I first dissolved the drug in 100% DMSO to 25mM and then further diluted in RPMI to 10mM and 1mM respectively. I realised that the 10mM concentration formed a lot of crystals and will not freeze. My final concentration use on cell lines was 1uM - 100uM and I can see the crystal in the 96 well plates. Will this affect the pharmacodynamics of the drug on the cell?
Hi,
If you require the concentration range to be between 1uM - 100uM, try preparing a stock soluion of 1mM in 100% DMSO and dilute straight down to 100uM using media instead of making a 1mM from diluting the 25mM using RPMI. That way you will still be able to maintain
Do you have any info about the solubility properties of your compound? Sometimes drugs are highly hydrophobic, so they easely precipitate in crystal forms. If it is a commercial compound, check the solubility in different solvents; if it's not commercial, try to prepare a DMSO stock solution more diluted and you don't need to dilute too much the compound. If your concentration range is between 1uM and 100 uM, I would try with Siva's approach: of course, you have to keep the DMSO % in culture in range according to your cells tolerance. Hope it helps!
I had the same problem with different drugs and compounds here in our lab.
In general, I would follow what Rosario and Siva said and not just add random co-solvents to your solution. Here are two ways to think about it:
1) The smart but time-consuming way: Take a look at your drug structure and check for at least some chemical properties including the pKa, the dielectric constant and hydrophobicity. If you know that you have an uncharged hydrophobic compound, go for non-polar solvents and start maybe with toluene or go up to cyclohexane/hexane. If you know that your compound is charged in your medium pH, keep the "egg-laying wool-milk pig" DMSO as the polar solvent of choice. If - in addition - you see some nice oxygen or nitrogen atoms hungry for hydrogens on your structure, try adding or mixing in a protic solvent like IPA or ethanol (as mentioned above).
2) The equally smart but boring way: Call the provider of your drug and ask them specifically for their solvent and the respective solubility limit. Another important question you may wanna ask is if this drug was purified by LC/MS and if they simply eluted it with a common solvent like acetonitrile/TFA prior to freeze-drying and shipping. If so, the freeze-dried drug may still contain decent amounts of TFA (trifluoroacetic acid) that needs to be buffered. TFA is also a perfect ion-pairing reagent and thus may "neutralize" any charge on your compound so that it may interact less with the polar solvent. Cheers!
Use a cosolvent together with RPMI, NOT with DMSO, where your drug has been disolved.
Lily,
the problem might not be the DMSO. Try dissolving the drug in DMSO and then add it to the same solution you use to culture your cells and use the same conditions (except no cells). Your drug might be precipitating out of solution because it is not very polar. This will happen no matter what concentration of DMSO you use. If the drugs do not precipitate out of solution (form crystals) when you add them to your solution without the cells, then you might want to try to lower your DMSO concentration. Make your initial stock in the concentration that does not precipitate out and then dilute it as needed.
Let me know if that helps.
Yes, it will affect the pharmacodynamics f the drug on the cell as the pharmacodynamic effect depends on the concentration of the drug in the well. If the crystals are formed, this is due to limited solubility of the drug in the media in the 96 well plates and the concentration in the media will be lower than the concentration added to the well. You may need to add other solubilizers to keep drug in solution in the well.
I agree with Maria, Moreover I would try to make always fresh stock solution of DMSO with the compound, make the dilution with KCl water solution or a mild buffer that helps with the ionic strengh of the sotution. If you have precitapitation you will be able to see it, Dilute the sample in order to use not higher final concentration than1% of DMSO
You can avoid the potential toxic effects of DMSO and make your drug bioavailable by formulating it into a cyclodextrin complex using a derivatized cyclodextrin like randomly methylated beta CD or hydroxypropyl beta CD. The complexes are stabile, soluble and do not crystalize out of solution. Theses cyclodextrin derivatives are inert in cell culture and do not affect the cells.
I would stick with your 25mM DMSO stock solution and do your intermediate dilutions in DMSO rather than media. With DMSO, you should aim for a final DMSO concentration on the cells of
what do these crystals look like? I m getting black needle like structures in my 96 well plates. I don't know if its the DMSO forming crystals or the peptide,... I have actually been using 4% DMSO. Would this form the black needle like structures?
I would suggest to avoid non-polar solvents (cylcoexane/hexane). They are not water miscible and they would probably affect cell viability.
Dear Lily
Your compound seems to be highly non polar. I would suggest you to first do a serial dilutions(2 fold) of your compound in RPMI and check for the precipitation/crystallization. The results will give you the min and maximum concentrations to be worked with your experiment. You cannot go beyond these concentrations....
Secondly try to use a cyclodextrin formulation or co-solvent method. Be sure to check the effect of the formulations on you cells.
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