Heparin sodium (Mn = 1.25 kDa, 150 U/mg) was obtained from Sinopharm Chemical Reagent Co. (Shanghai, China). Oregon green488 cadaverine and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were obtained from Invitrogen Co. (USA). 4-dimethylaminopyridine (DMAP), 1-ethyl-3,3-dimethylaminopropylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Medpep Co. (Shanghai, China). 732 cation-exchange resins were purchased from Shanpu Co., Ltd. (Shanghai, China). cRGD peptide (Cyclo-Arg-Gly-Asp-D-Tyr-Lys) was synthesized by BoXin Biology Co. Ltd. (Xiamen, China). Succinylated-heparin, Biotin-NH2 and Biotinylated MB were synthesized in our lab. All other chemicals and reagents were purchased from Sigma Co. (USA). Spectra/Por 3 Dialysis Membrane (MWCO 3,500) was purchased from Pharmacia (Piscataway, NJ). Ultrapure water (Mili-Q, 18 M • W ) was used in the experiment.
Preparation of cRGD Nano
Succinylated-heparin (0.050 g) was dissolved by dry DMSO with gentle heating. cRGD peptide (0.006 g), and Biotin-NH2 (0.004 g), EDC (0.04 g, 0.2 mmol) and NHS (0.025 g, 0.2 mmol) were mixed and reacted at room temperature for 24 h. Biotinylated cRGD-H conjugate in solution was then dialyzed with distilled water for 48 h in a dialysis membrane (MWCO 3500) to fabricate cRGD Nano. After lyophilization, biotinylated cRGD-H conjugate (0.22 g) obtained as white powder. Oregon green488-labled biotinylated cRGD-H was prepared by a similar method as that of biotinylated cRGD-H. The amount of cRGD in nanoparticles was measured by bicinchoninic acid (BCA) assay according to the manufacturer’s instruction. Folate content was measured by UV method at 366 nm. The loading efficiency of cRGD in cRGD Nano was calculated using the following equation: loading efficiency (%) = (weight of agent in nanoparticles/initial weight of agent) × 100%.
Preparation of cRGD Nano loaded microbubbles (cRGD Nano-MB) and characterization
cRGD Nano-MB was prepared by the described method with minor modifications.(Yin et al. 2013) The biotinylated MB reacted with 500 mL avidin (10 mg/mL) for 10 min at room temperature with gentle shaking. Free avidin were separated from the avidin-bound biotinylated MB by washing for 2 times. And then biotinylated cRGD Nano (50 mL) mixed with avidinylated MB (1 mL) for 15 min with gentle shaking. Free biotinylated cRGD Nano were removed by washing for 2 times. Oregon green488 labled cRGD Nano loaded MB was using similar method. DiI was mixed with MB for 20 min to prepare DiI labeled MB (MB-DiI). To investigate the binding efficiency and construction of NPs-loaded MB, Oregon green488 labled cRGD Nano loaded MB-DiI was visualized using a confocal laser scanning microscope (Olympus FV1000, Japan) with the excitation wavelength of 405 nm for blue channel, 488 nm for green channel and 559 nm for red channel. The 1H NMR spectra of cRGD-H were recorded on a Bruker-400 MHz NMR in D2O. Size distributions of cRGD Nano and cRGD Nano-MB were detected by dynamic lighting scattering (DLS) using a Zetasizer Nano-Zs (Malvern Instruments, UK) and Coulter Multisizer IIe (Beckman Coulter, USA), respectively. 1H NMR spectra of Biotinlyated cRGD-F-H in D2O were detected for structural characterization (Fig. S1).
Human umbilical cord vein endothelial cells (HUVEC), human ovarian carcinoma SKOV3 cells and human lung carcinoma A549 cells were come from ATCC. All cell lines were cultured in DMEM (Corning, USA) containing 10% fetal bovine serum (Bioind, Israel) and 100 units/mL penicillin/streptomycin. All cell lines were maintained in humid environment containing 5% CO2 at 37 ºC.
Ultrasound-waves were produced from an unfocused single element transducer (Vermon SA, Tours, France) with a center frequency of 1 MHz, driven by a waveform generator (Agilent, City, CA, USA). A power amplifier (ADECE, Artannes sur Indre, France) was generated a pulsed US at acoustic pressure amplitude determined by a calibrated PDVF needle hydrophone (0.2 mm diameter, Precision Acoustics, Dorchester, UK). cRGD Nano-MB were scanned under the transducer (30 s, a center frequency of 1 MHz, 50% duty cycle) and negative peak pressure amplitude for HUVEC cells, SKOV3 cells and A549 cells was 0.7 -1 MPa.
Cellular uptake of cRGD Nano and cRGD Nano-MB with and without US
Oregon green488-labeled cRGD Nano and cRGD Nano-MB were synthesized by the similar procedure. HUVEC, SKOV3 and A549 cells were respectively cultured in 6-well plates (3´ 105 cell/well) and incubated for 24 h at 37 oC. The culture medium was replaced by fresh medium with Oregon green488 labeled cRGD Nano and cRGD Nano-MB and then followed by US exposure. And the bottom of the plate was drowned in water for acoustic coupling without contaminating the sample in the well of the plate. Meanwhile, the US transducer was submerged in water upward aiming at the plates. HUVEC SKOV3 and A549 cells treated with US under 0.7 MPa-1 MPa for 30 s. The cells were incubated for 4 h and then were washed for three times with cold PBS, detached by 0.02% EDTA-PBS, and suspended in PBS containing 0.1% BSA. The collected cells were analyzed by a FACSort flow cytometer (Becton Dickinson, USA) with 488 nm and cells without any treatment were taken as control.
Confocal laser scanning microscope was used to visualize the cellular uptake of Oregon green488 labeled cRGD Nano and cRGD Nano-MB in HUVEC and SKOV3 cells. The cells were incubated in 6-well plate for 24 h and then the culture medium was replaced with medium containing Oregon green488-labeled cRGD Nano and cRGD Nano-MB with or without US. The ultrasound parameters were the same as the above procedure. The plates were incubated for 4 h at 37 oC after US treatment, and cells were washed by PBS and fixed by 4% (w/v) para-formaldehyde solution. The fluorescent images were observed by confocal microscope.
In vitro anti-angiogenesis (anti-EDV and anti-VM) assays
Endothelial tube formation in vitro was conducted by the described method.(Kheirolomoom et al. 2007) Matrigel (50 mL) was added to a 24-well plate and allowed to polymerize. HUVEC cells were suspended in medium with 3´ 105 cells/mL, and the cell suspension (0.1 mL) was added to each well coated with Matrigel. And then Cells were treated with cRGD Nano or cRGD Nano-MB (each at an equivalent cRGD dose of 600 mg/mL) with or without US and then incubated for 12 h at 37 oC. After that, the cells were photographed using an inverted light microscope (NiKon ECLPSE 80i system, Japan) and branch points from 4 to 6 high-power fields (200) were counted and averaged.
Matrigel (150 mL) was placed on 24-well plates and allowed to polymerize for 1 h at 37 oC. 1.5´ 104 number of MDA-MB-231 were seeded on the well to observe the ability of capillary-like structure formation. The mild dosage was selected to guarantee its cell viability around 80%. cRGD Nano or cRGD Nano-MB (each at an equivalent cRGD dose of 600 mg/mL) with or without US were added to the cell suspensions. After that, images were photographed using an inverted light microscope after 96 h.
In vivo assessment of angiogenesis using chicken chorioallantoic membrane (CAM)
White Leghorn chicken eggs (South China Agricultural University, Guangzhou) were incubated under routine conditions (constant humidity and 37 oC) in CAM assay, and a square window was opened in the egg shell at day 3 incubation which exposed the CAM. Gelatin sponges were cut to 1 cm3 size and placed on the top of the CAM under sterile conditions at day 8. The sponges were then absorbed with cRGD Nano or cRGD Nano-MB (each at an equivalent cRGD dose of 600 mg/mL) with or without US and then they were tested. Sponges with PBS were used as negative controls and each group consisted of three eggs. The blood vessel inhibition ratio was identified as the percentage of treated group vessels area (VA) occupied by the whole area of CAM under the microscopic field. CAMs were observed daily and photographed in vitro at 72 h. Image-Pro Plus 7.0 software was used to automatically assess and quantify the blood vessel areas.
Cell viability detected by MTT assay
HUVEC and SKOV3 cells were separately seeded in 6-well plates (3´ 105 cell per well) and incubated for 24 h at 37 oC. After incubation of 24 h, cRGD Nano or cRGD Nano-MB were added to the medium with drug concentration of 0.06, 0.12, 0.3 and 0.6 mg/mL. The bottom surface of the plate was drowned in water for irradiation and the US transducer was then submerged in water upward aiming at the plates. US condition were set to 0.7 MPa-1 MPa for 30 s and then cells were incubated for 4 h. After that, the cells were washed and trypsinized to transfer 96-well plates with fresh medium for 20 h incubation. The culture medium was replaced with MTT solution and then the cells were further incubated for another 4 h. The medium was replaced by DMSO and the plates were slightly shaken for 10 min. The plates were measured by a microplate reader (Labsystem, Multiskan, Ascent, Finland) at optical absorbance of 570 nm. Cells with PBS were used as control.
In vivo anti-tumor experiments
Athymic nude female nu/nu mice (4–5 weeks old) were injected with 100 mL of SKOV3 cells (1´ 106) on the right flank. The tumors were allowed to grow until their size reached between 9 and 25 mm3. Mice were randomly divided into three groups (PBS, cRGD Nano-MB and cRGD Nano-MB+US, n = 5) and injected tail intravenously. The cRGD Nano-MB and cRGD Nano-MB+US (each at an equivalent dose of 150 ug cRGD per mice) were injected at one day intervals. For the cRGD Nano-MB +US group, tumors were irradiated with US after every injection under the following settings: 1 MHz, 2 % duty cycle, duration of 1 min and intensity of 2 W/cm2.T umor length (a), width (b) measurements, and body weight were recorded at one day intervals. Tumor volume was calculated according to the formula: V= (a´b2)/2
Statistical analysis was conducted to determine the differences of the experiments. One-way analysis of variance was used to analyze by a statistical program (Statistical Package for the Social Sciences, Version 19.0, SPSS Inc., USA). All the data were performed in triplicate and showed as a mean value with its standard deviation indicated (mean ± SD). Differences were considered the significance at * P < 0.05, * *P < 0.01, * * * P < 0.001.