Evaluation and validation of in vitro assays to determine cell viability for HIV/AIDS expermentation with Pheroid TM technology

Abstract

The Southern parts of Africa have the highest prevalence of HIV-infected people and South Africa is the country with the highest number of infections in the world. There is still no cure for AIDS, but anti-HIV medicine can prolong and enhance the quality of life of an HIV infected person. Patient adherence with antiretroviral therapy is extremely low due to difficult dosing intervals, problematic dosage forms, instability of the antiretrovirals (ARVs) and the severe side-effects caused by these drugs; this leads to resistance of HIV to these drugs. Pheroid™ technology is a patented delivery system. Pheroid™ vesicles were used during this study. The entrapment of an active within the Pheroid™ would generally provide a safer, more effective formulation than the active alone. This could mean that the amount of drug needed for treatment of HIV can be decreased while producing fewer adverse effects and reducing the price of treatment. The main objectives of this study were to optimise and validate the cell viability and viral replication assays that can be used in an in vitro viral infection model. The MTT assay was used to asses the viability of the cells and to determine the toxicity of the antiretroviral drugs and Pheroid™ on the cells. HIV-1 assays were evaluated and used to determine the viral replication in the cells. Two different continuous cell lines were chosen for this study, an anchorage dependent GHOST cell line and suspended M7-Luc cells. Both these cell lines were best infected with the SWl virus. SWl is a subtype C, CXCR4 utilising virus. Subtype C is responsible for 60 % of the HIV infections worldwide and is the prevalent subtype in SUb-Saharan Africa .. Infection enhancers were not added to the cells to improve viral infection since it was observed that the Pheroid™ in combination with DEAE-dextran or Polybrene caused cytotoxicity probably by disrupting the cell's membrane. Antioxidants were added to the Pheroid ™ formulation since it was observed that the viability of the cells incubated with the Pheroid™ decreased as the Pheroid ™ matured. The added antioxidants had no significant effect on the cells. Abacavir (ABC) was chosen as the test substance for this study since it showed low cytotoxicity in cell cultures and is water soluble and would not present solubility issues in the media. It was entrapped within the Pheroid™ and its in vitro efficacy and toxicity was tested on HIV-infected and uninfected cell cultures. One directlHIV-specific (p24 antigen ELISA assay) and one indirect (Luciferase) assays were used to asses the inhibition of HIV replication caused by ABC. The p24 antigen ELISA (Enzyme-Linked ImmunoSorbent Assay) assay required a lot of washing steps and were rather expensive to use. The Luciferase assay was only used on the M7-Luc cells; this assay was sensitive, inexpensive and easy to use. The MTT (3-(4,5-demethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) viability assay was used to measure the toxicity caused by the Pheroid ™ and/or ABC on the cells. MTT is a widely used quantitative colorimetric assay to measure the viability of cells. The vitamin E and antioxidants contained in the Pheroid ™ reduced the MTT and produced results that were misinterpreted as enhanced viability when the Pheroid™ was present during MTT analysis. To prevent this problem an additional washing step should be introduced prior to analysis to reduce the interference of the Pheroid ™ with analytical methods. In conclusion, the efficacy of ABC entrapped within the Pheroid™ is still inconclusive and further studies will have to be done. MTT should be used with care for viability analysis of cells incubated in the presence of Pheroid TM.