Self-assembly of nanoparticles by human serum albumin and photosensitizer for targeted near-infrared emission fluorescence imaging and effective phototherapy of cancer

Article abstract of DOI:10.1039/c8tb03054e

Photodynamic therapy (PDT) and photothermal therapy (PTT) are effective cancer treatments, and photosensitizers play the most important role in the treatment. However, photosensitizers are insufficient for in vivo tumor treatment. Herein, we develop a small molecule fluorophore Cy-HPT as a novel photosensitizer, which possesses the advantages of near-infrared (NIR) emission, high photothermal conversion efficiency and high singlet oxygen generation efficiency. Moreover, a nanoplatform of HSA@Cy-HPT was synthesized by self-assembly of Cy-HPT and human serum albumin (HSA) in aqueous solution. Compared to Cy-HPT, HSA@Cy-HPT possesses more stable spectral properties, enhances the effect of PDT/PTT, and exhibits more satisfactory in vivo metabolism. HSA@Cy-HPT demonstrates outstanding tumor targeting in subcutaneous tumor xenograft models owing to its enhanced permeability and retention in tumor tissue. Furthermore, HSA@Cy-HPT was successfully utilized in tumor xenograft models and tumor tissue growth was clearly inhibited without any regrowth, extending survival rate of the models. Also, no distinct damage of the normal tissue of tumor xenograft models was observed using hematoxylin & eosin staining. This study presents a promising therapeutic agent for the synergetic PDT and PTT cancer treatment.

Full text of DOI:10.1039/c8tb03054e

View Article Online
View Journal
Journal of
Materials Chemistry B
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Y. Huang, N. He,
Y. Wang, D. Shen, Q. Kang, R. Zhao and L. Chen, J. Mater. Chem. B, 2019, DOI: 10.1039/C8TB03054E.
This is an Accepted Manuscript, which has been through the
Volume
4 Number 1 7 January 2016 Pages 1–178
Royal Society of Chemistry peer review process and has been
accepted for publication.
Journal of
Materials Chemistry B
Accepted Manuscripts are published online shortly after
Materials for biology and medicine
www.rsc.org/MaterialsB
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 2050-750X
PAPER
Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
micropattern features
rsc.li/materials-b

Please do not adjust margins
Page 1 of 11
Journal of Materials Chemistry B
View Article Online
DOI: 10.1039/C8TB03054E
Journal Name
ARTICLE
Self-assembly Nanoparticles by Human Serum Albumin and
Photosensitizer for Targeted Near-infrared Emission Fluorescence
Imaging and Effective Phototherapy of Cancer
Received 00th January 20xx,
Accepted 00th January 20xx
Yan Huang,a,b Na He,b Yunqing Wang,b Dazhong Shen,a Qi Kang,*a Rongfang Zhao,b Lingxin Chen*b,c
DOI: 10.1039/x0xx00000x
www.rsc.org/
Photodynamic therapy (PDT) and photothermal therapy (PTT) are effective treatment ways for cancer, and the
photosensitizer plays the most important role in the treatment. However, the ideal photosensitizers are insufficient for in
vivo tumor treatment. Herein, we develop a small molecule fluorophore Cy-HPT as a novel photosensitizer, which with the
advantages of near-infrared (NIR) emission wavelength, high photothermal conversion efficiency and high singlet oxygen
generation efficiency. Moreover, a nanoplatform of HSA@Cy-HPT is synthesized by self-assemble of Cy-HPT and human
serum albumin (HSA) and Cy-HPT in aqueous solution. Compared to Cy-HPT, HSA@Cy-HPT exhibits not only much stable
spectral properties, superior effect of PDT/PTT but also more satisfactory in vivo metabolism. HSA@Cy-HPT shows an
outstanding tumor targeting feature in subcutaneous tumor xenograft models due to its enhanced permeability and
retention effect for tumor tissue. Furthermore, HSA@Cy-HPT is successfully applied to treatment of tumor xenograft
models, the tumor tissue is obviously inhibited without any regrowth and extend survival rate of models. Also, the normal
tissue of tumor xenograft models is observed no distinct damage by hematoxylin & eosin staining. This work provides a
promising therapeutic agent for the synergetic PDT and PTT for cancer.
temperature than normal cells. The local temperature increasing
causes DNA damage and the apoptosis of tumor cells eventually.5
Considering the superiority of PDT and PTT, the synergetic PDT
and PTT treatments have been widely used in clinical treatment of
tumor.6-13
Recently, the design and synthesis of small molecule
photosensitizer has been widely reported.14-16 Some fluorescent dyes
have been demonstrated with photosensitization effects, which are
closely associated with their lipo-hydro partition coefficients (log P),
polarizabilities, and molar absorption coefficients.17,18 Most of the
reported photosensitizers having the excitation and emission
wavelengths in visible region, and are not suitable for the imaging
and PDT/PTT in deep organization.19-23 Indocyanine green (ICG) is
1. Introduction
As the incidence and death rates associated with cancer remain high,
the innovated therapeutic strategies are imperative. Conventional
treatment of cancers includes combinations of surgery, radiotherapy,
and chemotherapy. Photodynamic therapy (PDT) and photothermal
therapy (PTT) are the promising therapeutic techniques due to high
curative effect, low toxicity and negligible side effects.1-3
Photosensitizer plays the key role in both PDT and PTT. On one
hand, they are generally excited to generate singlet oxygen (1O2)
under light irradiation due to the photophysical conversion
phenomena.4 1O2 not only has an extremely oxidation for the
clipping react with protein, free radical and other bioactive
molecules in cytoplasm, but also can penetrate the nuclear
membrane and lead to DNA damage and some other physiological
changes.4 On the other hand, photosensitizer is also crucial for PTT,
which can transform the luminous energy to thermal energy under
the laser irradiation. The tumor cells are extremely sensitive to
Food
and
Drug
Administration
(FDA)-approved
NIR
25
photosensitizer,24,
for clinical application.26 However, there are
several drawbacks such as self-aggregation in physiological
solutions, nonspecific binding to proteins, and rapid renal
elimination from the body. To address these problems, researchers
developed a new ICG analog, IR-DBI, with simultaneous NIR
imaging and chemo-/PDT/PTT/multimodal anticancer activity,
which has the better treatment than ICG.18 However, the maximum
absorbance (near 780 nm) is still away from the wavelength of
common used 808 nm excitation laser, thus affecting the treatment
efficacy. Moreover, IR-DBI with a small-molecule structure is
difficult to accumulate in tumor tissue within the suitable treatment
time. Therefore, the development of ideal photosensitizer with a
suitable excitation and emission wavelengths for the effective PDT
a.College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory
of Molecular and Nano Probes, Ministry of Education, Shandong Normal
University, Jinan 250014, China. *E-mail: kangqi@sdnu.edu.cn.
b.CAS Key Laboratory of Coastal Environmental Processes and Ecological
Remediation, The Research Center for Coastal Environmental Engineering and
Technology, Yantai Institute of Coastal Zone Research, Chinese Academy of
Sciences, Yantai 264003, China. *E-mail: lxchen@yic.ac.cn.
c. College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu
273165, China.
†Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 2 of 11
ARTICLE
Journal Name
and PTT, which could be accumulated in tumor tissue with a Scientific NanoDrop 2000/2000C spectrophotometeVriewanAdrticlae ONnlIinRe
reasonable time is necessary.
fluorescence spectrometer (HORIBA ScientiDfiOcIF: 1lu0o.1r0o3m9/aCx8-T4Bs0p3e0c5t4rEo
Photosensitizer loaded, multifunctional nanoplatform also has fluorometer with a Xenon lamp and 1.0-cm quartz cells) with 810
great importance and interest in a variety of medical applications.27- nm excitation and scanning the wavelength from 830 to 900 nm (5-
32
The rapid development of nanomaterials with large surface areas, nm slit width at a scan speed of 600 nm/min).
tunable material properties, and strong signal output will have
significant impact on the future personalized oncology.33-35 2.2 Animals and Tumor Xenografts.
Compared with small-molecule photosensitizer, the nanoplatform
BALB/c mice (aged 5-6 weeks, weighed 20-25 g) and athymic nude
has some advantages, such as superior photostability, effective
mice (aged 5-6 weeks, weighted 20-25 g) were purchased from
targeting and long-time accumulation in tumor.36-41 To date, various
Binzhou Medical University. All experimental procedures were
inorganic and organic nanomaterials (NMs) made of carbon, gold
conducted in conformity with institutional guidelines for the care
and organic polymer have been applied to PDT and PTT for cancer.1,
and use of laboratory animals, and protocols were approved by the
35, 42-44 However, NMs with hydrodynamic diameter ranging from 10
Institutional Animal Care and Use Committee in Binzhou Medical
to 200 nm are easy to non-specific accumulation in
University, Yantai, China. For HepG2 tumor xenografts, 5 ×106
reticuloendothelial system (RES) organs for a long time, which
HepG2 cells suspended in 200 μL PBS were subcutaneously
implanted into the right flank of each athymic nude mouse. All
animals were maintained under aseptic conditions and were housed
raises concerns about the long-term risk of adverse effects.45-47
Recently, protein based theranostics nanoplatforms have been
extensively investigated. The self-assemblies between amphiphilic
in a group of five in standard cages with free access to food and
photosensitizers and endogenic proteins (such as human serum
water and a 12 h light/dark cycle.
albumin, HSA) exhibit high drug loading rates, good water-
solubility, biocompatiblity, and tumor selectivity.48-50 Meanwhile,
2.3 Tumor NIR Fluorescent Imaging and Thermal Imaging.
the size (nearly 10 nm) is suitable for renal clearance to eliminate
The animals with HepG2 tumor xenograft models were used for
possible accumulation toxicity.48
tumor-targeted NIR imaging after 48 h intravenously injected 10
Herein, we report a photosensitizer that benzo-heptamethine
mg/kg HSA@Cy-HPT with the Perkinelmer IVIS Lumina XRMS
cyanine dye triphenylphosphonio benzo cyanine (Cy-HPT), with a
Series III In Vivo Imaging System.5 NIR imaging of whole animals
longer emission wavelength (860 nm) than previous research,14
in vivo was performed at different times (0.5h, 6 h, 24 h, 48 h, 72 h)
post drug injection. After sacrifice of the mice at 0.5 h, 6 h, 24 h, 48
which is benefit for the application of PDT/PTT in vivo.
Furthermore, we synthesized a nanoplatform HSA@Cy-HPT by
h and 72 h, major organs and tumors were dissected for ex vivo NIR
self-assembling of Cy-HPT and HSA. This nanoplatform exhibits
fluorescent imaging, and for studying their tissue distribution. For in
vivo assessment of PTT property, mice with HepG2 tumor
xenografts were intravenously injected HSA@Cy-HPT with 10
mg/kg. After 24 h injection, the realtime temperature change of mice
excellent photostability, tumor cell-selectivity, and satisfactory
synergetic PDT/PTT effect in vitro and in vivo via the efficient
1
photothermal conversion and the generation of O2 under NIR laser
irradiation. Our study indicates that the synergetic PDT and PTT
was imaged by the infrared thermal camera (Testo 865, Germany)
have the distinguished treatment effect for cancer cells. Western blot
verified the HSA@Cy-HPT induced apoptosis by quantified the
apoptosis related protein. In vivo experiments reveal that HSA@Cy-
thermal imaging, mice were anesthetized by inhalation of isoflurane.
HPT has long-time accumulation in tumor site due to the EPR effect,
when the whole tumor tissue was exposed to the continuous NIR
laser beam (808 nm, 1.5 W/cm2, 5 min). Prior to NIR fluorescence or
followed by efficient renal clearance. Finally, the tumor tissue has
been obviously inhibited without any regrowth and extend survival
2.4 Synthesis of Compound Cy-HPT
rate of models by the PDT and PTT of HSA@Cy-HPT.
Compound
4 (46.5 mg, 0.05 mmol) and (4-Bromobutyl)
triphenylphosphonium bromide （ 47.8 mg ， 0.1 mmol ） were
dissolved in dry acetone (25 mL), then anhydrous potassium
carbonate (27.5 mg，0.2 mmol) was added. The mixture was stirred
under a dry argon atmosphere for 24 h and monitored by TLC. After
the reaction was finished, the mixture was cooled to room
temperature, evaporated under reduced pressure and partitioned with
CH2Cl2 and saturated KBr solution. Finally, the organic layer was
separated. Purification by column chromatography on silica eluting
with EtOAc/CH3OH (3:1) gave the product Cy-HPT as dark green
2. Experimental
2.1 Preparation and Characterization of HSA@Cy-HPT
HSA@Cy-HPT were prepared with the similar method previously
reported.48 Briefly,
5 mg as-synthesized Cy-HPT was firstly
dispersed in 10 ml methanol. HSA (Sigma-Aldrich, 200 mg) and 10
μL TEA (triethylamine) were mixed in 10 ml ultrapure water for 30
min. Then, the mixture was added with 10 ml above-mentioned
methanol solution of Cy-HPT for overnight vigorous stirring. After
24 h dialysis of the reaction solution with a dialysis bag (MWCO 10
KDa), HSA@Cy-HPT nanoparticles were obtained. The mean
particle size was determined by DLS Zetasizer Nano ZS90 (Malvern
Instruments Ltd, UK) and by TEM (JEOL, model JEM-1230, Japan)
operated at 100 kV. The optical properties of chemicals and
HSA@Cy-HPT nanoparticles were characterized by using Thermo
1
crystals (35.74 mg, 53%). H NMR (CH3DO, 500 MHz) δ (ppm):
8.47-8.45 (m, 2H), 7.87-7.76 (m, 6H), 7.63-7.50 (m, 5H), 7.36-7.28
(m, 19H), 7.05-6.91 (m, 5H), 6.37-6.10 (m, 1H), 5.33 (m, 1H), 4.57-
4.27 (m, 2H), 4.13-4.03 (m, 2H), 3.87-3.44 (m, 8H), 3.30 (m, 8H),
2.96-2.93 (m, 1H), 2.63-2.62 (m, 2H), 2.18-2.09 (m, 4H), 1.94-1.88
(m, 8H), 1.76-1.62 (m, 4H), 1.28 (m, 6H), 0.89 (m, 2H). 13C NMR
(CH3DO, 125 MHz) δ (ppm): 175.7, 171.5, 165.4, 165.1, 160.6,
137.5, 133.4, 132.0, 131.9, 131.8, 129.8, 129.0, 128.7, 128.6, 128.5,
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Page 3 of 11
Journal of Materials Chemistry B
Journal Name
ARTICLE
126.3, 117.3, 113.7, 113.6, 112.4, 112.3, 49.7, 49.6, 49.5, 49.2, 46.7, We then evaluated their photoconverted thermal effVeicewtsAritnicledOetnalinile.
2
DOI: 10.1039/C8TB03054E
46.5, 46.4, 46.2, 46.0, 45.9, 45.7, 45.5, 42.6, 30.1, 27.8, 27.5, 27.4, Under exposure to an 808 nm continuous NIR laser at 1.5 W/cm for
25.3, 25.1, 25.0, 24.8, 18.4. LC-MS (ESI+): m/z C88H86ClN4O7P2+ 5 min (Fig. 2a), the temperature of Cy-HPT (10 μM) and HSA@Cy-
calcd. 674.29, found [M+] 674.69.
HPT (200 μg/mL, containing equivalent 10 μM of Cy-HPT)
solutions rose from 24 °C to 45 °C and 55 °C, respectively. HSA@-
3. Results and discussion
3.1 Synthesis and Characterization of Cy-HPT and HSA@Cy-
HPT
A benzo-heptamethine cyanine dye by modification of N-alkyl side
chains based on the heptamethine core was designed and
synthesized, and was named Cy-HPT (Scheme 1). Structures of the
Cy-HPT were characterized by 1H NMR, 13C NMR, and mass
spectrometry (MS), as described in Supporting Information.
HSA@Cy-HPT nanoparticle (NP) was synthesized by the self-
assembly of Cy-HPT and HSA (Scheme 1). Cy-HPT can be
adsorbed onto the hydrophobic domain of HSA, the complex with
much higher hydrophobicity can act as an “adhesive” between
different proteins, and then induce assembly of more albumin
proteins to form a nanoparticle, inside which Cy-HPT are embedded.
We then carefully characterized the obtained HSA@Cy-HPT
nanoparticles. The HSA@Cy-HPT we obtained had the loading ratio
of 6.7% for Cy-HPT. Transmission electron microscopy (TEM)
imaging and DLS analysis revealed that the size of NP was around
10 nm, indicating that the Cy-HPT binding dose not induce severe
HSA aggregation (Fig. 1a and 1b). The NPs could be easily
dispersed in various physiological buffers. The different pH effects
on Cy-HPT and HSA@Cy-HPT were investigated in Supporting
Information (Fig. S1).
The spectroscopic properties of the Cy-HPT and HSA@Cy-HPT
were investigated under simulated physiological conditions (10 mM
HEPES pH 7.4). As shown in Fig. 1c, the maximum absorption of
Cy-HPT and HSA@Cy-HPT appeared at 830 nm (ε830 nm = 4.2 × 104
M-1 cm-1) and 833 nm (ε833 nm = 7.9 × 104 M-1 cm-1), respectively.
The Cy-HPT and HSA@Cy-HPT exhibited fluorescence emission
spectra centered at 860 nm and 863 nm, respectively, which
extended across the NIR region (Fig. 1d). The modification of N-
alkyl side chains based on the heptamethine core might make the
long emission wavelength. Due to extremely low tissue
autofluorescence and absorption in NIR spectral range (650 - 900
nm), optical imaging and phototherapy with high sensitivity and
deep tissue penetration could be realized. It should be noted that
HSA@Cy-HPT exhibited slightly red-shifted absorbance and
emission peak as well as obviously enhanced fluorescence compared
with Cy-HPT (Fig. 1d), which are likely owing to the slightly
changed molecular conformation after Cy-HPT is complexed with
the albumin protein. Cy-HPT can be adsorbed onto the hydrophobic
domain of HSA, the complex with much higher hydrophobicity can
act as an “adhesive” between different proteins, and then induce
assembly of more albumin proteins to form a nanoparticle, inside
which Cy-HPT are embedded.48 The satisfactory optical properties
lay the foundation for the dyes to be used for bioimaging and tumor
treatment.
Scheme 1. The synthetic route and the photoinduced treatment mechanism
of Cy-HPT and HSA@Cy-HPT.
Cy-HPT has larger photothermal conversion efficiency (54.98%)
than Cy-HPT (45.21%). This is probably because 1) the absorption
coefficient of HSA@Cy-HPT is 42% higher than that of Cy-HPT
owing to the complex effect. 2) The photostability of HSA@Cy-
HPT is dramatically increased. It has been reported that ICG
decomposition due to the break of the double bonds under
irradiation,51 such phenomenon also occurs to our Cy-HPT. As
shown in Fig. 2c, the color of Cy-HPT sample disappeared by five
cycles of irradiation 808 nm 1.5 W/cm2 10 min due to similar
double-bond breaking decomposition mechanism proved by
analyzing the mass spectra of sample solution (m/z = 403.46, 429.73
and 964.44) and characterizing the structures of the decomposition
products (Fig. S1). Cy-HPT is decomposed by the laser through the
reaction of the double bonds of the flexible polymethine chain with
1
photo-induced generated O2 by cycloaddition and finally thermal
decomposition into carbonyl compounds.52, 53 By contrast, as shown
as Fig. 2b and 2c, the heating ability of HSA@Cy-HPT greatly
increased, because the molecular conformation of Cy-HPT
embedded in hydrophobic region of HSA was stabilized, preventing
the occurrence of the oxidation-induced decomposition reaction.
Thus, the photo-stability and photothermal ability of Cy-HPT in
hybrid NPs are dramatically improved.
Fig. 1 a) TEM imaging of HSA@Cy-HPT. Scale bar: 20 nm. b) The
Hydrodynamic diameters of HSA@Cy-HPT measured by DLS. c) Absorption
spectra of Cy-HPT and HSA@Cy-HPT. d) Fluorescence emission spectra of Cy-
HPT and HSA@Cy-HPT.
3.2 Evaluation Photothermal Conversion and Singlet Oxygen
Generation Efficiency
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 4 of 11
ARTICLE
Journal Name
Similar results can be found in living cells. As shown in Fig. 2d, nontargeting clinically available heptamethine cyanVinieew Adrtyicele.5O4 nlAines
1
DOI: 10.1039/C8TB03054E
the fluorescence signal of HSA@Cy-HPT in HepG2 cells exhibited shown in Fig. 2e, Cy-HPT and HSA@Cy-HPT exhibited more O2
stability after the laser irradiation for 30 min. By contrast, the generation than that of ICG. This finding is consistent with previous
1
fluorescence from Cy-HPT labeled cells apparently decreased within studies.16 Next, O2 generation of Cy-HPT and HSA@Cy-HPT in
10 min and totally disappeared after 30 min irradiation. This result cells upon NIR laser irradiation was also investigated. SOSG was
suggested that HSA@Cy-HPT was suitable for long-term and employed to detect the generation of 1O2 in living cells by laser
repeated cancer treatment.
scanning confocal microscope. As shown in Fig. 2f, the control
Furthermore, the singlet oxygen (1O2) generated from Cy-HPT group showed weak fluorescence, indicating no 1O2 generated in
and HSA@Cy-HPT were also detected by using Singlet oxygen blank cells. Both laser groups emitted fluorescence with prolonged
sensor green (SOSG, Invitrogen co., USA) as a probe. As shown in irradiation time. And the signal from HSA@Cy-HPT treated cells is
1
Fig. 2e, the emission intensity indicated that O2 was generated by stronger. These imaging results convinced that HSA@Cy-HPT could
Cy-HPT and HSA@Cy-HPT in the solution. However, the 1O2 generate more 1O2 in living cells under laser irradiation. These above
production of HSA@Cy-HPT was higher than that in blank water results show the potential of Cy-HPT and HSA@Cy-HPT for
1
and Cy-HPT. We also compared the O2 generation of Cy-HPT and PDT/PTT therapy.
HSA@Cy-HPT with indocyanine green (ICG), which is
a
Fig. 2 a) Temperature curves of Cy-HPT and HSA@Cy-HPT with during 5 min NIR laser irradiation. b) The photothermal stability of HSA@Cy-HPT. c) The
photothermal stability of Cy-HPT. d) Confocal microscopy images of HepG2 cells for photothermal stability of Cy-HPT and HSA@Cy-HPT upon the continuous
laser irradiated for 30 min. Fluorescence channel: λex = 808 nm, λem = 830 - 880 nm. e) The fluorescence intensity of SOSG: 1. Blank, 2. ICG, 3. Cy-HPT, 4.
HSA@Cy-HPT. f) Confocal microscopy images of HepG2 cells for evaluating the generation of 1O2 by SOSG upon NIR laser irradiation. Fluorescence channel:
λex = 490 nm, λem = 510 - 560 nm. Images are representative of n = 5 independent experiments. Scale bar = 20 μm.
3.3 In Vitro Photoinduced Cytotoxicity and Organelle indicated the low cytotoxicity of Cy-HPT and HSA@Cy-HPT under
Localization
no laser irradiation.
On the basis of the above experiments, it is necessary to verify
whether Cy-HPT and HSA@Cy-HPT has great potential to
phototherapeutic effect in complex biological systems. First, we
tested Cy-HPT and HSA@Cy-HPT for its PTT and PDT ability in
tumor cells (HepG2 cells). MTT assay showed the cell viability in
the radiation groups decreased with the increased concentration of
Cy-HPT (0 - 10 μM) and HSA@Cy-HPT (0 - 200 μg/mL), and the
cell viability rate obviously decrease at 10 μM (Cy-HPT) and 200
μg/mL (HSA@Cy-HPT) (Fig. 3a-3b). In contrast, the cell viability
in the nonirradiation groups was not affected significantly. All these
data enabled Cy-HPT and HSA@Cy-HPT exhibited an efficient PTT
and PDT ability to kill tumor cells.
We next tested the in vitro cancer cell killing efficacy of Cy-HPT
and HSA@Cy-HPT. HepG2 cells originally obtained from the
Committee on Type Culture Collection of Chinese Academy of
Sciences (Shanghai, China) and cultured under recommended
conditions, were incubated with free Cy-HPT and HSA@Cy-HPT
for 72 h. The standard cell viability assay using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was
then conducted. As demonstrated in Fig. 3a and 3b, above 72% (Cy-
HPT) and 81% (HSA@Cy-HPT) of cells survived for tumor cells
(HepG2 cells) after the cells were incubated with 10 μM Cy-HPT or
200 μg/mL HSA@Cy-HPT for 72 h, then the 50% cell survival
concentration value (IC50) was predicted to be 32 μM (Cy-HPT) and
826 μg/mL (HSA@Cy-HPT) for HepG2 cells, respectively, which
Nanoparticles can selectively enter into cancer cells via
endocytosis by lysosomes.55 Therefore, organelle co-localization
experiment of HSA@Cy-HPT was investigated. As shown in Fig.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Page 5 of 11
Journal of Materials Chemistry B
Journal Name
ARTICLE
3c, HepG2 cells were able to uptake the HSA@Cy-HPT after 3 h of cells were trearted with chlorpromazine (15 µg/mL)VifeowrA1rtichle,Otnhlienne
DOI: 10.1039/C8TB03054E
incubation. A large amount of HSA@Cy-HPT was observed to HSA@Cy-HPT, as as described in above experiment, was added to
accumulate and colocalize with LysoTracker in the cells, indicating the cells for 3 h. As shown as Fig. 3d, an obviously faint
that the HSA@Cy-HPT could be taken up by cancer cells via fluorescence was exhibited in cells, ≈40% decreased in the cellular
endocytosis. Afterward, their endocytosis was further studied using uptake of HSA@Cy-HPT, indicated that the nanoparticles were
an inhibitor. Chlorpromazine, an inhibitor of clathrin-mediated mainly internalized via endocytosis.
endocytosis,15 was employed in HepG2 cells experiment. HepG2
Fig. 3 a), b) The cytotoxicity of a) Cy-HPT and b) HSA@Cy-HPT for HepG2 cells under no laser irradiation or laser irradiation. c) Lysosomes localization of
HSA@Cy-HPT was determined by co-stained HSA@Cy-HPT with lysosomes trackers (Lyso Tracker) in HepG2 cells, the co-localization areas of the red and
blue channels selected, the co-localization areas of the green and blue channels selected, the co-localization areas of the green and red channels selected
and the intensity profile of regions of interest (white line in Merge) across cells. Red channel: λex = 808 nm, λem = 830 - 880 nm). LysoTracker showing
lysosomes (green channel: λex = 559 nm, λem = 570 - 650 nm). Hoechst showing nuclei (blue channel: λex = 405 nm, λem = 425 - 525 nm). Images are
representative of n = 5 independent experiments. Scale bar: 10 µm. d) Confocal microscopy images of HepG2 cells for the way of cellular uptake of HSA@Cy-
HPT via endocytosis. Scale bar: 40 µm. e) Flow cytometry assay of HepG2 cells correspond with d.
3.4 Cellular Uptake Kinetic and Cellular Selectivity
The obvious difference in fluorescence intensities between cancer
cells and normal cells suggested the cancer targeting capability of
HSA@Cy-HPT. For comparison, the same experiments were
performed with 2 μM free Cy-HPT. There almost no obvious
differences between tumor cells and normal cells (Fig. 4b). This is
probably because the small-molecules enter cells mainly via non-
specific free diffusion. The enhanced endocytosis of HSA@Cy-HPT
by tumor cells was also verified by flow cytometry. The results were
consistent with the cell fluorescence imaging. These results indicated
that the nanoparticles HSA@Cy-HPT had a tumor targeting function
without any tumor targeting ligand and enhanced uptake of
HSA@Cy-HPT due to gp60 and SPARC receptor-mediated
transcytosis.26 This is beneficial to obtain ideal curative effect for
real tumor treatment applications.
On the basis of the above fluorescence imaging experiments, the
kinetic experiment of the cellular uptake of Cy-HPT and HSA@Cy-
HPT was studied. By choosing HepG2 cells as cell models, the cells
were treated with Cy-HPT (2 μM) and HSA@Cy-HPT (40 μg/mL,
containing equivalent 2 μM of Cy-HPT), then subjected to cell
fluorescence imaging by laser scanning confocal microscope at
different time points. As shown as Fig. 4a, the cells were treated
with Cy-HPT and HSA@Cy-HPT from 0 - 3 h, the fluorescence
signal was became stronger over time indicated that the cellular
uptake of Cy-HPT and HSA@Cy-HPT exhibited time-dependent
mode. However, the fluorescence signal that treated with HSA@Cy-
HPT for 3 h and Cy-HPT for 0.5 h reached the maximum value. This
result demonstrates that Cy-HPT and HSA@Cy-HPT had the most
intracellular accumulation after that treated for 0.5 h and 3 h,
respectively. Support data the best accumulation time point of Cy-
HPT and HSA@Cy-HPT for PDT and PTT in the tumor cells.
3.5 In Vitro Synergetic PDT, PTT Effect of HSA@Cy-HPT
To verify that the therapeutic effect of HSA@Cy-HPT were
It has been reported that HSA NPs possess both passive and active contributed by synergetic PDT and PTT treatments, laser irradiation
tumor-targeting abilities via enhanced permeability and retention toward HepG2 cells were performed under ice incubation for single
(EPR) effect and gp60 and SPARC receptor-mediated transcytosis, PDT treatment or pretreated with N-acetylcysteine (NAC) and NaN3
56
respectively.26 In our study, the cellular uptake selectivity of the to scavenge ROS and O2 for single PTT treatment. When treated
nanoparticles HSA@Cy-HPT was examined. HSA@Cy-HPT were with HSA@Cy-HPT at 0, 40, 80, 120, 160, 200 μg/mL, MTT assay
added to both tumor cells (HepG2 cells) and normal cells (L02 cells) showed that no cell viability was detected in both PTT and PDT
at 37 °C and incubated for 3 h. As shown as Fig. 4b, only weak treatment at 200 μg/mL (HSA@Cy-HPT), 20.3% cell viability in
fluorescence signal was observed in L02 cells after 3 h of incubation. PTT treatment alone, and 12.4% cell viability in PDT treatment
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 6 of 11
ARTICLE
Journal Name
alone (Fig. 5a). The results suggest that the photoinduced nm, 1.5 W/cm2), apoptotic ratios of synergetic PDT and PTT were
View Article Online
cytotoxicity of HSA@Cy-HPT is synergistically enhanced by PDT observed with obvious increase than the sinDgOleI:P10T.1T03o9r/Csi8nTgBl0e3P05D4TE
and PTT combinatorial treatment and the treatment effect of for HepG2 cells (Fig. 5c). Therefore, the cell death was mediated by
HSA@Cy-HPT for tumor cells is effective. The phototoxicity of the synergetic PDT and PTT of HSA@Cy-HPT manner. Heat shock
HSA@Cy-HPT was also intuitively confirmed through fluorescence protein 70 (Hsp70) is typically synthesized in response to heat stress
images of Calcine AM and propidium iodide (PI) co-stained HepG2 to protect the cell for thermal or oxidative stress.57 Thus, Hsp70
cells (Fig. 5b). The green from Calcine AM and the red from PI expression was monitored to evaluate the heat stress of cells. As
showed the live and dead cells respectively, confirmed the shown in Fig. 5d, the Hsp70 expression was significantly increased
synergistic effects of PDT and PTT. Flow cytometry with Annexin with the PTT group and synergetic PDT and PTT group. Further
V-PE was tested to evaluate the apoptosis rate of single PTT, single indicating that cell apoptosis was mediated by the synergetic PDT
PDT and synergetic PDT and PTT. After 3 h incubation with and PTT of HSA@Cy-HPT.
HSA@Cy-HPT at 200 μg/mL and 5 min NIR light irradiation (808
Fig. 4 a) Confocal microscopy images and flow cytometry assay of HepG2 cells for the kinetic experiment of the cellular uptake of Cy-HPT and HSA@Cy-HPT.
b) The selective cellular uptake of the HSA@Cy-HPT and free Cy-HPT in tumor cells (HepG2 cells) and normal cells (L02 cells). Fluorescence channel: λex = 808
nm, λem = 830 - 880 nm. Images are representative of n = 5 independent experiments. Scale bar = 20 μm.
3.6. In Vivo NIR Fluorescent Imaging and Tumor-bearing Mice
Imaging
with institutional guidelines for the care and use of laboratory
animals, and protocols were approved by the Institutional Animal
Care and Use Committee in Binzhou Medical University, Yantai,
China (Approval Number: No.BZ2014-102R). HSA@Cy-HPT (10
mg/kg) and Cy-HPT (5 mg/kg) were injected into BALB/c mice by
intravenous (i.v.) injection. NIR fluorescent imaging was performed
at different time points. As shown as Fig. 6a, a weak fluorescence
signal was obtained at the beginning, then the fluorescence signal
was becoming stronger and stronger with the passage of time. The
strongest fluorescence signal (mainly in the liver) was obtained after
HSA@Cy-HPT was chosen as the ideal PDT and PTT agent applied
to mice and tumor xenograft models due to its excellent
photothermal conversion ability and photodynamic properties. First,
the application of HSA@Cy-HPT for fluorescence imaging in vivo
was studied in BALB/c mice. Simultaneously, pharmacokinetics of
HSA@Cy-HPT in vivo also was investigated, and Cy-HPT as the
control. All experimental procedures were conducted in conformity
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Page 7 of 11
Journal of Materials Chemistry B
Journal Name
ARTICLE
HSA@Cy-HPT was injected for 12 h, and for Cy-HPT after injected accumulation of HSA@Cy-HPT due to the liver is a mVaijeowrAmrtieclteaObnollinice
for 6 h, next the fluorescence signal was getting weaker due to the site, and the obviously fluorescence signaDl OwI:a1s0.a10ls3o9/Cob8TtaBi0n3e0d54inE
mice metabolism. And the main tissue and organ were removed by kidney. The results revealed that the nanoparticles (HSA@Cy-HPT)
dissected, the fluorescence signal of ex vivo organs (heart, liver, not only could retain in vivo for a longer time than free Cy-HPT, but
spleen, lung, kidney) were analyzed by fluorescence imaging. High also had an efficient renal clearance to avoid the long-term risk of
fluorescent contrast between liver and other normal tissue in adverse effects.
BALB/c mice was observed, suggested that the liver preferential
Fig. 5 a) MTT assay of single PTT, single PDT and synergetic PDT and PTT of HSA@Cy-HPT for HepG2 cells under laser irradiation. b) Fluorescent images of
Calcein AM/PI co-stained HepG2 cells after PTT, PDT, or synergetic PDT and PTT treatments with HSA@Cy-HPT. Scale bar: 60 µm. c) Flow cytometry analysis
of Annexin V-PE to evaluate the apoptosis rate of single PTT, single PDT and synergetic PDT and PTT. d) Western blotting analysis of Hsp70 of single PTT,
single PDT and synergetic PDT and PTT, β-actin was used as a loading control.
Nanoparticles can preferentially accumulate in the tumor site
through enhanced permeability and retention (EPR) effect without
requiring chemical conjugation of tumor-targeting ligands have been
recently identifed and have shown unique properties for cancer in
58
vivo imaging.46,
In order to evaluate the cancer specificity of
HSA@Cy-HPT, nude mice bearing HepG2 cell xenografts were
established as described previously. HSA@Cy-HPT (10 mg/kg)
intravenously into the nude mice bearing HepG2 cell tumor
xenografts and then NIR fluorescent imaging was performed by
Perkinelmer IVIS Lumina XRMS Series III In Vivo Imaging
System. As shown as Fig. 6b, the fluorescence signal appeared in the
location of liver after intravenous injection 0.5 h, then HSA@Cy-
HPT was injected after 6 h, a strong fluorescence signal was
obtained in the tumor tissue site. As our expected, the fluorescence
signal in tumor site was continuous, and the effective fluorescence
signal was still existed after 72 h. To further confirm the
accumulation of HSA@Cy-HPT in tumor tissue site, the mice were
sacrificed and their main organs including tumors were dissected.
The ex vivo NIR imaging of the dissected organs from above models
further confirmed its tumor preferential accumulation (Fig. 6b). The
result demonstrated that HSA@Cy-HPT was an effective nano-
reagent for tumor-targeted in vivo. And the long-time accumulation
of HSA@Cy-HPT in tumor site was beneficial to the efficient of
PDT and PTT.
Fig. 6 a) Cy-HPT and HSA@Cy-HPT for fluorescence imaging in BALB/c mice
and ex vivo fluorescent imaging of dissected main organs in different time
points. b) HSA@Cy-HPT for fluorescence imaging in subcutaneous HepG2
tumor model and ex vivo fluorescent imaging of dissected main organs in
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 8 of 11
ARTICLE
Journal Name
different time points. He: heart; Li: liver; Sp: spleen; Lu: lung; Ki: kidney; Tu: HSA@Cy-HPT nonirradiation group, the tumor xenVoiegwraAfrtticmle oOdnleinles
tumor. Images displayed represent emission intensities collected window: were injected HSA@Cy-HPT and exposure tDoOaIn: 1800.1803n9m/Cc8oTnBt0in30u5o4uEs
830 - 880 nm, λex= 808 nm. Data are presented as mean ± SD (n = 5).
NIR laser at 1.5 W/cm2 for 5 min at 24 h postinjection as HSA@Cy-
HPT irradiation group. Then the tumor xenograft models were
thermal imaged by Infrared Thermal Camera (TESTO 865) to
monitor the real-time surface temperature of tumors at 24 h
postinjection. As shown in Fig. 7a, the temperature of PBS
irradiation group was increased to 43 °C after 5 min irradiation. As
expected, the temperature of HSA@Cy-HPT irradiation group was
reached up to near 60 °C after 5 min irradiation, which was high
enough to ablate tumors.59 Therefore, HSA@Cy-HPT has huge
potential as a nano-reagent for efficient PTT therapy in tumor
xenograft models.
3.7 Thermal Images and Phototherapy in Xenograft Model
On the basis of the above experiments, in vivo phototherapeutic
efficacy of HSA@Cy-HPT was initially elucidated in HepG2
subcutaneous tumor xenograft models. The tumor xenograft models
were divided into four groups, PBS buffer solution was into the
tumor xenograft models by intravenous injection as PBS
nonirradiation group, the tumor xenograft models were injected PBS
and exposure to an 808 nm continuous NIR laser at 1.5 W/cm2 for 5
min at 24 h postinjection as PBS irradiation group, the tumor
xenograft models were injected HSA@Cy-HPT (10 mg/kg) as
Fig. 7 a) IR thermal images of subcutaneous HepG2 tumor xenograft mice during 5 min NIR laser irradiation. b) The relative tumor volume of subcutaneous
HepG2 tumor xenograft mice in different groups. Statistical analysis was performed using the Student’s t-test: **p < 0.01. c) The survival rate of HepG2
tumor xenograft mice in different groups. d) The body weight of HepG2 tumor xenograft mice in different groups. e) H&E staining of HepG2 tumor in
different groups. Scale bar: 20 µm. f) H&E staining of the normal tissues in different groups. Scale bar: 20 µm. Magnification: × 400. Data are presented as
mean ± SD (n = 5).
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Page 9 of 11
Journal of Materials Chemistry B
Journal Name
ARTICLE
To evaluate the inhibitory effect of HSA@Cy-HPT for tumor
tissue in tumor xenograft models, the relative tumor volume were
recorded for 20 d after treatment. The PBS nonirradiation group and
PBS irradiation group all showed a ≈12-fold increase in the tumor
volumes, suggesting that the irradiation at 808 nm has no influence
on tumor growth (Fig. 7b). And HSA@Cy-HPT nonirradiation
group also exhibited a similar tumor growth in the absence of 808
nm irradiation, owing to its negligible cytotoxicity. Importantly, the
HSA@Cy-HPT irradiation group resulted in a obviously tumor
ablation without any regrowth under 808 nm irradiation (Fig. 7b). In
addition, the survival rate was recorded for 60 d after treatment.
Satisfactory, the survival rate of HSA@Cy-HPT irradiation group
was maintained at 100%. The result demonstrated that HSA@Cy-
HPT offers excellent PDT/PTT effect for inhibit tumor growth and
extend survival rate.
Conflicts of interest
There are no conflicts to declare.
View Article Online
DOI: 10.1039/C8TB03054E
Acknowledgements
We thank the National Nature Science Foundation of China
(Nos. 21575159, 81573393, 21575080), the program of Youth
Innovation Promotion Association, CAS (Grant 2017256), the
Instrument Developing Project of the Chinese Academy of
Sciences (YZ201662).
References
1
Cheng, L., Wang, C., Feng, L., Yang, K. and Liu, Z., Chem
Rev, 2014, 114, 10869-10939.
To verify the anticancer effcacy of HSA@Cy-HPT under 808 nm
laser irradiated, we further examined their tumor-damaging ability
using the body weight of tumor xenograft models, as well as the
hematoxylin & eosin (H&E) staining of tumor tissue and normal
tissues. The body weight was a subtle decrease in the PBS irradiation
groups in the first several days, and no significant difference for all
groups at the end of observation period (Fig. 7d), suggesting the
well-tolerance and unobvious photoinduced side effects of this
treatment. For H&E staining, HSA@Cy-HPT irradiation group
resulted in the destructive cell necrosis in the tumor at 6 h post-
irradiation (Fig. 7e), indicating a severe cell injury. By contrast, PBS
nonirradiation group, PBS irradiation group and HSA@Cy-HPT
nonirradiation group had no distinct tumor damage regardless of
irradiation. Moreover, HSA@Cy-HPT also caused no distinct
damage against the normal tissues such as heart, liver, spleen, lung
and kidney (Fig. 7f). Hence, it is clear that HSA@Cy-HPT precisely
achieve potent photothermal damage against the tumors, synergized
with photodynamic damage under 808 nm irradiated.
2
Jaque, D., Martinez, M.L., Del, R.B., Haro-Gonzalez, P.,
Benayas, A., Plaza, J.L., Martin, R.E. and Garcia, S.J.,
Nanoscale, 2014,
Dolmans, D.E., Fukumura, D. and Jain, R.K., Nat Rev
Cancer, 2003, , 380-387.
6, 9494-9530.
3
4
3
Agostinis, P., Berg, K., Cengel, K.A., Foster, T.H., Girotti,
A.W., Gollnick, S.O., Hahn, S.M., Hamblin, M.R., Juzeniene,
A., Kessel, D., Korbelik, M., Moan, J., Mroz, P., Nowis, D.,
Piette, J., Wilson, B.C. and Golab, J., CA Cancer J Clin,
2011, 61, 250-281.
5
6
Hilger, I., Rapp, A., Greulich, K.O. and Kaiser, W.A.,
Radiology, 2005, 237, 500-506.
Du Y, Jiang, Q., Beziere, N., Song, L., Zhang, Q., Peng, D.,
Chi, C., Yang, X., Guo, H., Diot, G., Ntziachristos, V., Ding,
B. and Tian, J., Adv Mater, 2016, 28, 10000-10007.
Wang, S., Li, X., Chen, Y., Cai, X., Yao, H., Gao, W., Zheng,
Y., An, X., Shi, J. and Chen, H., Adv Mater, 2015, 27, 2775-
2782.
7
8
9
Zheng, M., Yue, C., Ma, Y., Gong, P., Zhao, P., Zheng, C.,
Sheng, Z., Zhang, P., Wang, Z. and Cai, L., ACS Nano, 2013,
7
, 2056-2067.
Mauceri, H.J., Hanna, N.N., Beckett, M.A., Gorski, D.H.,
Staba, M.J., Stellato, K.A., Bigelow, K., Heimann, R., Gately,
S., Dhanabal, M., Soff, G.A., Sukhatme, V.P., Kufe, D.W.
and Weichselbaum, R.R., Nature, 1998, 394, 287-291.
4. Conclusions
In summary, we have developed a small molecule fluorophores
Cy-HPT as a photosensitizer and a nanoparticle HSA@Cy-HPT as a
tumor-targeted nano-photosensitive material. Cy-HPT and
HSA@Cy-HPT all exhibited the excellent PDT/PTT effect in vitro
and in vivo, and they could be metabolized in living mice within a
reasonable time. However, the photothermal stability, photothermal
conversion and generation of 1O2 of HSA@Cy-HPT were better than
free Cy-HPT, and HSA@Cy-HPT have the long-time accumulation
of in tumor site. These results suggestting that HSA@Cy-HPT could
be serve as a tumor-targeted NIR imaging reagents in vivo for
precise surgery. On the other hand, utilizing our formulated
HSA@Cy-HPT, highly effectively combined PDT and PTT under
the guidance of NIR imaging is conducted. Such treatment approach
could not only result in obviously tumor ablation without any
regrowth and extend survival rate of mice, but also offer significant
therapeutic benefit in its minimal side effects in our study. Based on
such biocompatible theranostic agent, it is hoped that imaging-
guided combined PDT plus PTT would bring new hope to the future
fight against cancer.
10 Zhang, W., Guo, Z., Huang, D., Liu, Z., Guo, X. and Zhong,
H., Biomaterials, 2011, 32, 8555-8561.
11 Park, H., Yang, J., Lee, J., Haam, S., Choi, I.H. and Yoo,
K.H., ACS Nano, 2009, 3, 2919-2926.
12 Lane, D., NAT Biotechnol, 2006, 24, 163-164.
13 Wang, N., Zhao, Z., Lv, Y., Fan, H., Bai, H., Meng, H., Long,
Y., Fu, T., Zhang, X. and Tan, W., Nano Res, 2014, 7, 1291-
1301.
14 Luo, S., Tan, X., Fang, S., Wang, Y., Liu, T., Wang, X.,
Yuan, Y., Sun, H., Qi, Q. and Shi, C., Adv Funct Mater,
2016, 26, 2826-2835.
15 He, H., Ji, S., He, Y., Zhu, A., Zou, Y., Deng, Y., Ke, H.,
Yang, H., Zhao, Y., Guo, Z. and Chen, H., Adv Mater, 2017,
29, 1606690-1606700.
16 Tan, X., Luo, S., Long, L., Wang, Y., Wang, D., Fang, S.,
Ouyang, Q., Su, Y., Cheng, T. and Shi, C., Adv Mater, 2017,
29, 1704196-1704205.
17 Delaey, E., van Laar, F., De Vos, D., Kamuhabwa, A.,
Jacobs, P. and de Witte, P., J Photochem Photobiol B, 2000,
55, 27-36.
18 Kassab, K., J Photochem Photobiol B, 2002, 68, 15-22.
19 Yang, K., Xu, H., Cheng, L., Sun, C., Wang, J. and Liu, Z.,
Adv Mater, 2012, 24, 5586-5592.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 10 of 11
ARTICLE
Journal Name
20 Meng, Z., Wei, F., Ma, W., Yu, N., Wei, P., Wang, Z., Tang, 47 Liu, J., Wang, P., Zhang, X., Wang, L., Wang, D., Gu, Z.,
View Article Online
Y., Chen, Z., Wang, H. and Zhu, M., Adv Funct Mater, 2016,
26, 8231-8242.
Tang, J., Guo, M., Cao, M., Zhou, H.,DLOiIu: ,10Y.1.03a9n/dC8CThBe0n30, 5C4.E,
ACS Nano, 2016, 10, 4587-4598.
21 Patel, N., Pera, P., Joshi, P., Dukh, M., Tabaczynski, W.A., 48 Chen, Q., Liang, C., Wang, C. and Liu, Z., Adv Mater, 2015,
Siters, K.E., Kryman, M., Cheruku, R.R., Durrani, F., 27, 903-910.
Missert, J.R., Watson, R., Ohulchanskyy, T.Y., Tracy, E.C., 49 Chen, Q., Liang, C., Wang, X., He, J., Li, Y. and Liu, Z.,
Baumann, H. and Pandey, R.K., J Med Chem, 2016, 59
,
Biomaterials, 2014, 35, 9355-9362.
9774-9787.
50 Huang, P., Rong, P., Jin, A., Yan, X., Zhang, M.G., Lin, J.,
Hu, H., Wang, Z., Yue, X., Li, W., Niu, G., Zeng, W., Wang,
W., Zhou, K. and Chen, X., Adv Mater, 2014, 26, 6401-6408.
22 Zheng, M., Li, Y., Liu, S., Wang, W., Xie, Z. and Jing, X.,
ACS Appl Mater Interfaces, 2016, , 23533-23541.
8
23 Tian, J., Ding, L., Ju, H., Yang, Y., Li, X., Shen, Z., Zhu, Z., 51 Chen, J., Sheng, Z., Li, P., Wu, M., Zhang, N., Yu, X.F.,
Yu, J.S. and Yang, C.J., Angew Chem Int Ed Engl, 2014, 53
9544-9549.
,
Wang, Y., Hu, D., Zheng, H. and Wang, G.P., Nanoscale,
2017, , 11888-11901.
9
24 Sevick-Muraca, E.M., ANNU REV MED, 2012, 63, 217-231.
25 Inoue, Y., Saiura, A., Arita, J. and Takahashi, Y., Ann Surg,
2015, 262, e98-e99.
52 Penha, F.M., Rodrigues, E.B., Maia, M., Meyer, C.H., Costa,
E.P., Dib, E., Bechara, E., Lourenco, A., Lima, F.A.,
Freymuller, E.H. and Farah, M.E., Ophthalmologica, 2013,
230, 59-67.
26 Sheng, Z., Hu, D., Zheng, M., Zhao, P., Liu, H., Gao, D.,
Gong, P., Gao, G., Zhang, P., Ma, Y. and Cai, L., ACS Nano, 53 Engel, E., Schraml, R., Maisch, T., Kobuch, K., Konig, B.,
2014,
8
, 12310-12322.
Szeimies, R.M., Hillenkamp, J., Baumler, W. and Vasold, R.,
27 Jang, H.L., Zhang, Y.S. and Khademhosseini, A.,
Nanomedicine (Lond), 2016, 11, 1495-1497.
28 Conte, C., Maiolino, S., Pellosi, D.S., Miro, A., Ungaro, F.
Invest Ophthalmol Vis Sci, 2008, 49, 1777-1783.
54 Polom, K., Murawa, D., Rho, Y.S., Nowaczyk, P., Hunerbein,
M. and Murawa, P., Cancer-AM Cancer Soc, 2011, 117,
and Quaglia, F., Top Curr Chem, 2016, 370, 61-112.
29 Lucky, S.S., Soo, K.C. and Zhang, Y., Chem Rev, 2015, 115
4812-4822.
,
55 He, D., Hai, L., He, X., Yang, X. and Li, H., Adv Funct
Mater, 2017, 27, 1704089-1704101.
1990-2042.
30 Chen, W., Ouyang, J., Liu, H., Chen, M., Zeng, K., Sheng, J., 56 Friday, E., Koshy, N., Bhanderi, V.K. and Torturro, F.,
Liu, Z., Han, Y., Wang, L., Li, J., Deng, L., Liu, Y. and Guo,
S., Adv Mater, 2017, 29, 1603864-1603871.
Blood, 2010, 116, 1475.
57 Chen, H., Adam, A., Cheng, Y., Tang, S., Hartung, J. and
31 Park, J., Jiang, Q., Feng, D., Mao, L. and Zhou, H.C., J Am
Chem Soc, 2016, 138, 3518-3525.
32 Kim, J., Kim, J., Jeong, C. and Kim, W.J., Adv Drug Deliv
Bao, E., Mol Med Rep, 2015, 11, 2276-2284.
58 Liu, J., Yu, M., Zhou, C., Yang, S., Ning, X. and Zheng, J., J
Am Chem Soc, 2013, 135, 4978-4981.
Rev, 2016, 98, 99-112.
33 Banerjee, S.R., Pullambhatla, M., Byun, Y., Nimmagadda, S.,
Foss, C.A., Green, G., Fox, J.J., Lupold, S.E., Mease, R.C.
59 Perez-Hernandez, M., Del, P.P., Mitchell, S.G., Moros, M.,
Stepien, G., Pelaz, B., Parak, W.J., Galvez, E.M., Pardo, J.
and de la Fuente, J.M., ACS Nano, 2015, 9, 52-61.
and Pomper, M.G., Angew Chem Int Ed Engl, 2011, 50
,
9167-9170.
34 West, J.L. and Halas, N.J., Annu Rev Biomed Eng, 2003,
5
,
285-292.
35 Huang, X., El-Sayed, I.H., Qian, W. and El-Sayed, M.A., J
Am Chem Soc, 2006, 128, 2115-2120.
36 Lim, E.K., Kim, T., Paik, S., Haam, S., Huh, Y.M. and Lee,
K., Chem Rev, 2015, 115, 327-394.
37 Chen, G., Roy, I., Yang, C. and Prasad, P.N., Chem Rev,
2016, 116, 2826-2885.
38 Huang, J., Guo, M., Ke, H., Zong, C., Ren, B., Liu, G., Shen,
H., Ma, Y., Wang, X., Zhang, H., Deng, Z., Chen, H. and
Zhang, Z., Adv Mater, 2015, 27, 5049-5056.
39 Song, X.R., Wang, X., Yu, S.X., Cao, J., Li, S.H., Li, J., Liu,
G., Yang, H.H. and Chen, X., Adv Mater, 2015, 27, 3285-
3291.
40 Wang, Y., Yang, T., Ke, H., Zhu, A., Wang, Y., Wang, J.,
Shen, J., Liu, G., Chen, C., Zhao, Y. and Chen, H., Adv
Mater, 2015, 27, 3874-3882.
41 Lv, G., Guo, W., Zhang, W., Zhang, T., Li, S., Chen, S.,
Eltahan, A.S., Wang, D., Wang, Y., Zhang, J., Wang, P.C.,
Chang, J. and Liang, X.J., ACS Nano, 2016, 10, 9637-9645.
42 Ng, K.K. and Zheng, G., Chem Rev, 2015, 115, 11012-11042.
43 Gao, D., Gao, L., Zhang, C., Liu, H., Jia, B., Zhu, Z., Wang,
F. and Liu, Z., Biomaterials, 2015, 53, 229-238.
44 Qian, C., Yu, J., Chen, Y., Hu, Q., Xiao, X., Sun, W., Wang,
C., Feng, P., Shen, Q.D. and Gu, Z., Adv Mater, 2016, 28
,
3313-3320.
45 Choi, H.S., Liu, W., Misra, P., Tanaka, E., Zimmer, J.P., Itty,
I.B., Bawendi, M.G. and Frangioni, J.V., Nat Biotechnol,
2007, 25, 1165-1170.
46 Zhou, C., Long, M., Qin, Y., Sun, X. and Zheng, J., Angew
Chem Int Ed Engl, 2011, 50, 3168-3172.
10 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 11 of 11
Journal of Materials Chemistry B
View Article Online
DOI: 10.1039/C8TB03054E
Table of Contents
A self-assembly nanoplatform of HSA@Cy-HPT for targeted near-infrared
emission fluorescence imaging and effective phototherapy of cancer