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1 Mgneto-erotctic cteri deliver drug-contining nnoliposomes to tumour hypoxic regions Oujdi Felfoul, Mhmood Mohmmdi, Smir Therkhni, Dominic de Lnuze, Yong Zhong Xu, Dumitru Loghin, Sherief Ess, Sylwi Jncik, Dniel Houle, Michel Lfleur, Louis Goury, Mrym Trizin, Neil Kou, Michel Atkin, Té Vuong, Gerld Btist, Nicole Beuchemin, Dnut Rdzioch, nd Sylvin Mrtel NATURE NANOTECHNOLOGY 1

2 Supplementry Figure 1. To relily detect cteri in the tumours c d Helicocter pylori PBS FITC Texs Red Supplementry Figure 1A. Assessment of ntiody specificity using gstric mucos nd xenogrfts injected with either PBS or., Section of gstric mucos hevily populted with Helicocter pylori (1), xenogrft injected with PBS(2), nd xenogrft injected with (3) were deposited on the sme slide. All tissue sections were incuted with the ntiody., Section of gstric mucos chroniclly infected with Helicocter pylori nd incuted with the ntiody. The rection ws reveled using either FITC (upper pnel) or Texs Red (lower pnel). No specific lelling ws found in the gstric mucos. c, Xenogrft injected with PBS nd incuted with ntiody. Asence of specific lelling in the hypoxic res using either FITC (upper pnel) or Texs Red (lower pnel) conjugted ntiodies. d, Xenogrft injected with nd lelled with ntiody. Both fluorophores (FITC & Texs Red) redily indicte the presence of in tumours. c Supplementry Figure 1B. Assessment of specificity., Gstric mucos densely populted with Helicocter pylori, common pthogen ssocited with gstritis nd gstric ulcer (Whrtin-Strry stin*). The Helicocter microorgnisms pper on silver stining s smll, curved rods on the surfce of the gstric mucos. This section ws used s specificity control for the ntiody., c, Section of gstric mucos hevily populted with Helicocter pylori () ws deposited on the sme slide next to section of xenogrft injected with (c). Both tissue sections were incuted with the ntiody. No specific lelling ws oserved with Helicocter pylori (). Section of xenogrft incuted with ntiody shown to e teeming with clusters of (c). *Histotechnology A self-instructionl Text. 3rd ed. FL Crson & C Hldik editors. Americn Society for Clinicl Pthology Press. (2009) p NATURE NANOTECHNOLOGY

3 Supplementry Figure 2. Hypoxic regions Hypoxic regions 500 µm c d Mgnetosomes 500 µm 200 µm 500nm Supplementry Figure 2A. cells re preferentilly locted in the hypoxic regions of xenogrfts. To determine the exct loction of with regrds to the locl oxygen tension in tissue, we took dvntge of the hypoxyproe specific ntiody ( nd ) nd the ntiody tht specificlly lelled.,, strnds nd islnds of remining hypoxic tumour tissue give positive rownish rection in the vicinity of necrotic res. c, Adjcent sections of the sme xenogrfts were incuted with ntiody nd FITC conjugted specific secondry ntiody to lel. We next extrcted smples from the prffin emedded mteril for TEM. d, the identity of the cell ws confirmed ccording to their typicl ultrstructurl fetures (mgnetosomes). in hypoxic regions Hypoxic region Supplementry Figure 2B. cells ccumulte in hypoxic regions of xenogrfts. lelled section () confirms the presence of undnt in res of lowered oxygen concentrtion s shown y the hypoxic proe rection nd the confirmtory histologic signs of oxygen deprivtion such s ghosts of cells nd poorly stined residul nuclei (). NATURE NANOTECHNOLOGY 3

4 Supplementry Figure 3. Cytotoxicity did not ffect the levels of inflmmtory cytokines produced in mice, while the positive control Pseudomons eruginos (PA) produced significnt increses in inflmmtory cytokines reltive to the negtive control (Vehicle, PBS) nd reltive to positive control (PA) Supplementry Figure 3. Injection of does not induce inflmmtory cytokine increses in mice. C57BL/6 mice were injected with 10 7, PA, or PBS. Blood ws hrvested t 6 h post injection, nd nlyzed vi ELISA on Luminex to ssess the levels of inflmmtory cytokines. * mens p A) IL-6 levels in infected mice re similr to the control, while significntly elevted in PA infected mice. B) MIP1α levels in infected mice re similr to the control, while significntly elevted in PA infected mice. C) There is not significnt increse in TNFα level in infected mice compred to the negtive control. D) IFN-γ levels in infected mice re similr to the control, while significntly elevted in PA infected mice. E) KC levels in infected mice re similr to the control, while significntly (*, p 0.05) elevted in PA infected mice. (Brs show mens ± SEM (*p 0.05, *). 4 NATURE NANOTECHNOLOGY

5 Supplementry Figure 4. Cytotoxicity Supplementry Figure 4. do not lter lood cell counts when injected into rts. Ten week old Sprgue-Dwley rts were injected intrvenously in the til with either 150 µl of PBS, or 150 µl 10 8 or Pseudomons eruginos (PA) resuspended in PBS. PA ws used s the positive control. Blood ws smpled t 6, 24 nd 72 h post infection. A) White lood cell counts of rts infected with t 24 nd 72 h do not significntly differ from the PBS negtive control. Rts infected with PA hve significntly elevted white lood cell counts t 24 h. B) Neutrophil levels in infected rts do not differ from the negtive control, while there is spike in PA infected rts t 24 h post infection. C) Pltelet counts dropped in PA infected rts t 24 h post infection, while pltelet counts in infected rts remin firly consistent similr to the levels of the PBS control. D) Red lood cell levels remin consistent in the infected rts nd the positive nd negtive control. E) Hemogloin levels lso remin constnt. F) Alumin levels slightly dip in the PA-infected rts t 72 h post infection, while levels remin constnt in the infected nd negtive control. Hemtologicl ssessment of the lood ws performed y McGill McIntyre Medicl Services. All niml work ws pproved y the McGill University Animl Cre Committee, in complince with the Cndin Council of Animl Cre guidelines. (Brs show mens ±SEM (*p 0.05, *) NATURE NANOTECHNOLOGY 5