Efficient Codelivery of Paclitaxel and Curcumin by Novel Bottlebrush

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Efficient Codelivery of Paclitaxel and Curcumin by Novel Bottlebrush Copolymer-based Micelles Qing Yao,†,§ David C. Gutierrez,‡ Ngoc Ha Hoang,†,∥,⊥ Dongin Kim,† Ruoning Wang,# Christopher Hobbs,*,‡ and Lin Zhu*,† †

Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M University Health Science Center, Kingsville, Texas 78363, United States ‡ Department of Chemistry, Texas A&M University-Kingsville, Kingsville, Texas 78363, United States § Department of Pharmaceutics, College of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 110016 Liaoning, People’s Republic of China ∥ Nanobiopharmaceutics laboratory, College of Pharmacy, Chung-Ang University, Seoul, South Korea ⊥ Department of Pharmaceutics, Hanoi University of Pharmacy, Ha Noi, Vietnam # Center for Childhood Cancer and Blood Disease, The Research Institute at Nationwide Children’s Hospital, Ohio State University, Columbus, Ohio 43025-2696, United States S Supporting Information *

ABSTRACT: The novel self-assembling bottlebrush polyethylene glycolpolynorbornene-thiocresol block copolymers (PEG-PNB-TC) were synthesized by the ring opening metathesis polymerization (ROMP), followed by functionalization of the polymer backbone via the thio-bromo “click” postpolymerization strategy. The PEG-PNB-TC copolymers could easily selfassemble into the nanoscale core−shell polymeric micelles. The hydrophobic anticancer drugs, such as paclitaxel (PTX), could be loaded into their hydrophobic core to form a stable drug-loaded micelle with a superior drug loading capacity of up to ∼35% (w/w). The sustained drug release behavior of the PEG-PNB-TC micelles was observed under a simulated “sink condition”. Compared with commercial PTX formulation (Taxol), the PTX-loaded PEGPNB-TC micelles showed the enhanced in vitro cellular uptake and comparable cytotoxicity in the drug-sensitive cancer cells, while the copolymers were much safer than Cremophor EL, the surfactant used in Taxol. Furthermore, curcumin (CUR), a natural chemotherapy drug sensitizer, was successfully coloaded with PTX into the PEG-PNB-TC micelles. High drug loading capacity of the PEG-PNB-TC micelles allowed for easy adjustment of drug doses and the ratio of the coloaded drugs. The combination of PTX and CUR showed synergistic anticancer effect in both the drug mixture and drug coloaded micelles at high CUR/PTX ratio, while low CRU/PTX ratio only exhibited additive effects. The combinatorial effects remarkably circumvented the PTX resistance in the multidrug resistant (MDR) cancer cells. Due to the easy polymerization and functionalization, excellent self-assembly capability, high drug loading capability, and great stability, the PEG-PNB-TC copolymers might be a promising nanomaterial for delivery of the hydrophobic anticancer drugs, especially for combination drug therapy. KEYWORDS: polynorbornene copolymer, polymeric micelles, drug delivery, combination drug therapy, drug resistance

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INTRODUCTION

of further functionalization also influence the applications of nanomaterials. The development of reliable and functionalized polymers is critical for constructing future drug delivery systems. Recently, research efforts have been focused on developing amphiphilic, self-assembling block copolymers for easy nano-

Thanks to the progress in materials science and engineering, various drug delivery systems have been developed and show advantages over the drug itself. Though the nanocarriers broaden the therapeutic index of the loaded drugs via improving bioavailability and disease specificity and reducing side effects,1 it remains challenging for most formulations/drug delivery systems to achieve both high drug loading and stable nanostructure.2 This is mainly due to the unfavorable physicochemical properties of the building materials of the drug carriers. In addition, the ease of preparation and possibility © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

April 4, 2017 May 17, 2017 June 12, 2017 June 12, 2017 DOI: 10.1021/acs.molpharmaceut.7b00278 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

through ROMP of a hydrophilic macromonomer (NB-PEG16) and (2) a postpolymerization thio-bromo “click” reaction17 for the installation of a highly hydrophobic moiety, thiocresol (TC). The resulting materials, polyethylene glycol-polynorbornene-thiocresol block copolymers (PEG-PNB-TC), could undergo self-assembly in aqueous environments to form polymeric micelles that could be loaded with hydrophobic drugs, such as PTX and CUR (Figure 1). The characteristics of

particle formation and drug loading. These polymer-based micelles are the most intriguing nanoparticles due to their simple preparations and efficient drug loading capabilities.3 The micelle structures and morphologies are highly dependent on polymers’ properties including the properties of monomers, degree of polymerization, and block ratios. A diverse set of block copolymers has become available due to advances in polymerization methodologies, providing a large library of selfassembly materials for biomedical applications.4,5 Among various polymerization methods, ring-opening metathesis polymerization (ROMP) is one of the most attractive strategies for the preparation of such polymers since it can deliver welldefined materials using the exceptionally functional groups tolerant to highly reactive Grubbs-type initiators.6 Because of these attributes, ROMP has been utilized for the preparation of efficient drug delivery vehicles that, mostly, are composed of self-assembling, bottlebrush copolymers, such as recently reported polymer-drug conjugates.7 The amphiphilic bottlebrush (statistical and block) copolymer-based micelles have also been reported.8 In all cases, these structural motifs can easily be accessed using a “grafting-through” ROMP technique of norbornene-terminated polymers (aka macromonomers). However, most of these self-assembling materials are covalently linked (or conjugated) to low molecular weight drugs that can only be released through decomposition reactions. This could possibly affect drug release due to slow and/or incomplete decomposition. So, utilizing these polymeric micelles as noncovalent, encapsulating carriers may be advantageous, as long as the micelles are sufficiently stable. Paclitaxel (PTX), a widely used antineoplastic agent, is able to stabilize microtubules and arrest cancer cells at the G2/M phase, causing cell apoptosis,9 while its outcome is compromised by its low solubility and severe side effects. Although Taxol [PTX in the mixture of Cremophor EL (polyoxyethylene castor oil, a nonionic surfactant)/ethanol] has been developed and widely used in cancer treatment, the side effects, such as the frequent life-threatening hypersensitivity and dose-limiting toxicity, limit its application.10 A lot of effort has been made to tackle the challenges of PTX. The development of polymeric micelles is one of the most straightforward and promising strategies. Due to the inherent characteristics, however, most conventional polymeric micelles have a relatively low drug loading capability, which hampers their clinic applications. It was reported that the long-term exposure of PTX induced drug resistance via the activation of nuclear factor-κB (NF-κB), resulting in cell proliferation, invasion, and metastasis, which could be suppressed by the natural product curcumin (CUR).11,12 CUR can also suppress major drug efflux transporters (Pgp, MRP-1, and ABCG2) to enhance the apoptosis of tumor cells.11 Many studies have shown that the combined use of PTX and CUR could overcome PTX resistance and potentiate PTX’s anticancer activity.11−14 However, like PTX, CUR is poorly water-soluble and its bioavailability needs to be improved. So far, various drug delivery systems have been developed for PTX or CUR, individually. These systems are usually sequentially administered or coadministered as a physical mixture.13−15 As a result, these molecules may not be delivered into the same tumor cell simultaneously, leading to low synergistic effects. For drug codelivery, the drug delivery system should possess high drug loading capability as well as stable micelle structure. In this study, we synthesized a series of amphiphilic, bottlebrush block copolymers using a combination of (1) grafting-

Figure 1. Scheme of the bottlebrush PEG-PNB-TC polymeric micelles.

the copolymers and micelles were studied in terms of the chemical structure, particle size, zeta potential, critical micelle concentration, stability, drug-polymer interaction, drug loading, and drug release. The cellular uptake and cytotoxicity of the drug-loaded micelles were studied in drug-sensitive cancer cells. Furthermore, the drug codelivery capability of the PEG-PNBTC micelles was analyzed on the multidrug resistant cancer cells.

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MATERIALS AND METHODS Materials. Grubbs second generation initiator (G-2) was donated by Materia Inc. Paclitaxol was purchased from LC Laboratories (Worburn, MA) and Curcumin was purchased from Sigma-Aldrich (St. Louis, MO). Cremophor EL was obtained from BASF SE (Ludwigshafen, German). 1,2Dioleoylsn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Rh-PE) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Dialysis tubings (MWCO 12000−14000 Da) were obtained from Spectrum Laboratories, Inc. (Houston, TX). Penicillin streptomycin (PS) solution (100×), trypsin-EDTA, and Hoechst 33258 were purchased from Invitrogen Corporation (Carlsbad, CA). Hank’s Balanced Salt Solution (HBSS) was obtained from Mediatech (Manassas,VA). Fetal bovine serum (FBS) was purchased from VWR International (Radnor, PA). Thiazolyl Blue Tetrazolium Blue (MTT) was purchased from ChemImpex International (Wood Dale, IL). Other chemicals and reagents were purchased from commercial sources (SigmaAldrich, Thermo Fisher Scientific, TCI, Alfa-Aesar) and used as received. 1H NMR spectra were obtained in CDCl3 on a Bruker 300 MHz (operating at 300.128). All peaks were reported in ppm and the CHCl3 peak was a reference. Gel permeation chromatography (GPC) experiments were obtained in THF at room temperature using a Shimadzu RID-20A refractive index detector and polystyrene standards were used as references. The human non-small cell lung cancer (A549) and cervical cancer (HeLa) cells were grown in the Roswell Park Memorial Institute 1640 medium (RPMI) (Invitrogen Corporation, Carlsbad, CA) supplemented with PS (1×) and 10% FBS at 37 °C in a 5% CO2. The multidrug resistant (MDR) ovarian B

DOI: 10.1021/acs.molpharmaceut.7b00278 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics cancer cells (NCI/ADR-RES) were grown in the Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen Corporation) supplemented with PS (1×) and 10% FBS at 37 °C in a 5% CO2. Synthesis, Purification, and Characterization of Bottlebrush Block Copolymers. Representative Procedure for Copolymerization. PEG-PNB/PNB-Br (50/50) was as an example. NB-PEG (2k Da) (0.3 g) and methylene chloride (2 mL) were added and stirred in a 5 mL flask equipped with a rubber septum. This solution was subsequently frozen in liquid nitrogen. At this point, G-2 (0.01 g) was added to the flask and this mixture was degassed under vacuum and refilled with N2 3 times. This mixture was then stirred at room temperature for 3 h. At this time, NB-Br (0.03 g) was added via syringe. The reaction was allowed to proceed for 1 h. The reaction was then allowed to cool to room temperature at which point, ethyl vinyl ether (1 mL) was added and the mixture was stirred for 30 min. The product polymer was isolated after being passed through silica gel followed by removal of solvent under reduced pressure. 1H NMR (CDCl3, 300 MHz): δ 5.54−5.13 (bs, 8H), 4.45−4.30 (bs, 1H), 4.29−3.83 (m, 8H), 3.66 (s, 180H), 3.39 (s, 9H), 3.32−2.29 (bs, 15H). Representative Procedure for Thio-bromo “Click” Functionalization. PEG-PNB/PNB-TC (50/50) was as an example. PNB-PEG/PNB-Br (50/50) (0.13 g) and methylene chloride (3 mL) were added and stirred in a 10 mL flask. To this stirring solution was added thiocresol (TC) (0.08 g) and triethylamine (0.08 mL). The reaction was allowed for 1 h at which the crude polymer was isolated after the solvent was removed under reduced pressure. The crude material was purified via dialysis against water for 48 h before use. The final product was named as PEG-PNB-TC. 1H NMR (CDCl3, 300 MHz): δ 7.33 (d, 2H), 7.14 (d, 2 H), 5.53−5.08 (bs, 2 H), 4.23−3.80 (bs, 2H), 3.67 (s, 64H), 3.57 (m, 1H), 3.39 (s, 1H). Determination of the Critical Micelle Concentration (CMC). The CMC of synthesized copolymers were measured by a pyrene based method.18,19 Briefly, 8 × 10−5 M of pyrene and various concentrations of copolymers (10−5 to 1 mg/mL) were added to the test tubes. The tubes were dried under vacuum and hydrated by HBSS. The fluorescence intensity was recorded on an Infinite M1000 PRO microplate reader at λex = 338 and 334 nm, λem= 390 nm. The ratios of fluorescence intensities at 338 nm over 334 nm (I338/I334) was plotted against the copolymer concentration. The value of the intersection of two tangents was considered as the CMC. Preparation of PEG-PNB-TC Polymeric Micelles. To prepare the PTX-loaded polymeric micelles, 100 μg PTX, and 2 mg copolymers in 1 mL methanol were dried under nitrogen flow. Then, the formed drug-polymer film was hydrated in 1 mL HBSS using vortex for 2 min. For loading of both PTX and CUR, the copolymers, PTX and CUR at various ratios were dissolved in methanol. Then, the same procedures were followed. The unentrapped drug was removed from the micelles by filtration through a 0.45 μm hydrophilic nylon filter (Agela Technologies Inc.).20−24 The drug in the filtrate was quantitated by HPLC. For the preparation of the fluorescent dye-labeled micelles, the drugs were replaced with Rh-PE. The following equations were used to calculate drug loading (DL) and encapsulation efficacy (EE). DL(%) =

EE(%) =

Weight of loaded drugs × 100% Weight of total added drugs

Drug Quantitation by HPLC. The micelles were dissolved in methanol25 and both PTX and CUR were quantitated on a C18 column (250 mm × 4.6 mm) by a Waters HPLC system at 25 °C. PTX was analyzed using acetonitrile/water (60/40, v/v) as the isocratic mobile phase at 1.0 mL/min and detected at 227 nm. CUR was analyzed using acetonitrile/water (90/10, v/ v) at 1.0 mL/min and detected at 420 nm. Particle Size and Zeta Potential. The particle size and zeta potential were determined in PBS by dynamic light scattering (DLS) on a NanoBrook 90Plus PALS Zeta Potential Analyzer. (Brookhaven Instruments). The experiments were performed at 25 °C. Morphological Observations. The morphology of the polymeric micelles was analyzed by transmission electron microscopy (TEM). Briefly, one drop of polymer solution was placed on a 400-mesh carbon-coated copper grid (Ted Pella, USA). The grid was then dried at room temperature for several minutes. The TEM images were taken using JEOL JEM-2010 TEM (JEOL, Japan). Differential Scanning Calorimetry (DSC). The samples including drug powder, PEG-PNB-TC (50/50) copolymers, the physical mixture of PTX and PEG-PNB-TC (50/50) (1/20, wt/wt), and the lyophilized powder of PTX-loaded PEG-PNBTC (50/50) micelles were weighted and sealed in the aluminum pans. The samples were scanned from 20 to 300 °C at a heating rate of 10 °C/min on the TA Instruments DSC Q-20 under a nitrogen gas atmosphere. In Vitro Drug Release. The drug release profile from the PEG-PNB-TC micelles was determined using a dialysis method.18,19 Briefly, one milliliter of the drug-loaded micelles were placed in the dialysis bag (MWCO 12000−14000 Da) and stirred in 30 mL PBS (pH7.4, 0.5% Tween 80) at 37 °C. The amount of the released PTX in the outside medium was determined by HPLC over a period of 24 h. Stability Study. The PTX-loaded micelles (lyophilized micelles or micelle solution) were stored in the dark at 4 °C for up to 28 days. Their stability was evaluated by the alteration in particle size and drug content of the micelles. For drug quantitation, the micelle solution or the reconstituted lyophilized micelles were filtered through a 0.45 μm filter, followed by HPLC. Cellular Uptake of PEG-PNB-TC Micelles. Rh-PE was used as a fluorescent indicator to prepare the polymeric micelles to study the in vitro cellular uptake of the micelles. The A549 cells (1 × 105 cells/well) were seeded in 24-well plates and incubated for 24 h. The cells were washed with PBS and the serum-free medium was added. The micelles were incubated with the cells for 2 h at 37 °C. Then, the medium was removed and the cells were washed with PBS for 3 times. The cells were harvested by trypsinization and centrifugation at 600 g for 2 min. The collected cells were washed with PBS and resuspended in PBS, followed by fluorescence-activated cell sorting (FACS) analysis on a BD Accuri C6 flow cytometer. The dead cells and cell debris were excluded from viable cells using forward vs side scatter gate. A total of 1.5 × 104 cell counts were acquired per sample. The fluorescence microscopy was used to confirm the cellular uptake and indicate the intracellular localization of the PEG-PNB-TC micelles. The treated cells were fixed with 4% paraformaldehyde for 10 min The cell nuclei were stained by Hoechst 33258 (2 μM) for 1

Weight of loaded drugs × 100% Weight of micelles C

DOI: 10.1021/acs.molpharmaceut.7b00278 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 2. Polymerization of bottle brush block copolymers and thio-bromo “click” functionalization.

min. The cells were observed on a Nikon ECLIPSE 80i fluorescence microscope system with a 400× magnification. Cellular Uptake of the Micellar Drugs. The A549 cells or HeLa cells (4 × 105 cells/well) were seeded in 6-well plates 24 h before the experiment. Equivalent PTX-loaded micelles or Taxol formulation [30 mg PTX dissolved in 5 mL of the Cremophor EL and dehydrated ethanol mixture (50/50, v/ v)26] were incubated with the cells for 4 h. The cells were washed and collected by centrifugation at 600 g for 2 min. The collected cells were washed with PBS four times to remove any uninternalized PTX. Then, the cells were lysed with PBS containing 1% Triton X-100. The cell lysate (150 μL) was mixed with acetonitrile (300 μL) by ultrasonication for drug extraction, followed by centrifugation at 10000 g for 10 min. The drug in the supernatant was quantitated by HPLC. The cellular protein in the sample was quantitated by the BCA protein assay. The intracellular PTX was normalized by the total cellular proteins. In Vitro Cytotoxicity. The cancer cells (2 × 103 cells/well) were seeded in 96-well plates 24 h before the treatment. After incubation with PTX formulations for 72 h, cell viability was determined by MTT assay.27 Briefly, the culture medium was removed and 200 μL fresh medium containing 0.5 mg/mL MTT was added. After 4 h incubation, the culture medium was carefully removed and the obtained blue formazan crystals were dissolved in 200 μL/well DMSO. The absorbance was measured at 570 nm on a microplate reader. Statistical Analysis. Data were expressed as mean ± standard deviation (SD). Differences between two groups were analyzed using an unpaired Student’s t-test (GraphPad Software). P-value smaller than 0.05 was considered statistically significant.

CMC; and potential of further functionalization of bottlebrush side-chains.28 In this study, we took advantage of these properties for synthesis of novel self-assembly copolymers and investigation of their application as a drug delivery carrier for hydrophobic drugs including combination drugs. Polymer Synthesis and Characterization. Our study commenced with the copolymerization of norbornene-terminated poly(ethylene glycol) macromonomer (NB-PEG16) and 5-norbornene-2-bromopropionate (NB-Br29) in the presence of Grubbs second generation initiator (G-2) at varying feed ratios ([NB-PEG]:[NB-Br]:[G-2] = 25:75:1, 50:50:1, and 75:25:1) (Figure 2). These reactions were carried out by adding G-2 to NB-PEG methylene chloride solution. The polymerizations were monitored by 1H NMR (Figure S-1) and, when complete (based on the disappearance of the olefin signals between 6.2 and 5.8 ppm and the appearance of a broad signal between 5.5 and 5.0 ppm), comonomer NB-Br was added. Once the polymerizations were complete, they were quenched by the addition of ethyl vinyl ether and each bottle brush block copolymer was isolated as a gummy solid after passing through silica gel (to remove residual Ru contaminants). In all cases, product formation was confirmed (using 1H NMR spectroscopy) by the complete disappearance of monomer olefinic signals (6.2−5.8 ppm) as well as the appearance of a broad signal corresponding to the polymer backbone (5.5−5.0 ppm) and a signal between 4.9 and 4.5 ppm (−CHBrCH3). Gel permeation chromatography (GPC) data are shown in Table 1. Table 1. GPC Data of the PEG-PNB-TC Copolymers

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RESULTS AND DISCUSSION Bottlebrush polymers, also known as molecular brushes, are branched or graft polymers with polymeric side chains attached to a linear polymer backbone. Compared to linear polymers, the architectures of bottlebrushes provide several unique properties, including easy self-assembly capability due to their highly entangled structure and large molecular weight; high stability of the self-assembled polymeric micelles due to low

copolymers

Mn

Mw

Đ

PNB-PEG/PNB-TC (25/75) PNB-PEG/PNB-TC (50/50) PNB-PEG/PNB-TC (75/25)

12900 16500 29500

14300 19600 36000

1.11 1.19 1.22

Once each copolymer was prepared, a thio-bromo “click” approach was utilized for the installation of thiocresol (TC) groups to serve as the hydrophobic moiety. This was achieved by the addition of TC and triethylamine to a solution of PEGNB-Br in methylene chloride, similar to our previous report.29 The success of functionalization was confirmed by the disappearance of the signal between 4.9 and 4.5 ppm D

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fluorescent probe (Figure 4). All tested PEG-PNB-TC copolymers could efficiently solubilize the pyrene and showed the significant inflection points on the curve of the I338/I334 ratio vs logarithm of polymer concentration, indicating the CMC of the polymers. The CMC was considered as the concentration at the crossover point of the two tangents and obtained as 0.081, 0.075, and 0.057 mg/mL for the PEG-PNBTC copolymers with the PEG/TC ratios of 25/75, 50/50, and 75/25, respectively. The increase in the PEG content slightly decreased CMC, while these CMC were typical for the PEGylated micelles.32,33 Their CMC values were also similar to that of the Cremophor EL (0.090 mg/mL).34 However, the molecular weight of PEG-PNB-TC was much higher than Cremophor EL, indicating that it might need fewer numbers of the amphiphilic monomers to form a PEG-PNB-TC micelle than form a Cremophor EL micelle. The mean particle size and zeta potential of the PEG-PNBTC micelles are shown in Figure 5A. All tested polymeric micelles had the particle size of 20−50 nm and zeta potential of −8 ∼ −10 mV in PBS. Loading the drug to the micelles just slightly increased their particle sizes. The change of the PEG/ TC ratio affected the particle size as well as zeta potential. The PEG-PNB-TC (50/50) micelles had the smallest size in both blank and drug-loaded micelles, probably due to their proper PEG/TC (hydrophilicity/hydrophobicity) ratio.35 Therefore, the special focus was given to the PEG-PNB-TC (50/50) copolymers/micelles. The TEM data (Figure 5B) showed that the PEG-PNB-TC (50/50) micelles were near spherical in shape with a narrow size distribution, which was confirmed by the dynamic light scattering data (Figure 5C). These data indicated that the PEG-PNB-TC copolymers at the tested PEG/TC ratios could easily self-assemble to the uniform tiny micellar nanoparticles for loading hydrophobic drugs. Drug Loading (DL) and Encapsulation Efficiency (EE). A thin-film hydration method was used to prepare the drugloaded PEG-PNB-TC micelles. The model drug, PTX, could easily be loaded into all three PEG-PNB-TC micelles. When the initial drug/polymer ratio was 5% (w/w), the encapsulation efficiency for all micelles was about 80%. After removal of the unentrapped drugs, the actual drug loading in the PEG-PNBTC micelles were 4.22 ± 0.07%, 4.14 ± 0.10%, and 4.02 ± 0.13%, for PEG-PNB-TC (25/75), PEG-PNB-TC (50/50), and PEG-PNB-TC (75/25), respectively (Figure 6A). The data indicated that all PEG-PNB-TC copolymers had the similar properties and the change of the PEG/TC ratio in the polymer did not significantly influence the drug encapsulation. As a micellar drug delivery system, the drug loading (maximum) capacity and stability of the drug-loaded micelles

(−CHBrCH3) and the appearance of signals at 7.4 and 7.2 ppm (corresponding to the aromatic protons of TC), as shown in Figure 3b. Purification of resulting polymers by solvent

Figure 3. (a) Partial 1H NMR spectrum of PNB-PEG/PNB-Br (50/ 50) and (b) partial, crude 1H NMR spectrum of PNB-PEG/PNB-TC (50/50). The crossed out singlet at 5.35 is due to residual methylene chloride. Full 1H NMR spectra are listed in Supporting Information.

precipitation was difficult. Instead, each crude polymer was isolated in quantitative yield after removal of solvent and subjected to dialysis against water directly before use. Several PNB polymers have been recently reported and most of them contained both hydrophilic (mostly PEG) and hydrophobic parts, such as polystyrene, polylactide, and poly(ε-caprolactone),30,31 to possess the self-assembly capability. Here, our resulting polymer, i.e., polyethylene glycolpolynorbornene-thiocresol block copolymers (PEG-PNB-TC), contained PNB as its backbone, PEG as its hydrophilic side chains, and TC as its hydrophobic side chains. Its amphiphilicity and self-assembly capability could be easily adjusted by the PEG/TC ratio. Micelle Formation. The CMC of PEG-PNB-TC copolymers were determined using pyrene as a hydrophobic

Figure 4. CMC of PEG-PNB-TC copolymers. (A) PEG/TC (25/75), (B) PEG/TC (50/50), and (C) PEG/TC (75/25). E

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Figure 5. Physicochemical characterization of the PEG-PNB-TC micelles. (A) Particle size and zeta potential of the PEG-PNB-TC micelles; (B) morphology of the PEG-PNB-TC (50/50) micelles; and (C) size distribution of the blank (a) and PTX-loaded (b) PEG-PNB-TC (50/50) micelles. Data were expressed as mean ± SD.

1.57%. The data were in agreement with the previous reports about drug encapsulation in micellar drug delivery systems.36,37 Moreover, the micelle solution did not show any visible aggregates at any tested drug/polymer ratio once preparation, indicating the drug-loaded micelles were stable. The detailed stability data were discussed below. High drug loading capacity of the bottlebrush PEG-PNB-TC polymer-formed micelles would favor the administration of high drug doses and dose adjustment. Stability of the PTX-Loaded PEG-PNB-TC Micelles. To study the stability of the drug-loaded micelles, the drug content and mean particle size of the PTX-loaded PEG-PNB-TC micelles were determined over a period of 28 days (Figure 7).

Figure 7. Stability of the PTX-loaded PEG-PNB-TC (50/50) micelles during storage at 4 °C for up to 28 days. (A) PTX content determined by HPLC; and (B) particle size determined by DLS. Data were expressed as mean ± SD. Figure 6. Drug loading (DL) and encapsulation efficiency (EE) of the PEG-PNB-TC polymers. (A) DL and EE of all three PEG-PNB-TC polymeric micelles at the initial drug/polymer ratio of 5%; and (B) the appearance, DL and EE of the PEG-PNB-TC (50/50) micelles with various initial drug/polymer ratios. Model drug: PTX. Data were expressed as mean ± SD.

The PTX content and particle size slowly went down in the micelle solution along with time after reconstitution in water, while the drug content and particle size showed almost no change in the initial 24 h. In contrast, the lyophilized forms of micelles were fairly stable even after 28 days at 4 °C. The data indicated that the PTX-loaded PEG-PNB-TC micelles had a great stability in both the solution and lyophilized form. It was also noted that the lyophilization process did not compromise the micellar properties and the lyophilized PEG-PNB-TC micelles had a great dispersibility (passing through the 0.45 μm filter), which would be beneficial in future drug development and clinical applications. Drug-Polymer Interaction. The DSC analysis was performed to determine if any interaction between the PTX and PEG-PNB-TC copolymers existed. To make the data comparable, the drug content was kept the same in all samples. The DSC thermograms are shown in Figure 8. The free PTX

are crucial. Herein, the PEG/TC (50/50) copolymers were chosen as the example to evaluate the drug loading capacity and other properties of the PEG-PNB-TC copolymers. As shown in Figure 6B, the PEG-PNB-TC micelles could efficiently load PTX at the initial drug/polymer ratios from 1 to 50% and their drug encapsulation efficiencies were above 70%. The increase in the initial drug/polymer ratio significantly increased the drug loading. The DL of about 34.02 ± 2.62% could be reached at the initial drug/polymer ratio of 50%. Further increase in the initial drug input from 50 to 100% destabilized the micelle structure and dramatically dropped the drug loading to 24.24 ± F

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Figure 9. Accumulative PTX release from the PEG-PNB-TC micelles under simulated “sink condition”. Data were expressed as mean ± SD.

micelles’ PEG shell and reduce the in vivo drug clearance, providing sufficient time for loaded drugs to accumulate in the tumor. It was also noted that the release of the micellar drugs seemed incomplete within 24 h in the in vitro test, in agreement with the previous reports about the micellar drugs.42−44 Here, the use of 0.5% Tween 80 to increase the drug “solubility” to simulate the “sink condition” in the release media was simple and widely accepted in the in vitro study. However, it might not fully represent the tumoral or intracellular conditions. Especially, upon cell internalization, the acidic pH and enzymes in the endosomes/lysosomes would facilitate drug release,43,45 which was confirmed by our in vitro efficacy data. Cellular Uptake of the PEG-PNB-TC Polymeric Micelles. Many anticancer drugs showed the limited tissue penetration and cellular uptake, due to low drug solubility and passive diffusion rather than the “active” cell internalization, while the nanoparticles usually have the improved tissue and cell penetration via the increased drug solubility (high drug concentration gradient across cell membrane) and various types of vesicular transport.46 To test the cellular uptake of the micelles, the PEG-PNB-TC micelles were labeled by Rh-PE. The cell association and uptake were investigated by the FACS and fluorescence microscopy (Figure 10). Since Rh-PE was highly hydrophobic/lipophilic and its release/leakage from the core−shell nanoparticles was negligible, it has been widely used as the hydrophobic dye for the visualization of nanoparticles.20,47 The FACS analysis showed that all PEG-PNBTC micelles could be efficiently internalized by the cancer (A549) cells compared to Cremophor EL micelles (∼16000 vs ∼5000) and different types of PEG-PNB-TC micelles had no significant difference. In the micrographs, strong red fluorescence was observed in the A549 cells after cell incubation with the Rh-PE labeled PEG-PNB-TC (50/50) micelles for 2 h, while the uptake of the Rh-PE labeled Cremophor EL micelles was not significant. After cell internalization, the micelles were mainly localized in the cytosol rather than cell nuclei. The observed high uptake of the PEG-PNB-TC micelles suggested that the nanoparticles were endocytosed by the cancer cells most likely as an intact entity. Drug Uptake. To study if the micelles could increase the cellular uptake of the loaded drugs, the A549 or HeLa cells were incubated with the Taxol or PTX-loaded PEG-PNB-TC micelles at different concentrations. The intracellular drugs were quantitated by HPLC and shown in Figure 11. The drug uptake of the PEG-PNB-TC micelles was 1.47 and 1.53 fold higher than those of Taxol in the A549 cells, and 1.28 and 1.65

Figure 8. DSC thermograms of PTX (a), PEG-PNB-TC (50/50) copolymers (b), physical mixture of PTX and PEG-PNB-TC (50/50) (c), and lyophilized powder of PTX-loaded PEG-PNB-TC (50/50) micelles (d).

(a) displayed a sharp endothermic peak at 222.20 °C, indicating that it was in a crystalline state. It was worth noting that the PTX used in this study might contain dihydrated PTX (paclitaxel·2H2O), as evidenced by a broad endotherm (50 to 100 °C) corresponding to water evaporation.38,39 The empty PEG-PNB-TC micelles (b) showed a single endothermic peak at 46.72 °C, which might be the melting point of the polymers. In the thermogram of the physical mixture of the empty PEGPNB-TC polymers and PTX (c), the PTX was shown as an exothermic peak at around 220 °C. The PEG-PNB-TC polymers in the physical mixture exhibited an endothermic peak at 45.83 °C. These changes observed might be the result of a weak interaction between the drug and polymer. In contrast, the PTX melting peak disappeared and the peak of PEG-PNB-TC polymers was shifted to the left further (41.60 °C) in the DSC thermogram of the drug-loaded PEG-PNB-TC micelles (d), suggesting that the drug-polymer interaction was strong and the recrystallization of the loaded drugs was completely inhibited. The PTX was most likely in an amorphous or molecularly dispersed state in the PEG-PNBTC matrix.40 In Vitro Drug Release Behavior. The drug release rate is an important parameter for evaluating a drug carrier. To maximize anticancer activity and minimize off-target toxicity in the normal tissue, the drug delivery systems should have a slow/sustained initial drug release in the normal physiological condition as well as a complete drug release in the tumor tissue/cells. Here, the in vitro PTX release from the PEG-PNBTC micelles was investigated by a dialysis method under the simulated “sink condition”41 and compared with free PTX for a period of 24 h (Figure 9). The free PTX had a rapid initial release and reached the plateau (∼85%) of its release curve at 6 h, while the PEG-PNB-TC micelles showed a sustained drug release pattern and it took more than 12 h to reach their plateau (∼70%) in the curves. The micelles formed by different PEG-PNB-TC copolymers had the similar drug release profile. The sustained PTX release pattern might be a result of the hydrophobic interaction between the PTX and TC, as evidenced by the drug-polymer interaction data (Figure 8). From the in vivo point of view, the sustained drug release would facilitate drugs’ prolonged blood circulation mediated by G

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Figure 12. Toxicity of the PEG-PNB-TC copolymers in the A549 cells. Data were presented as mean ± SD *p < 0.05; ***p < 0.001.

EL showed significant toxicity, while the viability of PEG-PNBTC-treated cells was much higher than that of Cremophor EL (60% vs 85%), while the Cremophor EL, the surfactant used in Taxol, showed slight toxicity (75%) at 500 μg/mL. At 1000 μg/mL, both the PEG-PNB-TC copolymers and Cremophor H

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Figure 13. In vitro anticancer activity of Taxol and PTX-loaded PEG-PNB-TC micelles in the A549 (A and B) and HeLa (C and D) cells. Data were presented as mean ± SD.

corresponds to an additive effect and when the CI is greater than 1.1, an antagonistic effect is observed. The MDR cancer (NCI/ADR-RES) cells were as the model and treated with various drug formulations. As shown in Figure 14 and Table 3, PTX was less effective in the NCI/ADR-RES cells than in the drug-sensitive cancer cells. The single drug-loaded micelles did not show a significant increase in their cytotoxicity compared to free drugs, which was similar to those in the drug-sensitive cancer cells (Figure 13). In contrast, the PTX and CUR coloaded micelles showed much higher cytotoxicity in the NCI/ADR-RES cells compared to the single drug or single drug-loaded micelles (Figure 14A,B and Table 3) The PTX/ CUR (1:20) showed a > 10-fold higher anticancer activity (of PTX) compared to the PTX/CUR (1:1) in both the physical mixture and PEG-PNB-TC micelles. After calculating the CI values (Figure 14 C), we found that the PTX/CUR ratio of 1:1 had a CI value of around 1.0, suggesting an additive effect of the two drugs, while the ratio of 1:20 had a CI value of 0.74, which showed a synergistic effect between the two drugs. It was known that the combinatory effects were highly associated with the drug ratios50,51 and the synergism was observed only at certain ratios of combination drugs, including PTX and CUR.52,53 Therefore, the drug codelivery systems should possess both high drug loading efficiency and capability of dose adjustment, especially for high drug ratios. The loading to the micelles did not influence the combinatorial effect of PTX and CUR. Actually, it was reasonable because these micelles were “plain”. We could not expect these plain PEG-PNB-TC micelles to dramatically improve the synergistic effects of the combined drugs. The similar effects between the free drug combination and micellar drug combination indicated that the PEG-PNB-TC micelles did not compromise drug efficacy. The use of these PEG-PNB-TC

Table 2. IC50 Values of the PTX-Loaded PEG-PNB-TC Micelles in the A549 and HeLa Cells IC50 (ng/mL) formulation

DL%

Taxol PEG-PNB-TC(25/75) PEG-PNB-TC(50/50) PEG-PNB-TC(50/50) PEG-PNB-TC(50/50) PEG-PNB-TC(75/25)

4.22 4.14 14.61 34.02 4.02

A549 60.13 74.43 68.72 73.71 75.29 68.12

± ± ± ± ± ±

0.90 1.12 0.96 1.03 1.05 0.96

HeLa 2.85 3.04 2.69 2.75 3.14 2.61

± ± ± ± ± ±

0.04 0.04 0.03 0.03 0.04 0.03

intracellular pathways could effectively conquer the biological and pathological barriers. Here, CUR, a widely investigated natural product, was employed as the PTX sensitizer for improving the anticancer activity of PTX as well as evaluating the drug codelivery capability of the PEG-PNB-TC polymeric micelles. The PTX and CUR pair has shown synergistic effects in treating various cancers11,12 via the interference of the antiapoptotic pathway, cell proliferation, metastasis, and drug efflux.11,13,14 However, CUR is also a poorly water-soluble drug and needs a drug delivery carrier. Although various drug delivery systems have been developed for PTX or CUR, individually,13−15 the effective drug codelivery systems were rarely reported. In this study, two different weight ratios of PTX/CUR, 1:1 and 1:20, were used to prepare the micelles. After cell incubation, the combination index (CI) was calculated by the classic isobologram equation (Chou and Talalay),50 CI = a/A + b/B, where a is the IC50 of PTX in combination with CUR while b is the IC50 of CUR in combination with PTX; A is the IC50 of free PTX and B is the IC50 of free CUR. When the CI is less than 0.9, a synergistic effect is observed; CI = 0.9−1.1 I

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Figure 14. Codelivery of PTX and CUR by the PEG-PNB-TC micelles. (A) Cytotoxicity of the free drugs; (B) cytotoxicity of the PTX and CUR coloaded PEG-PNB-TC micelles; and (C) evaluation of the combinatorial effects of PTX and CUR. Cell line: NCI/ADR-RES cancer cells. Data were presented as mean ± SD.

Table 3. IC50 and CI Values of the Treatments in the NCI/ADR-RES Cells IC50 (ng/mL) formulation

PTX

free PTX free CUR PTX/CUR(1/1) PTX/CUR(1/20) PTX/PEG-PNB-TC CUR/PEG-PNB-TC PTX/CUR(1:1)/CUR/PEG-PNB-TC PTX/CUR(1:20)/CUR/PEG-PNB-TC

3963.00 ± 46.67 1863.00 ± 23.49 146.60 ± 1.62 6764.00 ± 82.74 1781.00 ± 20.35 168.10 ± 1.86

CUR

CI

4175.00 ± 47.66 1863.00 ± 23.49 2932.00 ± 32.50

0.92 0.74

3534.00 ± 40.79 2279.68 ± 28.27 2894.17 ± 35.89

1.01 0.74

PNB-TC micelles at different ratios. The PTX and CUR coloaded PEG-PNB-TC micelles could significantly overcome the PTX resistance and sensitize the PTX treatment in the MDR cells at high CUR/PTX ratio. Owing to the ease of polymer preparation and functionalization, high drug loading capability, and great stability of their self-assembled micelles, the PEG-PNB-TC copolymers could be considered as a promising nanomaterial for efficient delivery of high doses of drugs or codelivery of multiple drugs, for treating various cancers including the drug-resistant ones.

micelles might be beneficial to current cancer treatments in two ways: (i) higher drug loading and stable micelle structure ensure the loading of multiple drugs and easy adjusting drug ratios; and (ii) the clinically used/well-investigated combination drugs could be directly applied to the PEG-PNB-TC micelles without further adjustment of drug ratios, to exert the uncompromised efficacy. Furthermore, the bottlebrush PEGPNB-TC polymers contained more reaction sites for side chain addition or modification by “click” reaction, providing engineering potential of micelles to further improve micelles’ efficacy and specificity.

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ASSOCIATED CONTENT

* Supporting Information

CONCLUSION In this study, we successfully utilized a combination of graftingthrough ROMP and a thio-bromo “click” reaction for the preparation of novel, amphiphilic bottlebrush self-assembling PEG-PNB-TC copolymers and evaluated their capability as the micellar nanocarriers for delivery of hydrophobic drugs. The self-assembled PEG-PNB-TC micelles were tiny, uniform, and near spherical. High drug loading (up to around 35% DL) could be achieved when the hydrophobic drugs, such as PTX, was loaded to the PEG-PNB-TC micelles. Loading of the drug to the micelles did not significantly influence the micelle structure and the drug-loaded micelles were fairly stable. The polymeric micelles as well as the loaded drugs could be efficiently taken up by cancer cells, leading to high cytotoxicity. Moreover, PTX and CUR could be coloaded into the PEG-

S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b00278. Full 1H NMR spectrum of PNB-PEG/PNB-BR (50/50) and full crude 1H NMR spectrum of PNB-PEG/PNBTC (50/50) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Phone: (361) 593-2667; E-mail: christopher.hobbs@tamuk. edu. *Phone: (361)221-0757; Fax: (361)221-0793; E-mail: lzhu@ tamhsc.edu. J

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Qing Yao: 0000-0003-0115-9075 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Ms. Qing Yao thanks the China Scholarship Council (CSC) (File No. CSC No.201508210197) for partial support of this project. Dr. Ruoning Wang thanks the National Institute of Health (R21AI117547 and 1R01AI114581), V-Foundation (V2014-001), and American Cancer Society (128436-RSG15-180-01-LIB) for financial support. Mr. David C. Gutierrez and Dr. Christopher Hobbs thank the NSF (1610307) and the Robert A. Welch Foundation (572006) for partial support of this project and Materia Inc. for donation of Grubbs 2nd generation initiator as well as Shimadzu for GPC analysis.

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