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1 Supporting Information Multifunctional Albumin-MnO 2 Nanoparticles Modulate Solid Tumor Microenvironment by Attenuating Hypoxia, Acidosis, Vascular Endothelial Growth Factor and Enhance Radiation Response Preethy Prasad 1 Ƣ, Claudia R. Gordijo 1 Ƣ, Azhar Z. Abbasi 1, Azusa Maeda 2, Angela Ip 1, Andrew Michael Rauth 3, Ralph S. DaCosta 2 and Xiao Yu Wu 1 *. 1- Department of Pharmaceutical Sciences, Leslie L. Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada, M5S 3M2 2- Princess Margaret Cancer Center, The Campbell Family Institute for Cancer Research, University Health Network, 610 University Avenue, Toronto, Ontario Canada, M5G 2M9 3- Department of Medical Biophysics, University of Toronto,, 610 University Avenue, Toronto, Ontario, Canada, M5G 2M9 ƢThese authors contributed equally to this work. * xywu@phm.utoronto.ca rdacosta@uhnresearch.ca
2 pka Calibration for SNARF in tissue like phantoms. SNARF-4F 5-(and-6)-carboxylic acid (Life Technologies, USA, #S23920) is dual imaging ph sensitive fluorophore that allows the measurement of ph values in solution as well as in biological tissue. The typical fluorescence emission spectra of SNARF shift from green-yellow to red when the ph changes from acidic to alkaline. The ratio between the two fluorescence intensities, typically at 580 nm and 640 nm, provides quantitative information about the ph values. For the quantitative measurement of ph using SNARF, calibration curves must be obtained under the similar conditions at which the ph values are to be determined as the pka values of the dye are sensitive to the local environment. In the present studies, the calibration of SNARF was performed using tissue-like phantoms prepared in ph range 4-10, following an established protocol. 1, 2 In details, buffers were prepared using the following buffering systems: potassium hydrogen phthalate/hydrochloric acid from ph 3-4, potassium hydrogen phthalate/sodium hydroxide from ph 4.5-6, potassium dihydrogen phosphate/sodium hydro oxide from ph 6-8 and disodium hydrogen phosphate/hydrochloric acid from ph ph values for all buffers were measured using Acumet AB15 ph meter (Fisher Scientific, US). Phantoms were prepared by heating 10% (wt/v) gelatine in deionised water at 50 C. Once gelatine melted, the temperature was reduced to 40 C and haemoglobin (Sigma #H2625) and intra lipid (Sigma #I141) were added to give final concentration of 42.5 µm and 1% (wt/v), respectively. After few minutes of stirring, one part of gelatin solution was mixed with three parts of 1 mm SNARF solution prepared in different ph buffers (ph 4-10) with a ph increment of 0.5 unit. 1 In a last step, 200 µl of the final mixture was transferred to a 96 wells microplate and placed in fridge for solidification. Biological phantom were prepared in triplicate for each ph value. Fluorescence images of the microplates were recorded using a Xenogen microscope (Xenogen IVIS Spectrum, Caliper Life Sciences Inc., USA) with two different filter channels; the excitation wavelength for each channel was 535 nm whereas the emission intensities were recorded at580 nm (green channel) and 640 nm (red channel). For the calculation of the pka values, the fluorescence intensities I g (580 nm) and I r (640 nm) were measured using Image-J program by drawing the region of interest (ROI) on the obtained image for each ph value (Fig. S2a). The ratio (R) of the intensities I g /I r was then calculated and plotted against the respective ph values (Fig. S2b), and the R curve was then fitted using the Boltzmann function (Fig. S2.c).
3 R R = ph ph 1+ exp ph a b R(pH) + inf R b Eq. 1 In the above equation (Eq.1), R is the measured ratio of fluorescence intensities at each ph value, R a is the value of the R curve at acidic ph which is considered as the acidic titration endpoint, and R b is the value of the R curve at alkaline ph which is considered as the basic titration endpoint. The parameter ph infl is the point of inflection of the R curve, i.e. the ph value at which the slope of the curve is maximum, ph is an indicator for the slope at the point of inflection. From the fitting of the R curve the fit parameters R a, R b, ph infl and ph were determined. In the next step pka value of SNARF was calculated using the following equation (Eq.2), ph= pk a R R I b log x R a R I b( λ ) aλ 2 (2) Eq. 2 The log.. term in equation 2 was calculated for all ph values from ph 4-10, the value R in log.. term correspond to each point in Boltzmann fitting curve, whereas parameter R a and R b were obtained from fitting of equation1. The two other parameters I a(λ2) and I b(λ2) in log.. term correspond to the fluorescence intensities of SNARF obtained from images using image-j program at 640 nm (red channel) at acidic and alkaline region respectively for the fitted R curve (in the present study we took intensities at ph4.5 and ph 8.5). At the end, graph was plotted for log.. versus ph that would give a straight line (see Eq.1) and intercept of linear fit is pka. The calculated pka value was 6.39.
4 Figure S2. Calculation of pka value for SNARF in biological phantoms. (a) Fluorescence images of SNARF containing phantoms at different ph values, recorded using Xenogen microscope with a excitation wavelength of 535 nm (row (i) green channel, row (ii) red channel and row(iii) is overlay of two channel). (b) The ratios (R=I g /I r) versus ph graph, the ratio R was calculated from the values I g and I r which were obtained from fluorescent image in (a) using image-j program. (c) The Boltzmann fit of data points R using Eq.1, the values for fit parameter were R a = 1.47, R b =0.60, ph infl = 6.25, and ph = 0.3.(d) Shows graph of - log(...) term versus ph, the ratio R was obtained from the Boltzmann fit in (c) and I a(λ2) and I b(λ2) are fluorescence intensities at 640nm (red channel) obtained from image in (a) at ph 4.5 and ph 8.5 using image- J program, I a(λ2) = and I b(λ2) = Finally the intercept of linear fit of data points in (d) is pka according to Eq. 2. The obtained pka of SNARF was 6.39.
5 Consumption of A-MnO 2 Nanoparticles by H 2 O 2 : Figure S3. Upon reaction with hydrogen peroxide for the production of molecular oxygen MnO 2 nanoparticles are consumed. In the graph we show the consumption of the MnO 2 NPs (90 µm) by various endogenous concentrations of H 2 O 2 (up to 1 mm). For the experiment, H 2 O 2 was added to A-MnO 2 in saline, incubated for 5 min at room temperature and the absorbance of the MnO 2 was measured at 360 nm.
6 Nanoparticle structure and MnO2 quantification: Figure S4. Two different nanoparticle systems are described in the manuscript: NP #1 (MnO2) small (~15 nm) MnO2 NPs stabilized with a positively charged polyelectrolyte (PAH) and NP #2 (A-MnO2) - complex formed between NP #1 and BSA (~50 nm). For all in vitro and in vivo experiments, NP #2, named A-MnO2 NPs, were used. In NP #1 MnO2 is stabilized by PAH polymer, while in NP #2 several NP #1 particles are entrapped in a PHA/BSA complex due to strong electrostatic interaction between the protein and the polymer. The complex formation was confirmed by zeta potential analysis and TEM. As shown in the TEM picture above, several small MnO2 NPs can be identified within the protein/polymer complex. We have estimated 100% loading of the MnO2 NPs in NP #2. UV-Vis spectrophotometry analysis of the supernatant indicated the absence of free MnO2 NPs in the supernatant after centrifugation of NP #2 emulsion. The absolute concentration of MnO2 molecules in the emulsion was quantified by ICP analysis to determine the concentration of Mn2+ ions, and thereby the concentration of MnO2 in the emulsion. We then correlated the molar or weight ratios MnO2/PAH or MnO2/BSA in our formulation, as expressed in molar units or w/w ratios in the manuscript Zhang, F. et al. Ion and ph Sensing with Colloidal Nanoparticles: Influence of Surface Charge on Sensing and Colloidal Properties. Chemphyschem 11, del Mercato, L.L., Abbasi, A.Z. & Parak, W.J. Synthesis and Characterization of Ratiometric Ion-Sensitive Polyelectrolyte Capsules. Small 7,
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