PEGâPLA-Coated and Uncoated Radio-Luminescent CaWO4 Micro

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PEG-PLA-Coated and Uncoated Radio-Luminescent CaWO4 Micro/Nanoparticles for Concomitant Radiation/ UV-A and Radio-Enhancement Cancer Treatments Sung Duk Jo, Jaewon Lee, Min Kyung Joo, Vincenzo Pizzuti, Nicholas J. Sherck, Slgi Choi, Beom Suk Lee, Sung Ho Yeom, Sang Yoon Kim, Sun Hwa Kim, Ick Chan Kwon, and You-Yeon Won ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00119 • Publication Date (Web): 26 Feb 2018 Downloaded from http://pubs.acs.org on February 26, 2018

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ACS Biomaterials Science & Engineering

PEG-PLA-Coated and Uncoated Radio-Luminescent CaWO4 Micro/Nanoparticles for Concomitant Radiation/UV-A and RadioEnhancement Cancer Treatments

Sung Duk Jo,1,§ Jaewon Lee,2,§ Min Kyung Joo,1 Vincenzo J. Pizzuti,2 Nicholas J. Sherck,2 Slgi Choi,2 Beom Suk Lee,1 Sung Ho Yeom,3 Sang Yoon Kim,1 Sun Hwa Kim,1 Ick Chan Kwon,1 You-Yeon Won1,2,4,*

1

Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and

Technology (KIST), 5 Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, South Korea 2

School of Chemical Engineering, Purdue University, 480 Stadium Mall Drive, West Lafayette,

Indiana 47907, United States of America 3

Department of Biochemical Engineering, Gangneung-Wonju National University, 7 Jukheongil, Gangneung-si, Gangwon-do 25457, South Korea 4

Purdue University Center for Cancer Research, 201 S. University Street, West Lafayette,

Indiana 47907, Unites States of America §

Co-first authors

*

To whom correspondence should be addressed: [email protected]

1

ACS Paragon Plus Environment

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Abstract Currently, there is great interest in the development of ways to achieve the benefits of radiation treatment with reduced negative effects. The present study demonstrates the utilization of Radio-Luminescent Particles (RLPs) as a means to achieve radio-sensitization/enhancement and their ability to affect head and neck cancer cell cultures (in vitro) and xenografts (in vivo). Our approach utilizes a naturally-abundant radio-luminescent mineral, calcium tungstate (CaWO4) in its micro or nanoparticulate form for generating secondary UV-A light by γ ray/Xray photons. In vitro tests demonstrate that an un-optimized RLP materials (uncoated CaWO4 (CWO) microparticles (MPs) and PEG-PLA-coated CWO nanoparticles (NPs)) induce a significant enhancement of the tumor suppressive effect of X-rays/γ rays in both radio-sensitive and radio-resistant cancer models; uncoated CWO MPs and PEG-PLA-coated CWO NPs demonstrate comparable radio-sensitization efficacy in vitro. Mechanistic studies reveal that concomitant CaWO4 causes increased mitotic death in radio-resistant cells treated with radiation, whereas CaWO4 sensitizes radio-sensitive cells to X-ray-induced apoptosis/necrosis. Radiosensitization efficacy of intratumorally injected CaWO4 particles (uncoated CWO MPs and PEGPLA-coated CWO NPs) is also evaluated in vivo in mouse head and neck cancer xenografts. Uncoated CWO MPs suppress tumor growth more effectively than PEG-PLA-coated CWO NPs. Based on theoretical considerations, an argument is proposed that uncoated CWO MPs release sub-toxic levels of tungstate ions, which cause increased effects of photoelectric electron emissions. The effect of folic acid functionalization on in vitro radio-sensitization behavior produced by PEG-PLA-coated CWO NPs is studied. Surface folic acid results in a significant improvement in radio-sensitization efficiency of CaWO4.

Keywords Cancer; radiation therapy; radio-sensitization; radio-luminescence; nanoparticle

2


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Introduction Radiation therapy is one of the three pillars of cancer therapy alongside chemotherapy and surgery. About two-thirds of all cancer patients receive radiation therapy during their illness, and in the US annually nearly a million patients are treated with radiation therapy1. Unfortunately, radiation therapy generally carries significant side effects in patients, both acute and chronic, because of the damage high-energy ionizing radiation (such as X-ray or γ ray radiation) causes to normal cells and tissues. Due to the side effects, patients receive small amounts of radiation over a period of weeks which increases cost of treatment not only for the patients but also for hospitals and insurance companies. For these reasons, clinicians and researchers are pursuing ways of using radiation therapy to treat cancer more safely, effectively, and rapidly. A current active area of research attempts to develop chemical agents (“radio-sensitizers”) that make cancer cells easier to kill with radiation therapy2. Many anticancer drugs have radio-sensitization effects. Some drugs (such as doxorubicin, 5-fluorouracil, and cisplatin) sensitize cancer cells to radiation by intercalation with DNA, and others (e.g., paclitaxel, and etanidazole) produce sensitization effects by arresting the cell cycle, usually in the G2/M phase3-5. For this reason, “chemoradiotherapy” is now the standard of care for some cancers (e.g., glioblastoma/gliosarcoma6, locally advanced (Stages III – IV) squamous cell cancers of the head and neck7, locally advanced cervical cancer8-9, locally advanced non-small cell lung cancer10, etc.). However, chemotherapy agents, even if injected locally, typically diffuse out of the site of delivery after a relatively short time, causing systemic side effects. Nanoparticles (NPs) offer hope for improvements because, due to their larger sizes, nanoparticles are easier to maintain at the tumor sites for longer periods of time. Recently, significant attention has been paid to metal/metal oxide nanoparticles; these “high-Z” materials (particularly, those derived from gold, silver, iron, gadolinium, hafnium) produce strong secondary electron radiation due to the photoelectric, Compton and Auger effects, and thus cause localized augmentation of radiation damage;11-12 hereafter, these combined photoelectric, Compton and Auger effects will be referred to simply as photoelectric effects for simplicity’s sake. These currently available compounds are typically able to increase the radiation’s potency by factors typically in the range 1.0 – 2.0;13-14 radio-sensitizing (radio-enhancing) effects are 3



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typically quantified from in vitro clonogenic cell survival assay data in terms of the so-called Sensitizer Enhancement Ratio (SER) or Dose Enhancement Factor (DEF) defined as the ratio of the radiation dose at 10% clonogenic survival in the absence of nanoparticles relative to the radiation dose at 10% survival in the presence of nanoparticles. In in vivo situations, these photoelectric nanoparticles are limited because of the small mean free paths of electrons; nanoparticles must be internalized by cells to produce radio-sensitization/enhancement effects15. Also, current radio-sensitization methods based on photo-electric nanoparticles work best with X-rays of the order of 100 kVp in energy, but not as well with more clinically relevant radiations with MVlevel X-ray/γ-ray photon energies because of the significantly reduced absorption cross-sections at the higher energies14,

16

. Also, unfortunately, the most studied material in this regard, i.e.,

nanoparticulate gold, is known to cause genotoxic and mutagenic effects in exposed tissues in vivo17-19. It is notable that a European startup, called “Nanobiotix”, is currently attempting to commercialize

NP radio-sensitizers.

Nanobiotix

technology

uses

heavy

metal-based

“photoelectric” NPs which produce secondary electrons under X-ray/γ ray irradiation. Their first NP radio-sensitizer product (“NBTXR3”, 50-nm HfO2 NPs coated with tetra methyl ammonium hydroxide, named “NBTXR3” designed for direct intratumoral administration) is currently under Phase II/III clinical trials for treatment of soft tissue sarcoma and Phase I/II trials for head and neck cancer, hepatocellular cancer, liver metastases, prostate cancer, and rectal cancer20. Since 2013 our laboratory has been exploring the possibility of using radio-luminescent nanoparticles (calcium tungstate (CaWO4) nano crystals) as theranostic agents that allow enhancement of X-ray imaging and treatment of cancer21-24. CaWO4 is a naturally-abundant radio-luminescent mineral, and it exhibits a luminescence emission in the UV-A/blue range with a maximum at about 420 nm wavelength under high energy ionizing radiation such as γ rays, Xrays or short wavelength UV light at room temperature. During the course of this study, we found that the radio-enhancement effect of CaWO4 was, in fact, discovered more than a hundred years ago; in a report quoted in the June 1914 issue of the Journal of Advanced Therapeutics, it was mentioned that the combined CaWO4 and X-ray exposures significantly increased the necrosis of the tumor relative to the region exposed to X-rays only25. It is inferred that in this early study, the CaWO4 substance was tested in non-nanoparticle form, because at that time the method for synthesizing CaWO4 nanoparticles was not likely available. To our knowledge, the mechanism of action of CaWO4 as a radio-sensitizer/enhancer has never been studied. In theory, 4


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three different effects can be produced upon (cancer) cells by the use of CaWO4 under X-ray irradiation: (i) radio-luminescence, (ii) photoelectric and (iii) photocatalytic effects. CaWO4 crystals are radio-luminescent (“scintillation”) materials which emit long-wavelength UV (“UV-A”) light (320 – 400 nm) as a result of exposure to ionizing radiation (such as X-ray or γ ray). It is a largely unexplored question whether the combination of X-ray (or γ ray) and UV-A light would significantly enhance the cancer cell-killing effectiveness of X-rays. Of course, it is well known that lower-wavelength UV light (particularly, the “UV-C” light having wavelengths in the range of about 100 to about 280 nm) has genotoxic effects on cancer cells; it causes damage to DNA in cancer cells26-27. DNA damage by UV-C light initiates repair sequences, and arrests progression of the cell cycle from G2 to mitosis28. Therefore, it would be beneficial, for instance, to combine radiation therapy with UV-C (pre-)treatment, because cancer cells are most susceptible to radiation damage when they are in the process of separating the replicated chromosomes during cell division (i.e., in the G2/M phase)29. In fact, synergistic interactions of UV-C light with X-rays in killing cells have been known for more than 50 years30. However, the longer wavelength UV-A has never before been thought useful for X-ray/γ ray sensitization in clinical radiation therapy because UV-A light is 104 times less mutagenic even than the intermediate wavelength UV-B 290 – 320 nm31. As disclosed in the Supporting Information (SI) section, work by our laboratory demonstrates that UV-A lamp treatment (365 nm) itself suppresses tumor growth in a mouse xenograft model (Figure S1 of the Supporting Information (SI)). This preliminary data led us to explore the possibility of combining radiation therapy with UV-A treatment by utilizing radio-luminescent agents which emit UV-A light under ionizing radiation (Figure 1). Direct UV-A light would not be usable for X-ray/γ ray sensitization in clinical radiation therapy because UV-A has a very limited penetration distance in tissue (< 1 mm). However, secondary UV-A radiation can be generated even in deep tissue tumors by delivering radio-luminescent particles to the tumor and illuminating them with deep-penetrating γ rays or X-rays. This new method of radio-sensitization based on radio-luminescent materials has potential advantages over the conventional nanoparticle radio-sensitization approaches. For instance, unlike the conventional photoelectric nanoparticle radio-sensitization methods, this radio-luminescence therapy is expected to work equally well with clinically relevant MV-level X-rays/γ rays16. Radio-luminescent nanoparticles produce photons with X-ray exposure; because 5



photons have two to three orders of magnitude larger mean free paths than electrons32, these materials might not even need to be present inside cells to kill them. Unlike many heavy metal nano substances, CaWO4 nano/micro particles are largely non-toxic21, 23. Certainly, CaWO4 can also produce photoelectric effects to a certain extent, because it contains the heavy element tungsten (W) with an atomic number of 74. However, a recent Monte Carlo simulation study showed that nano/microparticles of CaWO4 are far inferior to conventional nanoparticle radiosensitizers/enhancers (such as those made of gold or hafnium oxide) in generating electrons under X-ray irradiation16. On the other hand, one interesting possible situation that might have occurred in the 1914 study25 is that CaWO4 particles (with its solubility product constant of the order of 10-10) 33 slightly dissolved and released a small amount of tungstate ions (WO42-) within the tumor (at a level of about 10-5 M), and these dissolved tungstate ions produced a significantly higher level of photoelectric effect than what could be achieved with CaWO4 in its particulate form. CaWO4 can also function as a photocatalyst, breaking apart water/oxygen molecules into reactive oxygen species when illuminated with UV light. Although it has not been previously studied, it is plausible to think that the use of ionizing radiation (X-rays/γ rays) can also cause the same photocatalytic effect because under ionizing radiation CaWO4 self-produces UV light. On the basis of limited information available, we are unable to even speculate which mechanism, among these three (i.e., radio-luminescence, photoelectric vs. photocatalytic), is responsible for the observed radio-sensitization/enhancement effect of CaWO425. The present manuscript reports results from our first set of study aimed at addressing this knowledge gap. Specifically, the study investigated the radio-sensitizing effects of three different types of CaWO4 materials: (1) biocompatible poly(ethylene glycol-block-D,L-lactic acid) (PEGPLA) block copolymer-coated CaWO4 (CWO) nanoparticles (NPs) with about 170 nm hydrodynamic diameter, (2) uncoated CWO microparticles (MPs) with 2 – 3 µm diameter, and (3) folic acid(FOL)-functionalized PEG-PLA-coated CWO NPs (again with about 170 nm hydronamic diameter). This study thus represents a first demonstration of the radio-sensitization effect of nanoparticulate CaWO4. In a previous experimental investigation, we have shown that (for some reason not yet clear) the overall luminescence intensity at a given mass concentration of CaWO4 particles generally increases with decreasing particle size, and polymer coating 6


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slightly decreases the UV-A flux22. FOL-PEG-PLA-coated CWO NPs were included in this study with the intention to elucidate the importance of NP cell internalization in producing the radio-sensitization/enhancement effects; it is well known that conjugation of folic acid to the surface of nanoparticles enhances the cell internalization efficiency of the nanoparticles34. PEGPLA-coated CWO NPs and uncoated CWO MPs were studied together to demonstrate the effects of the polymer coating; unlike gold or silica, CaWO4 does not have any reactive sites, and the PEG-PLA surface functionalization was achieved by a physical encapsulation method22. The cytotoxicities of the above three CaWO4 formulations (that is, PEG-PLA-coated CWO NPs, uncoated CWO MPs, and FOL-PEG-PLA-coated CWO NPs) were evaluated in vitro in squamous cell carcinoma cultures. A method for UV-A dosimetry was designed to quantitate the fluence of UV-A light at the surface of the CaWO4 particles under different energies and doses of X-ray/γ ray irradiation. The radio-sensitization/enhancement efficacies and mechanisms were assessed in cell cultures for various radiation (energy, dose and dose rate) conditions. The efficacies of PEG-PLA-coated CWO NPs and uncoated CWO MPs were also evaluated in mouse (head and neck squamous cell carcinoma) tumor xenografts. Theoretical analyses (modeling of clonogenic cell survival curves, predictions of dissolution times, and Monte Carlo simulations of dose enhancement for tungstate ions) were performed to better understand differences observed between the folic-acid-functionalized vs. non-functionalized CWO NP systems and also between the PEG-PLA-coated CWO NPs and uncoated CWO MPs.

Experimental Procedures Preparation of folate-functionalized and non-folate-functionalized PEG-PLA block copolymer(BCP)-encapsulated CaWO4 (CWO) nanoparticles (NPs). The CWO NPs were synthesized by a micro-emulsion method22-23. First, 20 ml of cyclohexane was mixed with 2 ml of 1-hexanol. CTAB (2 mmol) (≥ 99%, Sigma) was added to this solvent mixture, and then the solution was heated to 70 ℃ or until the solution became transparent (Solution 1). Meanwhile, 0.4 mmol of Na2WO4 (99%, Acros Organics) was dissolved in 0.6 ml of Milli-Q water (Solution 2). Next, 0.4 mmol of CaCl2 was dissolved in a mixture of 0.564 ml of Milli-Q water and 0.036 ml of 0.1 M HCl solution (Solution 3). Solutions 2 and 3 were immediately injected to Solution 1, and the resulting mixture was vigorously stirred. After about a minute, the mixture was 7



transferred into a Teflon-lined stainless steel autoclave, and the autoclave was heated to 160 ℃ and maintained at that temperature for 24 hours. Afterwards, the autoclave was gradually cooled to room temperature. The product was collected by centrifugation and washed twice with ethanol and chloroform to remove residual cyclohexane and excess CTAB and 1-hexanol. The PEG-PLA diblock copolymer was synthesized by 1,8-diazabicyclo[5.4.0] undec-7ene(DBU, 98%, Aldrich)-catalyzed ring-opening polymerization of lactide (LA, a racemic mixture) using α-methoxy-ω-hydroxy-terminated PEG (PEG-ME, Mn 5,000 g/mol, PDI 1.17, Polysciences, Inc.) as the starting material. 0.45 g of PEG-ME was dissolved in dichloromethane (DCM, anhydrous, ≥99.8%, Sigma-Aldrich) (22 ml) dried with molecular sieves. After a day LA (0.35 g) was added into the PEG-ME solution. The polymerization was initiated by adding 2 ml of a DBU solution (3.35 mmol of DBU dissolved in 30 ml of DCM) to the LA/PEG-ME mixture at room temperature. The polymerization reaction was run for 1 hour at room temperature. Afterward the reaction was terminated by adding 10 mg of benzoic acid (≥ 99.5%, SigmaAldrich). The polymerization mixture was added drop-wise to 1000 ml petroleum ether for precipitation. After the PEG-PLA product settled to the bottom, the supernatant was decanted. The polymer was dried in a vacuum oven. The folate-functionalized PEG-PLA (FOL-PEG-PLA) was prepared in three steps (as described in Figure S10 of the Supporting Information (SI)). First, allyl-PEG-PLA was synthesized by DBU-catalyzed ring-opening polymerization of D,L-LA using α-allyl-ω-hydroxyterminated PEG (allyl-PEG, Mn 5,000 g/mol, Creative PEGWorks, see Figure S10 for the chemical structure of the allyl-containing end group) as the starting material. 0.268 g of allylPEG and 0.188 g of LA were dissolved in anhydrous DCM (2.7 ml, dried with molecular sieves). After a day, the polymerization was initiated by adding 0.3 ml of a DBU solution (16.7 µl DBU/ml DCM) to the allyl-PEG/LA mixture at room temperature. After 1 hour, the polymerization was terminated by adding 50 mg of benzoic acid (≥ 99.5%, Sigma-Aldrich). The polymerization mixture was added drop-wise to 1000 ml petroleum ether for precipitation. After the PEG-PLA product settled to the bottom, the supernatant was decanted. The polymer was dried in a vacuum oven. In the second step, under argon atmosphere the allyl-PEG-PLA (0.4 g) was mixed with 2-aminoethanethiol hydrochloride (≥98%, Sigma) (0.046 g) and 2,2′-azobis(2methylpropionitrile) (AIBN, 98%, Aldrich) (4.86 mg) in anhydrous N,N-dimethylformamide 8


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(DMF, 99.8%, Sigma-Aldrich) (1.93 ml). The mixture was stirred at 60 ℃ for 24 h. The product, amine-terminated PEG-PLA, was isolated by precipitation in cold diethyl ether, filtration and drying in vacuum for 2 days. In the last (third) step, the carboxyl acid group of folic acid (≥97%, Sigma) (0.04 g) was activated with dicyclohexylcarbodiimide (DCC, 99%, Aldrich) (0.02 g) and N-hydroxysuccinimide (NHS, 98%, Aldrich) (0.01 g) in anhydrous dimethyl sulfoxide (DMSO, ≥99.9%, Sigma-Aldrich) (0.385 mL) in dark at room temperature for 12 h. Then, a solution of amine-PEG-PLA (0.3 g) and triethylamine (TEA, 99%, Sigma-Aldrich) (0.003 g) in DMSO (0.3 ml) was added to the activated folic acid solution. The reaction was allowed to proceed at room temperature for 24 h. The product was isolated by filtration to remove 1,3-dicyclohexylurea (DCU), dialysis against DMSO for 48 h and milli-Q water for additional 48 h (MWCO 1000 Da, Spectrum Lab), and freeze-drying. PEG-PLA-encapsulated (“PEGylated”) CWO NP samples were prepared as follows. 1.0 mg of CWO (purified by centrifugation) was dispersed in 1.0 g of DMF. 100.0 mg of PEG-PLA was added to 2.9 g of the above nanoparticle suspension. This mixture was stirred using a high speed overhead mechanical stirrer (at 15000 rpm) with simultaneous sonication. 2.1 ml of Milli-Q water was added to the DMF solution. The resulting mixture was emulsified with a mechanical stirrer and then ultrasonicated in a sonication bath for 30 minutes. This emulsion was placed in a dialysis bag (molecular weight cutoff 50 kDa) and dialyzed for 3 days against a total of 1.0 liter of Milli-Q water (regularly replaced with fresh Milli-Q water) to remove DMF35. Folate surface functionalized CWO NPs were prepared by encapsulating the as-synthesized CWO NPs with a mixture of folate-functionalized and non-functionalized PEG-PLA BCPs (mixed at 1:9 molar ratio); the detailed encapsulation procedure was same as that for the plain PEG-PLA CWO NPs. Sample preparation for CWO NP UV-A dosimetry under γ ray/X-ray irradiation. 3.4 mg of CWO NPs were dissolved in 1.0 g of DMF. 340.0 mg of PEG-PLA was dissolved in 1.9 g of DMF. These CWO NP and PEG-PLA solutions were mixed together. 1.0 g of a PpIX solution (0.5 mg of PpIX (Sigma-Aldrich) dissolved in 1.0 ml of DMF) was added to the mixed CWO NP/PEG-PLA solution. While the mixture solution was vigorously agitated using a mechanical stirrer at 15000 rpm under ultrasonication, 2.1 ml of Milli-Q water was added to it. Afterwards, mechanical stirring under ultrasonication was continued for 30 additional minutes. The resulting mixture was centrifuged at 5000 rpm for 60 minutes at room temperature to separate 9



unencapsulated PpIX molecules (supernatant) from PEG-PLA-coated CWO NPs loaded with PpIX (precipitate). The precipitates (PpIX-loaded PEG-PLA-encapsulated CWO NPs) were redispersed in Milli-Q water at a CaWO4 concentration of 0.56 mg/ml for UV-A dosimetry experiments. The supernatant solution containing unencapsulated PpIX was collected for determination of the encapsulated amount of PpIX. The concentration of PpIX in the supernatant fraction was determined by UV/Vis absorbance measurements (Cary 100 Bio UV-VIS Spectrometer, Varian) as follows. UV-Vis absorption spectra were obtained from 5 different DMF solutions of PpIX (at PpIX concentrations of 0.000625, 0.00125, 0.0025, 0.005, 0.01 and 0.05 mg/mL). From these data, a linear calibration plot was obtained relating UV/Vis absorbance to PpIX concentration. Based on this calibration curve, the PpIX concentration of the supernatant was estimated from its absorbance value. Knowing the volume of the supernatant solution, the total amount of PpIX in the supernatant could be calculated. Finally, the encapsulated PpIX amount could be calculated by subtracting the amount in the supernatant from the total amount in the entire system. A typical amount of encapsulated PpIX was estimated to be 4.28 × 10-18 moles (2.38 × 10-15 g) per PEGPLA-coated CWO NP; the PEG-PLA/CWO NP concentration was 3.59 × 109 particles per ml solution. As shown in Figure S3, PEG-PLA micelles encapsulating PpIX only (i.e., not encapsulating CWO NPs) were used as control (to confirm that PpIX does not photobleach under bare γ ray/Xray irradiation). PpIX-loaded PEG-PLA micelles were prepared as follows. 340.0 mg of PEGPLA was dissolved in 2.9 g of DMF, to which 1.0 g of a PpIX solution prepared by dissolving 0.5 mg of PpIX in 1.0 ml of DMF was added. Under mechanical stirring and ultrasonication, 2.1 ml of Milli-Q water was added to this mixture. Following additional mechanical stirring and ultrasonication for 30 minutes, the solution was dialyzed in a dialysis bag (1 kDa molecular weight cutoff) for 1 day against 300 liters of Milli-Q water to remove DMF and unencapsulated PpIX. UV-A irradiation of PpIX-loaded PEG-PLA-encapsulated CWO NPs. A UVP’s B100AP/R High Intensity UV Lamp (wavelength = 365 nm, power density = 8.9 mW/cm2 at 10inch distance) was used to illuminate PpIX-loaded PEG-PLA-encapsulated CWO NPs (for constructing a PpIX photobleaching vs. UV-A fluence calibration plot). UV-A illumination was 10


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performed using a custom-designed environmental chamber (to block light from the surrounding environment) and under continuous air ventilation at the bottom (to prevent heating during irradiation)36. PpIX-loaded PEG-PLA-coated CWO NPs were dissolved in Milli-Q water (at a CaWO4 concentration of 0.56 mg/ml); the concentration of PpIX in this sample was 0.008565 mg/ml. The degree of PpIX photobleaching (fluorescence decay) was measured as a function of UV-A fluence (i.e., UV-A exposure duration). The UV-A irradiation times used were 1, 2, 4, 8, 17 and 32 minutes, which corresponded to UV-A energy intensity (fluence) values of 0, 0.534, 1.068, 2.136, 4.272, 9.078 and 17.088 J/cm2, respectively. PpIX fluorescence intensity was measured using a Cary Eclipse Fluorescence Spectrophotometer (excitation: 406 nm, emission: 635 nm). X-ray/γ ray irradiation of PpIX-loaded PEG-PLA-encapsulated CWO NPs. (i) PpIXloaded PEG-PLA-encapsulated CWO NPs and (ii) PpIX-loaded PEG-PLA micelles (with no coencapsulated CWO NPs) were exposed to various doses of X-rays/γ rays (to determine the dosedependent degree of PpIX photobleaching caused (i) by UV-A generated by CWO NPs under Xray/γ ray irradiation or (ii) by direct X-rays/γ rays in the absence of CWO NPs). X-rays were obtained using various X-ray/γ ray sources: a 6 MV Varian 600 CD Linear Accelerator for 6 MeV X-rays, an XRAD 320 X-ray Irradiator for 320 keV X-rays, and a 60Co γ Irradiator for 1.17/1.33 MeV γ rays. The PpIX fluorescence intensities of these samples ((i) and (ii)) were measured (excitation: 406 nm, emission: 635 nm) following various doses of X-ray irradiation (0, 1, 2, 4 and 8 Gy). The fluorescence intensity of the PpIX-loaded PEG-PLA-encapsulated CWO NP sample was normalized by the fluorescence intensity of the PpIX-loaded PEG-PLA micelle sample in order to subtract any effect of direct X-rays/γ rays on PpIX fluorescence; in fact, as shown in Figure S3, X-rays/γ rays alone caused a slight decay in PpIX fluorescence. From experiments described in the previous paragraph, the amount of 365 nm UV-A necessary for producing a certain level of PpIX photobleaching could be estimated. From experiments described in the present paragraph, it could be estimated how much PpIX photobleaching occurred at a given dose of X-rays/γ rays. By comparison of these two results, we were able to calculate the amount of secondary UV-A light (365 nm equivalent) produced by CWO NPs as a function of X-ray/γ ray dose. Cell viability. HN31 cells were cultured in DMEM (Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS, 100 Units/ml penicillin and 100 µg/ml streptomycin in humidified 11



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atmosphere with 5% CO2 37oC. For cytotoxicity measurements HN31 cells were seeded on 96well culture plates at a density of 0.5 × 104 cells per well and incubated for 24 h prior to exposure to CaWO4 (CWO). The cells were treated with uncoated CWO microparticles (MPs) or PEGylated (PEG-PLA-encapsulated) CWO nanoparticles (NPs) for 24 h at the various CWO concentrations indicated in Figure 3. To quantitate the viability of the CWO-treated cells, Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Rockville, MD, USA) assays were performed at 24 h post-CWO treatment using the manufacturer’s procedures (n = 5). Flow cytometry. The mode of cell death (apoptosis, early apoptosis, necrosis) following γ radiation in the presence and absence of CWO particles was investigated by flow cytometry analysis. HN31 cells were seeded on 60-mm2 culture plates at various designated densities (see the figure captions of Figures 6 and 9 for details) with DMEM medium containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. After 24 h incubation the cells were exposed to various designated doses (see figure captions) of

137

Cs γ radiation (IBL 437C, CSI

Bio International, France) at a rate of 1 Gy per every 11 s. At designated times (see figure captions) following γ ray treatments in the presence and absence of various loadings of CWO MPs or NPs (see figure captions), the cells were collected by trypsin treatment, double-stained with Annexin V-FITC and PI (BD Biosciences, San Jose, CA, USA), and analyzed using the fluorescence-activated cell sorting (FACS) technique. In vitro clonogenic assay. HN31 cells were seeded on 60-mm2 culture plates at various designated densities for different radiation doses and incubated for 24 h prior to exposure to γ rays; see the figure captions for Figures 5, 8, 12, 13 and 14 for details of the radiation conditions. The irradiated cells were incubated for 14 days. Colonies resulting from radio-resistant cells were stained with Crystal Violet. Colonies of more than 50 daughter cells in culture were counted and the survival fraction was calculated based on the number of such colonies relative to that of the untreated or unradiated control (n = 4). In vivo tumor growth and mouse survival.

HN31

xenografts

were

prepared

by

7

implanting 7 × 10 cells (per mouse) to the left flank of female athymic nude mice (Balb/c, 5 weeks old, 20 to 26 g body weight, n = 6) (Orient Bio, Korea). In experiments presented in Figures 10(A) and 10(B), tumors were grown over a 21-day period to approximately 250 mm3, total 0.30 mg of uncoated CWO MPs or PEG-PLA-encapsulated CWO NPs were infused directly into the tumor (total 1.2 mg of CWO per cc of tumor); the procedure involved two 12


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injections of 120 µl CWO solution in PBS (CWO concentration: 1.23 mg/ml) over a two-day period. In experiments presented in Figures 10(C) and 10(D), tumors were grown over a 14-day period to approximately 85 mm3, total 1.00 mg of uncoated CWO MPs or PEG-PLAencapsulated CWO NPs were infused directly into the tumor (total 11.8 mg of CWO per cc of tumor); the procedure involved four injections of 50 µl CWO solution in PBS (CWO concentration: 5.00 mg/ml) over a two-day period. A few hours afterwards, unfractionated 60Co γ radiation with Leksell Gamma Knife Perfexion TM (Elekta Instrument, Sweden) (total dose: 5 Gy, dose rate: 2 Gy per minute, γ ray energy: 1.17 and 1.33 MeV) was applied to the tumor (defined to be “day 0”). Tumor volume was measured (using the formula (L×W2)/2 to give volume in ml) for each group only up to the time point at which any one mouse dies of the tumor, or any one mouse has a tumor greater than 2000 mm3, or any one mouse loses more than 20% of its original weight; in the latter two situations, the mouse was humanely sacrificed and counted as dead. The same criteria were used for the survival analysis shown in Figures 10(B) and 10(D). Statistical analysis.

All data values represent averages from independent series of

experiments. Error bars represent standard errors unless noted otherwise specifically. Student’s ttest was used to analyze statistical significance between control and treatment. Specifically, a two-tailed unpaired t-test was used to calculate p-values relative to controls using Graph-Pad Quickcalcs. p < 0.05 is considered statistically significant.

Results and Discussion Basic characteristics of CaWO4 micro/nanoparticles. In the present study, two types of CaWO4 (CWO) samples were investigated: (1) uncoated CWO microparticles (MPs) of about 2 – 3 µm diameter (purchased from Sigma-Aldrich, used with no further purification), and (2) poly(ethylene glycol-block-D,L-lactic acid) (PEG-PLA) block copolymer-encapsulated CWO nanoparticles (NPs) having a mean hydrodynamic diameter of about 170 ± 10 nm (prepared as described in our previous paper)22-23. The steps used for preparing the CWO NP sample can be summarized as follows. Primary CWO NPs of a nearly monodisperse diameter (10 nm) were prepared by the reaction of sodium tungstate dihydrate with calcium chloride salt. Cetyltrimethylammonium bromide (CTAB) surfactant was used to control the particle nucleation 13



and growth kinetics; thus, the as-synthesized primary CWO NPs were coated with CTAB. This original CTAB coating was replaced with an enclosure formed by PEG-PLA block copolymer (BCP) materials using the solvent exchange procedure developed in our laboratory37. This procedure was able to produce fully PEGylated CWO NP products that are essentially devoid of surfactant and are stable against aggregation under physiological salt concentrations for long periods of time (> 3 months at least). The mean hydrodynamic diameter of the resulting PEGPLA-encapsulated CWO NPs was determined by dynamic light scattering (DLS) to be about 170 nm (DLS data presented in Figure S2(A)); as depicted in Figure 2(A), CWO NPs were encapsulated within BCP capsules as clusters (see TEM micrograph in Figure 2(B)). In all studies discussed in this manuscript, the PEG-PLA-encapsulated CWO NP sample with 170 nm diameter has been used; of note, it is also possible to produce smaller-sized PEG-PLA-coated CWO NPs (e.g., having a diameter of about 50 nm), and a separate study is underway to investigate how size affects the radio-sensitization efficiency and also the pharmacokinetic behavior of CWO NPs. The crystallinity of the CWO NP/MP materials were confirmed by X-ray diffraction (Figure 2(C)). Also, as demonstrated in Figure 2(D), the polymer encapsulation does not alter the luminescence activity of CaWO4; a more detailed discussion can be found in the references22. To study the effect of folate functionalization on CWO NP radio-sensitization (discussed in the last subsection of this section), a folate-functionalized PEG-PLA-coated CWO NP sample was also prepared by encapsulating CWO NPs using a (9:1 molar) mixture of normal and folic acid-terminated PEG-PLA polymers. These folate PEG-PLA-coated CWO NPs had the same overall hydrodynamic size as that of the non-folate-functionalized PEG-PLA-coated CWO NPs (170 nm diameter, Figure S2(B)). The cytotoxicity of uncoated CWO MPs and PEG-PLA-coated CWO NPs were evaluated by CCK-8 assays. It was confirmed that both uncoated and PEG-coated CWO materials are nontoxic to cultured human cells (p53-mutant human head and neck cancer HN31 cells) (Figure 3). The measurements were performed up to sufficiently high CWO concentrations; the maximum concentrations tested were 5.0 mg/ml for uncoated CWO MPs, and 1.0 mg/ml for both folate and non-folate PEG-PLA-coated CWO NPs. It should be noted that the actual CaWO4 concentration that the cells experience is typically significantly higher than the nominal value of CaWO4 concentration because of the sedimentation of the CaWO4 particles; we estimate that it takes about 7 minutes for a 3-µm diameter CaWO4 sphere (and about 35 hours for a 170-nm diameter 14


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CaWO4 sphere) to sediment a distance of 1 cm (which is approximately the depth of the cell culture plate used), so it is expected that during the 24-hour period of cell incubation with CaWO4 materials, a significant number (if not all) of CaWO4 particles have accumulated at the bottom of the plate (the above sedimentation time values were estimated using the equation, τ ≈ 9lη/(2R2∆ρg) where l is the sedimentation distance, η is the viscosity of the medium, R is the radius of the particle, Δρ is the density difference between CaWO4 and water, and g is the gravitational acceleration). In a previous study, the maximum tolerated dose (MTD) of the 170nm diameter PEG-PLA-encapsulated CWO NPs in normal (BALB/c) mice has been determined to be 250 ± 50 mg per body weight following a single i.v. administration23; this material is thus safer than, for instance, commercially available dextran-coated iron oxide nanoparticles that are used clinically as MRI contrast agents (MTD in mice ≈ 168 mg/kg per dose i.v.)38. The observed non-toxicity of the CaWO4 material can be rationalized as follows. The solubility product constant for CaWO4 is Ksp ≈ 4.9 × 10-10 at neutral pH at 298 K33. Therefore, even if a mass of CaWO4 is confined in a closed environment, the local concentration of the liberated WO4-2 ions can never reach a value greater than 2.2 × 10-5 (≈ Ksp½) M. This theoretical maximum reachable WO4-2 concentration is still below the reported cytotoxic threshold for WO4-2 in mammalian cells (≈ 3.5 × 10-5 M)39 and is also about two orders of magnitude lower than the LD50 value of WO4-2 (≈ 4.6 × 10-3 M in mouse circulatory system) estimated from literature data; LD50 is the dose of a chemical that causes death of half of the tested animals. U.S. Department of Health & Human Services (HHS) literature also notes that there is no evidence of long-term accumulation of WO42

in human body40. The PEG-PLA encapsulating agent is a biocompatible and biodegradable

FDA-approved drug excipient, and is generally recognized as a safe chemical41-42. Taken together, it is reasonable to expect that both uncoated and PEG-PLA-coated CaWO4 particles are safe for clinical use. In order to validate that CaWO4 particles generate sufficient UV-A light (320 – 400 nm) for producing cellular biological effects, dosimetry measurements were performed using a fluorophore, protoporphyrin IX (PpIX), as a marker of UV exposure. PpIX degrades (photobleaches) under UV-A illumination in a dose-dependent manner, which allows for the 15



quantification of UV fluence on the surface of a CaWO4 particle. PpIX was co-encapsulated (along with CWO NPs) within the hydrophobic PLA domain of the BCP coating structure (Figure 4(A)); co-loading with PpIX did not alter the size of PEG-PLA/CWO NP assemblies (170 nm hydrodynamic diameter). As shown in Figures 4(B) through 4(D) (black data), when this encapsulated PpIX was exposed to 365 nm UV-A light from a UV lamp (UVP), photobleaching occurs, which could be determined by tracing the decrease of its fluorescence intensity at 635 nm (under 406 nm excitation) following varying periods of UV exposure (raw data not shown). The black curve shown in Figures 4(B) through 4(D) is the “calibration” curve demonstrating how the degree of PpIX photo-bleaching changes as a function of the extent of UV exposure. As shown in Figure S3 of the Supporting Information (SI), exposure of PEG-PLAencapsulated PpIX to X-rays/γ rays alone (that is, in the absence of co-encapsulated CWO NPs) did not induce a significant decay of PpIX fluorescence; these PpIX-containing PEG-PLA assemblies tested had a mean hydrodynamic diameter of 50 nm. However, in the presence of coencapsulated CWO NPs, X-ray/γ ray irradiation caused a decay in the fluorescence of PpIX, which suggests that CWO NPs indeed generated sufficient UV light to induce PpIX photobleaching under X-ray/γ ray radiation. The extent of CaWO4-mediated PpIX photobleaching was measured as a function of X-ray (γ ray) dose for several different X-ray (γ ray) energies, and these measured values were compared to the “calibration” curve (Figures 4(B) through 4(D)) in order to obtain the values of “equivalent” UV-A fluence produced by CWO NPs under various X-ray (γ ray) dose/energy conditions (Figure 4(E)); note that CaWO4 does not produce monochromatic 365 nm radiation, but it instead produces a spectrum of light over a range of wavelengths (Figure 2(D)), and here the “equivalent” UV-A fluence means the fluence of 365 nm UVP lamp light that would cause an equivalent level of photo-bleaching to PpIX as with CWO NPs at a specific X-ray/γ ray dose. As shown in Figure 4(E), the amount of UV-A radiation emitted by CWO NPs was on the order of 104 J/m2, which is far above, for instance, the minimum UV-A radiation known to induce skin cancer (≈ 1000 J/m2)43. Synergistic effect of concomitant γ ray and UV-A radiation: Proof of the concept in vitro. Synergistic interactions of UV-C light (200 – 290 nm) with X-rays in killing cells have been known for more than 50 years; preliminary UV-C exposure significantly sensitizes cells to X-rays, and vice versa (reversing the order does not alter the total cell kill for a given pair of UVC and X-ray doses)30. To our knowledge, similar investigations have not been conducted with 16


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UV-A radiation (320 – 400 nm). We suspect that the reason is simply that UV-A is known to be far less effective at destructing DNA; for instance, UV-A is known to be about 4 orders of magnitude less mutagenic in skin cells even relative to UV-B (290 – 320 nm)31. We first performed an in vitro clonogenic study to determine whether UV-A is indeed capable of inducing a significant enhancement of the tumor suppressive effect of γ rays/X-rays. For this purpose, a p53-mutant (radio-resistant44) human head and neck cancer cell line (HN31) was used. The p53 gene is responsible for inducing programmed cell death (apoptosis) in response to DNA damage from radiation, and thus tumors with p53 mutations are radioresistant45; therefore, HN31 can serve as a good model for testing the radio-enhancement effect of UV-A light. Also of note is that at about 25%, head and neck cancer accounts for the largest share of the radiotherapy market among all cancer types46. In one test, HN31 cells were first exposed to UV light (365 nm) for 20 minutes (the total UV-A energy transmitted to the cells was 10.68 J/cm2) and then subsequently irradiated with varying γ radiation doses. As shown in Figure 5(A) (see data labeled as “UV → γ”), this UV-A pre-treatment rendered the cells significantly more susceptible to γ radiation damage than non-UV-treated cells (see data labeled as “γ”). Even when the sequence of the two types of radiation was reversed (i.e., γ ray first, and then UV-A light), the combination radiation still produced a synergistic increase in cells undergoing cell death (see data labeled as “γ → UV” in Figure 5(A)). The effect of UV-A light was statistically significant; for instance, for both pre and post-UV-exposed cases the p-values relative to the control group (γ ray only) were estimated to be p < 0.05 at 9 Gy γ ray radiation (see Table S1 of the Supporting Information (SI)). We note, as shown in Figure 5(A), that the clonogenic survival curves for γ-irradiated HN31 cells (regardless of whether γ rays were used alone or in combination of preliminary or subsequent UV-A radiation) were seen to follow the standard exponential-quadratic decay formula. From the data shown in Figure 5(A), the values of the Sensitizer Enhancement Ratio or SER (defined as the ratio of the radiation dose at 10% clonogenic survival in the absence of UV exposure relative to the radiation dose at 10% survival in the presence of UV exposure) were estimated to be 1.09 for the pre-UV-treated group (UV → γ) and 1.20 for the post-UV-treated group (γ → UV) (Table S1). We then investigated whether concomitant CaWO4 particles could also increase the biological effectiveness of γ rays. Clonogenic survival was assessed in vitro in HN31 cultures 17



after various γ radiation doses in the absence and presence of either uncoated CWO MPs (2 – 3 µm diameter, 0.20 mg/ml CWO) or PEG-PLA-coated CWO NPs (170 nm diameter, 0.20 mg/ml CWO). The results are displayed in Figure 5(B). As shown in the figure, concomitant UV-A indeed produced a significant dose enhancement. The SER values were estimated to be 1.12 for the CWO MP-treated cells (“MP + γ”) and 1.12 for the CWO NP-treated cells (“NP + γ”). For MP + γ, the p-value relative to control (“γ”) was estimated to be, for instance, 0.04 at 9 Gy. For NP + γ, the p-value was 0.03 at 9 Gy. See Table S1 for p-values for other dose conditions. What is remarkable is that this concomitant UV-A of CaWO4 (Figure 5(A)) produced a similar level of radio enhancement relative to the sequential UV-A exposure (Figure 5(B)), even though the intensity of the concomitant UV-A radiation (< 1 J/cm2 as shown in Figure 4(F)) was at least an order of magnitude lower than that of the sequential UV-A radiation used (10.68 J/cm2). Therefore, it is concluded that the concurrent UV-A and γ ray treatment offers a greater synergistic relationship between the two types of radiation than their sequential combinations. Mechanism of CaWO4-induced radio-sensitization. It is known that in cancer cells with p53 mutations (such as HN31 cells), unchecked cell division occurs despite DNA damage following ionizing radiation, and for this reason, X-rays/γ rays typically cause cell death predominantly via a mechanism known as mitotic catastrophe—rather than p53-regulated active cell death mechanisms (such as apoptosis, necrosis or senescence)47. However, a question that arises in our situation is whether the radio-sensitization effect of CaWO4 involves any change in the mode of cell death (apoptosis, necrosis, senescence vs. mitotic death) in the HN31 cell line. To address this question, the cell death pathways were investigated in HN31 cells after γ ray irradiation in the absence and presence of CaWO4 particles. First, the extent of apoptotic and necrotic death in irradiated HN31 cells was analyzed by fluorescence-activated cell sorting (FACS) after dual staining with annexin V (to detect phosphatidylserine, an apoptosis marker on the plasma membrane) and propidium iodide (PI) (a plasma-membrane impermeable DNA intercalating agent). As shown in Figure S4, the apoptotic and necrotic populations increased with increasing γ radiation dose. Figure 6 displays how the extent of apoptotic and necrotic cell death changed in response to concurrent or sequential UV-A exposure at a constant γ ray dose of 5 Gy; regardless of the method of combination of the two types of radiation (i.e., concurrent vs. sequential), the additional UV-A exposure caused a similar marginal increase in the overall 18


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apoptosis plus necrosis levels (for instance, at 72 h post γ irradiation the “MP + γ”, “NP + γ”, “UV → γ” and “γ → UV” treatments resulted in, respectively, 18, 18, 18 and 33 percent increase in apoptotic/necrotic cell death relative to γ rays alone (“γ”)). Cytochemical assays (for detecting a biomarker of cellular senescence, senescence-associated beta-galactosidase) were also carried out in order to determine whether senescent cells48-51 were generated by γ radiation and also, if so, whether the senescent population increased under the influence of UV-A co-radiation. As shown in the optical micrographs of Figure 7, only negligible numbers of γ-irradiated HN31 cells were found to be blue-stained regardless of adjuvant/concomitant use of UV-A light; in all cases examined (γ rays alone, and γ rays in the presence of concurrent CaWO4 or in sequential combination with UV-A), no evidence of senescence (i.e., no evidence of production of a bluedyed precipitate that would result from the cleavage of the chromogenic substrate, X-Gal, in the event of senescence) was detected. Taken together, it could be concluded that the apoptosis and necrosis mechanisms (but not the senescence mechanism) contribute (at least) non-negligibly to the overall cell death in HN31 cells in response to combined γ ray/UV-A irradiation. However, these results did not provide sufficient information as to whether the mitotic catastrophe pathway52-54 also plays a role in the UV-A-induced radio-sensitization of HN31 cells. In an attempt to elucidate this issue, a similar set of measurements (clonogenic, annexin V/PI and beta-galactosidase assays) were also performed with a more radio-sensitive (p53-functional) cancer model, SCC7 (a murine oral squamous cell carcinoma cell line). As shown in Figure 8, it was confirmed that the effect of γ radiation on cell death was more pronounced in SCC7 cells than in HN31 cells; for instance, a 9-Gy γ ray dose resulted in a survival fraction of 0.005 for SCC7 (Figure 8), whereas the survival fraction of HN31 cells was 0.02 at the same γ ray dose (Figure 5). However, the dose enhancement effects of CaWO4 particles were comparable for both SCC7 and HN31 cells; from the data shown in Figure 8, the SER values were estimated to be 1.11 for the CWO MP-treated SCC7 cells (“MP + γ”) and 1.09 for the CWO NP-treated SCC7 cells (“NP + γ”) (Table S1). Flow cytometric annexin V/PI staining analysis, on the other hand, showed that concurrent CaWO4 increased apoptosis and necrosis in SCC7 cells to a far greater extent than what was observed in HN31 cells (Figure 9). At 72 h post γ irradiation, for instance, the “MP + γ” treatment resulted in about 60% increase in apoptotic/necrotic cell death relative to γ rays alone; the “UV → γ” and “γ → UV” treatments also showed similarly high 19



levels of increase (i.e., 103% and 70%, respectively) in apoptotic/necrotic cell death relative to γ rays alone at 72 h post-treatment. We note that concurrent UV-A had similar overall radiosensitization effects between HN31 and SCC7 cells (see Figures 5(B) and 8, and Table S1). Therefore, it is reasonable to deduce that the mitotic catastrophe pathway indeed plays a significant role in the UV-A-induced radio-sensitization of HN31 cells. In vivo feasibility study. A preliminary investigation was performed to assess the radiosensitization efficacy of intratumorally injected PEG-PLA-encapsulated CWO NPs and uncoated CWO MPs with mouse HN31 xenografts. We tested whether UV-A light generated by CaWO4 particles under γ ray radiation increases the biological effectiveness after radiation by repeated measurements of tumor volume. The volume of the tumor was measured by physical examination using calipers. For survival curves, surviving mice were counted daily up to a certain date, using the following criteria; a mouse was counted as dead when the tumor volume reached 2,000 mm3, or the mouse lost > 20% of its original body weight, or the mouse actually physically died. Two different CaWO4 dose levels were tested, 1.2 and 11.8 mg of CaWO4 per cc of tumor, and compared with a saline-treated group. The results are presented in Figure 10. It may be somewhat surprising to see that, unlike what was observed in vitro (Figure 5(B)), PEGylated CWO NPs appear to produce statistically insignificant radio-sensitization effects with 5-Gy γ ray radiation in terms of both tumor growth delay and mouse survival time. Currently, we suspect that this in vitro vs. in vivo discrepancy is due to the differences in nanoparticle distribution. Our recent study showed that 170-nm PEG-PLA-coated CWO NPs were completely confined at the site of injection (i.e., along the needle track) within the tumor following direct intratumoral injection in vivo for a long period of time (> at least one week)23; due to their large sizes, these nanoparticles were not able to diffuse away from the injection site. To the contrary, in an in vitro situation, (as discussed earlier in this section) a significant number of PEGylated CWO NPs are expected to be in direct contact with cells at the bottom of the culture plate, which makes the radio-sensitization effects of the nanoparticles more pronounced in the in vitro experiment. We note that an ideal nanoparticle radio-sensitizer would be the one that is sufficiently large to stay within the tumor boundary and at the same time small enough to spread out uniformly throughout inside the tumor tissue. In this regard, previous literature suggests that the ideal nanoparticle size should be about 30 – 60 nm55. Additionally, for a given total amount of CaWO4 material, smaller nanoparticles have generally been shown to produce higher UV-A 20


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emissions when measured macroscopically (perhaps because of larger surface areas)22. On the cellular level, smaller nanoparticles are expected to be generally easier internalized by cells. Based on these considerations, we have recently developed a PEG-PLA-coated CWO NP formulation having a diameter of about 50 nm. A study using this 50-nm nanoparticle sample is currently in progress. What is most remarkable in the data presented in Figure 10 is that uncoated CWO MPs showed almost complete suppression of tumor growth at the 11.8 mg/cc CaWO4 dose (Figure 10(C)). Improvement was also seen in mouse survival (Figure 10(D)). Based on the weaker effect of concomitant PEG-PLA-coated CWO NPs on in vivo tumor growth (Figure 10(C)) and the comparable in vitro radio-sensitization performances between PEG-PLA-coated CWO NPs and uncoated CWO MPs (Figures 5(B) and 8), it is reasonable to interpret that the great radioenhancement effect of uncoated CWO MPs observed in vivo is not due to the effect of UV-A light from CaWO4 generated under X-ray/γ ray irradiation. We suspect that this in vivo radioenhancement by uncoated CWO MPs is related to the liberation of WO42- ions from the CaWO4 material; PEG-PLA-coated CWO NPs do not release WO42- because of the PLA coating, which is stable in aqueous solution for at least three months22. It needs to be pointed out that there was a difference in the CaWO4 treatment protocol between the in vitro and in vivo tests; in the in vitro clonogenic measurements, the cells were incubated with CWO MPs/NPs for 3 hours prior to exposure to X-rays/γ rays, whereas in the in vivo experiments, CWO MPs/NPs were intratumorally infused over a 2-day period before γ radiation. As discussed earlier, the equilibrium concentration of WO4-2 ion in a solution of CaWO4 is 2.2 × 10-5 (≈ Ksp½) M; inside a tumor tissue the actual concentration of WO4-2 ion would be smaller than this maximum value because of continuous diffusion of these ions away from the tumor, and the rate of CaWO4 dissolution will determine the local concentration of the liberated WO4-2 ions within the tumor. To get an idea how fast CWO MPs liberate WO4-2 ions, a simple reaction kinetic analysis was conducted as follows. The species mass balance for a control volume around a spherical CWO particle in the reaction-controlled limit is written below: −

= 4 .

(1)

Here, is the radius of the spherical CWO particle, is the density, is the 21



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formula weight of CaWO4, and is the dissolution rate constant. The final equation for the total dissolution time is obtained by solving the above differential equation: =

!

"#$

( & − )

(2)

where & is the initial radius of the particle. The is fit from CaWO4 dissolution data in the literature56. (see Figure S9 of the SI). Using the above equations, estimates of the dissolution time for bare CaWO4 particles under different conditions were obtained. The results are summarized in Table 1. The 3-µm particle lifetime is estimated to be significantly longer than that of the 10-nm particle regardless of conditions. These results indeed support the working hypothesis that the uncoated CWO MPs release WO42- anions over an extended period of time, and therefore there likely exist some amount (a non-toxic amount) of WO4-2 within the tumor. Considering that in non-γ-irradiated groups the treatment with uncoated CWO MPs did not cause any effect on tumor growth relative to the PBS-treated cohort (Figure 10(C)), WO4-2 itself alone does not appear to have any direct destructive influence on cancer. Our current hypothesis is that although the dissolved tungstate (WO4-2) ions do not emit luminescence (the radio-luminescence of CaWO4 originates from the crystalline (tetrahedral) arrangement of the WO4 units), they are still able to produce secondary electrons under exposure to γ ray/X-ray radiation (similarly to crystalline CaWO4). As demonstrated in a recent paper16, however, the efficiency of electron emission from particulate CaWO4 (i.e., CWO NPs/MPs) is typically insufficient to produce radio therapy dose enhancement effects. We suspect that it becomes different when tungstates exist as dissolved ions because every W atom contributes to electron emission; in the particulate form of CaWO4, only W atoms in or near the surface layer contribute to emission of electrons to the outer surroundings (electrons generated from W atoms in the core region are absorbed by other W atoms before they escape from the particle territory), resulting in a significantly lower electron emission efficiency relative to its dissolved ionic state of WO4-2 for an identical amount of W atoms. To test this hypothesis we performed a simulation study using the procedure published previously16. Specifically, to determine the dissolved WO4-2 ions effect on the secondary particle spectrum inside a cell, a spherical 10 µm diameter “cell” was positioned inside a cylindrical geometry; note in this discussion we use the terminology “particles” to refer to both electrons (positrons) 22


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and photons. The WO4-2 ions were distributed uniformly at an assumed concentration of 2.2 × 105

M throughout the surrounding medium and inside the cell. Incident upon the spherical cell was

a monoenergetic, photon (() beam at 393 keV (corresponding to the maximum energy in the spectrum of a 1.2 MeV ( beam). Particle fluence was used as the metric to assess the effect of ions on the secondary electrons. The particle fluence is estimated by tracking the distance a particle, at a specific energy, travels before undergoing an interaction that alters the particles energy. The simulations were conducted using the Monte Carlo software package, Penetration and Energy Loss of Positrons and Electrons (PENELOPE 2014), developed by Francesc Salvat57. The steering program for the PENELOPE physics model was PenEasy developed by Brualla and coworkers58. The cutoff absorption values were set to 100 eV for both electrons and positrons, and 50 eV for photons. The simulation parameters C1, C2, WCC and WCR were set to the recommended 0.1, 0.1, 10 and 10, respectively. Maximum step sizes for the particles were set to < 1/10th the overall thickness of the material. Please refer to the material from Salvat for more details on the meaning of these parameters58-60. For more on the simulation procedure please see the prior study16, where the spherical nanoparticles are a scaled down version of the micrometer scale, macroscopic simulations conducted herein. As shown in Figure 11, the simulation results fully support this hypothesis, suggesting that uncoated CWO MPs offer another different approach to achieving radio-therapy dose enhancement—namely, through release of a small (non-toxic) amount of radio-enhancing heavy metal-bearing ions in situ (locally) within the tumor. This is another new concept that deserves further examination. Effect of folic acid (FOL) functionalization. We have been exploring how we can further improve the radio-enhancement effectiveness of PEG-PLA-coated CWO NPs. One approach we have been pursuing is to functionalize the nanoparticles with folic acid; the folic acid (or folate) receptor is overexpressed in many cancer cell types including head and neck cancer cells61 and therefore, the conventional wisdom is that folate ligands enhance the internalization of nanoparticles in cancer cells34. Folate-functionalized PEG-PLA-encapsulated CWO NPs were prepared as described in the Experimental Procedures section; also see Figures S10 through S12 of the SI for the molecular characteristics of the FOL-PEG-PLA polymer used in this study. HN31 cells treated with folate-functionalized versus non-folate-functionalized PEG-PLAencapsulated CWO NPs were examined with an in vitro clonogenic survival assay to determine their sensitivity to killing by X-rays/γ rays. The results are displayed in Figure 12. As shown in 23



Figure 12(A), the test with 320 keV X-ray source (at a dose rate of 1.9 Gy/min) demonstrated that surface-functionalization with folate indeed remarkably increased the radio-sensitization effects of CWO NPs. The value of the SER was estimated to be 1.55 for FOL-PEG-PLA CWO NPs; it is notable that this value is quite comparable to the best reported value of SER (≈ 1.5) for hafnium oxide (HfO2) nanoparticle radio-sensitizers15 that have been FDA-registered in the US as of December 31, 2015, and are currently under Phase II/III clinical trials in Europe and Asia for several different indications (Nanobiotix, nanobiotix.com). Note that folate functionalization does not produce any effect on the (non-)toxicity of PEG-PLA-coated CWO NPs in HN31 cells (Figure 3(C)). What was also notable is that the effect of folic acid depended on radiation energy and/or dose rate (Figure 12(B)). As shown in Figure 12(B), when the test was performed using a γ ray beam at an energy of 1.18 MeV and a dose rate of 10.3 Gy/min, folic acid did not produce any enhancement in CWO NP radio-sensitization; we suspect that it is the radiation dose rate (rather than the radiation energy) that is crucial to the functioning of folic acid, because at this dose rate even PEG-PLA CWO NPs and uncoated CWO MPs did not show any significant dose enhancement (Figure 12(B)), which is in contrast to what was observed at the same γ ray energy (1.18 MeV) but at a lower dose rate of 5.5 Gy/min (Figure 5(B)). Further study is needed to clarify the mechanisms involved in this phenomenon. Nevertheless, this result appears to suggest that increased nanoparticle cell internalization, at least, may not be the (main) cause of the folic acid-induced increased radio-sensitization, because otherwise the effect of folic acid should also be visible regardless of radiation dose rate, i.e., at the 10.3 Gy/min dose rate (Figure 12(B)). On the basis of the data shown in Figure 12(A), one may question whether folic acid itself alone has the ability to sensitize cells to X-ray/γ ray radiation. To answer this question, we performed an in vitro clonogenic survival assay in which HN31 cells were irradiated with 320 keV X-rays after respective treatments with either folate-functionalized or non-folatefunctionalized PEG-PLA (empty) micelles that do not contain CWO NPs. As shown in Figure 13, in the absence of CaWO4, neither folic acid-functionalized nor non-functionalized PEG-PLA micelles produced any radio-sensitization effects, which suggests that CWO NPs (that is, the UV-A light produced by them) were indeed the critical factor for the observed radio-sensitization of HN31 cells by FOL-PEG-PLA CWO NPs (Figure 12(A)). As an initial step toward better understanding how folic acid helps in the process of CaWO4 24


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radio-sensitization, we performed further quantitative analysis of the clonogenic survival data. Firstly, be reminded that we know what percentage of HN31 cells survive various doses of 320 keV X-ray radiation treatment (Figure 12(A), and also reproduced in Figure 14(B) for the reader’s convenience). We also know how much UV-A light is produced by PEG-PLA-coated CWO NPs under various doses of 320 keV X-rays (Figure 4(F)). In order to be able to predict how much dose enhancement occurs due to PEG-PLA CWO NPs (i.e., how much the original clonogenic survival curve shown in black in Figure 14(B) would be shifted downward for cells treated with PEG-PLA CWO NPs), an additional clonogenic survival assay was performed on HN31 cells directly irradiated with a UV-A lamp at various UV-A doses; the results are presented in Figure 14(A) (in black). Then, by combining this UV-A clonogenic survival data (Figure 14(A)) with the 320 keV X-ray clonogenic survival data shown in Figure 14(B), for each UV-A dose value we calculated an equivalent 320 keV X-ray dose value that would produce the same level of HN31 cell killing (red data in Figure 14(A)). Using this information and also the data shown in Figure 4(F), at each X-ray dose level (i.e., at each point on the original clonogenic survival curve shown in black in Figure 14(B)) we estimated the magnitude of dose enhancement in the units of X-ray dose. For instance, the magnitude of dose enhancement calculated at an original dose of 3 Gy is marked with a red horizontal arrow pointing to the right in Figure 14(B). For this enhanced X-ray dose level, we can calculate the expected cell survival percentage value (marked with a red vertical arrow pointing downward in Figure 14(B)). This adjusted survival percentage value actually corresponds to the original X-ray dose applied (3 Gy), as indicated with the red horizontal arrow pointing to the left. This algorithm allowed us to predict how much the original clonogenic survival curve shown in black in Figure 14(B) would be shifted if HN31 cells were treated with concomitant PEG-PLA CWO NPs; the resulting prediction is shown as a red dotted curve in Figure 14(B). As shown in the figure, this theoretical prediction is in excellent agreement with experimental data (red points/solid curve). This close agreement between prediction and experiment supports that in in vitro situations, even these non-folatefunctionalized PEG-PLA CWO NPs were, if not completely internalized, at least located at close proximity to HN31 cells (due to the sedimentation of the nanoparticles as discussed earlier); this could be inferred because the UV-A dosimetry measurements shown in Figure 4(F) (from which the predicted radiation survival curve for HN31 cells treated with PEG-PLA CWO NPs shown in Figure 14(B) was derived) were performed at the surfaces of PEG-PLA-coated CWO NPs. TEM 25



images taken from biopsies of mouse HN31 xenografts have demonstrated that non-folatefunctionalized PEG-PLA CWO NPs were indeed capable of being efficiently internalized by the tumor cells; see Supporting Information Figure S13 of Reference 23. Most importantly, it is now obvious from Figure 14(B) that the dramatic enhancement of the radio-enhancement effect of CaWO4 induced by folate functionalization (green data) cannot be explained by the dose enhancement effect of the concurrent UV-A radiation; folic acid appears to function not by the conventional dose enhancement mechanism. Further study is underway. Conclusion Although radiation therapy is a key component of cancer treatment, there are significant side effects. Thus there is great interest in the development of ways to achieve the benefits of radiation treatment with reduced negative effects. Our laboratory is developing a new type of radio-sensitizers (namely, CaWO4 Radio-Luminescent Particles (RLPs)) that make cancer cells easier to kill with less radiation. Data obtained with two different types of RLP formulation (uncoated CWO MPs of 2 – 3 µm diameter and PEG-PLA-encasulated CWO NPs of about 170 nm hydrodynamic diameter) demonstrated that CaWO4 RLPs are non-toxic and effective in producing radio-sensitization effects. Specifically, in in vitro clonogenic tests using radiationresistant cells (p53-mutant human head and neck cancer HN31 cells) both concomitant uncoated CWO MPs and polymer-coated CWO NPs induced about a factor of 1.1 enhancement of the cellkilling effect of 1.18-MeV γ rays (i.e., SER (at 10% cell survival) ≈ 1.1). Detailed radioluminescence dosimetry measurements and quantitative modeling analysis indicated that UV-A light generated by CWO MPs/NPs under γ ray/X-ray irradiation was indeed responsible for the observed enhancement of the tumor suppressive effect of γ rays/X-rays in HN31 cells. Concomitant CWO MPs/NPs also induced a similar level of decrease in the clonogenicity of 1.18-MeV γ ray-treated radiation-sensitive cells (a murine squamous cell sarcoma SCC7 cell line) (SER ≈ 1.1). The modes of HN31 and SCC7 cell death following γ ray/X-ray radiation in the presence and absence of uncoated CWO MPs and PEGylated CWO NPs were analyzed in vitro by FACS with Annexin V/PI double staining (apoptosis, early apoptosis, necrosis) and βgalactosidase assays (senescence). The combined results appear to suggest that the mitotic catastrophe pathway played a significant role in the CaWO4-induced radio-sensitization of p53mutant HN31 cells, which was in contrast to the more conspicuous role played by the apoptosis 26


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and necrosis mechanisms in increasing cell death of radio-sensitive SCC7 cells in response to concomitant CaWO4 treatment. Radio-sensitization efficacy of intratumorally injected CaWO4 particles (uncoated CWO MPs and PEG-PLA-caoted CWO NPs) was assessed in vivo in mouse HN31 xenografts. Interestingly, uncoated CWO MPs (at 11.8 mg CaWO4 per cc of tumor) almost completely suppressed tumor growth for almost 3 months, whereas the tumor suppression effect of PEG-PLA-coated CWO NPs was significantly lower at the identical CaWO4 dose (11.8 mg/cc). These data along with PENELOPE dosimetry simulation results suggest that non-toxic WO42- anions released from uncoated CWO MPs generated significant amounts of secondary electrons and photons under γ ray/X-ray irradiation, which therefore can account for the dramatic dose enhancement observed with uncoated CWO MPs. This finding suggests another completely new mechanism by which radiotherapy dose enhancement can be achieved by using CaWO4 compounds (and also by using other metal tungstate compounds with similar or better solubility characteristics). An in vitro clonogenic study showed that folic acid (FOL) functionalization dramatically improved the radio-enhancement effectiveness of PEG-PLA-coated CWO NPs. Further study is required to investigate this preliminary finding. Acknowledgments Funding for this research was provided by Purdue Office of the Executive Vice President for Research and Partnerships (OEVPRP) (New NIH R01 Program), Indiana Clinical and Translational Sciences Institute (CTSI) (Collaboration in Translational Research (CTR) Pilot Grant Program), Purdue University Center for Cancer Research (PCCR, NIH Grant P30 CA023168) (SIRG Graduate Research Assistantship, Shared Resource Biological Evaluation Project, and Phase I Concept Award), LoDos Theranostics LLC (Gift Grant), the School of Chemical Engineering at Purdue University, the US National Science Foundation (NSF, CBET1264336), the Global Innovative Research Center (GiRC) Program of the National Research Foundation of Korea (2012k1A1A2A01056095), and the “Global RNAi Carrier Initiative” Intramural Research Program of the Korea Institute of Science and Technology (KIST). We would like to thank Dr. Marc Mendonca in the Department of Radiation Oncology at the Indiana University School of Medicine for allowing us to use his 160 kV X-ray irradiator for obtaining the 160 kV dosimetry data shown in Figure 4. The HN31 cell line was generously provided by Dr. Jeffrey N. Myers at MD Anderson Cancer Center. Supporting Information (SI) Available: Summaries of clonogenic cell survival assay results (Tables S1 - S3); tumor growth suppression by UV-A light (Figure S1); DLS correlation functions for PEG-PLA-coated CWO NPs (Figure S2); PpIX fluorescence decays due to exposure to UV-A generated by CWO NPs under X-ray irradiation (Figure S3); apoptotic and necrotic populations in HN31 cells following γ ray treatment (Figure S4); effect of γ irradiation on clonogenic survival of HN31 cells in the presence of uncoated CWO MPs and PEG-PLAcoated CWO NPs (Figure S5); FACS analysis of γ/UV-A-irradiated HN31 cells with Annexin 27



V/PI staining (Figure S6); β-galactosidase assay visualization of senescent HN31 cells following γ irradiation in the presence of CaWO4 (Figure S7); FACS analysis of γ/UV-A-irradiated SCC7 cells with Annexin V/PI staining (Figure S8); kinetic model fits of CaWO4 dissolution data (Figure S9); synthesis scheme for FOL-PEG-PLA (Figure S10); 1H NMR spectra of the PEGPLA polymers used in this study (Figure S11); GPC traces for the PEG-PLA polymers used in this study (Figure S12). This information is available free of charge via the Internet at http://pubs.acs.org.

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Table 1. Estimated lifetimes of CaWO4 particles. Conditions

10 nm particle

3 µm particle

T = 23 °C, pH 7.0

2.2 hours

660 hours

T = 30 °C, pH 6.2

3.9 hours

1180 hours

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Figure 1. Schematic illustration of the concept of combining γ ray/X-ray radiation therapy with UV-A treatment through the use of radio-luminescent agents (“Radio Luminescence Therapy”). UV-A light can be generated in deep tissue tumors by delivering radio-luminescent (micro/nano) particles (RLPs) to the tumor and illuminating them with deep-penetrating γ rays/X-rays. UVinduced G2/M arrest makes cancer cells more susceptible to γ/X-ray radiation damage.

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Figure 2. (A) Schematic illustration, (B) a representative TEM micrograph, (C) the X-ray diffraction pattern, and (D) luminescence emission spectra (under 200 nm excitation) of poly(ethylene glycol-block-D,L-lactic acid)(PEG-PLA)-encapsulated CaWO4 (CWO) nanoparticles (NPs). Luminescence measurements were performed at an identical CWO concentration of 0.1 mg/ml.

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Figure 3. In vitro cytotoxicities of (A) uncoated CaWO4 (CWO) microparticles (MPs), (B) PEGylated (i.e., poly(ethylene glycol-block-D,L-lactic acid) or PEG-PLA-encapsulated) CaWO4 (CWO) nanoparticles (NPs) and (C) folate(FOL)-functionalized PEG-PLA-encapsulated CWO NPs (FOL functionality: 10%) in HN31 cells assessed by Cell Counting Kit-8 (CCK-8) (n = 5). The diameters of the CWO MPs were in the range of about 2 – 3 µm. The average diameter of the pristine CWO NPs was 10 nm. The mean hydrodynamic diameter of the PEG-PLA-coated CWO NPs was about 170 nm. The number-average degrees of polymerization of the PEG and PLA blocks of the PEG-PLA block copolymer used were determined by 1H NMR spectroscopy to be 113 and 44, respectively. In these cytotoxicity measurements, HN31 cells were seeded on 96-well culture plates at a density of 0.5 × 104 per well and incubated for 24 h prior to exposure to CWO. The cells were treated with uncoated CWO MPs or PEGylated CWO NPs for 24 h at the various CWO concentrations indicated above. CCK-8 assays were performed at 24 h postCWO treatment. Error bars represent standard deviations.

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Figure 4. (A) A schematic diagram illustrating the method used to measure the amount of UV-A radiation emitted by PEG-PLA-coated CWO NPs under X-ray/γ ray irradiation; fluorophores (PpIX) that photo-bleach under UV-A illumination were encapsulated within the hydrophobic PLA domain of the PEG-PLA (micelle) coating structure. (B, C, D, E) The degree of photobleaching of the encapsulated PpIX measured as a function of UV-A dose. Black squares were obtained by exposing the encapsulated PpIX to direct UV-A light (365 nm) using a UVP lamp. Red circles represent data obtained with (B) 160 keV X-rays (X-RAD 160, 0.738 Gy/min), (C) 320 keV X-rays (X-RAD 320, 2.3 Gy/min), (D) 60Co γ rays (1.17 and 1.33 MeV, 2.525 Gy/min), and (E) 6 MeV X-rays (Clinac 6EX, 0.4 Gy/min); under X-ray irradiation CWO NPs produce secondary UV-A photons. The degree of UV-A photo-bleaching was determined by measuring the PpIX fluorescence intensity at 635 nm emission wavelength under 406 nm excitation. The original PpIX fluorescence photo-bleaching data (from which the UV-A photo-bleaching degrees shown in red circles were calculated) are presented in Figure S3. As shown in the figure, X-ray/γ ray itself causes a slight decay in PpIX fluorescence intensity; therefore, the degree of PpIX photo-bleaching (caused by the secondary UV-A emission) was calculated by normalizing the fluorescence intensity obtained from X-ray/γ-ray-exposed PEG-PLA/CWO NP/PpIX NPs by the fluorescence intensity obtained from X-ray/γ-ray-exposed PEG-PLA/PpIX NPs (i.e., NPs that did not contain CWO NPs). (F) estimated from (B, C, D, E), the total fluence of UV-A radiation at the surfaces of PEG-PLA/CWO NP/PpIX NPs was determined as a function of X-ray/γ ray dose.

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Figure 5. Effect of γ ray irradiation on clonogenic survival of HN31 cells at various radiation doses. γ: γ radiation. UV → γ: 365 nm wavelength UV-A irradiation (8.9 mW/cm2 power density at 10-inch distance) for 20 minutes followed by γ radiation. γ → UV: γ radiation followed by 20minute UV-A irradiation. MP + γ: γ ray radiation in the presence of added 0.20 mg/ml uncoated CaWO4 (CWO) radio-luminescent microparticles (MPs) (2 – 3 µm diameter). NP + γ: γ ray radiation in the presence of added 0.20 mg/ml PEG-PLA-encapsulated CWO nanoparticles (NPs) (170 nm hydrodynamic diameter). HN31 cells were seeded on 60-mm2 culture plates at densities of 0.2 × 103 (0 Gy), 1.0 × 103 (3 Gy), 2.0 × 103 (6 Gy) and 5.0 × 103 (9 Gy) cells per plate. After 3 h incubation with CWO NPs/MPs the cells were exposed to various doses of 1.176-MeV 137Cs γ radiation (IBL 437C, CSI Bio International, France) at a rate of 5.455 Gy per minute. Irradiated cells were cultured for 14 days. Medium was replaced at day 1 post-radiation and then every 3 – 4 days afterwards. Colonies resulting from radio-resistant cells were stained with Crystal Violet. Colonies of more than 50 daughter cells in culture were counted (n = 4). The survival fraction was calculated based on the number of such colonies relative to that of the respective nonirradiated subgroup for each group. The solid curves represent fits to the linear-quadratic exponential survival function, S = exp(a + bD + cD2) where S denotes the survival fraction, D denotes the radiation dose, and a, b and c are fitting parameters. The results of the fitting and statistical analysis of these data are presented in Table S1. The cell line types, the γ ray energy levels and the types of radio enhancers used for different experiments can be summarized as follows: (A) HN31, 1.18 MeV γ ray, UV; (B) HN31, 1.18 MeV γ ray, CWO MP/CWO NP. Error bars represent standard errors.

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Figure 6. Apoptotic and necrotic populations measured by FACS with Annexin V/PI double staining in HN31 cells following exposure to γ radiation (5 Gy) in the absence and presence of concomitant UV-A light generated by CaWO4 (CWO). Control: no γ ray, UV-A light or CWO treatment. MP: in the presence of added 0.125 mg/ml uncoated CWO radio-luminescent microparticles (MPs) (2 – 3 µm diameter) with no γ radiation. NP: in the presence of added 0.125 mg/ml PEG-PLA-encapsulated CWO radio-luminescent nanoparticles (NPs) (170 nm hydrodynamic diameter) with no γ radiation. γ: γ radiation. MP + γ: γ radiation in the presence of added 0.125 mg/ml uncoated CWO MPs. NP + γ: γ radiation in the presence of added 0.125 mg/ml PEG-PLA-encapsulated CWO NPs. UV: 365 nm wavelength UV-A irradiation (8.9 mW/cm2 power density at 10-inch distance) for 20 minutes with no γ radiation. UV → γ: 365 nm wavelength UV-A irradiation for 20 minutes followed by γ radiation. γ → UV: γ radiation followed by 20-minute UV-A irradiation. HN31 cells were seeded on 60-mm2 culture plates at densities of 4 × 105, 2 × 105 and 1 × 105 cells per plate (for 24, 48 and 72-hour experiments, respectively) with DMEM medium containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin). After 24 h incubation the cells were exposed to a total 5-Gy dose of 137Cs γ radiation (IBL 437C, CSI Bio International, France) at a rate of 1 Gy per every 11 s. At different times (24, 48 and 72 hours) after these radiation treatments, the apoptotic, early apoptotic and necrotic populations were measured as the percentages of total cell populations by FACS (fluorescence-activated cell sorting) with Annexin V/PI double staining. The original twodimensional dot plots demonstrating the fluorescence gating criteria used are presented in Figure S6.

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Figure 7. Visualization of senescent HN31 cells by β-galactosidase assay following exposure to γ radiation (5 Gy) in the absence and presence of concomitant UV-A light generated by CaWO4 (CWO). Control: no γ ray, UV-A light or CWO treatment. γ: γ radiation. MP: in the presence of added 0.125 mg/ml uncoated CWO radio-luminescent microparticles (MPs) (2 – 3 µm diameter) with no γ radiation. MP + γ: γ radiation in the presence of added 0.125 mg/ml uncoated CWO MPs. UV: 365 nm wavelength UV-A irradiation (8.9 mW/cm2 power density at 10-inch distance) for 20 minutes with no γ radiation. UV → γ: 365 nm wavelength UV-A irradiation for 20 minutes followed by γ radiation. γ → UV: γ radiation followed by 20-minute UV-A irradiation. HN31 cells (total 4 × 105 cells) were seeded on a 4-well plate. After 24 h incubation the cells were exposed to a total 5-Gy dose of 137Cs γ radiation (IBL 437C, CSI Bio International, France) at a rate of 1 Gy per every 11 s. Irradiated cells were cultured for 3 or 6 days. Afterward the cells were stained with X-Gal Staining Solution (Sigma-Aldrich). The cells were imaged using a microscope in order to count blue-stained and unstained cells.

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Figure 8. Effect of γ ray irradiation on clonogenic survival of SCC7 cells at various radiation doses. γ: γ radiation. MP + γ: γ ray radiation in the presence of added 0.20 mg/ml uncoated CaWO4 (CWO) radio-luminescent microparticles (MPs) (2 – 3 µm diameter). NP + γ: γ ray radiation in the presence of added 0.20 mg/ml PEG-PLA-encapsulated CWO nanoparticles (NPs) (170 nm hydrodynamic diameter). SCC7 cells were seeded on 60-mm2 culture plates at densities of 0.2 × 103 (0 Gy), 1.0 × 103 (3 Gy), 2.0 × 103 (6 Gy) and 5.0 × 103 (9 Gy) cells per plate. After 3 h incubation with CWO NPs/MPs the cells were exposed to various doses of 1.176-MeV 137Cs γ radiation (IBL 437C, CSI Bio International, France) at a rate of 5.455 Gy per minute. Irradiated cells were cultured for 14 days. Medium was replaced at day 1 post-radiation and then every 3 – 4 days afterwards. Colonies resulting from radio-resistant cells were stained with Crystal Violet. Colonies of more than 50 daughter cells in culture were counted (n = 4). The survival fraction was calculated based on the number of such colonies relative to that of the respective nonirradiated subgroup for each group. The solid curves represent fits to the linear-quadratic exponential survival function, S = exp(a + bD + cD2) where S denotes the survival fraction, D denotes the radiation dose, and a, b and c are fitting parameters. The results of the fitting and statistical analysis of these data are presented in Table S1.

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Figure 9. Apoptotic and necrotic populations measured by FACS with Annexin V/PI double staining in murine squamous cell carcinoma SCC7 cells following exposure to γ radiation (5 Gy) in the absence and presence of concomitant UV-A light generated by CaWO4 (CWO). SCC7 cells (grown to about 70% confluency in RPMI 1640 medium (Invitrogen) containing 10% FBS and 100 mg/ml penicilin/streptomycin) were exposed to various radiation environments. Control: no γ ray, UV-A light or CWO treatment. γ: 137Cs γ radiation (IBL 437C, CSI Bio International, France) for 55 seconds (5 Gy total dose). MP: in the presence of added 0.5 mg/ml uncoated CWO radio-luminescent microparticles (MPs) (2 – 3 µm diameter) with no γ radiation. MP + γ: 6-MV γ radiation at 5 Gy total dose in the presence of 0.5 mg/ml uncoated CWO MPs. UV: 365 nm wavelength UV-A irradiation (8.9 mW/cm2 power density at 10-inch distance) for 20 minutes. UV → γ: 20-minute UV-A irradiation followed by 5-Gy γ radiation. γ → UV: 5-Gy γ radiation followed by 20-minute UV-A irradiation. At different times (24, 48 and 72 hours) after these treatments, the apoptotic, early apoptotic and necrotic populations were measured as the percentages of total cell populations by FACS with Annexin V/PI double staining. The original two-dimensional dot plots demonstrating the fluorescence gating criteria used are presented in Figure S8.

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Figure 10. Assessment of the radio-sensitization efficacy of intratumorally injected CWO MPs and NPs in mouse HN31 xenografts in terms of their effects on (A, C) tumor growth and (B, D) mouse survival. HN31 xenografts were prepared by implanting 1 × 107 cells (per mouse) to the left flank of male athymic nude mice (Balb/c, 5 weeks old, 20 to 26 g body weight, n = 6). When tumors were grown over a (A, B) 21- or (C, D) 14-day period to approximately (A, B) 250 or (C, D) 85 mm3, total (A, B) 0.30 or (C, D) 1.00 mg of uncoated CWO MPs or PEG-PLAencapsulated CWO NPs were infused directly into the tumor (total (A, B) 1.2 or (C, D) 11.8 mg of CWO per cc of tumor); the procedure involved (A, B) two injections of 120 µl CWO solution in PBS (CWO concentration: 1.25 mg/ml) over a two-day period or (C, D) four injections of 50 µl CWO solution in PBS (CWO concentration: 5.00 mg/ml) over a two-day period. After unfractionated 60Co γ radiation with Gamma Knife (total dose: 5.0 Gy, dose rate: 2.0 Gy per minute, γ ray energy: 1.17 and 1.33 MeV) (defined to be “day 0”), tumor volume was measured for (A, B) 73 or (C, D) 83 days (using the formula (L×W2)/2 to give volume in ml). Error bars represent standard errors. (A) and (C) show tumor size data for each group only up to the time point at which any one mouse dies of the tumor, or any one mouse has a tumor greater than 2000 mm3, or any one mouse loses more than 20% of its original weight; in the latter two situations, the mouse was humanely sacrificed and counted as dead. The same criteria were used for the survival analysis shown in (B) and (D).

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Figure 11. Computational comparison of the secondary electron and photon fluence (nm/eV/100 incident () within the cellular volume with and without WO4-2 ions. Both the electron and photon fluence is significantly enhanced (>1000) in the presence of the WO4-2 ions, at the simulated WO4-2 ion concentration of 2.2 × 10-5 M. This is especially true in the region of low energy electrons (< 1000 eV), that are expected to be the primary inducers of cellular stress. The grey bar indicates the ( beam energy, 393 keV. Error bars are on the order of or smaller than the symbol and correspond to 1 standard error. 102 Particle Fluence (nm/eV/100 photons)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


101 100 10-1

e + WO42-

10-2

e- baseline 2γ + WO4

10-3

γ baseline

10-4 10-5 10-6 10-7 10-8 2 10

103 104 Particle Energy (eV)

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105


Figure 12. Effect of X-ray (or γ ray) irradiation on clonogenic survival of HN31 cells at various radiation doses. X: X-ray radiation. γ: γ radiation. MP + X (or γ): X-ray (or γ ray) radiation in the presence of added 0.20 mg/ml uncoated CaWO4 (CWO) radio-luminescent microparticles (MPs) (2 – 3 µm diameter). NP + X (or γ): X-ray (or γ ray) radiation in the presence of added 0.20 mg/ml PEG-PLA-encapsulated CWO nanoparticles (NPs) (170 nm hydrodynamic diameter). NP-FOL + X (or γ): X-ray (or γ ray) radiation in the presence of added 0.20 mg/ml folic acid-functionalized PEG-PLA-encapsulated CWO nanoparticles (NPs) (170 nm hydrodynamic diameter). HN31 cells were seeded on 60-mm2 culture plates at densities of 0.2 × 103 (0 Gy), 1.0 × 103 (3 Gy), 2.0 × 103 (6 Gy) and 5.0 × 103 (9 Gy) cells per plate. After 3 h incubation with CWO NPs/MPs the cells were exposed to various doses of 320-keV X-ray radiation (XRAD 320, Precision X-Ray, USA) at a rate of 1.875 Gy per minute or 1.176-MeV 137 Cs γ radiation (Gammacell 1000 Elite, Best Theratronics, Canada) at a rate of 10.274 Gy per minute. Irradiated cells were cultured for 14 days. Medium was replaced at day 1 post-radiation and then every 3 – 4 days afterwards. Colonies resulting from radio-resistant cells were stained with Crystal Violet. Colonies of more than 50 daughter cells in culture were counted (n = 4). The survival fraction was calculated based on the number of such colonies relative to that of the respective non-irradiated subgroup for each group. The solid curves represent fits to the linearquadratic exponential survival function, S = exp(a + bD + cD2) where S denotes the survival fraction, D denotes the radiation dose, and a, b and c are fitting parameters. The results of the fitting and statistical analysis of these data are presented in Table S1. The cell line types, the Xray/γ ray energy levels and the types of radio enhancers used for different experiments can be summarized as follows: (A) HN31, 320 keV X-ray, CWO MP/CWO NP/CWO NP-FOL; (B) HN31, 1.18 MeV γ ray, CWO MP/CWO NP/CWO NP-FOL.

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Figure 13. Effect of X-ray irradiation on clonogenic survival of HN31 cells at various radiation doses. X: X-ray radiation. Micelle + X: X-ray radiation in the presence of added 0.20 mg/ml PEG-PLA micelles (63.0 nm hydrodynamic diameter) with no encapsulated CWO NPs. MicelleFOL + X: X-ray radiation in the presence of added 0.20 mg/ml folic acid-functionalized PEGPLA micelles (60.4 nm hydrodynamic diameter) with no encapsulated CWO NPs. HN31 cells were seeded on 60-mm2 culture plates at densities of 0.2 × 103 (0 Gy), 1.0 × 103 (3 Gy), 2.0 × 103 (6 Gy) and 5.0 × 103 (9 Gy) cells per plate. After 24 h incubation with PEG-PLA/FOL-PEGPLA micelles the cells were exposed to various doses of 320-keV X-ray radiation (XRAD 320, Precision X-Ray, USA) at a rate of 1.875 Gy per minute. Irradiated cells were cultured for 14 days. Medium was replaced at day 1 post-radiation and every 3 – 4 days afterwards. Colonies resulting from radio-resistant cells were stained with Crystal Violet. Colonies of more than 50 daughter cells in culture were counted (n = 4). The survival fraction was calculated based on the number of such colonies relative to that of the respective non-irradiated subgroup for each group. The solid curves represent fits to the linear-quadratic exponential survival function, S = exp(a + bD + cD2) where S denotes the survival fraction, D denotes the radiation dose, and a, b and c are fitting parameters. The results of the fitting and statistical analysis of these data are presented in Table S3.

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Figure 14. (A) Effect of UV-A irradiation (365 nm wave length, 8.9 mW/cm2 power density at 10-inch distance) on clonogenic survival of HN31 cells at various UV-A doses (black points). HN31 cells were seeded on 60-mm culture plates at a density of 1.0 × 102 cells per plate. After 24 h incubation with DMEM medium the cells were exposed to various doses of 365 nm UV-A radiation (B-100AP lamp, UVP). UV-irradiated cells were cultured for 14 days. Medium was replaced at day 1 post-radiation and then every 3 – 4 days afterwards. Colonies resulting from UV-resistant cells were stained with Crystal Violet. Colonies of more than 50 daughter cells in culture were counted (n = 4). The survival fraction was calculated based on the number of such colonies relative to that of the non-UV-irradiated group. The black solid curve represents a fit to the linear-quadratic exponential survival function, S = exp(a + bD + cD2) where S denotes the survival fraction, D denotes the UV-A dose, and a, b and c are fitting parameters; the best fit values were: a = -8.73 × 10-3, b = -0.494, and c = 0.132. Based on the X-ray-only colonogenic data shown in Figure 12(A) (“X”), 320-keV X-ray dose levels that produce equivalent effects on HN31 cell survival were estimated for various UV-A fluence values (red points/curve). (B) Comparison of prediction (red dotted curve, based on parameters obtained from (A)) to experimental results for the X-ray dose enhancement effects of NP-FOL (green points/curve, redrawn from Figure 12(A)). The prediction was made assuming that the X-ray dose enhancement is mainly due to the UV-A radiation produced by CWO NPs. The detailed procedure used to produce the predicted result is discussed in the text.

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For Table of Contents (TOC) Use Only

PEG-PLA-Coated and Uncoated Radio-Luminescent CaWO4 Micro/Nanoparticles for Concomitant Radiation/UV-A and RadioEnhancement Cancer Treatments Sung Duk Jo,1,§ Jaewon Lee,2,§ Min Kyung Joo,1 Vincenzo J. Pizzuti,2 Nicholas J. Sherck,2 Slgi Choi,2 Beom Suk Lee,1 Sung Ho Yeom,3 Sang Yoon Kim,1 Sun Hwa Kim,1 Ick Chan Kwon,1 You-Yeon Won1,2,4,* 1

Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, South Korea

2

School of Chemical Engineering, Purdue University, West Lafayette, Indiana 47907, United States of America 3

Department of Biochemical Engineering, Gangneung-Wonju National University, Gangneungsi, Gangwon-do 25457, South Korea 4

Purdue University Center for Cancer Research, West Lafayette, Indiana 47907, Unites States of America §

Co-first authors

*

To whom correspondence should be addressed: [email protected]

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PEGâPLA-Coated and Uncoated Radio-Luminescent CaWO4 Micro

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