polymers

Article
Synthesis of Well-Defined Gold Nanoparticles Using
Pluronic: The Role of Radicals and Surfactants in
Nanoparticles Formation
Marina Sokolsky-Papkov 1,† and Alexander Kabanov 1,2, *
1 Center for Nanotechnology in Drug Delivery Eshelman School of Pharmacy, UNC-Chapel Hill, Chapel Hill,
NC 27514, USA; msokolsk@email.unc.edu
2 Laboratory of Chemical Design of Bionanomaterials, Faculty of Chemistry, M.V. Lomonosov Moscow State
University, Moscow 119992, Russia
* Correspondence: kabanov@email.unc.edu; 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
(HAuCl4 ·3H2 O), PEO (Mw 8000), sodium chloride (NaCl), hydrochloric acid (HCl), sodium hydroxide
(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
range of 4000–400 cm−1 .

2.5.6. NMR
The dried reaction products or GNP were dissolved in D2O. 1 H-NMR and 13 C-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 Me4 Si 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
25 ◦ C, the temperature was ramped at 10 ◦ C min−1 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 KH2 PO4 , 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 HAuCl4 ·3H2 O with Pluronic
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 HAuCl4 ·3H2 O and incubated at
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 (Figure 1). 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
240 nm corresponding to Au0 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 (Figure 1). 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 (Figure 2). Therefore, we purified the GNP from the
excess of F127. The purified GNP represent isolated and relatively uniform structures (Figure 3A,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
purified particles
polymers 2019, 11, x;revealed some
doi: FOR PEER changes in double distilled water (DDW)
REVIEW and PBS (Supplemental
www.mdpi.com/journal/polymers
S3). Thus,
polymers 2019,the
11, particle
x; doi: FORsize
PEERdecreased
REVIEW over time from about 70 nm to about 60 nm in both DDW and
www.mdpi.com/journal/polymers
DataS3).
Figure
Thus, S3).
the Thus,
particlethesize
particle size decreased
decreased over time over about
from time from about
70 less
nm to 70 nm
about 60 to
nmabout
in both60 DDW
nm in and
both
PBS. The PDI
S3). and
Thus, theThe values
particle increased
size decreasedin PBSover (to
timenearly
from 0.2),
about with
70 nm change
toless
about observed
60 nm in DDW.
in both DDW No
and
DDW PBS. The PDI
PBS.
precipitation was values
PDI
observedincreased
values
during in
increased
the PBSin (to nearly 0.2),
PBS
experiment.
(to nearly with less
0.2), with change observed
change observed in DDW. in
in DDW. NoNo
PBS. The PDI values increased inthe
PBS (to nearly The0.2),purified
with less particles
changealso did not in
observed precipitate
DDW. No in
precipitation
precipitation
10%
was
NaCl (1.72 was observed they
observed
M)observed
although during
during
did the experiment.
experiment. The purified
The purified particles
particles also did
also did not ±precipitate
not precipitate in
precipitation was during theexhibit some changes
experiment. in effective
The purified diameters
particles also did(83.49 3.94 nm vs.
not precipitate in
10% 10%
NaCl
67.18 NaCl
(1.72(1.72
± 0.32(1.72
M)
nm)M) M) although
although
and
theythey
polydispersity
diddid exhibit
exhibit some
some changes
changes in
in effective
effective diameters (83.49
(83.49 ±±3.94 nm
3.94 nm vs.
vs.
10% NaCl although they did(PDI 0.278
exhibit ± 0.012
some vs. 0.12
changes ± 0.008). diameters (83.49 ± 3.94 nm vs.
in effective
67.18
67.18 ± 0.32
± 0.32 nm) nm)andand polydispersity(PDI
polydispersity (PDI0.278
0.278±±0.012
0.012 vs.vs. 0.12
0.12 ±±0.008).
0.008).
67.18 ± 0.32Anm) and polydispersity (PDI 0.278 ± 0.012 B 2.5vs. 0.12 ± 0.008).
2.5
A 2.5 B 0 min 1min
A 2.52 0 min 1min B 2.522.5 3min
0 min 5min
1min

(a.u) (a.u)
3min 5min 10min 15min
(a.u) (a.u)

0 min
10min 1min
15min 2 3min
0 min 5min
1min
2

Absorbance (a.u)
3min
0 min 5min
1min 30min
10min 45 min
15min
3min 5min
Absorbance (a.u)

Absorbance
1.5
2 30min 45 min 1.5
2
10min 15min 60 min 120
45min
Absorbance

3min
60 min 5min
120 min 30min
10min 15minmin
1.5 30min
10min 45 min
15min 1.5
60 min 60 min
30min 45120
minmin
45120
minmin
Absorbance
1
1.5 30min 1
1.5 60 min 120 min
Absorbance

60 min 120 min

1 1
0.5
1 0.5
1
0.5 0.5
0
0.5 0
0.5
200
0 300 400 500 600 700 200
0 300 400 500 600 700
0 200 300 Wavelength
400 (nm)
500 600 700 0 200 300 Wavelength
400 (nm)
500 600 700
200 300 400
Wavelength 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
Figure
mM
time
1. The
HAuCl timeevolution
evolutionof
of the UV–Vis spectra
the UV–Vis spectraininthe
4 during incubation at 25 °C (A) or 45 °C (B).
the mixtures
mixtures containing
containing w/w w/w
6.3%6.3% F127 F127 and
and 0.25
Figure 1. The time evolution of the UV–Vis spectra
◦C in the
◦ Cmixtures containing 6.3% w/w F127 and 0.25
0.25mM HAuCl4 during
mM HAuCl 4 incubation at 25 °C (A) or 45 °C (B).
during incubation at 25 (A) or 45 (B).
mM HAuCl4 during incubation at 25 °C (A) or 45 °C (B).
Size (reaction mixture)
A Size
Size(purified
(reactionparticles)
mixture)
B Dilution factor
A Size (reaction mixture) B N.D. Dilution2 factor 4
A 200
200
PDI
Size
PDI
PDI
(reaction
Size (purifiedmixture)
(purified
(purified
(reaction
particles)
particles)
particles)
mixture)
0.3
0.3
B 0 N.D. Dilution 2factor 4
160 0 N.D. 2 4
200 PDI
PDI(reaction
(purifiedmixture)
particles) 0.3
0
(mV)(mV)

160 PDI (purified particles) 0.2 -10

(nm)(nm)

120
160
z-potential (mV)

0.2
PDI PDI

-10
Deff (nm)

120 0.2
z-potential

80 -10
PDI

120
Deff Deff

0.1 -20
80 -20
40 0.1
z-potential

80 -20
40 0.1 -30
0
40 0
-30
0 N.D. 2 4 0 -30
0 0 -40
N.D. 2 4 Reaction mixture
N.D. Dilution2 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
2. Changes
Figure in the
2. Changes (A)(A)
in the particle effective diameter (columns)
(columns)andand PDI
PDI (lines) and (B)
(B)ζ-potential
ofparticle effective diameter (lines) and
Figure ζ-potential
potential
2. following
Figure following indilution
Changesdilution
the (A) reaction mixture and purification
(columns)ofand
GNPs.
potential following
potential ofparticle
dilution reactioneffective
of reaction
diameter
mixture
mixture andpurification
and PDI (lines) and (B) ζ-potential
purificationofofGNPs.
GNPs.
potential following dilution of reaction mixture and purification of GNPs.

A B
A B
A B

100 nm
100 nm
100 nm

Figure3.3.(A)
Figure (A)TEM
TEMand
and(B)
(B) AFM
AFM images
images of
of purified
purifiedgold
goldnanoparticles.
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. Effect of HAuCl4 Concentration
The above-described reactiononconditions
the GNP Formation
resulted and Characteristics
in formation of relatively uniform spherical
The above-described
nanoparticles. reaction
We further examined conditions
the effect ofresulted
HAuClin in formation of relatively uniform spherical
4·3H2O concentration on the size, ζ-potential
The above-described reaction conditions resulted formation of relatively uniform spherical
nanoparticles. We further examined the effect of HAuCl 4·3H2O concentration on the size, ζ-potential
and shape of the unpurified aggregates and purified nanoparticles. The concentration of
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 HAuCl4 ·3H2 O concentration on the size, ζ-potential
and shape of the unpurified aggregates and purified nanoparticles. The concentration of HAuCl4 ·3H2 O
polymers 2019, 11,
was varied in x;
the doi: FOR PEER
range REVIEW
of 0.05 to 0.25 mM while keeping F127 concentration www.mdpi.com/journal/polymers
constant (6.3% w/w). The
(6.3%
PR peakw/w). The PR peak
absorbance absorbance
increased linearlyincreased
suggesting linearly
that moresuggesting that more
nanoparticles werenanoparticles
formed as HAuCl were4
formed as HAuCl
concentration 4 concentration
increased increased
(Figure 4A,B). (Figure
This was 4A and B).by
accompanied This
the was accompanied
increase in the sizeby
andthe increase
decrease in
in
thethe
PDIsize andunpurified
of the decrease aggregates
in the PDIuntil of the0.15unpurified
mM HAuClaggregates
4 ·3H 2 O until 0.15
concentration mM
was HAuCl
reached. 4·3H
Above
2O
concentration was reached.
this concentration, Above
the size and PDIthis concentration,
remained constantthe size and
(approx. 200PDInmremained constant
and PDI 0.22). (approx.
Interestingly
200 nm and
between 0.1PDI
and0.22).
0.15 mMInterestingly
HAuCl4 ·3H between
2 O the 0.1 and 0.15
position of themM PRHAuCl 4·3H2O from
peak shifted the position
530 nm ofto the
540 PRnm
peak shiftedthe
indicating from
change530 of
nmthetoparticle
540 nmshape.
indicating the change
Analysis of the particle
of the purified particlesshape.
by TEM Analysis
revealed of that
the
purified particles
their shape by TEMaffected
was indeed revealed that 1their
(Table andshape was indeed
Supplemental Data affected
Figure(Table
S4). At1 low
and HAuCl
Supplemental
4 ·3H2 O
Data Figure S4).
concentrations At lowmM)
(0.05–0.1 HAuCl 4·3H2O concentrations
the particles were mostly spherical,(0.05–0.1 mM)and
small, therelatively
particlesuniform
were mostly
by size
spherical,
(TEM). Assmall, and relativelyincreased
the concentration uniform by size (TEM).
(0.15–0.2 mM) As the concentration
consistent increased
with the change (0.15–0.2
of the PR peak mM)the
consistent
triangles andwith rodstheappeared
change of the PR
among thepeak
sphericalthe triangles
particles. and
At the rods appeared
highest among the
concentration (0.25spherical
mM) the
particles. At the highest
rods disappeared concentration
and mostly spherical (0.25 mM) the
particles rods
with disappeared
some triangles and weremostly
formed spherical particles
again. However,
with some triangles
in contrast were formed
to the effective diameters again.
of the However,
aggregates in the
contrast
effectiveto the effective
diameters of diameters
the purified ofGNP
the
aggregates
practically didthe not effective
depend diameters
on the HAuCl of the purified
4 ·3H GNP practically
2 O concentration (Table 1). didAsnot depend
already on their
noted, the
HAuCl 4·3H2O
ζ-potential was concentration (Tablecompared
strongly negative 1). As already
to the noted,
ζ-potentialtheirofζ-potential was strongly
the aggregates, which was negative
low in
compared
the absolute to the ζ-potential
value. of the aggregates,
The ζ-potential which particles
of the purified was low in or the absolutedid
aggregates value.
notThe ζ-potential
depend on the
of the purified
HAuCl 4 ·3H2 Oparticles or aggregates did not depend on the HAuCl4·3H2O concentrations.
concentrations.

A B 560 1.5

Maximal absorption at
3 0.05mM
0.1mM 1.2

PR center (a.u)
PR peak center
Absorbance (a.u)

0.15mM 540
2 0.2mM 0.9
0.25mM
(nm)

0.6
520
1 0.3
500 0
0 0.05 0.1 0.15 0.2 0.25
200 300 400 500 600 700
Wavelength (nm) HAuCl4 concentration (mM)
C 0.25 mM Au+3 0.1% D 580 1.6
Maximal absorption at

3 0.5% 1%
Absorbance (a.u)

560 1.2
PR peak center

PR center (a.u)

2% 3.15%
5% 6.3%
2 7.5% 10% 540 0.8
(nm)

520 0.4
1
500 0
0 1 2 3.15 5 6.3 7.5 10
200 400 600
Wavelength (nm) F127 concentration (%w/w)
E 3 0.25 mM Au+3 pH 2.2 F
Maximal absorption

560 1.6
at PR center (a.u)

pH 4.5 pH 6.6
Absorbance (a.u)

pH 7.2 pH 9 1.2
PR peak center

2 540
pH 11
0.8
(nm)

1 520
0.4

0 500 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

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

HAuCl4 DLS (Reaction Mixture) DLS (Purified Particles)
Concentration Morphology by TEM
(mM) Deff PDI ζ-Potential Deff PDI ζ-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 HAuCl4 ·3H2 O concentration constant
(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 (Figure 4C,D). The effective diameter of unpurified
aggregates also increased from about 100 nm to about 300 (Table 2). 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 (Table 2, 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
(% w/w) Deff PDI ζ-Potential Deff PDI ζ-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
1Particles 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 Table 1. 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 (Figure 5). However, the reduced Au0 formed some GNP as exhibited by a well-defined albeit
 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
Polymers 2019,concentration
11, 1553 used [16]. At 1–2% F127 there was little reduction of Au3+ during 9 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 as reducing agent at 45
using non-micelle-forming 10% PEO 8000 as reducing agent at 45 ◦ C. Albeit Au3+ 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 ·3H2 O (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 ·3H2 O 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 ·3H2 O 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

Absorbance (a.u)

3 1%
2% 0.3
5%
7.50%
10%
Absorbance (a.u)

0.15
2 15%

0
450 550 650
1 Wavelength (nm)

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. Table 3
presents the results of the GNP synthesis for different pH values before and after mixing the F127 and
HAuCl4 ·3H2 O solutions.
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

(Table 3, 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 (Table 3).

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 Deff ζ-Potential Deff ζ-Potential Deff
PDI PDI 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
4 −5.20 ±
11.5 (7.76) 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
and 0.25 mM, respectively) followed by incubation of the mixture for 2 h at 45 ◦ C. 1 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
solution (pH 6.7) was adjusted with 2 1N HCl, 3 not adjusted, 4 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
incubated at 25 ◦ C revealed that Au3+ 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 (Figure 6A) and the PR of the GNP (Figure 6B). 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 (Figure 6B). 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
 M NaCl a peak at 320 nm corresponding to Au 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
presence
of the salt.ofConsistent
the salt the PR the
with peak wasof
shape considerably
the PR peakbroader and shifted
in the presence towards
of the higher
salt these GNPwavelengths
represented
athan that in the absence
heterogeneous mixture of
of the salt.
larger andConsistent with theof
smaller particles shape of the
different PR peak shapes
polygonal in the presence of the
(Supplemental
salt these GNP represented a heterogeneous mixture of larger and smaller
Data Figure S9B) compared to much more homogeneous and closer to spherical particles formedparticles of different
polygonal
without shapes
added NaCl(Supplemental
(SupplementalData Figure
data S9B)
Figure compared to much more homogeneous and closer
S5D).
to spherical particles formed without added NaCl (Supplemental data Figure S5D).
A 2

Absorbance intensity at 1.5

240 nm (a.u)
1

0.5

1
01
04
05

1
2
3
4
5

25
5

0.
0.
0.
0.
0.
00

0.
0.

1.
0.
0.

NaCl concentration (M)

B 1.2 25C, 6h
25C, 24h
Absorbance intensity at

0.8 45C, 6h
PR (a.u)

0.4

0
01
04
05

1
2
3
4
5

25
5

1
0.
0.
0.
0.
0.
00

0.
0.
0.

1.
0.

NaCl concentration (M)

Figure 6.
Figure 6. (A)
(A) Absorbance intensity at
Absorbance intensity at 240
240 nm
nm and
and (B)
(B) the
the maximal absorbance intensity
maximal absorbance intensity of
of the
the PR
PR peak
peak
in the mixtures of 0.25 mM HAuCl44 and 6.3% w/w F127 as a function of the NaCl concentration after
in the mixtures of 0.25 mM HAuCl and 6.3% w/w F127 as a function of the NaCl concentration after
incubation of
incubation of the
the reaction
reaction mixture
mixture at
at 25
25 ◦°C or 45
C or 45 °C at 6 h and 24 h.
◦ C at 6 h and 24 h.

3.8.
3.8. Chemical
Chemical Conversion
Conversion of
of the
the Block
Block Copolymer
Copolymer During
During thethe Reaction
Reaction
Soluble
Soluble reaction
reaction products
products and
and GNP GNP were
were separated
separated by by centrifugal
centrifugal filtration,
filtration, lyophilized,
lyophilized, and
and
analyzed
analyzed by NMR (Figure 7A and B) and IR (Figure 7C). When the reaction was atcarried
by NMR (Figure 7A,B) and IR (Figure 7C). When the reaction was carried out considerable
out at
excess of F127excess
considerable both the IR and
of F127 NMR
both thespectra
IR andofNMR the separated
spectra ofpolymer were nearly
the separated polymeridentical to the
were nearly
initial F127,
identical albeit
to the theF127,
initial spectra of the
albeit purifiedofGNP
the spectra were markedly
the purified GNP were different.
markedly However,
different.when F127
However,
and HAuCl ·3H O were mixed in 1:1 molar ratio (0.25 mM) the spectra of the
when F127 4and 2HAuCl4·3H2O were mixed in 1:1 molar ratio (0.25 mM) the spectra of the soluble soluble products were
drastically.
products wereThisdrastically.
suggested that
Thissome small amount
suggested that some of F127
smallwas undergoing
amount of F127chemical conversion
was undergoing during
chemical
the reaction. 1 H-NMR (Figure 6A) and 13 C-NMR (Figure 7B) revealed considerable differences between
conversion during the reaction. 1H-NMR (Figure 6A) and 13C-NMR (Figure 7B) revealed considerable
the soluble reaction
differences betweenproducts andreaction
the soluble the nanoparticles
products on andthe one
the hand and theoninitial
nanoparticles F127hand
the one on the
andother.
the
Specifically, 1
initial F127 the
on theH-NMR
other.revealed that the
Specifically, the O–CH
1H-NMR 2 peak at 3.75 that
revealed ppmthe
wasO–CH
attenuated while several new
2 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 13 C-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
 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)
of PPO,
PPO was suggesting
not present that PPODetailed
in GNP. was not analysis
present in of GNP. Detailed
IR spectra analysis
confirms theseofobservations
IR spectra confirms
(Figure these
7C).
observations
Specifically, in(Figure 7C). Specifically,
the purified GNP threeinnew the purified
IR peaksGNP three new
appeared IR peaks
at 3300, 1030,appeared
and 920 at cm3300, 1030,
−1 along

and the
with 920decrease
cm−1 along with
of the the decrease
peaks of the peaks
in the 1000–1500 cm−1inrange.
the 1000–1500 cm−1 range.
This combination of This combination
IR spectra changesof
IR spectra
suggests changes
that C–O bondsuggests
of F127thatwas
C–O bond ofcleaved
partially F127 was andpartially
the OH cleaved
groups wereand the OH groups
formed duringwere the
formed during
reaction. Notablythethereaction.
IR spectra Notably
did notthe IR spectra
support did not support
the formation the formation
of the carboxylic groups:of the
even carboxylic
though
groups:
some evenexhibited
samples though some a minorsamples
peak atexhibited
1645 cm−1 a minor peak at
when these 1645 cm
samples −1 when these samples were
were carefully dried this peak
carefully dried
disappeared this peak
completely disappeared
while the peak at completely
3300 cm while
−1 remainedthe peak at 3300 It
unchanged. cm −1 remained unchanged.
was previously reported
that such behavior can be explained by the presence of water in the copolymer [18].ofFurthermore,
It was previously reported that such behavior can be explained by the presence water in the
copolymer
the CH3 bends [18]. Furthermore,
peak at 1337 cm thewas
−1 CHobserved
3 bends peak atinitial
in the 1337 cm F127was
−1 observed
as well in the products
as in soluble initial F127 butas
well
not in as
theinpurified
solubleGNP.
products but not in the
The degradation purified
of the GNP. in
F127 chain The
thedegradation
process of the of reaction
the F127was chain in the
directly
process of thebyreaction
demonstrated was directlyData
GPC (Supplemental demonstrated
Figure S10). byBefore
GPC (Supplemental
the reaction oneData majorFigure
peak wasS10).seen
Before
in
theinitial
the reaction one major
0.5 mM peak was
F127 sample withseen
theinretention
the initial 0.5 of
time mM 7.03F127
min,sample
whichwith the retention
corresponded to Mwtime=of 7.03
9600
min,
Da. The which
lowercorresponded to Mw = 9600
value of the molecular massDa. The lower
compared value
to the of for
listed the F127
molecular
copolymermass (Mwcompared
12,600to the
Da),
was probably due to compaction of the PPO chains of F127. There was also a peak at 7.59 min (3500of
listed for F127 copolymer (Mw 12,600 Da), was probably due to compaction of the PPO chains
F127.
Da), There was
probably also a peaktoata 7.59
corresponding mincopolymer
diblock (3500 Da),PEO-PPO
probably and/or
corresponding to a diblockadmixtures.
PPO homopolymer copolymer
PEO-PPO and/or PPO homopolymer admixtures. After the reaction,
After the reaction, the main polymer fraction (7 min) was dramatically decreased. New peaks the main polymer fraction
were(7
min) wasatdramatically
observed 10 min (major), decreased.
and 12 min New peaks which
(minor), were observed
were below at 10themin (major),
effective and 12 range
separation min (minor),
of the
which
GPC were below
column, the effective
suggesting that theirseparation
Mw values range
wereof below
the GPC 700column, suggesting
Da. Analysis of thethat their Mw
RI areas under values
the
were below 700 Da. Analysis of the RI areas under the curve suggested
curve suggested that under the optimized conditions used to produce GNPs 55% of the F127 chains are that under the optimized
conditionsFinally,
degraded. used to producethe
although GNPs
GNP55% did of notthe F127presence
reveal chains are degraded.
of intact F127,Finally, although
they definitely the GNP
contained
some organic component(s). This was confirmed by TGA analysis demonstrating that the This
did not reveal presence of intact F127, they definitely contained some organic component(s). portionwas
confirmed by TGA analysis demonstrating that the portion of this
of this component was approximately 13% w/w and it consisted of two compounds with the boiling component was approximately
13% w/w andofit242
temperatures consisted
◦ C and 348of two compounds with
◦ C (Supplemental Datathe boiling
Figure 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

C1

C2

C3

Figure 7. (A) 1 H-NMR, (B) 13 C-NMR, and (C) the IR spectra of (A1, B1, C1) original F127, (A2, B2, C2)
Figure 7. (A)
separated
1H-NMR, (B) 13C-NMR, and (C) the IR spectra of (A1, B1, C1) original F127, (A2, B2, C2)
reaction mixture and (A3, B3, C3) the purified gold nanoparticles.
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 these studies
and F127_3). we testedinseveral
The polymers different stock
these solutions solutions the
had essentially of F127
same(markedchemicalasstructure
F127_1,
F127_2 and F127_3). The polymers in these solutions had essentially the same chemical structure and
and molecular weight as confirmed by IR, NMR, and GPC of lyophilized aliquots. It is important to
molecular
note that duringweightlyophilization
as confirmed by IR, NMR,
volatile molar and GPCdegradation
mass of lyophilized aliquots.
products, It is important
which might have to been
note
that during
present in thelyophilization
solution, were volatile
lost and molar
could massnotdegradation
be detected products,
by any of whichthe appliedmightmethods.
have been present
However,
in
onethe solution,between
difference were lostthese and batches
could not was be indetected
the extentsby any of the applied
of polymer oxidation methods. However, data
(Supplemental one
difference between these
Table S2). Specifically, thebatches was in thelevel
hydroperoxides extents of polymer
in F127_3 was 2–3 oxidation (Supplemental
times higher than thesedata Table
in F127_1
S2).
and Specifically,
F127_2 (Supplemental the hydroperoxides
data Table S2). levelThe in F127_3
free radicalwaslevels
2–3 times
werehigher
comparablethan these in F127_1
in all three and
solutions.
F127_2
Comparison (Supplemental data Table
of these batches S2). Theconsiderable
discovered free radical levels were comparable
batch-to-batch variation inin allthe
three solutions.
formation of
Comparison
GNP. Thus, reacting of theseHAuCl
batches 4 ·3H discovered
2 O with considerable
F127_1 resulted batch-to-batch
in the GNP withvariation
UV in the
characteristics formation
and of
sizes
GNP.
similar Thus, reacting
to these of theHAuCl ·3H2O withabove.
GNP4described F127_1However,
resulted inreacting
the GNP with UV
HAuCl 4 withcharacteristics
F127_2 resulted and sizes
in a
similar
relatively to broader
these of PR the peak
GNPcentered
described at above.
600 nmHowever,
and a larger reacting HAuCl4 with
hydrodynamic size F127_2
(156 nm) resulted in a
of purified
relatively
GNP (Figure broader PR peak
8A, Table 4). Incentered
the caseatof600 nm and
F127_3, the aUVlarger hydrodynamic
spectra of the reaction sizemixture
(156 nm) of purified
exhibited 1) a
GNP (Figure
shift in 8A, Tablefrom
the absorbance 4). In220theto case
240of nmF127_3, the UV
indicative spectra
of Au 3+
of the reaction
reduction 0
to Au ; and mixture exhibited 1)
2) appearance of aa
shift
peakin at the
330absorbance from 220to
nm, corresponding toformation
240 nm indicative of Au3+ (Figure
of Au0 clusters reduction 8A). toHowever,
Au0; and 2) noappearance
PR peak was of
aobserved
peak at 330 nm,case,
in this corresponding
suggestingtothat formation
GNP did of not
Au0form.
clusters (Figure
Indeed, 8A).DLS
albeit However, no PR peak
of unpurified was
reaction
observed
mixture showedin this presence
case, suggesting
of 500 nm that GNP did(Supplemental
aggregates not form. Indeed, Dataalbeit
TableDLS S3), no of unpurified
GNPs were reaction
isolated
mixture showed
after standard presence ofprocedure
purification 500 nm aggregates
(Table 4). (Supplemental
Among notableData Table S3),
differences no GNPs
between thewere
batchesisolated
were
after standard purification procedure (Table 4). Among notable differences between the batches were
the larger particle sizes (60 nm) and more acidic pH of the F127_3 stock solution (pH 5.1) compared
the larger
to the particle
solutions of sizes (60 nm)
two other and more
polymer samplesacidic(pH pH6.8–6.9)
of the F127_3 stock solution
(Supplemental Data Table(pH 5.1)S2).compared
Since we
to the solutions
previously of twothat
established other thepolymer
formation samples
of the GNP (pH 6.8–6.9)
is dependent(Supplemental
on the pH of Data the Table S2). Since
copolymer we
solution,
previously
we adjustedestablished
the pH of the that the formation
F127_3 solution toofeither the GNPweakly is acidic
dependentpH 6.5onorthe pH of
alkaline pHthe copolymer
11.6. Notably,
solution,
pH adjustment we adjusteddid notthedecrease
pH of thethe F127_3 solution
particle size (74to either
nm atweaklypH 6.5acidic
and pH 81 nm 6.5 orat alkaline
pH 11.6), pHwhich
11.6.
Notably,
remainedpH much adjustment
larger than did not decrease
normally expected the for
particle size (74 nm
F127 micelles at pH
(27 nm) 6.5 and 81 nm
(Supplemental at pH
Data Table11.6),
S2).
which
However, remained
reacting much
HAuCllarger
4 ·3H than normally
2 O with expected
the F127_3 for F127
solutions micelles
with adjusted (27 pHnm)proceeded
(Supplemental Data
differently
Table
and, inS2).both However, reacting
cases, resulted HAuCl4·3H
in formation of2O withThus,
GNP. the F127_3
UV spectra solutions with adjusted
suggested that after pH proceeded
mixing F127_3
differently
solutions, pH and, 6.5inwith
bothHAuCl
cases, 4resulted
·3H2 O the in formation
reduction of of Au
GNP. Thus, UV spectra suggested that after
3+ was decelerated as was evident by the

mixing
presenceF127_3of a largesolutions,
peak ofpH Au 6.5atwith
3+ 220 nmHAuCl 4·3H8A).
(Figure 2O theAt reduction
the same time, of Au 3+ was
the peakdecelerated as was
of the Au0 clusters
evident
at 330 nm by disappeared.
the presence ofHowever,a large peak there ofwasAu3+aatsmall
220 nm and(Figure
broad 8A). At the
PR peak, same time,formation
suggesting the peak of of
the Au The
GNPs. 0 clusters
DLSat 330
has nm disappeared.
shown a presence of However, there was ainsmall
280 nm aggregates and broad
the reaction PR peak,
mixture suggesting
(Supplemental
formation
Data TableofS3), GNPs.
which The DLS
after has shownproduced
purification a presence 107ofnm280GNPs
nm aggregates
(Table 4). in When the reaction
the reactionmixture
was
(Supplemental
carried out using DatatheTable
alkaliS3), whichofafter
solution F127_3,purification
pH 11.6 the produced
reduction 107 ofnm AuGNPs (Table 4). When the
3+ was complete, and the

reaction
large PR was peakcarried
of GNPout was using
observed.the alkali solution
Overall, the UV of F127_3,
spectra werepH 11.6 the reduction
similar of Au3+ after
to these observed 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

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

conducting the reaction under basic conditions (Figure 4E,F). Moreover, the particle sizes were also
observed after conducting the
nearly identical (compare Tables 2 and 3). reaction 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

Absorbance (a.u)
F127_3
2 F127_3, pH 6.5
F127_3, pH 11
1

0
200 400 600 800
Wavelenght (nm)
B 3 F127_1L
F127_2L
Absorbance (a.u.)

F127_3L
2

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
and (B) after lyophilization. The HAuCl
was 0.25 mM and F127 concentration was 6.3%.
4 concentration was 0.25 mM and F127 concentration was
The reaction mixtures were incubated for 2 h (unless shown 1 h) at
6.3%. The reaction mixtures were incubated for 2 h (unless shown 45at◦ C.
1 h) 45 °C.

3.10. 3.10.
EffectEffect
of Lyophilization of the Copolymer Solutions on GNP Formation
of Lyophilization of the Copolymer Solutions on GNP Formation
In anInattempt to remove
an attempt hydroperoxides
to remove the polymer
hydroperoxides solutions
the polymer werewere
solutions lyophilized and re-dissolved
lyophilized and re-
in the dissolved in the same volume of bidistilled water using a previously described protocol [19].There
same volume of bidistilled water using a previously described protocol [19]. There was
little was
changelittle(less
change (less
than thanin
15%) 15%)
the in the hydroperoxides
hydroperoxides in F127_1,
in F127_1, approximately2-fold
approximately 2-fold increase
increaseinin the
the hydroperoxides
hydroperoxides in F127_2, in and
F127_2,
nearlyand nearly
6-fold 6-fold in
decrease decrease in the hydroperoxides
the hydroperoxides in F127_3
in F127_3 (Supplemental
data (Supplemental
Table S2). Moreover, data Table S2). Moreover,
lyophilization hadlyophilization had little
little if any effect if any
on the freeeffect on the
radical free which
levels, radical were
levels, which were similar in all studied samples. Although there was no effect
similar in all studied samples. Although there was no effect on the pH in F127_1 and F127_2 solutions,on the pH in F127_1
and F127_2 solutions, the pH of the reconstituted F127_3 solution increased from pH 5.1 to pH 6.7
the pH of the reconstituted F127_3 solution increased from pH 5.1 to pH 6.7 (Supplemental 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 were distinct
before lyophilization theand profound.
lyophilized and Specifically,
reconstituted in contrast
F127_3 to F127_3
solution was ablesolution
to reducebefore
lyophilization the lyophilized and reconstituted F127_3 solution was able to reduce Au 3+ and promote
Au and promote formation of GNP when mixed with HAuCl4 (Figure 8B and Table 4).
3+

formation of GNP when mixed with HAuCl4 (Figure 8B and Table 4).
Table 4. Characteristics of purified GNP synthesized using non-lyophilized and lyophilized F127
Tablesamples 1.
4. Characteristics of purified GNP synthesized using non-lyophilized and lyophilized
F127 samples 1 .
GNP synthesized using GNP synthesized using
F127 batch GNP non-lyophilized
Synthesized UsingF127 lyophilized F127 Using
GNP Synthesized
F127 Batch Non-Lyophilized F127 Lyophilized F127
Deff (nm) PDI Deff (nm) PDI
Deff (nm) PDI Deff (nm) PDI
F127_1 F127_1 62 ± 1 62 ± 1 0.16
0.16 ± 0.01
± 0.01 4747± ±1 1 0.30 ± 0.30
0.004± 0.004
F127_2 F127_2 157 ± 2157 ± 2 0.06
0.06 ± 0.02
± 0.02 7070± ±1 1 0.07 ± 0.07
0.01± 0.01
F127_3 Not formed Not formed 57 ± 1 0.30 ± 0.04
F127_3
F127_3 (pH 6.5) 108 ±Not
2 formed Not
0.17 formed
± 0.003 57 ±n/a1 0.30 ± 0.04n/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
1The 12.6% stock solutions of F127 solutions were prepared by dissolving the copolymer in bidistilled water. These
solutions were lyophilized and restored to the same volume by bidistilled water. GNP weren/a
F127_3 (pH 11.6) 39 ± 3 0.51 ± 0.12 n/a synthesized by mixing
The 12.6%
equal 1volumes of stock solutions ofand
non-lyophilized F127 solutions were
lyophilized F127 prepared by dissolving
solutions and the copolymer
HAuCl4 solution in bidistilled 6.3%
(final concentrations
w/w F127 and 0.25 mM HAuCl4 ) followed by incubation of the mixture for 2 h at 45 ◦ C. Particles were purified
water. These solutions were lyophilized and restored to the same volume by bidistilled water. GNP
as described in Table 1. The purified GNPs were restored to the original volume using bidistilled water (particles
were synthesized
concentration remained bythe mixing equaland
same before volumes of non-lyophilized and lyophilized F127 solutions and
after purification).
 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 study
of F127 measured
solution withchanges
HAuClin the content of free radicals and hydroperoxides before and after
4 ·3H2 O (Figure 9A). At 2 h the content of free radicals remained
mixing of F127 solution with HAuCl 4·3H2O (Figure 10A). At 2 h the content of free radicals remained
relatively small, however, there was a very considerable increase in the hydroperoxides. To elucidate
relatively
the small, however,
role of oxygen there was
in the formation a very
of the considerable in
hydroperoxides increase in the
the course ofhydroperoxides. To elucidate
the reaction we purged the
the role of oxygen in the formation of the hydroperoxides in the course of the reaction
copolymer solution with nitrogen for 5 min before initiating the reaction. This resulted in attenuation we purged the
ofcopolymer
formation of solution with nitrogenin
the hydroperoxides for
the 5 min before
reaction initiating
mixture the 9A).
(Figure reaction. This
In this resulted
case, we didinnot
attenuation
register
of UV
the formation
spectra ofto the
avoidhydroperoxides
exposure of the insolutions
the reaction mixture
to the (Figureoxygen.
atmospheric 9A). In However,
this case, the
we visual
did not
register the UV spectra to avoid exposure of the solutions to the atmospheric oxygen.
observation of the reaction mixtures at 2 h revealed that the color of the solutions was different—pink However, the
visual observation of the reaction mixtures at 2 h revealed that the color of the solutions was different
in before purging (Figure 9B) and purple after purging (Figure 9C). This suggested that the PR has
—pink in after
red-shifted before purgingwhich
purging, (Figure 9B) and purple
is indicative after purging
of the alteration (Figure
of the 9C).
particle This
size andsuggested
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
EPR arbitrary units (*10^3)

C
400

300

200

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 HAuCl4 ·3H2 O Reduction by Pluronic
3.12. Mechanism of HAuCl4·3H2O Reduction by Pluronic
Analysis of the data allows to propose the following schematic for HAuCl4 ·3H2 O reduction in the
presence Analysis of the data allows to propose the following schematic for HAuCl4·3H2O reduction in
of Pluronic:
the presence of Pluronic:
1. The decomposition of the hydroperoxides present in Pluronic leads to Pluronic chains degradation
1. The
anddecomposition
formation of of the hydroperoxides
lower molecular mass present in Pluronic
alcohols. leads toresults
This process Pluronicinchains degradation
the release of a
andhydroxonium
formation of lower
ion that molecular mass
contributes toalcohols. This of
acidification process results
the media andinformation
the releaseofofthe
a hydroxonium
superoxide
−• and hydroxyl radical HO• or hydrogen peroxide that can participate in further
ion O
that
2 contributes to acidification of the media and formation of the superoxide O2- and hydroxyl redox
radical HO orHere

reactions. hydrogen
we present peroxide that canand
the oxidation participate in further
decomposition redox
of the PPOreactions. Hereitwe
block because present
is known
the oxidation and decomposition of the PPO block because it is known to degrade faster than
to degrade faster than the PEO [20]. However, similar processes can also proceed at the PEO the
PEO [20].
block However,
and
polymers 2019, 11, similar
PEG homopolymer. processes
x; doi: FOR can also proceed at the PEO block and PEG
PEER REVIEW homopolymer.
www.mdpi.com/journal/polymers
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 reactiveion
oxygen
[AuClspecies formedacid
4]− undergoes in (1),
and(2), and hydrolysis
alkalli (3) promoteand
thegradually
reductiontransforms
of Au3+. Since
into
Polymers 2019, 11, 1553 16 of 21

2HO• → H2 O2 (3)

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
O2 −• + H3 O+ → HO2 • + H2 O (4)

[AuCl4 ]− + 3HO2 • + 3H2 O → Au0 + 3O2 + 3H3 O+ + 4Cl− (5)

[AuCl4 ]− + 3O2 −• → Au0 + 3O2 + 4Cl− (6)

2[AuCl4 ]− + 3H2 O2 + 6H2 O → 2Au0 + 3O2 + 6H3 O+ + 8Cl− (7)

In alkaline conditions
Au(OH)4 − + 3O2 −• → Au0 + 3O2 + 4OH− (8)

H2 O2 + OH− → H2 O + HO2 − (9)

2Au(OH)4 − + 3HO2 − → 2Au0 + 3O2 + 5OH− + 3H2 O (10)

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

(11)

4. Discussion
4. Discussion
Previous studies have demonstrated formation of GNPs in the presence of Pluronics without
Previous
use studies have
of any standard demonstrated
reducing formation
agent such as sodiumof GNPs
citrateinorthe
NaBHpresence of Pluronics without use
4 [22]. The mechanism postulated
of any standard reducing agent such as sodium citrate or NaBH [22]. The mechanism
in these works includes: 1) reduction of [AuCl4]− ions by PEO4 chains leading to the formation postulatedofinAu0
these works2)includes: 1) reduction ofchains
[AuCl4of −
] the
ions by PEO chains leading toclusters
the formation of Au0
clusters; adsorption of the PPO block copolymers on these with subsequent
clusters; 2) adsorption
reduction [AuCl4]− on of the
thecluster
PPO chains of the
surface; andblock copolymers
3) growth on these
of the GNPs clusters
stabilized bywith subsequent
the adsorbed block
reduction [AuCl ] − on the cluster surface; and 3) growth of the GNPs stabilized by the adsorbed
copolymers [9]. In this mechanism known as ‘three-step reduction and particles formation’ the first
4
block
twocopolymers [9]. In to
stages correspond this
themechanism
nucleation known
of GNPs, asand
‘three-step
the thirdreduction
stage to theand particles
growth formation’
or the nuclei into
themature
first twonanoparticles.
stages correspond The mechanism suggests that the copolymer plays a crucial roleorinthe
to the nucleation of GNPs, and the third stage to the growth both
nuclei into mature nanoparticles. The mechanism suggests that the copolymer
processes. However, to the best of our knowledge, the specific steps in the mechanism and the plays a crucial role in
both processes.
resulting However,
products havetonot thebeen
best not
of our knowledge,
precisely definedtheand/or
specific steps in the
separated frommechanism
the reactionand the
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
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
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
HAuCl4 ·3H2 O 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 (1 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 13 C-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, 1 H-NMR, or 13 C-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 HAuCl4 ·3H2 O by Pluronic,
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 • , H2 O2 , 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 HAuCl4 ·3H2 O concentration appears to be more
complex. We observed formation of small spheres at low concentrations of HAuCl4 ·3H2 O, followed by
larger spheres, multiple shapes, and spheres again as the HAuCl4 ·3H2 O concentration increased. Since
HAuCl4 ·3H2 O contained some amount of Au0 upon change of its concentration there was likely a
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.

References
1. Saha, K.; Agasti, S.S.; Kim, C.; Li, X.; Rotello, V.M. Gold nanoparticles in chemical and biological sensing.
Chem. Rev. 2012, 112, 2739–2779. [CrossRef] [PubMed]
2. Nash, M.A.; Lai, J.J.; Hoffman, A.S.; Yager, P.; Stayton, P.S. Smart Diblock Copolymers as Templates for
Magnetic-Core Gold-Shell Nanoparticle Synthesis. Nano Lett. 2010, 10, 85–91. [CrossRef] [PubMed]
3. Oishi, M.; Hayashi, H.; Uno, T.; Ishii, T.; Iijima, M.; Nagasaki, Y. One-pot synthesis of pH-responsive PEGylated
nanogels containing gold nanoparticles by autoreduction of chloroaurate ions within nanoreactors. Macromol.
Chem. Phys. 2007, 208, 1176–1182. [CrossRef]
4. Sun, X.P.; Jiang, X.; Dong, S.J.; Wang, E.K. One-step synthesis and size control of dendrimer-protected gold
nanoparticles: A heat-treatment-based strategy. Macromol. Rapid. Commun. 2003, 24, 1024–1028. [CrossRef]
5. Toroz, D.; Corni, S. Peptide synthesis of gold nanoparticles: The early steps of gold reduction investigated by
density functional theory. Nano Lett. 2011, 11, 1313–1318. [CrossRef] [PubMed]
6. Longenberger, L.; Mills, G. Formation of Metal Particles in Aqueous Solutions by Reactions of Metal
Complexes with Polymers. J. Phys. Chem. 1995, 99, 475–478. [CrossRef]
7. Sakai, T.; Alexandridis, P. Spontaneous formation of gold nanoparticles in poly (ethylene oxide)-poly
(propylene oxide) solutions: Solvent quality and polymer structure effects. Langmuir 2005, 21, 8019–8025.
[CrossRef] [PubMed]
8. Sakai, T.; Alexandridis, P. Size- and shape-controlled synthesis of colloidal gold through autoreduction of
the auric cation by poly (ethylene oxide)-poly (propylene oxide) block copolymers in aqueous solutions at
ambient conditions. Nanotechnology 2005, 16, S344–S353. [CrossRef] [PubMed]
9. Sakai, T.; Alexandridis, P. Mechanism of gold metal ion reduction, nanoparticle growth and size control in
aqueous amphiphilic block copolymer solutions at ambient conditions. J. Phys. Chem. B 2005, 109, 7766–7777.
[CrossRef]
10. Shou, Q.; Guo, C.; Yang, L.; Jia, L.; Liu, C.; Liu, H. Effect of pH on the single-step synthesis of gold
nanoparticles using PEO-PPO-PEO triblock copolymers in aqueous media. J. Colloid Interface Sci. 2011, 363,
481–489. [CrossRef]
11. Chen, S.; Guo, C.; Hu, G.H.; Wang, J.; Ma, J.H.; Liang, X.F.; Zheng, L.; Liu, H.Z. Effect of hydrophobicity
inside PEO-PPO-PEO block copolymer micelles on the stabilization of gold nanoparticles: Experiments.
Langmuir 2006, 22, 9704–9711. [CrossRef] [PubMed]
12. Khullar, P.; Singh, V.; Mahal, A.; Kaur, H.; Banipal, T.S.; Kaur, G.; Bakshi, M.S. Tuning the Shape and Size of
Gold Nanoparticles with Triblock Polymer Micelle Structure Transitions and Environments. J. Phys. Chem. C
2011, 115, 10442–10454. [CrossRef]
13. Polte, J.; Emmerling, F.; Radtke, M.; Reinholz, U.; Riesemeier, H.; Thunemann, A.F. Real-Time Monitoring of
Copolymer Stabilized Growing Gold Nanoparticles. Langmuir 2010, 26, 5889–5894. [CrossRef] [PubMed]
14. Ray, D.; Aswal, V.K.; Kohlbrecher, J. Synthesis and Characterization of High Concentration Block
Copolymer-Mediated Gold Nanoparticles. Langmuir 2011, 27, 4048–4056. [CrossRef] [PubMed]
15. Dikalov, S.I.; Dikalova, A.E.; Bikineyeva, A.T.; Schmidt, H.; Harrison, D.G.; Griendling, K.K. Distinct roles of
Nox1 and Nox4 in basal and angiotensin II-stimulated superoxide and hydrogen peroxide production. Free
Radic. Biol. Med. 2008, 45, 1340–1351. [CrossRef] [PubMed]
16. Bohorquez, M.; Koch, C.; Trygstad, T.; Pandit, N. A study of the temperature-dependent micellization of
pluronic F127. J. Colloid Interface Sci. 1999, 216, 34–40. [CrossRef] [PubMed]
17. Nukolova, N.V.; Oberoi, H.S.; Cohen, S.M.; Kabanov, A.V.; Bronich, T.K. Folate-decorated nanogels for
targeted therapy of ovarian cancer. Biomaterials 2011, 32, 5417–5426. [CrossRef]
18. Innocenzi, P.; Malfatti, L.; Piccinini, M.; Marcelli, A. Evaporation-Induced Crystallization of Pluronic F127
Studied in Situ by Time-Resolved Infrared Spectroscopy. J. Phys. Chem. A 2010, 114, 304–308. [CrossRef]
Polymers 2019, 11, 1553 21 of 21

19. Kumar, V.; Kalonia, D.S. Removal of peroxides in polyethylene glycols by vacuum drying: Implications in
the stability of biotech and pharmaceutical formulations. Aaps Pharmscitech 2006, 7, E47. [CrossRef]
20. Erlandsson, B. Stability-indicating changes in poloxamers: The degradation of ethylene oxide-propylene
oxide block copolymers at 25 and 40 ◦ C. Polym. Degrad. Stab. 2002, 87, 571–575. [CrossRef]
21. Mironov, I.V.; Makotchenko, E.V. The Hydrolysis of AuCl4- and the Stability of Aquachlorohydroxocomplexes
of Gold(III) in Aqueous Solution. J. Solut. Chem. 2009, 38, 725–737. [CrossRef]
22. Abdullin, T.I.; Bondar, O.V.; Shtyrlin, Y.G.; Kahraman, M.; Culha, M. Pluronic Block Copolymer-Mediated
Interactions of Organic Compounds with Noble Metal Nanoparticles for SERS Analysis. Langmuir 2010, 26,
5153–5159. [CrossRef] [PubMed]
23. Kim, J.U.; Cha, S.H.; Shin, K.; Jho, J.Y.; Lee, J.C. Preparation of gold nanowires and nanosheets in bulk block
copolymer phases under mild conditions. Adv. Mater. 2004, 16, 459–464. [CrossRef]
24. Paclawski, K.; Fitzner, K. Kinetics of Gold (III) Chloride Complex Reduction Using Sulfur (IV). Metall. Mater.
Trans. B 2004, 35, 1071–1085. [CrossRef]
25. Paclawski, K.; Fitzner, K. Kinetics of Reduction of Gold(III) Complexes Using H2O2. Metall. Mater. Trans. B
2006, 37, 703–714. [CrossRef]
26. Rahme, K.; Gauffre, F.; Marty, J.D.; Payre, B.; Mingotaud, C. A systematic study of the stabilization in
water of gold nanoparticles by poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide) triblock
copolymers. J. Phys. Chem. C 2007, 111, 7273–7279. [CrossRef]
27. Straney, P.J.; Andolina, C.M.; Millstone, J.E. Seedless Initiation as an Efficient, Sustainable Route to Anisotropic
Gold Nanoparticles. Langmuir 2013, 29, 4396–4403. [CrossRef] [PubMed]
28. Murphy, C.J.; Thompson, L.B.; Chernak, D.J.; Yang, J.A.; Sivapalan, S.T.; Boulos, S.P.; Huang, J.; Alkilany, A.M.;
Sisco, P.N. Gold nanorod crystal growth: From seed-mediated synthesis to nanoscale sculpting. Curr. Opin.
Colloid Interface Sci. 2011, 16, 128–134. [CrossRef]
29. Goy-Lopez, S.; Taboada, P.; Cambon, A.; Juarez, J.; Alvarez-Lorenzo, C.; Concheiro, A.; Mosquera, V.
Modulation of Size and Shape of Au Nanoparticles Using Amino-X-Shaped Poly (ethylene oxide)-Poly
(propylene oxide) Block Copolymers. J. Phys. Chem. B 2010, 114, 66–76. [CrossRef]
30. Genc, R.; Clergeaud, G.; Ortiz, M.; O’Sullivan, C.K. Green Synthesis of Gold Nanoparticles Using
Glycerol-Incorporated Nanosized Liposomes. Langmuir 2011, 27, 10894–10900. [CrossRef]
31. Lee, Y.L.; Kim, D.W.; Shin, S.I.; Oh, S.G. Preparation of Au Colloids by Polyol Process Using NaHCO3 as a
Buffering Agent. Mater. Chem. Phys. 2006, 100, 85–91. [CrossRef]
32. Steinfeldt, N. In Situ Monitoring of Pt Nanoparticle Formation in Ethylene Glycol Solution by SAXS-Influence
of the NaOH to Pt Ratio. Langmuir 2012, 28, 13072–13079. [CrossRef]
33. Treguer-Delapierre, M.; Majimel, J.; Mornet, S.; Duguet, E.; Ravaine, S. Synthesis of non-spherical gold
nanoparticles. Gold Bull. 2008, 41, 195–207. [CrossRef]
34. Pandit, N.K.; Kisaka, J. Loss of gelation ability of Pluronic(R) F127 in the presence of some salts. Int. J. Pharm.
1996, 145, 129–136. [CrossRef]
35. Su, Y.L.; Liu, H.Z.; Wang, J.; Chen, J.Y. Study of salt effects on the micellization of PEO-PPO-PEO block
copolymer in aqueous solution by FTIR spectroscopy. Langmuir 2002, 18, 865–871. [CrossRef]
36. Boisselier, E.; Astruc, D. Gold nanoparticles in nanomedicine: Preparations, imaging, diagnostics, therapies
and toxicity. Chem. Soc. Rev. 2009, 38, 1759–1782. [CrossRef] [PubMed]
37. Carabineiro, S.A.C. Applications of Gold Nanoparticles in Nanomedicine: Recent Advances in Vaccines.
Molecules 2017, 22, 857. [CrossRef] [PubMed]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).