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Bifunctional Chelating Supramolecular Polymer and Its Application in Down-Regulation of Cellular Iron Uptake Fan Xu, Jing Jing, Xiuman Zhou, and Yanwu Zhang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00765 • Publication Date (Web): 04 Aug 2017 Downloaded from http://pubs.acs.org on August 9, 2017
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Bifunctional Chelating Supramolecular Polymer and Its Application in Down-Regulation of Cellular Iron Uptake
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Fan Xua, Jing Jinga, Xiuman Zhoub and Yanwu Zhanga,*
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a
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450001, P.R. China
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b
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*
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School of Chemical Engineering and Energy, Zhengzhou University, 100# Science Road, Zhengzhou,
School of Life Sciences, Zhengzhou University, 100# Science Road, Zhengzhou , 450001, P.R. China
E-mail:
[email protected] 9
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ABSTRACT: Bifunctional chelating supramolecular polymer (SP-Ch) is constructed from brush-like
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macromolecule (P-Ch) through hydrogen bonds. Two kinds of norbornene derivatives are used to
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synthesize P-Ch, in which phosphonic acid as side-group of polynorbornene can act as chelating group
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and ascorbic acid as side-chain capper of polynorbornene can reduce Fe3+ to Fe2+. It can attach to cell
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membranes and form two kinds of “barriers” to hinder cells from iron uptake by virtue of phosphonic
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acid and ascorbic acid. Higher monomer conversion and polymerization degree of P-Ch are achieved
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when the ratio among M1, M2 and G2 is set as 50:10:1 and SP-Ch particles reach to submicron level
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(mean size 147.5 nm). The best chelating capacity and reducing capacity of SP-Ch for Fe3+ are 0.034
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mg/mg and 0.047 mg/mg respectively. After treated with SP-Ch, the amount of iron in MCF-7 cells is
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reduced from 3.376 ng/105 cells to 1.784 ng/105 cells after 48h, which confirms that the cellular iron
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uptake is down-regulated. As a result, iron deprivation induces growth inhibition of MCF-7 cells.
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KEYWORDS: Chelating Supramolecular Polymer, ROMP, Down-Regulation of Cellular Iron Uptake,
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Ascorbic Acid
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1. INTRODUCTION
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As an essential metal element of living body, iron plays an important role in the implementation of
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diverse physiological function. However, a high iron level can lead to physiological disorder of
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organism and result in multifarious illness, such as hepatic disease1 and cardiovascular disease.2
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Moreover, a high level of iron is associated with the generation and metastasis of tumors.3 Since
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cancer cells demand significantly more iron than normal cells to cope with their rapid proliferation,
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iron-deprivation becomes a promising therapeutic strategy.4,5
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Since iron chelators are able to inhibit the growth and induce the apoptosis of cancer cells, their
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usage as anti-cancer agents has attracted increasing interest.6 Nowadays, iron chelators in clinical
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treatment are mainly small molecules, including deferoxamine, deferiprone and deferasirox,7 but they
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usually have side effects or unsatisfactory pharmacokinetic parameters resulting from their
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low-molecular-weight characteristics.8,9 Recently, macromolecular iron chelators have drawn more
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and more attention by virtue of their noncytotoxicity and unique pharmaceutical properties.10,11
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Especially, compared with conventional polymeric macromolecular, supramolecular polymers that
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are constructed through non-covalent connections of blocks can combine noncytotoxicity of
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macromolecules and superior biodegradability of building blocks.12 However, although recent years
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have witnessed the an upsurge of research in supramolecular polymers for biomedical applications,
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there is few report about supramolecular polymers which are used in down-regulation of cellular iron
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uptake.
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Besides iron chelators, ascorbic acid has been demonstrated to decrease iron uptake of cells because
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ascorbic acid of reducibility initiates a reduction of transferrin receptor (TfR) expression and this
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function is only effective in the extracellular compartments.13-14 When ascorbic acid exists in cells,
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transferrin (Tf) expression is enhanced and cellular iron uptake is significantly increased.15 Therefore,
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it is important to keep ascorbic acid from getting into cells to realize down-regulate iron uptake.
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Anchoring ascorbic acids onto supramolecular polymers is an effective method to restrict them in
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extracellular compartments, because molecules with great size are difficult to permeate through a cell
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membrane.
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Herein, one bifunctional supramolecular polymer (SP-Ch) is designed to realize chelating iron and
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reduction of TfR expression simultaneously. SP-Ch is constructed from brush-like macromolecule
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(P-Ch) through hydrogen bonds. In various synthetic methods of well-defined macromolecules,
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ring-opening metathesis polymerization (ROMP) is superior to others because of higher tolerance to
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functional groups and independence on reactivity ratios faced by free radical polymerization.16-23 In
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P-Ch, phosphonic acid as chelating group is incorporated in the backbone of polynorbornene and
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ascorbic acid is linked with the backbone as side-chain capper through polyethylene oxide (PEO)
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bridge (Scheme 1). This structure will form two kinds of barriers hinder cells from uptaking iron.
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When Fe3+ ions approach cells, some of them are chelated by phosphonic acid and others are reduced
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to Fe2+ by ascorbic acid to diminish Fe3+ binding with apo-transferrin (Apo-Tf).
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Scheme 1. Structure and function for SP-Ch.
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In the synthesis of P-Ch (Scheme 2), ( (1R,4R)-bicyclo[2.2.1]hept-5-en-2-yl) phosphonate (M1) and
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dimethyl 2- ( ( (1R,4R)-bicyclo[2.2.1] hept-5-ene-2-carbonyl)oxy)ethyl (2-
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(3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl)-2-hydroxyethyl) glutarate (M2) were synthesized as
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monomers. Their copolymerization was initiated by the second generation ruthenium-based Grubbs
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catalyst (G2). Finally, dimethyl phosphonate onto the backbone was transformed to phosphonic acid by
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methanolysis after reacting with trimethylbromosilane (Scheme 2).
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Scheme 2. Synthesis of P-Ch.
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In this study, monomer conversion and polymerization degree of copolymers are investigated. The
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size and morphology of SP-Ch particle of single molecule is confirmed. Both chelating capacity and
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reducing capacity of SP-Ch are measured at normal saline. Human breast cancer cells (MCF-7) are
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used as a model to study the impact of SP-Ch on cellular iron uptake. Amount of iron in MCF-7 cells
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is quantified. In addition, the growth of cancer cells and the ultrastructure morphological changes of
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cell membranes are observed. Finally, the fluorescent group (fluorescein isothiocyanate FITC) was
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added to SP-Ch and the locations of fluorescent SP-Ch (FITC-P-Ch), fluorescent homopolymer of
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M1 (FITC-P-M1) as well as fluorescent homopolymer of M2 (FITC-P-M2) was observed under a
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fluorescence microscope. The process of adding the fluorescent group is shown as scheme 3.
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Scheme 3. The process of adding the fluorescent group.
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2. Experimental section
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Chemicals and reagents, synthesis and characterization of monomers and polymers, as well as cell
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culture are provided in Supporting Information.
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2.1. Characterization.
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1
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internal standard. Gel permeation chromatography (GPC) measurements were carried out with
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Agilent 1100 series equipped with a RI-G1362A RI detector and a PL gel Mixed-C column using
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DMF as the mobile phase at a flow rate of 1.0 mL min-1at 25 °C. Dynamic light scattering (DLS)
H NMR and 13C NMR spectra were recorded on Bruker AV 400 spectrometer with Si(CH3)4 as an
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measurements were performed on Malvern Zetasizer Nano equipped with a 4.0 mW He-Ne laser7
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operating at λ = 633 nm. Scanning electron microscope (SEM) images were recorded by JEOL
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JSM-7500F instrument operating at 15 kV. UV-visible absorption measurements were carried out on
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Shimadzu UV-2450 spectrometer. Fourier transform-infrared (FT-IR) spectra were recorded on an
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FT-IR Nicole spectrometer over the range of 4000-500 cm-1. Coupled plasma mass spectrometry
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(ICP-MS) measurements were carried out with OptiMass 9500 NWR-213. Fluorescence microscopic
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images were recorded by Olympus U-RFL-T DP73.
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2.2. Determination of iron content of cells.
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Triplicate wells were treated with control or 10 µg/mL SP-Ch for 48h. 48 hours later, cells were
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harvested and washed with phosphate buffered saline (PBS) for three times to absolutely remove
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extracellular irons and counted using cell counting chamber. Then cells digested by HNO3 and H2O2 in
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a 3:1 ratio. Iron content of cells was measured by ICP-MS. Values indicated are the mean ± S.D. of
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three independent experiments. The statistical significance was determined by using ANOVA at P
