Our research is centered around our intestinal expansion sleeves and vaginal expansion sleeves, which both provide a distracted force that has been shown to significantly lengthen the intestinal and vaginal canals. We are hoping to optimize our devices with the addition of protein-based therapeutics to help minimize the local inflammation and fibrosis following the deployment of these devices. By binding these therapeutic agents directly to our devices, we can bypass the need for systemic administration directly targeting our areas of interest with a localized dose.
This localization will allow for lower drug dosing, decreasing the risk of systemic side effects, and also lowering the cost of treatments. In the future, we want to determine the release rate of these protein-based therapeutics that are bound to our PVA-coated devices, which will be necessary for determining drug dosages for different types of treatments. To begin, prepare the 5%polyvinyl alcohol or PVA solution by adding five grams of PVA into 100 milliliters of deionized water.
Stir the mixture on a hot plate to aid in dissolution. Next, prepare the one milligram per milliliter of glucagon-like peptide 2 or GLP-2 solution by adding one milligram of GLP-2 into one milliliter of deionized water. Pre-contract the intestinal expansion sleeves and vaginal expansion sleeves over a glass pipette, stabilizing the ends with cut plastic pipettes fitted tightly around the glass pipette and sleeves.
Paint the pre contracted sleeves with the 5%PVA solution using a small paintbrush. Allow the sleeves to dry at room temperature for 10 minutes between each coat. Once the fifth coat is dry, place the pre contracted sleeves into a desiccate under a chemical safety hood.
Add three milliliters of 25%glutaraldehyde and three milliliters of 94 to 98%sulfuric acid into separate open beakers in the desiccator to allow vapor reaction and cross-link the PVA membranes onto the sleeves. After cross-linking for 48 hours, remove the sleeves from the chamber and allow the glutaraldehyde to evaporate for 15 minutes. Pipette 50 microliters of GLP-2 solution onto each cross-linked sleeve for later use.
To make the standard curve of GLP-2 bound to the PVA, pipette 50 microliters of 5%PVA onto the bottom of 18 wells of a 24 well plate. Leave six wells blank to hold the intestinal expansion sleeves and vaginal expansion sleeves with three wells for each type of device. Allow the PVA layer to dry overnight in an oven at 120 degrees Celsius.
The next day, place the 24 well plate into a desiccate under a chemical safety hood. Add three milliliters of 25%glutaraldehyde and three milliliters of 94 to 98%sulfuric acid into separate open beakers inside the desiccator to allow vapor reaction and cross-link the PVA membranes onto the 24 well plate. After 48 hours of cross-linking, remove the 24 well plate from the desiccator and allow the glutaraldehyde to evaporate for 15 minutes.
Next, create a concentration gradient by adding 250, 150, 25 and zero microliters of one milligram per milliliter GLP-2 solution into their respective labeled wells. Then, place the drug coated intestinal and vaginal expansion sleeves into their respective wells. After adding the respective GLP-2 concentration and drug coated intestinal expansion sleeves and vaginal expansion sleeves to the wells, refrigerate the 24 well plate overnight at four degrees Celsius to allow for drug binding.
The next day, add two milliliters of one milligram per milliliter of milk powder in a deionized water blocking solution to each well for one hour. After blocking, wash each well three times with PBS for five minutes per wash. Prepare the rabbit anti GLP-2 antibody solution by adding 40 microliters of rabbit anti GLP-2 antibody into 50 milliliters of deionized water.
Then, add two milliliters of the antibody solution to each well of the plate. Refrigerate the plate overnight at four degrees Celsius. The next day, wash each well three times with PBS for five minutes per wash.
Prepare the anti rabbit IgG alkaline phosphatase secondary antibody solution by adding 20 milliliters of antibody into 40 milliliters of deionized water. Add two milliliters of the secondary antibody solution to each well and incubate at room temperature for two hours. After washing the wells with PBS, add two milliliters of the alkaline phosphatase blue micro well substrate to each well and incubate for 20 minutes.
During incubation, monitor the reaction as the solution changes color from yellow to blue. Stop the reaction by pipetting 200 microliters of the solution from each well into a labeled 96 well plate. Read the 96 well plate using an absorbance microplate reader at a wavelength of 620 nanometers.
Generate the standard curve and calculate the concentration of GLP-2 on the PVA-coated sleeves. The GLP-2 concentration of the intestinal expansion sleeves and vaginal expansion sleeves devices average 22.69 micrograms per square centimeter similar to the concentration in the 50 microgram wells on the 24 well plate with 23.7 micrograms per square centimeter.
