The authors acknowledge the Saudi Arabia-funded scholarship (I821) for making the research possible. The authors are grateful to Dr Xianwei Liu for expert support in TEM analysis at Cranfield University.

1. Preparation of Oil-in-water Emulsion
<ol>
	<li>Weigh glyceryl palmitostearate (120 mg), isopropyl myristate (60 mg), d-&#945;-tocopheryl polyethylene glycol succinate (30 mg) and soybean lecithin (30 mg), and add them to 12.5 ml of organic solvents (6.25 ml acetone and 6.25 ml ethanol). Quickly dissolve the mixture at the temperature 70&#160;&#176;C (5 &#176;C above the melting point of the solid lipid) in water bath.</li>
	<li>Add either 125, 250 or 375 mg of Poloxamer188 in 12.5 ml of ddH2O to achieve 1...

<ol>
	<li>Loo, T. L., Housholder, G. E., Gerulath, A. H., Saunders, P. H., Farquhar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+action+and+pharmacology+studies+with+DTIC+(NSC+45388).">Mechanism of action and pharmacology studies with DTIC (NSC 45388).</a> Cancer Treat Rep. 60 (2), 149-152 (1976).</li><li>Behringer, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Omission+of+dacarbazine+or+bleomycin,+or+both,+from+the+ABVD+regimen+in+treatment+of+early-stage+favourable+Hodgkin's+lymphoma+(GHSG+HD13):+An+open-label,+randomised,+non-inferiority+trial.">Omission of dacarbazine or bleomycin, or both, from the ABVD regimen in treatment of early-stage favourable Hodgkin's lymphoma (GHSG HD13): An open-label, randomised, non-inferiority trial.</a> The Lancet. 385 (9976), 1418-1427 (2014).</li><li>Carvajal, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+2+randomised+study+of+ramucirumab+(IMC-1121B)+with+or+without+dacarbazine+in+patients+with+metastatic+melanoma.">A phase 2 randomised study of ramucirumab (IMC-1121B) with or without dacarbazine in patients with metastatic melanoma.</a> Eur J Cancer. 50 (12), 2099-2107 (2014).</li><li>Jiang, G., Li, R., Sun, C., Liu, Y., Zheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dacarbazine+combined+targeted+therapy+versus+dacarbazine+alone+in+patients+with+malignant+melanoma:+A+meta-analysis.">Dacarbazine combined targeted therapy versus dacarbazine alone in patients with malignant melanoma: A meta-analysis.</a> PLoS ONE. 9 (12), (2014).</li><li>Lazar, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorafenib+plus+dacarbazine+in+solid+tumors:+A+phase+i+study+with+dynamic+contrast-enhanced+ultrasonography+and+genomic+analysis+of+sequential+tumor+biopsy+samples.">Sorafenib plus dacarbazine in solid tumors: A phase i study with dynamic contrast-enhanced ultrasonography and genomic analysis of sequential tumor biopsy samples.</a> Invest New Drugs. 32 (2), 312-322 (2014).</li><li>Niemeijer, N. D., Alblas, G., Van Hulsteijn, L. T., Dekkers, O. M., Corssmit, E. P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+with+cyclophosphamide,+vincristine+and+dacarbazine+for+malignant+paraganglioma+and+pheochromocytoma:+Systematic+review+and+meta-analysis.">Chemotherapy with cyclophosphamide, vincristine and dacarbazine for malignant paraganglioma and pheochromocytoma: Systematic review and meta-analysis.</a> Clin Endocrinol (Oxf). 81 (5), 642-651 (2014).</li><li>Bedikian, A. Y., Garbe, C., Conry, R., Lebbe, C., Grob, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dacarbazine+with+or+without+oblimersen+(a+Bcl-2+antisense+oligonucleotide)+in+chemotherapy-naive+patients+with+advanced+melanoma+and+low-normal+serum+lactate+dehydrogenase:+'The+AGENDA+trial'.">Dacarbazine with or without oblimersen (a Bcl-2 antisense oligonucleotide) in chemotherapy-naive patients with advanced melanoma and low-normal serum lactate dehydrogenase: 'The AGENDA trial'.</a> Melanoma Res. 24 (3), 237-243 (2014).</li><li>Daponte, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+III+randomized+study+of+fotemustine+and+dacarbazine+versus+dacarbazine+with+or+without+interferon-a+in+advanced+malignant+melanoma.">Phase III randomized study of fotemustine and dacarbazine versus dacarbazine with or without interferon-a in advanced malignant melanoma.</a> J Trans Med. 11 (1), (2013).</li><li>Jiao, J., Rhodes, D. 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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymoquinone-loaded+nanostructured+lipid+carrier+exhibited+cytotoxicity+towards+breast+cancer+cell+lines+(MDA-MB-231+and+MCF-7)+and+cervical+cancer+cell+lines+(HeLa+and+SiHa).">Thymoquinone-loaded nanostructured lipid carrier exhibited cytotoxicity towards breast cancer cell lines (MDA-MB-231 and MCF-7) and cervical cancer cell lines (HeLa and SiHa).</a> BioMed Research International. , (2015).</li><li>Sun, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quercetin-nanostructured+lipid+carriers:+Characteristics+and+anti-breast+cancer+activities+in+vitro.">Quercetin-nanostructured lipid carriers: Characteristics and anti-breast cancer activities in vitro.</a> Colloids Surf., B. 113, 15-24 (2014).</li><li>Savla, R., Garbuzenko, O. 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Lipid-based nanostructured particles have been utilized to provide a highly lipophilic carrier for delivery of hydrophobic drugs. A NLC is the second generation of solid lipid nanostructured carrier, which are solid at room and body temperature. The incorporation of a solid lipid into a liquid lipid in a NLC results in a less perfect crystallization, thus increasing the drug loading efficiency and also reducing the expulsion of encapsulated drugs during storage.
For NLC synthesis, the most com...

Dacarbazine (Dac) is an alkylating agent that exerts anti-tumor activity through nucleic acids methylation or direct DNA damage, leading to cell cycle arrest and cell death 1.
As a first line chemotherapeutic agent, Dac has been used alone or in combination with other chemotherapy drugs for treating various cancers 2-6. It is the most active agent so far used in treating cutaneous and metastatic melanoma, which is the most aggressive form of skin cancer 3,7,8. The response rate, however, is only 20% at best, and the therapeutic effects are often accompanied with severe systemic side effects.
In its natural form, Dac is hydrophilic and is unstable due to its photosensitivity 9. The only available formula for clinical use currently is a sterile powder to be used in suspension for intravenous infusion 7,8. The low response rate and high systemic toxicity rate of the drug is largely attributable to its poor water solubility, therefore low availability at target site, and high distribution at non-target sites, which limits the maximum dose of the drug 10. The rapid degradation and metabolism after intravenous admission together with the development of drug resistance limit the clinical application and therapeutic effect of the drug 11. Therefore, there is an urgent need to develop alternative Dac formulations for treating malignant melanoma.
Colloidal systems containing liposomes, micelles or nanostructured particles have been intensively investigated for their use in drug delivery as reviewed by Marilene et al. 12 Nanostructured particles as potential drug carriers have been attracting increasing attention in the last decade due to their ability to increase drug loading efficiency, control drug release, improve drug pharmacokinetics and biodistribution, and therefore reduce drug systemic toxicity 13. Only a few nanoformulations, however, have been investigated so far for Dac delivery, showing protection of the drug from photo degeneration, increased drug solubility, and improved therapeutic effect 10,14,15. However these formulations suffered from low encapsulating efficiency while some also using synthetic polymer nanoparticles that are not cost effective.
Nanostructured lipid carriers (NLC), made of a mixture of solid and liquid lipids, have been developed for drug delivery 16,17. The drugs to be encapsulated are often soluble in both the liquid lipids and solid lipids phases 18, resulting in a high loading and controlled release 19. This study aims to develop a new Dac formulation based on NLC-encapsulation using glyceryl palmitostearate and isopropyl myristate as lipids. The preparation involved oil-in-water emulsion, evaporation, solidification, and homogenization. The preparations have been characterized for NLC size, shape, ultrastructure, and dispersity, drug encapsulation efficiency and drug loading 20.

<table border="0" cellpadding="0" cellspacing="0" width="968">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dacarbazine (DAC)</td>
			<td style="width:467px;">Sigma Aldrich (Gillingham Dorset, UK)</td>
			<td style="width:119px;">D2390-100MG</td>
			<td style="width:167px;">drug used for uploading</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glyceryl palmitostearate&#160;</td>
			<td>Gattefoss&#233; (Saint_Priest_c&#233;dex, France)</td>
			<td style="width:119px;">85251-77-0</td>
			<td>solid lipid&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">d-&#945;- Tocopherol polyethylene glycol succinate (TPGS)</td>
			<td>Sigma Aldrich (Gillingham Dorset, UK)</td>
			<td style="width:119px;">57668</td>
			<td>lipid phase surfactant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poloxamer 188</td>
			<td>Sigma Aldrich (Gillingham Dorset, UK)</td>
			<td style="width:119px;">15759-1KG</td>
			<td>liqiud phase surfactant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetone&#160;</td>
			<td>Sigma Aldrich (Gillingham Dorset, UK)</td>
			<td style="width:119px;">650501-1L</td>
			<td>organic solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol&#160;</td>
			<td>Sigma Aldrich (Gillingham Dorset, UK)</td>
			<td style="width:119px;">459836-1L</td>
			<td>organic solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Soybean lecithin (SL)</td>
			<td>Cuisine Innovation (Dijon, France)</td>
			<td style="width:119px;">SLL1402</td>
			<td>lipid phase surfactant</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Double-distilled water was collected in our laboratory from</td>
			<td>Millipore-Q Gradient A10 ultra-pure water system (Millipore, France)</td>
			<td style="width:119px;">SAS - 67120&#160;</td>
			<td>aqueous phase&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T 25 digital ULTRA-TURRAX</td>
			<td style="width:467px;">IKA</td>
			<td style="width:119px;">3725000</td>
			<td>as high shear disperser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hotplate Magnetic Stirrer</td>
			<td>Scientific Support, Inc</td>
			<td style="width:119px;">1454&#160;</td>
			<td>emulsion homogenization</td>
		</tr>
	</tbody>
</table>

encapsulation of cancer therapeutic agent dacarbazine using nanostructured lipid carrier

The only formula of dacarbazine (Dac) in clinical use is intravenous infusion, presenting a poor therapeutic profile due to the low dispersity of the drug in aqueous solution. To overcome this, a nanostructured lipid carrier (NLC) consisting of glyceryl palmitostearate and isopropyl myristate was developed to encapsulate Dac. NLCs with controlled size were achieved using high shear dispersion (HSD) following solidification of oil-in-water emulsion. The synthesis parameters, including surfactant concentration, the speed and time of HSD were optimized to achieve the smallest NLC with size, polydispersion index and zeta potential of 155 &#177; 10 nm, 0.2 &#177; 0.01, and -43.4 &#177; 2 mV, respectively. The optimal parameters were also employed for Dac-loaded NLC preparation. The resultant NLC loaded with Dac possessed size, polydispersion index and zeta potential of 190 &#177; 10 nm, 0.2 &#177; 0.01, and -43.5 &#177; 1.2 mV, respectively. The drug encapsulation efficiency and drug loading reached 98% and 14%, respectively. This is the first report on encapsulation of Dac using NLC, implying that NLC could be a new potential candidate as drug carrier to improve the therapeutic profile of Dac.

The preparations of the NLC and NLC-Dac using glyceryl palmitostearate and isopropyl myristate with different parameters were characterized for PS, PDI, morphology and ultrastructure 20. The PS and PDI of the NLCs were surfactant concentration, HSD speed and duration dependent. As judged by PS and PDI of the NLCs, the best results were achieved with 1% of surfactant and a sheer dispersion speed of 15,000 rpm for 30 min (Figure 1A, B and <strong...

Watch this Scientific Journal Video about Encapsulation of Cancer Therapeutic Agent Dacarbazine Using Nanostructured Lipid Carrier at JoVE.com

Encapsulation of Cancer Therapeutic Agent Dacarbazine Using Nanostructured Lipid Carrier

The most commonly used method for nanostructured lipid carrier (NLC) synthesis involves oil-in-water emulsion, homogenization and solidification. This was modified here by applying high shear dispersion after solidification to achieve a NLC with desirable size, improved drug encapsulation and drug loading efficiency as a potential carrier for dacarbazine delivery.

The only formula of dacarbazine (Dac) in clinical use is intravenous infusion, presenting a poor therapeutic profile due to the low dispersity of the drug ...

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.

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JoVE Journal

Chemistry

11.0K Views.  Cranfield University. 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 le...