Hi everyone!
This past week, I started another release study using the same polymers I have used before mixed with a drug and salt/acid solution. I intended to begin the study on Monday, but something unexpected happen: when I mixed the polymer and drug phases together, the following mixture turned white! (they're usually clear after mixing). This occured for each of my samples (acid mixtues, salt mixtures, and neither). This is problematic because this means one of two things: 1) the mixture became a gel or 2) there was simply a very high concentration of salt or drug that could not be dissolved. 1) is problematic because it prevents the gel from being placed into vials where the release studies take place. 2) is problematic because that means that maybe not all the drug or salt can dissolve with that much gel.
To test which of these hypotheses are true, I simply placed the mixtures back into the refrigerator. After a couple of hours, the mixtures became clear again. Thus, 1) was shown true as a decrease in temperature caused the mixture to transition back to liquid. After the mixtures became completely clear, I began the release study and took time points throughout the week.
One possibility for this is the high concentration of the drug (I doubled the drug concentration from previous release studies). The drug may react with the polymer in some way that causes the polymer to become a gel. However, this is just speculation. Furthermore, in the past, this has happened in several cases with different molecules, and cooling the "gel-ed" mixture does not affect results. However, it does affect how I plan release studies for the future as I may need an extra day, or I may need to stay very late some days (it can take around 4-6 hours for the gel to transition back).
With that extra time though, I can analyze how much drug was released by using the plate reader. Furthermore, I will help out another project in the lab.
This project I am helping on concerns the degradation time of the polymer. The degradation time is the time it takes for the polymer to break down to a certain point. This is measured by seeing the temperature at which the gel transitions from liquid to solid (that temperature is called the Lower Critical Solution Temperature or LCST). The higher the LCST, the more the polymer has degraded (this is true because of the properties of the molecules that compose of the polymer).
To find the LCST of a gel, I have to run a cloud point test. This test is actually the first test I learned when I went to lab over the summer. Simply, I heat a water bath to a certain temperature. Place the gels in the water bath at that temperature for a minute. Then dry of the container and place it in a spectrophotometer. This time, I measure the absorbance for the color white. Thus, if the gel is clear, the spectrophotometer will read 0. At multiple temperatures, I measure the absorbance. With this data, I can approximately determine where the LCST is.
Hopefully this week I can attain lots of data. If I do, I will share them with you next week.
See you then!
Russell
Sunday, March 27, 2016
Friday, March 18, 2016
Second Try
Hi everyone,
This week, I began another release study. As mentioned in my last post, the goal of this study was to see the differences in drug release between different masses of gels (related to the surface area to volume ratio as the gels are placed into a vial) and to double check my technique as my results from
my last test looked wonky.
Last Monday (the week during Spring Break), I actually went to lab and prepared for this release study, so that I could start it the following week. Because there are time points one day and two days after the start of the study, my mentor (who takes time points for me when I am not there), I prefer to start release studies on Mondays and so I need to prefer much beforehand.
So, this Monday, with preparation finished, I was able to start the release study quickly and take a couple of time points before I left. Later the night and Tuesday, my mentor took more time points. On Wednesday, I took another time point and ran a test to see amount of drug released so far from the gels.
Here is a graph of the cumulative release over these four days (Sorry for the poor quality, I had to take a screenshot since I don't have paint on this laptop and I cannot find how to copy and paste from Excel or attach files on Blogger):
This week, I began another release study. As mentioned in my last post, the goal of this study was to see the differences in drug release between different masses of gels (related to the surface area to volume ratio as the gels are placed into a vial) and to double check my technique as my results from
my last test looked wonky.
Last Monday (the week during Spring Break), I actually went to lab and prepared for this release study, so that I could start it the following week. Because there are time points one day and two days after the start of the study, my mentor (who takes time points for me when I am not there), I prefer to start release studies on Mondays and so I need to prefer much beforehand.
So, this Monday, with preparation finished, I was able to start the release study quickly and take a couple of time points before I left. Later the night and Tuesday, my mentor took more time points. On Wednesday, I took another time point and ran a test to see amount of drug released so far from the gels.
Here is a graph of the cumulative release over these four days (Sorry for the poor quality, I had to take a screenshot since I don't have paint on this laptop and I cannot find how to copy and paste from Excel or attach files on Blogger):
If you cannot see it well, here is a summary: the top three lines are from the gels without the salt with the topmost line being the gel with the lowest mass. The bottom three lines are from the gels with the salt with the highest of the those three (the purple one) being the lowest mass as well.
Overall, I am mostly satisfied with this data/graph because the difference between the release profiles of the gels with and without the salt are as large as expected (my mentor performed a similar procedure previously without the various masses). Furthermore, that difference is mostly constant with the different masses in the gel. This is especially seen with the lowest mass curves as they are both significantly higher than the other two masses (I used 200 mg, 300 mg, and 400 mg).
However, one reason for doubt is the flatness of the curve after two days. As the curve becomes more flat, the more likely it is that the release is ending. In other words, the slope of the line between each time point is always decreasing. Thus, do the bottom three lines, or any really, reach 100% release? If not, I may have made a mistake somewhere. To check this, I plan to take a time point next Monday (7 days after the study has started and usually around the time when release ends) and see the release.
Nevertheless, the positives outweigh the negatives so far in this case, and so the following week, I want to start a new release study, going back to the first release study I did in which I tested various acids and salts and their effects on release. Hopefully, this time my results make more sense and say something more meaningful.
See you next week!
Russell Llave
Friday, March 11, 2016
Spring Break
Hi everyone,
I'm on spring break this week, so I will not be posting.
See you next week!
Russell
I'm on spring break this week, so I will not be posting.
See you next week!
Russell
Friday, March 4, 2016
First Results
This week, I processed the data from my previous release study, and after seeing the results, I planned for how to go forward with my project.
As mentioned in my previous post, the way I measure the amount of drug released from the gel, I take aliquots of the solvent in the vial into tubes at different times. Each time I place some solvent in a tube, I replace the solvent in the vial. After a certain time, I place a small sample of each tube onto a plate reader. The plate reader is then placed into a spectrometer which measures the absorbance of the sample of a particular wavelength of light. The results from the spectrometer transferred to an excel document look like this
The bottom line of the results does not have samples from the release study. Rather, they are standards I created. Standards are samples in which the concentration of a particular drug is known (because I made them). They are necessary because they show the relationship between absorbance and concentration. When I plotted the absorbance of the standards against their calculated concentrations, I created the following graph
Firstly, as shown by the high R2 value, a linear approximation is appropriate, and so the equation listed in the graph is used to calculate the concentration of each of the samples from the absorbance in the plate reader. After a series of unit conversions, I was able to calculate the mass released from each gel at each time point. Furthermore, a calculated the cumulative release of each gel. To calculate this, I add the mass of a particular time point with the masses of all the time points before it. This is done because the solvent is replaced at each time point. Then, I calculated the theoretical amount of drug within each gel. This can be calculated because the percentage of drug in the gel is known by how I created the gels.
After all of these calculations, I created two graphs. The first graph shows the cumulative amount of drug divided by the total amount of drug for each gel at each time point. The second graph shows the mass at each time point divided by the cumulative release of that gel. “Average” is used because for each type of gel, there were three samples for repetition. In the graph, rather than having twenty-seven different lines, I averaged each sample of each gel. “Control” is a gel without a salt or acid.
One trend shown in these graphs is that a higher concentration of a specific salt/acid has a faster release than the lower concentration of the same salt/acid. Furthermore, the salt/acid mostly had slower releases then the control group. However, my data suggests that the gels were not mixed very well. When I mixed the gels, I connected two syringes and pushed the contents of one syringe into another multiple times. If the gel in the syringe was not mixed well, then some parts of the syringe will have large concentrations of drug, while other parts would have lower concentrations of drug. As one syringe is used for three samples of a gel (for example, one syringe has ~800 mg of gel and so ~200 mg of gel is placed into each vial), the release would be different between the samples. This is shown in the table below.
In addition to this bad mixing, the gels also finished releasing relatively quickly then previous studies done in the lab. Usually, it takes approximately 4-5 days for the gel to finish releasing, while my gels finished in about 1-2 days. A gel is finished releasing when the lines become horizontal in the graph. This may not be a mistake in procedure, but possibly because I used smaller gels and smaller vials, which no one has really used before.
And so, next week, I plan to start a new release study with one control and another compound. In addition to seeing the drug release from these new drugs, I will test another variable: size, specifically surface area and volume. To do this, for each gel, I will have samples of different masses. With this, I can my technique (particularly mixing), see if my variability in results was caused by size of my gels, and hopefully find a relationship between size and release.
Thanks for reading!
Russell Llave
As mentioned in my previous post, the way I measure the amount of drug released from the gel, I take aliquots of the solvent in the vial into tubes at different times. Each time I place some solvent in a tube, I replace the solvent in the vial. After a certain time, I place a small sample of each tube onto a plate reader. The plate reader is then placed into a spectrometer which measures the absorbance of the sample of a particular wavelength of light. The results from the spectrometer transferred to an excel document look like this
Firstly, as shown by the high R2 value, a linear approximation is appropriate, and so the equation listed in the graph is used to calculate the concentration of each of the samples from the absorbance in the plate reader. After a series of unit conversions, I was able to calculate the mass released from each gel at each time point. Furthermore, a calculated the cumulative release of each gel. To calculate this, I add the mass of a particular time point with the masses of all the time points before it. This is done because the solvent is replaced at each time point. Then, I calculated the theoretical amount of drug within each gel. This can be calculated because the percentage of drug in the gel is known by how I created the gels.
After all of these calculations, I created two graphs. The first graph shows the cumulative amount of drug divided by the total amount of drug for each gel at each time point. The second graph shows the mass at each time point divided by the cumulative release of that gel. “Average” is used because for each type of gel, there were three samples for repetition. In the graph, rather than having twenty-seven different lines, I averaged each sample of each gel. “Control” is a gel without a salt or acid.
One trend shown in these graphs is that a higher concentration of a specific salt/acid has a faster release than the lower concentration of the same salt/acid. Furthermore, the salt/acid mostly had slower releases then the control group. However, my data suggests that the gels were not mixed very well. When I mixed the gels, I connected two syringes and pushed the contents of one syringe into another multiple times. If the gel in the syringe was not mixed well, then some parts of the syringe will have large concentrations of drug, while other parts would have lower concentrations of drug. As one syringe is used for three samples of a gel (for example, one syringe has ~800 mg of gel and so ~200 mg of gel is placed into each vial), the release would be different between the samples. This is shown in the table below.
In addition to this bad mixing, the gels also finished releasing relatively quickly then previous studies done in the lab. Usually, it takes approximately 4-5 days for the gel to finish releasing, while my gels finished in about 1-2 days. A gel is finished releasing when the lines become horizontal in the graph. This may not be a mistake in procedure, but possibly because I used smaller gels and smaller vials, which no one has really used before.
And so, next week, I plan to start a new release study with one control and another compound. In addition to seeing the drug release from these new drugs, I will test another variable: size, specifically surface area and volume. To do this, for each gel, I will have samples of different masses. With this, I can my technique (particularly mixing), see if my variability in results was caused by size of my gels, and hopefully find a relationship between size and release.
Thanks for reading!
Russell Llave
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