Thermoresponsive Delivery of Paclitaxel by β-Cyclodextrin-Based

Department of Biomedical Engineering, Faculty of Engineering, Faculty of Engineering, National University of Singapore, 7 Engineering Drive 1, Singapo...
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Thermoresponsive Delivery of Paclitaxel by #-Cyclodextrin-based Poly(N-isopropylacrylamide) Star Polymer via Inclusion Complexation Xia Song, Yuting Wen, Jing-Ling Zhu, Feng Zhao, Zhongxing Zhang, and Jun Li Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01344 • Publication Date (Web): 24 Oct 2016 Downloaded from http://pubs.acs.org on October 26, 2016

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Thermoresponsive Delivery of Paclitaxel by βCyclodextrin-based Poly(N-isopropylacrylamide) Star Polymer via Inclusion Complexation Xia Song,† Yuting Wen,† Jing-ling Zhu,† Feng Zhao,† Zhong-Xing Zhang,‡ and Jun Li*,† †

Department of Biomedical Engineering, Faculty of Engineering, Faculty of Engineering,

National University of Singapore, 7 Engineering Drive 1, Singapore 117574, Singapore ‡

Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology

and Research), 2 Fusionopolis Way, Singapore 138634, Singapore

ABSTRACT

Paclitaxel (PTX), a hydrophobic anticancer drug, is facing several clinical limitations such as low bioavailability and drug resistance. To solve the problems, a well-defined β-cyclodextrinpoly(N-isopropylacrylamide) star polymer was synthesized and used as a nanocarrier to improve the water solubility and aim to thermoresponsive delivery of PTX to cancer cells. The star polymer was able to form supramolecular self-assembled inclusion complex with PTX via hostguest interaction at room temperature, which is below the low critical solution temperature 1 ACS Paragon Plus Environment

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(LCST) of the star polymer, significantly improving the solubilization of PTX. At body temperature (above LCST), the phase transition of poly(N-isopropylacrylamide) segments induced formation of nanoparticles, which greatly enhanced the cellular uptake of the polymerdrug complex, resulting in efficient thermoresponsive delivery of PTX. Particularly, the polymer-drug complex exhibited better antitumor effects than the commercial formulation of PTX in overcoming the multidrug resistance in AT3B-1 cells.

KEYWORDS β-cyclodextrin, poly(N-isopropylacrylamide), paclitaxel, star polymer, host-guest interaction.

1. INTRODUCTION Paclitaxel (PTX) is a natural anticancer drug that was first isolated from the bark of the western yew, Taxus brevifolia, by Wani et al., and has been widely used to treat various cancers such as head, neck, breast and ovarian cancers.1,

2

PTX works by promoting tubulin

polymerization and microtubules stabilization against depolymerization whereby the normal tubule dynamics essential for cell division was interfered.3, 4 One of the main clinical limitations for PTX is its very poor water solubility. The formulation for clinical administration of PTX contains Cremophor EL (polyoxyethylated castor oil) in a 1:1 v/v mixture with absolute alcohol. Cremophor EL is associated with many serious side effects including bronchospasms, hypotension and hypersensitivity which were observed at almost every phase in both preclinical and clinical trials. Therefore, its dosage and infusion period were both restricted.5 Moreover, same as other anticancer drugs, PTX also faces the problem of multidrug resistance (MDR), the ability of the cancer cells to become resistant to different drugs simultaneously.6, 7 One potential 2 ACS Paragon Plus Environment

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strategy to overcome MDR is to encapsulate anticancer drugs into carriers that deliver them to cancer cells. As a result, there have been urgent needs to develop different formulations as effective carriers for PTX to improve its physiological availability, reduce its side effects and overcome MDR in cancer cells. Cyclodextrins (CDs), a series of cyclic oligosaccharides, are composed of 6, 7, or 8 D(+)glucose units linked by α-1,4-linkages, named α-, β-, or γ-CD, respectively. CDs have a hydrophobic interior and hydrophilic exterior, being capable of forming supramolecular inclusion complexes with various molecules which can fit into the cavities of CDs.8-11 CDs are water soluble with low toxicity and are widely used to improve the solubility and stability of many small molecules including hydrophobic drugs.12, 13 In the past two decades, there have been many investigations on the inclusion complexes formed between PTX and various CDs and CD derivatives.14-21 α-CD was proven to have relatively poor complexation capacity with the anticancer drugs due to its smaller cavity than β- and γ-CD.22 The enhancement of PTX solubility by β-CD and its derivatives has been widely studied. The apparent 1:1 stability constants for β-CD/PTX complex in water was found to be 337 M-1.23 Dimethyl-β-cyclodextrin (DM-β-CD) showed remarkable enhancement of PTX solubility with satisfactory antitumor activity compared to β-CD and other β-CD derivatives due to its more efficient partitioning into the hydrophobic cluster site of PTX.15,

24

Inclusion complexation between β-CD dimers of

different spacer lengths and PTX was also investigated by Moser and Liu’s groups, demonstrating improved solubility of PTX with good stability, high antitumor activity and low toxicity.14,

25

However, their applications are limited by the difficulty of synthesis and

purification of the β-CD dimers. Recently, our group also investigated the solubilization of PTX

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by star-shaped oligoethylenimine (OEI) with a γ-CD core.26 However, γ-CD is relatively expensive. Stimuli-responsive polymers with incorporated stimuli-responsive moieties have attracted a lot of interest and were used to form drug delivery systems that were sensitive to environmental changes.27-29 Poly(N-isopropylacrylamide) (PNIPAAm) is one of the most popular and extensively-studied thermoresponsive polymers. It exhibits reversible phase transition behavior in aqueous solutions, with its lower critical solution temperature (LCST) at 32 °C in water.30 PNIPAAm is water soluble below its LCST, and undergoes a reversible phase transition above its LCST.31, 32 PNIPAAm and its copolymers have been studied and analyzed as “intelligent” systems for their potential applications in biotechnology, nanotechnology and biomedicine areas, such as for controlled drug and gene delivery, biosensor, bioaffinity separation and cell and enzyme immobilization.33-35 There have been increasing studies to take advantage of the thermoresponsive properties of PNIPAAm for enhanced cellular uptake in drug delivery therapy.36-39 Generally, the PNIPAAm chains formed the corona of the micelles or the core-shell nanoparticles. The increase in temperature above its LCST triggered its phase transition, and the PNIPAAm chains became hydrophobic and collapsed, resulting in enhanced cellular uptake and cytotoxicity. In this work, a well-defined 4-arm PNIPAAm star polymer with a β-CD core, which was previously synthesized by our group,40, 41 has been studied and characterized as a supramolecular nanocarrier for drug solubilization enhancement and thermoresponsive delivery of PTX. The ability of this nanocarrier to solubilize PTX by the β-CD core via host-guest interactions in aqueous solutions at room temperature was studied. We hypothesize that the temperatureinduced formation of nanoparticles by the inclusion complexes at 37 °C could promote cellular 4 ACS Paragon Plus Environment

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uptake and then enhance the delivery of PTX. Moreover, the enhanced antitumor effect of the inclusion complexes in overcoming MDR was also evaluated in cell culture systems.

2. MATERIALS AND METHODS 2.1. Materials. β-cyclodextrin (β-CD, ≥ 98.0%) and methyl-β-cyclodextrin (Me-β-CD) were purchased from Tokyo Chemical Industry Co. Ltd (TCI). 2-Hydroxypropyl-β-cyclodextrin (HP-β-CD) was purchased from Alfa Aesar. N-Isopropylacrylamide (NIPAAm, > 98.0%, TCI) was purified by recrystallization from hexane and dried at 100 °C under vacuum before use. Copper(I) bromide (CuBr, ≥ 98.0%), tris[2-(dimethylamino)ethyl]amine (Me6-TREN, 97%), rhodamine B (RhB, ≥ 95.0%) and fluorescein isothiocyanate (FITC, ≥ 90.0%) were purchased from Sigma-Aldrich. Poly(ethylene glycol) with average molecular weight of 20000 Da (PEG20k) was purchased from Sigma-Aldrich. Cremophor EL was purchased from Sigma-Aldrich. Paclitaxel (PTX, 99.5%) was purchased from LC Laboratories. DMSO-d6, D2O and CDCl3 used as solvents in the NMR measurements were obtained from Aldrich. All other reagents were used as received without further purification. 2.2. Synthesis of β-CD-core PNIPAAm Star Polymer (β β CD-(PNIPAAm)4) via ATRP Method. βCD-4Br macroinitiator and βCD-(PNIPAAm)4 4-arm star polymer were synthesized according to our previous report.40, 41 The feed ratio of [NIPAAm]:[CD-Br]:[CuBr]:[Me6-TREN] was increased to 40:1:1:1.5 to obtain star polymer with higher degree of polymerization per arm, where [NIPAAm], [CD-Br], [CuBr] and [Me6-TREN] represented the concentrations of NIPAAm monomers, initiation sites of the macroinitiator, CuBr and Me6-TREN, respectively.

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2.3. Solubilization of PTX by β CD-(PNIPAAm)4 via Inclusion Complexation. The following is the general procedure for preparation of inclusion complexes between βCD(PNIPAAm)4 and PTX. To a solution of βCD-(PNIPAAm)4 (86 mg, 0.005 mmol) in DI water (4 mL) was added a solution of PTX (0.85 mg, 0.001 mmol) in ethanol (4 mL). The mixture was stirred at room temperature for 5 days under dark, and then centrifuged to remove the insoluble PTX. After evaporating the solvent to around 4 mL under vacuum by a rotary evaporator, the residue was centrifuged again to remove the precipitate and lyophilized to yield 71.2 mg of white solid βCD-(PNIPAAm)4/PTX (yield, 81.9%). 1H NMR (400 MHz, D2O): δ 7.22-8.13 (m, 15 H, ArH of PTX), 4.80-5.65 (m, 39 H, C(1)H of β-CD, H of PTX), 2.87-4.55 (m, 867 H, C(2)HC(6)H of β-CD, -CH(CH3)2 of PNIPAAm, H of PTX), 0.80-2.00 (m, 5778 H, -CH2-CH(CONH)-, -CH2-CH(CONH-)- and -CH(CH3)2 of PNIPAAm, H of PTX). β-CD/PTX, methyl-β-CD/PTX (Me-β-CD/PTX), and 2-hydroxypropyl-β-CD/PTX (HP-βCD/PTX) were prepared in the same way as described above, using β-CD (5.7 mg, 0.005mmol), Me-β-CD (7.1 mg, 0.005mmol) and HP-β-CD (7.0 mg, 0.005mmol), respectively. The yield of β-CD/PTX, Me-β-CD/PTX and HP-β-CD/PTX was 3.5 mg (53%), 6.1 mg (77%) and 4 mg (51%), respectively. Linear PNIPAAm of Mn 11750 (58.8 mg) without β-CD was also tested in the same way as a control experiment, and 48.6 mg of PNIPAAm was recovered. 2.4. Fluorescence Labeling. FITC was conjugated to the hydroxyl group of cyclodextrin. βCD-(PNIPAAm)4 polymer (100 mg) was dissolved in 4 mL of anhydrous DMSO, and 23 mg of FITC was added to the solution. The reaction solution was allowed to stir for 24 h at room temperature. After that, the solution was directly dialyzed against water (MWCO 2000) to remove the unreacted FITC and the product was then obtained by freeze-drying. All the experiments were conducted in the dark. Yield: 90 mg, 74%. 6 ACS Paragon Plus Environment

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PEG-20k was labeled with FITC following the same protocol as described above. Yield: 104 mg, 87%. 2.5. Nuclear Magnetic Resonance (NMR) Spectroscopy. 1H and 13C NMR spectra were recorded on a Bruker AV-400 NMR spectrometer at 400 MHz and 100 MHz at room temperature, respectively. The 1H NMR measurements were carried out with an acquisition time of 3.2 s, a pulse repetition time of 2.0 s, a 30° pulse width, 5208 Hz spectral width, and 32 K data points. Chemical shifts were referenced to the solvent peak (δ = 4.70 ppm for D2O, and δ = 2.50 ppm for DMSO, δ = 7.26 ppm for CDCl3). 2.6. Gel Permeation Chromatography (GPC). Gel permeation chromatography (GPC) measurement was performed with a Shimadzu SIL-10A and LC-20AD system equipped with two Phenogel 10 µm 100 and 10000 Å columns (size: 300 × 7.8 mm) connected in series and a Shimadzu RID-10A refractive index detector. Tetrahydrofuran (THF) was used as the mobile phase at a flow rate of 0.6 mL/min at 40 °C. The system was calibrated using monodispersed PEG standards. 2.7. Fourier Transform Infrared Spectroscopy (FT-IR). FT-IR spectrum of βCD(PNIPAAm)4 in potassium bromide (KBr) was measured on a Shimadzu IRPrestige-21 spectrometer in the region of 4000-400 cm-1. 2.8. Ultraviolet-visible Spectroscopy. All absorption spectra were measured with a UVvis spectrophotometer (Shimadzu UV-2600) against PBS or 1:1 v/v mixture of methanol and PBS as blank. Absorption of βCD-(PNIPAAm)4 (2.0 mg/mL), βCD-(PNIPAAm)4/PTX inclusion complexes (2.0 mg/mL) and PTX (0.02 mg/mL) were measured in quartz cuvettes (frosted wall, 0.7 mL).

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2.9. Differential Scanning Calorimetry (DSC). Thermal analysis of the samples was performed via differential scanning calorimetry (Mettler Toledo DSC 1 STARe System). Samples (approximately 10 mg) were heated in sealed aluminium pans at 10 °C/min from 25 °C to 180 °C and cooled at -10 °C/min from 180 °C to 25 °C twice under N2 purge. The samples were heated again at 10 °C/min from 25 °C to 230 °C. The thermogram shows the last heating curve. Both inclusion complexes and physical mixtures of βCD-(PNIPAAm)4 and paclitaxel were analyzed. 2.10. Lower Critical Solution Temperature (LCST) Determination. The LCST of βCD(PNIPAAm)4 (2.0 mg/mL) and βCD-(PNIPAAm)4/PTX inclusion complexes (2.0 mg/mL) in deionized water (DI water) or PBS buffer solutions were measured with a UV-vis spectrophotometer (Shimadzu UV-2600) equipped with a Julabo F12-ED refrigerated/heating circulator. Temperature was increased from 25 °C to 45 °C. The temperature was increased by 1 °C for every 30 min at the temperature range between 32 °C and 38 °C. Transmittance of the sample polymers (in a 1 cm sample cell referenced against DI water or PBS) at 500 nm were measured after equilibrating at each temperature for 30 min. LCST was defined as the temperature at which the optical transmittance of an aqueous solution of the polymer decreases to 50% of total decrease at 500 nm.42 2.11. Dynamic Light Scattering (DLS) Measurement. The hydrodynamic size was measured on a Zetasizer Nano ZS (Malvern Instruments Ltd., MA, USA), with a laser light wavelength of 633 nm at a 173° scattering angle. The polymer solutions or inclusion complex solutions of various concentrations were prepared by dissolving predetermined amount of the polymers or inclusion complexes in PBS. The measurements were performed at 25 °C and 37 °C. The sample solution was equilibrated at the particular temperature for 30 min before the 8 ACS Paragon Plus Environment

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measurement was taken. Average hydrodynamic sizes based on number percentage were given by the instrument. 2.12. Inclusion Complexation between βCD-(PNIPAAm)4-FITC and Rhodamine B. βCD-(PNIPAAm)4-FITC (30 mg, 0.002 mmol) and Rhodamine B (RhB) (0.8 mg, 0.002 mmol) were dissolved in DI water (1.5 mL), followed by sonication for 10 min and stirring at room temperature for 24 h. The inclusion complex solution was dialysed (MWCO 1000 Da) for 1 day, and then lyophilized (Yield: 19 mg, 61%). The inclusion complex formed between βCD(PNIPAAm)4-FITC and Rhodamine B was named as βCD-(PNIPAAm)4-FITC/RhB. All the experiments were conducted in the dark. To determine the loading level, βCD-(PNIPAAm)4FITC/RhB was dissolved in PBS (1 mg/mL). The concentration of RhB was determined by measuring the absorbance of PBS with a microplate reader (Infinite M200 PRO, TECAN) at a wavelength of 554 nm, with reference to calibration plot obtained from RhB working standards in PBS. 2.13. In Vitro Cellular Uptake of βCD-(PNIPAAm)4-FITC/RhB. Briefly, AT3B-1 (MDR+) cells were seeded on cover slips in 24-well plates (8 × 104 cells/well) in 0.4 mL growth medium and allowed to attach and grow for 20 to 24 h. The cells were then incubated with βCD(PNIPAAm)4-FITC/RhB (0.1 mg/mL), prepared with FITC-labeled βCD-(PNIPAAm)4 in DMEM medium at 25 °C and 37 °C for different durations. After different pre-determined time (1 h, 4 h, 8 h and 24 h), βCD-(PNIPAAm)4-FITC/RhB solution was removed. The cells were washed with PBS for three times and fixed with 4% paraformaldehyde. The cover slips with fixed cells were mounted onto glass slide with FluoroshieldTM with DAPI, then imaged using an Olympus Fluoview FV1000 confocal laser scanning microscope (Olympus, Japan) equipped with a 100 × 1.35 NA oil immersion objective lens. Green fluorescence was excited by 473 nm 9 ACS Paragon Plus Environment

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laser line and detected using a 519 nm filter. The excitation wavelength for red fluorescence was set at 559 nm with an emission at 619 nm. The excitation wavelength for blue fluorescence was set at 405 nm with an emission at 461 nm. FITC-labeled PEG-20k (PEG-20k-FITC) (0.1 mg/mL) was used as control, and cellular uptake of PEG-20k-FITC in AT3B-1 (MDR+) cells was carried out following the same protocols as described above. 2.14. In Vitro Cytotoxicity of β CD-(PNIPAAm)4/PTX. In vitro cytotoxicity of βCD(PNIPAAm)4/PTX

was

evaluated

by

using

3-(4,

5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium bromide (MTT) viability assay against AT3B-1-N (MDR-) and AT3B-1 (MDR+) cell lines. Both cells (1.5 × 104 cells/well) were seeded in 96-well plates in 0.1 mL DMEM medium and incubated for 20-24 h prior to the addition of sample solutions. The cells were incubated with 0.1 mL growth medium containing βCD-(PNIPAAm)4/PTX (Cinclusion complex = 0.1 mg/mL, CPTX = 0.001 mg/mL), Taxol (CPTX = 0.001 mg/mL) or βCD-(PNIPAAm)4 host polymer (Chost polymer = 0.1 mg/mL) at 25 °C and 37 °C for 24 h or 48 h. Then 10 µL of sterile MTT stock solution (5 mg/mL in PBS) was added to each well. After incubation for 4 h, the unreacted dye was removed by aspiration. The formazan crystals were dissolved in DMSO (150 µL/well), and the absorbance was measured with a microplate reader (Infinite M200 PRO, TECAN) at a wavelength of 570 nm. The cell viability (%) was calculated according to the following equation: Cell viability (%) = OD570(sample)/OD570(control) × 100, where sample was the well treated with βCD-(PNIPAAm)4/PTX, Taxol or βCD-(PNIPAAm)4 host polymer, and control represented the well treated with DMEM medium only. Paclitaxel (PTX) is a natural anticancer drug that was derived from the bark of the western yew, Taxus brevifolia, by Wani et al., and has been widely used to effectively treat various 3. RESULTS AND DISCUSSION 10 ACS Paragon Plus Environment

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3.1. Formation of Inclusion Complexes. The βCD-(PNIPAAm)4 4-arm star polymer was synthesized according to our previous reported protocols,40, 41 as shown in Figure S1. The star polymer has a degree of polymerization of 32 per arm, i.e. 32 units of NIPAAm per arm, thus is denoted by βCD-(N32)4. βCD-(N32)4 and all control compounds β-CD, Me-β-CD, and HP-β-CD were dissolved with PTX in 5:1 molar ratio in a mixture solution of water/ethanol (1:1 v/v) for five days in the dark. PTX molecules were encapsulated into the hydrophobic β-CD core of these compounds. The

1

H NMR spectroscopy provides direct evidence for the successful inclusion

complexation between βCD-(N32)4 and PTX (Figure 1). As PTX is poorly soluble in water,43 it gives no signal in the 1H NMR spectrum in D2O solvent. Therefore, the appearance of the phenyl signals from PTX in the 1H NMR spectrum of βCD-(N32)4/PTX strongly supports the formation of inclusion complexes, which further proves the star polymer’s ability to solubilize PTX in aqueous solution. The stoichiometric information could be determined by comparing the integration of the phenyl signals (δ 7.2-8.2 ppm) derived from PTX to those for the 1-positioned protons (-C(1)H) of β-CD in the region of 4.93 ppm. It is calculated that the ratio of βCD-(N32)4 to encapsulated PTX is 5:1, corresponding to 1.0 wt% loading level and 83% of encapsulation efficiency of PTX in the inclusion complexes. However, the 1H NMR spectra of β-CD/PTX, Meβ-CD/PTX, and HP-β-CD/PTX show negligible phenyl signals, much lower than that of βCD(N32)4/PTX in D2O, indicating low solubilization efficiency of PTX by β-CD and other derivatives. The significantly improved solubilization of PTX by βCD-(N32)4 star polymer may be attributed to the multi-substituted long arms of hydrophilic PNIPAAm chains on β-CD core that promoted the formation of inclusion complexes with PTX. Moreover, negligible phenyl

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signals are observed in the spectrum when linear PNIPAAm without β-CD was used for solubilizing PTX, indicating that PTX cannot be solubilized by the PNIPAAm chains alone.

Figure 1. 1H NMR spectra of (a) PTX in CDCl3, (b) PNIPAAm/PTX, (c) β-CD/PTX, (d) Me-βCD/PTX, (e) HP-β-CD/PTX, and (f) βCD-(N32)4/PTX in D2O at room temperature.

UV-vis spectra were also measured to further prove the inclusion complexation. PTX in aqueous solution did not show absorption signal due to its very low water solubility. In contrast, the βCD-(N32)4/PTX inclusion complexes showed higher absorption than βCD-(N32)4 host 12 ACS Paragon Plus Environment

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polymer at λ = 230 nm, the typical signal of PTX (Figure 2). The higher absorption confirmed the solubilization of PTX by βCD-(N32)4 via inclusion complexation.

Figure 2. UV-vis spectra of PTX (CPTX = 0.02 mg/mL) in PBS and in methanol/PBS (1:1) mixture, βCD-(N32)4 (Chost polymer = 2 mg/mL) and βCD-(N32)4/PTX (Cinclusion

complex

= 2 mg/mL)

in PBS.

DSC was performed to further support the successful formation of inclusion complexes in the lyophilized powder (Figure 3). The DSC curve of PTX shows an endothermic peak at 220 °C, due to the melting of the drug. Different from a physical mixture of βCD-(N32)4 and PTX, there was no endothermal melting peak of PTX (at 220 °C) in the βCD-(N32)4/PTX inclusion complexes. The disappearance of the peak confirmed that the successful formation of inclusion complexes between the βCD-(N32)4 and PTX.

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Figure 3. DSC thermograms of (a) PTX, (b) βCD-(N32)4, (c) βCD-(N32)4/PTX inclusion complex, and (d) physical mixture of βCD-(N32)4 and PTX.

3.2. Thermoresponsive Behavior of Inclusion Complexes. The change in the optical properties of βCD-(N32)4 host polymers and βCD-(N32)4/PTX inclusion complexes as a function of temperature in DI water and PBS were monitored by UV-vis spectrophotometer and their LCST were determined from the transmittance in Figure 4. The LCST of βCD-(N32)4 is around 37.3 °C in water and 33.8 °C in PBS. Below LCST, PNIPAAm chains were hydrophilic and soluble in water, and the transmittance was around 100%. When the temperature was increased to above its LCST, the transmittance decreased sharply as PNIPAAm chains became hydrophobic and started to aggregate in the aqueous solution. It was observed that the introduction of PTX into βCD-(N32)4 host polymer did not alter the LCST much. It is noted that there was a 3.5 °C decrease of LCST for both βCD-(N32)4 and βCD-(N32)4/PTX in PBS than in DI water, probably due to the destabilizing effects of the salt ions in the PBS buffer solution.44, 45 These results indicate that, βCD-(N32)4 is hydrophilic and soluble in PBS at 25 °C, and may 14 ACS Paragon Plus Environment

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enhance the solubility of PTX through complexation of PTX with the β-CD core. When temperature increases from room temperature to body temperature (37 °C), βCD-(N32)4 will undergo phase transition and form nanoparticles, which may enhance the cellular uptake and lead to thermoresponsive delivery of PTX.

Figure 4. Turbidity variations of aqueous solutions of βCD-(N32)4 and βCD-(N32)4/PTX in DI water and PBS as a function of temperature. C = 2.0 mg/mL.

The phase transition of PNIPAAm chains from hydrophilic to hydrophobic with increasing temperature triggers the concentration-dependent formation of particles of βCD-(N32)4 or βCD(N32)4/PTX. The size distribution of the polymers was measured by DLS, and the hydrodynamic diameters of the particles based on number percentage given by the instrument were summarized in Table 1. It was found that at room temperature (25 ºC) below its LCST, PNIPAAm chains were hydrophilic and the polymers existed as single molecules. As the temperature is increased to body temperature (37 ºC) above its LCST, onset of phase transition of PNIPAAm chain triggered the formation of particles of different sizes depending on the polymer concentration in PBS. At 37 ºC, 0.1 mg/mL of βCD-(N32)4 host polymer formed particles with size ca. 622 nm. 15 ACS Paragon Plus Environment

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As the concentration increased, the size of the particles measured at 37 ºC by DLS also increased. At a concentration of 0.5 mg/mL, the particle size was increased to over 1000 nm. βCD-(N32)4-FITC/RhB at 0.1 mg/mL also formed nanoparticles with size ca. 572 nm. For 0.1 mg/mL of βCD-(N32)4/PTX inclusion complexes, it formed nanoparticles with size ca. 274 nm which is suitable as drug carriers for delivery of drugs.

Table 1. Average hydrodynamic diameters of βCD-(N32)4 host polymer and its inclusion complexes of various concentrations in PBS at 25 ºC and 37 ºC measured by DLS.

Sample

Concentration (mg/mL)

βCD-(N32)4

Hydrodynamic diametera (nm) 25 ºCb

37 ºCb

0.50

4.7 ± 0.6

1452 ± 63

βCD-(N32)4

0.20

4.5 ± 0.5

841 ± 58

βCD-(N32)4

0.10

3.0 ± 1.8

622 ± 47

βCD-(N32)4/PTX

0.10

3.6 ± 2.0

274 ± 34

0.10 4.9 ± 0.7 572 ± 26 βCD-(N32)4-FITC/RhB Mean diameters and standard deviations based on number percentage from three individual DLS measurements. b Sample was equilibrated at the measurement temperature for 30 min before measurement was taken. a

3.3. Thermoresponsive Cellular Uptake of βCD-(N32)4-FITC/RhB inclusion complex. In the study of cellular uptake of the thermoresponsive inclusion complex, Rhodamine B (RhB) was used as a reporter dye,46-48 complexed with FITC-labeled βCD-(N32)4 (βCD-(N32)4-FITC). The βCD-(N32)4-FITC/RhB inclusion complexes were formed by physical interactions in water for 24 h in the dark. The RhB loading level of the final inclusion complex was calculated to be 16 ACS Paragon Plus Environment

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1%. Figure 5 shows the cellular uptake of inclusion complexes between βCD-(N32)4-FITC (green) and RhB (red), as well as FITC-labeled PEG-20k (PEG-20k-FITC) in multidrug resistant AT3B-1 (MDR+) cells at different time points at 25 °C and 37 °C. β-CD of the star polymer was labelled with FITC. Cell nuclei were stained with DAPI (blue). Yellow color indicates the colocalization of RhB and star polymer in merged images, indicating RhB was complexed with the β-CD of the host polymer.49 The PEG-20k-FITC was labeled in the same way as βCD-(N32)4FITC and used as a control. The PEG-20k-FITC was hydrophilic and was often used as the shielding polymer due to its low affinity for cellular uptake. As shown in Figure 5, there was very little uptake of PEG-20kFITC at different incubation time at both temperatures. Similarly, the PNIPAAm chains of βCD(N32)4-FITC were hydrophilic at room temperature, and were less prone to be taken up by the cells due to its hydrophilic nature, as evident by its little cellular uptake and the complexed RhB at 25 °C. In comparison, much higher cellular uptake of βCD-(N32)4-FITC and the complexed RhB was observed at different time points at 37 °C while the cellular uptake of PEG-20k-FITC remained low. This was probably due to the phase transition of the PNIPAAm chains triggered by the temperature increase. The PNIPAAm chains became hydrophobic and formed nanoparticles at body temperature, favoring the cellular uptake of the inclusion complexes, and thus, the enhanced drug delivery ability.

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Figure 5. Confocal microscope images of cellular distribution of FITC-labeled star polymer (green) and RhB (red) in AT3B-1 (MDR+) cells at different time points at 25 °C and 37 °C. βCD of the star polymer was labelled with FITC. Cell nuclei were stained with DAPI (blue). Yellow color indicates the colocalization of RhB and star polymer in the merged images, indicating RhB was inclusion complexed with β-CD. Scale bar is 20 µm.

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3.4. Thermoresponsive Drug Delivery of βCD-(N32)4/PTX Inclusion Complex. In vitro cytotoxicity of βCD-(N32)4/PTX was evaluated by using the MTT viability assay against AT3B1-N (MDR-) and AT3B-1 (MDR+) cell lines. Taxol was prepared by dissolving PTX (6 mg/mL) in a 1:1 v/v mixture of absolute ethanol and Cremophor EL, and was used as a control. As shown in Figure 6, the very low cytotoxicity of βCD-(N32)4 host polymer alone at both temperatures in the two cell lines is indicative of the good biocompatibility of the polymer carriers. Taxol showed antitumor effect in the non-multidrug resistant AT3B-1-N (MDR-) cells at both temperatures. However, taxol had very low cytotoxicity in the multidrug resistant AT3B-1 (MDR+) cells at both temperatures. On the other hand, the βCD-(N32)4/PTX was able to have antitumor effect in both AT3B-1-N (MDR-) and AT3B-1 (MDR+) cell lines at 37 ºC. This may be because, unlike free drug molecules, the PTX was complexed with the host polymer and delivered to the cells by the polymer carrier, evading the recognition and resistant mechanisms by the MDR+ cells. Furthermore, the thermoresponsive properties of the polymer are also illustrated in Figure 6. At low temperatures, the low cellular uptake of the inclusion complexes (due to the hydrophilic nature) resulted in low cytotoxicity in both cell lines. At body temperature, the higher cytotoxicity of the inclusion complex observed is attributed to the higher cellular uptake due to the phase transition of the thermoresponsive PNIPAAm chains of βCD(N32)4 and the formation of nanoparticles.

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(a) 24 h

(b) 48 h

AT3B-1-N (MDR-) 25 °C AT3B-1-N (MDR-) 37 °C 120

***

AT3B-1 (MDR+) 25 °C AT3B-1 (MDR+) 37 °C ***

***

***

*** P < 0.001

100

AT3B-1 (MDR+) 25 °C AT3B-1 (MDR+) 37 °C

AT3B-1-N (MDR-) 25 °C AT3B-1-N (MDR-) 37 °C 120

*** ***

***

***

*** P < 0.001

100 Cell viability (%)

Cell viability (%)

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80 60 40 20

80 60 40 20

0

0

βCD-(N32)4

Taxol

βCD-(N32)4/PTX

βCD-(N32)4

Taxol

βCD-(N32)4/PTX

Figure 6. Cell viability assay in AT3B-1-N (MDR-) and AT3B-1 (MDR+) cells for βCD(N32)4/PTX (Cinclusion

complex

= 0.1 mg/mL, CPTX = 0.001 mg/mL), Taxol (CPTX = 0.001 mg/mL)

and βCD-(N32)4 (Chost polymer = 0.1 mg/mL) host polymer at 25 ºC and 37 ºC for (a) 24 h and (b) 48 h. Each value represents the mean value ± SD (*** p < 0.001, n = 4).

4. CONCLUSIONS In this study, a well-defined β-CD-based PNIPAAm 4-arm star polymer with a degree of polymerization of 32 per arm (βCD-(N32)4) was synthesized as a thermoresponsive nanocarrier to encapsulate and deliver the hydrophobic anticancer drug paclitaxel (PTX). This host polymer greatly improved the water solubility of PTX via host-guest inclusion complexation than β-CD and other β-CD derivatives. The investigation of the thermoresponsive behavior of βCD-(N32)4 host polymer and βCD-(N32)4/PTX inclusion complexes showed that at room temperature (25 ºC), the PNIPAAm chains were hydrophilic, and contributed to the improved solubilization of PTX. When temperature was increased to body temperature (37 ºC), the PNIPAAm chains became hydrophobic and formed nanoparticles which are more favorable for cellular uptake. The 20 ACS Paragon Plus Environment

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in vitro cellular uptake test of βCD-(N32)4-FITC/RhB in AT3B-1 (MDR+) cells demonstrated its enhanced cellular uptake and delivery at body temperature than at room temperature, as a result of the phase transition of PNIPAAm and temperature-induced formation of nanoparticles. Subsequently, this thermoresponsive property of the polymer carrier also contributed to the higher cytotoxicity at 37 ºC than 25 ºC, in both AT3B-1-N (MDR-) and AT3B-1 (MDR+) cells. These results proved this thermoresponsive βCD-(PNIPAAm)4 star polymer a “smart” and effective drug carrier, with improved solubilization of PTX in aqueous solutions, enhanced cellular uptake and therapeutic effect, and the ability to overcome the multidrug resistance in cancer cells, showing great potential for drug delivery applications.

ASSOCIATED CONTENT Supporting Information. Synthesis procedures for βCD-(N32)4 and its molecular characterizations by 1H NMR, 13C NMR, GPC and FT-IR. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Tel.: +65 6516 7273. Fax: +65 6872 3069. E-mail: [email protected]. Notes The authors declare no competing financial interests. ACKNOWLEDGMENT 21 ACS Paragon Plus Environment

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We acknowledge the support from Ministry of Education, Singapore (Grant No. R-397-000-188112 and R-397-000-136-112), National University of Singapore (Grant No. R-397-000-136-731), and Agency for Science, Technology and Research (A*STAR), Singapore (Grant No. 132 148 0007). REFERENCES (1) Schiff, P. B.; Fant, J.; Horwitz, S. B. Nature 1979, 277 (5698), 665-667. (2) Slichenmyer, W. J.; Von Hoff, D. D. J. New Drugs 1990, 30 (9), 770-788. (3) Cragg, G. M. Med. Res. Rev. 1998, 18 (5), 315-331. (4) Slichenmyer, W. J.; Von Hoff, D. D. Anti-Cancer Drugs 1991, 2 (6), 519-530. (5) Surapaneni, M. S.; Das, S. K.; Das, N. G. ISRN Pharmacol. 2012, 2012, 15. (6) Gottesman, M. M.; Fojo, T.; Bates, S. E. Nat. Rev. Cancer 2002, 2 (1), 48-58. (7) Szakacs, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.; Gottesman, M. M. Nat. Rev. Drug. Discov. 2006, 5 (3), 219-234. (8) Li, J.; Loh, X. J. Adv. Drug Delivery Rev. 2008, 60 (9), 1000-1017. (9) Szejtli, J. Chem. Rev. 1998, 98 (5), 1743-1754. (10) Zhu, J. L.; Liu, K. L.; Wen, Y. T.; Song, X.; Li, J. Nanoscale 2016, 8 (3), 1332-1337. (11) Higashi, T.; Li, J.; Song, X.; Zhu, J. L.; Taniyoshi, M.; Hirayama, F.; Iohara, D.; Motoyama, K.; Arima, H. ACS Macro Lett. 2016, 5 (2), 158-162. (12) Brewster, M.; Davis, M. E. Nat. Rev. Drug. Discov. 2004, 3 (12), 1023-1035. (13) Uekama, K.; Hirayama, F.; Irie, T. Chem. Rev. 1998, 98 (5), 2045-2076. (14) Liu, Y.; Chen, G.-S.; Chen, Y.; Cao, D.-X.; Ge, Z.-Q.; Yuan, Y.-J. Bioorg. Med. Chem. 2004, 12 (22), 5767-5775. (15) Hamada, H.; Ishihara, K.; Masuoka, N.; Mikuni, K.; Nakajima, N. J. Biosci. Bioeng. 2006, 102 (4), 369-371. (16) Bouquet, W.; Ceelen, W.; Fritzinger, B.; Pattyn, P.; Peeters, M.; Remon, J. P.; Vervaet, C. Eur. J. Pharm. Biopharm. 2007, 66 (3), 391-397. (17) Fenyvesi, F.; Kiss, T.; Fenyvesi, É.; Szente, L.; Veszelka, S.; Deli, M. A.; Váradi, J.; Fehér, P.; Ujhelyi, Z.; Tósaki, Á.; Vecsernyés, M.; Bácskay, I. J. Pharm. Sci. 2011, 100 (11), 47344744. (18) Matsumura, Y. Adv. Drug Delivery Rev. 2011, 63 (3), 184-192. (19) Namgung, R.; Mi Lee, Y.; Kim, J.; Jang, Y.; Lee, B.-H.; Kim, I.-S.; Sokkar, P.; Rhee, Y. M.; Hoffman, A. S.; Kim, W. J. Nat. Commun. 2014, 5, 3702. (20) Cheng, J.; Khin, K. T.; Jensen, G. S.; Liu, A.; Davis, M. E. Bioconjugate Chem. 2003, 14 (5), 1007-1017. (21) Davis, M. E. Adv. Drug Delivery Rev. 2009, 61 (13), 1189-1192. (22) Cserháti, T.; Forgács, E.; Holló, J. J. Pharm. Biomed. Anal. 1995, 13 (4–5), 533-541. (23) Alcaro, S.; Ventura, C. A.; Paolino, D.; Battaglia, D.; Ortuso, F.; Cattel, L.; Puglisi, G.; Fresta, M. Bioorg. Med. Chem. Lett. 2002, 12 (12), 1637-1641. (24) Bouquet, W.; Ceelen, W.; Adriaens, E.; Almeida, A.; Quinten, T.; Vos, F.; Pattyn, P.; Peeters, M.; Remon, J. P.; Vervaet, C. Ann. Surg. Oncol. 2010, 17 (9), 2510-2517. 22 ACS Paragon Plus Environment

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