may be cytotoxicity of PEG. there is narrow window b/w fusion and cytottoxicity induced by PEG.

Do your liposome have any surface charge? Do have measure their zeta potential?

i had this problem before

you have to increase your evaporation time beacause solvent didnt evaprate well and make your formulasion toxic, good luck

As any drug or compund, empty liposomes can yield toxicity, it is normal and I have encountered before.

You can optimize the lipid conposition of empty liposomes in order to find the less toxic one and then, try to increase toxicity by adding your compound into the liposomes, the objetive is minimize the toxicity of empty ones and increase the activity of loaeded liposomes. 

Thank you for all the answers!

Regarding the evaporation, I create the film in a rotavapor and then leave overnight in exsicator connected to a pump. But maybe I need to do it longer?

Enrique, thank you for your tips! However, the HSPC-composition is the same as used in Doxil which should not yield any toxicity...but I will try this same approach for my other composition

This depends on liposome lipid composition and concentration.

      Liposomes usually composed of phospholipids and cholesterol (biocompatible and biodegradable lipids) have no toxic effect in cell culture in the concentration up to 200 µM final lipid concentration in the cell culture medium. In order to use liposomes as  drug delivery system , they must be first tested for in vitro cytotoxicity on cell cultures and, subsequently, in vivo in pre-clinical and clinical trials. In vitro tests for biocompatibility evaluation could serve to establish the optimal concentration and formulation of liposomal systems in primary cultures or stabilized lines. According to the International Standard ISO 10993-5, there are several specific methods to analyze the cytotoxicity of medical devices, which involve different aspects of cell function, such as viability and proliferation, cell morphology, loss of membrane integrity, decrease in cell adhesion.

In general, those neutral lipids applied at doses mentioned above shouldn't show cellular toxicity.

what is the cell line you are using?

In my case, i measure cellular viability of lipid Nanoparticles (NP) using the WST assay, which is fundamentally similar to the MTT one. I am facing problems where absorbance is highly variable depending on the location of wells on a 96 well-plate. leading to over/under estimation of  empty NP effect on cellular viability. 

It is better to avoid edge wells and randomize the distribution of treated and non-treated wells through out the plate in order to get accurate results. 

I studied DPPC:DSPE-PEG:Nanoparticle:Drug composition and checked the liposomal toxicity. Liposomes showed a little bit higher toxicity than Lipids:NP composition.

I dont think it happened because of organic solvent but your lipid concentration looks high per well or if you use same wells for same samples in 96 well plate change the wells so you can check the spectro.whether it's reading correct. Also hydrodynamic size and zeta potential should be considered.

Try lower concentrations of lipid up to 25 µg/ml. Should be no toxicity!

The materials of the liposome must be non toxic and biocompatible. Probably, you need to test  the raw materials separately on cells. Try to add the phospholipid and the cholesterol separately to specify which of them causing this problem and then change the supplier.

good luck 

check your material and concentration. if not all, most of the liposome show cytotoxity at higher concentration. 

Lower concentrations of lipids may help. I agree with Sarmad's comment about testing the individual reagent for toxicity.

In addition to what all mentioned above. Please check you lipid purity and confirm that

Thanks for your question. My opinion is test for sterility of your product after filtration . If it confirms sterlity then go cell toxicity studies .If   possible go for irradiation  type sterlization to avoid this problem 

Have you removed the non-encapsulated drug from the formulation? Check lipids purity. thx