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...

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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. ...

The overall goal of this procedure is to provide a simple and effective protocol for synthesizing drug loaded disulfide cross-linked micelles for targeted drug delivery. This method can help answer questions in the nano-medicine field. Such as how to effectively produce stable nano-formulations.The main advantage of this technique is that it allows us to easily synthesize disulfide cross-linked micelles on a large scale, which is extremely desirable for clinical studies in patients. To begin this procedure, add two grams of monomethyl terminated polyethylene glycol monoamine to a round bottom flask. Add ten milliliters of anhydrous DMF to dissolve it.Chill on ice. Add three equivalents of hydroxy benzotriazole, three equivalents of DIC, and three equivalents of di-eth-moc protected lysine to a round bottom flask. Next, add ten milliliters of anhydrous DMF.Stir the mixture for 20 minutes on a magnetic stir plate. Then add the mixture to the round bottom flask containing monomethyl terminated polyethylene glycol. Remove the round bottom flask from the ice bath, then stir overnight at room temperature.Once the stirring is complete confirm the completion of the reaction as detailed in the text protocol. Add 200 milliliters of ice cold ether to the reaction flask. Centrifuge at 6, 000 x g for six minutes at four degrees Celsius To separate the precipitated polymer.Repeat the precipitation and centrifugation process twice redissolving the product in 10 milliliters of DMF each time before precipitation. Then wash the polymer three times with ice cold ether. Connect the tube to a high vacuum source to remove the residual ether.After this add 20 milliliters of 20%4-Methylpiperidine in DMF to the polymer. Stir until dissolution is complete. Allow the reaction to fun for three hours.After confirming the completion of the reaction, dry the polymeric product. Now polymer intermediate number two under a vacuum, then perform additional polymer synthesis steps as outlined in the text protocol. Once polymer intermediate number 10 is produced transfer it into a reaction flask.Add 30 milliliters of anhydrous DMF. In a new flask dissolve 24 equivalents of activated cholic acid in 20 milliliters of DMF. Then add 48 equivalents of N, N-Diisopropylethylamine.Stir for 10 minutes. After the stirring is complete transfer the mixture to the reaction flask containing polymer intermediate number 10. Allow the reaction to run overnight.The next day confirm the completion of the reaction and precipitate polymer intermediate number 11, as outlined in the text protocol. Then dialize the polymer intermediate in deionized water. Lyophilize the sample yielding a white powder.After preparing the micelle dissolve 20 milligrams of telodendrimer and five milligrams of PTX in one milliliter of chloroform. Remove the solvent using a rotary evaporator to obtain a homogenous dry polymer film. Reconstitute the film with one milliliter of PBS by vortexing.Then sonicate for 30 minutes at 40 kilohertz. Add 3%hydrogen peroxide to oxidize the thiol groups on the telodendrimmer. Perform additional preparation and verification steps as outlined in the text protocol.After micelle preparation is complete measure the size and size distribution of the micelles with a dynamic light scattering instrument. Perform the measurements at room temperature and keep the micelle concentration at one milligram per milliliter. Next prepare a 7.5 milligram per milliliter stock solution of sodium dodecyl sulfate in PBS.Then prepare a 1.5 milligram per milliliter stock solution of disulphide cross-linked micells in PBS. Using these stock solutions create a mixture in which the final SDS concentration is 2.5 milligrams per milliliter. And the micelle concentration is 1.0 milligrams per milliliter.Load the sample and measure the size and size distribution of the micelle solution at two minute intervals. After collecting fresh citrated blood from nude mice centrifuge a one milliliter blood sample at 1, 000 x g for 10 minutes to collect red blood cells. Wash them three times with PBS then resuspend the cell pellet with PBS to a final concentration of 2%Mix 200 microliters of erythrocyte suspension with a 1.0 milligram per milliliter solution of PTX NCMs.Then mix 200 microliters of erythrocyte suspension with a 1.0 milligram per milliliter solution of PTX DCMs. Incubate these mixtures in a incubator shaker for four hours at 37 degrees Celsius. After incubation is complete centrifuge the mixtures at 3, 000 x g for five minutes.Transfer 100 microliters of the supernatant of each sample to a 96 well plate. After this use a micro plate reader to measure the absorbance of free hemoglobin at 540 nanometers. As measured by Ellman's Test the conversion rate from free thiol groups to disulfide bonds reached 85%after 48 hours of oxygen mediated oxidation.Here a hydrogen peroxide mediated oxidation method is employed as an alternative method. The conversion rate reaches 88%in 30 minutes. Which is 96 times faster than the oxygen mediated approach.Using this more efficient approach over 50 grams of nanoparticles have been produced with a measured particle size of 27 nanometers with a narrow size distribution. The stability of PTX loaded disufide cross-linked micelles is also investigated under severe micelle disrupting conditions. The particle size of the micelles remained steady over time, indicating that they stayed intact.After the introduction of GSH at an intracellular concentration the size of the drug loaded disulfide cross-linked micelles remain intact for 30 minutes then abruptly decreased to one nanometer. This signifies the reduction of a critical number of disulfide bonds a prerequisite of the rapid disassociation of the micelles. The representative results of the hemolytic activity of PTX loaded micelles is shown here.The PTX NCMs have a dose dependent red blood cell lysis count. However PTX DCMs that have disulfide cross-linking show no observable hemolytic activities. Once mastered this technique can be done in another if it is performed properly.While attempting this procedure it's important to remember to add an equal amount of 3%hydrogen peroxide for disulfide formation. Following this procedure Hemolysis Assay can be performed in order to evaluate the blood compatibility of the disulfide cross-linked micelles. After watching this video you should have a good understanding of how to effectively synthesize drug loaded disulfide cross-linked micelles.Don't forget that some of these chemicals can be extremely hazardous and precautions such as wearing personal protective equipment should always be taken while performing this procedure.

a-facile-efficient-approach-for-production-reversible-disulfide-cross

Research

JoVE Journal

Biochemistry

8.7K Views.  University of California, Davis. The overall goal of this procedure is to provide a simple and effective protocol for synthesizing drug loaded disulfide cross-linked micelles for targeted drug delivery. This method can help answer questions in the nano-medicine field. Such as how to effectively produce stable nano-formulations.The main advantage of this technique is that it allows us to easily synthesize disulfide cross-linked micelles on a large scale, which is extremely desirable for clinical studies in patients. To...