase in the intensities of the peaks after the incorporation. The surface morphology of Cu (G2PPI) was confirmed by the use of HRSEM which showed a difference in the surface morphology of the dendrimer moiety with the addition of the copper metal. The HRSEM images after Cu incorporation resulted in the change from rough surface to smooth surface with open cavities which were essential for the entrapment of the biological systems (CYP3A4). Energy dispersive spectrometry (EDS) and HRTEM were used to confirm the presence of spherically shaped copper nanoparticles in the Cu (G2PPI) and were found to have a size distribution of 12-17 nm with an average particle size of 15nm. The star copolymer (Cu(G2PPI)-co-PPy) was characterised using cyclic voltammetrywhere it was confirmed that the material was electroactive and conducting due to electron movement along the polymer chain. A diffusion co-efficient (D₀) value of 8.64 x 10⁻⁵ cm²/s was determined for the material indicating a slow electron transfer kinetics within the diffusion layer. The constructed nanobiosensor was developed using copper poly (propylene imine) – polypyrrole star copolymer, bovine serum albumin and glutaraldehyde coupled to the enzyme CYP3A4. The resultant nanobiosensor parameters include a dynamic linear range (DLR) of 0.01-0.06 nM, a limit of detection (LOD) of 0.025 nM and a sensitivity value of0.379 μA/nM.