Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

The overall goal of this procedure is to functionalize polyethylene glycol and peptides with meth acrylate groups for subsequent chain polymerizations and hydrogel synthesis. This is accomplished by first combining meth acrylic anhydride and the UN functionalized precursor in a scintillation vial. The second step is to use microwave energy to functionalize the peg or peptide.

Next, the PEG DM is dissolved in chloro methane, or the peptide is cleaved from the resin. And all side chain protecting groups are removed in an acid-based cocktail. The final step is to precipitate the PEG DM or meth acrylamide functionalized peptide in ether.

Ultimately proton NMR or Maldi time of flight mass spectrometry is used to show successful functionalization of the peg or peptide macro. Hi, my name is Amy Van Hove. I'm a PhD student in the department of Biomedical Engineering at the University of Rochester.

The main advantage of this technique over existing methods like reacting PEG with meth acrylic fluoride is that the reaction is significantly faster and more environmentally friendly. Hi, my name is Brandon Wilson. I'm an undergraduate in chemical engineering at the University of Rochester.

This method can help answer key questions in the biomaterials field, such as how hydrogels produce using peg dimethyl can be employed to control the delivery of therapeutic cells in molecules. Hi, I am Danielle Benoit, an assistant professor in the Department of Biomedical Engineering at the University of Rochester. The implications of this technique are for developing therapies for a variety of diseases as the synthesized compounds can be used for cell and drug delivery applications.

Prior to starting this procedure, pred dried the required glassware in an oven for one hour to prevent contamination with water in a large whey boat. Way out five grams of peg with a molecular weight between 1000 and 100, 000 daltons. If present, remove the plastic piece from the lid of a scintillation vial in the chemical hood.

Dispense 10 molar excess of meth acrylic anhydride into a teared scintillation vial. Then add PEG to the vial. Next loosely, twist the cap onto the vial and place the vial in the microwave.

Set the microwave to five minutes on maximum power wearing heat resistant gloves. Remove the vial from the microwave every 30 seconds. Then fully tighten the cap and vortex the sample for 30 seconds.

Loosen the cap before returning the vial to the microwave. Repeat these steps until the solution has been microwaved for the full five minutes. After removing the vial from the microwave, loosen the cap and allow the PEG DM to cool to room temperature.

Then dissolve the PEG DM in 10 to 15 milliliters of di chloro methane. Precipitate the PEG DM in 10 x excess of pre dathyl ether for 20 minutes. Using a seven centimeter Buckner funnel and a 500 milliliter flask.

Collect the PEG DM by vacuum filtration. Next, transfer the filtered PEG DM to a 50 milliliter conical tube with a large gauge needle pierced through the cap for ventilation. Place the tube in a vacuum chamber and dry the PEG DM overnight.

After removing the tube from the vacuum chamber, red dissolve the PEG DM in DI chloro methane precipitate with ice cold dathyl ether. To remove unreactive meth acrylic anhydride following filtration and collection, dry the PEG DM in a vacuum chamber in the same manner as before. For proton NMR analysis, place approximately 10 milligrams of PEG DM in a scintillation vial and add approximately one milliliter of deuterated chloroform.

Once the sample is dissolved, transfer it to a clean NMR tube. Next, collect the proton NMR spectra and run the samples at room temperature for at least 64 scans to obtain sufficient data resolution. After synthesizing the peptide of interest by solid phase synthesis, store it on the resin in DMF at four degrees Celsius until ready for use.

Then collect the peptide resin via filtration using a seven centimeter buckner funnel with filter paper and a 250 milliliter flask. Remove the plastic piece from the lid of a scintillation vial and transfer the resin to the vial. Using a disposable pipette add just enough meth acrylic anhydride to cover the resin After loosely twisting the cap onto the vial, place it in the microwave and set the timer to three minutes.

On maximum power wearing heat resistant gloves, remove the vial from the microwave every 15 to 20 seconds. After fully tightening the cap, vortex the sample for 15 seconds. Loosen the cap before returning the vial to the microwave.

Repeat these until the solution has been microwaved for the full three minutes. With the vial cap loosened, allow the peptide solution to cool to room temperature using a small amount of DMF and a seven centimeter buckner funnel. With filter paper and a 250 milliliter flask.

Collect the peptide resin from the vial for cleavage and deep protection. Transfer the peptide resin to a fresh scintillation vial for 0.25 millimoles of arginine containing peptide. Add 0.25 milliliters each of tri ISO propylene three six dioxide one eight octane diol thiol deionized water, and 18.0 milliliters of TFA.

Then rotate the reaction mixture on a lab quake rotator for four hours at room temperature. When finished, precipitate the peptide in 10 x excess of pre chill ethyl ether. Divide the solution evenly amongst four 50 milliliter conical tubes, centrifuge the four samples at 3, 200 G for 10 minutes to collect the peptide following centrifugation, decant off the ether resuspend the peptide in 100 milliliters of fresh ethyl ether divided between two 50 milliliter conical tubes.

Repeat the centrifugation process. Resus suspending the peptide in 50 milliliters of fresh ether twice for a total of four ether washes after the last centrifugation step decant the waste ether and dry the peptide overnight under vacuum. To prepare the sample for Maldi time of flight analysis, place one to two milligrams of the peptide in a 1.5 milliliter einor tube and dissolve the sample in one milliliter of maldi solvent.

Following this, prepare a 10 milligram per milliliter matrix solution of Alpha Sano four hydroxy ceramic acid in the multis solvent. Combine the peptide and matrix solutions in a one-to-one ratio spot one microliter of the combined solution onto three separate locations on the MALDI sample plate. Dry the spots using a heat gun.

Then re-spot and dry each sample. Finally, collect the MALDI time of flight data and confirm a 68 gram per mole increase in molecular weight due to the addition of the meth acrylamide group to the end terminus of the peptide. Shown here is the NMR spectrum of a two kilodalton linear PEG DM functionalized using the microwave assisted method for this reaction.

Proton NMR analysis can be used to calculate the percent functionalization from the observed to theoretical ratio of the terminal methacrylate protons to the central PEG protons. The overall percent functionalization is 91%and this PEG DM is adequately functionalized for use in hydrogel synthesis. See the text protocol for details on calculating the percent functionalization due to the multitude of proton peaks that arise in proton NMR analysis of pep peptides.

Peptide functionalization is more easily investigated using maldi time of flight mass spectrometry. This is demonstrated here where the peptide G-K-R-G-D-S-G was synthesized and subjected to meth acrylamide functionalization, a small fraction of the peptide was cleaved for pre functionalization molecular weight assessment, which showed the observed molecular weight peak occurring at 676 grams per mole, the expected molecular weight of the peptide, the remainder of the peptide underwent meth acrylamide functionalization prior to cleavage after meth acrylamide functionalization. The observed molecular weight peak occurs at 744 grams per mole, the expected weight of the meth acrylamide functionalized peptide.

To demonstrate the functionality of both the PEG DM and the meth acrylamide functionalized peptides PEG hydrogels were produced with and without 0.5 millimolar meth. Acrylamide functionalized GK R-G-D-S-G shown here are representative phase contrast and live dead fluorescent images of MSCs on peg gels alone and on peg gels containing the meth functionalized peptide. The MSCs were unable to adhere to the UN functionalized peg hydrogels, but upon inclusion of the cell adhesion peptide RGD, they were able to adhere to and spread on the hydrogel surface.

The live dead images do not represent the viability of the seeded MSC population. Rather, the fluorescent images are intended to demarcate between adherence cells and small variations in the hydrogel topology, which can be difficult Under phase contrast alone. Interestingly, the non spread cells seeded on the PEG only gels stain positive for both calcium AM and AUM Homodimer indicating that cells were dying at the time of imaging.

One of the many advantages to PEG hydrogels is their highly tunable nature modifying the specific makeup of the peg. Hydrogel affords researchers a high degree of control over properties such as modulus of elasticity as illustrated here. Both PEG molecular weight and weight percentage are known to control hydrogel mesh size, resultant hydrogel, stiffness, and release rate of encapsulated drugs.

This was demonstrated by producing hydrogels with varying molecular weight PEG dm and investigating the resultant hydrogel modulus and mesh size as hypothesized increasing the molecular weight of the peg. Macer caused an increase in hydrogel mesh size and a decrease in hydrogel stiffness, hydrogel mesh size and the resulting gel stiffness can also be controlled by altering the weight. Percentage of PEG hydrogels were produced with varying weight.

Percent of linear 10 kilodalton PEG DM and hydrogel stiffness and mesh size were determined as illustrated here, increasing the weight percent of PEG causes a significant decrease in mesh size and an increase in hydrogel stiffness. Hydrogels containing encapsulated bovine serum albumin were formed using varying molecular weight peg as shown here. Bovine serum albumin release occurs more rapidly from hydrogels formed using higher molecular weight PEG DM as a result of the larger mesh size within the hydrogel.

After watching this video, you should have a good understanding of how to synthesize PEG DME accolade using the microwave assisted method and how to assess functionalization using proton NMR. Once mastering this procedure, one could also utilize ring opening of hydrolytic degradable groups in order to answer additional questions about how hydrogel degradation affects cell function. You should also have a good understanding of how this technique can be used to selectively incorporate meth acrylamide functionalities onto the end termini peptide sequences and how to assess this functionalization using multi time of flight mass spectrometry.

Thanks for watching.