To investigate the influence of ligand clustering on receptor mediated uptake , a series of eight micelle formulations were used : 0%F-100%mix , the untargeted control containing unfunctionalized LDP ( 0%F ) used as 100 % of the micelle and 10%F-100%mix , 20%F-60%mix , 30%F-40%mix , 40%F-30%mix , 60%F-20 % , 70%F-20%mix and 100%F-10%mix are formulations presenting a similar amount of folate in total . UV - Vis data show statistically similar numbers of folate per micelle by one way ANOVA analysis between the different groups at the 95 % confidence interval ( Figure 2c ) . We used FR overexpressing KB cells to evaluate targeting and binding of the micelles to receptors on the cell surface . After a 24 h period of incubation , the highest cell associated fluorescence was observed for cells incubated with the 20%F-60%mix formulation ( Figure 3a ) . The measured EC50 ( concentration producing 50 % binding ) was observed to be the lowest for the 20%F-60%mix micelle . To facilitate discussion , we approximated the apparent dissociation constants of the tested micelles ( K D ) by fitting the experimental data to a 1:1 binding model for site - specific binding . The apparent micelle K D is given in Table 1 . EC50 and K D values are similar , indicating that there is a direct relationship between binding and the measured fluorescence . The measured dissociation rate constant ( k off ) of the different micelles also show that the optimal 20%F-60%mix formulation had the longest dissociation time ( 2×10 −5 s −1 ) ( Figure 2d , Table 1 ) . To examine the apparent rate of association ( K on ) relationship between the micelles , we used the following equation : k on = k off /K D and found that the calculated values were not significantly different ( less than 1 order of magnitude apart , Table 1 ) . Confocal analysis and competitive binding experiments confirmed that our observations for binding and targeting are FR mediated and that the mechanism for internalization of targeted LDP is dependent on both energy driven endocytosis and the presence of folate receptors ( Supplemental Figure 5 ) .