Materials
Cs2CO3 (99%, Sigma, Cat#441902), I2 (≥ 99.8%, Sigma, Cat#207772), NaI (≥ 99.5%, Sigma, Cat#383112), magnesium acetylacetonate dihydrate, (Mg(acac)2, 98%, Sigma, Cat#129577), 1-Octadecene (C18H36, technical grade, 90%, Sigma, Cat#0806), oleic acid (C18H34O2, technical grade, 90%, Sigma, Cat#364525), oleylamine (C18H35NH2, technical grade, 70%, Sigma, Cat#07805), 1,2-tetradecanediol (technical grade, 90%, Sigma, Cat#260290), 1,2-hexadecanediol (technical grade, 90%, Sigma, Cat#213748), hexane (C6H14, ≥ 99%, Sigma, Cat#139386), ethanol (anhydrous, Sigma, Cat#443611), benzyl ether (98%, Sigma, Cat#108014), chloroform (CHCl3, ≥ 99.8%, Fisher Scientific), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (16:0 Liss Rhod PE) (Avanti, Cat#810,158), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (sodium salt) (DSPE-PEG(2000) Carboxylic Acid) (Avanti, Cat#880135), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000] (ammonium salt) DSPE-PEG(2000) Folate (Avanti, Cat#880124), methylene blue (C16H18ClN3S·xH2O, powder, ≥ 82%, Sigma), phosphate buffer saline (PBS, pH 7.2), Milli-Q Water (H2O, 18.2 MΩ.cm@25 °C).
Synthesis of CsI(Na) nanoparticles
0.203 g Cs2CO3 was mixed with 10 mL 1-octadecene and 1 mL oleic acid in a 100 mL flask. The solution was heated to 150 °C and maintained at this temperature for 30 min with magnetic stirring. Next, 1 mL oleylamine, 0.02954 g 1,2-hexadecanediol, and 0.01 g NaI were added into the mixture. After reacting for 10 min, the solution was cooled to room temperature, and 0.3165 g I2 were added. The flask was then sealed and stirred for another 3 h. CsI nanoparticles were collected by centrifugation and washed with a 1:1 hexane and ethanol mixture 3 times.
Synthesis of CsI(Na)@MgO nanoparticles
In a typical reaction, 20 mg CsI(Na) nanoparticles in 5 mL hexane was mixed with 0.02 g Mg(acac)2, 0.053 g 1,2-tetradecanediol, and 20 mL benzyl ether in a 100 mL three-neck flask. The solution was heated to 120 °C under Argon protection and maintained at this temperature for 20 min. The solution was further heated to 310 °C before the addition of 1 mL pre-heated oleic acid. The reaction continued for 10 min before being cooled to room temperature. The product was collected by centrifugation and washed with a 1:1 hexane and ethanol mixture 3 times.
Synthesis of CIS@M-F
20 mg CsI(Na)@MgO nanoparticles were dispersed in 2 mL chloroform. Into the solution, 75 µL of DSPE-PEG(2000)-COOH in chloroform (10 mg mL−1) and 25 µL DSPE-PEG(2000)-Folate in chloroform (10 mg mL−1) were added. The mixture was stirred at room temperature overnight, and the solvent was removed by rotary evaporation. The nanoparticles were dispersed in PBS and passed through a desalting column before use.
Synthesis of CIS@M-F or CIS@M-C with rhodamine B labelling
20 mg CsI(Na)@MgO nanoparticles were dispersed in 2 mL chloroform. Into the solution, 50 µL of DSPE-PEG(2000)-COOH in chloroform (10 mg mL−1) and 25 µL DSPE-PEG(2000)-Folate in chloroform (10 mg mL−1) together with 25 µL of 16:0 Liss Rhod PE in chloroform (10 mg mL−1) were added. The mixture was stirred at room temperature overnight, and the solvent was removed by rotary evaporation. The nanoparticles were dispersed in PBS and passed through a desalting column before use.
Nanoparticle characterizations
Nanoparticle crystallinity was assessed using the Bruker D8-Advance X-ray diffraction (XRD) diffractometer with Cu Kα radiation (λ = 1.5418 Å) at a scanning rate of 10° min−1. The hydrodynamic sizes and surface charges of the particles were characterized on a Malvern Zetasizer Nano ZS system. Nanoparticle size, morphology, and elemental analysis was characterized using a Scanning Electron Microscope (FE-SEM Thermo Fisher Teneo) which was equipped with an EDX system and Transmission Electron Microscope (FEI Tecnai20 and FEI Tecnai G2 F30 Hi-Res TEM). Nanoparticle composition was analyzed by Inductively Coupled Plasma Atomic Emission Spectroscopy using an Xseries II ICP/MS system (Thermo Electron Corporation). An iodide-selective electrode was used to conduct release experiments in PBS solutions of nanoparticles at room temperature (Mettler Toledo perfectION™).
Radical production
100 µL PBS control, 100 µg mL−1 CIS@M-F, 0.04 M PpIX, and 100 µg mL−1 CIS@M-F plus 0.04 M PpIX solutions were prepared, distributed into a 96-well plate, and irradiated with 5 Gy X-ray (X-RAD 320). 80 µL 1 µм methylene blue was added to each well immediately following radiation, and the plate was shaken and kept in darkness at room temperature for 5 min before testing. A UV–vis spectrometer was then used to record absorbances (664 nm).
Cell culturing
4T1 breast cancer cells were used for in vitro and in vivo studies. Cells were grown in RPMI1640 medium which was supplemented with 10% FBS and 100 units mL−1 of penicillin (ATCC). Cells were maintained in a humidified, 5% carbon dioxide (CO2) atmosphere at 37 °C.
ATP viability assay to test nanoparticle and 5-ALA toxicity
The ATP viability assay was performed according to the manufacturer’s protocol (PerkinElmer, ATPlite 1step Luminescence Assay Cat#6016736)[61]. 4T1 cells were seeded at 5000 cells/well in a white 96-well plate. After 24 h of incubation, CIS@M-F and 5-ALA (18.75, 37.5, 75, 150, 300, 600 µg mL-1 for CIS@M-F;19.5, 78.1, 312.5, 1250 µg mL−1 for 5-ALA) were added to each well for another 24 h of incubation. The ATP kit solution was then added to each well, and a 96-well microplate reader was used to measure total luminescence. Cell viability was calculated as a percentage of the luminescence of the untreated control.
ATP viability assay to test X-PDT efficacy
The ATP viability assay was performed according to the manufacturer’s protocol (PerkinElmer, ATPlite 1step Luminescence Assay Cat#6016736). 4T1 cells were seeded at 5000 cells/well in a white 96-well plate and incubated for 24 h before the addition of 20 µg 5-ALA to each well. Cells were incubated for 1 h before the addition of 10 µg CIS@M-F. Cells were then incubated for 2 h before being irradiated (5 Gy). The plate was returned to the incubator and maintained in darkness for 24 h before the ATP luminescence test.
Intracellular PpIX analysis
PpIX extraction was performed according to a published protocol [62]. Cells were seeded in a 96-well plate at 5000 cells/well. After 24 h of incubation, cells were trypsinized, harvested by centrifugation, and redispersed in 5% HCl at 37 °C for an hour. Following incubation in acid, the supernatant was collected and fluorescence signals (ex/em: 406/604 nm) were recorded.
APF assay
ROS (reactive oxygen species) were measured with the APF assay (Invitrogen™ Cat#A36003) [63]. 4T1 cells were seeded in 96-well plates at 5000 cells/well. After 24 h, nanoparticles in 100 µL RPMI medium or medium only were added to each well and incubated for 2 h before being irradiated (5 Gy by X-Rad 320). Thereafter, the plate was incubated with 100 µL APF solution (2 µм) for 30 min at room temperature in the dark. Lastly, the medium was diluted with an equal volume of fresh PBS, and fluorescence signals (ex/em: 490/515 nm) were analyzed on a microplate reader (Biotek).
SOSG (singlet oxygen) assay
The SOSG assay was conducted following the vendor’s protocol (Invitrogen™ Cat#S36002) [64]. 4T1 cells were seeded in 96-well plates at 5000 cells/well. Following 24 h of incubation, the medium was aspirated and 20 µL SOSG solution (5 µм) with 100 µg mL−1 CIS@M-F in 100 µL medium were added to each well. After 1 h, 20 µL 5-ALA (1 mg mL−1) was added. 5 Gy radiation was delivered following another 2 h of incubation. Fluorescence signals (ex/em: 504/525 nm) were recorded on a microplate reader (Biotek).
Cell uptake studies
Cell uptake of CIS@M-C and CIS@M-F nanoparticles were analyzed on a CytoFLEX flow cytometer. 4T1 cells were seeded at 0.5 × 106 cells/well into 6-well plates. Then, nanoparticles were incubated with cells at a final nanoparticle concentration of 100 µg mL−1. Both CIS@M-F and CIS@M-C were labeled with Rhodamine B following a published protocol [65]. Following either 2 or 6 h of incubation, cells were harvested for flow cytometer analysis, and the MFI was recorded.
SOD activity
SOD activity was assessed following the vendor’s protocol (Cayman Chemical Cat#706002). 4T1 cells were seeded into 6-well plates at 1 million cells/well. After 24 h, 2 mL RPMI medium containing 100 µg mL−1 CIS@M-F and 200 mL−1 5-ALA were added to each well. 5 Gy X-ray irradiation was delivered after 4 h. Immediately following irradiation cells were washed with PBS three times and collected with a rubber scraper. Cell pellets were subjected to differential centrifugation at 4 °C and 12,000 rpm for 20 min to separate the mitochondrial and cytosolic fractions. Both the supernatant and mitochondrion were collected, aliquoted, sonicated and transferred into a 96-well plate. Test kit solution was added to each well, and the 96-well plate was shaken for 10 min in the dark at room temperature before measurement. Absorbance (450 nm) was measured on a microplate reader (Biotek).
Lipid peroxidation
The Image-iT Lipid Peroxidation Kit (Invitrogen™ Cat#C10445) was used to assess lipid peroxidation. 4T1 cells were seeded into 96-well plates at 5000 cells/well. Following 24 h of incubation, 200 µg mL−1 5-ALA or 100 µg mL−1 CIS@M-F were added to each well. Following 4 h of incubation, the plate was irradiated (5 Gy). The Image-iT Lipid Peroxidation dye was added to each well, and the plate was incubated at 37 °C and 5% CO2 for 30 min. Green (ex/em: 488/510 nm) fluorescence intensity was used to quantitate lipid peroxidation.
Caspase-3 activity
4T1 cells were incubated with CIS@M-F (100 µg mL−1) and 5-ALA (200 µg mL−1) for 2 h prior to receiving 5 Gy X-ray irradiation. Control treatments included CIS@M-F, 5-ALA, or PBS. Following 24 h of incubation, cells were stained with the FAM-FLICA® Caspase-3/7 kit (Immunochemistry, Cat#94) following the manufacturer’s protocol. The caspase-3 activity was evaluated by measuring fluorescence signals (ex/em: 488/530 nm) on a microplate reader (Synergy Mx, BioTeK).
Mitochondrial membrane potential (ΔΨm)
Mitochondrial potential was assessed using the TMRE staining kit following the vendor’s protocol (Abcam Cat#ab113852). 4T1 cells were incubated with CIS@M-F (100 µg mL−1) for 2 h followed by 5-ALA (200 µg mL−1) for 3 h before receiving 5 Gy irradiation. Control treatments included CIS@M-F, 5-ALA, or PBS with or without irradiation. The medium was aspirated after 24 h, and cells were incubated in TMRE staining solution for 15 min. Fluorescence signals (ex/em: 549/575 nm) were measured on a microplate reader.
γH2AX
DNA damage was evaluated using anti-rH2AX (Alexa 647 labeled) antibodies (Millipore Sigma, Cat# 07-164-AF647). Briefly, 4T1 cells were seeded onto a 4-well imaging chamber at a density of 10,000 cells/well and incubated for 24 h. After washing, the cells were incubated with CIS@M-F (100 µg mL−1) for 2 h followed by 5-ALA (200 µg mL−1) for 3 h before receiving 5 Gy irradiation. Control treatments included CIS@M-F, 5-ALA, or PBS with or without irradiation. After 1 h, cells were collected, fixed, permeabilized, and stained with anti-rH2AX antibodies following the vendor’s protocol. Fluorescent images were acquired on a Zeiss LSM 710 confocal microscope. ImageJ was used to count the number of foci per cell.
Clonogenic assay
Clonogenic assays were performed using a modified protocol [66]. 4T1 cells were pre-seeded into 6-well plates. After 24 h, cells were incubated with CIS@M-F (100 µg mL−1) for 2 h followed by 5-ALA (200 µg mL−1) for 3 h before receiving 0, 1, 3, 5, 7, and 9 Gy of radiation. Treated cells were trypsinized, replanted in petri dishes (100 * 15 mm), and incubated at 37 °C with 5% CO2. After 14 days, cells were rinsed carefully with PBS, fixed in 2–3 mL of 6.0% glutaraldehyde solutions, and treated with 1 mL of 0.5% crystal violet. After 10 min, cells were rinsed with D.I. water and dried before colony counting. Colonies containing at least 50 stained cells were included in survival fraction (SF) calculations.
In vivo therapy studies
Animal studies were performed according to a protocol (A2020 06-004-R1) approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Georgia. The animals were maintained under pathogen-free conditions. 4T1 tumors were established by subcutaneously injecting 2 × 105 cells in 50 µL PBS into the right flanks of 5–6-week old female BALB/c mice (Charles River). When tumor volume reached 50 mm3, the animals were randomly divided into 6 groups (n = 5) and received the following treatments (Day 1): PBS plus ionizing radiation (PBS + IR), 5-ALA with irradiation (5-ALA + IR), CIS@M-F plus irradiation (CIS@M-F + IR), PBS only (PBS), CIS@M-F plus 5-ALA, no irradiation (CIS@M-F + 5-ALA), or CIS@M-F plus 5-ALA plus irradiation (CIS@M-F + 5-ALA + IR). 5-ALA (50 mg kg−1 in PBS) was administered intraperitoneally. CIS@M-F in PBS (1.25 mg kg−1) were intratumorally injected 2 h after the 5-ALA injection. A 320 KV cabinet irradiator (X-RAD 320, Precision X-ray, Inc.) was used to irradiate (3 Gy) tumors 1 h after CIS@M-F administration, while the rest of the animal body was protected with lead. Animals underwent two additional treatment sessions on Days 3 and 5. Tumor size and body weight were inspected every 3 days. Tumor dimensions were measured with a caliper. Tumor volume was estimated by calculating (length) × (width)2/2. Animals were euthanized after 22 days. Tumors were dissected and sliced for H&E and Ki67 staining. Organs including the heart, spleen, liver, brain, intestine, kidney, and lung were also harvested for H&E staining.
Biodistribution studies
5–6 week-old female BALB/c mice (Charles River) were intravenously injected with CIS@M-F (50 µL, 1.25 mg kg−1) or PBS (control) via the tail vein. All mice were sacrificed after 2 weeks. Blood was collected through cardiac puncture for complete blood count (CBC), BUN, and ALT measurements. Major organs, including the heart, spleen, liver, brain, intestine, kidney, and lung were harvested. Half of the tissues were weighted, homogenized, and digested in hot nitric acid. Supernatants were subjected to ICP-MS analysis to measure tissue concentrations of cesium and iodine in tissues (µg/g of tissue). The remaining tissues were fixed and sliced for H&E staining.
Statistical analysis
All quantitative data were shown as mean ± SD. Statistical analysis was conducted using student’s t or ANOVA test. *p < 0.05, **p < 0.01, ***p < 0.001.