ARTICLE pubs.acs.org/Biomac
Redox-Responsive Polyphosphate Nanosized Assemblies: A Smart Drug Delivery Platform for Cancer Therapy Jinyao Liu,† Yan Pang,† Wei Huang,* Zhaoyang Zhu, Xinyuan Zhu, Yongfeng Zhou, and Deyue Yan* School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, P. R. China
bS Supporting Information ABSTRACT: Novel redox-responsive polyphosphate nanosized assemblies based on amphiphilic hyperbranched multiarm copolyphosphates (HPHSEP-star-PEPx) with backbone redoxresponsive, good biocompatibility, and biodegradability simultaneously have been designed and prepared successfully. The hydrophobic core and hydrophilic multiarm of HPHSEP-starPEPx are composed of hyperbranched and linear polyphosphates, respectively. Benefiting from the amphiphilicity, HPHSEP-star-PEPx can self-assemble into spherical micellar nanoparticles in aqueous media with tunable size from about 70 to 100 nm via adjusting the molecular weight of PEP multiarm. Moreover, HPHSEP-star-PEPx micellar structure can be destructed under reductive environment and result in a triggered drug release behavior. The glutathione-mediated intracellular drug delivery was investigated against a HeLa human cervical carcinoma cell line, and the results indicate that doxorubicin-loaded (DOX-loaded) HPHSEP-star-PEPx micelles show higher cellular proliferation inhibition against glutathione monoester pretreated HeLa cells than that of the nonpretreated ones. In contrast, the DOX-loaded micelles exhibit lower inhibition against buthionine sulfoximine pretreated HeLa cells. These results suggest that such redox-responsive polyphosphate micelles can rapidly deliver anticancer drugs into the nuclei of tumor cells enhancing the inhibition of cell proliferation and provide a favorable platform to construct excellent drug delivery systems for cancer therapy.
’ INTRODUCTION Driven by the aim to develop smart nanomaterials for biomedical applications, a great number of stimuli responsive polymers have been synthesized and investigated in detail.18 Among them, amphiphilic copolymers with stimuli-responsive elements are of fundamental interest in drug delivery because they can selfassemble into nanosized micelles in water.915 These micelles possess an ability to provide interiors for physical encapsulation of hydrophobic drugs, and the loaded drugs can be released from them triggered by external stimuli. In particular, redox-responsive amphiphilic copolymers and their nanosized assemblies are interesting to construct drug delivery systems because of the existence of redox potential gradient between the extra- and intracellular space.16 To date, various redox-responsive polymeric micelles have been successfully obtained;1726 however, most of them fail in preclinical studies because of their high toxicity. To decrease the toxicity of micellar drug delivery systems, the biocompatible polymers (such as poly(ethylene oxide), sugar-derivative, and polyglycerol) were frequently used as hydrophilic segments, and the biodegradable polymers (such as polyester, polycarbonate, and polypeptide) were often employed as hydrophobic segments. However, these polymers did not show any redox responsiveness owing to the inherent structure of polymeric backbone. To endow them with redox responsiveness, the disulfide functionality is always introduced by the postpolymerization modification.16 Little r 2011 American Chemical Society
attention has been paid to develop nanosized assemblies based on amphiphilic copolymers with backbone redox responsiveness, good biocompatibility, and biodegradability, simultaneously. Polyphosphates are an important class of biomaterials with good biocompatibility, biodegradability, and structural similarity to nucleic and teichoic acids.2732 They can degrade naturally into harmless low-molecular-weight products through hydrolysis or enzymatic digestion of phosphate linkages under physiological conditions and also exhibit good flexibility in adjusting the pendant groups as well as the physicochemical properties by the convenient functionalization of pentavalent phosphorus. Recently, polyphosphate with different topological structures has been extensively designed and prepared for biomedical application, concluding drug delivery, gene (DNA and siRNA) delivery, and tissue engineering.31,3335 In this Article, we intend to design an amphiphilic copolyphosphate with backbone redox responsiveness and further construct a nanosized micellar drug delivery system with smart redox responsiveness and good biocompatibility and biodegradability, simultaneously. Nanosized micelles self-assembled from amphiphilic hyperbranched multiarm copolymers are widely explored and recognized Received: April 15, 2011 Revised: May 8, 2011 Published: May 10, 2011 2407
dx.doi.org/10.1021/bm2005164 | Biomacromolecules 2011, 12, 2407–2415
Biomacromolecules
ARTICLE
Scheme 1. Structure of Amphiphilic Hyperbranched Multiarm Copolyphosphates of HPHSEP-star-PEPx
as promising nanovehicles for drug delivery.3642 These micelles possess several unique features, including a large number of terminal functional groups for further conjugation of drug or targeting/imaging ligands, various micellar structures with different drug-loaded routes, sensitive environmental responsiveness for controlled drug release, and relative small hydrodynamic volume for efficient cellular uptake.3 Herein, a novel kind of amphiphilic hyperbranched multiarm copolyphosphates (HPHSEPstar-PEPx) with disulfide bonds in the backbone was synthesized and utilized to construct a nanosized intelligent drug delivery system (Scheme 1). The self-assembly behavior of HPHSEPstar-PEPx in aqueous media as well as the redox responsiveness of the resulting micelles under an extra- and intracellular reductive environment were further investigated in detail. Meanwhile, the glutathione (GSH)-mediated intracellular drug delivery was also evaluated against a HeLa human cervical carcinoma cell line by pretreating these cells with glutathione monoester (GSH-OEt) or buthionine sulfoximine (BSO), which could increase or decrease the concentration of GSH in the cytoplasm, respectively.
’ EXPERIMENTAL SECTION Materials. 2-Chloro-2-oxo-1,3,2-dioxaphospholane (COP) and 2-ethoxy-2-oxo- 1,3,2-dioxaphospholane (EP) were synthesized according to the method previously described and distilled under reduced
pressure just before use.33 N,N-dimethylformamide (DMF) was dried over calcium hydride and then purified by vacuum distillation. Triethylamine (TEA) was refluxed with phthalic anhydride, potassium hydroxide, and calcium hydride in turn and distilled just before use. Tetrahydrofuran (THF) and toluene were dried by refluxing with the fresh sodium-benzophenone complex under N2 and distilled just before use. Tin(II) octoate (Sn(Oct)2), D,l-1,4-dithiothreitol (DTT), GSH-OEt, BSO, acridine orange (AO), ethidium bromide (EB), and 3-(4,5dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma and used as received. Doxorubicin hydrochloride (DOX 3 HCl) was purchased from Beijing Huafeng United Technology Corporation and used as received. Clear polystyrene tissue-culturetreated 12- and 96-well plates were obtained from Corning Costar. All other reagents and solvents were purchased from the domestic suppliers and used as received. Measurements. Nuclear magnetic resonance (NMR) analyses were recorded on a Varian Mercury Plus 400 MHz spectrometer with deuterated dimethyl sulfoxide (d6-DMSO) as solvent. The numberaverage molecular weight (Mn), weight-average molecular weight (Mw), and polydispersity index (PDI) were measured by gel permeation chromatography (GPC). GPC was performed on a Perkin-Elmer series 200 system (10 μm PL gel 300 7.5 mm mixed-B and mixed-C column, linear polystyrene calibration) equipped with a refractive index (RI) detector. DMF containing 0.01 mol L1 lithium bromide was used as the mobile phase at a flow rate of 1 mL min1 at 70 °C. Dynamic light scattering (DLS) measurements were performed in aqueous solution 2408
dx.doi.org/10.1021/bm2005164 |Biomacromolecules 2011, 12, 2407–2415
Biomacromolecules
ARTICLE
Table 1. Characterization Data of the Resulting Polymers and Self-Assembled Micelles Mw/
diameter
sample
Mn (g mol1) (104)a
Mn
(nm)b
PDI
HPHSEPOH HPHSEP-star-PEP1
1.12 1.99
1.72 2.66
104
0.316
HPHSEP-star-PEP2
2.21
2.86
85
0.282
HPHSEP-star-PEP3
2.52
2.44
70
0.245
a
Molecular weight and polydispersity index were measured by GPC. b Diameter and PDI of self-assembled micelles were analyzed by DLS. using a Malvern Zetasizer Nano S apparatus equipped with a 4.0 mW laser operating at λ = 633 nm. All samples of 1 mg mL1 were measured at 20 °C and at a scattering angle of 173°. Transmission electron microscopy (TEM) studies were performed with a JEOL 2010 instrument operated at 200 kV. The samples were prepared by directly dropping the solution of micelles onto carbon-coated copper grids and dried at room temperature overnight without staining before measurement.
Synthesis of 2-[(2-Hydroxyethyl)disulfanyl]ethoxy-2-oxo1,3,2-dioxaphospholane (HSEP). COP (18.0 g, 0.126 mol) in 100 mL of THF was added dropwise to a solution over 2 h, which was composed of 2-hydroxyethyl disulfide (19.4 g, 0.126 mol) and TEA (12.8 g, 0.126 mol) in 100 mL of THF under the magnetic stirring at 20 °C in a low temperature thermostatic bath. Then, the mixture was stirred at 20 °C overnight, and the precipitate was filtered off by a Schlenk funnel with the dried silica gel. The filtrate was evaporated under vacuum to produce some transparent and colorless oil, yield 93.1%. 1H NMR (d6-DMSO, ppm): 4.40 (4H, -POCH2CH2OP-), 4.21 (2H, -POCH2CH2S-), 3.59 (2H, SCH2CH2OH), 2.97 (2H, -POCH2CH2S-), 2.77 (2H, -SCH2CH2OH). 13 C NMR (d6-DMSO, ppm): 67.22 (-POCH2CH2OP-), 66.12 (-POCH2CH2S-), 60.21 (-SCH2CH2OH), 41.73 (-SCH2CH2OH), 38.44 (-POCH2CH2S-).
Synthesis of Hyperbranched PolyHSEP (HPHSEPOH). The self-condensing ring-opening polymerization (SCROP) of HSEP was carried out in bulk in a glovebox with the water content
