Deltamethrin was obtained as a gift sample from Tagros India Ltd. Mumbai, India. Polyvinylpyrrolidone (PVP K-30), Dibutylphthalate (DBT), and Ethylcellulose (EC, ethoxy content 48-49.5% w/w) were obtained from Himedia Laboratories Pvt. Ltd. Mumbai, India. Chloroform and water (for HPLC) were obtained from Spectrochem Pvt. Ltd. Mumbai, India. Acetonitrile (for HPLC) was purchased from Merck Specialities Pvt. Ltd. Mumbai, India. Dimethylsulfoxide (DMSO) was purchased from Loba Cheime Pvt. Ltd. Mumbai, India. All reagents and solvents used were of analytical grade and used as received without any further purification.
Preparation of deltamethrin loaded patches
Deltamethrin loaded mosquito repellent patches were prepared by solvent evaporation method using varying ratios of different blends of polymers. DBT was used as plasticizer at a fixed concentration of 20% w/w of dry weight of polymer. Initially, the polymers at a varied ratio were dissolved in chloroform and then deltamethrin and plasticizer were added to it. This mixture was moulded into rings with defined surface area and thickness over the backing membrane on a horizontal surface followed by solvent evaporation at an ambient temperature. The rate of evaporation was controlled by inverting the funnel. The patches formed were separated from the rings for further processing.
Physicochemical characterization of deltamethrin loaded patches
All the deltamethrin loaded patches were visually inspected for shape, smoothness, stickiness, clarity, homogeneity, flexibility and uniformity.
Uniformity of weight
Weight was determined by individually weighing three randomly selected patches from each batch of formulation. In the experiment, attention was given so that the individual weight was uniform as compared to the average weight. The weight was expressed by mean and standard deviation.
Surface area of patches was studied by using a millimeter scale and expressed by mean and standard deviation.
Surface pH determination
The patches were allowed to remain in contact with chloroform for 2 h at room temperature, and pH was determined using pH paper.
Percent moisture uptake
Weighed patches were kept in a dessicator at room temperature for 48 h. These were exposed to 75.5% relative humidity over a saturated solution of aluminium chloride in a dessicator until a constant weight is achieved. The percent moisture uptake was calculated using the following formula;
Percent moisture content
Three patches from each formulation were weighed and kept in dessicators containing fused calcium chloride at 37°C until the notable weight change was observed. This weight was noted as the final weight. The percent moisture content is calculated using following formula;
A patch should possess a smooth surface and should not constrict with time. This can be demonstrated in a flatness study. For flatness determination, one patch is cut from the centre (2×1 cm) and applied on the skin. The length of patch is measured and variation in length is measured by determining percent constriction. It is assumed that zero percent constriction refers to 100 percent flatness.
Where, I2 = Final length of each strip, I1 = Initial length of each strip.
Measurement of thickness
The thickness of the patch was measured using a digital micrometer (Mitotousu, Tokyo, Japan) at three different points of each patch and was expressed in mean and standard deviation.
Scanning electron microscopy (SEM)
The surface morphology of the blank and deltamethrin loaded patches was studied using scanning electron microscopy (JEOL, JSM-6390 LV, England). The samples were mounted onto stubs using double sided adhesive tape and sputter coated with gold palladium. The coated patches were observed and photographed at the required magnification at room temperature.
Estimation of deltamethrin by HPLC
The estimation of the deltamethrin was performed using HPLC (Analytical technologies limited, Gujarat, India) with a UV/visible detector and C18 column (Chromosil, particle size 5 μm, 250 mm × 4.6 mm). Deltamethrin was separated by isocratic elution technique with a mixture of mobile phase containing acetonitrile: water (80: 20,% v/v) at a flow rate of 1.0 ml/min and UV detection at 245 nm.
The patch of specified diameter was extracted with chloroform and kept for about 2 h at room temperature in order to extract the deltamethrin completely from the polymeric matrix and centrifuged at 3000 × g for 15 min. A fixed amount of resulting supernatant solution from above was analyzed for deltamethrin content using HPLC (Analytical technologies limited, Gujarat, India) as method described elsewhere .
The infrared data are helpful to confirm the identity of the component and to detect the interaction of the components with the polymer. Infrared spectra of deltamethrin and polymer, alone and in physical mixtures were obtained and investigated for any possible interaction between polymer and deltamethrin by FT-IR spectrophotometer (Bruker, α Alpha-E, Germany).
Differential scanning calorimetry (DSC)
The physicochemical compatibility between the components and polymer used in the formulations of deltamethrin-loaded mosquito repellent patches were evaluated by DSC analysis. The DSC thermograms (Perkin Elmer, Jade DSC, USA) obtained for pure deltamethrin, pure polymers, their physical mixtures and formulated patches were compared to ascertain the interactions. Samples were heated at a temperature range between 50-250°C at a heating rate of 10°C/min.
The release study was carried out under accelerated conditions of higher temperature. Patches were kept in an oven at 40°C and withdrawn at different time intervals from each batch and then extracted with chloroform and kept for about 2 h at room temperature. The solution was centrifuged at 3000 rpm for 15 min to remove the polymeric remnants and the supernatant liquid was collected and analyzed using HPLC.
Mathematical modelling of release kinetics
To understand the mechanism of deltamethrin permeation kinetics from the developed patches, the release data were fitted to various release kinetic equations;
(cumulative percentage drug permeated vs. time) [24
Where, Qt is the amount of deltamethrin release in time t, Q
is the initial amount of deltamethrin in the solution (most times, Q
= 0) and k
is the zero order release rate.
(log cumulative percentage drug remaining to be permeated vs. time)
is the amount of deltamethrin released in time t, Q
is the initial amount of deltamethrin in the solution and k1 is the first order release rate constant.
Higuchi’s model equation
Where, Q is the amount of deltamethrin release at time t, and k
is the Higuchi diffusion rate constant.
Where, Mt is the amount of deltamethrin released at time t, M α is the amount of deltamethrin released after infinite time, K is a kinetic constant incorporating structural and geometric characteristics of the formulation and n is the diffusional exponent indicative of the drug release mechanism.
Mosquitoes and repellency test
Ae. albopictus mosquitoes are regularly maintained in the laboratory under controlled temperature (28 ± 2°C) and relative humidity (75-80%). For the present study, about 80-100 adult (3 days old) female Ae. albopictus mosquitoes were introduced into the customised repellent trial chamber (46 × 37 × 36 cm) through the hole on top with the help of sucking tube. Prior to testing, the mosquitoes were starved by providing them with only water for 12 h. Both hands of the test volunteers were used for testing the repellent activity. The Right hand applied with a blank patch was the placebo control, while the left hand applied with a deltamethrin loaded patch was taken as the test. The repellent activity was evaluated by inserting the hand into the test chamber for one minute at the start of the trial (0 min), after 30 min interval for the first hour of application and after an interval of 60 min for the rest of testing duration. Therefore, mosquito landing and biting was recorded eleven times during each trial. The hand was placed inside the repellent chamber through a hole up to wrist and plugged with cotton to prevent escape of mosquitoes. The test chamber had clear glass sides and front (for viewing) and a sheet aluminium bottom. Sucrose solution (10%) was available to the mosquitoes at all the times during the trial. Repellency was evaluated up to 9 h and the test mosquitoes were replaced with a new set of mosquitoes after 4 h of the trial. Commercially available DEPA (N, N-diethyl phenylacetamide) based mosquito repellent cream Mosqshield®, applied at 1.0 mg/cm2 was taken as positive control for comparison with the test patch. Each trial was replicated at least three times on different days using three volunteers. The volunteers were assigned randomly for each trial. All the volunteers selected were non smokers, non alcoholic and had no known history of allergic reactions to mosquito bites. The trials were randomised between the investigators in order to minimise the bias. Written informed consent was obtained from all volunteers used in the present study. Number of mosquitoes attempted to settle for the blood feed were scored as landing, whereas those landed successfully and attempted to suck blood were scored as biting.
The percent repellency was calculated using equation–5
Where, C = Number of mosquitoes on control
N = Number of mosquitoes on test
The repellency index was calculated using the equation–6
The values obtained in the physiochemical characterisation were expressed as mean ± standard deviations. PR and RI obtained at different time intervals of the trials were compared using two-way ANOVA followed by Tukey-Krammer test of multiple comparisons at 95% confidence interval. Further, the PR and RI of deltamethrin loaded patch (test) and Mosqshield® cream (positive control) at different time intervals of test were compared using Student’s ‘t’ test.
The study project was approved by the Institutional Review Committee of Defence Research Laboratory, Tezpur and Ethical Review Committee of LGB Regional Institute of Mental Health, Tezpur. Informed written consents were obtained from the volunteers participated in the repellent trials.