Controlled Release and Delivery Systems

Low-temperature Trigger Nitric Oxide Nanogenerators
for Enhanced Mild Photothermal Therapy
Chaoqun You, Yao-Jia Li, yixin Dong, Like Ning, Yu Zhang, Liyang Yao, and Fei Wang
ACS Biomater. Sci. Eng., Just Accepted Manuscript
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1
Low-temperature Trigger Nitric Oxide Nanogenerators for
Enhanced Mild Photothermal Therapy
Chaoqun You a

College of Chemical Engineering, Nanjing Forestry University; Jiangsu Key Lab for the
Chemistry and Utilization of Agro-Forest Biomass, Nanjing 210037, PR China Fax: +86 25
85427649, Tel: +86 25 85427649, E-mail address: [email protected]
b: School of Chemistry and Chemical Engineering, Southeast University, Nanjing 210089, PR
China
Abstract
Surmounting the restriction issues of nitric oxide (NO) delivery for realizing their
precious on-demand release are highly beneficial for widespread deployments of gas
therapy to build network in biomedicine. Herein, by employing core-shell structure
Au@SiO2 nanomaterials with high photothermal performance, a novel strategy was
proposed by integrating photothermal conversion nanomaterials and heat-triggered NO
donors (RSNO) into a nanoplatform, which achieved photothermal therapy (PTT)-
enhanced NO gas therapy under near infrared (NIR) radiation. Specially, 2-
phenylethynesulfonamide (PES), an inhibitor of Heat Shock Protein 70 (HSP-70), was
loaded into the NO nanogenerators to realize effective low-temperature (~45 ℃) PTT.
The obtained results exhibited that the near infrared radiation (NIR) mediated mild PTT
and gas therapy by releasing NO showed substantially improved synergistic effect
based on in vitro and vivo results in breast cancer (MCF-7) models. Our study points
out a strategy to realize mild photothermal therapy by inhibiting the expression of HSP-
70, and simultaneously providing an avenue to achieve controllable release of NO.
More important, this research highlights the great potential of multifunctional
therapeutic agent in synergistic treatment of cancer.
Keywords: Nitric oxide; Mild photothermal therapy; Nanomaterials; Heat Shock
Proteins; NIR Trigger
Introduction

Gas-dependent tumor therapy, including sulfuretted hydrogen (H2S) 1,2, carbonic
oxide (CO) 3–6, or nitric oxide (NO) 7–9, has been emerging into a novel anticancer
strategy over the last few years. Specifically, extensive researches have revealed NO
molecule involved in divers cellular physiological and pathophysiological activities as
secondary messengers10,11, which clearly related with multiple diseases, such as
cardiovascular disease, liver cirrhosis, neurological disease, and even cancer 12,13
.
According to the published study results, tumor inhibition behavior of NO is
concentration dependent, and high dose of NO could directly and effectively induce
apoptosis or necrosis 14, nevertheless low-dose NO only efficient in killing cancer cells
with an assist from other assault mode 15–17. However, the further development of NO
in biomedicine were greatly limited owing to the intrinsic nature with respect to highly
reactive activity, poor selectivity and short half-life (less than 3 s) 18. Therefore,
maximum utilization of strongly reactive ability of NO for combatting cancer will be
realized by selective delivery of NO molecule or NO donor based on nanoplatform to
diseased region 19,20, and then precisely mediating intracellular sudden release under
certain stimuli, such as pH 21,22, heat 21,23, or light 24,25
.
Photothermal therapy (PTT), employing near-infrared light responsive molecules
or inorganic nanomaterial to produce localized heat in lesion site, have drawn
increasing attention in recent years 26–28. The generated hyperthermia directly ablates
cells and causes unrecoverable destroy to tumor tissues. However, Complete ablation
of tumor cell commonly necessitate higher temperature under strong power laser
(>50 ℃), which will have to compromise heat injury to healthy tissue and painful
treatment experience of patient 29,30. Existing research results indicated that cell self￾repair process was based on heat shock proteins (HSP) 31, a crucial protein to prevent
cells from heat damage, when encountering relatively low level of heat (e.g., 45 ℃).
Therefore, developing a novel strategy to inhibit the activities of HSP and
simultaneously generate mild heat (e.g., 45℃) nearby the tumor will be extremely
valuable in the clinical application of PTT 31–33
.
Interestingly, the generated mild heat is also a superior stimulus to spatio￾temporally control the release of NO. Thus, based on the obtained research results that

have been published so far, an NIR-responsive NO nanogenerators with the
combination of mild PTT and gas therapy were rationally designed and reported.
Specifically, high-performed photothermal golden rod (Au) were coated with
mesoporous silica to afford multifunctional Au@SiO2 nanocomposites, on whose
surface allowed for the further conjugation with S-nitrosothiols, a thermosensitive
donor of NO. Then, 2-phenylethynesulfonamide (PES), a low-toxic and stable HSP-70
inhibitor, were encapsulated into the Au@SiO2 to reduce the heat resistance of tumor
cells. Sequentially, the polyethylene glycol (PEG) were decorated on the Au@SiO2,
endowing them with enhanced stability and biocompatibility.
When the PES/Au@SiO2 were internalized into tumor cells, under exposure of
near infrared light (NIR), the generated mild heat not only induce apoptosis or necrosis
in relatively low temperature with assist of HSP-70 inhibitor, but also promote the NO
release for practical synergy of mild PTT and gas therapy. The vitro and vivo
experimental results revealed the great collaborative efficiency, leading to effective
inhibition of tumor growth.
Scheme 1. Schematic illustration of preparation of PEG-PAu@SiO2-SNO nanocomposites
and the process of mild heat-enhanced gas therapy under NIR irradiation in MCF-7 cells.
Results and Discussion
Synthesis and characterization of Au@SiO2

In this paper, AuNPs were synthesized by a classic seed growth method. The TEM
image clearly showed the rodlike morphology with length-diameter ratio of 5 in Figure
1a. And the selected area electron diffraction of AuNPs revealed each crystal faces,
which are agreed with the reported work. Then, the Au@SiO2 were fabricated using
spatially confined growth method 34. As showed in Figure 1b, the TEM image of
Au@SiO2 showed the successful condensation of SiO2 on the surface of Au, which
possessed a narrow size distribution of about 120 nm. And the weaker bright spot in
SAED image of Au@SiO2 revealed the amorphous structure of SiO2. Meanwhile,
according to Figure 1d, the surface plasmon resonance (SPR) band observed in UV-Vis
spectrum had approximately 30 nm red shift owing to the changed the surface refractive
index from the existence of SiO2 shell. Sequentially, sulfhydryl groups (SH) were
introduction to the surfaces of Au@SiO2 shell by further grafting using silane coupling
agent 3-methoxysilylpropanethiol (MPTES). Then, the S-NO was further synthesized
on the surface of Au@SiO2-SH through reacting SH with tert-butyl nitrite (TBN). To
further improve the water solubility and biological compatibility of Au@SiO2-SNO,
PEG-MAL were linked to the surface of nanocomposites by Michael addition reaction
between SH groups and MAL groups. As showed in Figure 1c, dynamic light scattering
(DLS) of PEG-Au@SiO2 possessed the hydrodynamic dimension size about 140 nm
with polymerization degree index (PDI) of 0.156, which was slightly more than the
results of TEM (Figure S1). The thermal gravity analysis (TGA) in Figure 1e reflected
about 10% PEG and RSNO in the PEG-Au@SiO2. The stability of the prepared PEG￾Au@SiO2-SNO were evaluated by the size and PDI change of nanocomposites
dispersed in PBS (pH=7.4) in 80 h. The Figure 1f showed PEG-Au@SiO2-SNO has
negligible changes at size and PDI, indicated the superior stability in physiological
environment. Sequentially, the Zeta potential of each step treatment showed mild
changes according Figure S2, which indicated the successful preparation of PEG￾Au@SiO2-SNO nanocomposites.
Sequentially, benefited from porous properties of PEG-Au@SiO2-SNO, 2-
phenylethynesulfonamide (PES), a low-toxic and stable HSP-70 inhibitor, were loaded
into the cavity. The loading rate was determined by the ninhydrin method on the basis

of the amino group on PES, giving a ~5% loading rate. Then, the release profiles of
PES in different pH PBS with or without NIR irradiation were investigated. As showed
in Figure S3, compared with incubation with PBS (pH=7.4), PEG-Au@SiO2-SNO
showed obviously enhanced release of PES in PBS (pH=5.0). However, upon exposure
to NIR laser for 5 min, the temperature of PEG-Au@SiO2-SNO solution elevated, and
the release rate of PES reached up to 67% after 24 h of incubation with PBS (pH=5.0).
It could be concluded that the NIR-induced increased temperature effectively facilitated
the release of loaded cargos.
Figure 1. TEM image of AuNPs (a) and (b) Au@SiO2: inset images were the corresponding selected
electron diffraction pattern (SAED). (c) Size distribution of PEG-Au@SiO2-SNO in aqueous
solutions; d) the UV-vis spectra of AuNPs and Au@SiO2. (e) Thermogravimetric analysis (TGA)
curves of AuNPs and PEG-Au@SiO2-SNO. (f) The change of size and PDI of PEG-Au@SiO2-SNO
in PBS (pH=7.4)
Controlled release of NO and detection of NO in vitro
Prior to assess the heat-triggered controlled release of NO, we investigated the
photothermal effects of Au@SiO2 under NIR exposure. Figure 2a and 2b showed the
temperature change of Au@SiO2 in PBS at different concentration and laser power,
respectively, indicating that the temperature response of Au@SiO2 solution is
positively correlated with concentration and power. Despite the relatively low
concentration at 50 mg/mL, Au@SiO2 solution under NIR exposure (1 W/cm2
, 10 min)
could increase to 50.0 ℃, allowing for induced NO release from S-NO. The results of
photothermal stabilities of PEG-Au@SiO2-SNO also indicated that the prepared
nanomaterials showed excellent light stability with uniform temperature changes in 5
cycles (Figure S4). As shown in Figure 2c, the NO release from PEG-Au@SiO2-SNO
solution under different power of NIR laser (10 min) was monitored. After 10 min of
irradiation at 1 W/cm2
, the accumulated NO concentration reached up to 14.6 μM
(Figure 2d), when the temperature of solution was only 40.8 ℃ according the previous
photothermal data. However, the results of direct heating in 70.0 ℃ was only 11.7 μM,
which was mainly attributed to the quite high surface temperature of PEG-Au@SiO2-
SNO was much larger than the apparent temperature in surroundings. The above results
revealed that the PEG-Au@SiO2-SNO could efficiently induce the breakdown of S-NO,
whereby achieving the controlled release of NO at relatively low temperature.
Sequentially, the qualitatively and quantitatively detection of NO in vitro were
performed using confocal laser scanning microscopy (CLSM) and flow analysis,
respectively. A NO indicator 3-amino, 4-aminomethyl-2′,7′ difluorescein diacetate
(DAFDA) was employed to monitor the intracellular NO. As showed in Figure 2e, the
bright green fluorescence, belonging to DAFDA, was observed when MCF-7 cells were
incubated with PEG-Au@SiO2-SNO plus NIR laser. While cells incubated with only
PEG-Au@SiO2-SNO in the absence of NIR irradiation showed weaker green
fluorescence, indicating the prominent effect of NIR-triggered heat on NO release. The
flow analysis in Figure 2f showed that cells incubated with PEG-Au@SiO2-SNO plus
NIR laser obviously had enhanced mean fluorescence than that groups of lacking NIR
irradiation, indicating NIR-induced enhanced NO generation were by drastic
temperature rise.

Figure 2. Temperature rise profiles of Au@SiO2 solution as a function of different nanocomposites
concentrations (a), and different power densities of NIR irradiation (b, the irradiation wavelength
was 808 nm). (c) NIR-induced NO generation from PEG-Au@SiO2-SNO at different power
densities. (d) NO generation from PEG-Au@SiO2-SNO induced by different conditions. (e)
Evaluation of NO generation capacity from different formation of nanocomposites using NO
indicator DAFDA; Scale bar: 40 μm. (f) Quantitative analysis of generated NO in MCF-7 cells by
flow analysis; the red was control group, the orange was PEG-Au@SiO2-SNO group, and the green
was PEG-Au@SiO2-SNO +NIR group.
In vitro evaluation of PEG-PAu@SiO2-SNO
The uptake of PEG-PAu@SiO2 were assessed using confocal laser scanning
microscopy (CLSM), with dying PEG-Au@SiO2 and cell nucleus into red and blue,
respectively. As showed in Figure 3a, with the extension of incubation time with RhB￾labelled PEG-Au@SiO2, MCF-7 cells exhibited gradually enhanced red fluorescence,
indicating that the PEG-Au@SiO2 were effectively internalized and spread to the
cytoplasm and even the nucleus of cells. The quantitative results further showed the
fluorescence intensity increased obviously (Figure 3b).
To further assess the anticancer efficiency of PEG-Au@SiO2-SNO, the In vitro
cytotoxicity of different formation of nanocomposites were investigated based on MTT

assay. Firstly, MCF-7 cells incubated with PES-loaded PEG-PAu@SiO2 showed quite
low toxicity even though the concentration reached up to as high as 200 μg/mL (Figure
S5), indicating the good biocompatibility of the nanocomposites. However, MCF-7
cells treated with PEG-PAu@SiO2 under heating condition, not exceeding 45.0 ℃ from
NIR irradiation, showed enhanced death ratio than that of PEG-Au@SiO2, which was
mainly attributed to inhibition effect of PES to HSP-70 (Figure 3c). More importantly,
the PEG-PAu@SiO2-SNO plus NIR groups possess the highest inhibition effect, which
was 3-fold as high as the PEG-PAu@SiO2 plus NIR groups owing to the synergistic
effect of NO and PES-mediated low temperature photothermal therapy.
Sequentially, the underlying mechanism about the low-temperature PTT was
investigated by employing western blot analysis to determine express levels of HSP-
70, a key heat stress protein to protect cells from heat damage, with glyceraldehyde 3-
phosphate dehydrogenase as an internal reference (Figure 3f and 3g). As expected,
PEG-Au@SiO2 under NIR irradiation caused the enhanced intracellular express levels
of HSP-70, while PES-loaded PEG-PAu@SiO2 at the same conditions significantly
impair the HSP-70, proving the inhibiting effect of PES to HSP-70. Thus, the tumor
cells had a higher probability to encounter apoptosis or necrosis under PES-assisted low
temperature photothermal therapy, which were also verified by the increased apoptosis
rate of cells treated with PEG-PAu@SiO2 than that of PEG-Au@SiO2 (Figure 3c).
Meanwhile, the PEG-PAu@SiO2-SNO groups under NIR irradiation caused about 70%
apoptosis ratio, 2-fold more than PEG-PAu@SiO2 at the same condition (Figure 3e).
The above results the integration of low-temperature phototherapy and NO gas therapy
held promising potential in combating malignant tumor.
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Figure 3. The cell uptake of RhB-labelled PEG-Au@SiO2-SNO at different time interval by CLSM
(a) and Flow cytometer (b): the cell nucleuses were stained using DAPI; Scale bar: 60 μm. (c)
Evaluation of cytotoxicity of different nanocomposite formations (1: PEG-Au@SiO2; 2: PEG￾Au@SiO2-SNO; 3: PEG-Au@SiO2 + NIR; 4: PEG-PAu@SiO2 + NIR; 5: PEG-Au@SiO2-SNO +
NIR; 6: PEG-PAu@SiO2-SNO + NIR). (d) Cell apoptosis of MCF-7 cells treated with different
nanocomposites (a, b, c, d, e) using flow analysis and quantitative analysis (e). (f) Western blot and
its analysis for the detection of HSP-70 expression; standard reference: glyceraldehyde-3-phosphate
dehydrogenase (GADPH). (g) The corresponding ratio between HSP-70 and GAPDH. Data were
shown as mean ± S.D. (n=3). Significance is defined as * P < 0.05, ** P < 0.01.
Evaluation of in vivo anticancer activity
To investigate the anticancer efficiency in vivo of PEG- PAu@SiO2-SNO for
combination of low temperature photothermal therapy and NO gas therapy, Tumor￾bearing female mice were randomly divided into five groups: (1) PBS control; (2) PEG￾AuSiO2; (3) PEG-Au@SiO2 plus NIR; (4) PEG-Au@SiO2-SNO (5) PEG-Au@SiO2-
SNO plus NIR; (6) PEG-PAu@SiO2-SNO plus NIR. When the tumor volume reached
to 300 mm3
, each group were treated with above different agents by intratumor injection.
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Firstly, after certain time (0 h, 8 h, 12 h, 24 h) of injection, mice treated with PEG￾Au@SiO2 received 8 min of NIR irradiation, and the tumor temperatures were plotted
against irradiation time (Figure 4a). As showed in Figure 4c, the temperature changes
from 31.2 ℃ to 46.8 ℃, which allowed for the low-temperature photothermal therapy.
Meanwhile, the PBS control groups only increase less than 6.0 ℃ (Figure 4c),
demonstrating the heating ability of PEG-Au@SiO2 and minor side effects of NIR laser
alone. Then, the inhibition of the six groups to tumor tissues were further studied by
monitoring and measuring the monitoring tumor volume every two days. As showed in
Figure 4b, in the absence of NIR irradiation, the PBS control and PEG-Au@SiO2 had
negligible influence on the tumor tissues, while moderate heating to tumor tissues not
beyond 46.9 ℃ from PEG-Au@SiO2 plus NIR groups exhibited moderate inhibition to
tumor. With the assist of NO donor, the PEG-Au@SiO2-SNO plus NIR groups caused
relatively higher tumor growth inhibition efficiency, which was attributed to the heat￾triggered rapid release of NO promoted the anticancer efficiency. Also of note is the
comparable inhibition effect of PEG-Au@SiO2-SNO without NIR irradiation
compared with PEG-Au@SiO2 plus NIR groups. However, with the help of HSP-70
inhibitor PES, the PEG-Au@SiO2-SNO groups showed superior anticancer activity
than that of the others, giving an about 85% of tumor inhibition rate owing to effective
synergistic effect between NO and HSP-mediated photothermal therapy (Figure 4e).
Meanwhile, no obvious change on mice body weight was observed (Figure 4d),
demonstrating the negligible side effect of the PEG-Au@SiO2-SNO.
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Figure 4. (a) NIR thermal imagery of tumor tissues after certain time (0 h, 8 h, 12 h, 24 h) of
injection. (c) The corresponding temperature rise curve treated with PEG-PAu@SiO2-SNO at power
density of 1W/cm2
with different irradiation time. (b) Tumor volume, (d) body weight, and (e) tumor
tissue weight of mice treated with different formations of nanocomposites. (f) H&E stained images
of extracted tumor tissues treated with different nanocomposites (a: Control, b: PEG-Au@SiO2, c:
PEG-Au@SiO2 + NIR, d: PEG-Au@SiO2-SNO, e: PEG-Au@SiO2-SNO + NIR, f: PEG￾PAu@SiO2-SNO + NIR groups); Scale bar: 40 μm. Data were shown as mean ± S.D. (n=3).
Significance is defined as * P < 0.05, ** P < 0.01, *** P < 0.001 and n.s. P > 0.05.
Sequentially, the H&E histology analysis of excised tumor tissues and major
organs including heart, liver, spleen, lung, and kidney was performed. As showed in
Figure 4f, compared with other groups, significantly enhanced the cell shrinkage and
nuclear condensation indicated the large-scale apoptosis, which was agree with the
previous results. However, no obvious histopathological lesions to major organs were
observed after administration of various nanocomposites (Figure 5). Thereby, effective
anticancer efficiency of PEG-Au@SiO2-SNO were realized accompanying high
biosafety.
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Figure 5. H&E stained images of extracted tumor tissues treated with different nanocomposites;
Scale bar: 40 μm.
Conclusion
In this study, we successfully fabricated a novel low-temperature triggered NO
release for sensitized mild PTT by integrating photothermal agents, and NO donors
RSNO into a multifunctional nanocomposite. Under NIR irradiation, this PEG￾PAu@SiO2-SNO could cause controlled increase of temperature, which not only
caused dramatic release at specific site, but als realized low-temperature PTT with the
help of HSP-70 inhibitor. The generated NO could effectively induce cell apoptosis or
necrosis, thereby strengthening the damage of PES-mediated mild PTT for combating
tumor. The in vitro and vivo results confirmed the powerful dominance of PTT￾enhanced NO gas therapy with minimized side effects. Consequently, our study sheds
light on the rational design of the controlled NO release nanoplatform for synergistic
antitumor therapy and other NO-relevant diseases.
Supporting Information
The experimental section in details; TEM image of PEG-Au@SiO2-SNO
nanocomposites; Zeta potential change of different nanocomposites; The cumulative
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release kinetics of PEG-SNO nanocomposites in different pH with or
without NIR irradiation; The temperature variation of PEG-Au@SiO2-SNO
nanocomposites suspensions (50 μg/mL) with 5 cycles of NIR laser irradiation; MCF-
7 cell viability HSP inhibitor incubated with different concentration PEG-PAu@SiO2 nanocomposites.
Author Information
Corresponding Author: [email protected]
Acknowledgements
We are highly grateful to the financial support from the National Natural Science
Foundation of China (Grant Nos. 21905138, 31200564), the National Natural Science
Foundation of Jiangsu Province (Grant No. BK20190756), the project funded by China
Postdoctoral Science Foundation (No. 2019M651841), the National Key Research and
Development Program of China (2016YFD0600801), the Top-notch Academic
Programs Project of Jiangsu Higher Education Institutions (TAPP, Grant Nos.
PPZY2015C221) and Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD).
Statement
All animal experiments in this research work complied with the Regulations on
the Administration of Laboratory Animals and the relevant national laws and
regulations, and were performed under the guidance approved by the Laboratory
Animal Ethics Committee at School of Medicine, Southeast University (Nanjing,
China).
References
(1) Módis, K.; Bos, E. M.; Calzia, E.; van Goor, H.; Coletta, C.; Papapetropoulos, A.;
Hellmich, M. R.; Radermacher, P.; Bouillaud, F.; Szabo, C. Regulation of
mitochondrial bioenergetic function by hydrogen sulfide. Part II. Pathophysiological
and therapeutic aspects. Brit. J. Pharmacol. 2014, 171, 2123–2146.
(2) He, Q. Precision gas therapy using intelligent nanomedicine. Biomater. Sci. 2017,
5, 2226–2230.
(3) He, Q.; Kiesewetter, D. O.; Qu, Y.; Fu, X.; Fan, J.; Huang, P.; Liu, Y.; Zhu, G.;
Page 13 of 17
ACS Paragon Plus Environment
ACS Biomaterials Science & Engineering

Liu, Y.; Qian, Z. et al. NIR-Responsive On-Demand Release of CO from Metal
Carbonyl-Caged Graphene Oxide Nanomedicine. Adv. Mater. 2015, 27, 6741–6746.
(4) García-Gallego, S.; Bernardes, G. J. L. Carbon-monoxide-releasing molecules for
the delivery of therapeutic CO in vivo. Angew. Chem. 2014, 53, 9712–9721.
(5) Wegiel, B.; Gallo, D.; Csizmadia, E.; Harris, C.; Belcher, J.; Vercellotti, G. M.;
Penacho, N.; Seth, P.; Sukhatme, V.; Ahmed, A. et al. Carbon monoxide expedites
metabolic exhaustion to inhibit tumor growth. Cancer Res. 2013, 73, 7009–7021.
(6) Motterlini, R.; Otterbein, L. E. The therapeutic potential of carbon monoxide.
Nat. Rev. Drug Discovery. 2010, 9, 728–743.
(7) Guo, R.; Tian, Y.; Wang, Y.; Yang, W. Near-Infrared Laser-Triggered Nitric
Oxide Nanogenerators for the Reversal of Multidrug Resistance in Cancer. Adv.
Funct. Mater. 2017, 27, 1606398.
(8) Tan, L.; Huang, R.; Li, X.; Liu, S.; Shen, Y.-M. Controllable release of nitric
oxide and doxorubicin from engineered nanospheres for synergistic tumor therapy.
Acta Biomater. 2017, 57, 498–510.
(9) Jia, X.; Zhang, Y.; Zou, Y.; Wang, Y.; Niu, D.; He, Q.; Huang, Z.; Zhu, W.; Tian,
H.; Shi, J. et al. Dual Intratumoral Redox/Enzyme-Responsive NO-Releasing
Nanomedicine for the Specific, High-Efficacy, and Low-Toxic Cancer Therapy. Adv.
Mater. 2018, 30, e1704490.
(10) Duong, H. T. T.; Kamarudin, Z. M.; Erlich, R. B.; Li, Y.; Jones, M. W.;
Kavallaris, M.; Boyer, C.; Davis, T. P. Intracellular nitric oxide delivery from stable
NO-polymeric nanoparticle carriers. Chem. Commun. 2013, 49, 4190–4192.
(11) Fukumura, D.; Kashiwagi, S.; Jain, R. K. The role of nitric oxide in tumour
progression. Nat. Rev. Cancer. 2006, 6, 521–534.
(12) Fan, W.; Yung, B. C.; Chen, X. Stimuli-Responsive NO Release for On-Demand
Gas-Sensitized Synergistic Cancer Therapy. Angew. Chem. 2018, 57, 8383–8394.
(13) Sharma, K.; Chakrapani, H. Site-directed delivery of nitric oxide to cancers.
Nitric Oxide. 2014, 43, 8–16.
(14) Kim, J.; Yung, B. C.; Kim, W. J.; Chen, X. Combination of nitric oxide and drug
delivery systems: tools for overcoming drug resistance in chemotherapy. J. Controlled
Release. 2017, 263, 223–230.
(15) Jin, Z.; Wen, Y.; Hu, Y.; Chen, W.; Zheng, X.; Guo, W.; Wang, T.; Qian, Z.; Su,
B.-L.; He, Q. MRI-guided and ultrasound-triggered release of NO by advanced
nanomedicine. Nanoscale. 2017, 9, 3637–3645.
(16) Fan, W.; Bu, W.; Zhang, Z.; Shen, B.; Zhang, H.; He, Q.; Ni, D.; Cui, Z.; Zhao,
K.; Bu, J. et al. X-ray Radiation-Controlled NO-Release for On-Demand Depth￾Independent Hypoxic Radiosensitization. Angew. Chem. 2015, 54, 14026–14030.
(17) Choi, H. W.; Kim, J.; Kim, J.; Kim, Y.; Song, H. B.; Kim, J. H.; Kim, K.; Kim,
W. J. Light-Induced Acid Generation on a Gatekeeper for Smart Nitric Oxide
Delivery. ACS Nano. 2016, 10, 4199–4208.
(18) Huerta, S. Nitric oxide for cancer therapy. Future Sci. OA. 2015, 1, FSO44.
(19) Rink, J. S.; Sun, W.; Misener, S.; Wang, J.-J.; Zhang, Z. J.; Kibbe, M. R.;
Dravid, V. P.; Venkatraman, S.; Thaxton, C. S. Nitric Oxide-Delivering High-Density
Lipoprotein-like Nanoparticles as a Biomimetic Nanotherapy for Vascular Diseases.
Page 14 of 17
ACS Paragon Plus Environment
ACS Biomaterials Science & Engineering

ACS Appl. Mater. Interfaces. 2018, 10, 6904–6916.
(20) Ravikumar, G.; Bagheri, M.; Saini, D. K.; Chakrapani, H. A small molecule for
theraNOstic targeting of cancer cells. Chem. Commun. 2017, 53, 13352–13355.
(21) Méndez, J.; Monteagudo, A.; Griebenow, K. Stimulus-responsive controlled
release system by covalent immobilization of an enzyme into mesoporous silica
nanoparticles. Bioconjugate chemistry. 2012, 23, 698–704.
(22) Li, Y.; Gao, Z.; Chen, F.; You, C.; Wu, H.; Sun, K.; An, P.; Cheng, K.; Sun, C.;
Zhu, X. et al. Decoration of Cisplatin on 2D Metal-Organic Frameworks for
Enhanced Anticancer Effects through Highly Increased Reactive Oxygen Species
Generation. ACS Appl. Mater. Interfaces. 2018, 10, 30930–30935.
(23) Gao, Z.; Li, Y.; You, C.; Sun, K.; An, P.; Sun, C.; Wang, M.; Zhu, X.; Sun, B.
Iron Oxide Nanocarrier-Mediated Combination Therapy of Cisplatin and Artemisinin
for Combating Drug Resistance through Highly Increased Toxic Reactive Oxygen
Species Generation. ACS Appl. Bio Mater. 2018, 1, 270–280.
(24) Lee, H. J.; Kim, D. E.; Park, D. J.; Choi, G. H.; Yang, D.-N.; Heo, J. S.; Lee, S.
C. pH-Responsive mineralized nanoparticles as stable nanocarriers for intracellular
nitric oxide delivery. Colloids Surf., B. 2016, 146, 1–8.
(25) Zhang, X.; Guo, Z.; Liu, J.; Tian, G.; Chen, K.; Yu, S.; Gu, Z. Near infrared
light triggered nitric oxide releasing platform based on upconversion nanoparticles for
synergistic therapy of cancer stem-like cells. Sci. Bull. 2017, 62, 985–996.
(26) You, C.-Q.; Wu, H.-S.; Gao, Z.-G.; Sun, K.; Chen, F.-H.; Tao, W. A.; Sun, B.-
W. Subcellular co-delivery of two different site-oriented payloads based on multistage
targeted polymeric nanoparticles for enhanced cancer therapy. J. Mater. Chem. B.
2018, 6, 6752–6766.
(27) You, C.; Gao, Z.; Wu, H.; Sun, K.; Ning, L.; Lin, F.; Sun, B.; Wang, F. Reactive
oxygen species mediated theranostics using a Fenton reaction activable lipo￾polymersome. J. Mater. Chem. B. 2019, 7, 314–323.
(28) Gao, Z.; You, C.; Wu, H.; Wang, M.; Zhang, X.; Sun, B. FA and cRGD dual
modified lipid-polymer nanoparticles encapsulating polyaniline and cisplatin for
highly effective chemo-photothermal combination therapy. J. Biomater. Sci-polymer.
E. 2018, 29, 397–411.
(29) Zhou, J.; Li, M.; Hou, Y.; Luo, Z.; Chen, Q.; Cao, H.; Huo, R.; Xue, C.;
Sutrisno, L.; Hao, L. et al. Engineering of a Nanosized Biocatalyst for Combined
Tumor Starvation and Low-Temperature Photothermal Therapy. ACS Nano. 2018, 12,
2858–2872.
(30) Zhang, H.; Tian, X.-T.; Shang, Y.; Li, Y.-H.; Yin, X.-B. Theranostic Mn￾Porphyrin Metal-Organic Frameworks for Magnetic Resonance Imaging-Guided
Nitric Oxide and Photothermal Synergistic Therapy. ACS Appl. Mater. Interfaces.
2018, 10, 28390–28398.
(31) Zhang, K.; Meng, X.; Cao, Y.; Yang, Z.; Dong, H.; Zhang, Y.; Lu, H.; Shi, Z.;
Zhang, X. Metal-Organic Framework Nanoshuttle for Synergistic Photodynamic and
Low-Temperature Photothermal Therapy. Adv. Funct. Mater. 2018, 28, 1804634.
(32) Liu, D.; Ma, L.; Liu, L.; Wang, L.; Liu, Y.; Jia, Q.; Guo, Q.; Zhang, G.; Zhou, J.
Polydopamine-Encapsulated Fe3O4 with an Adsorbed HSP70 Inhibitor for Improved
Page 15 of 17
ACS Paragon Plus Environment
ACS Biomaterials Science & Engineering

Photothermal Inactivation of Bacteria. ACS Appl. Mater. Interfaces. 2016, 8, 24455–
24462.
(33) Shevtsov, M.; Stangl, S.; Nikolaev, B.; Yakovleva, L.; Marchenko, Y.; Tagaeva,
R.; Sievert, W.; Pitkin, E.; Mazur, A.; Tolstoy, P. et al. Granzyme B Functionalized
Nanoparticles Targeting Membrane Hsp70-Positive Tumors for Multimodal Cancer
Theranostics. Small. 2019, 15, e1900205.
(34) Zhang, Z.; Wang, L.; Wang, J.; Jiang, X.; Li, X.; Hu, Z.; Ji, Y.; Wu, X.; Chen,
C. Mesoporous silica-coated gold nanorods as a light-mediated multifunctional
theranostic platform for cancer treatment. Adv. Mater. 2012, 24, 1418–1423.