Protocol for preparation of Quantum dot loaded dissolvable microneedle patches

Protocol for preparation of Quantum dot loaded dissolvable microneedle patches

Preparation of negative PDMS molds from positive master-molds 

• Prepare PDMS mixture with PDMS: Crosslinker ratio of 9:1 and mix with a spatula until homogeneous.
• Degas this mixture by pouring it in 50ml Falcon tubes and centrifuging for 3 min at 300G.
• Meanwhile, attach the master-molds onto a small petri-dish with a double-sided tape making sure it is uniformly stuck to the base.
• Spray the petri-dish with mold-release spray from a distance (make sure there is just a fine one coat layer of mold release on the petri-dish)
• Pour the PDMS mixture onto the center of the petri-dish until it forms a flat layer on the master-mold covering all the needles and making sure not to entrap any air bubbles if possible. 
• Make sure you don’t pour PDMS too much. There should be about 1-2mm of PDMS over the needles.
• Place these petri-dishes under vacuum for about 40 mins to remove any air bubbles
• Transfer them further to an oven at 60°C overnight or at least 3hrs to cure
• Once cured, break the petri-dish around the cured PDMS mold and carefully detach master-mold from the negative PDMS mold. 
• Wash the PDMS negatives by sonicating in ultrapure water to ensure efficient filling during patch fabrication (decrease hydrophobicity introduced due to use of mold release spray)

Preparation of STERILE 30% PVA/PVP polymer solution (to make a total of 100ml of solution)

• Sterilize a glass bottle with a magnetic stirrer.
• Add 70 ml of sterile water to this bottle followed by 15g of PVA and 15g of PVP.
• Manually mix the solution by agitating the bottle and place it on a magnetic stirring plate at 60°C with appropriate stirring. For about 30 min.
• Put the solution in the sonicator for about 30 min.
• After this, put it back onto the plate at the same setting overnight to get a homogeneous polymer solution. 

Microneedle Patch Fabrication

• Lay a PDMS negative mold into each holder of a clean centrifuge (make sure to balance the centrifuge)
• Weigh Quantum dots (encapsulated in PMMA) in a tube and mix with water to make the desired concentration of QD solution.
• Sonicate and vortex this mixture alternately for about 15 minutes until a uniform solution is obtained without any undissolved QD particles
• Vortex the quantum dots to resuspend any settled particles before pipetting into the mold. 

(The amounts of QD solution and polymer to be used at different steps vary depending on the different microneedle patch geometries. Specific volumes to be used for different patches should be optimized and chosen for each geometry.)

• Pipette A μL of Quantum dots solution into the mold in the centrifuge and spread it evenly to cover the entire patch. Re-aspirate the QDs IF left for a while to resuspend any QDs settled on the patch before centrifuging.
• Centrifuge for 3 min at 4200rpm
• When the cycle is over, flip the centrifuge units 180 degrees to aid uniform patch filling. 
• Add B μL of QD solution to the mold, spread and resuspend the entire volume onto the negative thoroughly by carefully aspirating it repeatedly.
• Repeat the centrifugation cycle at the same settings again.
• After this cycle is over, carefully remove the molds from the centrifuge and examine the lengths of the needles filled with QDs. 
• If all the needles are uniformly filled, approximately up to ⅓^rd their entire length, wipe the mold with a dry VWR wipe to remove any water left. 
• If the needles are not appropriately filled (some needles have less than ⅓), add C μL of water and D μL quantum dots to the mold, make sure that the side of the patch with less QDs is facing outward in relation to the centrifuge and repeat the cycle.
• If none of the needles are filled appropriately, add E μL more of QDs and repeat the cycle. 
• When the needles are filled correctly and the mold is cleaned, slowly add F μL of 30% PVA/PVP to the mold and place it back into the centrifuge for another cycle of 3 minutes at 4200 rpm
• After the cycle is over, remove the molds from the centrifuge and check for any spilled polymer. If there is any spillage onto the mold, use a razor to scrape the polymer off of the edges of the negative pit of the mold so that the polymer doesn’t flow off in the subsequent steps. 
• Place the molds in the hood and add G μL of the polymer making sure it doesn’t flow out of the negative pit. Wait for about 7-10 mins until all the bubbles accumulate at the center of the polymer bulge. Aspirate these bubbles off to get a cleaner patch. Let these molds sit for about 30-45 min.
• After 30 minutes, add another H μL of the polymer and let them dry for around 4-5 days before removing the patches from the molds.