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Drug delivery system design and development for boron neutron capture therapy on cancer treatment
Applied Radiation and Isotopes ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Contents lists available at ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Drug delivery system design and development for boron neutron capture therapy on cancer treatment Lin-Chiang Sherlock Huang a,d, Wen-Yuan Hsieh b, Jiun-Yu Chen c, Su-Chin Huang c, Jen-Kun Chen c, Ming-Hua Hsu d,n a
Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC Material and Chemical Research Laboratories, Industrial Technology Research Institute, Hsinchu 31040, Taiwan, ROC c Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli 35053, Taiwan, ROC d Nuclear Science & Technology Development Center, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC b
H I G H L I G H T S
Herein, we have synthesized boron-modified diblock copolymer. Bpin-PLA-PEOz, which will be served as new boron containing vehicle for transporting the boron drug. This boron containing Bpin-PLA-PEOz micelle was low toxicity can be applied to drug delivery.
art ic l e i nf o
a b s t r a c t
Article history: Received 24 December 2012 Received in revised form 20 December 2013 Accepted 21 December 2013
We have already synthesized a boron-containing polymeric micellar drug delivery system for boron neutron capture therapy (BNCT). The synthesized diblock copolymer, boron-terminated copolymers (Bpin-PLA-PEOz), consisted of biodegradable poly(D,L-lactide) (PLA) block and water-soluble polyelectrolyte poly(2-ethyl-2-oxazoline) (PEOz) block, and a cap of pinacol boronate ester (Bpin). In this study, we have demonstrated that synthesized Bpin-PLA-PEOz micelle has great potential to be boron drug delivery system with preliminary evaluation of biocompatibility and boron content. & 2013 Elsevier Ltd. All rights reserved.
Keywords: BNCT PLA PEOz Drug delivery Micelle
1. Introduction Cancer is currently the most dangerous threat on human being, and causes one of the biggest casualties among people of all races across the world. Countless resources and efforts have been devoted into this World War against cancer, in order to annihilate this tough enemy. In 1936, Locher proposed the concept of boron neutron capture therapy (BNCT) (Locher, 1936) just after Chadwick found the neutron in 1932 (Chadwick, 1932): a binary therapeutic strategy that deploys the selective delivery of non-radioactive boron-10 to cancer cells and irradiation with low energy (0.0025 eV) thermal neutrons. Combining this two elements in the same time will cause a nuclear capture and fission reactions,
n
Corresponding author. Tel.: +886 3 573 1180; fax: +886 3 572 5974. E-mail addresses: [email protected], [email protected] (M.-H. Hsu).
10 B(n,α)7Li, which produce α particles (4He) and recoiling lithium-7 (7Li) with high linear energy transfer (LET) properties, and precisely cause cytotoxic effect to boron containing cells with single cell range (5–9 μm). Therefore, the key to make BNCT more reliable cancer therapy is the effective delivery and accumulation of boron compounds to the cancer cells. The functionally important requirements of boron deliver agent for BNCT are as follows: (1) low toxicity; (2) high tumor tissue uptake with a tumor:normal tissue and tumor/blood boron concentration ratios of 3; (3) sufficient boron deposit in tumor tissue with 20 μg 10B/g tumor; and (4) rapid clearance from blood circulation and normal tissues but persistence in tumor (Barth et al., 2012). From 1950s to present, lots of boron drugs for clinical trials have been developed, form boronic acid derivatives, to sodium mercaptoundecahydro-closododecaborate (BSH) and boronophenylalanine (BPA); still, no clinical boron reagents could, specifically and efficiently, deliver boron to tumor sites. New forms of potential boron deliver systems are urgently needed to be invented for therapeutic investigation.
0969-8043/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.apradiso.2013.12.025
Please cite this article as: Sherlock Huang, L.-C., et al., Drug delivery system design and development for boron neutron capture therapy on cancer treatment. Appl. Radiat. Isotopes (2014), http://dx.doi.org/10.1016/j.apradiso.2013.12.025i
2
L.-C. Sherlock Huang et al. / Applied Radiation and Isotopes ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Recently, nanotechnology based drug delivery systems, such as liposomes (Ueno et al., 2010), and polymeric micelles (Sumitani et al., 2011) have emerged as attractive boron carriers for BNCT. Thanks to the passive tumor-targeting property of nanoparticles, the nano-scale particles can be accumulated in tumors through the enhanced permeability and retention (EPR) effect, which is caused by leaky neovascular walls and uneffective lymphatic drainage (Iyer et al., 2006). To our interest, micelles are one of the beneficial drug carriers for the delivery of hydrophobic drugs or highly cytotoxic chemotherapeutics; the modified functional polymer micelles have some impressive features such as targeting delivery, elevating drug efficiency, minimizing therapeutics cytotoxicity, and lower cost comparing to other drug carriers. Furthermore, the nanoscopic polymeric micelles can escape the surveillance of mononuclear phagocyte system (MPS) and reticular epithelial system (RES), leading to prolonged blood circulation time (Oku et al., 1992; Kwon and Okano, 1996). In this research, the poly(2-ethyl-2-oxazoline)-poly(D,L-lactide) (PEOz-PLA) diblock copolymer was served as core structure of micelle, which was shown as potential drug carrier with pH sensitivity, low cytotoxicity, low cytotoxicity, and a pKa value near neutral pH that can facilitate drug release in an acid environment (Hsiue et al., 2006; Wang et al., 2005; (Shieh et al., 2010). This diblock copolymer consists of two primary components, one is biodegradable polylactide (PLA), and the other is water-soluble polyelectrolyte polyoxazoline (PEOz); both are FDA approved biomaterials for application in clinical trial. In this study, we synthesized boron-terminated PLA-PEOz diblock copolymer (Bpin-PLA-PEOz) by ring-opening polymerization with a pinacol boronate ester-containing (Bpin) as initiator of reaction. Procedure of Bpin-PLA-PEOz was modified form synthesis of diblock copolymer, PLA-PEOz (Syu et al., 2012). After micellelation, the boron moiety of copolymers was embedded into the core of the micelle, and the hydrophilic PEOz section of copolymers was exposed to the water solution. The micellar structure could also encapsulate hydrophobic boron compound to increase boron content; in this study, we selected and synthesized poor water soluble phenylboric acid derivative (PBAD), which was already evaluated with lipiodol entrapping (Liao et al., 2010).
2. Materials and methods 2.1. Materials Acetone and toluene were purchased from Merck GmbH; Dichloromethane, ethyl acetate, hexane, and tetrahydrofuran were purchased from Mallinckrodt Chemical Co. Acetonitrile, anhydrous ether, and methanol were bought from J.T. Baker Reagent Chemicals. Acetontrile, and dichloromethane were dried and distilled from CaH2. Tetrahydrofuran was dried by distillation from sodium and benzophenone under an atmosphere of nitrogen. Benzyl alcohol, pinacol, D,L-lactide, triethylamine, mesyl chloride, potassium carbonate (K2CO3), and 2-ethyl-2-oxazoline were purchased from Alfar Aesar Company. 4-Hydroxylphenylboronic acid was bought from luminescence technology corporation, Taiwan. Tin (II) octanoate [Sn(Oct)2] was acquired from Sigma-Aldrich. Potassium hydroxide and magnesium sulfate (MgSO4) was gained from Showa, Japan. The ultrapure water employed in all experiments was obtained from a Millipore-Milli-Q system. Membrane for dialysis (MW 3000) was acquired from Spectrum Inc. 2.2. Synthesis and characterization of Bpin-PLA-PEOz diblock copolymer For Bpin-PLA-PEOz diblock copolymer synthesis (Fig. 1), first, pinacol (2.33 g, 19.70 mmol, 1.2 equiv) and 4-hydroxylphenylboronic acid (2.50 g, 16.45 mmol, 1.0 equiv) were dissolved in THF (50 mL) and stirred until homogeneous. MgSO4 (1.00 g) was added and the reaction was heated at 50 1C overnight. The reaction was allowed to cool to room temperature and the MgSO4 was removed with a syringe filter. The filtrate was vaporized to dryness and the crude material was purified using a solvent gradient of 7–25% ethyl acetate in hexane with Teledyne Isco CombiFlashs Rf flash chromatography system. The desired product, pinacol boronate ester (3.9 g, 98%), was isolated as a transparent viscous liquid. D-L Lactide (30.0 g, 208 mmol) was added to a two-necked round-bottle flask with a condenser, and was treated with three times nitrogen replacement. After toluene (94 mL) as solvent was injected with syringe, the ambient temperature was elevated to 140 73 1C, and then pinacol boronate ester (2.47 g, 11.97 mmol)
Fig. 1. Synthsis of Bpin-PLA-PEOz.
Please cite this article as: Sherlock Huang, L.-C., et al., Drug delivery system design and development for boron neutron capture therapy on cancer treatment. Appl. Radiat. Isotopes (2014), http://dx.doi.org/10.1016/j.apradiso.2013.12.025i
L.-C. Sherlock Huang et al. / Applied Radiation and Isotopes ∎ (∎∎∎∎) ∎∎∎–∎∎∎
was added. The reaction was held in 30 min after the addition of toluene-solvated (3 mL toluene) stannous octoate (0.31 g, 0.765 mmol). The mixture was cool down to the room temperature, and concentrated to the volume of 50 mL. Mixed solvent of n-hexane/ether (2/1, v/v, 900 mL) was slowly poured into the condensed mixture with stirring for 10 min in the ambient temperature of 4 1C, and after the whole system was stood for 10 min, the supernatant was removed and refilled with the mixed n-hexane/ether solvent, and the purified procedure was repeat again. The precipitate, Bpin-PLA-OH was dried with vacuum for 24 h. Bpin-PLA-OH (30.0 g) was placed in the two-necked roundbottle flask, and kept the flask under vacuum with nitrogen replacement. Dry THF (250 mL) was added by syringe, and then triethylamine (7.21 mL) was added by the same way. Mesyl chloride (3.89 mL, 50.16 mmol) presolved in THF (50 mL) injected dropwise into the flask, the reaction was held in 1 h at room temperature. The white crude product was filtrated through the celite column with 100 mL THF as eluent. The solvent was removed by reduced pressure, the residue was dissolved by 100 mL methanol at 50 1C, cooled down to 90 1C by liquid nitrogen, and placed at the room temperature for 25 min. After methanol was removed, the product was once again dissolved by 100 mL methanol at 50 1C, and held with the same procedures as mentioned. The product, Bpin-PLA-mesylate, was vacuumed for 24 h. Bpin-PLA-mesylate (3.00 g) was added to a two-necked roundbottle flask with a condenser. The vacuum line was connected to the flask, and three times nitrogen replacement were carried out. Dry aceotonitrile (27 mL, fresh distilled), as the solvent, was injected with syringe, and the ambient temperature was elevated to 100 7 2 1C. Dry 2-ethyl-2-oxazoline (6.11 mL, 60.47 mmol, fresh distilled) was injected into the flask to begin the 17 h reaction. While the reaction was finished, the ambient temperature was cooled down to room temperature. The 0.1 N potassium hydroxide (KOH) in methanol was injected into the flask with stirring for 2 h, and potassium carbonate (K2CO3) was added into the flask with stirring for overnight. The crude product was filtrated through the flash column filled with celite 525 to eradicate the salt formed during the reaction with the elution solvent THF (50 mL). The solvent was remove by reduce pressure, 20 mL DMSO was added to dissolve the product, and the solution was dialyzed with MWCO 3500 dialysis membrane in ultrapure water for 2 days. The dialyzed solution was then lyophilized 2 days to get the dry diblock copolymer, Bpin-PLA-PEOz. The composition of the copolymer was analyzed by 1H NMR (Bruker Avance-500 MHz FT-NMR) in CDCl3. The critical micelle concentration (CMC) of copolymer was determined by a fluorescence technique with pyrene as a hydrophobic probe. 2.3. Synthesis of PBAD For synthesis of PBAD, boron compound for micelle encapsulation (Fig. 2), a suspension of the phenyl boronic acid in toluene (50 mL) was stirred at reflux for 6–18 h with a Dean-Stark trap. After reaction finished, toluene was removed, and the crude
3
material was purified using a solvent gradient of 5–20% ethyl acetate in hexane with Teledyne Isco CombiFlashs Rf flash chromatography system. The boroxines, PBAD, were obtained in quantitative yield (5–15% boronic acid remaining). Mass of product was confirmed with gas chromatography and low resolution mass spectral analysis system (Agilent Technology 6890 N Network GC System equipped with an Agilent 5973 Network Mass Selective Detector and capillary HP–1 column). 2.4. Preparation of PBAD loaded micelles Bpin-PEOz-b-PLA and PBAD were dissolved in a minimum volume of THF and added dropwise to vigorously stirred deionized water (10 mL). The organic solvent was completely evaporated with a rotorvapor. After the organic solvent was removed, the micelle solution was sonicated for 1 h using a sonicator at 25 1C. The resultant solution was filtered through a 0.45 μm filter to remove unencapsulated PBAD aggregates. 2.5. Boron concentration measurement In this study, we measured B concentration in Bpin-PLA-PEOz micelles with and without PBAD encapsulation. Concentrated HNO3 (300 μL) was added to each sample in a sealed Teflon vessel prior to digestion in a microwave oven (CEM MARS Xpress). All digested solutions were diluted up to a 30 mL of volume. The boron concentrations were determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent Technologies, 7500cx, Tokyo, Japan). Calibration was performed using a boron standard solution (1000 ppm; High-Purity Standard from Uni-onward CORP., Charleston, SC). 2.6. Cell Lines and culture Conditions Human HeLa cells were grown and maintained in Eagle0 s minimum essential medium (EMEM) supplemented with 10% feta calf serum (FCS), 100 units/ml Penicillin and 100 units/ml Streptomycin under 5% CO2 atmosphere at 37 1C. Cells were maintained in 75 cm2 flasks by passage every 3–4 days. 2.7. Cell viability assay For cell viability evaluation, HeLa cells were seeded at a density of 3 103 cells with 200 μL of EMEM medium and 10% FBS in 96wells plates, and incubated for 24 h till the 70% confluence. The cells were respectively treated with Bpin, PBAD, Bpin-PLA-PEOz micelle and Bpin-PLA-PEOz/PBAD micelle, 100 μL of serum-free EMEM medium, containing various concentrations (0.2–4.0 μM). After 24 h co-incubation, the medium was replaced and the cells were cultured of vital dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), which were followed by incubation at 37 1C for 3 h. Subsequently, MTT solution was removed and 100 μL of DMSO was added to dissolve the blue formazan crystal produced by proliferating cells. The plate was incubated for an additional 10 min before determination at absorbance at 590 nm and was recorded using a microplate reader (VersaMax, Molecular Devices Inc.).
3. Results and discussions 3.1. Preparation and characterization of Bpin-PLA-PEOz diblock copolymer
Fig. 2. Synthesis of Phenyl Boronic Acid Derivatives (PBAD).
For developing potential water-soluble micellar boron delivery system, the Bpin-PLA-PEOz copolymer was synthesized and
Please cite this article as: Sherlock Huang, L.-C., et al., Drug delivery system design and development for boron neutron capture therapy on cancer treatment. Appl. Radiat. Isotopes (2014), http://dx.doi.org/10.1016/j.apradiso.2013.12.025i
L.-C. Sherlock Huang et al. / Applied Radiation and Isotopes ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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characterized with modification of previous published methods of PLA-PEOz diblock copolymer synthesis (Syu et al., 2012). In order to add the boron entry to the copolymer for boron-containing micelle production, 4-hydroxymethylphenylboronic acid was chosen for the alcohol initiator of the following PLA synthesis. Since the boronic acid has two hydroxyl groups attached to the boron, which might interfere the following ring-opening polymerization, the borate ester (Bpin) used as initiator was obtained by condensation with pinacol and boronic acid. First, the PLA polymers were synthesized by cationic ring-opening polymerization with Bpin intiator, D,L-lactide and stannous octanoate as a catalyst. Subsequently, the diblock copolymers consisting of the biodegradable PLA block and the water-soluble PEOz block were synthesized by ring-opening polymerization from mesylated PLA block and 2ethyl-2-oxazoline (Fig. 1). The chemical structure and molecular weight of each copolymer were verified by 1H NMR (Avance 400, Bruker) and GPC using THF as an elution solvent. Characterizations of a series of Bpin-PLA-PEOz diblock copolymers were summarized in Table 1. The composition and molecular weight of BpinPLA-PEOz were determined by comparing the integral peak area associated with the Methylene bridge of PEOz (3.42 ppm) and methanetriyl groups of PLA (5.15 ppm). The number average molecular weight (Mn) of Bpin-PLA-PEOz as determined by 1H NMR was 14247, and the polydispersity index was 1.24 as determined by GPC (Table 1). The CMC of the Bpin-PLA-PEOz was measured by a fluorescence spectrophotometer (Thermo NanoDrop 2000 UV–vis Spectrophotometer) with pyrene as a hydrophobic probe, and shown in Table 1.
3.2. Boron contents of Bpin-PLA-PEOz Micelles Boron contents obtained from synthesized Bpin and PBAD, were 51.38 mg/g and 24.77 mg/g, respectively. In this study, water suspensions of Bpin-PLA-PEOz micelle and PBAD loaded Bpin-PLAPEOz micelle (Bpin-PLA-PEOz/PBAD) showed 6.06 70.3, and 15.7 70.6 μg B/ml, corresponding to boron/vehicle ratios for 0.06 and 0.15, respectively. Bpin-PLA-PEOz diblock copolymer itself has boron component, additional boron compound, PBAD, could be capsulated by Bpin-PLA-PEOz formed micelle, and increased the boron content about two fold.
3.3. Cytotoxicity Formulating drugs into biocompatible carriers could be a good chance to reduce cytotoxicity and/or improve water solubility/ suspension of BNCT drug candidates. The HeLa cells were incubated with various concentrations of Bpin, PBAD, Bpin-PLA-PEOz and Bpin-PLA-PEOz/PBAD for 48 h to investigate cell viability by MTT assay (Fig. 3). The analogous experiments with Bpin, BpinPLA-PEOz and Bpin-PLA-PEOz/PBAD indicated very high biocompatibility at boron concentration over the range of 0.2–4.0 μM. The cell viability of PBAD alone (92.36%) demonstrated significantly lower than Bpin-PLA-PEOz/PBAD (104.13%), which demonstrated
Table 1 Characterizations of synthesized polymers. Polymer
Mna
Polydispersity index (PDI)b
Yield (%)
CMC (wt%)
Bpin-PLA Bpin-PLA-PEOz
7218 14247
1.21 1.24
98 62
7 10 4
a b
Estimated by 1H NMR. Estimated by GPC.
Fig. 3. Cell viability after 48 h of incubation with the Bpin, PBAD, Bpin-PLA-PEOz and Bpin-PLA-PEOz/PBAD; error bars are mean 7 SD (n ¼6). *Significantly different between PBAD and Bpin-PLA-PEOz/PBAD at the indicated concentration (p o 0.05).
the Bpin-PLA-PEOz/PBAD formulation not only maintain excellent suspension but also reduce cytotoxicity of PBAD.
4. Conclusions In conclusion, we have synthesized boron-modified diblock copolymer, Bpin-PLA-PEOz, which will be served as new boron containing vehicle for transporting the boron drug. The production of Bpin-PLA-PEOz was able to comply with the similar procedure of the PLA-PEOz formation, i.e., the living polymerization by sequential addition of monomers and the synthesis of functionalended polymers by selective termination of living ends with appropriate reagents. Also, the preliminary biocompatibility evaluation with MTT assay showed that boron containing Bpin-PLA-PEOz micelle has low toxicity compared to the phenyl boronic acid derivatives used in this study, indicating Bpin-PLA-PEOz could be potential drug delivery platform.
Acknowledgment For financial support, we thank the National Science Council (NSC 100–2113-M-007–015-MY2 and 102–2113-M-007–010) and National Tsing Hua University. For the cell viability data, authors are grateful of financial support from National Health Research Institutes (NM-100-PP-07 for JKC) and National Science Council (101-2113-M-400-001-MY3 for JKC). References Barth, R.F., Vicente, M.G., et al., 2012. Current status of boron neutron capture therapy of high grade gliomas and recurrent head and neck cancer. Radiat. Oncol. 7, 146. Chadwick, J., 1932. The existence of a neutron. Proceedings of the Royal Society of London Series A – Containing Papers of a Mathematical and Physical Character 136 (830), 692–708. Hsiue, G.H., Wang, C.H., et al., 2006. Environmental-sensitive micelles based on poly (2-ethyl-2-oxazoline)-b-poly(L-lactide) diblock copolymer for application in drug delivery. Int. J. Pharm. 317 (1), 69–75. Iyer, A.K., Khaled, G., et al., 2006. Exploiting the enhanced permeability and retention effect for tumor targeting. Drug Discov. Today 11 (17–18), 812–818. Kwon, G.S., Okano, T., 1996. Polymeric micelles as new drug carriers. Adv. Drug Deliv. Rev. 21 (2), 107–116. Liao, A.H., Chou, F.I., et al., 2010. Biodistribution of phenylboric acid derivative entrapped lipiodol and 4-borono-2-18F-fluoro-L-phenylalanine-fructose in GP7TB liver tumor bearing rats for BNCT. Appl. Radiat. Isot. 68 (3), 422–426. Locher, G.L., 1936. Biological effects and therapeutic possibilities of neutrons. Am. J. Roentgenol. Radium Ther. 36, 1–13. Oku, N., Namba, Y., et al., 1992. Tumor accumulation of novel res-avoiding liposomes. Biochim. Et Biophys. Acta 1126 (3), 255–260. Shieh, M.J., Peng, C.L., et al., 2010. Reduced skin photosensitivity with meta-tetra (hydroxyphenyl)chlorin-loaded micelles based on a poly(2-ethyl-2-oxazoline)b-poly(D,L-lactide) diblock copolymer in vivo. Mol. Pharm. 7 (4), 1244–1253.
Please cite this article as: Sherlock Huang, L.-C., et al., Drug delivery system design and development for boron neutron capture therapy on cancer treatment. Appl. Radiat. Isotopes (2014), http://dx.doi.org/10.1016/j.apradiso.2013.12.025i
L.-C. Sherlock Huang et al. / Applied Radiation and Isotopes ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Sumitani, S., Oishi, M., et al., 2011. Carborane confined nanoparticles for boron neutron capture therapy: improved stability, blood circulation time and tumor accumulation. React. Funct. Polym. 71 (7), 684–693. Syu, W.J., Yu, H.P., et al., 2012. Improved photodynamic cancer treatment by folateconjugated polymeric micelles in a KB xenografted animal model. Small 8 (13), 2060–2069.
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Ueno, M., Ban, H.S., et al., 2010. Dodecaborate lipid liposomes as new vehicles for boron delivery system of neutron capture therapy. Bioorganic Med. Chem. 18 (9), 3059–3065. Wang, C.H., Fan, K.R., et al., 2005. Enzymatic degradation of PLLA-PEOz-PLLA triblock copolymers. Biomaterials 26 (16), 2803–2811.
Please cite this article as: Sherlock Huang, L.-C., et al., Drug delivery system design and development for boron neutron capture therapy on cancer treatment. Appl. Radiat. Isotopes (2014), http://dx.doi.org/10.1016/j.apradiso.2013.12.025i
Contents lists available at ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Drug delivery system design and development for boron neutron capture therapy on cancer treatment Lin-Chiang Sherlock Huang a,d, Wen-Yuan Hsieh b, Jiun-Yu Chen c, Su-Chin Huang c, Jen-Kun Chen c, Ming-Hua Hsu d,n a
Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC Material and Chemical Research Laboratories, Industrial Technology Research Institute, Hsinchu 31040, Taiwan, ROC c Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli 35053, Taiwan, ROC d Nuclear Science & Technology Development Center, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC b
H I G H L I G H T S
Herein, we have synthesized boron-modified diblock copolymer. Bpin-PLA-PEOz, which will be served as new boron containing vehicle for transporting the boron drug. This boron containing Bpin-PLA-PEOz micelle was low toxicity can be applied to drug delivery.
art ic l e i nf o
a b s t r a c t
Article history: Received 24 December 2012 Received in revised form 20 December 2013 Accepted 21 December 2013
We have already synthesized a boron-containing polymeric micellar drug delivery system for boron neutron capture therapy (BNCT). The synthesized diblock copolymer, boron-terminated copolymers (Bpin-PLA-PEOz), consisted of biodegradable poly(D,L-lactide) (PLA) block and water-soluble polyelectrolyte poly(2-ethyl-2-oxazoline) (PEOz) block, and a cap of pinacol boronate ester (Bpin). In this study, we have demonstrated that synthesized Bpin-PLA-PEOz micelle has great potential to be boron drug delivery system with preliminary evaluation of biocompatibility and boron content. & 2013 Elsevier Ltd. All rights reserved.
Keywords: BNCT PLA PEOz Drug delivery Micelle
1. Introduction Cancer is currently the most dangerous threat on human being, and causes one of the biggest casualties among people of all races across the world. Countless resources and efforts have been devoted into this World War against cancer, in order to annihilate this tough enemy. In 1936, Locher proposed the concept of boron neutron capture therapy (BNCT) (Locher, 1936) just after Chadwick found the neutron in 1932 (Chadwick, 1932): a binary therapeutic strategy that deploys the selective delivery of non-radioactive boron-10 to cancer cells and irradiation with low energy (0.0025 eV) thermal neutrons. Combining this two elements in the same time will cause a nuclear capture and fission reactions,
n
Corresponding author. Tel.: +886 3 573 1180; fax: +886 3 572 5974. E-mail addresses: [email protected], [email protected] (M.-H. Hsu).
10 B(n,α)7Li, which produce α particles (4He) and recoiling lithium-7 (7Li) with high linear energy transfer (LET) properties, and precisely cause cytotoxic effect to boron containing cells with single cell range (5–9 μm). Therefore, the key to make BNCT more reliable cancer therapy is the effective delivery and accumulation of boron compounds to the cancer cells. The functionally important requirements of boron deliver agent for BNCT are as follows: (1) low toxicity; (2) high tumor tissue uptake with a tumor:normal tissue and tumor/blood boron concentration ratios of 3; (3) sufficient boron deposit in tumor tissue with 20 μg 10B/g tumor; and (4) rapid clearance from blood circulation and normal tissues but persistence in tumor (Barth et al., 2012). From 1950s to present, lots of boron drugs for clinical trials have been developed, form boronic acid derivatives, to sodium mercaptoundecahydro-closododecaborate (BSH) and boronophenylalanine (BPA); still, no clinical boron reagents could, specifically and efficiently, deliver boron to tumor sites. New forms of potential boron deliver systems are urgently needed to be invented for therapeutic investigation.
0969-8043/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.apradiso.2013.12.025
Please cite this article as: Sherlock Huang, L.-C., et al., Drug delivery system design and development for boron neutron capture therapy on cancer treatment. Appl. Radiat. Isotopes (2014), http://dx.doi.org/10.1016/j.apradiso.2013.12.025i
2
L.-C. Sherlock Huang et al. / Applied Radiation and Isotopes ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Recently, nanotechnology based drug delivery systems, such as liposomes (Ueno et al., 2010), and polymeric micelles (Sumitani et al., 2011) have emerged as attractive boron carriers for BNCT. Thanks to the passive tumor-targeting property of nanoparticles, the nano-scale particles can be accumulated in tumors through the enhanced permeability and retention (EPR) effect, which is caused by leaky neovascular walls and uneffective lymphatic drainage (Iyer et al., 2006). To our interest, micelles are one of the beneficial drug carriers for the delivery of hydrophobic drugs or highly cytotoxic chemotherapeutics; the modified functional polymer micelles have some impressive features such as targeting delivery, elevating drug efficiency, minimizing therapeutics cytotoxicity, and lower cost comparing to other drug carriers. Furthermore, the nanoscopic polymeric micelles can escape the surveillance of mononuclear phagocyte system (MPS) and reticular epithelial system (RES), leading to prolonged blood circulation time (Oku et al., 1992; Kwon and Okano, 1996). In this research, the poly(2-ethyl-2-oxazoline)-poly(D,L-lactide) (PEOz-PLA) diblock copolymer was served as core structure of micelle, which was shown as potential drug carrier with pH sensitivity, low cytotoxicity, low cytotoxicity, and a pKa value near neutral pH that can facilitate drug release in an acid environment (Hsiue et al., 2006; Wang et al., 2005; (Shieh et al., 2010). This diblock copolymer consists of two primary components, one is biodegradable polylactide (PLA), and the other is water-soluble polyelectrolyte polyoxazoline (PEOz); both are FDA approved biomaterials for application in clinical trial. In this study, we synthesized boron-terminated PLA-PEOz diblock copolymer (Bpin-PLA-PEOz) by ring-opening polymerization with a pinacol boronate ester-containing (Bpin) as initiator of reaction. Procedure of Bpin-PLA-PEOz was modified form synthesis of diblock copolymer, PLA-PEOz (Syu et al., 2012). After micellelation, the boron moiety of copolymers was embedded into the core of the micelle, and the hydrophilic PEOz section of copolymers was exposed to the water solution. The micellar structure could also encapsulate hydrophobic boron compound to increase boron content; in this study, we selected and synthesized poor water soluble phenylboric acid derivative (PBAD), which was already evaluated with lipiodol entrapping (Liao et al., 2010).
2. Materials and methods 2.1. Materials Acetone and toluene were purchased from Merck GmbH; Dichloromethane, ethyl acetate, hexane, and tetrahydrofuran were purchased from Mallinckrodt Chemical Co. Acetonitrile, anhydrous ether, and methanol were bought from J.T. Baker Reagent Chemicals. Acetontrile, and dichloromethane were dried and distilled from CaH2. Tetrahydrofuran was dried by distillation from sodium and benzophenone under an atmosphere of nitrogen. Benzyl alcohol, pinacol, D,L-lactide, triethylamine, mesyl chloride, potassium carbonate (K2CO3), and 2-ethyl-2-oxazoline were purchased from Alfar Aesar Company. 4-Hydroxylphenylboronic acid was bought from luminescence technology corporation, Taiwan. Tin (II) octanoate [Sn(Oct)2] was acquired from Sigma-Aldrich. Potassium hydroxide and magnesium sulfate (MgSO4) was gained from Showa, Japan. The ultrapure water employed in all experiments was obtained from a Millipore-Milli-Q system. Membrane for dialysis (MW 3000) was acquired from Spectrum Inc. 2.2. Synthesis and characterization of Bpin-PLA-PEOz diblock copolymer For Bpin-PLA-PEOz diblock copolymer synthesis (Fig. 1), first, pinacol (2.33 g, 19.70 mmol, 1.2 equiv) and 4-hydroxylphenylboronic acid (2.50 g, 16.45 mmol, 1.0 equiv) were dissolved in THF (50 mL) and stirred until homogeneous. MgSO4 (1.00 g) was added and the reaction was heated at 50 1C overnight. The reaction was allowed to cool to room temperature and the MgSO4 was removed with a syringe filter. The filtrate was vaporized to dryness and the crude material was purified using a solvent gradient of 7–25% ethyl acetate in hexane with Teledyne Isco CombiFlashs Rf flash chromatography system. The desired product, pinacol boronate ester (3.9 g, 98%), was isolated as a transparent viscous liquid. D-L Lactide (30.0 g, 208 mmol) was added to a two-necked round-bottle flask with a condenser, and was treated with three times nitrogen replacement. After toluene (94 mL) as solvent was injected with syringe, the ambient temperature was elevated to 140 73 1C, and then pinacol boronate ester (2.47 g, 11.97 mmol)
Fig. 1. Synthsis of Bpin-PLA-PEOz.
Please cite this article as: Sherlock Huang, L.-C., et al., Drug delivery system design and development for boron neutron capture therapy on cancer treatment. Appl. Radiat. Isotopes (2014), http://dx.doi.org/10.1016/j.apradiso.2013.12.025i
L.-C. Sherlock Huang et al. / Applied Radiation and Isotopes ∎ (∎∎∎∎) ∎∎∎–∎∎∎
was added. The reaction was held in 30 min after the addition of toluene-solvated (3 mL toluene) stannous octoate (0.31 g, 0.765 mmol). The mixture was cool down to the room temperature, and concentrated to the volume of 50 mL. Mixed solvent of n-hexane/ether (2/1, v/v, 900 mL) was slowly poured into the condensed mixture with stirring for 10 min in the ambient temperature of 4 1C, and after the whole system was stood for 10 min, the supernatant was removed and refilled with the mixed n-hexane/ether solvent, and the purified procedure was repeat again. The precipitate, Bpin-PLA-OH was dried with vacuum for 24 h. Bpin-PLA-OH (30.0 g) was placed in the two-necked roundbottle flask, and kept the flask under vacuum with nitrogen replacement. Dry THF (250 mL) was added by syringe, and then triethylamine (7.21 mL) was added by the same way. Mesyl chloride (3.89 mL, 50.16 mmol) presolved in THF (50 mL) injected dropwise into the flask, the reaction was held in 1 h at room temperature. The white crude product was filtrated through the celite column with 100 mL THF as eluent. The solvent was removed by reduced pressure, the residue was dissolved by 100 mL methanol at 50 1C, cooled down to 90 1C by liquid nitrogen, and placed at the room temperature for 25 min. After methanol was removed, the product was once again dissolved by 100 mL methanol at 50 1C, and held with the same procedures as mentioned. The product, Bpin-PLA-mesylate, was vacuumed for 24 h. Bpin-PLA-mesylate (3.00 g) was added to a two-necked roundbottle flask with a condenser. The vacuum line was connected to the flask, and three times nitrogen replacement were carried out. Dry aceotonitrile (27 mL, fresh distilled), as the solvent, was injected with syringe, and the ambient temperature was elevated to 100 7 2 1C. Dry 2-ethyl-2-oxazoline (6.11 mL, 60.47 mmol, fresh distilled) was injected into the flask to begin the 17 h reaction. While the reaction was finished, the ambient temperature was cooled down to room temperature. The 0.1 N potassium hydroxide (KOH) in methanol was injected into the flask with stirring for 2 h, and potassium carbonate (K2CO3) was added into the flask with stirring for overnight. The crude product was filtrated through the flash column filled with celite 525 to eradicate the salt formed during the reaction with the elution solvent THF (50 mL). The solvent was remove by reduce pressure, 20 mL DMSO was added to dissolve the product, and the solution was dialyzed with MWCO 3500 dialysis membrane in ultrapure water for 2 days. The dialyzed solution was then lyophilized 2 days to get the dry diblock copolymer, Bpin-PLA-PEOz. The composition of the copolymer was analyzed by 1H NMR (Bruker Avance-500 MHz FT-NMR) in CDCl3. The critical micelle concentration (CMC) of copolymer was determined by a fluorescence technique with pyrene as a hydrophobic probe. 2.3. Synthesis of PBAD For synthesis of PBAD, boron compound for micelle encapsulation (Fig. 2), a suspension of the phenyl boronic acid in toluene (50 mL) was stirred at reflux for 6–18 h with a Dean-Stark trap. After reaction finished, toluene was removed, and the crude
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material was purified using a solvent gradient of 5–20% ethyl acetate in hexane with Teledyne Isco CombiFlashs Rf flash chromatography system. The boroxines, PBAD, were obtained in quantitative yield (5–15% boronic acid remaining). Mass of product was confirmed with gas chromatography and low resolution mass spectral analysis system (Agilent Technology 6890 N Network GC System equipped with an Agilent 5973 Network Mass Selective Detector and capillary HP–1 column). 2.4. Preparation of PBAD loaded micelles Bpin-PEOz-b-PLA and PBAD were dissolved in a minimum volume of THF and added dropwise to vigorously stirred deionized water (10 mL). The organic solvent was completely evaporated with a rotorvapor. After the organic solvent was removed, the micelle solution was sonicated for 1 h using a sonicator at 25 1C. The resultant solution was filtered through a 0.45 μm filter to remove unencapsulated PBAD aggregates. 2.5. Boron concentration measurement In this study, we measured B concentration in Bpin-PLA-PEOz micelles with and without PBAD encapsulation. Concentrated HNO3 (300 μL) was added to each sample in a sealed Teflon vessel prior to digestion in a microwave oven (CEM MARS Xpress). All digested solutions were diluted up to a 30 mL of volume. The boron concentrations were determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent Technologies, 7500cx, Tokyo, Japan). Calibration was performed using a boron standard solution (1000 ppm; High-Purity Standard from Uni-onward CORP., Charleston, SC). 2.6. Cell Lines and culture Conditions Human HeLa cells were grown and maintained in Eagle0 s minimum essential medium (EMEM) supplemented with 10% feta calf serum (FCS), 100 units/ml Penicillin and 100 units/ml Streptomycin under 5% CO2 atmosphere at 37 1C. Cells were maintained in 75 cm2 flasks by passage every 3–4 days. 2.7. Cell viability assay For cell viability evaluation, HeLa cells were seeded at a density of 3 103 cells with 200 μL of EMEM medium and 10% FBS in 96wells plates, and incubated for 24 h till the 70% confluence. The cells were respectively treated with Bpin, PBAD, Bpin-PLA-PEOz micelle and Bpin-PLA-PEOz/PBAD micelle, 100 μL of serum-free EMEM medium, containing various concentrations (0.2–4.0 μM). After 24 h co-incubation, the medium was replaced and the cells were cultured of vital dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), which were followed by incubation at 37 1C for 3 h. Subsequently, MTT solution was removed and 100 μL of DMSO was added to dissolve the blue formazan crystal produced by proliferating cells. The plate was incubated for an additional 10 min before determination at absorbance at 590 nm and was recorded using a microplate reader (VersaMax, Molecular Devices Inc.).
3. Results and discussions 3.1. Preparation and characterization of Bpin-PLA-PEOz diblock copolymer
Fig. 2. Synthesis of Phenyl Boronic Acid Derivatives (PBAD).
For developing potential water-soluble micellar boron delivery system, the Bpin-PLA-PEOz copolymer was synthesized and
Please cite this article as: Sherlock Huang, L.-C., et al., Drug delivery system design and development for boron neutron capture therapy on cancer treatment. Appl. Radiat. Isotopes (2014), http://dx.doi.org/10.1016/j.apradiso.2013.12.025i
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characterized with modification of previous published methods of PLA-PEOz diblock copolymer synthesis (Syu et al., 2012). In order to add the boron entry to the copolymer for boron-containing micelle production, 4-hydroxymethylphenylboronic acid was chosen for the alcohol initiator of the following PLA synthesis. Since the boronic acid has two hydroxyl groups attached to the boron, which might interfere the following ring-opening polymerization, the borate ester (Bpin) used as initiator was obtained by condensation with pinacol and boronic acid. First, the PLA polymers were synthesized by cationic ring-opening polymerization with Bpin intiator, D,L-lactide and stannous octanoate as a catalyst. Subsequently, the diblock copolymers consisting of the biodegradable PLA block and the water-soluble PEOz block were synthesized by ring-opening polymerization from mesylated PLA block and 2ethyl-2-oxazoline (Fig. 1). The chemical structure and molecular weight of each copolymer were verified by 1H NMR (Avance 400, Bruker) and GPC using THF as an elution solvent. Characterizations of a series of Bpin-PLA-PEOz diblock copolymers were summarized in Table 1. The composition and molecular weight of BpinPLA-PEOz were determined by comparing the integral peak area associated with the Methylene bridge of PEOz (3.42 ppm) and methanetriyl groups of PLA (5.15 ppm). The number average molecular weight (Mn) of Bpin-PLA-PEOz as determined by 1H NMR was 14247, and the polydispersity index was 1.24 as determined by GPC (Table 1). The CMC of the Bpin-PLA-PEOz was measured by a fluorescence spectrophotometer (Thermo NanoDrop 2000 UV–vis Spectrophotometer) with pyrene as a hydrophobic probe, and shown in Table 1.
3.2. Boron contents of Bpin-PLA-PEOz Micelles Boron contents obtained from synthesized Bpin and PBAD, were 51.38 mg/g and 24.77 mg/g, respectively. In this study, water suspensions of Bpin-PLA-PEOz micelle and PBAD loaded Bpin-PLAPEOz micelle (Bpin-PLA-PEOz/PBAD) showed 6.06 70.3, and 15.7 70.6 μg B/ml, corresponding to boron/vehicle ratios for 0.06 and 0.15, respectively. Bpin-PLA-PEOz diblock copolymer itself has boron component, additional boron compound, PBAD, could be capsulated by Bpin-PLA-PEOz formed micelle, and increased the boron content about two fold.
3.3. Cytotoxicity Formulating drugs into biocompatible carriers could be a good chance to reduce cytotoxicity and/or improve water solubility/ suspension of BNCT drug candidates. The HeLa cells were incubated with various concentrations of Bpin, PBAD, Bpin-PLA-PEOz and Bpin-PLA-PEOz/PBAD for 48 h to investigate cell viability by MTT assay (Fig. 3). The analogous experiments with Bpin, BpinPLA-PEOz and Bpin-PLA-PEOz/PBAD indicated very high biocompatibility at boron concentration over the range of 0.2–4.0 μM. The cell viability of PBAD alone (92.36%) demonstrated significantly lower than Bpin-PLA-PEOz/PBAD (104.13%), which demonstrated
Table 1 Characterizations of synthesized polymers. Polymer
Mna
Polydispersity index (PDI)b
Yield (%)
CMC (wt%)
Bpin-PLA Bpin-PLA-PEOz
7218 14247
1.21 1.24
98 62
7 10 4
a b
Estimated by 1H NMR. Estimated by GPC.
Fig. 3. Cell viability after 48 h of incubation with the Bpin, PBAD, Bpin-PLA-PEOz and Bpin-PLA-PEOz/PBAD; error bars are mean 7 SD (n ¼6). *Significantly different between PBAD and Bpin-PLA-PEOz/PBAD at the indicated concentration (p o 0.05).
the Bpin-PLA-PEOz/PBAD formulation not only maintain excellent suspension but also reduce cytotoxicity of PBAD.
4. Conclusions In conclusion, we have synthesized boron-modified diblock copolymer, Bpin-PLA-PEOz, which will be served as new boron containing vehicle for transporting the boron drug. The production of Bpin-PLA-PEOz was able to comply with the similar procedure of the PLA-PEOz formation, i.e., the living polymerization by sequential addition of monomers and the synthesis of functionalended polymers by selective termination of living ends with appropriate reagents. Also, the preliminary biocompatibility evaluation with MTT assay showed that boron containing Bpin-PLA-PEOz micelle has low toxicity compared to the phenyl boronic acid derivatives used in this study, indicating Bpin-PLA-PEOz could be potential drug delivery platform.
Acknowledgment For financial support, we thank the National Science Council (NSC 100–2113-M-007–015-MY2 and 102–2113-M-007–010) and National Tsing Hua University. For the cell viability data, authors are grateful of financial support from National Health Research Institutes (NM-100-PP-07 for JKC) and National Science Council (101-2113-M-400-001-MY3 for JKC). References Barth, R.F., Vicente, M.G., et al., 2012. Current status of boron neutron capture therapy of high grade gliomas and recurrent head and neck cancer. Radiat. Oncol. 7, 146. Chadwick, J., 1932. The existence of a neutron. Proceedings of the Royal Society of London Series A – Containing Papers of a Mathematical and Physical Character 136 (830), 692–708. Hsiue, G.H., Wang, C.H., et al., 2006. Environmental-sensitive micelles based on poly (2-ethyl-2-oxazoline)-b-poly(L-lactide) diblock copolymer for application in drug delivery. Int. J. Pharm. 317 (1), 69–75. Iyer, A.K., Khaled, G., et al., 2006. Exploiting the enhanced permeability and retention effect for tumor targeting. Drug Discov. Today 11 (17–18), 812–818. Kwon, G.S., Okano, T., 1996. Polymeric micelles as new drug carriers. Adv. Drug Deliv. Rev. 21 (2), 107–116. Liao, A.H., Chou, F.I., et al., 2010. Biodistribution of phenylboric acid derivative entrapped lipiodol and 4-borono-2-18F-fluoro-L-phenylalanine-fructose in GP7TB liver tumor bearing rats for BNCT. Appl. Radiat. Isot. 68 (3), 422–426. Locher, G.L., 1936. Biological effects and therapeutic possibilities of neutrons. Am. J. Roentgenol. Radium Ther. 36, 1–13. Oku, N., Namba, Y., et al., 1992. Tumor accumulation of novel res-avoiding liposomes. Biochim. Et Biophys. Acta 1126 (3), 255–260. Shieh, M.J., Peng, C.L., et al., 2010. Reduced skin photosensitivity with meta-tetra (hydroxyphenyl)chlorin-loaded micelles based on a poly(2-ethyl-2-oxazoline)b-poly(D,L-lactide) diblock copolymer in vivo. Mol. Pharm. 7 (4), 1244–1253.
Please cite this article as: Sherlock Huang, L.-C., et al., Drug delivery system design and development for boron neutron capture therapy on cancer treatment. Appl. Radiat. Isotopes (2014), http://dx.doi.org/10.1016/j.apradiso.2013.12.025i
L.-C. Sherlock Huang et al. / Applied Radiation and Isotopes ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Sumitani, S., Oishi, M., et al., 2011. Carborane confined nanoparticles for boron neutron capture therapy: improved stability, blood circulation time and tumor accumulation. React. Funct. Polym. 71 (7), 684–693. Syu, W.J., Yu, H.P., et al., 2012. Improved photodynamic cancer treatment by folateconjugated polymeric micelles in a KB xenografted animal model. Small 8 (13), 2060–2069.
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Ueno, M., Ban, H.S., et al., 2010. Dodecaborate lipid liposomes as new vehicles for boron delivery system of neutron capture therapy. Bioorganic Med. Chem. 18 (9), 3059–3065. Wang, C.H., Fan, K.R., et al., 2005. Enzymatic degradation of PLLA-PEOz-PLLA triblock copolymers. Biomaterials 26 (16), 2803–2811.
Please cite this article as: Sherlock Huang, L.-C., et al., Drug delivery system design and development for boron neutron capture therapy on cancer treatment. Appl. Radiat. Isotopes (2014), http://dx.doi.org/10.1016/j.apradiso.2013.12.025i