Signals obtained from in vitro assay included

1) ICSI,0

2) ICSI,t

3) IMPE,0

4) IMPE,t

5) ICKDt

6) ICC

Overall, raw fluorescence descriptive of cell uptake (ICKD,t) was taken relative to raw fluorescence of unwashed cells at time t (ICSI,t) to obtain a calibrated fraction of uptake (fcell,c)fcell,c=ICKDtICSIt(2)

Raw fluorescence descriptive of cell uptake (ICKD,t) was also taken relative to raw fluorescence of unwashed cells at time 0 (ICSI,0) to obtain a raw fraction of uptake (fcell,r)fcell,r=ICKDtICSI0(3)

These two fractions were then used to obtain concentration of NM uptake using the general equation[Uptake]c,t=fcell,x*[Dose](4)where fcell,x was the fraction of uptake for x = raw or corrected, [Uptake]t was the concentration of NM taken up by cells (nM), and [Dose] was the applied dose in nM. To determine if cell-induced degradation was present, a two-tailed t test was performed between unwashed CSI and MPE compartments at time t (ICSIt and IMPEt, respectively). To determine if medium-induced degradation was present, a two-tailed t test was performed between unwashed MPE at time 0 and time t. Cell-induced degradation (Icdegt) was taken as the difference between unwashed without (IMPEt) and with (ICSIt) cell exposureIcdegt=IMPEtICSIt(5)

If medium degradation was present, the intensity of this degradation type was taken as the difference between unwashed wells without cell exposure from time 0 to time t.Imdegt=IMPE0IMPEt(6)

Together, the sum of these values equals the total degradation that an NM can undergo for the in vitro assay (Idegt)Idegt=Icdegt+Imdegt(7)

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