Received: 1 January 2022 Accepted: 7 March 2022

DOI: 10.1002/jccs.202200001

ARTICLE

Conquering multidrug resistant lung cancer by

upconversion nanoparticles-mediated photodynamic
therapy and gene silencing

Poliraju Kalluru1 | Munusamy Shanmugam1 | Raviraj Vankayala2 |

Chi-Shiun Chiang3 | Kuo Chu Hwang1

1
Department of Chemistry, National Tsing
Hua University, Hsinchu, Taiwan, R.O.C.
Abstract
2
Department of Bioscience and Multidrug resistance (MDR) problem is a challenging task in cancer treat-
Bioengineering, Indian Institute of ments. Among various types of cancers, lung cancer is considered incurable, as
Technology Jodhpur, Jodhpur, Rajasthan,
it is diagnosed very often at the advanced metastatic stage, where surgical
India
3
Department of Biomedical Engineering
operations and radiation treatments become highly ineffective. In addition,
and Environmental Sciences, National lung cancers are multidrug resistant either intrinsically or upon continuous
Tsing Hua University, Hsinchu, Taiwan, administration and stimulation of chemotherapeutic drugs. To conquer the dif-
R.O.C.
ficult MDR problem, in this study, we developed a simple and effective thera-
Correspondence peutic strategy to achieve complete destruction of multidrug resistant lung
Kuo Chu Hwang, Department of
cancer by an unprecedented upconversion nanoparticles (UCNPs)-mediated
Chemistry, National Tsing Hua
University, Hsinchu 30013, Taiwan, photodynamic therapy (PDT) combined with effective (~70%) superoxide dis-
R.O.C. mutase 1 (SOD1) gene silencing using ultra-low doses (320 mW/cm2) of NIR
Email: kchwang@mx.nthu.edu.tw
light (980 nm) excitation. To the best of our knowledge, this is the first success-
Funding information ful literature example in achieving complete destruction of multidrug resistant
Frontier Research Center on Fundamental cancers. Overall, the current work points out a new direction to the clinicians
and Applied Sciences of Matters, National
Tsing Hua University, Taiwan; Ministry of
for the therapeutic design and effective treatments of multidrug resistant
Science and Technology, Taiwan cancers.

KEYWORDS
gene silencing, multidrug resistance, near infrared light, photodynamic therapy,
upconversion nanoparticles

1 | INTRODUCTION before it reaches the target intracellular organelle.[2,3]

Multidrug resistance (MDR) is one of the major obstacles
by which many cancers develop resistance toward molec-
ular anticancer drugs, leading to the failure of chemo-
therapeutic treatments and deaths.[1–4] MDR generally
arises due to stimulation of chemotherapeutic drugs and
subsequent overexpression of cellular membrane pro-
teins, such as, ATP-binding cassette (ABC) transporter, p-
glycoprotein (p-gp), and multidrug resistance associated
proteins (MRPs), that can mediate molecular drug efflux
MDR problem is commonly observed in various types of
cancers. Among them, lung cancer is considered incur-
able, because the survival rate of lung cancer patients is
very low (<15%). The majority of the lung cancer patients
were very often diagnosed only at the advanced meta-
static stage, where the surgical operation and radiation
treatments are usually ineffective.[4,5] The non-small cell
lung cancers (NSCLC) was found to be intrinsically
multidrug resistant with overexpression of MRP family
proteins; but not the p-gp.[6] Previously, a lot of efforts

J Chin Chem Soc. 2022;1–13. http://www.jccs.wiley-vch.de © 2022 The Chemical Society Located in Taipei & Wiley-VCH GmbH 1
2 KALLURU ET AL.
have been devoted to overcoming the MDR problem of
lung tumors, but only partial suppression or delay tumor
growths were achieved.[7–10] Therefore, it leaves a grand
challenge to develop a noninvasive therapeutic modality
that can effectively conquer the MDR problem, and facili-
tate complete destruction of lung tumors.
In addition to the chemotherapy, radiotherapy and pho-
totherapy are two treatment modalities that can cure cancer
effectively. Among them, phototherapeutic approach has
many advantages such as, noninvasiveness,[11] excellent
spatial–temporal selectivity, low side effects,[12] and
cost-effective as compared to other treatment modalities.[13]
Photodynamic therapy (PDT) requires the use of organic
photosensitizers (PS) to absorb and transfer the photon
energy to molecular oxygen (3O2) in tumor tissues, to gener-
ate cytotoxic singlet oxygen (1O2) and kill cancer cells.[14]
Conventional organic PS-mediated PDT has many limita-
tions, such as, poor water solubility, severe photo-bleaching
problem, restriction to visible light activation, which has
very short tissue-penetration depths, and therefore not
applicable to deep tissue-buried tumors. To treat deep
tissue-buried tumors, near infrared (NIR) light with far
larger tissue penetration depths must be used. However,
NIR light activatable organic PS molecules are very rare.[15]
Currently, the use of nanomaterials to absorb NIR light to
exert NIR PDT is becoming the major trend of photother-
apy for cancer treatment.[16] Previously, upconversion
nanoparticles (UCNPs) were used to absorb and upconvert
NIR light to visible/uv light for indirect sensitization of
organic photosensitizers for treatments of non-multidrug
resistant tumors.[17–21] However, only partial suppression or
delay the growth of tumors was achieved, albeit very high
laser powers (0.33 ~ 0.78 W/cm2) of NIR (980 nm) light was
used to photochemically excite the UCNPs, which is proba-
bly due to multiple-step sensitization and therefore
extremely low overall efficiencies of singlet O2
generation.[22–26] Multiple-step photosensitization processes
always suffer from very low overall quantum efficiency for
singlet oxygen generation. Recently, we have reported sev-
eral examples of near infrared light initiated photodynamic
therapy (NIR-PDT) for effective treatments for non-
multidrug resistant cancers, such as, gold nanorods,
nanoshells, nanoechinus, and tungsten oxide nanowires,
can sensitize formation of singlet oxygen directly by NIR
light irradiation, and simultaneously exert PDT effects for
the destruction of tumors without drug resistant capability
under low laser doses (<0.4 W/cm2).[27–40]
In the literature, it is known that the SOD1 gene is an
essential enzyme that eliminates superoxide radical
(O2
.) and thus protects cancer cells from ROS-induced
damages. Silencing of the SOD1 gene of cancer cells is
expected to suppress the antioxidation ability of cancer
cells and thus trigger apoptosis efficiently, leading to the
effective cancer cell deaths.[41–43] In addition to the SOD1
gene, there are still many other genes available, such as
survivin, polo-like kinase (Plk1), Bcl-2, tumor necrosis
factor (TNF-α), HSP70, and HSPA1A, which can help the
cancer cells to prevent themselves from undergoing oxi-
dation stress-induced deaths.[44–47] These genes are
potential targets for gene silencing to suppress cancer
cells' ability against oxidation stress.
In the current work, we present an unprecedented,
simple, but effective therapeutic strategy to achieve the
complete destruction of multidrug resistant cancers/
tumors. We develop nanoparticles prodrug to generate
reactive oxygen species (ROS) to kill cancer cells having
MDR proteins. We foresee that multidrug resistant pro-
teins cannot pump out large (relative to molecular drugs)
nanoparticles-based nanomedicines,[40] which are very
different from small chemodrugs in terms of sizes, sur-
face functionalities and polarities. Therefore, cancer cells
with MDR proteins can be easily killed the same as those
without multidrug resistant abilities. Our in vivo lung
cancer experiments demonstrate that such a therapeutic
design works very well and solid lung tumor with intrin-
sic MDR proteins can be completely destroyed.
2 | EXPERIMENTAL SECTION
2.1 | Materials or reagents
Yttrium oxide (99.9%), ytterbium oxide (99.9%), and erbium
oxide (99.9%), hydrochloric acid, oleic acid, octadecene,
sodium hydroxide, 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC), and N-Hydroxysulfosuccinimide (Sul-
NHS) were purchased from Sigma-Aldrich, USA. Ammo-
nium fluoride was purchased from Alfa-Aesar, USA,
whereas methanol is from Echo Chemicals Co. Ltd.,
Taiwan. Anti-epidermal growth factor receptor antibody
(anti-EGFR) was obtained from R&D systems, USA.
40 ,6-Diamidino-2-phenylindole-dihydrochloride (DAPI) was
obtained from Thermo Fisher Scientific, USA.
2.2 | Synthesis of UCNPs
In a typical experiment, Y-oleate (0.68 mmol), Yb-oleate
(0.3 mmol), and Er-oleate (0.02 mmol) complexes were
mixed with 6 ml of oleic acid and 15 ml 1-octadecene in a
100 ml three neck round bottomed flask. The reaction
mixture was refluxed at 150 C in an inert Ar atmosphere
until getting clear solution and then cooled down to room
temperature. Then, a 10 ml of a methanol solution con-
taining NaOH (2.5 mmol) and NH4F (4 mmol) was gently
added to the reaction mixture and stirred for 20 min at
KALLURU ET AL.
RT. Then, methanol was removed by vacuum distillation.
The reaction mixture was heated at 315 C for 90 min.
The resulting reaction mixture was then immediately
cooled down to room temperature. Upon addition of eth-
anol into the reaction mixture, the UCNPs were precipi-
tated and then collected upon ultra-centrifugation. The
contents were centrifuged for 3 ~ 5 times to remove
excess surfactant and solvent. The purified UCNPs were
then dispersed in hexane.
2.3 | Synthesis of citrate-capped UCNPs
Thirty milligrams of hydrophobic UCNPs (NaYF4: Yb,
Er) was mixed with 3 ml of chloroform and 2 ml of tolu-
ene, followed by ultrasonication. This suspension solu-
tion was slowly injected into the three-necked flask
containing 150 mg of citric acid and 15 ml of diethylene
glycol, and heated at 110 C within 1.5 hr under argon
atmosphere. Then, air was introduced into the solution,
which was further heated at 160 C for another 1.5 hr
until the solution becomes clear. The reaction was cooled
down to room temperature and the solid contents were
washed thoroughly with ethanol and deionized
(DI) water. The citrate-capped UCNPs were finally re-
suspended in DI water to obtain a solution with a final
UCNP concentration of 1.0 mg/ml.
2.4 | Synthesis of anti-EGFR conjugated
UCNPs
One milligram of citrate-capped UCNPs were mixed in
500 μl phosphate-buffered saline (PBS, pH ~ 7.4), followed
by the addition of a solution containing 1.5 mg of 1-ethyl-
3-(3-dimethylaminopropyl) carbodiimide (EDC) and 1 mg
of N-hydroxysulfosuccinimide (Sul-NHS), and vortex for
1 min. Further, 20 μl (10 μg) of anti- EGFR was added and
stirred for overnight at 4 C. Later, 1 ml PBS was added
and then centrifuged at 10000 rpm for 5–6 min. The anti-
EGFR conjugated UCNPs settle down to the bottom of the
eppendorf tube, where the supernatant contains the
unreacted anti-EGFR. Upon washing for 3 times, the
anti-EGFR conjugated UCNPs were used for further
experiments.
2.5 | Singlet oxygen phosphorescence
emission measurements
Citrate capped-UCNPs (1 mg/ml) were dispersed in D2O
solvent, followed by photo excitation to generate singlet
O2 phosphorescence emission, which was recorded using
3
a luminescence spectrometer (FLS920, Edinburgh Instru-
ments Ltd. Scotland, UK). A 1000 nm long pass filter
(Iszu Optics, Taiwan) was placed in-between the light
source and detector to avoid the stray and scattering light
from the light source which is a 450 W Xe lamp.
2.6 | DPBF quenching experiment
0.08 mM DPBF (1,3-diphenyl isobenzofuran) and
1 mg/ml of citrate-UCNPs were thoroughly mixed and
irradiated with 980 nm laser with the power intensity of
320 mW/cm2 for 30 min. The absorbance of the solution
at 410 nm was measured for every 5 min using a
UV–visible spectrophotometer. The decrease in the
absorbance of DPBF caused by the photobleaching was
measured in all experiments.
2.7 | EPR measurements
For the detection of 1O2 using EPR, 500 μl of 1 mg/ml
citrate-UCNPs in D2O suspension was added to the 40 μl
of 0.3 M TEMP in D2O solution, and the resulting solu-
tion was mixed well. The mixture was then bubbled with
O2 for 5 min and then irradiated with 980 nm laser with
the power intensity of 320 mW/cm2 right in front of the
ESR spectrometer during the measurements. 10 mM his-
tidine was used as a scavenger for 1O2 in these experi-
ments. Parameter settings: microwave power, 10.12 mW;
frequency, 9.8 GHz; time constant, 40.96 ms; scan
width, 100 G.
2.8 | Cell culture, materials, and
reagents
Non-small cell lung cancer (NCI-H23) cells were grown in
Roswell Park Memorial Institute medium (RPMI-1640)
(Gibco, USA) supplemented with 10% heat-inactivated
fetal bovine serum (Gibco, USA), 2 mM L-glutamine,
100 μg/ml penicillin, and 100 U/ml streptomycin.
The cells were grown in a humidified incubator at 37 C
(95% humidity, 5% CO2).
2.9 | In vitro uptake studies
For confocal microscopy experiments, NCI-H23 cells
(2  105 cells/ml) were cultured in 6-well dish with
RPMI-1640 medium, and then added different concentra-
tions of anti-EGFR-UCNPs and incubated for 24 hr. After
that 1x phosphate buffered saline (PBS) solution was
4 KALLURU ET AL.
used to wash the cells. The cells were then further treated
by 4% paraformaldehyde solution on to the glass slide
and then followed by PBS wash and once again treated
with 5% Tween-20 in PBS solution onto a glass slide and
then further two times washed with PBS. The clean
washed NCI-H23 cells were stained with DAPI
(40 ,6-diamidino-2-phenylindole) and observed under a
multi-photon confocal scanning microscope (Leica TCS
SPX5, Germany), which was equipped with a 404 nm uv
laser and a tunable NIR laser. For all the uptake experi-
ments, 980 nm excitation light was used with a laser
power intensity of 5 mW/cm2. The emission was col-
lected from the PI (propidium iodide) channel
(λem = 600–700 nm).
2.10 | Preparation of siRNA-anti-EGFR-
UCNPs
A stock solution of 0.5 μM of superoxide dismutase
1 (SOD1) siRNA (siGENOME Human SOD1 [6647],
siRNA SMART pool, Thermo Scientific, USA) was
diluted to 100 nM using RNAse free water. Then the
stock solution of anti-EGFR-UCNPs (1 mg/ml) was
added to the dilute siRNA solution and vortexed for few
minutes and kept undisturbed for 15 min at room tem-
perature to form a stable complex before use.
2.11 | Size and zeta-potential
measurements
The siRNA-anti-EGFR-UCNPs complexes were prepared
accordingly from the abovementioned procedures. The
surface charge and average particle size was measured
using Zeta-sizer instrument (Malvern, UK).
2.12 | Isolation of total RNA from
siRNA-anti-EGFR-UCNPs internalized
NCI-H23 cells
NCI-H23 cells were cultured in a 6-well plate with a
seeding density of 2.0  105 cells per well and incubated
for 24 hr. The cells were treated with different concentra-
tions of siRNA-anti-EGFR-UCNPs nanoconjugates for
24 hr. The cells were then washed with a phosphate
buffer solution (PBS, pH 7.4) and incubated for an addi-
tional time of 24 hr. Further, the cells were washed with
PBS for one more time, trypsinized and followed by cen-
trifugation to make a cell pellet. Then 1 ml of TRIZOL
(Sigma-Aldrich, USA) reagent was added to the cell pellet
and the contents were kept at
80 C for 24 hr. The
solution was further centrifuged for 10 min
(at 12000 rpm) at 4 C. 200 μl of chloroform was then
added into it, vortexed for 20 s, and then kept it stand by
in ice for 10 min. The solution was then centrifuged at
12000 rpm for 20 min at 4 C and RNA was obtained in
the supernatant which was collected into another clean
eppendorf tube. Now equal volume of 2-propanol was
added to precipitate the RNA and kept at
20 C for
24 hr. Later, the contents were centrifuged once again at
12000 rpm for 20 min at 4 C and the supernatant was
removed. To the bottom pellet, 1 ml of 75% ethanol was
added, vortexed, and centrifuged for 20 min at 4 C.
Finally, an appropriate volume of RNAse free water was
added to the air-dried RNA pellet and stored at
20 C
for further experiments. The final concentration of total
RNA was measured using Nanodrop.
2.13 | Polymerase chain reaction (PCR)
of total RNA to complementary DNA
Two micrograms of total RNA was taken in a neat
eppendorf tube and various PCR components were added
in a sequence as follows. First 5 mM dNTP mix
(Promega, USA) was added, followed by the addition of
10 μM oligo-dT primer (Thermo Fisher Scientific, USA)
and vortexed gently. In the next step, 10 units/μl of
RNAse inhibitor (Promega, USA) was added, which was
followed by the addition of Omniscript Reverse Tran-
scriptase (Promega, USA). The eppendorf tube was kept
at 37 C for 1 hr to complete the PCR reaction. The as
obtained complementary DNA (cDNA) concentration
was measured using Nanodrop Lite spectrophotometer
(ThermoFisher Scientific, USA) and used for further
experiments.
2.14 | Real-time quantitative PCR (RT-
qPCR) experiment
The cDNA was served as a template and the manufacturer
protocol was followed for the RT-qPCR experiment (SYBR
Green Master Mix, Thermo Scientific, USA). In a typical
experiment, an appropriate concentration of cDNA
(~500 ng) was added into the PCR tube. In the next step,
the forward and reverse primers (~0.3 μM each) for the
target gene (SOD1, 50 -TGTGGCCGATGTGTCTATTG-30
(forward) and 50 -GCGTTTCCTGTCTTTGTACTTTC-30
(reverse)) and house-keeping gene (GADPH, 50 -TGAA
GGTCGG-AGTCAACGGATTTGGT-30 (forward) and 50 -
CATGTGGGCCATGAGGTCCACCAC-30 (reverse)) were
also added and gently pipetted to ensure thorough mixing
of the components. During the last step, 12.5 μl of SYBR
KALLURU ET AL.
Green dye was added, followed by the addition of RNAse
free water to make a total volume of 25 μl. The tubes were
kept in the RT-qPCR machine (Thermo Scientific, USA)
with the following steps: (a) Uracil-DNA glycolase (UDG)
pre-treatment at 50 C for 2 min; (b) 1 cycle of initial dena-
turation at 95 C for 10 min; (c) 1 cycle of denaturation at
95 C for 15 s; and (d) 40 cycles of annealing/extension at
60 C for 60 s. The results were analyzed using the ΔΔCT
method (comparative CT method) where CT is the thresh-
old cycle. The blank controls for these experiments are the
primers alone without the template cDNA.
2.15 | Cytotoxicity assays
To the 24-well plate, NCI-H23 cells-containing solution
(2.0  104 cells/ml) were added and then incubated
with different amounts of anti-EGFR-UCNPs. The cell
solution was incubated at 37 C for 24 h. NCI-H23 cells
were exposed to a 980 nm laser (320 mW/cm2; 30 min)
in order to determine the photo-toxicities of anti-
EGFR-UCNPs. Then the cells were incubated for 12 h,
followed by addition of 50 μl of a MTT reagent
(0.5 mg/ml). The cells were further incubated for
another 4 h. The upper layer was discarded, and 1 ml
of dimethyl sulfoxide (DMSO) was added to each well.
After that the solution was centrifuged at 13000 rpm,
followed by ELISA measurement of optical density at
570 nm. The optical absorbances were converted to cell
viabilities based on (absorbance vs. cell numbers) stan-
dard curve.
2.16 | ROS experiments
NCI-H23 cancer cells were seeded in 6-well plates with
a density of 2  105 cells per well. After 24 hr of incuba-
tion, anti-EGFR-UCNPs and siRNA-anti-EGFR-UCNPs
were added to the cancer cells. The cell solution was
further incubated for 24 hr, followed by photo-
irradiation (980 nm, 320 mW/cm2, 30 min). Then, a
20 ,70 -dichlorofluorescien diacetate (DCFH-DA) solution
(5 μM in cell culture medium) was used to replace
the cell culture medium immediately. DCFH-DA
is nonfluorescent and converts to fluorescent 20 ,70 -
dichlorofluorescien diacetate (DCF) upon reaction with
the highly active oxygen species, which were generated
upon photo-irradiation of UCNPs. The cell solution was
further incubated for 30 min at 37 C. Cells were then
measured using flow cytometry with the FITC channel.
50 mM NaN3 solution was used for the quenching
experiments.
5
2.17 | Singlet oxygen sensor green
(SOSG) experiments
NCI-H23 cells were loaded into the 6-well dishes with
the density of 2  105 cells per well. After that incubate
for 24 hr, anti-EGFR-UCNPs, and siRNA-anti-EGFR-
UCNPs stock solutions were added to the cells, followed
by incubation for 24 hr. Then, the medium was added
2 μl of singlet oxygen sensor probe SOSG (1 mM, Invi-
trogen, USA), and then further incubated for 30 min.
After 30 min incubation and subsequent 980 nm laser
irradiation (320 mW/cm2; 30 min), the cells were then
trypsinized, aspirated, and suspended in a 1 ml PBS, and
further proceeded to analysis using flow cytometry.
2.18 | In vitro apoptosis detection using
Annexin V
NCI-H23 cells were cultured in a 6-well plate with the
cell number of 2  105 cells per well. After 24 hr of incu-
bation, different amounts of siRNA-anti-EGFR-UCNPs
were added and incubated for another 24 hr. After 24 hr,
the cells were washed with the PBS, followed by addition
of serum medium and incubation for additional 24 hr.
The cells were then subjected to photo irradiation with
980 nm laser (320 mW/cm2, 30 min), trypsinized, and
suspended in 200 μl of 1 binding buffer. Then FITC-
Annexin-V (5 μl) antibody (BD Biosciences, USA) and
propidium iodide PI (5 μl) were fed to the cell solution
and incubated for 5 min and resuspended in fresh PBS
(1 ml), followed by analysis using flow cytometry.
2.19 | In vivo tumor growth study
The BAlB/c male nude mice were purchased from the
National Laboratory Animal Center, Taiwan. The mice
were raised according to the protocol approved by the
institutional animal care and use committee (IACUC) of
National Tsing Hua University. Thirty Mice were divided
into 8 groups on day 0, and used in the in vivo tumor
growth study. Within them, 2 groups (siRNA and anti-
EGFR-UCNPs injected) were treated without photo-
irradiation and other 6 groups were irradiated by 980 nm
laser (320 mW/cm2; 30 min), once every day. During irra-
diation, anti-EGFR-UCNPs (15 mg/kg) and siRNA-anti-
EGFR-UCNPs (15 mg/kg) were injected thrice (day
1, day 5, and day 9) intravenously. One group of mice
was injected with anticancer drug (doxorubicin) with
another group as control without injection of doxorubi-
cin. Every day, the tumor volumes and body weights
6 KALLURU ET AL.
were measured. The tumor volume (mm3) was calculated
as d2  D/2, where d is the shortest diameter of the
tumor and D is the longest diameter of the tumor. The
relative tumor volume (V/Vo) is calculated by dividing
the volume of the tumor (V) measured on that day to that
of the initial tumor volume (Vo) measured at day 0.
2.20 | HE and caspase 3 staining
Tumor, liver, and spleen were dissected and then sub-
jected to the HE (hematoxylin and eosin) staining. For
caspase-3 staining, only tumor was stained. For tissue
sectioning, 10 μm slice depth was used.
3 | R ES U L T S A N D D I S C U S S I O N
3.1 | Synthesis, characterization, and
upconversion luminescence property of
UCNPs
The UCNPs (NaYF4: Yb(30%) and Er(2%)) used in the
current work were prepared according to the literature
procedures.[48] Further, the as-synthesized UCNPs were
processed by ligand exchange with the citrate ligands to
render water dispersibility. The structure of UCNPs was
characterized using powder x-ray diffraction (see
Figure S1(a)). The energy dispersive X-ray analysis of
UCNPs reveals the presence of Y, Yb, and Er, respectively
(Figure S1(b)). To facilitate the combination of UCNPs-
mediated photodynamic therapy and gene silencing in
treating multidrug resistant lung cancer cells, anti-
epidermal growth factor receptor (anti-EGFR) was
chemically linked onto the surface of citrate capped
UCNPs via EDC amine-acid coupling process (see
Scheme 1).[49,50] The transmission electron microscopy
(TEM) images of citrate-capped UCNPs exhibits an aver-
age particle size of 10 ~ 15 nm. The high resolution TEM
image in the inset reveals a lattice d-spacing value of
0.292 nm (see Figure 1a). Dynamic light scattering (DLS)
measurements of citrate-capped UCNPs also reveals an
average particle size of ~10 nm with a polydispersibility
index (PDI) value of 0.08 (see Figure S2). The successful
chemical conjugation of anti-EGFR onto the surface of
UCNPs was confirmed by the UV–vis absorption spec-
trum, which reveals an absorption peak at ~280 nm, a
characteristic absorption of the anti-EGFR (Figure 1b).
The citrate capping and subsequent anti-EGFR linking
on the surface of the UCNPs were also confirmed by
FTIR spectroscopic analysis (see Figure S3).[50] Upon
excitation of 980 nm light, citrate-capped UCNPs reveals
three upconversion emission peaks centered at 525 nm
S C H E M E 1 Schematic representation for the synthesis of
siRNA-anti-EGFR-UCNPs to facilitate the UCNPs-mediated
photodynamic therapy combined with SOD1 gene silencing
(2H11/2 – 4I15/2), 550 nm (4S3/2 – 4I15/2), and 660 nm (4F9/2
– 4I15/2), respectively. Since upconverted emission is a
two-photon process, both the upconversion emission
intensity and quantum yield will become higher at
higher incident laser power intensities.[41] Previously, it
was reported that 45 nm sized NaYF4:Yb3+/Tm3+ hex-
agonal nanocrystals exhibit an upconverted lumines-
cence quantum yield of ~0.032 upon 980 nm light
excitation with a very high laser power intensity of
78 W/cm2. Notice that in our system, large upconverted
emission signals were observed upon exceptionally low
intensity (25 ~ 150 mW/cm2) of 980 nm NIR excitation
(Figure 1c), of which the incident laser power is 1 ~ 3
orders lower than the previously reported threshold
laser intensity of 1 ~ 1,000 W/cm2 required to observe
upconversion emission signals.[51] The upconversion
emission quantum yields of the as-synthesized and
citrate-capped UCNPs upon low intensity (0.32 W/cm2)
980 nm laser light excitation are determined to be
0.0005% and 0.0002%, respectively (see Figures S4 and
S5).[46,52] Although the upconversion luminescence
quantum yields are very low under ultra-low laser
power excitation, the upconversion emission intensities,
however, are still large enough to be detected and visu-
alized (see Figure 1c).
The UV–vis–NIR spectrum in Figure 2a shows that
citrate-capped UCNPs exhibit flat absorption extending
up to the NIR region (1,100 nm) along with a small peak
at 976 nm, which is characteristic for the absorption of
Yb(III) ions.[53] The molar extinction coefficient for
citrate-capped UCNPs was determined for the first time
to be ~2.0 x 107 M
1/cm at 980 nm, which is 3 ~ 4 orders
KALLURU ET AL. 7

F I G U R E 1 Characterization of UCNPs. (a) Transmission electron microscopy (TEM) image of bare UCNPs. The inset represents the
high-resolution TEM image of citrate-capped UCNPs. (b) UV–vis absorption spectra for citrate-capped UCNPs, anti-EGFR alone, and
siRNA-anti-EGFR-UCNPs, respectively. The green and red dashed arrows indicate their respective absorption peak at 280 nm for anti-EGFR
alone and siRNA-anti-EGFR-UCNPs. (c) Upconverted emission spectra measured using a 980 nm laser with different power intensities. Inset
is the digital luminescence photograph of citrate-capped UCNPs

F I G U R E 2 Optical properties of UCNPs. (a) UV–vis–NIR absorption spectrum (black solid line) and excitation spectrum (black dashed
line) for singlet oxygen phosphorescence of citrate-capped UCNPs (λem = 1,261 nm). (b) Singlet oxygen phosphorescence emission spectra
sensitized by citrate capped UCNPs, in the absence of organic photosensitizers, at 980 and 1,008 nm excitation wavelengths, respectively

higher than the conventional organic dyes, but 1 ~ 2
orders smaller than those for spherical gold nanoparticles
and short aspect ratio gold nanorods.[27,54] In addition to
the upconverted emission, amazingly we also found that
the citrate-capped UCNPs can sensitize formation of
singlet O2 by itself upon 980 nm light excitation in the
absence of organic photosensitizers. The excitation spec-
trum for singlet O2 phosphorescence from UCNPs reveals
three characteristic peaks centered at 947, 980, and
1,008 nm (see dashed lines in Figure 2a). Interestingly,
8 KALLURU ET AL.
singlet O2 can only be formed when excited by NIR light
(875 ~ 1,100 nm), but not by visible and short NIR light.
Upon excitation at 980 nm and 1,008 nm, very clear-cut
singlet O2 phosphorescence emission signals were
detected, with an emission maximum at ~1,261 nm
(Figure 2b). The quantum yield for the sensitization of
singlet O2 by UCNPs was determined to be 0.353 at
980 nm (at an incident laser intensity of 320 mW/cm2,
see Data S1 for more details). The generation of singlet
O2 by photo excitation of UCNPs was also confirmed
using a chemical trapping agent, 1,3-diphenyl iso-
benzofuran (DPBF). DPBF reacts with singlet O2 via
1,4-cycloaddition to form an endoperoxide product,
which leads to a decrease in the absorption intensity at
410 nm.[55] As shown in Figure S6(a), the 410 nm absorp-
tion intensities of the solution containing UCNPs and
DPBF decreases gradually upon prolonged 980 nm
photo-irradiation, whereas no or negligible change was
observed at 410 nm from two control solutions: (a) a
DPBF-containing solution (without UCNPs) under
photo-irradiation, and (b) a DPBF-UCNPs solution in the
dark. In addition to the observation of singlet O2 phos-
phorescence emission and DPBF experiments upon
980 nm light excitation of citrate-UCNPs, we have also
detected 1O2 using electron paramagnetic resonance
(EPR) spectroscopy. To detect 1O2 using EPR, we have
used 2,2,6,6,-tetramethyl-4-piperidone (TEMP) as a 1O2
spin-trapping reagent. The EPR signal for the photoex-
cited citrate-UCNPs dispersion clearly displayed a charac-
teristic 1:1:1 triplet due to the formation of a TEMP-1O2
spin adduct. Further, the TEMP-1O2 spin adduct EPR sig-
nal was remarkably suppressed in the presence of a spe-
cific singlet oxygen scavenger, L-histidine (see Figure S6
(b)). Overall, it is very clear that upon photo-excitation
using 980 nm light, citrate-UCNPs alone in the absence
of additional organic photosensitizers can effectively sen-
sitize the formation of singlet oxygen. All these results
clearly point to the same conclusion that citrate-capped
UCNPs alone in the absence of organic photosensitizers
can sensitize formation of singlet O2 as well as
upconverted emission upon 980 nm light excitation. The
photo-sensitization of singlet O2 from UCNPs was based
on single photon excitation and subsequent energy trans-
fer process from the excited state of UCNPs to molecular
O2. To have energy transfer occur to generate singlet O2,
molecular O2 most probably adsorbs on the surface of the
UCNPs. In other words, the singlet excited state energy
of UCNPs was estimated to be ~1.26 eV (i.e., E = 1,239/
λex = 1239/980 nm = 1.26 eV or 29.17 kcal/mol), which
is much larger than the formation energy of singlet O2,
0.97 eV (i.e., 22.5 kcal/mol). Overall, an energy transfer
mechanism from UCNPs to molecular O2 can lead to the
generation of singlet oxygen.
3.2 | In vitro combined UCNPs-mediated
PDT and gene silencing
To evaluate whether combination of UCNP-mediated
PDT and gene silencing can kill multidrug resistant non-
small lung cancer cells, anti-EGFR surface-modified
UCNPs were fed to the NCI-H23 cells, and the cellular
uptake was monitored. The confocal optical images in
Figure S7 and S8 clearly show that the anti-EGFR
surface-modified UCNPs were successfully uptaken by
the NCI-H23 cells, as evidenced by the observation of
upconverted red fluorescence emission image upon
980 nm laser light excitation. In contrast, citrate-UCNPs
internalized NCI-H23 cells as well as the control (without
any pre-treatment) did not show any noticeable
upconverted emission. Further, we have loaded SOD1
siRNA together with the anti-EGFR-UCNPs, aiming at
gene therapy to kill non-small lung cancer cells. The
zeta-potential measurements in Figure 3a confirm the
complexation, via electrostatic interactions, of poly-
anionic siRNA onto the surface of cationic anti-EGFR-
coated UCNPs. The gene silencing efficiencies of SOD1
using siRNA-anti-EGFR coated UCNPs as nanocarriers
were evaluated using quantitative polymerase chain reac-
tion (qPCR) in NCI-H23 cells under serum free condi-
tions (see Figure 3b and c). Overall, ~70% gene silencing
efficiency of SOD1 was achieved in the NCI-H23 cells
after pre-treatment with siRNA-anti-EGFR-coated
UCNPs (at 100 nM siRNA concentration). Therefore, it is
anticipated that upon silencing of SOD1 gene, the
siRNA-anti-EGFR-UCNPs might be able to mediate pho-
todynamic therapeutic effects, upon NIR light irradiation,
on killing the NC1H23 lung cancer cells effectively. To
prove such a possibility and to evaluate the cancer cell-
killing efficiency, NCI-H23 cells were incubated with dif-
ferent concentrations of anti-EGFR-UCNPs and siRNA-
anti-EGFR-UCNPs and irradiated with 980 nm laser. As
shown in Figure 3d, neither anti-EGFR-UCNPs nor
siRNA-anti-EGFR-UCNPs induce any noticeable cellular
deaths in the dark. However, upon exposure to 980 nm
laser (320 mW/cm2; 30 min), the percentage of cellular
deaths from the siRNA-anti-EGFR-UCNPs group
increases dramatically, which is ~3.8 fold higher than
that observed from the anti-EGFR-UCNPs group without
silencing gene. In contrast, siRNA (100 nM concentra-
tion) alone did not induce any significant cellular deaths
both in the dark and siRNA (100 nM concentration)
upon photo irradiation condition. To provide deeper
insights into the cellular deaths by anti-EGFR-UCNPs
and siRNA-anti-EGFR-coated UCNPs internalized NCI-
H23 cells using 980 nm laser irradiation, several path-
ways were investigated to understand the mechanisms
which lead to high cellular deaths. From Figure 4a, it
KALLURU ET AL. 9

F I G U R E 3 Combined in vitro UCNPs-mediated photodynamic therapy and gene silencing. (a) Zeta-potential measurements for citrate
UCNPs, anti-EGFR-UCNPs and various concentrations of siRNA-anti-EGFR-UCNPs solutions. (b) Percentage of gene silencing efficiencies
measured using RT-qPCR for SOD1 gene at various concentrations of siRNA. (c) Western blotting result for the SOD1 gene at various siRNA
concentrations with β-Actin as an internal control. (d) Percentages of cellular deaths of siRNA, anti-EGFR-UCNPs and siRNA-anti-EGFR
UCNPs internalized NCI-H23 cells in the dark and under photo irradiation (980 nm, 320 mW/cm2; 30 min) conditions at 37 C

clearly reveals that upon 980 nm laser irradiation
(320 mW/cm2; 30 min), the ROS levels for anti-EGFR
UCNPs and siRNA-anti-EGFR UCNPs treated NCI-H23
cells were ~2.0 and ~2.6-fold, respectively, higher than
that for the dark control group. In contrast, in the
absence of photo irradiation the ROS levels for both
anti-EGFR modified UCNPs and siRNA-anti-EGFR
UCNPs are about the same as that of the control cells
(see Figure S9 for confocal images). To further confirm
the role of singlet O2 on cellular deaths, NCI-H23 cells
were pre-treated with a singlet O2-specific quencher,
that is, sodium azide (NaN3).[27–31] The ROS levels were
significantly suppressed by addition of sodium azide for
both anti-EGFR-UCNPs and siRNA-anti-EGFR-UCNPs
groups in the case of 980 nm photo irradiation (see
Figure 4b). In a few previous studies, significant
amounts of ROS levels and its subsequent cellular
deaths were observed for the UCNPs alone when
exposed to 980 nm NIR light irradiation.[55–57] However,
the origin for these elevated levels of ROS were never
addressed in these previous reports.[55–57] Our results
can help rationalize these previous observations, which
are, in fact, due to the direct sensitization of singlet oxy-
gen by photochemically excited UCNPs. The fluores-
cence intensity levels of singlet O2 sensor green (SOSG)
in Figure S10 exhibit ~2.2-fold higher, as compared to
that in the dark, upon 980 nm laser irradiation for both
anti-EGFR-UCNPs and siRNA-anti-EGFR-coated
UCNPs systems. From Figure 4c, we also observed that
the amounts of apoptotic and necrotic cells generated
from siRNA-anti-EGFR-coated UCNPs-internalized
NCI-H23 cells upon 980 nm light irradiation are far
higher than those for anti-EGFR-UCNPs treated group
in the dark. Overall, our in vitro experiments clearly
show that a combination of UCNPs-mediated PDT and
gene silencing can conquer the MDR problem and exerts
effective killing of the NCI-H23 lung cancer cells in
in vitro experiments.
10 KALLURU ET AL.

F I G U R E 4 In vitro ROS generation and apoptosis. (a) ROS generation monitored by the mean fluorescence intensities of 20 ,70 -
dichlorofluorescein (DCF) using flow cytometry for anti-EGFR-UCNPs and siRNA-anti-EGFR-UCNPs internalized NCI-H23 cells followed
by photo irradiation. (b) ROS levels observed after 50 mM NaN3 pretreatment. (c) Percentages of apoptotic and necrotic cell populations
obtained by the NCI-H23 cells treated with anti-EGFR-UCNPs and siRNA-anti-EGFR-UCNPs in the dark and under photo irradiation
(980 nm, 320 mW/cm2; 30 min) conditions

3.3 | In vivo combination of UCNPs- Although less than 5% of siRNA-anti-EGFR UCNPs

mediated PDT and gene silencing on
tumor destruction
Based on the effective killing of the intrinsically
multidrug resistant NCI-H23 lung cancer cells in the
in vitro experiments, we took one step further to deter-
mine, as a proof-of-concept study, the effectiveness of the
combined UCNPs-mediated PDT and gene silencing on
in in-vivo NCI-H23 non-small cell lung tumor destruc-
tion in a BALB/c nude mice model. The NCI-H23 tumors
were implanted subcutaneously on the leg muscle region,
and allowed to grow until it reaches a diameter of
~5 mm. The siRNA-anti-EGFR-UCNPs (15 mg/kg)
nanoconjugates were then injected intravenously. At
24 hr after iv injection, the distribution of UCNPs in vari-
ous organs, including tumor, liver, spleen, and kidney,
were examined using inductively coupled plasma mass
spectrometry (ICP-MS) analysis (see Figure S11).
nanoconjugates reach the tumor site, the amount is
enough to sensitize formation of singlet oxygen efficiently
and achieve complete destruction of lung tumors under
our experimental conditions (vide infra). At 24 hr after
intravenous injection of siRNA alone or anti-EGFR-
UCNPs or siRNA-anti-EGFR-UCNPs, the NCI-H23
tumor bearing mice was subjected to 980 nm NIR light
irradiation. As shown in Figure 5a, the mice treated with
siRNA-anti-EGFR-UCNPs and exposed to 980 nm laser
have complete destruction of tumors at day 20th, which
is in sharp contrast to the much larger tumor sizes of 2.0
and 26-fold (relative to the initial tumor size) for anti-
EGFR-UCNPs (without silencing gene) and siRNA alone
(without UCNPs) plus exposure to 980 nm laser light.
The sizes (regions indicated in the dashed white circles)
of the NCI-H23 tumors on mice treated with different
conditions at day 20th were presented in Figure 5b. It is
also interesting to note that the tumor site of the mice
KALLURU ET AL. 11

F I G U R E 5 Combined in vivo UCNPs-mediated photodynamic therapy and gene silencing. (a) Tumor growth curves of different groups
of tumor-bearing mice after treatment. The tumor volumes were normalized to the initial size at day 0. Asterisks indicate statistically
significant differences (p < .001) between all the pairs. (b) Representative mice images showing the size of the tumors indicated by dashed
white circles at day 20 after treatment with siRNA alone or anti-EGFR-UCNPs or siRNA-anti-EGFR-UCNPs exposure to 980 nm
(320 mW/cm2; 30 min) light, whereas the blank controls receive no any treatment. The representative mice images showing the scar
formation at day 20th and day 30th, followed by a new skin formation at day 40th for siRNA-anti-EGFR-UCNPs treated mice and exposure
to 980 nm light. (c) In vivo fluorescence microscopy images for the caspase 3 staining of the tumor tissue dissected from various treatment
groups

treated with siRNA-anti-EGFR-UCNPs and exposure to
980 nm laser light forms a scar at day 20th, which got
peeled off later and a new skin was then gradually devel-
oped at day 40th (see Figure 5b). The results unambigu-
ously demonstrate that the multidrug resistant NCI-H23
non-small cell lung tumor could be destroyed completely
by the combined UCNPs-mediated PDT and SOD1 gene
silencing. Besides the tumor growth data presented in the
Figure 5a and b, other measurements, such as, caspase
activity and mice survival rate, are also consistent with
each other. The caspase activity of tumor tissues dissected
from those mice treated with siRNA-anti-EGFR-UCNPs
and exposure to 980 nm laser exhibit higher expression of
caspase 3 than those treated with anti-EGFR-UCNPs or
siRNA alone and exposure to photo irradiation (see
Figure 5c). The average half-life span for the mice treated
with siRNA-anti-EGFR-UCNPs plus exposure to 980 nm
laser is longer than 40 days (4 mice completely survive
out of 6 mice monitored at day 40th in the combined
UCNPs-mediated PDT and gene silencing with photo
irradiation group), which is much longer than the mice
treated with anti-EGFR-UCNPs and exposure to 980 nm
laser group (28 days), doxorubicin treated mice group
(15 days) as well as PBS-treated and photo irradiated
mice group (17 days), respectively (see Figure 6a). During
the therapy period, there are no noticeable changes in
the body weights for all the mice groups (see Figure 6b).
Hematoxylin & Eosin (HE) staining in Figure S12 from
siRNA alone-treated mice followed by photo irradiation.
In contrast, severe necrosis was observed for the tumor
tissues dissected from the siRNA-anti-EGFR-UCNPs or
anti-EGFR-UCNPs treated mice and exposure to 980 nm
12
light. Overall, our in vivo results, consistent with the
in vitro cellular data, clearly demonstrate a successful
example of complete destruction of multidrug resistant
lung tumors by combination of unprecedented NIR light
activatable UCNPs-mediated PDT and SOD1 gene
silencing.
4 | C ON C L U S I ON S
In summary, we have presented a successful therapeutic
design, via combination of UCNPs-based PDT and gene
silencing, to conquer the tumor multidrug resistance
problem. As a proof-of-concept experiment, we have
demonstrated that multidrug resistant NCI-H23 lung
tumor in a mice model can be completely destructed by
an unprecedented UCNPs-mediated photodynamic ther-
apy combined with efficient SOD1 gene silencing (~70%)
using ultra-low doses (320 mW/cm2) of NIR light
(980 nm). The multi-functional UCNPs act as an unique
theranostic reagent to provide the following functions,
including (a) in vivo tumor fluorescence imaging probe
(via its NIR-activatable upconverted emission property),
where we report for the first time that upconversion
emission can be observed under an exceptionally low
intensity of 980 nm (25 ~ 50 mW/cm2) light excitation;
(b) ROS can be generated directly by NIR light photo-
excitation of UCNPs in the absence of additional organic
photosensitizers, and (c) combination of NIR-I PDT with
SOD1 gene silencing for eradication of multidrug resis-
tant lung tumor.
In the in vitro cellular experiments, upon photo exci-
tation of 980 nm light, significantly higher amounts of
cellular deaths were observed from the siRNA-anti-EGFR
UCNPs treated NCI-H23 cells, as compared to that
treated with anti-EGFR UCNPs alone without silencing
gene (79% vs. 33% cellular deaths at 50 μg/ml concentra-
tions). In the in vivo experiments, combination of the
unprecedented UCNPs-mediated PDT and gene silencing
was demonstrated to completely destruct the multidrug
resistant non-small cell NCI-H23 lung tumors in mice
KALLURU ET AL.
F I G U R E 6 (a) Survival rates of the
mice bearing NCIH23 lung tumors after
various treatments monitored for
40 days. (b) Body weight measurements
of the mice bearing NCIH23 lung
tumors for various treatment groups
models. The average lifespan (>40 days) of tumor bearing
mice treated with combined UCNPs-mediated PDT and
gene silencing is ~2.5 times longer than that (15 days) of
anticancer drug (doxorubicin)-treated group. To the best
of our knowledge, this is the first successful literature
example of achieving complete destruction of multidrug
resistant cancers. Overall, our work has pointed a new
direction for effective therapeutic design to completely
conquer multidrug resistance problems commonly associ-
ated in many cancers.
ACKNOWLEDGMENTS
The authors are grateful to prof. Hsing-Wen Sung,
Department of Chemical Engineering, National Tsing
Hua University, Hsinchu, Taiwan for providing the RT-
qPCR instrumentation facility, and also to the financial
support from Frontier Research Center on Fundamental
and Applied Sciences of Matters, National Tsing Hua
University, and Ministry of Science and Technology,
Taiwan.
ORCID
Kuo Chu Hwang https://orcid.org/0000-0003-1814-9869
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SU PP O R TI N G I N F O RMA TI O N
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How to cite this article: P. Kalluru, M.
Shanmugam, R. Vankayala, C.-S. Chiang, K. C.
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