Hydrochloric acid (HCl, 37%) was purchased from Sinopharm Chemistry. Foetal bovine serum (FBS), RPMI Media 1640, Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin and trypsin were supplied by Gibco Invitrogen. HS-RGD was purchased from GL Biochem (Shanghai), Ltd. FITC-conjugated anti-mouse CD11b and PE/Cy7-conjugated anti-mouse Ly-6G/Ly-6C were purchase from Tonbo Biosciences. CCK8 and 4,6-diamidino-2-phenylindole (DAPI) were obtained from Beyotime Institute of Biotechnology. Paraformaldehyde (4%) was obtained from DingGuo Chang Sheng Biotech. Gold (III) chloride trihydrate (HAuCl4•3H20), 4-(N-Maleimidomethyl) cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-smcc) and bovine serum albumin (BSA) was purchased from Sigma-Aldrich (Shanghai) Trading. Phosphate and phosphate buffered saline (PBS, pH=7.2, 10 mM) was obtained from Hyclone. Hexadecyl trimethyl ammonium bromide (CTAB), silver nitrate concentrate (AgNO3), ascorbic acid, sodium borohydride (NaBH4), dimethyl sulfoxide (DMSO), Fluorescein5(6)-isothiocyanate (FITC) and formylmethionylleucylphenylalanine (fMLP) were supplied by Aladdin. The BODIPY NHS Ester (succinimidyl ester) and DiD were supplied by Thermo Fisher. Lewis cell line was purchased from American Type Culture Collection (Manassas, VA) and maintained according to American Type Culture Collection-recommended conditions.
Fluorescence spectra were recorded by a Hitachi F2500 luminescence spectrometer (Hitachi, HongKong). UV-Vis spectra were characterised by an UV-vis spectrophotometer (Varian, Hong Kong). Transmission electron microscope (TEM, Tecnai-12 Bio-Twin, FEI, Netherlands) was used to observe the nanoparticle morphology. The zeta potential was determined by Zeta Sizer Nano ZS (ZS90, Malvern). Cell viability assay was conducted by using a microplate reader. The cellular uptake was detected using confocal laser scanning microscopy (CLSM, Nikon A1R, Japan). Flow cytometer (FCM, BD FACSVerse) was performed to quantitatively analyse the cellular uptake of nanoparticles. The infrared thermal camera (DALI TECHNOLOGY, LT3-P) was used to record the temperature of the dispersions. The inductively coupled plasma-atomic emission spectroscopy (ICP-AES, iCAP 7400) was used to determine Au concentration.
Synthesis of AuNR, AuNRB and AuNRBR
AuNR was synthesised by the seeded growth according to the reported the literature with little modification . AuNRB was got by modifying AuNR with BSA based on the method described by Moustafa et al . Briefly, 1 mL BSA solution dissolved in the PBS (pH = 7.2-7.4) at a concentration of 0.25 mM was added to 10 mL of the AuNR solution (0.3 nM), and the mixed solution was stirred vigorously for 12 h at room temperature. Then, the reaction was stopped by removing the excess BSA by centrifugation at 12,000 rpm for 5 min and the precipitate was resuspended in ultrapure water to gather AuNRB.
AuNRB was suspended in PBS (2 mg/mL) were respectively functionalised by sulfo-smcc (150 μL, 5 mg/mL) to endow AuNRB with Mal for further conjugation with HS-RGD. The functionalisation was performed at room temperature (RT) for 1 h. Then, the excessive Sulfo-SMCC was removed by centrifugation with speed of 10000 rpm for 10 min thrice. The purified solution was collected and 100 μL HS-RGD (25 mg/mL) was added by stirring at 350 rpm at RT for 2 h. Afterwards, the reaction was stopped by removing the excess RGD by centrifugation with speed of 10000 rpm for 10 min for 3 times and the solution was resuspended in ultrapure water with final volume of 2 mL. The sample was stored at 4 °C for further study.
FITC DMSO solution was reacted with BSA at a molar ratio of 3: 1 (FITC: BSA) for 6 h. After the ultrafiltration with water to remove free FITC, FITC labelled BSA was collected and used to synthsize FITC-AuNRBR and FITC-AuNRB.
In vitro photothermal effect
AuNRBR dispersed in PBS with the concentration of 10, 20, 40 and 80 μg/mL in a tube was irradiated with an 808 nm laser at the power density of 1 W/cm2 for 8 min. The temperature increase was recorded at 1, 2, 3, 4, 5, 6, 7 and 8 min, and the thermal images of the dispersions were recorded at 8 min by an infrared thermal camera, respectively. PBS was used as control treated under the same conditions.
Isolation and purity of neutrophils from mice
Neutrophils were isolated from femurs and tibias of 3-week-old to 4-week-old C57BL/6 mice. Cells isolated from femurs and tibias were suspended in PBS and centrifuged at 1000 rpm for 3 min. The collected cells were resuspended with 2 mL separation mixed solution containing 55%, 65% and 78% (v:v) Percoll in PBS, followed by centrifugation for 30 min at 500 g. Afterwards, neutrophils were collected at the interface of the 65% and 78% fractions and further washed by ice-cold PBS thrice.
To identify the purity of neutrophils, they were doubly stained with FITC-conjugated anti-mouse CD11b and PE/Cy7-conjugated anti-mouse Ly-6G/Ly-6C to analyse by flow cytometry. Neutrophils were also directly observed by microscope.
AuNRBR loaded into neutrophils (AuNRBR/N)
AuNRBR was incubated with neutrophils to produce AuNRBR/N. To measure the loading efficiency, AuNRBR was labelled with FITC (1 μg/105 cells) and incubated with neutrophils for 10, 30 and 60 min at 37 °C with slight shaking. Afterwards, the cells were centrifuged at 1200 rpm for 5 min to remove excess AuNRBR-FITC. Finally, the cells were analysed by flow cytometry and observed by fluorescence microscope.
AuNRBR release from AuNRBR/N
The release behaviour of AuNRBR from AuNRBR/N was investigated by determining the amount of AuNRBR in supernatant. AuNRBR/N was resuspended in RPMI 1640, followed by irradiation with 808 nm laser. Then, the cell suspension was cultured with 5% CO2 at 37°C. At the interval time point (0, 1, 2, 4, 8 and 12 h), the cell suspension was centrifuged at 2000 g for 5 min. The supernatant was collected, and the AuNRBR content was determined by ICP-AES. Additionally, AuNRBR/N suspension with no laser irradiation was treated with the same process used in the control.
Cellular uptake of AuNRB and AuNRBR
Lewis cells were planted in 6-well plate with DMEM containing 10% FBS and incubated at 37°C and 5% CO2. They were allowed to grow until about 70% confluency. Then, the medium was removed and replaced with 2.5 mL serum-free DMEM with FITC labelled AuNRB and AuNRBR (equivalent FITC, quantified by fluorescence spectra) and incubated for 4 h, respectively. After incubation, Lewis cells were washed twice with PBS and collected for uptake analysis by flow cytometry. To more directly observe the uptake of FITC labelled AuNRB and AuNRBR by Lewis cells, the same operation as above was performed except for the planting of Lewis cells in laser confocal dishes. The cells washed with PBS were fixed with 4% paraformaldehyde and counterstained with DAPI for further observation by confocal laser scanning microscopy (CLSM).
The permeability was determined by using a transwell system (5 μm pore size, EMD Millipore Corporation). Briefly, human umbilical vein endothelial cells (HUVECs) were seeded in the upper chamber of the transwell in the 24-well plate and cultured with complete medium. The tightness of the cell monolayer was determined by measuring the trans-endothelial electrical resistance (TEER) value. When the TEER value arrive at or beyond 200 Ω cm2, the cell monolayer was used for the transmigration studies.
AuNRBR/N was added into upper chamber to co-incubate with HUVECs for 4 h, as well as serum-free medium added into the lower chamber in the presence or absence of fMLP. Then, the media in the upper and lower chambers were collected, and the cell monolayer on the transwell membrane was harvested. The AuNRBR in the supernatant, intracellular and filtrate compartments was measured by ICP-AES to further calculate the ratio by comparing with the input amount of AuNRBR.
Lewis cells were planted in 96-well plate with DMEM containing 10% FBS and incubated for 24 h. Afterwards, the medium was removed and replaced with 100 μL serum-free DMEM containing AuNRBR/N (Au concentration of 10, 20, 40, 60, 80 μg/mL) and incubated for 6 and 12 h, respectively. Then, the medium was removed and washed with PBS twice. Another 200 μL of DMEM was added prior to irradiation with or without an 808 nm laser for 5 min (0.5 W/cm2). Following incubation overnight at 37 °C, the cell viability was determined by the above mentioned CCK-8 protocol.
In vivo tumour targeting
The mice (C57BL/6, 5-6 weeks, female) were injected subcutaneously with Lewis cells to build tumour-bearing mice model and divide into 3 groups (3 mice per group). When the tumour volume reached about 200 mm3, the fluorescence dye (BODIPY)-labelled AuNRBR and AuNRBR/N (equivalent fluorescence dye) were injected intravenously to the mice. At 4, 8, 12, 24 and 48 h post-injection, the images were taken. For further analysis of targeting efficiency, the major organs and tumours were taken out to image at 12 h post-injection. Additionally, the tumour tissues were fixed with paraformaldehyde (4%) and cut into 20 μm sections, untreated tumour-bearing mice tissue used as negative control. The slides were observed by CLSM only after staining with DAPI.
To further analyse the ratio of the administered neutrophils to total neutrophils in tumour, the AuNRBR/N stained with DiD was injected intravenously into tumour-bearing mice. Then, the mice were sacrificed at each individual time point (4, 8, 12, 24 and 48 h), and the tumour tissues were collected. Subsequently, the tumour tissues were digested to make a cell suspension, followed by staining with PE/Cy7-conjugated anti-mouse Ly-6G/Ly-6C and analysis by flow cytometry.
In vivo photothermal effect
AuNRBR/N and AuNRBR dispersed in PBS at a concentration of 100 μg/mL were injected intravenously into tumour-bearing mice. After 12 h, the mice were irradiated with an 808 nm laser at the power density of 0.5 W/cm2 for 5 min. The temperature increase was recorded at 0, 1, 3 and 5 min, and the thermal images of the AuNRBR/N-treated mice were recorded. PBS was used as control and treated under the same conditions.
In vivo antitumour therapy
The tumour-bearing mice models were built as described above. Twenty tumour-bearing mice were randomly divided into four groups with five mice per group and intravenously injected with AuNRBR/N, AuNRBR and AuNR with equivalent doses of Au (100 μg) every 3 days for 3 times per day. The mice injected with PBS were used as the control. The tumours of mice were exposed to 808 nm laser irradiation (0.5 W/cm2) for 5 min at 12 h post-injection. The tumour size was measured, and body weight was recorded every other day along with the day the mice died. The tumour volume was calculated through the formula: V=(L×W2)/2 (L, tumour length; W, tumour width). On day 15, the mice were sacrificed, and tumours were extracted for imaging and further fixed in 4% paraformaldehyde for H&E assay according to the protocol.
The statistical significance of the differences between sphere groups was calculated using a one-way ANOVA test with SPSS 19.0 (SPSS Inc., USA). Significant differences between or among the groups are implied by *p < 0.05, **p < 0.01, ***p < 0.001.