closo-Dodecaborate-conjugated human serum albumins: preparation and in vivo selective boron delivery to tumor

Chemistry

1,4-Dioxane-closo-dodecaborate complex 1 [29] is an important intermediate for both synthesis of MID and ISD. In our previous report, amine 2 was synthesized by the ring opening of 1,4-dioxane-closo-dodecaborate complex 1 of closo-dodecaborate with tetrabutylammonium azide followed by Staudinger reduction [28], [30]. We found that the ring opening of 1,4-dioxane-closo-dodecaborate 1 with ammonia was more efficient to synthesize amine 2 [31], [32]. Indeed, the tetrabutylammonium (TBA) salt of amine 2 was simply purified by recrystallization from methanol. It was converted to its di-TBA salt in 10 % TBAOH in methanol and then reacted with 4-maleimidebutylic acid in the presence of EDCI using NaHCO₃ as a base in acetonitrile to afford di-TBA salt of MID. The counter cation of TBA was sequentially converted to the di-tetramethylammonium followed by the di-sodium form according to our previously reported procedure [28] (Scheme 1).

Scheme 1:

Synthesis of MID.

On the other hand, ISD was also synthesized from amine 2 as shown in Scheme 2. The tetrabutylammonium (TBA) salt of amine 2 was treated with 1,1′-thiocarbonyldiimidazole (TCDI) in the presence of triethylamine (TEA) in dichloromethane to give the di-TBA salt of ISD, which was converted to di-sodium salt through tetramethylammonium (TMA) counter cation.

Scheme 2:

Synthesis of ISD.

Conjugation efficacy of MID, ISD, and 1,4-dioxane-closo-dodecaborate complex 1 to BSA

It has been reported that 1,4-dioxane-closo-dodecaborate complex 1 undergoes the ring-opening reaction with various nucleophiles including amino acids. Thus, we first examined the conjugation efficacy of MID, ISD, and 1 to BSA. The conjugation reactions of MID, ISD, and 1 to BSA were carried out in PBS buffer (pH 7.4) at room temperature for 1 h and the resulting albumin-closo-dodecaborate conjugates were detected by Western blot analysis using anti-BSH antibody. The results are shown in Fig. 2. The conjugation efficacy of MID to BSA was higher than that of ISD, although an isothiocyanate group is widely used for protein modification at Lys residue. We previously reported that the maleimide group of MID was conjugated not only to SH free Cys residue but also an amino group of Lys residue [28]. In general, common maleimide compounds do not react with a nucleophilic amino group of Lys residue that is protonated under physiological conditions (pH 7.4). In the case of MID, MID contains negatively charged boron cluster in the molecule associated with sodium counter cations. In the local environment in proteins, the protonated amino group of Lys is probably deprotonated with the negatively charged boron cluster to induce the nucleophilicity. Therefore, the Michael reaction proceeded between the amino group of Lys residue and the maleimide moiety of MID. Interestingly, conjugation of 1,4-dioxane-closo-dodecaborate complex 1 to BSA was also observed under the physiological conditions although the conjugation efficacy was not very high. These results indicate that MID possesses the most promising conjugation efficacy among three closo-dodecaborate derivatives synthesized.

Fig. 2:

Western blot analysis of the conjugation efficacy of MID, ISD, and 1,4-dioxane-closo-dodecaborate complex 1 to BSA. BSA (0.1 mM) was reacted with MID (1 mM), ISD (1 mM), or 1 (1 mM) in PBS buffer at pH 7.4 for 1 h, and the resulting mixture was subjected to PAGE. Western blot analysis of BSA-closo-dodecaborate conjugates was carried out using anti-BSH antibody.

SEM image of albumin-MID conjugates

Nanoparticles albumin-bound technology using high-pressure homogenizers [33] has been used to make albumin nanoparticles of 100–200 nm. In particular, Abraxane^® was reported to form albumin-paclitaxel nanoparticles with a mean particle size of 130 nm. In the current study, a self-assembly method was used to make BSA-MID nanoparticles. As shown in Fig. 3, albumin-MID conjugates were found to be almost spherical with size distribution from 36 to 52 nm in diameter by field-emission scanning electron microscopy (FE-SEM) analysis.

Fig. 3:

FE-SEM images of albumin-MID conjugates.

Conjugation of MID to BSA and HSA

We compared the conjugation efficacy of MID to BSA and human serum albumin (HSA) by Western blot analysis. The conjugation reactions of MID to BSA and HSA were carried out in PBS buffer (pH 7.4) at room temperature for 1 h and the resulting albumin-closo-dodecaborate conjugates were detected by Western blot analysis using anti-BSH antibody. Expectedly, as shown in Fig. 4, the conjugation reaction of MID to HSA proceeded in a similar manner to BSA under PBS buffer conditions (pH 7.4). In addition, the use of 10 equiv. of MID against HSA showed more effective conjugation than the use of 1 equiv. of MID.

Fig. 4:

Western blot analysis of the conjugation efficacy of MID to BSA and HSA. MID (1 mM) was reacted with BSA (0.1 mM) or HSA (0.1 mM) in PBS buffer at pH 7.4 for 1 h, and the resulting mixture was subjected to PAGE. Western blot analysis of BSA- and HSA-closo-dodecaborate conjugates was carried out using anti-BSH antibody.

Biodistribution of HSA-MID conjugates in tumor-bearing mice

Boron distribution of HSA-MID was examined in colon 26 tumor-bearing mice. Previously, we examined the time-dependent boron distribution of BSA-MID in colon 26 tumor-bearing mice and observed that the significant boron accumulation in tumor was achieved 12 h after administration at the dose of 30 mg[B]/kg. In addition, Soloway et al. reported that BSH was appeared to bind to serum albumin strongly under physiological condition enough to prevent the cleavage under dialysis conditions, suggesting selective accumulation of BSH into subcutaneously transplanted tumor [5]. Therefore, we conducted the biodistribution study toward the following HSA-closo-dodecaborate conjugates: HSA-Na₂B₁₂H₁₂, HSA-Na₂BSH, HSA-NaB₁₂H₁₁NH₃, and HSA-MID. Na₂B₁₂H₁₂, was used as a control [34]. The boron agents were freshly prepared by mixing HSA and each closo-dodecaborates in PBS for 12 h at 37°C before use. The mice were injected intravenously with 200 μL of boron agent in saline at 30 mg[B]/kg. At 12 h after injection, boron concentration in blood, liver, kidney, spleen, and tumor was measured by inductively coupled plasma optical emission spectroscopy (ICP-OES). The results are summarized in Fig. 5. HSA-MID was highly accumulated in tumor (23 ppm B), whereas boron concentrations in other organs such as liver, kidney and spleen were relatively low (3~8 ppm B), indicating that the tendency of biodistribution of HSA-MID in tumor-bearing mice is similar to that of BSA-MID. In contrast, rapid clearance from organs were observed in the case of HSA-Na₂B₁₂H₁₂, HSA-Na₂BSH, and HSA-NaB₁₂H₁₁NH₃ similar to Na₂B₁₂H₁₂, suggesting that the bindings of HSA-closo-dodecaborate conjugates except HSA-MID are not strong enough to induce EPR effect during their circulation in blood.

Fig. 5:

Boron distribution of HSA-Na₂B₁₂H₁₂, HSA-Na₂BSH, HSA-NaB₁₂H₁₁NH₃, and HSA-MID in colon 26 tumor-bearing mice 12 h after administration at the dose of 30 mg[B]/kg. Each boron agent was injected into tumor-bearing mice (Balb/c, female, six weeks old, 14–16 g) via the tail vein. Na₂B₁₂H₁₂ (30 mg[B]/kg), was used as a control. Data are expressed as means±SD (n=4).

Chemicals

Analytical thin layer chromatography (TLC) was performed on glass plates of silica gel 60 GF₂₅₄ (Merck & Co., Inc., Kenilworth, New Jersey, USA), which were visualized by an aqueous alkaline KMnO₄ solution or an acid PdCl₂ solution followed by heating. Column chromatography was conducted on silica gel (Merck Kieselgel 70–230 mesh). Chemicals purchased from commercial sources were used without further purification. (Et₃NH)₂[B₁₂H₁₂] was purchased from Katchem Ltd. (Prague, Czech Republic). ¹H- and ¹³C-NMR spectra were recorded on a Bruker AVANCE-400 (400 MHz) and ¹¹B-NMR spectra were recorded on a ASCEND-500 (160 MHz). Synthetic procedures of compound 3 and MID were reported previously.

Synthesis of di-tetrabutylammonium 4-aminoethoxyethoxy-closo-dodecaborate 2

This compound was synthesized according to the literature procedure. Briefly, to 1,4-dioxane-closo-dodecaborate complex 1 (1 g, 2.12 mmol) in CH₃CN (8 mL) was added 25 % NH₃ aq. (4 mL NH₃, 105 mmol), and the mixture was stirred at 50°C for 11 h. The reaction mixture was cooled to room temperature and then concentrated under reduced pressure. The corresponding ring-opening product was obtained as a white solid. The resulting product was suspended in MeOH and 10 % (w/w) TBAOH in MeOH (6.73 mL, 2.54 mmol) was added. The mixture was stirred until the white solid was dissolved into the MeOH solution. After removing the solvent under reduced pressure, the residue was dissolved CH₂Cl₂ and washed with distillated water 3 times. The CH₂Cl₂ phase was dried over MgSO₄ anhydride and concentrated to give amine 2 as a white solid (1.53 g, 2.10 mmol, 99 %). Identification of 2 was accomplished in comparison with the reported ¹H NMR data: ¹H NMR (400 MHz; CD₃CN): δ 3.60 (t, J=5.2 Hz, 2H), 3.45 (m, 4H), 3.12 (t, J=5.2 Hz, 16H), 2.78 (t, J=8.4 Hz, 2H), 1.63 (tt, J=7.2, 8.4 Hz), 1.38 (sext, J=7.2 Hz, 16H), 0.99 (t, J=7.2 Hz, 24H); m.p. 134–135°C; Anal. Calcd for C₃₆H₉₃N₃O₂B₁₂(H₂O): C, 57.82; H, 12.90; N, 5.62. Found: C, 58.27; H, 13.36; N, 5.89.

Synthesis of di-tetramethylammmonium salt of isothiocyanate-conujugated dodecaborate (4)

To a solution of amine 2 (0.40 g, 0.55 mmol) in CH₂Cl₂ were added 1,1′-thiocarbonyldiimidazole (TCDI; 0.130 g, 0.730 mmol) and tetraethylamine (0.152 mL, 1.10 mmol) and the mixture was stirred at room temperature for overnight. The organic solvent was removed under reduced pressure and the residue was purified by column chromatography on silica gel (CH₂Cl₂:MeOH=15/1) to give the ditetrabutylammmonium salt of isothiocyanate-conujugated dodecaborate as a colorless oil. A MeOH solution of tetramethylammmonium chloride (TMACl; 0.227 g, 2.07 mmol) was added to a solution of the ditetrabutylammmonium salt of isothiocyanate-conujugated dodecaborate (0.312 g, 0.410 mmol) in MeOH dropwise and the mixture was stirred for 3 h at room temperature. The product 4 was obtained by filtration of white solid precipitated from the reaction mixture (0.152 g, 2 steps 63 %): m.p. >300°C; ¹H NMR (400 MHz; CD₃CN): δ 3.71–3.69 (m, 2 H), 3.66–3.64 (m, 2 H), 3.54 (m, 4 H), 3.10 (s, 24 H); ¹³C NMR (100 MHz; CD₃CN): δ 72.56, 68.53, 67.78, 55.35, 45.42; ¹¹B NMR (160 MHz; CD₃CN): δ 6.29, −16.56, −17.99, −22.96; Anal. Calcd for C₁₃H₄₃N₃O₂SB₁₂(H₂O): C, 34.45; H, 10.01; N, 9.27. Found: C, 34.23; H, 9.62; N, 9.57.

Synthesis of di-sodium salt of isothiocyanate-conujugated dodecaborate (ISD)

Compound 4 (0.522 g, 1.20 mmol) was dissolved in water (50 mL) and Amberlite Na⁺ form (20 g) was added. The mixture was stirred at ambient temperature for 12 h then the resin was removed by filtration. The filtrate was concentrated and freeze-dried to give ISD (0.329 g, 83 %) as a hygroscopic white solid: ¹H NMR (400 MHz; D₂O): δ 3.79 (m, 4 H), 3.70 (m, 4 H); ¹³C NMR (100 MHz; D₂O): δ 71.14, 68.67, 67.66, 45.00; ¹¹B NMR (160 MHz; CD₃CN): δ 5.61, −16.96, −17.61, −22.74.

FE-SEM analysis of MID-albumin conjugates

The morphology of MID-albumin conjugates was measured by FE-SEM (Hitachi SEM S-5500, Tokyo, Japan) at an accelerating voltage of 5.0 kV. Dried MID-albumin conjugates were placed on double-side carbon tape and then sputter-coated with gold-palladium in an argon atmosphere using a Hummer I sputter coater (Anatech Ltd. St. Alexandria, VA, USA).

Conjugation of closo-dodecaborates (MID, ISD, and 1,4-dioxane-closo-dodecaborate complex 1) to selum albumin and Western blot analysis

BSA (0.1 mM, Sigma-Aldrich, St. Louis, USA) was reacted with MID (1.0 mM), ISD (1.0 mM), or 1 (1.0 mM) to BSA in PBS (50 μL) at ambient temperature for 1 h. The mixture was subjected to SDS-polyacrylamide gel electrophoresis (PAGE), transferred to polyvinyliden difluoride (PVDF) membrane (GE Healthcare, Buckinghamshire, UK), and immunoblotted with anti-B₁₂H₁₁SH (BSH) antibody. After further incubation with horseradish peroxidase (HRP)-conjugated secondary antibody, the membrane was treated with ECL kit (GE Healthcare, Buckinghamshire, UK) and closo-dodecaborate-conjugated BSAs were visualized with a Molecular Imager ChemiDoc XRS System (Bio-Rad, Hercules, CA, USA). Total proteins (closo-dodecaborate-conjugated and unreacted BSA) were visualized by coomassie brilliant blue (CBB) staining. Conjugation of MID to HSA (Sigma-Aldrich, St. Louis, USA) was also carried out in a similar to conjugation to BSA.

Biodistribution of HSA-Na₂B₁₂H₁₂, HSA-Na₂BSH, HSA-NaB₁₂H₁₁NH₃, and HSA-MID in the colon 26 cancer-bearing mice

Each HSA-Boron cluster conjugates (MID:HSA=10:1, 3000 ppm B) was prepared from HSA (245.6 mg) and Na₂B₁₂H₁₂ (6.95 mg), Na₂BSH (8.40 mg), Na₂BNH (6.69 mg), MID (16.8 mg) in PBS (1.6 mL) at room temperature for 1 h respectably.

Tumor-bearing mice (Balb/c female, 5–6 weeks old, 16–20 g, Sankyo Labo Service Co., Japan) were prepared by injecting subcutaneously (s.c.) a suspension (1.0×10⁶ cells/mouse) of colon 26 cells directly into the right thigh. The mice were kept on a regular chow diet and water and maintained under 12 h light/dark cycle in an ambient atmosphere. When the tumor diameter was 7–9 mm, the mice were injected via the tail vein with a 200 μL solution of HSA-Na₂B₁₂H₁₂, HSA-Na₂BSH, HSA-NaB₁₂H₁₁NH₃, HSA-MID (HSA: closo-dodecaborate derivatives=1:10 mixture; 30 mg[B]/kg) or Na₂B₁₂H₁₂ (30 mg[B]/kg). The mice were lightly anesthetized and blood samples were collected from the retro-orbital sinus at 12 h after administration. The mice were then sacrificed by cervical dislocation and dissected. Liver, kidney, spleen, brain, and tumor were excised, washed with 0.9 % NaCl solution, and weighed. The excised organs were digested with 2 mL of conc. HNO₃ (ultratrace analysis grade, Wako, Japan) at 90°C for 1–3 h, and then the digested samples were diluted with distilled water. After filtering through a hydrophobic filter, boron concentration was measured by ICP-OES (Thermo Fisher Scientific Inc. Waltham, MA, USA). All protocols were approved by the Institutional Animal Care and Use Committee of Tokyo Institute of Technology.