The overall goal of this procedure is to prepare a nanostructured lipid carrier for encapsulating the cancer therapeutic agent dacarbazine. This procedure is simple and well-optimized. Allowing for the preparation of nanostructured lipid carrier with a well-controlled capacity.
The main advantages of the new formulation of dacarbazine include increased drug dispersity in aqueous solution, prolonged historic stability and improved drug-release control. This improvement can potentially lead to prolonged half-life in vivo as well. Begin by adding 6.25ml each of acetone and ethanol to a flask.
To the solvents, add glyceryl palmitosterate isopropyl myristate, D-a-tocopheryl polyethylene glycol succinate and soybean lecithin. Dissolve the mixture at 70 degrees Celsius in a water bath. Into an additional flask, add 12.5ml of water.
Create a solution of 1%poloxamer 188 by dissolving 125mg into the water. Heat the flask to 70 degrees Celsius to dissolve the chemical fully. While stirring the organic solution at 400 rpm dropwise add the 1%poloxamer 188 solution to form an emulsion.
Stir the emulsion for an additional four hours to evaporate the organic solvent. After four hours, place the emulsion in a fridge at four degrees Celsius for two hours to solidify. To obtain nanostructured lipid carriers or NLCs, use a homogenizer to subject the solidified emulsions to high shear dispersion.
Mix the solution at 15000 rpm for 30 minutes to disperse the emulsion. Sample the solution in 10 minute increments. Take 14 microliters of emulsion in a dynamic light scattering or DLS cuvette.
And dilute to 1ml with deuterated water for measurement. Read the cuvette sample in the instrument. Examine the samples to assess which samples have the smallest size and smallest polydispersion index.
Therefore, to determine the optimal mix speed and mix time for NLC creation. Observe samples for morphology and ultrastructure of the NLC. Apply the same dilution of samples to the carbon fiber-coated copper grid of a transmission electron microscope instrument.
Allow the sample to air-dry and then run the sample per the manufacturer's instructions. Now that conditions have been optimized, prepare a solution as performed for the initial emulsions. After combining the glyceryl, isopropyl, polyethylene glycol succinate and soybean lecithin reagents add 70mg of dacarbazine and then heat the solution to 70 degrees Celsius.
Prepare 12.5ml of an aqueous 1%poloxamer 188 solution. Heat the flask to 70 degrees Celsius. After dissolving the poloxamer 188 add the solution dropwise to the organic solution, stirring at 400 rpm to form an emulsion.
Stir the emulsion for an additional four hours to evaporate the organic solvent. As before, place the emulsion in a cold room at four degrees Celsius for two hours to solidify. Use the optimized high dispersion speed of 15000 rpm for 30 minutes to homogenize the emulsion.
The preparations of the NLC and the dacarpazine-loaded NLC with different parameters were characterized for particle size and polydispersion index. The particle size and polydispersion index of the NLCs were surfactant concentration, high shear dispersion speed and duration dependent. As judged by particle size and polydispersion index, the best results were achieved with 1%of surfactant and the shear dispersion speed of 15000 rpm for 30 minutes.
Which were selected as the optimal parameters for NLC preparation in this study. When the optimal parameters were used for dacarbazine-loaded NLC preparation, the smallest size achieved was 155 nanometers for NLC and 190 nanometers for dacarbazine-loaded NLC. Both with a polydispersion index of 0.2, indicating good uniformity.
The uploading and encapsulation of dacarbazine in NLC is indicated by the size and structure changes. Where dacarbazine-loaded NLC show larger size and altered internal structures compared with NLC. Once mastered, this technique can be done in eight hours if it is performed perfectly.
While attempting this procedure, it's very important to remember that the lines of the evaporation the solidification is very critical. As too long or too short will have negative effect on the generation of nanostructured lipid carriers. Following this procedure, the reduced nanostructured lipid carrier are stable for six months at four degrees Centigrade.
Allowing characterizations such as pharmacology and toxicology to be performed. This technique paved the way for those in constant research to explore nanostructured carrier-based formulation of dacarbazine for a variety of cancer treatments. To date, the dacarbazine is the most active agent in treating cutaneous and metastatic melanoma.
After watching this video you should have a good understanding of how to prepare nanostructured lipid carrier and use nanostructured lipid carrier to encapsulate dacarbazine. While working with hazardous materials such as dacarbazine, personal protection equipment, including lab coat, protective gloves, and eye protection should always be used. Access to is also needed for working with the hazardous solvent.
