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Supplementary Materials and Methods
S1. Materials and synthesis of MDNPs
All chemicals were of analytical grade and used without further purification and purchased from
Sigma-Aldrich (Canada) if not indicated otherwise. The precursor polyelectrolyte coated MnO2
(PMD) NPs (< 30 nm diameter, zeta potential (ζ) + 30 mV) were prepared by strictly following
our previous protocol (1). The precursor MnO2 NPs (PMD) were prepared by directly mixing 18
mL of KMnO4 solution (3.5 mg mL-1) with 2 mL of poly(allylamine hydrochloride) (PAH, 15
kDa) solution (37.4 mg mL-1). The mixture was left for 15 min at room temperature until all
permanganate was converted to MnO2. NP formation was confirmed by recording ultravioletvisible (UV-vis) absorption spectrum (Fig. S1B). NPs were washed three times with doubly
distilled (DDI) water using ultracentrifugation (50 k rpm for 1 h). This step produced small (~15
nm) MnO2 NPs stabilized with PAH (Fig. S1C). The molar concentration of NPs was determined
by ICP-AES analysis. The calibration curve from the stock solution was then obtained by taking
the (UV-Vis) spectra and collecting the absorbance value at 325 nm wavelength from the series
of samples prepared by diluting the stock solution. The calibration curve was used to obtain the
PMD NPs concentration in a given mass.
The synthesis procedure of TMD and LMD NPs are shown schematically in Fig. S1A. Graft
terpolymer (poly(methacrylic acid)-polysorbate 80-starch) was prepared as previously described
without further modifications (2,3). Potassium permanganate (KMnO4), poly(allylamine
hydrochloride) Mw ~15,000 (PAH), polyvinyl alcohol (PVA), N-(3-dimethylaminopropyl)-N′ethylcarbodiimide
hydrochloride
(EDC),
N-hydroxysuccinimide
(NHS),
N,N'
dicyclohexylcarbodiimidecarbodiimide (DCC), bovine serum albumin (BSA) lyophilized
powder, oleic acid, myristic acid, polyoxyethylene (40) stearate, and hydrogen peroxide (H2O2)
30 wt.% solution were purchased from Sigma-Aldrich (Canada). Myrj® 59 polyoxyethylene
(100) stearate was from Spectrum Chemicals (USA). Dimethylformamide (DMF) and
tetrahydrofuran (THF) were from Caledon (Canada). Indocyanine green (ICG) was purchased
from MP Biomedcals (USA). Distilled and deionized (DDI) water were obtained from a Milli-Q
water purifier (Milli-Pore Inc.). Normal saline (0.9% NaCl, pH 5.5) was obtained from sterile IV
bags (Baxter Corp, Canada), and the pH was adjusted to 6.8 and 7.4 with 0.1M HCl and 0.1M
NaOH solutions.
Covalent conjugation of oleic acid to the surface of PMD NPs (OMD NPs): In a glass vial a
DCC solution in DMF (5 mL, 10 mg mL-1) was poured under constant stirring to an oleic acid
solution also in DMF (5 mL, 15 mg mL-1), the mixture was left under stirring for 15 min at room
temperature, and a PMD aqueous solution (500 µL, 30 mM) was then added slowly under
vigorous stirring. Big aggregates were observed after 10 min reaction and chloroform (5 mL)
was added to clear the dispersion and the mixture was left under stirring overnight at room
temperature. For NP separation, the reaction solution was mixed with acetone in a volume ratio
of 1:3 (reaction mixture: acetone), and big aggregates of NPs were formed and then separated by
centrifugation (4 k rpm, 5 min). The pellet was re-dispersed in chloroform and the washing
procedure repeated to remove reaction by-products. Finally, the precipitate was re-dispersed in
chloroform and stored at 4 °C.
Preparation of TMD NPs: In a first step, terpolymer (50 mg) was dissolved in DDI water (1
mL) and both EDC and NHS (15 mg each) were added and the solution was stirred for 30 min at
room temperature for activation of the carboxylic groups of the TER. Meanwhile, in a 14 mL
conical polypropylene tube, PMD NPs (10 mL, 10 mM in DDI water) were mixed with a BSA
solution (1.2 mL, 10 mg mL-1 in normal saline) and Pluronic F-68 solution (50 µL, 100 mM in
DDI water). The tube was added to a water bath with temperature set to 80 °C and kept under
stirring and sonicated for 60 sec with an ultrasonic processor probe (100 Hz, 5 mm probe depth,
Heischer UP100H, Germany). The mixture was removed from heat, and activated terpolymer
(500 µL) was added and the dispersion was sonicated for another 5 min. To obtain NPs with a
PEG corona, a mixture of melted polyoxyethylene (40) stearate (8 mg) and polyoxyethylene
(100) stearate (4 mg) was added during NP preparation prior to adding the crosslinker TER. The
obtained brown dispersion was purified by ultracentrifugation (14 k rpm, 20 min, 3) and the
pellet was dispersed with 1 mL of sterile saline solution by ultrasonication at room temperature.
The resultant emulsion of TMD NPs was washed and centrifuged. The remaining PMD NPs in
the supernatant were analyzed by UV-Vis spectrophotometry by measuring the absorbance at
325 nm and using the calibration curve for MnO2 concentration obtained using the ICP-AES
(Fig. S1B), which indicated that the TMD NPs contained about 85-90% PMD NPs. A typical
preparation led to a ~7 mM MnO2 emulsion. TMD NPs were stored at 4 °C and further diluted
with cell medium or sterile saline for in vitro and in vivo studies, respectively.
Preparation of LMD NPs: In a 14 mL polypropylene conical tube, a mixture of myristic acid (7
mg), polyoxyethylene (40) stearate (1 mg) and polyoxyethylenes(100) stearate (2 mg) were
dissolved in chloroform (50 µL) and mixed with OMD NPs (50 µL, 75 mM) at room
temperature. PVA (1 mL, 0.1 wt%) was added, and the tube was immersed in a water bath with
temperature set to 54°C and sonicated for 3 min with an ultrasonic processor probe (100 Hz, 5
mm probe depth, Heischer UP100H, Germany). The obtained emulsion was immediately
transferred to ice cold DDI water (0.5 mL) under stirring and gently purged with N2 for 30 min to
remove chloroform. The emulsion was left under low vacuum overnight to confirm the complete
removal of the organic solvent. It was estimated 100% loading of the OMD NPs occurred in the
PEG-lipid matrix, as determined by measuring remaining OMD NPs in the supernatant after
ultracentrifugation (14 k rpm, 1 h) of the NPs by UV-Vis spectrophotometry analysis (Fig. S1B).
A typical preparation led to a ~2.5 mM MnO2 emulsion. LMD NPs were stored at 4 °C and
further diluted with cell medium or sterile saline for in vitro and in vivo studies, respectively.
Near-Infrared LMD NPs were obtained by adding an ICG solution (20 µL, 50 mM in methanol)
to the OMD, myristic acid and polyoxyethylene stearate mixture and NPs were prepared as
described above.
S4. Immunohistochemistry analysis
IHC image quantifications were conducted using ImageJ software. The IHC plugin was used to
select the positive color pixels in diaminobenzidine (DAB) stained images and to eliminate the
background color pixels. Finally, the standard ImageJ pixel intensity threshold plugin was used
to select the positively stained areas and minimize background signal.
S5. Higher H2O2 produced by tumor cells
The exact concentration of H2O2 in tumors is not well defined as it is difficult to quantify
accurately. However, there is a wealth of published literature showing a high level of H 2O2
produced by different breast cancer cells in vitro (1-6 pmole/h/103 cells, taken from (4) and 0.20.5 nmole/h /104 cells, taken from (5). If it is assumed that a 1 cm3 tumor contains ~109 tumor
cells (6-9) then a 100 mm3 tumor would contain 108 cells. Using the above published data the
H2O2 concentration produced by these cells in 100 mm3 tumor volume are in the range of 160-
1200 μM/h for the different breast cancer cell lines (based on (4), and from 2000-10000 μM/h for
the different tumor cells (based on (5) (see more details in Table S1). To mimic the in vivo tumor
condition, where only a portion of tumor cells are viable, as we observed in our tumor models
(Fig. S2 and S3), we have assumed only a 20% viable region, i.e. 2 × 107 cells, 5 times lower
than the number of cells contained in the 100 mm3 size tumor. Moreover, in the referenced
manuscripts the H2O2 levels were not obtained under hypoxic conditions where H2O2 production
by cells would be even higher.
S6. Study Design
This study reports the use of hybrid manganese dioxide nanoparticles (MDNPs) to enhance RT
outcomes by modulation of TME. We have previously demonstrated the ability of MDNPs to
reduce tumor hypoxia, acidosis and downstream genes regulated by hypoxia such as HIF-1α and
VEGF (1,10). Based on these previous experimental observations, we hypothesized that MDNPs
would enhance the RT by generating oxygen in the TME thus reducing tumor hypoxia and
modulating TME. Previous proof-of-principle study by our laboratory using a murine breast
tumor model demonstrated the increase in DNA DSBs when MDNPs were delivered IT prior to
irradiation (1). The overall goal of this study was to examine in detail the effect of MDNPs
treatment on the efficacy of RT, and further examine the changes in tumor cell proliferation,
apoptosis, VEGF expression and tumor vasculature using IHC methods. Quantification of IHC
images was performed using ImageJ program. For each tissue 6 to 8 viable tumor areas were
quantified. Two different MDNP formulations with different oxygen production rates were
utilized, and the effect of local and systemic administration of MDNPs on RT was investigated.
In the present study BALB/c and SCID mice bearing murine and human breast tumors were
used, respectively, to determine the anti-tumor potential of MDNPs treatment delivered prior to
RT. Mice were randomized at the same age and into four groups 1) saline, 2) saline+RT, 3)
MDNPs and 4) MDNPs+RT for both IT and IV treatments. For the survival studies 5 to 10
animals were in each group. For the IHC studies 3 animals were used for the each treatment
group.
S7. Local treatment with MDNPs plus RT results in tumor regression in short term
The experimental design for the IT treatment studies is shown schematically in Fig. S3A and
described in Table S2. To assess the anti-tumor efficacy of the combination MDNPs+RT
treatment, we measured tumor volume over time for up to 5 days post-treatments and recorded
tumor weights of resected tumors at the end of the experiment (n = 5/ group). For both breast
tumor model significant regression in tumor volume was observed, while RT alone resulted in
tumor growth delay. In EMT6 tumors, TMD+RT and LMD+RT treated groups exhibited 31%
and 87% regression in tumor volume, respectively, compared to day zero, whereas, tumor weight
decreased by 43% and 85%, respectively, compared to saline+RT (P < 0.05) at day 5 (Fig. S5B,
and Table S3). In MDA-MB-231 tumors, tumor regression by 21% and 27% was observed
relative to time zero, and tumor weight decreased by 11% and 15% compared to saline+RT
group (P < 0.05) in the TMD+RT and LMD+RT treatment groups at day 5, respectively (Fig.
S5C, Table S3). Not unexpectedly, the saline+RT treatment only reduced tumor growth rate by
40% in EMT6 tumor and by 17% in MDA-MB-231 tumor compared to saline alone at day 5.
Interestingly, IT treatment with MDNPs alone showed a measureable effect on % change in
tumor volume at day 5. Compared to saline groups with 140% tumor volume increase in EMT6
tumor and 53% in MDA-MB-231 tumor, the tumor volume only increased 83% (TMD) and 8%
(LMD) in EMT6 tumor, 39% (TMD) and 34% (LMD) in MDA-MB-231 tumor at day 5 relative
to time zero (Fig. S5B and S5C). These results correspond to TGD of 41%, 94%, 26%, and 37%,
respectively, in the MDNP treatment groups compared with tumor-bearing mice treated with
saline alone.
S8. Biodistribution and early effect on tumor growth of IV administrated MDNPs alone or
in combination with RT
The experimental design for the IV treatment studies is shown schematically in Fig. S7A and
described in Table S2. Our previous study has demonstrated an excellent accumulation of LMD
NPs in tumors after IV administration in mice bearing murine EMT6 breast tumors (10). Here,
for the first time, the tumor uptake of MDNPs was observed for a human MDA-MB-231 breast
tumor model after their IV administration. The in vivo biodistribution and tumor accumulation of
LMD NPs were determined using SCID mice bearing human MDA-MB-231 breast tumors.
Slightly larger tumors (200 – 300 mm3) were used for better imaging during the biodistribution
studies. The LMD NPs were loaded with NIR dye indocyanine green (ICG) by strictly following
our previously published protocol (10) to enable visualization of their biodistribution by
fluorescence imaging. Once tumors reached the required size, 200 µL of labeled LMD NPs (1
mM MnO2) were injected IV, and the biodistribution and tumor uptake characteristics of the
LMD NPs were monitored non-invasively for up to 24 h using the Xenogen IVIS Spectrum
Imaging System (Caliper Life Sciences Inc. MA, USA). During imaging, mice were
anaesthetized with 1.8% isoflurane and whole body fluorescence images were acquired using
745 nm excitation and 820 nm emission filters. Shortly after injection, a rapid increase of the
fluorescence intensity of the LMD NPs was observed throughout the body. Accumulation of
LMD NPs in the tumors was observed within 30 min and remained for at least 24 h.
Fluorescence images of ex vivo organs and tumor acquired at 4 h and 24 h after injection of LMD
NPs also confirmed the tumor uptake and retention of LMD NPs.
Next, we performed the assessment of tumor response to MDNPs and RT treatments in both
EMT6 and MDA-MB-231 models by measuring percent change in tumor volumes from time
zero to day 5 post-treatment, and measuring the tumor weights of various groups (n = 3/group) at
day 5. We observed that IV administered LMD NPs alone led to a significant reduction in EMT6
tumor growth by 78% at day 5 post-treatment, compared to the saline control (P < 0.05) (Fig.
S7B), whereas, the same treatment resulted in 16% decrease in tumor growth in the MDA-MB231 tumor model, compared to saline control (Fig. S7C). Moreover, consistent with the IT study,
the combination of IV LMD NPs and RT treatment led to 32% and 16% regression (negative
percent change) in EMT6 and MDA-MB-231 tumors, respectively, relative to the time zero
control (Figs. S7B and C). A similar trend was observed for the weight of ex vivo tumors
measured at day 5, where the LMD+RT group showed a decrease in tumor weight by 46% and
14% for EMT6 and MDA-MB-231 tumors, respectively, compared to saline+RT group (P <
0.05) (Fig. S7B and C). Compared with IT treatments, the IV treatments showed a reduced
percent change in tumor growth during the short-term even for the saline control (140% for IT
versus 88% for IV treatment in EMT6 tumors and 54% for IT versus 35% for IV treatment in
MDA-MB-231 tumors) (Fig. S5 and Fig. S7). This difference in tumor growth rates could be
ascribed to local inflammation and edema within the tumor volume resulting from the inherent
tissue injury during IT injections (11), however, we did not confirm this explicitly.
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