Name: encapsulation of cancer therapeutic agent dacarbazine using nanostructured lipid carrier
Uploaded: 2016-04-26T14:00:00
Description: Watch this Scientific Journal Video about Encapsulation of Cancer Therapeutic Agent Dacarbazine Using Nanostructured Lipid Carrier at JoVE.com

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. G., Burgess, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Emulsion+Stability:+Pressure+Balance+and+Interfacial+Film+Strength.">Multiple Emulsion Stability: Pressure Balance and Interfacial Film Strength.</a> J Colloid Interface Sci. 250 (2), 444-450 (2002).</li><li>Kakumanu, S., Tagne, J. B., Wilson, T. A., Nicolosi, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nanoemulsion+formulation+of+dacarbazine+reduces+tumor+size+in+a+xenograft+mouse+epidermoid+carcinoma+model+compared+to+dacarbazine+suspension.">A nanoemulsion formulation of dacarbazine reduces tumor size in a xenograft mouse epidermoid carcinoma model compared to dacarbazine suspension.</a> Nanomedicine. 7 (3), 277-283 (2011).</li><li>Xie, T., Nguyen, T., Hupe, M., Wei, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug+resistance+decreases+with+mutations+of+melanosomal+regulatory+genes.">Multidrug resistance decreases with mutations of melanosomal regulatory genes.</a> Cancer Res. 69 (3), 992-999 (2009).</li><li>Estanqueiro, M., Amaral, M. H., Concei&#231;&#227;o, J., Lobo, J. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnological+carriers+for+cancer+chemotherapy:+the+state+of+the+art.">Nanotechnological carriers for cancer chemotherapy: the state of the art.</a> Colloids Surf., B. 126, 631-648 (2015).</li><li>Koziara, J. M., Whisman, T. R., Tseng, M. T., Mumper, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vivo+efficacy+of+novel+paclitaxel+nanoparticles+in+paclitaxel-resistant+human+colorectal+tumors.">In-vivo efficacy of novel paclitaxel nanoparticles in paclitaxel-resistant human colorectal tumors.</a> J Controlled Release. 112 (3), 312-319 (2006).</li><li>Ding, B., 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=Biodegradable+methoxy+poly+(ethylene+glycol)-poly+(lactide)+nanoparticles+for+controlled+delivery+of+dacarbazine:+Preparation,+characterization+and+anticancer+activity+evaluation.">Biodegradable methoxy poly (ethylene glycol)-poly (lactide) nanoparticles for controlled delivery of dacarbazine: Preparation, characterization and anticancer activity evaluation.</a> Afr J Pharm Pharacol. 5 (11), 1369-1377 (2011).</li><li>Ding, B., 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=Anti-DR5+monoclonal+antibody-mediated+DTIC-loaded+nanoparticles+combining+chemotherapy+and+immunotherapy+for+malignant+melanoma:+target+formulation+development+and+in+vitro+anticancer+activity.">Anti-DR5 monoclonal antibody-mediated DTIC-loaded nanoparticles combining chemotherapy and immunotherapy for malignant melanoma: target formulation development and in vitro anticancer activity.</a> Int J Nanomedicine. 6, 1991-2005 (2011).</li><li>Jenning, V., Th&#252;nemann, A. F., Gohla, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+a+novel+solid+lipid+nanoparticle+carrier+system+based+on+binary+mixtures+of+liquid+and+solid+lipids.">Characterisation of a novel solid lipid nanoparticle carrier system based on binary mixtures of liquid and solid lipids.</a> Int J Pharm. 199 (2), 167-177 (2000).</li><li>M&#252;ller, R. H., Radtke, M., Wissing, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructured+lipid+matrices+for+improved+microencapsulation+of+drugs.">Nanostructured lipid matrices for improved microencapsulation of drugs.</a> Int J Pharm. 242 (1-2), 121-128 (2002).</li><li>Pouton, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+formulations+for+oral+administration+of+drugs:+Non-emulsifying,+self-emulsifying+and+'self-microemulsifying'+drug+delivery+systems.">Lipid formulations for oral administration of drugs: Non-emulsifying, self-emulsifying and 'self-microemulsifying' drug delivery systems.</a> Eur. J. Pharm. Sci. 11 (Suppl. 2), S93-S98 (2000).</li><li>Jores, K., Mehnert, W., Drechsler, M., Bunjes, H., Johann, C., M&#228;der, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigations+on+the+structure+of+solid+lipid+nanoparticles+(SLN)+and+oil-loaded+solid+lipid+nanoparticles+by+photon+correlation+spectroscopy,+field-flow+fractionation+and+transmission+electron+microscopy.">Investigations on the structure of solid lipid nanoparticles (SLN) and oil-loaded solid lipid nanoparticles by photon correlation spectroscopy, field-flow fractionation and transmission electron microscopy.</a> J Controlled Release. 95 (2), 217-227 (2004).</li><li>Almousallam, M., Zhu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+cancer+therapeutic+agent+dacarbazine+using+nanostructured+lipid+carrier.">Encapsulation of cancer therapeutic agent dacarbazine using nanostructured lipid carrier.</a> Int nano lett. , (2015).</li><li>Ng, W. 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. B., Chen, S., Rodriguez-Rodriguez, L., Minko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-Targeted+Responsive+Nanoparticle-Based+Systems+for+Magnetic+Resonance+Imaging+and+Therapy.">Tumor-Targeted Responsive Nanoparticle-Based Systems for Magnetic Resonance Imaging and Therapy.</a> Pharm Res. 31 (12), 3487-3502 (2014).</li><li>Chen, Y., 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=Formulation,+characterization,+and+evaluation+of+in+vitro+skin+permeation+and+in+vivo+pharmacodynamics+of+surface-charged+tripterine-loaded+nanostructured+lipid+carriers.">Formulation, characterization, and evaluation of in vitro skin permeation and in vivo pharmacodynamics of surface-charged tripterine-loaded nanostructured lipid carriers.</a> Int J Nanomedicine. 7, 3023 (2012).</li><li>Sanna, V., Caria, G., Mariani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+lipid+nanoparticles+containing+fatty+alcohols+having+different+chain+length+on+the+ex+vivo+skin+permeability+of+Econazole+nitrate.">Effect of lipid nanoparticles containing fatty alcohols having different chain length on the ex vivo skin permeability of Econazole nitrate.</a> Powder Technol. 201 (1), 32-36 (2010).</li><li>Brigger, I., Dubernet, C., Couvreur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticles+in+cancer+therapy+and+diagnosis.">Nanoparticles in cancer therapy and diagnosis.</a> Adv Drug Deliv Rev. 54 (5), 631-651 (2002).</li><li>Visaria, R. 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=Enhancement+of+tumor+thermal+therapy+using+gold+nanoparticle-assisted+tumor+necrosis+factor-a+delivery.">Enhancement of tumor thermal therapy using gold nanoparticle-assisted tumor necrosis factor-a delivery.</a> Mol Cancer Ther. 5 (4), 1014-1020 (2006).</li><li>Tripathi, A., Gupta, R., Saraf, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLGA+nanoparticles+of+anti+tubercular+drug:+Drug+loading+and+release+studies+of+a+water+in-soluble+drug.">PLGA nanoparticles of anti tubercular drug: Drug loading and release studies of a water in-soluble drug.</a> Int J PharmTech Res. 2 (3), 2116-2123 (2010).</li><li>Joshi, M., Patravale, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructured+lipid+carrier+(NLC)+based+gel+of+celecoxib.">Nanostructured lipid carrier (NLC) based gel of celecoxib.</a> Int J Pharm. 346 (1-2), 124-132 (2008).</li><li>Lim, W. M., Rajinikanth, P. S., Mallikarjun, C., Kang, Y. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formulation+and+delivery+of+itraconazole+to+the+brain+using+a+nanolipid+carrier+system.">Formulation and delivery of itraconazole to the brain using a nanolipid carrier system.</a> Int J Nanomedicine. 9 (1), 2117-2126 (2014).</li><li>Bei, D., Zhang, T., Murowchick, J. B., Youan, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formulation+of+dacarbazine-loaded+cubosomes.+Part+III.+physicochemical+characterization.">Formulation of dacarbazine-loaded cubosomes. Part III. physicochemical characterization.</a> AAPS PharmSciTech. 11 (3), 1243-1249 (2010).</li><li>Lei, 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=Dual+drug+encapsulation+in+a+novel+nano-vesicular+carrier+for+the+treatment+of+cutaneous+melanoma:+Characterization+and+in+vitro/in+vivo+evaluation.">Dual drug encapsulation in a novel nano-vesicular carrier for the treatment of cutaneous melanoma: Characterization and in vitro/in vivo evaluation.</a> RSC Advances. 5 (26), (2015).</li></ol>

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...

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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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Research

JoVE Journal

Chemistry

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB&#8217;s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is ...

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential nutrients. The objective of this work is to demonstrate the qualitative and quantitative analysis of encapsulated fish oil supplements using high-resolution 1H and 13C NMR spectroscopy utilizing two different NMR instruments; a 500 MHz and an 850 MHz instrument. Both proton (1H) and carbon (13C) NMR spectra can be used for the quantitative determination of the major constituents of fish oil supplements. Quantification of the lipids in fish oil supplements is achieved through integration of the appropriate NMR signals in the relevant 1D spectra. Results obtained by 1H and 13C NMR are in good agreement with each other, despite the difference in resolution and sensitivity between the two nuclei and the two instruments. 1H NMR offers a more rapid analysis compared to 13C NMR, as the spectrum can be recorded in less than 1 min, in contrast to 13C NMR analysis, which lasts from 10 min to one hour. The 13C NMR spectrum, however, is much more informative. It can provide quantitative data for a greater number of individual fatty acids and can be used for determining the positional distribution of fatty acids on the glycerol backbone. Both nuclei can provide quantitative information in just one experiment without the need of purification or separation steps. The strength of the magnetic field mostly affects the 1H NMR spectra due to its lower resolution with respect to 13C NMR, however, even lower cost NMR instruments can be efficiently applied as a standard method by the food industry and quality control laboratories.

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

From Molecules to Materials: Engineering New Ionic Liquid Crystals Through Halogen Bonding

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid crystals (ILCs). Taking advantages of the high specificity of XB for haloperfluorocarbons and the ability of anions to act as XB-acceptors, we obtained supramolecular complexes based on 1-alkyl-3-methylimidazolium iodides and iodoperfluorocarbons, overcoming the well-known immiscibility between hydrocarbons (HCs) and perfluorocarbons (PFCs). The high directionality of the XB combined with the fluorophobic effect, allowed us to obtain enantiotropic liquid crystals where a rigid, non-aromatic, XB supramolecular anion acts as mesogenic core.
X-ray structure analysis of the complex between 1-ethyl-3-methylimidazolium iodide and iodoperfluorooctane showed the presence of a layered structure, which is a manifestation of the well-known tendency to segregation of perfluoroalkyl chains. This is consistent with the observation of smectic mesophases. Moreover, all the reported complexes melt below 100 &#176;C, and most are mesomorphic even at room temperature, despite that the starting materials were non-mesomorphic in nature.
The supramolecular strategy reported here provides new design principles for mesogen design allowing a totally new class of functional materials.

A protocol for the synthesis of a new type of mesogens, based on the halogen-bonded supramolecular anion [CnF2n+1-I&#183;&#183;&#183;I&#183;&#183;&#183;I-CnF2n+1]&#8722;, is reported.

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid ...

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and &#946;-glycerophosphate (&#946;-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM).
Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions.
The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

Organic Structure-directing Agent-free Synthesis for *BEA-type Zeolite Membrane

Membrane separation has drawn attention as a novel-energy saving separation process. Zeolite membranes have great potential for hydrocarbon separation in petroleum and petrochemical fields because of their high thermal, chemical, and mechanical strength. A *BEA-type zeolite is an interesting membrane material because of its large pore size and wide Si/Al range. This manuscript presents a protocol for *BEA membrane preparation by a secondary growth method that does not use an organic structure-directing agent (OSDA). The preparation protocol consists of four steps: pretreatment of support, seed preparation, dip-coating, and membrane crystallization. First, the *BEA seed crystal is prepared by conventional hydrothermal synthesis using OSDA. The synthesized seed crystal is loaded on the outer surface of a 3 cm long tubular &#945;-Al2O3 support by a dip-coating method. The loaded seed layer is prepared with the secondary growth method using a hydrothermal treatment at 393 K for 7 days without using OSDA. A *BEA membrane having very few defects is successfully obtained. The seed preparation and dip-coating steps strongly affect the membrane quality.

A *BEA seed crystal was loaded on a porous &#945;-Al2O3 support by the dip-coating method, and hydrothermally grown without using an organic structure-directing agent. A *BEA-type zeolite membrane having very few defects was successfully prepared by the secondary growth method.

Membrane separation has drawn attention as a novel-energy saving separation process. Zeolite membranes have great potential for hydrocarbon separation in ...

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging.
Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.