Article pubs.acs.org/Biomac
Platinum-Incorporating Poly(N‑vinylpyrrolidone)-poly(aspartic acid) Pseudoblock Copolymer Nanoparticles for Drug Delivery Xikuang Yao, Chen Xie, Weizhi Chen, Chenchen Yang, Wei Wu, and Xiqun Jiang* Department of Polymer Science and Engineering, College of Chemistry and Chemical Engineering, and Jiangsu Provincial Laboratory for Nanotechnology, Nanjing University, Nanjing 210093, People’s Republic of China S Supporting Information *
ABSTRACT: Cisplatin-incorporating pseudoblock copolymer nanoparticles with high drug loading efficiency (ca. 50%) were prepared built on host−guest inclusion complexation between β-cyclodextrin end-capped poly(N-vinylpyrrolidone) block and admantyl end-capped poly(aspartic acid) block, followed by the coordination between cisplatin and carboxyl groups in poly(aspartic acid). The host−guest interaction between the two polymer blocks was examined by two-dimensional nuclear overhauser effect spectroscopy. The size and morphology of nanoparticles formed were characterized by dynamic light scattering, zeta potential, transmission electron microscopy, and atomic force microscopy. The size control of nanoparticles was carried out by varying the ratio of poly(N-vinylpyrrolidone) to poly(aspartic acid). The nanoparticles were stable in the aqueous medium with different pH values but disintegrated in the medium containing Cl− ions. The in vitro and in vivo antitumor effects of cisplatin-loaded nanoparticles were evaluated. The biodistribution of the nanoparticles in vivo was studied by noninvasive nearinfrared fluorescence imaging and ion-coupled plasma mass spectrometry. It was found that cisplatin-loaded nanoparticles could effectively accumulate in the tumor site and exhibited significant superior in vivo antitumor activity to the commercially available free cisplatin by combining the tumor volume, body weight, and survival rate measurements.
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INTRODUCTION In recent decades, significant progress has been made in using nanotechnology in drug delivery for cancer therapy based on passive targeting and active targeting routes.1 Due to the chaotic and twisty tumor endothelium termed the enhanced permeability and retention (EPR) effect,2−4 many types of nanometer-scale polymer vehicles have been developed to accumulate selectively in solid tumors.5,6 To obtain effective tumor accumulation of polymer nanoparticles via EPR effect, poly(ethylene glycol) (PEG) is often used to modify the carriers’ surface to gain prolonged blood circulation time for receiving preferable “passive targeting”.7−9 While, with aborative research and feedback from clinical trials, some shortcomings of PEG are also being discovered. It has been demonstrated that PEG-modified nanocarriers preferentially accumulate in the spleen.10 Furthermore, it was published that plasma concentration and residue time of time after time injected PEGylated nanoparticles massively decreased when administrating the nanosystem with a couple of days gap, known as accelerated blood clearance (ABC) phenomenon.11,12 In addition, anti-PEG antibody can be activated upon repeated © XXXX American Chemical Society
administration of PEGylated nanoparticles and PEG additives, subsequently leading to an increase in phagocytosis by the reticuloendothelial system (RES).13,14 These may finally impact the injected dose, treatment efficiency, or even cause side effects and toxicities to patients.15 A substitution for PEG as surface modification agent and stabilizing agent is the employment of highly water-soluble poly(N-vinylpyrrolidone) (PVP).16,17 PVP is a well-known lowfouling, hydrophilic, biocompatible polymer and is widely employed in bioapplication,18,19 such as polymer micelles and liposomes.20 It was demonstrated that PVP integrated with tumor necrosis factor-α (TNF-α) revealed longer blood circulation time than PEG-conjugated TNF-α.21 Also, PVPTNF-α integration showed a more potent antitumor activity than PEGylated TNF-α.22 However, the synthesis of PVP homopolymers and block copolymers with narrow molecular weight distribution is impossible until utilizing reversible Received: April 11, 2015 Revised: May 27, 2015
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DOI: 10.1021/acs.biomac.5b00479 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
Figure 1. Schematic diagram of the preparation of PVP−PASP-CDDP nanoparticles.
vehicles based on host−guest inclusion complexation between β-CD and adamantyl moieties are very limited. In the present study, we synthesized β-CD end-capped PVP by RAFT polymerization using β-CD modified xanthate (βCD-CTA), and AD end-capped poly(aspartic acid) based on ring opening polymerization (ROP) initiated by 1-adamantanamine. By taking advantage of host−guest inclusion complexation between β-CD-terminated poly(N-vinylpyrrolidone) and AD-terminated poly(aspartic acid), the noncovalently connected poly(N-vinylpyrrolidone)-poly(aspartic acid) block copolymers (PVP−PASP) were prepared in complete aqueous solution. The host−guest interaction between the two polymer blocks was examined by two-dimensional (2D) nuclear overhauser effect spectroscopy (NOESY). Subsequently, PVP−PASP copolymers were coordinated with antitumor agent, cisplatin (CDDP), to form CDDP-loaded PVP−PASP nanoparticles (Figure 1). The dynamic diameter and morphology of CDDP-loaded PVP−PASP nanoparticles were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM). The biologic effects of nanoparticles including drug release, cellular uptake and biodistribution were investigated. The antitumor efficiency of the CDDP-loaded PVP−PASP nanoparticles was evaluated in vivo against solid tumor bearing mice.
addition−fragmentation chain transfer (RAFT) polymerization recently.17,23−25 For example, poly(ε-caprolactone)-b-poly(Nvinylpyrrolidone) (PCL-b-PVP) copolymers with controllable molecular weight were recently synthesized by RAFT polymerization using xanthate-terminated PCL.17,23 The influence of PVP block length on cytotoxicity, antiprotein adsorption, circulation time, biodistribution, and antitumor efficiency was systematically examined.23 On the other hand, the nanometer-scale polymer assemblies made of pseudo copolymers (also termed as noncovalently linked copolymers) based on H-bonding, metal−ligand bond, and host−guest interactions have been paid much attention recently.26−28 The various supramolecular vehicles including micelles and vesicles have been developed through host−guest interactions.29 The stimuli-responsive functions were also incorporated into these vehicles.30−33 Compared to the conventional strategy, the noncovalently linking among different components of copolymers provides great easement and flexibility in the preparation of amphiphilic copolymers and their assemblies. Every added synthetic route may sacrifice the purity and productivity of desired copolymers, which can be avoided through separate syntheses. Also, the ratio of every component in covalently linked block copolymers is fixed, while in noncovalently linked copolymers we can modulate the ratio of each part easily. It is found that cyclodextrins (CDs) having both a hydrophilic exterior surface and a hydrophobic interior cavity can accommodate a wide range of guests to form inclusion complexes.34 Quantitative results indicate that βcyclodextrin (β-CD) and adamantane (AD) have strong binding competence with an association constant around 1 × 105 M−1 in water.26 During these decades, numerous stimulisensitive noncovalently connected micelles (NCCMs) based on β-cyclodextrin inclusion complexation have been reported.26 However, the investigations on biologic effects, drug delivery, and antitumor activity in vitro and in vivo for supramolecular
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MATERIALS AND METHODS
Materials. N-Vinypyrrolidone (VP) and potassium O-ethyl xanthate were obtained from Acros. L-Aspartic acid β-benzyl ester, 2bromine propionic acid, N,N′-diisopropylcarbodiimide (DIC), propargyl alcohol, 1-adamantanamine hydrochloride, fluorescein isothiocyanate isomer I (FITC), NIR-797 isothiocyanate, and sodium ascorbate were purchased from Sigma-Aldrich. Cisplatin (CDDP) was purchased from shandong boyuan Co., Ltd. (Jinan, China). Azobis(isobutyronitrile) (AIBN), β-cyclodextrin (β-CD), sodium azide (NaN3), phosphorus pentoxide (P2O5), copper(II) sulfate B
DOI: 10.1021/acs.biomac.5b00479 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
1.0 mL/min at 35 °C. H2O/CH3OH (8:2, v/v) with 0.1 M NaNO3 was used as eluent. Synthesis of Adamantane-Ended Poly(aspartic acid) (ADPASP). AD-ended poly(β-benzyl L-aspartate) (AD-PBLA) was prepared by the ring-opening polymerization of N-carboxy anhydride of β-benzyl L-aspartate (BLA-NCA) initiated by 1-adamantanamine as previously reported.38 Briefly, BLA-NCA (0.75 g, 3.00 mmol) was dissolved in 10 mL of DCM/DMF (9:1, v/v), and 1-adamantanamine (7.6 mg, 0.05 mmol) in DCM (2 mL) was added. The mixture solution was stirred for 48 h at 35 °C under an argon atmosphere and then precipitated in diethyl ether. The product was dried under reduced pressure overnight to obtain admantyl-ended PBLA (ADPBLA). The polymerization degree (DP) of the AD-PBLA product was calculated to be 57 from the peak intensity ratio of the admantyl protons (C10H15-, δ = 1.50−2.00 ppm) at the polymer chain end to the benzyl protons (C6H5-, δ = 7.3 ppm) at the side chain in the 1H NMR spectrum. AD-PASP was prepared from AD-PBLA by removal of the benzyl groups in 0.1 N NaOH at room temperature. Gel permeation chromatography (GPC) was utilized to determine the molecular weight distribution (MWD) of AD-PASP using a PL-GPC 50 integrated GPC system equipped with PL aquagel−OH 30 + 40 columns and an internal refractive index (RI) detector at a flow rate of 1.0 mL/min at 35 °C. Distilled water with 0.2 M NaNO3 was used as eluent. Preparation of CDDP-Loaded PVP−PASP Nanoparticles (PVP−PASP-CDDP). AD-PASP and β-CD-PVP were dissolved in distilled water ([AD]/[β-CD] = 1:1.2, [Asp] = 5 mmol/L) and stirred for 2 days at 37 °C to allow complete host−guest inclusion interaction. Then an appropriate amount of CDDP was added ([Asp]/[CDDP] = 1). The reaction mixture was stirred for 72 h at 37 °C in the dark and CDDP-loaded PVP−PASP nanoparticles were formed spontaneously. The prepared nanoparticles were purified by ultrafiltration (molecular weight cutoff size (MWCO): 100000). Finally, the CDDP-loaded PVP−PASP nanoparticles were designed to PVP−PASP-CDDP nanoparticles (PVP−PASP-CDDP NPs). Characterization of PVP−PASP-CDDP Nanoparticles. 1H NMR 2D NOESY measurements of PVP−PASP block copolymers and PVP−PASP-CDDP nanoparticles were carried out on a Bruker DRX 400 NMR spectrometer. The average hydrodynamic diameter and size distribution of PVP−PASP-CDDP nanoparticles were determined by dynamic light scattering (DLS) using a Brookheaven BI9000AT system (Brookheaven Instruments Corporation, U.S.A.). Zeta potentials of the PVP−PASP-CDDP nanoparticles were measured by Zetaplus analyzer (Brookheaven Instruments Corporation, U.S.A.). All DLS measurements were done with a laser wavelength of 660.0 nm at 25 °C, and each sample was analyzed three times. The morphology of PVP−PASP-CDDP nanoparticles was observed by transmission electron microscopy (TEM, JEOLTEM-100, Japan). A 200-mesh nitrocellulose-covered copper grid was used to place the PVP−PASP-CDDP sample and allowed to dry at room temperature without negative staining and was then examined with the TEM. The morphology of PVP−PASP-CDDP nanoparticles could also be investigated by atomic force microscopy (AFM, SPI3800, Seiko Instruments Inc., Japan). The nanoparticles suspension was diluted to an appropriate concentration, placed on the surface of a clean silicon wafer, and air-dried at room temperature. Afterward, the silicon wafer was observed by atomic force microscopy (AFM, SPI3800, Seiko Instruments, Japan) with a 20 μm scanner in the tapping mode. CDDP-Loading Content and Encapsulation Efficiency. The drug loading content of the PVP−PASP-CDDP nanoparticles was determined by ion-coupled plasma mass spectrometry (ICP-MS, Perkine-Elmer Corporation, U.S.A.). Briefly, after the PVP−PASPCDDP nanoparticles were prepared, the PVP−PASP-CDDP nanoparticle solution was filtrated by ultrafilter (MWCO: 100000). Then the filtrate was diluted and measured by ICP-MS. The amount of the drug in the nanoparticles could be calculated by addition amount of CDDP subtracting CDDP one in supernatant. The following equations were utilized to determine the CDDP loading content and encapsulation efficiency.
pentahydrate, 4-dimethylaminopyridine (DMAP), anhydrous magnesium sulfate (MgSO4), acetone, N,N-dimethylformamide (DMF), dichloromethane (DCM), tetrahydrofuran (THF), petroleum ether, and ethyl acetate were purchased from Nanjing Wanqing Co., Ltd. (Nanjing, China). AIBN was purified by recrystallization before use. DMF and THF were newly distilled before use. All other reagents were of analytical grade from Sigma Chemical Co. and were used without further purification. Human derived lung cancer cell line (A549 cells) and murine hepatic H22 cell line were obtained from Shanghai Institute of Cell Biology (Shanghai, China). Male ICR mice (18−22 g) were supplied from Animal Center of Drum-Tower Hospital (Nanjing, China). N-Carboxy anhydride of β-benzyl Laspartate (BLA-NCA) and mono(6-azido-6-desoxy)-β-cyclodextrin (βCD-N3) were prepared as previously reported.35,36 Synthesis of Alkyne-Ended Chain Transfer Agent (AlkyneCTA). Alkyne-ended chain transfer agent was prepared as previously reported with slight modification.37 2-Bromine propionic acid (2 mL, 22 mmol) was dissolved in 40 mL of acetone, and potassium ethyl xanthate (5.4 g, 33 mmol) was added. The mixture solution was stirred at room temperature for 1 day, followed by filtration. The acetone was removed by rotary evaporation. Then the attained crude yellow liquid was diluted by chloroform, washed 3× with water, and dried overnight by anhydrous magnesium sulfate to obtain acid-ended chain transfer agent (acid-CTA). Second, acid-CTA (1.5 g, 7.7 mmol) and propargyl alcohol (0.5 mL, 8.7 mmol) were dissolved in 30 mL of dichloromethane. Then DMAP (40 mg, 0.3 mmol) and DIC (1.5 mL, 9.7 mmol) were added sequentially. The reaction mixture was stirred in an ice−water bath for 2 days. Then the mixture was filtrated, washed 3×, and dried overnight by anhydrous magnesium sulfate to get alkyne-ended chain transfer agent (alkyne-CTA). The obtained crude yellow oily liquid was purified by column chromatography on silica-gel using petroleum ether/ethyl acetate (95/5, v/v) as eluent. The product was obtained as light yellow liquid and characterized by proton nuclear magnetic resonance (1H NMR, Bruker DPX-300). 1 H NMR (CDCl3): δ 1.42 (−OCH2CH3, 3H, t), 1.60 (CHCH3, 3H, d), 2.54 (CCH, 1H, s), 3.23 (CHCH3, 1H, q), 4.39 (OCH2CH3, 2H, q), 4.61 (OCH2CCH, 2H, s) ppm. Synthesis of β-Cyclodextrin-Modified Xanthate (β-CD-CTA). Alkyne-CTA (160 mg, 0.7 mmol) and mono(6-azido-6-desoxy)-βcyclodextrin (β-CD-N3) (800 mg, 0.7 mmol) were dissolved in 8 mL of DMF. Then copper(II) sulfate (10 mg, 0.04 mmol) and sodium ascorbate (80 mg, 0.4 mmol) were added. The reaction mixture was stirred at 30 °C for 3 days under the protection of an argon atmosphere. After precipitating the reaction mixture in 150 mL of diethyl ether, the product was washed with water and dried in vacuum overnight over phosphorus pentoxide. The product was obtained as a white powder and characterized by proton nuclear magnetic resonance (1H NMR, Bruker DPX-300) and Fourier transform infrared (FT-IR) spectroscopy (Bruker VERTEX80 V, Germany). The molecular structure of the β-CD-CTA was confirmed by comparing the peak intensity ratio of the alkyne-CTA protons (−OCH2CH3, 3H, δ = 1.28 ppm, CHCH3, 3H, δ = 1.45 ppm) with the peak intensity ratio of the β-CD-N3 protons (C-2, −3 OH, 14H, δ = 5.73 ppm) in the 1H NMR spectrum. Electrospray ionization mass spectrometry (ESI-MS) was performed on a Finnigan LCQ mass spectrometer (ThermoFinnigan, San Jose, CA). ESI-MS: m/z = 1414.50 [M + Na]+. Synthesis of β-Cyclodextrin-Ended Poly(N-vinylpyrrolidone) (β-CD-PVP). β-CD-PVP was synthesized by RAFT polymerization. Briefly, β-CD-CTA (140 mg, 0.1 mmol), AIBN (3.2 mg, 0.02 mmol), and fresh distilled VP (2.5 g, 23 mmoL) were placed in the Schlenk flask and degassed via three freeze−pump−thaw cycles. The VP monomer was polymerized in oil bath at 60 °C for 3 days. After the polymerization, the product was precipitated in diethyl ether and dried under reduced pressure overnight to obtain β-CD-PVP. Gel permeation chromatography (GPC) was used to determine the molecular weight distribution (MWD) of β-CD-PVP using a PL-GPC 50 integrated GPC system equipped with PL aquagel−OH 30 + 40 columns and an internal refractive index (RI) detector at a flow rate of C
DOI: 10.1021/acs.biomac.5b00479 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules Scheme 1. Synthesis of β-CD-PVP Homopolymer
drug loading content% =
5% CO2 incubator at 37 °C. The culture was replaced every other day until 80% confluence was reached. Then A549 cells were seeded on a 96-well plate with a density around 5000 cells/well and allowed to adhere for 24 h before the assay. A series of doses of free CDDP, PVP−PASP inclusion complexes and PVP−PASP-CDDP nanoparticles based on CDDP amount were added into the plate at 37 °C for 48 h. Afterward, 50 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) solution (5 mg/mL in PBS, pH = 7.4) was added into each well, and the plate was incubated for another 2 h at 37 °C in the dark. The medium was taken out and 200 μL of acidified isopropanol (0.33 v/v HCl in isopropanol) was added into each well and shaken completely to dissolve the formazan crystals. The absorbance of a single well was measured at 490 nm by a microplate reader (Huadong, DG-5031, NJ). The values were expressed as a percentage of the control cells to the blank cells. Cellular Uptake of Nanoparticles. To trace the cellular uptake of nanoparticles, the PVP−PASP-CDDP nanoparticles were labeled with FITC. The FITC-labeled nanoparticles were prepared as follows: ADPBLA (124.5 mg, 0.01 mmol) was dissolved in appropriate anhydrous DMSO solution, and FITC (0.72 mg, 0.01 mmol) and one drop of triethylamine were added, and then the mixture was stirred for 24 h at 60 °C in the dark. After precipitating the product in diethyl ether, FITC-labeled AD-PBLA was obtained. Then, FITC-labeled AD-PASP was prepared from FITC-labeled AD-PBLA by removal of the benzyl groups in 0.1 N NaOH at room temperature. Through utilizing FITC-
wt of the drug in nanoparticles × 100% wt of the nanoparticles
Encapsulation efficiency% Weight of the drug in nanoparticles = × 100% Weight of the feeding drug In Vitro CDDP Release from the Nanoparticles. The release of CDDP from PVP−PASP-CDDP nanoparticles in phosphate buffered saline (PBS, 0.01 M phosphate buffer containing 150 mM NaCl) at 37 °C on different conditions were evaluated by the dialysis method, as reported previously.39 Briefly, an appropriate amount of PVP−PASPCDDP nanoparticles solution was placed inside a dialysis bag (MWCO, 3.5 kDa). Then, the dialysis bag was placed in an appropriate amount of PBS buffer and gently shaken at 37 °C in a swing bed at 80 rpm. The released Pt PBS solution outside of the dialysis bag was sampled at a specified time point, properly diluted and measured by ion-coupled plasma mass spectrometry (ICP-MS, Hewlette-Packard 4500). In Vitro Cytotoxicity. The cytotoxicity of PVP−PASP-CDDP nanoparticles against human derived lung cancer cell line (A549 cells) was evaluated by MTT assay. Briefly, the A549 cells were cultured in DMEM (Dulbecco’s modified Eagle essential medium) supplemented with 10% (v/v) inactivated FBS (fetal bovine serum) in a humidified D
DOI: 10.1021/acs.biomac.5b00479 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules Table 1. Molecular Weight and Polydispersity of β-CD-PVP Homopolymer
a
sample
AIBN (mg)
β-CD-CTA (mg)
NVP (g)
temp (°C)
reaction time (h)
Mna (g/mol)
Mwa (g/mol)
PDIa
1 2 3 4 5 6
3.2 3.2 3.2 3.2 3.2 3.2
140 140 140 140 140 140
2.5 2.5 2.5 2.5 2.5 2.5
60 60 60 60 60 60
30 36 48 60 72 75
4600 4400 6300 8500 11000 14000
7900 8100 9700 13000 16000 21000
1.72 1.84 1.54 1.53 1.45 1.50
Measured by the GPC in 0.1 M NaNO3 CH3OH/H2O (v/v; 2/8) with PEG calibration.
labeled AD-PASP instead of AD-PASP, the FITC-labeled PVP−PASPCDDP nanoparticles were prepared and purified by ultrafiltration (MWCO: 100000). The A549 cells were seeded in a six-well plate at a density of 1 × 105 cells per well containing a cover glass and allowed to adhere for 24 h in a humidified atmosphere of 5% CO2 at 37 °C. After 24 h incubation, 200 μL of the FITC-labeled PVP−PASP-CDDP nanoparticles suspension was added into the plate. With 6 h further incubation, the cover glass containing adherent cells was taken out, washed thrice with PBS to remove free FITC-labeled PVP−PASP-CDDP nanoparticles, and fixed through inversely putting the cover glass onto the glass slide. DAPI was used to dye the nucleus zone of the cells. Cells were observed by confocal laser scanning microscopy (CLSM, Zeiss LSM 710, Germany). Real-Time Near-Infrared Fluorescence (NIRF) Imaging. All animal experiments were carried out in keeping with the guidelines for animal experiments and were approved by the Animal Care Committee at Drum Tower Hospital. To establish the experimental tumor model, murine hepatic H22 tumor cells (5−6 × 106 cells per mouse) were inoculated subcutaneously at the left flank of male ICR mice (6−8 weeks, provided by Central Animal Laboratory of Nanjing Medical University). Tumor growth was monitored everyday until it reached an acceptable size. The NIR-797-labeled PVP−PASP-CDDP NPs were prepared as follows: first, the dithioesters group of β-CDPVP was converted to the azide group by using thiol−ene click reaction between β-CD-PVP with azidoethyl acrylate, which was prepared as previously reported.40 And utilizing Cu(I) catalyzed azide−alkyne click reaction between the azide group modified β-CDPVP with propargylamine, then the amino group ended β-CD-PVP was synthesized. The impurities were removed by dialysis in deionized water. Then NIR-797 was conjugated to β-CD-PVP by reaction in DMSO in the dark at room temperature for 24 h. Finally, the NIR797-labeled PVP−PASP-CDDP NPs were prepared by using NIR797-labeled β-CD-PVP instead, and purified by ultrafiltration as described above. The NIR-797-labeled PVP−PASP-CDDP NPs were then injected into the H22 tumor-bearing mice via tail vein. The mice were anesthetized with isoflurane at various time points postinjection and imaged using Maestro in vivo near-infrared (NIR) fluorescence imaging system (CRi, Inc., U.S.A.). Once the in vivo imaging was completed, the mice were sacrified, and tumor, heart, liver, spleen, lung, kidney, intestine, and stomach were harvested for isolated organ imaging. Biodistribution Examination In Vivo. To establish H22 tumorbearing mouse model, ICR mice were inoculated on the left flank with H22 tumor cells (4−6 × 106 cells per mouse) as described above. The PVP−PASP-CDDP nanoparticles were injected intravenously at a dose of 6 mg/kg on a CDDP basis 7 days after the inoculation. The mice were sacrificed at specified time points after intravenous administration (n = 3 at each time point). Then, the tumor, liver, spleen, kidney, lung, and heart were harvested, and the blood was collected. Each of the organs and the blood were decomposed in hot perchloric acid and nitric acid. After being evaporated to dryness, resulting precipitates were dissolved in 5 mL of 3% (v/v) nitric acid solution. The concentration of Pt solution was measured by ICP-MS. Finally, the data were normalized to the tissue weight. In Vivo Antitumor Efficacy. H22 bearing mice with the mean tumor size about 20 mm3 were separated into four groups for different formations. Each group has 10 mice. Then, saline, PVP−PASP
inclusion complexes, free CDDP (3 mg/kg), and PVP−PASP-CDDP nanoparticles (3 mg/kg on a CDDP basis) were injected into the mice via the tail vein, respectively. This day was designated as day 1. The tumor volumes were measured in two dimensions every other day using a vernier caliper for 15 days. The tumor volume was calculated as V = a × b2/2, where a and b corresponded to the longest and the shortest diameter of tumor in mm, respectively. Also, the body weight and survival of the mice was supervised every other day as the manifestation of side effects. Statistical Analysis. Student’s t test was employed to determine the differences of tumor inhibition among the groups treated with saline, PVP−PASP inclusion complexes, free CDDP, and PVP−PASPCDDP NPs, and P values less than 0.05 were considered statistically significant.
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RESULTS AND DISCUSSION Preparation of β-Cyclodextrin-Ended Poly(N-vinylpyrrolidone) (β-CD-PVP). Although PVP has highly hydrophilic and great biocompatible properties, the preparation of well-defined PVP with functionalized end group has only recently become possible by RAFT polymerization.17,23 In present work, to ensure only one PVP chain grafted on β-CD, we first synthesized alkyne end-functionalized CTA (alkyneCTA) in two steps (Scheme 1). Then through copper(I)catalyzed azide−alkyne cycloaddition between β-CD-N3 and alkyne-CTA, β-CD-CTA was prepared and used as initiator for the RAFT polymerization of N-vinylpyrrolidone. After the click reaction, the FT-IR absorption peak of 2104 cm−1 corresponding to azide group disappeared completely (Figure S1). The molecular structure of β-CD-CTA was confirmed by 1H NMR spectrum (Figure S2) and ESI-MS spectrum (Figure S3). From Figure S2, we could see the proton peaks from β-CD-N3 and alkyne-CTA, and the proton peak of triazol cycle of β-CDCTA. By comparing the integral intensities of these peaks, we were sure that only one Alkyne-CTA molecule was clicked to one β-CD-N3 molecule, which was consistent with ESI-MS result. Based on RAFT method by using β-CD-CTA as chain transfer agent, different molecular weights of β-CD-PVP were prepared by controlling the reaction time (Table 1). The gel permeation chromatography (GPC) measurements of six βCD-PVP samples all displayed a single and symmetric peak with a small PDI, indicating the controllable RAFT polymerization of NVP by β-CD-CTA (Figure S4). In our previous work, it was found that the longer PVP block had the better antiprotein absorption property and a longer circulation time in vivo than shorter one.23 In this study, β-CD-PVP (Mn = 14000, PDI = 1.50) was chosen as the hydrophilic block to prepare CDDP-loaded nanoparticles. Preparation of Adamantyl End-Capped Poly(aspartic acid) (AD-PASP). Adamantyl end-capped poly(aspartic acid) (AD-PASP) was synthesized by a previously reported procedure with slight modification. Initially, N-carboxy anhydride of α-benzyl L-aspartate (BLA-NCA) was synthesized E
DOI: 10.1021/acs.biomac.5b00479 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules by the Fuchs-Farthing method.35,41−43 Adamantyl-terminated poly(α-benzyl L-aspartate) (AD-PBLA) was then prepared by polymerization of BLA-NCA using 1-adamantanamine as initiator (Scheme 2). The composition of AD-PBLA was
of 1.2:1, were dissolved in distilled water and stirred for 2 days for complete host−guest inclusion complexation between β-CD and AD moieties. The formation of host−guest structures between β-CD-PVP and AD-PASP was proven by 2D nuclear overhauser effect spectroscopy (NOESY) in D2O (Figure 2a).
Scheme 2. Synthesis of AD-PASP Homopolymer
determined by 1H NMR (Figure S5). Next, AD-PASP was prepared by removal of the benzyl groups from AD-PBLA in alkaline aqueous solution. Complete deprotection of AD-PBLA was confirmed by 1H NMR measurement (Figure S6). Because of intramolecular isomerization and racemization of the aspartic acid units in Ad-PASP, β-aspartic units were formed during the deprotection process. As reported previously,44 the ratio of α to β units in the deprotected AD-PASP was 1:3. By modulating the ratio of the monomer BLA-NCA to the initiator 1adamantanamine, we synthesized different polymerization degrees of AD-PASP (Table 2). From the results of 1H Table 2. Molecular Weight and Polydispersity of AD-PASP Homopolymer sample
[monomer]/ [initiator]
temp (°C)
reaction time (h)
Mideala (g/mol)
M1HNMRb (g/mol)
PDIc
1 2 3 4 5
15 30 60 90 245
35 35 35 35 35
48 48 48 48 48
2200 4300 8400 13000 34000
5400 6600 8500 15000 40000
1.05 1.18 1.07 1.09 1.28
Figure 2. 1H NMR NOESY spectra of PVP−PASP (a) and PVP− PASP-CDDP (b) in D2O.
NOE correlation signals arising from the β-CD cavity protons in 3.0−4.0 ppm and the protons of the admantyl moiety in 1.5−2.0 ppm were observed, clearly indicating the formation of PVP−PASP pseudo block copolymers through the inclusion complexation between β-CD-PVP and AD-PASP. After CDDP was added to the solution, the two chloride ligands in CDDP were gradually substituted with H2O molecules and eventually substituted with the carboxylate ligands of Asp units. When the molar ratio of [CDDP]/[Asp] was 1, as the reaction proceeded, the mixture solution became light blue from colorless, suggesting the formation of PVP−PASP-CDDP NPs. The drug loading content was determined to be about 50%, and the encapsulation efficiency was about 66%, indicating the high CDDP payload in the nanoparticles. In addition, when the lyophilized PVP−PASP-CDDP NPs were redispersed in D2O, and measured by NOESY (Figure 2b). NOE correlation signals of the β-CD cavity protons between 3.0 and 4.0 ppm and the protons of the adamantyl moiety in 1.5−2.0 ppm were observed again, clearly proving the good stability of host− guest inclusion complexation in PVP−PASP-CDDP NPs. The dynamic diameter of PVP−PASP-CDDP NPs was determined to be about 115 nm by DLS (Figure 3a). From TEM and AFM
a
Calcd by the [monomer]/[initiator] ratio. bCalcd by comparing the peak intensity of unit protons with the peak intensity of the terminal group protons. cCalcd by the GPC in 0.2 M NaNO3 water with PEG calibration.
NMR spectra (Figure S6) and GPC measurements (Figure S7), it seemed that low polymerization degree (
Platinum-Incorporating Poly(N‑vinylpyrrolidone)-poly(aspartic acid) Pseudoblock Copolymer Nanoparticles for Drug Delivery Xikuang Yao, Chen Xie, Weizhi Chen, Chenchen Yang, Wei Wu, and Xiqun Jiang* Department of Polymer Science and Engineering, College of Chemistry and Chemical Engineering, and Jiangsu Provincial Laboratory for Nanotechnology, Nanjing University, Nanjing 210093, People’s Republic of China S Supporting Information *
ABSTRACT: Cisplatin-incorporating pseudoblock copolymer nanoparticles with high drug loading efficiency (ca. 50%) were prepared built on host−guest inclusion complexation between β-cyclodextrin end-capped poly(N-vinylpyrrolidone) block and admantyl end-capped poly(aspartic acid) block, followed by the coordination between cisplatin and carboxyl groups in poly(aspartic acid). The host−guest interaction between the two polymer blocks was examined by two-dimensional nuclear overhauser effect spectroscopy. The size and morphology of nanoparticles formed were characterized by dynamic light scattering, zeta potential, transmission electron microscopy, and atomic force microscopy. The size control of nanoparticles was carried out by varying the ratio of poly(N-vinylpyrrolidone) to poly(aspartic acid). The nanoparticles were stable in the aqueous medium with different pH values but disintegrated in the medium containing Cl− ions. The in vitro and in vivo antitumor effects of cisplatin-loaded nanoparticles were evaluated. The biodistribution of the nanoparticles in vivo was studied by noninvasive nearinfrared fluorescence imaging and ion-coupled plasma mass spectrometry. It was found that cisplatin-loaded nanoparticles could effectively accumulate in the tumor site and exhibited significant superior in vivo antitumor activity to the commercially available free cisplatin by combining the tumor volume, body weight, and survival rate measurements.
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INTRODUCTION In recent decades, significant progress has been made in using nanotechnology in drug delivery for cancer therapy based on passive targeting and active targeting routes.1 Due to the chaotic and twisty tumor endothelium termed the enhanced permeability and retention (EPR) effect,2−4 many types of nanometer-scale polymer vehicles have been developed to accumulate selectively in solid tumors.5,6 To obtain effective tumor accumulation of polymer nanoparticles via EPR effect, poly(ethylene glycol) (PEG) is often used to modify the carriers’ surface to gain prolonged blood circulation time for receiving preferable “passive targeting”.7−9 While, with aborative research and feedback from clinical trials, some shortcomings of PEG are also being discovered. It has been demonstrated that PEG-modified nanocarriers preferentially accumulate in the spleen.10 Furthermore, it was published that plasma concentration and residue time of time after time injected PEGylated nanoparticles massively decreased when administrating the nanosystem with a couple of days gap, known as accelerated blood clearance (ABC) phenomenon.11,12 In addition, anti-PEG antibody can be activated upon repeated © XXXX American Chemical Society
administration of PEGylated nanoparticles and PEG additives, subsequently leading to an increase in phagocytosis by the reticuloendothelial system (RES).13,14 These may finally impact the injected dose, treatment efficiency, or even cause side effects and toxicities to patients.15 A substitution for PEG as surface modification agent and stabilizing agent is the employment of highly water-soluble poly(N-vinylpyrrolidone) (PVP).16,17 PVP is a well-known lowfouling, hydrophilic, biocompatible polymer and is widely employed in bioapplication,18,19 such as polymer micelles and liposomes.20 It was demonstrated that PVP integrated with tumor necrosis factor-α (TNF-α) revealed longer blood circulation time than PEG-conjugated TNF-α.21 Also, PVPTNF-α integration showed a more potent antitumor activity than PEGylated TNF-α.22 However, the synthesis of PVP homopolymers and block copolymers with narrow molecular weight distribution is impossible until utilizing reversible Received: April 11, 2015 Revised: May 27, 2015
A
DOI: 10.1021/acs.biomac.5b00479 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
Figure 1. Schematic diagram of the preparation of PVP−PASP-CDDP nanoparticles.
vehicles based on host−guest inclusion complexation between β-CD and adamantyl moieties are very limited. In the present study, we synthesized β-CD end-capped PVP by RAFT polymerization using β-CD modified xanthate (βCD-CTA), and AD end-capped poly(aspartic acid) based on ring opening polymerization (ROP) initiated by 1-adamantanamine. By taking advantage of host−guest inclusion complexation between β-CD-terminated poly(N-vinylpyrrolidone) and AD-terminated poly(aspartic acid), the noncovalently connected poly(N-vinylpyrrolidone)-poly(aspartic acid) block copolymers (PVP−PASP) were prepared in complete aqueous solution. The host−guest interaction between the two polymer blocks was examined by two-dimensional (2D) nuclear overhauser effect spectroscopy (NOESY). Subsequently, PVP−PASP copolymers were coordinated with antitumor agent, cisplatin (CDDP), to form CDDP-loaded PVP−PASP nanoparticles (Figure 1). The dynamic diameter and morphology of CDDP-loaded PVP−PASP nanoparticles were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM). The biologic effects of nanoparticles including drug release, cellular uptake and biodistribution were investigated. The antitumor efficiency of the CDDP-loaded PVP−PASP nanoparticles was evaluated in vivo against solid tumor bearing mice.
addition−fragmentation chain transfer (RAFT) polymerization recently.17,23−25 For example, poly(ε-caprolactone)-b-poly(Nvinylpyrrolidone) (PCL-b-PVP) copolymers with controllable molecular weight were recently synthesized by RAFT polymerization using xanthate-terminated PCL.17,23 The influence of PVP block length on cytotoxicity, antiprotein adsorption, circulation time, biodistribution, and antitumor efficiency was systematically examined.23 On the other hand, the nanometer-scale polymer assemblies made of pseudo copolymers (also termed as noncovalently linked copolymers) based on H-bonding, metal−ligand bond, and host−guest interactions have been paid much attention recently.26−28 The various supramolecular vehicles including micelles and vesicles have been developed through host−guest interactions.29 The stimuli-responsive functions were also incorporated into these vehicles.30−33 Compared to the conventional strategy, the noncovalently linking among different components of copolymers provides great easement and flexibility in the preparation of amphiphilic copolymers and their assemblies. Every added synthetic route may sacrifice the purity and productivity of desired copolymers, which can be avoided through separate syntheses. Also, the ratio of every component in covalently linked block copolymers is fixed, while in noncovalently linked copolymers we can modulate the ratio of each part easily. It is found that cyclodextrins (CDs) having both a hydrophilic exterior surface and a hydrophobic interior cavity can accommodate a wide range of guests to form inclusion complexes.34 Quantitative results indicate that βcyclodextrin (β-CD) and adamantane (AD) have strong binding competence with an association constant around 1 × 105 M−1 in water.26 During these decades, numerous stimulisensitive noncovalently connected micelles (NCCMs) based on β-cyclodextrin inclusion complexation have been reported.26 However, the investigations on biologic effects, drug delivery, and antitumor activity in vitro and in vivo for supramolecular
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MATERIALS AND METHODS
Materials. N-Vinypyrrolidone (VP) and potassium O-ethyl xanthate were obtained from Acros. L-Aspartic acid β-benzyl ester, 2bromine propionic acid, N,N′-diisopropylcarbodiimide (DIC), propargyl alcohol, 1-adamantanamine hydrochloride, fluorescein isothiocyanate isomer I (FITC), NIR-797 isothiocyanate, and sodium ascorbate were purchased from Sigma-Aldrich. Cisplatin (CDDP) was purchased from shandong boyuan Co., Ltd. (Jinan, China). Azobis(isobutyronitrile) (AIBN), β-cyclodextrin (β-CD), sodium azide (NaN3), phosphorus pentoxide (P2O5), copper(II) sulfate B
DOI: 10.1021/acs.biomac.5b00479 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
1.0 mL/min at 35 °C. H2O/CH3OH (8:2, v/v) with 0.1 M NaNO3 was used as eluent. Synthesis of Adamantane-Ended Poly(aspartic acid) (ADPASP). AD-ended poly(β-benzyl L-aspartate) (AD-PBLA) was prepared by the ring-opening polymerization of N-carboxy anhydride of β-benzyl L-aspartate (BLA-NCA) initiated by 1-adamantanamine as previously reported.38 Briefly, BLA-NCA (0.75 g, 3.00 mmol) was dissolved in 10 mL of DCM/DMF (9:1, v/v), and 1-adamantanamine (7.6 mg, 0.05 mmol) in DCM (2 mL) was added. The mixture solution was stirred for 48 h at 35 °C under an argon atmosphere and then precipitated in diethyl ether. The product was dried under reduced pressure overnight to obtain admantyl-ended PBLA (ADPBLA). The polymerization degree (DP) of the AD-PBLA product was calculated to be 57 from the peak intensity ratio of the admantyl protons (C10H15-, δ = 1.50−2.00 ppm) at the polymer chain end to the benzyl protons (C6H5-, δ = 7.3 ppm) at the side chain in the 1H NMR spectrum. AD-PASP was prepared from AD-PBLA by removal of the benzyl groups in 0.1 N NaOH at room temperature. Gel permeation chromatography (GPC) was utilized to determine the molecular weight distribution (MWD) of AD-PASP using a PL-GPC 50 integrated GPC system equipped with PL aquagel−OH 30 + 40 columns and an internal refractive index (RI) detector at a flow rate of 1.0 mL/min at 35 °C. Distilled water with 0.2 M NaNO3 was used as eluent. Preparation of CDDP-Loaded PVP−PASP Nanoparticles (PVP−PASP-CDDP). AD-PASP and β-CD-PVP were dissolved in distilled water ([AD]/[β-CD] = 1:1.2, [Asp] = 5 mmol/L) and stirred for 2 days at 37 °C to allow complete host−guest inclusion interaction. Then an appropriate amount of CDDP was added ([Asp]/[CDDP] = 1). The reaction mixture was stirred for 72 h at 37 °C in the dark and CDDP-loaded PVP−PASP nanoparticles were formed spontaneously. The prepared nanoparticles were purified by ultrafiltration (molecular weight cutoff size (MWCO): 100000). Finally, the CDDP-loaded PVP−PASP nanoparticles were designed to PVP−PASP-CDDP nanoparticles (PVP−PASP-CDDP NPs). Characterization of PVP−PASP-CDDP Nanoparticles. 1H NMR 2D NOESY measurements of PVP−PASP block copolymers and PVP−PASP-CDDP nanoparticles were carried out on a Bruker DRX 400 NMR spectrometer. The average hydrodynamic diameter and size distribution of PVP−PASP-CDDP nanoparticles were determined by dynamic light scattering (DLS) using a Brookheaven BI9000AT system (Brookheaven Instruments Corporation, U.S.A.). Zeta potentials of the PVP−PASP-CDDP nanoparticles were measured by Zetaplus analyzer (Brookheaven Instruments Corporation, U.S.A.). All DLS measurements were done with a laser wavelength of 660.0 nm at 25 °C, and each sample was analyzed three times. The morphology of PVP−PASP-CDDP nanoparticles was observed by transmission electron microscopy (TEM, JEOLTEM-100, Japan). A 200-mesh nitrocellulose-covered copper grid was used to place the PVP−PASP-CDDP sample and allowed to dry at room temperature without negative staining and was then examined with the TEM. The morphology of PVP−PASP-CDDP nanoparticles could also be investigated by atomic force microscopy (AFM, SPI3800, Seiko Instruments Inc., Japan). The nanoparticles suspension was diluted to an appropriate concentration, placed on the surface of a clean silicon wafer, and air-dried at room temperature. Afterward, the silicon wafer was observed by atomic force microscopy (AFM, SPI3800, Seiko Instruments, Japan) with a 20 μm scanner in the tapping mode. CDDP-Loading Content and Encapsulation Efficiency. The drug loading content of the PVP−PASP-CDDP nanoparticles was determined by ion-coupled plasma mass spectrometry (ICP-MS, Perkine-Elmer Corporation, U.S.A.). Briefly, after the PVP−PASPCDDP nanoparticles were prepared, the PVP−PASP-CDDP nanoparticle solution was filtrated by ultrafilter (MWCO: 100000). Then the filtrate was diluted and measured by ICP-MS. The amount of the drug in the nanoparticles could be calculated by addition amount of CDDP subtracting CDDP one in supernatant. The following equations were utilized to determine the CDDP loading content and encapsulation efficiency.
pentahydrate, 4-dimethylaminopyridine (DMAP), anhydrous magnesium sulfate (MgSO4), acetone, N,N-dimethylformamide (DMF), dichloromethane (DCM), tetrahydrofuran (THF), petroleum ether, and ethyl acetate were purchased from Nanjing Wanqing Co., Ltd. (Nanjing, China). AIBN was purified by recrystallization before use. DMF and THF were newly distilled before use. All other reagents were of analytical grade from Sigma Chemical Co. and were used without further purification. Human derived lung cancer cell line (A549 cells) and murine hepatic H22 cell line were obtained from Shanghai Institute of Cell Biology (Shanghai, China). Male ICR mice (18−22 g) were supplied from Animal Center of Drum-Tower Hospital (Nanjing, China). N-Carboxy anhydride of β-benzyl Laspartate (BLA-NCA) and mono(6-azido-6-desoxy)-β-cyclodextrin (βCD-N3) were prepared as previously reported.35,36 Synthesis of Alkyne-Ended Chain Transfer Agent (AlkyneCTA). Alkyne-ended chain transfer agent was prepared as previously reported with slight modification.37 2-Bromine propionic acid (2 mL, 22 mmol) was dissolved in 40 mL of acetone, and potassium ethyl xanthate (5.4 g, 33 mmol) was added. The mixture solution was stirred at room temperature for 1 day, followed by filtration. The acetone was removed by rotary evaporation. Then the attained crude yellow liquid was diluted by chloroform, washed 3× with water, and dried overnight by anhydrous magnesium sulfate to obtain acid-ended chain transfer agent (acid-CTA). Second, acid-CTA (1.5 g, 7.7 mmol) and propargyl alcohol (0.5 mL, 8.7 mmol) were dissolved in 30 mL of dichloromethane. Then DMAP (40 mg, 0.3 mmol) and DIC (1.5 mL, 9.7 mmol) were added sequentially. The reaction mixture was stirred in an ice−water bath for 2 days. Then the mixture was filtrated, washed 3×, and dried overnight by anhydrous magnesium sulfate to get alkyne-ended chain transfer agent (alkyne-CTA). The obtained crude yellow oily liquid was purified by column chromatography on silica-gel using petroleum ether/ethyl acetate (95/5, v/v) as eluent. The product was obtained as light yellow liquid and characterized by proton nuclear magnetic resonance (1H NMR, Bruker DPX-300). 1 H NMR (CDCl3): δ 1.42 (−OCH2CH3, 3H, t), 1.60 (CHCH3, 3H, d), 2.54 (CCH, 1H, s), 3.23 (CHCH3, 1H, q), 4.39 (OCH2CH3, 2H, q), 4.61 (OCH2CCH, 2H, s) ppm. Synthesis of β-Cyclodextrin-Modified Xanthate (β-CD-CTA). Alkyne-CTA (160 mg, 0.7 mmol) and mono(6-azido-6-desoxy)-βcyclodextrin (β-CD-N3) (800 mg, 0.7 mmol) were dissolved in 8 mL of DMF. Then copper(II) sulfate (10 mg, 0.04 mmol) and sodium ascorbate (80 mg, 0.4 mmol) were added. The reaction mixture was stirred at 30 °C for 3 days under the protection of an argon atmosphere. After precipitating the reaction mixture in 150 mL of diethyl ether, the product was washed with water and dried in vacuum overnight over phosphorus pentoxide. The product was obtained as a white powder and characterized by proton nuclear magnetic resonance (1H NMR, Bruker DPX-300) and Fourier transform infrared (FT-IR) spectroscopy (Bruker VERTEX80 V, Germany). The molecular structure of the β-CD-CTA was confirmed by comparing the peak intensity ratio of the alkyne-CTA protons (−OCH2CH3, 3H, δ = 1.28 ppm, CHCH3, 3H, δ = 1.45 ppm) with the peak intensity ratio of the β-CD-N3 protons (C-2, −3 OH, 14H, δ = 5.73 ppm) in the 1H NMR spectrum. Electrospray ionization mass spectrometry (ESI-MS) was performed on a Finnigan LCQ mass spectrometer (ThermoFinnigan, San Jose, CA). ESI-MS: m/z = 1414.50 [M + Na]+. Synthesis of β-Cyclodextrin-Ended Poly(N-vinylpyrrolidone) (β-CD-PVP). β-CD-PVP was synthesized by RAFT polymerization. Briefly, β-CD-CTA (140 mg, 0.1 mmol), AIBN (3.2 mg, 0.02 mmol), and fresh distilled VP (2.5 g, 23 mmoL) were placed in the Schlenk flask and degassed via three freeze−pump−thaw cycles. The VP monomer was polymerized in oil bath at 60 °C for 3 days. After the polymerization, the product was precipitated in diethyl ether and dried under reduced pressure overnight to obtain β-CD-PVP. Gel permeation chromatography (GPC) was used to determine the molecular weight distribution (MWD) of β-CD-PVP using a PL-GPC 50 integrated GPC system equipped with PL aquagel−OH 30 + 40 columns and an internal refractive index (RI) detector at a flow rate of C
DOI: 10.1021/acs.biomac.5b00479 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules Scheme 1. Synthesis of β-CD-PVP Homopolymer
drug loading content% =
5% CO2 incubator at 37 °C. The culture was replaced every other day until 80% confluence was reached. Then A549 cells were seeded on a 96-well plate with a density around 5000 cells/well and allowed to adhere for 24 h before the assay. A series of doses of free CDDP, PVP−PASP inclusion complexes and PVP−PASP-CDDP nanoparticles based on CDDP amount were added into the plate at 37 °C for 48 h. Afterward, 50 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) solution (5 mg/mL in PBS, pH = 7.4) was added into each well, and the plate was incubated for another 2 h at 37 °C in the dark. The medium was taken out and 200 μL of acidified isopropanol (0.33 v/v HCl in isopropanol) was added into each well and shaken completely to dissolve the formazan crystals. The absorbance of a single well was measured at 490 nm by a microplate reader (Huadong, DG-5031, NJ). The values were expressed as a percentage of the control cells to the blank cells. Cellular Uptake of Nanoparticles. To trace the cellular uptake of nanoparticles, the PVP−PASP-CDDP nanoparticles were labeled with FITC. The FITC-labeled nanoparticles were prepared as follows: ADPBLA (124.5 mg, 0.01 mmol) was dissolved in appropriate anhydrous DMSO solution, and FITC (0.72 mg, 0.01 mmol) and one drop of triethylamine were added, and then the mixture was stirred for 24 h at 60 °C in the dark. After precipitating the product in diethyl ether, FITC-labeled AD-PBLA was obtained. Then, FITC-labeled AD-PASP was prepared from FITC-labeled AD-PBLA by removal of the benzyl groups in 0.1 N NaOH at room temperature. Through utilizing FITC-
wt of the drug in nanoparticles × 100% wt of the nanoparticles
Encapsulation efficiency% Weight of the drug in nanoparticles = × 100% Weight of the feeding drug In Vitro CDDP Release from the Nanoparticles. The release of CDDP from PVP−PASP-CDDP nanoparticles in phosphate buffered saline (PBS, 0.01 M phosphate buffer containing 150 mM NaCl) at 37 °C on different conditions were evaluated by the dialysis method, as reported previously.39 Briefly, an appropriate amount of PVP−PASPCDDP nanoparticles solution was placed inside a dialysis bag (MWCO, 3.5 kDa). Then, the dialysis bag was placed in an appropriate amount of PBS buffer and gently shaken at 37 °C in a swing bed at 80 rpm. The released Pt PBS solution outside of the dialysis bag was sampled at a specified time point, properly diluted and measured by ion-coupled plasma mass spectrometry (ICP-MS, Hewlette-Packard 4500). In Vitro Cytotoxicity. The cytotoxicity of PVP−PASP-CDDP nanoparticles against human derived lung cancer cell line (A549 cells) was evaluated by MTT assay. Briefly, the A549 cells were cultured in DMEM (Dulbecco’s modified Eagle essential medium) supplemented with 10% (v/v) inactivated FBS (fetal bovine serum) in a humidified D
DOI: 10.1021/acs.biomac.5b00479 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules Table 1. Molecular Weight and Polydispersity of β-CD-PVP Homopolymer
a
sample
AIBN (mg)
β-CD-CTA (mg)
NVP (g)
temp (°C)
reaction time (h)
Mna (g/mol)
Mwa (g/mol)
PDIa
1 2 3 4 5 6
3.2 3.2 3.2 3.2 3.2 3.2
140 140 140 140 140 140
2.5 2.5 2.5 2.5 2.5 2.5
60 60 60 60 60 60
30 36 48 60 72 75
4600 4400 6300 8500 11000 14000
7900 8100 9700 13000 16000 21000
1.72 1.84 1.54 1.53 1.45 1.50
Measured by the GPC in 0.1 M NaNO3 CH3OH/H2O (v/v; 2/8) with PEG calibration.
labeled AD-PASP instead of AD-PASP, the FITC-labeled PVP−PASPCDDP nanoparticles were prepared and purified by ultrafiltration (MWCO: 100000). The A549 cells were seeded in a six-well plate at a density of 1 × 105 cells per well containing a cover glass and allowed to adhere for 24 h in a humidified atmosphere of 5% CO2 at 37 °C. After 24 h incubation, 200 μL of the FITC-labeled PVP−PASP-CDDP nanoparticles suspension was added into the plate. With 6 h further incubation, the cover glass containing adherent cells was taken out, washed thrice with PBS to remove free FITC-labeled PVP−PASP-CDDP nanoparticles, and fixed through inversely putting the cover glass onto the glass slide. DAPI was used to dye the nucleus zone of the cells. Cells were observed by confocal laser scanning microscopy (CLSM, Zeiss LSM 710, Germany). Real-Time Near-Infrared Fluorescence (NIRF) Imaging. All animal experiments were carried out in keeping with the guidelines for animal experiments and were approved by the Animal Care Committee at Drum Tower Hospital. To establish the experimental tumor model, murine hepatic H22 tumor cells (5−6 × 106 cells per mouse) were inoculated subcutaneously at the left flank of male ICR mice (6−8 weeks, provided by Central Animal Laboratory of Nanjing Medical University). Tumor growth was monitored everyday until it reached an acceptable size. The NIR-797-labeled PVP−PASP-CDDP NPs were prepared as follows: first, the dithioesters group of β-CDPVP was converted to the azide group by using thiol−ene click reaction between β-CD-PVP with azidoethyl acrylate, which was prepared as previously reported.40 And utilizing Cu(I) catalyzed azide−alkyne click reaction between the azide group modified β-CDPVP with propargylamine, then the amino group ended β-CD-PVP was synthesized. The impurities were removed by dialysis in deionized water. Then NIR-797 was conjugated to β-CD-PVP by reaction in DMSO in the dark at room temperature for 24 h. Finally, the NIR797-labeled PVP−PASP-CDDP NPs were prepared by using NIR797-labeled β-CD-PVP instead, and purified by ultrafiltration as described above. The NIR-797-labeled PVP−PASP-CDDP NPs were then injected into the H22 tumor-bearing mice via tail vein. The mice were anesthetized with isoflurane at various time points postinjection and imaged using Maestro in vivo near-infrared (NIR) fluorescence imaging system (CRi, Inc., U.S.A.). Once the in vivo imaging was completed, the mice were sacrified, and tumor, heart, liver, spleen, lung, kidney, intestine, and stomach were harvested for isolated organ imaging. Biodistribution Examination In Vivo. To establish H22 tumorbearing mouse model, ICR mice were inoculated on the left flank with H22 tumor cells (4−6 × 106 cells per mouse) as described above. The PVP−PASP-CDDP nanoparticles were injected intravenously at a dose of 6 mg/kg on a CDDP basis 7 days after the inoculation. The mice were sacrificed at specified time points after intravenous administration (n = 3 at each time point). Then, the tumor, liver, spleen, kidney, lung, and heart were harvested, and the blood was collected. Each of the organs and the blood were decomposed in hot perchloric acid and nitric acid. After being evaporated to dryness, resulting precipitates were dissolved in 5 mL of 3% (v/v) nitric acid solution. The concentration of Pt solution was measured by ICP-MS. Finally, the data were normalized to the tissue weight. In Vivo Antitumor Efficacy. H22 bearing mice with the mean tumor size about 20 mm3 were separated into four groups for different formations. Each group has 10 mice. Then, saline, PVP−PASP
inclusion complexes, free CDDP (3 mg/kg), and PVP−PASP-CDDP nanoparticles (3 mg/kg on a CDDP basis) were injected into the mice via the tail vein, respectively. This day was designated as day 1. The tumor volumes were measured in two dimensions every other day using a vernier caliper for 15 days. The tumor volume was calculated as V = a × b2/2, where a and b corresponded to the longest and the shortest diameter of tumor in mm, respectively. Also, the body weight and survival of the mice was supervised every other day as the manifestation of side effects. Statistical Analysis. Student’s t test was employed to determine the differences of tumor inhibition among the groups treated with saline, PVP−PASP inclusion complexes, free CDDP, and PVP−PASPCDDP NPs, and P values less than 0.05 were considered statistically significant.
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RESULTS AND DISCUSSION Preparation of β-Cyclodextrin-Ended Poly(N-vinylpyrrolidone) (β-CD-PVP). Although PVP has highly hydrophilic and great biocompatible properties, the preparation of well-defined PVP with functionalized end group has only recently become possible by RAFT polymerization.17,23 In present work, to ensure only one PVP chain grafted on β-CD, we first synthesized alkyne end-functionalized CTA (alkyneCTA) in two steps (Scheme 1). Then through copper(I)catalyzed azide−alkyne cycloaddition between β-CD-N3 and alkyne-CTA, β-CD-CTA was prepared and used as initiator for the RAFT polymerization of N-vinylpyrrolidone. After the click reaction, the FT-IR absorption peak of 2104 cm−1 corresponding to azide group disappeared completely (Figure S1). The molecular structure of β-CD-CTA was confirmed by 1H NMR spectrum (Figure S2) and ESI-MS spectrum (Figure S3). From Figure S2, we could see the proton peaks from β-CD-N3 and alkyne-CTA, and the proton peak of triazol cycle of β-CDCTA. By comparing the integral intensities of these peaks, we were sure that only one Alkyne-CTA molecule was clicked to one β-CD-N3 molecule, which was consistent with ESI-MS result. Based on RAFT method by using β-CD-CTA as chain transfer agent, different molecular weights of β-CD-PVP were prepared by controlling the reaction time (Table 1). The gel permeation chromatography (GPC) measurements of six βCD-PVP samples all displayed a single and symmetric peak with a small PDI, indicating the controllable RAFT polymerization of NVP by β-CD-CTA (Figure S4). In our previous work, it was found that the longer PVP block had the better antiprotein absorption property and a longer circulation time in vivo than shorter one.23 In this study, β-CD-PVP (Mn = 14000, PDI = 1.50) was chosen as the hydrophilic block to prepare CDDP-loaded nanoparticles. Preparation of Adamantyl End-Capped Poly(aspartic acid) (AD-PASP). Adamantyl end-capped poly(aspartic acid) (AD-PASP) was synthesized by a previously reported procedure with slight modification. Initially, N-carboxy anhydride of α-benzyl L-aspartate (BLA-NCA) was synthesized E
DOI: 10.1021/acs.biomac.5b00479 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules by the Fuchs-Farthing method.35,41−43 Adamantyl-terminated poly(α-benzyl L-aspartate) (AD-PBLA) was then prepared by polymerization of BLA-NCA using 1-adamantanamine as initiator (Scheme 2). The composition of AD-PBLA was
of 1.2:1, were dissolved in distilled water and stirred for 2 days for complete host−guest inclusion complexation between β-CD and AD moieties. The formation of host−guest structures between β-CD-PVP and AD-PASP was proven by 2D nuclear overhauser effect spectroscopy (NOESY) in D2O (Figure 2a).
Scheme 2. Synthesis of AD-PASP Homopolymer
determined by 1H NMR (Figure S5). Next, AD-PASP was prepared by removal of the benzyl groups from AD-PBLA in alkaline aqueous solution. Complete deprotection of AD-PBLA was confirmed by 1H NMR measurement (Figure S6). Because of intramolecular isomerization and racemization of the aspartic acid units in Ad-PASP, β-aspartic units were formed during the deprotection process. As reported previously,44 the ratio of α to β units in the deprotected AD-PASP was 1:3. By modulating the ratio of the monomer BLA-NCA to the initiator 1adamantanamine, we synthesized different polymerization degrees of AD-PASP (Table 2). From the results of 1H Table 2. Molecular Weight and Polydispersity of AD-PASP Homopolymer sample
[monomer]/ [initiator]
temp (°C)
reaction time (h)
Mideala (g/mol)
M1HNMRb (g/mol)
PDIc
1 2 3 4 5
15 30 60 90 245
35 35 35 35 35
48 48 48 48 48
2200 4300 8400 13000 34000
5400 6600 8500 15000 40000
1.05 1.18 1.07 1.09 1.28
Figure 2. 1H NMR NOESY spectra of PVP−PASP (a) and PVP− PASP-CDDP (b) in D2O.
NOE correlation signals arising from the β-CD cavity protons in 3.0−4.0 ppm and the protons of the admantyl moiety in 1.5−2.0 ppm were observed, clearly indicating the formation of PVP−PASP pseudo block copolymers through the inclusion complexation between β-CD-PVP and AD-PASP. After CDDP was added to the solution, the two chloride ligands in CDDP were gradually substituted with H2O molecules and eventually substituted with the carboxylate ligands of Asp units. When the molar ratio of [CDDP]/[Asp] was 1, as the reaction proceeded, the mixture solution became light blue from colorless, suggesting the formation of PVP−PASP-CDDP NPs. The drug loading content was determined to be about 50%, and the encapsulation efficiency was about 66%, indicating the high CDDP payload in the nanoparticles. In addition, when the lyophilized PVP−PASP-CDDP NPs were redispersed in D2O, and measured by NOESY (Figure 2b). NOE correlation signals of the β-CD cavity protons between 3.0 and 4.0 ppm and the protons of the adamantyl moiety in 1.5−2.0 ppm were observed again, clearly proving the good stability of host− guest inclusion complexation in PVP−PASP-CDDP NPs. The dynamic diameter of PVP−PASP-CDDP NPs was determined to be about 115 nm by DLS (Figure 3a). From TEM and AFM
a
Calcd by the [monomer]/[initiator] ratio. bCalcd by comparing the peak intensity of unit protons with the peak intensity of the terminal group protons. cCalcd by the GPC in 0.2 M NaNO3 water with PEG calibration.
NMR spectra (Figure S6) and GPC measurements (Figure S7), it seemed that low polymerization degree (
