Dual-functional protein for one-step production of a soluble and targeted fluorescent dye

Article abstract of DOI:10.7150/thno.24613

Low water solubility and poor selectivity are two fundamental limitations that compromise applications of near-infrared (NIR) fluorescent probes. Methods: Here, a simple strategy that can resolve these problems simultaneously was developed by using a novel hybrid protein named RGD-HFBI that is produced by fusion of hydrophobin HFBI and arginine-glycine-aspartic acid (RGD) peptide. This unique hybrid protein inherits self-assembly and targeting functions from HFBI and RGD peptide respectively. Results: Boron-dipyrromethene (BODIPY) used as a model NIR dye can be efficiently dispersed in the RGD-HFBI solution by simple mixing and sonication for 30 min. The data shows that self-assembled RGD-HFBI forms a protein nanocage by using the BODIPY as the assembly template. Cell uptake assay proves that RGD-HFBI/BODIPY can efficiently stain αvβ3 integrin-positive cancer cells. Finally, in vivo affinity tests fully demonstrate that the soluble RGD-HFBI/BODIPY complex selectively targets and labels tumor sites of tumor-bearing mice due to the high selectivity of the RGD peptide. Conclusion: Our one-step strategy using dual-functional RGD-HFBI opens a novel route to generate soluble and targeted NIR fluorescent dyes in a very simple and efficient way and may be developed as a general strategy to broaden their applications.

Full text of DOI:10.7150/thno.24613

Theranostics 2018, Vol. 8, Issue 11
3111
Ivyspring
International Publisher
Theranostics
2018; 8(11): 3111-3125. doi: 10.7150/thno.24613
Research Paper
Dual-functional protein for one-step production of a
soluble and targeted fluorescent dye
Yunjie Xiao1, *, Qian Zhang2, *, Yanyan Wang3, *, Bin Wang1, Fengnan Sun2, Ziyu Han3, Yaqing Feng4,


Haitao Yang1, 5, Shuxian Meng2 and Zefang Wang1
1. School of Life Sciences, Tianjin University, Tianjin 300072, China.
2. School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
3. College of Precision Instrument and Opto-electronics Engineering, Tianjin University, Tianjin 300072, China.
4. Collaborative Innovation Center of Chemical Science and Engineering, Tianjin University, Tianjin 300072, China.
5. Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China.
* These authors contributed equally to this work.

Corresponding authors: Prof. Zefang Wang; School of Life Sciences, Tianjin University, Tianjin 300072, China; Tel: +86-22-27403096; E-mail:
zefangwang@tju.edu.cn; Prof. Shuxian Meng, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China; Tel: +86-1303433353;
E-mail: msxmail@tju.edu.cn.
©
Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license
(https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.
Received: 2017.12.28; Accepted: 2018.03.17; Published: 2018.04.30
Abstract
Low water solubility and poor selectivity are two fundamental limitations that compromise
applications of near-infrared (NIR) fluorescent probes.
Methods: Here, a simple strategy that can resolve these problems simultaneously was developed
by using a novel hybrid protein named RGD-HFBI that is produced by fusion of hydrophobin HFBI
and arginine-glycine-aspartic acid (RGD) peptide. This unique hybrid protein inherits self-assembly
and targeting functions from HFBI and RGD peptide respectively.
Results: Boron-dipyrromethene (BODIPY) used as a model NIR dye can be efficiently dispersed in
the RGD-HFBI solution by simple mixing and sonication for 30 min. The data shows that
self-assembled RGD-HFBI forms a protein nanocage by using the BODIPY as the assembly template.
Cell uptake assay proves that RGD-HFBI/BODIPY can efficiently stain αvβ3 integrin-positive cancer
cells. Finally, in vivo affinity tests fully demonstrate that the soluble RGD-HFBI/BODIPY complex
selectively targets and labels tumor sites of tumor-bearing mice due to the high selectivity of the
RGD peptide.
Conclusion: Our one-step strategy using dual-functional RGD-HFBI opens a novel route to
generate soluble and targeted NIR fluorescent dyes in a very simple and efficient way and may be
developed as a general strategy to broaden their applications.
Key words: NIR fluorescent probes, RGD peptide, self-assembly, protein nanocage
Introduction
Near-infrared (NIR) fluorescent probes have
become powerful tools for non-invasive monitoring of
various biologically important processes in vivo [1-7].
NIR probes have the advantages of deep tissue
penetration, minimum photodamage and minimum
interference from auto-fluorescence by biomolecules
present in living systems [8, 9]. Accordingly, there is
considerable interest in developing novel NIR
fluorescent probes to satisfy the increasing demands
of their applications including diagnostics and
therapeutics [10-12]. However, NIR fluorescent
probes do suffer from several major limitations
including low water solubility and poor selectivity,
which dramatically compromise their applications in
diagnostics and therapeutics [6, 13, 14].
Up to now, tremendous efforts have been made
http://www.thno.org

Theranostics 2018, Vol. 8, Issue 11
3112
to overcome the drawbacks mentioned above of NIR
fluorescent probes, but in two separate steps [15-18].
In the first step, numerous polar groups like sulfonate,
glycol, and carboxylate are linked to the core
structures of NIR dyes to gain solubility in aqueous
solutions [19, 20]. Alternatively, a hydrophilic shell
pastoris (P. pastori). Then we systemically investigated
the solubility and targeting abilities of RGD-HFBI
fusion protein by studying its interaction with a
synthesized NIR BODIPY derivative in different
environments, including cell culture and animal
models. Our results show that self-assembled
RGD-HFBI fusion protein can readily modify NIR
fluorescent probes with high solubility and selectivity
in one step by simple mixing and sonication. To our
knowledge, this is the first demonstration of
modification of NIR fluorescent dyes by biomolecules
with dual functions. Furthermore, our one-step
strategy using a self-assembled hydrophobin hybrid
opens a novel route to generate solubilized and
targeted NIR fluorescent dyes in a very simple and
efficient way and may be developed as a general
strategy for broadening their applications.
such as
a
phospholipid monolayer could be
assembled onto the surface of NIR probes to increase
their water solubility [21, 22]. After solving the
solubility problem, the next step is to improve the
selectivity of NIR fluorescent probes. The current
predominant strategy is through conjugation of
specific ligands to the NIR probes, such as proteins,
peptides and small molecules [23-27]. These ligands
have enough binding affinity to recognize and bind
the target molecules resulting in high selectivity of the
conjugated NIR probes. After these two-step
modifications, NIR fluorescent probes do obtain high
water solubility and selectivity. However, we have to
admit that this two-step strategy is not only
somewhat complicated and time-consuming, but also
requires careful selection of conjugation or labeling
techniques to prevent any disruption of the intrinsic
structure of the NIR fluorescent probes [28, 29],
thereby compromising their exciting properties.
Therefore, a one-step strategy that can render NIR
fluorescent probes soluble and selective is highly
desired to facilitate their applications.
Methods
Production of recombinant RGD-HFBI fusion
protein
RGD-HFBI protein was constructed by fusion of
RGD peptide and hydrophobin HFBI with a flexible
linker (GGGGSGGGGS). The plasmid pPIC9-rgd-hfbI
was transformed into P. pastoris GS115 His- cells
(Invitrogen, Beijing, China) by electroporation. To
obtain positive clones with high-level protein
expression, selected P. pastoris positive clones were
cultured in 25 mL of buffered minimal glycerol at 300
rpm at 30 °C until the OD600 reached 6.0. Then, the
medium was exchanged with 100 mL buffered
minimal methanol, and protein expression was
induced by 0.5% (v/v) methanol at 28 °C for 96 h at
250 rpm. The supernatant was collected immediately
after 96 h induction. Finally, 16% Tricine-SDS-PAGE
was employed to analyze the protein content in the
supernatant.
Large-scale cultivation (1 L) was performed after
the clone with the highest level of RGD-HFBI
expression was selected. When the cultivation was
over, the culture was centrifuged at 7500 × g to get the
supernatant. The resulting supernatant was
concentrated by ultrafiltration with a 3 kDa molecular
weight cut-off (Millipore, China). After ultrafiltration,
the supernatant was lyophilized for reversed-phase
high-performance liquid chromatography (RP-HPLC)
purification. A Vydac C4 reversed-phase column (4.6
× 250 mm, GRACE, China) was used for purification
of RGD-HFBI. Lyophilized protein powder was
dissolved in 40% acetonitrile containing 0.1% TFA.
Then, the supernatant was loaded and eluted with a
20-70% (v/v) acetonitrile gradient containing 0.1%
TFA with a flow rate of 1 mL/min. The elution was
monitored by UV absorption at 215 nm. All the
To meet the requirement of a one-step strategy,
one has to find a material that can render NIR
fluorescent
probes
soluble
and
selective
simultaneously. Hydrophobins are small-secreted
proteins produced by filamentous fungi [30-32].
Strikingly, they expose most of their hydrophobic
amino acids on the protein surface, forming a
“hydrophobic patch” [33, 34], rather than burying
their hydrophobic amino acids into their inner
structures like normal proteins. This novel property
makes hydrophobins natural amphiphilic molecules
that can self-assemble at various hydrophobic/
hydrophilic interfaces producing,
a stable and
well-ordered protein film 10 nm in thickness and
reversing the features of the interface coated by them
[35-40]. Moreover, hydrophobins with new properties
and functionalities can be produced in a controlled
manner by protein engineering [41, 42]. Therefore, we
can easily produce an engineered hydrophobin with
target selectivity by adding a specific ligand through
recombinant DNA techniques. We intentionally chose
the RGD tripeptide as the ligand in our study. We
believe the hybrid hydrophobin-RGD fusion protein
preserving dual abilities can render NIR fluorescent
probes soluble and selective simultaneously.
To test our hypothesis, we firstly produced and
purified hybrid RGD-HFBI fusion protein in Pichia
http://www.thno.org

Theranostics 2018, Vol. 8, Issue 11
3113
fractions from RP-HPLC were collected. The purified
RGD-HFBI was identified by 16% Tricine-SDS-PAGE.
atmosphere was removed when the raw material 2,
4-dimethylpyrrole disappeared (about 4 h). DDQ (2,
3-dichloro-5, 6-dicyano-1, 4-benzoquinone) (1.2 g, 5.28
mmol) was dissolved in 5 mL CH2Cl2 and 5 mL THF.
The resultant mixture was added to the reaction flask
with constant-voltage funnels and was stirred in the
dark for 1 h at room temperature. Then, 8.6 mL
triethylamine was added by dropping, and the
reaction was kept for 5 min under the condition of the
ice bath. Next, 8.6 mL boron trifluoride etherate was
added by dropping, and the reaction liquid was
stirred in the dark for 3 h without the ice bath. After
that, the mixture was washed with water and
extracted with dichloromethane. The product was
Characterization of recombinant RGD-HFBI
fusion protein
The self-assembly ability of RGD-HFBI was
characterized by transmission electron microscopy
(TEM) and water contact angle (WCA) measurements.
For the TEM, 3 μL of RGD-HFBI (or native HFBI)
solution in water (200 μg/mL) was placed onto the
surface of a carbon-coated formvar film on a copper
grid (Zhongjingkeyi Technology, Beijing, China) and
left to dry overnight at room temperature. Then, the
grid was examined by field emission TEM at 200 kV
(JEM-2100F, JEOL, Japan).
isolated
by
column
chromatography
using
WCA measurements of purified RGD-HFBI (or
native HFBI) on hydrophobic and hydrophilic
surfaces were performed using pieces of polystyrene
and mica sheets, respectively. Both surfaces were
coated with 20 μL (0.02 mg/mL) RGD-HFBI solution
and incubated at room temperature for 30 min. After
removing the solution gently, all the sheets were dried
in a nitrogen stream and kept at room temperature
overnight. WCA was measured with a 5 μL water
droplet on the modified surfaces at room temperature.
At least three water droplet readings were analyzed
on different areas of the sample surfaces. The surface
rinsed with water was also under the evaluation of
WCA measurements.
CH2Cl2/hexane = 1/2 as eluent. The second orange-
yellow band was taken to recrystallize with CH2Cl2
and C6H12. Finally, BOD (210 mg) was obtained in the
above reaction. 1H NMR (400 MHz, CDCl3): δ 8.18 (d, J
= 8.0 Hz, 2H), 7.41 (d, J = 8.0 Hz, 2H), 5.99 (s, 2H), 3.97
(s, 3H), 2.56 (s, 6H), 1.36 (s, 6H). MS (ESI): [M]+ calcd
for C21H21BF2N2O2, 382.1660, found [M + H] = 383.1,
[M + Na] = 405.1, [M + K] = 421.1. 13C NMR (400 MHz,
CDCl3) δ 166.44, 155.96, 142.86, 139.80, 130.76, 130.35,
128.34, 121.48, 52.39, 14.60, 14.50.
BOD (20 mg, 0.0524 mmol) and N-ethyl-3-
formyl-dibenzyl (42.7 mg, 0.17 mmol) were dissolved
in benzene. 0.1 mL catalyst piperidine and 0.1 mL
acetic acid were injected with a syringe at the same
time. The mixture was heated to 90 °C and stirred for
12 h. The product was isolated by column
chromatography using CH2Cl2/hexane = 2/1 as the
eluent. The blue band was taken to recrystallize with
CH2Cl2 and C6H12. Finally, the dark green solid (18.7
Synthesis and characterizations of the
BODIPY derivative
All solvents and starting materials were
commercially available and were used without
further purification (unless specially mentioned).
Silica gel for column chromatography (CC) was
300-400 mesh. 13C and 1H NMR spectra were recorded
on a Bruker AV400 MHz spectrometer in CDCl3 or
with tetramethylsilane as a reference. Electrospray
1
mg) was in the above reaction. H NMR (400 MHz,
CDCl3): δ 8.18 (d, J = 8.0 Hz, 2H), 7.57-7.60 (m, 2H),
7.36-7.44 (m, 6H), 7.14-7.19 (m, 6H), 7.07-7.10 (m, 4H),
6.96-6.97 (m, 2H), 6.60 (s, 2H), 3.97 (s, 3H), 3.82-3.87 (q,
J = 6.8 Hz, 4H), 3.20 (s, 8H), 1.40 (s, 6H), 1.18 (t, J = 6.8
Hz, 6H). MS (ESI): [M]+ calcd for C55H51BF2N4O2
848.4230, found 848.0673. 13C NMR (400 MHz, CDCl3)
δ 166.56, 153.04, 148.78, 136.43, 135.50, 133.03, 130.21,
129.89, 129.59, 129.25, 129.02, 126.45, 126.09, 122.98,
120.90, 119.88, 116.97, 52.39, 45.63, 33.02, 31.77, 14.77,
13.95.
ionization-MS spectra were determined with
a
Shimadzu LCMS-2020 instrument. The UV-Vis
spectra of dyes in DMSO solution were measured
using Shimadzu UV-1800 in a 10 mm quartz cell
spectrometer.
Methyl 4-formylbenzoate (0.864 g, 5.27 mmol)
and 32 mL CH2Cl2 were added to a 100 mL
round-bottom flask, and 1 mL (10.0 mmol) 2,
4-Dimethylpyrrole was added. The mixture was
stirred in the dark for 3 h at room temperature.
Nitrogen gas was introduced for 30 min to remove
dissolved oxygen in the flask. Then, 0.08 mL
CF3COOH was added with a syringe slowly under the
nitrogen atmosphere. Samples were taken every 30
min during the reaction process for thin-layer
chromatography (TLC) analysis. The nitrogen
Preparation of RGD-HFBI/BODIPY complex
Four concentrations of RGD-HFBI solution (50,
100, 150 and 200 μg/mL) were prepared by using
phosphate buffers (pH 7.4). Then, 1 mg of BODIPY
was dissolved in four concentrations of RGD-HFBI
solutions respectively. The resultant mixtures were
dispersed by ultrasonic agitation (120 W) for 30 min in
iced water, and then nitrogen gas was introduced into
the dispersion for 5 min to remove the free
http://www.thno.org

Theranostics 2018, Vol. 8, Issue 11
3114
HFBI-RGD. Finally, the resultant dispersion was
subjected to ultra-centrifugation at 9000 × g for 30 min
at 4 °C, and 80% of the supernatant was collected and
lyophilized for further analysis.
The concentration of all the probes was 10 μM.
Afterwards, the plates were washed with 1 mL of PBS
(pH 7.4) per well three times to remove probes that
were still in the medium, and then the cells
underwent digestion with trypsin. Next, the cells
were collected by centrifugation and resuspended in 1
mL of PBS, which was repeated 3 times. Finally, 2 mL
of PBS was used to resuspend the cells. The cellular
uptake of the fluorescence was measured on a BD
FACSCanto II flow cytometer (BD Biosciences, USA).
Approximately 10,000 events were counted for each
sample.
Characterization of RGD-HFBI/BODIPY
complex
Dynamic light scattering and zeta potential
measurements were carried out using a Zetasizer
Nano ZS (Malvern Instrument, Malvern, Worcester-
shire, UK). TEM images were obtained by field
emission TEM at 200 kV (JEM-2100F, JEOL, Japan).
The chemical compositions of BODIPY and
HFBI/BODIPY were analyzed using X-ray photo-
electron spectroscopy (XPS) Apparatus (PHI-5300).
The experiment conditions were as follows: the
energy of excitation source monochromatic Mg-Kα
radiation was 1253.6 eV, and the survey scan range
was 0-1100 eV. Fourier-transform infrared (FTIR)
spectra were obtained on a BRUKER IFS 55 FTIR
system using the KBr disk method. The transmittance
spectra were recorded at a resolution of 2 cm-1
between 4000 and 400 cm-1.
Confocal microscopy analysis
The MCF-7 (αvβ3 integrin negative), U-87MG
(high αvβ3 integrin expression) and HeLa (low αvβ3
integrin expression) cells were grown separately on
glass coverslips in 48-well plates with a seeding
concentration of 1 × 103 cells per well for 24 h. After
that, cells were incubated with RGD-HFBI/BODIPY,
HFBI/BODIPY and BODIPY at a concentration 10
μM, and then cultured for 4 h. Afterwards, the plates
were washed with 100 μL of PBS per well three times
to remove probes that were still in the medium. Before
confocal microscopy (UltraVIEW VoX, PerkinElmer)
imaging, all the samples were stained with DAPI for 5
min and washed with 100 μL of PBS per well three
times. The excitation wavelengths for RGD-HFBI/
BODIPY, HFBI/BODIPY and BODIPY were 405 nm
and 633 nm.
Cytotoxicity measurement
Cell viability measurements were performed by
MTT analysis. MCF-7 (8 × 103 cells per well) and HeLa
(7 × 103 cells per well) cells were cultured in DMEM
medium with 10% fetal bovine serum. U-87MG (8 ×
103 cells per well) cell was cultured in RPMI 1640
medium with 10% fetal bovine serum. Cells were
seeded in 96-well flat-bottomed plates and incubated
for 24 h at 37 °C under 5% CO2. After 24 h of cell
attachment, the plates were washed with 100 µL of
PBS per well. The cells were then cultured in a
medium with ten different concentrations (0, 0.1, 1, 5,
10, 25, 50, 100, 250 and 500 µM) of
RGD-HFBI/BODIPY, HFBI/BODIPY and BODIPY for
48 h. Cells in a culture medium without fluorescent
dyes were used as the control. MTT (10 µL, 5 mg/mL)
in PBS was subsequently added to each well. The
plates were then incubated at 37 °C for 4 h in a 5%
CO2 humidified incubator. The medium was carefully
removed, and the products were lysed in 200 µL of
DMSO. The plate was shaken for 10 min, and the
absorbance was measured at 490 nm and 570 nm
using a microplate reader (EnSpire Multilabel Reader,
PerkinElmer).
Stability assays of RGD-HFBI/BODIPY
complex in vivo and in vitro
For the in vivo assay, blood samples from rats
were directly drawn by venipuncture at different time
points (2, 4, 6, 8, 24, 48 and 72 h) after administration
of RGD-HFBI/BODIPY complex. Then, the amounts
of RGD-HFBI/BODIPY complex and free RGD-HFBI
in serum were measured by RP-HPLC. The ratio
between the amount of RGD-HFBI/BODIPY complex
and free RGD-HFBI was considered an indicator of
the serum stability of the complex. For the in vitro
assay, the RGD-HFBI/BODIPY complex was added to
rat serum directly. The remaining protocol was the
same as the in vivo assay.
In vivo imaging in animal tumor models
Female nude mice (4-6 weeks) were obtained
from the Institute of Radiation Medicine Chinese
Academy of Medical Sciences. The U-87MG tumor
and HeLa tumor models were generated via
subcutaneous injection of 5.0 × 106 tumor cells in the
left forelimb of the mice. Caliper measurements of the
perpendicular axes of the tumor were performed to
monitor the tumor growth. The mouse weights were
also measured. The treatment was initiated when the
Flow cytometry analysis
The cell binding and internalization were
analyzed by flow cytometry. U-87MG, MCF-7 and
Hela cells were grown separately in 6-well plates with
a seeding concentration of 3 × 105 cells per well for 24
h. After that, cells were incubated with RGD-
HFBI/BODIPY, HFBI/BODIPY and BODIPY for 4 h.
http://www.thno.org

Theranostics 2018, Vol. 8, Issue 11
3115
tumor reached a diameter of approximately 8.0 mm
(3-4 weeks after inoculation). The HFBI-RGD/
BODIPY and HFBI/BODIPY fluorescent probes (17.7
nM, 200 μL) were injected via the tail vein. The mice
were anesthetized with oxygen/air mixture
containing 2% isoflurane and the in vivo images were
captured at different points of time (2, 4, 6, 8, 24, 48
and 72 h). All images were collected with a Cri
Maestro In vivo imaging system (Cri, Woburn, MA),
with an excitation wavelength of 605 nm and emission
wavelengths from 615 nm to 800 nm. To eliminate
interference from the background, ex vivo imaging
procedures were carried out. Thereafter, tumor-
bearing mice were sacrificed, and then tumor and
main organs (heart, liver, spleen, lung and kidneys)
were harvested for isolated organ imaging that was
performed with the same instrument under the same
conditions as the in vivo imaging. All experimental
procedures were reviewed and approved by the
Animal Use and Care Committee for Research and
Education of the Institute of Radiation Medicine
Chinese Academy of Medical Sciences (Tianjin,
China) due to the lack of animal facility in Tianjin
University, and were in accordance with the
guidelines provided by the National Institute of
Health.
angiogenic endothelial cells [45, 46]. Based on those
two functions, we proposed that RGD-HFBI has the
abilities to impart hydrophobic materials with
solubility and targeting behaviors. To test our
hypothesis, we firstly produced the RGD-HFBI
protein in P. pastoris system. After several rounds of
selection, a clone with the highest amount of protein
production was picked to produce RGD-HFBI at large
scale (Figure S1). Figure 1C shows the purification
results of RGD-HFBI and native HFBI with RP-HPLC
system. The retention volume of RGD-HFBI was quite
close to that of native HFBI, indicating the similar
polarities of those two proteins. Fractions corres-
ponding to the predominant peak were lyophilized
and analyzed by Tricine-SDS-PAGE.
A
single
predominant band suggested the high purity of
RGD-HFBI after RP-HPLC purification (Figure 1D).
After the production and purification process,
the self-assembly ability of RGD-HFBI was
characterized by TEM and WCA measurements [47,
48]. Figure 1E shows a self-assembled RGD-HFBI film
on a TEM copper grid. Like the native HFBI film, the
RGD-HFBI film could form wrinkles or folds,
implying the flexibility or elasticity of this film [49-51].
In our study,
a surface is considered to be
hydrophobic if its WCA is more than 60° while
hydrophilic if it is lower. The WCA of bare and
modified polystyrene and mica are shown in Figure
1F. The WCA of the bare polystyrene was about 87°,
suggesting its natural hydrophobic property.
However, the WCA of the polystyrene sharply
decreased to 11° after modification with RGD-HFBI.
The reversal of the surface property of polystyrene
from hydrophobic to hydrophilic confirmed the
self-assembly of RGD-HFBI onto the polystyrene
surface. Several studies have proposed that when
Statistical analysis
The assays were performed at least in triplicate
on separate occasions. The data collected in this study
are expressed as the mean value ± standard deviation.
In all cases, p < 0.05 was considered significant.
Results and discussion
Production and characterization of
recombinant RGD-HFBI fusion protein
amphiphilic hydrophobin self-assembles on
a
Figure 1A shows the amphiphilic structure of
native hydrophobin HFBI. The sun-yellow part
represents the “hydrophobic patch” composed of
about 80% of the hydrophobic residues of this protein
[33, 43]. This hydrophobic patch is the basis for
binding of HFBI to various hydrophobic surfaces by
strong hydrophobic interactions [44]. In Figure 1B, a
RGD motif is linked with the N terminus of HFBI
protein via a flexible linker, and the resultant hybrid
protein was named RGD-HFBI. Accordingly, we
proposed that RGD-HFBI containing two distinct
biological domains owns dual functions based on its
structural properties. The first function derived from
the HFBI part was the ability to self-assemble on
different hydrophobic surfaces to make them
hydrophilic. Another one inherited from the RGD
motif was to facilitate recognition and binding to the
target αvβ3 integrin protein on the surface of tumor
hydrophobic surface, the “hydrophobic patch”
exposed on its surface adheres onto the hydrophobic
surface via strong hydrophobic interactions [52, 53].
Therefore, the hydrophilic part of hydrophobin is
displayed to the outside, converting the hydrophobic
surface into a hydrophilic one [54]. When RGD-HFBI
covered the mica surface, the WCA increased from 4°
to 17°. The hydrophobicity of mica surface was
slightly improved. These results were very similar
with those obtained from the native HFBI.
Furthermore, we found that the WCAs on the
polystyrene and mice surfaces little changed after
extensive washing by pure water, indicating the
stability of the assembled RGD-HFBI films (Figure
S2). In summary, we concluded that RGD-HFBI
fusion protein has a self-assembly ability like native
HFBI and, therefore, can potentially modify
hydrophobic materials.
http://www.thno.org

Theranostics 2018, Vol. 8, Issue 11
3116
Figure 1. (A) X-ray crystal structure of the amphiphilic hydrophobin HFBI [PDB ID 2FZ6]. The sun-yellow part in the surface of HFBI represents the hydrophobic patch that
is the basis for binding to various hydrophobic surfaces by strong hydrophobic interactions. (B) A RGD motif is linked with the N terminus of HFBI protein via a flexible linker
and the resultant hybrid protein was named RGD-HFBI, which owns dual functions based on its structural properties. The first function derived from the HFBI part is the ability
to self-assemble and bind onto different hydrophobic surfaces. The second function inherited from the RGD motif is to facilitate recognition and binging to the target αvβ3
integrin protein on the surface of tumor angiogenic endothelial cells. (C) Purification results of RGD-HFBI and native HFBI with an RP-HPLC system. (D) Tricine-SDS-PAGE
results of the purified RGD-HFBI and native HFBI. (E) Self-assembled RGD-HFBI film and native HFBI film on TEM copper grids. (F) WCA measurements of polystyrene and mica
before and after modification with RGD-HFBI and native HFBI.
spectral band with a major peak in the visible region
at 671 nm. This BODIPY derivative in DMSO shows
an emission spectrum (Figure 2C) with a maximum at
The synthesis and characterization of BODIPY
dye
After the production of RGD-HFBI, we then
designed and synthesized a new long-wavelength
BODIPY dye that would be used as the model
fluorescent dye in our following studies to test if
RGD-HFBI could impart solubility and targeting
abilities to hydrophobic NIR fluorescent probes. The
design and synthesis of the BODIPY derivative are
shown in Figure 2A. Briefly, the benzyl derivative
was introduced to the 3- and 5-methyl sites through
the Knoevenagel reaction. Then, an ethyl group was
incorporated into BODIPY through the benzyl to
enhance the solubility of the dye in non-polar solvents
to facilitate preparation and characterization of this
derivative. With a few steps, the BODIPY dye was
obtained with a reasonable yield that was about 50%
732 nm (excitation wavelength), with what could be
Rayleigh and Tyndall scattering in the emission
spectrum. The photophysical data of this BODIPY dye
are summarized in Figure 2D.
RGD-HFBI disperses BODIPY in a
concentration dependent manner
Considering that RGD-HFBI protein owns a
self-assembly ability, we proposed that RGD-HFBI
could modify hydrophobic BODIPY dye to gain
solubility. To test this idea, we tried to disperse
BODIPY using RGD-HFBI solutions at different
concentrations. Figure 3A-B show results of
absorption and emission spectra of the BODIPY in
RGD-HFBI solutions at different concentrations.
BODIPY gave the highest intensity both in the
absorption and emission spectra when the protein
concentration was at 0.15 mg/mL. The photophysical
data of RGD-HFBI-treated BODIPY dye are sum-
marized in Table 1. Both the fluorescence quantum
yield and lifetime were increased to the maximum
1
and was characterized by 13C NMR (Figure S3), H
NMR (Figure S4) and MS (ESI) (Figure S5). We then
dissolved this BODIPY derivative in DMSO at
different concentrations to investigate its absorption
and emission spectra. As shown in Figure 2B, the
absorption spectrum of this dye contains a narrow
http://www.thno.org

Theranostics 2018, Vol. 8, Issue 11
3117
when BODIPY was dispersed in 0.15 mg/mL
RGD-HFBI solution. Although we could not
understand the detailed mechanism by which
RGD-HFBI acted at this stage, these results give us
clues that protein concentration might have a crucial
impact on the dispersion of BODIPY. Therefore, the
long-term stability of each sample was examined over
20 days. Figure S6 shows that the protein
concentration had an obvious influence on the
stability of the dispersed solutions. We quantified the
stability of each sample by measuring its absorption at
699 nm. As shown in Figure 3C, there was almost no
change in absorbance of the 0.15 mg/mL
RGD-HFBI-treated sample within 20 days, indicating
that a stable dispersion was formed immediately after
sonication. For other samples, we found that the
normalized absorptions kept on decreasing and
finally stabilized after two weeks. The zeta potential
of 0.15 mg/mL RGD-HFBI-treated BODIPY was
around 40 mV indicating its relatively high stability
[55] (Table 2). Regarding the other groups, the low
zeta potential values suggested the instability of those
solutions. Finally, the size of each sample was
measured. As shown in Figure 3D, the smallest size of
BODIPY was about 100 nm when BODIPY was
dispersed in 0.15 mg/mL RGD-HFBI solution. The
size of nanoparticles has a great effect on their
behaviors like cell uptake and tumor-targeting ability.
Normally, smaller sizes obtain better biodistribution
and targeting results in vivo [56, 57]. Taking all the
results together, we demonstrated that 0.15 mg/mL
HFBI-RGD could disperse BODIPY effectively. When
the protein concentration was less than 0.15 mg/mL,
we believed that there was not enough hydrophobin
to disperse the excessive BODIPY. However, when
protein concentrations were above 0.15 mg/mL, the
extra RGD-HFBI protein might compromise the
stability of the resultant dispersions [58]. Therefore,
we chose 0.15 mg/mL-treated BODIPY in the
following studies. In addition, the best dispersion
result of BODIPY was obtained by using 0.1 mg/mL
native HFBI in the control group. This result
strengthened the fact that hydrophobin concentration
was a key factor influencing the outcome of the
dispersion result as well.
Table 1. Photophysical properties of RGD-HFBI- and native
HFBI-treated BODIPY dye
-1
-1
Dye
Dispersant
λabs (nm) ε (M cm ) λem (nm) Фf τ (ns)
BODIPY 0.05 mg/mL HFBI
BODIPY 0.10 mg/mL HFBI
BODIPY 0.15 mg/mL HFBI
BODIPY 0.20 mg/mL HFBI
BODIPY 0.05 mg/mL
RGD-HFBI
699
699
699
699
699
75385
77796
78793
77302
76842
753
753
753
753
753
0.32 1.04
0.39 1.36
0.37 1.34
0.33 1.28
0.34 1.07
BODIPY 0.10 mg/mL
RGD-HFBI
BODIPY 0.15 mg/mL
RGD-HFBI
BODIPY 0.20 mg/mL
RGD-HFBI
699
699
699
79861
78181
76762
753
753
753
0.38 1.33
0.41 1.38
0.32 1.31
Table 2. Zeta potential of RGD-HFBI- and native HFBI-treated
BODIPY dye
Formulation
Concentration
(mg/mL)
Zeta Potential
(mV)
RGD-HFBI/BODIPY
0.05
0.10
0.15
0.20
0.05
0.10
0.15
0.20
18.7 ± 1.51
28.4 ± 2.13
39.9 ± 2.42
22.5 ± 1.12
17.8 ± 1.25
32.3 ± 3.15
26.9 ± 2.08
8.93 ± 0.23
HFBI/BODIPY
Figure 2. (A) Synthetic procedure for the synthesis of the BODIPY derivative. (B-C) Absorption and fluorescence spectra of the BODIPY dye at different concentrations (10,
20, 30 and 40 μM) in DMSO. (D) Photophysical properties of the BODIPY dye.
http://www.thno.org

Theranostics 2018, Vol. 8, Issue 11
3118
Figure 3. (A-B) Absorption and fluorescence spectra of the BODIPY dye solubilized in RGD-HFBI and native HFBI solution at different concentrations (0.05, 0.1, 0.15 and 0.2
mg/mL). (C) Stability of RGD-HFBI- and native HFBI-dispersed BODIPY solution over 20 days. (D) Particles size of RGD-HFBI- and native HFBI-treated BODIPY dye.
the size of RGD-HFBI-treated BODIPY was reduced
by 50 times when compared with the huge aggregate
in water. These results showed clearly that RGD-HFBI
Characterization of RGD-HFBI/BODIPY
complex
After solubilizing the BODIPY in RGD-HFBI
solution, we used TEM to see if there were
morphological changes of BODIPY. As shown in
Figure 4A, we saw an irregular aggregate of BODIPY
in pure water with a dimension of about 5 μm, which
illustrated its intrinsic hydrophobic property. When
BODIPY was dissolved in DMSO, the shape sharply
changed to round, and the size was dramatically
decreased to 600 nm (Figure S7). However, we found
that BODIPY formed well-dispersed particles with a
round shape and uniform size of about 108 nm
(Figure S7) when solubilized in RGD-HFBI solution.
This result was very similar to that obtained from the
dynamic light scattering experiment. Furthermore,
could disperse BODIPY efficiently. Moreover, we
found that RGD-HFBI/BODIPY formed a core-shell
heterostructure with BODIPY dye in the core area and
the RGD-HFBI layer taking the shape of the BODIPY
template. As shown in Figure 4A, a clear interface
could be observed between the BODIPY core and the
RGD-HFBI shell or film. The average thickness of the
RGD-HFBI film was about 10 nm, which was similar
to that of HFBI film formed on the BODIPY core,
suggesting that RGD motif did not disturb the
assembly of HFBI protein [59]. In addition, we found
that there were plenty of small holes in the RGD-HFBI
shell. Those holes obviously made the RGD-HFBI
http://www.thno.org

Theranostics 2018, Vol. 8, Issue 11
3119
shell like an empty hollow “protein nanocage” with
an embedded BODIPY core. Unlike the classic protein
nanocages composed of multiple protein subunits
that self-assemble with excellent precision forming a
hollow caged structure of nanometer size, RGD-HFBI
itself could self-assemble into a protein nanocage by
using the BODIPY as the assembly template [60, 61].
So far, protein-based nanocages have been
functionalized and applied into many applications
including drug delivery, imaging and theranostics in
cancer [62-64]. RGD-HFBI protein presents numerous
reactive groups such as hydroxyls, amines, thiols,
carboxylic acids and others. Therefore, it could
provide active sites for easy further surface
modification for different purposes. Figure 4B shows
the RGD-HFBI film self-assembled on the mica
surface that was observed with atomic force
microscopy. We could see clearly small holes in the
RGD-HFBI film, indicating that the self-assembly of
RGD-HFBI protein was independent of the BODIPY
dye. Therefore, RGD-HFBI has the potential to form
protein nanocages by using other hydrophobic cores
as the self-assembly template.
4C) showed an overall change in the F1s, O1s, N1s,
C1s and B1s of the BODIPY, HFBI-modified BODIPY
and RGD-HFBI-modified BODIPY. The F1s (685 eV)
peak was characteristic for BODIPY dye according to
its chemical composition. The intensity of this
elemental peak was remarkably decreased in the
narrow spectrum of RGD-HFBI-modified BODIPY,
suggesting that RGD-HFBI protein self-assembled
and covered the surface of BODIPY. Furthermore,
increased content of oxygen (Figure 4C) also
confirmed the self-assembly of RGD-HFBI protein
onto the surface of BODIPY. Next, FTIR (Figure 4D)
was used to characterize the RGD-HFBI/BODIPY
complex as the shape and frequency of the amide I
band was modulated by the secondary structure of a
protein [65]. The FTIR spectrum of RGD-HFBI protein
showed a sharp peak at 1637 cm-1 involved in the
vibration bands of amide I, which was caused by C=O
stretching vibrations of peptide linkages. This result
indicated RGD-HFBI contained mainly beta-sheet
(1637 cm-1) structure. Hakanpaa et al. reported that
native HFBI protein contains four beta-sheets based
on its three-dimensional crystal structure [33]. The
coherence between the secondary structures of
RGD-HFBI and native HFBI illustrated that the RGD
motif did not affect the correct protein folding of
HFBI. For the BODIPY dye, strong peaks at 2848 and
To further confirm the self-assembly of
RGD-HFBI onto the BODIPY, chemical compositions
of BODIPY before and after RGD-HFBI modification
were analyzed by XPS. The full XPS spectra (Figure
Figure 4. (A) TEM images of BODIPY dissolved in different solvents (H2O, DMSO, HFBI and RGD-HFBI). The red dashed line represents the RGD-HFBI shell or film. (B)
Atomic force microscopy images of self-assembled RGD-HFBI (native HFBI) film on a mica surface. (C) Wide XPS spectra of RGD-HFBI/BODIPY, HFBI/BODIPY and BODIPY,
and high-resolution spectra of F1s, B1s and O1s. (D) FTIR spectra of RGD-HFBI/BODIPY, HFBI/BODIPY and BODIPY.
http://www.thno.org

Theranostics 2018, Vol. 8, Issue 11
3120
2919 cm-1 were attributed to the C-H vibrations of the
benzene bonds, and a peak at 1728 cm-1 resulted from
C=O vibration. Moreover, sharp peaks at 1486, 1538
and 1594 cm-1 corresponded to v (C=C) stretching
vibrations in benzene. Regarding the RGD-HFBI/
BODIPY complex, the FTIR spectrum consisted of
both characteristic peaks of HFBI/BODIPY and
BODIPY without detectable chemical bond formation.
This result suggested that RGD-HFBI adsorbed onto
the surface of BODIPY dye non-covalently without
protein structure changes. Furthermore, This finding
was quite consistent with the previous reports that
there was no secondary structure change during the
binding process when class II hydrophobin assembled
physically onto hydrophobic surfaces [66]. The FTIR
results also strengthened the idea that physical
hydrophobic force was the major driving force for the
interaction between the hydrophobic part of HFBI and
BODIPY dye, resulting in the formation of RGD-
HFBI/BODIPY complex.
immune system. Therefore, we considered that
RGD-HFBI might mask most of the immunogenicity
of BODIPY, resulting in its low cytotoxicity. This
consideration will be addressed in future studies
using immunocompetent animals. Nevertheless, we
proved that the RGD-HFBI/BODIPY complex has the
potential to be applied in the living cell system for
bioimaging due its good biocompatibility.
Next, flow cytometry analysis (Figure 5B) was
performed to quantify the cell binding and cellular
uptake. The uptakes of RGD-HFBI/BODIPY complex
were investigated by using the same three cell lines
used in the MTT assays. As expected, the cell-
associated fluorescence was highest in U-87MG cells.
The fluorescence in HeLa cells was decreased about
one-fold compared with the U-87MG cells. Regarding
the negative control MCF-7 cells, we observed the
lowest fluorescence that might come from nonspecific
cellular uptake of RGD-HFBI/BODIPY complex. The
relatively small size (about 100 nm) of RGD-
HFBI/BODIPY complex might contribute to this
phenomenon [70, 71]. These results demonstrated that
integrin expression did have an obvious effect on the
cellular uptake of RGD-HFBI/BODIPY complex and
also suggested the biological activity of the RGD motif
in the hybrid RGD-HFBI was preserved very well. In
our flow cytometry analysis, HFBI-modified BODIPY
and DMSO-dissolved BODIPY were used as controls.
All three types of cells treated by those two BODIPYs
with different solvents showed much lower cell-
associated fluorescence than that obtained from
RGD-HFBI/BODIPY. These results indicated the
functionality of RGD motif of RGD-HFBI protein was
a key factor in regulating the specific cell binding and
cellular uptake of BODIPY as well. In order to further
evaluate the cellular uptake of RGD-HFBI/BODIPY
complex, we used confocal microscopy analysis to
directly observe the interactions between RGD-HFBI/
BODIPY complex and target cells. As shown in Figure
5C, we got very similar results as that obtained from
flow cytometry analysis. U-87MG cells and HeLa cells
with αvβ3 integrin expression showed higher
fluorescence than that from the MCF-7 cells that did
not have any αvβ3 integrin expression on their cell
surfaces. All cells treated with HFBI-modified
BODIPY and DMSO-dissolved BODIPY showed low
fluorescence at the same level due to the lack of RGD.
Moreover, the overall fluorescence was obviously
decreased by adding the synthetic RGD peptide both
in the U-87MG and HeLa cells. These results
obviously demonstrated that the RGD motif
remarkably improved the specific cell binding and
cellular uptake of RGD-HFBI/BODIPY complex.
Analysis of biocompatibility, cell binding and
cellular uptake in vitro
After we obtained the RGD-HFBI/BODIPY
complex, we were eager to know whether this
complex could be used in biolabeling or bioimaging
from the safety point of view. Therefore, biological
compatibility of this complex was an important issue
we should consider in the next step. The
biocompatibility of RGD-HFBI/BODIPY was evalu-
ated by classical MTT assay. Three different cancer
cell lines were used in our assays, including U-87MG
(high αvβ3 integrin expression), HeLa (low αvβ3
integrin expression) and MCF-7 cells (without αvβ3
integrin expression) [67, 68]. As shown in Figure 5A,
the viabilities of the three cell lines were not affected
by the exposure of different concentrations of
RGD-HFBI/BODIPY complex for 48 h, indicating its
high biocompatibility and low cytotoxicity.
HFBI/BODIPY showed low cytotoxicity towards
all the tested cell lines as well. However,
DMSO-dissolved BODIPY showed slight cytotoxicity
at high concentrations (250 and 500 μM), indicating
BODIPY itself had detectable cytotoxicity at high
concentrations. From the above results, we saw
clearly that hydrophobin modification could increase
the biocompatibility of BODIPY dye. Aimanianda et
al. reported that surface hydrophobin prevented
immune recognition of airborne fungal spores in the
human body [69]. Their results implied that people
could generate hydrophobin-based nanoparticles
containing embedded diagnostic and therapeutic
molecules that have to be transported to a specific
body location without being recognized by the host
http://www.thno.org

Theranostics 2018, Vol. 8, Issue 11
3121
Figure 5. (A) MTT assay results of three different cancer cell lines including U-87MG (high αvβ3 integrin expression), HeLa (low αvβ3 integrin expression) and MCF-7 cells
(without αvβ3 integrin expression). Those three cell lines were treated with different concentrations (0, 0.1, 1, 5, 10, 25, 50, 100, 250 and 500 µM) of RGD-HFBI/BODIPY,
HFBI/BODIPY and BODIPY for 48 h. (B) Flow cytometry analysis of U-87MG, Hela and MCF-7 cells incubated with RGD-HFBI/BODIPY, HFBI/BODIPY and BODIPY for 4 h. The
concentration of all probes was 10 μM and cells without treatments were used as the blank controls (red lines). (C) Confocal microscopy images of U-87MG, Hela and MCF-7
cells treated with RGD-HFBI/BODIPY, HFBI/BODIPY, BODIPY and synthetic RGD peptide.
http://www.thno.org

Theranostics 2018, Vol. 8, Issue 11
3122
detected only in the liver due to nonspecific uptake
and retention of HFBI/BODIPY.
In vivo imaging in the tumor model
Before we did in vivo imaging using tumor
models, we evaluated the stability of RGD-HFBI/
BODIPY complex in vitro and in vivo. The stability of
RGD-HFBI/BODIPY complex was determined by
whether the RGD-HFBI on the shell of the complex
could resist protease degradation in serum both in
vivo and in vitro. Figure S8 shows that the
RGD-HFBI/BODIPY complex was relatively stable
both in vivo and in vitro. For example, only about 11%
of RGD-HFBI/BODIPY was degraded after 72 h in
vivo. This result was consistent with the previous
reports that self-assembled hydrophobin could resist
protease degradation both in vitro and in vivo [72-75].
After the stability of RGD-HFBI/BODIPY complex
was confirmed, we did in vivo imaging using
different tumor models. Figure 6A shows typical NIR
fluorescence images of athymic nude mice bearing
subcutaneous U-87MG glioblastoma tumor after
intravenous injection of 17.7 nmol of RGD-HFBI/
BODIPY. The subcutaneous U-87MG tumor could be
clearly delineated from the surrounding background
tissue at all the time points. We saw limited but
distinguishable fluorescence at 2 h, indicating that
RGD-HFBI/BODIPY reached the tumor site at this
time point. Accumulation of fluorescence was
observed with time, increasing until the maximum
fluorescence occurred at 24 h. After that, the
fluorescence in the tumor site went down, suggesting
the washing-out of RGD-HFBI/BODIPY, but still
remained distinguishable at 72 h. For the control
HFBI/BODIPY group, we did not observe any
fluorescence with high intensity in the tumor sites at
all the time points (Figure 6C). The weak fluorescence
in this group could be attributed to the enhanced
permeability and retention (EPR) effect. However, we
believed that effective accumulation of fluorescence in
the RGD-HFBI/BODIPY group not only depended on
the EPR effect, but also relied on the active targeting
effect of the RGD motif of the RGD-HFBI protein.
Therefore, the above results did reveal the
We also did NIR fluorescence analysis of
athymic nude mice bearing subcutaneous HeLa
tumors. As shown in Figure 6E-H, the results were
quite similar with those obtained from the U-87MG
tumor cell. The only difference was the fluorescence
obtained from HeLa cells was relatively weaker than
that obtained from the U-87MG tumor cell at all the
time points. This result is quite consistent with the
results obtained from in vitro cell culture assay. Taken
together, the in vivo affinity tests fully demonstrated
the high specificity of RGD-HFBI/BODIPY due to the
high selectivity and strong affinity of the RGD
molecules.
Conclusion
In this paper, we showed a simple and reliable
one-step strategy to produce water-soluble and
targeted BODIPY dye by designing a novel hybrid
protein named RGD-HFBI. This novel protein has
dual biological functions according to our design. The
first function of this protein is to self-assemble on
hydrophobic surfaces including the hydrophobic
BODIPY we used in this work. The outcome of the
self-assembly of RGD-HFBI on different hydrophobic
surfaces is a change in surface properties. In our
study, RGD-HFBI could readily self-assemble on the
BODIPY dye by simple mixing and sonication for 30
min, resulting in the efficient solubilization of this
hydrophobic dye. It was notable that a protein
nanocage was formed during the self-assembly
process, which means the performance of this protein
could be improved by further reasonable
modifications. We also found that physical
hydrophobic interaction could be the major driven
force for the interaction of RGD-HFBI and BODIPY.
The second function of this protein is to target specific
cell lines by using the classic RGD motif in the protein.
In our study, we proved that RGD-HFBI could
efficiently guide the BODIPY dye to specifically label
the U-87MG cancer cell in cell culture and in animal
experiments. Furthermore, the good biocompatibility
specific affinity between the RGD-HFBI/BODIPY and
the U-87MG glioblastoma tumor. To further verify the
accumulation of RGD-HFBI/BODIPY, the mice were
sacrificed at three time points (24, 48 and 72 h), and
dominant organs were selected for in vitro imaging
(Figure 6B). For the RGD-HFBI/BODIPY-treated
mice, the tumor tissue showed distinct fluorescence at
all three time points, suggesting BODIPY
accumulation in the tumor due to specific targeting of
the RGD-HFBI/BODIPY (Figure 6D). Regarding the
control group, limited fluorescence was observed in
the tumor tissue, and strong fluorescence signals were
and
prolonged
circulation
might facilitate
time
of
future
RGD-HFBI/BODIPY
application of this dye. In our study, we only used a
synthesized BODIPY dye to prove the dual functions
of RGD-HFBI. We believed that RGD-HFBI could be
applied to other hydrophobic fluorescent probes or
even other hydrophobic materials that suffer from
solubility and targeting problems. Therefore, our
one-step strategy may be developed as a general
strategy for biolabeling and bioimaging to broaden
their applications.
http://www.thno.org

Theranostics 2018, Vol. 8, Issue 11
3123
Figure 6. (A) NIR fluorescence images of nude mice bearing U-87MG tumor after intravenous injection of RGD-HFBI/BODIPY and HFBI/BODIPY. (B) The ex vivo optical
images of organs (heart, liver, spleen, lung and kidney) and tumors of the U-87MG tumor-bearing mice sacrificed at three time points (24, 48 and 72 h) after administration. (C)
Quantitation of in vivo fluorescence intensity of U-87MG tumor after administration of RGD-HFBI/BODIPY and HFBI/BODIPY in the tumor-bearing mice at different time points
(2, 4, 6, 8, 24, 48 and 72 h). (D) Quantitation of ex vivo fluorescence intensity of harvested tumors of the U-87MG tumor-bearing mice sacrificed at three time points (24, 48 and
72 h) after administration. (E) NIR fluorescence images of nude mice bearing HeLa tumor after intravenous injection of RGD-HFBI/BODIPY and HFBI/BODIPY. (F) The ex vivo
optical images of organs (heart, liver, spleen, lung and kidney) and tumors of the HeLa tumor-bearing mice sacrificed at three time points (24, 48 and 72 h) after administration.
(G) Quantitation of in vivo fluorescence intensity of HeLa tumor after administration of RGD-HFBI/BODIPY and HFBI/BODIPY in the tumor-bearing mice at different time
points (2, 4, 6, 8, 24, 48 and 72 h). (H) Quantitation of in vitro fluorescence intensity of harvested tumors of the HeLa tumor-bearing mice sacrificed at three time points (24, 48
and 72 h) after administration.
http://www.thno.org

Theranostics 2018, Vol. 8, Issue 11
3124
13. Amiot CL, Xu S, Liang S, Pan L, Zhao J. Near-infrared fluorescent materials for
sensing of biological targets. Sensors (Basel). 2008; 8: 3082-105.
14. Yuan L, Lin W, Zhao S, Gao W, Chen B, He L, et al. A unique approach to
development of near-infrared fluorescent sensors for in vivo imaging. J Am
Chem Soc. 2012; 134: 13510-23.
15. Verwilst P, Kim HR, Seo J, Sohn NW, Cha SY, Kim Y, et al. Rational design of
in vivo tau tangle-selective near-infrared fluorophores: expanding the
BODIPY universe. J Am Chem Soc. 2017; 139: 13393-403.
16. Han C, Yang H, Chen M, Su Q, Feng W, Li F. Mitochondria-targeted
near-infrared fluorescent off-on probe for selective detection of cysteine in
living cells and in vivo. ACS Appl Mater Interfaces. 2015; 7: 27968-75.
17. Zhang J, Yang M, Li C, Dorh N, Xie F, Luo F, et al. Near-infrared fluorescent
probes based on piperazine-functionalized BODIPY dyes for sensitive
detection of lysosomal pH. J Mater Chem B. 2015; 3: 2173-84.
18. Wang D, Fan J, Gao X, Wang B, Sun S, Peng X. Carboxyl BODIPY dyes from
bicarboxylic anhydrides: one-pot preparation, spectral properties,
photostability, and biolabeling. J Org Chem. 2009; 74: 7675-83.
19. Kim J, Kim Y. A water-soluble sulfonate-BODIPY based fluorescent probe for
selective detection of HOCl/OCl- in aqueous media. Analyst. 2014; 139:
2986-9.
Abbreviations
BODIPY: Boron-dipyrromethene; CC: column
chromatography; DDQ: 2, 3-Dichloro-5, 6-dicyano-1,
4-benzoquinone; EPR: enhanced permeation and
retention; FTIR: Fourier-transform infrared; NIR:
near-infrared; RGD: arginine-glycine-aspartic acid;
RP-HPLC: reversed-phase high-performance liquid
chromatography;
TEM:
transmission
electron
microscopy; TLC: thin-layer chromatography; WCA:
water contact angle; XPS: X-ray photoelectron
spectroscopy.
20. Zhu S, Zhang J, Vegesna G, Luo F, Green SA, Liu H. Highly water-soluble
neutral BODIPY dyes with controllable fluorescence quantum yields. Org Lett.
2011; 13: 438-41.
21. Hu R, Law WC, Lin G, Ye L, Liu J, Liu J, et al. PEGylated phospholipid
micelle-encapsulated near-infrared PbS quantum dots for in vitro and in vivo
bioimaging. Theranostics. 2012; 2: 723-33.
22. Liu F, Deng D, Chen X, Qian Z, Achilefu S, Gu Y. Folate-polyethylene glycol
conjugated near-infrared fluorescence probe with high targeting affinity and
sensitivity for in vivo early tumor diagnosis. Mol Imaging Biol. 2010; 12:
595-607.
23. Ha Y, Choi HK. Recent conjugation strategies of small organic fluorophores
and ligands for cancer-specific bioimaging. Chem Biol Interact. 2016; 248:
36-51.
24. Patalag LJ, Jones PG, Werz DB. BOIMPYs: rapid access to a family of
red-emissive fluorophores and NIR dyes. Angew Chem Int Ed Engl. 2016; 55:
13340-4.
25. Becker A, Hessenius C, Licha K, Ebert B, Sukowski U, Semmler W, et al.
Receptor-targeted optical imaging of tumors with near-infrared fluorescent
ligands. Nat Biotechnol. 2001; 19: 327-31.
26. Kanduluru AK, Srinivasarao M, Low PS. Design, synthesis, and evaluation of
a neurokinin-1 receptor-targeted near-IR dye for fluorescence-guided surgery
of reuroendocrine cancers. Bioconjug Chem. 2016; 27: 2157-65.
27. Haque A, Faizi MS, Rather JA, Khan MS. Next generation NIR fluorophores
for tumor imaging and fluorescence-guided surgery: a review. Bioorg Med
Chem. 2017; 25: 2017-34.
28. Achilefu S. The insatiable quest for near-infrared fluorescent probes for
molecular imaging. Angew Chem Int Ed Engl. 2010; 49: 9816-8.
29. Zhong Y, Ma Z, Zhu S, Yue J, Zhang M, Antaris AL, et al. Boosting the
down-shifting luminescence of rare-earth nanocrystals for biological imaging
beyond 1500ꢀnm. Nat Commun. 2017; 8: 737.
Acknowledgements
Zefang Wang acknowledges the support of the
National Natural Science Foundation of China (No.
81601593). Shuxian Meng acknowledges the support
of National Natural Science Foundation of China (No.
21676187). Yanyan Wang acknowledges the support
of the National Natural Science Foundation of China
(No. 61501320). Yunjie Xiao, Qian Zhang and Yanyan
Wang contributed equally to this work.
Supplementary Material
Supplementary figures and tables.
http://www.thno.org/v08p3111s1.pdf
Competing Interests
The authors have declared that no competing
interest exists.
References
1. Guo Z, Park S, Yoon J, Shin I. Recent progress in the development of
near-infrared fluorescent probes for bioimaging applications. Chem Soc Rev.
2014; 43: 16-29.
30. Bayry J, Aimanianda V, Guijarro JI, Sunde M, Latge JP. Hydrophobins-unique
fungal proteins. PLoS Pathog. 2012; 8: e1002700.
31. Wosten HA. Hydrophobins: multipurpose proteins. Annu Rev Microbiol.
2. Sato R, Kozuka J, Ueda M, Mishima R, Kumagai Y, Yoshimura A, et al.
Intracellular protein-labeling probes for multicolor single-molecule imaging of
immune receptor-adaptor molecular dynamics. J Am Chem Soc. 2017; 139:
17397-404.
2001; 55: 625-46.
32. Linder MB, Szilvay GR, Nakari-Setala T, Penttila ME. Hydrophobins: the
protein-amphiphiles of filamentous fungi. FEMS Microbiol Rev. 2005; 29:
877-96.
3. Zhong Y, Ma Z, Zhu S, Yue J, Zhang M, Antaris AL, et al. Boosting the
down-shifting luminescence of rare-earth nanocrystals for biological imaging
beyond 1500 nm. Nat Commun. 2017; 8: 737.
4. Evans CL. New near-infrared dyes light up deep tissue imaging. Sci Transl
Med. 2017; 9: eaam6065.
33. Hakanpaa J, Szilvay GR, Kaljunen H, Maksimainen M, Linder M, Rouvinen J.
Two crystal structures of Trichoderma reesei hydrophobin HFBI-the structure of
a protein amphiphile with and without detergent interaction. Protein Sci. 2006;
15: 2129-40.
34. Sunde M, Kwan AH, Templeton MD, Beever RE, Mackay JP. Structural
analysis of hydrophobins. Micron. 2008; 39: 773-84.
5. Frangioni JV. In vivo near-infrared fluorescence imaging. Curr Opin Chem
Biol. 2003; 7: 626-34.
35. Laaksonen P, Kainlauri M, Laaksonen T, Shchepetov A, Jiang H, Ahopelto J, et
al. Interfacial engineering by proteins: exfoliation and functionalization of
graphene by hydrophobins. Angew Chem Int Ed Engl. 2010; 49: 4946-9.
36. Qin M, Wang L, Feng X, Yang Y, Wang R, Wang C, et al. Bioactive surface
modification of mica and poly (dimethylsiloxane) with hydrophobins for
protein immobilization. Langmuir. 2007; 23: 4465-71.
37. Valo HK, Laaksonen PH, Peltonen LJ, Linder MB, Hirvonen JT, Laaksonen TJ.
Multifunctional hydrophobin: toward functional coatings for drug
nanoparticles. ACS Nano. 2010; 4: 1750-8.
38. Pigliacelli C, Maiolo D, Nonappa N, Haataja JS, Amenitsch H, Michelet C, et
al. Efficient encapsulation of fluorinated drugs in the confined space of
water-dispersible fluorous supraparticles. Angew Chem Int Ed Engl. 2017; 56:
16186-90.
39. Hahl H, Vargas JN, Griffo A, Laaksonen P, Szilvay G, Lienemann M, et al.
Pure protein bilayers and vesicles from native fungal hydrophobins. Adv
Mater. 2017; 29: 1602888.
6. Lou Z, Li P, Han K. Redox-responsive fluorescent probes with different design
strategies. Acc Chem Res. 2015; 48: 1358-68.
7. Yu F, Li P, Wang B, Han K. Reversible near-infrared fluorescent probe
introducing tellurium to mimetic glutathione peroxidase for monitoring the
redox cycles between peroxynitrite and glutathione in vivo. J Am Chem Soc.
2013; 135: 7674-80.
8. Yuan L, Lin W, Zheng K, He L, Huang W. Far-red to near infrared
analyte-responsive fluorescent probes based on organic fluorophore platforms
for fluorescence imaging. Chem Soc Rev. 2013; 42: 622-61.
9. Luo S, Zhang E, Su Y, Cheng T, Shi C. A review of NIR dyes in cancer
targeting and imaging. Biomaterials. 2011; 32: 7127-38.
10. Li Y, Xu D, Ho SL, Li H, Yang R, Wong M. A theranostic agent for in vivo
near-infrared imaging of beta-amyloid species and inhibition of beta-amyloid
aggregation. Biomaterials. 2016; 94: 84-92.
11. Hyun H, Owens EA, Wada H, Levitz A, Park G, Park MH, et al.
Cartilage-specific near-infrared fluorophores for biomedical imaging. Angew
Chem Int Ed Engl. 2015; 54: 8648-52.
12. Hyun H, Park MH, Owens EA, Wada H, Henary M, Handgraaf HJ, et al.
Structure-inherent targeting of near-infrared fluorophores for parathyroid and
thyroid gland imaging. Nat Med. 2015; 21: 192-7.
40. Gravagnuolo AM, Morales-Narváez E, Matos CRS, Longobardi S, Giardina P,
Merkoçi A. On-the-spot immobilization of quantum dots, graphene oxide, and
proteins via hydrophobins. Adv Funct Mater. 2015; 25: 6084-92.
41. Malho JM, Arola S, Laaksonen P, Szilvay GR, Ikkala O, Linder MB. Modular
architecture of protein binding units for designing properties of cellulose
nanomaterials. Angew Chem Int Ed Engl. 2015; 54: 12025-8.
http://www.thno.org

Theranostics 2018, Vol. 8, Issue 11
3125
42. Janssen MI, van Leeuwen MB, Scholtmeijer K, van Kooten TG, Dijkhuizen L,
Wosten HA. Coating with genetic engineered hydrophobin promotes growth
of fibroblasts on a hydrophobic solid. Biomaterials. 2002; 23: 4847-54.
43. Kallio JM, Linder MB, Rouvinen J. Crystal structures of hydrophobin HFBII in
the presence of detergent implicate the formation of fibrils and monolayer
films. J Biol Chem. 2007; 282: 28733-9.
72. Zhao L, Xu H, Li Y, Song D, Wang X, Qiao M, et al. Novel application of
hydrophobin in medical science: a drug carrier for improving serum stability.
Sci Rep. 2016; 6: 26461.
73. Askolin S, Nakari-Setälä T, Tenkanen M. Overproduction, purification, and
characterization of the Trichoderma reesei hydrophobin HFBI. Appl Microbiol
Biotechnol. 2001; 57: 124-30.
44. Goldian I, Jahn S, Laaksonen P, Linder M, Kampf N, Klein J. Modification of
interfacial forces by hydrophobin HFBI. Soft Matter. 2013; 9: 10627-39.
45. Mas-Moruno C, Fraioli R, Rechenmacher F, Neubauer S, Kapp TG, Kessler H.
αvβ3- or α5β1-integrin-selective peptidomimetics for surface coating. Angew
Chem Int Ed Engl. 2016; 55: 7048-67.
74. Wang X, Permentier HP, Rink R, Kruijtzer JA, Liskamp RM, Wösten HA, et al.
Probing the self-assembly and the accompanying structural changes of
hydrophobin SC3 on a hydrophobic surface by mass spectrometry. Biophys J.
2004; 87: 1919-28.
75. Wang X, Graveland-Bikker JF, de Kruif CG, Robillard GT. Oligomerization of
hydrophobin SC3 in solution: from soluble state to self-assembly. Protein Sci.
2004; 13: 810-21.
46. Avraamides CJ, Garmy-Susini B, Varner JA. Integrins in angiogenesis and
lymphangiogenesis. Nat Rev Cancer. 2008; 8: 604-17.
47. Grunér MS, Szilvay GR, Berglin M, Lienemann M, Laaksonen P, Linder MB.
Self-assembly of class II hydrophobins on polar surfaces. Langmuir. 2012; 28:
4293-300.
48. Macindoe I, Kwan AH, Ren Q, Morris VK, Yang W, Mackay JP, et al.
Self-assembly of functional, amphipathic amyloid monolayers by the fungal
hydrophobin EAS. Proc Natl Acad Sci USA. 2012; 109: E804-11.
49. Stanimirova RD, Gurkov TD, Kralchevsky PA, Balashev KT, Stoyanov SD,
Pelan EG. Surface pressure and elasticity of hydrophobin HFBII layers on the
air-water interface: rheology versus structure detected by AFM imaging.
Langmuir. 2013; 29: 6053-67.
50. Pham CLL, Rey A, Lo V, Soulès M, Ren Q, Meisl G, et al. Self-assembly of
MPG1, a hydrophobin protein from the rice blast fungus that forms functional
amyloid coatings, occurs by a surface-driven mechanism. Sci Rep. 2016; 6:
25288.
51. Magarkar A, Mele N, Abdel-Rahman N, Butcher S, Torkkeli M, Serimaa R, et
al. Hydrophobin film structure for HFBI and HFBII and mechanism for
accelerated film formation. PLoS Comput Biol. 2014; 10: e1003745.
52. Moldovan C, Thompson D. Molecular dynamics of the "hydrophobic patch"
that immobilizes hydrophobin protein HFBII on silicon. J Mol Model. 2011; 17:
2227-35.
53. Peng C, Liu J, Zhao D, Zhou J. Adsorption of hydrophobin on different
self-assembled monolayers: the role of the hydrophobic dipole and the electric
dipole. Langmuir. 2014; 30: 11401-11.
54. Lumsdon SO, Green J, Stieglitz B. Adsorption of hydrophobin proteins at
hydrophobic and hydrophilic interfaces. Colloids Surf B Biointerfaces. 2005;
44: 172-8.
55. Ostolska I, Wisniewska M. Application of the zeta potential measurements to
explanation of colloidal Cr2O3 stability mechanism in the presence of the ionic
polyamino acids. Colloid Polym Sci. 2014; 292: 2453-64.
56. Hao Y, Huang Y, He Y, Peng J, Chen L, Hu X, et al. The evaluation of cellular
uptake efficiency and tumor-targeting ability of MPEG-PDLLA micelles: effect
of particle size. RSC Adv. 2016; 6: 13698-709.
57. He C, Hu Y, Yin L, Tang C, Yin C. Effects of particle size and surface charge on
cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials.
2010; 31: 3657-66.
58. Wang Z, Wang Y, Huang Y, Li S, Feng S, Xu H, et al. Characterization and
application of hydrophobin-dispersed multi-walled carbon nanotubes.
Carbon. 2010; 48: 2890-8.
59. Hakanpaa J, Paananen A, Askolin S, Nakari-Setala T, Parkkinen T, Penttila M,
et al. Atomic resolution structure of the HFBII hydrophobin, a self-assembling
amphiphile. J Biol Chem. 2004; 279: 534-9.
60. Bhaskar S, Lim S. Engineering protein nanocages as carriers for biomedical
applications. NPG Asia Mater. 2017; 9: e371.
61. Votteler J, Ogohara C, Yi S, Hsia Y, Nattermann U, Belnap DM, et al. Designed
proteins induce the formation of nanocage-containing extracellular vesicles.
Nature. 2016; 540: 292-5.
62. Schoonen L, van Hest JC. Functionalization of protein-based nanocages for
drug delivery applications. Nanoscale. 2014; 6: 7124-41.
63. Lin X, Xie J, Niu G, Zhang F, Gao H, Yang M, et al. Chimeric ferritin nanocages
for multiple function loading and multimodal imaging. Nano Lett. 2011; 11:
814-9.
64. Truffi M, Fiandra L, Sorrentino L, Monieri M, Corsi F, Mazzucchelli S. Ferritin
nanocages: a biological platform for drug delivery, imaging and theranostics
in cancer. Pharmacol Res. 2016; 107: 57-65.
65. Kong J, Yu S. Fourier transform infrared spectroscopic analysis of protein
secondary structures. Acta Biochim Biophys Sin (Shanghai). 2007; 39: 549-59.
66. Meister K, Bäumer A, Szilvay GR, Paananen A, Bakker HJ. Self-assembly and
conformational changes of hydrophobin classes at the air-water interface. J
Phys Chem Lett. 2016; 7: 4067-71.
67. Ye Y, Chen X. Integrin targeting for tumor optical imaging. Theranostics. 2011;
1: 102-26.
68. Taherian A, Li X, Liu Y, Haas TA. Differences in integrin expression and
signaling within human breast cancer cells. BMC Cancer. 2011; 11: 293.
69. Aimanianda V, Bayry J, Bozza S, Kniemeyer O, Perruccio K, Elluru SR, et al.
Surface hydrophobin prevents immune recognition of airborne fungal spores.
Nature. 2009; 460: 1117-21.
70. Win KY, Feng S. Effects of particle size and surface coating on cellular uptake
of polymeric nanoparticles for oral delivery of anticancer drugs. Biomaterials.
2005; 26: 2713-22.
71. Shang L, Nienhaus K, Nienhaus GU. Engineered nanoparticles interacting
with cells: size matters. J Nanobiotechnology. 2014; 12: 5.
http://www.thno.org