Boron encapsulated in a liposome can be used for combinational neutron capture therapy

All animal experiments were performed according to the Animal Protection Guidelines of Peking University, China. All animal care and experimental procedure were performed by following the animal protocols (CCME-LiuZB-2) approved by the ethics committee of Peking University.

Chemical synthesis of thiocarboranyl triethylamine

The substrate (o-carborane, 1.00 g, 6.93 mmol) was dissolved in THF (30 mL) and cooled at 0 °C in dry and anaerobic conditions. Then n-BuLi (4.4 mL, 6.93 mmol) was added to the solution dropwise for 15 min, and stirred for 30 min. Then the solution was warmed up to r.t. for another 30 min. Then sulfur(α) (225 mg, 6.93 mmol) was dissolved in the solution above at 0 °C to react for 30 min. Then the solution was warmed up to r.t. for another 30 min. The solution was then evaporated in vacuo to produce a yellow liquid. Then HCl (1 M, 7 mL) was added to quench the remaining n-BuLi. The solution was extracted with hexane and washed with water and brine, then evaporated in vacuo. In a solution of 1-mercapto-1,2-dicarba-closo-dodecaborane in hexane (6 mL) at room temperature, Et₃N (1 mL, 7.18 mmol) was added dropwise and stirred for 15 min. The white precipitate formed was filtered, washed with 3 × 6 mL of hexane and dried in air (compound 3, 1.26 g, 66% yield).

Chemical synthesis of carboranyl fatty acids

Synthesis of 1-C₂H₅OC(O)(CH₂)_nBr (compound 8–11) (n = 3, 7, 11, 15)

To a solution of 1-HOOC(CH₂)_nBr (9.3 mmol) in ethanol (50 mL), 5 mL of concentrated sulfuric acid was added dropwise, stirred for 15 min at room temperature and heated under reflux for 12 h. The reaction mixture was cooled and evaporated to dryness in vacuo. The residue was treated with ethyl acetate (30 mL) and water (30 mL). The organic layer was separated, washed with water and saturated brine, dried over Na₂SO₄ and evaporated in vacuo. The crude product was purified using column chromatography on silica with petroleum ether and ethyl acetate as eluent. The solvent was evaporated under a vacuum to yield a yellow oil.

Synthesis of 1-C₂H₅OC(O)(CH₂)_nS-1,2-C₂B₁₀H₁₁ (compound 12–15) (n = 3, 7, 11, 15)

To a solution of 1 (1.86 mmol) in ethanol (42 mL), Br(CH₂)_nC(O)OC₂H₅ (1.86 mmol) was added, stirred for 15 min at room temperature and heated under reflux for 16 h. The reaction mixture was cooled and evaporated to dryness in vacuo. The residue was treated with ethyl acetate (30 mL) and water (30 mL). The organic layer was separated, washed with water and saturated brine, dried over Na₂SO₄ and evaporated in vacuo. The crude product was purified using column chromatography on silica with petroleum ether and ethyl acetate as eluent. The solvent was evaporated under a vacuum to yield a yellow oil.

Synthesis of 1-HOOCC(O)(CH₂)_nS-1,2-C₂B₁₀H₁₁ (compound 16–19) (n = 3, 7, 11, 15)

1-C₂H₅OC(O)(CH₂)_nS-1,2-C₂B₁₀H₁₁ was dissolved in glacial acetic acid (29 mL) forming a clear colourless solution. After stirring for 15 min at room temperature, water (9.8 mL) and concentrated sulfuric acid (9.8 mL) were added. The reaction mixture was heated at reflux for 16 h. The reaction mixture was cooled and poured into cold water (30 mL). The white precipitate was extracted with petroleum ether. The organic phase was collected and evaporated in vacuo to yield products as white solid^36,37.

Chemical synthesis of BoPs

To a solution of 0.1 M carboranyl fatty acid (2.0 eq.) in chloroform was added N,N-dicyclohexylcarbodiimide (2.6 eq.) and N,N-dimethyl-4-aminopyridine (4.0 eq.). The solution was stirred at r.t. for 5 min. Lyso-PPC (1.3 eq.) was then added and the reaction was stirred for 24 h. The reaction mixture was concentrated using rotary evaporation. Purification by silica flash chromatography (65:25:4 CH₂Cl₂:MeOH:H₂O) yielded BoP species (compound 21–24)³⁸.

Boronsome preparation

Boronsomes were prepared by the same method as previously described³⁹. To generate 1 mL of boronsomes (total lipids 4 mg/mL) of the indicated formulations (BoPs: DPPC: Cholesterol: DSPE-PEG2k = 50:10:40:1, mol%), lipids were dissolved in 0.2 mL ethanol at 60 °C, followed by injection of 0.8 mL of 250 mM ammonium sulfate (pH 5.5) buffer at 60 °C. The boronsome solutions were then passed 10 times at 60 °C through sequentially stacked polycarbonate membranes of 0.2 and 0.1 µm pore size using an extruder (Avanti Polar Lipids). Free ammonium sulfate was removed by dialysis in a 500 mL solution composed of 10% sucrose and 10 mM histidine (pH 6.5) with at least three buffer exchanges.

Cargo loading

For SRB loaded boronsomes, lipids of the indicated formulations were dissolved in ethanol and hydrated with 50 mM SRB, sonicated at 45 °C for 30 min. Doxorubicin (Dox) or Olaparib (PARPi) was loaded via the ammonium the ammonium sulfate gradient method. Dox or Olaparib with a drug to lipid molar ratio of 1:8 was added into the liposome solution and incubated at 60 °C for 1 h. Stability and encapsulation ability were evaluated by SRB release which was measured by absorbance using the formula: %SRB release = (A_final − A_initial)/(A_Triton-X-100 − A_initial) × 100%.

Characterisation

Intensity-average hydrodynamic diameters and size distributions (PDI) were measured by Dynamic laser light scattering (DLS) which were conducted on a Zetasizer Nano ZS system (Malvern Instruments Ltd, England). The scattered light was collected at a fixed angle of 173° for duration of ~5 min. All data were averaged over three consecutive measurements. The morphological examination of boronsome was performed with TEM operated at 200 kV (JEM-2100, JEOL, Japan). A drop of boronsome solution was added to a copper mesh covered with an ultrathin carbon film and dried at room temperature for 10 min. The excess solution was removed before negatively staining with uranyl acetate solution (4% w/v) followed by washing with water.

Preparation of [⁶⁴Cu]Cu-NOTA-boronsome

⁶⁴CuCl₂ in 0.01 M hydrochloric acid was provided from Peking University Cancer Hospital. NOTA-boronsome was prepared as described at the indicated formulation (BoP-3: DPPC: Cholesterol: DSPE-PEG2k-NOTA = 50:10:40:1, mol%) before use. Briefly, boronsome solution (0.5 mg/mL) was made up to 1 mL of NaAc buffer (pH 5.5, 0.2 M), and incubated with ⁶⁴CuCl₂ (185 MBq) at 37 °C for 2 h⁴⁰. [⁶⁴Cu]Cu -NOTA-boronsome was purified through PD-10 chromatography with PBS as an eluent. Radiochemical yield was up to 90.3%.

Radio-TLC

Stability of [⁶⁴Cu]Cu-NOTA-boronsome was determined by thin-layer chromatography with the eluant as EDTA solution. Radio-signal of [⁶⁴Cu]Cu-NOTA-boronsome on paper stripes at various time points were collected by Scan-RAM (LabLogic).

3D structures modelling

Spartan 14 software was used to predict the 3D structure of BoPs after optimised at Density Functional Theory/Becke, 3-parameter, Lee-Yang-Parr, and 6-31 G* basis set (DFT/B3LYP/6-31 G*). Parameters including I_c, carbon arm length (Å); I_b, boron arm length (Å); Δl, length difference between lipid arms (Å); d, the distance between lipid arms (Å) and θ, the dihedral angle between lipid arms (°) were measured and evaluated.

MD simulation

We simulated the dynamics trajectory of lipid bilayers systems consisting of 128 lipid molecules (BoP-1, BoP-2, BoP-3, BoP-4 and DPPC). For BoP-n lipid bilayers systems which are composed of 108 BoP-n molecules, 20 DPPC molecules and 3200 water molecules. The dynamics simulations were carried out in an NPT ensemble (298.15 K, 1 atm), and we took 10 ns simulations per systems.

The Molecular Dynamics simulations were performed by GROMACS 2018 software⁴¹. We used Automated force field Topology Builder (ATB, 3.0) tool⁴² to generate the force field parameter and topology information. The GROMOS 54a7 force field modified by ATB^43,44 ware utilised to describe this system. VMD 1.9.3 was used in the computational study for visualisation.

We used the chain order parameter (Szz) to estimate the degree of regularity of hydrocarbon chain. It was defined as

({{{{{{rm{S}}}}}}}_{{{{{{rm{ZZ}}}}}}}=frac{{3 < {cos },{{{{{{rm{theta }}}}}}}_{{{{{{rm{i}}}}}}} > }^{2}-1}{2}{{mbox{,}}}) θi represents the angle between bilayer normal and the vector of Ci-1 to Ci+1. We chose z axis as bilayer normal and Szz indicates the orientation of hydrocarbon chain with respect to the bilayer normal. When Szz is equal to 1, the hydrocarbon chain parallel to bilayer normal. We calculated the average chain order parameter of hydrocarbon chain in 5 to 10 ns (See Fig. 1f). Detailed information about the parameters when performing the MD simulation was listed (Supplementary Tables 1–3).

Irradiation of cells and animals

Cells or animals were irradiated at In-Hospital Neutron Irradiator (IHNI) based on miniature neutron source reactor. For cell irradiation, 4T1 cells (3101MOUSCSP5056, obtained from National Infrastructure of Cell Line Resource) cultured in 96-well plates were incubated with boronsome (5 mg/mL) or PRAPi-boronsome (same boron dose) for 24 hours, and were irradiated for 10 min. For animal irradiation, thermal neutron irradiation experiments were performed when the tumour volume of 4T1 tumour-bearing mice reached about 70 mm³. Each mouse in the experimental group was intravenously injected with liposome (500 mg/kg), boronsome (500 mg/kg), Dox-boronsome (same boron dose) or PRAPi-boronsome (same boron dose) while each mouse in the control group was intravenously injected with PBS. After 12 hours, mice anaesthetised with isoflurane (5%, 100 μL) were fixed onto the special-made module (Fig. 5b), and were irradiated for 30 min.

Determination of minimal boron requirement

The neutron capture cross-sections (σ) vary significantly between nuclides. Because the thermal neutrons are preferentially captured by ¹⁰B nuclei (3837 barn, capture cross-section many times greater than that of C (0.0037 barn), H (0.332 barn), O (0.0002 barn), and N (1.75 barn) of which tissues are mainly composed of), and cell damage is mainly caused by high-LET particles generated from ¹⁰B(n,α)⁷Li reaction, concentrations of ¹⁰B in targeted tissues sufficient to produce therapeutic doses is of vital for BNCT treatment.

Neutron flux (ϕ) of IHNI-1 = 1.9 × 10⁹ cm⁻² s⁻¹

Irradiation time (T) = 10 min (cells) or 30 min (animals)

To ensure at least one successful ¹⁰B(n,α)⁷Li reaction within one cell, N_reaction ≥ 1, 10⁹ of ¹⁰B (N_B) is usually considered to be the minimum requirement.

Dosimetry by SERA

The magnitude of the radiation dose of BNCT is complicated due to the radiation field in BNCT consists of several separate radiation dose components including B-10 dose (from ¹⁰B(n,α)⁷Li reaction), gamma dose (from hydrogen ¹H(n,γ) in tissue), N-14 dose (from ¹⁴N(n,p)¹⁴C in tissue) and hydrogen dose (from ¹H(n,n′)p in tissue) with different physical properties and biological effectiveness. The Monte Carlo computation-based dose calculation was performed with SERA (Simulation Environment for Radiotherapy Applications; Idaho National Laboratory, Idaho Falls, ID).

Cell culture method

The 4T1 cell lines (3101MOUSCSP5056) were obtained from National Infrastructure of Cell Line Resource (Beijing, China). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All the cells were cultured in a humidified 5% CO₂ incubator at 37 ˚C and the medium was replaced every 1–2 days.

Cell viability assays

4T1 cells were seeded in 96-well plates at a concentration of 5 × 10³ cells per well and incubated overnight in a cell culture incubator. Up to 400 kBq/mL [⁶⁴Cu]Cu-NOTA-boronsome, boronsome (5 mg/mL) and PARPi-boronsome (same boron dose) were added to the wells (100 μL final volume per well) with different concentrations. After 24 h incubation at 37 °C, cells were irradiated by thermal neutron beam. And after 24 h post irradiation, cell viability was assessed with the CCK-8, following the manufacturer’s protocol.

Colony formation assay

To determine cell reproductive death after treatment, 10,000 cells are seeded out in a 10-cm culture dish to form colonies in 7 days. Colonies are fixed with methanol and stained with crystal violet (0.02% w/v).

Cell uptake studies

Cell uptake studies were performed on 4T1 cells. Briefly, 4T1 cells (1 × 10⁵ per well) were seeded in 24-well plates and incubated overnight at 37 °C. A series of concentration gradient solutions with 0.1–10 mg/mL boronsome (Fig. 3g), boronsome (5 mg/mL), FBY (same boron dose) and BPA (same boron dose) (Fig. 3h) were added to wells, and the mixture was incubated at 37 °C for 24 h. The cells were then carefully washed three times with ice-cold PBS and lysed with 0.1 M HNO₃. We determined the cellular boron concentration by ICP-OES: the crudely treated cells obtained in the previous step were digested using a microwave accelerated reaction system (Mars; CEM), and then diluted with deionized water. The boron concentration was determined by ICP-OES on a PerkinElmer Optima 7000 DV according to the published method.

γ-H2AX Immunofluorescence staining

The cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X 100 for 20 min and blocked with 10% goat serum for 30 min at room temperature (RT). After blocking, the cells were incubated with anti-γH2AX antibody (1:200, abcam, ab81299) at 4 °C overnight and then incubated with the secondary antibodies, goat anti-rabbit IgG H&L (Alexa Fluor® 488, 1:1000, abcam, ab150077) for 1 h at room temperature in the dark. The cells were stained in 4′,6-diamidino-2-phenylindole (DAPI, Santa Cruz Biotechnology) for 15 min at RT, covered with glycerol-PBS (1:1) and coverslips, sealed with clear nail polish and examined under a laser scanning confocal microscope (Nikon, Japan). Quantification of fluorescence intensities was analysed by ImageJ 1.53a.

Animal studies

In all, 6–8-weeks female BALB/c mice were obtained from Beijing Vital River Laboratory Animal Technology Co, Ltd. and maintained under specific pathogen-free facility conditions with a 12 light/12 dark cycle, and free access to food and water. Mice were housed under temperature of 24 ± 2 °C, and humidity of 50 ± 10%.

Xenografts implantation

Subcutaneous 4T1 tumour model was established by injection of 10⁶ 4T1 cells in 100 µL PBS into the right flank of female BALB/c mice. After 1 week, all drugs were administered to mice by intravenous (i.v.) injection 12 hours before neutron irradiation.

Therapeutic evaluation

4T1 tumour diameters were measured by caliper, and the tumour volume (V_t, mm³) was calculated by V_t = 0.5 × a × b², where a was the long axis and b was the short axis. Body weight was recorded every 2 days. Data were collected and the relationship between tumour volume and time was plotted, the therapeutic effects of different groups were evaluated from the experimental results. When the tumour size reached 2000 mm³ or the loss was >20% of total body weight, the mice were removed from the experimental group and euthanized.

Blood routine and biochemical test

Mice bearing 4T1 tumour were injected with 5 mg/kg of [⁶⁴Cu]Cu-NOTA-boronsome intravenously (7.4 MBq) or 500 mg/kg boronsome, and then blood routine and serum biochemical were analysed according to the standard method at different time.

Small-animal PET/CT imaging

Mice bearing 4T1 tumour were injected with 5 mg/kg of [⁶⁴Cu]Cu-NOTA-boronsome intravenously (7.4 MBq) and anaesthetised by inhalation of 2% isoflurane/oxygen mixture and placed on a scanner bed approximately 10 minutes prior to PET/CT image acquisition. PET scans were performed on Nanoscan PET-CT 122 s (Mediso Medical Imaging Systems). PET acquisitions for static reconstructions were performed 0.5 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h after injection. For each acquisition, 3D regions of interest (ROI) were acquired by Nucline NanoScan software (InterViewTM FUSION, Mediso Medical Imaging Systems) on the decay-corrected images. The radioactivity in relevant tissues was obtained from mean and maximum standard uptake values (SUVs) of the ROIs and then converted to the percentage injected dose per gram (% ID/g, assuming a tissue density of 1 g/mL).

Pharmacokinetics study

We determined the cellular boron concentration by ICP-OES: mice that were injected intravenously with 500 mg/kg boronsome or BPA were sacrificed at different time points, then obtained tumour, blood, heart, liver, spleen, lung, muscle. kidney and brain. These organs were lysed with HNO₃ and were digested using a microwave accelerated reaction system (Mars; CEM), then diluted with deionized water. The boron concentration was determined by ICP-OES on a PerkinElmer Optima 7000 DV according to the published method.

H&E staining

The major organs including the heart, liver, spleen, lung and kidney were excised from mice of different groups 21 days after treatment for the histopathologic study. Organs were fixed in 4% paraformaldehyde, embedded with paraffin, sectioned into slices, and stained with hematoxylin and eosin. Samples were chosen at random, and the slices were photographed by 3Dhistech.

Statistics analysis

Statistical analyses were performed using GraphPad Prism 8. All data were expressed as mean ± SEM.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.