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Combination Loading of Doxorubicin and Resveratrol in Polymeric Micelles for Increased Loading Efficiency and Efficacy Katherine E. Washington, Ruvanthi N. Kularatne, Michael C. Biewer, and Mihaela C. Stefan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00972 • Publication Date (Web): 20 Feb 2018 Downloaded from http://pubs.acs.org on March 1, 2018
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ACS Biomaterials Science & Engineering
Combination Loading of Doxorubicin and Resveratrol in Polymeric Micelles for Increased Loading Efficiency and Efficacy Katherine E. Washington, ‡ Ruvanthi N. Kularatne, ‡ Michael C. Biewer, and Mihaela C. Stefan* Department of Chemistry and Biochemistry, University of Texas at Dallas, 800 W Campbell Road, Richardson, Texas 75080, United States ‡These authors contributed equally. KEYWORDS: Resveratrol, doxorubicin, combination loading, polymeric micelles, drug delivery
ABSTRACT
Combined loading of doxorubicin (DOX) and resveratrol (RSV) in polymeric micelles enabled an increased loading of DOX into a micellar drug delivery system. Herein, we report the coloading of DOX and RSV in amphiphilic diblock copolymer micelles of poly(ethylene glycol)-bpoly(ε-caprolactone) (PEG-b-PCL) and poly(ethylene glycol)-b-poly(γ-benzyl-ε-caprolactone) (PEG-b-PBCL) for which an increase in the loading efficiency and increased in vitro cytotoxicity was observed. The increased loading was attributed to the favorable interactions of DOX and
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RSV, as well as to the interaction with benzyl substituents of PEG-b-PBCL diblock copolymer micelles. Combination loaded micelles made of PEG-b-PBCL diblock copolymer showed a dramatic improvement in DOX loading in comparison to DOX only loaded PEG-b-PBCL, with an increase in encapsulation efficiency of DOX from 31.0% to 87.7%. Combination loaded micelles also showed increased cytotoxicity to HeLa cells as compared to DOX only loaded micelles. Optimization of the combination loading, size and morphology, drug release, and cellular studies are also reported here. Combination loading was shown to improve the loading capacity, and efficiency of both systems and shows promise to improve loading of DOX in polymer micelle systems regardless of the polymer used.
INTRODUCTION Doxorubicin (DOX) is used for the treatment of several cancers including breast, ovarian, and lymphoma. DOX is regarded as one of the most potent chemotherapeutic drugs permitted by the Food and Drug Administration (FDA).1 However, DOX is generally known to have toxic side effects to non-cancerous cells, and its use is limited by its cardiotoxicity.1-2 In addition, DOX can induce drug resistance in cancer cells due to high dosage and repeated treatment. In an effort to combat these drawbacks, attention has been focused towards developing treatment with reduced side-effects from DOX. Recently, studies have shown that the adverse reactions of DOX can potentially be mitigated with the co-administration of antioxidants such as polyphenols.3-6 One such polyphenolic compound, resveratrol (RSV), has received attention due to its cardioprotective properties.7-8 RSV is derived from natural sources such as grapes, peanuts, and blueberries, with trans-RSV as the biologically active form.7 The “French Paradox” in which
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cardiovascular disorders in France are low even though their diet has a high content of fat, has been attributed to the consumption of red wine which contains a significant amount of RSV.7 RSV has shown health benefits in addition to its antioxidative nature, including acting as an antiinflammatory and anti-tumorigenic.9-10 DOX, paclitaxel, curcumin, and cis-platin are several anti-cancer drugs where RSV has been studied through co-administration to mitigate their toxic side effects, while also helping to overcome multi-drug resistance.11-13 Combination therapy using RSV and DOX has been shown to reduce cardiotoxicity. RSV can act as a chemosensitizer, providing a synergistic effect against cancer cells in vitro and in vivo.14-19 The cardioprotective properties of RSV have been largely attributed to reduced oxidative stress, the inhibition of apoptosis, and modulated autophagy process.6 Research has been moving toward the combined loading of DOX and RSV into nanoparticles for controlled delivery to mitigate toxic side effects as well as cancer cell resistance to DOX, but few drug delivery systems have been reported.20-21 Combination drug delivery using DOX and RSV shows an encouraging new path towards more efficient cancer treatment by combining polyphenols with anticancer drugs which have the potential to increase loading in these systems through hydrogen bonding and πstacking interactions. Developing a drug delivery system for the combined delivery of DOX and RSV through polymeric micelles allows delivery of the drugs in a controlled and less toxic manner. Polymeric micelles are advantageous for drug delivery systems due to their small size (typically 10-100 nm) and long circulation times allowing for accumulation in tumor sites due to the enhanced permeability and retention (EPR) effect.22 Amphiphilic block copolymers that have a hydrophilic poly(ethylene glycol) (PEG) block, as well as a hydrophobic polyester block are commonly used for drug encapsulation.23-24
Biodegradable and biocompatible polyesters, in particular,
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poly(caprolactone)s, are often used as the hydrophobic block due to their tunability through the addition of substituents to the polyester backbone.25-28 However, polymeric micelles have been known to suffer from low drug loading, therefore finding ways to increase loading efficiencies of these drug carriers is beneficial and will improve the effectiveness of these systems.29-30 In this report, the combined drug loading of RSV and DOX is investigated for polymeric micelles using poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-b-PCL) and poly(ethylene glycol)-b-poly(γ-benzyl-ε-caprolactone) (PEG-b-PBCL) amphiphilic diblock copolymers. These two amphiphilic diblock copolymers were compared for their drug loading ability, based on different interactions they may have with the drugs for loading. Increased drug loading was anticipated for PEG-b-PBCL over PEG-b-PCL because of the likelihood of π-stacking of the DOX and RSV molecules with the benzyl substituents. In addition, differing ratios of DOX and RSV were examined to optimize the encapsulation efficiency of the polymeric drug delivery systems. EXPERIMENTAL Materials. All commercially available chemicals were acquired from Sigma Aldrich or Fisher Scientific and used without additional purification unless stated.
Tin (II)-ethyl hexanoate
(Sn(Oct)2) was purified through vacuum distillation before use. All polymerization reactions were performed in a purified nitrogen atmosphere in glassware heated to 120 oC for 24 hours to remove moisture and cooled in a desiccator prior to use. Analysis. 1H NMR spectra of the monomer and polymers were determined using a Bruker AVANCE III 500 MHz NMR instrument at 25 °C in CDCl3. 1H NMR data is shown in parts per million as chemical shifts relative to tetramethylsilane (TMS) as the internal standard. Molecular
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weight and polydispersity of the resulting polymers were determined using SEC analysis on an OMNISEC multi-detector system equipped with Viscotek columns (T6000M), equipped with refractive index (RI) detector with HPLC grade THF as eluent and polystyrene standard calibration. Fluorescence spectra of the synthesized polymers were collected using a PerkinElmer LS 50 BL luminescence spectrometer at 25 °C with emission wavelength set at 390 nm. Size measurements were conducted using dynamic light scattering (DLS) with a Malvern Zetasizer Nano ZS instrument equipped with a He–Ne laser (633 nm) and 173° backscatter detector, limits of detection for the instrument are from 1 nm to 1 µm. Transmission electron microscopy (TEM) imaging was performed on the empty and combination loaded micelles using a Tecnai G2 Spirit Biotwin microscope by FEI and images were analyzed using Gatan Digital Micrograph software. Samples were prepared through a staining method where the copper mesh grid was exposed to 1 mg mL-1 aqueous polymer micelle solution for 2 minutes, dried, and then stained with 2% phosphotungstic acid for 30 seconds. Absorbance spectra for DOX and RSV loading was recorded using an Agilent UV/Vis spectrophotometer. Cytotoxicity and cellular uptake measurements were completed with a Biotek Cytation 3 imaging reader. Statistical analysis was performed with Mann-Whitney U-test (α= 0.05) using MS Excel. A P-value
