Figure 6. Evaluation of MLNPs in vivo.

A. Biodistribution of C6 MLNPs. MLNPs accumulated primarily in the liver and tumor. Three h after a single dose, there was minimal signal in most tissues. However, after four repeated doses over 48 h, there was significantly greater C6 signal in the tumor and liver. *P<0.5 comparing weight normalized C6 signal after four doses was significantly greater than the signal after one dose. **P<0.5 comparing weight normalized C6 signal between the tumor and liver is statistically similar, and significantly greater than signal from other tissue sites. B. Antitumor evaluation of MLNPs in vivo. Antitumor effects of MLNP treatment groups can be seen after five tail vein injections, and 16 days of treatment. Mice treated with CT2 MLNPs had significantly reduced tumor growth compared to all other treatment groups after 15 days. Gray arrows designate timing of tail vein injections. *P<0.5 compared to all other treatment groups. Representative gross tumor resections are displayed as images I–V (I. PBS, II. BL MLNP, III. CL2 MLNP, IV. BT MLNP, V CT2 MLNP). Scale bar set to 1 cm. C–E. TUNEL staining of representative tumor sections after five injections, and 16 days of growth at 10× magnification. CT2 MLNP treated mice displayed greater proportion of apoptotic cells. (C. BL MLNP, D. CL2 MLNP, E. BT MLNP, F. CT2 MLNP). BL MLNPs – MLNPs delivering only pLuc, BT MLNPs - MLNPs delivering only pTRAIL, CI – combination index, CL2 MLNP - MLNPs delivering CPT and pLuc, CPT - camptothecin, C6 – coumarin 6, CT2 MLNP - MLNPs delivering CPT and pTRAIL, MLNP – multi-layered nanoparticles, PBS – phosphate buffered saline, pLuc – luciferase encoding plasmid, pTRAIL – TRAIL encoding plasmid, TRAIL – TNF related apoptosis inducing ligand. TUNEL - terminal deoxynucleotidyl transferase dUTP nick end labeling.