Green anchors: Chelating properties of ATRP-click curcumin-polymer conjugates

Reactive and Functional Polymers 102 (2016) 47–52

Contents lists available at ScienceDirect

Reactive and Functional Polymers journal homepage: www.elsevier.com/locate/react

Green anchors: Chelating properties of ATRP-click curcumin-polymer conjugates Amram Averick a, Sukanta Dolai a, Ashish Punia a, Kamia Punia a, Sara R. Guariglia b, William L'Amoreaux b, Kun-lun Hong c, Krishnaswami Raja a,⁎ a b c

Department of Chemistry, College of Staten Island, The City University of New York, 2800 Victory Boulevard, Staten Island, NY 10314, USA Department of Biology, College of Staten Island, The City University of New York, 2800 Victory Boulevard, Staten Island, NY 10314, USA Center for Nanophase Materials Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA

a r t i c l e

i n f o

Article history: Received 10 November 2015 Received in revised form 4 March 2016 Accepted 7 March 2016 Available online 10 March 2016 Keywords: Curcumin Curcumin-polymer conjugates Metal complexes Polymer ATRP Metal poisoning

a b s t r a c t The development of environmentally friendly and biologically benign effective systems to chelate and remove toxic heavy metal ions is of utmost importance. Curcumin, the active ingredient in the spice Turmeric is a known chelator of transition metals. The metal chelation ability of curcumin is severely limited by its hydrophobicity. The efficient synthesis of water soluble curcumin and sugar brush polymer conjugate via atom-transfer radical polymerization (ATRP) and click chemistry is reported here. The polymer conjugate selectively binds and precipitates a range of highly toxic metals that include cadmium, lead, and copper, under physiologically relevant conditions. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Curcumin, (1E,6E)1,7-bis(4-hydroxy-3-methoxyphenyl)1-6-heptadiene-3-5dione, is a phytochemical isolated from the Turmeric plant (Curcuma longa), and it has been identified as a potential therapeutic agent for the treatment of cancer [1–4], Alzheimer's Disease [5,6], inflammation [7], and metal poisoning [8]. Curcumin is Generally Recognized as Safe (GRAS) by the FDA. It chelates several metal ions via keto-enol tautomerism of the β-diketone moiety to form metal complexes [8]. It is an attractive candidate for the development of environmentally friendly and biologically benign effective systems to chelate and remove toxic heavy metal ions. Heavy metal poisoning is generally considered as the accumulation of heavy metals, in toxic amounts, in the soft tissues of the body [9]. The heavy metals (Hg, Cd, As, Pb, Cr, Bi, etc.) usually bind with one or more reactive functional groups (\\SH, \\NH2, \\SS\\, etc.), which are essential for normal physiological functions [10]. Symptoms and physical findings associated with heavy metal poisoning vary according to the metal accumulated. Metal poisoning directly and/or indirectly affects various parts of the human body and organs. The symptoms could be categorized as cardiovascular, gastrointestinal, genitourinary, musculoskeletal, nervous, reproductive, respiratory, skin, eyes, ear, nose, and throat. Many of the heavy metals, such as: zinc (Zn), copper (Cu), ⁎ Corresponding author. E-mail address: [email protected] (K. Raja).

http://dx.doi.org/10.1016/j.reactfunctpolym.2016.03.009 1381-5148/© 2016 Elsevier B.V. All rights reserved.

chromium (Cr), iron (Fe), and manganese (Mn) are essential to body functions in very small amounts, preferably at ppm levels. However, if these metals accumulate in the body in high concentrations sufficient to cause poisoning, then serious damage may occur. Common treatments for metal poisoning include chelation using DMSA (dimercaptosuccinic acid), EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), BAL (2,3-dimercaptopropanol), and penicillamine (2-amino-3-mercapto-3-methylbutanoic acid) [11,12]. These substances form stable soluble complexes with metals, and thus prevent or reverse the binding of the metal ions to the biological receptors. However, the current standard of treatment for metal poisoning, chelation therapy, has a number of significant limitations including: toxicity, potential damage to the liver and kidney, and limited scope of individual chelating agents [11]. Therefore, there is an urgent need to develop new biologically benign and environmentally friendly techniques to remove toxic metals. It has been previously reported that curcumin could effectively chelate several metal ions including cadmium (Cd) [13], copper (Cu) [14], iron (Fe) [15], lead (Pb) [16], mercury (Hg) [8], and selenium (Se) [17]. A major problem associated with employing curcumin as a metal chelating agent is its lack of solubility in water [2,18]. In our previous studies, we have demonstrated that chemical manipulations of curcumin can render it water soluble [19]. We also reported a method for the synthesis of water soluble brush polymers polyvalently displaying several copies of dye and sugar molecules for imaging applications [20]. Herein, for the first time,

48

A. Averick et al. / Reactive and Functional Polymers 102 (2016) 47–52

we synthesized water soluble curcumin glucose neoglycopolymers to enhance the activity of this highly biocompatible and environmentally friendly compound (curcumin), and successfully demonstrated the applicability of these Curcumin-Glucose Polymer Conjugates (CGPC) to selectively chelate and precipitate toxic metals with a high degree of efficiency. The polymers were synthesized via a one pot, two step amidation of polyacrylic acid using novel monoamine derivative of curcumin followed by glucose amine in accordance with Scheme 1, and characterized via 1H NMR spectroscopy and Gel permeation chromatography. 2. Materials and methods

were purchased from Fisher Scientific and used as received. tert-Butyl acrylate (with 10–20 ppm of monomethyl ether hydroquinone as stabilizer) was purified using an alumina (Al2O3) column prior to use. Barium sulfate (99%), ammonium aluminum sulfate dodecahydrate (98%), chromium (III) potassium sulfate dodecahydrate (99%), mercury (II) chloride (99.5%), manganese (II) chloride (98%), magnesium chloride (98%), potassium antimony (III) tartrate hydrate (99%), cadmium sulfate (99%), silver nitrate (99%), cobalt (II) acetate (99%), cesium carbonate (99%), copper(II) sulfate pentahydrate (98%), sodium chloride (99%), lithium bromide (99%), calcium chloride (98%), lead(II) acetate trihydrate (99%), potassium acetate (99%), iron (III) chloride (97%), zinc nitrate hexahydrate (98%), and nickel chloride (98%) were purchased from Sigma-Aldrich and used as received.

2.1. Materials Curcumin, potassium carbonate (K2CO3), copper sulfate pentahydrate (CuSO4·5H2O), sodium ascorbate, and tert-butanol (99.5%) were purchased from Acros Organics. N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (98%), tert-butyl acrylate (98%), N-hydroxybenzotriazole (HOBt, 99%), and propargyl bromide (80 wt% in xylene) were purchased from Sigma-Aldrich. N,Ndimethylformamide (DMF, HPLC grade), dichloromethane (ACS grade), hexanes (ACS grade), and tetrahydrofuran (THF, HPLC grade)

2.2. Synthesis of (1E,6E)-1-(4-hydroxy-3-methoxyphenyl)-7-(3-methoxy-4(prop-2-ynyloxy)phenyl)hepta-1,6 diene-3,5-dione, curcumin mono-alkyne Curcumin mono-alkyne was synthesized according to previously reported procedure developed by our group (Scheme 1A (1a)) [13]. Briefly, curcumin (5 g, 13.57 mmol) was reacted with 1.62 g (13.61 mmol) of propargyl bromide in presence of K2CO3 (1.88 g, 13.62 mmol) in 60 ml DMF under N2 atmosphere for 48 h at room temperature. The reaction was diluted with distilled water to stop the reaction and the solvent

Scheme 1. A. Synthesis of curcumin mono-amine from curcumin. B. Synthesis of curcumin functionalized CGPC copolymer.

A. Averick et al. / Reactive and Functional Polymers 102 (2016) 47–52

was removed under reduced pressure. The product was purified by column chromatography using dichloromethane/hexane (50/50 v/v) as eluent. Yield: 47%. 1 H NMR (CDCl3), δ (ppm): 2.54 (s, 1H), 3.94 (d, 6H), 4.81 (d, 2H), 5.82 (d, 1H), 5.93 (d, 1H), 6.47–6.52 (t, 2H), 6.93–7.15 (m, 6H), 7.59– 7.61 (d, 2H). 13CNMR (CDCl3), δ (ppm): 30.73, 30.77, 31.38, 31.40, 36.50, 55.70, 55.72, 55.76, 56.35, 101.44, 109.97, 110.01, 110.30, 113.39, 113.40, 115.14, 115.17, 121.22, 121.27, 121.94, 122.00, 122.25, 122.30, 122.85, 122.86, 127.13, 127.18, 128.93, 139.90, 139.92, 140.83, 140.86, 147.23, 148.33, 148.40, 148.46, 149.44, 162.74, 162.80, 182.57, 183.73. MS (ESI) calculated for C24H22O6: 406.43, found: 407.2 [M + H]+, 445.2 [M+ + K]. 2.3. Synthesis of (1E,6E)-1-(4-((1-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)-3-methoxyphenyl)-7(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione, curcumin mono-amine The curcumin mono-amine was synthesized via ‘click’ chemistry as per the following procedure (Scheme 1A (1b)). Curcumin monoalkyne (Scheme 1a) (1.00 g, 2.45 mmol) and N3-PEG3-NH2 (480 μl, 2.45 mmol) were dissolved in 40 ml mixture of t-BuOH and THF (1:1). Freshly prepared solutions of CuSO4·5H2O and sodium ascorbate in water were added to the reaction mixture. Final concentrations of CuSO4·5H2O and sodium ascorbate were kept at 4 mM. The mixture was stirred for 12 h at room temperature. Once the reaction was stopped, solvent was evaporated and finally the dark brown colored product was isolated via column chromatography using chloroform/ methanol (90:10 v/v) as eluent. Yield: 1.22 g (78%). 1 H NMR (600 MHz, CDCl3), δ (ppm): 3.36–3.88 (m), 4.51–4.52 (t), 5.27 (s), 5.77 (s), 6.44 (t), 6.87 (d), 7.01–7.06 (m), 5.52–7.56 (q), 7.83 (s). 13C NMR (150 MHz, CDCl3), δ (ppm): 50.28, 50.63, 55.82, 55.88, 62.79, 68.10, 69.33, 69.98, 70.12, 70.21, 70.36, 70.46, 70.48, 70.56, 70.59, 70.63, 101.24, 109.87, 110.36, 113.60, 115.25, 121.36, 122.18, 122.29, 122.97, 124.37, 127.12, 128.69, 128.75, 130.84, 139.97, 140.81, 143.41, 147.33, 148.67, 149.51, 149.54, 182.66, 183.70. ESI-MS calculated for C32H40N4O9: 624.28, found: 625.6 [M + H]+, 647.5 [M+ + Na]. 2.4. Synthesis of azide terminated poly(tert-butyl acrylate) A 100 ml flask fitted with a stopcock was flame-dried under vacuum and allowed to cool at ambient temperature under Ar. The flask was charged with CuBr (115 mg, 0.8 mmol). Under positive pressure of Argon, a solution of 2-bromo-2-methylpropionic acid 2-[2-(2Azidoethoxy)ethoxy] ethyl ester (118 mg, 0.36 mmol) dissolved in tertbutyl acrylate (4 ml, 27.55 mmol) was added via syringe, followed by the addition of 1,1,4,7,7-pentamethyldiethylenetriamine (PMDETA) (0.17 ml, 0.8 mmol). Following three freeze-pump-thaw degassing cycles, the reaction was allowed to stir for 2 h 10 min at 60 °C. Submerging the flask in liquid nitrogen quenched the polymerization, the mixture was allowed to warm to ambient temperature, and diluted with tetrahydrofuran. CupriSorb® was added and stirred ca. 30 min for removing copper complex. After filtration, the mixture was poured into a 15% methanol, deionized water solution, white solid product was precipitated. The polymer was further purified via reprecipitation and dried under vacuum, isolated weight was 2.18 g. Mn = 8586 g/mol, Mw/Mn = 1.21. 1H NMR (600 MHz, CDCl3): δ (ppm) 4.19 (s, 2H), 3.55–3.75 (m, 6H), 3.39 (t, 2H), 2.18–2.30 (bm), 1.85 (s), 1.28–1.62 (bm), 1.15 (s, 6H), 0.89 (m). FTIR (film, cm−1): 3433, 2977, 2289, 2113 (azide), 1727, 1619, 1450, 1367, 1255, 1153, 845, 752.

49

mixture was dialyzed using 3500 MWCO membrane in distilled water with several exchanges and finally the lyophilization yielded the deprotected polymer as white powder. Yield: 97%, Mn = 42.423 Mw = 50.570 PDI = 1.19; 1H NMR (600 MHz, D2O), δ (ppm): 1.14– 1.18 (m, 8H), 1.40 (s, 4H), 1.61 (s, 46H), 1.73 (s, 112H), 1.90 (s, 40H), 2.36 (s, 104H), 3.67–4.17 (m, 12H). FT-IR (cm− 1): 3022, 2117 (N3), 1681, 1454. 2.6. Synthesis of azide terminated poly(curcumin)-poly(glucosamine) copolymer (CGPC copolymer) The water-soluble curcumin-glucose copolymer was produced via EDC·HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) amide coupling through a one-pot synthetic strategy previously developed by our group to synthesize dye-incorporated copolymers [20]. Poly(acrylic acid) required for this synthesis was prepared following the procedure reported above [20]. Azide terminated poly(acrylic acid) (140 mg, 0.006 mmol), Curcumin mono-amine (100 mg, 0.16 mmol), EDC·HCl (60 mg, 0.3 mmol) and HOBt (50 mg, 0.3 mmol) were dissolved in 9 ml of DMF in a round bottom flask, degassed with N2 and followed by addition of triethylamine (0.3 ml, 2 mmol). The reaction mixture was stirred at room temperature for 72 h. After 72 h, 248 mg (1.15 mmol) of D(+)-glucosamine (dissolved in 1 ml of distilled water) was injected via a syringe and stirred for 24 h at room temperature. The reaction mixture was dialyzed extensively using a 10 kDa MWCO membrane, and then purified by passing through a Sephadex LH20 size-exclusion column. Finally, the CGPC copolymer product was isolated as light-orange colored fluffy powder after lyophilization. Typical yield for this synthesis is ~ 325 mg. 1H NMR (600 MHz, DMF-d7), δ (ppm): 1.03 (t), 1.18–1.21 (t), 1.31–1.33 (d), 1.39–1.44 (t), 1.59–1.87 (bt), 2.11 (bs), 2.66 (s), 3.27–4.32 (m), 4.61–5.55 (bm), 6.25 (s), 7.02–7.85 (bm). GPC (H2O): Mn = 14.358, Mw = 18.953, PDI = 1.32. 2.7. Precipitation test for CGPC copolymer-metal binding A 5 mg/ml CGPC solution was prepared by dissolving CGPC in ultrapure water, and 50 mM solutions of each metal ion were prepared by dissolving the required mass of metal in pure water. For each metal binding experiment, 100 μl of the metal salt solution and 100 μl of the CGPC solution were combined in a centrifuge tube, mixed, and centrifuged at 12 k RPM for 5 min. If a precipitate was observed, chelation was indicated. If no precipitate was observed, it was implied that curcumin did not chelate that metal. 2.8. Control experiments using NIRF-dye-glucose copolymer, poly(acrylic acid), curcumin, and glucose Poly(acrylic acid) (pAA) and a near infrared dye labelled glucose copolymer(NDGPC) were synthesized and used as controls for the metal-chelation experiment. NDGPC (5% NIRF dye loading), made in accordance with our reported procedure (see supporting information for structure) [19], and pAA were dissolved in water in 5 mg/ml concentrations and used for the metal-chelation experiments as mentioned above. Similarly, if a precipitate was observed, chelation was indicated and if no precipitate was observed, it was implied that the pAA or NIRF-dye-glucose copolymer did not chelate the metal. The experiments were also repeated with glucose and curcumin separately, although the curcumin had to be dissolved in DMSO before being mixed with the metal solution.

2.5. Synthesis of azide terminated poly(acrylic acid)

2.9. Scanning electron microscopy (SEM) analysis

Trifluro acetic acid (2 ml, 27 mmol) of was added drop-wise to a round bottom flask containing azido-poly(tert-butyl acrylate) (0.3 g, 0.02 mmol). The reaction was stirred under N2 for 4 h. The reaction

Samples were placed on the surface of two-sided carbon adhesive tabs, which covered aluminum SEM specimen holders. Samples were placed inside the vacuum chamber of a Med20 Sputter coater (Baltec-

50

A. Averick et al. / Reactive and Functional Polymers 102 (2016) 47–52

Leica, Buffalo Grove, IL) and were coated with palladium for 120 s. Samples were then analyzed using an Amray 1910 Field Emission Scanning Electron Microscope at the operating voltage of 5 kV. Digital images were obtained using SEM Image Display software (SEMTech Solutions, North Billerica, MA). 3. Results and discussion 3.1. Synthesis of water soluble curcumin loaded copolymer CGPC We have recently reported a convenient route to synthesize water soluble polymers loaded with multiple copies of near infrared fluorescent dye molecules [20]. In the current study, we first synthesized a welldefined living polymer containing functional side chain pendant groups, and then attached a number of water soluble, biocompatible moieties, and curcumin to the functional polymer side chains. The synthesis of water soluble polyvalent curcumin conjugate was achieved via the synthesis of novel mono-functional curcumin derivatives in which one of the phenolic groups of curcumin has been chemically modified with reactive functional group (Scheme 1A). The mono-alkyne derivative of curcumin (Scheme 1A (1a)) was synthesized according to the published procedure [13], where curcumin was etherified with propargyl bromide in presence of K2CO3 at room temperature in DMF. To functionalize curcumin with amine group, curcumin mono-alkyne was reacted with commercially available azido-triethylene glycolamine in presence of ‘click’ reagents (copper (II) sulfate and sodium ascorbate), and the product was purified via column chromatography. The 7.83 ppm peak (triazole proton) in the 1H NMR and ESIMS data m/z = 624.28 confirms the conjugation of curcumin with azido-triethylene glycolamine. Curcumin monoamine and Glu-NH2 (D-(+)-glucosamine), were grafted to the poly(acrylic acid) (pAA) polymer 2 using the standard amide coupling reagents: 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxybenzotriazole (HOBt), in DMF to produce Curcumin-Glucose Polymer Conjugate, as depicted in Scheme 1B. The pAA polymer was synthesized according to the procedure published earlier [20], where synthesis involved the polymerization of tertbutyl acrylate via ATRP to produce poly(tert-butyl acrylate) and subsequent deprotection with trifluoracetic acid. The polymers synthesized were characterized via 1H NMR, Gel Permeation Chromatograph (GPC) and FT-IR spectroscopy (see supporting information). The percentage of the curcumin in the polymer 3 was determined to be ~5% per polymer chain, based on the GPC and 1H NMR analysis. Briefly, the percent composition (~5%) of curcumin in the polymer was calculated by comparing the molecular weights of 2 and 3 and by calculating the ratios between the aromatic protons of curcumin (at 7.02–7.85 ppm) and the aliphatic protons of glucose moieties (at 3.27–4.32 ppm). We synthesized copolymers with 5% curcumin loading for metal chelation studies because higher curcumin loading leads to substantially lower water solubility. 3.2. Metal chelation and precipitation with CGPC copolymer The chelation properties of curcumin via its 1,3-diketo unit towards a wide range of metals are well documented in literature [8,13–17]. Curcumin forms chelate-complexes with certain metal ions but its water insolubility severely restricts its use as a metal decontaminant. Since curcumin is polyvalently attached in a brush architecture in case of Curcumin Glucose Polymer Conjugates, we postulated that upon CGPC addition to metal solutions a precipitate would form due to inter- and intra-polymer binding of metals (Fig. 1). This cross-linking/ precipitation behavior of the CGPC facilitated a rapid assay of metalchelation activity. CGPC chelation was tested by mixing equivolumetric aliquots of CGPC (5 mg/ml) and metal salt (50 mM) and incubating for 5 min, followed by centrifugation (12,000 RPM, 5 min.). The CGPC chelation properties were found to be pH independent (from pH ≤ 1 to pH = 7) in the acidic pH range tested. This is of importance since acidic conditions destabilize

Fig. 1. Schematic approximation of cross-linking. Curcumin is shown in orange, the polymer in blue, and the metal in grey (glucose not shown, not to scale). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the chelation complex of commonly used metal chelators, which limits the use of traditional agents. In the stomach or the kidney and bladder, the pH can be as low as 1 and 4.5, respectively, which is sufficient to substantially destabilize or even prevent the binding of many chelating agents to metal ions. However, the CGPC formed stable complexes with metal ions at a range of pH values, which are substantially lower than the pH values that occur under physiological conditions. The results are as shown below in Table 1 and Fig. 2. Table 1 also includes the results of the control experiments which are discussed later on. The results of the CGPC metal chelation indicate that the CGPC has high specificity for toxic metals (Cd, Cr, Cu, Pb, Ag, etc.), with no observed precipitation for most of the macro metals (calcium, magnesium, potassium, and sodium) with the exception of iron. The mass of precipitate after lyophilization revealed approximately complete precipitation of polymer and metal ions through complex formation. These observations support the application of using CGPC or similar strategies to purify the heavy metal containing wastewaters as well as a potential method for the treatment of heavy metal poisoning. 3.3. Control experiments We preformed several control experiments to show that metal chelation was exclusively due to the presence of curcumin moieties in the

Table 1 Results of metal chelation with CGPC polymer and control experiments in the range of pH ≤ 1 to pH = 7 (P = complete precipitation; N = no precipitation).

Metal ions

CGPC

Curcumin

pAA

NDGPC

Glucose

Aluminum

P

N

N

N

N

Anitmony

N

N

N

N

N

Barium

P

N

P

N

N

Cadmium

P

N

N

N

N

Calcium

N

N

N

N

N

Cesium

N

N

N

N

N

Chromium

P

N

N

N

N

Cobalt

P

N

P

N

N

Copper

P

N

N

N

N N

Iron

P

P

N

N

Lead

P

N

P

N

N

Lithium

N

N

N

N

N

Magnesium

N

N

N

N

N

Manganese

N

N

N

N

N

Mercury

N

N

N

N

N

Nickel

P

N

N

N

N

Potassium

N

N

N

N

N

Silver

P

N

N

N

N

Sodium

N

N

N

N

N

Zinc

P

N

N

N

N

A. Averick et al. / Reactive and Functional Polymers 102 (2016) 47–52

Fig. 2. Digital photograph of chelation test results for (from left) water (negative control), aluminum, barium, cadmium, chromium, cobalt, copper, iron, lead, nickel, silver, and zinc.

CGPC and not the glucose or unreacted carboxylates. First, a CGPC mimic was prepared with a 5% loading of a near-infrared fluorescent dye (NIRF) replacing the curcumin moieties using a previously reported method [20]. The NIRF dye conjugated neoglyocoplymer control is not expected to have any metal chelation properties. This mimic was tested against the same metal ions employed to evaluate CGPC and no precipitation was observed (Table 1). Secondly, an assay was developed to determine if unreacted carboxylic acid groups on the CGPC were present in the final polymer which could bind with the metal ions. In this assay, sodium bicarbonate was added to a 5 mg/ml solution of either to pure pAA or CGPC. In the presence of carboxylic acids, bubbling would be observed due to release of CO2. Bubbling was observed for the pAA but not for the CGPC, indicating that carboxylic acid moiety was not present on the CGPC copolymer. Third, we performed an assay where pure pAA was tested for its metal chelation properties against the metals (see results in Table 1). For the solutions with low pH (pH b 2) no precipitation was observed, presumably because the carboxylic acid is protonated under these conditions. Although crosslinking and precipitation was observed for some metal salts that produced less acidic solutions or under buffered conditions, the lack of activity at low pH indicates pAA does not contain the chelating moiety responsible for our observations. Finally, the chelating properties of free curcumin and glucose were tested with the above mentioned metal ions under similar conditions as the CGPC copolymer. (Please note that in case of curcumin, it had to be dissolved in DMSO prior to the mixing with the solutions containing metal ions.) CGPC binds to a much wider range of heavy metal ions compared to the controls. From these findings we concluded that CGPC is superior in its metalbinding, cross-linking and precipitation properties compared to the control entities evaluated.

3.4. Characterization of chelation To determine the extent of polymer precipitated via metal chelation, the absorbance at 370 nm of the CGPC was measured in the solution (pH b 2) before and after metal chelation. An absorbance calibration curve of CGPC was generated by measuring a solution of 2.5 mg/ml CGPC (mimicking 0% precipitation), a 0.025 mg/ml CGPC solution (mimicking 99% CGPC precipitation), and other intermediate concentrations. The degree of precipitation was calculated by dividing the A370 of the sample by that of the 0.025 mg/ml CGPC solution (which was found to be 0.189) and subtracting that number from 100. As there are several factors which may have led to errors in the measurements, including the absorption of the metal salt itself, the results reported here should be interpreted as the approximate lower bound of the degree of precipitation. Fig. 3 shows the resulting metal chelation results as measured by CGPC precipitation.

51

Fig. 3. Degree of metal chelating effectiveness with CGPC copolymer for each metal ion based on UV–Vis absorption. Error bars represent standard deviation.

We believe the intrinsic chelation behavior is being accentuated by two factors. First, by polyvalent effect of multiple curcumin units in the CGPC allows for multiple cross-links, i.e. inter-polymer and intrapolymer forming insoluble adducts. Second, since the cross-linked CGPC precipitates, following Le Chatalier's principle, the equilibrium shifts further for more CGPC-metal binding and cross-linkage/ precipitation. Several precipitates were also studied using Scanning Electron Microscopy (SEM) for their surface topography. Fig. 3 compares CGPC obtained by lyophilizing 5 mg/ml solution to the CGPC precipitated with lead, silver, and nickel salts that have been washed and lyophilized. The fluffy, extended structure of the lyophilized CGPC (Fig. 4a) is in contrast to the blocky, dense Pb-CGPC and Ag-CGPC precipitates (Fig. 4b and c, respectively). The structure of the metal ion-CGPC precipitates is markedly different for the various metals indicating that the kinetics of metal-polymer complex precipitation and other factors are metal dependent. 4. Conclusions Curcumin is an important phytochemical with metal binding properties, and in this work we succeeded in substantially enhancing these properties by attaching curcumin to a polymer and rendering it watersoluble. The resulting polymer conjugate was found to have a high specificity for the removal of toxic metals, notably lead and cadmium. The complexes formed were found to be stable at any physiologically relevant pH. The high stability and specificity of the curcumin-polymer conjugate implies that it has potential to improve the treatment of toxic metal poisoning as well as for applications in heavy metal containing wastewater treatment. The unique azide chain end present in the curcumin neoglycopolymers will be employed for metal free click bioconjugation to produce novel metal binding biomaterials in future. Acknowledgments The authors would like to thank College of Staten Island at the City University of New York and the Oakridge National Lab for support. We would like to acknowledge Dr. Alan Lyons and Dr. Probal Banerjee for their advice. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.reactfunctpolym.2016.03.009.

52

A. Averick et al. / Reactive and Functional Polymers 102 (2016) 47–52

Fig. 4. SEM images of a) GCPG, b) Pb-GCPG, c) Ag-CGPC, and d) Ni-CGPC.

References [1] J. Ravindran, S. Prasad, B.B. Aggarwal, Curcumin and cancer cells: how many ways can curry kill tumor cells selectively? AAPS J. 11 (2009) 495–510. [2] K.S. Raja, R. Balambika, S. Dolai, W. Shi, The concept of a green drug, curcumin and its derivatives as a model system, Mini-Rev. Org. Chem. 6 (2009) 152–158. [3] S. Debnath, D. Saloum, S. Dolai, C. Sun, S. Averick, K. Raja, J.E. Fata, Dendrimercurcumin conjugate: a water soluble and effective cytotoxic agent against breast cancer cell lines, Anti Cancer Agents Med. Chem. 13 (2013) 1531–1539. [4] P. Langone, P.R. Debata, J.D.R. Inigo, S. Dolai, S. Mukherjee, P. Halat, K. Mastroianni, G.M. Curcio, M.R. Castellanos, K. Raja, P. Banerjee, Coupling to a glioblastomadirected antibody potentiates anti-tumor activity of curcumin, Int. J. Cancer 135 (2014) 710–719. [5] F. Yang, P.L.G.P. Lim, A.N. Begum, O.J. Ubeda, M.R. Simmons, S.S. Ambegaokar, P. Chen, R. Kayed, C.G. Glabe, S.A. Frautschy, G.M. Cole, Curcumin inhibits formation of amyloid β oligomers and fibrils, binds plaques, and reduces amyloid in vivo, J. Biol. Chem. 280 (2005) 5892–5901. [6] S. Dolai, W. Shi, C. Corbo, C. Sun, S. Averick, D. Obeysekera, M. Farid, A. Alonso, P. Banerjee, K. Raja, “Clicked” sugar–curcumin conjugate: modulator of amyloid-β and tau peptide aggregation at ultralow concentrations, ACS Chem. Neurosci. 2 (2011) 694–699. [7] A.N. Nurfinal, M.S. Reksohadiprodjo, H. Timmerman, U.A. Jenie, D. Sugiyant, H. van der Goot, Synthesis of some symmetrical curcumin derivatives and their antiinflammatory activity, Eur. J. Med. Chem. 32 (1997) 321–328. [8] R. Agarwal, S.K. Goel, J.R. Behari, Detoxification and antioxidant effects of curcumin in rats experimentally exposed to mercury, J. Appl. Toxicol. 30 (2010) 457–468. [9] A. Adal, S.W. Wiener, Heavy Metal Toxicity, Medscape, 2013 (http://emedicine. medscape.com/article/814960-overview). [10] S.C. Harvey, in: L.S. Goodman, A. Gilman (Eds.),The Pharmacological Basis of Therapeutics 1975, pp. 924–945. [11] S. Margel, A novel approach for heavy metal poisoning treatment, a model. Mercury poisoning by means of chelating microspheres: hemoperfusion and oral administration, J. Med. Chem. 24 (1981) 1263–1266.

[12] S. Flora, V. Pachauri, Chelation in metal intoxication, Int. J. Environ. Res. Public Health 7 (2010) 2745–2788. [13] S. Daniel, J.L. Limson, A. Dairam, G.M. Watkins, S. Daya, Through metal binding, curcumin protects against lead- and cadmium-induced lipid peroxidation in rat brain homogenates and against lead-induced tissue damage in rat brain, J. Inorg. Biochem. 98 (2004) 266–275. [14] J. Nair, S. Strand, N. Frank, J. Knauft, H. Wesch, P.R. Galle, H. Bartsch, Apoptosis and age-dependant induction of nuclear and mitro-chondrial etheno-DNA adducts in Long–Evans Cinnamon (LEC) rats: enhanced DNA damage by dietary curcumin upon copper accumulation, Carcinogenesis 26 (2005) 1307–1315. [15] H. Manjunatha, K. Srinivasan, Protective effect of dietary curcumin and capsaicin on induced oxidation of low-density lipoprotein, iron- induced hepatotoxicity and carrageenan-induced inflammation in experimental rats, FEBS J. 273 (2006) 4528–4537. [16] A. Dairam, J.L. Limson, G.M. Watkins, E. Antunes, S. Daya, Curcuminoids, curcumin and demethoxycurcumin reduce lead-induced memory deficits in male Wistar rats, J. Agric. Food Chem. 55 (2007) 1039–1044. [17] S. Padmaja, T.N. Raju, Antioxidant effect of curcumin in selenium induced cataract of Wistar rats, Ind. J. Exp. Biol. 42 (2004) 601–603. [18] A.L. Cheng, C.H. Hsu, J.K. Lin, M.M. Hsu, Y.F. Ho, T.S. Shen, J.Y. Ko, J.T. Lin, B.R. Lin, M.S. Wu, H.S. Yu, S.H. Jee, G.S. Chen, T.M. Chen, C.A. Chen, M.K. Lai, Y.S. Pu, M.H. Pan, Y.J. Wang, C.C. Tsai, C.Y. Hsieh, Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant lesions, Anticancer Res. 21 (2001) 2895–2900. [19] W. Shi, S. Dolai, S. Rizk, A. Hussain, H. Tariq, S. Averick, W. L'Amoreaux, A. El Idrissi, P. Banerjee, K. Raja, Synthesis of monofunctional curcumin derivatives, clicked curcumin dimer, and a PAMAM dendrimer curcumin conjugate for therapeutic applications, Org. Lett. 9 (2007) 5461–5464. [20] W. Shi, S. Dolai, S. Averick, S.S. Fernando, J.A. Saltos, W. L'Amoreaux, P. Banerjee, K. Raja, A general methodology toward drug/dye incorporated living copolymer−protein hybrids: (NIRF dye-glucose) copolymer−avidin/BSA conjugates as prototypes, Bioconjug. Chem. 20 (2009) 1595–1601.