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 nanocarriers for drug delivery. Their unique core-shell structure &#39;&#39;solubilize&#39;&#39; the hydrophobic drug molecules under aqueous conditions by physical encapsulation. Polymeric micelles composed of well-defined TDs prepared in our lab form monodispersed nanoparticles (10-50 nm particle size) with a long shelf-life and high efficiency in encapsulating drugs. One of the many advantages of the robust TD platform is the versatility of the polymer. Numerous drugs, fluorescent dyes, and targeting ligands can easily be incorporated into the TD platform13,15,24.
Critical Steps within the Protocol
We have developed a robust, reversible, disulfide cross-linked micelle system for PTX delivery, which is well-defined, with a particle size around 27 nm, and has a narrow size distribution. The TDs were synthesized via stepwise solution phase peptide chemistry. The well-defined chemical structure of these TDs is the key factor for generating nanoparticles with the desired properties. Therefore, confirming the completion of the reaction with TLC and Kaiser&#39;s test is very critical. The H2O2 oxidization is also a critical step, the thiol groups on the TD are oxidized to form disulfide cross-links, which greatly enhance the stability of the micelles. Compared to our previous oxygen-based oxidation method, the H2O2-based oxidization method is 96 times faster, thus providing cross-linked micelles in shorter time periods. Furthermore, GSH, a thiol-containing tripeptide, is an important anti-oxidant produced by the cells, and it plays an important role in scavenging free radicals and reactive oxygen compounds. It makes major contributions by maintaining the cellular redox homeostasis. The cellular concentration of GSH is an interesting aspect of drug design. The intracellular concentration of GSH is now firmly established to be substantially higher than the extracellular concentration (10 mM versus 2 &#181;M, respectively)25. More importantly, an elevated intracellular GSH level has often been reported in many drug-resistant human and murine tumor cells26. The high intracellular distribution of GSH facilitates the breakage of the disulfide cross-linking of the micelles and results in the release and accumulation of the drug payload. The disulfide cross-linked nanoparticles are stable in the presence of SDS. However, a GSH-rich intracellular environment triggers the release of the drug by breaking down the disulfide bonds, thereby destabilizing the system. Polymer-induced hemolysis resulted from the detrimental interaction of polymeric micelles with blood constituents, such as red blood cells. Cross-linked micelles showed much less hemolysis compared to non-cross-linked analogues.
Modifications and Troubleshooting
Utilizing H2O2 as an oxidizing agent allows for a more efficient disulfide cross-link formation when compared to the previously reported, oxygen-based method13. H2O2 is a strong oxidizing agent27 that is inexpensive and highly soluble in water and gives high-quality products compared to some other known oxidizing agents (e.g., chromate or permanganate). However, the oxidization time may need to be further optimized based on the scale of the synthesis.
Limitations of the Technique
H2O2 is a strong oxidizer. Even though a low concentration of H2O2 is used for carrying out the disulfide cross-link formation, care has to be taken when using with TDs loaded with &#34;oxidation-sensitive&#34; drugs to prevent undesirable drug degradation.
Significance of the Technique with Respect to Existing/Alternative Methods
The technique described is an easy experimental setup that can be readily replicated in a standard laboratory. For instance, drug loading using a standard evaporation method is less time consuming compared to the dialysis method. The disulfide formation in the core using H2O2 as the oxidizing agent is 96 times faster than the previously reported oxidization method using oxygen13. Reproducibility of the drug-loading step can be easily assessed by measuring the particle size (DLS method) and drug content (HPLC).
Future Applications or Directions after Mastering this Technique
Compared to small-molecule drugs, one of the major obstacles that needs to be overcome before nano-medicine can enter mainstream cancer care settings is the technical challenges of scaling up the production28. After mastering this newly developed technique, one can easily synthesize disulfide cross-linked micelles on a large scale. This is extremely desirable for clinical studies of this nano-formulation in human patients.

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

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 5K) through a branched poly(lysine-cysteine-Ebes) backbone. There are several advantages of using these block copolymers over other reported micellar systems. First, it can be readily synthesized via stepwise solution phase condensation reactions. Second, compared to several other reported amphiphilic polymers, the thiolated polymer system, prepared through well-established solution phase stepwise Fmoc peptide chemistry, has a well-defined structure. It can be stored in lyophilized powder form and has an extended shelf life.
Several techniques can be utilized to characterize the end product. The amount of cholic acid attached to TD can be detected by comparing the signal ratio of the protons on PEG to those on the three methyl groups of cholic acid in the 1H NMR spectra. The molecular weights of the TD can be confirmed by MALDI-TOF mass spectrometry and gel permeation chromatography (GPC). The quantitative Ellman&#39;s test can be used to decipher the number of free cysteine residues present per molecule.
PEG5K-Cys4-Ebes8-CA8 can self-assemble to form micelles in aqueous media. Using a standard solvent evaporation method, a variety of hydrophobic drugs, such as PTX and vincristine, have been successfully encapsulated in the micelles13,22. Oxygen was previously utilized to oxidize the free thiol groups of PEG5K-Cys4-Ebes8-CA8 and to form intra-micellar disulfide cross-linkages. As monitored by Ellman&#39;s test, the conversion rate from free thiol groups to disulfide bonds reached 85% after 48 hr of oxidation. A H2O2-mediated oxidization was recently employed as an alternative method. The conversion rate reached 88% in 30 min (Figure 2), which was 96 times faster than the previous approach. Fifty grams of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced using this more efficient approach (Figure 3). The particle size was around 27 nm, with a narrow size distribution (Figure 3). The drug loading efficiency approached 100% with PTX loading of 4.0 mg/ml. Disulfide cross-linking had a dramatic effect on the CMC value. Compared to standard TD PEG5K-CA8 that lack disulfide bonds, the cross-linked TD showed a 10-fold decrease in the observed CMC values (5.53 &#181;M versus 0.67 &#181;M)13.
Stability Studies
We further investigated the stability of PTX-loaded, disulfide cross-linked micelles against severe micelle-disrupting conditions. Sodium dodecyl sulfate (SDS) is a strong ionic detergent that is routinely used for assessing micellar stability, as it can efficiently break down polymeric micelles. The stability of drug-loaded, disulfide cross-linked micelles (1.0 mg/ml) was monitored by DLS in the presence of the micelle-disrupting SDS (2.5 mg/ml). The particle size of the micelles remained steady over time, indicating that such cross-linked micelles remained intact (Figure 4, left panels). Reductive conditions are expected to cleave disulfide bonds in the hydrophobic core, thereby making micelles susceptible to destabilization. Glutathione (GSH), an endogenous reducing agent, is often employed for such studies. There is stark contrast in the intracellular level of GSH compared to the extracellular level (10 mm versus 2 &#181;m). This difference in concentrations is often used to generate stimuli-responsive systems. After the addition of GSH at an intracellular concentration (10 mm)23, the size of the drug-loaded, disulfide cross-linked micelles remained intact for 30 min. They abruptly decreased to 1 nm, signifying the reduction of a critical number of disulfide bonds, a prerequisite of the rapid dissociation of the micelles (Figure 4, right panel). On the contrary, the system remained stable in the presence of extracellular concentrations of GSH (data not shown).
Hemolysis Study
Figure 5 shows differences in the observed hemolytic activity of PTX-loaded micelles, with or without disulfide cross-linking. Hemolysis of blood cells must be avoided when administering these particles into the blood stream, as it undermines their therapeutic advantages. Hemato-compatibility is very crucial for the in vivo application of polymer-based drug carriers, such as the amphiphilic TDs that have the potential to solubilize lipids or to insert themselves into phospholipid membranes, leading to the rupturing of plasma membranes. As shown in Figure 5, the PTX-NCMs were found to have a dose-dependent red blood cell (RBC) lysis count, with the percentage of hemolysis increasing from 8.1% to 14.2% as the concentration of PTX-NCMs increased from 0.2 mg/ml to 1.0 mg/ml. However, PTX-DCMs that have disulfide cross-linking showed no observable hemolytic activities (&#60; 1.0%) in the RBCs under the same experimental conditions. This difference in the hemolysis trend can be attributed to the intra-micellar disulfide bridges present at the hydrophobic core, which prevent PTX-DCMs from dissociating to form amphiphilic TDs.
<img alt="Figure 1" src="/files/ftp_upload/54722/54722fig1.jpg" /> 
Figure 1: Steps for forming cross-linked micelles. Schematic representation of the disulfide cross-linked micelles formed by oxidization of the thiolated telodendrimer PEG5K-Cys4-Ebes8-CA8 after self-assembly. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54722/54722fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54722/54722fig2.jpg" /> 
Figure 2: Oxidation methods for cross-linked micelle formation. The conversion rate of the thiol groups on the TDs to disulfide bonds as a function of oxidation time for the two oxidation methods. The total concentration of the micelles was kept at 20 mg/ml. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54722/54722fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54722/54722fig3.jpg" /> 
Figure 3: Drug-loaded, cross-linked micelle. Left panel: a picture of a batch of PTX-loaded, disulfide cross-linked micelles. Scale bar: 1 inch. Right panel: the particle size, as determined by dynamic light scattering (DLS). The total concentration of micelles was kept at 20 mg/ml. The loading of PTX was 4 mg/ml. The particle size data was shown as the average particle size &#177; SD based on three measurements. SD: standard deviation, describes the width of the measured particle size distribution. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54722/54722fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54722/54722fig4.jpg" /> 
Figure 4: Stability study. The particle size of PTX-loaded, disulfide cross-linked micelles in the presence of 2.5 mg/ml SDS without (left) or with (right) GSH (10 mM), as measured by DLS. The particle size data was shown as the average particle size &#177; SD based on three measurements. SD: standard deviation, describes the width of the measured particle size distribution. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54722/54722fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54722/54722fig5.jpg" /> 
Figure 5: Hemolysis assay. The in vitro hemolytic activities of PTX-loaded, disulfide cross-linked micelles in comparison to non-crosslinked micelles on red blood cells (RBCs). Triton-100 (2%) and PBS were used as the positive and negative controls, respectively. The reported values are the mean &#177; SD of triplicate samples. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54722/54722fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.8K Views.  University of California, Davis. 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.

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

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

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.

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Chemistry

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

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (&#955; = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

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.

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

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

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

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

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.

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

Immunology and Infection

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

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-based structure control. This method involves first using electrospray to generate uniform ultrafine liquid droplets, each of which functions as a micro-reactor in which self-assembly reaction occurs forming micellar nanocrystals, with the structures (micelle shape and nanocrystal encapsulation) controlled by the organic solvent used. This method is largely continuous and produces high quality micellar nanocrystal products with an inexpensive structure control approach. By using a water-miscible organic solvent tetrahydrofuran (THF), worm-shaped micellar nanocrystals can be produced due to solvent-induced/facilitated micelle fusion. Compared with the common spherical micellar nanocrystals, worm-shaped micellar nanocrystals can offer minimized non-specific cellular uptake, thus enhancing biological targeting. By co-encapsulating multiple nanocrystals into each micelle, multifunctional or synergistic effects can be achieved. Current limitations of this fabrication method, which will be part of the future work, primarily include imperfect encapsulation in the micellar nanocrystal product and the incompletely continuous nature of the process.

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.

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of ...

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

The structures of many proteins are stabilized through covalent disulfide linkages. In recent work, this bond has also been classified as a post-translational modification. Thus, it is important to be able to study this modification in living cells. A simple method to analyze these cysteine-stabilized multimeric complexes is through a two-step method of non-reducing SDS-PAGE analysis and formaldehyde cross-linking. This two-step method is advantageous as the first step to uncovering multimeric complexes stabilized by disulfide linkages due to its technical ease and low cost of operation. Here, the human bone osteosarcoma cell line U-2 OS is used to illustrate this method by specifically analyzing the nuclear isoform of dUTPase.

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.

The structures of many proteins are stabilized through covalent disulfide linkages. In recent work, this bond has also been classified as a ...

Developmental Biology

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

Bioengineering

Synthesis of Stimuli-responsive Nanogels using Aqueous One-step Crosslinking and Co-nanopolymerization

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to their versatile bottom-up synthesis and biocompatibility. While various methods have been employed for nanogel synthesis to date, very few have achieved it without the use of harsh organic solvents or high temperatures that can damage the integrity of the biological payload. In contrast, the methodology presented here accomplishes the synthesis of sub-100 nm sized, protein-loaded nanogels using mild reaction conditions. Here, we present a method for the non-covalent encapsulation of protein-based payloads within nano-gels that were synthesized using an aqueous-based, single-step, crosslinking copolymerization technique. In this technique, we initially electrostatically bind a protein-based payload to a cationic quaternary ammonium monomer and simultaneously cross-link and co-polymerize it using ammonium persulfate and N,N,N&#39;,N&#39;-tetramethylethylenediamine to form nanogels that entrap the protein payload. The size and polydispersity index of the nanogels is determined using dynamic light scattering (DLS), while the surface morphology is assessed by transmission electron microscopy (TEM). The mass of protein entrapped within nanogels is determined by calculating the encapsulation efficiency. Furthermore, the controlled-release ability of the nanogels via the gradual degradation of redox-responsive structural elements is also assessed in bioreduction assays. We provide examples of nanoparticle optimization data to demonstrate all caveats of nanogel synthesis and characterization using this technique. In general, uniformly sized nanogels were obtained with an average size of 57 nm and a polydispersity index value of 0.093. A high encapsulation efficiency of 76% was achieved. Furthermore, the nanogels exhibited controlled release of up to 86% of the encapsulated protein by gradual degradation of novel redox-responsive components in the presence of glutathione over 48 h.

Nanogels are an excellent and versatile nanoparticle platform for the delivery of biologics. Stimuli-responsive poly(ethylene) glycol-based polymeric nano-gels, capable of encapsulating protein-based payloads, were synthesized using a one-step cross-linking co-nanopolymerization strategy in aqueous conditions. The optimal fabrication and characterization of these novel nanoparticles are presented here.

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

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.

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...

Preparation and Characterization of Targeted Microbubbles

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of microbubble preparation and targeting ligand attachment are cumbersome, complicated, and lengthy. Therefore, there is a need to simplify the targeted microbubble preparation procedure to bring it closer to clinical translation. The purpose of this publication is to provide a detailed description and explanation of the steps necessary for targeted microbubble preparation, functional characterization and testing. A sequence of the optimized and simplified procedures is presented for two systems: a biotin-streptavidin targeting pair model, and a cyclic RGD peptide targeting the recombinant &#945;v&#946;3 protein, which is overexpressed on the endothelial lining of the tumor neovasculature.
Here, we show the following: covalent coupling of the targeting ligand to a lipid anchor, assessment of the reagent quality, and tests that confirm the successful completion of the reaction; preparation of the aqueous precursor medium containing microbubble shell components, followed by microbubble preparation via amalgamation; assessment of the efficacy of lipid transfer onto the microbubble stabilizer shell; adjustment of microbubble size distribution by flotation at normal gravity to remove larger microbubbles that might be detrimental for in vivo use; assessment of microbubble size distribution by electrozone sensing; evaluation of targeted binding of the microbubbles to receptor-coated surface in a static binding assay test (in an inverted dish); and evaluation of targeted binding of the microbubbles to receptor-coated surface in a parallel plate flow chamber test.

The goal of this protocol is to prepare, purify and characterize gas-filled microbubbles (targeted contrast agents for ultrasound molecular imaging). Two targeting systems are described: biotinylated bubbles adherent to streptavidin, and cyclic RGD peptide microbubbles that bind to &#945;v&#946;3, a known tumor neovasculature biomarker.

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of ...

Developing Sodium Alginate Hydrogel Microspheres for Biomedical Use

Preparation of Cross-Linked Sodium Alginate Microspheres with Different Metal Ions Using the Microfluidic Electrospray Technology

Microspheres are micrometer-sized particles that can load and gradually release drugs via physical encapsulation or adsorption onto the surface and within polymers. In the field of biomedicine, hydrogel microspheres have been extensively studied for their application as drug carriers owing to their ability to reduce the frequency of drug administration, minimize side effects, and improve patient compliance. Sodium alginate (ALG) is a naturally occurring linear polysaccharide with three backbone glycosidic linkages. There are two auxiliary hydroxyl groups present in each of the moieties of the polymer, which have the characteristics of an alcohol hydroxyl moiety. The synthetic ALG units can undergo chemical cross-linking reactions with metal ions, forming a cross-linked network structure of polymer stacks, ultimately forming a hydrogel. Hydrogel microspheres can be prepared using a simple process involving the ionic cross-linking properties of ALG. In this study, we prepared ALG-based hydrogel microspheres (ALGMS) using a microfluidic electrodeposition strategy. The prepared hydrogel microspheres were uniformly sized and well-dispersed, owing to accurate control of the microfluidic electrospray flow. ALGMS cross-linked with different metal ions were prepared using a microfluidic electrospray technique combining microfluidic and high electric field, and its antimicrobial properties, slow drug release ability, and biocompatibility were investigated. This technology holds promise for application in advanced drug development and production.

Author Spotlight: Advancing Therapeutics with Biocompatible Sodium Alginate Hydrogel Microspheres

The protocol describes the preparation of sodium alginate microspheres cross-linked with different metal ions using a microfluidic device for drug carrier design. The antimicrobial properties and slow drug release of these microspheres were also investigated.

Microspheres are micrometer-sized particles that can load and gradually release drugs via physical encapsulation or adsorption onto the surface and within ...

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

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