Dengue Fever: A Continuous Threat

Dengue fever is a vector borne disease and is caused by DEN Virus. This virus has four different serotypes. The vectors are two mosquitoes known as Aedesaegypti (the yellow fever mosquito) and Aedesalbopictus(the Asian tiger mosquito).  First case of dengue fever was reported back in 1994 in Karachi. A complete outbreak of this epidemic shook the whole nation in 2012. Uptill now, Lahore a city full of culture, witnessed about 16,580 confirmed cases and 257 deaths. About 5000 confirmed cases with 60 deaths were reported from the rest of the provinces. Under guidelines of WHO, Government has made efforts to combat this epidemic. Although the overall efforts have minimized the outbreak on controllable levels but dengue fever is a continuous threat. Since no permanent cure is available, the transmission of DEN virus is controlled indirectly. So the prime focus is to control mosquito population and decrease the possible hot spots i.e. Mosquito breeding sites in human habitations. Every year, the country witnesses monsoon season which brings vast areas full of clear standing waters providing breeding sites for mosquitoes which ultimately leads to increased number of patients suffering from dengue fever. Efforts have been made to fight against dengue including formation of dengue wards in hospitals, vector surveillance, community education, reactive vector control etc. A study has shown prevalence of four mosquito genera in Pakistan including Aedes, Culex, Armigeresand Anopheles. All of the above mentioned genera are associated with disease transmissions as they are the vectors of different viruses and parasites. It is the need of hour to do a collaborative effort stressing the community mobilization and management in war against dengue.

Solid Lipid Nanoparticles for Thermoresponsive Drug Delivery: Fabrication and Evaluation

Thermoresponsive drug delivery systems (DDS) are designed for the controlled and targeted release of therapeutic payload by exploiting the hyperthermic temperature (>39°C), which may be applied by some external means or an encountered symptom in inflammatory diseases such as cancer, arthritis etc. However, available thermoresponsive DDS, including liposomes, have complex method of preparation involving toxic solvents and reagents. Recently, we have shown for the first time that melting point of solid lipid nanoparticles (SLN) can be optimized for thermoresponsive drug release by tuning their melting point (MP). The objective of this study was to provide some strong evidence in support of hypothesis that thermoresponsive solid lipid nanoparticles (TSLN) undergo solid-liquid phase transition at their melting point (>39ºC) leading to faster drug release. Thermoresponsive lipid mixtures (TLM) were prepared by mixing solid (lauric, myristic, palmitic and stearic acid) and liquid (oleic and linoleic acid) natural fatty acids in different ratios (0.1:1 to 1:2) and melting point was measured by differential scanning calorimetry (DSC). A graph was plotted between liquid content in TLM and the MP, and TLM that would melt at 39°C were identified by using straight line equation of the graph. The solidliquid phase transition was assessed by determination of temperature dependent change in viscosity (low at 39°C) and light transmission (higher at 39°C) that are characteristic of liquids. TSLN containing a chemotherapeutic drug, either hydrophilic 5-fluorouracil (5-FU) or lipophilic paclitaxel, were synthesized by hot melt encapsulation method. It should be noted that the TLM and the TSLN were made by physical interaction of materials and no chemical reaction was needed. The TSLN showed desirable spherical shape (TEM), size (100-300 nm), physicochemical stability (FTIR analysis), high yield (>85%) and encapsulation efficinecy (5-FU >40% and paclitaxel >90%). In 5-FU loaded TSLN, drug release studies were first performed by USP type II dissolution apparatus in PBS (7.4) at 37°C and 39°C. A sustained release pattern was observed at 37°C and 22-34% 5-FU was released in 5 hrs. On the other hand, >90% drug was released at 39°C suggesting that the SLN show thermoresponsive drug release in agreement with our hypothesis. Drug release from SLN at 39°C was similar to model oleic acid and linoleic acid nanoemulsions which further supports our hypothesis. Next, a quick and real-time differential pulse voltammetry (DPV) based electrochemical chemical detection method was developed using a graphite electrode to detect change in current with 5-FU concentration while increasing voltage was applied on reference and counter electrodes. This method also showed that sustained release pattern of 5-FU at 37°C was converted to an immediate drug release when heated to 39°C, thus, confirming the thermoresponsive drug release. In case of paclitaxel loaded TSLN, drug release was minimum at 37°C and 70-100% drug release achieved after 60 hrs. On the other hand, whole drug was released in 4-7 hours at 39°C. This 15-20 time higher drug release at hyperthermic conditions confirmed the thermoresponsive drug release from the TSLN. Blank SLN were found to be biocompatible with human gingival fibroblast cells (PCS- 201-108) although and breast cancer cells (MDA-MB-231). However, 5-FU loaded SLN showed some cytotoxicity after 24 hours which was due to the release of drug. 5-FU loaded SLN showed thermoresponsive cytotoxicity to breast cancer cells (MDA-MB-231) as cytotoxicity was higher at 39°C (22-28%) compared to 37°C (<10%) within 1 hour. Similarly, paclitaxel loaded TSLN showed higher cytotoxicity to glioblastoma cells at 39°C (31% cell viability after one hour) compared to 37°C (18% cell viability). The higher cytotoxicity at 39°C was due to the higher drug release. Finally, the TSLN were evaluated for brain targeting across blood brain barrier (BBB) and an in vitro BBB model was used consisting astrocytes (CRL-2541) and endothelial cells (b.End3). The BBB model was optimized at 39°C for 1 hour duration due to retention of semipermeable nature and lack of paclitaxel and heat related toxicity. The TSLN showed higher permeability across BBB at 39°C which may be attributed to the deformable liquid state that squeezes through the tight junctions of BBB without any damaging effects. In conclusion, the novel TSLN reported in this thesis may serve as safe and effective platform of thermoresponsive targeting of cancer.