Synthesis and characterization of Dox@HAuNS
PEGylated hollow gold nanoshells (PEG-HAuNS) were obtained from Ocean NanoTech (San Diego, CA, USA). Briefly, HAuNS were synthesized by the cobalt NP-mediated reduction of chlorauric acid followed by PEGylation as described previously (You et al. 2010). The resulting HAuNS were coated with MeO-PEG5000-SH to yield PEG-HAuNS. For Dox loading, 0.5 mL of Dox (OChem Inc., Des Plaines, IL, USA) in water (1 mg/mL) was added to 0.5 mL of PEG-HAuNS (80 OD). The mixture was incubated at 4 °C overnight to form Dox@HAuNS. Before injection, 1 mL of Dox@HAuNS solution was thoroughly mixed with 0.4 mL of Lipiodol oil by using two syringes connected to a 3-way Hi-Flo disposable stopcock.
For characterization, the UV–visible spectroscopy was recorded on a Beckman Coulter DU-800 UV–visible spectrometer (Fullerton, CA). The hydrodynamic sizes of PEG-HAuNS and Dox@HAuNS were determined using dynamic light scattering on a Brookhaven 90 plus particle size analyzer (Holtsville, NY). The size of HAuNS in dry state was examined by a JEM-1400 transmission electron microscope operated at 80 kV (JEOL Ltd., Tokyo, Japan).
All animal experimentation was approved by the Institutional Animal Care and Use Committee. Animals were housed in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care International and in accordance with regulations and standards of the US Department of Agriculture, the US Department of Health and Human Services, and the National Institutes of Health.
A total of 25 adult New Zealand white rabbits were included in this study. All procedures were performed under anesthesia with isoflurane (5%) in oxygen (1.5 L/min) and intramuscular buprenorphine (0.15 mg, Buprenex, Bedford Laboratories, Bedford, OH). For tumor inoculation procedures, the abdomen was shaved and was subsequently prepped and draped in standard sterile fashion. VX2 tumors were implanted into the left hepatic lobe, as previously described (Tian et al. 2013). Tumors were allowed to grow for 2 weeks, a duration that we have found to consistently result in ~ 1.0–1.5 cm diameter tumors. The rabbits were then randomized to 4 groups: sham surgery (control group, n = 5), intra-arterial Dox@HAuNS only (Dox@HAuNS group, n = 5), intra-arterial Dox@HAuNS followed by laser (Dox@HAuNS + laser, n = 7), and intra-arterial HAuNS followed by laser (HAuNS + laser, n = 5). Three rabbits were randomized to receive laser treatment only as an additional negative control group.
Intra-arterial delivery of nanoparticles
Transarterial intrahepatic delivery of the nanoparticles was performed via a femoral approach. After achieving adequate anesthesia, the right inguinal region was shaved and then prepped and draped in standard sterile fashion. A 1-cm vertical incision was made in the proximal right femur, and after blunt dissection, the right common femoral artery was isolated. Proximal and distal control was obtained with 0-silk vessel loops. Intra-arterial access was obtained with a 21 g micropuncture needle followed by a 0.016″ microwire. The access was then serially dilated to allow for the placement of a 4Fr sheath (Cook Medical, Bloomington, IN). Through this sheath, a 2.8Fr microcatheter (EmboCath Plus; BioSphere Medical, Rockland, MA, USA) was advanced over a 0.014″ microwire (Transcend, Boston Scientific, Natick, MA) into the celiac trunk under fluoroscopic guidance. Intermittent digital subtraction angiography was performed to assist in navigating the microcatheter to the proper hepatic artery. The nanoparticle solution (1 mL total volume, 40 OD, 1 mg Au/mL, 0.5 mg Dox/mL) was emulsified in 0.4 mL ethiodized oil (Lipiodol, Guerbet, Aulnay-sous-Bois, France). The emulsion was delivered through the microcatheter at a rate of 0.2 mL per minute under fluoroscopic visualization. After removal of the microcatheter and sheath, the femoral artery was ligated proximally and distally with the previously placed vessel loops.
Photothermal ablation of hepatic tumors
Immediately following the delivery of the nanoparticles, the skin overlying the abdomen was prepped and draped in standard sterile fashion. A midline incision was made, and the left hepatic lobe was elaborated to the skin. A fiberoptic catheter with a 1 cm diffusing tip (BioTex) was advanced into the tumor under direct visualization. Photothermal ablation was performed with an 808 nm laser at 1.5 W for 3 min. Continuous thermography was performed with an infrared camera (FLIR). The abdomen was then closed with both deep and subcuticular absorbable sutures.
Contrast-enhanced CT scans were performed using a helical CT scanner (HiSpeed Advantage; GE Medical Systems, Milwaukee, WI). Imaging was performed on operative day to establish baseline tumor volumes, as well as on post-operative day 7 (POD7) and POD14. Tumor volumes were measured using a standard three-dimensional image analysis software package (iNtuition; TeraRecon, Foster City, CA) based on 1.5-mm slices.
On POD14, the rabbits were euthanized with an overdose of Beuthanasia-D (1.0 mL/10 lb). Tumor and adjacent liver from control (n = 3), Dox@HAuNS (n = 4), laser alone (n = 2), HAuNS + laser (n = 4), and Dox@HAuNS + laser (n = 5) groups were harvested for analysis. Tissues for histopathology were fixed in 10% neutral buffered formalin, processed using standard protocols, embedded in paraffin, sectioned at 5 microns and stained with hematoxylin and eosin or for nanoparticle visualization. Nanoparticles in tissue sections were highlighted using a silver enhancer kit (ab170733, Abcam), with neutral red (ab146365, Abcam) counterstain to identify lysosomes in phagocytes. Stained tissue sections were evaluated microscopically by a board-certified veterinary pathologist using a Leica DM2500 microscope equipped with a Leica DFC495 digital camera. Residual tumor was quantified by measuring the longest diameter of viable tumor. Each tumor was sampled in triplicate to minimize the risk of sampling bias.
Quantitative analysis of doxorubicin
Dox levels in each tumor, liver tissue adjacent to tumors, and in plasma were measured by high performance liquid chromatography (HPLC). Plasma samples were collected at 1-h post-operation, POD1, POD7 and POD14. Tumor and liver tissues were harvested on POD14. Blood and tissues collected were kept at − 80 °C until the time of process. For sample preparation, dissected tissues were weighed and homogenized in water (0.2 g tissue/mL of water). Dox in the homogenate (0.1 mL) was extracted by using Waters’ Oasis HLB solid-phase extraction cartridge (Milford, MA. USA) with the following steps: (1) loading of 100 μL homogenates mixed with 1 mL 0.1 N HCl to the cartridge; (2) washing the cartridge with 1 mL water to remove impurities; (3) elution of analytes by 1 mL of acetonitrile, and (4) collection of eluate and evaporation to dryness followed by reconstitution with 20% acetonitrile in water (100 μL). Plasma samples (100 μL) were extracted by adding 0.5 mL of ethyl acetate. After vortexing and centrifugation (15,000g for 15 min), the supernatants were evaporated to dryness and reconstituted with 20% acetonitrile in water (100 μL).
To construct the calibration curve, stock solutions of Dox HCl (1 mg/mL) and daunorubicin HCl (Cayman Chemical, Ann Arbor, MI. USA) (1 mg/mL) were dissolved in water. Working solutions of the analytes were obtained by further diluting the corresponding stock solution with 20% acetonitrile in water. Calibration standard solutions were prepared freshly by spiking in Dox to plasma or homogenized liver/tumor tissues and extracted before each analytical run. At least eight calibration concentrations in the range of 1.5–500 ng/mL for plasma and 1.5–500 ng/mL for liver/tumor (7.5–2500 ng/gram of tissue) were used to generate the calibration curves for quantifications of unknown samples. All samples and calibration standards contained 100 ng/mL of daunorubicin HCl as the internal standard.
HPLC separation was achieved on a Waters HPLC system equipped with a Model 2475 multi λ Fluorescent Detector and an Eclipse XDB-C18, 5 μm, 4.6 × 150 mm column. Mobile phase was delivered at a flow rate of 1 mL/min with 16 min run time. The mobile phase consisted of water containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B). The following gradient was used: 80% of A (0–1 min); 80–65% of A (1–2 min); 65% of A (2–8 min); 65–10% of A (8–9 min); 10% of A (9–13 min); 10–80% of A (13–14 min); 80% of A (14–16 min). The wavelengths of excitation and emission were 480 nm and 560 nm, respectively, for fluorescent detection. Injection volume was 80 μl for extracts of liver/tumor homogenates and plasma samples.
All statistical analyses were performed with R (The R Foundation), a free software environment for statistical computing and graphics or Prism 7.03 for Windows (GraphPad Software, La Jolla, CA). Univariate analysis was performed using the Wilcoxon rank sum test. The Kruskal–Wallis test was used to compare percent change in tumor volumes across the treatment groups. One-way ANOVA with Tukey’s multiple comparisons test was performed to compare tumor diameters in histologic section. A cutoff value of P < 0.05 was used for statistical significance.