Synthesis of Well-Defined Gold Nanoparticles Using Pluronic

polymers

Article Synthesis of Well-Deﬁned Gold Nanoparticles Using Pluronic: The Role of Radicals and Surfactants in Nanoparticles Formation

1, 1,2, Marina Sokolsky-Papkov † and Alexander Kabanov * 1 Center for Nanotechnology in Drug Delivery Eshelman School of Pharmacy, UNC-Chapel Hill, Chapel Hill, NC 27514, USA; [email protected] 2 Laboratory of Chemical Design of Bionanomaterials, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119992, Russia * Correspondence: [email protected]; Tel.: +1-919-962-537-3800 The work was partially conducted at the Center for Drug Delivery and Nanomedicine, University of † Nebraska Medical Center, Omaha, USA. Received: 2 July 2019; Accepted: 17 September 2019; Published: 24 September 2019

Abstract: Synthesis of gold nanoparticles (GNP) by reacting chloroauric acid (HAuCl4) and Pluronic F127 was thoroughly investigated. The rate of reduction of HAuCl4 and the yield and morphology of GNP strongly depended on the concentration of the reactants and sodium chloride, as well as pH and temperature. Upon completion of the reaction heterogeneous mixtures of small GNP of defined shape and Pluronic aggregates were formed. GNP were separated from the excess of Pluronic by centrifugal filtration. Under optimized conditions the GNP were small (ca. 80 nm), uniform (PDI ~0.09), strongly negatively charged (ζ-potential 30 mV) and nearly spherical. They were stable in distilled water − and phosphate-buffered saline. Purified GNP contained ~13% by weight of an organic component, yet presence of polypropylene oxide was not detected suggesting that Pluronic was not adsorbed on their surface. Analysis of the soluble products suggested that the copolymer undergoes partial degradation accompanied by cleavage of the C–O bonds and appearance of new primary hydroxyl groups. The reaction involves formation of free radicals and hydroperoxides depends on the oxygen concentration. GNP did not form at 4 ◦C when the micellization of Pluronic was abolished reinforcing the role of the copolymer self-assembly. In conclusion, this work provides insight into the mechanism of HAuCl4 reduction and GNP formation in the presence of Pluronic block copolymers. It is useful for improving the methods of manufacturing uniform and pure GNP that are needed as nanoscale building blocks in nanomedicine applications.

Keywords: gold nanoparticles; Pluronic block copolymers; polymeric micelles

1. Introduction Gold nanoparticles (GNP) have attracted considerable attention due to their potential applications in biology and medicine as drug delivery and sensory devices. Control of size, shape, and colloidal stability of the GNP is very important for these applications. A main synthetic route for GNP involves reduction of chloroauric acid (HAuCl4) using sodium citrate or sodium borohydrate [1]. Although tuning the feed ratio of gold and sodium salts can control the size of GNP to some extent, the resulting GNP usually are not stable in aqueous solution. Their colloidal stabilization can be further achieved by surface functionalization with various SH-containing species and represents an additional synthetic task. As alternative, stable GNP can be synthesized in the presence of certain water-soluble polymers (polyethyleneimine, poly(N-isopropyl-acrylamide-N,N-dimethylaminoethyl-acrylamide [2–5], or polyethers, such as poly(ethylene oxide) (PEO) or Pluronic, a triblock copolymer of PEO and

Polymers 2019, 11, 1553; doi:10.3390/polym11101553 www.mdpi.com/journal/polymers Polymers 2019, 11, 1553 2 of 21 poly(propylene oxide) (PPO) (PEO-PPO-PEO) that can adsorb on the GNP surface and form a protective stabilizing layer in situ [6–10]. Furthermore, the polymer layer around the GNP can be utilized for additional biomedical applications such as incorporation of drugs and utilized as theranostic drug delivery systems. Interestingly, these polymers concurrently act as reducing agents. As true for any synthetic process of practical signiﬁcance, identifying the role of all reaction components is extremely important upon synthesis of GNP.For amine containing polymers the reducing ability of the polymer can be clearly attributed to the amines. However, in the case of polyethers the question is more complicated and despite extensive investigations remains uncertain. Numerous studies characterized interaction of Pluronic block copolymers with metal colloids as well as the role of the block copolymer in HAuCl4 reduction and colloidal particles formation. The correlations between the Pluronic composition and the characteristics of the formed Au nanoparticles were evaluated to tune the size and the shape of the nanoparticles as well as to determine the role of unimers and/or micellar assemblies in the process of the reduction reaction and nanoparticles formation [7,8,10–12]. However, analysis of the previously reported data suggested no straightforward correlation between various Pluronic characteristics, reduction kinetics and GNP formation. For example, increase in polymer concentration was reported to result in the formation of either more heterogeneous [11] or more uniform GNP [9]. Sakai and Alexandridis postulated a three-step reduction and GNP formation process attributing the driving force for nanoparticles formation to block copolymers-Au0 interaction and the nanoparticles growth to reduction of Au3+ on the surface of the nanoparticle’s seeds. Polte et all [13], reported on the GNP formation in the presence of Pluronic F127 showing burst reduction of Au3+ followed by GNP nucleation and growth due to Au0 coalescence with the nuclei. Kohlbrecher et al. [14] showed that GNP formation in the presence Pluronic P85 was dictated by HAuCl4/polymer ratio, however most block copolymer remained unassociated with GNPs. The objective of this work was to: 1) establish a robust and repeatable synthetic procedure route for uniform and well deﬁned GNP; 2) advance understanding of the role of the block copolymers in the reduction reaction; 3) examine interactions between Pluronic and GNP; and 4) delineate environmental factors (pH, NaCl concentration, temperature) governing the particle formation. The ultraviolet–visible (UV–vis) spectroscopy and dynamic light scattering (DLS) were used to examine the rates of HAuCl4 reduction and particle formation. Formation of free radicals and hydroperoxides during the reaction was analyzed by electron paramagnetic resonance (EPR) spectroscopy. The GNP morphology was analyzed by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The chemical composition and structure of the products of the reaction were analyzed by Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), gel permeation chromatography (GPC), and thermo-gravimetric analysis (TGA). Under optimized conditions we produced small, uniform, negatively charged, and nearly spherical GNP that were fully separated from the block copolymer and were stable in aqueous media. The study provides a valuable insight into the mechanism of HAuCl4 reduction and GNP formation and could be useful for improving methods of manufacturing of uniform and pure nanoparticles.

2. Materials and Methods

2.1. Materials

Pluronic F127 ((PEO)200(PPO)65(PEO)200, Mw 12600), Pluronic P104 ((PEO)54(PPO)63(PEO)54, Mw 5900), Pluronic P85 ((PEO)52(PPO)40(PEO)52, Mw 4600) and Pluronic F88 ((PEO)208(PPO)39(PEO)208, Mw 11400), were supplied by BASF corporation. Hydrogen tetrachloroaurate (III) trihydrate (HAuCl 3H O), PEO (Mw 8000), sodium chloride (NaCl), hydrochloric acid (HCl), sodium hydroxide 4· 2 (NaOH), 4-acetamidophenol (AAP), horseradish peroxidase (HRP), and diethylenetriaminepentaacetic acid (DTPA) were purchased from Sigma-Aldrich, St. Louis, MO. 1-Hydroxy-3-methoxycarbonyl- 2,2,5,5-tetramethylpyrrolidine (CMH), EPR buﬀer, diethyldithiocarbamic acid sodium salt (DETC), and deferoxamine (DF) were purchased from Noxygen Science Transfer and Diagnostics, Elzach, Germany Polymers 2019, 11, 1553 3 of 21

All reagents were used as received without farther puriﬁcation. All aqueous solutions were prepared using bi-distilled water (Millipore, Billerica, MA, USA).

2.2. Synthesis of GNP Particles were synthesized as described by Sakai8 with slight modification. Briefly, 1 mL of aqueous stock solution of HAuCl4 was added to 1 mL of Pluronic stock solutions, vortexed for 10 s and incubated at desired temperatures until the reaction was completed (the PR band of the optical spectra attained plateau level). In the typical experiment, 1 mL of 12.6% w/w Pluronic F127 aqueous solution was mixed with 1 mL of 0.5 mM HAuCl4 aqueous solution and the reaction mixture was incubated for 2 h at 45 ◦C. Pluronic F127 solutions with different pH values were prepared by adding 1M NaOH or 1M HCl to stock 12.6% w/w aqueous solution of Pluronic F127. pH of the solutions before and after mixing with of HAuCl4 aqueous solution was measured using Oakton three-point calibration pH meter. Pluronic F127 solutions with various NaCl concentrations were prepared by mixing different volumes of 20% w/w aqueous solution of Pluronic F127 and 5M NaCl aqueous solutions. Pluronic F127 concentration in the final stock solution was 12.6% w/w.

2.3. GNP Purification Particles were purified from excess of the copolymer by centrifugal filtration at 1500 rpm for 30 min at 4 ◦C (repeated three times). The purified nanoparticles were restored to original volume with distilled water. Particles concentration remained the same before and after purification (no GNP were detected in the filtrate).

2.4. UV–Vis Spectroscopy

HAuCl4 reduction and GNP formation in reaction mixtures as well as puriﬁed GNP solutions were analyzed by UV–Vis spectroscopy. The measurements were carried out using PerkinElmer Lambda 25 UV–Vis multi-chamber spectrometer. The instrument was operated at spectrum mode with the wavelength interval of 2 nm, and the samples were held in quartz cuvettes of path length 1 cm. Pluronic F127 aqueous solutions of the same concentration were used as reference.

2.5. Physico-Chemical Analysis

2.5.1. DLS Analysis

Effective hydrodynamic diameter (Deff) and ζ-potential of F127 solutions, reaction mixtures and purified GNP were determined by DLS using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). All measurements were performed in automatic mode at 25 ◦C. Software provided by the manufacturer was used to calculate the size, polydispersity indices (PDI), and ζ-potential. All measurements were performed at least in triplicate to calculate mean values SD. The reaction ± mixtures were analyzed as prepared without further dilutions unless specified otherwise.

2.5.2. TEM Analysis Twenty microliters of a concentrated aqueous solution of the puriﬁed GNP (synthesized under optimal conditions as previously described) were deposited on the top of carbon-coated grid for 1 min, the excess of the solution was removed, and the grid was dried for 2 min at RT. In some samples, negative staining (2% of methylamine tungstate solution) was applied (one drop was deposited on the dried grid for 1 min, removed and the grid was dried at RT for 2 min). TEM imaging was performed using a Tecnai GZ Spirit TW device (FEI, Hillsboro, OR, USA) equipped with an AMT digital imaging system (Danvers, MA, USA) operating at 80 kV. Polymers 2019, 11, 1553 4 of 21

2.5.3. AFM Analysis Five microliters of an aqueous solution of the puriﬁed GNP were deposited onto positively charged aminopropylytriethoxy silane (APS) mica surface for 2 min, washed with water and dried under argon atmosphere. AFM was carried out with MFP-3D microscope (Asylum Research, Santa Barbara, CA, USA) mounted on inverted optical microscope (Olympus, Center Valley, PA, USA) operated in tapping mode. The imaging in air was performed with regular etched silicon probes (TESP) with a spring constant of 42 N/m.

2.5.4. Surface Analysis of GNP and Reaction Products To analyze the reaction products, the particles were synthesized by mixing 1 mL of Pluronic F127 aqueous solution (0.63% w/w) and 1 mL of aqueous HAuCl4 solution (0.5 mM)-1:1 molar ratio. Reaction mixture was incubated at 45 ◦C for 24 h. The nanoparticles were separated from the reaction mixture as described above and the separated ﬁltrate was lyophilized to dryness. To analyze the GNP the particles were synthesized, separated from the reaction mixture and lyophilized to dryness.

2.5.5. FT-IR The dried reaction products or GNP were analyzed in solid state using a Nicolet 380 FT-IR spectrometer (Thermo Scientiﬁc, Waltham, MA, USA). FTIR spectra were recorded in the wavelength 1 range of 4000–400 cm− .

2.5.6. NMR The dried reaction products or GNP were dissolved in D2O. 1H-NMR and 13C-NMR spectra were acquired at 25 ◦C using a Bruker AVANCE III spectrometer (400 MHz, Billerica, MA, USA). Chemical shifts are expressed in parts per million downﬁeld from Me4Si as internal standard.

2.5.7. GPC The number-average molecular mass (Mn), weight-averaged molecular mass (Mw), and molecular mass distribution (Mw/Mn) of Pluronic F127 before and after nanoparticles synthesis were determined by GPC, using Viscotek GPC system equipped with G3000PWXL column and refractive index detector. All measurements were performed at 25 ◦C with a ﬂow rate of 1 ml/min using distilled water containing 0.02% NaN3 as an eluent. The calibration of column was performed using PEO standards (Mw 2–22 kDa, Viscotek, TX, USA).

2.5.8. TGA TGA was carried out on the puriﬁed dried GNP (synthesized under optimal conditions as described above) using a TA Instruments TGA Q500 instrument. After equilibrating the samples at 1 25 ◦C, the temperature was ramped at 10 ◦C min− to a maximum temperature of 650 ◦C under a nitrogen purge. Char yields (the mass remaining at the end of the experiment) were recorded at the maximum temperature.

2.5.9. Free Radical and Hydroperoxide Assays The free radicals and hydroperoxides in the samples were assayed by EPR spectroscopy. These studies used a ‘KDD buffer’ containing EPR Krebse HEPES buffer (99 mM NaCl, 4.69 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 25 mM NaHCO3, 1.03 mM KH2PO4, 5.6 mM D-glucose, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)) and metal chelators, 4.34 mM DETC and 1.85 mM DF. The following samples were studied: 1) 6.3% solutions of F127 (before and after lyophilization); 2) 0.25 mM HAuCl4; 3) reaction mixture containing 1) and 2); 4) unpurified GNP dispersion (2 h, 45 ◦C). To determine free radicals 68.52 µL of sample solutions in bi-distilled water were mixed with 26.6 µL of KDD buffer and 4.88 µL of 4.1 mM CMH spin probe in KDD buffer. To determine Polymers 2019, 11, 1553 5 of 21 hydroperoxides 68.52 µL of sample solutions were mixed with 13.74 µL of 7.28 mM AAP, 7.87 µL of 2.54 mM DTPA, 4.99 µL of 20 U/ml HRP and 4.88 µL of 4.1 mM CMH spin probe (all in KDD buffer) [15]. The final concentrations of the reagents were as follows: 5 mM DETC, 25 mM DF, 1 mM AAP, 0.2 mM DTPA, 1U/mL HRP, and 0.2 mM CMH. Mixtures were incubated for 5 min at room temperature and then 50 µL of these mixtures was loaded into a glass capillary tube and analyzed in Bruker Biospin e-scan EPR spectrometer spectrometer (Bruker). The CMH radical signal was recorded at 37 ◦C and EPR spectrum amplitude was quantified in arbitrary EPR units. The net hydroperoxide signal was calculated as a difference between the EPR spectrum amplitudes in AAP/DTPA/HRP containing and free KDD buffers.

2.5.10. In Vitro Stability of Purified GNP Stability of purified GNP was evaluated in distilled water or phosphate-buffered saline (PBS). Particles were synthesized by mixing 1 mL of F127 aqueous stock solution (12.6% w/w) and 1 mL HAuCl4 aqueous stock solution (0.5 mM) followed by incubation of the mixture for 2 h at 45 ◦C. Reaction mixture was purified from excess of the polymer as described above and the concentrated solution was restored into 2 mL with distilled water or PBS respectively. The solutions were incubated at 37 ◦C and effective hydrodynamic diameter was measured by DLS as described above.

3. Results

3.1. Preliminary Considerations and Choice of a Lead Copolymer GNP were synthesized in the aqueous media through a reaction of HAuCl 3H O with Pluronic 4· 2 block copolymers. Several block copolymers (Supplementary Material Table S1) were evaluated in order to select one, which can: 1) effectively reduce the Au3+ and 2) facilitate the formation of the uniform, stable GNP. These copolymers had different PEO and PPO content, HLB and CMC values. Reactions involving the reduction of Au3+ to Au0 and formation of the GNP were monitored by the changes in the UV absorption spectra (Figure S1). The greatest differences between the copolymer samples were observed in the position and shape of the PR peak (ca. 530–580 nm.). The particles formed in the presence of F127 displayed relatively narrow PR peak centered between 530 and 550 nm. Moreover, only these particles were stable, while for the rest of the copolymers, the particles precipitated in several hours. Hence, we selected F127 as the lead copolymer in this study.

3.2. Effect of the Temperature on Au3+ Reduction and GNP Formation To evaluate the effects of the temperature, F127 was mixed with HAuCl 3H O and incubated at 4· 2 25 ◦C or 45 ◦C. Immediately after mixing the absorbance intensity at 220 nm decreased sharply (from 3 to 1.9) suggesting that the initial phase of Au3+ reduction to Au0 proceeded very rapidly at both temperatures. This rapid phase was followed by subsequent slower Au3+ reduction as evidenced by a decrease of intensity at 220 nm (Figure1). The slower phase was temperature dependent and clearly faster at 45 ◦C than at 25 ◦C. Altogether, at 45 ◦C the initial AuCl4− peak center shifted to 0 240 nm corresponding to Au after 30 min of the incubation, compared to 180 min for 25 ◦C. This was followed by the phase corresponding to the formation of GNP exhibited by a decrease in the absorbance intensity at 240 nm and increase of absorbance intensity in the PR region. This process was also faster at 45 ◦C compared to 25 ◦C. However, despite the differences in the rates of reduction and the particles formation, the final absorbance intensity values both at 240 nm and PR region were the nearly same (Figure1). Following completion of the reaction the aqueous dispersions of the obtained GNP were characterized by DLS. This method confirmed the formation of the nanoparticles, which were slightly negatively charged. Interestingly, when the reaction mixture was diluted by at least two-fold the particle size (here and below particle sizes are reported as effective diameters, Deff), PDI and ζ-potential values sharply decreased (Figure2). Therefore, we purified the GNP from the excess of F127. The purified GNP represent isolated and relatively uniform structures (Figure3A,B). Polymers 2019, 11, 1553 6 of 21

Moreover, TEM analysis of the particles did not show any difference between the particles synthesized at 25 ◦C and 45 ◦C in either size or shape (Supplemental Data Figure S2). In both cases the particles were either close to spherical or have hexagon-like projections. Based on these studies, subsequent synthesis of GNP was carried out at 45 ◦C, unless otherwise specified. Importantly, under the reaction conditions reported above we were able to produce nearly spherical particles of ca 80 nm (by DLS), which were relatively uniform with a PDI as low as 0.09. The hydrodynamic size and PDI values of polymers 2019, 11, x; doi: FOR PEER REVIEW www.mdpi.com/journal/polymers purifiedpolymers particles 2019, 11, x; revealed doi: FOR PEER some REVIEW changes in double distilled water (DDW) www.mdpi.com/journal/polymers and PBS (Supplemental S3).polymers Thus, 2019 the, 11, particlex; doi: FOR size PEER decreased REVIEW over time from about 70 nm to about www.mdpi.com/journal/polymers 60 nm in both DDW and DataS3). Figure Thus, S3). the Thus,particle the size particle decreased size decreasedover time from over about time from 70 nm about to about 70 nm 60 tonm about in both 60 DDW nm in and both PBS.S3). Thus, The PDIthe particle values size increased decreased in PBS over (to time nearly from 0.2),about with 70 nm less to changeabout 60 observed nm in both in DDW.DDW and No DDWPBS. and The PBS. PDI The values PDI values increased increased in PBS in (toPBS nearly (to nearly 0.2), 0.2), with with less less change change observed observed in in DDW. DDW. No No precipitationPBS. The PDI was values observed increased during in the PBS experiment. (to nearly The 0.2), purified with less particles change also observed did not inprecipitate DDW. No in precipitationprecipitation was was observed observed during during the the experiment. experiment. The The purifiedpurified particlesparticles also did did not not precipitate precipitate in in 10%precipitation NaCl (1.72 was M) observed although during they did the exhibit experiment. some changes The purified in effective particles diameters also did (83.49 not precipitate ± 3.94 nm vs. in 10%10% NaCl NaCl (1.72 (1.72 M) M) although although they they did did exhibit exhibit some some changes changes inin eeffectiveffective diametersdiameters (83.49 (83.49 ± 3.943.94 nm nm vs. vs. 67.1810% NaCl ± 0.32 (1.72 nm) M) and although polydispersity they did (PDI exhibit 0.278 some ± 0.012 changes vs. 0.12 in effective± 0.008). diameters (83.49 ± 3.94± nm vs. 67.1867.180.32 ± 0.32 nm) nm) and and polydispersity polydispersity (PDI (PDI 0.278 0.278 ±0.012 0.012 vs.vs. 0.12 ± 0.008).0.008). 67.18± ± 0.32A nm) and polydispersity (PDI 0.278 ± 0.012B vs. 0.12 ± ±0.008). 2.5 B 2.5 A 2.5 2.5 0 min 1min A 0 min 1min B 2 3min 5min 2.52 3min 5min 2.5 0 min 1min 0 min 1min 10min 15min 2 10min 15min 2 0 min3min 1min5min 0 min3min 1min5min 30min10min 4515min min 1.52 30min 45 min 1.52 3min 5min 603min10min min 1205min15min min 6010min30min min 12015min45 min min 30min 45 min 1.5 10min 15min 1.5 30min60 min 45120 min min 1.51 30min60 min 45120 min min 1.51 60 min 120 min 60 min 120 min

1 (a.u) Absorbance 1 Absorbance Absorbance (a.u) 0.51 0.51 Absorbance (a.u) Absorbance Absorbance Absorbance (a.u)

0.5 (a.u) Absorbance 0.5 Absorbance Absorbance (a.u) 0.50 0.50 2000 300 400 500 600 700 2000 300 400 500 600 700 0 200 300Wavelength 400 (nm) 500 600 700 0 200 300Wavelength 400 500(nm) 600 700 200 300Wavelength 400 500(nm) 600 700 200 300 400Wavelength 500 (nm) 600 700 Wavelength (nm) Wavelength (nm) Figure 1. The time evolution of the UV–Vis spectra in the mixtures containing 6.3% w/w F127 and 0.25 Figure 1. The time evolution of the UV–Vis spectra in the mixtures containing 6.3% w/w F127 and Figure 1. The time evolution of the UV–Vis spectra in the mixtures containing 6.3% w/w F127 and 0.25 mMFigure HAuCl 1. The4 duringtime evolution incubation of the at 25UV–Vis°C (A) spectra or 45 °C in ( Bthe). mixtures containing 6.3% w/w F127 and 0.25 0.25mM mM HAuCl HAuCl4 during4 during incubation incubation at 25 at °C 25 (◦AC() orA )45 or °C 45 (B◦C(). B). mM HAuCl4 during incubation at 25 °C (A) or 45 °C (B). Size (reaction mixture) Dilution factor A Size (purified particles) B A Size (reaction mixture) B N.D.Dilution 2 factor 4 200 PDISizeSize (reaction (reaction (purified mixture) mixture) particles) 0.3 Dilution factor A Size (purified particles) B 0 N.D. 2 4 200 PDIPDI (purified (reaction particles) mixture) 0.3 N.D. 2 4 160200 PDIPDI (reaction (purified mixture) particles) 0.3 0 160 0.2 0 120 PDI (purified particles) -10 160 0.2 120 -10 80 0.2 PDI -10 120 0.1 -20 PDI

Deff (nm) 80

PDI -20 4080 0.1 Deff (nm) -20 40 0.1 -30 Deff (nm)

0 0 z-potential (mV) 40 -30 0 N.D. 2 4 0 z-potential (mV) -30

0 0 z-potential (mV) -40 N.D. 2 4 Reaction mixture N.D.Dilution 2 factor 4 -40 Dilution factor -40 Reaction mixture Dilution factor Reaction mixture Figure 2. Changes in the (A) particle effective diameter (columns) and PDI (lines) and (B) ζ-potential FigureFigure 2. Changes 2. Changes in thein the (A ()A particle) particle eﬀ effectiveective diameter diameter (columns) (columns) andand PDIPDI (lines) and and ( (BB)) ζ-potentialζ-potential potentialFigure 2. followingChanges in dilution the (A) ofparticle reaction effective mixture diameter and purification (columns) of and GNPs. PDI (lines) and (B) ζ-potential potentialpotential following following dilution dilution of reactionof reaction mixture mixture and and puriﬁcation purification of of GNPs. GNPs. potential following dilution of reaction mixture and purification of GNPs. A B A B A B

100 nm 100 nm 100 nm

FigureFigure 3. 3.( A(A)) TEM TEM and and ((BB)) AFMAFM images of of purified puriﬁed gold gold nanoparticles. nanoparticles. Figure 3. (A) TEM and (B) AFM images of purified gold nanoparticles. Figure 3. (A) TEM and (B) AFM images of purified gold nanoparticles. 3.3. Effect of HAuCl4 Concentration on the GNP Formation and Characteristics 3.3. Effect of HAuCl4 Concentration on the GNP Formation and Characteristics 3.3. EffectThe above-describedof HAuCl4 Concentration reaction on conditions the GNP Formation resulted andin formation Characteristics of relatively uniform spherical The above-described reaction conditions resulted in formation of relatively uniform spherical nanoparticles.The above-described We further examinedreaction conditions the effect ofresulted HAuCl in4·3H formation2O concentration of relatively on the uniform size, ζ-potential spherical nanoparticles. We further examined the effect of HAuCl4·3H2O concentration on the size, ζ-potential andnanoparticles. shape of We the further unpurified examined aggregates the effect of and HAuCl purified4·3H 2O nanoparticles. concentration Theon the concentration size, ζ-potential of and shape of the unpurified aggregates and purified nanoparticles. The concentration of HAuCland shape4·3H 2O of was the varied unpurified in the range aggregates of 0.05 to and 0.25 purified mM while nanoparticles. keeping F127 Theconcentration concentration constant of HAuCl4·3H2O was varied in the range of 0.05 to 0.25 mM while keeping F127 concentration constant HAuCl4·3H2O was varied in the range of 0.05 to 0.25 mM while keeping F127 concentration constant

Polymers 2019, 11, 1553 7 of 21

3.3. Effect of HAuCl4 Concentration on the GNP Formation and Characteristics The above-described reaction conditions resulted in formation of relatively uniform spherical nanoparticles. We further examined the effect of HAuCl 3H O concentration on the size, ζ-potential 4· 2 and shape of the unpurified aggregates and purified nanoparticles. The concentration of HAuCl 3H O 4· 2 polymerswas varied 2019, 11 in, x; the doi: range FOR PEER of 0.05 REVIEW to 0.25 mM while keeping F127 concentration www.mdpi.com/journal/polymers constant (6.3% w/w). The (6.3%PR peak w/w absorbance). The PR peak increased absorbance linearly increased suggesting linearly that more suggesting nanoparticles that more were nanoparticles formed as HAuCl were4 formedconcentration as HAuCl increased4 concentration (Figure4A,B). increased Thiswas (Figure accompanied 4A and B). by This the increasewas accompanied in the size by and the decrease increase in inthe the PDI size of the and unpurified decrease aggregates in the PDI until of the 0.15 unpurified mM HAuCl aggregates3H O concentration until 0.15 was mM reached. HAuCl4·3H Above2O 4· 2 concentrationthis concentration, was reached. the size andAbove PDI this remained concentration, constant the (approx. size and 200 PDI nm remained and PDI 0.22).constant Interestingly (approx. 200between nm and 0.1 PDI and 0.22). 0.15 mM Interestingly HAuCl 3HbetweenO the 0.1 position and 0.15 of the mM PR HAuCl peak shifted4·3H2O fromthe position 530 nm of to the 540 PR nm 4· 2 peakindicating shifted the from change 530 ofnm the to particle540 nm shape. indicating Analysis the change of the purifiedof the particle particles shape. by TEM Analysis revealed of thatthe purifiedtheir shape particles was indeedby TEM a ffrevealedected (Table that1 their and shape Supplemental was indeed Data affected Figure (Table S4). At 1 lowand HAuClSupplemental3H O 4· 2 Dataconcentrations Figure S4). (0.05–0.1 At low mM) HAuCl the particles4·3H2O concentrations were mostly spherical, (0.05–0.1 small, mM) and the relatively particles uniform were mostly by size spherical,(TEM). As small, the concentration and relatively increased uniform by (0.15–0.2 size (TEM). mM) As consistent the concentration with the change increased of the (0.15–0.2 PR peak mM) the consistenttriangles andwith rods the appeared change of among the PR the peak spherical the triangles particles. and At the rods highest appeared concentration among the (0.25 spherical mM) the particles.rods disappeared At the highest and mostlyconcentration spherical (0.25 particles mM) the with rods some disappeared triangles and were mostly formed spherical again. However,particles within contrast some triangles to the eff ective were formeddiameters again. of the However, aggregates in the contrast effective to the diameters effective of diametersthe purified of GNP the aggregatespractically didthe not effective depend diameters on the HAuCl of the 3H purifiedO concentration GNP practically (Table1 ). did As not already depend noted, on their the 4· 2 HAuClζ-potential4·3H2O was concentration strongly negative (Table compared 1). As already to the noted,ζ-potential their of ζ-potential the aggregates, was strongly which was negative low in comparedthe absolute to the value. ζ-potential The ζ-potential of the aggregates, of the purified which particleswas low in or the aggregates absolute didvalue. not The depend ζ-potential on the ofHAuCl the purified3H O particles concentrations. or aggregates did not depend on the HAuCl4·3H2O concentrations. 4· 2

560 1.5 A 3 0.05mM B 0.1mM 1.2 0.15mM 540 2 0.2mM 0.9 0.25mM 0.6 520 1 (nm) 0.3 PR center (a.u) center PR

PR peak center center peak PR 500 0

Absorbance (a.u) Absorbance 0 0.05 0.1 0.15 0.2 0.25 at absorption Maximal 200 300 400 500 600 700 Wavelength (nm) HAuCl4 concentration (mM) C 0.25 mM Au+3 0.1% D 580 1.6 3 0.5% 1% 2% 3.15% 560 1.2 5% 6.3% 2 7.5% 10% 540 0.8 (nm) 520 0.4 1 PR center (a.u) center PR PR peak center center peak PR Absorbance (a.u) Absorbance 500 0 0 1 2 3.15 5 6.3 7.5 10 at absorption Maximal 200 400 600 Wavelength (nm) F127 concentration (%w/w) E 3 0.25 mM Au+3 pH 2.2 F 560 1.6 pH 4.5 pH 6.6 1.2 2 pH 7.2 pH 9 540 pH 11 0.8 1 520 (nm) 0.4 at PR (a.u) atcenter PR Maximal absorption absorption Maximal Absorbance (a.u) Absorbance

0 peak PR center 500 0 200 300 400 500 600 700 2.2 4.5 6.7 7.5 9 11 Wavelength (nm) pH Figure 4. (A,C,E) Absorbance spectra, and (B,D,F) the position of the PR peak maxima (columns) Figure 4. (A, C, E) Absorbance spectra, and (B, D, F) the position of the PR peak maxima (columns) and the maximal PR absorbance intensity (N) in the mixtures containing HAuCl4 and F127 after 2 h and the maximal PR absorbance intensity () in the mixtures containing HAuCl4 and F127 after 2 h incubation at 45 ◦C. (A,B) HAuCl4 concentration is constant (0.25 mM) and F127 concentration varied incubation at 45 °C. (A, B) HAuCl4 concentration is constant (0.25 mM) and F127 concentration varied from 0.1 to 10 % w/w. The pH of the F127 solution before HAuCl4 addition was pH 6.5. In all cases from 0.1 to 10 % w/w. The pH of the F127 solution before HAuCl4 addition was pH 6.5. In all cases reaction is complete based on the absence of a peak at 220 nm or shift of the pick to 240 nm for 0.25 mM. reaction is complete based on the absence of a peak at 220 nm or shift of the pick to 240 nm for 0.25 (C,D) F127 concentration is constant (6.3% w/w), HAuCl4 concentration is varied from 0.05 to 0.25 mM. mM. (C, D) F127 concentration is constant (6.3% w/w), HAuCl4 concentration is varied from 0.05 to The pH of the F127 solution before HAuCl4 addition was pH 6.5. (E,F) 0.25 mM HAuCl4 and 6.3% w/w 0.25 mM. The pH of the F127 solution before HAuCl4 addition was pH 6.5. (E, F) 0.25 mM HAuCl4 F127 mixture was adjusted to various pH. and 6.3% w/w F127 mixture was adjusted to various pH.

Polymers 2019, 11, 1553 8 of 21

1 Table 1. Eﬀect of HAuCl4 concentration on the characteristics of the GNP .

HAuCl4 DLS (Reaction Mixture) DLS (Purified Particles) Concentration Morphology by TEM ζ ζ (mM) Deff PDI -Potential Deff PDI -Potential (nm) (mV) (nm) (mV) 0.05 50 1 0.53 0.02 3.76 0.11 77 16 0.18 0.04 30.23 0.05 Multi-sized spheres ± ± − ± ± ± − ± 0.10 122 1 0.34 0.001 3.52 0.03 62 1 0.25 0.01 28.13 1.29 Multi-sized spheres ± ± − ± ± ± − ± 0.15 216 1 0.23 0.01 3.18 0.01 79 1 0.19 0.01 28.70 3.41 Triangles, rods, spheres ± ± − ± ± ± − ± 0.20 231 1 0.22 0.02 2.45 0.02 87 1 0.14 0.03 27.47 0.06 Triangles, plates, spheres ± ± − ± ± ± − ± 0.25 229 2 0.23 0.01 3.01 0.02 72 1 0.12 0.02 37.73 0.25 Spheres, triangles ± ± − ± ± ± − ± 1 Particles were synthesized by mixing equal volumes of F127 solution (final concentration 6.3 % w/w, pH 6.5) and HAuCl4 solutions of different concentrations, followed by incubation of the mixture for 2 h at 55 ◦C. Particles were purified from excess of the polymer by centrifugal filtration at 1500 rpm for 30 min at 4 ◦C (repeated three times). The purified nanoparticles were restored to original volume with distilled water (particles concentration remained the same before and after purification).

3.4. Effect of F127 Concentration on the GNP Formation and Characteristics Next, we varied the concentration of F127 while keeping the HAuCl 3H O concentration constant 4· 2 (0.25 mM). The absorbance at the PR peak increased and the position of the PR peak shifted to 530 to 550 nm. as the F127 concentration increased (Figure4C,D). The e ffective diameter of unpurified aggregates also increased from about 100 nm to about 300 (Table2). However, once the GNP were purified their effective diameters did not depend on the copolymer concentration and ranged from 60 to 80 nm while PDI values gradually decreased from about 0.3 to 0.09. Notably, the ζ-potential of the purified particles was strongly negative ( 30 to 45 mV) and much lower than of any unpurified − − particles ( 11 to 2.5 mV). TEM analysis revealed some alterations in the shape and heterogeneity of − − the particles (Table2, and Supplemental data Figure S5). At lower concentrations of the copolymer (1–3.15 wt %) the purified particles were heterogeneous by both size and shape, representing a mixture of smaller and larger spheres, triangles and rods. As the concentration of F127 increased, the rods disappeared, followed by disappearance of triangles; at 7.5% and 10% F127 relatively homogeneous by size and shape and nearly spherical or cubic particles were formed.

Table 2. Eﬀect of F127 concentration on the characteristics of the GNP 1.

F127 DLS (Reaction Mixture) DLS (Purified Particles) Concentration Morphology by TEM ζ ζ (% w/w) Deff PDI -Potential Deff PDI -Potential (nm) (mV) (nm) (mV) 1.0 121 1 0.28 0.01 12.13 0.30 80 1 0.29 0.01 25.57 0.40 Multi-sized spheres, rods, ± ± − ± ± ± − ± hexagons 2.0 91 1 0.43 0.01 6.45 0.30 54 1 0.42 0.03 30.20 0.66 Multi-sized spheres, rods, ± ± − ± ± ± − ± hexagons, triangles 3.15 101 2 0.28 0.01 5.52 0.02 62 1 0.29 0.003 28.20 0.20 Spheres, triangles, rods ± ± − ± ± ± − ± 5.0 133 2 0.22 0.01 4.04 0.20 63 1 0.19 0.02 32.23 1.85 Spheres, triangles ± ± − ± ± ± − ± 6.3 153 12 0.23 0.01 3.84 0.06 58 1 0.17 0.01 36.50 0.38 Spheres, triangles ± ± − ± ± ± − ± 7.5 227 6 0.17 0.02 3.76 0.20 75 1 0.15 0.01 41.80 0.56 Spheres ± ± − ± ± ± − ± 10.0 305 3 0.11 0.02 2.83 0.30 86 1 0.09 0.01 45.30 0.15 Uniform spheres ± ± − ± ± ± − ± 1 Particles were synthesized by mixing equal volumes of F127 and HAuCl4 solutions (final concentrations 1–10% w/w, pH 6.5, and 0.25 mM respectively) followed by incubation of the mixture for 2 h at 55 ◦C. Particles were purified as described in Table1. The purified nanoparticles were restored to original volume with distilled water (particles concentration remained the same before and after purification).

3.5. Relationship between Polymer Aggregation and GNP Formation Notably, the concentrations of the copolymer in this study were above the critical micelle concentration (CMC) at the given temperatures of the experiment [16]. Under these conditions the copolymer exists in solution in two forms: 1) the micelle aggregates and 2) the single chains (unimers) that are in equilibrium with the micelles [16]. To elucidate the role of the self-assembly of F127 in GNP formation we carried out same reactions but at 4 ◦C when the micellization of F127 is abolished at every concentration used [16]. At 1–2% F127 there was little reduction of Au3+ during the observation time of 2 h (Figure5). However, the reduced Au 0 formed some GNP as exhibited by a well-deﬁned albeit polymers 2019, 11, x; doi: FOR PEER REVIEW www.mdpi.com/journal/polymers 3.5. Relationship between Polymer Aggregation and GNP Formation Notably, the concentrations of the copolymer in this study were above the critical micelle concentration (CMC) at the given temperatures of the experiment [16]. Under these conditions the copolymer exists in solution in two forms: 1) the micelle aggregates and 2) the single chains (unimers) that are in equilibrium with the micelles [16]. To elucidate the role of the self-assembly of F127 in GNP formation we carried out same reactions but at 4 °C when the micellization of F127 is abolished Polymersat every2019 concentration, 11, 1553 used [16]. At 1–2% F127 there was little reduction of Au3+ during9 of the 21 observation time of 2 h (Figure 5). However, the reduced Au0 formed some GNP as exhibited by a well-defined albeit small PR peaks. At higher F127 concentrations the reduction of Au3+ was nearly small PR peaks. At higher F127 concentrations the reduction of Au3+ was nearly complete judging by complete judging by the shift of the absorption peak to 240 nm. However, the PR peaks were much the shift of the absorption peak to 240 nm. However, the PR peaks were much smaller and less deﬁned smaller and less defined compared to the PR peaks observed at the same F127 concentrations at compared to the PR peaks observed at the same F127 concentrations at higher temperatures (25 ◦C and higher temperatures (25 °C and 45 °C), when the copolymer formed micelles. Another evidence for 45 ◦C), when the copolymer formed micelles. Another evidence for the role of the micelles was seen

the role of the micelles was seen using non-micelle-forming 10% PEO 8000 as3+ reducing agent at 45 using non-micelle-forming 10% PEO 8000 as reducing agent at 45 ◦C. Albeit Au reduction appeared °C. Albeit Au3+ reduction appeared to be slower compared to the F127 systems, the GNP still formed. to be slower compared to the F127 systems, the GNP still formed. However, these GNP were highly However, these GNP were highly heterogeneous representing assembly of sizes and shapes in heterogeneous representing assembly of sizes and shapes in contrast to the nearly spherical particles contrast to the nearly spherical particles observed at similar concentrations of F127 (Supplemental observed at similar concentrations of F127 (Supplemental data Figure S6). This suggests that the F127 data Figure S6). This suggests that the F127 micelles play some role in the GNP formation, perhaps micelles play some role in the GNP formation, perhaps serving as the templates for nucleation and/or serving as the templates for nucleation and/or growth of Au0 atoms favoring formation of the growth of Au0 atoms favoring formation of the spherical particles. Interestingly, we obtained evidence spherical particles. Interestingly, we obtained evidence suggesting that the size of the particles and suggesting that the size of the particles and the self-assembly of the copolymer change in the course of the self-assembly of the copolymer change in the course of the reaction. First, the DLS suggested that the reaction. First, the DLS suggested that addition of HAuCl4 3H2O (0.25 mM) to F127 solution (5 mM, addition of HAuCl4·3H2O (0.25 mM) to F127 solution (5 mM,· 6.3%) resulted in the immediate increase 6.3%) resulted in the immediate increase of the particle size from 27 nm corresponding to F127 micelles of the particle size from 27 nm corresponding to F127 micelles to 102 nm. Subsequently, the particle to 102 nm. Subsequently, the particle size continued to increase in the course of the reaction to 160 nm size continued to increase in the course of the reaction to 160 nm (Supplemental Data Figure S7). (Supplemental Data Figure S7). Moreover, when the HAuCl4 3H2O solution was carefully added as a Moreover, when the HAuCl4·3H2O solution was carefully added· as a layer on top of the previously layer on top of the previously formed concentrated F127 gel (30 wt%, room temperature), the reaction formed concentrated F127 gel (30 wt%, room temperature), the reaction proceeded at the interface proceeded at the interface with the GNP forming in the mixed layer, which spread frontally through the with the GNP forming in the mixed layer, which spread frontally through the entire gel volume. entire gel volume. Notably in course of this reaction the gel structure of F127 was disrupted ultimately Notably in course of this reaction the gel structure of F127 was disrupted ultimately producing the producing the homogeneous liquid dispersion containing GNP. The color of the formed GNP solution homogeneous liquid dispersion containing GNP. The color of the formed GNP solution was purple was purple suggesting formation of large, irregular particles. This observation is very interesting since suggesting formation of large, irregular particles. This observation is very interesting since it shows it shows that the HAuCl4 3H2O is reduced with copolymer present in the upper layer of the gel and that the HAuCl4·3H2O is· reduced with copolymer present in the upper layer of the gel and the the reaction disrupts the interaction between the copolymer chains and destroys the gel matrix. reaction disrupts the interaction between the copolymer chains and destroys the gel matrix.

0.25 mM Au+3 0.45 3 1% 2% 0.3 5% 7.50% 10% 0.15 2 15%

Absorbance (a.u) Absorbance 0 450 550 650 1 Wavelength (nm) Absorbance (a.u) Absorbance

0 200 300 400 500 600 700 Wavelength (nm) Figure 5. UV–Vis absorbance spectra and (insert) absorbance in PR region in the mixtures of 0.25 mM Figure 5. UV–Vis absorbance spectra and (insert) absorbance in PR region in the mixtures of 0.25 mM HAuCl4 and F127 as a function of the copolymer concentration 2 h after incubation at 4 ◦C. Copolymer HAuCl4 and F127 as a function of the copolymer concentration 2 h after incubation at 4 °C. Copolymer concentration varied from 1–15% w/w. concentration varied from 1–15% w/w. 3.6. Eﬀect of pH on the GNP Formation and Characteristics

The size and morphology of GNP depended on the pH of the F127 initial solution. Table3 presents the results of the GNP synthesis for diﬀerent pH values before and after mixing the F127 and HAuCl 3H O solutions. 4· 2 When the reaction was carried out at acidic conditions (initial pH 2.2 to 4.5) the formed GNP particles were heterogeneous by size and shape as seen by TEM analysis of the puriﬁed particles. Moreover, at the lowest pH 2.2 the reduction reaction was considerably decelerated and required 24 h for completion based on the UV spectra analysis (data not shown). On the contrary, as the pH of the initial F127 solution increased and approximated neutral (pH 6.7 to 7.5) and then became alkali (pH 9.0 to 11.5) the particles became more homogeneous by size and shape as determined by TEM. Furthermore, the particle size considerably decreased as suggested by both DLS and TEM analyses Polymers 2019, 11, 1553 10 of 21

(Table3, Supplemental data Figure S8). It is well known that the TEM particle size is usually less than the effective diameters measured by DLS, which is commonly explained by the hydration and swelling of the particle polymer coatings during the DLS studies [17]. However, the differences between the TEM and DLS diameters in the present case were too strong especially for the purified GNP produced using alkali solutions of the copolymer—e.g., ca. 12 nm by TEM vs. 40–50 nm by DLS. Most likely these differences were due to aggregation of the GNP in aqueous solution during the DLS study. Notably, as determined by DLS the PDI indexes of the purified GNP synthesized using alkali solutions of the copolymer were relatively high while the TEM study suggested that the particles were rather homogeneous by size and shape. Such discrepancy in polydispersity assessments by DLS and TEM are consistent with the particle aggregation in solution. At the same time this aggregation appeared to be restricted as the particle effective diameters measured by DLS remained relatively small. Moreover, for the GNP synthesized using alkali solutions of the copolymer, no significant difference in the size of the purified and non-purified particles was observed and these particles remained stable in dispersion showing no aggregation or precipitation for several weeks. In contrast, the particles formed using acidic solutions of the copolymer exhibited significant differences in size before and after purification and had a tendency to precipitate during first 24 h after their synthesis. Interestingly, the ζ-potentials of both purified and unpurified particles were nearly the same regardless the initial pH used in the synthesis (Table3).

Table 3. Eﬀect of pH of F127 solution on the GNP characteristics 1.

pH of the DLS DLS TEM F127 (Reaction Mixture) (Purified Particles) (Purified Particles) Solution a D ζ-Potential D ζ-Potential D eff PDI eff PDI eff Morpho-Logy (nm) (mV) (nm) (mV) (nm) Spheres, rods, 2.2 (1.24) 2 375 3 0.11 0.3 2.7 0.05 114 1 0.17 0.02 25.8 3.13 n.d. ± ± − ± ± ± − ± triangles 3.07 4.5 (1.44) 2 219 3 0.20 0.24 − ± 85 1 0.16 0.01 32.53 0.58 77 6 Spheres ± ± 0.13 ± ± − ± ± 3.98 6.7 (3.14) 3 160 2 0.19 0.04 − ± 72 1 0.12 0.02 37.70 0.25 54 13 Spheres ± ± 0.18 ± ± − ± ± 4.50 7.5 (5.5) 4 115 3 0.36 0.04 − ± 57 1 0.26 0.01 33.77 0.66 23 11 Spheres ± ± 0.66 ± ± − ± ± 4.45 9.0 (6.35) 4 56 1 0.56 0.01 − ± 53 1 0.36 0.02 35.40 1.37 12 6 Spheres ± ± 0.21 ± ± − ± ± 5.20 11.5 (7.76) 4 65 4 0.44 0.02 − ± 41 1 0.36 0.03 32.06 1.45 12 4 Spheres ± ± 0.62 ± ± − ± ±

Particles were synthesized by mixing equal volumes of F127 and HAuCl4 solutions (ﬁnal concentrations 6.3% w/w 1 and 0.25 mM, respectively) followed by incubation of the mixture for 2 h at 45 ◦C. The pH values of the initial 12.6% Pluronic solution and ﬁnal reaction mixture at 2 h (in the brackets) are presented. The pH in the Pluronic 2 3 4 solution (pH 6.7) was adjusted with 1N HCl, not adjusted, 1N NaOH. After mixing this solution with HAuCl4 the pH value rapidly (mins) decreased and then did not change in the course of the reaction. n.d.—not determined.

3.7. Effect of NaCl on the GNP Formation Addition of NaCl to the reaction medium profoundly affected the formation of GNP. However, the results depended on the temperature. Thus, the analysis of the UV spectra of the reaction mixture 3+ incubated at 25 ◦C revealed that Au was reduced in the entire range of NaCl concentrations (0.005 M to 1.25 M). However, below 0.05 M NaCl this was accompanied both by the appearance of the Au0 absorbance at 240 nm (Figure6A) and the PR of the GNP (Figure6B). In contrast, at and above 0.001 M NaCl a peak at 320 nm corresponding to Au0 clusters was observed while the PR peak was attenuated. In this case, no PR of GNP was observed even after 24 h of incubation of the reaction mixture at 45 ◦C suggesting that the GNP formation was completely inhibited. Au3+ was reduced but Au0 absorbance at 240 nm was low compared to the reaction at 45 ◦C in the salt-free system (Supplemental Data Figure S9A). The GNPs were formed at NaCl concentrations below 0.05 M. Like in the case of the lower temperature, at 45 ◦C the formation of GNP was nearly completely abolished at higher NaCl concentrations (Figure6B). At all temperatures, when the GNP were formed in the presence of the salt the PR peak was considerably broader and shifted towards higher wavelengths than that in the absence polymers 2019, 11, x; doi: FOR PEER REVIEW www.mdpi.com/journal/polymers M to 1.25 M). However, below 0.05 M NaCl this was accompanied both by the appearance of the Au0 absorbance at 240 nm (Figure 6A) and the PR of the GNP (Figure 5B). In contrast, at and above 0.001 M NaCl a peak at 320 nm corresponding to Au0 clusters was observed while the PR peak was attenuated. In this case, no PR of GNP was observed even after 24 h of incubation of the reaction mixture at 45 °C suggesting that the GNP formation was completely inhibited. Au3+ was reduced but Au0 absorbance at 240 nm was low compared to the reaction at 45 °C in the salt-free system (Supplemental Data Figure S9A). The GNPs were formed at NaCl concentrations below 0.05 M. Like Polymers 2019, 11, 1553 11 of 21 in the case of the lower temperature, at 45 °C the formation of GNP was nearly completely abolished at higher NaCl concentrations (Figure 6B). At all temperatures, when the GNP were formed in the ofpresence the salt. of Consistent the salt the with PR thepeak shape was ofconsiderably the PR peak broader in the presence and shifted of the towards salt these higher GNP wavelengths represented athan heterogeneous that in the absence mixture of of the larger salt. and Consistent smaller particleswith theof shape different of the polygonal PR peak shapesin the presence (Supplemental of the Datasalt these Figure GNP S9B) represented compared to a heterogeneousmuch more homogeneous mixture of largerand closer and to smaller spherical particles particles of different formed withoutpolygonal added shapes NaCl (Supplemental (Supplemental Data data Figure Figure S9B) S5D). compared to much more homogeneous and closer to spherical particles formed without added NaCl (Supplemental data Figure S5D).

A 2

1.5

0.5 240 nm (a.u) nm 240

Absorbance intensity at intensity Absorbance 0 5 1 5 .01 0.1 0.2 0.3 0.4 0. .005 0 0.04 0.05 1.2 0 NaCl concentration (M)

B 1.2 25C, 6h 25C, 24h 0.8 45C, 6h

0.4 PR (a.u) PR

0 Absorbance intensity at intensity Absorbance 1 5 1 4 1 5 05 .0 .04 0 0. 0.2 0.3 0. 0.5 .2 0 0 0 0. 1 0. NaCl concentration (M) FigureFigure 6. (A) Absorbance intensity at 240 nm and (B) the maximal absorbance intensityintensity ofof thethe PR peak inin thethe mixturesmixtures ofof 0.250.25 mMmM HAuClHAuCl44 and 6.3% w/w F127 as a functionfunction ofof thethe NaClNaCl concentrationconcentration afterafter incubationincubation ofof thethe reactionreaction mixturemixture atat 2525 ◦°CC or 45 °C◦C at 6 h and 24 h.

3.8. Chemical Conversion of the Block Copolymer During the Reaction 3.8. Chemical Conversion of the Block Copolymer During the Reaction Soluble reaction products and GNP were separated by centrifugal filtration, lyophilized, and Soluble reaction products and GNP were separated by centrifugal filtration, lyophilized, and analyzed by NMR (Figure7A,B) and IR (Figure7C). When the reaction was carried out at considerable analyzed by NMR (Figure 7A and B) and IR (Figure 7C). When the reaction was carried out at excess of F127 both the IR and NMR spectra of the separated polymer were nearly identical to the considerable excess of F127 both the IR and NMR spectra of the separated polymer were nearly initial F127, albeit the spectra of the purified GNP were markedly different. However, when F127 identical to the initial F127, albeit the spectra of the purified GNP were markedly different. However, and HAuCl4 3H2O were mixed in 1:1 molar ratio (0.25 mM) the spectra of the soluble products were when F127 and· HAuCl4·3H2O were mixed in 1:1 molar ratio (0.25 mM) the spectra of the soluble drastically. This suggested that some small amount of F127 was undergoing chemical conversion during products were drastically. This suggested that some small amount of F127 was undergoing chemical the reaction. 1H-NMR (Figure6A) and 13C-NMR (Figure7B) revealed considerable di fferences between conversion during the reaction. 1H-NMR (Figure 6A) and 13C-NMR (Figure 7B) revealed considerable thedifferences soluble reactionbetween products the soluble and reaction the nanoparticles products onand the the one nanoparticles hand and the on initial the one F127 hand on the and other. the 1 Specifically, the H-NMR revealed that the O–CH2 peak at 3.75 ppm was attenuated while several new initial F127 on the other. Specifically, the 1H-NMR revealed that the O–CH2 peak at 3.75 ppm was peaks in the 3.6–3.9 ppm range were observed in both soluble products and nanoparticles. However, while the soluble products displayed the signal of the CH3 group of PPO at 1.18 ppm this signal was slightly shifted in the nanoparticles (1.17 ppm) and there was a new peak at 1.30 ppm that was not seen in neither the initial F127 nor soluble products. The differences were even more pronounced in the 13C-NMR spectra. Both the soluble products and the nanoparticles displayed new peak at 62 (not observed in initial F127), which can be related to C–OH carbon atoms. However, the nanoparticle spectrum was lacking the peak at 14 ppm (CH3) as well as at 73 (O–CCH3) of PPO, suggesting that polymers 2019, 11, x; doi: FOR PEER REVIEW www.mdpi.com/journal/polymers attenuated while several new peaks in the 3.6–3.9 ppm range were observed in both soluble products and nanoparticles. However, while the soluble products displayed the signal of the CH3 group of PPO at 1.18 ppm this signal was slightly shifted in the nanoparticles (1.17 ppm) and there was a new peak at 1.30 ppm that was not seen in neither the initial F127 nor soluble products. The differences were even more pronounced in the 13C-NMR spectra. Both the soluble products and the nanoparticles Polymers 2019, 11, 1553 12 of 21 displayed new peak at 62 (not observed in initial F127), which can be related to C–OH carbon atoms. However, the nanoparticle spectrum was lacking the peak at 14 ppm (CH3) as well as at 73 (O–CCH3) PPOof PPO, was suggesting not present that in GNP. PPO Detailed was not analysispresent in of GNP. IR spectra Detailed confirms analysis these of observationsIR spectra confirms (Figure 7theseC). 1 Specifically,observations in (Figure the purified 7C). Specifically, GNP three in new the purified IR peaks GNP appeared three new at 3300, IR peaks 1030, appeared and 920 at cm 3300,− along 1030, −1 1 −1 withand the920 decrease cm along of thewith peaks the decrease in the 1000–1500 of the peaks cm− inrange. the 1000–1500 This combination cm range. of This IR spectra combination changes of suggestsIR spectra that changes C–O bond suggests of F127 that was C–O partially bond of cleaved F127 was and partially the OH cleaved groups wereand the formed OH groups during were the reaction.formed during Notably the the reaction. IR spectra Notably did not the support IR spectra the formation did not support of the carboxylic the formation groups: of the even carboxylic though 1 −1 somegroups: samples even exhibitedthough some a minor samples peak atexhibited 1645 cm −a minorwhen thesepeak at samples 1645 cm were when carefully these dried samples thispeak were 1 −1 disappearedcarefully dried completely this peak while disappeared the peak atcompletely 3300 cm− whileremained the peak unchanged. at 3300 Itcm was remained previously unchanged. reported thatIt was such previously behavior canreported be explained that such by behavior the presence can ofbe waterexplained in the by copolymer the presence [18]. of Furthermore, water in the 1 −1 thecopolymer CH3 bends [18]. peak Furthermore, at 1337 cm −thewas CH observed3 bends peak in the at initial1337 cm F127 was as well observed as in soluble in the productsinitial F127 but as notwell in as the in purified soluble GNP. products The degradationbut not in the of thepurified F127 chainGNP. inThe the degradation process of the of reactionthe F127 was chain directly in the demonstratedprocess of the by reaction GPC (Supplemental was directly Datademonstrated Figure S10). by Before GPC (Supplemental the reaction one Data major Figure peak wasS10). seen Before in thethe initial reaction 0.5 mMone major F127 sample peak was with seen the in retention the initial time 0.5 ofmM 7.03 F127 min, sample which with corresponded the retention to Mw time= of9600 7.03 Da.min, The which lower corresponded value of the molecular to Mw = 9600 mass Da. compared The lower to the value listed of forthe F127molecular copolymer mass (Mwcompared 12,600 to Da), the waslisted probably for F127 due copolymer to compaction (Mw of12,600 the PPODa), chainswas probably of F127. due There to wascompaction also a peak of the at 7.59 PPO min chains (3500 of Da),F127. probably There was corresponding also a peak to at a 7.59 diblock min copolymer (3500 Da), PEO-PPOprobably andcorresponding/or PPO homopolymer to a diblock admixtures. copolymer AfterPEO-PPO the reaction, and/or thePPO main homopolymer polymer fraction admixtures. (7 min) After was dramaticallythe reaction, decreased.the main polymer New peaks fraction were (7 observedmin) was at dramatically 10 min (major), decreased. and 12 min New (minor), peaks whichwere observed were below at 10 the min effective (major), separation and 12 rangemin (minor), of the GPCwhich column, were below suggesting the effective that their separation Mw values range were of belowthe GPC 700 column, Da. Analysis suggesting of the that RI areastheir Mw under values the curvewere suggestedbelow 700 that Da. under Analysis the optimizedof the RI conditionsareas under used the tocurve produce suggested GNPs 55%that of under the F127 the chainsoptimized are degraded.conditions Finally, used to although produce the GNPs GNP 55% did of not the reveal F127 presence chains are of intactdegraded. F127, Finally, they definitely although contained the GNP somedid not organic reveal component(s). presence of intact This F127, was they confirmed definitely by contained TGA analysis some demonstrating organic component(s). that the This portion was ofconfirmed this component by TGA was analysis approximately demonstrating 13% w /thatw and the it portion consisted of ofthis two component compounds was with approximately the boiling temperatures13% w/w and of it 242 consisted◦C and 348of two◦C (Supplementalcompounds with Data the Figure boiling S11). temperatures of 242 °C and 348 °C (Supplemental Data Figure S11).

Figure 7. Cont. Polymers 2019, 11, 1553 13 of 21 polymers 2019, 11, x; doi: FOR PEER REVIEW www.mdpi.com/journal/polymers

Figure 7. (A) 1H-NMR, (B) 13C-NMR, and (C) the IR spectra of (A1, B1, C1) original F127, (A2, B2, C2) Figureseparated 7. (A reaction) 1H-NMR, mixture (B) 13 andC-NMR, (A3, B3,and C3) (C) the the purified IR spectra gold of nanoparticles.(A1, B1, C1) original F127, (A2, B2, C2) separated reaction mixture and (A3, B3, C3) the purified gold nanoparticles. 3.9. Batch to Batch Variation in the Formation of GNP 3.9. Batch to Batch variation in the Formation of GNP During these studies we tested several different stock solutions of F127 (marked as F127_1, F127_2During and F127_3).these studies The polymers we tested inseveral these solutionsdifferent stock had essentially solutions theof F127 same (marked chemical as structure F127_1, F127_2and molecular and F127_3). weight The as polymers confirmed in by these IR, NMR,solutions and had GPC essentially of lyophilized the same aliquots. chemical It is structure important and to molecularnote that duringweight lyophilizationas confirmed by volatile IR, NMR, molar and mass GPC degradation of lyophilized products, aliquots. which It is important might have to beennote thatpresent during in the lyophilization solution, were volatile lost and molar could mass not degradation be detected products, by any of which the applied might methods. have been However, present inone the di solution,fference betweenwere lost these and batchescould not was be indetected the extents by any of polymerof the applied oxidation methods. (Supplemental However, dataone differenceTable S2). Specifically,between these the batches hydroperoxides was in the level extents in F127_3 of polymer was 2–3 oxidation times higher (Supplemental than these data in F127_1 Table S2).and Specifically, F127_2 (Supplemental the hydroperoxides data Table S2).level The in F127_3 free radical was levels2–3 times were higher comparable than these in all threein F127_1 solutions. and F127_2Comparison (Supplemental of these batchesdata Table discovered S2). The considerablefree radical levels batch-to-batch were comparable variation in inall the three formation solutions. of ComparisonGNP. Thus, reacting of these HAuCl batches3H discoveredO with F127_1 considerable resulted batch-to-batch in the GNP with variation UV characteristics in the formation and sizes of 4· 2 GNP.similar Thus, to these reacting of the HAuCl GNP4 described·3H2O with above. F127_1 However, resulted in reacting the GNP HAuCl with UV4 with characteristics F127_2 resulted and sizes in a similarrelatively to broaderthese of PRthe peakGNP centereddescribed at above. 600 nm However, and a larger reacting hydrodynamic HAuCl4 with size F127_2 (156 nm) resulted of purified in a relativelyGNP (Figure broader8A, Table PR peak4). In centered the case ofat F127_3,600 nm theand UV a larger spectra hydrodynamic of the reaction size mixture (156 nm) exhibited of purified 1) a GNPshift in(Figure the absorbance 8A, Table from4). In 220 the to case 240 of nm F127_3, indicative the UV of Au spectra3+ reduction of the reaction to Au0; andmixture 2) appearance exhibited 1) of a shiftpeak in at the 330 absorbance nm, corresponding from 220 toto formation240 nm indicative of Au0 clusters of Au3+ (Figurereduction8A). to However, Au0; and no2) appearance PR peak was of aobserved peak at 330 in this nm, case, corresponding suggesting to that formation GNP did of notAu0 form. clusters Indeed, (Figure albeit 8A). DLSHowever, of unpurified no PR peak reaction was observedmixture showed in this presencecase, suggesting of 500 nm that aggregates GNP did (Supplementalnot form. Indeed, Data albeit Table DLS S3), noof unpurified GNPs were reaction isolated mixtureafter standard showed purification presence of procedure 500 nm aggregates (Table4). Among(Supplemental notable Data di fferences Table S3), between no GNPs the were batches isolated were afterthe larger standard particle purification sizes (60 procedure nm) and more (Table acidic 4). Among pH of notable the F127_3 differences stock solution between (pH the 5.1) batches compared were theto the larger solutions particle of sizes two other(60 nm) polymer and more samples acidic (pH pH 6.8–6.9) of the F127_3 (Supplemental stock solution Data Table(pH 5.1) S2). compared Since we topreviously the solutions established of twothat other the polymer formation samples of the GNP(pH 6.8–6.9) is dependent (Supplemental on the pH ofData the Table copolymer S2). Since solution, we previouslywe adjusted established the pH of the that F127_3 the formation solution to of either the GNP weakly is acidicdependent pH 6.5 on or the alkaline pH of pH the 11.6. copolymer Notably, solution,pH adjustment we adjusted did not thedecrease pH of the the F127_3 particle solution size (74to either nm at weakly pH 6.5 acidic and pH 81 nm6.5 or at alkaline pH 11.6), pH which 11.6. Notably,remained pH much adjustment larger than did normally not decrease expected the forparticle F127 micellessize (74 nm (27 nm)at pH (Supplemental 6.5 and 81 nm Data at pH Table 11.6), S2). whichHowever, remained reacting much HAuCl larger3H thanO withnormally the F127_3 expected solutions for F127 with micelles adjusted (27 pHnm) proceeded (Supplemental differently Data 4· 2 Tableand, in S2). both However, cases, resulted reacting in formationHAuCl4·3H of2O GNP. with Thus, the F127_3 UV spectra solutions suggested with adjusted that after pH mixing proceeded F127_3 3+ differentlysolutions, pHand, 6.5 in with both HAuCl cases, 4resulted3H2O the in formation reduction of AuGNP. wasThus, decelerated UV spectra as suggested was evident that by after the 3+· 0 mixingpresence F127_3 of a large solutions, peak of pH Au 6.5at with 220 nmHAuCl (Figure4·3H82A).O the At thereduction same time, of Au the3+ peakwas decelerated of the Au clustersas was evidentat 330 nm by disappeared.the presence of However, a large peak there of wasAu3+ a at small 220 nm and (Figure broad 8A). PR peak, At the suggesting same time, formation the peak of theGNPs. Au0 Theclusters DLS at has 330 shown nm disappeared. a presenceof However, 280 nm aggregatesthere was a in small the reaction and broad mixture PR peak, (Supplemental suggesting formationData Table of S3), GNPs. which The after DLS purification has shown produced a presence 107 of nm 280 GNPs nm aggregates (Table4). Whenin the thereaction reaction mixture was (Supplementalcarried out using Data the Table alkali S3), solution which of after F127_3, purification pH 11.6 theproduced reduction 107 ofnm Au GNPs3+ was (Table complete, 4). When and the reactionlarge PR was peak carried of GNP out was using observed. the alkali Overall, solution the UV of F127_3, spectra were pH 11.6 similar the reductionto these observed of Au3+ after was complete, and the large PR peak of GNP was observed. Overall, the UV spectra were similar to these

Polymers 2019, 11, 1553 14 of 21 conductingpolymers 2019 the, 11 reaction, x; doi: FOR under PEER REVIEW basic conditions (Figure4E,F). Moreover, www.mdpi.com/journal/polymers the particle sizes were also nearlyobserved identical after (compare conducting Tables the 2reaction and3). under basic conditions (Figure 4E, F). Moreover, the particle sizes were also nearly identical (compare Tables 2 and 3).

A 3 F127_1 F127_2 F127_3 2 F127_3, pH 6.5 F127_3, pH 11 1 Absorbance (a.u) Absorbance 0 200 400 600 800 Wavelenght (nm) B 3 F127_1L F127_2L F127_3L 2

Absorbance (a.u.) Absorbance 0 200 300 400 500 600 700 800 Wavelength (nm)

Figure 8. Absorbance spectra in the mixtures of 0.25 mM HAuCl4 and different F127 batches (A) before Figure 8. Absorbance spectra in the mixtures of 0.25 mM HAuCl4 and different F127 batches (A) before and (B) after lyophilization. The HAuCl4 concentration was 0.25 mM and F127 concentration was 6.3%. and (B) after lyophilization. The HAuCl4 concentration was 0.25 mM and F127 concentration was The reaction6.3%. The mixtures reaction mixtures were incubated were incubated for 2 h for (unless 2 h (unless shown shown 1 h) at1 h) 45 at◦ C.45 °C. 3.10. Effect of Lyophilization of the Copolymer Solutions on GNP Formation 3.10. Effect of Lyophilization of the Copolymer Solutions on GNP Formation In anIn attempt an attempt to remove to remove hydroperoxides hydroperoxides the polymer the polymer solutions solutions were were lyophilized lyophilized and re-dissolvedand re- in thedissolved same volume in the same of bidistilledvolume of bidistilled water using water a using previously a previously described described protocol protocol [19 [19].]. ThereThere was littlewas change little (lesschange than (less 15%) than in 15%) the in hydroperoxides the hydroperoxides in F127_1, in F127_1, approximately approximately 2-fold2-fold increase in in the hydroperoxidesthe hydroperoxides in F127_2, in and F127_2, nearly and 6-fold nearly decrease 6-fold in decrease the hydroperoxides in the hydroperoxides in F127_3 (Supplemental in F127_3 data(Supplemental Table S2). Moreover, data Table lyophilization S2). Moreover, had lyophilization little if any ehadffect little on theif any free effect radical on the levels, free whichradical were similarlevels, in all which studied were samples. similar in Although all studied there samples. was Although no effect onthere the was pH no in effect F127_1 on andthe pH F127_2 in F127_1 solutions, the pHand of F127_2 the reconstituted solutions, the pH F127_3 of the solution reconstituted increased F127_3 from solution pH increased 5.1 to pH from 6.7 (SupplementalpH 5.1 to pH 6.7 Data (Supplemental Data Table S2). At the same time the effects of lyophilization of F127_3 samples on the Table S2). At the same time the effects of lyophilization of F127_3 samples on the Au3+ reduction Au3+ reduction and GNP formation were distinct and profound. Specifically, in contrast to F127_3 and GNPsolution formation before lyophilization were distinct the and lyophilized profound. and Specifically,reconstituted inF127_3 contrast solution to F127_3 was able solution to reduce before 3+ lyophilizationAu3+ and promote the lyophilized formation and of GNP reconstituted when mixed F127_3 with solutionHAuCl4 (Figure was able 8B toand reduce Table 4). Au and promote formation of GNP when mixed with HAuCl4 (Figure8B and Table4). Table 4. Characteristics of purified GNP synthesized using non-lyophilized and lyophilized F127 Tablesamples 4. Characteristics1. of purified GNP synthesized using non-lyophilized and lyophilized F127 samples 1. GNP synthesized using GNP synthesized using F127 batch GNPnon-lyophilized Synthesized Using F127 lyophilizedGNP Synthesized F127 Using F127 Batch Non-Lyophilized F127 Lyophilized F127 Deff (nm) PDI Deff (nm) PDI Deff (nm) PDI Deff (nm) PDI F127_1F127_1 62 1 62 ± 1 0.16 0.16 0.01± 0.01 47 47 ± 1 1 0.30 ± 0.300.004 0.004 ± ± ± ± F127_2 157 2 0.06 0.02 70 1 0.07 0.01 F127_2 ± 157 ± 2 0.06± ± 0.02 70 ± ±1 0.07 ± 0.01± F127_3 Not formed Not formed 57 1 0.30 0.04 ± ± F127_3 (pH 6.5)F127_3 108 Not2 formed 0.17 Not 0.003formed 57 ± n /1a 0.30 ± 0.04 n /a ± ± F127_3 (pH 11.6) 39 3 0.51 0.12 n/a n/a F127_3 (pH 6.5) ± 108 ± 2 0.17± ± 0.003 n/a n/a 1 The 12.6% stock solutions of F127 solutions were prepared by dissolving the copolymer in bidistilled water. These solutions were lyophilizedF127_3 (pH and 11.6) restored to 39 the ± 3 same volume 0.51 ± by0.12 bidistilled water. n/a GNP were n/a synthesized by mixing equal1 volumes The 12.6% of stock non-lyophilized solutions of and F127 lyophilized solutions were F127 prepared solutions andby dissolving HAuCl4 solution the copolymer (final concentrations in bidistilled 6.3% w/w F127 and 0.25 mM HAuCl ) followed by incubation of the mixture for 2 h at 45 C. Particles were purified water. These solutions were4 lyophilized and restored to the same volume by bidistilled◦ water. GNP as described in Table1. The purified GNPs were restored to the original volume using bidistilled water (particles concentrationwere synthesized remained by the mixing same before equal and volumes after purification). of non-lyophilized and lyophilized F127 solutions and

polymers 2019, 11, x; doi: FOR PEER REVIEW www.mdpi.com/journal/polymers

HAuCl4 solution (final concentrations 6.3% w/w F127 and 0.25 mM HAuCl4) followed by incubation of the mixture for 2 h at 45 °C. Particles were purified as described in Table 1. The purified GNPs were Polymers 2019, 11, 1553 15 of 21 restored to the original volume using bidistilled water (particles concentration remained the same before and after purification). 3.11. Formation of Reacting Oxygen Species during the Reaction 3.11. Formation of Reacting Oxygen Species during the Reaction This study measured changes in the content of free radicals and hydroperoxides before and after mixingThis of F127 study solution measured with changes HAuCl in3H the Ocontent (Figure of9 A).free At radicals 2 h the and content hydroperoxides of free radicals before remained and after 4· 2 relativelymixing of small, F127 however,solution with there HAuCl was a very4·3H2O considerable (Figure 10A). increase At 2 h inthe the content hydroperoxides. of free radicals To elucidate remained therelatively role of oxygen small, however, in the formation there was of thea very hydroperoxides considerable inincrease the course in the of hydroperoxides. the reaction we purgedTo elucidate the copolymerthe role of solution oxygen within the nitrogen formation for of 5 minthe hydroperoxides before initiating in the the reaction. course of This the resulted reaction in we attenuation purged the ofcopolymer formation ofsolution the hydroperoxides with nitrogen in for the 5 min reaction before mixture initiating (Figure the9 reaction.A). In this This case, resulted we did in not attenuation register theof UVformation spectra of to the avoid hydroperoxides exposure of the in solutionsthe reaction to the mixture atmospheric (Figure oxygen. 9A). In However,this case, thewe visualdid not observationregister the of UV the spectra reaction to mixtures avoid exposure at 2 h revealed of the solutions that the color to the of atmospheric the solutions oxygen. was diﬀ However,erent—pink the invisual before observation purging (Figure of the9 reactionB) and purple mixtures after at 2 purging h revealed (Figure that9 theC). color This suggestedof the solutions that thewas PR different has red-shifted—pink in afterbefore purging, purging which (Figure is indicative9B) and purple of the alterationafter purging of the (Figure particle 9C). size This and suggested shape. that the PR has red-shifted after purging, which is indicative of the alteration of the particle size and shape. A B 600 1 2 3 4 500 C 400

300

200 EPR arbitrary units(*10^3) arbitrary EPR

100

0 Free radicals Hydroperoxides

Figure 9. (A) Free radicals and hydroperoxides content in the regular (1,2) and N2 purged (3,4) Figure 9. (A) Free radicals and hydroperoxides content in the regular (1,2) and N2 purged (3,4) stock stock solutions of 6.3% w/w F127 (1,3) and the reaction mixtures of these solutions with HAuCl4 (2,4). solutions of 6.3% w/w F127 (1,3) and the reaction mixtures of these solutions with HAuCl4 (2,4). (B, C) (B,C) Images of GNP dispersions formed in (B) regular and (C)N2 purged solutions of 6.3% w/w (5 mM) Images of GNP dispersions formed in (B) regular and (C) N2 purged solutions of 6.3% w/w (5 mM) F127. The F127 solution was purged with N2 for 5 min before mixing with HAuCl4, and then the F127. The F127 solution was purged with N2 for 5 min before mixing with HAuCl4, and then the reaction was carried in a closed vessel. In all cases, the reaction mixtures were incubated for 2 h (unless reaction was carried in a closed vessel. In all cases, the reaction mixtures were incubated for 2 h (unless shown 1 h) at 45 ◦C. shown 1 h) at 45 °C. 3.12. Mechanism of HAuCl 3H O Reduction by Pluronic 4· 2 3.12. Mechanism of HAuCl4·3H2O Reduction by Pluronic Analysis of the data allows to propose the following schematic for HAuCl 3H O reduction in the 4· 2 presenceAnalysis of Pluronic: of the data allows to propose the following schematic for HAuCl4·3H2O reduction in the presence of Pluronic: 1. The decomposition of the hydroperoxides present in Pluronic leads to Pluronic chains degradation 1. Theand decomposition formation of of lower the hydroperoxides molecular mass present alcohols. in Pluronic This process leads to resultsPluronic in chains the release degradation of a andhydroxonium formation of lower ion that molecular contributes mass to alcohols. acidiﬁcation This ofprocess the media results and in formationthe release of of the a hydroxonium superoxide  ion Othat2−• contributesand hydroxyl to acidification radical HO •ofor the hydrogen media and peroxide formation that of can the participatesuperoxide inO2 further- and hydroxyl redox radicalreactions. HO or Here hydrogen we present peroxide the oxidation that can and participate decomposition in further of the redox PPO reactions. block because Here it we is known present the tooxidation degrade and faster decomposition than the PEO of [ 20the]. PPO However, block similarbecause processes it is known can to also degrade proceed faster at the than PEO the PEOblock [20].polymers andHowever, 2019 PEG, 11 homopolymer., similarx; doi: FOR processes PEER REVIEW can also proceed at the PEO block and PEG www.mdpi.com/journal/polymers homopolymer. polymers 2019, 11, x; doi: FOR PEER REVIEW www.mdpi.com/journal/polymers

(1)

(2)

2. The reactive oxygen species formed in (1), (2), and (3) promote the reduction of Au3+. Since 2.tetracloroauric The reactive ion oxygen [AuCl species4]− undergoes formed acid in (1), and (2), alkalli and hydrolysis(3) promote and the graduallyreduction transformsof Au3+. Since into tetracloroaurictetrahydroxoaurate ion [AuCl ion [Au(OH)4]− undergoes4]− at basic acid conditions and alkalli [21] hydrolysis the reduction and graduallyreactions proceed transforms through into tetrahydroxoauratedifferent mechanisms ion [Au(OH) depending4]− at on basic pH. conditions Most likely [21] these the reduction reactions reactions are complex proceed and through involve differentmultiple elementary mechanisms steps depending and intermediates. on pH. Most Their likely formal these description reactions is arefurther complex complicated and involve by the multiplecoexistence elementary of several steps forms and of intermediates. aquachlorohydroxo Their formalcomplexes description of gold(III) is further that can complicated participate by in the the coexistencereducton with of several varying forms reactivity. of aquachlorohydroxo Therefore, we present complexes only ofthe gold(III) simplified that schemes can participate of the overallin the reductonreactions with with varying either tetracloroauricreactivity. Therefore, or tetrahydroxoaurate we present only the ions simplified at acidic schemes or alkaline of the conditions, overall  reactionsrespectively. with We either also tetracloroauricwould like to point or tetrahydroxoaurate out that the reactivities ions of at the acidic superoxide or alkaline radical conditions, O2- and  respectively.hyrogen peroxide We also can would depend like to on point pH. out The that superoxide the reactivities radical of at the low superoxide pH exists radical mainly O 2- in and the  hyrogenprotonated peroxide form of canthe hydroperoxyl depend on pH. radical The HO superoxide2 (pKa 4.8), radical while at the low hyrogen pH exists peroxide mainly (pKa in  11.6) the  protonatedat alkaline formconditions of the canhydroperoxyl fom the hydroperoxide radical HO2 (pKa anion  4.8),HO2 −while. the hyrogen peroxide (pKa 11.6) at alkaline conditions can fom the hydroperoxide anion HO2−.

Reactions (5), (6), (7), (8), and (10) are reversible, but we assume that their equilibria are shifted to theReactions right due (5), to (6), the (7), rapid (8), and consumption (10) are reversible, of the molecular but we assume oxygen that in theirsubsequent equilibria reactions are shifted with toPluronic. the right The due latter to themay rapid be accelerated consumption by the of thecomplexation molecular ofoxygen [AuCl 4in]− andsubsequent related goldreactions complexes with Pluronic.with the The block latter copolymer may be accelerated chains, as by in the this complexation case the polymer of [AuCl oxidation4]− and related may gold proceed complexes either withconcurrently the block or immediately copolymer chains, after the as reduction in this caseof the the gold polymer ions. oxidation may proceed either concurrently or immediately after the reduction of the gold ions. 3. Finally, the molecular oxygen formed in the reduction reactions (5), (6), (7), (8), and (10) 3.propagates Finally, hydroxyperoxidation the molecular oxygen of either formed PPO in or the PEO reduction chains in reactions Pluronic. (5), (6), (7), (8), and (10) propagates hydroxyperoxidation of either PPO or PEO chains in Pluronic.

Polymers 2019, 11, 1553 16 of 21

2HO• H O (3) → 2 2 2. The reactive oxygen species formed in (1), (2), and (3) promote the reduction of Au3+. Since tetracloroauric ion [AuCl4]− undergoes acid and alkalli hydrolysis and gradually transforms into tetrahydroxoaurate ion [Au(OH)4]− at basic conditions [21] the reduction reactions proceed through diﬀerent mechanisms depending on pH. Most likely these reactions are complex and involve multiple elementary steps and intermediates. Their formal description is further complicated by the coexistence of several forms of aquachlorohydroxo complexes of gold(III) that can participate in the reducton with varying reactivity. Therefore, we present only the simpliﬁed schemes of the overall reactions with either tetracloroauric or tetrahydroxoaurate ions at acidic or alkaline conditions, respectively. We also would like to point out that the reactivities of the superoxide radical O2−• and hyrogen peroxide can depend on pH. The superoxide radical at low pH exists mainly in the protonated form of the hydroperoxyl radical HO2• (pKa ~4.8), while the hyrogen peroxide (pKa ~11.6) at alkaline conditions can fom the hydroperoxide anion HO2−. In acidic conditions + O −• + H O HO • + H O (4) 2 3 → 2 2 0 + [AuCl ]− + 3HO • + 3H O Au + 3O + 3H O + 4Cl− (5) 4 2 2 → 2 3 0 [AuCl ]− + 3O −• Au + 3O + 4Cl− (6) 4 2 → 2 0 + 2[AuCl ]− + 3H O + 6H O 2Au + 3O + 6H O + 8Cl− (7) 4 2 2 2 → 2 3 In alkaline conditions 0 Au(OH) − + 3O −• Au + 3O + 4OH− (8) 4 2 → 2 H O + OH− H O + HO − (9) 2 2 → 2 2 0 2Au(OH) − + 3HO − 2Au + 3O + 5OH− + 3H O (10) 4 2 → 2 2 Reactions (5), (6), (7), (8), and (10) are reversible, but we assume that their equilibria are shifted to the right due to the rapid consumption of the molecular oxygen in subsequent reactions with Pluronic. The latter may be accelerated by the complexation of [AuCl4]− and related gold complexes with the block copolymer chains, as in this case the polymer oxidation may proceed either concurrently or immediately after the reduction of the gold ions. 3. Finally, the molecular oxygen formed in the reduction reactions (5), (6), (7), (8), and (10) propagates

polymershydroxyperoxidation 2019, 11, x; doi: FOR PEER of either REVIEW PPO or PEO chains in Pluronic. www.mdpi.com/journal/polymers

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4. Discussion 4. Discussion Previous studies have demonstrated formation of GNPs in the presence of Pluronics without Previous studies have demonstrated formation of GNPs in the presence of Pluronics without use use of any standard reducing agent such as sodium citrate or NaBH4 [22]. The mechanism postulated of any standard reducing agent such as sodium citrate or NaBH [22]. The mechanism postulated in in these works includes: 1) reduction of [AuCl4]− ions by PEO4 chains leading to the formation of Au0 0 theseclusters; works 2) includes: adsorption 1) reduction of the PPO of chains [AuCl 4of]− theions block by PEO copolymers chains leading on these to clusters the formation with subsequent of Au clusters; 2) adsorption of the PPO chains of the block copolymers on these clusters with subsequent reduction [AuCl4]− on the cluster surface; and 3) growth of the GNPs stabilized by the adsorbed block reductioncopolymers [AuCl [9].4]− Inon this the mechanism cluster surface; known and as 3)‘three-step growth ofreduction the GNPs and stabilized particles byformation’ the adsorbed the first blocktwo copolymers stages correspond [9]. In to this the mechanism nucleation knownof GNPs, as and ‘three-step the third reduction stage to the and growth particles or the formation’ nuclei into themature ﬁrst two nanoparticles. stages correspond The mechanism to the nucleation suggests of GNPs, that the and copolymer the third stage plays to a the crucial growth role or in the both nucleiprocesses. into mature However, nanoparticles. to the best The of mechanism our knowledge, suggests the that specific the copolymer steps in theplays mechanism a crucial role and in the bothresulting processes. products However, have to not the been best not of our precisely knowledge, defined the and/or speciﬁc separated steps in thefrom mechanism the reaction and mixture. the The current GNPs formation theory suggests that the first stage of the process involves formation of the ‘crown ether-like structures’ between [AuCl4]− ions and PEO chains followed by reduction of Au3+ and oxidation of PEO. This is based on an early study published by Longenberger and Mills on HAuCl4·3H2O reduction by PEO homopolymer [6]. They reported that the formation of GNPs was accelerated, and the particles became more stable as the PEO molar mass increased. Therefore, it was proposed that, for full reduction, Au3+ ion should migrate through several ether crown structures. Further work suggested that metal ions interact with the hydrated PEO blocks of the block copolymers but are immiscible with their PPO blocks [23]. Thus it was suggested that PPO blocks cannot directly reduce metal ions but are essential for well-defined shape controlled synthesis of gold nanostructures in mesophase media with low water content [23]. Sakai and Alexandridis compared PEO homopolymer to various Pluronics and concluded that the reaction activity of the latter is much higher than that of the PEO [7,9]. While these authors noted that the PPO block may be sufficiently hydrated to interact with metal ions, they stopped short of suggesting that PPO can actually participate as a reagent in the reduction reaction. However, they suggested that PPO blocks play significant role in the later stages of the process by absorbing at the particle surface and limiting the particle growth. In this work, we separated the GNPs and soluble polymer products and analyzed them by several physicochemical methods. The results clearly suggested that the copolymer undergoes chemical decomposition in the process of the reaction including cleavage of the C–O bonds (IR), depletion of the O–CH2 signal (1H-NMR), and decrease of the molecular mass of the copolymer (GPC). Although the purified nanoparticles contained about 13% by weight of the organic component (by TGA), we did not find any presence of PPO groups in them suggesting that PPO chains or intact Pluronic molecules are not adsorbed on the particles surface. Interestingly, the analysis of soluble products did reveal presence of the CH3 group of PPO. The product analysis also indicated presence of newly formed OH groups both in the purified GNPs and in the soluble products (IR), which according to 13C-NMR correspond to primary hydroxyl C–(OH) groups. In contrast to previous reports suggesting that oxidation of the PEO proceeds all the way to the carboxylate groups [6], IR, 1H-NMR, or 13C-NMR did not confirm the presence of these groups in the purified GNPs or reaction products. Hence, the purified GNPs appear to contain lower molecular mass alcohols produced as a result of Pluronic degradation that are likely attached to the nanoparticle surface rather than embedded within their volume. A very interesting phenomenon was observed when the reaction was carried out at the surface of F127 gel, with the gel being dissolved in the course of the frontal reaction. This suggests that cleavage of Pluronic chains during the reaction can disrupt self-assembly of the block copolymer aggregates (in the specific case—the copolymer gel). We also discovered that the reaction of the block copolymer and HAuCl4 involves formation of free radicals and hydroperoxides as their concentration is sharply increased. Interestingly, we found

Polymers 2019, 11, 1553 17 of 21 resulting products have not been not precisely defined and/or separated from the reaction mixture. The current GNPs formation theory suggests that the first stage of the process involves formation of the ‘crown ether-like structures’ between [AuCl4]− ions and PEO chains followed by reduction of Au3+ and oxidation of PEO. This is based on an early study published by Longenberger and Mills on HAuCl 3H O reduction by PEO homopolymer [6]. They reported that the formation of GNPs was 4· 2 accelerated, and the particles became more stable as the PEO molar mass increased. Therefore, it was proposed that, for full reduction, Au3+ ion should migrate through several ether crown structures. Further work suggested that metal ions interact with the hydrated PEO blocks of the block copolymers but are immiscible with their PPO blocks [23]. Thus it was suggested that PPO blocks cannot directly reduce metal ions but are essential for well-defined shape controlled synthesis of gold nanostructures in mesophase media with low water content [23]. Sakai and Alexandridis compared PEO homopolymer to various Pluronics and concluded that the reaction activity of the latter is much higher than that of the PEO [7,9]. While these authors noted that the PPO block may be sufficiently hydrated to interact with metal ions, they stopped short of suggesting that PPO can actually participate as a reagent in the reduction reaction. However, they suggested that PPO blocks play significant role in the later stages of the process by absorbing at the particle surface and limiting the particle growth. In this work, we separated the GNPs and soluble polymer products and analyzed them by several physicochemical methods. The results clearly suggested that the copolymer undergoes chemical decomposition in the process of the reaction including cleavage of the C–O bonds (IR), depletion of the 1 O–CH2 signal ( H-NMR), and decrease of the molecular mass of the copolymer (GPC). Although the purified nanoparticles contained about 13% by weight of the organic component (by TGA), we did not find any presence of PPO groups in them suggesting that PPO chains or intact Pluronic molecules are not adsorbed on the particles surface. Interestingly, the analysis of soluble products did reveal presence of the CH3 group of PPO. The product analysis also indicated presence of newly formed OH groups both in the purified GNPs and in the soluble products (IR), which according to 13C-NMR correspond to primary hydroxyl C–(OH) groups. In contrast to previous reports suggesting that oxidation of the PEO proceeds all the way to the carboxylate groups [6], IR, 1H-NMR, or 13C-NMR did not confirm the presence of these groups in the purified GNPs or reaction products. Hence, the purified GNPs appear to contain lower molecular mass alcohols produced as a result of Pluronic degradation that are likely attached to the nanoparticle surface rather than embedded within their volume. A very interesting phenomenon was observed when the reaction was carried out at the surface of F127 gel, with the gel being dissolved in the course of the frontal reaction. This suggests that cleavage of Pluronic chains during the reaction can disrupt self-assembly of the block copolymer aggregates (in the specific case—the copolymer gel). We also discovered that the reaction of the block copolymer and HAuCl4 involves formation of free radicals and hydroperoxides as their concentration is sharply increased. Interestingly, we found that the amount of the hydroperoxides decreases in the solutions purged with nitrogen and therefore appears to be dependent on the concentration of the oxygen. At the same time the nitrogen-purged solutions reveal some increase in the free radicals suggesting that the oxidation mechanisms are complex and involve multiple intermediates. It is noteworthy that the reaction proceeding under the nitrogen atmosphere resulted in the formation GNPs with different spectral characteristics than the reaction carried out under the air. Moreover, we found that F127 batches having different levels of the free radicals and hydroperoxides behave differently in the reaction. This is an important result for all such studies suggesting that the underlying processes are affected by the initial degree of oxidation of the polymers. These processes are hard to control and at the same time they can contribute to Pluronic batch-to-batch variation. Taken together, we propose a multi-stage process for the reduction of HAuCl 3H O by Pluronic, 4· 2 see reactions (1)–(11). It is well known that polyethers including poloxamers can undergo oxidation and formation of the free radicals and hydroperoxides [19]. Hence, we posit that at the first, initiation stage the Pluronic hydroperoxides decompose in reactions (1) and (2) resulting in polymer degradation Polymers 2019, 11, 1553 18 of 21 to lower molecular mass primary alcohols and formation of the reactive oxygen species (1–3), that drive the subsequent reduction of various gold(III) forms. In the process of polymer degradation the hydronium ions are also released, which explains a pH decrease (by about 2 pH units) observed experimentally. At the second stage, gold(III) is reduced to Au0 in complex processes (4–10) involving multiple elementary steps also leading to formation of molecular oxygen. Some of these reactions are probably similar to previously reported reactions of reduction of transition metals by the reactive oxygen species [24]. These processes are pH dependent due to the presence of several aquachlorohydroxo complexes of gold(III) and various forms of reactive oxygen species, O2−•, HO2•,H2O2, and HO2−, with different reactivity [25]. The pH dependence of the reaction was observed experimentally in this study as well as in previous work that reported that formation of GNPs in the presence Pluronic P123 was inhibited at acidic conditions ( pH 2.0) and could be reversed by pH adjustment [10]. Finally, at ≈ the third stage, the molecular oxygen formed during the gold(III) reduction propels further oxidation of Pluronic to hydroperoxides (11) and propagation of the block copolymer degradation. This explains a burst in the hydroperoxides observed experimentally. It is possible that reaction (11) proceeds in gold-Pluronic complexes either simultaneously (intramolecularly) or right after elementary steps of the gold ion oxidation and the O2 formation. The effect of the Pluronic self-assembly on the formation of GNPs observed in this study is also of considerable interest. Even though Pluronic molecules do not appear to strongly bind with the GNPs (as they can be separated by centrifugal filtration), the GNPs formation kinetics and final properties are strongly affected by the block copolymer self-assembly. First, there was a clear dependence of the shape of GNPs on Pluronic concentration. As the concentration increased the particles became more homogeneous and spherical. Second, at low temperature when the self-assembly of Pluronic was suppressed the formation of GNPs was impaired although the reduction of Au3+ proceeded. Hence, we believe that Pluronic aggregates serve as templates for the GNPs growth. Finally, formation of aggregates that are larger than both individual GNPs and Pluronic micelles was observed. These aggregates represented clusters of GNPs grains interconnected by the block copolymer and they disintegrated upon dilution. Finally, our study suggests that F127 having the longest PEO and PPO chains forms the most stable nanoparticles. Colloidal stabilization of pre-formed GNPs by Pluronics has been previously reported [26]. The stabilization effect enhanced as the PPO or PEO chain length increased. This behavior was attributed to the formation of the polymer layers surrounding the GNPs rather than adsorption of PPO chains directly at the GNPs surface. Finally, of interest are the effects of various reaction conditions on the shape of the GNPs. It is known that the rate of production of the reduced metal precursors—i.e., Au0—can influence the nanoparticle shape. Rapid production of Au0 facilitates formation of thermodynamically favored polyhedral (spherical) GNPs, while slower production of Au0 usually results in heterogeneous GNPs shapes. For example, reacting HAuCl4 with a strong reducing agent such as NaBH4 can lead to a rapid formation of the Au0 nuclei followed by the fast growth of spherical GNPs [27,28]. In contrast, when a mild reductant, such as phenylenediamine, PVP or glucose, is used the GNPs growth is kinetically controlled resulting in the formation of hexagonal and triangular particles. This consideration helps to explain the effect of Pluronic F127 concentration on the GNPs shape observed in this study. As the concentration of the copolymer increases Au0 production becomes faster and the particles become more uniform and homogeneous. The effect of HAuCl 3H O concentration appears to be more 4· 2 complex. We observed formation of small spheres at low concentrations of HAuCl 3H O, followed by 4· 2 larger spheres, multiple shapes, and spheres again as the HAuCl 3H O concentration increased. Since 4· 2 HAuCl 3H O contained some amount of Au0 upon change of its concentration there was likely a 4· 2 change of both the nuclei and metal precursor. Another factor, which affects the GNPs shape, is pH. In the present study at low pH we observed slow formation of heterogeneous multiform GNPs, which is explained by slower Au0 production due to a decrease of the reduction potential of the reactive oxygen species [25]. As the pH increased the particles became more homogeneous, spherical, smaller, and stable. Prior studies concluded that pH had similar effect on the size and shape of GNPs regardless of Polymers 2019, 11, 1553 19 of 21 the type of the reductant, such as Tetronic T904 [29], Pluronics [10], or polyols [30]. Moreover, similar effects of pH were observed for silver [31] and platinum nanoparticles [32]. Previously, chloride ions were reported to promote formation of triangular GNPs [33]. We also observed that addition of small amounts of NaCl to the reaction medium results in the formation of heterogeneous, polygonal GNPs. Surprisingly however, as the NaCl concentration further increased, there was complete inhibition of the GNP formation. This phenomenon may be explained by the effects NaCl on the interactions of the block copolymer with Au0 clusters or GNPs, since NaCl is known to promote dehydration of PEO and PPO chains, enhance crystallization of PEO chains, and affect the micelle structure [34,35]. Therefore, by varying the concentration of NaCl in the system, one can modify the process parameters and the shape of the formed particles.

5. Conclusions In conclusion, this work presents a detailed study on the formation of the GNP as a result of reduction of chloroauric acid with Pluronic block copolymers. We demonstrated that in the course of this reaction the copolymer chains undergo degradation and become enriched with primary hydroxyl groups, although the oxidation of the polymer does not go all the way down to the carboxylic acids. The purified nanoparticles also contain about 13% by weight of the organic component but can be fully separated from the excess of the copolymer. The reaction involves formation of free radicals and hydroperoxides and appears to be dependent on the concentration of the oxygen in the reaction mixture. We elucidated various environmental factors such as pH, temperature, the sodium chloride concentration, as well as the concentrations of the reactants that affect the rate of the reaction and the yield and morphology of the resulting GNP. The findings advance understanding of the reaction mechanisms and could be useful for improving the methods of manufacturing uniform and pure nanoparticles for various technical applications including nanomedicine, where GNPs could be used in numerous medical applications, from diagnostics to therapy [36,37].

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4360/11/10/1553/s1, Table S1: Characteristics of Pluronic block copolymers used in the experiment; Table S2: Physicochemical characteristics and ROS levels in F127 aqueous solutions; Table S3: Characteristics of the unpurified GNP formed using non-lyophilized and lyophilized F127 samples; Figure S1: Absorbance spectra recorded in the initial HAuCl4 aqueous mixtures containing 5 mM Pluronic P104, F127, F88, and P85 and 0.25 mM HAuCl4 2 h after incubation at 25 ◦C. The copolymer and HAuCl4 concentrations were kept constant with the copolymer concentration being well above its CMC and at the 20-fold molar excess compared to HAuCl4; Figure S2: TEM images of the purified gold nanoparticles (GNP) synthesized at (A) 25 ◦C or (B) 45 ◦C; Figure S3: Stability of purified GNP in double distilled water (DDW) or PBS; Figure S4: TEM images of the purified GNP synthesized at 45 ◦C in the presence of 6.3% F127 at various concentrations of HAuCl4; Figure S5: TEM images of the purified GNP synthesized at 45 ◦C in 0.25 mM HAuCl4 solution in the presence of various concentrations of F127; Figure S6: (A) Absorbance spectra of the reaction mixtures of 0.25 mM HAuCl4, and various concentrations of PEO 8000 6 h after incubation at 45 ◦C, (B) TEM images of the purified GNP formed at 10% w/w PEO 8000; Figure S7: DLS analysis of the mixtures containing 6.3% w/w F127 and 0.25mM of HAuCl4 at 25 ◦C: (A) 0.25mM HAuCl4 (B) 6.3% w/w F127 (C) 20 min, (D) 60 min, (E) 120 min; Figure S8: T EM images of the purified GNP synthesized in the presence of 6.3% w/w F127 and 0.25 mM HAuCl4 at different pH: (A) pH 2.2, (B) pH 4.5, (C) pH 6.7, (D) pH 7.5, (E) pH 9.0, and (F) pH 11.5; Figure S9: (A) Absorbance spectra of the reaction mixtures of 6.3% F127 and 0.25 mM HAuCl4, 6.3% with various concentrations of NaCl 6 h after incubation at 45 ◦C, (B) TEM image of the purified GNP synthesized in the solutions of 6.3% w/w F127 and 0.25 mM HAuCl4 in the presence of 0.005 M NaCl at 45 ◦C; Figure S10: Gel permeation chromatography of (A) original F127 (0.5 mM) and (B) separated reaction products; Figure S11: TGA of purified GNP. Author Contributions: M.S.-P. conducted the experiments, collected and analyzed the data and wrote the manuscript (original draft preparation and editing). A.K. conceptualized the study, provided the resources and wrote the manuscript (review and editing). Funding: This work was funded by the United States National Institutes of Health (NIH) grant no. RR021937—the Center for Biomedical Research Excellence (COBRE) Nebraska Center for Nanomedicine (now Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the NIH under grant P20GM103480). Polymers 2019, 11, 1553 20 of 21

Acknowledgments: We would like to thank Hemant M. Vishwasrao for assistance with “the effect of pH” studies, Matt Zimmerman and Josalin Jones for assistance in EPR studies, UNMC Nanonimaging core facility and L. Shlyakhtenko for carrying our AFM imaging, UNMC Electron microscopy facility and T. Bargar for training and assistance in TEM imaging and Ed Ezell, M.S UNMC Nuclear Magnetic Resonance (NMR) Facility for assistance in NMR studies, data analysis, and interpretation. Conflicts of Interest: The authors declare no conflict of interest.

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