Journal of Pharmaceutical Sciences 109 (2020) 1780-1788

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Journal of Pharmaceutical Sciences

journal homepage: www.jpharmsci.org

Pharmaceutical Nanotechnology

Optimizing a Novel Au-Grafted Lipid Nanoparticle Through

Chelation Chemistry for High Photothermal Biologic Activity
Yu Gao 1, 2, Qingxin Mu 2, Lisheng Zhu 1, Ziying Li 1, Rodney J.Y. Ho 2, 3, *
1
Cancer Metastasis Alert and Prevention Center, College of Chemistry, Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and
Chemotherapy, Fuzhou University, Fuzhou 350108, China
2
Department of Pharmaceutics, University of Washington, Seattle, Washington 98195
3
Department of Bioengineering, University of Washington, Seattle, Washington 98195

a r t i c l e i n f o a b s t r a c t

Article history: Gold nanoparticles through nucleation of Au clusters have been extensively studied. However, due to low
Received 4 November 2019 potency, prolonged tissue retention, and irreversible accumulation, the safety considerations have limited
Revised 13 January 2020 their therapeutic and diagnostic applications. Novel gold nanostructures with retained physical properties
Accepted 11 February 2020
and higher biodegradability could be prepared by alternative approaches. Previously, a lipid nanoparticle
Available online 18 February 2020
(LNP) platform carrying gadolinium (Gd3þ) has been reported to eliminate through the biliary without
accumulation in the liver or kidney within 24 h. Inspired by this discovery, we investigated a new approach
Keywords:
lipid nanoparticles of forming gold nanoparticles using preformed LNPs grafting diethylenetriamine-pentaacetic acid as a
gold nanoparticles chelating agent. Tiny Au nanoparticles are formed by simply mixing Au3þ with preformed
photothermal diethylenetriamine-pentaacetic acideLNP. The Au3þ associates stably to these LNPs after a systematic
clinical translation
optimization. The Au-grafted LNPs are scalable and showed excellent photothermal effects when subjected
to near-infrared light irradiation. They exhibit enhanced light-induced tumor cell killing at higher effi-
ciency, compared with that of classical gold nanoparticles (citrated reduced). Given an additional small
dose (2 Gy) of gamma irradiation, Au-grafted LNP could produce synergistic photothermal and radio-
therapeutic effects under reduced light dose. The simple and adaptive nanoparticle design may enhance
the margin of safety of gold nanoparticles in the treatment of cancers and other diseases.
© 2020 Published by Elsevier Inc. on behalf of the American Pharmacists Association.

Introduction polyethylene glycol (PEG) polymers with surface hydration prop-

Gold in metallic state is inert, and colloidal gold is often used as
a tracer for molecular and cellular studies because of its unique
photophysical properties for tracing single-molecule localization
and trafficking within the cells and tissues.1,2 The nanoparticulate
gold nanoparticles of about 10-30 nm were commonly synthesized
using classical Turkevich method from gold (III) chloride interac-
tion with sodium citrate via nucleation reactions. Addition of other
reducing agents to gold nucleation reactions has been investigated
in great detail with respect to size, charge, and stability and means
to reduce aggregations in suspension.3 Some additional chemistry
has been developed to modify the gold nanoparticle surface to
allow gold particle surface modification via thio-linkages using
Conflicts of interest: There are no conflicts to declare.
The authors Yu Gao and Qingxin Mu contributed equally.
* Correspondence to: Rodney J.Y. Ho (Telephone: þ1 206 543 3796).
E-mail address: rodneyho@uw.edu (R.J.Y. Ho).
erty to reduce gold particle aggregation.4,5 Gold-thiol chemistry
also allows conjugation of gold nanoparticles to DNA aptamer,
peptide, and carbohydrate for target-selective gold particle docking
(binding).6-9 Also, drug-gold particle conjugates have been re-
ported with other innovative chemistry.10,11
With available gold nanoparticles, a number of biomedical ap-
plications exploited the photothermal properties. Some reports
demonstrated the potential of gold nanoparticles in heat-activated
cell killing for use in photothermal therapy (PTT),12 whereas others
demonstrated gold particleemediated DNA damage due to the
enhancement of cell’s sensitivity to gamma or X-ray irradiation for
use as an adjuvant to radiation therapy (RT).13 These reports based
on cell studies have provided promising results.
However, in animal model studies, long resident time and slow
elimination of gold nanoparticles in tissues become a major chal-
lenge.14 With a single intravenous IV dose of 20 nm citrate-gold
nanoparticle (~0.7 mg Au/kg) in rats, the liver retained significant
amount of gold particles at day 28 (3920 ± 592 ng/g; ~1/3 retention
compared with 13,205 ± 1291 ng/g at 30 min).15 The high gold

https://doi.org/10.1016/j.xphs.2020.02.010
0022-3549/© 2020 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
 Y. Gao et al. / Journal of Pharmaceutical Sciences 109 (2020) 1780-1788
concentrations in the liver may be related to mild anemia and
spleen atrophy detected at day 28. In addition, gold nanoparticles
(d ¼ 8-37 nm) (8 mg/kg/wk) injected intraperitoneally were re-
ported to induce severe sickness in mice, and most of these mice
died within 21 days.16 Histopathological analysis revealed that the
accumulation of gold in mouse tissues caused an increase of Kupffer
cells in the liver, loss of structural integrity in the lungs, and
diffused white pulp in the spleen. Collectively, the data suggest that
nonpreferential uptake and retention of gold nanoparticles have led
to systemic toxicity. Inability of clearance of gold nanoparticles
limits the therapeutic potential of otherwise attractive photo-
thermal and radiation-adjuvant property of the gold nanoparticles.
Recently, we have reported a biocompatible and biodegradable
lipid nanoparticle (LNP) expressing gadolinium (lanthanide) that
exhibits limited liver uptake, and a majority of the particles are
cleared within 24 h. These LNPs contained diethylenetriamine-
pentaacetic acid (DTPA) with negative tetravalency that allows
stable association of gadolinium with positive trivalency (Gd3þ).17
When given to rats, a majority of these particles (d ¼ 60~70 nm)
were cleared from the blood through the bile and eliminated in
feces within 24 h. If DTPA particles could stably chelate Au3þ and
exhibit photodynamic response, it could overcome long resident
time of gold nanoparticles that often prevents clinical translation
because of toxicity concern. Binding onto these LNPs could promote
clearance of Au from the body and minimize systemic toxicity.
In this report, we evaluated the interaction of Au and DTPA-
conjugated LNPs and formation of stable Au-grafted LNPs (Au-
LNPs) based on the Gd-LNP platform, which has been proven to be
safe in preclinical studies. We found that Au is stably loaded on the
DTPA chelate that is stably inserted into lipid excipients in the LNPs.
The Au-LNPs are scalable and stable in suspension. Furthermore,
Au-LNPs showed excellent photothermal effects and photothermal
stability and exhibit enhanced light-induced tumor cell killing at
much higher potency than that of classical citrate-coated gold
nanoparticles.
Materials and Methods
Materials
1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-
distearoyl-sn-Glycero-3-phosphoethanolamine-N-DTPA (DSPE-
DTPA) were purchased from Avanti Polar Lipids (Alabaster, AL). 1,2-
Distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly (ethylene
glycol) 2000] (mPEG-DSPE) was obtained from Genzyme Pharma-
ceuticals (Cambridge, MA). Gold (III) chloride trihydrate
(HAuCl43H2O) and sodium citrate were purchased from Sigma
Chemical Co. (St. Louis, MO). The RPMI 1640 medium, fetal bovine
serum (FBS), and trypsin were purchased from Invitrogen Corporation.
Alamar Blue obtained from Sigma was used as a 10% solution unless
otherwise stated. Other ingredients were of analytical grade or higher.
Preparation and Characterization of Au-LNPs
The Au-DTPA LNPs were prepared by mixing LNPs with
HAuCl43H2O. DSPC, DTPA-DSPE, and mPEG-DSPE (18:5:2 molar
ratio) were dissolved in chloroform/ethanol, dried into a thin film
under N2, and placed in vacuum overnight. The phosphate-buffered
saline (PBS, pH 7.4) was added to the film and either sonicated or
extruded through 50 nm polycarbonic filter at 60 C to obtain the
LNPs. To prepare Au-bound LNPs, the LNPs in suspension were
mixed with Au3þ (1:1 DTPA-PE:Au3þ mole ratio) for at least 6 h. The
Au-LNP particle diameter was determined by laser light scattering
using a Nicomp 380/ZLS zeta potential analyzer (Particle Sizing
System) and expressed as mean ± SD. A transmission electron
1781
microscope (HT7700, Hitachi, Japan) image was detected after
negative staining with uranyl acetate (UA, 1%, w/v). The stability of
Au-LNPs was studied by checking the mean particle size of the
samples at different conditions. Each experiment was indepen-
dently performed at least 3 times. Citrate-stabilized gold nano-
particles were synthesized using the Turkevich reduction method.18
Photothermal Measurement for Au-LNPs
To study the photothermal properties of Au-LNPs, Au-LNP aqueous
solutions with different concentrations were irradiated with 808-nm
laser at different power density for different times. The temperature of
the solution samples for photothermal conversion measurement was
recorded by an IR thermal camera (E50, Arlington, VA).
Cell Culture
MDA-MB-231 cell line was obtained from the American Type
Culture Collection (ATCC, Middlesex, UK). The MDA-MB-231 cells
were grown in normal RPMI 1640 medium containing 10% FBS. The
cells were maintained in a humidified cell incubator at 37 C with
95% humidity and 5% CO2.
Photothermal Effects
Cell viability in terms of mitochondrial integrity was measured by
the Alamar Blue assay. MDA-MB-231 cells were seeded onto 96-well
plates at a density of 2  103 cells per well and cultured for 24 h.
Various concentrations of Au-LNPs were added into each well. After
being cultured for 4 h, the irradiation groups were exposed to 3.0 W/
cm2 light irradiation (heat lamp) for 2 min followed by cultivation for
another 5 days. The cells were then washed by PBS twice and then
treated with Alamar Blue solution. The plates were again incubated
for 4 h at 37 C. Fluorescence readings of the wells were recorded by a
PerkinElmer 1420 VICTOR 3V multi-well plate reader (PerkinElmer,
Waltham, MA) with excitation at 530 nm and emission at 580 nm.
Synergistic Photothermal Therapy and Radiotherapy
A total of 2  103 MDA-MB-231 cells were seeded in a 96-well
plate and cultured overnight. Various concentrations of Au-LNPs
were added into each well. After 4 h of incubation, the irradiation
groups were exposed to either 3.0 W/cm2 light irradiation for 1 min
(light bulb), or 2 Gy irradiation dose (Cobalt-60 Gamma-Ray Irra-
diator), or both, followed by cultivation for another 7 days. The cell
viability was then measured by Alamar Blue assay.
Statistical Analysis
Data are presented as the mean ± SD. Statistical analysis was
performed based on unpaired Student's t-tests (2-sided) or one-
way ANOVA with the SigmaPlot software (Systat, San Jose, CA).
Results
Interactions of Au and DTPA Expressed in Lipid Nanoparticles
We first evaluate the role of reaction order and DTPA/Au ratio on
nanoparticle formation. Three different orders used are schemati-
cally presented in Scheme 1. The first method (A) is mixing Au3þ
ions with DTPA-DSPE (molar ratio 1:1) to allow chelation in the
solution. The transparent solution of DTPA-DSPE turned to gray
suspension after the addition of Au3þ ions. Then the Au/DTPA-DSPE
suspension was mixed with DSPC/DSPE-PEG lipid suspension to
form Au LNPs. This product, designated as Au-LNPs1, exhibited
1782 Y. Gao et al. / Journal of Pharmaceutical Sciences 109 (2020) 1780-1788

Scheme 1. Preparation orders and appearance of differently formed Au-LNP. Au3þ ions in the solution were reacted with DTPA-DSPE before addition into the DSPC/DSPE-PEG lipid
solution (method a), directly added into the DSPC/DSPE-PEG/DTPA-DSPE lipid in suspension (method b), or added into the preformed lipid nanoparticles made from DSPC/DSPE-
PEG/DTPA-DSPE lipids (method c). The final products were monitored for color and suspension behavior at 25 C for 24 h. The molar ratio of DSPC/DSPE-PEG2000/DTPA-DSPE in all
preparations was 18:2:5. Au-LNPs1 and Au-LNPs2 readily aggregate. Au-LNPs3 demonstrates good suspension and chromogenic stability.

gray-to-whitish color in suspension. However, after overnight
storage, black precipitates appeared in solution of Au-LNPs1, sug-
gesting dissociation of gold or gold aggregates from Au-LNPs1. A
second method (B) directly adds Au3þ ions in solution to the DSPC/
DSPE-PEG/DTPA-DSPE lipid solution (molar ratio of DTPA-DSPE/
Au3þ ions 1:1). Then, the mixture was subjected to size-reduction
by sonication to form Au-LNPs (Au-LNPs2). The resulting purple-
colored Au-LNPs2 exhibited a mean diameter of ~30 nm. As the
color of gold nanoparticles, prepared by the nucleation method,
corresponds to the size of gold particles,19 it is possible that the
purple color might be a result from the gold aggregation. The Au-
LNPs2 was also found to be unstable, and precipitants were found
at the bottom of the purple suspension. Thus, a third method (C)
was deployed. Au3þ ions in the solution were added to preformed
LNPs containing DTPA-DSPE (with DSPC and mPEG-DSPE); (the
preformed LNPs were reported previously17). After 1.5 h incubation
of this mixture in suspension at 25 C, the LNPs (Au-LNPs3) turned
from yellow to green. Compared with the gold nanoparticles pre-
pared from the method A or B, Au-LNPs3 show good stability and
distinct color of the solution. In addition, the final method C is
simple and could be easily scalable. Therefore, we chose Au-LNPs3
for further optimization studies. A schematic representation of the
formation of Au-LNPs3 is shown in Scheme 2.
Optimizing Composition and Methods to Prepare Gold-Grafted Lipid
Nanoparticles Au-LNPs3
In initial studies, the stability, color appearance, and particle size
of Au-LNPs3 were closely related to the DSPE-DTPA-to-Au3þ molar
ratio and lipid concentration in preformed DTPA-LNPs. With the
total lipid concentration maintained at 7.75 mM, the color of so-
lution, the maximum absorption wavelength, and particle size
distribution of Au-LNPs3 are closely related to the DSPE-DTPA/Au3þ
molar ratios (Fig. 1). The color changed from transparent to pink
when decreasing the DSPE-DTPA/Au3þ molar ratio from 10 to 5
(Fig. 1a). When the DSPE-DTPA/Au3þ molar ratio decreased to 1, the
color of the aqueous solution became green. When the DSPE-DTPA/
Au3þ molar ratio reached 0.2, the preformed LNPs were unable to
provide adequate stabilization capacity for gold nanoparticles and
solid gold could be found. The maximum absorption wavelengths
of the Au-LNPs3 increased from about 530 nm to 650 nm when the
DSPE-DTPA/Au3þ molar ratio decreased from 10 to 0.5 (Fig. 1b).
The particle size distribution of Au-LNPs3 varied with increasing
DSPE-DTPA/Au3þ molar ratio. Using the dynamic light scattering
method, 2 populations of Au-LNPs3 were found, similar to that of
blank LNPs, albeit, at much smaller size distribution. Two-thirds of
the particle size exhibited at 16.8 nm, and one-third of the particle

Scheme 2. The schematic representation of the formation of Au-LNPs3.

 Y. Gao et al. / Journal of Pharmaceutical Sciences 109 (2020) 1780-1788
Figure 1. Effects of the DSPE-DTPA/Au3þ molar ratio on the properties of Au-LNPs3. The color (a), the maximum absorption wavelength (b), and the particle size distribution (c) of
Au-LNPs3 changed as the DSPE-DTPA/Au3þ molar ratio changed.
size was about 31.7 nm. The Au3þ solution was also tested for particle
size detection, but no readout could be obtained. Compared to the
size distribution spectra of blank LNPs, Au-LNPs3 showed larger but
different particle size and size distribution (Fig. 1c), suggesting the
apparent size increase is due to Au associated to and grafted on the
LNPs. With an increase of the DSPE-DTPA/Au3þ molar ratio, the
percentage of the size in the range of 11.0-11.4 nm decreased. When
the DSPE-DTPA/Au3þ molar ratio was higher than 2, particles size in
the range of 100-250 nm was found, indicating the aggregation of the
Au-LNPs3 due to the strong reduction of Au3þ by excessive lipid.
Based on the data collected with varying DSPE-DTPA/Au3þ, the
optimal DSPE-DTPA/Au3þ ratio range was within 0.5~1.5.
We next determined the effects of initial lipid concentration on
the formation of Au-LNPs3. We found that the particle size distri-
bution vary with the total lipid concentrations during the Au addi-
tion to form Au-LNPs3. As shown in Figure 2 at a fixed DSPE-DTPA/
Au3þ molar ratios of 0.5, 1, or 5, the proportion of particles with
larger size appeared to increase with lower lipid concentration.
Thus, to obtain smaller particle size, total lipid concentration should
be 1 mM or higher at 25 C to avoid the formation of large aggregates.
Through observation of the color of mixed solution of LNPs and
Au3þ, we found that the color turned from yellow to blue at about
1 h incubation time at 25 C room temperature, and the color turned
from blue to green in about 1.5 h. The color continued to darken,
suggesting larger aggregate formation up to about 6 h. At DTPA-
Figure 2. Effects of the initial lipid concentration on the particle size distribution of Au-LNPs3. The particle size distribution varied with the total lipid concentrations when the
DSPE-DTPA/Au3þ molar ratios were fixed at 0.5 (a), 1 (b), and 5 (c).
1783
DSPE/Au3þ molar ratio of 1 and lipid concentration of 7.75 mM, we
found time-dependent effects on the particle size distribution of
Au-LNPs3 at 25 C (Fig. 3). It was reported that the formation of gold
nanoparticles by the reduction of Au(III) complex ions is a very
complex process, and the reaction mechanism constitutes several
steps.20 We speculate that the formation of the Au-LNPs3 is a
multistage dynamic process. Thus, the particle size distribution of
Au-LNPs3 did not seem to follow a particular trend over the reac-
tion time. To provide sufficient time to complete Au3þ and LNP
interactions, each mixture at 25 C was allowed to react overnight.
We further investigated the effect of the temperature on the
characteristic of Au-LNPs3. Increasing the temperature from 25 C to
45 C appeared to accelerate the formation of particles as indicated
by color change. However, the color of the suspension turned from
yellow to purple, which suggests Au dissociation from the nano-
particles or aggregation of the gold nanoparticles. When we kept the
mixture of LNPs and Au3þ in the 4 C, the reaction was much slower
than higher temperature. Only slight green (an indicator of Au-
LNPs3 formation) was detectable only after 2-week incubation.
Based on these data, we found that DTPA-DSPE-to-Au3þ optimal
ratio is 1:1, and total lipid concentration is 7.75 at 25 C for 12-18 h to
provide Au-LNPs3 preparation in a reproducible manner. Au-LNPs3
showed superior stability. There are no obvious changes in the
particle size of Au-LNPs3 even in the presence of 10% FBS for 48 h
(Fig. 4). Therefore, this optimal condition is used for further studies.
1784 Y. Gao et al. / Journal of Pharmaceutical Sciences 109 (2020) 1780-1788
Figure 3. The effects of the reaction time on particle size distribution of Au-LNPs3. Au-
LNPs3 were prepared with DSPE-DTPA/Au3þ molar ratio of 1 and lipid concentration of
7.75 mM.
Characterization of Au-LNPs3 Morphology and Photothermal
Properties
To evaluate the morphology of Au-LNPs3 and compare to typical
citrate stabilized gold nanoparticles, we analyzed these particles
with electron microscopy (Fig. 5). To improve contrast resolution of
lipids in electron micrograph, we stained LNPs with 1% UA. As
shown in Figure 5, we found that LNPs exhibit an elongated and
rice-like shape with dimensions of ~25  15 nm (Fig. 5a). Blank
LNPs without staining (Fig. 5b) did not show any discernible par-
ticle. Au-LNPs3 stained with 1% UA showed size and shape similar
to those of LNPs (Fig. 5c), indicating that the chelation of Au did not
change the morphologies of LNPs. Small black dots could be clearly
seen in the image of Au-LNPs3 without staining (Fig. 5d), which
could be attributed to the formation of the ultra-small gold nano-
particles bound on the LNPs. The morphologies of the Au-LNPs3 at
different DTPA-DSPE/Au3þ molar ratios were also investigated.
Because of low Au concentration, it is difficult to find gold dots
when the DTPA-DSPE/Au3þ molar ratio was higher than 1. Inter-
estingly, Au-LNPs3 contained snowflake-shaped gold nano-
structure when DTPA-DSPE/Au3þ molar ratio was 0.5 (Fig. 5e). This
could be due to the insufficiency of the DTPA-DSPE in LNPs to
stabilize ultra-small gold nanoparticles. The shape of the Au-LNPs3
Figure 4. The stability of Au-LNPs3 in RPMI 1640 þ 10% FBS medium measured by the
changes of the particle sizes. Au-LNPs3 were prepared with DSPE-DTPA/Au3þ molar
ratio of 1 and lipid concentration of 7.75 mM. The particle size was shown as Z-average
hydrodynamic diameter.
at DTPA-DSPE/Au3þ molar ratio 1 was entirely different from that of
citrate-coated gold nanoparticles (Fig. 5f).
To evaluate the photothermal properties of Au-LNPs3, the
temperature changes of each solution were recorded at various Au
concentrations (0, 0.15, 0.5, and 1.5 mM) under near infrared (NIR)
laser irradiation (808 nm, 0.3~0.9 W/cm2). As illustrated in
Figure 6a, it was observed that with increase of Au-LNPs3 con-
centration, the solution temperatures increases greatly. After 4 min
laser irradiation (0.9 W), the solution temperature of Au-LNPs3 (1.5
mM) shows an increase of around 25 C, suggesting good photo-
thermal effect of Au-LNPs3. In contrast, PBS showed only a slight
temperature increase of 4 C. Moreover, the temperature changes
under different power intensity (0.3, 0.5, 0.7, and 0.9 W/cm2) were
measured, as well at a concentration of 1.5 mM (Fig. 6b). It is
evident that the increase of NIR light intensity led to faster increase
of Au-LNPs3 suspension’s temperature.
We further evaluated the photothermal stability of Au-LNPs3,
under NIR laser irradiation (808 nm, 0.9 W/cm2), for 5 alternate
ON/OFF cycles (Fig. 6c). The temperature changes of Au-LNPs3 (1.5
mM) were consistent in each cycle, indicating the excellent pho-
tothermal stability of Au-LNPs3. We also evaluated the photo-
thermal effect of citrate-stabilized gold nanoparticles. Au-LNPs3
demonstrated excellent photothermal effects compared with
citrate-stabilized gold nanoparticles (Fig. 6d).
Effects of Au-LNPs3 Mediated Breast Cancer Cell Sensitivity to
Infrared and Gamma Irradiation
The photothermal effects of Au-LNPs3 were investigated in
MDA-MB-231 human breast cancer cells. Cell viabilities were
detected with or without light irradiation (3.0 W/cm2, 2 min) after
5 days of treatment. When Au concentrations were initially fixed at
16 mM, both Au-LNPs3 and citrate-stabilized gold nanoparticles
showed negligible toxicity toward MDA-MB-231 cells (Fig. 7). We
then performed a dose-dependent study to explore the ability of
Au-LNPs3 to enhance tumor cell killing under light exposure. With
light irradiation, the citrate-goldetreated group showed no cell
growth inhibition with over 90% cell viability. However, Au-LNPs3
with 2 min light irradiation could significantly inhibit cancer cell
growth, and the cell viability was lower than 10% with an equiva-
lent 15.6 mM Au concentration.
The synergistic photothermal and radiotherapeutic effects of
Au-LNPs3 were then investigated. Cells treated with Au-LNPs3
were exposed to a very low dose of irradiation including 3.0 W/
cm2 light irradiation for 1 min and 2 Gy gamma irradiation, fol-
lowed by cultivation for another 7 days for observation of the high-
energy, irradiation-induced cell death. Compared with cells treated
with Au-LNPs3 for 5 days, prolonging the incubation time to 7 days
could inhibit certain cell proliferation to about 80%, suggesting the
potential long-term toxicity of gold nanoparticles (Fig. 8). Cells after
treatment with Au-LNPs3 exposed to either 3.0 W/cm2 light irra-
diation for 1 min or 2 Gy gamma irradiation showed no obvious cell
proliferation inhibition effects. The combination of light and
gamma irradiation could significantly increase the anticancer ef-
fects of Au-LNPs3, indicating the synergistic photothermal and
radiotherapeutic effects of Au-LNPs3.
Discussion
Gold nanoparticles have been widely studied for biomedical
application because of their intrinsic optical, electronic, and phys-
icochemical properties.21 Gold nanoparticles have the ability to
absorb light in the visible or NIR region and generate heat.22 They
also have the ability to absorb X-rays and effectively enhance the
radiation cytotoxicity.23 For cancer treatment, gold nanoparticles
 Y. Gao et al. / Journal of Pharmaceutical Sciences 109 (2020) 1780-1788 1785

Figure 5. TEM images of the nanoparticles. (a) Blank LNPs stained with 1% UA. (b) Blank LNPs without staining. (c) Au-LNPs3 with DTPA-PE/Au3þ molar ratio of 1 stained with 1%
UA. (d) Au-LNPs3 with DTPA-PE/Au3þ molar ratio of 1 without staining. (e) Au-LNPs3 with DTPA-PE/Au3þ molar ratio of 0.5 stained with 1% UA. (f) Citrate-coated gold nanoparticles
without staining.

Figure 6. Photothermal heating curves for Au-LNPs3 suspension under NIR laser irradiation (808 nm, 0.9 W/cm2) at different concentrations (a) or under NIR laser irradiation (808 nm, 1.5
mM) at different laser power densities (b). (c) Photothermal stability of Au-LNPs3 aqueous dispersion under irradiation for alternate 5 ON/OFF cycles (808 nm, 0.9 W/cm2). (d) Comparison
of the photothermal heating curves for Au-LNPs3 and the citrate-stabilized gold nanoparticle aqueous solution under NIR laser irradiation (808 nm, 0.9 W/cm2, 1.5 mM).
1786 Y. Gao et al. / Journal of Pharmaceutical Sciences 109 (2020) 1780-1788
Figure 7. Photothermal effects of Au-LNPs3 in MDA-MB-231 cells. Cells were treated
with different concentrations of Au-LNPs3 or citrate-stabilized gold nanoparticles.
After being cultured for 4 h, the irradiation groups were exposed to 3.0 W/cm2 light
irradiation for 2 min, followed by cultivation for another 5 d.
have been extensively studied for PTT to cause direct antitumor
effects via photothermal ablation or for a combination of hyper-
thermia and RT by inducing mild hyperthermia that sensitizes cells
to RT. The optical and electrical properties of gold nanoparticles are
closely related with size, thickness, and shape. Au nanorods (~50
nm) and nanoshells (~130 nm) with longitudinal plasmon bands
(LSPR) in the NIR region (>800 nm) demonstrate good photo-
thermal and radiation therapeutic effects.24 The size of spherical
solid Au nanoparticles larger than 50 nm is more suitable for PTT
because of strong NIR absorption.25 However, particles with larger
size are likely to accumulate in the liver and lung and prove difficult
for elimination from the body through excretion, which may lead to
long-term toxicity and immunogenic response.14,26 Mice exposed
to commercially available 15-nm gold nanoparticles developed
granulomas in the liver, which transiently increased the proin-
flammatory cytokine interleukin-18 in serum level.27 Currently, due
to the safety concern, no gold nanoparticle products are available
for clinical application.2,28 The high degree of tissue retention of
gold nanoparticles, regardless of the size and shape, and long-term
safety on human health and reproduction concerns have prevented
the otherwise attractive physical and biological properties of gold
nanoparticles to enhance tumoricidal activities in vivo.29
To reduce tissue retention of gold particles, a number of reports
have attempted to reduce self-aggregation and surface modifica-
tion to enhance particle clearance from the tissues while retaining
Figure 8. Synergistic photothermal and radiotherapeutic effects of Au-LNPs3 in MDA-
MB-231 cells. Cells were treated with different concentrations of Au-LNPs3. After being
cultured for 4 h, the irradiation groups were exposed to either 3.0 W/cm2 light irra-
diation for 1 min, or 2 Gy gamma irradiation, or both, followed by cultivation for
another 7 d.
their photodynamic properties for application in photothermal
therapy. In one report, 15-nm spherical Au nanoparticles are mixed
with PEG shell to form stable particles at physiological pH; these
particles were able to absorb light in the visible region. Under acidic
condition, the Au nanoparticles containing lipoid acids caused
aggregation-dependent red shift into absorption optimum peak at
the NIR region.30 However, the irreversible aggregation might
cause severe toxicity reported with these particles. Other 4-5 nm
spherical Au nanoparticles with pH- or thermal-responsive
reversible aggregation properties were also developed.31,32 But
their reversible aggregation properties under the physiological
conditions were not studied. In addition, small Au nanoparticles
(<7 nm) may undergo rapid renal clearance; however, these
smaller particles are unable to accumulate in the tumor, thus
limiting their potential for use as photothermal cancer therapy.
Another approach to overcoming this limitation is to generate
hybrid systems by encapsulating small Au nanoparticles in larger
nanoparticle platforms such as liposomes33 and silica nano-
particles.34 These larger nanoparticle platforms can also provide
loading space for other imaging or therapeutic agents which could
provide an opportunity for multimodal treatment. However, the
difficulty of large-scale preparation, and their storage stability, may
impede the clinical translation of these hybrid systems. In our
laboratory, LNPs based on similar compositions were produced at a
large greater than 1 L scale, and they have been proved to be safe in
multiple preclinical species including in monkeys.35,36 Au-LNPs
developed similarly could be prepared through a very simple pro-
cess with easy scalability (Scheme 1). Within the Au-LNP hybrid
system, the ultra-small Au nanoparticles could bind on the LNPs
without changing the morphologies of LNPs (Fig. 5). The Au-LNPs3
showed excellent photothermal effects and photothermal stability
(Figs. 6a-6c). The photothermal effect of Au-LNPs was proved in
cancer cells (Fig. 7). Therefore, Au-LNPs3 might overcome the size
dilemma of gold nanoparticles and show great potential for further
development for clinical use.
Several techniques such as microwave, radiofrequency, laser, or
ultrasound are usually implemented in the clinic for delivering heat
to generate hyperthermia. However, the difficulty of heating deep
tumors impeded the clinical implementation of hyperthermia. The
optimal wavelength for best tissue penetration is in the NIR region
or further. Spherical Au nanoparticles absorb UV and visible light but
do not absorb NIR light significantly. So, it is generally a poor choice
to use small spherical Au nanoparticles for PTT. Gold nanoshells and
nanorods with large particle sizes were found to have strong
absorbance at ~800 nm and were used to treat subcutaneous tumors
in mice.22,37 However, gold nanoshells and nanorods with a particle
size larger than 50 nm showed poor tumor penetration. Consistent
with previous reports, the gold nanoparticles (produced by the
citrate nucleation method) showed poor photothermal effects un-
der 808-nm laser irradiation (Fig. 6d). By contrast, Au-LNPs3 showed
excellent photothermal effects and photothermal stability under
808-nm laser irradiation (Figs. 6a-6c). The results suggested that Au-
LNPs3 integrate the advantages of LNPs and gold, which allow for a
good tumor penetration/accumulation (mediated by LNPs) and
absorb NIR light to generate hyperthermia for tumor-killing effects.
Several studies have demonstrated that the sensitivity of radio
(or gamma irradiation) therapy (RT) could be enhanced by hyper-
thermia. Gold nanoparticles with unique optical properties have
been studied extensively in combined PTT and RT for synergistic
effects. It was reported that gold nanoparticles have the ability to
reduce the dose of X-rays and increase sensitivity to radiation in a
very radioresistant subcutaneous squamous cell carcinoma
(SCCVII) in mice during radiotherapy.38 Au-LNPs3 exposed to a very
low dose of irradiation, including 3.0 W/cm2 light irradiation for
1 min and 2 Gy gamma irradiation, could significantly increase the
 Y. Gao et al. / Journal of Pharmaceutical Sciences 109 (2020) 1780-1788
anticancer effects (Fig. 8). The results suggest the potential of Au-
LNPs3 to be used in combined PTT and RT. Although the exact
mechanism of synergy is not clear, it is possible that the mild hy-
perthermia may reduce the radioresistant hypoxic cell death,
leading to enhanced cell toxicity to gamma irradiation.39
Chelation therapy based on a metal chelator such as disodium
ethylenediaminetetraacetic acid (EDTA) and an oral iron chelator
FBS0701 has been used to treat heavy metal ion poisoning, neu-
rodegeneration, cancer,40 and cardiovascular diseases.41 Some
natural and synthetic antioxidants which possess metal-chelating
properties have been used in disease prevention and clinical re-
covery against heavy metal intoxication.42 Based on the proven
property of LNPs containing DTPA to chelate Gd3þ and the DTPA-
LNPs ability to clear from the blood and liver within 24 h,17 we
used LNPs in this study to form an optimal Au3þ chelated to LNPs
(referred to as Au-LNPs3 particles). Although the ionic form of gold
(Au3þ) without chelation is toxic, the nanoparticulate form of gold
has been shown to reduce nonspecific toxicity. The optimized
formulation Au-LNPs3 can provide a stable, reproducible, and
potentially scalable product. In addition, the approach of producing
LNPs with DTPA expressed on the surface may be useful to chelate
gold (III) and other metals to detoxify metal toxicity in humans.
Conclusion and Future Direction
Au/LNPs were synthesized by simply mixing Au3þ with pre-
formed LNPs with DTPA. As the LNPs loaded with Gd3þ have been
reported to clear from the liver and blood within 1 day, we expect
that the Au-loaded LNPs or Au-LNPs3 would likely be cleared in
similar manner, thus overcoming high degree of Au nanoparticle
retention (for months to years). The properties of Au-LNPs3 were
closely related with the DSPE-DTPA/Au3þ molar ratio and the lipid
concentration during reaction. Ultra-small gold nanoparticles
bound on the LNPs or Au-LNPs3 (~25  15 nm) could be prepared
reproducibly with DTPA-DSPE/Au3þ molar ratio of 1 and lipid
concentration of 7.75 mM at 25 oC for 18 h. Au-LNPs3 demonstrate
excellent stability and are highly scalable. Au-LNPs3 exhibit
enhanced light irradiationeinduced killing of tumor cells compared
with that of classical gold nanoparticles (prepared with citrate) and
show synergistic photothermal and radiotherapeutic effects. The
simple and adaptive nanoparticle design as well as increased safety
margin of these gold nanoparticles may have great potential for the
treatment of cancers and other diseases.
Acknowledgments
This work was supported by the National Institutes of Health
(UM1AI120176), Washington Life Science Discovery Fund (3983577
and 20079493), United States. This work also received support from
the National Natural Science Foundation of China (81871481 and
81571802), the Natural Science Foundation of Fujian Province
(2016J06020), the Fujian Provincial Youth Top-notch Talent Support
Program, and the China Scholarship Council (No. 201706655015),
China.
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