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Control of Surface Ligand Density on PEGylated Gold
Nanoparticles for Optimized Cancer Cell Uptake
Hongying Liu, Tennyson L. Doane, Yu Cheng, Feng Lu, Shriya Srinivasan, Jun-Jie Zhu,*
and Clemens Burda*
Polyethylene glycol (PEG), one of the most
widely used surface ligands, is a neutral
amphiphilic polymer that provides “stealth”
character to nanostructures.[8,9] PEGylation
of nanoparticles is a widespread approach
to improve therapeutic efficacy since being
first demonstrated by Ballou et al.,[10] generally increasing blood circulation, decreasing
immunogenicity, and slowing the clearance
by the reticuloendothelial system.[11,12] Daou
et al.[13] have correlated the behavior of PEG
length on in vivo processing of quantum
dots in mice via fluorescence microscopy,
demonstrating that optimizing PEGylation is critical for biological applications.
Recently, it was reported that PEGylated
hollow mesoporous silica nanoparticles and
PEGylated cationic polymers could be used
in drug delivery[14] and gene delivery,[15]
respectively. Up-conversion nanoparticles with unique multiphoton excitation
photoluminescence properties were also
grafted with PEG and then used for drug
delivery.[16] However, it has been reported
that PEGylated nanoparticles had reduced
interaction with cellular surface proteins
and consequently reduced cellular uptake.[17,18] Significant attention has been directed toward surface ligand design to improve
the cancer cell uptake. It is crucial to better understand the role of
PEG on the interaction between the Au NP and cancer cells.
Recent studies revealed that the biofunctionality of Au NPs
were related to surface ligand density and structure, which are
dependent on the total quantity of grafted ligand molecules,
ligand mass, and binding moieties.[19,20] These properties include
hydrodynamic size, ligand density, charge density, dispersity, and
stability. PEGylation is especially sensitive to the grafting density,
with the transition from a loose polymer organization (the socalled mushroom coating) to a more rigid structure (a brush conformation), which can effect nanoparticle properties including
protein adsorption resistance.[21] Effective control of nanoparticle surface properties is essential for their application in cancer
therapy, where Au NPs must reach specific site of interest
without aggregation and nonspecific protein adsorption, while
maintain the ability to be taken up by the tumor cancer cells.
Here, we present a robust quantitative approach for the optimization of such Au NP surface properties for in vivo applications.
Several methods have been used to measure the surface
properties of nanoparticles.[22,23] Dynamic light scattering
Surface chemistry plays a critical role in the solution phase behavior of gold
nanoparticles (Au NPs) for applications such as in situ diagnostics and drug
delivery. Polyethylene glycol (PEG), a hydrophilic polymer with low immunogenicity, is most commonly used for protecting Au NPs for biomedical
applications. The ligand density and molecular weight of PEG on the gold
nanoparticle surface are key factors that control the particles’ behavior.
Specifically, the total density of PEG ligands gives rise to a transition from a
disorganized, deformable polymer “mushroom” orientation to a more rigid
“brush” orientation. Here, it is investigated how to rationally control this
transition for Au NPs coated with PEG-SH molecules within the weight range
of 0.55 to 5 kDa, and evaluate their subsequent interaction with cancer cells.
Several complementary methods are used to evaluate the effect of PEGylation
on biologically relevant aspects, including surface ligand density, hydrodynamic size, dispersity, and cellular toxicity. In this work, the optimal synthesis
ratios of PEG:Au NPs for achieving stability and maximum dispersity with
0.55, 1, 2, and 5 kDa PEG are determined to be 2500, 700, 500, and 300,
respectively. Importantly, ratios that exceed those necessary for maximum
dispersion of the Au NPs as determined by UV–vis and DLS are found to be
the best ratios for highest cell viability.
1. Introduction
Gold nanoparticles (Au NPs) have attracted significant attention
in the past decade as a multifunctional platform for various bioapplications,[1–6] including drug and gene delivery, photothermal
therapy, biosensing, and radiosensitization, owing to their tunable
size, morphology, adjustable surface chemistry, and low toxicity.[7]
Dr. H. Liu, Dr. T. L. Doane, Dr. Y. Cheng, F. Lu,
S. Srinivasan, Prof. C. Burda
Department of Chemistry
Center for Chemical Dynamics
and Nanomaterials Research
Case Western Reserve University
Cleveland, OH 44106, USA
E-mail: [email protected]
Dr. H. Liu, F. Lu, Prof. J.-J. Zhu
State Key Laboratory of Analytical Chemistry for Life Science
School of Chemistry and Chemical Engineering
Nanjing University
Nanjing 210093, P. R. China
E-mail: [email protected]
DOI: 10.1002/ppsc.201400067
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(DLS)[24] is used for determination of hydrodynamic radius,
translational diffusion coefficient, and polydispersity of polymers, proteins, and nanoparticles.[25–28] Gel electrophoresis
(GEP) is an effective approach for studying nanoparticle surface chemistry and mobility.[29–32] However, no nondestructive
method exists to quantify the PEG ligand density on nanoparticle surfaces. Thermal gravimetric analysis (TGA) has been
previously used to estimate the density of PEG molecules on the
particle surfaces,[33] which determines the average total number
of PEG molecules on nanoparticle surface. TGA requires milligram amounts of functionalized particles for accuracy and is a
destructive analytical method, which makes it impractical to be
utilized as a routine analytic approach. Here, we present work
that aims to determine the optimal PEG ligand to nanoparticle
synthesis ratio for drug delivery applications, so that TGA analysis in the future can be minimized and eventually spared.
PEG ligand density also has a profound role on the interaction
between Au NPs and macrophages.[34] In this work, UV–vis spectroscopy, DLS, TGA, cell viability assays, and atomic absorption
spectroscopy (AAS) are used to study the aggregation, dispersion,
ligand grafting density, cytotoxicity, and cellular uptake, respectively. For this purpose, we made Au NPs of identical core size
(6 ± 2.5) with varying PEG ligand density, simply by adjusting the
PEG:Au NP synthesis ratio. In the following, we provide a systematic correlation between optimal grafting density of PEG and
its molecular weight, which is verified by multiple analytic techniques. We show that the resulting biofunctionality of Au NPs,
specifically cellular uptake and toxicity, critically depend on the
PEG surface ligand density and molecular weight. The results
of this study provide a quantitative approach to optimizing the
PEGylation of nanostructures for cancer cell targeting.
2. Results and Discussion
The PEGylation of Au NPs is typically conducted using a PEG
ligands functionalized with strongly binding end groups, which
are often composed of one or more sulfur ligands. While it has
been demonstrated that PEG-based ligands can be used directly
for gold nanoparticle synthesis,[35] the more general route is to
synthesize Au NPs via reduction of auric acid (AuCl3) using citrate in aqueous media (Turkevich method)[36] or sodium borohydride in inverse micelles (Brust–Shiffrin method)[37] and then
postfunctionalizing the nanoparticles with the functionalized PEG
ligands. In this work, Au NPs with an average diameter of 6 ±
2.5 nm (n = 200) were synthesized via the Brust–Shiffrin method
with dodecylamine (DDA) utilized as the weak-capping ligand
(see Supporting Information for full details). The Au NPs cores
were crystalline and adopt the shape of a regular-truncated octahedron,[38,39] and retained a similar morphology after PEGylation
(Figure S1, Supporting Information). The hydrophobic Au NPs
were then mixed directly with thiol-terminated PEG (HS-mPEG)
in chloroform over a period of 2 d to ensure maximum surface
coverage. The above methodology allows for facile control of (A)
maximum polymer layer thickness through PEG length (specifically 0.55, 1, 2, and 5 kDa PEG) and (B) maximum surface density through the control of the reaction ratio (100–3000:1 PEG:Au
NP ratios). Subsequent extraction with water, vacuum drying, and
purification through membrane filters results in a purified end
product.
2.1. Determination of the Stability and Hydrodynamic Diameter
The stability of PEGylated conjugates can be assessed both
qualitatively (by eye) and quantitatively (via UV–vis). Due to
the characteristic surface plasmon resonance (SPR) of Au NPs,
colorimetric visualization is a simple method to determine
colloidal dispersity and stability.[40,41] The color of Au NPs is
wine red in the well-dispersed stable state, while purple or blue
color corresponds to an aggregated state. Figure 1A shows the
photographs of Au NPs coated with different ligand ratios of
various PEG (0.55, 1, 2, and 5 kDa) as prepared in solution. The
color gradually changes from purple to red with increased PEG
Figure 1. A) Photographs and B)normalized UV–vis spectra of Au NPs after PEGylation with different ratios of PEG 0.55, 1, 2, and 5 kDa. For
0.55 kDa PEG capping, the curves assigned as 1–6 correspond to PEG:Au NP ratios of 1000, 1500, 2000, 2500, 3000, and 4000, respectively; for 1 kDa
PEG capping, the curves assigned as 1–8 correspond to PEG:Au NP ratios of 400, 500, 600, 700, 800, 900, 1000, and 2000, respectively; for 2 and 5 kDa
PEG capping, the curves assigned as 1–10 correspond to PEG:Au NP ratios of 200, 300, 400, 500, 600, 700, 800, 900, 1000, and 2000, respectively.
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Figure 2. A) Plots of surface plasmon resonance (SPR) wavelengths maxima and B) hydrodynamic radius versus different ligand:particle reaction ratios
for 0.55, 1, 2, and 5 kDa HS-mPEG. Inset shows 0.55 kDa PEG alone due to significant larger scale. C) Best PEG:Au NP ratios for stable dispersions
(rstab) ratios versus PEG molecular weight obtained from UV–vis absorption spectra and DLS. D) Plots of the hydrodynamic radius (black) and PEG
layer thickness (red) as function of PEG molecular weight at the PEG:Au NP synthesis ratio rstab.
density (PEG:Au NPs ratio) until a critical stability ratio (rstab,
the optimal ratio of PEG:Au NPs for maximum dispersion) is
reached.
At rstab, the PEG:Au NPs are stably dispersed and do not
aggregate. When the ratio of PEG:Au NPs is increased above
rstab, the color of the solution became purple, indicating additional interparticle interactions. The effect of ligand density
was also monitored quantitatively using UV–vis spectroscopy,
as shown in Figure 1B. The Au NP plasmon resonance blueshifted with increasing ratio of PEG:Au NPs, followed by a
slight red-shift with excess PEG for the 0.55 kDa PEG sample
(Table S1, Supporting Information). A possible reason is aggregation of Au NPs in the 0.55 kDa sample, which is due to intertwining of the PEG layers between to particles during PEGylation, which we have observed in recent work on the ligand
exchange dynamics on Au NPs,[42] and which has also been
observed during other PEGylation studies.[43] For all investigated samples, stability of the Au NPs improved with increased
PEG concentration, resulting in a blue-shift of the SPR and a
corresponding color change. The optimal ratio of PEG:Au NPs
for achieving stability with PEG 0.55, 1, 2, and 5 kDa was determined by spectroscopic means to be 2500, 700, 500, and 300,
respectively.
The above observations are in accordance with the Flory–
Krigbaum theory[44] that predicts that longer surface-bound
ligands and higher ligand coverage favor conjugate stability
because of increased steric repulsion. Au NPs are prone to
aggregation due to van der Waals forces and surface interactions when they outweigh the electrostatic and steric repulsion
Part. Part. Syst. Charact. 2015, 32, 197–204
provided by surface-bound ligands. Thus, it is important to balance the effects of PEG length and PEG:Au NPs ratio to achieve
stability for PEGylated Au NPs. Noteworthy, three trends can
be determined from plotting the SPR wavelength maxima
versus reaction ratio as shown in Figure 2A: (i) for 6 nm Au
NPs, as the PEG length increases, the stability of PEGylated
Au NPs increases, (ii) for any given PEG length, the stability of
PEGylated Au NPs increases when PEG:Au NP ratio increases
until the critical stability ratio, rstab, is reached, and (iii) the critical stability ratio increases with decreasing PEG length.
The correlation of the maximum SPR absorption wavelength to Au NP dispersity was further tested by examining the
hydrodynamic radius of the resulting particles for each PEGylation reaction ratio. Figure 2B showed an increase in hydrodynamic diameter (dhydronamic) of Au NPs from ≈15 to ≈38. This
was expected due to the protective PEG layer formation.[45]
Importantly, as with the SPR maxima shift, the stabilization of
the hydrodynamic diameters correlated to increasing reaction
ratios until reaching a plateau (Figure 2C). The hydrodynamic
diameter versus PEG length showed a gradual growth. Given
that the core size was 6 nm, the thickness of the PEG corona
(L) was calculated to be 4.4, 7.3, 9.4, and 16.4, for PEG 0.55, 1,
2, and 5 kDa, respectively. Fitting these values according to the
PEG molecular weight (MPEG) in kDa yielded L = 6.6 (MPEG)0.56,
which is in good agreement with previous work.[46] These values
are larger than the hydrodynamic size for the corresponding
free PEG molecules,[47] indicating that the PEG polymers can
be mutually aligned and elongated on the Au NPs surface
when the grafting density is high enough. Here, the change
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In each gel, the ratio of PEG:Au NPs
increases from left to right. As the PEG:Au
NP ratio increases, the distance traveled in
the gel also increased until the critical stability ratio was reached. The last lanes of
2 and 5 kDa PEG showed a slightly decreased
mobility, which can be interpreted to be
the influence of excess PEG on the Au NP
corona, possibly leading to entanglement of
particles and thus slower mobility.
Based on the GEP results, the electrophoretic mobility (µ) of the PEGylated Au
NPs can be calculated.[46] Table S3 (Supporting Information) shows the results of the
mobility for the studied Au NPs with PEG
from 0.55 to 5 kDa at different PEG:Au NP
ratios.
The mobility is obtained by Equation (1)
μ=
V distance/time
=
E voltage/length
(1)
Figure 3. GEP of Au NP solutions after the addition of different ratios of PEG 0.55, 1, 2, and
5 kDa, respectively. The ratios used in the insets were the same as in Figure 2 with increasing
PEG:Au NP ratios from left to right. The critical stability ratios for 0.55, 1, 2, and 5 kDa PEG
were 3000, 900, 800, and 400, respectively.
in diameter clearly confirmed the formation of a dense PEG
monolayer. The larger hydrodynamic diameter at low grafting
densities is due to the formation of Au NP aggregates in the
absence of sufficient PEG. The critical stability ratios assessed
through DLS for PEG 0.55, 1, 2, and 5 kDa were 2000, 400, 400,
and 300, respectively (Figure 3). These ratios are very similar to
those obtained via UV–vis spectroscopy.
2.2. Determination of Au NP Mobility
Since ligand coating changes the mobility of NPs, GEP was used
to determine the effect of PEG coating on the transport properties of Au NPs.[48] GEP has been shown to be a highly sensitive
technique for discerning the binding of PEG to nanoparticle
surfaces.[49] A standard GEP experiment for PEGylated-Au NP
conjugates using a 1% agarose gel (80 V, 4 h) was performed
as shown in Figure 3. The PEG 0.55 and 1 kDa capped Au NPs
moved toward the positive terminal, while those capped with
2 and 5 kDa PEG moved toward the negative terminal. While
the most direct interpretation is a difference in nanoparticle
charge, previously it has been shown that the balance of electrophoretic, frictional, and drag forces ultimately dictates the
movement of uncharged polymer-coated Au NPs in agarose
gels.[46] The use of vitamin B12 as an uncharged electroosmotic
flow tracer (which is also small enough to have negligible steric
interactions with the gel) indicates that electroosmotic forces
play a substantial role in the different migration behaviors of
PEGylated Au NPs. Based on Figure 3, the role of frictional and
drag forces on the PEGylated Au NPs increases with increasing
PEG length, resulting in a reversed mobility for significantly
large polymer brushes.
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where time is 4 h, voltage is 80 V, and length
is 22 cm. The measured mobility increased
with increasing PEG molecular weight and
increasing PEG:Au NP ratios until it leveled off, as shown in Table S3 (Supporting Information). The
optimal grafting ratios determined from gel electrophoresis for
0.55, 1, 2, and 5 kDa were 3000, 900, 800, and 400, respectively.
Interestingly, the transition from a loosely organized polymer
brush at reaction ratios below rstab causes a decrease in mobility
for all nanoparticles. Similar behavior has been observed for
PEGylation of 20 nm Au NPs with different length PEG,[50] and
most likely reflects the deformability of the polymer coating
leading to stronger interactions with the agarose hydrogel.
Moreover, the smearing of the Au NP bands diminishes with
increasing reaction ratios, indicating the formation of more
rigid polymer brushes. The results from GEP confirm saturation of the Au NP surface as in the previous study with 20 nm
Au NPs,[50] and help support the conclusions drawn from UV–
vis and DLS measurements.
2.3. Determination of the Ligand Density
The calculation of the ligand density via Au:S ratio is based
on the fact that each ligand on the Au NPs surface carries a
single S atom, while Au atoms constitute the Au NP core. The
PEG on the surface of Au NPs decomposes thermally, releasing
volatile S and leaving behind Au. For simplicity of calculation, the geometry of Au NPs is assumed to be spherical. The
ligand density of each Au NP is calculated from the number
of Au NPs and PEG chains, as previously described.[46,51–53]
Figure 4A compares the TGA results of Au NPs capped with
PEG 1 kDa and 5 kDa, demonstrating that the decomposition
temperature increases as the PEG molecular weight increases,
and confirming that the monolayer bound on Au NPs is thermally stable up to ≈400 °C. Increasing PEGylation reaction
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Figure 4. A) TGA of PEG 1, 5, and 10 kDa capped Au NPs at the critical stability ratio monitored as a function of temperature under dry nitrogen
atmosphere. B) TGA for PEG 5 kDa capped Au NPs with PEG:Au NP ratios of 400, 600, 800, and 1000. C) Relationship between PEG/particle (black
curve) and PEG nm−2 (red curve) for 5 kDa PEG capping at different PEG:Au NP ratios. D) TGA analysis of the critical ratios (3000, 900, 800, and 400)
for 0.55,1,2, and 5 kDa, respectively.
ratios generally lead to more 5 kDa PEG ligands on the surface (Figure 4B,C), but the actual changes were slight relative
to the large reaction ratio increases. Importantly, as with both
the SPR wavelength and dhydrodynamic plots, the trend of PEG:Au
NP versus PEG length can be described by an allometric function (Figure 4D). The actual PEG coverage at rstab from GEP
was found to be 2950, 917, 800, and 484, PEG/particle for PEG
0.55, 1, 2, and 5 kDa, with corresponding ligand densities of
28.5, 8.9, 7.7, and 4.7 PEG nm−2, respectively. When PEG of
a higher molecular weight is used, ligand density decreases
because the larger PEG molecules consume more space and
surface area around the anchoring thiol group, decreasing
the number of PEG molecules needed to saturate the surface
and to achieve a stable dispersion.[54] Thus, when using PEG
of decreasing length, a greater quantity is required to achieve
stable PEGylated Au NPs, increasing the critical stability ratio.
2.4. Determination of Toxicity and Cellular Uptake
The cytotoxicity of these Au NPs was investigated in relation
to PEG molecular weight and PEG:Au ratio. The biocompatibility of PEG is well-established, but little information is available regarding cytotoxicity of PEGylated Au NPs prepared at
different PEG:Au NPs ratios with PEG of different molecular
weights.[32] The cytotoxicity of PEGylated Au NPs to the HeLa
cervical cancer cells was quantified using a colorimetric assay
with 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), as shown in Figure 5. In general, the cytotoxicity
Part. Part. Syst. Charact. 2015, 32, 197–204
for PEGylated Au NPs increased as the concentration increased
from 5 to 200 × 10−9 M (Figure 5A). Additionally, the cytotoxic
effects of PEGylated Au NPs decreased with increasing PEG
molecular weight (Figure 5B). The cell viability of PEGylated
Au NPs was greater than 90% when the molecular weight of
PEG increases up to 5 kDa, showing that PEGylation was an
effective strategy for reducing cytotoxicity of NPs. The biocompatibility of PEGylated Au NPs increased as the PEG:Au NP
ratio increased up to the critical stability ratio, at which point
PEGylated Au NPs with the lowest cytotoxicity were obtained.
Figure 5C,D highlights 2 kDa PEG as an example. Interestingly,
the synthesis ratios which are ideal for dispersion and solubility for 2 kDa PEG (400–500), while having relatively low toxicity, could be further improved by the addition of further PEG
ligands. Recently, work by Yang et al.[55] has suggested that even
in brush conformations, the formation of dense brush coverage
helps further protect the core material from intermittent gaps
in the polymer coating. Our results here seem to indicate that
at higher grafting densities the transition to a dense brush can
occur, even if the relative number of PEG ligands remains relatively similar (see Figure 4C).
NP interactions with cancer cells are a major focus in nanomedicine and are an important aspect for designing drug
delivery systems utilizing nanotechnology platforms, since particles need to be optimized for efficient cellular uptake. Using
AAS, it was determined that the molecular weight of PEG
affected the cell uptake. As shown in Figure 6, the content of
Au in the cells decreased as the molecular weight of the PEG
ligand increased, which is in good agreement with work by
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Figure 5. Cell viability of Au NPs capped with PEG 0.55, 1, 2, 5, 10 kDa at A) different NP concentrations and B) plotted at a constant NP concentration
of 50 × 10−9 M. All samples in (A) and (B) are at the best PEG:Au NP ratios determined by GEP. Cell viability of C) Au NPs capped with PEG 2 kDa at
different PEG:Au NP ratios, and D) cell viability of Au NPs capped with PEG 2 kDa at 5 × 10−9 M constant concentration with different PEG:Au NP ratios.
Brandenberger et al.[56] Previous studies have reported that nonspecific binding to cells also decreased with increasing molecular weight of PEG,[57] and subsequent half-lives in vivo can be
improved by using larger PEG molecular weight. Conversely,
this also increases the hydrodynamic size[58] and decreases the
uptake of PEGylated Au NPs into cells. Thus, while it is important to that although rstab should always be optimized to ensure
Figure 6. Dependence of cellular uptake of AuNPs on the molecular
weight of the surface PEG ligand molecules at rstab as attained by AAS.
The estimated error of the AAS analysis is ±5%.
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the best dispersity and least macrophage detection, the choice
of which PEG length to use is more dependent on the specific
application intended.
3. Conclusion
Au NPs have increasingly become important systems for
nanoscale drug delivery for cancer therapy. Recently, it has
become evident that in addition to particle size and shape, the
surface ligand composition and grafting density play a vital role
in fine-tuning nanoparticle-based drug delivery systems. However, to date, a systematic study to optimize their transport,
targeting, and toxicity properties as a function of PEG length
and surface density has not been performed. In this paper,
we presented a quantitative approach for controlling dispersity, stability, cell viability, and cellular uptake of PEGylated Au
NPs as a function of PEG molecular weight and grafting density. Controlling these properties is critical for designing Au
NPs that meet the need for increased specificity in biomedical
applications.
A model system of ≈6 nm Au NPs with different PEG molecular weight and ligand densities was fully characterized by
TEM, UV–vis spectroscopy, DLS, TGA, GEP, MTT, and AAS.
It was found that most properties correlate directly with PEG
molecular weight and similarly with PEG density. The optimal
synthesis ratio ranges of PEG:Au NPs for achieving critical stability (rstab) for PEG 0.55, 1, 2, and 5 kDa were determined to
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be 2000–3000, 400–900, 400–800, and 300–500, respectively.
An increase in the PEG:Au NP ratio led directly to increased
PEG density on the nanoparticle surface, up to the point of
saturation, occurring at rstab. As the PEG molecular weight
decreases, the critical stability ratio, rstab, increases, necessitating more PEG molecules to stabilize the Au NP. The
mobility of PEGylated Au NPs decreased as the surface density
increased due to increased frictional drag and increased steric
interactions.
In addition, the properties of Au NPs that are important to
in vivo applications were analyzed. Cell viability of HeLa cells
toward incubation with PEGylated Au NPs increased as the
PEG:Au NP ratio increased. Cell viability was optimal when
treated with PEGylated Au NPs synthesized at ratios exceeding
the rstab established from UV–vis and DLS. Additionally, cellular
uptake decreased as the molecular weight of the PEG increased.
Thus, by modulating the PEG:Au NP ratio or the molecular
weight of the PEG used in synthesis, the physical and physiological properties of the NPs can be adjusted and optimized
for biomedical applications.
and 1% penicillin/streptomycin at 37 ºC in 5% CO2/95% air atmosphere.
Cells were washed with phosphate-buffered saline (PBS) and 0.25%
trypsin–EDTA was added for 3 min to detach the cells, which were
counted by a hematocytometer. Dulbecco’s PBS was used to wash seeded
cells. After the addition of 1 mL trypsin solution, the cells were incubated
at 37 ºC for 3 min and subsequently 5 mL of the medium was added.
Detached cells were transferred into a new culture dish and incubated in
an incubator containing 5% CO2 at 37 ºC until reaching confluence.
Cell Viability Studies: Cell viability was determined with an MTT
colorimetric assay (3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium
bromide) supplied by Roche.
Quantification of Au NPs in Cells by AAS Experiments: HeLa cells (2 ×
106) were identically added to each culture dish (60 mm × 15 mm). A
conjugate solution (2 mL) with Au NPs concentration of 3.3 × 10−8 M was
added to the dishes at 100% confluence. After incubation for 24 h, the
dishes were rinsed with DPBS (2 mL × 2) and 2 mL of Hank’s balanced
salt solution. The cells were collected and digested in aqua regia for
2 h. The solution was diluted, and the concentration of Au in the solution
was obtained via AAS. Each sample was measured six times.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
4. Experimental Section
Materials: Methoxy PEG thiols (mPEG-SH) of different molecular
weights (0.55, 1, 2, and 10 kDa) were purchased from Laysan Bio.
Tetra-n-octylammonium bromide (TOAB) (98%) was purchased
from Alfa Aesar. 30 wt% solution of hydrogen tetrachloroaurate (III)
(HAuCl4) in dilute hydrochloric acid (99.99%), NaBH4, Vitamin B12, and
dodecylamine (DDA) (98%) were purchased from Sigma–Aldrich. 10×
TAE buffer (Fisher) was diluted to 1× using deionized water.
Characterization: UV–vis absorbance spectra were conducted
using a Cary 50 Bio spectrometer. DLS was obtained on a ZetaPals
dynamic light scattering instrument (Brookhaven Instruments Corp.).
Transmission electron microscopy (TEM) was performed using a JEOL
JEM 1200 EX electron microscope at 80 keV accelerating voltage. Images
of the GEP results were obtained using a digital camera (Olympus
D-595) on an optical bench with a fixed frame for consistent imaging.
Thermogravimetric analysis (TGA) was performed on a TA Instruments
Q500 TGA at a heating rate of 10 ºC min−1 from 25 to 800 ºC with
nitrogen purging at a flow rate of 40 mL min−1; 2–5 mg samples were
placed in an open platinum crucible for all tests.
Synthesis of PEGylated Au NPs: PEGylated Au NPs were synthesized
according to a previous report.[38] 0.25 mmol (0.137 g) of tetraoctly
ammonium bromide, 0.53 mmol of HAuCl4 (367 µL of 30% stock
solution), and 0.6 mmol (0.111 g) of dodecylamine were combined in 5 mL
of toluene and allowed to mix. 2 mmol (0.076 g) of sodium borohydride
dissolved in 1 mL of ice cold water was added dropwise until bubbling
subsided. The reaction was allowed to proceed for 20 min and then
quenched with excess ethanol in a 50-mL centrifuge tube (VTotal = 45 mL).
The solution was centrifuged at 6000 G for 10 min, decanted, and the
wash was repeated a second time. After the second wash, the pellet
was dried with an Ar stream and dissolved in chloroform, with the
exact concentration determined via UV–vis (ε = 1.5 × 107 cm−1M−1).
PEGylation was achieved by weighing out the desired mass of
HS-mPEG in a glove box and then added to the solution of Au NPs
in chloroform using 1 mL of the solvent to rinse out the vial. The
reaction was allowed to proceed under constant stirring for 48 h, and
subsequently the organic phase was washed six times with water and
then evaporated under vacuum. The particles were cleaned using a
30 kDa molecular-weight-cutoff filter (Sartorious) with deionized water.
The cleaning step is repeated 7–10 times to remove all excess PEG.
Cell Cultures: HeLa cells were cultured in Dulbecco’s Modified Eagle
Medium (DMEM, Sigma) supplemented with 10% fetal bovine serum
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Acknowledgements
J.-J.Z. thanks the support from NSFC (No. 21020102038) and National
Basic Research Program of China (No. 2011CB933502).
Received: April 2, 2014
Revised: May 27, 2014
Published online: September 2, 2014
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