The overall goal of this procedure is to form polyethylene glycol micro gels using a photo polymerized precipitation reaction to use as drug delivery vehicles and tissue engineering scaffolds. This is accomplished by first preparing the reagents, including a photo initiator, buffers, triethylamine, and PEG DL solution, and warming them to 37 degrees Celsius. The second step is to place the reagents into a microcenter fuge tube and vortex them while maintaining a temperature of 37 degrees Celsius.
Next sodium sulfate is mixed in and the solution is placed under UV light to cross-link for 30 seconds. Following cross-linking, a cloudy layer on top of the solution will form which contains the micro gels. The final step is to add PBS to the mixture and centrifuge to form a pellet.
Ultimately, microscopy and dynamic light scattering are used to measure the size of the micro gels and the density is measured using a dextrin density gradient. Additionally, the amount of swelling is calculated by measuring the wet and dry weights of the micro gels. The main advantage of this technique over the other methods such as emulsion, suspension and dispersion techniques, is that micro gels formed by precipitation reaction have a low poly dispersity index without the use of organic solvents or stabilizers, the mild conditions of the precipitation reaction, customizable properties of the micro gels and low viscosity for injections makes them suitable for NVivo applications.
This method can help answer key questions in the tissue engineering and regenerative medicine field, such as the fabrication of scaffolds with both uniform size and low poly dispersity index. The implications of this technique can extend towards drug delivery therapy because the growth factors in drugs can be easily encapsulated. To begin, weigh out 500 milligrams of the photo initiator and add it to 100 milliliters of deionized water to make a 0.5%solution.
Protect the solution from UV light and heat it to 40 degrees Celsius while staring constantly for approximately one hour to completely dissolve the photo initiator. After full dissolution solution, sterile filter the solution by passing it through a 0.22 micron filter and store it at four degrees Celsius in the dark until needed. Next, prewarm Triethylamine to 37 degrees Celsius to reduce its viscosity.
Then add 612 microliters of it along with 260 microliters of six molar hydrochloric acid to 39.4 milliliters of PBS and adjust the pH to 7.8 sterile. Filter the final solution through a 0.22 micron filter and store it at 37 degrees Celsius until it is needed. Following the work by Sony at all acro lte, the PEG precursors according to the published methods and store the finished product at minus 20 degrees Celsius under Argonne, then immediately before use, prepare one milliliter of a 200 milligram per milliliter Peg di Acrylate solution by slowly adding 0.8 milliliters of the PBS with added triethanolamine and hydrochloric acid to 200 milligrams of peg di acrylate.
Also, just before the experiment, weigh out 213 milligrams of sodium sulfate and dissolve it in one milliliter of deionized water. To prepare a 1.5 molar solution, heat the solution to 37 degrees Celsius for 20 minutes, and then vortex the solution until it is completely dissolved. Once dissolved, filter both the final sodium sulfate solution and PEG di Acrylate solution at 37 degrees Celsius through 0.22 micron filters.
Combine 127.5 microliters of PBS with triethylamine and hydrochloric acid, 10 microliters of the 0.5%photo initiator and 25 microliters of the 200 milligram per milliliter. Peg di acrylate solution into a 1.6 milliliter micro centrifuge tube. Then add any desired molecules for encapsulation.
Heat these tubes along with the 1.5 molar sodium sulfate solution to 37 degrees Celsius. Then add 87.5 microliters of the prewarm 1.5 molar sodium sulfate solution to the first tube of the PEG solution, and mix the tube by pipetting the solution three to five times. After mixing the salt concentration will be evenly distributed and the solution should become clear.
Place the tube under the UV light and cross-link for 30 seconds following cross-linking. There should be a cloudy layer on top of the solution containing the micro gels. Next, add 750 microliters of PBS to the tube and mix the solution well by vortexing.
Then centrifuge the mixture at 4, 000 times gravity for two minutes to form a pellet. Once pelleted, remove the SUP natin taken care not to disturb the pellet and suspend the micro gels in fresh PBS. Wash the micro gels a total of five times with fresh PBS, pelleting them in between each.
Following the final wash, remove the supernatant and pipette 30 microliters of micro gels from the pellet into one milliliter of deionized water. Then sonicate the micro gel solution in a water bath sonicate for one hour at 50 to 60 hertz and a power of 1.4 amps. Next pipette 15 microliters of the micro gel solution onto a clean acid etched glass slide and put a number 1.5 acid etched cover slip on top.
Flip the slide over onto a laboratory tissue and gently push down on the slide to remove excess solution and to ensure that the micro gels are in a single layer. Then using an optical microscope, capture several images of the micro gels using a DIC 100 x oil objective. Measure the micro gels to obtain a representative diameter.
Calculate the poly dispersity index using the volume based equation shown here in which N is the total number of micro gels and VI is the volume of the micro gel. In order to measure the average micro gel density first prepared dextrin solutions at 7, 6, 5, 4, 3, and 1%in deionized water, the densities of the dextrin solutions are shown here. Next, add two milliliters of the 7%dextrin solution to a 15 milliliter centrifuge tube.
Then slowly pipette two milliliters of the 6%dextrin solution to form a distinct layer atop the 7%dextrin. After five minutes, add two milliliters of the next layer consisting of 5%dextrin, and continue in this manner until each concentration of dextrin has been layered in order of decreasing dextrin concentration. Then carefully layer the micro gels above the 1%dextrin solution, centrifuge the gradient at five degrees Celsius in 4, 000 times gravity for 10 minutes.
Then visualize the location of the micro gels and calculate the density of the micro gels according to their position between the layers. To measure the swollen mass of the micro gels, first pre weigh a micro centrifuge tube and then add in one milliliter of the micro gels. Wash the micro gels five times in deionized water in between each wash.
Centrifuge the sample for 10 minutes at 4, 000 times gravity in order to pellet the micro gels after the last wash. Re suspend them in deionized water and allow them to swell for a minimum of 48 hours. After 48 hours, centrifuge the swollen micro gels for 10 minutes at 4, 000 times gravity and then remove all of the supernatant.
Then weigh the sample and record the wet mass. After an accurate wet weight is obtained, freeze the micro gels at minus 80 degrees Celsius, then ize them to remove the water and obtain the dry mass. Once all the water is sublimated, calculate the water and polymer content in the micro gels using the equations shown here where MS is the wet weight of the micro gels and MD is the dry weight.
The average diameter of PEG micro gels formed using various molecular weights of 20%PEG are shown here. Increasing the time of UV exposure also had an effect on the diameter of the micro gels. As UV exposure time increased, so did the diameters of the three formulations shown here.
The density as measured by dextrin gradients decreased with increasing molecular weight of the precursor peg molecules. This is also confirmed in the percent water content graph where higher molecular weight peg was found to have a higher amount of water per gram of polymer. Finally, in order to show the micro gels could be loaded with protein fluorescently conjugated, a albumin was added to the mixture prior to polymerization and became entrapped inside the spheres.
Increasing concentrations of the fluorescently labeled protein increased the poly dispersity index of the spheres, but the average size remained constant. While attempting this procedure, it's important to remember to heat all solutions to 37 degrees Celsius and vortex and micro gels well in order to break up any aggregates while washing and preparing the slides for imaging. Following this procedure, additional methods can be performed for drug encapsulation to answer additional questions regarding the effect of drug release.
After watching this video, you should have a good understanding of how to create peg DA micro gels under mild conditions via precipitation reactions.
