Figure 4. ZZ-CD14-GPI_rec function to associate anti-CD4 with liposomes. (A) Proposed model of liposome-resident ZZ-GPI_rec binding to antibodies. (B) The incorporation of ZZ-GPI_rec on proteoliposomes was analyzed by silver-stained SDS-polyacrylamide gels and western blot in pelleted proteoliposomes produced by co-incubation with ZZ-CD14-GPI_rec, or soluble ZZ as a control using the indicated antibodies. (C) To analyze whether a second incubation of proteoliposomes with antibodies is viable for linking antibodies to proteoliposomes, pellets of proteoliposomes produced with ZZ-GPI were placed with ZZ in a soluble version after incubation with anti-CD4 antibodies. The proteoliposomes of this incubation were submitted to a western blot using an antibody against the F_C domain of IgG. (D) Liposomes loaded with ZZ-CD14-GPI_rec and anti-CD4 were incubated with DiA, washed and exposed to splenocytes from BALB/c mice (n=5). The CD4⁺ and CD8⁺ splenocytes were differentiable, each being detected separately with anti-CD4 or anti-CD8 antibodies coupled to APC (APC fluorochrome emission at 660nm). Total splenocytes were analyzed by flow cytometry for each individual marker as CD4⁺ and CD8⁺ cells. The quantity of double-stained CD4⁺ cells with DiA⁺ and CD8⁺ cells with DiA⁺ were analyzed by a paired t test. A significantly higher number of immunoliposomes interacting with CD4⁺ cells compared to CD8⁺ cells was observed (*** is P<0.005). In (E) the capacity to mediate binding to CD4⁺ of ZZ-CD14-GPI_rec-loaded liposomes associated with anti-CD4 was verified. The soluble ZZ version associated with anti-CD4 was used as a control for passive transference/fusion of DiA-stained liposomes to cells. The amount of CD4/DiA-stained cells was significantly higher when the immunoliposomes contained the CD14 (and thus probably the GPI anchor) fused to the ZZ domain (paired t test, *** is p < 0.005). (F) The potential off-target labeling was checked by flow cytometry monitoring colocalization of ZZ-GPI_rec or ZZ pre-incubated with anti-CD4 plus DiA with APC-anti-CD8 labelled splenocytes. In this case, soluble ZZ-anti-CD4/DiA liposomes colocalized more with CD8⁺ cells than the GPI-anchored ZZ-antiCD4 complexes (paired t test, * is p < 0.05).