Tumor-targeting, enzyme-activated nanoparticles for simultaneous cancer diagnosis and photodynamic therapy

Article abstract of DOI:10.1039/c5tb02041g

Specific targeting towards tumors and the on-site activation of photosensitizers to diagnose tumors and reduce side effects for patients are currently the main challenges for photodynamic therapy (PDT) in the clinic. Herein, uniform diiodostyryl bodipy co

Full text of DOI:10.1039/c5tb02041g

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Materials Chemistry B
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Tumor-Targeting and Enzyme-Activated Nanoparticles for
Simultaneous Cancer Diagnose and Photodynamic Therapy
Huaxia Shi,a Wucheng Sun,a Changbing Liu,a Guiying Gu,c Bo Ma,c Weili Si,b Nina Fu,a
Qi Zhang,* c Wei Huang,*ab and Xiaochen Dong*b
aKey Laboratory for Organic Electronics & Information Displays (KLOEID), Nanjing University
of Posts and Telecommunications, Nanjing 210023, China.
bKey Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM),
Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), NanjingTech
University (NanjingTech), 30 South Puzhu Road, Nanjing 211816, China.
E-mail: iamxcdong@njtech.edu.cn, iamwhuang@njtech.edu.cn
cSchool of Pharmaceutical Science, Nanjing Tech University (NanjingTech), 30 South Puzhu Road,
Nanjing 211816, China. Email: zhangqi@njtech.edu.cn
Specific targeting towards tumors and onꢀsite activation of photosensitizers to
diagnose tumors and reduce side effects for patients are main challenges for current
photodynamic therapy (PDT), in clinic. Herein, uniform diiodostyryl bodipy
conjugated hyaluronic acid nanoparticles (DBHAꢀNPs) were successfully synthesized.
PDT effect evaluated in both cell level and animal models of tumorꢀbearing mice
shows that the DBHAꢀNPs present a remarkable suppression of tumorous growth due
to their specific targeting and enhanced permeability and retention (EPR) effect. More
importantly, enzymeꢀactivated “selfꢀassembly and disaggregation” behavior made by
tumors can lead DBHAꢀNPs onꢀsite activation, which can diagnose the tumor exactly
and reduce side effect for patients significantly. These findings confirm that
DBHAꢀNPs have a significant potential for photodynamically activated cancer
theranostics, in clinic.
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1. Introduction
Photodynamic therapy (PDT) is an effective therapeutic modality for a variety of
malignant and nonmalignant tumors.1ꢀ4 Mediated by photosensitizers (PSs), cancer
cells can be killed by reactive oxygen species (ROS), which are generated from
locally sensitizing oxygen upon light irradiation.5 As for cancer treatment, PDT has
emerged as an important method in preclinical research and clinical practice because
of its nonꢀinvasive property and selective light irradiation on tumor site, avoiding
normal tissues and organs being damaged.
However, there are still several problems to prevent the development of PDT in clinic.
First, traditional PSs suffer from their native hydrophobicity and poor
biocompatibility, limiting their potential application in biological field.6 At present,
two main strategies have been developed to increase their water solubility and
biocompatibility. On the one hand, PSs can be conjugated with hydrophilic molecule
through covalent bond.7 On the other hand, PSs can be incorporated into
biocompatible drug delivery platforms via noncovalent bond, such as liposomes,8ꢀ11
polymeric micelles,12ꢀ14 and inorganic nanoparticles.15ꢀ20 Second, how to enhance the
selectivity of highly effective PSs so that they can locate and function at tumor site
preferentially is another major challenge. A promising method is to directly conjugate
PSs with targeting ligands, including folates, antibodies, proteins, and so on.21ꢀ23 And,
the third obstacle for PDT in clinic is that activation of PSs at nonꢀtumor site would
damage normal tissues and organs of patients through blood circulation. Recently,
activatable photosensitizing systems have drawn a lot of attention, which can be
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activated selectively by tumorsꢀassociated stimuli. Commonly, activatable PSs are
photodynamic inactive in normal tissue environment and their fluorescence and ROS
generation are silent even with light irradiation. However, these PSs become
photodynamically active in tumor site through various kinds of activation mechanisms,
25
including enzymatic activation,24,
nucleic acidꢀactivation26,27 and tumor
environmental activation28 (e.g., remarkable decrease in pH value and significant
increased level of H2O2). The activatable PSs can achieve excellent treatment
selectivity and destroy targeted tumor cells, but not threat normal tissues and organs29.
Therefore, the development of new kinds of PSs to solve all these problems
mentioned above in PDT clinic is extremely important.
Fig. 1 Schematic illustration of DBHAꢀNPs as theranostic agents for PDT both in
vitro and in vivo.
In this study, diiodostyryl bodipy conjugated hyaluronic acid nanoparticles
(DBHAꢀNPs) are designed for activatable photodynamic theranostics in tumors. The
DBHAꢀNPs exhibit excellent hydrophilicity and biocompatibility, as well as specific
targeting towards CD44 receptor overexpressed on the surface of tumor cells (e.g.
HCTꢀ116 cells, human colon carcinoma cell line). And the DBHAꢀNPs reaching into
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the lysosomes inside HCTꢀ116 cells can be disaggregated, resulting in the restoration
of fluorescence and ROS with light irradiation, which is impossible in the native of
diiodostyryl bodipy due to its aggregation (Fig. 1). Additionally, both in vitro and in
vivo experiments demonstrate that DBHAꢀNPs can effectively label and
photodynamically suppress the growth of tumor.
2. Experimental Section
Synthesis of DBHA-NPs
Fig. 2 shows the synthetic route of DBHA. In brief, 4ꢀ hydroxybenzaldehyde was
treated with 3ꢀbromoꢀ1ꢀpropanol in the presence of K2CO3 in acetone to yield
Compound 1. And diiodo bodipy30, 31 (Compound 3) was prepared through iodination
33
of bodipy32,
(Compound 2), using Nꢀiodosuccinimide (NIS) as iodoꢀagent.
Furthermore, Compound 3 underwent Knoevenagel condensation with Compound 1
to yield diiodostyryl bodipy (Compound 4) through microwave reaction.34 As well
known, diiodostyryl bodipy has great potential as PS for PDT, mainly suffering from
its native hydrophobicity and bad biocompatibility, as well as targeting.35ꢀ37 It can be
concluded that the above diiodostyryl bodipy (Compound 4) is further conjugated
onto the hyaluronic acid (HA) through esterification reaction. Notably, DBHA shows
excellent waterꢀsolubility and good biocompatibility due to the existence of HA. And
finally, DBHA dissolved into deionized (DI) water was dropwise added into large
amount of DI water under the ultrasound to yield DBHAꢀNPs through selfꢀassembly.
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Fig. 2 Synthetic route of DBHA.
Characterization
Nuclear magnetic resonance (NMR) spectra were measured by using a Bruker Ultra
Shield Plus 400 MHz. GCꢀMS was recorded on a Shimadzu GCꢀMSꢀQP 2010 Plus
mass spectrometer. MALDIꢀTOFꢀMASS (Matrixꢀassisted laser desorption/ionization
timeꢀofꢀflight mass spectrometry) was performed on a Bruker autoflex under the
reflector mode (without the matrix) for data acquisition. The UVꢀvisible absorption
and photoluminescence (PL) spectra were recorded on a Shimadzu UVꢀ3600
UVꢀVISꢀNIR spectrophotometer and a RFꢀ5301 PC spectrofluorophotometer,
respectively. Transmission electron microscopy (TEM) images were obtained using a
JEOL JEMꢀ2100 transmission electron microscope. The hydrodynamic size of the
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nanoparticles (Dynamic light scattering, DLS) was measured by using a 90 Plus
particle size analyzer, Brookhaven Instruments.
Cell Lines and Culture Conditions
HCTꢀ116 and A2780 cell lines, provided by the Institute of Biochemistry and Cell
Biology, SIBS, CAS (China), were cultured in regular growth medium consisting of
Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal
bovine serum at 37 ℃ under 5% CO2 atmosphere. The cells were routinely harvested
by treating with a trypsinꢀethylene diamine tetraacetic acid (EDTA) solution (0.25%).
Cell Viability Assays
HA and DBHAꢀNPs were first dissolved in phosphate buffered saline (PBS) solution,
then diluted with DMEM to various concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4,
and 1.6 mg/ml). HCTꢀ116 cells and A2780 cells, rinsed with PBS solution, were
incubated with HA and DBHAꢀNPs solutions for 24 h at 37℃. Then the cells were
rinsed again with PBS solution and refilled with 100 µL of the culture medium before
being illuminated at ambient temperature. The light source was from Xenon lamp,
equipped with a color glass filter with cutꢀon at 600 nm. The fluence rate (λ > 600 nm)
was 40 mW/cm2 for 7 minutes.
Cell viability was determined by means of the colorimetric MTT assay. An MTT
(USB) solution in PBS solution (5 mg/mL, 20 µL) was added into each well followed
by incubation for 4 h under the same environment at 37 ℃. Then the medium was
discarded and 150 ꢁl DMSO was added. The plate was agitated on a BioꢀTek
microplate reader at ambient temperature before the absorbance at 490 nm for each
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well was taken. The average absorbance of the blank well, no cells, was subtracted
from the readings of the other wells. The cell viability was then determined by the
following equation: Viability (%) = {Ɖ [(Ai/A control) * 100]}/ n, where Ai is the
absorbance of ith data (i = 1, 2, 3 …, n), A control is average absorbance of the control
wells in which HA or DBHAꢀNPs was absent, and n is the number of the data points.
Flow Cytometric Study
Approximately 6*105 HCTꢀ116 cells in the medium (2 mL) were seeded on a 6ꢀwell
plate and incubated for 24 h under 5% CO2. The cells were treated with DBHAꢀNPs
at various concentrations (0.25, 0.5, 0.75, and 1.0 mg/ml) and incubated under the
same conditions for 4 h. The cells were then rinsed thrice with PBS solution and
refilled with 1 ml of the culture medium before being illuminated (λ > 600 nm, 40
mW/cm2, 7 min) at ambient temperature. After 24 h of incubation, the cell were rinsed
with PBS solution and harvested by 0.25% trypsin (Invitrogen, 500 µL) for 2 min,
followed by centrifugation at 1200 rpm. The cells were suspended in 100 µl of
binding buffer containing FITC annexinꢀV (5 µl) and PI (5 µl). After incubation in
darkness for 15 min at room temperature, the signals of FITC annexinꢀV and PI were
measured by a BD FACSCalibur flow cytometer (Becton Dickinson) with 104 cells
counted in each example. Both annexinꢀV and PI were excited by a 488 nm argon
laser. The emitted fluorescence was monitored at 500ꢀ560 nm for annexinꢀV and at >
670 nm for PI. The data collected were analyzed by using Cell QUEST Pro.
Fluorescence Microscopic Study
About 2*105 HCTꢀ116 cells or A2780 cells in the culture medium (2 ml) were seeded
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on a glass bottom Petri dish and incubated overnight at 37 ℃. Then the medium was
removed. The cells, rinsed with PBS solution, were incubated with solution of
DBHAꢀNPs (1 mg/ml, 100 ꢁl) in the medium for 4 h. The cells were rinsed with PBS
solution and viewed with an OlympusⅨ 70 inverted microscope. The excitation light
(588 nm) was provided by a multiꢀwavelength illuminator (PolychromeⅣ, TILL
Photonics). The emitted fluorescence at > 600 nm was collected, digitized, and
analyzed using MetaFluorⅤ6.3 (Universal imaging). The average intracellular
fluorescence intensities (24 cells for each sample) were determined.
To confirm the concept of CD44ꢀreceptor mediated tumor cell endocytosis, similarly,
about 2*105 HCTꢀ116 cells in the culture medium (2 ml) were seeded on a glass
bottom Petri dish and incubated overnight at 37 ℃ under 5% CO2. Then the medium
was removed. Firstly, the cells were incubated with HA solution (2 mg/ml, 100 ꢁl) for
4 h, further rinsed with PBS solution. Then the above cells were incubated with
DBHAꢀNPs solution (1 mg/ml, 100 ꢁl) for 4 h under the same concentrations. The
cells were rinsed with PBS solution and viewed with an OlympusⅨ 70 inverted
microscope. The excitation light (588 nm) was provided by a multiꢀwavelength
illuminator (PolychromeⅣ, TILL Photonics). The emitted fluorescence at > 600 nm
was collected, digitized, and analyzed using MetaFluorⅤ6.3 (Universal imaging).
Subcellular Location Study
About 1*105 HCTꢀ116 cells in culture medium (2 ml) were seeded on glass bottom
Petri dish and incubated overnight at 37 ℃ under 5% CO2. The medium was removed
and the cells were rinsed with PBS solution. The cells were incubated with
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DBHAꢀNPs solution in the medium (1 mg/ml, 100 ꢁl) for 24 h. For the study using
MitoꢀTracker, the cells were incubated with MitoꢀTracker Green FM (Molecular
Probes, 0.2 µM in the medium) for further 15 min. For the study using LysoꢀTracker,
green DND 26 (Molecular Probes, 0.075 µM in the medium) or ERꢀTracker Green
(Molecular Probes, 1.0 µM in PBS solution) for further 15 min. For all the cases, the
cells were rinsed with PBS solution and viewed with a Leica SP5 confocal
microscope equipped with a 488 nm argon laser and a 588 nm helium neon laser. All
trackers were excited at 488 nm and monitored at 510ꢀ560 nm, while DBHAꢀNPs
were excited at 588 nm and monitored at 600ꢀ700 nm. The images were digitized and
analyzed using the Leica Application Suite Advanced Fluorescence software. The
subcellular location of DBHAꢀNPs was revealed by comparing the intracellular
fluorescence images caused by the MitoꢀTracker, LysoꢀTracker, ERꢀTracker and
DBHAꢀNPs.
Real-time monitoring the fluorescence in vitro
About 1*105 HCTꢀ116 cells in culture medium (2 ml) were seeded on glass bottom
Petri dish and incubated overnight at 37 ℃ under 5% CO2. The medium was removed,
and the cells were rinsed with PBS solution, followed adding a solution of
DBHAꢀNPs (1 mg/ml, 100 ꢁl) into fresh medium for incubation at 37 ℃. Meantime,
the fluorescence of DBHAꢀNPs was observed by Confocal Laser Scanning
Microscope (CLSM) and the confocal images were obtained by using Zeiss confocal
laser microscope (ZEISS LSM 700) at different time.
Fluorescence Studies for ROS probe
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About 2*105 HCTꢀ116 cells in the culture medium (2 ml) were seeded on glass
bottom Petri dish and incubated overnight at 37 ℃ under 5% CO2. The medium was
removed. The cells, rinsed with PBS solution, were incubated with a solution of
DBHAꢀNPs or Porphyrin in PBS solution (1 mg/ml, 100 ꢁl) for 24 h. The cells were
rinsed with PBS solution and further incubated with ROS probe, DCFHꢀDA (50
ꢁg/ml, 10 ꢁl) for 1 h. And then, the cells were rinsed thrice with PBS solution and
refilled with 1 ml of the culture medium before being illuminated at ambient
temperature using a Xenon lamp light (λ > 600 nm, 40 mW/cm2, 7 min). After that,
the cells were viewed with Olympus IX70 inverted microscope. The excitation light
(488 nm) was provided by a multiꢀwavelength illuminator (PolychromeIV, TILL
Photonics). The emitted fluorescence from 500 to 600 nm was collected, digitized,
and analyzed using MetaFluorⅤ6.3 (Universal imaging).
For comparison without cells, no cells were added and only DBHAꢀNPs or Porphyrin
with DCFHꢀDA at the same concentrations.
In vivo fluorescence and Cancer treatment
All animal experiments were performed complying with the NIH guidelines for the
care and use of laboratory animals. The xenograft tumors were established
subcutaneously injecting HCTꢀ116 cells into the armpit of nude mice. After tumors of
30ꢀ40 mm3 were palpable, whole body images of mice were acquired respectively by
a tail vein injection of 200 µl of DBHAꢀNPs at a concentration of 10 mg/ml. All
images were analyzed and collected at the indicated time points with a MaestroTM
InꢀVivo Imaging System (Cri, Woburn, MA, USA).
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For the tumor treatment, the mice were divided into four groups with six mice in each
group, randomly. The mice in the first to the third group were injected with saline, HA
and DBHAꢀNPs solution (10 mg/ml, 200 ꢁl) by a tail vein injection, respectively, with
light irradiation (λ > 600 nm, 40 mW/cm2, 15 min). And the mice in the fourth group
were injected with DBHAꢀNPs solution (10 mg/ml, 200 ꢁl) by a tail vein injection
without light irradiation while under the same conditions.
3. Results and discussion
Preparation and characterization of DBHA-NPs
1
Fig. 3a shows the HꢀNMR spectrum of DBHA in D2O. The appearance of chemical
shifts between 7.0ꢀ8.0 in the spectra compared with that of HA38, 39 suggests that HA
has successfully conjugated onto the diiodostyryl bodipy. The TEM image in Fig. 3b
demonstrates that the selfꢀassembled DBHAꢀNPs have spherical morphology. The
dynamic light scattering (DLS) measurement indicates the average size of the
particles is around 50 nm (Inset in Fig. 3b). Fig. 3c shows the optical absorption
properties of DBHA, DBHAꢀNPs and HA dissolved in PBS, respectively. An obvious
absorption peak at 600 nm arising from the Qꢀband of diiodostyryl bodipy can be
observed. On the contrary, there are no peaks for pure HA, indicating that diiodostyryl
bodipy has been successfully conjugated onto the HA. Interestingly, after the addition
of hyaluronidase which can degrade HA polymer, the fluorescence of diiodostyryl
bodipy (at 678 nm) recovers in a timeꢀdependent manner (Fig. 3d and S1), confirming
the enzymatic degradation process of HA. With the degradation of HA, DBHA NPs
can also be deꢀaggregated and the fluorescence intensity increases.
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1
Fig. 3 (a) HꢀNMR spectrum of DBHA in D2O. (b) TEM image of DBHAꢀNPs. The
inset shows the size distribution of DBHAꢀNPs. (c) UVꢀVis absorption of DBHA,
DBHAꢀNPs and HA (5 mg/ml) in PBS. (d) Fluorescence spectra of DBHAꢀNPs (5
mg/ml) incubated with hyaluronidase (0.1 mg/ml) in PBS for different time, excited at
600 nm.
NIR fluorescence images and specific targeting of DBHA-NPs
CD44, a receptor for HA, is overexpressed in some types of cancer cells (e.g.,
HCTꢀ116 cells, corresponding to colon cancer).40 As expected in Fig. 4a, the
DBHAꢀNPs can abundantly stain the HCTꢀ116 cells41 and uniformly dispersed in the
cytoplasm of HCTꢀ116 cells. On the contrary, A2780 cells (human ovarian carcinoma
cell line), known to have low expression of CD44 receptors, are not wellꢀstained by
DBHAꢀNPs and the fluorescence intensity is greatly decreased compared with that of
HCTꢀ116 cells (Fig. 4b). It is well known that the staining can be eliminated by the
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previous addition of excess free HA molecules because of the competitive inhibition.
As shown in Fig. 4b, with the existence of HA, the fluorescence intensity of the
HCTꢀ116 cells decreases greatly, further indicating DBHAꢀNPs have specific
targeting properties towards CD44 overexpressed HCTꢀ116 cells. And all the results
suggest that DBHAꢀNPs are internalized via receptor mediated endocytosis triggered
by the binding of DBHAꢀNPs with CD44 receptor.42 What’ more, the parallel filter
evaluation indicates that the DBHAꢀNPs are mainly delivered into lysosomes, as
shown in Fig. 4c and Fig. S2.
Fig. 4 (a) Fluorescence images of DBHAꢀNPs in HCTꢀ116 cells, A2780 cells and
HA+DBHAꢀNPs in HCTꢀ116 cells, respectively. Red color represents the
fluorescence of DBHAꢀNPs. (Left panel, fluorescence images. Middle panel, bright
field. Right, merged images). (b) The intracellular fluorescence intensity of
DBHAꢀNPs in HCTꢀ116 cells, in A2780 cells and HA+DBHAꢀNPs in HCTꢀ116 cells.
All data were expressed as the mean ± standard deviation (number of experiments,
n=6). (c) Visualization of the intracellular fluorescence of HCTꢀ116 cells using filter
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sets specific for DBHAꢀNPs (in red, column 1) and lysosome tracker (in green
column 2). Column 3 is the merged images in bright field.
NIR fluorescent enzyme-activated of DBHA-NPs on tumorous site both in vitro
and in vivo
To investigate the onꢀsite activation properties of the DBHAꢀNPs in tumors, in vitro
and in vivo fluorescence images of DBHAꢀNPs entering into HCTꢀ116 cells at
different time are measured. Fig. 5a presents the fluorescence images of DBHAꢀNPs
incubating with HCTꢀ116 cells with time observed by confocal laser scanning
microscopy (CLSM). It can be found that the fluorescence becomes stronger and
stronger with time from 0 to 60 minutes. Similar phenomena has also been observed
in vivo after injection of DBHAꢀNPs in mice bearing HCTꢀ116 cells, as shown in Fig.
5b. There is no obvious fluorescence observed before 8h, even in heart, liver, lung,
brain and other organs and tissues.43ꢀ45 And the strong fluorescence only appears in
the tumor site after 24 h after administration of DBHAꢀNPs. Both results indicate that
DBHAꢀNPs only can be decomposed in cells or tumors, which is an effective onꢀsite
activated PSs and beneficial for diagnosing the tumor site exactly. Furthermore, this
result is also coincidence well with the previous characterization (Fig. 3d).
Enzyme-activated generation of ROS
Reactive oxygen species (ROS) generated from PSs can cause an oxidative stress and
membrane damage in the treated cells and eventual cell death, which is an important
factor for PDT. 2’,7’ꢀdichlorofluorescin diacetate (DCFHꢀDA), nonꢀfluorescence in
its native, can react with ROS to produce fluorescent 2′,7′ꢀdichlorofluorescein
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(DCF),46 which is selected as ROS probe to identify the produced ROS by
DBHAꢀNPs upon light irradiation. As shown in Fig. 5c, after DCFHꢀDA incubated
with DBAHꢀNPs, the fluorescence intensity of DCF becomes stronger and stronger
along with time from 0 to 60 min under light irradiation, indicating the concentration
of ROS increases greatly with the incubated time prolonging. More interestingly, no
fluorescence of DCF can be detected before DBHAꢀNPs enter into HCTꢀ116 cells. It
is quite different from the commercial PSs (porphyrin), which shows strong
fluorescence both out of and inside HCTꢀ116 cells (Fig. 5d). This phenomenon further
demonstrates that DBHAꢀNPs only can react in tumor cells and realize the onꢀsite
activation.
As is described above in Fig. 4c, DBHAꢀNPs mainly locate in the lysosomes after
entering into HCTꢀ116 cells. And lysosomes are full of enzymes, such as
hyaluronidase, which can make HA degradation and increase the fluorescence
intensity of DBHAꢀNPs (Fig. 3d). It can be speculated that HA, the backbone of
DBHA, can be degraded by hyaluronidase after the DBHAꢀNPs reach into lysosomes
and realize fluorescence and ROS onꢀsite activation in tumor cells. In clinic, onꢀsite
activation of PSs can be used in cancer diagnose and also provide a significant
strategy for no avoiding light to reduce the side effects during the progress of PDT.47
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Fig. 5 (a) Fluorescence images of DBHAꢀNPs entering into HCTꢀ116 cells observed
by CLSM from 0 to 60 min. (b) In vivo fluorescence images of mice bearing
HCTꢀ116 cells in armpit after tailꢀvein injection of DBHAꢀNPs. (c) Fluorescence
intensity of ROS probe after incubating with DBHAꢀNPs and hyaluronidase with light
irradiation for different time. (d) Fluorescence images of DBHAꢀNPs and porphyrin
with DCFHꢀDA out of or in HCTꢀ116 cells upon light irradiation by CLSM. Column
1, DBHA NPs and porphyrin with DCFHꢀDA out of HCTꢀ116 cells. Column 2,
DBHAꢀNPs and porphyrin with DCFHꢀDA in HCTꢀ116 cells.
Photodynamic efficiency of DBHA-NPs in vitro and in vivo
Encouraged by the above measurements, photodynamic activities of DBHAꢀNPs were
further investigated against tumor cells. As shown in Fig. 6a, DBHAꢀNPs show higher
phototoxicity and lower dark toxic, as well as doseꢀdependent survival curves, which
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is in accord well with the flow cytometric assay of DBHAꢀNPs (Fig. S2). More
importantly, the DBHAꢀNPs exhibit highly selective phototoxicity towards HCTꢀ116
cells (CD44+receptor) compared with A2780 cells (CD44ꢀreceptor), indicating their
high targeting property towards HCTꢀ116 cells. In contrast, HA is nonꢀtoxic towards
HCTꢀ116 cells. It can be concluded that the photodynamic activities of DBHAꢀNPs
originate from diiodo styryl bodipy, instead of HA. What’s more, from the results of
flow cytometric assay (Fig. S3), it can be speculated that the death mechanism of
tumor cells is mainly lateꢀapoptotic.
Fig. 6 (a) Comparison of cell viabilities by MTT assays (λ > 600nm). (b) The
relationship between tumor volume and treated time of DBHAꢀNPs in the HCTꢀ116
tumor cells bearing mouse model via tail vein injection. Twentyꢀfour mice were
randomly assigned into four groups (6 mice/groups), including saline with light, HA
with light, DBHAꢀNPs without light and DBHAꢀNPs with light. (c) Typical
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photographs of tumorꢀbearing mice treated at different time. Note, *p < 0.05, **p <
0.01.
To validate the practical application of DBHAꢀNPs, cancer treatments in vivo have
been employed by using mice bearing HCTꢀ116 cells. These mice are divided into
four groups (6 mice each group), which were intravenously injected with saline, HA
and DBHAꢀNPs with or without light irradiation, respectively, and followed by
monitoring their tumor growth volume and body weight for fifteen days. Fig. 6b and
6c show the changes of tumor volume of mice during the treatment, respectively. The
results indicate that the tumor volume of mice treated with saline and HA followed by
light irradiation and DBHAꢀNPs without light irradiation experienced a rapid growth
through the entire treatment period. However, the tumor growth of mice treated with
DBHA NPs followed by light irradiation is obviously suppressed. It suggests that the
DBHAꢀNPs can selectively accumulate in the malignant tumor, then inhibit the tumor
growth upon light irradiation through the specific targeting, as well as the enhanced
permeability and retention effect (“EPR” effect).48ꢀ50 Furthermore, no weight loss for
therapeutic group was observed, just as same as the controlled group (Fig. S4),
indicating that all the treatments were not severely toxic. It can be concluded that
DBHAꢀNPs not only can serve as an efficient PS for cancer treatment, but also
provide a significant strategy in clinic to solve the problem of nonꢀtumor site
activation of PSs, further reducing side effect for patients.
4. Conclusions
In summary, DBHAꢀNPs for cancer photodynamic theranostics have been
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successfully synthesized. Originating from CD44 receptor overexpressed towards
tumor cells by HA, as well as EPR effect, DBHAꢀNPs present specific targeting
towards HCTꢀ116 cells. What’s more, onꢀsite activation of DBHAꢀNPs by tumors can
realize the tumor diagnose exactly and effectively overcome the problem of avoiding
lighting to reduce side effects for patients significantly. In particular, both in vitro and
in vivo experiments demonstrate that DBHAꢀNPs can effectively suppress tumor
growth, which exhibits a great potential in photodynamically activatable cancer
theranostics, in clinic.
Acknowledgements
The project was supported by 973 program (2014CB660808), Key University Science
Research Project of Jiangsu Province (15KJA430006), Jiangsu Provincial Founds for
Distinguished Young Scholars (BK20130046), the NNSF of China (61525402,
21275076, 61328401, 51103074), Program for New Century Excellent Talents in
University (NCETꢀ13ꢀ0853), Qing Lan Project, Synergetic Innovation Center for
Organic Electronics and Information Displays, the Priority Academic Program
Development of Jiangsu Higher Education Institutions (PAPD).
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Graphical Abstract
Specific targeting and on-site activated diiodostyryl bodipy conjugated hyaluronic acid
nanoparticles (DBHA-NPs) have been synthesized through self-assembly, which not only can
effectively suppress tumor growth, but also diagnose the tumor exactly and reduce side effect for
patients significantly.