The HSA, ICG, and perfluorotributylamine (PFTBA, 98%) were purchased from Sigma-Aldrich (St. Louis MO, USA). RPMI-1640 medium, phosphate buffer saline (PBS), trypsin and ethylenediaminetetraacetic-acid (EDTA), penicillin–streptomycin, and fetal bovine serum (FBS) were purchased from Gibco Life Technologies (Gaithersburg MD, USA). 4′,6-diamidino-2-phenylindole (DAPI), paraformaldehyde and Cell Counting Kit-8 (CCK-8) were purchased from Boster Biotechnology (Wuhan, China). Hypoxyprobe plus kit were purchased from North Pacific International Inc. Singlet Oxygen Sensor Green (SOSG) was purchased from Thermo Fisher Scientific Inc. (Shanghai, China). Reactive Oxygen Species Assay Kit (DCFH-DA) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). 18F-FMISO NITTP were purchased from Huayi Isotopes Co. (Jiangsu, China). All of the aqueous solutions were prepared using deionized water (DI water) purified with a purification system. The other reagents used in this work were purchased from Aladdin-Reagent (Shanghai, China).
Preparation of CCm–HSA–ICG–PFTBA
HSA (20 mg) was mixed in deionized water (1 mL) with stirring for 10 min. ICG dissolved in DI water (1 mg/mL) then dispersed in HSA solution and shake for 30 min at 37 ℃ to obtain HSA–ICG. The PFC (0.1 mL) was added gradually under sonication at 300 W in an ice bath for 8 min (ultrasonic for 7 s and rest for 3 s in every 10 s) to formulate HSA–ICG–PFTBA. Free ICG was removed by ultrafiltration centrifuge tube (Millipore molecular weight cutoff = 30 kDa).
Cancer cell membrane derivation could be achieved by emptying harvested 4T1 cells of their intracellular contents using a combination of hypotonic lysing, mechanical membrane disruption, and differential centrifugation according to the previous report . The CCm coated on the surface of HSA–ICG–PFC were fabricated by the approach used in our previous study as reported . HSA–ICG–PFC solution (1 mL) mixed with the prepared CCm–vesicles at different proportions. The mixture was subsequently extruded 11 times through 400 nm porous polycarbonate membrane. The resulting CCm–HSA–ICG–PFC were kept in PBS at 4 ℃ for further use.
Characterization of CCm–HSA–ICG–PFC
The hydrodynamic diameter and zeta potential were measured by dynamic light scattering (DLS; Man 0486, Malvern, UK). The morphology and structure of HSA–ICG–PFC, CCm–HSA–ICG–PFC and CCm–vesicles will be characterized by transmission electron microscope (TEM; Talos F200X, FEI, Netherlands). The TEM samples were prepared by contacting the droplet containing HSA–ICG–PFC, CCm–HSA–ICG–PFC or CCm-vesicles with the copper grids for 60 s, negatively stained with 1% phosphotungstic acid for 30 s and dried with absorbent paper before the characterization. The stability experiments were carried out by measuring HSA–ICG–PFC and CCm–HSA–ICG–PFC in 1× PBS for 5 days using DLS for monitoring dynamic diameter.
The fluorescence of ICG was measured by the multifunctional microplate reader. The photoexcitation wavelength was 710 nm and the emission wavelength was 740–850 nm. The photostability of ICG was measured by 808 nm laser irradiation (1 W/cm2) to different samples (ICG, 2 μg/mL), and recording the absorption every 10 s for 1 min. The storage stability of ICG in different samples was performed by UV–vis spectra under dark condition till 60 h. The release of ICG in CCm–HSA–ICG–PFTBA and HSA–ICG–PFTBA (80 μg/mL) was determined by putting two samples into the dialysis bag (MWCO10k), and the dialysis bag was put into 15 mL of plasma, as release medium. The release of ICG in plasma was detected at 2, 4, 8, and 12 h by the UV–vis spectra and calculated based on the standard curve.
Oxygen release experiment was performed with a dissolved oxygen meter, to measure the oxygen concentrations in different solutions. Sample solutions (10 mL) were preoxygenated, and added into 50 mL deoxygenated water. The oxygen concentration in the water was monitored and recorded every 5 s for 800 s with a dissolved oxygen meter.
In vitro 1O2 and ROS evaluation
SOSG was applied to detect the 1O2 generation of these samples. 100 μL different samples with the same concentration of ICG (50 μg/mL) and 20 μL SOSG (50 μM) were added into a black 96-well plate. With 808 nm laser irradiation, the fluorescence of oxidized SOSG (Ex/Em = 504/525 nm) was recorded every 10 s by multifunctional microplate reader.
DCFH-DA (Ex/Em = 495/529 nm) was used to indicate the ROS by confocal laser scanning microscope (CLSM). The 4T1 cells were seeded in confocal glass bottom dish with a density of 1 × 104 cells. After incubated for 24 h, medium containing CCm–HSA–ICG–PFTBA, HSA–ICG–PFTBA, HSA–ICG and PBS were added to the dishes at the concentration of 10 μg/mL ICG for 3 h incubation. After washing for 3 times by PBS, the medium containing DCFH-DA (25 μM) was added to incubate with cells for 30 min. After washing for 3 times by PBS, cells were divided into two lines, with or without 808 nm laser irradiation (2 W/cm2) for 20 s (30 s pause after each 10 s irradiation). Then the cells were fixed by 4% polymer formaldehyde and the cell nucleus were labeled with 4′,6-diamidino-2-phenylindole (DAPI). CLSM was used to detect the green fluorescence of DCF.
Flow cytometry was applied to quantitatively reflect ROS generation. The procedure was similar to that for fluorescence imaging. The 4T1 cells were seeded in 6-well plates at the density of 1 × 105 cells and stained by DCFH-DA (25 μM) for 30 min. After 808 nm laser irradiation (2 W/cm2) for 20 s (30 s pause after each 10 s irradiation), the cells were centrifuged, re-suspended in 300 mL PBS and analyzed by flow cytometry. The green fluorescence was detected on FL1 channel (Ex/Em = 488/525 nm).
In vitro cytotoxicity
A CCK-8 assay was used to evaluate the enhanced PDT efficacy of CCm–HSA–ICG–PFTBA. 4T1 cells were seeded in 96-well plates at a density of 5 × 103 cells per well and cultured for 12 h. CCm–HSA–ICG–PFTBA, HSA–ICG–PFTBA, and HSA–ICG were added to incubate with cells for 3 h at various concentrations of ICG (i.e., 1.25, 2.5, 5, 7.5, 10, 20, and 40 μg/mL). The saline group was used as control. Then the cells were irradiated by 808 nm laser (2 W/cm2) for 20 s (30 s pause after each 10 s irradiation). After 2 h co-incubation, cells were washed by PBS, and fresh culture medium was added. After further 24 h incubation, the fresh culture medium without serum (90 μL) mixed with CCK-8 (10 μL) was added into wells and the plates were incubated for another 2 h. Finally, the absorbance values of the cells per well were determined with a microplate reader (Bio-rad, Hercules CA, USA) at 450 nm for analyzing the cell viability. The background absorbance of the well plate was measured and subtracted.
Animals and tumor models
Animals received care under the instruction of the Guidance Suggestions for the Care and Use of Laboratory Animals. Balb/c female mice (6 weeks) were purchased (Beijing HuaFuKang Bioscience Co. Ltd, China). To obtain tumor-bearing mice, hairs on the upper limb were removed. Then, 1 × 107 4T1 cells were subcutaneously injected into the right upper limb of each mouse. The tumor bearing mice was used for further experiments when the tumor volume reached 60–250 mm3.
In vivo fluorescence imaging
When the volumes of tumor reached 100–150 mm3, the BALB/c mice were divided into four groups randomly. CCm–HSA–ICG–PFTBA, HSA–ICG–PFTBA, HSA–ICG (200 μL, 0.8 mg/kg for ICG), and saline were intravenously injected into tumor-bearing mice via the tail vein. All mice were anesthetized by isoflurane. The fluorescence images of mice at different time points (0, 3, 6, 12, 24, 36, and 48 h) were obtained by imaging system (Ex/Em = 710/790 nm). Then all mice were sacrificed to obtain the major organs (including heart, lung, liver, spleen, and kidney) and tumors to conduct the ex vivo fluorescence imaging.
In vivo micro PET/CT imaging
PET/CT imaging was performed on a micro PET/CT (Trans-PET Discoverist 180, Raycan Technology Co., Ltd., Suzhou, China). 18F-FMISO and 18F-FDG were produced by PET Center, Union Hospital (Wuhan, China). For 18F-FMISO PET/CT imaging, on Day 1, each mouse was injected with 5.55 MBq (150 μCi) of 18F-FMISO via the tail vein. Then on Day 2, mice were divided into four groups and injected with CCm–HSA–ICG–PFTBA, HSA–ICG–PFTBA, HSA–ICG (200 μL, 0.8 mg/kg for ICG), and saline, respectively. 24 h later, on Day 3, mice were again injected with 5.55 MBq (150 μCi) of 18F-FMISO via the tail vein. Static scans of 10 min duration were acquired starting at 1 h post injection with 18F-FMISO, and the mice were maintained under isoflurane anesthesia during the scanning period.
For 18F-FDG PET imaging, mice in each group were randomly selected and injected with 5.55 MBq (150 μCi) of 18F-FDG via the tail vein. 10 min static scans were acquired at 1 h post injection. All the mice for 18F-FDG PET imaging were fasted overnight prior to the probe injection, maintained under isoflurane anesthesia and kept warm during the injection, waiting phase, and scanning periods.
The images were reconstructed using the orderedsubset expectation maximization (OSEM) algorithm. For each micro PET image, 3.0 mm diameter spherical regions of interest (ROIs) were drawn over the liver, tumor, and the contralateral muscle on the decay-corrected images using Amide to obtain the percentage of injected dose per gram-tissue (%ID/g) and measure the SUVmax of tumor, liver, and calculate the tumor to contralateral muscle (T/M) ratio. The highest uptake point of the entire tumor and liver was included in the ROI, and no necrosis area was included.
Ex vivo immunofluorescence staining
Hypoxyprobe plus kit was used to stain tissues and detect hypoxia. Tumor-bearing mice were injected with CCm–HSA–ICG–PFTBA, HSA–ICG–PFTBA, and HSA–ICG (200 μL, 0.8 mg/kg for ICG) via tail vein, and divided into six groups (0, 6, 12, 24, 36, and 48 h). Then pimonidazole hydrochloride (60 mg/kg, Hypoxyprobe plus kit) was injected into the mice via tail vein. After 90 min later, all mice were sacrificed to obtain tumors for immunofluorescence staining following the protocols . Hypoxia were stained with green fluorescence, cell nucleus were stained with DAPI and showed blue fluorescence, and blood vessels were stained with anti-CD31 and showed red fluorescence. All slices were examined by CLSM.
In vivo photodynamic therapy and systematic toxicity
When tumor size reached about 60 mm3, the mice were randomly divided into eight groups (n = 6). The treatment groups were as follows: CCm–HSA–ICG–PFTBA (NIR), CCm–HSA–ICG–PFTBA, HSA–ICG–PFTBA (NIR), HSA–ICG–PFTBA, HSA–ICG (NIR), HSA–ICG, saline (NIR), and saline. On Day 0, all groups were injected with different samples (200 μL, 0.8 mg/kg for ICG) via tail veins, respectively. After 24 h later, namely on Day 1, all NIR groups were treated with 808 nm laser irradiation (2 W/cm2) for 2 min (1 min pause after each 30 s irradiation). 18F-FDG PET imaging and photograph taken were performed on Day 2, 7, and 14 to evaluate the tumor burden. The length and width of the tumor and mice body weight were recorded every 2 days over 14 days. The tumor volumes were calculated according to this formula: V = D × d2/2 (D is the longest diameter of tumor, and d is the shortest diameter of tumor). Relative tumor volume was calculated as V/V0 (V0 is the original tumor volume on Day 0). On Day 14, mice were sacrificed and tumors were weighted and photographed.
For evaluating systematic toxicity, on Day 14, all mice were euthanized and their blood and major organs (heart, lung, liver, spleen, and kidney) were collected for blood biochemistry test (red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cells (WBC), lymphocytes percentage (Lymph%), monocyte percentage (Mon%), neutrophil percentage (Neu%), and platelets (PLT)), hematology tests [alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine transaminase (ALT), creatinine (CRE) and blood urea nitrogen (BUN)], and histology analysis (hematoxylin and eosin (H&E)-stained slices).
Results are expressed as mean ± standard error of the mean. Data analyses were conducted using the software GraphPad Prism 6.0 (GraphPad Software, San Diego CA, USA). The differences among groups were analyzed using one-way ANOVA analysis followed by Tukey’s post-test. P value of < 0.05 indicates statistical significance.