There are many possibilities:

1- First, is the effect of these two treatment groups you mentioned similar to free drug alone (without free liposomes)?

2- The drug may get incorporated within liposomes upon mixing (you need to provide some more information aobut the drug physicochemical properties)? This is still a less likely possibility. 

3- More likely, the liposomal formulation itself is not successful; i.e. the drug is not actually loaded into the liposomes, and it exists outside of it all the time. Again, some more info about the drug would help. If the drug is hydrophobic, then this problem may be easier to detect than when the drug is hydrophilic. In all cases, try to use ultrafiltration (at a low MW cutoff, e.g. 3 or even 10K Amicon tubes) to separate free drug from liposomal drug, and compare that with ultrafiltered free drug alone (to make sure the free drug is not retained for any reason). Make sure that the drug is a small molecule (< 1K) to be able to pass through the membrane.

4- The liposomes may be successful, but the are unstable. They break apart once they get internalized. In this case, endosomal cell uptake need to be compared to uptake of the drug in the free form (via diffusion if hydrophobic or transporter if hydrophilic).   

thanks a lot for your answer, the drug alone didnot give the same effect ( less effective on cancer cells) compared to liposomal drug or liposome+ free drug 

and yes the drug is hydrohilic

Then try the ultrafiltration method.

Also, do the liposomes alone give an effect? If you add the effect of liposomes alone (if any) to the effect of drug alone, will this be similar to the effect of the combination?

No it is totally different, the effect is not the same.

So does this mean there is an interaction between the free liposome and free drug in the cells but it will not be stable as the combination form ( liposomal drug)

I'm thinking it is either the drug may get bound or attached to the free liposomes when they are mixed together, or the liposomes are not stable in the cell.  

another question, how can I be sure that the drug is successfully loaded into the liposomes. we have measured the encapsulation efficiency is that enough? 

Usually when you use hydrophilic drug and apply the gradient remote loading method (like in the case of doxorubicin in Doxil) you can still see the drug crystals inside liposomes using TEM.

You have to find a way to separate the unencorporated drug from the liposomes. Ultrafiltration method (e.g. Amicon tubes) is a feasible way to tell you.  

If you have the same effect from the liposome-drug combinations but no effect when using only free drug it seems to me that the effect that you're seeing is from the liposomes themselves.

I suggest that you treat your cells with empty liposomes at the same lipid concentrations as when you're combiing with drug, at least then you'll be able to see or exclude if the effect is due to the liposomes themselves.

Actually, we already treated the cells with empty liposomes and no effect was observed with low concentrations.

Hello, u have some options to prove u have entrapped the drug in your liposome successfuly and study the stablity of your particle. 1st step check the entrapment% using ultracentrifuge method. 2) check the stability of your particle using zeta potential, if you havent considered, it can cause sometimes sever leakage of drug. the best is having - or+ 15 and above mv for electrical charge the the more u get the best. after all, if every thing is fine u need to consider you are doing a drug delivery study. which mainly affects on tissue rather than indvidual cell, u can get better idea if you try your sample in an ex vivo or in vivo study. and by the way in vitro drug release study can give u easier idea about your drug release from your liposome.sometimes some surfactants if u dont consider proper ratio can inhibit releasing the drug at specific time that we want. for example PEG has this potential specifically if your drug is protein. All the best

I agree with Dr. Nazari

regards