We next made quantitative measurements of the cellular uptake of different PEG-CS-NPs formulations using flow cytometry. The mean fluorescence intensities (MFIs) of the cells after 4 h of incubation with different PEG-CS-NPs formulations were shown in Figure 7. The MFI should be directly correlated with the mean

number of NPs taken up per cell. The MFI of HeLa cells treated with the FITC-(FA + PEG)-CS-NPs was significantly higher than the FITC-PEG-CS-NPs, and even the MFI of HeLa cells treated with the FITC-(MTX + PEG)-CS-NPs was also significantly higher than the FITC-(FA + PEG)-CS-NPs. These results also supported Vorinostat the idea of the targeting effect of both the FITC-(FA + PEG)-CS-NPs and FITC-(MTX + PEG)-CS-NPs to HeLa cells. The presence of excess of the free FA efficiently inhibited the cellular uptake of FITC-(MTX + PEG)-CS-NPs, which confirmed that the (MTX + PEG)-CS-NPs enter the cells through the FA receptor-mediated endocytosis. Figure 6 In vitro cellular uptake of the (MTX + PEG)-CS-NPs. Laser scanning confocal microscopy images of (A) HeLa cells incubated with the FITC-PEG-CS-NPs. (B)

HeLa cells incubated with the FITC-(FA + PEG)-CS-NPs. Selleck CRT0066101 (C) HeLa cells incubated with the FITC-(MTX + PEG)-CS-NPs. (D) HeLa cells blocked with excess of the free FA and then incubated with the FITC-(MTX + PEG)-CS-NPs. Incubation was carried out at 37°C for 6 h. The https://www.selleckchem.com/products/z-devd-fmk.html concentration of FITC was equivalent in all formulations. All images were taken using identical

instrumental conditions and presented at the same intensity scale. Figure 7 Cellular uptake of FITC-PEG-CS-NPs, FITC-(FA + PEG)-CS-NPs, and FITC-(MTX + PEG)-CS-NPs (equivalent FITC concentration) on HeLa cells by flow cytometry (mean ± SD, n  = 3). Statistical significance: *P <0.05. These quantitative results were consistent with those qualitative results, giving a further proof of high targeting efficacy of the (MTX + PEG)-CS-NPs to HeLa cells. The possible reason is that the integral binding avidity of the (MTX + PEG)-CS-NPs towards FA receptor presents a great advantage of targeting efficacy outperformed that of the (FA + PEG)-CS-NPs towards FA receptor. As mentioned above, MTX has a suboptimal affinity to FA receptor compared Oxymatrine with FA and may be less efficient to target to FA receptor than FA. Nevertheless, it was reported that multivalent binding avidity can be kinetically limited if the binding affinity of an individual receptor-ligand pair is too tight [44, 45]. Well consistent with the above theoretical analysis, our result further suggested that the targeting specificity of the nanoscaled drug delivery systems for a particular cell type can be enhanced by the weaker binding affinity of each individual receptor-ligand pair. Indeed, the integral binding avidity plays a predominant role in the targeting efficacy; the higher integral binding avidity increases the targeting efficacy.