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PDF file - 762K, (A) Immunoliposomes bind specifically to their targets in vitro. Specific binding capacity was evaluated in MDA-MB468, a human breast cancer cell line, with endogenous overexpession of EGFR. Cells were incubated with DiIC18(3)- DS-labeled immunoliposomes containing DC101-Fab'(anti-mVEGFR2), C225-Fab' (anti-hEGFR) or with control liposomes, where the Fab' regions of the antibodies were omitted. A shift of two log was detected in anti-hEGFR-ILs, while the anti-mVEGFR2-ILs were comparable with the DiIC18(3)- DS-labeled liposomes. This confirmed target specificity. (1) filled black histogram: untreated MDA-MB468 cells; (2) dark grey open histogram: control non-targeted DiIC18(3)- DS-labeled liposomes; (3) light grey open histogram: anti-VEGFR2-ILs; (4) filled light grey histogram: anti-EGFR-ILs. (B) Anti-VEGFR2-ILs specifically bind to VEGFR2 positive cells in vivo (immunofluorescence). Vessel in the endocrine pancreas of a Rip1Tag2 mouse treated with anti-VEGFR2 ILs-dox or PLD. The Fab` fragment of DC101, a rat antibody, was coupled to the liposomal surface. VEGFR2 was detected with a goat-derived antibody. Detection with anti-rat-Fab`-FITC labeled antibody showed specific binding of VEGFR2 in the tumor tissue in anti-VEGFR2-ILs treated mice, but not in PLD treated mice. Red = staining for anti-VEGFR2, green = staining for anti-rat-Fab', blue = DAPI. (C) Anti-VEGFR2-ILs specifically bind to CD31-positive tumor-associated vascular cells in vivo (FACS). For uptake studies in vivo, liposomes were labeled with 0.1-0.3 mol% DiIC18(3)-DS (DiI-Ls). 12 weeks old Rip1Tag2 mice were injected i.v. with DiI-ILs or anti-VEGFR2 liposomes labeled with DiI (anti-VEGFR2-DiI-ILs). 36h after injection mice were sacrificed and a single cell suspension of pancreatic tumors was prepared by Dispase digestion. For subsequent FACS analysis cells were stained with PerCp-CD45 and APC-CD31 Ab. The CD31 positive cells were gated and the incorporated DiI was measured. Mice injected with anti-VEGFR2-DiI-ILs showed an increase in DiI uptake compared to control mice (70.1% and 24.8%, respectively). The mean fluorescence augmented from 30.4 to 61.1 in anti-VEGFR2-DiI-ILs treated mice. |
