Article pubs.acs.org/Biomac
Doxorubicin-Loaded Carborane-Conjugated Polymeric Nanoparticles as Delivery System for Combination Cancer Therapy Hejian Xiong,†,‡ Dongfang Zhou,† Yanxin Qi,† Zhiyun Zhang,†,‡ Zhigang Xie,† Xuesi Chen,† Xiabin Jing,† Fanbo Meng,*,§ and Yubin Huang*,† †
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China ‡ Graduate School of Chinese Academy of Sciences, Beijing 100039, P. R. China § The Cardiology Department of China-Japan Union Hospital of Jilin University, No. 126 Xiantai Str., Changchun 130033, P. R. China S Supporting Information *
ABSTRACT: Carborane-conjugated amphiphilic copolymer nanoparticles were designed to deliver anticancer drugs for the combination of chemotherapy and boron neutron capture therapy (BNCT). Poly(ethylene glycol)-b-poly(L-lactide-co-2methyl-2(2-dicarba-closo-dodecarborane)propyloxycarbonylpropyne carbonate) (PLMB) was synthesized via the versatile reaction between decaborane and side alkynyl groups, and selfassembled with doxorubicin (DOX) to form drug-loaded nanoparticles. These DOX@PLMB nanoparticles could not only suppress the leakage of the boron compounds into the bloodstream due to the covalent bonds between carborane and polymer main chains, but also protect DOX from initial burst release at physiological conditions because of the dihydrogen bonds between DOX and carborane. It was demonstrated that DOX@PLMB nanoparticles could selectively deliver boron atoms and DOX to the tumor site simultaneously in vivo. Under the combination of chemotherapy and BNCT, the highest tumor suppression efficiency without reduction of body weight was achieved. This polymeric nanoparticles delivery system could be very useful in future chemoradiotherapy to obtain improved therapeutic effect with reduced systemic toxicity.
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INTRODUCTION Carboranes have showed significant applications in medical chemistry due to its unique physical and chemical properties.1,2 With a high content of 10B atoms, carobranes have been primally studied as potential candidates for boron neutron capture therapy (BNCT) in treatment of cancer. BNCT is a binary cancer treatment based upon the nonradioactive nuclide 10 B to capture thermal neutrons and yield two high energy particles (4He and 7Li nucleus). The particles are capable of destroying cells because of their high linear energy transfer characteristics. However, they possess very short path lengths (4.5−10 μm) in tissues and so their cytotoxicity is largely confined to the cell where 10B agent is located.3−5 As reported, nanoparticles based on enhanced permeability and retention (EPR) effect or active targeting allow delivered boron atoms to accumulate in high concentration within the tumor, while minimal accumulation occurs in adjacent and systemic normal tissues.6 To make the compounds more soluble and selective, boranes and carboranes have been conjugated to large entities to form nanoparticles, such as liposomes,7,8 dendrimers,9 polyethylene glycol,10,11 and so on. Polymeric nanoparticles have emerged as one of the most promising delivery systems in nanomedicine due to their facile preparation, high stability, solubilization of a water insoluble drug, and enhanced accumulation in tumor.12 Taking advantage of the hydro© 2015 American Chemical Society
phobicity of carboranes, polymeric nanoparticles can be formed with hydrophilic segments and carboranes on a single polymer backbone. As the concurrent administration of chemotherapy and radiotherapy is part of the standard of care and curative treatment of many cancers, nanoparticles-based delivery systems have been exploited to enhance efficacy and reduce toxicity.13−15 However, few works have combined BNCT with chemotherapy. Carborane-conjugated polymeric nanoparticle is potential to meet this need, because the formed hydrophobic core is efficient to load another poor water-soluble drug. What is more, owing to the presence of hydridic B−H units, carboranes are involved in particular kinds of interactions, such as dihydrogen bonding.1,16,17 As a result, carboranes are able to interact with anticancer drugs to increase loading contents and provide synergistic effect through the bond of B−Hδ‑···Hδ+−X where X is N, O, or S. Doxorubicin (DOX) is an anthracycline antitumor antibiotic with multiple of hydroxyl and amine groups in the structure.18 Although it is widely used to treat many types of cancer, its severe cardiac toxicity decreases the patients’ life quality.19 To Received: September 30, 2015 Revised: November 11, 2015 Published: November 12, 2015 3980
DOI: 10.1021/acs.biomac.5b01311 Biomacromolecules 2015, 16, 3980−3988
Article
Biomacromolecules
Scheme 1. Synthetic Scheme of PEG-b-P(LA-co-MPCB) and Schematic Illustration of Chemoradiation through DOX@PLMB Nanoparticles with Thermal Neutron Irradiation
Figure 1. 1H NMR spectrum of (A) PLM, (B) PLMB, in CDCl3, and (C) PLMB nanoparticles in D2O (400 MHz). prior to use. Cyclic carbonate monomer 5-methyl-5-propargyloxycarbonyl-1, 3-dioxan-2-one (MPC) was synthesized according to ref 23. Decaborane (B10H14) was purchased from Changchun Randall Technology Co., LTD and used without further purification. 1(Hydroxymethyl)-1,2-dicarba-closo-carbrane (HM-carborane) was synthesized and purified with a detailed procedure given in the Supporting Information. Doxorubicin (DOX) in the form of a hydrochloride salt was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd. All other chemicals were purchased from Sigma-Aldrich and used as received. Methods. 1H NMR and 13C NMR spectra were measured by a Unity-400 NMR spectrometer at room temperature. 1H and 13C chemical shifts are reported in ppm with tetramethylsilane as an internal reference. Inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectroscopy (ICP-OES) were used to determine the boron contents. Fourier transform infrared (FTIR) spectra were recorded on a Bruker Vertex 70 spectrometer. Gel permeation chromatography (GPC) measurements were operated on a TOSOH HLC-8220 SEC instrument (column: Super HZM-H × 3) at 40 °C using THF as eluent with a flow rate of 0.35 mL/min. Size and size distribution of nanoparticles were determined by dynamic light scattering (DLS) with a vertically polarized He−Ne laser (DAWN EOS, Wyatt Technology). The morphology of the polymer−drug nanoparticles was measured by transmission electron microscopy (TEM) performed on a JEOL JEM-
improve the therapeutic efficacy and reduce the toxicity, various drug delivery systems based on nanoparticles are reported.20−22 Herein, carborane-conjugated amphiphilic copolymer PEG-bP(LA-co-MPCB) (PLMB) nanoparticles were designed to deliver DOX for the combination of BNCT and chemotherapy. PLMB was synthesized through the versatile reaction between the alkynyl groups of PEG-b-P(LA-co-MPC) and decaborane (Scheme 1). More stable nanosized polymeric nanoparticles were formed in water because of the hydrophobicity of obtained pendant icosahedral carborane. DOX was encapsulated in nanoparticles due to hydrophobic interaction and dihydrogen bonding between DOX and carborane. It was demonstrated that DOX@PLMB nanoparticles could selectively deliver boron atoms and DOX to tumor site simultaneously in vivo. Accompanied by thermal neutron irradiation at the right time, this facile combination treatment exhibited better therapeutic effects and fair toxicity than sole chemotherapy or radiotherapy in tumor-bearing mice.
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EXPERIMENTAL SECTION
Materials. Monomethyloxy poly(ethylene glycol) (mPEG2000) was purchased from Sigma-Aldrich. L-Lactide (LA) was prepared in our own laboratory and recrystallized from ethyl acetate three times 3981
DOI: 10.1021/acs.biomac.5b01311 Biomacromolecules 2015, 16, 3980−3988
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Biomacromolecules 1011 electron microscope. TEM samples were prepared from micellar solution of the nanoparticles dropped onto amorphous carbon coated copper grids. Synthesis of PEG-b-P (LA-co-MPCB) Amphiphilic Copolymer. Poly(ethylene glycol)-b-poly(L-lactide-co-2-methyl-2(2-propargyloxycarbonyl)-propylene carbonate) (PEG-b-P(LA-co-MPC), PLM) was prepared via a macroinitiator route. First, 2 g of PEG2000 (1 mmol) was placed in a flask and dried via toluene azeotropic distillation for 2 h. Then 1.58 g of MPC monomer (8 mmol) and 1 g of LA monomer (7 mmol) were added simultaneously in the flask followed by addition of 0.3 mL of ZnEt2 (0.08 mmol) in toluene solution as catalyst under argon atmosphere. The polymerization system was stirred and heated to 110 °C. After 24 h, the polymerization was stopped by cooling the flask to room temperature. The resultant copolymer PLM was purified by dissolving in CHCl3 and precipitating in cold diethyl ether, and finally dried under vacuum (yield 3.5 g, 76.2%). 1H NMR (400 MHz, CDCl3) (Figure 1A): δ (ppm) 3.64 (s, 180H, H of PEG2K), 1.59 (d, 30H, −OCH(CH3)CO−), 5.16 (m, 10H, −OCH(CH3)CO−), 4.30 (s, 24H, −OCH 2−), 1.30 (s, 18H, −CH3 ), 4.72 (s, 12H, −OCH2C), 2.50 (s, 6H, −CCH). From the integration in the 1 H NMR spectrum, the conversion of LA and MPC was calculated as 71.4% and 75%, respectively. PEG-b-P(LA-co-MPCB) (PLMB) was synthesized through the reaction between decaborane and the pendant propargyl group. Briefly, a flask containing B10H14 (611 mg, 5 mmol), CH3CN (25 mL, 500 mmol) and 40 mL of dried toluene was warmed at reflux for 1 h. After cooling, PLM (1.56 g, 0.4 mmol) was added and the mixture was refluxed at 100 °C for 24 h. The mixture was cooled and the solvent evaporated under reduced pressure. Then the crude mixture was dissolved in CH2Cl2, filtered, and finally precipitated in cold diethyl ether. The resultant copolymer was dried under vacuum (75%). 1H NMR (400 MHz, CDCl3) (Figure 1B): δ (ppm) 3.64 (s, 180H, H of PEG2K), 1.59 (d, 30H, −OCH(CH3)CO−), 5.16 (m, 10H, −OCH(CH3)CO−), 4.30 (s, 24H, −OCH2−), 1.30 (s, 18H, −CH3), 4.63 (s, 12H, −OCH2C), 3.91 (s, 6H, CcageH). Critical Aggregation Concentration (CAC) Measurements. The CACs of the block copolymer PLM and PLMB in aqueous solution were determined according to reported procedure,24 employing hydrophobic pyrene as the probe. Steady state fluorescence spectra were obtained by a PerkinElmer LS55 luminescence spectrometer. The polymer aqueous solutions with various concentrations from 10−5 to 1.0 mg/mL were added to a series of volumetric flasks, and the pyrene concentration in the final solution was fixed at 6 × 10−7 mol/L (the saturation solubility of pyrene in water at 22 °C). Then the flasks were thermostated at 25 °C for about 2 h to equilibrate pyrene partition between water and polymeric nanoparticles. The emission wavelength was set at 391 nm for fluorescence excitation spectra. The spectra were recorded at a scan rate of 600 nm/ min. With the increase in the polymer concentration, a red shift was observed. Preparation of PLMB Nanoparticles. The solvent displacement method was used to prepare nanoparticles from PLMB copolymer. In brief, PLMB (100 mg) was dissolved in THF (5 mL), and then the solution was added into deionized water (20 mL) dropwise under stirring to form a micellar solution. The solution was dialyzed against water to remove THF and then freeze-dried. Stability Studies of PLMB Nanoparticles in PBS. The leakage of caborane from the polymeric nanoparticles was evaluated at 37 °C in phosphate-buffered saline (PBS) (pH 7.4 and pH 5.0). A solution of PLMB nanoparticles (2 mg/mL, 1 mL) was poured into dialysis bags (molecular weight cutoff: 3500), and each bag was immersed in 49 mL of PBS 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 sample was carried out to determine the amount of carborane released from nanoparticles based on the concentration of boron atoms. DOX Loading and Release Experiment. Typically, 20 mg of DOX·HCl and triethylamine (14 μL) were dissolved in DMSO (200 μL) and added to a THF solution containing PLMB (100 mg). The mixture was allowed to stir at room temperature for 2 h. Then, this
solution was added dropwise to 20 mL of deionized water under gentle stirring. After being stirred for additional 4 h, the solution was transferred to a dialysis bag (MWCO: 3500) and dialyzed for more than 24 h to get rid of free DOX and organic solvent. The final solution in the dialysis bag was freeze-dried to give red sponge-like nanoparticles. To measure the amount of DOX loaded into the nanoparticles, the nanoparticles were dissolved in DMF and the amount of DOX was determined by UV−vis spectrophotometer (UV2450PC, Shi-madzu, Japan), with the help of a standard curve obtained from DOX−DMF solutions at a series of DOX concentrations. Drug loading content (DLC) and drug loading efficiency (DLE) was calculated according to the following formula:
DLC (wt %) = (weight of loaded drug/weight of drug loaded nanoparticles) × 100% DLE (%) = (weight of loaded drug/weight of feeding drug) × 100% In vitro release profiles of DOX from the polymer nanoparticles were investigated in PBS (10 mM, pH 7.4, pH 5.0). The weighted DOX@PLMB nanoparticles were suspended in PBS (2 mL) and introduced into a dialysis bag (MWCO: 3500). The release experiment was initiated by placing the end-sealed bag in 18 mL of PBS at 37 °C with continuous shake at 100 rpm. At selected intervals, 0.5 mL of buffer was collected for UV−vis measurement and an equal volume of fresh buffer was replenished. The amount of DOX was determined by spectrophotometry at 480 nm using the standard curve method. Pharmacokinetics of PLMB Nanoparticles. Female Sprague− Dawley rats (weighing ∼300 g) and KM mice (weighing 18−20 g) were purchased from the Animal Center of Jilin University. Animals were maintained under standard conditions with free access to food and water. All studies were performed in accordance with the Guide for Care and Use of Laboratory Animals, as proposed by the Committee on Care Laboratory Animal Resources, Commission on Life Sciences and National Research Council. To develop the tumor U14 xenografts, U14 mice cervical tumor cells were implanted subcutaneously into the right legs of the mice (1 × 106 cells/mL saline). The blood persistence properties of PLMB nanoparticles were determined using female Sprague−Dawley rats. As a comparison, the pharmacokinetics of 1-(hydroxymethyl)-1,2-dicarba-closo-carbrane (HM-carborane) with similar structure to the pendant carborane on the side chain of PLMB was also evaluated. The animals, three per group, were injected in the tail vein with PLMB nanoparticles (1 mg B/kg) or HM-carborane (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 ICP-MS. In order to evaluate the biodistribution of boron species, U14 tumor-bearing female KM mice were randomly divided into PLMB nanoparticles (1 mg B/kg) (n = 3) and HM-carborane (1 mg B/kg) (n = 3) groups. The mice were sacrificed at 1, 24, 48, and 72 h after intravenous injection. Major tissues and organs including brain, heart, liver, spleen, lung, kidney, tumor, and blood were collected and washed with 0.9% saline before being weighted. The concentration of boron atoms in each tissue was measured using the procedure described above. Biodistribution Study of DOX Based on Fluorescence Measurements. For ex vivo biodistribution studies, U14 tumorbearing female mice were randomly divided into two groups, followed by intravenous injection with DOX@PLMB nanoparticles and free DOX at an equivalent DOX dose of 2 mg/kg body weight. This study used groups of four mice per time point. At specific time intervals (1, 12, 24, and 48 h), blood samples were collected via eye puncture. Plasma was obtained by centrifuging whole blood samples at 3000 rpm for 5 min. Subsequently, the six organs (heart, liver, spleen, lung, 3982
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Biomacromolecules kidney, and tumor) of the carcasses were collected and imaged under the same parameters using a CRI Maestro Imaging System (Cambridge Research & Instrumentation, Inc.), which consisted of a light-tight box equipped with a 150 W halogen lamp and an excitation filter (503−555 nm) to excite DOX, and the excitation time was set as 5000 ms. Fluorescence was detected using a CCD camera equipped with a C-mount lens and an emission filter (580 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 autofluorescence in analyzed images by the commercial software (Maestro 2.10). In order to get the quantitative information on DOX concentration in each tissue, fluorospectrophotometry was carried out according to the literature25 with minor modifications. After being imaged, the tissues and plasma were weighted and suspended in 70% ethanol with 0.3 M HCl and vigorously homogenated, followed by a further centrifugation step. DOX fluorescence intensity in the supernatant was measured with a fluorescence spectrometer at an excitation wavelength of 480 nm and an emission wavelength of 590 nm. The DOX concentrations was determined according to standard curves, establish by adding a certain amount of DOX to the respective tissues and plasma harvested form untreated mice, followed by procedures described above. In Vivo Anticancer Efficacy. Neutron irradiation was carried out by an accelerator-based neutron source in Department of Physics of Northeast Normal University in Changchun, China. U14 tumorbearing KM female mice were randomly divided into eight groups (n = 9, each group) when the tumor grew up to 60−90 mm3. The four groups among them were injected with DOX@PLMB nanoparticles, PLMB nanoparticles, DOX and saline, at a dose of 10 mg B/kg and 5 mg DOX/kg. And this day was designated as day 1. For DOX@PLMB group and PLMB group, PLMB nanoparticles were injected again at a dose of 10 mg B/kg on day 2. Then on day 3, mice of these four groups were irradiated for 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 above injection and irradiation treatments were repeated on days 4−6. The boron atoms concentrations in each tissue (heart, liver, spleen, lung, kidney, and blood) of tumor-bearing mice on day 3 after injection of PLMB nanoparticles on day 1 and day 2 were determined using ICP-MS. Other mice of the remaining four groups were treated according to similar procedures except for the thermal neutron irradiation. The tumor size was measured by using a caliper every other day, and the volume (V) was calculated as V = W2 × L/2, where W and L are the width and length of the tumor, respectively. Body weight was measured as an indicator of systemic toxicity. The tumor suppression rate was calculated with the following formulas:
involved insertion of carbon into a polyborane framework, as boranes furnish the required electron-delocalized matrix. Alkynes are the most commonly employed insertion reagents, with the CC unit ending up as vicinal carbon atoms in a C2Bn carborane skeleton.26 In order to obtain carborane-conjugated amphiphilic block copolymer, PEG-b-P(LA-co-MPC) (PLM) containing pendant propargyl groups on the hydrophobic chain was designed and synthesized through random polymerization of LA and MPC using PEG as macroinitiator for less steric hindrance during carborane conjugation. Decaborane (B10H14) could react with suitable derivatives with acetylenic compounds in the presence of Lewis bases to produce a variety of 1,2dicarba-closo-dodecarboranes.27 Hence, the one-pot reaction between PLM and commercially available B10H14 was then carried out to obtain carborane-conjugated block copolymer PLMB (Scheme 1).
Figure 2. FT-IR spectra of (A) DOX·HCl, (B) PLM, (C) PLMB, and (D) DOX@PLMB.
PLM and PLMB were characterized by 1H NMR (Figure 1), FT-IR (Figure 2), and GPC (Table 1). As shown in Figure 1A, the characteristic peaks of PEG (−OCH2CH2−, δ = 3.64 ppm), LA repeating units (−OCH(CH3)CO−, δ = 5.16 ppm) and MPC repeating units (−OCH2CCH3COOCH2−, δ = 4.30 ppm) appeared in the 1H NMR spectrum of PLM. The polymerization degree of LA and MPC was then calculated to be 5 and 6, respectively. Compared with PLM, the peak of propargyl group (δ = 2.5 ppm) disappeared and the peak of CH group in carborane (δ = 3.84 ppm) was observed in the 1H NMR spectrum of PLMB (Figure 1B). The number of formed carboranes was also calculated to be five from integration ratio between CH group in carborane and CH2 group in PEG. Typical FT-IR peaks of alkyl group in PLM at 2132 cm−1 (ν(−CC−)) and 3285 cm−1 (ν(C−H)) disappeared after conjugation, while the peak of carborane at 2591 cm−1 (ν(B− H)) was obvious (Figure 2). Successful conjugation of decaborane was also demonstrated by the increased molecular weight in GPC measurements. The boron content in PLMB was measured as 9.6% by ICP-OES. Self-Assembly of PLMB and Drug Loading. The obtained PEG-b-(LA-co-MPCB) was dissolved in THF and self-assembled into spherical nanoparticles in aqueous solution, as evidenced by TEM (Figure 3B). In the 1H NMR spectrum of PLMB in D2O (Figure 1C), only signal of methylene of PEG (3.64 ppm) appeared. It indicated that PLMB formed core− shell nanoparticles and carborane were protected in the core. The hydrophobic interactions between the grafted carborane
tumor growth rate (TGR, %) = (Vt/V0) × 100% tumor suppression rate (TSR, %) = [(TGR c − TGR x)/TGR c] × 100% where V0 means the volume of tumor on day 1, Vt means the volume at certain time, c means the control group (saline, without irradiation), and x means the other groups. Determination of Cell Apoptosis. At the end of in vivo study, the tumors (n = 3, per group) were excised from sacrificed mice and fixed in 10% formalin solution followed by paraffin embedding and TUNEL staining. Embedding and staining were done in Norman Bethune Health Science Center of Jilin University. Images were acquired by OLYMPUS CX31microscope and analyzed by Image Analysis System 10.0.
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RESULTS AND DISCUSSION Synthesis of Carborane-Conjugated Poly(ester-carbonate) Amphiphilic Block Copolymers. Since the first report in the early 1960s, the majority of carborane synthesis has 3983
DOI: 10.1021/acs.biomac.5b01311 Biomacromolecules 2015, 16, 3980−3988
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Biomacromolecules Table 1. Characteristics of Block Copolymers molecular formula PEG-b-P(LA-co-MPC) PEG-b-P(LA-co-MPCB)
f PEG:LA:MPCa
DPLAb
1:7:8 1:7:8
5 5
DPMPCb 6 6
CBb (number)
Mnb
Mnc
0 5
3.9 × 10 4.5 × 103
PDIc
5.4 × 10 5.9 × 103
3
3
1.22 1.16
a
Molar ratio of PEG, LA, and MPC in feed. bDetermined by 1H NMR spectra; CB is abbreviation for carborane. cAcquired from GPC using THF as the eluent.
Figure 3. (A) UV−vis absorption spectra of copolymers and DOX-loaded copolymers (DOX 0.02 mg/mL) in 0.01 M PBS (pH7.4) and (B) TEM images of PLMB nanoparticles (left) and DOX@PLMB nanoparticles (right).
Table 2. Properties of Polymeric Nanoparticles
a
molecular formula
CAC (mg/L)
Dh (nm)a
PDIa
DLC (wt %)b
DLE (%)b
PEG-b-P(LA-co-MPC) PEG-b-P(LA-co-MPCB)
5.8 ± 0.5 5.3 ± 0.3
103.5 107.8
0.21 0.22
2.7 ± 0.9 9.9 ± 1.2
16 ± 4.5 60 ± 7.6
Determined by DLS. bDrug loading content (DLC) and drug loading efficiency (DLE) calculated by UV−vis spectrophotometry.
Figure 4. (A) Time-dependent leakage of carborane from PLMB nanoparticles and (B) in vitro DOX release profiles from DOX@PLMB nanoparticles and DOX@PLM nanoparticles in PBS at pH 7.4 and 5.0.
504 nm because of the self-association of DOX involving in intermolecular hydrogen bonds or hydrophobic interaction.30 However, λm of DOX in the core of PLMB was 485 nm, which could be the result of balance between the intermolecular interactions of DOX and dihydrogen bonds of B−H···H−O (N). As a result, the interaction between DOX and carboranes increased the drug loading efficiency from 16% to 60%. The DOX@PLMB nanoparticles were around 118.1 nm in water, which was a little bigger than blank PLMB nanoparticles (SI Figure S4 and Figure 3B). The loading content of DOX could reach as high as 9.9 wt % at the feed ratio of 1:5 (DOX: PLMB). Stability of PLMB Nanoparticles and in vitro Drug Release. It is reported that the leakage of the boron compounds is potentially toxic to the normal tissues after the irradiation of thermal neutrons in BNCT.31 In order to confirm the stability of PLMB nanoparticles under physiological conditions, the leakage of carborane from the nanoparticles was evaluated by dialysis at 37 °C in phosphate buffer solution
facilitated the lower CMC values compared with PEG-b-(LAco-MPC) (Table 2). The average particle size of PLMB nanoparticles was around 107.8 nm with PDI of 0.258. There was no significant difference on size between PLMB and PLM nanoparticles. It is reasonable that the increased molecular weight after conjugation was not too much compared with the weight of polymer. Doxorubicin (DOX) was loaded into nanoparticles via a membrane dialysis method. As shown in Figure 2, the peak of B−H group of carborane was shifted to 2526 cm−1 in the spectrum of DOX@PLMB. As the interaction involving a close approach of protonic and hydridic hydrogens has been reported especially in boron hydrides,28,29 the shift indicated that the existence of unconventional dihydrogen bonding between B−H groups of carborane and O−H or N−H groups in the structure of DOX. This was further demonstrated by the red shift of maximum absorption wavelength (λ m ) of DOX after encapsulation in Figure 3A. The λm of DOX in PBS (pH7.4) was 480 nm, while λm of DOX in the core of PLM shifted to 3984
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Biomacromolecules (PBS) at pH 7.4 and 5.0 (Figure 4A). The leaked carborane was determined by ICP-OES, and the results were based on the concentration of boron atoms in the solution outside of the dialysis bags. PLMB nanoparticles showed limited leakage of boron species in the initial 2 h because of the nonspecific borane adsorption, while had high stability during the next 2 days. The in vitro release of DOX from the nanoparticles was determined under the similar conditions as mentioned above (Figure 4B). The release of DOX from DOX@PLM nanoparticles exhibited no obvious differences at different pHs, while DOX@PLMB nanoparticles showed much faster DOX release at endolysosomal pH (∼5.0) than that at physiological pH (7.4). For DOX@PLMB nanoparticles, the release profiles showed no significant initial burst release, and 27.9 ± 3.2% of the encapsulated DOX was released in a sustained manner over 12 h at pH 7.4. When the pH decreased to 5.0, the higher solubility of DOX and disruption of the dihydrogen bonds between DOX and carborane led to the faster release profile. The in vitro DOX release profiles indicated that the DOX@ PLMB nanoparticles are expected to maintain the core−shell structure integrity at physiological pH and prevent DOX from releasing by the interaction between carboranes and DOX, but induce rapid DOX release in endolysosomes after endocytosis. For clinical application, a boron delivery agent should have low systemic toxicity.32 As seen in Figure S5, PLMB nanoparticles showed no toxicity even at high concentration ([B] = 10 mM). PLMB nanoparticles were further injected to normal female KM mice at a series of boron doses to examine the in vivo toxicity. Nearly no death and decrease of body weight was observed, and a tolerable receiving dose up to 50 mg B/kg was determined. This dose is approximated to the average dosage administered in clinic (Table S3 and Figure S6). The cytotoxicity of DOX@PLMB nanoparticles tested against A549 and Hela cell lines in comparison with free DOX and blank nanoparticles (SI Table S1). DOX@PLMB nanoparticles and free DOX were able to inhibit cancer cell proliferation efficiently in a concentration-dependent manner. DOX@PLMB nanoparticles had relatively higher IC50 than free DOX. The sustained release of DOX from the nanoparticles could be the main reason. Pharmacokinetics Studies of Boron Species. One of the most important requirements for a successful boron delivery agent is the rapid clearance from blood and normal tissues, but persistence in tumor during BNCT.3 Unlike small molecules, nanoparticles-based drug delivery system can have a good targeting efficiency to ensure selective deposition of drug in the target site but low concentrations in healthy tissues or organs.33 In our system, the conjugated carborane on polymer backbone could be well protected in the core of nanoparticles during circulation, and selectively delivered to tumor site via EPR effect. The blood persistence properties of PLMB nanoparticles and HM-carborane were determined using female Sprague−Dawley rats at an intravenous dose of 1 mg B/kg. As shown in Figure 5A, the boron concentration both of PLMB and HM-carborane in plasma showed a biphasic kinetic profile. It is because that the excretion of most drugs from body belongs to first-order elimination.34 The removal rate of boron from blood in PLMB nanoparticles group was slower than that of small molecule HM-carborane. After administration of PLMB nanoparticles for 1.25 h, the remained boron in systemic circulation was 44.1 ± 12.5%, that was 3.6-fold higher than that of HM-carborane.
Figure 5. Variation of boron species (A) in blood with time following intravenous injection to rats (mean ± SD, n = 3), and (B) in tissues and organs of tumor-bearing mice after injection of PLMB nanoparticles and HM-carborane (mean ± SD, n = 3). The data was expressed as percentage of injected dose (ID)/g of wet tissues (% ID/g tissue).
Individual pharmacokinetics parameters were presented in SI Table S2. The mean area under curve (AUC) for PLMB nanoparticles was 1.7-fold higher than that of HM-carborane. It implied that the conjugation of borane resulted in a significant prolongation of boron presence in blood and more opportunity to reach the target tumor sites. In order to confirm the pharmacokinetics of the boron species in the tumor-bearing mice, PLMB nanoparticles and HM-carborane were injected in vein, and the concentrations of boron atoms in tissues and organs were evaluated using ICPMS (Figure 5B). The boron species of PLMB nanoparticles in the bloodstream retained at a high level (>2% ID/g) during 48 h after injection, while the value for HM-carborane decreased to 2.04 ± 0.62% ID/g only after 24 h. It may be inferred that most injected HM-carborane was immediately eliminated from the bloodstream by renal clearance, because the amounts of HM-carborane accumulated in the kidney 1 h after the injection (16.20 ± 4.95% ID/g) were apparently higher than those of PLMB nanoparticles (6.64 ± 1.63% ID/g). Unlike small molecules mainly cleared by kidney, most exogenous nanoparticles larger than 10 nm would be captured by the mononuclear phagocyte system (MPS).33 As a result, accumulation of PLMB was much higher than that of HMcarobrane in liver. It should be noted that the boron species in tumor increased as time passed by and reached the maximum (30.32 ± 9.24% ID/g) at 48 h after injection, whereas HMcarborane was excreted from the tumor rapidly. The higher tumor targeting efficiency of PLMB was attributed to the longer blood circulation time and EPR effect of tumors on PLMB nanoparticles. This indicates that there is enough time to precisely determine the boron concentration and implement the neutrons irradiation with adequate dose. Biodistribution of DOX@PLMB Nanoparticles. Compared to boron species accumulation, anticancer drug accumulation in tumor played a similarly important role. For biodistribution of DOX@PLMB nanoparticles and free DOX, 3985
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Article
Biomacromolecules
DOX for free DOX intravenous injection decreased significantly after 12 h. This could be further proven by fluorescence quantitative analysis of the concentration of DOX in plasma and tissues (Figure 6C). In blood circulation, DOX concentration at 1 h for DOX@PLMB nanoparticles and DOX were 10.09 ± 1.35% and 6.19 ± 1.27%, respectively. Within 24 h, the DOX concentration for DOX@PLMB nanoparticles (1.90 ± 0.12% ID/g) was 4.4-fold higher than that of free DOX (0.43 ± 0.05% ID/g). A high level of DOX for DOX@PLMB nanoparticles (5.06 ± 0.91% ID/g) in liver at 1 h after injection was detected and the value dropped dramatically to 2.61 ± 0.22% ID/g at 12 h. Compared with Figure 5B, it was interesting to find that DOX accumulation in the liver for DOX@PLMB nanoparticles was not as significant as that of boron species for PLMB nanoparticles. This different accumulation was ascribed to the faster release and clearance of encapsulated DOX than conjugated carborane. On the contrary, the DOX levels in heart and kidney for nanoparticles were significantly lower than that of free DOX group, which could reduce the cardiotoxicity and renal toxicity of DOX. The DOX concentration in tumor for DOX@PLMB nanoparticles group showed a continuous increase within 24 h period after administration and reached the maximum of 4.24 ± 1.34% ID/g, which was 2.5-fold higher than that of free DOX (1.71 ± 0.11% ID/g). On the basis of these data, it was clearly demonstrated that the carboraneconjugated amphiphilic polymer not only could effectively and selectively deliver boron atoms to the tumor site, but also improve the tumor accumulation of DOX encapsulated in the hydrophobic core of PLMB nanoparticles. Combination Treatment Effect for Tumor-Bearing Mice. The antitumor efficacy of DOX@PLMB nanoparticles was investigated with thermal neutrons irradiation against subcutaneous cervical U14 tumor-bearing mice, compared with the BNCT group (PLMB + irradiation), the chemotherapy group (DOX@PLMB, DOX), and other control groups. When
Figure 6. Fluorescent images of excised organs after different time periods post administration of DOX@PLMB nanoparticles (A) and free DOX (B), and (C) fluorescent quantitative analysis of the DOX concentration in plasma and tissues.
imaging of the excised organs at specific time intervals was carried out through DOX fluorescence signals collected from the isolated visceral organs (heart, liver, spleen, lung, kidney, and tumor). As seen in Figure 6A and B, more nanoparticles accumulated in liver than free DOX, due to the hepatic uptake of nanoparticles. On the contrary, DOX was retained at a high concentration in tumor during 48 h after the injection for DOX@PLMB nanoparticles, whereas the concentration of
Figure 7. (A) Boron atom concentration in each tissue and organ of tumor-bearing mice on day 3 after PLMB nanoparticles injection on day 1 and day 2. (B) Tumor growth curve, (C) tumor suppression percentage (n means the amount of mice on day 27 per group), and (D) body weight over 27 days for tumor-bearing mice that received different treatments as indicated. 3986
DOI: 10.1021/acs.biomac.5b01311 Biomacromolecules 2015, 16, 3980−3988
Article
Biomacromolecules the tumor of mice grew up to 60−90 mm3 in average, they were treated by intravenous injection of DOX@PLMB nanoparticles (DOX, 5 mg/kg; B, 10 mg/kg), PLMB nanoparticles, free DOX, and saline, respectively, and this day was designated as day 1. For the DOX@PLMB irradiation group and PLMB irradiation group, a second injection of PLMB nanoparticles was performed to improve the concentration of boron atoms at the tumor site at a dose of 10 mg B/kg on day 2. And then thermal neutrons irradiation was carried out on day 3. The above treatment cycle (drug injection, PLMB nanoparticles injection, and neutrons irradiation) was repeated on days 4−6. The boron atoms concentrations in each tissue and organ of tumor-bearing mice on day 3 after injection of PLMB nanoparticles on day 1 and day 2 were determined using ICP-MS (Figure 7A). The result showed that the boron atoms concentration in the tumor reached 50.3 ppm on day 3. It suggested that the sufficient 10B atoms (9.5 ppm) would capture thermal neutrons to produce lethal effect on cancer cells.4,35,36 For drug groups without neutrons irradiation, fastest growth of tumors was confirmed in negative groups administrated with saline and PLMB nanoparticles, indicating that the blank PLMB nanoparticles did not have antitumor capacity (Figure 7B). It was interesting to find that free DOX had notable tumor growth suppression on day 9, but the inhibition did not last for long. The mice treated with DOX@PLMB nanoparticles showed a better inhibition on tumor growth than free DOX after 13 days, and obtained a tumor suppression of 83.8 ± 15.5% on day 27 (n = 7) (Figure 7C). This improved antitumor capacity can be attributed to the EPR effect, delayed release and accumulation of DOX in the tumor to a higher extent for DOX@PLMB nanoparticles (Figure 6). For the tumor-bearing mice just treated with thermal neutrons irradiation (saline + irradiation group), the significant inhibition was not observed. However, in PLMB + irradiation group, the therapeutic effect was comparable to that of DOX@ PLMB nanoparticles (without irradiation) within 9 days, and the tumor suppression rate was 60.9 ± 18.6% on day 27 (n = 9). The above results strongly indicated that the carboraneconjugated PLMB nanoparticles could effectively deliver enough boron atoms to accumulate in tumor, capture thermal neutrons and produce energetic particle to kill cancer cells. Moreover, when mice were injected with the DOX@PLMB nanoparticles followed by thermal neutrons irradiation, the volume of tumor on day 27 was only 95 ± 31 mm3 (n = 7), which was nearly one-third of that for DOX@PLMB group without irradiation (250 ± 69 mm3). And the final tumor suppression rate was calculated as high as 92.9 ± 18.9%. As shown in Figure S7, the tumors disappeared on the legs of three out of seven mice on day 27. This superior antitumor activity was obviously attributed to the combination of chemotherapy from DOX and BNCT effect from PLMB with irradiation. The body weight of mice with different treatments was also monitored as it reflects general toxicity of different delivery formulations (Figure 7D). The body weight of free DOXtreated mice decreased dramatically to about 85% on day 7, and then gradually increased to the starting level. In contrast, the mice treated with PLMB nanoparticles, DOX@PLMB nanoparticles with irradiation showed a slight weight gain on day 27. In order to further compare the systematic toxicities, alterations of clinical chemical parameters were determined, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), uric acid (UA), urea (UREA), and creatinine (CREA).
It is well-known that these parameters are associated with the function of liver (AST, ALT) and kidney (UA, UREA, CREA) for human beings. The group of DOX displayed higher AST/ ALT ratios than other groups, and the levels for other parameters were proximity to those of the saline group (SI Figure S8). These results demonstrated that the DOX@PLMB nanoparticles could reduce the toxicity caused by the nonspecificity of DOX as mentioned above. Since DOX inhibits tumor growth through apoptosis mechanism,37 TUNEL staining was conducted to examine the apoptosis level in the tumors from different treatments. By TUNEL staining, the presence of apoptotic cells in tissues can be visualized by brown regions. As shown in Figure 8, there
Figure 8. TUNEL staining of U14 tumors from mice that received different treatments on day 27.
were significantly high apoptotic cells in the tumors from the mice that received chemotherapy, radiotherapy, and the concurrent chemoradiation treatment. On the contrary, the tumor from the mice treated by saline and PLMB nanoparticles without any irradiation showed few apoptotic cells. This was in line with no therapeutic effects of blank nanoparticles (Figure 7B). On the basis of these results, it could be inferred that chemotherapy of DOX and BNCT showed synergetic therapeutic effect through a cytotoxic enhancement mechanism. It is reported that the nuclear reaction of BNCT can induce alpha particles and lithium-7 nuclei, and induce specific cell death through the double strand break of DNA.38,39 When DOX is present in tumor at the time of irradiation, the treatment might enhance cell death by modulating the induction or processing of DNA damage. The action mode and mechanism of these two therapies should be further proven.
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CONCLUSION We fabricated DOX-loaded carborane-conjugate polymeric nanoparticles to combine chemotherapy and BNCT. PLMB was synthesized through the versatile reaction between decaborane and alkynyl groups of PLM. Due to the hydrophobicity of carborane, PLMB self-assembled into nanoparticles in water. Taking advantage of stable conjugation and excluded-volume effect of PEG, PLMB nanoparticles showed few leakages of carborane in PBS (pH 7.4), prolonged blood circulation time and enhanced accumulation of boron species at tumor site. Owing to the dihydrogen bonds between carborane and DOX, DOX was readily encapsulated into the core of PLMB nanoparticles, and DOX@PLMB nanoparticles exhibited a pH-dependent release behavior in vitro. Ex vivo imaging and fluorescence quantitative analysis showed that DOX@PLMB nanoparticles showed a greater and long-lasting DOX accumulation in the tumor than free DOX. It was worth noticing that the DOX@PLMB nanoparticles with thermal 3987
DOI: 10.1021/acs.biomac.5b01311 Biomacromolecules 2015, 16, 3980−3988
Article
Biomacromolecules
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neutrons irradiation showed the highest therapeutic efficacy than that of individual chemotherapy or BNCT in vivo. The selective and noninvasive polymeric nanoparticles delivery system can be an important exploration for chemoradiotherapy and represents a promising approach to develop highly efficient and low toxicity therapeutics.
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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.biomac.5b01311. Additional experimental protocols, characterization data for new compounds, and additional results (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Tel.: +86-13159757035. E-mail:
[email protected]. *Tel.: +86-0431-85262769. 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 (51321062 and 21174143) and Ministry of Science and Technology of China (863 Project, No. SS2012AA020603). This work was performed by using facilities of Department of Physics of Northeast Normal University and assistance of Pro. Shiwei Jing.
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REFERENCES
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DOI: 10.1021/acs.biomac.5b01311 Biomacromolecules 2015, 16, 3980−3988
