Name: a facile and efficient approach for the production of reversible disulfide cross-linked micelles
Uploaded: 2016-12-23T12:00:00
Description: Watch this Scientific Journal Video about A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles at JoVE.com

The authors thank Ms.Alisha Knudson for the editorial help. They would also like to acknowledge the financial support from the NIH/NCI (3R01CA115483, to K.S.L.), the DoD PRMRP Award (W81XWH-13-1-0490, to K.S.L.), the NIH/NCI (1R01CA199668, to Y.L.), and the NIH/NICHD (1R01HD086195, to Y.L.).

Ethics statement: Female athymic nude mice (Nu/Nu strain), 6-8 weeks old, were purchased and then kept under pathogen-free conditions according to AAALAC guidelines and were allowed to acclimatize for at least 4 days prior to any experiments. All animal experiments were performed in compliance with institutional guidelines and according to protocol No. 07-13119 and No. 09-15584, approved by the Animal Use and Care Administrative Advisory Committee at the University of California, Davis.
1. Synth...

<ol>
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Several nanoparticles have been investigated for their potential use in drug delivery. Liposomal doxorubicin and paclitaxel (PTX)-loaded human serum albumin nano-aggregates are among the nanotherapeutics approved by the FDA for cancer treatment. However, although clinically effective, both of these nanotherapeutics are relatively &#34;large&#34; in size, and they tend to accumulate in the liver and lungs. Polymeric micelles with relatively smaller particle sizes and higher drug loading capacities are emerging nanocarrier...

Nanotechnology is a fast-emerging field that has benefited a number of biomedical areas1. Nanoparticles provide opportunities for designing and tuning properties that are not feasible with other types of conventional therapeutics. Nano-carriers enhance the stability of drugs against biodegradation, prolong drug circulation time, overcome drug solubility issues, and can be fine-tuned for targeted drug delivery and for co-delivering imaging agents1,2. Nanoparticle-based delivery systems hold promise in cancer imaging and treatment. Tumor vasculatures are leaky to macromolecules and can lead to preferential accumulation of circulating nanoparticles at tumor sites via the enhanced permeability and retention (EPR) effect3. Among the several nano-carriers (e.g., liposomes, hydrogels, and polymeric micelles) that are being actively pursued as carriers for anti-cancer drugs, polymeric micelles have gained wide popularity over the last decade4,5.
Polymeric micelles are a thermodynamic system that, on intravenous administration, can potentially be diluted below the critical micelle concentration (CMC), leading to their dissociation into unimers. Cross-linking strategies have been employed to minimize micellar dissociation into unimers. However, excessively stabilized micelles may prevent the drug from releasing at the target sites, thereby reducing the overall therapeutic efficacy. Several chemical approaches have been explored to make the cross-linking degradable in response to redox or to external stimuli, such as reducible disulfide bonds6,7 and pH-cleavable8 or hydrolysable ester bonds9,10.
We have previously reported the design and synthesis of micellar nanoparticles consisting of dendritic cholic acid (CA) blocks and linear polyethylene glycol (PEG) copolymers, referred to as telodendrimers (TD)11-15. These TDs are represented as PEGnK-CAy (where n = molecular weight in kilodaltons (K), y = number of cholic acid (CA) units). They are characterized by their small size, long shelf life, and high efficiency in encapsulating drugs such as paclitaxel (PTX) and doxorubicin (DOX) in the hydrophobic core. The building blocks of TD, such as PEG, lysine, and CA, are biocompatible, and the presence of a PEG corona can impart a &#34;stealth&#34; nanoparticle character, preventing non-specific uptake of micellar nanoparticles by the reticuloendothelial systems.
Thiolated linear-dendritic polymers can easily be generated by introducing cysteines into the dendritic oligo-lysine backbone of our standard TDs. This article presents a facile protocol for the production of a reversibly cross-linked micellar drug delivery system by introducing disulfide cross-links into the hydrophobic core of TDs (Figure 1).

<table border="0" cellpadding="0" cellspacing="0" width="795">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">MeO-PEG5K-NH2</td>
			<td style="width:105px;">Rapp Polymere</td>
			<td style="width:119px;">125000-2</td>
			<td style="width:355px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fmoc-Lys(Fmoc)-OH</td>
			<td style="width:105px;">Aaptec</td>
			<td style="width:119px;">AFK107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fmoc-Lys(Boc)-OH</td>
			<td style="width:105px;">Anaspec</td>
			<td style="width:119px;">AS-20132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fmoc-Cys(Trt)-OH</td>
			<td>Aapptec</td>
			<td>AAC105</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dimethylformamide</td>
			<td style="width:105px;">Fisher Scientific</td>
			<td style="width:119px;">BP1160-4</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethyl ether</td>
			<td style="width:105px;">Fisher Scientific</td>
			<td style="width:119px;">E134-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N-Diisopropylethylamine</td>
			<td style="width:105px;">Sigma Aldrich</td>
			<td style="width:119px;">D125806</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trifluoroacetic acid</td>
			<td style="width:105px;">Sigma Aldrich</td>
			<td style="width:119px;">T6508</td>
			<td>Corrosive, handle with care</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4-methyl piperidine</td>
			<td style="width:105px;">Alfa-Aesar</td>
			<td style="width:119px;">L-02709</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ebes linker</td>
			<td style="width:105px;">Anaspec</td>
			<td>AS-61924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cholic acid</td>
			<td style="width:105px;">Sigma Aldrich</td>
			<td style="width:119px;">C1129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1,2-Ethanedithiol</td>
			<td style="width:105px;">Sigma Aldrich</td>
			<td style="width:119px;">02390</td>
			<td>Handle inside fume hood. Bleach gloves after usage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triisopropylsilane</td>
			<td style="width:105px;">Sigma Aldrich</td>
			<td style="width:119px;">233781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform (anhydrous)</td>
			<td style="width:105px;">Sigma Aldrich</td>
			<td style="width:119px;">288306</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hydrogen peroxide solution 30%</td>
			<td style="width:105px;">Aaron Industries</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HoBt-Cl</td>
			<td style="width:105px;">Aaptec</td>
			<td style="width:119px;">CXZ096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DIC</td>
			<td style="width:105px;">Sigma Aldrich</td>
			<td style="width:119px;">D125407</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Female athymic nude mice (Nu/Nu strain), 6&#8211;8 weeks age</td>
			<td colspan="2">Harlan (Livermore, CA)</td>
			<td></td>
		</tr>
	</tbody>
</table>

a facile and efficient approach for the production of reversible disulfide cross-linked micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

Preparation and Characterization of Drug-loaded, Disulfide Cross-linked Micelles
Amphiphilic polymer PEG5K-Cys4-Ebes8-CA8 is a dendritic polymer capable of forming a disulfide cross-linked micellar system for cancer drug delivery. Structurally, it is defined as a dendritic oligomer of cholic acids (hydrophobic domain) linked to one end of the linear PEG molecule (hydrophilic domain, molecular weight 5...

Watch this Scientific Journal Video about A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles at JoVE.com

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

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