Amphiphilic Polycarbonates from Carborane ... - ACS Publications

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Amphiphilic Polycarbonates from Carborane-Installed Cyclic Carbonates as Potential Agents for Boron Neutron Capture Therapy Hejian Xiong,†,‡ Xing Wei,†,‡ Dongfang Zhou,*,† Yanxin Qi,† Zhigang Xie,† Xuesi Chen,§ Xiabin Jing,† and Yubin Huang*,† †

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China ‡ University of Chinese Academy of Sciences, Beijing 100049, PR China § Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China S Supporting Information *

ABSTRACT: Carboranes with rich boron content have showed significant applications in the field of boron neutron capture therapy. Biodegradable derivatives of carboraneconjugated polymers with well-defined structure and tunable loading of boron atoms are far less explored. Herein, a new family of amphiphilic carborane-conjugated polycarbonates was synthesized by ring-opening polymerization of a carborane-installed cyclic carbonate monomer. Catalyzed by TBD from a poly(ethylene glycol) macroinitiator, the polymerization proceeded to relatively high conversions (>65%), with low polydispersity in a certain range of molecular weight. The boron content was readily tuned by the feed ratio of the monomer and initiator. The resultant amphiphilic polycarbonates self-assembled in water into spherical nanoparticles of different sizes depending on the hydrophilic-tohydrophobic ratio. It was demonstrated that larger nanoparticles (PN150) were more easily subjected to protein adsorption and captured by the liver, and smaller nanoparticles (PN50) were more likely to enter cancer cells and accumulate at the tumor site. PN50 with thermal neutron irradiation exhibited the highest therapeutic efficacy in vivo. The new synthetic method utilizing amphiphilic biodegradable boron-enriched polymers is useful for developing more-selective and -effective boron delivery systems for BNCT.

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INTRODUCTION

new boron neutron capture therapeutic agents for moreselective and -effective BNCT. The success of BNCT requires sufficient boron atoms to be delivered to the targeted cancer cells.8 Taking advantage of the enhanced permeability and retention (EPR) effect of tumors, carboranes have been conjugated to hydrophilic nanocarriers including liposomes,9,10 dendrimers,11,12 and polymers.13−16 Except for the above “conjugate-to” strategy, carboranes have also been incorporated in monomers to tailor dendrimers and polymers.17−19 For instance, Coughlin and co-workers have reported the ring-opening metathesis polymerization of an oxonorbornene-functionalized o-carborane, which showed the potential application in BNCT but without further in vitro and in vivo evaluation.20 What’s more, to be biocompatible, it is necessary to introduce biodegradable linkers to the polymer instead of conventional linkers, such as arylene, alkylyl, siloxy, and so on.21,22 Owing to their low toxicity, aliphatic polycarbonates have gained increasing credibility for biomedical

Dicarba-closo-dodecarboranes are a class of boron-rich compounds with globular structure that possess unusual properties, including high symmetry and remarkable thermal and oxidative stability.1,2 Due to their rich boron content, carboranes have showed significant application in the field of boron neutron capture therapy (BNCT). BNCT is a cancer treatment especially suited to radio-resistant and highly invasive tumors.3 This therapy is based on the capture reaction of thermal neutrons using nonradioactive 10B, which produces two highenergy particles (4He and 7Li nucleus). These high linearenergy-transfer (LET) particles dissipate their energy before traveling across the diameter of cells (4.5−10 μm) within tissues, resulting in cytotoxic effects.4,5 To date, two small molecules, sodium mercaptoundecahydro-closo-dodecaborate (BSH) and boronophenylalanine (BPA), have been utilized as boron neutron capture therapeutic agents for clinical trials. Nevertheless, clinical results from the two compounds are not universally attractive because of their rapid clearance from blood.6,7 There is correspondingly a clear incentive to develop © XXXX American Chemical Society

Received: August 15, 2016

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DOI: 10.1021/acs.bioconjchem.6b00454 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry application. 23 Lately, we have reported a amphiphilic biodegradable carborane-conjugated polycarbonate (PLMB) nanoparticle system for co-delivery of boron atoms and doxorubicin, demonstrating enhanced cancer chemoradiotherapy in vivo.16 However, the residual decaborane adsorption from excess dosage during conjugation likely lead to the initial leakage of boron species under physiological conditions, and the maximal loading of boron atoms in PLMB nanoparticles (9.6%) was not high in boron delivery systems. Hence, new synthetic methods are needed to prepare amphiphilic carborane-conjugated polycarbonates with well-defined structures and tunable loading of boron atoms. Herein, we developed a new method of synthesizing biodegradable amphiphilic carborane-conjugated polycarbonates by ring-opening polymerization (ROP) of a carboraneinstalled cyclic carbonate monomer (Scheme 1). Different from

our previous work,16 decaborane (B10H14) could react with pendant propargyl groups on the side chains of polycarbonate to obtain carborane-conjugated copolymer in high yield. Inspired by this, carborane-installed cyclic carbonate monomer (MPCB) was synthesized through the one-pot reaction between propargyl functional cyclic carbonate (MPC) and commercially available B10H14 (Scheme 1A). The monomer was purified by column chromatography and subsequently recrystallized from diethyl ether−tetrahydrofuran (4:1 v/v) and isolated in ∼50% yield. Successful synthesis of MPCB was confirmed by 1H, 13C, and 11B nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR), and electrospray ionization mass spectrometry (ESI-MS) analysis. As shown in Figure 1, the characteristic peak of alkynyl group

Scheme 1. Synthetic Scheme of (A) Carborane-Installed Cyclic Carbonate (MPCB), (B) Amphiphilic CarboraneConjugated Polycarbonates (mPEG-b-PMPCB), and (C) Self-Assembly of mPEG-b-PMPCB in Water

Figure 1. 1H NMR spectra of (A) MPC, (B) MPCB, and (C) mPEGb-PMPCB in CDCl3 (400 MHz).

of MPC (δ = 2.5 ppm) disappeared, and the peaks of CH group (δ = 3.84 ppm) and BH groups (δ = 1.5−3 ppm) in carborane were observed in the 1H NMR spectrum of MPCB. The resonances of two alkynyl carbon atoms in MPC shifted to high field in MPCB in the 13C NMR spectrum (Figure S1). Typical FT-IR peaks of alkynyl group in MPC at 2137 cm−1 (ν(−C C−)) and 3304 cm−1 (ν(C−H)) disappeared after reaction, while a characteristic peak of carborane at 2593 cm−1 (ν(B− H)) was obvious (Figure 2). The molecular ion peak at m/z = 339.3 (MPCB + Na+) in the ESI-MS spectrum further demonstrated the successful synthesis of MPCB with high purity (Figure S3). Ring-opening polymerization of MPCB monomer was carried out using 1,5,7-triazabicyclo-[4.4.0]dec-5-ene (TBD)

conventional approaches, carboranes were directly incorporated into the polymers, avoiding additional conjugation after polymerization. The carborane-installed cyclic carbonate (MPCB) was synthesized by a versatile reaction between decaborane (B10H14) and propargyl-functional cyclic carbonate. ROP of MPCB was further catalyzed by 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) from a poly(ethylene glycol) macroinitiator. The polymerization proceeded to relatively high conversions with low polydispersity in a certain range of molecular weight and enabled easy purification of products. The boron content was readily tuned by the feed ratio of monomer and initiator. The resultant amphiphilic carboraneconjugated polycarbonates (mPEG-b-PMPCB) self-assembled in water to spherical nanoparticles with different sizes, depending on the hydrophilic-to-hydrophobic ratio. The assembled morphology was respectively characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Size-dependent cellular uptake, biodistribution, and in vivo BNCT efficacy of mPEG-b-PMPCB nanoparticles were further investigated.

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RESULTS AND DISCUSSION Synthesis and Ring-Opening Polymerization of Carborane-Installed Cyclic Carbonates. As confirmed in

Figure 2. FT-IR spectra of (A) MPC, (B) MPCB, and (C) mPEG-bPMPCB. B


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Bioconjugate Chemistry Table 1. Characteristics of mPEG-b-PMPCB polycarbonates f

mPEG44-b-PMPCB6 mPEG113-b-PMPCB3 mPEG113-b-PMPCB10

M/Ia (feed)

Mnb/( × 103)

Mnc /( × 103)

Mw/Mnc

conversiond (%)

B wt %e

8.0 4.5 23.5

4.1 6.0 8.2

4.3 6.3 7.9

1.17 1.05 1.05

75 67 43

15.7 ± 0.8 5.3 ± 0.3 9.7 ± 0.4

a

Monomer-to-initiator feed ratios. bDetermined by 1H NMR spectra. cAcquired from GPC measurement. dMonomer conversion. eDetermined by inductively coupled plasma optical emission spectroscopy. fThe subscript represents the number of repeating units.

Figure 3. TEM images and size distributions (inset) of (A) PN50, (B) PN90, and (C) PN150, respectively.

Table 2. Properties of Size-Controlled Polymeric Nanoparticles

a

polycarbonates

particle type

CACa (mg/L)

Dhb (nm)

PDIb

ζ (mv)c

mPEG44-b-PMPCB6 mPEG113-b-PMPCB3 mPEG113-b-PMPCB10

PN50 PN90 PN150

2.1 ± 0.2 6.4 ± 0.4 3.6 ± 0.3

46.5 91.2 150.1

0.299 0.265 0.241

0 ± 0.27 −0.14 ± 0.21 0.18 ± 0.16

Critical aggregation concentration. bDetermined by DLS. cζ potential measured in water.

Figure 4. Changes of (A) particle sizes and (B) surface charge of mPEG-b-PMPCB nanoparticles in the presence of bovine serum albumin (BSA, 45 g/L) at 37 °C for different time periods. (C) Time-dependent leakage of carborane from mPEG-b-PMPCB nanoparticles in the presence of PBS (pH 7.4, 0.01 M) at 37 °C.

as the organocatalyst and mPEG as the macroinitiator (Scheme 1B). MPEG was chosen because the resultant diblock polymer is amphiphilic and likely to self-assemble into long circulating nanoparticles by virtue of PEGs’ stealthlike property (Scheme 1C).24 TBD is a proven versatile organocatalyst that offers excellent control over the polymerization of numerous cyclic carbonates, yielding polymers with relatively high monomer conversion and low molar-mass dispersity.25,26 The polymerization was implemented in chloroform at room temperature with different monomer-to-initiator feed ratios (Table 1). The resultant block polymers mPEG-b-PMPCB were characterized by 1H NMR (Figure 1C), FT-IR (Figure 2C), and gel permeation chromatography (GPC; Figure S4). The double doublet peaks (δ = 4.2, 4.7 ppm) of CH2O in cyclic carbonate combined into the broad peak (δ = 4.3 ppm) in mPEG-bPMPCB. GPC analysis showed monomodal distributions for all the block polymers. As shown in Table 1, the degree of polymerization and molecular weight could be adjusted conveniently by tuning the monomer-to-initiator feed ratio. The boron content of the amphiphilic polycarbonates was adjusted from 5% to 16% by increasing the MPCB-to-mPEG

feed ratio and decreasing the molecular weight of mPEG. The decrease of monomer conversion may be attributed to highly steric impediments when the molecular weight increased. Similar results have been reported for the polymerization of other sterically hindered cyclic carbonates by the catalysis of organicbase.27,28 Preparation and Characterization of Size-Controlled Polymeric Nanoparticles. The self-assembly of the obtained amphiphilic carborane-conjugated polycarbonates was examined by method of solvent displacement. As seen in Figure 3, spherical polymeric nanoparticles (PN50, PN90, and PN150) were formed in aqueous solution with relatively narrow size distributions in all cases. In the 1H NMR spectrum of mPEG-bPMPCB in D2O (Figure S5), only signal of methylene of PEG (3.64 ppm) appeared. It indicated that the hydrophobic segment collapsed and formed a solid core, and the outer shell of nanoparticles was PEG segments. The aggregate morphology of different carborane-conjugated polycarbonates was the same, while the size varied with hydrophilic and hydrophobic lengths (Table 2). Smaller nanoparticles (PN50) were formed from the amphiphiles with shorter PEG length, C


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Bioconjugate Chemistry

Figure 5. (A) Viability of L929 cells after incubation with MPCB, PN50, PN90, and PN150 for 48 h at 37 °C. (B) Confocal images and (C) flow cytometry analysis of size-dependent cellular uptake of RhB-PN by HeLa cells. Cell nucleus stained with Hoechst 33258 (blue). Scale bar: 20 μm.

Figure 6. (A) Variation of boron species in blood with time following intravenous injection to SD rats (means ± SD, n = 3, *p < 0.05); (B) fluorescent imaging of KM mice (n = 4) treated with NIR-PN50, NIR-PN90, and NIR-PN150 at different time intervals.

shown in Figure 5A, the 50% inhibitory concentration (IC50) of MPCB was 1.8 mM. The cytotoxicity of MPCB was much lower than the nido-carborane derivative (IC50 = 0.5 mM) used in the 10B-compound-conjugated liposomes.34 Moreover, all of the mPEG-b-PMPCB nanoparticles showed no toxicity, even at high concentration ([B] = 10 mM), because carboranes were carefully protected in the core of nanoparticles. It demonstrated that mPEG-b-PMPCB nanoparticles showed good biocompatibility for safer BNCT. In addition, a similar result was acquired for human cervical cancer HeLa cells (Figure S6), suggesting that mPEG-b-PMPCB nanoparticles could not kill cancer cells without thermal neutron irradiation. Interaction of biodegradable nanoparticles with cancer cells is of great interest in cancer therapy because they can selectively and efficiently deliver various anticancer drugs to cancer cells.35,36 It is reported that most nanoparticles enter the cancer cells through energy-dependent endocytosis, and the size of nanoparticles plays an important role in endocytosis.37−39 In our work, rhodamine B (RhB), a fluorescent imaging agent, was labeled on the hydrophobic terminal of mPEG-b-PMPCB for cellular image. The characterization of RhB-PN was shown in Table S1. The polymeric nanoparticles became a little bigger after RhB conjugation, while marked difference between the three nanoparticles still existed. We first compared the intracellular uptake of polymeric nanoparticles with different sizes in HeLa cells using CLSM. Figure 5B showed that the cellular uptake of nanoparticles by HeLa cells was sizedependent. As nanoparticle size decreased, the fluorescence signal of RhB enhanced, suggesting the increased cellular uptake by tumor cells. Uptake of the nanoparticles was also quantified by flow cytometry analysis (Figure 5C). The average fluorescence intensity showed an order of RhB-PN50 > RhBPN90 > RhB-PN150. The quantification result was in agreement with the CLSM results. The same rule for human lung cancer A549 cells was also observed (Figure S7). These

and the size of nanoparticles increased with longer PMPCB block length. When compared PN50 with PN90, it seemed that the longer PEG length of PN90 showed a more significant effect on the size than shorter PMPCB block. With the highest ratio of hydrophobic block length to hydrophilic block length for mPEG44-b-PMPCB6 (1.15) than those of mPEG113-bPMPCB3 (0.26) and mPEG113-b-PMPCB10 (0.58), PN50 had the lowest CAC value. This suggested that PN50 showed the strongest dilution stability. The stability of mPEG-b-PMPCB nanoparticles was evaluated in the presence of bovine serum albumin (BSA) at 37 °C.29,30 As shown in Figure 4A,B, there were no significant changes in size and surface charge, especially for PN50 and PN90 over 48 h. This was because the outer PEG segments of nanoparticles showed a steric repulsion effect to resist protein adsorption.31,32 It was reported that the leakage of boron compounds is potentially toxic to normal tissues under the irradiation of thermal neutrons in BNCT.33 To confirm the stability of polymeric nanoparticles under physiological conditions, the leakage of borane from nanoparticles was examined by a dialysis method at 37 °C in the presence of PBS (pH 7.4, 0.01 M) (Figure 4C). The leaked borane was determined by ICP-MS, and the results were based on the concentration of boron atoms in the solution outside of the dialysis bags. All of the three mPEG-b-PMPCB nanoparticles showed absent burst leakage and very limited leakage ( PN90 > PN150, and the boron concentration for PN50 nanoparticles reached 57.4 ppm. It suggested that sufficient 10B atoms (11.5 ppm) would capture thermal neutrons to produce lethal effect on cancer cells.15,48 The changes in the tumor volume of mice bearing U14 (mouse cervical cancer cell line) tumors after treatment showed in Figure 8C. For mice group without neutrons irradiation, a fastest growth of tumors was confirmed in the negative control group administrated with PN50, indicating that PN50 alone did not have antitumor capacity. No significant inhibition of tumor growth was observed for tumor-bearing mice just treated with thermal neutron irradiation (saline + irradiation group). In sharp contrast, tumors grew much slower for the mice injected with all polymeric nanoparticles and treated with the irradiation of thermal neutron. Especially after 11 days, the tumors of mice treated with PN50, PN90, and irradiation did not grow at all. Moreover, PN50 showed highest tumor growth suppression.

results demonstrated that the RhB-PN50 with the smallest size exhibited the highest efficiency for internalization by cancer cells via endocytosis. Size-Dependent Biodistribution. Increasing evidence indicated that nanoparticle size plays a vital role in the pharmacokinetic, biodistribution, and anticancer efficacy of drug delivery systems.40−42 The blood persistence properties of different mPEG-b-PMPCB nanoparticles were determined using Sprague−Dawley rats at an intravenous dose of 1 mg B/kg. As shown in Figure 6A, PN50 nanoparticles showed a relatively slower removal rate from blood than that of PN150 nanoparticles. It was reasonable that larger nanoparticles with larger surface areas were more easily subjected to the proteins in plasma and clearance from blood by mononuclear phagocyte system (MPS).43 To observe the biodistribution of different polymeric nanoparticles, near-infrared dye (NIR) was encapsulated in mPEG-b-PMPCB nanoparticles, and there was not much change in diameters (Table S2). Female KM mice with subcutaneously implanted cervical cancer U14 were injected with NIR-PN50, NIR-PN90, or NIR-PN150, respectively. At specific time intervals, the mice were anesthetized and imaged with CRI Maestro 500FL system. As shown in Figure 6B, the fluorescence signal was mainly in the liver due to the hepatic uptake of polymeric nanoparticles in the first several hours after injection. Meanwhile, the fluorescent signal in liver region was stronger for the larger nanoparticles, which would be ascribed to the increase of the opsonin-mediated phagocytotic uptake by Kupffer cells.44,45 As time increased, the fluorescent signal in the liver decreased, while the fluorescent signal in the tumor increased and reached a high level at 24 h. Compared with NIR-PN90 and NIR-PN150, NIR-PN50 had the highest and longest accumulation at the tumor site. These results indicated that the particle size played a vital role in tumor EPR effects. Biodegradation. To best minimize toxicity and collateral effects, a nanoparticle should either be degraded in situ into noncytotoxic products or be excreted from the body once it is used for diagnostic or therapeutic purposes.46 It is well-known that aliphatic polycarbonates degrade via an enzymatic process and that the degradation of polycarbonate produces an alcohol and carbon dioxide through hydrolysis.25,47 Lipase solution from Thermomyces lanuginosus was used to investigate the enzymatic degradation of carborane-conjugated polycarbonates in vitro. mPEG113-b-PMPCB10 was taken as an example. Compared with the 1H NMR spectrum of mPEG113-bPMPCB10 without treatment (Figure 7A), the peaks of polycarbonate disappeared and only the resonance at 3.64 ppm was observed except for the signals (indicated by E



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EXPERIMENTAL SECTION Materials. Monomethyloxy poly(ethylene glycol) (mPEG, average Mn 2000 and 5000) and rhodamine B (RhB, 95%) were purchased from Sigma-Aldrich. Decaborane (B10H14) was purchased from Changchun Randall technology Co., Ltd. and used without further purification. 1,5,7-Triazabicyclo[4.4.0]dec5-ene (TBD) was purchased from Tokyo Chemical Industry Co., Ltd., Shanghai, China. Dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP) were from Aladdin Chemistry Co. Ltd. Shanghai, China. Bovine serum albumin (BSA) powder was obtained from Kangyuan Biotechnology Co., LTD, Tianjing, China. Cyanine near-infrared dye was offered by Dalian University of Technology, China. All other chemicals were purchased from Sigma-Aldrich and used as received. Methods. 1H, 13C, and 11B NMR spectra were measured by a Unity-400 NMR spectrometer at room temperature. 1H and 13 C chemical shifts are reported in ppm with tetramethylsilane as an internal reference. 11B chemical shifts are reported in ppm relative to an external standard of BF3·Et2O. Fourier transform infrared spectra were recorded on a Bruker Vertex 70 spectrometer. Gel permeation chromatography measurements were operated on a TOSOH HLC-8220 SEC instrument (column: Super HZM-H × 3) at 40 °C using THF as eluent with a flowing rate of 0.35 mL/min. The size and size distribution of nanoparticles were determined by DLS with a vertically polarized He−Ne laser (DAWN EOS, Wyatt Technology, Santa Barbara, CA). ζ potential measurements were determined on a Malvern Zetasizer Nano ZS. The morphology of the polymeric nanoparticles was measured by TEM performed on a JEOL JEM-1011 electron microscope. TEM samples were prepared from micellar solution of the nanoparticles dropped onto amorphous carbon coated copper grids. Inductively coupled plasma mass spectrometry and inductively coupled plasma optical emission spectroscopy (ICP-OES) were used to determine the boron contents. Synthesis of 5-Methyl-5-[2-(1,2-dicarba-closododecaborane)]methyleneoxycarbonyl-1,3-dioxan-2one (MPCB). 5-Methyl-5-propargyloxycarbonyl-1,3-dioxan-2one (MPC), the cyclic carbonate with propargyl group, was synthesized according to reference.49 MPCB was synthesized with a similar method reported in our previous work.16 In brief, a dried flask containing decaborane B10H14 (820 mg, 6.7 mmol), CH3CN (34 mL, 0.67 mmol), and toluene (60 mL) was warmed at reflux for 1 h. After cooling, MPC (1.1 g, 5.6 mmol) was added, and the mixture was kept at 100 °C for 24 h. The mixture was cooled and the solvent was evaporated under reduced pressure. The crude mixture was dissolved in CH2Cl2 and filtered, and the filtrate was purified by chromatography (ethyl acetate−petroleum ether 1:1, Rf = 0.3). Finally, the white solid was recrystallized from tetrahydrofuran and ethyl ether (1:4, v/v). Yield: 1.01 g, 48%. 1H NMR (400 MHz, CDCl3, δ, ppm) (Figure 1B): 3.79 (s, 1H, CcageH), 4.66 (d, 2H, −OCH2Ccage), 1.37 (s, 3H, −CH3), 4.20 (d, 2H, -OCH2−), 4.70 (d, 2H, -OCH2−), 1.5−3 (m, 10H, BH). 13C NMR (100 MHz, CDCl3, δ, ppm) (Figure S1): 17.1, 40.5, 59.8, 65.8, 70.9, 72.1, 147.1, 170.2. 11B NMR (400 MHz, CDCl3, δ, ppm) (Figure S2): −1.2, −3.4, −8.5, −11.0, −12.4. FTIR (KBr, cm−1) (Figure 2B): ν 3031, 2593,1765. MS m/z (ESI+) (Figure S3): [M + Na+] calcd for C9H20B10O5Na, 339.2; found: 339.3. ROP of MPCB (mPEG44-b-PMPCB6). First, mPEG (0.3 g, 0.15 mmol) was placed in a flask and dried via toluene

Figure 8. (A) Boron atom concentration in tumors on day 3 after PN50, PN90, and PN150 nanoparticles injection on day 1 and day 2. (B) Pictures of excised tumors on day 21 (dividing rule: cm). (C) Tumor growth curve and (D) relative body weight change of U14 tumor-bearing KM mice that received different treatments as indicated (*p < 0.05; #p < 0.01).

This was directly demonstrated from the representative pictures of tumors on day 21 from all the groups (Figure 8B). The superior antitumor activity for PN50 nanoparticles was obviously attributed to the higher 10B atoms accumulation in the tumor. The body weight of mice with different treatments was also monitored because it reflects general systemic toxicity of different drug formulations (Figure 8D). The body weight of all the mice decreased slightly on day 3 because of the anesthesia before irradiation. However, the weight kept increasing after that. The mice treated with both nanoparticles and irradiation gained a small amount of weight on day 21. To further compare the systemic toxicity, alterations of clinical chemical parameters associated with the function of liver (AST and ALT) and kidney (UA, UREA, and CREA) were determined. The group of PN150 displayed highest AST-to-ALT ratios than did the PN50 and PN90 groups (Figure S8). This suggested that the smaller nanoparticles showed negligible hepatotoxicity because of the low accumulation in liver as mentioned above.

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CONCLUSIONS In this work, we synthesized amphiphilic biodegradable carborane-conjugated polycarbonates for boron neutron capture therapy by ring-opening polymerization of a carborane-installed cyclic carbonate monomer. The boron content was conveniently tuned by varying the monomer-to-initiator ratio and molecular weight of macroinitiator (mPEG). The obtained amphiphilic polycarbonates with different hydrophilicto-hydrophobic ratios could self-assemble into nanoparticles of different sizes (PN50, PN90, and PN150). Larger nanoparticles (PN150) were more easily subjected to protein adsorption, clearance from the blood, and capture by liver. In sharp contrast, smaller nanoparticles (PN50) were more likely to be internalized into cancer cells and accumulated at the tumor site. PN50 with thermal neutron irradiation also exhibited the highest therapeutic efficacy and lowest systemic toxicity. Therefore, the size-optimized amphiphilic biodegradable carborane-conjugated polycarbonate nanoparticles represent as promising boron carriers for cancer BNCT. F


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The leakage of caborane from the polymeric nanoparticles under physiological conditions was evaluated at 37 °C in the presence of PBS (pH 7.4, 0.01 M). A solution of PN50, PN90, or PN150 with PBS (2 mg/mL, 2 mL) was poured into dialysis bags (MWCO: 3500), and each bag was immersed in 18 mL of PBS (pH 7.4, 0.01 M) at 37 °C. At a definite time interval, 0.5 mL of the solution outside the dialysis bag was sampled and replaced by corresponding PBS solutions. ICP-MS measurement of the samples was carried out to determine the amount of carborane released from nanoparticles based on the concentration of boron atoms. Cell Lines. L929 (mouse fibroblasts cells), A549 (human pulmonary carcinoma cells), and HeLa (human cervical cancer cell line) cells were chosen for cell tests and supplied by the Medical Department of Jilin University, China. These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) supplied with 10% heat-inactivated fetal bovine serum (FBS, GIBCO), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma). Biocompatibility. The biocompatibility of MPCB and polymeric nanoparticles was assessed with a methyl tetrazolium (MTT) viability assay against L929 cells and HeLa cells. The cells were seeded in 96 well plate at a density of 4000 cells per well and incubated in 100 μL of DMEM overnight. Then, the medium was replaced by MPCB or polymeric nanoparticles (PN50, PN90, and PN150) at a final boron concentration from 0.5 to 10 mM. After coincubation for 48 h, 20 μL of MTT solution in PBS (5 mg/mL) was added, and the plates were incubated for another 4 h at 37 °C, followed by removal of the culture medium and addition of DMSO (150 μL) to each well to dissolve the formazan crystals. Finally, the plates were shaken for 5 min, and the absorbance of formazan product was measured at 490 nm by a microplate reader. Cellular Uptake. A549 and HeLa cells were seeded in a glass bottomed 6 well plate at 1 × 105 cells per well in 2 mL of culture medium and allowed to adhere for 24 h. Then, the cells were treated with RhB-PN50 (50 nm), RhB-PN90 (90 nm), and RhB-PN150 (150 nm) (1.5 μg/mL equivalent RhB concentration). After incubation for 2 h at 37 °C, the supernatant was removed and the cells were washed with icecold PBS and fixed with 4% formaldehyde. After the nucleus was stained with Hoechst 33258, the slides were mounted and imaged with a Zeiss LSM 700 confocal laser scanning microscope imaging (CLSM) system. For flow cytometry analysis, the coincubation of cells and RhB-PN was the same as the above procedure. Then, a single cell suspension was prepared consecutively by trypsinization, washing with PBS, and filtration through 200 μm nylon mesh. Finally, 10 000 cells were lifted using a cell stripper (Media Tech. Inc.) and analyzed using a FACS Calibur flow cytometer (BD Biosciences) for RhB. Blank cells without the addition of any RhB-PN were analyzed, and their fluorescent intensity was designated as the threshold value. Only the fluorescent intensity that exceeded the threshold value can be considered as the uptake signal. Subcutaneous Cervical Cancer Xenografts. Female Sprague−Dawley rats (weighing ∼300g) and female KM mice (weighing 20−25 g) were purchased from the Animal Center of Jilin University. Animals were maintained under standard conditions with free access to food and water. To develop the tumor U14 (mouse cervical cancer cell line) xenografts, 1 × 105 U14 tumor cells in 0.1 mL saline were implanted subcutaneously into the right legs for each KM

azeotropic distillation for 2 h. After the removal of toluene, MPCB monomer (0.4 g, 1.26 mmol) and TBD (1.8 mg, 0.0126 mmol) were added and dissolved in CHCl3 (10 mL). After stirring at room temperature for 24 h, the reaction was quenched with excess acetic acid, and the crude product was purified by double precipitation from chloroform into diethyl ether. Yield: 0.52 g, 74%. 1H NMR (400 MHz, CDCl3, δ, ppm) (Figure 1C): 3.64 (s, 180H, H of PEG2K), 4.30 (s, 24H, −OCH2−), 1.32 (s, 18H, −CH3), 4.60 (s, 12H, −OCH2Ccage), 3.86 (s, 6H, CcageH). FTIR (KBr, cm−1) (Figure 2): ν 2885, 2593, 1765. The molecular weight and conversion of resultant polymers were calculated by 1H NMR, referencing PEG methyl protons (δ = 3.64 ppm) with those of the CH2O protons (δ = 4.30 ppm) of the MPCB repeating unit. The other polymers based on different monomer-to-initiator feed ratios were prepared in the same way. Preparation of Polymeric Nanoparticles with Different Sizes. The solvent displacement method was used to prepare different sizes of nanoparticles from mPEG-b-PMCB. mPEG-b-PMCB (15 mg) was dissolved in THF (1 mL), and then the solution was added dropwise into water (3 mL). After that, the suspension was stirred under ambient conditions until most of THF was evaporated. Finally, the solution was dialyzed against water to remove residual THF and then freeze-dried. The critical aggregation concentrations (CAC) of different molecular weight mPEG-b-PMCB nanoparticles in aqueous solution were determined according to reported procedure, employing hydrophobic pyrene as the probe.50 Preparation of Fluorescence-Labeling Nanoparticles. To facilitate the fluorescence-labeling for cell image and biodistribution study, fluorescent imaging agents RhB and NIR were introduced into the polymeric nanoparticles in different ways. RhB-conjugated mPEG-b-PMCB was synthesized through typical DCC−DMAP condensation reaction between the terminal hydroxyl of mPEG-b-PMCB and the carboxyl of RhB. For example, mPEG44-b-PMCB6 (50 mg, 0.0125 mmol) and RhB (60 mg, 0.125 mmol) were dissolved in 10 mL of dried CH2Cl2 in an ice bath. Then, DCC (52 mg, 0.25 mmol) and DMAP (15 mg, 0.125 mmol) were added, and the ice bath was kept on for 0.5 h. After stirring at room temperature for 24 h, the mixture was precipitated in diethyl ether and then dialyzed in water. RhB-conjugated mPEG44-bPMCB6 was obtained after freeze-drying. The amount of RhB conjugated in polymer was determined by UV−vis spectrometer with the help of a standard curve obtained from RhB-DMF solutions at a series of RhB concentrations. RhB-conjugated mPEG-b-PMCB nanoparticles (RhB-PN) were prepared by the procedures described above. NIR was encapsulated into the core of polymeric nanoparticles due to hydrophobic interaction. NIR was mixed with an mPEG-b-PMCB−THF solution, and then the solution was added dropwise into water, followed by the procedures described above. The amount of NIR in NIR-PN was determined by UV−vis spectrometer with the help of a standard curve obtained from NIR acetronitrile−methanol (1:1) solutions at a series of NIR concentrations. Stability Studies. The protein adsorption to polymeric nanoparticles was evaluated in the presence of BSA. The solutions of different sizes of polymeric nanoparticles were mixed with equal volumes of PBS solution (pH 7.4, 0.01 M) containing 90 g/L BSA and incubated at 37 °C. At predetermined time points, the solutions were analyzed by DLS (n = 3). G


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1 h in a polyethylene mouse holder after being anesthetized with 10% chloral hydrate (3 mL/kg) at a rate of 1.58 × 108 neutrons/s. The tumor size was measured by a caliper every other day, and the volume (V) was calculated as V = W2 × L/2, where W and L were the width and length of the tumor, respectively. Body weight was measured as an indicator of systemic toxicity. Statistical Analysis. All experiments were performed at least three times and expressed as means ± SD. Data were analyzed for statistical significance using Student’ s test. A value of p < 0.05 was considered statistically significant, and p < 0.01 was considered highly significant.

mouse. All of the in vivo study protocols were approved by the local institution review board and performed according to the Guidelines of the Committee on Animal Use and Care of Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Biodistribution. For near-infrared fluorescence imaging, 12 U14 tumor-bearing mice were injected in the tail veins with NIR-PN50 (50 nm), NIR-PN90 (90 nm), and NIR-PN150 (150 nm) at a dose of 0.5 mg NIR/kg (n = 4, per group), respectively. At specific time intervals, the mice were anesthetized and exposed to the CRI Maestro In-vivo Imaging System from Cambridge Research and Instrumentation, Inc., MA, which consisted of a light-tight box equipped with a 150 W halogen lamp and an excitation filter (671−705 nm) to excite NIR. Fluorescence was detected by a CCD camera equipped with a C-mount lens and an emission filter (750 nm long pass). A spectral data “cube” was created by acquiring a series of images at different wavelengths. In this cube, a spectrum is associated with every pixel. The resulting data can be used to identify, separate, and remove the contribution of auto fluorescence in analyzed images by the commercial software (Maestro 2.10). Pharmacokinetics. The blood persistence properties of nanoparticles were determined using female Sprague−Dawley rats. The animals, three per group, were injected in the tail vein with PN50, PN90, or PN150 (1 mg B/kg), respectively. At predetermined time intervals, blood samples were collected and weighed. Then, the blood samples were treated with concentrated nitric acid on heating to obtain clear solution. The boron contents in the solutions were determined by ICPMS. Biodegradation. In vitro enzymatic degradation of carborane-conjugated polycarbonates was conducted in lipase solution (lipase from T. lanuginosus, EC 3.1.1.3, minimum 100 000 units/g, Sigma-Aldrich, Shanghai, China) at 37 °C with constant gentle shaking. mPEG113-b-PMPCB10 (100 mg) was dissolved in a solution of deionized water−lipase (2:1, 30 mL). After days 0, 1, 3, 5 and 7, 6 mL of solution was taken out every time. The lipase was removed by heating and centrifuging, and the supernatant was lyophilized for NMR and GPC analysis. Excretion from Body. MPEG113-b-PMPCB10 was taken as example to investigate the excretion of carborane-conjugated polycarbonates from body. A total of three Sprague−Dawley rats were injected in the tail vein with mPEG113-b-PMPCB10 (5 mg B/kg). The treated rats were housed in metabolic cages and provided with sufficient food and water. Urine and feces were collected daily and treated with concentrated nitric acid on heating, to obtain clear solution. The boron contents in the solutions were determined by ICP-MS. Antitumor Efficacy. A total of 45 tumor-bearing mice were randomly divided into five groups (n = 9 for each group): (1) PN50 + irradiation, (2) PN90 + irradiation, (3) PN150 + irradiation, (4) saline + irradiation, and (5) PN50. When the tumor grew to 100−120 mm3, treatments began, and the day was designated as day 1. For groups 1, 2, and 3, PN50, PN90, and PN150 were injected twice via tail vein at 24 and 48 h before irradiation (10 mg B/kg). For groups 4 and 5, the mice were injected with an equivalent volume of saline and the equivalent dose of PN50. Then, thermal neutrons irradiation was carried out on day 3 by an NG-9 accelerator-based neutron source (accelerating voltage: 90 kV, current: 0.1 mA) in Department of Physics of Northeast Normal University in Changchun, China. Mice in the four groups were irradiated for

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00454. Additional figures including NMR spectra, measured and theoretical mass spectra, GPC traces, visibility of HeLa cells after incubation, conforcal images and flow cytometry analysis, and blood biochemical analysis results. A table showing characteristics of fluorescence labeling nanoparticles. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (51273194, 51321062, and 51403198) and performed by using facilities of Department of Physics of Northeast Normal University and assisted with Prof. Shiwei Jing.

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REFERENCES

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