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

Our protocol offers a significant advancement in synthesizing sub 100-nanometer protein-loaded nanogels in a single step cross-linking and co-polymerization technique under mild aqueous-based reaction conditions. The main advantage of this technique is in its ability to synthesize protein-loaded nanogels under mild conditions, avoiding harsh organic solvents and high temperatures, thereby preserving the integrity of the biological payload. Begin by washing the glassware with filtered deionized water.

To dry the glassware, attach a secure nozzle to the compressed air outlet located on the side of the fume hood. Hold the vial firmly, position the nozzle inside and activate the compressed air to emit a controlled stream. Next, fill a 10-milliliter glass vial with deionized water and seal it with a rubber septum.

To deoxygenate the water, fill a balloon with nitrogen gas and attach a needle to the balloon. Place it on the septum and pierce the rubber septum to allow the nitrogen flow. Also, attach an exit needle to the flask to maintain a consistent nitrogen flow.

Next, dissolve BSA or Cy7-tagged BSA in deoxygenated deionized water. Then add this solution to a clean 10-milliliter glass vial. Add AETC solution to the flask and seal it with another rubber septum.

Dissolve acrylamide and deoxygenated deionized water and add this solution to the vial's mixture. Then dissolve the disulfide cross linker in deionized water and add it to the mixture in the vial. Flush the mixture with nitrogen gas for 20 minutes.

Afterward, dissolve SDS in deoxygenated deionized water. Using a nitrogen gas-flushed syringe, introduce it into the mixture. Next, add TEMED to the reaction mixture.

Let the mixture deoxygenate for three minutes under a continuous flow of nitrogen gas. Dissolve ammonium persulfate in one milliliter of deoxygenated deionized water and introduce it to the reaction mixture. Then seal the flask under a nitrogen atmosphere and stir the mixture for three hours and 30 minutes at room temperature.

To terminate the reaction, remove the seal from the flask. The reaction concludes once the mixture becomes clear and colorless. To purify nanogels, add the reaction mixture to a 15-milliliter centrifugal filter unit.

Fill the unit with deionized water and centrifuge to remove unreactive chemicals or unencapsulated payload. Add two milliliters of deionized water in the centrifugal unit to dilute the nanogel sample. Store the final sample at two to eight degrees Celsius to prevent degradation of the encapsulated protein.

Take a cuvette or a disposable folded capillary cell for analysis. Clean it with filter deionized water, followed by blowing compressed air to remove any large dust particles. Then fill the cuvette with 50 to 100 microliters of the sample, diluting it with 750 to 1, 000 microliters of filtered deionized water.

To insert the cuvette into the DLS instrument, open the sample compartment cover and hold the cuvette by its top edges to avoid leaving fingerprints to the optical windows. Place the cuvette properly aligned with the instrument's optical path. Then close the sample compartment to block ambient light.

Initiate the particle size measurement using the relevant software on the DLS machine. After the measurement, select the results relevant to the sample, including the Z average, average PI, count rate per minute, zeta potential and related graphics. Confirm that the correlation function displays a smooth sigmoidal curve indicative of a uniform sample.

Once the analysis is complete, carefully removed the cuvette from the DLS instrument. A gradual decrease in the intensity of the peak associated with the BSA protein was observed, suggesting encapsulation of the protein within the nanogel structure. The physical chemical characterization of the Cy7-tagged BSA-loaded nanogels showed a single defined peak at 50 to 100 nanometers.

However, empty nanogels demonstrated inconsistent and larger sizes. Up to 86%of encapsulated Cy7-tagged BSA was released within 48 hours. On the other hand, control experiments with no added glutathione only led to 15%protein released.

Fourier transform infrared analysis showed that BSA isolated during nanogel synthesis and post glutathione incubation retained its structure, indicated by the peak at 1, 662 centimeters inverse representing intact beta turns. However, BSA from nanogels stored for 30 days showed a peak at 1, 613 centimeters inverse, indicative of intermolecular beta sheet structures and protein aggregation. Circular dichroism showed unaltered spectra like native BSA, indicating preserved alpha helical structure.

However, minor aberrations in the characteristic peaks for BSA isolated after 30 days suggested some aggregation. Maintaining a continuous nitrogen flow is critical to prevent oxygen from inhibiting the free radical polymerization. Additionally, the initiating system components require swift addition for a successful synthesis.

After this procedure, nanogel-sized distribution, morphology and payload release kinetics can be further characterized through DLS, TEM and redox responsiveness assessment via incubation with the physiological reducing agent glutathione.