Name: A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles (Video) | JoVE Journal
Uploaded: 2016-12-23T12:00:00.000+00:00
Duration: 9 min 57 s
Description: Learn to synthesize disulfide cross-linked micelles using hydrogen peroxide-mediated oxidation for targeted cancer drug delivery. This scalable method enables stable nano-formulations suitable for clinical studies.

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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. Synthesis of TD PEG5K-Cys4-Ebes8-CA8
<ol>
	<li>In a round-bottom flask, dissolve MeO-PEG5K-NH2 (2 g, 0.4 mmol) in 10-20 ml of anhydrous dimethylformamide (DMF) and chill on ice.</li>
	<li>In a glass beaker, dissolve 3 equiv. of 1-hydroxy-6-chloro-benzotriazole (HOBt), 3 equiv. of N,N&#39;-diisopropylcarbodiimide (DIC), and 3 equiv. of (Fmoc)2Lys-OH in anhydrous DMF (10-15 ml). Stir for 15-20 min on a magnetic stir plate.</li>
	<li>Add the mixture to the reaction flask containing the MeO-PEG5K-NH2. Remove the ice bath and stir the reaction mixture overnight at room temperature.</li>
	<li>Confirm the completion of the reaction with thin layer chromatography (TLC)16 and Kaiser&#39;s test17 (a yellow color indicates the absence of free NH2). Precipitate the polymeric product 1 (MeO-PEG5K-Lys(NH-Fmoc)2) by adding approximately 200 ml of ice-cold ether to the reaction flask. Separate the precipitated polymer via centrifugation (6 min at 6,000 x g and 4 &#176;C).
	<ol>
		<li>To perform TLC, spot the samples on silica gel-coated TLC plates. Use dichloromethane/methanol (9:1) as the mobile phase. Observe spots under a UV lamp after developing the TLC plates. One can also use ninhydrin staining reagent to visualize amine spots on a hot plate.</li>
		<li>For Kaiser&#39;s test, place a little bit of the sample in glass tube containing Kaiser&#39;s reagent. Heat it at 100 &#176;C for 5 min and look for the color change (if the color of the solution remains yellow, then the reaction is complete).</li>
	</ol>
	</li>
	<li>Re-dissolve the product in anhydrous DMF (10-20 ml) and repeat the precipitation and centrifugation (see step 1.4).</li>
	<li>Repeat step 1.5, and then wash the polymer precipitate three times with ice-cold ether.</li>
	<li>Transfer the polymer precipitate 1 to a clean reaction flask and connect the flask to a high vacuum source to remove the residual ether.</li>
	<li>Prepare and add approximately 20-30 ml of 20% (v/v) 4-methylpiperidine in DMF to polymer intermediate 1. Stir until complete dissolution. Run the reaction for 3 hr.</li>
	<li>Perform TLC16 and Kaiser&#39;s test (a blue color confirms the presence of free NH2)17 (see step 1.4) to confirm the completion of the reaction. If the reaction is complete, proceed to the ether precipitation, as mentioned for product 1 (steps 1.4-1.6).</li>
	<li>Dry the polymeric product 2 (MeO-PEG5K-Lys(NH2)2) under a vacuum.</li>
	<li>Carry out one more round of (Fmoc)2Lys-OH-coupling onto intermediate 2 (steps 1.1-1.7) to generate product 3 (MeO-PEG5K-Lys(Lys(NH-Fmoc)2)2). De-protect (steps 1.8-1.10) the Fmoc groups (4, MeO-PEG5K-Lys(Lys(NH2)2)2) and couple (steps 1.1-1.7) the (Fmoc)Lys(Boc)-OH to generate a third-generation dendritic polylysine (5, MeO-PEG5k-Lys-Lys2-((Fmoc)Lys(Boc))4) terminated with four Boc and Fmoc groups on one end of the PEG chain.</li>
	<li>Transfer the resulting polymer intermediate 5 to a reaction flask. In a separate reaction flask, prepare 1:1 (v/v) trifluoroacetic acid (TFA) in dichloromethane (DCM). Add 15-20 ml of a 1:1 TFA/DCM (v/v) mixture to the polymer intermediate 5. Stir the mixture until the polymer is completely dissolved. Stir for an additional 3 hr.</li>
	<li>Perform TLC16 and Kaiser&#39;s test (a blue color confirms the presence of free NH2)17 (see step 1.4) to confirm the completion of the reaction. If the reaction is complete, evaporate the polymer-in-TFA/DCM mixture with air until a viscous solution is obtained. Proceed to the ether precipitation, as mentioned for product 1 (steps 1.4-1.6). Dry the polymeric product 6 (MeO-PEG5k-Lys-Lys2-((Fmoc)Lys(NH2))4) under a vacuum.</li>
	<li>Transfer polymer intermediate 6 into a reaction flask. Use approximately 40 ml of anhydrous DMF containing 8 equiv. of N,N-diisopropylethylamine (DIEA) to dissolve the polymer intermediate 6. In a glass beaker, dissolve 12 equiv. of HOBt, 12 equiv. of DIC, and 12 equiv. of (Fmoc)Cys(Trt)-OH in 20-25 ml of anhydrous DMF. Shake for 10-15 min, and then add the reaction mixture to the reaction flask containing 6. Run the reaction overnight.</li>
	<li>Confirm the completion of reaction with TLC16 and Kaiser&#39;s test (see step 1.4; a yellow color indicates the absence of free NH2)17. If the reaction is complete, proceed to the ether precipitation, as mentioned for product 1 (step 1.4-1.6), to isolate product 7 (MeO-PEG5K-Lys-Lys2-((Fmoc)Lys-((Fmoc)Cys(Trt)))4.</li>
	<li>Perform Fmoc de-protection on 7, as outlined in step 1.8, to obtain product 8 (PEG5K-Lys-Lys2-(Lys(NH2)-(Cys(NH2)(Trt)))4). Couple (Fmoc)-PEG2-Suc-OH (&#34;Ebes&#34; linker, 24 equiv.) on a polymer intermediate using the procedure outlined above for HOBt/DIC-mediated coupling to obtain intermediate 9 (MeO-PEG5K-Lys-Lys2-Lys4-(Cys(Trt))4(Ebes(NH-Fmoc))8).</li>
	<li>Perform one more round of Fmoc de-protection (steps 1.6-1.8) to get intermediate 10 (MeO-PEG5K-Lys-Lys2-Lys4-(Cys(Trt))4(Ebes(NH2))8).</li>
	<li>Transfer the polymer intermediate 10 into a reaction flask and add anhydrous DMF (approximately 30-40 ml) to dissolve it. In another reaction flask, dissolve 24 equiv. of CAOSu (prepared according to the previously published procedure) in anhydrous DMF (20-30 ml)18. Add 48 equiv. of N,N-diisopropylethylamine and let it stir for 10-15 min. Transfer the contents into the reaction flask containing 10 and let the reaction run overnight.</li>
	<li>Confirm the completion of the reaction with TLC16 and Kaiser&#39;s test (see step 1.4; a yellow color indicates the absence of free NH2)17. If the reaction is complete, proceed to the ether precipitation, as mentioned for product 1 (steps 1.4-1.6) to isolate product 11 (MeO-PEG5K-Lys-Lys2-Lys4-(Cys(Trt))4-Ebes8-CA8). Dialyze it in de-ionized water and lyophilize the sample to yield a white powder.</li>
	<li>Place the polymer intermediate 11 into a reaction flask. Prepare and add 20 ml of the TFA/1,2-ethanedithiol (EDT)/triethylsilane (TIS)/H2O (94/2.5/1/2.5, v/v) mixture into the polymer solution. Stir the mixture until complete dissolution. Run the reaction for 4 hr. Confirm the completion of the reaction by TLC16.</li>
	<li>Under the fume hood, blow air into the polymer-TFA/EDT/TIS/H2O mixture until the solution becomes viscous. Proceed to the ether precipitation, as mentioned for product 1 (steps 1.4-1.6), to isolate the final product, 12 (PEG5K-Cys4-Ebes8-CA8). Dissolve it in acetonitrile and lyophilize it to yield a white powder.</li>
</ol>
2. Preparation of PTX-loaded Micelles
<ol>
	<li>Prepare a PTX-loaded PEG5K-Cys4-Ebes8-CA8 micelle using the standard evaporation method.
	<ol>
		<li>Dissolve 20 mg of TD with a different amount of PTX (1-9 mg) in 1 ml of chloroform (CHCl3). Remove the solvent using a rotary evaporator to obtain a homogeneous, dry polymer film. Reconstitute the film with 1 ml of phosphate-buffered saline (PBS) by vortex, followed by sonication for 30 min at 40 kHz, if necessary, to allow the formation of drug-loaded micelles.</li>
		<li>Add 6 &#181;l of 3% (w/w) H2O2 (1 equiv. to free the thiol groups) to oxidize the thiol groups on the TD. Use the micelle solution for further characterization once the level of free thiol groups remains at constant low values, as indicated by Ellman&#39;s test19.</li>
		<li>Filter the solution with a 0.22-&#181;m filter to sterilize the sample. Analyze the amount of drug loaded in the micelles on a HPLC20 system after releasing the drugs from the micelles by adding 9 times acetonitrile and performing 10 min of sonication. Use a C18 column for HPLC with acetonitrile/water as the mobile phase.</li>
		<li>Calculate the drug loading according to the calibration curve between the HPLC area values and the concentrations of the drug standard11. 
		NOTE: The loading efficiency is defined as the ratio of drug loaded into the micelles to the initial drug content.</li>
	</ol>
	</li>
</ol>
3. Characterizations of Micelles
<ol>
	<li>Measure the size and size distribution of the micelles with a dynamic light scattering (DLS) instrument29. Perform the measurements at room temperature and keep the micelle concentration at 1 mg/ml. 
	NOTE: To perform particle size analysis, use PBS as blank, and then record the particle size for actual samples. Take the readings in triplicate for samples, and then average the readings.</li>
	<li>Use fluorescence spectra to measure the critical micellar concentration (CMC) of PEG5K-Cys4-Ebes8-CA8 before and after cross-linking with pyrene as a hydrophobic fluorescent probe, as described previously13,21. 
	NOTE: Typically, the micelle concentration ranges from 5&#215;10-7 to 5&#215;10-4 M.</li>
</ol>
4. Stability of Micelles in SDS with or without Reducing Agents
<ol>
	<li>Prepare stock solutions of sodium dodecyl sulfate (SDS) solution (7.5 mg/ml) and disulfide cross-linked micelles (1.5 mg/ml) in PBS. Next, using the stock solutions, make a solution mixture in which the final SDS concentration is at 2.5 mg/ml and the micelle concentration is at 1.0 mg/ml.</li>
	<li>Measure the size and size distribution (as mentioned in step 3.1) of the micelle solutions at predetermined time intervals with or without the presence of 10 mM glutathione (GSH).</li>
</ol>
5. Hemolysis Assay
<ol>
	<li>Evaluate the hemolytic potential of PTX-loaded, non-cross-linked micelles (PTX-NCMs) prepared according to previously published procedure11 and PTX-loaded cross-linked micelles (PTX-DCMs) using fresh citrated blood from nude mice collected from the tail veil.
	<ol>
		<li>Collect red blood cells by centrifugation of blood sample (1.0 ml) at 1,000 x g for 10 min, wash them three times with PBS, and then re-suspend the cell pellets with PBS to a final concentration of 2%.</li>
	</ol>
	</li>
	<li>Mix 200 &#181;l of erythrocyte suspension with different concentrations (0.2 and 1.0 mg/ml) of PTX-NCMs and PTX-DCMs, respectively, and incubate for 4 hr at 37 &#176;C in an incubator shaker.</li>
	<li>Centrifuge the micelle-erythrocyte mixtures at 3,000 x g for 5 min. Transfer 100 &#181;l of the supernatant of all samples to a 96-well plate. Measure the absorbance of free hemoglobin in the supernatant at 540 nm using a micro-plate reader. 
	NOTE: RBCs incubated with Triton-100 (2%) and PBS are to be used as the positive and negative controls, respectively. The percent hemolysis of the RBCs is calculated with the following formula: RBC hemolysis = (ODsample - ODnegative control)/(ODpositive control - ODnegative control) &#215; 100%.</li>
</ol>

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Y. Li and K.S.L. are the inventors of a pending patent on reversibly crosslinked micelle systems (US patent application 61/485,774). K.S.L. is the founding scientist of LamnoTherapeutics Inc., which plans to develop the nanotherapeutics described in the manuscript. The remaining authors declare no competing financial interests.

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><tbody><tr><td>MeO-PEG&lt;sup&gt;5K&lt;/sup&gt;-NH&lt;sub&gt;2&lt;/sub&gt;</td><td>Rapp Polymere</td><td>125000-2</td><td></td></tr><tr><td>Fmoc-Lys(Fmoc)-OH</td><td>Aaptec</td><td>AFK107</td><td></td></tr><tr><td>Fmoc-Lys(Boc)-OH</td><td>Anaspec</td><td>AS-20132</td><td></td></tr><tr><td>Fmoc-Cys(Trt)-OH</td><td>Aapptec</td><td>AAC105</td><td></td></tr><tr><td>Dimethylformamide</td><td>Fisher Scientific</td><td>BP1160-4</td><td></td></tr><tr><td>Ethyl ether</td><td>Fisher Scientific</td><td>E134-20</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Diisopropylethylamine</td><td>Sigma Aldrich</td><td>D125806</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma Aldrich</td><td>T6508</td><td>Corrosive, handle with care</td></tr><tr><td>4-methyl piperidine</td><td>Alfa-Aesar</td><td>L-02709</td><td></td></tr><tr><td>Ebes linker</td><td>Anaspec</td><td>AS-61924</td><td></td></tr><tr><td>Cholic acid</td><td>Sigma Aldrich</td><td>C1129</td><td></td></tr><tr><td>1,2-Ethanedithiol</td><td>Sigma Aldrich</td><td>02390</td><td>Handle inside fume hood. Bleach gloves after usage.</td></tr><tr><td>Triisopropylsilane</td><td>Sigma Aldrich</td><td>233781</td><td></td></tr><tr><td>Chloroform (anhydrous)</td><td>Sigma Aldrich</td><td>288306</td><td></td></tr><tr><td>Hydrogen peroxide solution 30%</td><td>Aaron Industries</td><td>NA</td><td></td></tr><tr><td>HoBt-Cl</td><td>Aaptec</td><td>CXZ096</td><td></td></tr><tr><td>DIC</td><td>Sigma Aldrich</td><td>D125407</td><td></td></tr><tr><td>Female athymic nude mice (Nu/Nu strain), 6&amp;ndash;8 weeks age</td><td>Harlan (Livermore, CA)</td><td></td><td></td></tr></tbody></table>

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

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...

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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Research

JoVE Journal

Biochemistry

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

A simple, robust and scalable technique to functionalize and self-assemble macroscopic nanoparticle-ligand monolayer films onto template-free substrates is described in this protocol.

A Technique to Functionalize and Self-assemble Macroscopic Nanoparticle-ligand Monolayer Films onto Template-free Substrates

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Here, we present a one-pot, transition-metal-free synthesis of thiols and thioesters from aromatic halides and sodium thiomethoxide, followed by the preparation of single crystals of a metal-dithiolene network using thiol species generated in situ from the more stable and tractable thioester.

Synthesis of a Thiol Building Block for the Crystallization of a Semiconducting Gyroidal Metal-sulfur Framework

Disulfide linkages have long been known to stabilize the structure of many proteins. A simple method to analyze multimeric complexes stabilized by these linkages is through non-reducing SDS-PAGE analysis. Here, this method is illustrated by analyzing the nuclear isoform of dUTPase from the human bone osteosarcoma cell line U-2 OS.

Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

Learn to synthesize disulfide cross-linked micelles using hydrogen peroxide-mediated oxidation for targeted cancer drug delivery. This scalable method enables stable nano-formulations suitable for clinical studies.

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

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