MOF-Mediated Destruction of Cancer Using the Cell's Own Hydrogen

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MOF Mediated Destruction of Cancer Using the Cell’s Own Hydrogen Peroxide Hadi Ranji-Burachaloo, Fatemeh Karimi, Ke Xie, Qiang Fu, Paul Andrew Gurr, David Edwin Dunstan, and Greg G. Qiao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07981 • Publication Date (Web): 08 Sep 2017 Downloaded from http://pubs.acs.org on September 8, 2017

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ACS Applied Materials & Interfaces

MOF Mediated Destruction of Cancer Using the Cell’s Own Hydrogen Peroxide

†‡

†

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†

†

‡*

Hadi Ranji-Burachaloo , Fatemeh Karimi , Ke Xie , Qiang Fu , Paul A. Gurr , David Edwin Dunstan , †*

Greg G. Qiao

†Polymer Science Group and ‡Complex Fluids Group, Department of Chemical & Biomedical Engineering, The University of Melbourne, Parkville, VIC 3010, Australia.

*Corresponding authors: [email protected]; [email protected] KEYWORDS. Metal–organic frameworks, Iron, Fenton, Cancer, Treatment. ABSTRACT. A novel reduced iron metal–organic framework nanoparticle with cytotoxicity specific to cancer cells is presented. This nanoparticle was prepared via a hydrothermal method, then reduced using hydroquinone and finally conjugated with folic acid (namely, rMOF-FA). The synthesized nanoparticle shows the controlled release of iron in an acidic ex-vivo environment. Iron present on the rMOF-FA, and released into solution can react with high levels of hydrogen peroxide found specifically in cancer cells to increase the hydroxyl radical concentration. The hydroxyl radicals oxidize proteins, lipids and/or DNA within the biological system to decrease cell viability. In vitro experiments demonstrate that this novel •

nanoparticle is cytotoxic to cancer cells (HeLa) through generation of OH inside the cells. At low concentrations of rMOF-FA, the cancer cell viability decreases dramatically, with no obvious reduction of normal cell (NIH-3T3) viability. The calculated half-maximum inhibitory concentration value (IC50) was 43 µg/mL for HeLa cells, which was significantly higher than 105 µg/mL for NIH-3T3. This work thus demonstrates a new type of agent for controlled hydroxyl radical generation using the Fenton reaction to kill the tumor cells.

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INTRODUCTION. Cancer encompasses a large number of diseases in which abnormal cells rapidly 1

divide and spread to other parts of the body. There are many differences between normal and cancer 2

cells, the most important of which is there uncontrollable reproductive nature. Normal cell growth is 3

controlled while cancer cells’ grow in a rapid and unregulated manner. A further difference between 4

normal and cancer cells is the high level of reactive oxygen species (ROS) in cancer cells. ROS are 5

produced from the partial reduction of molecular oxygen (O2), and consist of superoxide anion radical •−

1

• 6

(O2 ), singlet oxygen ( O2), hydrogen peroxide (H2O2) and hydroxyl radicals (OH ). Although ROS levels can be controlled by cells under normal physiologic conditions, they can be increased dramatically by 7

exogenous sources such as UV, light and/or heat exposure. High concentrations of ROS have an ability 8

to damage cellular constituents effectively. Due to the metabolic activity and mitochondrial malfunction, cancer cells generate more ROS stress than normal cells.

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Mitochondria are membrane-bound

organelles which are found in most cells and are considered to be a major source of hydrogen peroxide.

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Cancer cells exhibit much higher levels of H2O2, than normal cells caused during DNA alteration, cell proliferation and apoptotic resistance. II

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III

In the presence of iron (Fe and Fe ) ions, hydrogen peroxide can be converted to the hydroxyl radical •

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•

-9

(OH ) according to the Fenton and Fenton-like reactions. Since OH has a short half-life (10 s) and high reactivity, they do not diffuse from the generation site and instead oxidize any surrounding 6

•

biomacromolecules rapidly. Specific examples caused by high OH include the oxidation of 14

polyunsaturated fatty acids in lipids (lipid peroxidation) , oxidation of amino acids in proteins damage.

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and DNA

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Recent studies have shown that nanoparticles can take part as catalysts in the generation of free radicals 17

by heterogeneous reaction in the biological systems. Iron oxide nanoparticles, Fe3O4 and α-Fe2O3 can •

react with H2O2 and produce OH at low pH.

18

•

However, it is concluded that no OH was detected at

neutral pH suggesting a different mechanism for the catalase mimicking activity. Besides iron oxide, other 19

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particles such as silver , gold , MnFe2O4 , and FeOx-MSNs

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•

were reported to produce OH from H2O2 •

in the acidic lysosomes. The drawback of these studies is that these nanoparticles only produced OH at


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the surface via a heterogeneous reaction, and hence were unable to treat the cancer cells using the endogenous H2O2. As a result, exogenous sources are needed to increase H2O2 concentration in cancer cells. Other researchers used ascorbic acid for this aim.

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Ascorbic acid, known an antioxidant,

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is

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able to produce endogenous H2O2 to result in the oxidative stress by the generation of ROS. In addition, β-lapachone

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and cinnamaldehyde

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were shown to undergo redox cycles to generate high H2O2

levels. The combination of these agents with iron oxide

30

particles and ferrocene

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were used as a new

source of ROSs manipulating anticancer drugs. Very recently an amorphous iron nanoparticle was used •

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to generate OH from H2O2. This nanoparticle was ionized in acidic tumours and released iron, which in •

II

turn induced localized Fenton reactions. The rate of OH generation using free Fe ions was found to be II

faster than Fe on the surface of Fe3O4 nanoparticles.

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Moreover, it has been shown that amorphous

iron nanoparticles were not able to release sufficient amounts of iron to treat the cancer cells in in vitro study, so in this case exogenous H2O2 was added.

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To the best of our knowledge, there has not seen a

successful nanoparticle which can treat cancer cells using the Fenton reaction without the need for external H2O2 sources. Attempts of using FePt nanoparticles cause issues of biocompatibility, stability and low selectivity because of potential metal (Pt) contamination.

35-37

Herein, we report a reduced iron metal-organic framework conjugated with folic acid (rMOF-FA) as a new type of therapeutic agent for cancer treatment. This nanoparticle is completely stable at neutral pH, but rapidly releases iron ions at low pH (pH=5.0). It exhibits high peroxidase-like activity, which can catalyze •

tetramethylbenzidine as a chromogenic reaction for use in the detection and sensing of OH in the presence of H2O2. HeLa (cervical cancer cells) and NIH-3T3 (noncancerous fibroblasts) cell lines are used for in-vitro cytotoxicity and cellular uptake studies. The results show that rMOF-FA NPs have the •

ability to treat cancer cells by generation of OH inside cancer cells using the cell’s own H2O2 without the need for external hydrogen peroxide sources. EXPERIMENTAL SECTION

Materials: FeCl3.6H2O (97%, Sigma), 2-aminoterephtalic acid (H2N-BDC; 99%, Sigma), Hydroquinone (99%, Sigma), Pluronic F-127 (Sigma), Folic acid (97%, Sigma), 1-[3-(dimethylamino)propyl]-3ethylcarbodiimide hydrochloride (EDC; 98%, Acros), N- hydroxysuccinimide (NHS; 98%, Sigma), dimethyl sulfoxide (DMSO; AR, Ajax Finechem), trimethylamine (TEA; 99.5%, Sigma), Acetic acid (AcOH; Chem-


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Supply), Phosphate buffered saline tablets (Sigma), filtration (Dismic, 0.2 µm), Hydrogen peroxide(H2O2; 30 wt.%,,Chem-Supply), 3,3´,5,5´-Tetramethylbenzidine (TMB; 99%, Sigma), Terephthalic Acid (97%, Sigma), Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Invitrogen, U.S.A.), Fetal Bovine Serum (FBS; Gibco, Invitrogen, U.S.A.), GlutaMAXTM (Gibco, Invitrogen, U.S.A.), Penicillin (Gibco, Invitrogen, U.S.A.), trypsin-EDTA (1×, Gibco, Invitrogen, U.S.A.), 96-well plate (black, white), Hydrogen Peroxide Assay (Sigma), 2′,7′-Dichlorofluorescin diacetate (DCFH-DA; 97%, Sigma), cell counting kit-8 (CCK-8; Sigma), Ethanol (AR, Chem-Supply), Methanol (AR, Chem-Supply), was used as received.

Instrumentation: X-ray diffraction (XRD) patterns of the samples were recorded on a Bruker D8 Advance instrument with Cu Kα radiation (40 kV, 40 mA) and a nickel filter, and the samples were exposed at a °

°

scanning rate of 2θ = 0.020 s-1 in the range of 5-45 . X-ray photoelectron spectroscopy (XPS) analysis -9

was performed on a VG ESCALAB 220i-XL spectrometer under ultra-high vacuum (6 × 10 mbar) to reveal the surface composition of the polymer coating. A fixed photon energy (Al Kα 1486.6 eV) was used. A survey scan was performed between 0 and 1200 eV with a resolution of 1.0 eV and pass energy of 100 eV. High resolution scans for Fe2p (699 to 739 eV) were also conducted with a resolution of 0.1 eV and a pass energy of 20 eV. Attenuated Total Reflectance Fourier Transform Infrared (ATR FT-IR) −1

was performed on dried samples using a Bruker Tensor 27 with mid-infrared range (400−4000 cm ). The instrument was equipped with OPUS 6.5 software. Measurements were made in transmittance mode. Scanning Electron Microscopy (SEM) measurements were conducted on a Quanta FEG 200 ESEM. Samples were coated with a gold using a Dynavac Mini Sputter Coated prior to imaging. Dynamic Light Scattering (DLS) measurements were conducted on a Wyatt DynaPro NanoStar DLS/SLS instrument with °

a GaAs laser (658 nm) at an angle of 90º and a temperature of 25 ± 0.1 C. Stable spectra were -1

determined at sample concentrations of 1 mg mL . Zeta-potentials of the nanoparticles were analyzed -1

with a Zetasizer Nano ZS (Malvern Instruments). Eight hundred microliters of particles (0.1 mg mL . solution) were placed into a disposable zeta cell, and zeta-potential measurements were conducted at room temperature. The Inductively coupled plasma optical emission spectrophotometer (ICP-OES) was performed on a Perkin Elmer Optima 4300 DV using calibration curves generated from standard solutions (0.01 – 5 ppm). UV-vis spectrometry (UV-Vis) was performed on a Shimadzu UV-1800 spectrometer


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using quartz cuvettes with a 1 cm path length. Finally, fluorometric measurement was carried out by on a Varian eclipse fluorescence spectrophotometer using quartz cuvettes with a 1 cm path length.

Preparation of NH2-MIL-88B(Fe) MOF Nanoparticles: NH2-MIL-88B(Fe) MOF nanoparticles were synthesized using a hydrothermal method.

38

Briefly, Pluronic F-127 (640 mg) was dissolved in Milli-Q

water (60 ml) and, an aqueous solution of FeCl3.6H2O (714mg, 2.64 mmol) was added into this surfactant solution. The reaction mixture stirred for 2h. Acetic acid (2 ml) was added to the solution, and the mixture was stirred for further 1 h before H2N-BDC (240 mg, 1.32 mmol) was injected. Finally, the reaction mixture was stirred for a further 2 h and transferred into an autoclave and heated at 110 °C for 16 h. The mixture was washed 3 times with ethanol, centrifuged (7000 rpm, 1 h) and then dried under vacuum to afford raw MOF nanoparticles as a brown powder. (Yield ~ 50%) Hydrothermal Reduction of MOF to rMOF nanoparticles: According to our previously study, preformed raw MOF (100 mg) and hydroquinone (1 g) were dissolved in Milli-Q water (10 ml).

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The

mixture was transferred into an autoclave and heated at 150 °C for 16 h. After the autoclave had cooled to R.T., the black precipitate (rMOF) was washed with methanol, centrifuge (7000 rpm, 1 h) and dried under vacuum to afford rMOF as a black powder (Yield ~ 32%).

Modification of rMOF with folic acid: EDC/NHS chemistry is used for functionalization of the rMOF with 40

folic acid (FA).

Briefly, FA (70.6 mg, 0.16 mmol) was dissolved in Milli-Q water -DMSO mixture (1:1v/v,

10mL) and solution was adjusted to pH 8 using TEA. EDC (65.92 mg, 0.32 mmol) and NHS (36.83 mg, 0.32 mmol) were added and the pH of the solution was maintained 7−8 by TEA and. The reaction mixture stirred for 4h at RT under dark conditions. An aqueous dispersion of rMOF (20 mg) was added to the activated FA solution, and the mixture was stirred for 24 h at RT under dark conditions. A black precipitate was washed with ethanol, centrifuged (7000 rpm, 1 h) and then dried under vacuum (Yield = 75%). Iron Release Experiment: rMOF-FA (50 µg/ml) was incubated in AcOH (0.1 M, pH 5.0) and phosphate buffers (0.01 M, pH 7.4) at 37 °C for a predefined time period.

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The samples were centrifuged for 10 min

at 10000 rpm, and the supernatant was isolated by filtration (Dismic™, 0.2 µm) to remove the solids. Iron concentration of each sample was determined by ICP-OES at room temperature.


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Peroxidase-Like Activity Assay: Measurements were carried out in 3 mL AcOH buffer solution (0.1 M, pH 5.0) containing different concentrations of rMOF-FA (0, 5, 10, 20, 50 and 100 µg/mL), H2O2 (1 mM) and TMB (0.25 mM) at 37 °C for 5 min. The absorbance spectra were observed using a UV-vis spectrometer.

42

In addition, peroxidase-like activity of rMOF-FA (50 µg/ml) was determined while varying the pH from 2.5 to 8 (2.5-6 AcOH buffer and 6-8 phosphate buffer) and the temperature from 25 °C to 60 °C at an absorbance of 652 nm. Terephthalic Acid Probing Technique: Solution of different concentration of rMOF-FA (0, 5, 10, 20 and 50 µg/mL), H2O2 (1 mM), terephthalic acid (2 mM) was incubated in buffer (pH 5.0) at 37 °C. The photoluminescence spectra under the excitation wavelength of 315 nm were recorded after 2 hours using a spectrofluorophotometer.

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Cell Culture: HeLa and NIH-3T3 cells were maintained in DMEM supplemented with 10% FBS, GlutaMAXTM (2 mM), and Penicillin (100 units/mL). Cells were passaged every 3−4 days using 0.25% trypsin-EDTA at subconfluence and incubated at 37 °C, 5% CO2, and 90% humidity. Cell passages 5−15 were used for cell experiments.

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Hydrogen Peroxide Assay: HeLa and NIH-3T3 cells were seeded into a black 96-well plate (1 × 10

4

cells per well) in cell culture medium (supplemented DMEM). The medium was replaced with serum free medium (100 µL) containing red peroxidase substrate (1 µL), horseradish peroxide (4 µL, 20 Uint/mL) and different concentrations of rMOF-FA (0, 10, 20, 30, 40 and 50 µg/mL) after 12 h and incubated for a predefined time period. Finally, the medium was removed and the fluorescence intensity (λex=540/λem=590 nm) was measured using a fluorescence plate reader (TECAN M200 infinite Pro).

45

The

fluorescence intensity was converted to H2O2 concentration using the standard curve. All experiments were conducted in triplicate, and error bars shown represent the standard error of independent experiments.

Reactive Oxygen Species Assay: HeLa and NIH-3T3 cells were seeded into 8-well chamber slide with 4

glass bottom (Lab-Tek, Chambered #1.0 borosilicate coverglass) (2.5 × 10 cells per well) in cell culture


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medium (supplemented DMEM). The medium was replaced with serum free medium (300 µL) containing DCFH-DA (20 µM) after 12 h and incubated for a 30 min. The cells were further rinsed twice with PBS and incubated with 40 µg/mL rMOF-FA for 2 h. The cells were carefully rinsed again and the intracellular reactive oxygen species levels were evaluated by detecting the fluorescence of DCF (λex = 488 /λem= 525 nm) with confocal laser scanning microscopy (Nikon A1R).

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Images were generated by optical

sectioning in the z-direction and were analyzed using Image J software.

Cell Viability Assay: The cell viability tests were analyzed by the standard cell counting kit-8 (CCK-8) 4

assay method. HeLa and NIH-3T3 cells were seeded into a 96-well plate (1 × 10 cells per well) in cell culture medium. After 12 h, the medium was replaced with 100 µL of fresh medium containing various concentrations of rMOF-FA (0-120 µg) and incubated for a further 24 h. The cells were then washed twice with PBS and incubated with 110 µL fresh medium containing 10 µL CCK-8 solutions for a further 3 h. Finally, the medium was removed and the absorbance at 460 nm was measured using a microplate reader (TECAN M200 infinite Pro). Note that all experiments were conducted in triplicate, and error bars shown represent the standard error of independent experiments. The cell viability (%) was calculated by the following formula, where [A] is the average absorbance:

Cell Viability(%) =

[]() []() []( !" ) []()

Selectivity Index: The degree of selectivity of the nanoparticles against cancer cells was calculated by the selectivity index as follows:

Selectivity Index =

IC*+ of normal cells IC*+ of cancer cells

Statistical Analysis: Data are shown as averages and standard deviations. The student’s t tests were used to analyze the statistical differences between samples for cytotoxicity and hydrogen peroxide concentration measurements and were considered significant at p < 0.05.

RESULTS AND DISCUSSION


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Material Synthesis and Characterization.

The rMOF-FA was prepared via a 3 step process shown in Scheme 1. Initially, NH2-MIL-88B(Fe) was 38

synthesized according to previously reported procedure.

The raw MOF was then reduced in the 39

presence of hydroquinone to produce rMOF as a black crystal.

Finally, the obtained rMOF was 40

conjugated with folic acid (FA) through EDC/NHS chemistry (rMOF-FA, Scheme S1).

Scheme 1. Schematic Illustration of Synthetic Procedure for the Preparation of rMOF-FA

XRD patterns analysis were utilized to verify the formation of MOF and rMOF. As shown in Figure 1A(i), the diffraction profile of the MOF with the characteristic peaks at 2θ of 9.2, 10.5, 13.2, 17.2, 18.2 and 20.5 degree corresponds to NH2-MIL-88B(Fe) with a octahedral structure,

38, 47

indicating successful MOF

crystals formation. In addition, the diffraction profile of the rMOF shows that the crystalline structure with the characteristic peaks at 2θ of 9.2, 10.8, 12.1, 14.4, 18.2, 18.8 and 19.8 degree changed after reduction reaction (Figure 1A(ii)).

39

XPS analysis was carried out to verify the oxidation states of iron in MOF and

rMOF crystals. The XPS peaks of Fe 2p3/2 and Fe 2p1/2 for the MOF and rMOF are shown in Figure 1B. In the case of the MOF (Figure 1B(i)), the peak positions of Fe 2p3/2 and Fe 2p1/2 are 712.2 and 725.6 eV , III

respectively, which indicate Fe as a previously reported.

48

After reduction, the Fe 2p1/2 peak shift to II

727.7 eV, and the Fe 2p3/2 satellite peak at 716.6 eV, which is attributed to Fe appear, II

48

demonstrating

III

the co-existence of Fe and Fe in the rMOF (Figure 1B(ii)). Deconvolution of rMOF signal in the Fe 2p3/2


8

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II

III

reign was used to determine the portion of Fe (45%) and Fe (55%) in the sample (Figure 1B(ii)). SEM measurement of the MOF and rMOF was carried out. Figure 1C shows that MOF has a needle-shaped morphology with an average length of 430 nm and a width of 100 nm. In contrast, rMOF (Figure 1D) presented an irregular morphology compared to MOF. This may be attributed to partial structural damage during the reduction process. After modification, UV-Vis and FTIR spectroscopic tools were utilized to confirm the introduction of folic acid (FA) groups on the rMOF nanoparticles. Normalized UV-vis spectra of FA, rMOF and rMOF-FA are shown in Figure 1E. The FA suspension displays one strong peak at λ=285 nm, corresponding to π - π* transition of pterin ring.

49

A slight increase in the relative intensity of

the peak at 285 nm after the modification of rMOF surface using FA indicates the successful formation of rMOF-FA. As shown in Figure 1E insert, baseline subtraction of rMOF spectrum from the rMOF-FA spectrum presents an intensive peak at 285 nm, which due to the FA conjugation. The FTIR spectra of FA, rMOF and rMOF-FA (Figure S1) shows FA conjugation onto the surface of rMOF by a decrease of -1

-1

the absorbance at 1088 cm and the appearance of the characteristic band at 1606 cm corresponding to the N–H amide stretching vibration of rMOF-FA.

20, 50

Dynamic light scattering (DLS) results for rMOF and

rMOF-FA are displayed in Figure 1F. The average hydrodynamic diameter (DH) of MOF (961.9 nm), rMOF (297.5 nm) and rMOF-FA (244.6 nm) indicate that the reduction and conjugation steps help rMOFFA to be well-dispersed in aqueous solution. Due to the interparticle interaction of MOF particles, they aggregate in aqueous solution and hence have a larger apparent hydrodynamic diameter. However, the 39

partial structure of MOF particles is damaged in the reduction step as demonstrated by previous reports.

As a result, rMOF particles have a smaller size with an irregular morphology. Noteworthy, the molar ratio of clusters (Iron) to organic ligand (H2N-BDC) changed after the reduction, leading to an increased dispersity of rMOF in water. Finally, when rMOF was modified with folic acid, the particles are further isolated from each other and thus such interaction is decreased. The well dispersed nature of the nanoparticles is also verified by zeta potential (Figure S2). The higher negative surface charge of rMOF and rMOF-FA nanoparticles prevented them from aggregation in aqueous solution.


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Figure1: (A) XRD patterns of MOF (i) and rMOF (ii) samples. (B) High resolution Fe 2p XPS spectra for MOF (i) and rMOF (ii). SEM images of the (C) MOF and (D) rMOF. (E) Normalized UV−vis absorbance of FA, rMOF and rMOF-FA. (F) DLS data of MOF, rMOF and rMOF-FA. Hydroxyl Radicals Generation 3,3´,5,5´-Tetramethylbenzidine (TMB) is a classic chromogenic reagent which is often used with peroxidase enzymes for the reduction of H2O2 to H2O. Similarly, various nanoparticles

18, 20-21, 23, 51-52

have

been demonstrated to catalyze the oxidation of TMB using H2O2. Mechanistic studies demonstrated that


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•

these nanoparticles initially catalyze H2O2 to produce hydroxyl radicals (OH ), which then oxidase TMB to •

form ox-TMB. However, these nanoparticles only produce OH at the surface and do not release any ions in the solution. Importantly in this study, we demonstrated that the rMOF-FA nanoparticles with a •

mesoporous structure can produce OH both by reacting at the surface (heterogeneous catalysis) and by releasing iron in acidic environment (homogeneous catalysis). The reaction rate constant of H2O2 II

decomposition using Fe ions was thus found to be around 1000 times faster than iron on the surface of Fe3O4 nanoparticles.

33-34

As a result, our rMOF-FA nanoparticles are able to generate much more OH

•

•

than the other nanoparticles under similar condition. Generated OH can oxidize TMB to produce the blue product ox-TMB with a λmax = 652nm (Scheme 2). Scheme 2. Peroxidase-like activity of rMOF-FA

Figure 2A shows the release profile of iron from the rMOF-FA (50 µg/ml) at two different pH (5.0 and 7.4) values. The rMOF-FA nanoparticles are stable at pH 7.4 and release only 0.05 ppm Fe(II/III) after 8 h. In contract, nanoparticles are able to release iron under acidic condition very rapidly. The amount of released ions from rMOF-FA at pH 5.0 after 10, 60 and 480 min, respectively, are 0.48, 0.84 and 1 ppm, which are much higher than the values at pH 7.4. These results show that rMOF-FA nanoparticles are stable at neutral pH (physiological pH) but rapidly release iron under an acidic environment such as that found in cancer tissue. This result also indicates the significant degradation of rMOF-FA, which can be attributed to the less stability of pristine NH2-MIL-88B(Fe) under acidic condition. The iron(II) ions, both on •

the rMOF-FA and released, can react with H2O2 and produce OH according to Fenton reaction, which is verified using terephthalic acid probing technique (Figure S3).

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The terephthalic acid can easily reacted


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with OH to form a highly fluorescent 2-hydroxy terephthalic acid with the maximum emission at 435 nm.

43

As shown in Figure S3, the fluorescence intensity increases significantly with rMOF-FA concentration, while it shows a weak emission peak in the absence of nanoparticles. We further conducted the measurements of relative peroxidase-like activity of rMOF-FA (50 µg/mL) vs. various pH and temperature (Figures 2B and 2C). As expected, it dramatically increases when the pH decreases from 8.0 to 3.2. Also, the peroxide-like activity increases significantly with temperature increase. Its relative activity increases by °

65.2%, when the temperature increases from 25 °C to 60 C (Figures 2C). The absorbance spectra of the solution containing rMOF-FA, H2O2 and TMB relative to rMOF-FA concentration is shown in Figure 2D. In the absence of rMOF-FA, no obvious absorption is observed, suggesting that rMOF-FA is necessary to oxidize TMB. In addition, there is a direct correlation between nanoparticle concentration and relative absorbance of ox-TMB at 370 and 652 nm (Figures 2D insert) which is associated with an increase in OH

•

generation and hence oxidation of TMB.


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Figure 2: (A) Iron ion release from rMOF-FA in buffer at different pH (5 and 7.4). Effects of pH (B) and Temperature (C) on peroxidase-like activity of rMOF-FA. The maximum absorbance was set as 100%. (D) Absorbance spectra changes with addition of various concentrations of rMOF-FA at pH 5.0, 37 °C, and 1 mM H2O2 after 10 min. The inset shows the relative absorbance of ox-TMB at 370 and 652 nm as a function of rMOF-FA concentration. Cancer Studies. Cell Hydrogen Peroxide Studies. Time dependent generation of hydrogen peroxide in HeLa and NIH-3T3 cells was studied, and the results are shown in Figure 3. The level of [H2O2] increases steadily in a time dependent manner in both Hela and NIH-3T3 cells. Hydrogen peroxide is the byproduct of biological and chemical processes and is 54

generated by the incomplete reduction of oxygen in living cells. Importantly, HeLa cells produce higher concentration of H2O2 than NIH-3T3 cells due to the metabolic activity and mitochondrial malfunction of cancer cells.

4, 9

-1

-1

For NIH-3T3 cells, the rate of H2O2 generation is 0.0131(µmol.L .min ); however, it -1

-1

increases to 0.0631(µmol.L .min ) for HeLa cells at the same conditions. The significant difference in [H2O2] generation between HeLa and NIH-3T3 cells can facilitate selective killing of the cancer cells.

Figure 3: Time dependent generation of hydrogen peroxide in HeLa and NIH-3T3 cells. *P

MOF-Mediated Destruction of Cancer Using the Cell's Own Hydrogen

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