Lipid Nanocapsules Loaded with Rhenium-188 Reduce Tumor Progression in a Rat Hepatocellular Carcinoma Model

Background Due to their nanometric scale (50 nm) along with their biomimetic properties, lipid nanocapsules loaded with Rhenium-188 (LNC188Re-SSS) constitute a promising radiopharmaceutical carrier for hepatocellular carcinoma treatment as its size may improve tumor penetration in comparison with microspheres devices. This study was conducted to confirm the feasibility and to assess the efficacy of internal radiation with LNC188Re-SSS in a chemically induced hepatocellular carcinoma rat model. Methodology/Principal Findings Animals were treated with an injection of LNC188Re-SSS (80 MBq or 120 MBq). The treated animals (80 MBq, n = 12; 120 MBq, n = 11) were compared with sham (n = 12), blank LNC (n = 7) and 188Re-perrhenate (n = 4) animals. The evaluation criteria included rat survival, tumor volume assessment, and vascular endothelial growth factor quantification. Following treatment with LNC188Re-SSS (80 MBq) therapeutic efficiency was demonstrated by an increase in the median survival from 54 to 107% compared with control groups with up to 7 long-term survivors in the LNC188Re-SSS group. Decreased vascular endothelial growth factor expression in the treated rats could indicate alterations in the angiogenesis process. Conclusions/Significance Overall, these results demonstrate that internal radiation with LNC188Re-SSS is a promising new strategy for hepatocellular carcinoma treatment.


Introduction
Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor worldwide. The prognosis of HCC remains extremely poor, and a curative treatment (liver transplantation, surgical resection, and radiofrequency ablation) can only be carried out in approximately 25% to 30% of cases [1]. The use of conventional external beam radiation therapy in HCC treatment has been limited by the low radiation tolerance of the cirrhotic liver that often resulted in radiation-induced liver disease (RILD) [2]. Selective internal radiotherapy (SIRT) aims to deliver high tumoricidal doses while limiting the development of RILD. This locoregional strategy is defined as the infusion of radioactive carrier including microsphere of Yttrium-90, Iodine-131 iodized oil or similar agent into the hepatic artery [3]. Currently, 90 Ymicrospheres are the most SIRT technique used. Given the hypervascularity of HCC, 90 Y-microspheres injected into the hepatic artery will spread throughout the liver or confined to certain areas, where they can stop blood supply of the tumor by the embolisation process.
Progress in pharmaceutical research field has been exploited in the design of tumor-targeting nanoscale vectors able to deliver radionuclides. Among them, lipid nanocapsules (LNC), a nanovector with biomimetic properties [4], appear to be a useful therapeutic option for HCC treatment. Composed of a liquid lipidic core surrounded by a tensioactive shell, LNC lead to the encapsulation of a lipophilic complex of Rhenium-188 (c = 155 keV; b -= 2 MeV; T 1/2 = 16.9 h). The formulation, based on a fully automated phase-inversion process, is simple and results in nanoparticles solution presenting a mean diameter between 20 and 100 nm, depending on the quantity of excipients. The nanometric scale of LNC 188 Re-SSS (50 nm) could be highly advantageous, as LNCs may penetrate more deeply inside the tumor blood vessels, the mean diameter of microsphere devices are varying between 20 to 500 mm. Moreover, enhanced permeability retention effect (EPR), the main strategy for the delivery of nanoparticulate systems, may improve therapeutic efficiency. Indeed, it has been shown that small particles can passively cross the sinusoidal endothelium of the liver through fenestrations with a size of approximately a few hundred nanometers [5,6].
We report a study of LNC 188 Re-SSS as a new radiopharmaceutical carrier for internal radiotherapy of rats presenting hepatocellular carcinoma induced by diethylnitrosamine. No early mortality and no intolerance following LNC 188 Re-SSS intraarterial injection were observed. Our results provide evidences of therapeutic efficiency of LNC 188 Re-SSS with a reduction in tumor progression which could be combinated with an altered angiogenesis process as indicated by VEGF quantifications in plasmatic samples in a rat HCC model.

Ethics Statement
This study was carried out in strict accordance with the French Minister of Agriculture and the European Communities Council Directive of 24 November 1986 (86/609/EEC). The protocol was approved by the Committee on the Ethics of Animal Experiments of the ''Pays de la Loire'' (Permit Number: CEEA.2009.6). All surgery was performed under ketamine/xylazine anesthesia, and all efforts were made to minimize suffering.

Materials
LipoïdH S75-3 (soybean lecithin with 69% of phosphatidylcholine) and SolutolH HS15 (a mixture of polyethylene glycol 660 and polyethylene glycol 660 hydroxystearate) were kindly donated by Lipoïd Gmbh. (Ludwigshafen, Germany) and BASF (Ludwigshafen, Germany), respectively. NaCl and dichloromethane were provided by Sigma (St-Quentin, Fallavier, France). Deionized water was obtained from a Milli-Q plus system (Millipore, Paris, France). Lipophilic LabrafacH CC (caprylic-capric acid triglycerides) was provided by Gattefosse S.A. (Saint-Priest, France).  [7]. In brief, the 188 Re-SSS complex was obtained by the reaction of the ligand sodium dithiobenzoate (organic synthesis platform, Rennes, France) with a freeze-dried formulation kit containing 30 mg sodium gluconate, 30 mg ascorbic acid, 40 mg potassium oxalate, and 4 mg SnCl 2 .2H 2 O reconstituted in 0.5 mL of physiological serum. 1 110 MBq of 188 Re-perrhenate ( 188 ReO 4 -, in 0.5 mL) was added, and the solution was mixed for 15 minutes at room temperature. Then, 20 mg of sodium dithiobenzoate (in 0.5 mL; pH = 7) was added before being heated at 100uC for 30 minutes, which allowed for the formation of the 188 Re-SSS complex. Due to its precipitation in aqueous media, the 188 Re-SSS complex was extracted with dichloromethane (1 mL) and washed three times with 1 mL of deionized water. The radiochemical purity (RCP) of the complex was checked by thin-layer chromatography as the ratio of migrated radioactivity to total radioactivity. Thin-layer chromatography was carried out using silica gel 60-F 254 alumina plates (Merck) and a solution of petroleum ether/dichloromethane (6/4; v/v) as an eluant. Radioactivity was assessed with a phosphor-imaging machine (Packard, Cyclone storage phosphor system).

Nanocapsule formulation and characterization
The overall study was performed on 50 nm-diameter LNCs which were prepared according to a phase-inversion process described by Heurtault et al. [4]. In brief, 25 mg Lipoïd H S75-3, 282 mg SolutolH HS15, 342.7 mg LabrafacH, 29.7 mg NaCl, and 987.5 mg deionized water were mixed by magnetic stirring. The 188 Re-SSS complex extracted with dichloromethane (1 mL) was then added to the other components of the emulsion. The organic solvent was removed by being heated at 60uC for 15 minutes. Three cycles of progressive heating and cooling between 85uC and 60uC were then carried out and followed by an irreversible shock, induced by dilution with 4.16 mL of 0uC deionized water, which was added to the mixture at 70uC leading to 50 nm-lipid nanocapsules solution. Afterwards, slow magnetic stirring was applied to the suspension for 5 minutes. LNC 188 Re-SSS were dialyzed during 2 hours with deionized water at room temperature by magnetic stirring. The mean diameter and polydispersity index were then determined using a Malvern ZetasizerH Nano Serie DTS 1060 (Malvern Instruments S.A., Worcestershire, UK). The encapsulation yield was assessed with a gamma counter (Packard Auto-Gamma 5,000 series) according to the equation below.

|100
HCC model and treatment 49 Male Wistar rats weighting 150-180 g were obtained from the animal house of the Angers University Hospital. The animals were kept in polycarbonate cages in a room with controlled temperature (20-22uC), humidity (50-70%), and light (12-hour light/dark cycles). Room air was renewed at the rate of 10 vol/ hour. Tap water and food were provided ad libitum.
All experiments were performed on 6-week-old male Wistar rats. Hepatic carcinogenesis was induced chemically by adding diethylnitrosamine (DENA) to drinking water (100 mg/L) for 8 weeks. Each animal underwent hepatic artery catheterization on Day 10 (D10) after the end of tumor induction. Two LNC 188 Re-SSS groups were performed with one intra-hepatic artery injection of 80 MBq and 120 MBq of LNC 188 Re-SSS (LNC 188 Re-SSS -80 MBq, n = 12; LNC 188 Re-SSS -120 MBq, n = 11) as described in the ''Hepatic artery catheterization'' section below. Three control groups, the 188 ReO 4 group (80 MBq, n = 4), the sham group (n = 12) and the blank LNC group (n = 10) were assessed with the same procedure. We set the therapeutic activity at 80 MBq and 120 MBq, corresponding to an absorbed dose to the liver of 40Gy, which proved to be effective in the treatment of human HCC [8], and of 65Gy in order to appreciate the effect of a higher dose after LNC 188 Re-SSS internal radiation.

Hepatic artery catheterization
Hepatic artery catheterization was carried out according to the Garin et al. method [9]. The experiment was continued under a binocular magnifying glass. The first duodenal loop was pulled down to expose the gastroduodenal artery which was, after identification of the celiac and hepatic arteries, carefully dissected. A final distal ligature of the gastroduodenal artery was performed, and the celiac artery was clamped to temporarily stop arterial flow. After perforation using a 30 G needle, a 32 G catheter (CS-32, Bioseb, Vitrolles, France) was placed inside the gastroduodenal artery, and a volume of 400 mL of LNC 188 Re-SSS, 188 ReO 4 -, or blank LNC solution was then injected. After washing the syringe with saline solution, the upstream end of the gastroduodenal artery was tied off and arterial flow was restored. Concerning the sham group, the same procedure was performed except the injection which was not realized and a final ligature of the hepatic artery. This control group led to an appreciation of the collateral revascularization which could occur after injection.

Dosage of plasmatic vascular endothelium growth factor (VEGF)
Blood samples were collected from the tail vein using heparinized tubes in each group at D12, D20, D40, D55, D65, D80, and D105 after the end of tumor induction. After centrifugation at 1 000 g for 20 minutes, the rat VEGF ELISA test (R&D Systems Europe, Lille, France) was immediately performed according to the manufacturer's instructions.

Magnetic resonance imaging
Six animals from the LNC 188 Re-SSS -80 MBq and four animals from the 188 Re-perrhenate groups were explored by MRI in order to acquire images of the treatment-induced morphological changes at day 100 after the end of tumor induction. MRI was performed with a 1.5T Signa Excite HD device (General Electric Medical System Milwaukee, Ilinois). Rapid T2-weighted images were obtained using a fast spin echo (FSE) sequence with the following parameters: TR = 2500 s, TE = 102 s, matrix 3206320, twelve slices, thickness 3 mm, gap 0.5 mm, FOV = 15 cm, Nex = 4, acquisition time 69300). The following day, the animals were sacrificed for macroscopic and histopathological analyses.

Whole-body planar c-scintigraphy
Three animals from the LNC 188 Re-SSS -80 MBq and three animals from the 188 Re-perrhenate groups were explored by whole-body planar c-scintigraphy. Static 15-minutes scintigraphy acquisitions were obtained using a gamma camera (SOPHA DSX c camera, 155 keV615%, 128 2 matrix, HRLE collimator). 188 Re-perrhenate and LNC 188 Re-SSS solutions (80 MBq) were injected into the hepatic artery, and images were taken at 1.5 h, 3 h, and 24 h. A scan projection radiograph (Scout View) was acquired on a GE lightspeed system (General Electric Medical System Milwaukee, Ilinois) and fused with planar scintigraphy of the same animal for the localization of 188 ReO 4 and LNC 188 Re-SSS.

Tissue distribution study
A tissue distribution study was carried out on an extra group of 24 male Wistar rats with chemically-induced HCC. They were divided into two groups: one injected with a 188 ReO 4 solution following hepatic artery catheterization (n = 12), and the other with LNC 188 Re-SSS -80 MBq (n = 12). In both groups, the animals were sacrificed at 1.5 h (n = 4), 3 h (n = 4), and 24 h (n = 4) post-injection. The organs were removed, washed, and weighed (blood, liver, spleen, kidneys, heart, lung, stomach, small intestine, large intestine, bladder, bone, muscle, brain, tail, and carcass). The content activity of each organ was determined using a gamma counter (Packard Auto-Gamma 5,000 series).

Statistical analysis
The Kaplan-Meier method was carried out to plot animal survival. Statistical significance was assessed using the log-rank test (Mantel-Cox Test). StatView software was used for this purpose, and the tests were considered significant at p values less than 0.05. The different groups were compared in terms of survival time, increase in median survival time (IMST %), maximal survival time, and long-term survivors (rats were considered to be longterm survivors if they survived twice the median survival time of control groups [11]).

Encapsulation of the 188 Re-SSS complex in LNCs
The 188 Re-SSS complex presents a good purity as satisfactory RCP of more than 98% was obtained. Physico-chemical characteristics of the LNC are provided in Table 1

Biodistribution study
Planar scintigraphy led to the monitoring of the radioactivity distribution of LNC 188 Re-SSS and 188 ReO 4 following hepatic artery injection (Figure 1a). Results highlighted a liver uptake following LNC 188 Re-SSS injections and a stomach uptake after 188 ReO 4 injection up to 24 hours. LNC 188 Re-SSS were essentially confined to the liver, with an increased uptake up to 24 h (1.

Survival study
The descriptive and statistical data from the survival study are summarized in Table 2.
As shown in Figure 2, all animals of the control groups died due to tumor progression at D105, with a median survival time of 51619 days, 77619 days, and 57624 days for blank LNC, sham, and 188 ReO 4 groups, respectively. There was no significant difference between the three control groups (p.0.05).
Following treatment with LNC 188 Re-SSS (80 MBq) therapeutic efficiency was demonstrated by an increase in the median survival from 54 to 107% as compared to control groups with up to 7/12 long-term survivors in the LNC 188 Re-SSS group (80 MBq). Even if a slight difference in median survival time was observed between LNC 188 Re-SSS -80 MBq (118627 days) and LNC 188 Re-SSS -120 MBq (97618 days), comparison with these two groups was not significant (p = 0.4431).
Tumor volume assessment was performed using two different evaluation methods: MRI and macroscopic study. Figure 3 shows MRI and macroscopic views of control groups (sham, 188 ReO 4 and blank LNC) and LNC 188 Re-SSS -80 MBq rats at D100 (MRI) and D101 (macroscopy) after the end of tumor induction. Liver tumors represented approximately 100% of the liver parenchyma for control rats, while they occupied only 50% of the liver tissue for LNC 188 Re-SSS -80 MBq rats. Therefore, 50% of healthy liver tissue appeared to be preserved following LNC 188 Re-SSS -80 MBq treatment. This observation was confirmed by histopathological analysis (data not shown).

Transaminases assessment
Transaminases were assessed in each group and compared to healthy rat data. As shown in Tables 3 and 4

VEGF quantification
Results showed major differences between LNC 188 Re-SSS -80 MBq and the control groups (Figure 4)

Discussion
The use of LNCs, with a structure similar to lipoproteins, could represent a promising therapeutic modality for HCC management, as they modify the biodistribution of entrapped therapeutic agents [12,13,14]. These nano-objects include only FDA-approved excipients and are composed of a lipidic core leading to the entrapment of lipophilic molecules such as 188 Re-SSS [12], surrounded by a tension-active shell which induce physicochemical properties different from those of the drug. Additionally, their nanometric scale (LNC 188 Re-SSS mean diameter: 50 nm), and their low polydispersity index but also, the reduced viscosity of the drug, represent real advantages. It could likely avoid the excessive embolization process observed with chemoembolization (DC-beads TM -mean diameter: 300-500 mm [15]) and may penetrate more deeply inside the tumor blood vessels in comparison with 90 Y-microspheres devices (mean diameter around: 20-40 mm).
The first step in this study was to demonstrate the relevance of the encapsulation of Rhenium-188 for selective internal radiotherapy on a HCC rat model. The observed liver uptake following LNC 188 Re-SSS injection and 188 Re-perrhenate accumulation in the stomach, which have been already reported in the literature [13,14], validate the interest of the encapsulation of Rhenium-188. Organ biodistribution results indicated that LNC 188 Re-SSS clearance from the blood was mainly ascribed to the liver. The enhancement permeability retention effect (EPR) may account for these findings [6]. This phenomenon could be explained by the size of LNC 188 Re-SSS but Küpffer cells could be another explanation for these observations. In fact, it has been demonstrated that Küpffer cells and some macrophages are involved in nanoparticle capture [16]. Thus, the physicochemical properties of particulate systems improve the liver uptake of Rhenium-188.
The internal radiotherapy study by intra-arterial injection of LNC 188 Re-SSS was carried out on a chemically induced (DENA) HCC rat model. 100% tumor take was observed, with a median survival time ranging from 51 to 77 days after the end of tumor. Three control groups were performed: 188 Re-perrhenate, blank LNC and sham groups. Neither the injection of the 188 Reperrhenate solution nor the injection of blank LNC significantly modified rat survival, as the median survival time was 58 and 51 days respectively. The median survival time of the sham group (77 days) was higher than that of the 188 Re-perrhenate and blank LNC groups. This could be explained by a less invasive surgery, as neither hepatic artery catheterization nor injection, which could modify the hemodynamic parameters, were performed. As a consequence, treated groups (LNC 188 Re-SSS -80 MBq; LNC 188 Re-SSS -120 MBq) were compared with both 188 Reperrhenate or blank LNC groups and sham group.
The LNC 188 Re-SSS -80 MBq treatment was the most effective with 7/12 or 2/12 rats being long-term survivors and an IMST ranging from 107% to 54% according to the control group ( 188 Reperrhenate groups or sham, respectively). These results correlated  Increasing the absorded dose to the liver has shown no interest in terms of survival efficacy as LNC 188 Re-SSS -120 MBq group gave rise to the worse increase in median survival (IMST% ranging from 26% to 70% according to the control group). Hepatotoxicity demonstrated by higher levels of transaminases, could explain its less efficiency in term of survival.
Angiogenesis plays a key role in the pathogenesis of many cancers [17]. HCC, a hypervascular tumor, is mainly supplied by hepatic artery, whereas normal liver parenchyma and dysplastic nodules are largely supplied by the portal vein [18]. HCCs have been shown to express many angiogenic factors including VEGF [19]. Moreover, VEGF expression by the tumor and VEGF levels in patients' blood have been shown to correlate with the size, invasiveness, metastases, and prognosis of HCC [20]. As a consequence, the assessment of VEGF concentrations in plasma samples was carried out in each group except for LNC 188 Re-SSS -120 MBq group, which demonstrated worse results in terms of survival and hepatotoxicity. The lower VEGF levels, in LNC 188 Re-SSS -80 MBq group in comparison with control groups, could reflected an altered angiogenesis in the rat HCC model, which could reduced tumor progression confirmed by MRI.
Our results pointed out the advantage in using lipid nanocapsules, a drug delivery system for SIRT. In that field, an important breakthrough for HCC treatment has been done, with drug eluting beads (DEB, DC-beads TM ), 90 Y-microspheres (TheraS-phereH, SIR-SphereH), 188 Re-microspheres and nanocarriers. Dhanasekaran et al. demonstrated that doxorubicin DEB therapy with unresectable HCC provides a survival advantage over treatment with conventional chemoembolization [21]. However, micrometric size of DC-beads (300 to 500 mm) could generate excessive embolisation leading to Post-Embolization Syndrome (PES) [22]. Meanwhile DC-beads, SIRT with 90 Y-microspheres is another palliative HCC treatment option, with up to 50% in tumor HCC size reduction [23,24,25,26]. 188 Re-microspheres were also developed for HCC treatment with decrease in tumor growth after 188 Re-microsphere injection [27,28].
Nanocarriers [29,30] enable to load chemotherapeutic agents such as docetaxel [31] or oligonucleotides [32] were developed in order to reduce PES observed with DC-beads. Results demonstrated a slowdown in tumor progression but most of these preclinical studies assessed their efficacy on a subcutaneous HCC model.
We have developed 50 nm-lipid nanocapsules loaded with Rhenium-188 for selective internal radiotherapy and have assessed their efficiency on a chemically induced HCC rat model, known for its physiological properties similar to human hepatocarcinoma. Due to their nanometric scale, no embolisation process was possible. This could represent a real advantage as tumoral hypoxia areas could be reduced allowing a better efficiency of ionizing radiations.  Recently, sorafenib, an inhibitor of the VEGF receptor, has been shown to prolong the median survival time by 3 months in patients with advanced HCC [33]. As LNC 188 Re-SSS seems to alter angiogenic process, their combination with sorafenib as an adjuvant therapy could be a valuable approach in the treatment of advanced HCC.
In conclusion, 188 Re-loaded LNCs appear to be an encouraging new radiopharmaceutical carrier for HCC internal radiotherapy which could penetrate more deeply inside the tumor blood vessels. A comparative study of LNC 188 Re-SSS and 90 Y-microspheres SIRT, already used in clinical application, will probably provide informations of the effect of the size of particle systems and also in term of dosimetry.