Surface interactions of gold nanorods and polysaccharides: From clusters to individual nanoparticles

Gold nanorods (AuNRs) are suitable for constructing self-assembled structures for the development of biosensing devices and are usually obtained in the presence of cetyltrimethylammonium bromide (CTAB).

Carbohydrate Polymers 152 (2016) 479–486 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Surface interactions of gold nanorods and polysaccharides: From clusters to individual nanoparticles Heloise Ribeiro de Barros a , Leandro Piovan a , Guilherme L Sassaki b , Diego de Araujo Sabry b , Ney Mattoso c , Ábner Magalhães Nunes d , Mario R Meneghetti d , Izabel C Riegel-Vidotti a,∗ a Grupo de Pesquisa em Macromoléculas e Interfaces, Departamento de Química, Universidade Federal Paraná—UFPR, CxP 19081, CEP 81531-980, Curitiba, PR, Brazil b Departamento de Bioquímica e Biologia Molecular, Universidade Federal Paraná—UFPR, CxP 19046, CEP 81531-980, Curitiba, PR, Brazil c Departamento de Física, Universidade Federal Paraná—UFPR, CxP 19044, CEP 81531-980, Curitiba, PR, Brazil d Grupo de Catálise e Reatividade Química, Instituto de Química e Biotecnologia, Universidade Federal de Alagoas, Av Lourival de Melo Mota s/n, CEP 57072-970, Maceió, AL, Brazil a r t i c l e i n f o Article history: Received 14 April 2016 Received in revised form 28 June 2016 Accepted July 2016 Available online July 2016 Keywords: Gold nanorods Sulfated chitosan Surface interactions Self-assembling a b s t r a c t Gold nanorods (AuNRs) are suitable for constructing self-assembled structures for the development of biosensing devices and are usually obtained in the presence of cetyltrimethylammonium bromide (CTAB) Here, a sulfated chitosan (ChiS) and gum arabic (GA) were employed to encapsulate CTAB/AuNRs with the purpose of studying the interactions of the polysaccharides with CTAB, which is cytotoxic and is responsible for the instability of nanoparticles in buffer solutions The presence of a variety of functional groups such as the sulfate groups in ChiS and the carboxylic groups in GA, led to efficient interactions with CTAB/AuNRs as evidenced through UV–vis and FTIR spectroscopies Electron microscopies (HR-SEM and TEM) revealed that nanoparticle clusters were formed in the GA-AuNRs sample, whereas individual AuNRs, surrounded by a dense layer of polysaccharides, were observed in the ChiS-AuNRs sample Therefore, the presented work contributes to the understanding of the driving forces that control the surface interactions of the studied materials, providing useful information in the building-up of gold self-assembled nanostructures © 2016 Elsevier Ltd All rights reserved Introduction Gold nanoparticles (AuNPs) have increasingly been given extensive attention due to their unique properties making those materials useful in catalysis, nanoelectronics and, more interestingly, in optical sensing and diagnostics in the biomedical field (Garabagiu & Bratu, 2013; Kopwitthaya et al., 2010; Mitamura, Imae, Saito, & Takai, 2007; Pierrat, Zins, Breivogel, & Sonnichsen, 2007) Among the AuNPs, considerable attention has been dedicated to gold nanorods (AuNRs) The coherent oscillation of the electrons along the short axis (transversal SPR) and the long axis (longitudinal SPR) of the nanorods causes two surface plasmon resonance (SPR) bands At least one of these bands can be found in the visible spectra The transversal SPR band has a maximum absorption around ∗ Corresponding author E-mail addresses: izabel.riegel@ufpr.br, iriegel@gmail.com (I.C Riegel-Vidotti) http://dx.doi.org/10.1016/j.carbpol.2016.07.018 0144-8617/© 2016 Elsevier Ltd All rights reserved 520 nm (Rayavarapu et al., 2010), whereas the longitudinal SPR band is observed in the range from 650 nm (shorter rods) to 950 nm (longer rods) (Eutis & El-Sayed, 2006; Rayavarapu et al., 2010) As transversal and longitudinal SPR are shape and size dependent (Murphy & Jana, 2002; Xie et al., 2011), the AuNRs are particularly suitable for building up self-assembled structures for the development of biosensors (Yu et al., 2014), nanodevices (Xie et al., 2011), and non-invasive probes (Charan et al., 2012) Regarding the applications of AuNPs in biological environments, some important issues arise concerning the maintenance of their morphological stability, cytotoxicity, and interactions with different organisms or their components Therefore, the choice for appropriate stabilizing agents is of the utmost importance in obtaining AuNRs that are stable in different environmental conditions (pH and ionic strength) and that exhibit low toxicity The seed mediated method in the presence of the surfactant cetyltrimethylammonium bromide (CTAB) is the most commonly employed procedure to obtain AuNRs, although some other methods have been recently proposed (da Silva, Nunes, Meneghetti, 480 H.R de Barros et al / Carbohydrate Polymers 152 (2016) 479–486 & Meneghetti, 2013; Pérez-Juste, Pastoriza-Santos, Liz-Marzán, & Mulvaney, 2005) In general, the method initially proposed by Murphy (Gole, Orendorff & Murphy, 2004; Jana, Gearheart & Murphy, 2001a; Jana, Gearheart, & Murphy, 2001b; Jana, Gearheart, & Murphy, 2001c; Johnson, Dujardin, Davis, Murphy & Mann, 2002; Murphy & Jana, 2002) and El-Sayed groups (Nikoobakht & El-Sayed, 2001) consists of the formation of the AuNRs from small sized spherical AuNPs (seed solution), which then acts as nucleation centers in the AuNRs synthesis In the presence of CTAB, the AuNRs growth is mainly unidirectional since the interactions between the polar head groups of the surfactant, i.e the quaternary ammonium bromide moiety, and the crystallographic {110} facet of the growing particle is preferential, causing the growth in the longitudinal direction, parallel to the {001} planes (da Silva, Meneghetti, Denicourt-Nowicki, & Roucoux, 2014; Meena & Sulpizi, 2013; Nikoobakht & El-Sayed, 2003) Therefore, the different growth rates of the facets are the factors that determine the final shape of the nanoparticle (short versus long NRs) In addition, CTAB is responsible for maintaining the colloidal stability since the bilayer structure, which is formed by the self-interaction of the alkyl groups of CTAB, promotes the suitable protection against particle agglomeration in aqueous media through electrostatic and steric interactions (Boca & Astilean, 2010) However, CTAB is cytotoxic and causes AuNRs instability in buffer solutions, which then restricts their use in biological applications (Boca & Astilean, 2010; Hamon, Bizien, Artzner, Even-Hernandez, & Marchi, 2014; Rayavarapu et al., 2010) Therefore, when focusing on the obtention of AuNRs that safely can be used in the biomedical field, it is fundamental to replace part of CTAB or encapsulate the CTAB/AuNRs to obtain particles with reduced toxicity that are also stable under different environments Kopwitthaya et al (2010) and Rayavarapu et al (2010) synthetized CTAB/AuNRs and showed that the replacement of CTAB by thiolated poly(ethylene glycol) (PEGSH) produced particles with lower cytotoxicity compared to the as-prepared AuNRs (Kopwitthaya et al., 2010; Rayavarapu et al., 2010) In addition, other molecules have been used to replace CTAB intending to reduce the cytotoxicity, such as polystyrene sulfonate, polyethylene glycol (Rayavarapu et al., 2010), 1Mercaptoundec-11-yl)hexa(ethylene glycol) (EG6 OH) (Xie et al., 2011), thio-polyethylene glycols (Bogliotti et al., 2011), polyacrylic acid, poly(allylamine) hydrochloride (PAH) (Huang, Jackson & Murphy, 2012), and 3-mercaptopropionic acid (MPA) (Garabagiu & Bratu, 2013) Polysaccharides are part of a very promising family of naturally occurring molecules that have also been described to interact with gold nanoparticles The presence of a variety of functional groups in their structure assists the favorable interactions between the AuNRs and the surrounding media, which is responsible for the AuNRs stabilization and also provides sites for further chemical modifications (Erathodiyil & Ying, 2011; Liu et al., 2013) In addition to being natural products, polysaccharides have inherent properties such as biodegradability, biocompatibility, and low toxicity (Liu et al., 2013) Surprisingly, there is a relatively low number of scientific works reporting the use of polysaccharides as stabilizing agents of AuNRs (Wang, Chang & Peng, 2011; Yu et al., 2014), although many works have reported the efficient stabilization of spherical gold nanoparticles by polysaccharides Chitosan (Chi) is a linear polysaccharide extracted from the exoskeleton of crustaceans, obtained by deacetylation of chitin Medium and high molar mass chitosan is only soluble in water at pH lower than 6.0 (Williams & Phillips, 2000, Chp 21) Chitosan (Boca et al., 2011) and its derivatives have been successfully employed to cap AuNPs for photothermal therapy (Wang, Chang & Peng, 2011; Yang et al., 2015), for optoacoustic tomography (Wang et al., 2015), among other applications Gum arabic (GA) is a highly branched natural polysaccharide exuded from the trunks and barks of acacia trees This polysaccharide has been extensively used for the stabilization of spherical AuNPs (Chanda et al., 2010; Kattumuri et al., 2007; Wu & Chen, 2010), displaying optimal performance in a wide pH range (Barros et al., 2016) In order to reduce the cytotoxicity inherent to CTAB stabilized AuNRs and simultaneously improve the AuNRs stability in physiological media, we used a sulfated chitosan (ChiS) or GA to encapsulate CTAB/AuNRs The non-toxicity and biocompatibility of Chi and GA were evaluated in previous works (Bicho, Roque, Cardoso, Domingos, & Batalha, 2009; Boca et al., 2011) The chemical modification of Chi to obtain ChiS is of interest because it does not only keep the Chi main chain backbone intact, but it also improves its solubility in aqueous media (Jayakumar, Nwe, Tokura, & Tamura, 2007) Moreover, it can potentially infer new functionalities to the modified Chi since sulfated polysaccharides, like heparin for example, are known to present important biological functions such as anticoagulant and/or antithrombotic actions (Asif et al., 2016; Jayakumar et al., 2007; Maas et al., 2012) Herein we demonstrate by UV–vis (Ultraviolet–visible) and FTIR (Fourier Transform Infrared) spectroscopies, and also by transmission and scanning electron microscopies that ChiS and GA interact differently with the AuNRs The differences are discussed in terms of the different functional groups present in each polysaccharide that leads to distinct polysaccharide/AuNRs structures Therefore, this study contributes to understanding and controlling the selfassembling behavior of AuNRs, mediated by the capping agent Materials and methods 2.1 Materials Tetrachloroauric acid (HAuCl4 ·3H2 0, 30% in dilute HCl, 99,9%), CTAB (≥98%), GA (Mw = 9.3 × 105 g mol−1 ; uronic acid content of 17%) (Grein et al., 2013), chitosan (≥75% deacetylated), and silver nitrate (AgNO3 , > 99%) were purchased from Sigma-Aldrich Sodium borohydride (NaBH4 , ≥ 98%) was purchased from Nuclear (São Paulo, Brasil) and ascorbic acid (AA, >99%) was purchased from Dinâmica (São Paulo, Brasil) Milli-Q grade water (18.2 M cm, Millipore, USA) was used in the preparation of all solutions Prior to use, GA powder was solubilized in water, left overnight at ◦ C and subsequently dialyzed for 48 h against distilled water through a dialysis membrane (12–14 kDa cut-off) and freeze-dried 2.2 Sulfation of a commercial chitosan Commercial chitosan (Chi) underwent the sulfation reaction according to Terbojevich, Carraro, and Cosani (1989)’s sulfuric acid:chlorosulfonic acid method Briefly, 1.00 g of commercial chitosan was added to the pre-cooled (4 ◦ C) reaction mixture (40 mL of sulfuric acid (H2 SO4 95%) and 20 mL of chlorosulfonic acid (HClSO3 98%)) Then, the reaction was carried out at room temperature under stirring for h The sulfation was stopped by pouring 250 mL of cold diethyl ether (Et2 O) into the reaction mixture The precipitate formed was washed with cold Et2 O, then suspended in distilled water, neutralized with saturated NaHCO3 , dialyzed against tap water through a 3500 kDa cut-off membrane, and freeze-dried The final product was fully characterized (ChiS, Mw = 1.4 × 104 g mol−1 ; SO4 = 48%; SO3 O–3 = 6.8% and SO3 O–6 = 41.2%) (Supplementary material—Fig S1 and Table S1) and resulted in a pale yellow powder that was stored in a moisture free environment The surface charge was obtained using a Zetasizer Nano ZS instrument by solubilizing the polysaccharides (ChiS and GA) using Milli-Q water H.R de Barros et al / Carbohydrate Polymers 152 (2016) 479–486 2.3 Synthesis of the gold nanorods (AuNRs) 2.4 Preparation of GA-AuNRs and ChiS-AuNRs The AuNRs stabilized by the polysaccharides, either GA-AuNRs or ChiS-AuNRs, were prepared by a simple method First, the asprepared AuNRs were centrifuged (10.000 rpm, 15 min) and the supernatant was discarded to remove the excess CTAB The precipitate was dispersed in 0.5 mL of water Then, it was added 4.5 mL of GA or ChiS solution 0.1 wt% The final solution was kept under gentle magnetic stirring at room temperature (∼25 ◦ C) for 24 h Afterwards, the sample was again centrifuged and the supernatant was discarded to remove the unbound GA or ChiS from the solution The precipitate was dispersed in 2.0 mL of Milli-Q water (18.2 M cm at 25 ◦ C) and used thereafter UV–vis spectroscopy (Agilent, model 8453) was used to verify any changes in the SPR bands as a consequence of the interactions of the AuNRs with the polysaccharides 2.5 Microscopic and spectroscopic investigation of AuNRs, GA-AuNRs and ChiS-AuNRs Transmission electron microscopy (TEM) was performed using a JEOL 1200EX-II microscope working at an acceleration voltage of 80 kV A drop (∼10 ␮L) of the colloidal solution was deposited onto 400 mesh carbon-coated grids and air-dried High resolution scanning electron microscopy (HR-SEM) was performed using a FEI Quanta 450 FEG microscope working at an acceleration voltage of 10 kV An aliquot of 80 ␮L of the colloidal solution was deposited onto a sample support and air-dried FTIR measurements were performed using a BIORAD FTS-3500 GX FTIR spectrometer The measurements were made in the transmission mode in a spectral domain ranging from 400 to 4000 cm−1 , using KBr pellets Results and discussion In order to ensure the full surface coverage of the AuNRs, the concentration of the polysaccharides (GA or ChiS) was kept much higher than the concentration of the AuNRs in the preparation of GA-AuNRs and ChiS-AuNRs Both, GA and ChiS are negatively charged in aqueous solution with pH ∼5 The zeta potential values (␨) of GA and ChiS are −36.3 mV and −28.6 mV, respectively The negative charge of GA is due to the presence of the COO− groups, whereas the negative charge of ChiS corresponds to the presence of OSO3 − However, due to the cationic quaternary ammonium headgroup of CTAB, the as-prepared AuNRs bear positive surface (c) 0,3 Absorbance The AuNRs were synthetized by the seed-mediated method according to da Silva et al (2013) and previously described by Sau and Murphy (2004) In a typical procedure, the seed solution was prepared by 5.0 mL of aqueous solution 0.5 × 10−3 mol L−1 HAuCl4 added to 2.5 mL of 0.20 mol L−1 CTAB solution Then, 0.6 mL of ice-cold 0.01 mol L−1 NaBH4 solution was added The color of the solution immediately changed from dark to brownish yellow Next, the solution was kept under gentle mixing for and left to rest for at least h prior to use Afterwards, the growth solution was prepared by gentle mixing of a 2.5 mL of 0.20 mol L−1 CTAB solution, 5.0 mL of aqueous solution × 10−3 mol L−1 HAuCl4 and 150 ␮L of 4.0 × 10−3 mol L−1 AgNO3 solution Then, 70 ␮L of 80 × 10−3 mol L−1 ascorbic acid solution was added and the color changed immediately from dark yellow to colorless Lastly, 12 ␮L of seed solution was added by mixing gently for 10 s The color changed slowly from colorless to purple The final solution was kept undisturbed for at least h UV–vis spectroscopy (Agilent, model 8453) was used to monitor the AuNRs formation through the observation of the SPR bands 481 0,2 0,1 (b) (a) 0,0 400 500 600 700 800 900 1000 Wavelength (nm) Fig UV–vis absorption spectra of (a) as-prepared AuNRs, (b) GA-AuNR and (c) ChiS-AuNRs charge (Rayavarapu et al., 2010) Therefore, the choice of using negatively charged polysaccharides aids the interactions between the AuNRs and the polysaccharides through electrostatic attraction The as-synthesized AuNRs present typical SPR bands centered around ␭1 = 515 nm and ␭2 = 740 nm (Fig 1) that allow the determination of the particle concentration using the extinction coefficient (Garabagiu & Bratu, 2013; Orendorff & Murphy, 2006) The estimated concentration of particles of the as-prepared AuNRs is × 10−10 mol L−1 The profiles of the SPR bands of GA-AuNRs and ChiS-AuNRs are also shown in Fig Slight shifts at the maximum wavelengths are observed since the presence of GA and ChiS changes the dielectric constant of the surrounding environment In the case of ChiS-AuNRs, it is evident the appearance of a strong absorption at longer wavelengths, possibly caused by the changes in the surrounding environment, which will be clarified in the TEM images Furthermore, the maintenance of the position of the SPR bands after the polysaccharides adsorption indicates that aggregation has not taken place, preserving the morphology of the particles Through TEM images the AuNRs were observed to have an average size of 45 nm × 15 nm (aspect ratio = 3) Although some spherical particles are seen, most of the objects are rod-like structures, characterizing a high yield synthesis (Fig 2a) Also, the shape and size of the AuNRs were preserved in the presence of GA or ChiS (Fig 2b–e) The GA-AuNRs and ChiS-AuNRs exhibited average sizes of 47 nm × 15 nm and 43 nm × 14 nm, respectively, corroborating what was observed by UV–vis spectroscopy It is noticeable from the TEM images that the GA-AuNRs sample resulted in nanoparticle clusters (Fig 2b) The high magnification image revealed that the GA adsorbed molecules are not distinguishable from the substrate However, the nanoparticles in the ChiS-AuNR sample are separated from each other by a dense structure that is suggested to be composed of the ChiS molecules This behavior could be associated with the differences observed in the shape of the UV–vis absorption spectra For a better understanding of the organization of the polysaccharides around the AuNRs, HR-SEM images were obtained using secondary and backscattered electrons The comparative analysis of the images provides a deeper insight into the interactions between the polysaccharides and the AuNRs Fig 3a and b clearly shows the differences in the AuNRs surrounding medium due to the presence of GA or ChiS, respectively Both images were obtained using secondary electron signal that provides topographic contrast As observed by TEM, nanoparticle clusters were seen in Fig 3a (GA-AuNRs), whereas in Fig 3b (ChiS-AuNRs) the AuNRs were individually surrounded by ChiS in 482 H.R de Barros et al / Carbohydrate Polymers 152 (2016) 479–486 Fig TEM images of (a) as-prepared AuNR, (b-c) after the adsorption of GA (GA-AuNRs) and (d-e) of ChiS (ChiS-AuNRs) on AuNR surface a cloud-like arrangement As a result, when GA is used as the stabilizing agent, an increased particle density (number or particles per unit volume) is attained when compared with ChiS Fig 3c and e corresponds to low magnification images that were collected at the same region and obtained by secondary and backscattered electrons, respectively The contrast obtained by backscattered electron signal is related to the electron density of the material As can be seen, no important differences were observed supposedly because GA is arranged as a thin layer on the SEM support Sample ChiSAuNRs was analyzed similarly (Fig 3d and f) and in this case, striking differences were observed In the case of Fig 3d, which was obtained by secondary electrons, the edges of the structure are highlighted, providing a volume perspective (topographic contrast) In Fig 3f, obtained by backscattered electrons, the electron density and contrast of the organic matrix of ChiS with the metallic support are strongly evidencing that the particles are surrounded by ChiS molecules, confirming that ChiS acts as an efficient encapsulating/wrapping agent for individual AuNRs At low magnification, the sharp difference of contrast in this type of sample is very useful to quickly find the area to be studied at greater magnifications The differences observed by comparing the images of GA-AuNRs and ChiS-AuNRs can be ascribed to the differences between the electron densities of GA and ChiS and to the different arrange- ment of the polysaccharides around the AuNRs ChiS has higher electron density when compared with GA due to the presence of sulfate groups Additionally, the molecules are densely packed around the nanoparticles due to their lower molar mass and chain linearity, favoring the formation of a three-dimensional structure On the other hand, GA, which is a highly branched, high molar mass polysaccharide, bears atoms with low electron density (mainly C, O, H), forming a thin layer on the support Some other aspects can be discussed to clarify the HR-SEM observations, as follows It is widely known that GA presents surfactant-like properties and is highly soluble in water (Grein et al., 2013) Therefore, when GA-AuNRs were washed to remove the excess of GA, the molecules that were weakly bounded on the AuNRs surface most likely were removed, lowering the resulting final concentration of GA around the gold surface Conversely, it is known that Chi exhibits agglutinative properties (Lehr, Bouwstra, Schacht, & Junginger, 1992) and since Chi is soluble only in acidic media, the sulfation process improves its solubility in water However, GA is more water soluble than ChiS, resulting in a higher concentration of ChiS than GA molecules around the AuNRs Thus, the adequate selection of the stabilizing agent can efficiently tune the self- aggregation of AuNRs Gold nanoparticle clusters have applications in photothermal therapy (Zharov, H.R de Barros et al / Carbohydrate Polymers 152 (2016) 479–486 483 Fig HR-SEM images of GA-AuNRs (a, c, e) and ChiS-AuNRs (b, d, f) obtained by secondary electrons (a, b, c, d) and backscattered electrons (e, f) AuNR 960 669 962 719 731 1487 1431 1487 1431 2918 2850 CTAB 2918 2850 Mercer, Galitovskaya, & Smeltzer, 2006), whereas individual functionalized gold nanoparticles can perform important biological functions via specific signaling pathways (Li, Kawazoe & Chen, 2015; Nethi et al., 2014) FTIR spectroscopy was chosen to evaluate the nature of the interactions between the polysaccharides and the AuNRs The displacement, appearance or disappearance of bands in the FTIR spectra may be attributed to the interactions that occur in these assemblies First, the spectra of the as-prepared AuNRs and neat CTAB is presented (Fig 4) The assignments of the main bands are in Table It was observed that the characteristic bands present in CTAB are preserved in the AuNRs (Tang, Huang, & Man, 2013) The maintenance of the bands, relative to symmetric and asymmetric stretching vibration of CH2 of CTAB chain (2918 and 2850 cm−1 ), indicates that the hydrophobic tails of CTAB are not interacting with the AuNRs surface It is suggested that the alkyl tails are selfinteracting, forming a bilayer on the gold surface that does not restrain the stretching vibrational modes According to this proposition, unbound and bound surfactant headgroups are found in the gold surroundings (Nikoobakht & El-Sayed, 2001), thus rendering some C N+ groups free for further interactions (Gole et al., 2004) However, the bands corresponding to symmetric and asymmetric C H scissoring vibrations of H3 C N+ moiety (1487, 1473, 1462 and 1431 cm−1 ), and the band corresponding to C N+ stretching (960 cm−1 ) are relatively less intense and slightly shifted in AuNRs when compared to pure CTAB This decrease in intensity indicates 4000 3500 3000 1500 1000 500 -1 Wavenumber (cm ) Fig FTIR spectra of as-prepared AuNRs and neat CTAB that the hydrophilic portion of CTAB bound to the AuNRs surface Additionally, the bands corresponding to the CH2 chain rocking mode demonstrate important differences Pure CTAB shows two bands at 719 and 731 cm−1 whereas AuNRs show only one band at 669 cm−1 This fact is clear evidence of the constrainments that the CTAB alkyl chains are subjected to due to the interactions with the particles, resulting in the formation of a compact layered structure 484 H.R de Barros et al / Carbohydrate Polymers 152 (2016) 479–486 ChiS-AuNRs 1004 1062 AuNRs 1647 1542 2918 2850 AuNRs 1060 2918 2850 1153 GA-AuNRs 3500 3000 1500 960 1153 1062 1004 1647 1541 1487 1431 2918 2850 960 1253 1143 1064 1031 1608 4000 1419 GA 1487 1431 2918 2850 ChiS 1000 500 4000 3500 3000 1500 1000 500 -1 -1 Wavenumber (cm ) Wavenumber (cm ) Fig FTIR spectra of GA-AuNR, as-prepared AuNR and GA Fig FTIR spectra of ChiS-AuNR, as-prepared AuNR and ChiS around the particles Therefore, it was confirmed by FTIR spectroscopy that CTAB remained on the AuNRs surface even after the washing procedure The FTIR spectrum of GA-AuNRs is presented in Fig Important differences can be seen when comparing with the as-prepared AuNRs spectrum The assignment of the main bands for both samples is shown in Table The characteristic bands of AuNRs, relative to the symmetric and asymmetric stretching vibrations of −CH2 − of CTAB (2918 and 2850 cm−1 ) were maintained in the GA-AuNRs spectrum Thus, it was evidenced that CTAB is still present on the AuNRs surface Additionally, the absence of some bands attributed to C N+ moiety (1473, 1462, 1433 and 960 cm−1 ) and the absence of the two strong bands in the GA spectrum attributed to the asymmetric and symmetric stretching vibration of the COO− group (1608 and 1419 cm−1 ) The profile change of the bands corresponding to the stretching of the C O (1253, 1143, 1064 and 1031 cm−1 ) in the GA-AuNRs spectrum is an indication of the interaction between the C N+ headgroup of CTAB with the negatively charged carboxylate groups on GA structure Furthermore, the observation of new bands around 1060 and 700–400 cm−1 (finger print region) in the GA-AuNR spectrum could be associated to the interactions that occur through GA adsorbed on the AuNR surface The presence of CTAB in ChiS-AuNRs samples was likewise observed, as seen by the FTIR spectra in Fig The bands at 2918 and 2850 cm−1 are present, whereas the bands at 1487–1433 and in 960 cm−1 disappeared in the ChiS-AuNRs spectrum in comparison to the AuNRs spectrum Furthermore, the main bands observed in the ChiS spectrum not disappear when ChiS is associated with the AuNRs, but instead exhibit an intensity decrease in the ChiSAuNRs spectrum (1647, 1542, 1153, 1072, 1062 and 1004 cm−1 ) The assignment of the main bands is shown in Table Therefore, the interaction between the C N+ from CTAB and ChiS occurs through a mutual interaction of the diverse negative charge functional groups present in its structure It is widely known that alkanethiols exhibit a preferential binding on the surface of gold nanoparticles, promoted by thermodynamically favored covalent bonds (Karpovich, & Blanchard, 1994; Leff, Brandt, & Heath, 1996; Templeton, Pietron, Murray, & Mulvaney, 2000; Zakaria et al., 2013) Yet, the sulfur present in sulfate functional groups does not present the same characteristics as alkanethiols since the stabilization and the absence of free electron pairs promoted by the electron delocalization between the oxygen atoms hinders the occurrence of new bindings However, sulfate groups may stabilize nanoparticles by electrostatic interaction like hydroxyl, carbonyl and amino groups that are intrinsically inhibitory to particle aggregation It was confirmed through FTIR analyses that CTAB was not entirely removed from the AuNRs surface since sulfate and carboxyl groups from ChiS and GA, respectively, exhibit preferential interactions with the positively charged headgroups of CTAB The interactions of the polysaccharides and the AuNRs surface, therefore, occur via the C N+ of CTAB and carboxylate or sulfate groups, of GA and ChiS, respectively, as depicted in Fig Conclusions With a simple, straightforward methodology we demonstrated that GA and ChiS efficiently interact with the surface of CTAB coated AuNRs The resulting self-assembled structures were fully characterized Microscopy images showed that GA produced AuNRs irregular clusters and ChiS acted as an efficient encapsulating/wrapping agent resulting in individual AuNRs, which were well separated by the ChiS molecules FTIR analyses clearly showed that GA and ChiS interact with the AuNRs via charged groups of CTAB by electrostatic interactions, leaving the CH2 groups intact Combining our results with data already available in the literature, the toxicity of ChiS-AuNRs and GA-AuNRs is expected to decrease in relation to CTAB-AuNRs Therefore, by using a new polysaccharide we present an interesting strategy to produce individually wrapped Fig Schematic representation of the interaction of CTAB/AuNRs with (a) ChiS (CTA+ ROSO3 − /CTA+ /CTAB/AuNR) and (b) GA (CTA+ RCOO− /CTA+ /CTAB/AuNR) H.R de Barros et al / Carbohydrate Polymers 152 (2016) 479–486 485 Table FTIR band assignments of CTAB and as-prepared AuNRs Assignmenta Wavenumber (cm−1 ) CTAB AuNR Symmetric and assymetric stretching of CH2 of CTAB chain Asymmetric and symmetric C H scissoring of H3 C N+ moiety C N+ stretching Rocking mode of the CH2 chain ((CH2 )n , n > 4) 2918 and 2850 1487, 1473, 1462 and 1431 962 719 and 731 2918 and 2850 1487, 1473, 1462 and 1431 960 669 a Based on Nikoobakht & El-Sayed, 2001; Sui et al., 2006; Tang et al., 2013; Campbell et al., 2004; Innocenzi, Falcaro, Grosso & Babonneau 2006 Table FTIR band assignments of as-prepared AuNRs, GA, GA-AuNR, ChiS and ChiS-AuNRs Assignmenta AuNR GA GA-AuNR ChiS ChiS-AuNR Symmetric and asymmetric stretching of C CH2 of CTAB chain Assymetric and symetric stretching of the carboxilic acid salt COO Asymmetric and symmetric C H scissoring vibrations of CH3 N+ moiety C O stretching Could be attributed to the interactions that take place by GA-AuNRs interactions C N+ stretching C O Amide band Band associated to the NH3 + Symmetric stretching of C O C bands 2918 and 2850 – 2918 and 2850 – 2918 and 2850 – 1608 and 1419 – – – 1487, 1473, 1462 and 1431 – 1487 – – – – 1253, 1143, 1064 and 1031 – – 1060, 700–400 1062 and 1004 – 1062 and 1004 – 962 – – – – – – – – – – – – 1647 1541 1153 – 1647 1542 1153 a Based on Davidovich-Pinhas et al., 2014; Espinosa-Andrews et al., 2010; Tang et al., 2013 AuNRs, applicable when well dispersed nanoparticles are required In addition, the observation of clusters or individual AuNRs adds information to the proper manipulation and usage of these polysaccharide functionalized nanoparticles Acknowledgements The authors acknowledge the support given by the Brazilian National Counsel of Technological and Scientific Development (CNPq) mainly through the grants 577232/2008-8, 477467/20105 and 564741/2010-8 H R Barros, D A Sabry and A M Nunes express their gratitude to CAPES for their fellowships The authors are very grateful to the Electron Microscopy Center of UFPR (CMEUFPR) for the TEM images and to SENAI PR- Institute of Innovation in Electrochemistry for the zeta potential measurements Appendix A Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbpol.2016.07 018 References Asif, S., Ekdahl, K N., Fromell, K., Gustafson, E., Barbu, A., Le Blanc, K., et al (2016) Heparinization of cell surfaces with short peptide-conjugated PEG-lipid regulates thromboinflammation in transplantation of human MSCs and hepatocytes Acta Biomaterialia, 35, 194–205 Barros, H R., Cardoso, M B., de Oliveira, C C., Franco, C R C., Belan, D L., Vidotti, M., et al (2016) Stability of gum 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acid-terminated... the AuNRs surface Additionally, the absence of some bands attributed to C N+ moiety (1473, 1462, 1433 and 960 cm−1 ) and the absence of the two strong bands in the GA spectrum attributed to the