Photochemically-assisted synthesis of non-toxic and biocompatible gold nanoparticles

Accepted Manuscript Title: Photochemically-Assisted Synthesis of Non-toxic and Biocompatible Gold Nanoparticles Author: Priscila R. Teixeira Mayara S.C. Santos Ana Lu´ısa G. Silva Sˆonia N. B´ao Ricardo B. Azevedo Maria Jos´e A. Sales Leonardo G. Paterno PII: DOI: Reference:

S0927-7765(16)30644-0 http://dx.doi.org/doi:10.1016/j.colsurfb.2016.09.002 COLSUB 8134

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

14-5-2016 1-9-2016 2-9-2016

Please cite this article as: Priscila R.Teixeira, Mayara S.C.Santos, Ana Lu´ısa G.Silva, Sˆonia N.B´ao, Ricardo B.Azevedo, Maria Jos´e A.Sales, Leonardo G.Paterno, Photochemically-Assisted Synthesis of Non-toxic and Biocompatible Gold Nanoparticles, Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2016.09.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Photochemically-Assisted Synthesis of Non-toxic and Biocompatible Gold Nanoparticles Priscila R. Teixeiraa†, Mayara S. C. Santos

b,c

†, Ana Luísa G. Silvab, Sônia N. Báob,

Ricardo B. Azevedod, Maria José A. Salesa and Leonardo G. Paternoa,* a

Laboratório de Pesquisa em Polímeros e Nanomateriais, Instituto de Química,

Universidade de Brasília, Brasília DF 70904-970, Brazil. b

Laboratório de Microscopia Eletrônica, Departamento de Biologia Celular,

Universidade de Brasília, Brasília DF 70919-970, Brazil. c

Boston Children’s Hospital, Boston, MA 02115, USA.

d

Departamento de Genética e Morfologia, Universidade de Brasilia, Brasília DF 70919-

900, Brazil. *corresponding author: [email protected] (L.G. Paterno) † These authors contributed equally to the work.

Graphical Abstract

Highlights Gold nanoparticles produced without hazardous and toxic chemical reductants. Methodology capable of fine-tune the size of nanoparticles from 100 nm to 8 nm. Gold nanoparticles coated with biocompatible polymer produced at short times.

Abstract This contribution describes the photochemically-assisted synthesis of aqueous colloidal suspensions of non-toxic and biocompatible spherical gold nanoparticles stabilized by branched polyethylenimine, or else Au-np-PEI. The method consists on 30 minutes of photoexcitation (254 nm, 16 W) at room temperature of an aqueous diluted solution of chloroauric acid (HAuCl4) containing PEI. While the UV irradiation forms the [Au(3+)Cl4-]* excited species that succesively transforms into zero valent Au, PEI controls the nucleation step of nanoparticles formation. Varying the PEI to Au molar ratio permits one to tune the size of nanoparticles between 100 nm to 8 nm. The obtained colloidal suspensions display an intense plasmonic absorption band at 520-530 nm and positive zeta potentials greater than +20 mV. The cells viability for in vitro tests performed with human connective tissues and human breast adenocarcinoma (MCF-7) cell lines is over 80% and 90%, respectively, when they are incubated with Au-np-PEI formulations (25 g.mL-1). The present photochemically-assisted synthesis is advantageous because it is fast and does not require for either hazardous or cytotoxic reductant agents and additional purification procedures.

Keywords: gold nanoparticles, photochemically-assisted synthesis, photochemical reduction, biocompatible nanoparticles, branched polyethyleimine

1. Introduction Gold nanoparticles (Au-np) of different sizes and geometrical shapes are nowadays playing a major role in the nanomedicine field. The localized surface plasmons of Au-np interact strongly with visible and near infrared wavelengths depending upon the size and geometrical shape of particles. This feature can be used to tune selectively the radiative (absorption and scattering) or the nonradiative (conversion of light to heat) processes on Au-np and thus, to make feasible the use of Au-np as a theranostic agent for the tasks of diagnosis and therapy [1-3]. The synthesis of Au-np is easily performed by direct reduction of the AuCl4ion from chloroauric acid, HAuCl4, with common reductant chemicals such as citric acid and sodium borohydride to name a few [4,5]. The size and distribution of Au-np depend on the reaction parameters, mainly on the Au to reductant molar ratio, order of reactants addition, use of seeds, temperature, and use of surfactants and other stabilizers. More recently, different strategies have been proposed to produce biocompatible Au-np in a more environmentally friendly way. For example,

biocompatible

and

water-soluble

block

copolymers

such

as

poly(ethylene oxide)–poly(propyleneoxide)–poly(ethylene oxide) (PEO–PPO– PEO) (commercially available as Pluronic) or even gelatine are reductant agents with enough strength to reduce at room temperature AuCl4- ions to form hybrid polymer/Au-np systems [6,7]. These polymers are able to coordinate on metal surfaces and can provide sufficient colloidal stability to nascent nanoparticles.6 Some nitrogen-containing polymers, such as polyacrylates and polyamines exhibit the same ability [8,9]. This synthetic approach is quite simple since the polymeric system acts simultaneously as reductant and stabilizer and, therefore, requires no other reactant. As a drawback, the synthesis performed in this way occurs at quite slow rates that demand for several hours or even days to produce sizeable amounts of Au-np. Another harmless strategy developed for the preparation of Au-np is the photochemical synthesis that uses UV irradiation instead of a chemical reductant. UV light is capable of producing metallic nanoparticles directly by excitation of a metal source or photosensitization [10,11]. The synthesis can be performed in the presence of stabilizers so that very stable colloidal nanoparticles can be prepared

in a single one-pot step [12,13]. In addition, the experimental setup needed for the photochemical synthesis is of low cost and can be implemented to provide a better control of the dynamics of (photo)reduction of metallic precursors, which in summary permits one the careful tailoring of nanoparticles’ properties (size, shape, surface chemistry and so on). Herein, we describe a very clean and straightforward one-pot photochemicallyassisted methodology to obtain at times as short as 30 min., spherical, non-toxic and biocompatible Au-np coated by branched polyethylenimine (PEI), or else Au-np-PEI. Our proposed methodology consists on the direct photoexcitation (254 nm, 16 W) of an aqueous diluted solution of HAuCl 4 containing PEI. Since PEI amino groups reduce Au ions at a very low rate, the reduction of Au ions to produce Au-np is mainly driven by the UV light. Nonetheless, PEI plays its role on the nucleation step of nanoparticles formation so that the size of generated nanoparticles is tuned by the [PEI]/[Au] molar ratio. In addition, PEI amine groups coordinate strongly to the surface of metallic nanoparticles and provide for their colloidal stability [14-16]. Fortunately, PEI is a biocompatible polymer and already tested in nanomedicine devices, for example as a synthetic vector for DNA transfer in gene therapy [17,18]. The electronic structure of Au-np-PEI and the kinetics of the nanoparticles formation were accessed by UV-vis absorption spectroscopy, while the morphology and colloidal properties were investigated by Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS) and zeta potential measurements. In addition, cyclic voltammetry of aqueous diluted solutions of HAuCl4, PEI, and the mixture HAuCl4 + PEI was performed to support the proposition of a mechanism of nanoparticle formation. The non-toxic and biocompatible features of Au-np-PEI were assessed by in vitro cell viability tests using human connective tissue cell and human breast adenocarcinoma cell lines (MCF-7).

2. Materials and Methods

2.1. Materials Gold (III) chloride trihydrate (HAuCl4.3H2O), branched polyethyleneimine (PEI, Mn 60,000 g.mol-1), trisodium citrate dehydrate, gold standard solution for ICP (1000 mg.L-1 Au) and analytical grade ethanol were all purchased from Sigma-

Aldrich Brazil and used as received. All solutions were prepared with ultrapure water (resistivity: 18 Mohm.cm) provided by a Millipore Milli-Q water purification system.

2.2. Photochemically-Assisted Synthesis of Au Nanoparticles All glassware used for the preparation of solutions and photochemically-assisted reactions were thoroughly cleaned with aqua regia solution (HCl:HNO 3, 3:1, v/v) and rinsed with ultrapure water. Afterwards, the glassware were wrapped with PVC plastic film and stored until the experiments were carried out. In a typical run, 5 mL of aqueous HAuCl4.3H2O solution (0.88 mmol.L-1) and different volumes of PEI solution (1.0 g.L-1) were mixed in a 25 mL borosilicate beaker and the volume completed to 10 mL with ultrapure water. The reaction mixture in the beaker was then exposed for a definite period of time to UV irradiation (254 nm, 16 W, Osram) provided by a lab-made reaction chamber (Figure S1, Supporting Information). The Au-np-PEI-X samples were prepared at three different [PEI]/[Au] molar ratios, X = 5, 10, and 20. For the formulation X = 10, the synthesis was also carried out under ambient light and in dark room. We attempted to synthesize a control Au-np sample, in ultrapure water and in the absence of PEI under the same irradiation conditions but this approach was inefficient since no particles could be formed. We then replaced ultrapure water to a mixture of water and analytical grade ethanol (1:1, v/v), which seemed to be more effective. Nonetheless, the colloidal stability of these nanoparticles, named Au-np, were inferior to those stabilized by PEI or citrate. A second control sample of Au nanoparticles, absent of PEI, was prepared by reduction of HAuCl4.3H2O with trisodium citrate dihydrate, named Au-np-Cit. This sample was prepared according to the procedure originally developed by Turkevich et al. [4], which is described in detail in the supporting information along with the respective UV-vis spectrum and microscopic features (Figure S2).

2.3. Structural Characterizations All UV-vis spectra were recorded with an Agilent Cary 8454 UV-Visible spectrophotometer. The kinetics of formation of nanoparticles was investigated for the samples Au-np-PEI-10 prepared under UV irradiation, ambient light, and dark room conditions. In that experiment, a UV-vis spectrum of the reaction

mixture was recorded after different periods of time until absorbance and maximum wavelength stopped to changing. Zeta potential and hydrodynamic diameter (dH) were determined with Malvern Zeta Sizer Nano ZS. The nanoparticles morphology was assessed by TEM images acquired with a JEOL JEM-1011 (JEOL, Japan) microscope. The colloidal samples were diluted in water, then dropped onto 300 mesh Cu-grids pre-coated with Formvar® and left dry in room temperature. Number and size of nanoparticles were assessed by inspecting TEM images with the Image-Pro Plus 5 (Media Cybernetics, Inc., EUA) software. The size distribution fitted by a lognormal function and the average diameter of nanoparticles were determined with the counting of at least 300 particles. The concentration of the colloidal samples evaluated in the biological tests was expressed in terms of the concentration of Au-np (in micrograms of gold per mililiter). It was determined by the difference between the amount of HAuCl4 initially added for the nanoparticle synthesis and the amount of free Au 3+ found by differential pulse voltammetry (DPV) in the filtered of colloidal samples. The DPV analysis was conducted with a Metrohm Autolab PGSTAT 204 potentiostat in a three electrode cell (50 mL) provided with an Ag/AgCl reference electrode, Pt wire as the counter electrode and an ITO slide (1.00 cm2 active area) as the working electrode. Initially, a calibration curve was prepared with determinations performed by DPV with the gold standard solution diluted in KCl 0.1 mol.L -1 as the support electrolyte. The optimized conditions for DPV analysis were as follows: scan range: -0.4 to 1.2 V; scan rate: 0.01 V.s-1; step potential: 0.5 V; purging time with N2: 20 min; waiting time: 5 s. The as-synthesized colloidal suspensions of gold nanoparticles were centrifuged at 10,000 rpm in Amicon ultra-15 centrifugal tubes provided by a 100 kDa cutoff spin filter. The clear filtered was submitted to DPV analysis with successive additions of 150 micro liters of the gold standard solution (0.3 g.L-1). The concentration of free Au3+ was then determined by linear regression of the curves (Figure S3) obtained after DPV analysis. In order to access information to support the proposition of a mechanism of nanoparticle formation cyclic voltammetry of aqueous diluted solutions of HAuCl4, PEI, and the mixture HAuCl4 + PEI at 1:10 molar ratio was conducted. Cyclic voltammograms were recorded at 50 mV.s-1 scan rate in KCl 0.1 mol.L-1

as support electrolyte, previously purged with N2 for 20 min. For this particular experiment, the pH of all solutions was set at 3.6 since this is the value measured in the mixture of HAuCl4/PEI at 1:10 molar ratio.

2.4. Cell Culture Human connective tissue cells were obtained from dental pulp of normal teeth. These cells were maintained in primary culture and called as non-tumor cells control or ''normal cells" [19]. Human breast adenocarcinoma cells (MCF-7) were purchased from American Type Culture Collection, USA. Dulbecco’s Modified Eagle’s Medium and RPMI 1640 medium were purchased from Invitrogen, USA. Fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco, USA. Normal and tumor cells were cultured with DMEM and RPMI 1640 medium, respectively. The mediums were supplemented with 10% (v/v) FBS and 0.5% (v:v) antibiotic solution (100 units/mL penicillin and 100 mg/mL streptomycin). Cells were cultured in a 5% CO2 and 80% of humidified atmosphere at 37 °C.

2.5. Treatments Design with Au nanoparticles Normal and tumor cells were seeded in 96-well plates (3x103 cells/well) using DMEM and RPMI 1640, respectively, and grown in complete medium for 24 hours. Afterwards, the culture medium was removed and cells were incubated with 200 µL of growth mediums with Au nanoparticle samples (Au-np-PEI, Aunp-Cit and Au-np) samples at concentrations of 12.5, 25, 50, 100 and 200 µg of Au/mL. The same treatments were conducted with free PEI and trisodium citrate. The pH of nanoparticle samples after proper dilution ranged between 6.7-7.1. No further pH adjustment was made. The cells were maintained in a humid incubator (80%) at 37 °C with 5% of CO2 for a period of 72 hours. Each treatment was performed in quadruplicate.

2.6. Cell Viability Assays The cells viability was assessed by the colorimetric assay (MTT) using a SpectraMax M2 spectrophotometer (Molecular Devices, USA) at 595 nm. Firstly, the treatment medium was removed and the cells were incubated 150 µL of medium

containing

0.5

mg.mL-1

of

3-(4,5-dimethylthiazol-2-yl)-2,5

diphenyltetrazolium bromide (MTT) dye solution for 3 h at 37 °C in 5% CO 2 and

80% humidity environment. Thereafter, the formazan crystals were dissolved with 150 µL dimethylsulfoxide (DMSO) and the absorbance was measured. The experiments were performed in quadruplicate and repeated three times. The results are represented as the means ± standard deviation. Significant differences were assessed by Anova two-way analyses of variance followed by Bonferroni’s post-tests using the GraphPad Prism 5.0 software (GraphPad Software, Inc, EUA).

3. Results and Discussion The full utilization of nanomaterials such as Au-np in medicine will depend on the availability of synthetic approaches capable of precise controlling of different variables, including size, shape, and surface chemistry of nanoparticles. The latter is by far one of the most important since it affects the biocompatibility and the biodistribution of nanoparticles in the organism. Therefore, the surface chemistry of Au-np may imply on undesirable toxicity or else can lead to the efficacy or inefficacy of their respective nanomedicine applications. Herein, we provide a synthetic route for preparation of Au-np with size control in the range between 8100 nm. Since UV irradiation is used as a reductant and PEI as a stabilizer, no other chemical or additional step is needed to obtain in a very clean fashion biocompatible and non-toxic Au-np-PEI. Figure 1 provides the optical characterization by UV-vis spectroscopy of reaction mixtures and Au-np-PEI colloidal samples. In Figure 1a one sees the spectra of all reaction mixtures and solutions before exposition to UV irradiation. While the spectrum of PEI solution does not show any absorption band, the spectrum of HAuCl4 exhibits a well-defined absorption band peaking at 295 nm, which is ascribed to the ligand to metal charge transfer transition. This band gradually disappears as the [PEI]/[Au] molar ratio increases. For the largest [PEI]/[Au] ratio (20:1), the spectrum displays a subtle absorption at 357 nm, which is ascribed to the ionic pair PEI-H+:AuCl4-. Figure 1b shows the spectra of HAuCl4 + PEI mixtures after being exposed to UV irradiation by 30 min. All spectra share the common feature of colloidal gold nanoparticles, the plasmonic band centered around 520-530 nm. The exact positions of the corresponding peaks are highlighted in the graphic. These spectra resemble the one registered for Au-np-Cit sample (Figure S2a, supporting information). After UV irradiation,

the spectrum of plain PEI solution (not shown) remained equal to that acquired before irradiation. As-prepared, all mixtures are yellow and turn into a reddish wine color after UV irradiation. All these information together confirms the formation of Au-np-PEI colloidal suspensions. The obtained suspensions are highly stable and can stand for months without apparent sedimentation.

Figure 1. UV-vis spectra of reaction mixtures before (a) and after (b) UV irradiation (254 nm, 16 W) by 30 min.

The kinetics of formation of Au-np is relatively fast when HAuCl4 is reduced by citrate under boiling conditions. About 30 minutes are sufficient to lead to the reaction completion [4,5]. Instead, when block copolymers or UV irradiation alone are used as reductants, the process takes several hours to be

accomplished [7,8,10]. Under the present methodology this process is speeded up and can be as short as 10 min. In order to address this point, UV-vis spectra of the reaction mixture HAuCl4 + PEI-10, where the [PEI]/[Au] molar ratio is 10:1, were recorded in different time intervals. The results are shown in Figure 2.

Figure 2. UV-vis spectra acquired at different time intervals of HAuCl 4+PEI-10 reaction mixtures exposed to ambient light (a) and UV irradiation (b).

As it can be noted, in the spectra of the mixture exposed to ambient light (Figure 2a), the plasmonic band ascribed to Au-np is only observable after 5 h of reaction. After this time, the maximum absorbance of such a band is about 0.5 (in arbitrary units) at 542 nm. In dark conditions, the process is even slower and the first signature of Au-np is detected only after 30 h, as seen in Figure S4. When

the reaction is assisted by UV irradiation (Figure 2b), the plasmonic band is already seen in 5 minutes. The maximum absorbance is 2.0 (in arbitrary units) at 528 nm and is reached after 30 min. of reaction. The inserted graphics in Figure 2 show that in both the conditions, ambient light and UV irradiation, the generation of Au-np follows a sigmoid behavior. This behavior is often observed in autocatalytic processes divided into induction (nucleation) and growth steps [20]. It is noteworthy that the UV irradiation shortens the induction step to less than 2 min. (Figure 2b). Despite of that, the UV irradiation alone (in the absence of PEI) could not run the formation of Au-np, as it was already mentioned at the experimental section. Previous contributions have shown that certain types of amines as well as nitrogen-containing polymers are capable of reducing Au ions to produce metallic Au-np provided that their oxidation potential lies between the oxidation of Au(0) to Au(I) and the reduction of Au(III) to Au(0) [8,21]. In order to verify whether the PEI oxidation occurs in this range, cyclic voltammograms of plain PEI and HAuCl4 solutions and the mixture of these substances were recorded at 50 mV.s -1 scan rate. KCl 0.1 mol.L-1 was used as support electrolyte. The pH of all samples was set in 3.6 because this is the pH achieved after mixing PEI and HAuCl4 at 10:1 PEI/Au molar ratio. Also, the potential scanning started at +0.5V towards the cathodic direction. The respective voltammograms are shown in Figure 3.

Figure 3. Cyclic voltammograms of PEI and HAuCl4 plain solutions and HAuCl4+PEI-10 mixture, as indicated. Voltammograms acquired at 50 mV.s -1, in KCl 0.1 mol.L-1, pH 3.6. The voltammogram of plain HAuCl4 solution (in red) displays the oxidation of Au(0) to Au(III) at +0.99 V along with the reduction of Au(III) to Au(0) at +0.55 V and the reduction of Au(I) to Au(0) at –0.27 V. At the same potential range, the voltammogram of PEI (in black) does not show any electrochemical reaction. When HAuCl4 is mixed with PEI, the respective voltammogram (in green) shows a significant decrease of the cathodic peaks, specially the one ascribed to the Au(III) to Au(0) reduction located between 0V and +0.5V. This result suggests that PEI inhibits the reduction of HAuCl4 and it is not surprising since it explains why the formation of Au-np-PEI under ambient light or in the dark is so slow. As an additional point, the pHs of plain HAuCl 4 solution and the HAuCl4 + PEI mixture were 3.6. Since the pKa of amino groups in PEI is 8.8, the PEI chains become positively charged when mixed with HAuCl4. In fact, at pH < 4, all PEI amino groups (primary, secondary and tertiary) will be protonated [22]. Non-bonding electrons of initially neutral amino groups become unavailable for reducing Au ionic species when PEI is positively charged after mixing with HAuCl4. In a previous investigation, Scaravelli et al. [8] have reported that under ambient light Au-np could not be formed by the assistance of amino-functionalized amphiphilic block copolymers when the pH was below the pKa of amino groups. Therefore, it is concluded that in the present experimental conditions PEI is not directly involved on the reduction of Au ionic species, which is thus mainly driven by UV irradiation. While the importance of the UV irradiation for the formation of Au-npPEI is evident, up to now the role played by PEI remains unclear. The Au-np-PEI samples prepared by the photochemically-assisted method are spherical and depending upon the [PEI]/[Au] molar ratio, nanoparticles of different diameters are obtained. TEM images of Au-np-PEI samples are provided in Figure 4 (left panel) along with their diameter distribution (right panel).

Figure 4. TEM micrographs and respective diameter distribution of samples Aunp-PEI-05 (a,b), Au-np-PEI-10 (c,d), and Au-np-PEI-20(e,f).

The Au-np-PEI-05 sample, which was prepared with the lowest [PEI]/[Au] ratio (5:1), is composed by spherical particles along with large platelets of triangular and hexagonal shapes (Figure 4a), which implies a wider size distribution (Figure 4b). These large platelets result from a slow nucleation step as reported elsewhere [23,24]. As the [PEI]/[Au] molar ratio increases, samples become exclusively formed by smaller spherical particles, as shown by micrographs of samples Au-np-PEI-10 (Figure 4c) and Au-np-PEI-20 (Figure 4e). Consequently, these samples exhibit a narrower size distribution, 12.90.4 nm for Au-np-PEI-10 and 8.60.3 nm for Au-np-PEI-20. The high resolution TEM image of sample Au-np-PEI-10 depicted in Figure 5 confirms the presence of PEI coating the Au-np core.

Figure 5. High resolution TEM image of the Au-np-PEI-10 sample. Table 1 collects the average nanoparticles’ diameters estimated from TEM (dTEM) images along with zeta potentials and hydrodynamic diameters (dH). The data for Au-np-Cit sample are also reported. It is observed in the Au-np-PEI samples that zeta potential, dH, and dTEM decrease as the PEI/Au increases. Zeta potentials of all samples are positive likely due to protonation of PEI amino groups by HAuCl4 as discussed earlier. Meanwhile, Au-np-Cit sample exhibits negative zeta potential due to carboxylate groups from the citrate coating. The decrease of the zeta potential as the PEI/Au molar ratio increases simply occurs because the ratio of neutral to protonated amino groups increases. The d H values follow the same trend because neutral PEI chains assume a more compact, coillike conformation, that can contribute for preventing particles aggregation. In

addition, dH values of Au-np-PEI samples are greater than that of Au-np-Cit sample, due to the presence of PEI chains around the gold core, and corroborate the high resolution image provided in Figure 5.

Table 1. Colloidal and morphological parameters of Au-np-PEI samples. Data followed by standard deviation determined after three measurements.

a

Sample

Zeta Potential (mV)

dH (nm)

dTEM (nm)

Au-np-PEI-05

+39.7  3.1

109.3  0.4

90 and 30a

Au-np-PEI-10

+26.0  2.3

55.5  0.5

12.9  0.4

Au-np-PEI-20

+22.1  1.7

29.0  0.5

8.6  0.3

Au-np-Cit

-37.6  2.8

18.3  1.0

15.3  0.3b

According to the size distribution curve in Figure 4b, two size populations are

observed with average diameters of 90 nm and 30 nm. Nonetheless, the size standard deviations are omitted. bDetermined from TEM and distribution curve provided in Figure S2b.

The mixing of PEI with HAuCl4 before exposition to the UV irradiation forms strongly associated ionic pairs, PEI-H+:AuCl4-. This causes a change on the coordination environment of AuCl4- ions, as verified by UV-vis spectroscopy (Figure 1a), and hinders their reduction to zero valent Au, according to cyclic voltammetry (Figure 3). These ionic pairs are in fact the initial sites for the formation of Au-np-PEI. Obviously, as more PEI is added, more PEI-H+:AuCl4pairs are formed. Therefore, PEI enhances the nucleation step while the UV irradiation is responsible for generating the excited species PEI-H+:[AuCl4-]*. The latter undergoes redox and disproportionation reactions to finally form Aunp-PEI. Based on that argumentation and general mechanisms reported in the literature [10,13,25] a tentative of mechanism for the formation of Au-np-PEI is proposed by scheme 1, as follows. In step 1, before the UV irradiation is turned on, PEI-H+:AuCl4- ionic pairs are formed. In step 2, the UV irradiation produces the excited species PEI-H+:[AuCl4-]*, which decays to form a reduced divalent species in step 3. The subsequent steps, 4 to 6, feature disproportionation and reduction of ionic Au species until zero valent Au is formed. The step 7 represents the growth of zero valent Au nuclei into nanometric Au-np-PEI. The

superficial PEI chains coating the Au cores are protonated by H + released at steps 5 and 6, which gives rise to the positive zeta potentials observed in Table 1. PEI + HAuCl4  PEI-H+:AuCl4-

(1)

PEI-H+:AuCl4- + h  PEI-H+:[Au(3+)Cl4-]*

(2)

PEI-H+:[Au(3+)Cl4-]*  PEI-H+:Au(2+)Cl3- + Cl

(3)

2PEI-H+:Au(2+)Cl3-  PEI-H+:Au(1+)Cl2- + PEI-H+:Au(3+)Cl4-

(4)

4PEI-H+:Au(1+)Cl2-  2PEI-H+:Au(2+)Cl3- + 2PEI-Au0 + 2Cl- + 2H+

(5)

3PEI-H+:Au(1+)Cl2-  PEI-H+:Au(3+)Cl4- + 2PEI-Au0 + 2Cl- +2H+

(6)

nPEI-Au0  (PEI-Au)n (or else Au-np-PEI)

(7)

Scheme 1: Proposed mechanism for the formation of Au-np-PEI.

The toxicity of Au-np-PEI samples was assessed by the MTT assay performed in normal and tumor cells. The results are shown in Figure 6 in comparison to those achieved with control samples, Au-np (uncoated nanoparticle) and Au-np-Cit. Concentration of nanoparticles in formulations used for the MTT assay was determined by DPV curves, provided in Figure S3. Both cells exhibit similar viability trends, while showing no statistically significant difference between the three formulations (p>0.05). While Au-np-PEI samples affect both cell lines in a dose-dependent manner, Au-np-Cit affects only the normal cells in this way. No rational explanation is given for that difference, although Au-np-PEI and Au-np-Cit samples exhibit positive and negative zeta potential signals, respectively, which could play a role in this behavior. It is also observed that Aunp-PEI samples are more toxic than Au-np-Cit. Nonetheless, Au-np-PEI samples are much more biocompatible than uncoated Au-np and capable of providing, better than Au-np-Cit, anchoring sites for payloads, such as drugs and imaging probes. In normal human cells, there is a significant decrease in viability at concentrations of 100 and 200 g.mL-1 for the three Au-np-PEI formulations. After treatment with Au-np-PEI-20 at the concentration of 200 g.mL-1, the viability of normal cells is 32.1% ± 4.9 (p <0.001) when compared to control. However, with this same formulation in other concentrations (50, 25 and 12.5 g.mL-1) the viability of normal cells are 63.6% ± 6.2 (p <0.001), 87.3% ± 13 0 (p <0.05) and 105.2% ± 6.7,

respectively (Figure 6a). Additionally, one can note that although small, the gradual increase of PEI favors the maintenance of the cell viability. For example, at the nanoparticle dose of 12.5 g.mL-1, the viability of normal cells increases from approximately 90% (Au-np-PEI-05) to over 100% (Au-np-PEI-20). This effect is even greater in the viability of the MCF-7 lineage when treated with 50 g.mL-1 of Au-npPEI (Figure 6b). In that case, the viability starts at 55.7% ± 6.9, for Au-np-PEI-05, increases to 71.2% ± 6.4 with Au-np-PEI-10, and reaches 79.1% ± 3.4 with Au-np-PEI20 (Figure 6 b). These results suggest that the PEI coating layer plays a favorable role in Au-np-PEI toxicity. The lower tumor cell viability at 100 and 200 g.mL-1 can be assigned to the higher uptake of Au-np-PEI by these cells (p<0.001). This is likely due to the fact that the tumor cells exhibit higher endocytosis activity [26]. Therefore, the PEI coated Au-np are promising for use in biological systems. Since PEI is highly hydrophilic and biocompatible, the coating of Au-np with PEI readily enables one for anchoring payloads of interest and spans the range of biological applications.

Figure 6. Cell viability of human connective tissue (a) and human breast carcinoma cells (MCF-7) (b) incubated with Au-np-PEI, Au-np and Au-np-Cit at different concentrations, as indicated. Maintenance of viability in both treated cells at concentrations of 12.5, 25 and 50 g.mL-1. The bars represent the means ± standard error. * p < 0.05, **p < 0.01, and ***p < 0.001.

4. Conclusions The photochemically-assisted synthesis of gold nanoparticles coated by branched polyethylenimine, or else Au-np-PEI, is capable of producing stable colloidal

suspensions of spherical nanoparticles. While UV irradiation drives the excitation of the metal precursor, HAuCl4, and drastically reduces to few minutes the induction time for the nanoparticles formation, PEI enhances the nucleation step via formation of strongly associated ionic pairs with AuCl 4- ions. The [PEI]/[Au] molar ratio can be varied to tune the size of Au-np-PEI between 100 nm to 8 nm. PEI also plays the role of stabilizing agent avoiding aggregation and improvement of biocompatibility. Since the experimental apparatus employs low power UV lamps, the time expended for the nanoparticles synthesis is short and no additional experimental procedure is demanded, the present methodology is of low-cost and environmentally friendly and potentially promising for preparation of Au-np-PEI for future biomedical applications.

Acknowledgements The authors thank the financial support of Brazilian funding agencies CNPq (process n. 308038/2012-6), FAP-DF (process n. 0193.000829/2015) and CAPES.

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