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

Summary

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.

Abstract

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.

Introduction

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 agents^1,2. 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 ....

Protocol

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. Synthesis of TD PEG^5K-Cys₄-Ebes₈-CA₈

1. In a round-bottom flask, dissolve MeO-PEG^5K-NH

Results

Preparation and Characterization of Drug-loaded, Disulfide Cross-linked Micelles

Amphiphilic polymer PEG^5K-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.......

Discussion

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 "large" 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MeO-PEG5K-NH2	Rapp Polymere	125000-2
Fmoc-Lys(Fmoc)-OH	Aaptec	AFK107
Fmoc-Lys(Boc)-OH	Anaspec	AS-20132
Fmoc-Cys(Trt)-OH	Aapptec	AAC105
Dimethylformamide	Fisher Scientific	BP1160-4
Ethyl ether	Fisher Scientific	E134-20
N,N-Diisopropylethylamine	Sigma Aldrich	D125806
Trifluoroacetic acid	Sigma Aldrich	T6508	Corrosive, handle with care
4-methyl piperidine	Alfa-Aesar	L-02709
Ebes linker	Anaspec	AS-61924
Cholic acid	Sigma Aldrich	C1129
1,2-Ethanedithiol	Sigma Aldrich	02390	Handle inside fume hood. Bleach gloves after usage.
Triisopropylsilane	Sigma Aldrich	233781
Chloroform (anhydrous)	Sigma Aldrich	288306
Hydrogen peroxide solution 30%	Aaron Industries	NA
HoBt-Cl	Aaptec	CXZ096
DIC	Sigma Aldrich	D125407
Female athymic nude mice (Nu/Nu strain), 6–8 weeks age	Harlan (Livermore, CA)