Chemiluminescence-Generating Nanoreactor Formulation for Near- Infrared Imaging of Hydrogen Peroxide and Glucose Level In Vivo

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1 Supporting Information Advanced Functional Materials Chemiluminescence-Generating Nanoreactor Formulation for Near- Infrared Imaging of Hydrogen Peroxide and Glucose Level In Vivo Chang-Keun Lim, 1 Yong-Deok Lee, 1 Jinhee Na, 1 Jung Min Oh, 2 Song Her, 2 Kwangmeyung Kim, 1 Kuiwon Choi, 1 Sehoon Kim,*,1 and Ick Chan Kwon 1 1 Biomedical Science Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul (Korea) and 2 Division of Analytical Bio-imaging, Chuncheon Center, Korea Basic Science Institute, Hyoja-2-dong, Chuncheon, Gangwon-do (Korea). Supplementary Figures Normallized PL Cy5 in FBS FPOC in FBS 0 h 1 h 2 h 4 h 18 h Wavelength (nm) Figure S1. Normalized PL spectra of free Cy5 (20 g) and FPOC (11 mg, encapsulating 20 g Cy5) in 100% fetal bovine serum (FBS, 1 ml). The annealing time at 37 o C is indicated. The initial PL generated from the encapsulated Cy5 was spectrally retained over time with no emergence of free Cy5 PL. The result indicates that Cy5 does not leach out of the encapsulating nanoparticles and that the composite nanostructure is fairly stable in bodily fluid to maintaine the molecular environment of Cy5 in the inner matrix composed of poly(propylene oxide) block of F-127, CPPO and PLGA. It also

2 suggests that the biodegradability of PLGA has a minimal effect on the stability of FPOC NPs during the annealing of 18 h. Contl M H 2 O 2 30 sec 2 min 5 min 7 min 10 min CL intensity (a.u.) 1.6x x x x x x x x Time (min) Figure S2. Chemiluminescence (CL) images (top, captured in a 96-well plate with a Kodak Image Station 4000MM) and kinetic profile of FPOC NPs. For the kinetic profile, FPOC NP dispersion (1 ml, 11.1 mg/ml) was placed in a PL quartz cuvette, and then hydrogen peroxide (200 μl, 0.2 M) was added to the nanoparticle suspension. CL spectrum of the mixture was measured at every 5 min from 10 sec after hydrogen peroxide addition, by using a fluorescence spectrophotometer without photoexcitation. The CL intensities were calculated from the area under each spectral curve. The kinetic parameters were determined with nonlinear fitting according to the following equation, where I is the CL intensity and M is the maximum intensity. k r and k d are the rate constants for raising and decay, respectively. [ref] [ref] a) M. M. Rauhut, L. J. Bollyky, B. G. Roberts, M. Loy, R. H. Whitman, A. V. Iannotta, R. A. Clarke, J. Am. Chem. Soc. 1967, 89, b) M. Orlovic, R. L. Schowen, R. S. Givens, F. Alvarez, B. Matuszewaski, N.

3 Parekh, J. Org. Chem. 1989, 54, c) A. G. Hadd, A. Seeber, J. W. Birks, J. Org. Chem. 2000, 65, CL Spectral Area (a.u.) DNPO/Rubrene (CLQY = einsteins/mole) FPOC (CLQY = einsteins/mole) Time (min) Figure S3. Determination of CL quantum yield ( CL ) of FPOC NPs by using temporal profiles of CL intensity. As a reference POCL reaction system, a solution of bis(2,4-dinitrophenyl)oxalate (DNPO, TCI, 0.01 M) and rubrene (Aldrich, mm) in dimethyl phthalate (1 ml) was placed in a PL quartz cuvette, and then hydrogen peroxide (200 μl, 0.05 M) was added to the solution. [ref] CL spectrum of the mixture was measured at every 1 min from 10 sec after hydrogen peroxide addition until the spectral intensity reduced to 10% of the highest value, by using a fluorescence spectrophotometer without photoexcitation. The same procedure was carried out with a mixture of FPOC NPs (1 ml, 11.1 mg/ml, containing M CPPO and M Cy5) and hydrogen peroxide (200 μl, 0.05 M). The CL quantum yield of FPOC NPs was relatively calculated from the integrated areas under curves in the above figure according to the following equation 2 2 where is the quantum yield, n is the refractive index of solvent, and [DNPO] and [CPPO] are concentrations. Parameters with subscript r are values for the reference system.

4 [ref] M. M. Rauhut, L. J. Bollyky, B. G. Roberts, M. Loy, R. H. Whitman, A. V. Iannotta, A. M. Semsel, R. A. Clarke, J. Am. Chem. Soc. 1967, 89, (a) (b) FPOR (c) FPOR FPOC FPOC FPOR FPOC Figure S4. Visible and NIR CL imaging of exogenous hydrogen peroxide in vivo with an IVIS To elucidate the spectral advantage of NIR CL, visible CL nanoparticles (FPOR NPs, λ max.cl = 560 nm) were prepared as a control with rubrene instead of Cy5, following the same way for the preparation of FPOC NPs. Visible and NIR CL nanoparticles (147 μg), mixed with PBS containing 67 nmol of hydrogen peroxide, were injected (a) subcutaneously into the back, (b) intramuscularly into the thigh and (c) intra-articularly into the ankle. With the three times higher fluorescence quantum yield of rubrene compared to Cy5, FPOR NPs emitted much intenser CL signal from the shallow tissue (a), which was saturated under the same condition used for the unsaturated FPOC signal (acquisition time: 1 sec). With increasing injection depths (b, c; acquisition time: 1 min), however, NIR-luminescent FPOC NPs emitted stronger CL signals than those of visible FPOR NPs (by a factor of ca. 3), due to the improved tissue penetration property of NIR photons.

5 ~ M ~ M ~ M ~ M Figure S5. Triplicate NIR CL imaging to estimate the in vivo sensitivity of FPOC NPs toward hydrogen peroxide. Mice were injected with CL mixtures containing FPOC NPs (147 μg) and a varying amount of hydrogen peroxide ( M) into the thigh muscle and imaged by using an IVIS-200 with an acquisition time of 1 min. Red circles indicate the regions of interest (ROIs) for the integration of total flux of signal to obtain a correlation between CL intensity and hydrogen peroxide concentration (Fig. 3b).

6 5x CL Intensity, I (a.u.) 4x10 6 3x10 6 I = I max x [S] / (K m +[S]) 2x10 6 I max = (5.09 ± 0.07)x10 6 K m = 0.78 ± x10 6 R 2 = D-glu concentration, [S] (mm) Figure S6. Enzymatically assisted glucose sensitivity of FPOC NPs in vitro. Hydrogen peroxide, generated by the enzymatic reaction between glucose oxidase (GOx, 50 μl, 3 mg/ml in 0.1 M PBS) and a varying concentration of D-glucose (D-glu), was imaged by CL signals from FPOC NPs (50 μl, 11 mg/ml) in a 96-well plate with a Kodak Image Station 4000MM (right, n = 5). The correlation between D-glu concentration and CL intensity (estimated from the CL imaging) was fitted with a modified Michaelis-Menten equation (left). The equation, kinetic parameters and fitting reliability are indicated in the graph.