- Email: [email protected]
Preparation and characterization of doxorubicin functionalized gold nanoparticles
European Journal of Medicinal Chemistry 46 (2011) 1857e1860
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Preparation and characterization of doxorubicin functionalized gold nanoparticles Agha Zeeshan Mirza a, *, Hina Shamshad b a b
Department of Chemistry, Lab 9, University of Karachi, Karachi 75270, Pakistan Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, University of Karachi, Karachi 75270, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 July 2010 Received in revised form 15 February 2011 Accepted 18 February 2011 Available online 24 February 2011
In this paper, we have presented the demonstration of gold nanoparticles (Au NPs) functionalized with an anticancer drug, doxorubicin. Doxorubicin was assembled on gold via amino group. The reaction proceeded under mild acidic conditions. Au NPs could not be adsorbed on doxorubicin in alkaline solution because amino group was not protonated. However, under acidic conditions, protonation created a positively charged amino group thus adsorption was easier. The interaction between Au colloids and doxorubicin is believed to be electrostatic. High-resolution TEM was used for visualization of nanoparticles, which were found to retain their average size and shape. The method, demonstrated that doxorubicin could be attached to Au NPs in a controlled manner. Our research laid the foundation of a linking methodology through which hybrid multi drug and receptor labeled NPs could be created, which might serve as an alternative design for nanosized drug-delivery systems. Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords: Doxorubicin Drug-delivery systems Gold nanoparticles Raman spectroscopy TEM
1. Introduction In recent times, nanomaterials have gained significant attention due to their potential applications in drug delivery, sensing, imaging and chemotherapy [1e3]. Particularly for drug delivery, polymeric nanoparticles, dendrimers, liposomes and metal nanoparticles are being widely explored. Among the broad diversity of nanoparticles, iron oxide and Au NPs are the most intensively studied [4]. In clinical applications as well, Au NPs may have advantages over other metallic particles in terms of biocompatibility and non-cytotoxicity [5]. The size of Au NPs can be easily controlled during synthesis and their surfaces can be conveniently functionalized with different types of molecules [6e9] and can specifically interact with any physiological system [10,11]. Au NPs also have been exploited for their potential application in hyperthermia of cancer cells [12]. Doxorubicin (Structure 1), a cytotoxic drug, has a broad antitumor activity, widely used in the treatment of many malignant diseases and also intercalates DNA. The drug exerts its antitumor effects through interaction with the DNA replicative system in the cell nucleus and its activity is highly dependent upon intracellular levels [13]. Doxorubicin conjugated Au NPs have the potential to simultaneously enhance CT imaging contrast and facilitate
* Corresponding author. E-mail address: [email protected] (A.Z. Mirza). 0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2011.02.048
photothermal cancer therapy [14] and also showed enhanced cytotoxic effects on human glioma cell lines LN-18 and LN-229 [15]. Doxorubicin conjugated Au NPs may also be used to improve imaging contrast or for photothermal cancer therapy. In this paper, the functionalization of Au NPs surfaces with doxorubicin is described and the interaction sites were investigated. There have been several reports on drug nanoparticles interaction [16e19]. Some authors have also reported the complexes of doxorubicin with gold and iron nanoparticles [14,15,20,21], but they conjugated both compounds with the help of different linker molecules. This is the first report in which a doxorubicin molecule is used as the capping agent for Au NPs. The synthetic strategy used in this investigation is shown in Scheme 1. In citrate modified Au NPs, citrate anions self-assembled on the surface of Au NPs to form complex anions with negative charge. The protonated amine group of doxorubicin was positively charged and could bind with the complex anions. The attachment of the drug molecule through electrostatic or hydrogen bonds may be a better strategy than covalent linkages [16] and could be more beneficial in drug release applications. We studied the effects of pH on the adsorption of doxorubicin with gold colloidal nanoparticles and also investigated the optimum conditions of the reaction and influencing factors. It showed the nature of interactions between the colloidal Au NPs and amino groups. We used different buffers and different pH to observe the best condition for the interaction of Au NPs with doxorubicin molecule. The bare Au NPs and doxorubicin loaded Au
1858
A.Z. Mirza, H. Shamshad / European Journal of Medicinal Chemistry 46 (2011) 1857e1860
Scheme 1. Synthesis scheme for the preparation of DOX conjugated Au NPs.
NPs were characterized by transmission electron microscopy, UV/ Vis-NIR spectroscopy, IR and Raman spectroscopy and zeta potential measurements. These finding suggested that the Au NPs interaction with amino group of doxorubicin is electrostatic. 2. Experimental 2.1. Material and equipment Tetrachloroauric acid (HAuCl4), trisodium citrate and all other chemicals were purchased from Sigma (Milwaukee, WI, USA) and used as received. The anticancer drug doxorubicin hydrochloride was purchased from Tecoland Corporation, USA Ultra pure water (>17 MU cm) was used throughout the experiments. Other reagents were of analytical grade. Transmission electron microscopy (TEM), was a Philips CM-100 TEM (Philips, Eindhoven, Netherlands) were used. Absorption spectra were measured with a Jasco V570 UV/visible/NIR spectrophotometer (Jasco, Inc., Easton, MD), in the 400e600 nm wavelength range. FTIR spectra were recorded using a Digilab Excalibur FTS 6000 spectrometer fitted with a UMA 600 IR microscope (Digilab, Randolph, MA). SERS spectra were recorded using the Bruker microRaman spectrometer and excited using the 785 nm line. The incident laser beam was focused and the signal collected using a 20 long distance objective. Finally, zeta potential of nanoparticulate before and after loading of doxorubicin was determined using a Brookhaven Zeta Potential Analyzer (Brookhaven Instrument Corp., Holtsville, NY).
was brought to a boil with vigorous stirring. To this solution, 50 mL of 33.8 mM sodium citrate was added and the hot plate was turned off. A color change was observed from colorless to purple to ruby red within 10 min. The colloidal solution was stirred for an additional 20 min, cooled at room temperature, transferred to an amber bottle and stored at room temperature. AuNPs exhibit surface plasmon resonance at 520 nm (Fig. 2). The resulting particles were shown by TEM to be 13 1 nm. 2.3. Doxorubicin loading onto gold nanoparticles A calculated amount of doxorubicin with appropriate amount of different buffers was added in Au NPs solution to obtain the final doxorubicin concentration of 1.29 mM in solution. The solution was then incubated at room temperature and changes in surface plasmon resonance were monitored by UV/Vis/NIR spectroscopy measurements. IR spectra of doxorubicin bound to the Au NPs were recorded and surface charge of Au NPs before and after loading of
2.2. Synthesis of gold nanoparticles To complete removal of potential artificial nucleation sites, all glasswares were cleaned with an aqua regia solution (1:3 nitric acid/hydrochloric acid). An aqueous gold colloid, with an average particle size of 13 1 nm (determined from the TEM image) was synthesized by the method reported [22]. Briefly, 1 mM of 500 mL solution of hydrogen tetrachloroaurate trihydrate (HAuCl4.3H2O)
Fig. 1. TEM image of 13 nm Au NPs.
A.Z. Mirza, H. Shamshad / European Journal of Medicinal Chemistry 46 (2011) 1857e1860
Fig. 2. Representative UV/Vis spectra of (1) Au NPs and Au NPs Dox Complex in (2) MES, (3) 0.1 N HCl (pH 1), (4) acetic acid (pH 2.4), (5) PBS (pH 7.4).
doxorubicin was determined by measurement of zeta potential. SERS spectra of doxorubicin and Au NPs were recorded using 785 nm (40 mW) excitation in the range of 200e1800 cm1 with an integration time of 100 s.
3. Results and discussion Doxorubicin lacks tumor-targeting ability leading to poor biodistribution and therapeutic effects as well as serious undesirable side effects [14]. The novel approach presented in this paper was to conjugate doxorubicin via pH-sensitive bonds onto stabilized Au NPs, which may lead to much better tumor-targeting ability and improving the efficacy of cancer therapy. The doxorubicin-Au NPs complex was characterized by techniques including UV/vis/NIR
1859
spectroscopy, IR spectroscopy, Raman spectroscopy and zeta potential measurement. The TEM images recorded from as-synthesized Au NPs (Fig. 1) revealed that the nanoparticles were well dispersed with in a narrow size distribution and an average size of 13 1 nm. Absorption spectrum of Au NPs solution had maximum wavelength at 520 nm, which was in accordance with the plasmon resonance absorption wavelength of the Au NPs (Fig. 2) [22]. It has been reported that, during the formation of nanoparticles, citrate ion self-assembled on the surface of Au NPs to form negative charge. At pH near to 6 it would bind with some organic cations that coexisted in the solution to form new product through electrostatic attraction, which resulted in the change of some spectral characteristics and intensity [23]. Doxorubicin has a protonated amino group [24] and adsorption of nanoparticles has strong relations to the charging states of the amino groups [25]. When Au NPs combined with doxorubicin, the maximum absorption wavelength shifted to 528 nm (Fig. 2). The change of the absorption spectrum is due to the protonated nitrogen atom of doxorubicin molecule and combination of positive charged amine group on the surface of Au NPs. The zeta potential of citrate modified Au NPs was 27.44 mV and zeta potential of doxorubicin loaded Au NPs was 21.00 mV. This decrease in the zeta potential was approved to the presence of positively charged doxorubicin and also indicated that attractive forces including hydrogen bonding could be playing a major role facilitating the drug-loading process [15]. The bonding between protonated amine groups of the doxorubicin molecule with surface of Au NPs was also supported by FTIR (spectra not shown). Doxorubicin showed NeH stretching peak of amine at 3777 cm1 [15]. In case of doxorubicin-Au NPs complex the amine peak shifted to 3373 cm1, indicating the formation of electrostatic bond formation. Similar to infrared absorption spectroscopy, Raman scattering could be used for identification and quantification of molecule [13]. The surface-enhanced Raman scattering (SERS) has been used as a powerful method to get signal from
Fig. 3. Raman spectra of doxorubicin (Dox) and doxorubicin capped Au NPs (Dox þ Au NPs).
1860
A.Z. Mirza, H. Shamshad / European Journal of Medicinal Chemistry 46 (2011) 1857e1860
References
Structure 1. Doxorubicin.
fluorescent chromophores [26]. In SERS, the large increase of the near-field excitation intensity was observed at the location of the analyte, which is adsorbed to a noble-metal structure. The net surface enhancement exceeds ten orders-of-magnitude [27,28]. To determine the interaction of doxorubicin with Au NPs, Bruker Senterra Raman microscope (Bruker Optics, Inc., Billerica, MA) with 785 nm excitation laser at 25 mW was used to measure the SERS spectra. Fig. 3 shows the SERS spectra of doxorubicin and Au NPsdoxorubicin complex, represented the surface-enhanced Raman scattering (SERS) effect due to intense spectral signals. The comparison of the spectrum of free doxorubicin with the doxorubicin-complex revealed a small difference in wave number and Au NPs-doxorubicin complex spectra were similar to pure doxorubicin spectra (Fig. 3). These all above observations confirmed that the doxorubicin caped on the Au NPs. The pH-dependence of surface coverage indicated that these interactions were strongly affected by solution pH. It was observed that borohydride or citrate reduced Au NPs form aggregate at the slightest change in their pH and electrolyte environments [29] but it was also reported that gellan gum capped Au NPs were very stable between pH 4 to 8. Otherwise, the addition of doxorubicin solution to the AuNPs caused the particles to aggregate [15]. So, different pH buffers were used and it was found that in 2-(Nmorpholino)ethanesulfonic acid (MES) buffer at pH 6, interaction between doxorubicin and Au NPs was observed but in other buffer like 0.1 N HCl (pH 1), acetic acid (pH 2.4) and PBS (pH 7.4), nanoparticles form aggregates.
4. Conclusion Protonation of amino group present in doxorubicin molecule depended on the solution pH. Due to protonation, amino groups will be positively charged, which would promote the adsorption of negatively charged Au NPs by strong electrostatic interaction. In summary, it was concluded that, in alkaline solution, where the amino group was not protonated, Au NPs could not be adsorbed, while under acidic conditions, protonation made the amino group positively charged and the adsorption became easier and electrostatic interaction was believed to be between Au colloids and the substrate. In conclusion, we reported a novel method for successful loading of doxorubicin onto Au NPs. These nanoparticles might facilitate better transport of the loaded drug across bloodebrain barrier and this could be expanded to include the delivery of other drug molecules as well.
[1] D.D. Gutierrez, M. Surtchev, E. Eiser, C.J. Elsevier, Ru(II)-based metallosurfactant forming inverted aggregates, Nano Lett. 6 (2006) 145e147. [2] M. Everts, V. Saini, J.L. Leddon, R.J. Kok, M.S. Khalili, M.A. Preuss, C.L. Millican, G. Perkins, J.M. Brown, H. Bagaria, D.E. Nikles, D.T. Johnson, V.P. Zharov, D.T. Curiel, Covalently linked Au nanoparticles to a viral vector: potential for combined photothermal and gene cancer therapy, Nano Lett. 6 (2006) 587e591. [3] B.G.G. Lohmeijer, U.S. Schubert, Supramolecular engineering with macromolecules: an alternative concept for block copolymers, Angew. Chem., Int. Ed. 41 (2002) 3825e3829. [4] L. Li, M. Fan, R. Brown, L.J. Van, J. Wang, W. Wang, Y. Song, P. Zhang, Synthesis, properties, and environmental applications of nanoscale iron-based materials: a review, EnViron. Sci. Technol. 36 (2006) 405e431. [5] E.E. Connor, J. Mwamuka, A. Gole, C.J. Murphy, M.D. Wyatt, Gold nanoparticles are Taken up by human cells but do not cause acute cytotoxicity, Small 1 (2005) 325e327. [6] M. Daniel, D. Astruc, Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology, Chem. Rev. 104 (2004) 293e346. [7] K.K. Sandhu, C. McIntosh, J. Simard, S. Smith, V.M. Rotello, Gold nanoparticlemediated transfection of mammalian cells, Bioconjugate Chem. 13 (2002) 3e6. [8] A.C. Templeton, M.P. Wuelfing, R.W. Murray, Monolayer-protected cluster molecules, Acc. Chem. Res. 33 (2000) 27e36. [9] G.F. Paciotti, L. Myer, D. Weinreich, D. Goia, N. Pavel, R.E. McLaughlin, L. Tamarkin, Colloidal gold: a novel nanoparticle vector for tumor directed drug delivery, Drug Deliv. 11 (2004) 169e183. [10] V.S. Murthy, J.N. Cha, G.D. Stucky, M.S. Wong, Charge-driven flocculation of poly(l-lysine) e gold nanoparticle assemblies leading to hollow microspheres, J. Am. Chem. Soc. 126 (2004) 5292e5299. [11] S.J. Hurst, A.K.R. Lytton-Jean, C.A. Mirkin, Maximizing DNA loading on a range of gold nanoparticle sizes, Anal. Chem. 78 (2006) 8313e8318. [12] X. Huang, I.H. El-Sayed, W. Qian, M.A. El-Sayed, Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods, J. Am. Chem. Soc. 128 (2006) 2115e2120. [13] C. Eliasson, A. Lorén, K.V.G.K. Murty, M. Josefson, M. Kall, J. Abrahamsson, K. Abrahamsson, Multivariate evaluation of doxorubicin surface-enhanced Raman spectra, Spectrochimica Acta Part A 57 (2001) 1907e1915. [14] S. Aryal, J.J. Grailer, S. Pilla, D.A. Steeber, S. Gong, Doxorubicin conjugated gold nanoparticles as water-soluble and pH-responsive anticancer drug nanocarriers, J. Mater. Chem. 19 (2009) 7879e7884. [15] S. Dhar, E.M. Reddy, A. Shiras, V. Pokharkar, B.L.V. Prasad, Natural gum reduced/stabilized gold nanoparticles for drug delivery formulations, Chem. Eur. J. 14 (2008) 10244e10250. [16] J.D. Gibson, B.P. Khanal, E.R. Zubarev, Paclitaxel-functionalized gold nanoparticles, J. Am. Chem. Soc. 129 (2007) 11653e11661. [17] R.T. Tom, V. Suryanarayanan, P.G. Reddy, S. Baskaran, T. Pradeep, Ciprofloxacin-protected gold nanoparticles, Langmuir 20 (2004) 1909e1914. [18] H.X. Zhang, H. Zhao, J.X. Wang, J.F. Chen, Y.F. Lu, J. Yun, Facile preparation of monodisperse pharmaceutical colloidal spheres of atorvastatin calcium via self-assembly, Small 5 (2009) 1846e1849. [19] S. Santra, C. Kaittanis, J. Grimm, J.M. Perez, Drug/dye-loaded, multifunctional iron oxide nanoparticles for combined targeted cancer therapy and dual optical/magnetic resonance imaging, Small 5 (2009) 1862e1868. [20] M. Brzozowska, P. Krysinski, Synthesis and functionalization of magnetic nanoparticles with covalently bound electroactive compound doxorubicin, Electrochimica Acta 54 (2009) 5065e5070. [21] M. Prabaharan, J.J. Grailer, S. Pilla, D.A. Steeber, S. Gong, Gold nanoparticles with a monolayer of doxorubicin-conjugated amphiphilic block copolymer for tumor-targeted drug delivery, Biomaterials 30 (2009) 6065e6075. [22] K.C. Grabar, R.G. Freeman, M.B. Hommer, M.J. Natan, Preparation and characterization of Au colloid monolayers, Anal. Chem. 67 (1995) 735e743. [23] S.P. Liu, Y.Q. He, Z.F. Liu, L. Kong, Q.M. Lu, Resonance rayleigh scattering spectral method for the determination of raloxifene using gold nanoparticle as a probe, Anal. Chim. Acta 598 (2007) 304e311. [24] J. Bhattacharjee, G. Verma, V.K. Aswal, P.A. Hassan, Small angle neutron scattering study of doxorubicin-surfactant complexes encapsulated in block copolymer micelles, Pramana 71 (2008) 991e995. [25] T. Zhu, X. Fu, T. Mu, J. Wang, Z. Liu, pH-dependent adsorption of gold nanoparticles on p-aminothiophenol-modified gold substrates, Langmuir 15 (1999) 5197e5199. [26] I. Nabiev, I. Chourpa, M. Manfait, Comparative studies of antitumor DNA intercalating agents, aclacinomycin and saintopin, by means of surfaceenhanced Raman scattering spectroscopy, J. Phys. Chem. 98 (1994) 1344e1350. [27] H. Xu, E.J. Bjerneld, M. Kall, L. Borjesson, Spectroscopy of single hemoglobin molecules by surface enhanced Raman scattering, Phys. Rev. Lett. 83 (1999) 4357e4360. [28] E.J. Bjerneld, P. Johansson, M. Kall, Single molecule vibrational fine-structure of tyrosine adsorbed on Ag nano-crystals, Single Mol. 1 (2000) 239e248. [29] L.L. Rouhana, J.A. Jaber, J.B. Schlenoff, Aggregation-resistant water-soluble gold nanoparticles, Langmuir 23 (2007) 12799e12801.
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Preparation and characterization of doxorubicin functionalized gold nanoparticles Agha Zeeshan Mirza a, *, Hina Shamshad b a b
Department of Chemistry, Lab 9, University of Karachi, Karachi 75270, Pakistan Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, University of Karachi, Karachi 75270, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 July 2010 Received in revised form 15 February 2011 Accepted 18 February 2011 Available online 24 February 2011
In this paper, we have presented the demonstration of gold nanoparticles (Au NPs) functionalized with an anticancer drug, doxorubicin. Doxorubicin was assembled on gold via amino group. The reaction proceeded under mild acidic conditions. Au NPs could not be adsorbed on doxorubicin in alkaline solution because amino group was not protonated. However, under acidic conditions, protonation created a positively charged amino group thus adsorption was easier. The interaction between Au colloids and doxorubicin is believed to be electrostatic. High-resolution TEM was used for visualization of nanoparticles, which were found to retain their average size and shape. The method, demonstrated that doxorubicin could be attached to Au NPs in a controlled manner. Our research laid the foundation of a linking methodology through which hybrid multi drug and receptor labeled NPs could be created, which might serve as an alternative design for nanosized drug-delivery systems. Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords: Doxorubicin Drug-delivery systems Gold nanoparticles Raman spectroscopy TEM
1. Introduction In recent times, nanomaterials have gained significant attention due to their potential applications in drug delivery, sensing, imaging and chemotherapy [1e3]. Particularly for drug delivery, polymeric nanoparticles, dendrimers, liposomes and metal nanoparticles are being widely explored. Among the broad diversity of nanoparticles, iron oxide and Au NPs are the most intensively studied [4]. In clinical applications as well, Au NPs may have advantages over other metallic particles in terms of biocompatibility and non-cytotoxicity [5]. The size of Au NPs can be easily controlled during synthesis and their surfaces can be conveniently functionalized with different types of molecules [6e9] and can specifically interact with any physiological system [10,11]. Au NPs also have been exploited for their potential application in hyperthermia of cancer cells [12]. Doxorubicin (Structure 1), a cytotoxic drug, has a broad antitumor activity, widely used in the treatment of many malignant diseases and also intercalates DNA. The drug exerts its antitumor effects through interaction with the DNA replicative system in the cell nucleus and its activity is highly dependent upon intracellular levels [13]. Doxorubicin conjugated Au NPs have the potential to simultaneously enhance CT imaging contrast and facilitate
* Corresponding author. E-mail address: [email protected] (A.Z. Mirza). 0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2011.02.048
photothermal cancer therapy [14] and also showed enhanced cytotoxic effects on human glioma cell lines LN-18 and LN-229 [15]. Doxorubicin conjugated Au NPs may also be used to improve imaging contrast or for photothermal cancer therapy. In this paper, the functionalization of Au NPs surfaces with doxorubicin is described and the interaction sites were investigated. There have been several reports on drug nanoparticles interaction [16e19]. Some authors have also reported the complexes of doxorubicin with gold and iron nanoparticles [14,15,20,21], but they conjugated both compounds with the help of different linker molecules. This is the first report in which a doxorubicin molecule is used as the capping agent for Au NPs. The synthetic strategy used in this investigation is shown in Scheme 1. In citrate modified Au NPs, citrate anions self-assembled on the surface of Au NPs to form complex anions with negative charge. The protonated amine group of doxorubicin was positively charged and could bind with the complex anions. The attachment of the drug molecule through electrostatic or hydrogen bonds may be a better strategy than covalent linkages [16] and could be more beneficial in drug release applications. We studied the effects of pH on the adsorption of doxorubicin with gold colloidal nanoparticles and also investigated the optimum conditions of the reaction and influencing factors. It showed the nature of interactions between the colloidal Au NPs and amino groups. We used different buffers and different pH to observe the best condition for the interaction of Au NPs with doxorubicin molecule. The bare Au NPs and doxorubicin loaded Au
1858
A.Z. Mirza, H. Shamshad / European Journal of Medicinal Chemistry 46 (2011) 1857e1860
Scheme 1. Synthesis scheme for the preparation of DOX conjugated Au NPs.
NPs were characterized by transmission electron microscopy, UV/ Vis-NIR spectroscopy, IR and Raman spectroscopy and zeta potential measurements. These finding suggested that the Au NPs interaction with amino group of doxorubicin is electrostatic. 2. Experimental 2.1. Material and equipment Tetrachloroauric acid (HAuCl4), trisodium citrate and all other chemicals were purchased from Sigma (Milwaukee, WI, USA) and used as received. The anticancer drug doxorubicin hydrochloride was purchased from Tecoland Corporation, USA Ultra pure water (>17 MU cm) was used throughout the experiments. Other reagents were of analytical grade. Transmission electron microscopy (TEM), was a Philips CM-100 TEM (Philips, Eindhoven, Netherlands) were used. Absorption spectra were measured with a Jasco V570 UV/visible/NIR spectrophotometer (Jasco, Inc., Easton, MD), in the 400e600 nm wavelength range. FTIR spectra were recorded using a Digilab Excalibur FTS 6000 spectrometer fitted with a UMA 600 IR microscope (Digilab, Randolph, MA). SERS spectra were recorded using the Bruker microRaman spectrometer and excited using the 785 nm line. The incident laser beam was focused and the signal collected using a 20 long distance objective. Finally, zeta potential of nanoparticulate before and after loading of doxorubicin was determined using a Brookhaven Zeta Potential Analyzer (Brookhaven Instrument Corp., Holtsville, NY).
was brought to a boil with vigorous stirring. To this solution, 50 mL of 33.8 mM sodium citrate was added and the hot plate was turned off. A color change was observed from colorless to purple to ruby red within 10 min. The colloidal solution was stirred for an additional 20 min, cooled at room temperature, transferred to an amber bottle and stored at room temperature. AuNPs exhibit surface plasmon resonance at 520 nm (Fig. 2). The resulting particles were shown by TEM to be 13 1 nm. 2.3. Doxorubicin loading onto gold nanoparticles A calculated amount of doxorubicin with appropriate amount of different buffers was added in Au NPs solution to obtain the final doxorubicin concentration of 1.29 mM in solution. The solution was then incubated at room temperature and changes in surface plasmon resonance were monitored by UV/Vis/NIR spectroscopy measurements. IR spectra of doxorubicin bound to the Au NPs were recorded and surface charge of Au NPs before and after loading of
2.2. Synthesis of gold nanoparticles To complete removal of potential artificial nucleation sites, all glasswares were cleaned with an aqua regia solution (1:3 nitric acid/hydrochloric acid). An aqueous gold colloid, with an average particle size of 13 1 nm (determined from the TEM image) was synthesized by the method reported [22]. Briefly, 1 mM of 500 mL solution of hydrogen tetrachloroaurate trihydrate (HAuCl4.3H2O)
Fig. 1. TEM image of 13 nm Au NPs.
A.Z. Mirza, H. Shamshad / European Journal of Medicinal Chemistry 46 (2011) 1857e1860
Fig. 2. Representative UV/Vis spectra of (1) Au NPs and Au NPs Dox Complex in (2) MES, (3) 0.1 N HCl (pH 1), (4) acetic acid (pH 2.4), (5) PBS (pH 7.4).
doxorubicin was determined by measurement of zeta potential. SERS spectra of doxorubicin and Au NPs were recorded using 785 nm (40 mW) excitation in the range of 200e1800 cm1 with an integration time of 100 s.
3. Results and discussion Doxorubicin lacks tumor-targeting ability leading to poor biodistribution and therapeutic effects as well as serious undesirable side effects [14]. The novel approach presented in this paper was to conjugate doxorubicin via pH-sensitive bonds onto stabilized Au NPs, which may lead to much better tumor-targeting ability and improving the efficacy of cancer therapy. The doxorubicin-Au NPs complex was characterized by techniques including UV/vis/NIR
1859
spectroscopy, IR spectroscopy, Raman spectroscopy and zeta potential measurement. The TEM images recorded from as-synthesized Au NPs (Fig. 1) revealed that the nanoparticles were well dispersed with in a narrow size distribution and an average size of 13 1 nm. Absorption spectrum of Au NPs solution had maximum wavelength at 520 nm, which was in accordance with the plasmon resonance absorption wavelength of the Au NPs (Fig. 2) [22]. It has been reported that, during the formation of nanoparticles, citrate ion self-assembled on the surface of Au NPs to form negative charge. At pH near to 6 it would bind with some organic cations that coexisted in the solution to form new product through electrostatic attraction, which resulted in the change of some spectral characteristics and intensity [23]. Doxorubicin has a protonated amino group [24] and adsorption of nanoparticles has strong relations to the charging states of the amino groups [25]. When Au NPs combined with doxorubicin, the maximum absorption wavelength shifted to 528 nm (Fig. 2). The change of the absorption spectrum is due to the protonated nitrogen atom of doxorubicin molecule and combination of positive charged amine group on the surface of Au NPs. The zeta potential of citrate modified Au NPs was 27.44 mV and zeta potential of doxorubicin loaded Au NPs was 21.00 mV. This decrease in the zeta potential was approved to the presence of positively charged doxorubicin and also indicated that attractive forces including hydrogen bonding could be playing a major role facilitating the drug-loading process [15]. The bonding between protonated amine groups of the doxorubicin molecule with surface of Au NPs was also supported by FTIR (spectra not shown). Doxorubicin showed NeH stretching peak of amine at 3777 cm1 [15]. In case of doxorubicin-Au NPs complex the amine peak shifted to 3373 cm1, indicating the formation of electrostatic bond formation. Similar to infrared absorption spectroscopy, Raman scattering could be used for identification and quantification of molecule [13]. The surface-enhanced Raman scattering (SERS) has been used as a powerful method to get signal from
Fig. 3. Raman spectra of doxorubicin (Dox) and doxorubicin capped Au NPs (Dox þ Au NPs).
1860
A.Z. Mirza, H. Shamshad / European Journal of Medicinal Chemistry 46 (2011) 1857e1860
References
Structure 1. Doxorubicin.
fluorescent chromophores [26]. In SERS, the large increase of the near-field excitation intensity was observed at the location of the analyte, which is adsorbed to a noble-metal structure. The net surface enhancement exceeds ten orders-of-magnitude [27,28]. To determine the interaction of doxorubicin with Au NPs, Bruker Senterra Raman microscope (Bruker Optics, Inc., Billerica, MA) with 785 nm excitation laser at 25 mW was used to measure the SERS spectra. Fig. 3 shows the SERS spectra of doxorubicin and Au NPsdoxorubicin complex, represented the surface-enhanced Raman scattering (SERS) effect due to intense spectral signals. The comparison of the spectrum of free doxorubicin with the doxorubicin-complex revealed a small difference in wave number and Au NPs-doxorubicin complex spectra were similar to pure doxorubicin spectra (Fig. 3). These all above observations confirmed that the doxorubicin caped on the Au NPs. The pH-dependence of surface coverage indicated that these interactions were strongly affected by solution pH. It was observed that borohydride or citrate reduced Au NPs form aggregate at the slightest change in their pH and electrolyte environments [29] but it was also reported that gellan gum capped Au NPs were very stable between pH 4 to 8. Otherwise, the addition of doxorubicin solution to the AuNPs caused the particles to aggregate [15]. So, different pH buffers were used and it was found that in 2-(Nmorpholino)ethanesulfonic acid (MES) buffer at pH 6, interaction between doxorubicin and Au NPs was observed but in other buffer like 0.1 N HCl (pH 1), acetic acid (pH 2.4) and PBS (pH 7.4), nanoparticles form aggregates.
4. Conclusion Protonation of amino group present in doxorubicin molecule depended on the solution pH. Due to protonation, amino groups will be positively charged, which would promote the adsorption of negatively charged Au NPs by strong electrostatic interaction. In summary, it was concluded that, in alkaline solution, where the amino group was not protonated, Au NPs could not be adsorbed, while under acidic conditions, protonation made the amino group positively charged and the adsorption became easier and electrostatic interaction was believed to be between Au colloids and the substrate. In conclusion, we reported a novel method for successful loading of doxorubicin onto Au NPs. These nanoparticles might facilitate better transport of the loaded drug across bloodebrain barrier and this could be expanded to include the delivery of other drug molecules as well.
[1] D.D. Gutierrez, M. Surtchev, E. Eiser, C.J. Elsevier, Ru(II)-based metallosurfactant forming inverted aggregates, Nano Lett. 6 (2006) 145e147. [2] M. Everts, V. Saini, J.L. Leddon, R.J. Kok, M.S. Khalili, M.A. Preuss, C.L. Millican, G. Perkins, J.M. Brown, H. Bagaria, D.E. Nikles, D.T. Johnson, V.P. Zharov, D.T. Curiel, Covalently linked Au nanoparticles to a viral vector: potential for combined photothermal and gene cancer therapy, Nano Lett. 6 (2006) 587e591. [3] B.G.G. Lohmeijer, U.S. Schubert, Supramolecular engineering with macromolecules: an alternative concept for block copolymers, Angew. Chem., Int. Ed. 41 (2002) 3825e3829. [4] L. Li, M. Fan, R. Brown, L.J. Van, J. Wang, W. Wang, Y. Song, P. Zhang, Synthesis, properties, and environmental applications of nanoscale iron-based materials: a review, EnViron. Sci. Technol. 36 (2006) 405e431. [5] E.E. Connor, J. Mwamuka, A. Gole, C.J. Murphy, M.D. Wyatt, Gold nanoparticles are Taken up by human cells but do not cause acute cytotoxicity, Small 1 (2005) 325e327. [6] M. Daniel, D. Astruc, Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology, Chem. Rev. 104 (2004) 293e346. [7] K.K. Sandhu, C. McIntosh, J. Simard, S. Smith, V.M. Rotello, Gold nanoparticlemediated transfection of mammalian cells, Bioconjugate Chem. 13 (2002) 3e6. [8] A.C. Templeton, M.P. Wuelfing, R.W. Murray, Monolayer-protected cluster molecules, Acc. Chem. Res. 33 (2000) 27e36. [9] G.F. Paciotti, L. Myer, D. Weinreich, D. Goia, N. Pavel, R.E. McLaughlin, L. Tamarkin, Colloidal gold: a novel nanoparticle vector for tumor directed drug delivery, Drug Deliv. 11 (2004) 169e183. [10] V.S. Murthy, J.N. Cha, G.D. Stucky, M.S. Wong, Charge-driven flocculation of poly(l-lysine) e gold nanoparticle assemblies leading to hollow microspheres, J. Am. Chem. Soc. 126 (2004) 5292e5299. [11] S.J. Hurst, A.K.R. Lytton-Jean, C.A. Mirkin, Maximizing DNA loading on a range of gold nanoparticle sizes, Anal. Chem. 78 (2006) 8313e8318. [12] X. Huang, I.H. El-Sayed, W. Qian, M.A. El-Sayed, Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods, J. Am. Chem. Soc. 128 (2006) 2115e2120. [13] C. Eliasson, A. Lorén, K.V.G.K. Murty, M. Josefson, M. Kall, J. Abrahamsson, K. Abrahamsson, Multivariate evaluation of doxorubicin surface-enhanced Raman spectra, Spectrochimica Acta Part A 57 (2001) 1907e1915. [14] S. Aryal, J.J. Grailer, S. Pilla, D.A. Steeber, S. Gong, Doxorubicin conjugated gold nanoparticles as water-soluble and pH-responsive anticancer drug nanocarriers, J. Mater. Chem. 19 (2009) 7879e7884. [15] S. Dhar, E.M. Reddy, A. Shiras, V. Pokharkar, B.L.V. Prasad, Natural gum reduced/stabilized gold nanoparticles for drug delivery formulations, Chem. Eur. J. 14 (2008) 10244e10250. [16] J.D. Gibson, B.P. Khanal, E.R. Zubarev, Paclitaxel-functionalized gold nanoparticles, J. Am. Chem. Soc. 129 (2007) 11653e11661. [17] R.T. Tom, V. Suryanarayanan, P.G. Reddy, S. Baskaran, T. Pradeep, Ciprofloxacin-protected gold nanoparticles, Langmuir 20 (2004) 1909e1914. [18] H.X. Zhang, H. Zhao, J.X. Wang, J.F. Chen, Y.F. Lu, J. Yun, Facile preparation of monodisperse pharmaceutical colloidal spheres of atorvastatin calcium via self-assembly, Small 5 (2009) 1846e1849. [19] S. Santra, C. Kaittanis, J. Grimm, J.M. Perez, Drug/dye-loaded, multifunctional iron oxide nanoparticles for combined targeted cancer therapy and dual optical/magnetic resonance imaging, Small 5 (2009) 1862e1868. [20] M. Brzozowska, P. Krysinski, Synthesis and functionalization of magnetic nanoparticles with covalently bound electroactive compound doxorubicin, Electrochimica Acta 54 (2009) 5065e5070. [21] M. Prabaharan, J.J. Grailer, S. Pilla, D.A. Steeber, S. Gong, Gold nanoparticles with a monolayer of doxorubicin-conjugated amphiphilic block copolymer for tumor-targeted drug delivery, Biomaterials 30 (2009) 6065e6075. [22] K.C. Grabar, R.G. Freeman, M.B. Hommer, M.J. Natan, Preparation and characterization of Au colloid monolayers, Anal. Chem. 67 (1995) 735e743. [23] S.P. Liu, Y.Q. He, Z.F. Liu, L. Kong, Q.M. Lu, Resonance rayleigh scattering spectral method for the determination of raloxifene using gold nanoparticle as a probe, Anal. Chim. Acta 598 (2007) 304e311. [24] J. Bhattacharjee, G. Verma, V.K. Aswal, P.A. Hassan, Small angle neutron scattering study of doxorubicin-surfactant complexes encapsulated in block copolymer micelles, Pramana 71 (2008) 991e995. [25] T. Zhu, X. Fu, T. Mu, J. Wang, Z. Liu, pH-dependent adsorption of gold nanoparticles on p-aminothiophenol-modified gold substrates, Langmuir 15 (1999) 5197e5199. [26] I. Nabiev, I. Chourpa, M. Manfait, Comparative studies of antitumor DNA intercalating agents, aclacinomycin and saintopin, by means of surfaceenhanced Raman scattering spectroscopy, J. Phys. Chem. 98 (1994) 1344e1350. [27] H. Xu, E.J. Bjerneld, M. Kall, L. Borjesson, Spectroscopy of single hemoglobin molecules by surface enhanced Raman scattering, Phys. Rev. Lett. 83 (1999) 4357e4360. [28] E.J. Bjerneld, P. Johansson, M. Kall, Single molecule vibrational fine-structure of tyrosine adsorbed on Ag nano-crystals, Single Mol. 1 (2000) 239e248. [29] L.L. Rouhana, J.A. Jaber, J.B. Schlenoff, Aggregation-resistant water-soluble gold nanoparticles, Langmuir 23 (2007) 12799e12801.