The freezing-thawing method is used to produce chitosan-poly(vinyl alcohol) hydrogels without crosslinking agents. For this method, it is important to consider the freezing conditions (temperature, number of cycles) and polymer ratio, which can affect the properties and applications of the obtained hydrogels.
This protocol is significant because our freeze-thawing method is a suitable process to prepare biocompatible hydrogels for use in biomedical, pharmaceutical, or cosmetical applications. The main advantage of this method is that it doesn't use chemical crosslinking agents which can cause a birth defect. Also, the freezing condition utilized in this method control the final property of hydrogels such as degrees.
This method can be applied to hydrogels with other applications. This does use to treat water pollution. Indeed, it could be used to produce polymer beads used as a water container for a new culture.
It's important to completely homogenize the polymer solution it measures. Otherwise, the hydrogels will present cracking points. Also, you must be careful with the freezing times in each cycle.
To begin this procedure dissolve 0.2 grams of chitosan and 10 milliliters of 0.1 molar acetic acid at room temperature and maintain continuous mechanical stirring overnight to prepare a 2%chitosan solution. The next day, dissolve one gram of PVA and 10 milliliters of distilled water and stir at 80 degrees Celsius for one hour. Use a magnetic stirrer to mix an equal amount of both solutions at room temperature until they are homogenous.
Pour the mixtures on Petri dishes. Leave the samples at atmospheric pressure for two hours to degas. Freeze the hydrogels at minus four degrees Celsius, minus 20 degrees Celsius, or minus 80 degrees Celsius for 20 hours and four cycles.
Freeze another hydrogel at minus 80 degrees Celsius for 20 hours using either five or six freezing cycles. After the third freezing cycle, wash the hydrogels with deionized water. At the end of the last freezing cycle freeze dry the hydrogels at minus 50 degrees Celsius for 48 hours and stir for further characterization.
When ready to perform FT-IR characterization place a small piece of hydrogel in the FT-IR spectrophotometer in ATR mode. Take the FT-IR spectro from 4000 to 600 wavelengths. First, cut out discs from the hydrogel that are 13 millimeters in diameter and 10 millimeters in height.
Weigh them. Incubate the discs in 50 milliliters of deionized water at 25 degrees Celsius while shaking. Every 30 minutes remove the sample from the medium.
Blot the sample to eliminate any excess water and weigh it. Then calculate the swelling degree and perform electronic microscopy and pore asymmetry as outlined in the text protocol. Before loading, prepare four liters of Diflunisal solution at 15 milligrams per liter and stir overnight.
Confirm the concentration of the solution by UV-Vis Spectroscopy. Then swell 400 milligrams of freeze dried hydrogel samples and six milliliters of distilled water for 24 hours. For loading, fill a flask with 50 milliliters of Diflunisal solution and maintain at 25 degrees Celsius with constant stirring.
Submerge each swelled hydrogel in the flask. Next, take 2 milliliter aliquots of the remaining Diflunisal solution at different times in order to determine the plateau region of the curve. After 24 hours, replace the solution with a fresh one.
Measure the absorbance of each aliquot at 252 nanometers and determine the concentration of Diflunisal present in the solution using the calibration curve of Diflunisal. Determine the amount of Diflunisal retained in the hydrogel and the encapsulation efficiency as outlined in the text protocol. Then freeze the loaded hydrogels at minus 80 degrees Celsius and lyophilize them at minus 50 degrees Celsius.
For drug release, submerge 300 milligrams of freeze dried Diflunisal-loaded hydrogels and 50 milliliters of phosphate buffer at pH 7.4 and at 25 degrees Celsius. Maintain constant stirring. Withdraw two milliliter aliquots at different times and replace with fresh medium to keep a constant volume.
Determine the Diflunisal released spectrophotometrically at 252 nanometers according to a calibration curve. After this, deduce the predominant drug release mechanism in the hydrogels as outlined in the text protocol. Here CSPVA hydrogels are prepared without crosslinking agents using a freeze-thawing method.
The FT-IR spectro shows seven characteristic signals of both polymers. All CSPVA hydrogels show a highly porous surface and distinctive changes are observed according to the preparation conditions. Hydrogels prepared at minus four degrees Celsius present the largest pores.
The hydrogels prepared at minus 80 degrees Celsius appear to have a more porous network and the number of freezing cycles seems to promote more defined and circular pores. However, hydrogels prepared with six freezing cycles at minus 80 degrees Celsius show less permeability than those prepared with four freezing cycles at minus 80 degrees Celsius probably due to their high tortuosity, which was reflected in the lower total intrusion volume. In swelling behavior hydrogels quickly absorb large amounts of water, retaining ten times their weight for the first five hours and up to 15 times their weight after 20 hours.
When observing the effect of temperature it is seen that the hydrogel prepared with four freezing cycles at minus 80 degrees Celsius shows less swelling capacity in the first five hours. The number of freezing cycles are not seen to create any differences at any time. The releasing kinetics of Diflunisal from hydrogels is maintained for about 30 hours in all cases with the hydrogel prepared with four freezing cycles at minus 80 degrees Celsius, releasing the highest amount.
There is no difference in the release between the hydrogel prepared with four freezing cycles at minus 80 degrees Celsius and the hydrogel prepared with six freezing cycles at minus 80 degrees Celsius. The most important thing to remember when attempting this procedure are the freezing conditions. You must start from almost three freezing cycles in order to obtain a completed, formed hydrogels.
This method could be combined with cell culture to to evaluate in-vitro biocompatibility. It could also be combined with degradation studies. After the development of these procedures we realized that we could explore the possibility of incorporating other polymers to the mixture or even natural compounds such as aloe vera.
Doing so would allow us to incorporate new properties into these materials.
