Not knowing the specific composition of your NPs - one plausible explanation is that they interfere with the assay - consider taking the same mass of NPs and look at any color development with reagent alone.  It would not be the first time this has happened - that is, NPs facilitating e- transfer the the tetrazolium (MTT) dye.  Recall that MTT is a metabolic assay and assumed to correlate with cell viability - there can be instances where cells are still slowing metabolizing but not actively in division - be cautious in the interpretation of your findings,

MTT assays and related assays (i.e. MTS) rely on a mitochondrial reductase to convert the tetrazole to formazan. The assumption is that the conversion is dependent on the number of viable cells...but there is always the possibility that treatment of your cells may result with increased enzymatic activity without actually having an effect on cell number or cell viability. That is why performing your MTT experiment along with simple cell counts (Trypan blue exclusion assay) is a good idea to complement your observations.

Usually, as a control, cells are incubated with medium only. If your substance shows no toxicity at all, or no toxicity at a certain concentration, the viability can exceed 100%, since 100% is the viability of the control. Due to natural variations of cellular metabolism or because slightly more cells were seeded in that well due to small pipetting errors, is it totally normal to have samples  that exceed the 100% (maybe up to 110%) However, if it is far above 100% your substance may also have an influence on the cells.

lipid or polymeric nanoparticles affect liposomes, which helps extend the life of the cell, with the appropriate division (before reaching the Hayflick number).

The professionals on “cell surface engineering are often faced with this phenomenon, explaining that in contrast to the microbial cells are not protected by thick cell wall, which makes it difficult to modify their surface and prevents them from dividing, taking into account the fact that nanoparticles have colloidal stability in physiological environments

The simple reason I could think of is that you might have small lumps of cells. This might affect the number of cells so please make sure there are no lumps.

I agree with Dr. Pawel in this regard. To my experience, MTT assay is dependent on the number of cells (viable or early apoptotic) in your plate during measurement. If you have higher number of cells in your test sample than control, you will see OD higher than control. If you are sure your drug treatment should decrease viability, measure after the cells are completely dead and eliminated from the plate. Check whether your certain treatment condition increases cell proliferation as well.

I agree with Dr. Mohammad Mohammadzadeh, if you do your experiments many times and see the same, I think it not due to the seeding cells in the well but your nano-particle may have difference effect on cell-growth when apply the low or high dose. To my experiments, my compound at the low concentration mimic the action like a growth factor (ligand) at the cell surface and stimulate the growth of cells, but at high concentration it goes into the cells, interact with signalling pathways...and kill the cell.

Thanks every one for valuable suggestion and discussions. 

I think that if the nanoparticles did not have enough encapsuled drug, the viability percent would be the same as the control.

You could check the médiums in order to be sure that the variation on your experiments is made only by polimeric nanoparticles with the drug. But it depends on your experiment, if you are studing some tipe of pathology and the treated cells lived longer then you may have found some kind of successful treatment.

Personally I don't like very much MTT to check cell viability..why don't you try crystal violet instead?

In any case, I think that increase in cell viability with drugs can be due to hormetic effects...

Successful drug treatments often depend on (1) Effective drug concentrations (2) Ideal drug treatment times (3) Ideal cell numbers.

Increasing your drug concentration is one option (as suggested above). Increasing drug treatment duration is another option. This gives the cells enough time to respond to the drug, depending on the mechanism of action of the drug. In your case, you also need to consider the release profile of the drug from the nanoparticles. You can also try reducing the number of cells you plate (before drug treatment). This ensures that the cells dont grow too much in the interim period between the plating and the drug treatment. This rapid growth of cells in the interim period may result in little/no cell death due to the drug.

You could also re-look at your read outs. If your absorbance values are outside the linear range that is usually an indication of rapid cell growth. The rapid cell growth maybe attributed to any of the three causes mention above.

Also, you need to determine whether your drug of interest causes cell death or whether it slows down cell proliferation. If the drug only slows down cell proliferation, without causing too much cell death, then a proliferation assay maybe more useful. 

Hope this helps !

One thought will be changes in mitochondrial activity. If you treatment affect the mitochondrial function then you may get a >100% viability result.  

how much time after your treatment do you use the MTT assay?

when the cells are infected the MTT test  will show the viability of more than 100%, but you can see the microorganisms under microscope at least after 48 hours of incubation.

I do not rely on MTT alone as a "viability assay", its supposed activity being only in the mitochondria of living cells is not proven. Many reductases and compounds in cell free culture can convert MTT to formazan,. Very healthy cells can also have a pretty low basal metabolic rate and give you low MTT production. So to say it shows cell death/proliferation can be a bit presumptuous. That's not to say it isn't useful, I use the assay for purely what it is, comparing formazan production, and hence a particular measure of cell activity. Together with a more direct viability assay (LDH assay) it could be more useful.

The attached file is one found to obtain false results from an MTT assay when they measured the "real" viability.

Basically there are few reason why MTT assay give more than 100% viability. 

When the experiment performed in complete media i.e media with FBS/FCS , if the compound or drug is not active enough,,then cell grow in mean time if we do MTT for more than 12 hour give more 100% viabity.

Second some time because of drug, activity response of mitochondria increased compare to untreated cell leads to show more than 100% viability, as we know MTT assay is relative assay rather than absolute assay.

As others have suggested, I would avoid relying too much on MTT to test the impact of moieties on viability, especially if the assay is long enough for differences in cell growth to become apparent. Under such conditions there are too many ways to rationalise >100% 'viability'. You definitely need to include an assay for DNA to control for the cell numbers at different steps in the assay. I would agree with others and suggest there are limits to the value of the MTT assay. It simply informs you of the impact on conversion of tetrazole to formazan by the cultures. As such it can be useful to cross-compare similar moieties to look for gross improvements, (i.e. lower impact on MTT). You definitely need other tests though to attribute any changes to impact on viability, (e.g as suggested, crystal violet and LDH). 

Thanks all of you

What polymer and cell line are you using? 

We have found that void chitosan NPs could increase fibroblast cell viability for instance...

be careful about the infections... sometimes bacteria growth in the sample can cause this

You should take into consideration the pippetting error or how well you mixed the cell solution be plating.