Materials
Phosphatidyl choline (L-α-phosphatidylcholine, 95%, Avanti Polar Lipids, Cat# 131601), triolein (glyceryl trioleate, Sigma, Cat# T7140), 7-dehdrocholesterol (Sigma, Cat# 30800), cholesterol (Sigma, Cat# C3045), chloroform (Fisher Scientific, C298-4), methanol (Fisher Scientific, A452-4), 100-nm filter (Whatman, 800309), trypsin (0.25% with EDTA, Corning, Cat# 25-053-CI), DMSO (dimethyl sulfoxide, Fisher Scientific, Cat# BP231-1), trypan blue (0.4% in PBS, Corning, Cat# 25-900-CI), PBS (phosphate buffer saline, pH 7.4, Gibco, Cat# 10010023), Milli-Q Water (H2O, 18.2 MΩ.cm@25°C), crystal violet (Sigma, Cat# C0775), paraformaldehyde (4% in PBS, Chem Cruz, Cat# sc-281692), acetone (Fisher Scientific, A18-4), formalin (10% neutral buffered, Cancer Diagnostics, Cat# FX1000), sodium azide (Sigma, Cat# S8032), dynasore (Sigma, Cat# D7693), nystatin (Sigma, Cat# N6261), chlorpromazine (chlorpromazine hydrochioride, Sigma, Cat# C0982), tocopherol (α-tocopherol polyethylene glycol succinate, TCI, Cat# T3118), ascorbic acid (L-ascorbic acid, Sigma, Cat# A92902), ferrostatin-1 (Cayman, Cat# C816Z13), deferoxamine (deferoxamine mesylate, Sigma, Cat# PHR3411), glycine (Sigma, Cat# G7126), Z-VAD-FMK (Sigma, Cat# V116), 3-methyladenine (Sigma, Cat# M9281). Myristoylated NTSmut was custom-ordered from CSBio.
Nanoparticle Preparation
Phosphatidyl choline, triolein and 7-dehydrocholesterol (7DHC) were dissolved in a CHCl3/MeOH (v: v 2:1) solvent at a 3:2:1 molar ratio. For preparation of N-7DHC-lipos, myristoylated NTSmut (Lys-Pro-(NMe-Arg)-Arg-Pro-Tyr-Tle-Leu) at a 1:20 molar ratio to phosphatidylcholine was also added to the mixture. After removing the solvent via rotary evaporation, Tris buffer (pH = 8.0) was added to the flask and stirred for an hour at 55–65 °C to reconstitute the nanoparticles. The nanoparticles were then extruded at 55–65 °C through a 100-nm filter using a mini-extruder (Avanti Polar Lipids, Cat# 610020) for size uniformity. The resulting nanoparticles were stored at 4 °C. Synthesis of cholesterol encapsulated liposome counterpart followed the same procedure except replacing 7DHC with cholesterol.
To analyze targeting ligand and 7DHC contents, liposomes were lyophilized, weighted, and reconstituted in MeOH. The concentrations of myristoylated NTSmut and 7DHC was determined using LC-MS (Bruker Elute UHPLC and Bruker Impact II) with the following settings: Chromatography (Mobile Phase: 90% methanol, 10% water, 0.1% formic acid; Flow rate: 0.4 mL/min; 15 min isocratic gradient; Column Temperature: 40 °C); Column (Kinetex, Evo C18, 1 × 100 mm, 1.7 μm, 100 Å); Mass Spectrometry (Positive ionization mode (ESI); Voltage: 2.5 kV; Desolvation Temperature: 450 °C; Desolvation Gas Flow: 700 L/hr). Standards for 7DHC and myristoylated-NTSmut were analyzed to establish standard curves, which were used to quantify the concentrations of 7DHC and myristoylated-NTSmut in the samples.
Cryogenic TEM
Cryogenic TEM (cryo-TEM) grids were prepared using Vitrobot Mark IV (Field Electron and Ion Company, Hillsboro, OR) with the following settings: blot force of -10, wait time of 10 s, and blot times of 3,4 and 6 s. Quantifoil R1.2/1.3 400 Cu mesh grids were rendered hydrophilic with a TergeoEM (PIE Scientific LLC, Union City, CA) using indirect oxygen-argon plasm (25:75 ratio). 7DHC-Lipos or N-7DHC-Lipos solutions (~ 10 mg/mL) were applied to the carbon side of a TEM grid prior to vitrification by immersion in liquid ethane-propane (40:60 mixture). All images were analyzed using Image J software with at least 50 nanoparticle measurements to ensure a global representation of the assembled structure.
Dynamic light scattering and zeta potential
Zeta potential and size distribution measurements were carried out using a Malvern Zetasizer Nano ZS system. Prior to analysis, the solvent was exchanged for 1× PBS (pH 7.4) using a desalting column. To evaluate the stability of the liposomes, samples were stored at 4 °C for one week and the dynamic light scattering (DLS) was performed on days 1, 2, 3, and 7.
7DHC release
To measure 7DHC release, 7DHC-Lipos were loaded into a 10k MWCO dialysis tube and the tube was immersed in PBS solutions with pH values of 5.5, 6.5, and 7.4, respectively. The solutions were mounted on a shaker and the incubation temperature was maintained at 37 °C. Aliquots of the samples were taken at different time points (0.25, 0.5, 1, 2, 4, 8, 12, 24, 48 and 72 h), and the 7DHC content was quantified by LC-MS, as described above. For CHOL-Lipos, released cholesterol was quantified using a Cholesterol Quantification Assay kit (Sigma, Cat# CS0005) following the manufacturer’s instructions.
Cell culture
NCI-H1299, Hcc827, H460, and LLC1 cells were purchased from ATCC and cultured according to ATCC protocols. Typically, a complete growth medium was prepared by adding 50 mL fetal bovine serum (FBS, Atlanta Biologicals, Cat# S11150) and 5 mL penicillin-streptomycin (Corning Cat# 30-002-CI) to 445 mL of RPMI 1640 medium (Corning, Cat# 10-104-CV). Cells were subcultured every three days and maintained at 37 °C in a Thermo Scientific Heracell 150i incubator. One day before the experiment, the cells were washed with PBS, trypsinized (37 °C, 2 min), neutralized with cell culture medium, and centrifuged (1200 rpm, 5 min). The supernatant was removed, and the cells were resuspended in a fresh cell culture medium. For viability and other assays, cells were quantified using a hemocytometer (Hausser Scientific, Cat# 3200) before seeding the desired number of cells onto plates.
Cellular uptake (Florescence Microscopy)
DiR dye (Biotium, Cat# 60017) was added during the synthesis of 7DHC-Lipos and N-7DHC-Lipos, and was incorporated into the lipid layer of the nanoparticles. One day before the incubation, 1 × 105 H1299 cells were seeded on a 2-chamber glass slide (Nunc™ Lab-Tek™ II Chamber Slide™ System, ThermoFisher, Cat# 154534) and incubated at 37 °C overnight. The cell culture medium was removed, and the cells were incubated with 1 mL of serum-free cell culture medium containing DiR-labeled 7DHC-Lipos/N-7DHC-Lipos (50 µg/mL) for 4 h at 37 °C. After incubation, the cells were washed three times with PBS, fixed with 4% paraformaldehyde, and stained with DAPI. Fluorescence images were captured with a fluorescence microscope (Keyence, BZ-X800). To examine the ability of N-7DHC-Lipos to fuse with cell membranes, the nanoparticles were labeled with both calcein (Cayman, Cat# 16221) and DiL (ThermoFisher, Cat# D3911), which were loaded into the interior and the lipid layer of the nanoparticles, respectively. The nanoparticles were incubated with H1299 cells and the live cells were imaged by fluorescence microscopy. The images were analyzed using Image J.
Cellular uptake (flow cytometry)
16:0 Liss Rhod PE (Avanti, Cat# 810158 C) was added during the synthesis of 7DHC-Lipos and N-7DHC-Lipos to dye-label the nanoparticles. One day before incubation, 5 × 105 H1299 cells were seeded on a 6-well plate (Corning, Cat# 3516) and incubated at 37 °C overnight. The cell culture medium was removed, and the cells were incubated for with 2 mL of Rhod-labeled 7DHC-Lipos or N-7DHC-Lipos suspended in serum-free cell culture medium (50 µg/mL). For comparison, endocytosis inhibitors, including sodium azide (50 mM), dynasore (80 µM), nystatin (25 µM), and chlorpromazine (100 µM), were co-incubated with the nanoparticles. After 4 h, the cells were washed once with PBS, then incubated with DAPI-containing staining buffer (ThermoFisher, Cat# D1306) for 5 minutes. Next, the cells were collected by a cell lifter and washed once with PBS. Cells were then fixed with a 1:1 mixture of IC fixation buffer (Invitrogen, Cat# 00-8222-49) for 15 min, and resuspended in staining buffer. Trypan blue solution (20 µg/mL) was added to quench fluorescence from nanoparticles non-specifically bound to cell membranes. Flow cytometry was performed on NovoCyte Quanteon Flow Cytometer Systems (Agilent) and the mean fluorescence intensity (MFI) of Rhod was recorded. The uptake study with NTSR1 negative Hcc827 cells followed the same protocol.
Cell binding assay
The affinity of N-7DHC-Lipos for NTSR1 was assessed using neurotensin (NTS)-NOTA-Cu64 as a competitive ligand. Briefly, H1299 cells were seeded in a 24-well plate (1.5 × 105 cells per well) and incubated overnight at 37 °C in a 5% CO2 atmosphere. The medium was replaced with serum-free medium containing different concentrations of N-7DHC-Lipos (with NTSmut concentration of 400, 200, 50, 20, 2, and 0.2 nM, respectively) and 5 µCi of NT-NOTA-Cu64. For comparison, solutions containing free NTS (10000, 1000, 100, 10, and 1 nM, CSBio, CA) and 5 µCi of NT-NOTA-Cu64 were tested. All concentrations were tested in triplicate. After incubation for 1 h at 37 °C in a 5% CO2 atmosphere, the cells were rinsed three times with ice-cold PBS, and incubated with 1 N NaOH. Cells were collected and their radioactivity was measured using a γ-counter (PerkinElmer). Data were analyzed using Prism (GraphPad). Figures were plotted as counts per minute of radioactivity versus the concentration of NTS in nM on a log scale.
Cellular superoxide and hydroxyl radical levels
Superoxide levels were assessed using the Dihydroethidium Assay Kit (DHE, ThermoFisher, Cat# D11347). Briefly, H1299 cells (8,000 cells per well) were seeded on a black 96-well plate (Corning Costar, Cat# 3603) overnight. The next day, the cell culture medium was replaced with fresh serum-free medium containing either PBS or 50 µg/mL of N-7DHC-Lipos. After incubation for 4 h at 37 °C, the medium was removed and replaced with 5 µM DHE in FBS-free RPMI medium. After incubation in the dark for 30 min at room temperature, the cells were irradiated (5 Gy, X-Rad 320 Irradiator). DHE fluorescence (ex/em: 518/605 nm) was measured using a microplate reader (Synergy Mx, BioTeK). In control groups, nystatin (25 µM), tocopherol (100 µM), or ascorbic acid (100 µM) were added together with N-7DHC-Lipos to incubate with the cells.
Hydroxyl radical levels were assessed using the Aminophenyl Fluorescein Assay Kit (APF, Invitrogen™ Cat# A36003) by reading APF fluorescence (ex/em: 490/515 nm). Otherwise, the protocol was the same as for the DHE study.
Superoxide dismutase (SOD) activity
H1299 cells were seeded in a 6-well plate at a density of 1 × 106 cells per well (Corning, Cat# 3516) and incubated overnight at 37 °C. The next day, the cell culture medium was replaced with fresh serum-free medium containing either PBS or 50 µg/mL of 7DHC-Lipos. After incubation for 4 h at 37 °C, the cells were irradiated (5 Gy) using an X-RAD 320 irradiator. After incubation for an additional 1 h, the cells were washed 3 times with PBS, and collected with a cell scraper followed by centrifugation (1200 x g, 5 min). The cells were re-suspended in 1 mL of PBS and lysed by sonication with a probe sonicator (Fisherbrand™ Model 120 Sonic Dismembrator) in an ice bath (30% amplitude, 30 s, 5 s pause every 10 s). The supernatant was collected by centrifugation (1500 x g, 5 min) and analyzed with the Superoxide Dismutase Assay Kit (Cayman Chemical, Cat# 706002) according to the manufacturer’s protocol. Absorbance at 440 nm was measured using a microplate reader (Synergy Mx, BioTeK).
DNA damage
H1299 Cells (1 × 106) were seeded in a 6-well plate and incubated overnight. The next day, the cell culture medium was replaced with fresh serum-free medium containing PBS or 50 µg/mL of N-7DHC-Lipos. For the IR and 7DHC-Lipos plus IR groups, the cells received 5 Gy irradiation after 4 h. After further incubation for 1 h in full growth medium, cells were collected, fixed, permeabilized, and stained anti-γH2AX (Alexa 488) antibody (Biolegend, Cat# 613405) and DAPI according to the manufacturer protocol. The stained cells were analyzed using flow cytometry. In control groups, cells were co-incubated with nystatin (25 µM), tocopherol (100 µM), or ascorbic acid (100 µM) for comparison.
Lipid peroxidation
Lipid peroxidation was assessed using BODIPY™ 581/591 C11 (Lipid Peroxidation Sensor, ThermoFisher, Cat# D3861). Briefly, H1299 cells (8,000 cells per well) were seeded onto a black 96-well plate (Corning Costar, Cat# 3603). The next day, the cell culture medium was replaced with serum-free medium containing PBS or 50 µg/mL of N-7DHC-Lipos. After incubation at 37 °C for 4 h, the medium was replaced with 5 mM BIDOPY in serum-free RPMI medium, and the incubation continued in the dark for 30 min. Then, for the IR and 7DHC-Lipos + IR groups, cells were irradiated with 5 Gy. Red (ex/em: 581/590 nm) and green (ex/em: 488/510 nm) fluorescence was immediately measured using a microplate reader (Synergy Mx, BioTeK), and the green/red fluorescence ratio was calculated.
Cellular MDA and 4-HNE levels
Briefly, H1299 cells (106 cells per dish) were seeded onto 100 mm cell culture dishes (Corning, Cat# 353003). The next day, the cell culture medium was replaced with fresh serum-free medium containing either PBS or 50 µg/mL of 7DHC-Lipos and incubated at 37 °C for 4 h. For the IR and 7DHC-Lipos + IR groups, the wells were irradiated (5 Gy), then all cells were further incubated in full growth medium for 24 h. Cells were collected with a cell scraper, resuspended in 1 mL PBS, and lysed with a probe sonicator in an ice bath. The supernatant was collected by centrifugation (1500 x g, 5 min). The contents of 4,4’-methylenebisbenzenamine (MDA) and 4-Hydroxynonenal (4-HNE) were quantified using a TBARS Assay Kit (Cayman Chemical, Cat# 100009055) and a 4-HNE Assay Kit (abcam, Cat# ab238538), respectively.
Cellular sterol and oxysterol levels
Cell samples were prepared using the same protocol as for the MDA and 4-HNE studies. Lipid extraction and UHPLC-MS/MS were performed according to published protocols [55, 56]. Briefly, cell samples were lysed in an ice-cold ultrasonic bath for 30 min and vortexed. The protein content of each sample was quantified using the BioRad-DC Protein Assay Kit. Isotope-labeled internal standards (d7-cholesterol, d7-7-dehydrocholesterol, 13C3-desmosterol, and 13C3-lanosterol for sterols, and d7DHCEO, d7-7-keto-cholesterol, d6-24,25-epoxycholesterol, d7-24-hydroxycholesterol, and d7-4β-hydroxycholesterol for oxysterols) were added to each sample. 1 mL of 0.9% NaCl aqueous solution and 4 mL of Folch solution (2:1 v/v CHCl3: MeOH, with 1 mM BHT and 1 mM PPh3) were also added. The samples were vortexed for 30 s. After centrifugation, the lower organic layer was extracted and dried down in a speed vacuum concentrator. Samples were reconstituted in methylene chloride and stored at -80 °C until analysis. Sterol and oxysterol analysis was performed by UHPLC-MS/MS on an AB Sciex Triple Quad 6500 instrument. Samples were prepared at 2:1 concentration before injection. Data were analyzed using Analyst software. Protein content was used for data normalization. The mass spectroscopy studies were performed at the Mass Spectrometry Center, School of Pharmacy, University of Washington.
Cytotoxicity and clonogenicity
Cell viability was assessed with H1299 cells using the standard MTT assay. Briefly, H1299 cells (4,000 cells per well) were seeded on a 96-well plate (Corning Costar, Cat#3599). The next day, the cell culture medium was replaced with fresh serum-free medium containing either PBS or 50 µg/mL of 7DHC-Lipos and incubated at 37 °C for 4 h. For the IR and 7DHC-Lipos + IR groups, the wells were irradiated (5 Gy), then all cells were further incubated in full growth medium for 48 h. Twenty µL of 10 mg/mL 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well, followed by incubation at 37oC for 4 h. After incubation, the MTT solution was replaced with 100 µL of DMSO to solubilize the purple formazan crystals. Absorbance at 570 nm was measured using a microplate reader (Synergy Mx, BioTeK). For comparison, Ferr-1 (10 µM), DFO (5 µM), Z-VAD (0.1 mM), Gly (5 µM), or 3-MA (5 mM) were added together with the nanoparticles to incubate with cells.
For the LDH release assay, the cells were treated according to the same protocol as above, except that the incubation was stopped 24 h after the end of irradiation. For the total LDH group, 10 µL lysis buffer was first added to the incubation medium and incubated with cells at 37 °C for 30 min to release all the LDH. Then, for all treatment groups, 100 µL of supernatant was transferred to a new transparent 96-well plate and mixed with 100 µL of LDH kit working solution. The plate was incubated at room temperature for 30 min, followed by the addition of 50 µL of LDH kit working solution to stop the reaction. The absorbance at 570 nm was measured using a microplate reader (Synergy Mx, BioTek), and the percentage of LDH release was calculated by comparing the absorbance of each group to the total LDH group.
The ATP release was measured using the ATPlite 1step Luminescence Assay Kit (PerkinElmer, Cat# 6016731) according to the manufacturer’s protocol. Cells were treated according to the same protocol as for the LDH assay. After 24 h, 100 µL of supernatant from each well was transferred to a white 96-well plate (Corning Costar, Cat# 3610), followed by the addition of 70 µL of the ATP kit solution. Luminescence signals were measured immediately using a microplate reader (Synergy Mx, BioTek) and compared to a pre-established calibration curve to derive ATP concentrations.
For clonogenicity assay, H1299 cells were incubated with either PBS or 7DHC-Lipos for 4 h at 37 °C, and the dissociated cells were seeded onto a 100 mm cell culture plate (Corning, Cat# 353003) at a density ranging of 100 to 10,000 cells. The cells were then irradiated with a dose range of 0–10 Gy. After 14 days, cell colonies were stained with crystal violet and counted, and a survival fraction (S) relative to the untreated control was calculated. The data were fitted into the LQ model: \(\:S={e}^{-(\alpha\:D+\beta\:{D}^{2})}\). A clonogenic assay was also performed to evaluate ferroptosis. In that case, H1299, H460, and H226 cells were treated with 7DHC-Lipos plus 5 Gy irradiation, with or without Ferr-1 (10 µM).
Western blot
H1299 cells (0.5 M cells per well) were seeded in 6-wells plate overnight, then cell culture medium was replaced with fresh serum-free medium containing either PBS or 50 µg/mL of 7DHC-Lipos and incubated at 37 °C for 4 h. For the IR and 7DHC-Lipos + IR groups, the wells were irradiated (5 Gy), then all cells were further incubated in full growth medium for 24 h. The total cell proteins were extracted in RIPA lysis buffer (Thermo Scientific, Cat# 89901) supplemented with 1× proteinase inhibitor cocktail (Thermo Scientific, Cat# 78445), then quantified with BCA protein assay kit (Thermo Scientific, Cat# PI23225), resolved in a 10–12% SDS-PAGE gel, and then were incubated with appropriate primary antibodies. This is followed by incubation with secondary antibodies and exposed to X-ray films (Santa Cruz, Cat# 201696). The antibodies used are: KEAP1 (CST, Cat# 8047); GPX4 (CST, Cat# 52455); SLC7A11 (CST, Cat# 12691); SLC11A2 (CST, Cat# 15083); ASCL4 (Abcam, Cat# ab205197); GAPDH (CST, Cat# 2118); Apoptosis Western Blot Cocktail (Abcam, Cat# ab136812); Anti-rabbit IgG, HRP-linked Antibody (CST, Cat# 7074); Anti-mouse IgG, HRP-linked Antibody (CST, Cat# 7076).
TEM of cell samples
H1299 cells were treated with N-7DHC-Lipos (50 µg/mL) plus irradiation (5 Gy) as described above. Cells were then harvested and processed as previously described with some modifications [57]. Briefly, cells were fixed overnight with a solution containing 3% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate-HCl buffer, pH 7.25 at 4 °C. After being washed several times in 0.1 M cacodylate-HCl buffer solutions, the cells were agar-enrobed with 3% Noble Agar at 60 °C. After cooling, agar-cell pellets were extracted from Eppendorf tubes and placed in 0.1 M cacodylate-HCl buffer for further processing. Cells were then treated with 0.1% Millipore-filtered cacodylate-buffered tannic acid (30 min) and rinsed well in 0.1 M cacodylate-HCl buffer, pH 7.25. Cells were postfixed with 1% buffered osmium (1 h), rinsed well, and stained en bloc with 1% Millipore-filtered uranyl acetate (1 h in the dark). After rinsing well in deionized water, the cells were dehydrated in increasing concentrations of ethanol, infiltrated with propylene oxide, and embedded in an Epon-Araldite plastic [58]. Embedded cell pellets were polymerized in a 60 °C oven for 3 days. Ultrathin sections were cut on a Reichert Ultracut S ultramicrotome, placed on clean 200-mesh Cu Hex grids, and stained with uranyl acetate and lead citrate. Sections were examined on a JEOL JEM-1011 transmission electron microscope (JEOL USA, Inc.) at an accelerating voltage of 100 kV. Digital images were acquired using an AMT Imaging System (Advanced Microscopy Techniques). All processing, sectioning, and imaging was performed at the Georgia Electron Microscopy Core Facility on the campus of the University of Georgia.
In vivo whole-body fluorescence imaging
All animal experiments were conducted in accordance with an Animal Use Protocol (AUP) approved by the University of Georgia Institutional Animal Care and Use Committee (IACUC, PHS Assurance No. D16-00276). The in vivo imaging study was performed in nude mice bearing flank H1299 tumors. Briefly, 5 × 106 H1299 cells were injected subcutaneously into the right flank of a female 4–6-week-old female mouse (Charles River). When the tumor size reached 300 mm3, 10 mg/ml of DiR-labeled N-7DHC-Lipos or 7DHC-Lipos were injected intravenously into each mouse (n = 3 mice). Whole-body fluorescence images were acquired on a Vivo & In Vitro Imaging scanner (NEWTON 7.0) at 0.5, 4, and 24 h after injection. After 24 h, the tumors and major organs, including the liver, lung, brain, muscle, and kidney were harvested and scanned ex vivo. ROI analysis was performed to assess the distribution of nanoparticle in the tissues. Tumor samples were embedded in O.C.T. compound and then frozen at -80 °C. Tumor slices of 8 μm thickness were sectioned on a cryostat, which were then fixed with acetone, and stained with DAPI. Microscopic images were taken on a fluorescence microscope (Keyence, BZ-X 810).
Hematology and blood biochemistry
Healthy BALB/C mice (4–6 weeks old, Envigo) were injected intravenously with PBS or N-7DHC-Lipos (10 mg/kg, 50 µL) (n = 3 mice) and were euthanized after 14 days. Blood samples were collected by cardiac puncture. Major organs including liver, heart, lung, kidney, spleen, and intestine were harvested. Complete blood count (CBC) and histopathology were performed at Clinical Pathology Lab, College of Veterinary Medicine, University of Georgia. Liver and kidney functions were assessed using the Alanine Aminotransferase (ALT) Kit (Abcam, Cat# ab105134) and the Urea Nitrogen (BUN) Colorimetric Detection Kit (ThermoFisher, Cat# EIABUN) according to the manufactures’ protocols.
In vivo radiation therapy study
The efficacy study was evaluated in both H1299 and LLC-1 flank tumor models. The H1299 model was established by subcutaneous injection of 5 × 106 H1299 cells into the right flank of 4-week-old female nude mice. LLC-1 model was established by subcutaneous injection of 1 × 106 into the right flank of 4-week-old C57BL/6 mice. All animals were obtained from Envigo. When the tumor size reached 50 mm3, the mice were randomly divided into four groups (n = 5 mice). Animals in the 7DHC-Lipos + IR group were injected i.v. with N-7DHC-Lipos (10 mg/kg in 50 µL PBS). After 24 h, the animals received tumor irradiation (5 Gy), while the rest of the animal body was lead-shielded. In control groups, animals were treated with PBS alone, N-7DHC-Lipos alone, or IR alone. Two additional treatments were administered two days apart. Tumor size was measured every two days using a caliper, and tumor volume was calculated using the equation: \(\:Tumor\:volume=\frac{tumor\:length\:x\:{tumor\:width}^{2}}{2}\)), where tumor length ≥ tumor width. Mice were euthanized when they reached a humane endpoint such as length greater than 1.7 cm, weight loss more than 20%, or the presence of any tumor discharge. Tumors and major organs such as liver, heart, lung, kidney, spleen, and intestine were collected. Hematoxylin and eosin (H&E) and Ki67 staining were performed at the Histology Laboratory, College of Veterinary Medicine, University of Georgia. The microscopic images were captured with a digital microscope (Keyence, BZ-X 810).
In separate animals (H1299 bearing nude mice, n = 3 mice), animals from the four treatment groups were euthanized 24 h after single dose of treatment. Tumor tissue sections were stained with anti-4-hydroxynonenal antibody (Sigma, AB5605) according to a published protocol [59]. Microscopic images were taken under a fluorescence microscope (Keyence, BZ-X 810).
Statistical analysis
The means and standard deviations were calculated from at least three replicate groups in all the experiments. Statistical significance was calculated by one-way ANOVA with post-hoc Tukey-Kramer comparisons (for more than two groups) or two-tailed Student’s t test (for two groups). P values less than 0.05 were considered statistically significant. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, p > 0.05.
