| BackgroundRF ablation is one of the most promising minimally invasive medical procedures for the treatment of various localized malignant or benign tumors. In this procedure, the ions generated by alternating electric current oscillate and create friction within the tumor thus heat. And tumor necrosis is possible by heating the tissue beyond threshold temperature for tissue denaturation. However, tumor treatment by RF ablation produces highly variable results, with recurrence rates ranging from 4 to 78%. And the tumor recurrence rate is closely related to whether the tumor tissue is completely ablated. So expanding the ablated area and inactivating the tumor cell completely is an effective way to reduce tumor recurrence rate. Several strategies have been developed to increase the region of induced coagulation. These strategies can be classified as those that increase local heat deposition, those that improve tissue heat conduction, and those that decrease tumor tolerance to heat.Recently, a new type of nanoscale-particles (NPs) containing liquid perfluorocarbons (PFCs) has attracted much attention in diagnostic and therapeutic applications of ultrasound. These particles are stable in the blood stream and undergo an instant liquid-to-gas transition with the activation of three physical factors, namely heat, ultrasound, and injections through fine-gauge needles. In addition, these particles with diameter of 300-700 nm are able to pass through the endothelial gaps of defective blood vessels. These NPs remain in the vasculature as long as they are not recognized and cleared by the immune system, and even undergo an enhanced permeability and retention effect in neovascularized tumors.Recent studies have shown that high-intensity focused ultrasound (HIFU) combined with NPs can increase local heat deposition, enlarge coagulated volume, accelerate the local temperature increase and thus enhance the ablation effects. The main aim of HIFU is to destroy an entire tumor by heating beyond the tissue temperature threshold, which is similar to RF ablation. Therefore, it is reasonable to hypothesize NPs containing liquid PFCs as an exogenous enhancer to RF ablation treatment. To the best of the authors’knowledge, few published papers have reported the effect of NPs containing liquid PFCs on RF ablation in vivo.ObjectiveIn this paper, we describe the development and characterization of lipid-coated liquid perfluorohexane (PFH) NPs, we confirm the good biocompatibility in vitro, we validate the droplet-to-bubble transition of the NPs under thermal activation, and we also explore the effects of NPs on RF ablation in a rabbit tumor model. These NPs can be delivered to the tumor site by systemic administration. During a tumor RF ablation process, thermal energy at the lethal thermal dose will activate the NPs to shift, significantly enhance the effect of RF ablation.Materials and MethodsPreparation and characterization of NPsPhase-shift NPs were prepared using a homogenization/emulsion method. To prepare the lipids dispersion, lipids DPPA (1,2 Dipalmitoyl-sn-Glycero-3-Phosphate; MW,670.88), DPPC (1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine; MW,734.05), DPPE-PEG5000 (1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolaminen-N-[Methoxy (Polyethylene glycol)-5000]; MW,5745.04; Avanti Polar Lipids INC, Pelham, AL) with a weight ratio of 1:9.1:5.6 were dissolved in propylene glycol along with glycerol. The solvent was then mixed into 40 mL 8.5% sodium chloride (w/v) aqueous solution and placed in a thermostated bath maintained at 70℃ to ensure full miscibility. Then the premixed emulsions were prepared by adding the desired amount of Perfluorohexane (PFH, Apolo) into the lipids dispersion while emulsified using shears (ULTRA-TURRAX) at 12000-24000 rpm for 3 min. The emulsions were further subjected to high shear homogenizer (AH2010; ATS) for 10 min. Finally the NPs were stored at 4℃ until use.The NPs were pretreated by diluting a sample formulation 1:5 with deionized water. The surface morphology of the NPs was observed using Scan Electron Microscopy. The mean diameter, zeta potential of NPs and polydispersity of NPs were measured using Melvin laser particle size analyzer. Measurements were performed at 25℃.Cytotoxicity testWe selected the human colon cancer cell line SW-480 to evaluate the cytotoxicity of the NPs using the MTT assay. The cells were seeded in 96-well plates at a density of 5×104 cells/well and then cultured for 24 hours in 100ul of RPMI-1640 medium containing 10% fetal bovine serum (FBS) in a humidified atmosphere with 5% CO2. The cells were then incubated for 24 hours in the same volume of fresh medium with various NPs concentrations (0-1.2×109/mL); the medium was then replaced with 100ul of fresh medium containing 10 ul of MTT solution (5 mg/mL), and the cells were subsequently incubated for 4 hours. Dimethyl sulfoxide (150ul) was added to dissolve the substrate after the MTT-containing supernatant was discarded. After gentle agitation for 5 minutes, the absorbance of each well at 490 nm was recorded.Hemo lysis testNew Zealand White rabbit blood was used to test the hemo lysis effect of phase-shift NPs. Rabbit whole blood (10 mL) was heparinized and centrifuged at 1500 rpm for 15minutes, and the erythrocyte sediment was washed with physiological saline several times until the supernatant was no longer red in comparison to the color of normal saline. Erythrocyte pellets were added to physiological saline to prepare a 2% erythrocyte standard suspension. The NPs (0.2, 0.4,0.6,0.8, and 1.0 mL) were added to five tubes, each containing 2.5 mL of the 2% erythrocyte suspension. Physiological saline was added to each tube to a total volume of 5 mL. Physiological saline (2.5 mL) mixed with 2.5 mL erythrocyte suspension was used as negative control and the positive control was prepared by mixing 2.5 mL of purified water with 2.5 mL erythrocyte suspension. After blending, all the tubes were incubated at 37℃ and observed at baseline and after 15,45,90, and 180 minutes.Ultrasound images of NPs activation in water suspensionConventional grayscale Sonography (iU22; Philips Healthcare, Bothell, WA, using an L12-5 probe) was used to monitor the thermal activation of NPs dispersed in degassed water. NPs-dispersed water and degassed water were sealed in a special capsule respectively. Both capsules were heated up to 78℃ in a water bath. Comparative ultrasound images were acquired before and after the heating.Ultrasound images of NPs activation in solid gel phantomsRF ablation (RITA, US) was performed in vitro using agar-agar gel phantoms. To prepare the phantom,6 g agar-agar powder was fully dissolved in 400 mL distilled water (boiling) in a 500 mL beaker. As the temperature of the agar-agar gel dropped to 40℃, the NPs (1 ml) were added into the gel solution and stirred gently. The gel solution was then kept in room temperature until complete gelling. The control phantom was prepared following the same procedure except that no NPS were added. Both the sample and the control phantoms were ablated using the RF probe, which was placed at the center of the solid gel phantom. The ultrasound probe was placed on the top of the phantom to monitor the echo change.Animals and tumor modelsForty New Zealand White rabbits weighing 2.0 to 2.5 kg were purchased from the Laboratory Animal Center of Nanfang Hospital. Rabbits were handled according to a protocol approved by the Animal Care and Use Committee of the university. The VX2 tumor was obtained from the laboratory animal center of sun yat-sen University.First, VX2 tumors in the thigh of donor rabbits were harvested and cut into cubes of 1 mm3 in size under sterile conditions. Then, forty recipient rabbits were anesthetized with 3% carbrital. Thereafter, the right thigh of the rabbit was shaved and sterilized, and the rabbits were put in the lateral position on the table. The VX2 tumor tissue cube was inserted into the thigh muscle of rabbits through a 21-gauge needle about 0.5-cm deep from the skin. Gray-scale sonography was performed to monitor the tumor growth consecutively and measure the size of tumor.Evaluation of treatment efficacy in vivoTwo weeks after tumor implantation when the tumor diameter reached approximately 1-1.5 cm, rabbits were randomly divided into 3 groups (12 per group): RF ablation alone, RF ablation+NPs and Untreated control. An 18-gauge RF electrode with a 1-cm active tip and a RF generator (Cellon) were used for the RF ablation procedure. For RF ablation+NPs, RF ablation was performed 15 min after intravenous injection of NPs.To evaluate the ablated areas, contrast-enhanced sonography was performed 5 minutes after each ablation to avoid the influence of hyperechogenicity. The areas of the nonenhanced region on contrast-enhanced sonography were measured using a loci method. All images were taken from the largest section of the tumor before and after the treatment.Histological evaluation was processed by selecting three rabbits of each group. Immediately after the treatment, the tumor was harvested and the area of coagulation necrosis was observed.Gray-scale ultrasound was used to observe the tumors growth every 3 days until 27 days after the treatment. The tumor areas (using a loci method) were measured to assess the tumor relative growth rate using the following formula:X27= 100% x (A27-Ao)/Ao, where X27 is the tumor relative growth rate at day 27 after treatment (%), A27 is the tumor area at day 27 (cubic millimeters), A0 is the tumor area before processing (cubic millimeters). Statistical AnalysisAll data are presented as mean±STDEV (standard deviation). Statistical significance between groups was derived using one-way ANOVA and LSD tests. P<.05 was considered statistically significant. Data analysis was performed with SPSS version 20.0 software for MAC (SPSS Inc, Chicago, IL).ResultsNPs characterizationNPs had an average diameter of 288.7±36 nm with a polydispersity of 0.0622±0.014 measured by the Melvin laser particle size analyzer (n=12), and the zeta potential observed was-2.44±0.18 mV. The concentration observed by Coulter was 1.2×109/ml.In vitro NPs biocompatibilityThe MTT results indicated that the NPs had no obvious cytotoxicity toward the human colon cancer cell line SW-480 within the concentrations (0-1.2×109 particles/ml)used for in vivo experiments with NPs. The hemolysis results indicated that no hemo lysis and no erythrocyte agglutination occurred in the tubes contained samples at different concentrations of NPs, and the results were consistent with the negative control. Hemolysis only occurred in the positive control tube. The highest NPs concentration was 1.2×109 particles/mL, in which there was no occurrence of hemolysis and agglutination at 37℃. Thus, NPs were safe for the application of in vivo study.In vitro NPs activationThe ultrasonically echoic change in vitro was observed due to the activation of NPs. The ultrasound images of the NPs-dispersed water and the degassed water showed that before heating (room temperature), both of them showed no signal, the echo intensity was (0.18±0.04) dB and (0.23±0.09) dB respectively, while after heating up to 78℃, the NPs-dispersed water showed significant hyperechogenicity, while the degassed water showed no echoic change. The echo intensity was (26.49±0.71) dB and (0.25±0.05) dB respectively. The ultrasound images of the sample phantom and the control phantom showed that before ablation, both phantoms showed little signal, while after ablation, a significantly hyperechoic region was observed around the electrode tip, while the control phantom showed little to no hyperechoic region.In vivo NPs efficacyAmong the 40 rabbits that received VX2 fragments implantation,36 rabbits bearing the tumor were suitable for the experiment. Three rabbits died of diarrhea, and one died of the overdose of anesthetic. The tumors had an average area of 66.56±3.16 mm2 on the day of treatment, with no significant differences in tumor area between all groups. It was remarkable that the ablated area in RF ablation+NPs group was greatly increased compared with that in RF ablation alone and untreated control group (P<.05). Tumors in the untreated control group grew rapidly, reaching more than 250 mm2 at day 27. In contrast, tumor area increased slowly in RF ablation+NPs group and RF ablation alone group, reaching about 100 mm2 and 170 mm2 respectively at day 27. The RF ablation+NPs group had significantly suppressed tumor growth compared with the untreated control group (P<.05). In addition, the tumor growth inhibition efficacy of the RF ablation+NPs group was better than that of the RF ablation alone group (P<.05).Conclusions1. A homogenization/emulsion method was used to fabricate the NPs, which is consisting of liquid PFH cores and lipid shell. And the NPs have a smaller size that would be helpful in leaking into and accumulating at the tumor sites2. The NPs were fabricated using materials with no reported toxicity. We confirmed the good biocompatibility of NPs in vitro.3. The in vitro activation experiments demonstrated the heat-induced property of phase-shift NPs.4. According to the results of contrast-enhanced sonography and gross pathology, we confirmed that the treatment with NPs and RF ablation could enlarge the ablation area of the tumor and enhance the antitumor effect. |
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