- Email: [email protected]
Investigation on the interaction behavior between curcumin and PAMAM dendrimer by spectral and docking studies
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 108 (2013) 251–255
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Investigation on the interaction behavior between curcumin and PAMAM dendrimer by spectral and docking studies Jian Cao ⇑, Hongmei Zhang, Yanqing Wang ⇑, Jinming Yang, Fuguang Jiang Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City, Jiangsu Province 224002, People’s Republic of China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" Binding interactions of curcumin
The formation of non-covalent adducts between PAMAM-C12 25% and curcumin induced the fluorescence quenching of PAMAM-C12 25%. There were mainly five classes of binding sites at the interface of PAMAMC12 25% binding with curcumin by hydrophobic, hydrogen bonds, and van der Waals forces interactions.
with PAMAM dendrimer were investigated. " The non-covalent adducts of PAMAM-C12 25%@curcumin formed. " There were about five binding sites for curcumin in PAMAM-C12 25%. " The hydrophobic, hydrogen bonds, and van der Waals forces stabilized PAMAM-C12 25%@curcumin complex.
a r t i c l e
i n f o
Article history: Received 21 July 2012 Received in revised form 3 November 2012 Accepted 7 February 2013 Available online 16 February 2013 Keywords: PAMAM-C12 25% Curcumin Fluorescence Binding mode Binding sites
a b s t r a c t The interactions between PAMAM-C12 25% and curcumin were studied by UV/vis, fluorescence spectroscopy, and molecular modeling methods. The experimental results showed that the formation of PAMAM-C12 25%@curcumin non-covalent adduct induced the fluorescence quenching of PAMAMC12 25%; Curcumin entered the interface of PAMAM-C12 25% with mainly five classes of binding sites by hydrophobic, hydrogen bonds, and van der Waals forces interactions. The bigger values of binding constants indicated that PAMAM-C12 25% hold the curcumin tightly. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Dendrimers are three dimensional ‘‘ball-like’’ polymers with empty internal cavities and a high concentration of surface groups [1,2]. As relatively novel and the very promising biodegradable materials, they capture some small molecules and may potentially be used to deliver a large number of drug molecules [3–5]. They ⇑ Corresponding authors. Tel./fax: +86 515 88233188. E-mail addresses: [email protected] (J. Cao), [email protected] (Y. Wang). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.02.003
also have some other potential applications in catalysis, gene therapy, and nano-reactors [6,7]. Among these application, the dendrimers as a drug carrier has been of great interest. Polyamidoamine (PAMAM) dendrimers are based on an ethylenediamine core and branched units are built from methyl acrylate and ethylenediamine [1]. They are considered safe, nonimmunogenic and exhibit minimum cytotoxicity [8]. As such, in order to provide important insight into the interactions of PAMAM dendrimers with some drugs, the binding of small molecules to dendrimers have been investigated in recent years [3,9–12]. Ma
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J. Cao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 108 (2013) 251–255
et al. [11] have studied the evaluation of PAMAM dendrimers as drug carriers of anti-bacterial drug. Buczkowski and coworkers have investigated the incorporation of 5-fluorouracil into PAMAM dendrimers [3]. The interactions between gallic acid molecules and different types of PAMAM dendrimers with modified surfaces have been also studied by spectrofluorimetric methods [12]. Therefore, studies on the interactions between drugs and PAMAM dendrimers have an important utilization in biomedical applications. The parameters such as the mode of interaction, association constants and the number of binding sites are of course important from a pharmaceutical point of view. In the present work, the fourth generation (G4) PAMAM-C12 25% has been chosen as a dendrimer to study its ability to binding curcumin molecular. The surfaces of PAMAM-C12 25% were modified in 25% by attaching N-(2-hydroxydodecyl) groups. Curcumin (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiens-3,5-dione, Fig. S1) exhibits antioxidant activity, anti-inflammatory, antimicrobial and antiviral properties and is considered as cancer chemopreventive agent [13]. The aim of the present work was to study the binding efficiency of curcumin in new PAMAM dendrimer formulations, as well as to characterize the molecular interactions between curcumin and PAMAM-C12 25%. This is the first attempt on the incorporation of curcumin in PAMAM-C12 25% dendrimer, which is crucial of the pharmaceutical formulations. Materials and methods Materials Curcumin and PAMAM-C12 25% were all purchased from Sigma– Aldrich Chemical Company and used without further purification. The buffer Tris was purchased from Acros (Geel, Belgium), and NaCl, HCl, etc. were all of analytical purity. The curcumin (0.25 mM) was prepared in pH 7.40 Tris–HCl buffer containing 20% methanol. The PAMAM-C12 25% solution (3.0 lM) were prepared in pH 7.40 Tris–HCl buffer solution by diluting the 10 wt.% PAMAM-C12 25% menthol solution. Water was purified with a Millli-Q purification system. Equipments and spectral measurements The UV/vis spectra were recorded on a UV-3600 (SHIMADZU, Japan) equipped with 1.0 cm quartz cells at room temperature. The fluorescence spectra were carried out on LS-50B Spectrofluorimeter (Perkin–Elmer USA) equipped with 1.0 cm quartz cells and a thermostat bath. The excitation bandwidth was 5.0 nm and emission bandwidth was 5.0 nm with a nominal resolution of 0.5 nm for PAMAM-C12-25%.
MM2 and DFT/B3LYP/6-311 G. Grid maps were generated with 0.375 Å spacing using a grid box of 80–80–80 Å. The calculation process can be considered as blind docking and the docking parameters used were as follows: GA population size = 150; maximum number of energy evaluation = 25,000,00 and others used were default parameters. The PyMOL software was used to analyze the docking data with the lowest binding free energy.
Results and discussion The interaction between curcumin and PAMAM-C12 25% Fig. 1 showed that the absorption spectra of curcumin and PAMAM-C12 25% @curcumin system were obviously different. A weak peak at 256 nm and a strong peak at 427 nm with a shoulder at 367 nm characterize the absorbance curve of curcumin alone. The absorbance peak at 256 nm is due to the very weak electronic dipole forbidden n ? p transition of curcumin. The absorptions at 427 nm and 367 nm in the absorption spectrum of curcumin originate from the p ? p excitations of conjugated curcumin and feruloyl unit, respectively [16]. In PAMAM-C12 25%@curcumin system, the increased absorbance at 427 nm with a red shift and the almost disappeared characteristics at 367 nm can reveal that PAMAM-C12 25% can induce curcumin to remain the conjugated structure and can promote the absorption of solubilized curcumin [17]. The curcumin molecule enters into the pocket of PAMAM-C12 25%, which will increase the absorption at 427 nm. Fluorescence spectroscopy is a powerful method to investigate the interaction of small molecules with macromolecules, such as the binding mechanism, binding mode, and binding constants [18]. The effect of curcumin on PAMAM-C12 25% fluorescence intensity is shown in Fig. 2. Upon excitation at 325 nm, strong emission from PAMAM-C12 25% (kem, max = 391 nm) was registered in the emission range 350–500 nm. As the data shows, the fluorescence intensity of PAMAM-C12 25% decreased regularly and the maximum of emission wavelength had a small blue shift with the increasing curcumin concentration, indicating that there was interaction between curcumin and PAMAM-C12 25%. In the molecular interactions, ground-state complex formation, collision quenching, and energy transfer, etc. can result in fluorescence quenching. Fig. 3 shows the absorption spectrum of curcumin and the emission spectrum of PAMAM-C12 25%. The prominent overlap between the absorption spectrum of curcumin and the emission spectrum of PAMAM-C12 25% provides great probability of energy transfer from the excited dendrimer to curcumin, and hence the fluorescence quenching partly comes from energy transfer [19].
Procedures In fluorescence measurements, a 2.5 mL solution, containing appropriate concentration of PAMAM-C12 25% (3.0 lM) in a quartz cell, was titrated by successive additions of a 0.25 mM solution of curcumin (to give a final volume of 40 lL). Titrations were done manually by using micro-injector. All solutions were mixed thoroughly and kept 10 min before measurements. The fluorescence spectra were then measured (excitation at 325 nm and emission wavelengths of 350–500 nm) at two temperatures (292 and 310 K). The UV/vis absorbance spectra of PAMAM-C12 25%@curcumin system were recorded. The docking procedure calculations were carried out using Autodock 4.2.3 [14]. Gaussian 03, respectively [15], generated the three-dimensional structure of PAMAM-C12 25% and curcumin using the ChemBioOffice 2008 software suite and optimized using
Fig. 1. The absorption spectra of curcumin (a), PAMAM-C12 25% (b) and PAMAMC12 25%@curcumin (c). c (curcumin) = 6.0 lM, c (PAMAM-C12 25%) = 3.0 lM.
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Fig. 2. Effects of curcumin on the fluorescence spectra of PAMAM-C12 25% at 292 K, pH = 7.40; kex = 325 nm. c (PAMAM-C12 25%) = 3.0 lM in all cases; c (curcumin)/ (lM) from a to i: 0, 1.0, 2.0,3.0, 4.0, 5.0, 6.0, 7.0, and 8.0.
Fig. 4. The Stern–Volmer plots for the fluorescence quenching of PAMAM-C12 25% by curcumin at different temperature. pH = 7.40; kex = 325 nm. c (PAMAM-C12 25%) = 3.0 lM.
Table 1 The Stern–Volmer quenching constant of the PAMAM-C12 25%@curcumin system at different temperatures.
a b
T(K)
Equations
Ksv (L mol1)
Ra
SDb
292 310
F0/Fcor = 1.03 + 1.47 105 [Q] F0/Fcor = 1.01 + 1.27 105 [Q]
1.47 105 1.01 105
0.9985 0.9995
0.0232 0.0116
The correlation coefficient. The standard deviation.
quenching data according to the modified Scatchard’s procedure Eq. (2) [22,23]
F0 ½QF 0 nK A ½P ¼ KA F cor F 0 F cor Fig. 3. Overlap of the fluorescence emission spectrum of PAMAM-C12 25% (a) with the UV/vis spectrum of curcumin (b), c (PAMAM-C12 25%) = c (curcumin) = 3.0 lM.
In order to confirm the fluorescence quenching mechanism, the Stern–Volmer equation (Eq. (1)) is often used to analyze the fluorescence quenching data [20]:
F0 ¼ 1 þ K sv ½Q F cor
ð1Þ
where F0 is the fluorescence intensities of PAMAM-C12 25% in the absence of curcumin. Fcor is the fluorescence intensity of PAMAM-C12 25% corrected according to Ref [21]. Ksv and [Q] are the Stern–Volmer dynamic quenching constant and the concentration of curcumin, respectively. Hence, Eq. (1) was applied to determine Ksv by linear regression of a plot of F0/Fcor against [Q]. Fig. 4 displays the Stern–Volmer plots of the quenching of PAMAM-C12 25% fluorescence by curcumin. It shows that the Stern–Volmer plots are linear, which indicates that there may be a single type of quenching, either static or dynamic in the binding interaction of PAMAM-C12 25% with curcumin. As showed in Table 1., the quenching constants obtained by the Stern–Volmer method decreased with an increase in temperature, indicating that the probable quenching mechanism of fluorescence of PAMAM-C12 25% by curcumin is not initiated by dynamic collision but PAMAM-C12 25%@curcumin complex formation.
ð2Þ
where [P] is the molar concentration of the total PAMAM-C12 25%, [Q] is the molar concentration of total curcumin, F0 and Fcor are the corrected fluorescence intensities before and after the addition of curcumin, n is the number binding sites on each PAMAM-C12 25% molecule, and KA is the equilibrium binding constant. The plots of F0/Fcor versus [Q] F0/(F0 Fcor) are showed in Fig. 5 and the values of KA and n are list in Table 2. There are many active sites in a dendrimer macromolecule being capable of combining the drug and the PAMAM dendrimer can transfer many drug molecules in aqueous
Association capacity In this paper, the binding parameters for the PAMAM-C12 25%@curcumin system have been derived from fluorescence
Fig. 5. The plots of F0/Fcor versus [Q] F0/(F0 Fcor) at different temperatures for PAMAM-C12 25%@curcumin system. pH = 7.40; kex = 325 nm. c (PAMAM-C12 25%) = 3.0 lM.
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Table 2 The thermodynamic parameters of the PAMAM-C12 25%@curcumin system. KA (L mol1) 5
292 310 a b
1.18 10 1.04 105
n
Ra
SDb
DH° (kJ mol1)
DG° (kJ mol1)
DS° (J mol1 K1)
5.16 4.98
0.9875 0.9926
0.0599 0.0211
5.28
28.35 29.77
79.00
The correlation coefficient. The standard deviation.
solution [3,13,24]. Recent work by Klajnert and Bryszewska showed that the number of binding sites of gallic acid on PAMAM-C12 25% was calculated to be 7 [3]. Shcharbin et al. have reported that the number of molecules of ANS per one molecule of PAMAM G5 was approximately 0.68 [24]. The number of molecules of 5-fluorouracil per one molecule of PAMAM-NH2 G4 was approximately 30 calculated by Buczkowski et al. [13]. In the present study, the values of n approximately were about five indicating the existence of five binding sites for curcumin in PAMAM-C12 25% with higher binding affinity and selectivity. The bigger value of binding constants KA also indicated that curcumin molecules were incorporation into the dendrimer. At pH 7.40, the PAMAM-C12 25% holds the curcumin tightly. Hence, when the PAMAM-C12 25%@curcumin complex enters the human body, the release of curcumin can occur at a controlled rate into the bloodstream [25]. In order to get more insights into the host dendrimer and to discuss the occurrence of binding interaction between curcumin and PAMAM-C12 25%, molecular docking study has been implemented to recognize the probable binding location sites of curcumin in PAMAM-C12 25%. The mainly five sites with lower binding free energy are shown in Fig. S2. Our results suggested that the curcumin molecules were encapsulated into the host PAMAM-C12 25%. The calculated interaction energy of the five sites was found to be: site A, 7.28 kJ mol1, site B, 5.07 kJ mol1, site C, 4.65 kJ mol1, site D, 4.06 kJ mol1, and site E, 4.04 kJ mol1, respectively. The nature of the binding forces
ln
ðK A Þ2 DH ¼ ðK A Þ1 R
1 T 1 T12
! ð3Þ
DG ¼ RT ln K A
ð4Þ
DH DG T
ð5Þ
DS ¼
Conclusion In this work, we have carried out spectroscopic and molecular modeling methods to study the interaction of curcumin with PAMAM-C12 25. The analysis data revealed the presence of mainly five classes of binding sites of curcumin at the interface of PAMAM-C12 25%. PAMAM-C12 25% can induce curcumin to remain the conjugated structure by hydrophobic, hydrogen bonds, van der Waals forces interactions. The non-covalent adducts of PAMAM-C12 25% with curcumin have been formatted in the aqueous solution. As PAMAM dendrimer is one of drug carriers in delivery systems, our present findings could provides significant insight about the encapsulation and the release pattern of curcumin in the case of dendrimer systems. Acknowledgements We gratefully acknowledge financial support of the Natural Science Foundation of Jiangsu Province (Grant Nos. BK2011422 and BK2012671), the Educational Bureau (Grant No. 11KJB150019), the Jiangsu Fundament of ‘‘Qilan Project’’, and the Scientific Foundation of Yancheng Teachers University. Appendix A. Supplementary material
In the process of binding interaction of macromolecule with small molecule, hydrogen bonds, van der Waals forces, hydrophobic, electrostatic force, and steric interactions were often included in the binding sites [26]. To obtain above information, the thermodynamic parameters of PAMAM-C12 25% with curcumin were calculated from the following equations:
binding progress. Therefore, the hydrophobic, hydrogen bonds, van der Waals forces between curcumin and PAMAM-C12 25% is mainly binding force in their binding process.
From Table 2, it can be seen that DG° < 0, DH° < 0 and DS° > 0. The negative sign for DG° indicated the spontaneity of the binding of curcumin with PAMAM-C12 25%. The main source of DG° value was derived from a large contribution of DS° term, which indicated that the binding of curcumin with PAMAM-C12 25% was an entropically driven process; the hydrophobic interaction was involved in PAMAM-C12 25%@curcumin binding process. The negative DH° values maybe indicate the hydrogen bonds, van der Waals forces or electrostatic forces are involved in the binding progress [26]. The phenolic hydrogens of curcumin have pKa values of 8.38, 9.88 and 10.51 in aqueous solution [27]. At pH 7.40, curcumin is found in neutral form; hence, the electrostatic interaction between curcumin and PAMAM-C12 25% is not mainly binding force in their
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2013.02.003. References [1] D.M. Domanski, B. Klajnert, M. Bryszewska, Bioelectrochemistry 63 (2004) 193–197. [2] A.M. Caminade, R. Laurent, J.P. Majoral, Adv. Drug Delivery Rev. 57 (2005) 2130–2146. [3] B. Klajnert, M. Bryszewska, Bioelectrochemistry 70 (2007) 50–52. [4] J.F.G.A. Jansen, E.M.M. Brabander, E.W. Meijer, Science 266 (1994) 1226–1229. [5] R. Esfand, D.A. Tomalia, Drug Discov. Today 6 (2001) 427–436. [6] K.C. Wood, S.R. Little, R. Langer, P.T. Hammond, Angew. Chem. Int. Ed. Engl. 44 (2005) 6704–6708. [7] Y. Borodko, C.M. Thompson, W. Huang, H.B. Yildiz, G.A. Somorjai, J. Phys. Chem. C 115 (2011) 4757–4767. [8] N. Malik, R. Wiwattanapatapee, R. Klopsch, J. Control Release 65 (2000) 133– 148. [9] M.S. Refat, A.M. El-Didamony, I. Grabchev, Spectrochim. Acta A 67 (2007) 58– 65. [10] Y. Lei, Y. Su, J. Huo, Spectrochim. Acta A 83 (2011) 149–154. [11] M. Ma, Y. Cheng, Z. Xu, P. Xu, H. Qu, Y. Fang, T. Xu, L. Wen, Eur. J. Med. Chem. 42 (2007) 93–98. [12] A. Buczkowski, S. Sekowski, A. Grala, D. Palecz, K. Milowska, P. Urbaniak, T. Gabryelak, H. Piekarskia, B. Palecz, Internat. J. Pharm. 408 (2011) 266–272. [13] A.B. Kunnumakkara, S. Guha, S. Krishnan, P. Diagaradjane, J. Gelovani, B.B. Aggarwal, Cancer. Res. 67 (2007) 3853–3861. [14] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J. Olson, J. Comput. Chem. 30 (2009) 2785–2791. [15] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, J.A. Montgomery, T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian,
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Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Investigation on the interaction behavior between curcumin and PAMAM dendrimer by spectral and docking studies Jian Cao ⇑, Hongmei Zhang, Yanqing Wang ⇑, Jinming Yang, Fuguang Jiang Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City, Jiangsu Province 224002, People’s Republic of China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" Binding interactions of curcumin
The formation of non-covalent adducts between PAMAM-C12 25% and curcumin induced the fluorescence quenching of PAMAM-C12 25%. There were mainly five classes of binding sites at the interface of PAMAMC12 25% binding with curcumin by hydrophobic, hydrogen bonds, and van der Waals forces interactions.
with PAMAM dendrimer were investigated. " The non-covalent adducts of PAMAM-C12 25%@curcumin formed. " There were about five binding sites for curcumin in PAMAM-C12 25%. " The hydrophobic, hydrogen bonds, and van der Waals forces stabilized PAMAM-C12 25%@curcumin complex.
a r t i c l e
i n f o
Article history: Received 21 July 2012 Received in revised form 3 November 2012 Accepted 7 February 2013 Available online 16 February 2013 Keywords: PAMAM-C12 25% Curcumin Fluorescence Binding mode Binding sites
a b s t r a c t The interactions between PAMAM-C12 25% and curcumin were studied by UV/vis, fluorescence spectroscopy, and molecular modeling methods. The experimental results showed that the formation of PAMAM-C12 25%@curcumin non-covalent adduct induced the fluorescence quenching of PAMAMC12 25%; Curcumin entered the interface of PAMAM-C12 25% with mainly five classes of binding sites by hydrophobic, hydrogen bonds, and van der Waals forces interactions. The bigger values of binding constants indicated that PAMAM-C12 25% hold the curcumin tightly. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Dendrimers are three dimensional ‘‘ball-like’’ polymers with empty internal cavities and a high concentration of surface groups [1,2]. As relatively novel and the very promising biodegradable materials, they capture some small molecules and may potentially be used to deliver a large number of drug molecules [3–5]. They ⇑ Corresponding authors. Tel./fax: +86 515 88233188. E-mail addresses: [email protected] (J. Cao), [email protected] (Y. Wang). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.02.003
also have some other potential applications in catalysis, gene therapy, and nano-reactors [6,7]. Among these application, the dendrimers as a drug carrier has been of great interest. Polyamidoamine (PAMAM) dendrimers are based on an ethylenediamine core and branched units are built from methyl acrylate and ethylenediamine [1]. They are considered safe, nonimmunogenic and exhibit minimum cytotoxicity [8]. As such, in order to provide important insight into the interactions of PAMAM dendrimers with some drugs, the binding of small molecules to dendrimers have been investigated in recent years [3,9–12]. Ma
252
J. Cao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 108 (2013) 251–255
et al. [11] have studied the evaluation of PAMAM dendrimers as drug carriers of anti-bacterial drug. Buczkowski and coworkers have investigated the incorporation of 5-fluorouracil into PAMAM dendrimers [3]. The interactions between gallic acid molecules and different types of PAMAM dendrimers with modified surfaces have been also studied by spectrofluorimetric methods [12]. Therefore, studies on the interactions between drugs and PAMAM dendrimers have an important utilization in biomedical applications. The parameters such as the mode of interaction, association constants and the number of binding sites are of course important from a pharmaceutical point of view. In the present work, the fourth generation (G4) PAMAM-C12 25% has been chosen as a dendrimer to study its ability to binding curcumin molecular. The surfaces of PAMAM-C12 25% were modified in 25% by attaching N-(2-hydroxydodecyl) groups. Curcumin (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiens-3,5-dione, Fig. S1) exhibits antioxidant activity, anti-inflammatory, antimicrobial and antiviral properties and is considered as cancer chemopreventive agent [13]. The aim of the present work was to study the binding efficiency of curcumin in new PAMAM dendrimer formulations, as well as to characterize the molecular interactions between curcumin and PAMAM-C12 25%. This is the first attempt on the incorporation of curcumin in PAMAM-C12 25% dendrimer, which is crucial of the pharmaceutical formulations. Materials and methods Materials Curcumin and PAMAM-C12 25% were all purchased from Sigma– Aldrich Chemical Company and used without further purification. The buffer Tris was purchased from Acros (Geel, Belgium), and NaCl, HCl, etc. were all of analytical purity. The curcumin (0.25 mM) was prepared in pH 7.40 Tris–HCl buffer containing 20% methanol. The PAMAM-C12 25% solution (3.0 lM) were prepared in pH 7.40 Tris–HCl buffer solution by diluting the 10 wt.% PAMAM-C12 25% menthol solution. Water was purified with a Millli-Q purification system. Equipments and spectral measurements The UV/vis spectra were recorded on a UV-3600 (SHIMADZU, Japan) equipped with 1.0 cm quartz cells at room temperature. The fluorescence spectra were carried out on LS-50B Spectrofluorimeter (Perkin–Elmer USA) equipped with 1.0 cm quartz cells and a thermostat bath. The excitation bandwidth was 5.0 nm and emission bandwidth was 5.0 nm with a nominal resolution of 0.5 nm for PAMAM-C12-25%.
MM2 and DFT/B3LYP/6-311 G. Grid maps were generated with 0.375 Å spacing using a grid box of 80–80–80 Å. The calculation process can be considered as blind docking and the docking parameters used were as follows: GA population size = 150; maximum number of energy evaluation = 25,000,00 and others used were default parameters. The PyMOL software was used to analyze the docking data with the lowest binding free energy.
Results and discussion The interaction between curcumin and PAMAM-C12 25% Fig. 1 showed that the absorption spectra of curcumin and PAMAM-C12 25% @curcumin system were obviously different. A weak peak at 256 nm and a strong peak at 427 nm with a shoulder at 367 nm characterize the absorbance curve of curcumin alone. The absorbance peak at 256 nm is due to the very weak electronic dipole forbidden n ? p transition of curcumin. The absorptions at 427 nm and 367 nm in the absorption spectrum of curcumin originate from the p ? p excitations of conjugated curcumin and feruloyl unit, respectively [16]. In PAMAM-C12 25%@curcumin system, the increased absorbance at 427 nm with a red shift and the almost disappeared characteristics at 367 nm can reveal that PAMAM-C12 25% can induce curcumin to remain the conjugated structure and can promote the absorption of solubilized curcumin [17]. The curcumin molecule enters into the pocket of PAMAM-C12 25%, which will increase the absorption at 427 nm. Fluorescence spectroscopy is a powerful method to investigate the interaction of small molecules with macromolecules, such as the binding mechanism, binding mode, and binding constants [18]. The effect of curcumin on PAMAM-C12 25% fluorescence intensity is shown in Fig. 2. Upon excitation at 325 nm, strong emission from PAMAM-C12 25% (kem, max = 391 nm) was registered in the emission range 350–500 nm. As the data shows, the fluorescence intensity of PAMAM-C12 25% decreased regularly and the maximum of emission wavelength had a small blue shift with the increasing curcumin concentration, indicating that there was interaction between curcumin and PAMAM-C12 25%. In the molecular interactions, ground-state complex formation, collision quenching, and energy transfer, etc. can result in fluorescence quenching. Fig. 3 shows the absorption spectrum of curcumin and the emission spectrum of PAMAM-C12 25%. The prominent overlap between the absorption spectrum of curcumin and the emission spectrum of PAMAM-C12 25% provides great probability of energy transfer from the excited dendrimer to curcumin, and hence the fluorescence quenching partly comes from energy transfer [19].
Procedures In fluorescence measurements, a 2.5 mL solution, containing appropriate concentration of PAMAM-C12 25% (3.0 lM) in a quartz cell, was titrated by successive additions of a 0.25 mM solution of curcumin (to give a final volume of 40 lL). Titrations were done manually by using micro-injector. All solutions were mixed thoroughly and kept 10 min before measurements. The fluorescence spectra were then measured (excitation at 325 nm and emission wavelengths of 350–500 nm) at two temperatures (292 and 310 K). The UV/vis absorbance spectra of PAMAM-C12 25%@curcumin system were recorded. The docking procedure calculations were carried out using Autodock 4.2.3 [14]. Gaussian 03, respectively [15], generated the three-dimensional structure of PAMAM-C12 25% and curcumin using the ChemBioOffice 2008 software suite and optimized using
Fig. 1. The absorption spectra of curcumin (a), PAMAM-C12 25% (b) and PAMAMC12 25%@curcumin (c). c (curcumin) = 6.0 lM, c (PAMAM-C12 25%) = 3.0 lM.
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Fig. 2. Effects of curcumin on the fluorescence spectra of PAMAM-C12 25% at 292 K, pH = 7.40; kex = 325 nm. c (PAMAM-C12 25%) = 3.0 lM in all cases; c (curcumin)/ (lM) from a to i: 0, 1.0, 2.0,3.0, 4.0, 5.0, 6.0, 7.0, and 8.0.
Fig. 4. The Stern–Volmer plots for the fluorescence quenching of PAMAM-C12 25% by curcumin at different temperature. pH = 7.40; kex = 325 nm. c (PAMAM-C12 25%) = 3.0 lM.
Table 1 The Stern–Volmer quenching constant of the PAMAM-C12 25%@curcumin system at different temperatures.
a b
T(K)
Equations
Ksv (L mol1)
Ra
SDb
292 310
F0/Fcor = 1.03 + 1.47 105 [Q] F0/Fcor = 1.01 + 1.27 105 [Q]
1.47 105 1.01 105
0.9985 0.9995
0.0232 0.0116
The correlation coefficient. The standard deviation.
quenching data according to the modified Scatchard’s procedure Eq. (2) [22,23]
F0 ½QF 0 nK A ½P ¼ KA F cor F 0 F cor Fig. 3. Overlap of the fluorescence emission spectrum of PAMAM-C12 25% (a) with the UV/vis spectrum of curcumin (b), c (PAMAM-C12 25%) = c (curcumin) = 3.0 lM.
In order to confirm the fluorescence quenching mechanism, the Stern–Volmer equation (Eq. (1)) is often used to analyze the fluorescence quenching data [20]:
F0 ¼ 1 þ K sv ½Q F cor
ð1Þ
where F0 is the fluorescence intensities of PAMAM-C12 25% in the absence of curcumin. Fcor is the fluorescence intensity of PAMAM-C12 25% corrected according to Ref [21]. Ksv and [Q] are the Stern–Volmer dynamic quenching constant and the concentration of curcumin, respectively. Hence, Eq. (1) was applied to determine Ksv by linear regression of a plot of F0/Fcor against [Q]. Fig. 4 displays the Stern–Volmer plots of the quenching of PAMAM-C12 25% fluorescence by curcumin. It shows that the Stern–Volmer plots are linear, which indicates that there may be a single type of quenching, either static or dynamic in the binding interaction of PAMAM-C12 25% with curcumin. As showed in Table 1., the quenching constants obtained by the Stern–Volmer method decreased with an increase in temperature, indicating that the probable quenching mechanism of fluorescence of PAMAM-C12 25% by curcumin is not initiated by dynamic collision but PAMAM-C12 25%@curcumin complex formation.
ð2Þ
where [P] is the molar concentration of the total PAMAM-C12 25%, [Q] is the molar concentration of total curcumin, F0 and Fcor are the corrected fluorescence intensities before and after the addition of curcumin, n is the number binding sites on each PAMAM-C12 25% molecule, and KA is the equilibrium binding constant. The plots of F0/Fcor versus [Q] F0/(F0 Fcor) are showed in Fig. 5 and the values of KA and n are list in Table 2. There are many active sites in a dendrimer macromolecule being capable of combining the drug and the PAMAM dendrimer can transfer many drug molecules in aqueous
Association capacity In this paper, the binding parameters for the PAMAM-C12 25%@curcumin system have been derived from fluorescence
Fig. 5. The plots of F0/Fcor versus [Q] F0/(F0 Fcor) at different temperatures for PAMAM-C12 25%@curcumin system. pH = 7.40; kex = 325 nm. c (PAMAM-C12 25%) = 3.0 lM.
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Table 2 The thermodynamic parameters of the PAMAM-C12 25%@curcumin system. KA (L mol1) 5
292 310 a b
1.18 10 1.04 105
n
Ra
SDb
DH° (kJ mol1)
DG° (kJ mol1)
DS° (J mol1 K1)
5.16 4.98
0.9875 0.9926
0.0599 0.0211
5.28
28.35 29.77
79.00
The correlation coefficient. The standard deviation.
solution [3,13,24]. Recent work by Klajnert and Bryszewska showed that the number of binding sites of gallic acid on PAMAM-C12 25% was calculated to be 7 [3]. Shcharbin et al. have reported that the number of molecules of ANS per one molecule of PAMAM G5 was approximately 0.68 [24]. The number of molecules of 5-fluorouracil per one molecule of PAMAM-NH2 G4 was approximately 30 calculated by Buczkowski et al. [13]. In the present study, the values of n approximately were about five indicating the existence of five binding sites for curcumin in PAMAM-C12 25% with higher binding affinity and selectivity. The bigger value of binding constants KA also indicated that curcumin molecules were incorporation into the dendrimer. At pH 7.40, the PAMAM-C12 25% holds the curcumin tightly. Hence, when the PAMAM-C12 25%@curcumin complex enters the human body, the release of curcumin can occur at a controlled rate into the bloodstream [25]. In order to get more insights into the host dendrimer and to discuss the occurrence of binding interaction between curcumin and PAMAM-C12 25%, molecular docking study has been implemented to recognize the probable binding location sites of curcumin in PAMAM-C12 25%. The mainly five sites with lower binding free energy are shown in Fig. S2. Our results suggested that the curcumin molecules were encapsulated into the host PAMAM-C12 25%. The calculated interaction energy of the five sites was found to be: site A, 7.28 kJ mol1, site B, 5.07 kJ mol1, site C, 4.65 kJ mol1, site D, 4.06 kJ mol1, and site E, 4.04 kJ mol1, respectively. The nature of the binding forces
ln
ðK A Þ2 DH ¼ ðK A Þ1 R
1 T 1 T12
! ð3Þ
DG ¼ RT ln K A
ð4Þ
DH DG T
ð5Þ
DS ¼
Conclusion In this work, we have carried out spectroscopic and molecular modeling methods to study the interaction of curcumin with PAMAM-C12 25. The analysis data revealed the presence of mainly five classes of binding sites of curcumin at the interface of PAMAM-C12 25%. PAMAM-C12 25% can induce curcumin to remain the conjugated structure by hydrophobic, hydrogen bonds, van der Waals forces interactions. The non-covalent adducts of PAMAM-C12 25% with curcumin have been formatted in the aqueous solution. As PAMAM dendrimer is one of drug carriers in delivery systems, our present findings could provides significant insight about the encapsulation and the release pattern of curcumin in the case of dendrimer systems. Acknowledgements We gratefully acknowledge financial support of the Natural Science Foundation of Jiangsu Province (Grant Nos. BK2011422 and BK2012671), the Educational Bureau (Grant No. 11KJB150019), the Jiangsu Fundament of ‘‘Qilan Project’’, and the Scientific Foundation of Yancheng Teachers University. Appendix A. Supplementary material
In the process of binding interaction of macromolecule with small molecule, hydrogen bonds, van der Waals forces, hydrophobic, electrostatic force, and steric interactions were often included in the binding sites [26]. To obtain above information, the thermodynamic parameters of PAMAM-C12 25% with curcumin were calculated from the following equations:
binding progress. Therefore, the hydrophobic, hydrogen bonds, van der Waals forces between curcumin and PAMAM-C12 25% is mainly binding force in their binding process.
From Table 2, it can be seen that DG° < 0, DH° < 0 and DS° > 0. The negative sign for DG° indicated the spontaneity of the binding of curcumin with PAMAM-C12 25%. The main source of DG° value was derived from a large contribution of DS° term, which indicated that the binding of curcumin with PAMAM-C12 25% was an entropically driven process; the hydrophobic interaction was involved in PAMAM-C12 25%@curcumin binding process. The negative DH° values maybe indicate the hydrogen bonds, van der Waals forces or electrostatic forces are involved in the binding progress [26]. The phenolic hydrogens of curcumin have pKa values of 8.38, 9.88 and 10.51 in aqueous solution [27]. At pH 7.40, curcumin is found in neutral form; hence, the electrostatic interaction between curcumin and PAMAM-C12 25% is not mainly binding force in their
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