Finally, delivery of ALP-functionalized lipoproplexes (ALP-LPX) into B16 cells was investigated (Figure ). The cells were treated with ALP-LPX for 2 hours and washed to remove any remaining nanogels. The efficiency of delivery could not be assessed for the non-fluorescent NGs, instead we directly characterized the enzymatic activity of ALP inside the cells. For this purpose, DDAO-P substrate was added into the cell medium, and after 40 minutes of incubation the substrate was removed from the medium, cells were rinsed and intracellular fluorescence of the hydrolyzed DDAO was analyzed either by flow cytometry after detaching (Trypsin-EDTA) and fixing the cells (Figure ), or by fluorescence microscopy after rinsing and fixing the cells onto culture plate (Figure ,E, S14 and S15). We found that even in absence of added ALP, the B16 cells were able to slightly hydrolyze the DDAO-P substrate giving a basal level of alkaline phosphatase activity. Flow cytometry showed no effect of adding enzyme alone and enzyme in presence of liposomes on the fluorescence of the cells. The distribution of fluorescence of hydrolyzed substrate slightly increased when cells were incubated with ALP-functionalized nanogels (ALP-NG) without liposomes, but the increase in fluorescence was much stronger when the cells were incubated with ALP-LPX (Figure ). Figure shows fluorescence distributions for 3 independent experiments, and Figure gives the average fluorescence value of the triplicated ALP delivery experiments normalized by the basal level of ALP activity inside the B16 cells treated with phosphate buffer only. We systematically observed that fluorescence intensity corresponding to the hydrolyzed substrate inside the cells treated with ALP-LPX, was higher than for the controls containing phosphate buffer only, free enzyme (free ALP), ALP with liposomes or ALP-functionalized nanogels without liposomes, with significative difference for ALP-LPX vs ALP+liposomes and ALP-LPX vs. ALP alone (Kruskal-Wallis Test and Dunn's multiple comparison test). In an additional control, we compared ALP enzymatic activity inside cells incubated with functionalized and non-functionalized LPX. Figure demonstrates that no enhancement of ALP enzymatic activity after delivery of the enzyme-free LPX was observed, indicating that the increased enzymatic activity in ALP-LPX was due to the catalytic activity of delivered enzyme, and not a result of increasing the basal expression of alkaline phosphatase due to the interaction of lipoproplexes with cellular environment. All these results show that the ALP has been efficiently delivered inside the cells in its active state via functionalized lipoproplexes. To our knowledge, it is a first example of efficient delivery of a large local number of active enzymes.