Probing the binding of resveratrol, genistein and curcumin with chitosan nanoparticles

Accepted Manuscript Probing the binding of resveratrol, genistein and curcumin with chitosan nanoparticles

P. Chanphai, H.A. Tajmir-Riahi PII: DOI: Reference:

S0167-7322(17)33206-3 doi: 10.1016/j.molliq.2017.08.024 MOLLIQ 7732

To appear in:

Journal of Molecular Liquids

Received date: Revised date: Accepted date:

17 July 2017 2 August 2017 6 August 2017

Please cite this article as: P. Chanphai, H.A. Tajmir-Riahi , Probing the binding of resveratrol, genistein and curcumin with chitosan nanoparticles, Journal of Molecular Liquids (2017), doi: 10.1016/j.molliq.2017.08.024

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ACCEPTED MANUSCRIPT Revised manuscript MOLLIQ_2017_2936 for publication in J. Mol. Liquids Revisions made are shown in red color throughout the text and Table 1

Probing the binding of resveratrol, genistein and curcumin with

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chitosan nanoparticles

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P. Chanphai and H. A. Tajmir-Riahi*

Department of Chemistry-Biochemistry-Physics, Université du Québec à Trois-Rivières,

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C. P. 500, Trois-Rivières (Québec), Canada G9A 5H7

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Key words: polyphenols, chitosan, conjugation, loading efficacy, TEM, modeling

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Abbreviations: res, resveratrol; gen, genistein; cur, crucumin; LE, loading efficacy; FTIR, Fourier transform infrared; TEM. Transmission electron

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microscopy

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Running title : Polyphenol-chitosan conjugation

* Corresponding author: H. A. Tajmir-Riahi; Fax: 819-376-5084; Tel: 819-376-5011 (ext. 3310), e.mail: [email protected]

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Abstract The conjugation of antioxidant polyphenols resveratrol, genistein and curcumin with chitosan-15 and chitosan-100 kDa was studied in aqueous solution at physiological conditions,

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using multiple spectroscopic methods, TEM images and docking studies. Structural analysis

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showed that polyphenol bindings are via hydrophilic, hydrophobic and H-bonding contacts with

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resveratrol forming more stable conjugates. As chitosan size increased, the binding efficacy and stability of polyphenol-polymer adducts were increased. Polyphenol binding induced major

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alterations of chitosan morphology. Chitosan nanoparticles are capable of delivery of

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polyphenols in vitro.

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ACCEPTED MANUSCRIPT Introduction Studies have shown the important role of antioxidant polyphenols in human health and disease [1]. Epidemiological investigations suggest that high dietary intake of polyphenols is associated with decreased risk of several diseases such as cardiovascular diseases, cancer and

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neurodegenerative diseases [2-5]. Among polyphenols, resveratrol, genistein and curcumin

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(Scheme 1) shown major protection against cancer and cardiovascular diseases [6]. The

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antioxidant activity of these polyphenols consists of scavenging oxygen radicals and preventing DNA damage [7]. Resveratrol (3,4’,5’-trihydroxystilbene), a phytoalexin found in

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grapes, berries and wine, is one of the most interesting natural compound due to its role

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exerted in cancer prevention and therapy. In particular, resveratrol is able to delay cell cycle progression and to induce apoptotic death in several cell lines [8]. Genistien (4’,5,7-

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trihydroxy isoflavone) present in soybeans and chickpeas, has a wide spectrum of

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physiological and pharmacological functions and can inhibits DNA methylation and increase expression of tumor suppressor genes in human breast cancer cells [9]. Curcumin (diferuloyl

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methane) is a naturally occurring yellow pigment derived from the rhizome of Curcuma

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longa and exhibits a variety of pharmacologic effects including anti-inflammatory, antiinfectious and anticancer activities [10]. Curcumin has been shown to inhibit tumor

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promotion of skin, oral, intestinal and colon cancers in experimental animals [11]. It has been also shown that curcumin can inhibit proliferation and/or induce cell death in vitro experiments [12,13]. Despite the health benefits associated with polyphenols, the bioavailability of many polyphenol limits their effect. Problems with poor solubility fastmetabolism and food preparation techniques limit the bioavailability and bioactivity of these

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ACCEPTED MANUSCRIPT dietary micronutrients [14]. Encapsulation of polyphenols has shown to protect and increase bioavailability of these dietary compounds and to enhance their anticancer activity [15-19]. Chitosan and its derivatives have the desired properties for safe use as pharmaceutical drug delivery tools. This has prompted accelerated research activities worldwide on chitosan

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micro and nanoparticles as drug delivery vehicles. These systems have great utility in

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controlled release and targeting studies of almost all class of bioactive molecules. Chitosan

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is a natural polymer obtained by deacetylation of chitin [20]. It is non-toxic, biocompatible and biodegradable polysaccharide. Chitosan nanoparticles have gained more attention as

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drug delivery carriers because of their better stability, low toxicity, simple and mild

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preparation method and providing versatile routes of administration [21-25]. The deacetylated chitosan backbone of glucosamine units has a high density of charged amine

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groups, permitting strong electrostatic interactions with proteins and genes that carry an

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overall negative charge at neutral pH conditions [20,21]. The fast expanding research of the useful physicochemical and biological properties of chitosan has led to the recognition of the

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cationic polysaccharide, as a natural polymer for drug delivery [22-27]. Therefore it was of

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a major interest to study the encapsulation of polyphenols resveratrol, genistein and curcumin with chitosan of different sizes, in order to evaluate the efficacy of chitosan

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nanoparticles in polyphenol delivery. We report the conjugation of resveratrol, genistein and curcumin with chitosan

nanoparticles, using multiple spectroscopic methods, TEM analysis and molecular modeling. The binding of polyphenols to chitosan and the effects of polyphenol conjugation on polymer morphology are discussed here.

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2. Experimental 2.1. Materials Purified chitosan 15 and 100 KDa (90% deacetylation) were from Polysciences

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Inc. (Warrington, USA) and used as supplied. Highly purified resveratrol, genistein and

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curcumin were purchased from Sigma Chemical Company (St-Louis, MO) and used as supplied. Other chemicals were of reagent grade and used without further purification.

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2.2. Preparation of stock solutions

Polyphenol solution (1 mM) was first prepared in Tris-HCl/ethanol 50% and then diluted by

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serial dilution to different concentrations in Tris-HCl/ethanol.

An appropriate amount of

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chitosan was dissolved in 2% acetic acid and then the solution was adjusted to pH 5.5-6.5. Chitosan preparation was similar to our previous report [27].

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2.3. Fluorescence spectroscopy

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Fluorimetric experiments were carried out on a Perkin-Elmer LS55 Spectrometer. Stock solutions of polyphenol 1 mM were prepared at room temperature (24 ±1 °C). Various solutions

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of polyphenol (100 µM) were prepared from the above stock solutions at 24 ±1 °C. Solutions of

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chitosan (10 to 200 µM) were prepared in 2% acetic acid and diluted in Tris-HCl (pH. 7.4) at 24 ±1 °C. The above solutions were kept in the dark and used soon after. Samples containing 0.4 ml of the above polyphenol solution and various chitosan solutions (10 to 200 µM) were mixed to obtain final polyphenol concentration of 30 µM. The fluorescence spectra were recorded at λem = 420 nm (resveratrol), 375 nm (genistein ) and 365 nm (curcumin). The intensity of these bands were used to calculate the binding constant (K) [28-31].

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ACCEPTED MANUSCRIPT On the assumption that there are (n) substantive binding sites for quencher (Q) on protein (B), the quenching reaction can be shown as follows: nQ  B  Qn B

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The binding constant (KA), can be calculated as:

K A  Qn B/Q B

(2)

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n

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where, [Q] and [B] are the quencher and protein concentration, respectively, [QnB] is the concentration of non fluorescent fluorophore-quencher complex and [B0] gives total

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polymer concentration:

Qn B  B0   B K A   B0    B  / Q   B 

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n

(3) (4)

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The fluorescence intensity is proportional to the protein concentration as described: (5)

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B/B0   F / F0

Results from fluorescence measurements can be used to estimate the binding constant of

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drug-protein complex. From eq 4:

(6)

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log F0  F  / F   log K A  n log Q

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The accessible fluorophore fraction (f) can be calculated by modified Stern-Volmer equation:

F0 / F0  F   1 / fKQ  1 / f

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where, F0 is the initial fluorescence intensity and F is the fluorescence intensities in the presence of quenching agent (or interacting molecule). K is the Stern-Volmer quenching constant, [Q] is the molar concentration of quencher and f is the fraction of accessible fluorophore to a polar quencher, which indicates the fractional fluorescence contribution

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ACCEPTED MANUSCRIPT of the total emission for an interaction with a hydrophobic quencher [28-31]. The K will be calculated from F0/F= K[Q] +1. 2.4. FTIR spectroscopic measurements Infrared spectra were recorded on a FTIR spectrometer (Impact 420 model),

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equipped with deuterated triglycine sulphate (DTGS) detector and KBr beam splitter,

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using AgBr windows. Solution of polyphenol was added dropwise to the chitosan

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solution with constant stirring to ensure the formation of homogeneous solution and to reach the target polyphenol concentrations of 15 to 60 µM with a final chitosan

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concentration of chitosan 60 µM. Spectra were collected after 2h incubation chitosan and

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polyphenol solution at room temperature, using hydrated films. Interferograms were accumulated over the spectral range 4000-600 cm-1 with a nominal resolution of 2 cm-1

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and 100 scans. The difference spectra [(chitosan solution + polyphenol solution) –

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(chitosan solution)] were generated as reported [27]. 2.5. Transmission electron microscopy

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The TEM images were taken using a Philips EM 208S microscope operating at 180

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kV. The morphology of the polyphenol with chitosan conjugates was monitored in aqueous solution at pH 7.2, using transmission electron microscopy. One drop (5–10 µL)

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of the freshly-prepared mixture [polyphenol solution (60 µM) + chitosan solution (60 µM)] in Tris-HCl buffer (24 ± 1 °C) was deposited onto a glow-discharged carbon-coated electron microscopy grid. The excess liquid was absorbed by a piece of filter paper, and a drop of 2% uranyl acetate negative stain was was added before drying at room temperature.

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ACCEPTED MANUSCRIPT 2.6. Docking studies The docking studies were performed with ArgusLab 4.0.1 software (Mark A. Thompson, Planaria Software LLC, Seattle, Wa, http://www.arguslab.com). The chitosan structures were obtained from literature report [32] and the polyphenol three dimensional

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structures were generated from PM3 semi-empirical calculations using Chem3D Ultra

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6.0. The whole chitosan was selected as a potential binding site since no prior knowledge

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of such site was available. The docking runs were performed on the ArgusDock docking engine using regular precision with a maximum of 1000 candidate poses. The

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conformations were ranked using the Ascore scoring function, which estimates the free

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binding energy. Upon location of the potential binding sites, the docked complex conformations were optimized using a steepest decent algorithm until convergence, with

Results and discussion

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involved in the complexation [27].

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a maximum of 20 iterations within a distance of 3.5 Å relative to the polyphenol were

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3.1. Fluorescence spectra and stability of polyphenol-chitosan conjugates

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Since chitosan is a weak fluorophore, the titrations of polyphenols were done against various chitosan concentrations, using polyphenols emission bands at 425

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(resveratrol), 375 nm (genistein) and 365 nm (curcumin) [28-31].

When chitosan

interacts with polyphenol, fluorescence may change depending on the impact of such interaction on the polymer conformation, or via direct quenching effect [33-36]. The decrease of fluorescence intensity of polyphenols has been monitored at 365-425 nm for polyphenol-chitosan conjugates (Fig 1A-C). The plot of F0 / (F0 – F) vs 1 / [chitosan] is shown in Fig 1A’-C’. Assuming that the observed changes in fluorescence come from the

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ACCEPTED MANUSCRIPT interaction between the polyphenols and chitosan, the quenching constant can be taken as the binding constant of the complex formation. The K value obtained is the averages of four and six-replicate run for polyphenol-polymer systems (Table 1). The overall binding constants showing resveratrol forms more stable polymer conjugates than genistein and

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curcumin (Table 1). This indicates that polyphenol-polymer interactions involve both

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hydrophilic and hydrophobic contacts. The number of polyphenol molecules bound per

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polymer (n) is calculated from log [(F0 -F)/F] = logKS + n log [polyphenol] for the static quenching. The n values from the slope of the straight line plot showed 1.2 to 1 for

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polyphenol molecules that are bound per polymer molecule (Table 1).

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The loading efficacy for polyphenol-polymer conjugates was determined as reported [37]. The loading efficacy was estimated 35-50% for these polyphenol-chitosan

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conjugates. The loading efficacy enhanced as chitosan was size increased (Table 1). The

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number of loaded polyphenol molecule per protein was estimated to be 1 to 1.2 (Table 1).

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3.2. FTIR spectra and the bindings of polyphenol-chitosan conjugates

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The major spectral shifting and intensity variations of chitosan amide I band at 1633-1620 cm-1 (mainly C=O stretch) and amide II band at 1540-1520 cm-1 (C-N

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stretching coupled with N-H bending modes) [38,39] were used to characterize the nature of polyphenol-polymer interactions (Figures 3 and 4). At low and high polyphenol concentration (15-60 M), a minor decrease in the intensity was observed for the chitosan amide I at 1633-1620 cm-1 and amide II at 15401520 cm-1, in the difference spectra of the polyphenols-polymer complexes (Figs 3 and 4, diff., 15 and 60 M). The intensity variations were enhanced in the case of chitosan-100,

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ACCEPTED MANUSCRIPT upon polyphenol conjugation (compare Figs 3 and 4 difference spectra). The negative features are mainly located around 1650-1080 cm-1 in the spectra of polyphenol-chitosan conjugates (Figs 3 and 4, diff., 15 and 60 M). These negative features are related to the lose of intensity of the chitosan vibrational frequencies upon polyphenol complexation.

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The lose of intensity of the chitosan amide I and amide II bands is due to polyphenol

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3.3. TEM analysis of polyphenol-chitosan conjugates

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bindings to polymer C=O, C-N and N-H groups (hydrophilic interaction).

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The effect of polyphenol-chitosan interactions on the shape of chitosan nanoparticles was determined by using transmission electron microscopy. The shapes of uncomplexed

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chitosan alongside with their polyphenol conjugates are shown in the TEM micrographs (Fig. 5). TEM micrographs show that uncomplexed chitosan has a markedly different

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shape depending on its spherical-shaped size (Fig. 5A) with smooth surface and narrow

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size distribution of about 90 nm [40,41]. However, marked differences were observed in

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the morphology of the polyphenol–chitosan aggregates. TEM images clearly showed the appearance of the aggregates of irregular shapes dispersed in solution when conjugated

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with polyphenols (Fig. 5 B-D). In addition, the bound chitosan with polyphenol showed major changes of the polymer morphological shape (Fig. 5A-D). An increase of the

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spherical-shaped aggregates can be seen from TEM micrograph, suggesting that the spherical shapes were lost in favor of less spherical-shaped in the polyphenol–chitosan aggregates (Fig. 5 B-D).The loss of the spherical shape of chitosan nanoparticles after complex formation with polyphenol is likely to be the result of the polyphenol encapsulation. This is consistent with major particle size increase as encapsulation occurs (Fig. 5 B-D). Indeed, polyphenol binding to chitosan which is a linear polysaccharide

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ACCEPTED MANUSCRIPT with multiple sites of interaction and therefore should be regarded as core–shell system with polyphenol (core) and chitosan (shell) [42-46]. Therefore, if a tightly bound conjugate between polymer and polyphenol is formed, it can change the initial shape of polymer in favor of the polyphenol shape. The results suggest that the binding of

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polyphenol to chitosan may play a role in altering the predefined shape of the chitosan

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nanoparticles due to polyphenol encapsulation.

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3.4. Docking study and the bindings of polyphenol with chitosan

The spectroscopic data were combined with docking experiments in which

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resveratrol, genistein and curcumin molecules were docked to chitosan to determine the

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preferred binding sites on the chitosan. The models of the docking are shown in Fig. 6. The docking results showed that polyphenols are surrounded by several donor atoms of

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chitosan residue on the surface with a free binding energy of -3.97 kcal/mol for

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resveratrol-chitosan, -4.09 kcal/mol for genistein-chitosan and -3.39 kcal/mol for curcumin-chitosan conjugates (Fig. 6). It is evident that resveratrol, genistein and

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curcumin are not surrounded by similar donor groups showing different binding locations

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with genistein forming more stable adducts. This is in contrast with the spectroscopic results that showed resveratrol forms more stable conjugate than genistein and curcumin

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(Table 1).

4. Concluding remarks Bioavailability of antioxidant polyphenols causes limitation on the health benefits of these dietary micronutrients. Encapsulation of polyphenols enhances the bioavailability in solution [15,17]. Comparisons of binding efficacies of resveratrol, genistein and curcumin with chitosan nanoparticles show that polyphenol-polymer conjugation is via

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ACCEPTED MANUSCRIPT hydrophilic, hydrophobic and H-bonding contacts with the order of stability resveratrol >curcumin> genistein. The stability and loading efficacy of polyphenol-polymer conjugates increase as chitosan size increased. Polyphenol binding alters polymer morphology with an increase in chitosan aggregate diameter due to polyphenol

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encapsulation. Chitosan nanoparticles can transport polyphenols and enhance the

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bioavailability of these dietary micronutrients.

Acknowledgments

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The financial support of the Natural Sciences and Engineering Research Council

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of Canada (NSERC) to H.A. Tajmir-Riahi is highly appreciated .

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ACCEPTED MANUSCRIPT [45] V.P. Hoven, V. Tangpasuthadol, Y. Angkitpaiboon, N. Vallapa, S. Kiatkamjornwong, Surface-charged chitosan: preparation and protein adsorption, Carbohydr. Polymers 68 (2007) 44–53. [46] H. A. Tajmir-Riahi, Sh.Nafisi, S. Sanyakamdhorn, D. Agudelo, P.

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Chanphai, Applications of chitosan nanoparticles in drug delivery.

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Methods Mol. Biol. 1141 (2014) 65-184. doi: 10.1007/978-1-4939-

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Captions for Figures Scheme 1. Chemical structures of polyphenols Figure 1. Fluorescence emission spectra of polyphenols with chitosan-15 kDa for (A)

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resveratrol in 10 mM Tris-HCl buffer pH 7.2 at 24 C (resveratrol) (a) 20 µ M and (b-h)

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chitosan at 2, 5, 10, 15, 20 25 and 30 µM; B (genistein) (20 µM) (b-k) chitosan at 2, 5,

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10, 15, 20 25, 30, 35 and 40 µM and C (curcumin) (20 µM) (b-k) chitosan at 2, 5, 10, 15,

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20 25, 30, 35 and 40 µM. Inset The plot of F0/(F0- F) as a function of 1/chitosan concentration. The binding constant K being the ratio of the intercept and the slope for

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polyphenol-chitosan conjugates.

Figure 2. Fluorescence emission spectra of polyphenosl with chitosan-100 kDa for (A)

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resveratrol in 10 mM Tris-HCl buffer pH 7.2 at 24 C (resveratrol) (a) 20 µ M and (b-i)

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chitosan at 2, 5, 10, 15, 20 25, 30 and 40 µM; B (genistein) (20 µM) (b-k) chitosan at 2,

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5, 10, 15, 20 25, 30, 35 and 40 µM and C (curcumin) (20 µM) (b-k) chitosan at 2, 5, 10, 15, 20 25, 30, 35 and 40 µM. Inset The plot of F0/(F0- F) as a function of 1/chitosan

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concentration. The binding constant K being the ratio of the intercept and the slope for polyphenol-chitosan conjugates.

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Figure 3. FTIR spectra in the region of 1800-600 cm-1 of hydrated films (pH 7.2) for free chitosan-15 kDa for (A) resveratrol, (B) genistein and (C) curcumin), chitosan (60 µM) with difference spectra (diff.) of polyphenol-chitosan conjugates (bottom two curves) obtained at different polyphenol concentrations (indicated on the figure). Figure 4. FTIR spectra in the region of 1800-600 cm-1 of hydrated films (pH 7.2) for free chitosan-100 kDa for (A) resveratrol, (B) genistein and (C) curcumin), chitosan (60

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and polyphenols were 60 µM in all samples.

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Figure 6. Best conformations for polyphenols docked to chitosan with the free binding

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energy for A (resveratrol), B (genistein) and C (curcumin) (polyphenols is shown in

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green color) .

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Resveratrol- chitosan-15

4.2 (± 0.9) 0.021 (±0.01)

Curcumin-chitosan-15

0.086 (±0.04) 6.2 (±1)

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LE % 40

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0.21 (±0.01)

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Curcumin-chitosan-100

0.41(±0.2)

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Genistein-chitosan-100

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Scheme 1: Chemical structures of polyphenols

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Research Highlights ►Conjugation of antioxidant polyphenols with chitosan is investigated here.

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► Polyphenols bind chitosan via hydrophilic, hydrophobic and H-bonding contacts

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► As chitosan size increased more stable polyphenol conjugates were formed.

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► Resveratrol forms more stable conjugates than genistein and curcumin.

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► Major chitosan morphological changes occurred by polyphenol conjugation.

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