Cationized gelatin-HVJ envelope with sodium borocaptate improved the BNCT efficacy for liver tumors in vivo

Abstract

Background: Boron neutron capture therapy (BNCT) is a cell-selective radiation therapy that uses the alpha particles and lithium nuclei produced by the boron neutron capture reaction. BNCT is a relatively safe tool for treating multiple or diffuse malignant tumors with little injury to normal tissue. The success or failure of BNCT depends upon the 10B compound accumulation within tumor cells and the proximity of the tumor cells to the body surface. To extend the therapeutic use of BNCT from surface tumors to visceral tumors will require 10B compounds that accumulate strongly in tumor cells without significant accumulation in normal cells, and an appropriate delivery method for deeper tissues.Hemagglutinating Virus of Japan Envelope (HVJ-E) is used as a vehicle for gene delivery because of its high ability to fuse with cells. However, its strong hemagglutination activity makes HVJ-E unsuitable for systemic administration.In this study, we developed a novel vector for 10B (sodium borocaptate: BSH) delivery using HVJ-E and cationized gelatin for treating multiple liver tumors with BNCT without severe adverse events.

Methods: We developed cationized gelatin conjugate HVJ-E combined with BSH (CG-HVJ-E-BSH), and evaluated its characteristics (toxicity, affinity for tumor cells, accumulation and retention in tumor cells, boron-carrying capacity to multiple liver tumors in vivo, and bio-distribution) and effectiveness in BNCT therapy in a murine model of multiple liver tumors.

Results: CG-HVJ-E reduced hemagglutination activity by half and was significantly less toxic in mice than HVJ-E. Higher 10B concentrations in murine osteosarcoma cells (LM8G5) were achieved with CG-HVJ-E-BSH than with BSH. When administered into mice bearing multiple LM8G5 liver tumors, the tumor/normal liver ratios of CG-HVJ-E-BSH were significantly higher than those of BSH for the first 48 hours (p < 0.05). In suppressing the spread of tumor cells in mice, BNCT treatment was as effective with CG-HVJ-E-BSH as with BSH containing a 35-fold higher 10B dose. Furthermore, CG-HVJ-E-BSH significantly increased the survival time of tumor-bearing mice compared to BSH at a comparable dosage of 10B.

Conclusion: CG-HVJ-E-BSH is a promising strategy for the BNCT treatment of visceral tumors without severe adverse events to surrounding normal tissues.

Figure 1

Blood chemistry tests 24 hours after HVJ-E and CG-HVJ-E administration into normal mice. Blood markers (T.Bil, AST, ALT, LDH, BUN, and Cr) in normal mice tested 24 hours after intra-cardiac injection of PBS, HVJ-E or CG-HVJ-E. * p < 0.05. Results shown are the means ± SD (n = 4).

Figure 2

Affinity of CG-HVJ-E for tumor cells and the intracellular uptake of molecules incorporated into HVJ-E. A) Affinity of HVJ-E and CG-HVJ-E for tumor cells. LM8G5 cells were incubated alone (a), or with Qdot (b), HVJ-E-Qdot (c), or CG-HVJ-E-Qdot (d) for 60 min in a Lab-tek chamber slide and examined for Qdot (red) and Hoechst 33342 (blue) by fluorescence microscopy, Representative views are shown. B) Intracellular localization of Qdot transported by CG-HVJ-E. Tumor cells were incubated with CG-HVJ-E-Qdot (orange) and stained with Hoechst 33342 (blue) and Alexa Fluor 488 phalloidin (green). Image shows 3-dimensional analysis with confocal microscopy. C) Luciferase activity in tumor cells transfected with HVJ-E or CG-HVJ-E. Cells were cultured for 30 min with HVJ-E or CG-HVJ-E containing a luciferase-expressing plasmid. Luciferase activity was measured 24 hours later to evaluate the transfection efficiency. Results are shown as means ± SD (n = 4). Similar results were obtained in three experiments. * p < 0.05. D) ¹⁰B accumulation and retention in tumor cells in vitro. Cells were incubated with 20 μg boron/ml of BSH or CG-HVJ-E-BSH for 30 min, then washed twice with PBS, and the ¹⁰B concentration was measured by ICP-AES. Separately, cells were incubated in the same manner, but after washing, were incubated in medium without BSH for 24 or 48 hours before testing for ¹⁰B concentration as described above. The horizontal axis shows time after co-incubation. The vertical axis shows the percent of the administered dose (% dose) of CG-HVJ-E-BSH (open diamond) or BSH (solid square). Results shown are the means ± S.D. (n = 3). * p < 0.05.

Figure 3

Bio-distribution of ¹⁰B in mice with normal liver or with liver tumors. A) Time course of organ (lung, liver, kidney, and spleen) uptake of ¹⁰B delivered by 1.2 μg boron/g of BSH or CG-HVJ-E-BSH in normal mice. B) Time course of tumor accumulation (left panel), liver uptake (middle panel), and blood residence (right panel) of ¹⁰B delivered by 1.2 μg boron/g of BSH or CG-HVJ-E-BSH in tumor-bearing mice. The horizontal axis shows the time after administration. The vertical axis shows the percent of the administered dose per gram of tissue (% dose/g). C) Time course of the Tumor-to-Normal liver tissue (T/N) ¹⁰B concentration ratio for CG-HVJ-E-BSH (open diamond) or BSH solution (solid square). Data are expressed as the mean ± S.D. (n = 3). * p <0.05 compared with BSH.

Figure 4

Neutron capture radiographic image in murine liver sections after administration of BSH or CG-HVJ-E-BSH. Liver sections from tumor-bearing mice were prepared and frozen 1 hour after BSH (35 μg boron/g) injection or 24 hours after CG-HVJ-E-BSH (1.2 μg boron/g) injection. The sections were placed on CR-39 detector plates and exposed to thermal neutrons (2.1 × 10¹³ neutrons/m²·s¹) for 1 hour. A) The number of α particles per 10,000 μm² section was counted by VH Analyzer software after NaOH etching. B) The number of α particles per 10,000 μm² section of BSH or C) of CG-HVJ-E-BSH (n = 3). D) Tumor-to-normal liver tissue (T/N) ratio for the number of α particles

Figure 5

Anti-tumor efficacy of BNCT in mice with liver tumors. C3H/HeN mice were given an intra-portal injection of LM8G5 cells (1 × 10⁶ cells) on day 0. Mice were given a single intra-cardiac injection of CG-HVJ-E-BSH (1.2 μg boron/g) 24 hours before irradiation, or BSH (35 μg boron/g) 1 hour before irradiation. PBS and CG-HVJ-E-BSH solution were administered without neutron irradiation as a control. After irradiation on day 8, mice were sacrificed on day 14 to determine the BNCT efficacy on tumor metastasis. A) Macroscopic views (a) of the liver with tumors after administration of PBS; (b) normal liver; (c) liver with tumors after BNCT with BSH, and (d) liver with tumors after BNCT with CG-HVJ-E. B) Liver weight after BNCT. * p < 0.05 compared with PBS or CG-HVJ-E without irradiation. (each group n = 4). C) Representative light microscopic views of liver tumor tissue (upper panels, low magnification) or normal liver tissue (lower panels, high magnification) 6 days after BNCT with CG-HVJ-E-BSH (1.2 μg boron/g) (a, c) or BSH (35 μg boron/g) (b, d). Sections are stained with hematoxylin-eosin. Bar: 100 μm.