Trojan Horse for Light-Triggered Bifurcated Production of Singlet

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A Trojan Horse for Light-Triggered Bifurcated Production of Singlet Oxygen and Fenton-Reactive Iron Within Cancer Cells Daniela Cioloboc, Christopher Kennedy, Emily N Boice, Emily R. Clark, and Donald M. Kurtz Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01433 • Publication Date (Web): 01 Dec 2017 Downloaded from http://pubs.acs.org on December 1, 2017

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A Trojan Horse for Light-Triggered Bifurcated Production of Singlet Oxygen and Fenton-Reactive Iron Within Cancer Cells Daniela Cioloboc, Christopher Kennedy, Emily N. Boice, Emily R. Clark, and Donald M. Kurtz, Jr.* Department of Chemistry, University of Texas at San Antonio, San Antonio, TX 78249, United States KEYWORDS: zinc protoporphyrin IX, bacterioferritin, melanoma, Fenton reaction, singlet oxygen, photodynamic therapy .

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ABSTRACT: Traditional photodynamic therapy for cancer relies on dye-photosensitized generation of singlet oxygen. However, therapeutically effective singlet oxygen generation requires well-oxygenated tissues, whereas many tumor environments tend to be hypoxic. We describe a platform for targeted enhancement of photodynamic therapy that produces singlet oxygen in oxygenated environments and hydroxyl radical, which is typically regarded as the most toxic reactive oxygen species, in hypoxic environments. The 24-subunit iron storage protein bacterioferritin (Bfr) has the unique property of binding 12 heme groups in its protein shell. We inserted the isostructural photosensitizer, zinc(II) protoporphyrin IX (ZnP), in place of the hemes and extended the surface-exposed N-terminal ends of the Bfr subunits with a peptide targeting a receptor that is hyperexpressed on the cell surface of many tumors and tumor vasculature. We then loaded the inner cavity with ~2500 irons as a ferric oxyhydroxide polymer, and finally conjugated 2 kDa polyethylene glycol to the outer surface. We showed that the inserted ZnP photosensitizes generation of both singlet oxygen and the hydroxyl radical, the latter via the reaction of photo-released ferrous iron with hydrogen peroxide. This targeted ironloaded ZnP-Bfr construct was endocytosed by C32 melanoma cells and localized to lysosomes. Irradiating the treated cells with light at wavelengths overlapping the ZnP Soret absorption band induced photosensitized intracellular Fe2+ release and substantial lowering of cell viability. This targeted, light-triggered production of intracellular singlet oxygen and Fenton-reactive iron could potentially be developed into a phototherapeutic adjunct for many types of cancers.


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Introduction Targeted exogenous agents that elevate reactive oxygen species (ROS) above toxic threshold levels has been touted as a potentially effective, but largely untested cancer therapeutic strategy.1,2 The major clinically used ROS-mediated cancer therapy, referred to as photodynamic therapy (PDT), relies predominantly on dye-photosensitized generation of singlet oxygen.3-6 The cumulative damage resulting from reactions of singlet oxygen with cellular components can result in cell death via necrosis or apoptosis. PDT also damages tumor vasculature, leading to nutrient and oxygen starvation. An advantage of PDT is that the therapy is activated by exposure to intense light, which can be directed to the cancerous tissue. PDT is currently used to treat several types of cancer, and numerous clinical trials are underway.6 Up to now PDT has shown limited effectiveness in the clinic, which is attributed to, among other things, inefficient light penetration through tissues, and reliance on passive rather than targeted uptake of photosensitizers by tumors. The vast majority of clinically used PDT photosensitizers are porphyrins and porphyrinoid compounds; these suffer from low solubility in bodily fluids as well as aggregation, which lowers photosensitization efficiency. Hydrophilic nanoparticles as carriers for porphyrin-type photosensitizers have been used to ameliorate these limitations.7-9 Another, perhaps more fundamental limitation of PDT is that therapeutically effective singlet oxygen generation requires well-oxygenated tissues, whereas many tumor environments tend to be hypoxic.10-15 Hydroxyl radical (OH•) is typically regarded as the most toxic among ROS. In the presence of “free” intracellular iron, hydroxyl radical can be generated via the Fenton reaction: Fe2+ + H2O2 Fe3+ + OH- + OH•.2,16-18 Fenton-derived hydroxyl radicals damage lipids, proteins and DNA.2 The hydrogen peroxide for the intracellular Fenton reaction is produced as a


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byproduct of oxygenic respiration. The Fe3+ product of the Fenton reaction can be re-reduced to Fe2+ by intracellular reductants, which are relatively abundant in hypoxic environments. The free iron can, thus, catalyze intracellular generation of hydroxyl radicals even under hypoxic conditions and at relatively low (micromolar or lower) steady state levels of hydrogen peroxide.16,19 Hydrogen peroxide concentrations tend to be elevated in cancerous cells and tumor microenvironments.20-23 Excess intracellular ferrous iron can also reduce lipid hydroperoxides to lipid alkoxy radicals, contributing to a type of cell death referred to as ferroptosis.24,25 Strategies for delivery of Fenton-reactive iron within nanoparticles to cancer cells have shown some promise in mouse models,26-29 but we know of no previously reported cancer therapy that photosensitizes both generation of singlet oxygen and release of Fenton-reactive Fe2+. We describe proof-of-concept for a phototherapy that would overload cells with singlet oxygen in oxic environments, “free” Fenton-reactive Fe2+ in hypoxic environments, and do so

Scheme 1. Strategy for photosensitized targeting of singlet oxygen and Fenton reactive iron to cancer cells using the Bfr delivery platform.


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using a tumor-targeted protein nanocarrier. Our approach is illustrated in Scheme 1. We use the iron storage protein bacterioferritin (Bfr) as the platform for targeted delivery of both photosensitizer and iron.30 Relevant features of Bfr are shown in Figure 1. Bfr is structurally and functionally analogous to mammalian ferritins, consisting of 24 identical subunits forming an approximately spherical 12 nm shell (Figure 1A) surrounding an ~ 8 nm cavity (Figure 1B), which can store up to ~3,000 iron atoms as a ferric oxyhydroxide polymer ([FeO(OH)]n).31 The [FeO(OH)]n can be reduced to Fe2+ for facile release through pores in the protein shell.32

Figure 1. Structural features of Escherichia (E.) coli Bfr. (A) 24-subunit Bfr with hemes yellowhighlighted, and a head-to-tail subunit pair highlighted in blue and green. (B) Slice through the 24-mer showing the interior cavity and hemes embedded in the protein shell. (C) Surface view showing locations of exposed N-terminal ends (cyan) and Glu81 residues (red). (D) Heme. Protein structure drawings used coordinates from Protein Data Bank entry, 1bfr.33


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A key feature of Bfr for our approach is its binding of up to 12 hemes (Figure 1D) with each heme sandwiched between subunits of a head-to-tail dimer (Figure 1A). The hemes may be involved in the redox chemistry of iron uptake or release, although this function has not been clearly established in all Bfrs.32 As shown in Scheme 1, our proposed approach substitutes zinc(II)-protoporphyrin IX (ZnP) in place of the hemes in Bfr. ZnP is isostructural with heme (Figure 1D) but with Zn(II) substituted in place of Fe. ZnP is a well-established photosensitizer, and has been used for PDT in mouse models.34,35 The ZnP triplet excited state, 3ZnP*, can produce singlet oxygen in oxic environments.34-37 3ZnP* is also highly reducing.38-40 Two of us have recently shown that ZnP can be quantitatively substituted into the heme binding sites of E. coli Bfr, and that the ZnP-Bfr can photosensitize reduction of H+ to H2 on the surface of platinum nanoparticles in the presence of sacrificial electron donors upon irradiation with visible (white) light.41,42 We, therefore, reasoned that 3ZnP* in the Bfr protein shell could reduce Fe3+ in the enclosed [FeO(OH)]n core to the more labile, mobile, and Fenton-reactive Fe2+, which would diffuse out of the protein shell.32,43 ZnP is naturally present in blood44 and could, therefore, be less systemically toxic than currently used PDT dyes. Insertion into Bfr also effectively solubilizes and isolates ZnP molecules, which would otherwise aggregate in aqueous solution and lower photosensitization efficiency. Cancer cells generate high fluxes of both H2O2 and NAD(P)H due to increased glycolysis and mitochondrial metabolism.21,45-47 We, therefore tested NADH as a “sacrificial” electron donor in the photosensitized iron release. Mammalian ferritins do not bind porphyrins or metalloporphyrins in a stable fashion, and are, therefore, not suitable for our photochemical approach. Mammalian ferritins have, however, been used as nanocages for delivering both imaging and therapeutic cargoes to cancers.48-52 The


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12-residue disulfide-cyclized peptide, RGD4C (CDCRGDCFC), has been attached to the Nterminal ends of ferritin subunits to enhance uptake by cancer cells and tumors.50,53,54 RGD4C is well established to target αvβ3 integrins, which are hyperexpressed on cell surfaces of many cancers cells and tumor vasculature.55 We, therefore, extended the surface-exposed N-terminal ends of the Bfr subunits (Figure 1C) with the RGD4C peptide (RGD in Scheme 1) to create a multivalent cancer-targeted protein shell. In order to facilitate exposure of the RGD4C peptide on the outer surface of the 24-mer we inserted a 30-residue Ser/Gly-rich linker49 between the Nterminal RGD peptide and the Bfr N-terminus. E. coli Bfr does not contain any native Cys residues, which could conceivably interfere with the photochemistry. This absence also allowed us to introduce a Cys residue for sitespecific chemical modifications. We, therefore, substituted a Cys residue in place of Glu81, 24 of which protrude from the outer surface of the E. coli Bfr protein shell (Figure 1C). We hereafter refer to this engineered C (Cys81), R (RGD4C), L (linker) E. coli Bfr variant as CRL-Bfr, and the ZnP-containing CRL-Bfr as ZnP-CRL-Bfr. Coating with polyethylene glycol (PEG) is often used to mask foreign proteins from the immune system.49,56 We, therefore, conjugated a methoxy PEG MW 2000 (P2K) to Cys81 to obtain P2K-ZnP-CRL-Bfr. We chose to test our construct on a melanoma cell line known to express the RGD4C-targetable αvβ3 integrin.53 Skin cancers are among the most readily accessible to phototherapy.

Materials and Methods Reagents and general procedures. Reagents and buffers were of the highest grade commercially available. DMPO was purchased from Dojindo Molecular Technologies, Inc. ZnP was prepared by metallation of protoporphyrin IX (Frontier Scientific) with zinc acetate


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dihydrate in DMSO.41,57 Methoxy PEG-maleimide, MW 2K (P2K-maleimide) was purchased from Creative PEGWorks. All other chemicals were purchased from Fisher Scientific or SigmaAldrich. All reagents, protein, and media solutions were prepared using water purified with a Millipore ultrapurification system to a resistivity of 18MΩ to minimize trace metal ion contamination. Other than for light irradiation experiments, all manipulations of solutions, cell cultures, and proteins containing ZnP were conducted under reduced room light and/or in aluminum foil-wrapped containers. The Fe2+ imaging dye, Rhonox-1, was synthesized in the Medicinal Chemistry and Synthesis Core Facility of the Center for Innovative Drug Discovery using a published procedure.58 Bfr expression plasmids. The expression plasmid, pT7-7:Bfr, containing the gene encoding E. coli Bfr has been described previously.59 A polynucleotide encoding CRL-Bfr between codons for residues 2 and 3 and containing 5′ NdeI and 3′ BamHI restriction sites was synthesized and inserted into pT7-760 by GenScript (Piscataway, NJ). The resulting expression plasmid,

pT7-7:CRL-Bfr,

encoded

the

amino

acid

sequence:

MKCDCRGDCFCGGGGSGGGGSGGGGSGGGGSGGGGSGGGGDTKVINYLNKLLGNEV AINQYFLHARMFKNWGLKRLNDVEYHESIDEMKHADRYIERILFLEGLPNLQDLGKLNI GCDVEEMLRSDLALELDGAKNLREAIGYADSVHDYVSRDMMIEILRDEEGHIDWLETEL DLIQKMGLQNYLQAQIREEG, listed with RGD4C residues in bold, 30-residue glycine/serine linker italicized, and C81 bold/italicized. Protein overexpression and purification. E. coli BL21(DE3) competent cells (Invitrogen) were transformed with either pT7-7:Bfr or pT7-7:CRL-Bfr. ZnP-Bfr and ZnP-CRLBfr were expressed and purified as described previously except that 35 mg ZnP in 10 mL DMSO were added to 1 L cultures at the time of induction of protein expression. Typical yields of the


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purified proteins were 50 mg of ZnP-Bfr or 20 mg of ZnP-CRL-Bfr per 1 L culture. The corresponding heme-CRL-Bfr was expressed and purified analogously, except that 7 mg heme/L culture was substituted for ZnP. The concentrated purified proteins were stored in aluminum foilwrapped Eppendorf tubes at -80 0C until use. Protein, iron and ZnP analyses. ZnP in CRL-Bfr was quantified by UV-Vis absorbance using ε433

nm

= 120,000 M-1cm-1.41,57 Protein was quantified by the bicinchoninic acid

colorimetric assay (Pierce™ 660nm Protein Assay). Iron content was quantified using either a modified ferrozine assay61 or inductively coupled plasma-optical emission spectrometry. For the ferrozine assay 200 µL of protein solution containing ~20 µM iron was mixed with, in order, 250 µL of 0.2% sodium ascorbate, 25 µL 5 mg/mL ferrozine, and 30 µL of 6 M HCl. After vortex mixing for 30 seconds, 1 mL of 6 M guanidine hydrochloride and 200 µL of saturated ammonium acetate were added. The solution was mixed again for 30 seconds and allowed to react for either 10 minutes for as-isolated protein or overnight for iron-loaded protein. The iron was quantified by measuring the absorbance at 562 nm (ε562

nm

= 28,000 M-1cm-1).62

Oligomerization state of the Bfr in solution was determined by DLS, as described previously (50 nM Bfr 24-mer, 1 mL sample volume).41,42 Cancer cell line and culturing. Human amelanotic melanoma cell line, C32 (ATCC® CRL-1585™), was purchased from the American Type Culture Collection. Adherent C32 cells were cultured in Minimum Essential Medium Eagle (MEME, ATTC 30−2003) with L-glutamine supplemented with 10% fetal bovine serum (Gibco), 1.0 mM sodium pyruvate (Gibco), 0.1 mM non-essential amino acids (Gibco), 1.5 g/L sodium bicarbonate (complete MEME) at 37 °C in a 5% CO2 atmosphere. Cells were grown and passaged every 2-3 days in 25 mL cell culture flasks (Corning). Cells used for light irradiation experiments were cultured in phenol red-free complete


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MEME supplemented with antibiotic-antimycotic (Gibco) (100 µg/mL of penicillin, 100 µg/mL of streptomycin, and 0.25 µg/mL of Fungizone). PEGylation. ZnP-CRL-Bfr was incubated in 50 mM 2-(N-morpholino)ethanesulfonic acid (MOPS) pH 7.3 for 4 hours at room temperature with a 20-fold molar excess over Bfr monomer of P2K-maleimide. The excess unreacted reagent was removed by passing the mixture over a HiTrap desalting column (GE Healthcare Life Sciences) and monitoring UV-absorption of the eluate at 280 nm to confirm separation. The extent of P2K-maleimide conjugation was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% acrylamide) of PEGylated and non-PEGylated ZnP-CRL-Bfr from the same batch of purified protein. Iron loading. Incorporation of iron as a ferric oxyhydroxide polymer into the Bfr cavity was carried out by modification of a published protocol.31 An anaerobic stock solution of 0.02 mM ferrous ammonium sulfate was freshly prepared in deoxygenated ultrapure water under a N2 atmosphere in a glovebox (Vacuum Atmospheres). An aliquot of this Fe2+ stock solution was injected via gas tight syringe into a septum-capped vial containing an air-saturated solution of 1 µM Bfr 24-mer in 100 mM MOPS pH 6.5 at 25 °C to achieve 200 molar equivalents Fe2+/Bfr 24mer. This mixture was incubated at room temperature for 15-20 minutes, and then centrifuged (5,500 × g for 10 min) to remove any precipitate. This iron addition/centrifugation was repeated to achieve a total of 3000 added mol Fe/mol 24-mer in a final volume of 5 mL. The same protocol was used for iron loading of the empty ZnP-CRL-Bfr, P2K-ZnP-CRL-Bfr, or heme-Bfr and reproducibly resulted in an iron content of ~2500 iron per Bfr 24-mer, as determined by ferrozine, ICP-OES, and protein assays. Buffers were exchanged by passage of the empty or iron-loaded proteins over desalting columns.


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Transmission electron microscopy. A sample of iron-loaded P2K-ZnP-CRL-Bfr was diluted in 50 mM MOPS pH 7.4 to 50 nM in 24-mer. A small aliquot of this diluted sample was deposited onto an ultrathin holey carbon-coated copper grid (Ted Pella) and negatively stained with uranyl acetate. Transmission electron microscopy (TEM) was performed on a JEOL-2010F microscope operating at 200kV. The images shown in Figure 3 were obtained using a primary magnification of 150,000x. Light-triggered iron release. All experiments were conducted at room temperature on Bfrs loaded with 2000-2500 Fe/24-mer. Stock solutions of iron-loaded ZnP-Bfr, P2K-ZnP-CRLBfr, or heme-Bfr (0.1 µM Bfr 24-mer) containing 10 mM NADH and 150 mM NaCl (unbuffered, pH ~7) in deaerated ultrapure water were prepared in a Vacuum Atmospheres glove box under an N2 atmosphere. 1 mL of the stock solution was placed in a 1 cm pathlength semimicro quartz cuvette containing a 2 mm stir flea. The cuvette was then capped with a rubber septum, removed from the glove box, and placed on a stir plate 5 cm from the lens of a 300W 82V EXR halogen lamp in a Kodak Ektagraphic 3 AMT projector with wavelength cutoffs below 390 nm and above 700 nm (HOYA 62 mm UV-IR multi-coated filter). Either a 426±25 nm or a 550±25 nm band pass filter (90 mm by 40 mm, Optical Filter Shop) was placed between the projector lamp and cuvette. Irradiance was measured using a Konica Minolta CL-500A Illuminance spectrophotometer. Irradiance at the sample was adjusted to approximately 60mW/cm2 for each band pass filter by altering the distance between the sample and the light source. At selected irradiation times, the cuvette was removed from the projector beam, ferrozine was added from a concentrated stock solution to achieve a concentration of 10 mM, and the absorbance at 562 nm was measured to quantify the released iron.62 This procedure was repeated for separate 1 mL portions of the stock protein solution for each irradiation time listed in the


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results. Parallel “dark” control samples from the same stock solution were placed in aluminum foil-wrapped cuvettes, placed on a stir plate and assayed for iron release after 10 h. Reactive oxygen species assays. Singlet Oxygen Green Sensor Reagent (Invitrogen) was used to monitor singlet oxygen generation during aerobic irradiation of iron-loaded P2K-ZnPCRL-Bfr. The protein samples were prepared in a Vacuum Atmospheres glove box under an N2 atmosphere. All Bfr samples used for singlet oxygen measurements were in 150 mM NaCl (unbuffered, pH ~7) aqueous solution containing 50% deuterated water. Various volumes of airsaturated 150 mM NaCl solution 50% D2O solution were injected into the septum capped sample vials to attain the listed percentages of air saturation and a final concentration of 30 nM Bfr 24mer (0.36 µM ZnP). The singlet oxygen sensor reagent was added to each sample according to the manufacturer’s instructions. The samples were irradiated for 2 hours as described above for iron release. The fluorescence emission of the singlet oxygen sensor was recorded at 525 nm (excitation at 488 nm) with a SpectraMax5 plate reader. All experiments were performed in triplicate. Singlet oxygen percentages were normalized relative to the fluorescence response of the 100% air-saturated samples. A parallel set of iron-loaded P2K-ZnP-CRL-Bfr samples containing 10 mM NADH was irradiated and assayed for Fe2+ release at the same Bfr concentration and percentages of air saturation as used for the singlet oxygen assays. Production of hydroxyl radical was monitored by a standard EPR spin-trapping method.63,64 1 mL samples from a stock solution of 30 nM iron-loaded P2K-ZnP-CRL-Bfr in 150 mM NaCl (pH ~ 7) were added to a triplicate set of rubber septum-capped vials. The samples were made 2 mM in NADH and simultaneously irradiated under anaerobic conditions at the same irradiance (60 mW/cm2) used for measurement of photo-triggered iron release. Immediately following irradiation for the indicated time, to each protein sample was added via


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syringe 12.5 µL of a 9 M anaerobic stock solution of DMPO in water to achieve a concentration of 50 mM DMPO in the sample followed by addition of small volumes of an anaerobic stock solution of ~200 mM H2O2 to a achieve concentrations of 0.1, 1, or 10 mM H2O2 in individual samples. 700 µL of the DMPO/H2O2-treated sample was loaded anaerobically into a 4-bore AquaX EPR cell (Bruker). EPR spectra were recorded at room temperature on a Bruker EMX080 spectrometer equipped with a Bruker ER 041 XG microwave bridge. Spectra were also obtained of identically prepared irradiated samples except with no added H2O2 or “dark” samples containing 10 mM H2O2 in which the vials were covered with aluminum foil. Due to the limited lifetime (t1/2 ~ 55 min) of the DMPO hydroxyl radical adduct,65 we could not obtain reproducible signal intensities when the spin trap and H2O2 were added either prior to or during irradiation of the samples. EPR spectral parameters were: microwave frequency 9.86 GHz, microwave power 6.8 mW, modulation frequency 100 kHz, modulation amplitude 1 G, scan rate 40.96 G/s; total scan time 41.9 s, time constant 40.96 ms. Each EPR spectrum is the average of six scans. Fluorescent dye labeling of Bfr. Bfr, ZnP-CRL-Bfr, or P2K-ZnP-CRL-Bfr, (0.5 µM 24mer in 1 mL) in 50 mM MOPS, 250 mM NaCl, pH 7.4 was reacted with Alexa Fluor™ 488 Nhydroxysuccinimde ester (ThermoFisher) or Cy5® N-hydroxysuccinimide ester (Lumiprobe GmbH) at 20 mol equiv dye/Bfr monomer at room temperature for 4 hours as per the manufacturer’s instructions. Unbound fluorophore was removed by passage over several HiTrap desalting columns. Quantification of conjugated dye was determined using UV-vis absorbance at 488 or 650 nm, respectively and the manufacturers’ published extinction coefficients to be reproducibly 20-22 dyes molecules/24-mer. EC50. C32 cells were trypsinized, divided in 100000 cells/sample, incubated for 1 hour with various concentrations of Alexafluor 488-labeled-ZnP-Bfr, ZnP-CRL-Bfr, or P2K-ZnP-


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CRL-Bfr), then incubated for another 20 min with propidium iodide for quantification of cell viability. Cells were washed three times with cold phosphate buffered saline (PBS) pelleted by centrifugation and resuspended by vortexing in Dulbecco's phosphate buffered saline (DPBS) with 0.1% Triton X-100. For each sample 20000 cells were counted and analyzed using an LSRII flow unit in the University of Texas at San Antonio (UTSA) Flow Cytometry Core Facility. All measurements were done in triplicate. Compensation correction, gating, and statistical data were derived using the FlowJo software. The EC50 values were determined by fitting a single dose-response function available in Origin (OriginLab®) to the data. Confocal fluorescence microscopy. The binding of empty or iron-loaded, P2K-ZnPCRL-Bfr to live C32 cells were visualized on a Zeiss 710 confocal microscope in the University of Texas at San Antonio Biophotonics core. The C32 cells were grown in NuncTM glass bottom Petri dishes as described above (105 cells per dish). The cells were treated with either empty or iron-loaded the Cy5-labeled P2K-ZnP-CRL-Bfr to achieve a concentration of 100 nM (in 24mer) and incubated for 2 h. The Fe2+ imaging dye, Rhonox-1, was applied to the cells according to a published method,58 followed by treatment with immunostaining fluorescent probes (NucBlue® Live Cell Stain (Hoechst 33258) for nuclear membrane, and LysotrackDND99TM (ThermoFisher) for lysosomes, and CellMask Red™ (ThermoFisher) for plasma membrane according to manufacturer’s guidelines. The cells were washed three times with DPBS, followed by addition of Live Cell Imaging media (ThermoFisher). Cells were irradiated for 30 minutes with 426±25 nm light (60 mW/cm2) prior to imaging. Dishes were imaged on the Zeiss 710 microscope using a 63x objective. C32 cell killing. Cell viabilities were assayed by a standard fluorescence method. Approximately 20,000 C32 cells were seeded and cultured in complete MEME in 96-well black-


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sided, optically clear-bottom polystyrene microplates. After ~12 h incubation, the medium was replaced with 100 µL of complete MEME supplemented with antibiotic-antimycotic containing either empty or iron-loaded P2K-ZnP-CRL-Bfr to achieve concentrations within the ranges of either 0-200 or 0-1000 nM 24-mer, respectively. The plates were incubated at 37 0C in the 5% CO2 incubator for 3 hours. The plates were removed from the incubator and placed inside a fitted opening at the bottom of a closed Plexiglas® box and equilibrated for 15 minutes with the CO2 incubator atmosphere. The plate at the bottom of the box was then placed approximately 10 cm above the light source on top of either the 426±25 nm or the 550±25 nm band pass filter to obtain an emission irradiance of ~60 mW/cm2 on a 4 x 4 square of 16 wells. Half of the plate was covered with aluminum foil and served as the dark control. The wells were irradiated for 30 minutes, and the plate was then returned to the incubator. After 48 hours the medium was decanted, and cells were washed twice with DPBS. Cell viability was measured using the CyQuant NF® assay (Life TechnologiesTM). The fluorescent substrate was added per manufacturer’s instructions, the plate was incubated for 30 min at 37°C, and the fluorescence emission measured at 520 nm (excitation at 480 nm) on a SpectraMax® plate reader. All data points represent the average of viabilities measured on 4-by-4 squares of 16 simultaneously irradiated wells and on an analogous set of dark control wells. IC50 values were determined by fitting of a single dose-response function to the data in Origin. Significant differences in the fitted IC50 values were assessed using the Anova test (p < 0.05).

Results and Discussion Characterization of P2K-ZnP-CRL-Bfr. The UV-vis absorption spectrum of P2K-ZnPCRL-Bfr (Figure 2A) is essentially identical to that previously reported for ZnP-Bfr,41 showing a


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Soret absorption maximum at 433 and Q-band maxima at 586 and 592 nm. A mol ratio of ZnP/Bfr 24-mer within the range of 10-12 was consistently obtained for ZnP-CRL-Bfr, as previously reported for ZnP-Bfr.41 The ZnP spectrum in CRL-Bfr can be readily distinguished from that of the corresponding native heme spectrum, which shows a Soret absorption maximum at 418 nm.66 The narrow absorption bands indicate a homogenous ZnP environment, and the identical spectral features of the P2K-ZnP-CRL-Bfr to those of ZnP-Bfr are consistent with insertion of the ZnP into the 12 binding sites between the subunit dimers, i.e., at the same location as the native heme in Bfr (Figure 1). Neither extensive dialysis of P2K-ZnP-CRL-Bfr nor the other manipulations described below resulted in any detectable loss of ZnP or a significant change in its absorption spectrum, indicating that ZnP binding to CRL-Bfr is irreversible and stable under our conditions. The as-purified ZnP-CRL-Bfr reproducibly contained less than 10 irons/24-mer, and is referred to as “empty”. The UV-vis absorption spectrum of the P2K-ZnP-CRL-Bfr loaded with ~2,500 irons (Figure 2A) shows the expected relatively featureless absorption of the [FeO(OH)]n rising into the UV region underneath the ZnP spectrum, which is not detectably perturbed by the iron loading. Conjugation of P2K was quantitative (~1 P2K per ZnP-CRL-Bfr subunit), as assessed by SDS-PAGE (Figure S1). Dynamic light scattering (DLS) (Figure 2B) shows a homogeneous distribution of ZnPCRL-Bfr particles centered at a hydrodynamic radius of 13 nm, which was not significantly affected by PEGylation. TEM of the uranyl acetate-stained (Figure 2C) iron-loaded P2K-ZnPCRL-Bfr showed the expected ~12 nm spherical protein shells.67 The darker regions are due to [FeO(OH)]n within the protein shells, which are better visualized in the unstained TEM (Figure 2D).


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Figure 2. (A) UV-vis absorption spectra of 2.6 µM P2K-ZnP-CRL-Bfr (~7 µM in ZnP), and iron-loaded P2K-ZnP-CRL-Bfr in 50 mM MOPS pH 7.3. (B) DLS size distribution by volume of ZnP-CRL-Bfr, and P2K-ZnP-CRL-Bfr. (C) TEM of iron-loaded P2K-ZnP-CRL-Bfr negatively stained with uranyl acetate. (D) TEM of unstained iron-loaded P2K-ZnP-CRL-Bfr. In panels C and D, the scale bars indicate 60 nm and 50 nm, respectively.


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Photo-triggered iron release from iron-loaded ZnP-CRL-Bfr. Figure 3 shows that under anaerobic conditions in the presence of 10 mM NADH up to 95% of the iron in ironloaded P2K-ZnP-CRL-Bfr could be released as Fe2+ upon 3 hours of 426±25 nm light irradiation at 60mW/cm2. Comparable amounts of Fe2+ were released after 8 or 10 hours exposure to similar intensity 550±25 nm or white light irradiation, respectively. These experiments were conducted under anaerobic conditions so that the Fe2+ could be quantified by addition of ferrozine immediately after removal of the cuvette from the light source. Ferrozine rapidly forms a quantifiable colored complex with released Fe2+ but does not form a complex with Fe3+ of [FeO(OH)]n within the Bfr 24-mer. The rate and extent of photo-triggered release of Fe2+ from the iron-loaded ZnP-Bfr (not shown) was indistinguishable from that shown in Figure 3 for P2KZnP-CRL-Bfr, i.e., the iron release was unaffected by the fused RGD-linker peptide or by PEGylation. No precipitation was observed during these experiments. No significant iron release was observed under otherwise identical “dark” conditions. Under the same conditions the ironloaded heme-CRL-Bfr showed no significant photosensitized Fe2+ release. Reactive oxygen species. Figure 4A shows percentages of photosensitized singlet oxygen generation and iron released from P2K-ZnP-CRL-Bfr as a function of percentage air saturation. These results indicate a correlation between O2 concentration and photosensitization pathway, namely, either singlet oxygen generation at higher O2 or reduction of the iron core at lower O2. Figure 4B shows results of a standard EPR spin trapping assay using 5,5′-dimethyl-1pyrroline-N-oxide (DMPO),63,64 which demonstrated that the photo-released Fe2+ was capable of generating hydroxyl radical in the presence of hydrogen peroxide under anaerobic conditions, i.e., the photo-released iron was Fenton-reactive. The hydroxyl radical yield increased with increasing H2O2 concentration (Figure 4B) and also with increasing irradiation time (Figure S2).


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Figure 3. Time dependence of photosensitized iron release from P2K-ZnP-CRL-Bfr upon 60 mW/cm2 irradiation at the indicated wavelengths. Each bar represents the average of triplicate determinations, and error bars represent standard deviations. Solutions contained 100 nM Bfr 24mer and 10 mM NADH in 150 mM NaCl pH ~ 7.


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Figure 4. (A) Dependence of singlet oxygen generation and iron release on solution aeration. (B) hydroxyl radical generation monitored by EPR spectroscopy after irradiation and addition of hydrogen peroxide to the indicated millimolar concentrations listed near each spectrum. All solutions contained 30 nM iron-loaded P2K-ZnP-CRL-Bfr in 150 mM NaCl and for iron release also contained 10 mM (Panel A) or 2 mM (Panel B) NADH. Solutions were irradiated for 2 h with 426±25 nm light. Error bars in Panel A represent standard deviations from averages of


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triplicate determinations. For Panel B the solutions contained 10 mM DMPO (added after irradiation).

Binding to and localization of ZnP-CRL-Bfr in C32 melanoma cells. Flow cytometry results for binding affinities are plotted in Figure 5, and the fitted EC50s values are listed in the figure legend. They show a statistically significant (Anova, p < 0.05) three- to four-fold increase in binding affinity of the P2K-ZnP-CRL-Bfr over that of the ZnP-Bfr, which can be attributed to the RGD4C peptide. The lower fitted EC50 for the P2K-PEGylated vs non-PEGylated ZnP-CRLBfr is not statistically significant. That is, P2K conjugation does not significantly interfere with receptor binding.

Figure 5. EC50 plots obtained from flow cytometry of ZnP-Bfr (closed circles), ZnP-CRL-Bfr (closed squares), and P2K-ZnP-CRL-Bfr (open circles) with corresponding fitted curves (solid, dashed, and dotted), which gave respective EC50 values of 800, 300, and 200 nM.


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The confocal electron microscopy results in Figure 6 show that both empty and ironloaded P2K-ZnP-CRL-Bfr were endocytosed by C32 cells and localized to lysosomes. The Fe2+labeled row of Figure 6 shows that the empty Bfr does not absorb or transport significant amounts of iron from the medium into the cells. Photosensitized intracellular iron release and killing. The Fe2+-labeled row in Figure 6 shows that upon irradiation with 426 nm light, iron from endocytosed iron-loaded P2K-ZnPCRL-Bfr was released into the lysosomes and subsequently diffused through the cell in as little as 30 minutes after the start of irradiation. Figure 7 shows that, in the absence of light irradiation (“dark”), C32 melanoma cell viability after 48 h is essentially unaffected by treatment with up to 1 mM of either empty or iron-loaded P2K-ZnP-CRL-Bfr. At the lowest Bfr concentrations or in the absence of Bfr, light irradiation also did not affect cell viability. However, above ~20 nM Bfr (24-mer), irradiation with 426 nm light for 30 minutes resulted in a significant dose-dependent reduction in 48-h cell viability. Greater than 95% photosensitized cell killing was achieved at 200 nM iron-loaded P2K-ZnP-CRL-Bfr with little or no dark killing at the same concentration. Fitting of a single dose-response function to the irradiated cell viability data gave fitted IC50 values of 220 and 160 nM for the empty and iron-loaded P2K-ZnP-CRL-Bfr, respectively.

Although this difference in IC50 is relatively modest, the pairs of starred data points at 100, 150, and 200 nM Bfr in Figure 7 indicate statistically significant differences (Anova, p < 0.05) for Empty light vs Loaded light cell viabilities, which are each the average for 16 simultaneously irradiated wells.


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Figure 6. Live cell confocal fluorescence microscopy showing binding, localization and iron release of Cy5-labeled P2K-ZnP-CRL-Bfr on C32 melanoma cells. Cells were exposed to 100 nM (24-mer) of either the empty or iron-loaded protein in the dark incubator for 2 h, and then washed. Dishes were exposed to 426±25 nm irradiation for 30 min, while a parallel set was kept in the dark. Each column shows the same cells stained to visualize organelles, Bfr, or released Fe2+ according to row label. White bars indicate 20 µm.


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Figure 7. Photo-induced killing of C32 melanoma cells treated with varying concentrations (24mer) of empty or iron-loaded P2K-ZnP-CRL-Bfr. 48-h cell viabilities were measured after 30 minutes of 426±25 nm light exposure at 60 mW/cm2 (“Empty light” and “Loaded light”) and on a parallel set of dark controls (“Empty dark” and “Loaded dark”). Error bars represent standard deviations for the average of 16 determinations. Three pairs of open and closed stars represent statistically significant differences (Anova p < 0.05) for Empty Light vs Loaded light at 100, 150 and 200 nM Bfr. Fitted curves were derived using the Levenberg–Marquardt fitting algorithm based on the single dose-response equation.

Conclusions Our results provide proof-of-concept for a targeted cancer phototherapy operating according to the bifurcated photochemistry illustrated in Scheme 1. The photoexcited ZnP generates singlet O2, as expected. Additionally the ferric oxyhydroxide polymer “prodrug” hidden within the targeted Bfr “Trojan horse” can be activated by photosensitized reduction and release of Fenton reactive Fe2+. In the presence of NADH, essentially all of the ~2500 irons in the ferric oxyhydroxide polymer inside the P2K-ZnP-CRL-Bfr 24-mer could be released as Fe2+


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upon 3 h irradiation with light in a wavelength range that overlaps the Soret absorption of ZnP. The observation that little or no iron was released upon light irradiation of iron-loaded heme-Bfr further supports ZnP’s role as the photosensitizer in this process. In the cell free system no photosensitized iron release from iron-loaded P2K-ZnP-CRL-Bfr was observed in air, and photosensitized iron release occurred most efficiently under anaerobic conditions. We infer from these observations that the photosensitized Fe2+ release is not due to any process involving O2. The photosensitized iron release mechanism presumably involves either reductive or oxidative quenching of the 3ZnP* by either NADH or the core Fe3+, respectively.41,42 Light irradiation overlapping the ZnP Q band absorptions was less efficient in photosensitizing Fe2+ release. The more efficient iron release using irradiation overlapping the Soret absorption is consistent with the higher extinction coefficient of the Soret band, which could lead to higher steady state levels of ZnP photo-excited states. We observed substantial photosensitized Fe2+ release from iron-loaded ZnP-CRL-Bfr in solutions containing sub-aerobic concentrations of O2 (Figure 4A), which more closely mimic the hypoxic intracellular environment of tumors. Intracellular “free” ferrous iron was observed only in cells treated with the iron-loaded protein, and much more free Fe2+ was observed upon irradiation of these treated cells. These observations are consistent with the notion of photosensitized reduction of Bfr core iron by intracellular reducing equivalents and release of the ferrous iron from the protein, paralleling the cell free results. The higher acidity and protease activity of lysosomes could also promote the photosensitized reduction of Fe3+ and release of Fe2+. The observation that empty P2K-ZnP-CRL-Bfr was nearly as effective as the iron-loaded protein in lowering cell viability upon irradiation is consistent with our observation of photosensitized production of singlet oxygen by the empty P2K-ZnP-CRL-Bfr under aerobic and


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even sub-aerobic conditions. However, the statistically significant photosensitized lowering of cell viability by the iron-loaded vs empty protein is consistent with our observation of photosensitized Fe2+ release in cells treated with the iron-loaded protein, even when cultured under an essentially aerobic atmosphere (5% CO2). The collective results support the notion that both singlet oxygen and Fe2+ release contributed to cell killing and support a gradual shift from photosensitization of singlet oxygen generation in aerobic environments to Fenton-reactive ferrous iron release in hypoxic or anaerobic environments. Photosensitized killing of C32 cells treated with P2K-ZnP-CRL-Bfr was most efficient for irradiation wavelengths overlapping the ZnP Soret absorption. Chromophores with absorptions bands closer to the “phototherapeutic optical window” (650-850 nm) are thought to be optimal for light penetration through tissue.13,68 However, irradiation with 420 nm light of xenograft tumors in mice pre-treated with polymer-conjugated ZnP has also been reported to be an effective PDT.35 Higher yields of porphyrin photo-excited states and singlet oxygen when irradiating at wavelengths overlapping the Soret absorption may compensate for decreased depth of light penetration. The iron-loaded ZnP-CRL-Bfr is easy to produce in high yield. Other tumor-targeting peptides have been attached to ferritins,48,49 and numerous other cell receptor targeting peptides are known.55 Our Trojan horse protein nanoscaffold should be adaptable to phototherapeutic treatment of internal or resected tumors using optical fibers.69 This delivery platform could, thus, serve as a potential adjuvant phototherapy for many types of cancers.


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Supporting Information. SDS-PAGE of ZnP-CRL-Bfr and P2K-ZnP-CRL-Bfr (Figure S1) and time dependence of hydroxyl radical formation (Figure S2) (PDF). The Supporting Information is available free of charge on the ACS Publications website at DOI:

Acknowledgments This research was supported by a grant from the Cancer Prevention and Research Institute of Texas (RP110165 to D.M.K). The UTSA Biophotonics Core facility and Flow Cytometry Core facility are supported by a grant from the National Institute on Minority Health and Health Disparities from the National Institutes of Health (G12MD007591). H. Shipley in the UTSA Department of Civil and Environmental Engineering, and K. Nash in the UTSA Department of Physics and Astronomy provided access to ICP-OES and DLS instrumentation, respectively. Q. Cao conducted some initial experiments.

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63. Finkelstein, E.; Rosen, G. M.; Rauckman, E. J. Spin Trapping of Superoxide and Hydroxyl Radical - Practical Aspects. Arch. Biochem. Biophys. 1980, 200 (1), 1-16. 64. Hawkins, C. L.; Davies, M. J. Detection and Characterisation of Radicals in Biological Materials Using EPR Methodology. Biochim. Biophys. Acta 2014, 1840 (2), 708-721. 65. Villamena, F. A.; Hadad, C. M.; Zweier, J. L. Kinetic Study and Theoretical Analysis of Hydroxyl Radical Trapping and Spin Adduct Decay of Alkoxycarbonyl and Dialkoxyphosphoryl Nitrones in Aqueous Media. J. Phys. Chem. A 2003, 107 (22), 4407-4414. 66. Andrews, S. C.; Lebrun, N. E.; Barynin, V.; Thomson, A. J.; Moore, G. R.; Guest, J. R.; Harrison, P. M. Site-Directed Replacement of the Coaxial Heme Ligands of Bacterioferritin Generates Heme-Free Variants. J. Biol. Chem. 1995, 270 (40), 23268-23274. 67. Willies, S. C.; Isupov, M. N.; Garman, E. F.; Littlechild, J. A. The Binding of Haem and Zinc in the 1.9 Å X-Ray Structure of Escherichia coli Bacterioferritin. J. Biol. Inorg. Chem. 2009, 14 (2), 201-207. 68. Szacilowski, K.; Macyk, W.; Drzewiecka-Matuszek, A.; Brindell, M.; Stochel, G. Bioinorganic Photochemistry: Frontiers and Mechanisms. Chem. Rev. 2005, 105 (6), 2647-2694. 69. Shafirstein, G.; Bellnier, D.; Oakley, E.; Hamilton, S.; Potasek, M.; Beeson, K.; Parilov, E. Interstitial Photodynamic Therapy-a Focused Review. Cancers 2017, 9 (2), 12.


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Trojan Horse for Light-Triggered Bifurcated Production of Singlet

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