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.
