B.5. Research Approach and Methodology
The idea of the approach is to synthesize a topical ointment that the lawsone encapsulated into a nano-emulsion particle will be the active ingredient. The produced ointment will then be tested in viro on Hacat cell lines and in vivo on mice models suffering from the infection. This ointment is expected to produce a bactericidal effect on staphylococcus bacteria which causes the cellulitis skin infection. In vitro testing will be done on a negative control group which will receive saline treatment and an experimental group which involves using lawsone compound on the cells.
In vivo tests on infected mice models will also involve a negative control group adminsteted saline and a test group in which the ointment will be applied on infected rat skin that contains lawsone compound only and test group (2) which will involve applying the ointment encapsulated in nano-emulsion on infected rat.
B.5.B Detailed Research Approach and Methodology
Specific Objective 1: Sample collection.
The Lawsonia Inermis will be purchased online in the form of powder from amazon
Specific Objective 2: Extraction and purification of lawsone compound from lawsonia inermis.
Activity 1: Extraction of lawsone by using method of soxhlet extractor: In a big beaker 40 g of dried henna leaves powder will be placed and 1 L distilled n-hexane will be added with each other by using a magnetic stirring rod. On a magnetic stirrer the suspension will be stired for 1 week. The suspension will be then clarified and sited in a porous bag that made of very strong filter paper, that will be sited in chamber that found in the soxhlet apparatus.
In flask, 320 mL methanol (500 mL) will be heated, and in condenser, the vapor will be condensed. The henna powder that found in the condensed extractor will be dripped into the thimble, and by contact it will be extracted. The liquid fillings of chamber overflow into flask, when the level of liquid in chamber increases to the highest of siphon tube. Until a drop of solvent from the siphon pipe does not leave residue when evaporated, this procedure will be continued and will be carried out. By rotary evaporated, the solvent will be evaporated after two days. Then, in 100 mL toluene it will be dissolved. Into separatory funnel, the solution will be poured. Then, for minutes, 100 mL NaOH 0.2 M will be added to the solution and it will be shake. After the collection of the aqueous phase, it will be acidified to pH 3 by HCl 0.2 M. The brown extract experiences a clarification in this step and it will be turned a little cloudy. With diethyl ether (3 ? 100 mL), the filter will be ectracted. The ether turns to a very pale yellow in the final extraction that indicate the finish of the extraction. With 30 mL water and it will be dried over MgSO4, the collective ethereal phases will be washed. The ether is removed completely in vacuum to leave a reddish brown solid (720 mg)as crude product.
The crude lawsone is purified by thin layer chromatography and then the product will be chromatographed over silica gel by ethanol:ethyl acetate in a ratio of 1:2 v/v. The melting point of the isolated, pure lawsone will be in the range of 192-195?C which is same as the literature value, 195?C.
Specific Objective 3: Synthesis of nano-emulsion, Characterization and incorporation of active compound (lawsone) inside it.
3.1: Synthesis of nanoemulsions: Nanoemulsions main component will be evening primrose oil as a Nano-emulsion particle is a mixture of oil and water molecules stabilized by a surfactant molecule. Phase inversion temperature (PIT) technique will be used to synthesize the nanoemulsions as it is categorized by its low energy method. Then chremophor RH 40 which is biosurctant will be added to the heating vessel in the phase inversion temperature system. Raw oil evening primrose will be added near the end of the reaction. Finally, nano-emulsion particles of different sizes ranging from 20-100nm will be obtained.
3.2: Characterization of Nanoemulisions:
The stability of the synsthsized nanoemulsion will be measured by centrifugation at full speed followed by heating up to 80 °C for 30 minutes. Then the particle size will be assessed using dynamic laser light scattering method. Finally, the zeta potential of the nanoemulsion will be determined by incubation at 25 °C and applying a voltage of 3.9 V.
3.3: Incorporation of Lawson inside nanoemulsions:
High Pressure Homogenization will be used to encapsulate lawsone inside the nano emulsion.
Specific Objective 4: In vitro testing on cell lines infected with cellulitis.
The infected HaCat cell lines that will be used to undergo in vitro testing will be divided to be used in two test groups, the first group will be used as a negative control where the placebo component (saline) will be added to the cell lines with the aid of a dropper. The other group of the cell lines which will be used as a test group, will have the Lawsone which is considered the active ingredient to be introduced to the infected cell lines by dropping, then it will be left for incubation at 37°C, with continuous checkups for observations. Further testing will be performed in order to detect the efficacy and safety of the combination to be applied to infected murine models and if it will be safe enough as a product in order to be commercialized. The in vitro tests will include cytotoxicity assays as MTT and IC50 assays, moreover, the trypan blue assay will be used to aid in evaluating the cellular membrane integrity after the product application.
Specific Objective 5: Cytotoxicity assays.
i.The MTT-assay and dosage administration (IC50):
Infected HaCat cells will be added to 96-well microplate, then lawsone compound will be added in different concentrations ranging in size from 200 to 400 µm. After the incubation period, lawsone will then be removed and replaced with a growth media and left for a duration of 3 to 4 hours. Eventually, the IC50 value will be calculated and used to determine the final dose that will be used in the in vivo trials.
0.1 ml of trypan blue solution will be added to an equal amount of cells and loaded on a hemocytometer slide which will then be examined under the microscope to determine the number of stained cells versus the total number of the cells in order to calculate the % of viable cells.
Specific Objective 6: Ointment Preparation.
Ointments are prepared by using emulsification method. The ointment ingredients are emulsifiying wax, white soft paraffin, distilled water, liquid paraffin and the active ingredient of ointment is Lawson incorporated inside nanoemulsions. First the ointment emulsification will be prepared as follow 6gm of emulsifying wax, then 15g of white soft paraffin and 4g of liquid paraffin will be melted paraffin in an evaporating dish over a water bath, stir with a glass rod until the mixture is even. Finally we will be obtaining 40gm of ointment. Then, to incorporate the nanoemulsion with lawsone inside it into an ointment will be prepared by levigation technique in which 2 g of nanoemulsion powder will be incorporated into 30 g of cream. Then the 10 g rest of the ointment will be added and well homogenized together. Finally, the ointment will be ready to be packed inside the tube.
Specific Objective 7: In vivo testing on murine models (rats) infected with cellulitis.
In this step, the murine models will be divided in order to be used in three groups. The first group will be used as a negative control group, where the topical ointment that will be applied to the infected skin of murine model will contain placebo the active ingredient (saline).Then, the second group will be used as a test group (1) where the ointment that will applied to the infected skin of murine model wll contain the lawsone active ingredient only, while the third group, will be used as test group (2), where the topical ointment that will be applied, will conatin lawsone which will be encapsulated in nano-emulsion particles. Finally, the skin of the infected murine models will be visualized using the naked eye and microcamera after the applications of the ointmenmts. Following, discharges from the diseased murine models will be collected in order to be cultured in bacterial agar medium plates. Then the bacterial cells will counted to indicated the ointment efficacy in eliminating the bacteria.
Specific Objective 8: Commercialization of the product.
The product will go through FDA for seeking an approval to be commercialized.
B.10. Expected Project Outcomes and Impact:
The predicted outcome, of this approachs experimental design will be divided according to the in vivo and in vitro testings. The expected results of the in vitro testing on the infected HaCat cell lines, will be divided in 2 groups, where the cell lines of the negative group encompassing the placebo component(saline), will show no improvement, while according to the test group, the infected cell lines that contains the lawsone component will be expected to show slight improvemnt of the cellulitis.
The expected outcomes of the in vivo testing on murine models after the application of the ointment, will be that the negative control which contains the placebo component (saline) will show no improvement, while the test group (1) which will be containing the lawsone compound will show a slight treatment. In the test group (2), by the application of the ointment containing the encapsulated lawsone compound inside the nano-emulsions, will show an effective treatment of the cellulitis on the infected murine model.
B.10.B Financial feasibility & Socio-economic Impact:
This proposed research introduces a huge step in the field of therapeutics in the area of cosmetics, as its emphasis the role of nano-emulsions as a potent drug delivery tool to treat cellulitis skin disorder. The finalized product which is a cream is expected to be a popular product in the market due to its expected overall efficiency and accuracy in targeting the infection. Moreover, it will relatively have a low cost which will make it affordable to the patients. Eventually making it a good candidate to top the market in this area.