Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Binding analysis of antioxidant polyphenols with PAMAM nanoparticles P. Chanphai & H. A. Tajmir-Riahi To cite this article: P. Chanphai & H. A. Tajmir-Riahi (2017): Binding analysis of antioxidant polyphenols with PAMAM nanoparticles, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2017.1391124 To link to this article: http://dx.doi.org/10.1080/07391102.2017.1391124
Accepted author version posted online: 11 Oct 2017.
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Date: 12 October 2017, At: 03:38
Publisher: Taylor & Francis Journal: Journal of Biomolecular Structure and Dynamics
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DOI: http://doi.org/10.1080/07391102.2017.1391124
Accepted manuscript (with no mark) ID TBSD-2017-0584 for JBSD
Binding analysis of antioxidant polyphenols with PAMAM nanoparticles
P. Chanphai and H. A. Tajmir-Riahi*
Department of Chemistry-Biochemistry, Physics, University of Québec in TroisRivières, C. P. 500, Trois-Rivières (Québec), Canada G9A 5H7
Key words: polyphenols, dendrimers, conjugation, loading efficacy, TEM, modeling Abbreviations: res, resveratrol; gen, genistein; cur, crucumin; PAMAM, polyamidoamine, LE, loading efficacy; FTIR, Fourier transform infrared; TEM. Transmission electron microscopy Running title : Polyphenol-PAMAM 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 Dietary polyphenols are abundant micronutrients in our diet and paly major role in prevention of degenerative diseases. The binding efficacy of antioxidant polyphenols resveratrol, genistein and curcumin with PAMAM-G3 and PAMAM-G4 nanoparticles was investigated in aqueous solution at physiological conditions, using multiple spectroscopic methods, TEM images and docking studies. The polyphenol bindings are via hydrophilic, hydrophobic and H-bonding contacts with resveratrol forming more stable conjugates. As PAMAM size increased the loading efficacy and the stability of polyphenol-polymer conjugates were increased. Polyphenol binding induced major alterations of dendrimer morphology. PAMAM nanoparticles are capable of delivery of polyphenols in vitro.
Introduction High dietary intake of polyphenols is associated with decreased risk of several diseases such as cardiovascular diseases, cancer and neurodegenerative diseases (Manach et al., 2004; Tsao, 2010; Han et al., 2007; Watson et al., 2014; Vauzour et al., 2010; Scalbert et al., 2005; Visioli et al., 2011; Cao et al., 2016). Among polyphenols, resveratrol, genistein and curcumin (Scheme 1) have shown major protection against cancer and cardiovascular diseases (Thornthwaite et al.,
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2017). The antioxidant activity of these polyphenols consists of scavenging oxygen radicals and preventing DNA damage (Azqueta & Collins, 2016). Resveratrol (3,4’,5’-trihydroxystilbene), a phytoalexin found in grapes, berries and wine, is one of the most interesting natural compound due to its role exerted in cancer prevention and therapy. In particular, resveratrol is able to
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delay cell cycle progression and to induce apoptotic death in several cell lines (Leone et al., 2010). Genistein (4’,5,7-trihydroxy isoflavone) present in soybeans and chickpeas, has a wide spectrum of physiological and pharmacological functions and can inhibits DNA methylation and increase expression of tumor suppressor genes in human breast cancer cells (Xie et al., 2014). Curcumin (diferuloyl methane) is a naturally occurring yellow pigment derived from the rhizome of Curcuma longa and exhibits a variety of pharmacologic effects including anti-inflammatory, anti-infectious and anticancer activities (Sikora et al., 2006). Curcumin has been shown to inhibit tumor promotion of skin, oral, intestinal and colon cancers in experimental animals (Kroemer & Reed, 2006). It has been also shown that curcumin can inhibit proliferation and/or induce cell death in vitro experiments (Karunagaran et al., 2005; Sharma et al., 2005). Despite the health benefits associated with polyphenols, the bioavailability of many polyphenol limits their effect. Problems with poor solubility, fast-metabolism and food preparation techniques limit the bioavailability and bioactivity of these dietary micronutrients (Bishop et al., 2016). Encapsulation of polyphenols has shown to protect and increase bioavailability of these dietary compounds and to enhance their anticancer activity (Lu et al. 2016; Vitorio et al., 2017; Fan & Bhandari, 2010; Abderrezak et al. 2012; Munin & Edward-Levy, 2011). Among the dendrimers, polyamidoamines (PAMAM) are widely used in drug delivery (Samal et al., 2012; Patri et al., 2002; Sevenson, 2009; Maiti et al., 2004; Duncan & Izzo, 2005; Stiriba et al., 2002). These polymers can act as drug delivery tools, either through physical
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interactions (encapsulation) or through chemical bonding (Wolinsky & Grinstaff, 2008; Mandeville et al., 2013; Sanyakamdhorn et al., 2015; Sanyakamdhorn et al., 2016). Predicting binding affinity from structural models is of a major research interest because of its fundamental role in drug design and development. The structural analysis of drug-carriers is crucial for drug
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design as well as drug loading and unloading, thus the task of drug delivery to the target sites, while maintaining high efficacy combined with low systemic exposure is important here. Therefore it was of a major interest to study the conjugation of polyphenols resveratrol, genistein and curcumin by PAMAM dendrimers, in order to evaluate the efficacy of PAMAM nanoparticles in polyphenol delivery. Here, we report the conjugation of PAMAM dendrimers with resveratrol, genistein and curcumin, using multiple spectroscopic methods, TEM analysis and molecular modeling. The binding of polyphenols to PAMAM and the effects of polyphenol conjugation on polymer morphology are discussed here.
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Scheme 1: Chemical structures of polyphenols
Experimental Materials PAMAM-G3 (MW = 6909 (g/mol), PAMAM-G4 (MW = 14 242 g/mol) and highly purified resveratrol, genistein and 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. Preparation of stock solutions Polyphenol solution (1 mM) was first prepared in Tris-HCl/ethanol 50% and then diluted by serial dilution to different concentrations in Tris-HCl/ethanol. An appropriate amount of PAMAM was dissolved in water and adjusted to pH 7.2 in Tris-HCl. PAMAM preparation was similar to our previous report (Sanyakamdhorn et al., 2016). 5
Fluorescence spectroscopy Fluorimetric titrations 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 of polyphenol (100 µM) were prepared from the above stock solutions at 24 ±1 °C. Solutions of
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PAMAM (10 to 200 µM) were prepared in Tris-HCl (pH. 7.2) 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 PAMAM 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) (Liang et al., 2008; Usha et al., 2006; Usha et al., 2005; Chignell et al., 1994; Kanakis et al., 2013; N’ soukpoé-Kossi et al., 2006). 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 (1) The binding constant (KA), can be calculated as:
K A Qn B /Q B n
(2)
where, [Q] and [B] are the quencher and polymer concentration, respectively, [QnB] is the concentration of non fluorescent fluorophore-quencher complex and [B0] gives total polymer concentration:
Qn B B0 B
(3)
K A B0 B / Q B n
(4)
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The fluorescence intensity is proportional to the polymer concentration as described:
B/B0 F / F0
(5)
Results from fluorescence measurements can be used to estimate the binding constant of polyphenol-polymer complex. From eq 4:
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logF0 F / F log K A n logQ
(6)
The accessible fluorophore fraction (f) can be calculated by modified Stern-Volmer equation:
F0 / F0 F 1 / fK Q 1 / f (7) 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 of the total emission for an interaction with a hydrophobic quencher (Liang et al., 2008; Usha et al., 2006; Usha et al., 2005; Chignell et al., 1994). The K will be calculated from F0/F= K[Q] +1. FTIR spectroscopic measurements Infrared spectra were recorded on a FTIR spectrometer (Impact 420 model), equipped with deuterated triglycine sulphate (DTGS) detector and KBr beam splitter, using AgBr windows. Solution of polyphenol was added dropwise to the PAMAM 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 concentration of PAMAM 60 µM. Spectra were collected after 2h incubation chitosan and polyphenol 7
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 and 100 scans. The difference spectra [(PAMAM + polyphenol solution) – (PAMAM solution)] were generated as reported (Sanyakamdhorn et al., 2016).
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Transmission electron microscopy The TEM images were taken using a Philips EM 208S microscope operating at 180 kV. The morphology of the polyphenol with PAMAM conjugates was monitored in aqueous solution at pH 7.2, using transmission electron microscopy. One drop (5–10 µL) of the freshly-prepared mixture [polyphenol solution (60 µM) + PAMAM 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 added before drying at room temperature. Docking The docking studies were performed with ArgusLab 4.0.1 software (Mark A. Thompson, Planaria Software LLC, Seattle, Wa, http://www.arguslab.com). The structure of PAMAM was obtained from literature report (Mandeville et al., 2013) and the polyphenol three dimensional structures were generated from PM3 semi-empirical calculations using Chem3D Ultra 6.0. The whole PAMAM was selected as a potential binding site since no prior knowledge 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 conformations were ranked using the Ascore scoring function, which estimates the free binding energy. Upon location of the potential binding sites, the docked complex conformations were optimized using a steepest decent algorithm until
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convergence, with a maximum of 20 iterations within a distance of 3.5 Å relative to the polyphenol were involved in the conjugation (Mandeville et al., 2013). Results and discussion Stability of polyphenol-PAMAM conjugates by fluorescence spectroscopy
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Since PAMAM dendrimers are weak fluorophores, the titrations of polyphenols were done against various PAMAM concentrations, using polyphenols emission bands at 425 (resveratrol), 375 nm (genistein) and 365 nm (curcumin) (Liang et al., 2008; Usha et al., 2006; Usha et al., 2005; Chignell et al., 1994). When PAMAM interacts with polyphenol, fluorescence may change depending on the impact of such interaction on the polymer conformation, or via direct quenching effect (Lakowicz, 2006). The decrease of fluorescence intensity of polyphenols has been monitored at 365-425 nm for polyphenolPAMAM complexes (Fig 1A-C). The plot of F0 / (F0 – F) vs 1 / [PAMAM] is shown in Fig 1A’-C’. Assuming that the observed changes in fluorescence come from the interaction between the polyphenols and polymer, the quenching constant can be taken as the binding constant of the complex formation. The overall binding constants shown resveratrol forms more stable polymer conjugates with the order resveratrol>curcumin>genistein (Table 1). PAMAM-G4 forms more stable polyphenol adducts than PAMAM-G3 (Table 1). The extra stability of polyphenol-PAMAM-G4 conjugate is related to the presence of additional terminal charged NH2 groups (64 amino groups) compared to PAMAM-G3 (32 amino groups), that are involved in polyphenolpolymer complexation. This indicates that polyphenol-PAMAM interactions involve hydrophilic, hydrophobic and H-bonding contacts. The loading efficacy for polyphenol-PAMAM conjugates was determined as
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reported (Chandra et al., 2011). The loading efficacy was estimated 30-55% for these polyphenol-PAMAM conjugates. The loading efficacy enhanced as PAMAM size was increased from G3 to G4 (Table 1). The number of loaded polyphenol molecule per PAMAM was estimated to be 1 to 1.2 (Table 1). The number of polyphenol molecules
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bound per 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 polyphenol molecules that are bound per polymer molecule (Table 1). Table 1. Calculated binding constants (K) for the polyphenol-PAMAM conjugates with loading efficacy (LE) and the number of bound polyphenol (n) per polymer K
n
Complexes
(×106 M-1)
Resveratrol- PAMAM-G3
5.2 (±0.9)
1.2
45
Genistein-PAMAM-G3
0.31 (±0.1)
1
30
Curcumin-PAMAM-G3
0.41 (±0.2)
1
35
Resveratrol– PAMAM-G4
5.5 (±0.8)
1.1
55
Genistein-PAMAM-G4
0.32 (±0.1)
1
40
Curcumin-PAMAM-G4
0.5 (±0.2)
1
45
LE %
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Figure 1. Fluorescence emission spectra of polyphenols with PAMAM-G3 for (A) resveratrol in 10 mM Tris-HCl buffer pH 7.2 at 24 C (resveratrol) (a) 20 µM and (b-k) polymer at 2, 5, 10, 15, 20, 30, 40, 50, 60 and 70 µM; B (genistein) (20 µM) (b-k) polymer at 2, 5, 10, 15, 20, 30, 40, 50, 60 and 70 µM; C (curcumin) (20 µM) (b-k) polymer at 2, 5, 10, 15, 20, 30, 40, 50, 60 and 70 µM. Inset The plot of F0/(F0- F) as a function of 1/PAMAM-G3 concentration. The binding constant K being the ratio of the intercept and the slope for polyphenol-PAMAM conjugates.
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Figure 2. Fluorescence emission spectra of polyphenols with PAMAM-G4 for (A) resveratrol in 10 mM Tris-HCl buffer pH 7.2 at 24 C (resveratrol) (a) 20 µM and (b-k) polymer at 2, 5, 10, 15, 20, 30, 40, 50, 60 and 70 µM; (B) genistein) (20 µM) (b-k) polymer at 2, 5, 10, 15, 20, 30, 40, 50, 60 and 70 µM; (C) curcumin (20 µM) (b-k) polymer at 2, 5, 10, 15, 20, 30, 40, 50, 60 and 70 µM. Inset The plot of F0/(F0- F) as function of 1/PAMAM-G4 concentration. The binding constant K being the ratio of the intercept and the slope for polyphenol-PAMAM conjugates.
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Binding of polyphenol-PAMAM conjugates by FTIR spectroscopy Evidence regarding polyphenol-PAMAM interactions comes from infrared results presented in Figures 3 and 4. Spectral shifting and intensity variations were observed for the PAMAM C=O, C-N, C-O stretching and OH and NH bending (Chanphai & Tajmir-
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Riahi, 2016; Popesou et al., 2006, Froehlich et al., 2009), upon polyphenol hydrophilic contacts with polymer polar groups. In the free PAMAM-G3 and PAMAM-G4 infrared spectra, the bands at 1640 and 1560-1540 cm-1 (OH and NH bending), 1465, 1380, 1303, 1159 and 1060 cm-1 (C-O and C-C stretch), exhibited shifting and intensity decreases, upon polyphenol-polymer complexation (Figs 3 and 4, complexes, 60 µM). The observed spectral shifting was accompanied by a gradual decrease in the intensity of the above vibrational frequencies, in the difference spectra [(polymer + polyphenol solution) – (polymer solution)] of polyphenol-polymer complexes (Figs 3 and 4, diffs 15 µM). As polyphenol concentrations increased to 60 mM, major decreases in the intensity of polymer vibrational frequencies were observed (Figs 3 and 4, diffs 60 µM). The strong negative features in the difference spectra of polyphenol-PAMAM conjugates are due to the loss of intensity of polymer vibrational frequencies (Figs 3 and 4 diffs 60 µM). The spectral changes observed are attributed to the hydrophilic interactions of polyphenol polar groups with polymer OH, NH2, C-O and C-N groups. The hydrophilic interaction is more pronounced at high polyphenol concentrations (Figs 3 and 4, diffs 60 µM).
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Figure 3. FTIR spectra in the region of 1800-600 cm-1 of hydrated films (pH 7.2) for free PAMAM-G3 for (A) resveratrol, (B) genistein and (C) curcumin), PAMAM (60 µM) with difference spectra (diff.) of polyphenol-PAMAM conjugates (bottom two curves) obtained at different polyphenol concentrations (indicated on the figure).
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Figure 4. FTIR spectra in the region of 1800-600 cm-1 of hydrated films (pH 7.2) for free PAMAM-G4 for (A) resveratrol, (B) genistein and (C) curcumin), PAMAM (60 µM) with difference spectra (diff.) of polyphenol-PAMAM conjugates (bottom two curves) obtained at different polyphenol concentrations (indicated on the figure)
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TEM analysis and morphology of polyphenol-PAMAM conjugates TEM images showed major changes in the morphological aggregation of PAMAMnanoparticles upon conjugation with polyphenols. The TEM images of PAMAM-G4 and
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their polyphenol conjugates in aqueous solution at pH 7.2 are shown in Fig. 5. The TEM images of the native PAMAM (Fig. 5A) show a spherical morphology with mean dimensions of 10 ± 2 nm (Ottaviani et al., 1998; Yang et al., 2013; Ottaviani et al., 2000; Zhang et al., 2013). This is in agreement with the fact that dendrimers are globular polymers with nonpolar interior structure and polar surface, which can be viewed as unimolecular micelles (Tono et al., 2006; Reddy et al., 2012; Liu et al., 2000). The interaction between PAMAM and polyphenols leads to the transformation of the shape of the aggregates. Upon conjugation of PAMAM with resveratrol, genistein and curcumin, there is a shape transformation from spherical to a mixture composed of spherical micelles with an almost exclusive filamentous micelles (Fig. 5A-D). This transformation can be explained in terms of a strong electrostatic repulsion between the primary amino chain of PAMAM in the presence of polyphenols. Encapsulation of polyphenols by PAMAM results in noncovalent interaction such as hydrogen bonding, van der waals interaction, and electrostatic contact. In the case of polyphenol-PAMAM conjugates, the interactions of polyphenols with polymer occurs via hydrogen bonds between drug amide or OH group and dendrimer-NH groups (hydrogen donors) or vice versa (Ambade et al., 2005; Sanyakamdhorn et al., 2017). Dendrimers dissolved in polar solvents such as aqueous media can encapsulate polyphenol molecules in their internal cavities or by condensing them on the surface. If polyphenol is encapsulated in the interior of PAMAM,
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there is no disruption in the electrostatic repulsion between the primary amino chain ends of PAMAM. This indicates that the morphology of dendrimer (spherical shape) will not be changed. However, if the polyphenol interacts with the surface of dendrimer, the electrostatic repulsion between the primary amino chain of polymer will disappear.
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Consequently, this can induce a shape transformation from spherical to filamentous micelles. Polyphenols interact with the internal tertiary amine and surface amino groups through hydrogen bonding, that lead to a mixture of spherical and filamentous micelles (Fig. 5A-D).
Figure 5. TEM images showing the morphology of PAMAM (A) with resveratrol (B), genistein (C) curcumin (D) conjugates at pH 7.2 at 24°C. The concentrations of polymer and polyphenols were 60 µM in all samples
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Docking study and the bindings of polyphenol-PAMAM conjugates Docking results in which polyphenols docked to PAMAM are shown in Fig. 6. The results showed that resveratrol (A), genistein (B) and curcumin(C) are located on the dendrimer surface and internal cavities surrounded by hydrophilic and hydrophobic
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groups (Fig. 6). The free binding energy showed spontaneous interaction between polyphenols and polymer with genistein forming more stable complexes with free binding of -4.14 kcal/mol (resveratrol), and -4.29 kcal/mol (genistein) and -3.73 kcal/mol (curcumin) (Fig. 6A-C). This is in contrast with the spectroscopic results that showed resveratrol forms more stable polymer conjugate than genistein and curcumin (Table 1).
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Figure 6. Best conformations for polyphenols docked to PAMAM with the free binding energy for A (resveratrol), B (genistein) and C (curcumin) (polyphenols is shown in green color) 19
Conclusions Extensive research has been conducted on the effect of polyphenols on human health (Manach et al., 2004; Tsao, 2010; Han et al., 2007; Watson et al., 2014; Vauzour et al., 2010; Scalbert et al., 2005; Visioli et al., 2011; Cao et al., 2016). Bioavailability of
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polyphenols causes limitation on the health benefits of these dietary micronutrients. Encapsulation of polyphenols enhances the bioavailability in solution (Lu et al., 2016; Fang & Bhandari, 2010; Shadrack et al., 2015). Previously we reported the interactions of polyphenols with mPEG-PAMAM-G3, mPEG-PAMAM-G4 and PAMAM-G4 (Abderrezak et al., 2012). However, a more detailed structural analysis regarding the binding efficacy of polyphenols with PAMAM-G3 and pAMAM-G4 is carried out here. Comparisons of the binding efficacies of polyphenols with PAMAM dendrimers showed the presence of hydrophilic, hydrophobic and H-bonding contacts with the order of stability resveratrol >curcumin> genistein. As polymer size increased the stability and the biding efficacy of polyphenol-PAMAM were increased. Polyphenol binding alters PAMAM morphology leading to a major increase in the size of polymer aggregates. PAMAM nanoparticles can transport polyphenols and enhance the bioavailability of these dietary micronutrients. Acknowledgments The financial support of the Natural Sciences and Engineering Research Council of Canada (NSERC) to H.A. Tajmir-Riahi is highly appreciated.
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ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Binding analysis of antioxidant polyphenols with PAMAM nanoparticles P. Chanphai & H. A. Tajmir-Riahi To cite this article: P. Chanphai & H. A. Tajmir-Riahi (2017): Binding analysis of antioxidant polyphenols with PAMAM nanoparticles, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2017.1391124 To link to this article: http://dx.doi.org/10.1080/07391102.2017.1391124
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Date: 12 October 2017, At: 03:38
Publisher: Taylor & Francis Journal: Journal of Biomolecular Structure and Dynamics
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DOI: http://doi.org/10.1080/07391102.2017.1391124
Accepted manuscript (with no mark) ID TBSD-2017-0584 for JBSD
Binding analysis of antioxidant polyphenols with PAMAM nanoparticles
P. Chanphai and H. A. Tajmir-Riahi*
Department of Chemistry-Biochemistry, Physics, University of Québec in TroisRivières, C. P. 500, Trois-Rivières (Québec), Canada G9A 5H7
Key words: polyphenols, dendrimers, conjugation, loading efficacy, TEM, modeling Abbreviations: res, resveratrol; gen, genistein; cur, crucumin; PAMAM, polyamidoamine, LE, loading efficacy; FTIR, Fourier transform infrared; TEM. Transmission electron microscopy Running title : Polyphenol-PAMAM 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 Dietary polyphenols are abundant micronutrients in our diet and paly major role in prevention of degenerative diseases. The binding efficacy of antioxidant polyphenols resveratrol, genistein and curcumin with PAMAM-G3 and PAMAM-G4 nanoparticles was investigated in aqueous solution at physiological conditions, using multiple spectroscopic methods, TEM images and docking studies. The polyphenol bindings are via hydrophilic, hydrophobic and H-bonding contacts with resveratrol forming more stable conjugates. As PAMAM size increased the loading efficacy and the stability of polyphenol-polymer conjugates were increased. Polyphenol binding induced major alterations of dendrimer morphology. PAMAM nanoparticles are capable of delivery of polyphenols in vitro.
Introduction High dietary intake of polyphenols is associated with decreased risk of several diseases such as cardiovascular diseases, cancer and neurodegenerative diseases (Manach et al., 2004; Tsao, 2010; Han et al., 2007; Watson et al., 2014; Vauzour et al., 2010; Scalbert et al., 2005; Visioli et al., 2011; Cao et al., 2016). Among polyphenols, resveratrol, genistein and curcumin (Scheme 1) have shown major protection against cancer and cardiovascular diseases (Thornthwaite et al.,
2
2017). The antioxidant activity of these polyphenols consists of scavenging oxygen radicals and preventing DNA damage (Azqueta & Collins, 2016). Resveratrol (3,4’,5’-trihydroxystilbene), a phytoalexin found in grapes, berries and wine, is one of the most interesting natural compound due to its role exerted in cancer prevention and therapy. In particular, resveratrol is able to
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delay cell cycle progression and to induce apoptotic death in several cell lines (Leone et al., 2010). Genistein (4’,5,7-trihydroxy isoflavone) present in soybeans and chickpeas, has a wide spectrum of physiological and pharmacological functions and can inhibits DNA methylation and increase expression of tumor suppressor genes in human breast cancer cells (Xie et al., 2014). Curcumin (diferuloyl methane) is a naturally occurring yellow pigment derived from the rhizome of Curcuma longa and exhibits a variety of pharmacologic effects including anti-inflammatory, anti-infectious and anticancer activities (Sikora et al., 2006). Curcumin has been shown to inhibit tumor promotion of skin, oral, intestinal and colon cancers in experimental animals (Kroemer & Reed, 2006). It has been also shown that curcumin can inhibit proliferation and/or induce cell death in vitro experiments (Karunagaran et al., 2005; Sharma et al., 2005). Despite the health benefits associated with polyphenols, the bioavailability of many polyphenol limits their effect. Problems with poor solubility, fast-metabolism and food preparation techniques limit the bioavailability and bioactivity of these dietary micronutrients (Bishop et al., 2016). Encapsulation of polyphenols has shown to protect and increase bioavailability of these dietary compounds and to enhance their anticancer activity (Lu et al. 2016; Vitorio et al., 2017; Fan & Bhandari, 2010; Abderrezak et al. 2012; Munin & Edward-Levy, 2011). Among the dendrimers, polyamidoamines (PAMAM) are widely used in drug delivery (Samal et al., 2012; Patri et al., 2002; Sevenson, 2009; Maiti et al., 2004; Duncan & Izzo, 2005; Stiriba et al., 2002). These polymers can act as drug delivery tools, either through physical
3
interactions (encapsulation) or through chemical bonding (Wolinsky & Grinstaff, 2008; Mandeville et al., 2013; Sanyakamdhorn et al., 2015; Sanyakamdhorn et al., 2016). Predicting binding affinity from structural models is of a major research interest because of its fundamental role in drug design and development. The structural analysis of drug-carriers is crucial for drug
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design as well as drug loading and unloading, thus the task of drug delivery to the target sites, while maintaining high efficacy combined with low systemic exposure is important here. Therefore it was of a major interest to study the conjugation of polyphenols resveratrol, genistein and curcumin by PAMAM dendrimers, in order to evaluate the efficacy of PAMAM nanoparticles in polyphenol delivery. Here, we report the conjugation of PAMAM dendrimers with resveratrol, genistein and curcumin, using multiple spectroscopic methods, TEM analysis and molecular modeling. The binding of polyphenols to PAMAM and the effects of polyphenol conjugation on polymer morphology are discussed here.
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Scheme 1: Chemical structures of polyphenols
Experimental Materials PAMAM-G3 (MW = 6909 (g/mol), PAMAM-G4 (MW = 14 242 g/mol) and highly purified resveratrol, genistein and 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. Preparation of stock solutions Polyphenol solution (1 mM) was first prepared in Tris-HCl/ethanol 50% and then diluted by serial dilution to different concentrations in Tris-HCl/ethanol. An appropriate amount of PAMAM was dissolved in water and adjusted to pH 7.2 in Tris-HCl. PAMAM preparation was similar to our previous report (Sanyakamdhorn et al., 2016). 5
Fluorescence spectroscopy Fluorimetric titrations 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 of polyphenol (100 µM) were prepared from the above stock solutions at 24 ±1 °C. Solutions of
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PAMAM (10 to 200 µM) were prepared in Tris-HCl (pH. 7.2) 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 PAMAM 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) (Liang et al., 2008; Usha et al., 2006; Usha et al., 2005; Chignell et al., 1994; Kanakis et al., 2013; N’ soukpoé-Kossi et al., 2006). 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 (1) The binding constant (KA), can be calculated as:
K A Qn B /Q B n
(2)
where, [Q] and [B] are the quencher and polymer concentration, respectively, [QnB] is the concentration of non fluorescent fluorophore-quencher complex and [B0] gives total polymer concentration:
Qn B B0 B
(3)
K A B0 B / Q B n
(4)
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The fluorescence intensity is proportional to the polymer concentration as described:
B/B0 F / F0
(5)
Results from fluorescence measurements can be used to estimate the binding constant of polyphenol-polymer complex. From eq 4:
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logF0 F / F log K A n logQ
(6)
The accessible fluorophore fraction (f) can be calculated by modified Stern-Volmer equation:
F0 / F0 F 1 / fK Q 1 / f (7) 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 of the total emission for an interaction with a hydrophobic quencher (Liang et al., 2008; Usha et al., 2006; Usha et al., 2005; Chignell et al., 1994). The K will be calculated from F0/F= K[Q] +1. FTIR spectroscopic measurements Infrared spectra were recorded on a FTIR spectrometer (Impact 420 model), equipped with deuterated triglycine sulphate (DTGS) detector and KBr beam splitter, using AgBr windows. Solution of polyphenol was added dropwise to the PAMAM 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 concentration of PAMAM 60 µM. Spectra were collected after 2h incubation chitosan and polyphenol 7
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 and 100 scans. The difference spectra [(PAMAM + polyphenol solution) – (PAMAM solution)] were generated as reported (Sanyakamdhorn et al., 2016).
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Transmission electron microscopy The TEM images were taken using a Philips EM 208S microscope operating at 180 kV. The morphology of the polyphenol with PAMAM conjugates was monitored in aqueous solution at pH 7.2, using transmission electron microscopy. One drop (5–10 µL) of the freshly-prepared mixture [polyphenol solution (60 µM) + PAMAM 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 added before drying at room temperature. Docking The docking studies were performed with ArgusLab 4.0.1 software (Mark A. Thompson, Planaria Software LLC, Seattle, Wa, http://www.arguslab.com). The structure of PAMAM was obtained from literature report (Mandeville et al., 2013) and the polyphenol three dimensional structures were generated from PM3 semi-empirical calculations using Chem3D Ultra 6.0. The whole PAMAM was selected as a potential binding site since no prior knowledge 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 conformations were ranked using the Ascore scoring function, which estimates the free binding energy. Upon location of the potential binding sites, the docked complex conformations were optimized using a steepest decent algorithm until
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convergence, with a maximum of 20 iterations within a distance of 3.5 Å relative to the polyphenol were involved in the conjugation (Mandeville et al., 2013). Results and discussion Stability of polyphenol-PAMAM conjugates by fluorescence spectroscopy
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Since PAMAM dendrimers are weak fluorophores, the titrations of polyphenols were done against various PAMAM concentrations, using polyphenols emission bands at 425 (resveratrol), 375 nm (genistein) and 365 nm (curcumin) (Liang et al., 2008; Usha et al., 2006; Usha et al., 2005; Chignell et al., 1994). When PAMAM interacts with polyphenol, fluorescence may change depending on the impact of such interaction on the polymer conformation, or via direct quenching effect (Lakowicz, 2006). The decrease of fluorescence intensity of polyphenols has been monitored at 365-425 nm for polyphenolPAMAM complexes (Fig 1A-C). The plot of F0 / (F0 – F) vs 1 / [PAMAM] is shown in Fig 1A’-C’. Assuming that the observed changes in fluorescence come from the interaction between the polyphenols and polymer, the quenching constant can be taken as the binding constant of the complex formation. The overall binding constants shown resveratrol forms more stable polymer conjugates with the order resveratrol>curcumin>genistein (Table 1). PAMAM-G4 forms more stable polyphenol adducts than PAMAM-G3 (Table 1). The extra stability of polyphenol-PAMAM-G4 conjugate is related to the presence of additional terminal charged NH2 groups (64 amino groups) compared to PAMAM-G3 (32 amino groups), that are involved in polyphenolpolymer complexation. This indicates that polyphenol-PAMAM interactions involve hydrophilic, hydrophobic and H-bonding contacts. The loading efficacy for polyphenol-PAMAM conjugates was determined as
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reported (Chandra et al., 2011). The loading efficacy was estimated 30-55% for these polyphenol-PAMAM conjugates. The loading efficacy enhanced as PAMAM size was increased from G3 to G4 (Table 1). The number of loaded polyphenol molecule per PAMAM was estimated to be 1 to 1.2 (Table 1). The number of polyphenol molecules
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bound per 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 polyphenol molecules that are bound per polymer molecule (Table 1). Table 1. Calculated binding constants (K) for the polyphenol-PAMAM conjugates with loading efficacy (LE) and the number of bound polyphenol (n) per polymer K
n
Complexes
(×106 M-1)
Resveratrol- PAMAM-G3
5.2 (±0.9)
1.2
45
Genistein-PAMAM-G3
0.31 (±0.1)
1
30
Curcumin-PAMAM-G3
0.41 (±0.2)
1
35
Resveratrol– PAMAM-G4
5.5 (±0.8)
1.1
55
Genistein-PAMAM-G4
0.32 (±0.1)
1
40
Curcumin-PAMAM-G4
0.5 (±0.2)
1
45
LE %
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Figure 1. Fluorescence emission spectra of polyphenols with PAMAM-G3 for (A) resveratrol in 10 mM Tris-HCl buffer pH 7.2 at 24 C (resveratrol) (a) 20 µM and (b-k) polymer at 2, 5, 10, 15, 20, 30, 40, 50, 60 and 70 µM; B (genistein) (20 µM) (b-k) polymer at 2, 5, 10, 15, 20, 30, 40, 50, 60 and 70 µM; C (curcumin) (20 µM) (b-k) polymer at 2, 5, 10, 15, 20, 30, 40, 50, 60 and 70 µM. Inset The plot of F0/(F0- F) as a function of 1/PAMAM-G3 concentration. The binding constant K being the ratio of the intercept and the slope for polyphenol-PAMAM conjugates.
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Figure 2. Fluorescence emission spectra of polyphenols with PAMAM-G4 for (A) resveratrol in 10 mM Tris-HCl buffer pH 7.2 at 24 C (resveratrol) (a) 20 µM and (b-k) polymer at 2, 5, 10, 15, 20, 30, 40, 50, 60 and 70 µM; (B) genistein) (20 µM) (b-k) polymer at 2, 5, 10, 15, 20, 30, 40, 50, 60 and 70 µM; (C) curcumin (20 µM) (b-k) polymer at 2, 5, 10, 15, 20, 30, 40, 50, 60 and 70 µM. Inset The plot of F0/(F0- F) as function of 1/PAMAM-G4 concentration. The binding constant K being the ratio of the intercept and the slope for polyphenol-PAMAM conjugates.
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Binding of polyphenol-PAMAM conjugates by FTIR spectroscopy Evidence regarding polyphenol-PAMAM interactions comes from infrared results presented in Figures 3 and 4. Spectral shifting and intensity variations were observed for the PAMAM C=O, C-N, C-O stretching and OH and NH bending (Chanphai & Tajmir-
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Riahi, 2016; Popesou et al., 2006, Froehlich et al., 2009), upon polyphenol hydrophilic contacts with polymer polar groups. In the free PAMAM-G3 and PAMAM-G4 infrared spectra, the bands at 1640 and 1560-1540 cm-1 (OH and NH bending), 1465, 1380, 1303, 1159 and 1060 cm-1 (C-O and C-C stretch), exhibited shifting and intensity decreases, upon polyphenol-polymer complexation (Figs 3 and 4, complexes, 60 µM). The observed spectral shifting was accompanied by a gradual decrease in the intensity of the above vibrational frequencies, in the difference spectra [(polymer + polyphenol solution) – (polymer solution)] of polyphenol-polymer complexes (Figs 3 and 4, diffs 15 µM). As polyphenol concentrations increased to 60 mM, major decreases in the intensity of polymer vibrational frequencies were observed (Figs 3 and 4, diffs 60 µM). The strong negative features in the difference spectra of polyphenol-PAMAM conjugates are due to the loss of intensity of polymer vibrational frequencies (Figs 3 and 4 diffs 60 µM). The spectral changes observed are attributed to the hydrophilic interactions of polyphenol polar groups with polymer OH, NH2, C-O and C-N groups. The hydrophilic interaction is more pronounced at high polyphenol concentrations (Figs 3 and 4, diffs 60 µM).
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Figure 3. FTIR spectra in the region of 1800-600 cm-1 of hydrated films (pH 7.2) for free PAMAM-G3 for (A) resveratrol, (B) genistein and (C) curcumin), PAMAM (60 µM) with difference spectra (diff.) of polyphenol-PAMAM conjugates (bottom two curves) obtained at different polyphenol concentrations (indicated on the figure).
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Figure 4. FTIR spectra in the region of 1800-600 cm-1 of hydrated films (pH 7.2) for free PAMAM-G4 for (A) resveratrol, (B) genistein and (C) curcumin), PAMAM (60 µM) with difference spectra (diff.) of polyphenol-PAMAM conjugates (bottom two curves) obtained at different polyphenol concentrations (indicated on the figure)
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TEM analysis and morphology of polyphenol-PAMAM conjugates TEM images showed major changes in the morphological aggregation of PAMAMnanoparticles upon conjugation with polyphenols. The TEM images of PAMAM-G4 and
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their polyphenol conjugates in aqueous solution at pH 7.2 are shown in Fig. 5. The TEM images of the native PAMAM (Fig. 5A) show a spherical morphology with mean dimensions of 10 ± 2 nm (Ottaviani et al., 1998; Yang et al., 2013; Ottaviani et al., 2000; Zhang et al., 2013). This is in agreement with the fact that dendrimers are globular polymers with nonpolar interior structure and polar surface, which can be viewed as unimolecular micelles (Tono et al., 2006; Reddy et al., 2012; Liu et al., 2000). The interaction between PAMAM and polyphenols leads to the transformation of the shape of the aggregates. Upon conjugation of PAMAM with resveratrol, genistein and curcumin, there is a shape transformation from spherical to a mixture composed of spherical micelles with an almost exclusive filamentous micelles (Fig. 5A-D). This transformation can be explained in terms of a strong electrostatic repulsion between the primary amino chain of PAMAM in the presence of polyphenols. Encapsulation of polyphenols by PAMAM results in noncovalent interaction such as hydrogen bonding, van der waals interaction, and electrostatic contact. In the case of polyphenol-PAMAM conjugates, the interactions of polyphenols with polymer occurs via hydrogen bonds between drug amide or OH group and dendrimer-NH groups (hydrogen donors) or vice versa (Ambade et al., 2005; Sanyakamdhorn et al., 2017). Dendrimers dissolved in polar solvents such as aqueous media can encapsulate polyphenol molecules in their internal cavities or by condensing them on the surface. If polyphenol is encapsulated in the interior of PAMAM,
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there is no disruption in the electrostatic repulsion between the primary amino chain ends of PAMAM. This indicates that the morphology of dendrimer (spherical shape) will not be changed. However, if the polyphenol interacts with the surface of dendrimer, the electrostatic repulsion between the primary amino chain of polymer will disappear.
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Consequently, this can induce a shape transformation from spherical to filamentous micelles. Polyphenols interact with the internal tertiary amine and surface amino groups through hydrogen bonding, that lead to a mixture of spherical and filamentous micelles (Fig. 5A-D).
Figure 5. TEM images showing the morphology of PAMAM (A) with resveratrol (B), genistein (C) curcumin (D) conjugates at pH 7.2 at 24°C. The concentrations of polymer and polyphenols were 60 µM in all samples
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Docking study and the bindings of polyphenol-PAMAM conjugates Docking results in which polyphenols docked to PAMAM are shown in Fig. 6. The results showed that resveratrol (A), genistein (B) and curcumin(C) are located on the dendrimer surface and internal cavities surrounded by hydrophilic and hydrophobic
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groups (Fig. 6). The free binding energy showed spontaneous interaction between polyphenols and polymer with genistein forming more stable complexes with free binding of -4.14 kcal/mol (resveratrol), and -4.29 kcal/mol (genistein) and -3.73 kcal/mol (curcumin) (Fig. 6A-C). This is in contrast with the spectroscopic results that showed resveratrol forms more stable polymer conjugate than genistein and curcumin (Table 1).
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Figure 6. Best conformations for polyphenols docked to PAMAM with the free binding energy for A (resveratrol), B (genistein) and C (curcumin) (polyphenols is shown in green color) 19
Conclusions Extensive research has been conducted on the effect of polyphenols on human health (Manach et al., 2004; Tsao, 2010; Han et al., 2007; Watson et al., 2014; Vauzour et al., 2010; Scalbert et al., 2005; Visioli et al., 2011; Cao et al., 2016). Bioavailability of
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polyphenols causes limitation on the health benefits of these dietary micronutrients. Encapsulation of polyphenols enhances the bioavailability in solution (Lu et al., 2016; Fang & Bhandari, 2010; Shadrack et al., 2015). Previously we reported the interactions of polyphenols with mPEG-PAMAM-G3, mPEG-PAMAM-G4 and PAMAM-G4 (Abderrezak et al., 2012). However, a more detailed structural analysis regarding the binding efficacy of polyphenols with PAMAM-G3 and pAMAM-G4 is carried out here. Comparisons of the binding efficacies of polyphenols with PAMAM dendrimers showed the presence of hydrophilic, hydrophobic and H-bonding contacts with the order of stability resveratrol >curcumin> genistein. As polymer size increased the stability and the biding efficacy of polyphenol-PAMAM were increased. Polyphenol binding alters PAMAM morphology leading to a major increase in the size of polymer aggregates. PAMAM nanoparticles can transport polyphenols and enhance the bioavailability of these dietary micronutrients. Acknowledgments The financial support of the Natural Sciences and Engineering Research Council of Canada (NSERC) to H.A. Tajmir-Riahi is highly appreciated.
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