Folate and Heptamethine Cyanine Modified Chitosan-Based Nanotheranostics for Tumor Targeted Near-Infrared Fluorescence Imaging and Photodynamic Therapy

Article abstract of DOI:10.1021/acs.biomac.7b00466

Folate (FA) and heptamethine cyanine (Cy7)-modified chitosan (CF7) was synthesized by click chemistry and its self-assembled nanoparticles (CF7Ns) were developed for tumor-specific imaging and photodynamic therapy. The characterization spectrum confirmed CF7 had a good FA and Cy7 conjugation efficacy. The diameter of CF7Ns measured by DLS was about 291.6 nm, and the morphology observed with AFM showed filamentous clusters of particles. The results of targeting ability of CF7Ns demonstrated enhanced targeting behaviors of CF7Ns compared with non-FA-modified nanoparticles C7Ns in FA receptor-positive HeLa cells. The cytotoxicity and cell apoptosis assay showed that CF7Ns under near-infrared light irradiation led to more apoptotic cell death in HeLa cells to improve the therapeutic efficacy. The mechanisms of the photodynamic effects of CF7Ns were demonstrated through measurement of intracellular reactive oxygen species and the apoptosis-related cytokines. These results suggested that CF7Ns are promising tumor targeting carriers for simultaneous fluorescence imaging and photodynamic therapy.

Full text of DOI:10.1021/acs.biomac.7b00466

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Article
Folate and heptamethine cyanine modified chitosan-
based nanotheranostics for tumor targeted near infrared
fluorescence imaging and photodynamic therapy
Yingying Zhang, Tingting Lv, Huijuan Zhang, Xiaodong Xie, Ziying Li, Haijun Chen, and Yu Gao
Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 19 Jun 2017
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Folate and heptamethine cyanine modified
chitosan-based nanotheranostics for tumor
targeted near infrared fluorescence imaging
and photodynamic therapy
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Yingying Zhang1,2, Tingting Lv1,2, Huijuan Zhang1,2, Xiaodong Xie1,2, Ziying Li1,2,
Haijun Chen2, and Yu Gao1,2,*
1 Cancer Metastasis Alert and Prevention Center, and Biopharmaceutical Photocatalysis,
State Key Laboratory of Photocatalysis on Energy and Environment, Fuzhou University,
Fuzhou 350108, China.
2 Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and
Chemotherapy, Fuzhou University, Fuzhou 350108, China.
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KEYWORDS. Chitosan; Folate receptor; Targeted cancer therapy; NIR fluorescence
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imaging; Photodynamic therapy
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ABSTRACT. Folate (FA) and heptamethine cyanine (Cy7)ꢀmodified chitosan (CF7) was
synthesized by click chemistry and its selfꢀassembled nanoparticles (CF7Ns) were
developed for tumorꢀspecific imaging and photodynamic therapy. The characterization
spectrum confirmed CF7 had a good FA and Cy7 conjugation efficacy. The diameter of
CF7Ns measured by DLS was about 291.6 nm and the morphology observed with AFM
showed filamentous clusters of particles. The results of targeting ability of CF7Ns
demonstrated enhanced targeting behaviors of CF7Ns compared with nonꢀFAꢀmodified
nanoparticles C7Ns in FA receptorꢀpositive HeLa cells. The cytotoxicity and cell
apoptosis assay showed that CF7Ns under nearꢀinfrared light irradiation led more
apoptotic cell death in HeLa cells to improve the therapeutic efficacy. The mechanisms of
the photodynamic effects of CF7Ns were demonstrated through measurement of
intracellular reactive oxygen species and the apoptosisꢀrelated cytokines. These results
suggested that CF7Ns are promising tumor targeting carriers for simultaneous
fluorescence imaging and photodynamic therapy.
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INTRODUCTION
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Cancer is one of the most serious lifeꢀthreatening diseases worldwide. Operation,
radiotherapy, and chemotherapy are the main cancer treatment alternatives at present.
However, the clinical outcomes of radiotherapy and chemotherapy were impeded by the
lack of tumor selectivity and severe toxicity1. Photodynamic therapy (PDT) is a
nonꢀinvasive cancer therapy modality which allows the local creation of singlet oxygen
or other free radicals by the activation of photosensitizers (PSs) via a specific frequency
of light in the visible or nearꢀinfrared (NIR) region, thus selectively destroying tumor
cells in the targeted tumor tissues2. Hence, PDT could be a promising approach for cancer
treatment with comparable effectiveness to that of standard therapies without the
corresponding side effects. Although PSs used in clinic for PDT have been shown to be
both effective and safe in the treatment of a variety of human cancers3, they still suffered
from the drawback of poor selectivity in tumors with low PDT selectivity indexes4. PSs
by systemic administration could distribute all over the body especially the skin. Most
patients received PDT treatment exhibit cutaneous photosensitivity which might last for
up to 6ꢀ8 weeks, such that they have to stay away from the sunlight to avoid getting
sunburned5, 6. Therefore, it is great important to develop a kind of PSs which can target
and precisely accumulate in tumor cells, without affecting other normal cells.
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One of the most efficient ways to achieve precise tumor accumulation is the use of
tumorꢀtargeted drug delivery systems (TDDSs) with the help of nanomaterials7. Due to
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their small physical sizes, nanomaterials could passively accumulate in tumor sites by
enhanced permeability and retention (EPR) effects8. To obtain active targeting
characteristics, nanoparticles could be functionalized with ligands, such as peptides,
antibodies, antibody fragments, and nucleic acids9. Folic acid (FA) is one of the most
used ligands for recognition of folate receptors (FRs)10 which are highly expressed on a
variety of cancers11. FAꢀmodified nanoparticles for FR targeting could sharply enhance
the specificity, efficacy, and bioavailability of the loaded drugs or contrast agents in drug
delivery or tumorꢀimaging applications12 .
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In consideration of the multiꢀlevel complexities and variability of cancers, cancer
treatment is moving towards personalized medicine for individual patients.
Multifunctional nanoparticles which enable a combination of two or more functions, such
as cancer imaging and therapy, could facilitate the development of personalized medicine
for tailored cancer treatment. NIR fluorescence imaging has been used in cancer
diagnostics due to its distinct advantages including the convenient use in cell labeling, no
ionic radiation effect, low cost of the instrumentation, and especially, the deep tissue
penetration and negligible background autofluorescence for in vivo imaging
applications13. Heptamethine cyanines (Cy7s) are fluorescent dyes being used as
biological molecular markers in imaging and other biological analyses14. They have a
strong adsorption peak in the NIR region with minimal scattering and low tissue
absorption11. In addition, Cy7s can not only be selected as a kind of PSs for photothermal
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therapy (PTT), but also can generate cytotoxic reactive oxygen species (ROS) for PDT
under NIR irradiation. Among the Cy7s, indocyanine green (ICG) is the only optical
reagent approved by the Food and Drug Administration for use in human studies.
Because of the lack of group for additional functionalization of ICG, a great deal of study
on functionalization of the cyclohexenyl ring in the Cy7 dyes have been done to develop
probes for NIR imaging applications15. Due to the fragile structure of NIR dyes, only the
physical incorporation not the chemical conjugation of Cy7s into nanoparticles have been
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reported to facilitate tumor targeted NIR fluorescence imaging16, 17
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Among currently available nanoparticle platforms, chitosan (Cs), a natural
polysaccharide, is a very attractive biomaterial utilized widely in biological and
pharmaceutical field such as bioimaging18 and drug delivery19 for its high yield, low cost,
high biocompatibility, biodegradability, and low toxicity20. However, Cs is only soluble
in water containing organic acids. The insolubility in organic solvents extremely limits its
chemical modification21. In order to make regioselective modification of Cs, some
Nꢀsubstituted derivatives were synthesized as precursors for subsequent modification due
to its solubility in common aprotic polar solvents. Nꢀ(2ꢀphthaloylation) chitosan is
regarded as one of the most popular precursors for synthesis of novel Cs derivatives with
excellent solubility and bioactivities, especially for the synthesis of substituents at the
C6ꢀOH position22, 23. As a widely used coupling technology with many advantages of
high stereoꢀspecificity, high yield, easy separation of products, and mild reaction
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conditions with benign solvents, copperꢀcatalyzed azide–alkyne cycloaddition (click
chemistry) had been used to synthesize various Cꢀ6 substituent Cs derivatives based on
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the Nꢀ(2ꢀphthaloylation) Cs precursor24, 25
.
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We have interest in developing a new Csꢀbased nanotheranostics including folate as
a tumor targeting ligand and Cy7 derivative used both as an NIR fluorescent reporter and
a PS for PDT therapy. In this work, Cs was firstly modified with azide groups at Cꢀ6
position. Next the alkylated FA derivative (ALKꢀF) and the alkylated Cy7 derivative
(ALKꢀCy7) were interacted with Cs derivative bearing azide groups by click chemistry to
obtain a new polymer derivative, named as CF7. The CF7 selfꢀassembled nanoparticles
(CF7Ns) were prepared and the physiochemical properties of CF7Ns were investigated.
Then the in vitro drug release behavior of CF7Ns was characterized. Finally, the tumor
targeting efficiency and the PDT therapy efficacy of CF7Ns were studied in HeLa and
HepG2 cancer cells.
EXPERIMENTAL SECTION
Materials. Cs (Mw = 60 K, deacetylation degree ≥ 90.0%) was purchased from
BoAo Biotechnologies Co., Ltd. (Shanghai, China). Propargylamine, phenylhydrazine,
3ꢀmethylꢀ2ꢀbutanone, acetic acid (HAc), methyl iodide (MeI), methylbenzene (PhMe),
acetic anhydride (AA), piperazine, acetonitrile (ACN), propiolic acid,
4ꢀdimethylaminopyridine (DMAP), 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl) carbodiimide
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(EDCI), phthalic anhydride, dicyclohexylcarbodiimide (DCC), dichloromethane (DCM),
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Nꢀhydroxysuccinimide
(NHS),
N,Nꢀdiisopropylethylamine
(DIPEA),
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Nꢀbromosuccinimide (NBS), triphenylphosphine (TPP), dimethylformamide (DMF),
Nꢀmethylꢀ2ꢀpyrrolidone, copper sulfate (CuSO4), sodium azide, and FA were bought from
Sinopharm Chemical Reagent Co., Ltd. (China). Lysozyme was got from Genview Co.,
Ltd. (USA). Cy7 derivative was synthesized according to a previously reported method11.
3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT), propidium iodide
(PI), and 4',6ꢀdiamidinoꢀ2ꢀphenylindole (DAPI) were obtained from SigmaꢀAldrich.
Phosphate buffered saline (PBS), RNase A, and trypsinꢀEDTA were purchased from
GibcoꢀBRL (Burlington, ON, Canada). The RPMI 1640 medium, dulbeccos modified
eagle medium (DMEM), and fetal bovine serum (FBS) were purchased from Life
Technologies GmbH (Darmstadt, Germany). The rabbit antiꢀhuman Survivin, p21, and
GAPDH antibodies were purchased from Wanlei bio Co., Ltd. (Shenyang, China). The
secondary antibody goat antiꢀrabbit IgG horseradish peroxidase conjugate was obtained
from Promega.
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Synthesis of ALK-F and ALK-Cy7. ALKꢀF and ALKꢀCy7 were synthesized as
displayed in the Scheme 1. FA (1 g, 2.27 mmol, 1 eq) was added to DMSO (16.7 mL) and
ultrasound dissolved. Afterwards, DCC (560 mg, 2.27 mmol, 1 eq) and NHS (314 mg,
2.27 mmol, 1eq) were added in. The solution was stirred in the dark at the room
temperature for 18 h. After collecting the filtrate by suction filtration, propargylamine
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(207 mg, 2.84 mmol, 1.25 eq) and DIPEA (367 mg, 3.76 mmol, 1.66 eq) were put in the
filtrate. The solution was stirred in the dark at the room temperature for 48 h. The
reaction solution was poured in ether/acetone (v/v, 7/3) mixed solvent, and the yellow
solid was precipitated. ALKꢀF was collected by centrifugation as yellow solid26 (Scheme
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1A). H NMR (400 MHz, DMSO) : δ ꢀ8.64 (s, 1H), 8.41–8.20 (m, 1H), 8.12–7.89 (m,
1H), 7.77–7.52 (m, 2H), 7.19 (s, 1H), 6.93 (d, J = 5.8 Hz, 1H), 6.61 (dd, J = 38.4, 8.3 Hz,
2H), 4.66–4.38 (m, 2H), 4.36–4.12 (m, 1H), 3.78 (d, J = 40.7 Hz, 2H), 3.47–3.30 (m, 1H),
3.05 (dd, J = 14.6, 11.6 Hz, 1H), 2.94–2.81 (m, 1H), 2.34–2.08 (m, 2H), 2.02 (dd, J =
14.8, 7.1 Hz, 1H), 1.91 (dd, J = 14.5, 7.0 Hz, 1H).
ALKꢀCy7 was obtained as a deepꢀblue solid by the previously reported methods27,
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28(Scheme 1B). H NMR (400 MHz, CDCl3): δ ꢀ7.81–7.69 (m, 2H), 7.39–7.28 (m, 4H),
7.21–7.13 (m, 2H), 7.08–7.01 (m, 2H), 6.03–5.90 (m, 2H), 4.27–4.22 (m, 1H), 4.10–4.06
(m, 1H), 4.05–4.00 (m, 1H), 3.96–3.87 (m, 2H), 3.81 (s, 1H), 3.78–3.72 (m, 1H),
3.69–3.66 (m, 1H), 3.63 (s, 4H), 3.61–3.58 (m, 2H), 3.56–3.53 (m, 1H), 2.06–1.99 (m,
2H), 1.85 (s, 6H), 1.67 (s, 12H).
Synthesis of FA-Modified Cs (CF), Cy7-Modified Cs (C7), and Cs Conjugated
with both FA and Cy7 (CF7). CF7 was synthesized as displayed in the Scheme 2.
Firstly, Cs was in reaction with phthalic anhydride to obtain Nꢀphthaloylꢀchitosan (2).
NBS and TPP were reacted with (2) to introduce the bromide groups at the Cꢀ6 position
to
afford
6ꢀbromideꢀ6ꢀdeoxyꢀNꢀphthaloylꢀchitosan
(3).
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6ꢀAzidoꢀ6ꢀdeoxyꢀNꢀphthaloyl–chitosan (CsꢀN3, 4) was prepared by replacing the
bromide groups with azido groups in accordance with the previously reported methods by
Satoh et al21. Finally, CF7 (5) was synthesized as follows: CsꢀN3 (20 mg), ALKꢀF (10
mg), ALKꢀCy7 (10 mg), CuSO4 (2.5 mg predissolved in 100 µL ddH2O), and sodium
ascorbate (2 mg predissolved in 100 µL ddH2O) were added to DMSO (5mL). The
solution was stirred in the dark at 50 oC for 3 days. After the reaction was completed, the
solution was dialyzed in the ddH2O with dialysis bag (MWCO:14000) for 3 days. The
solution was lyophilized to get CF7 as the brown powder. CF and C7 were obtained by
reaction of CsꢀN3 with ALKꢀF and ALKꢀCy7, respectively, along with the method of
CF7.
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Characterization of ALK-F, ALK-Cy7 and Cs Derivatives. H NMR spectra
were obtained from a Bruker AVANCE 400 spectrometer (400MHz). Infrared spectra
were analyzed using a FTIR spectrometer (Intelligent, Nicolet 360, USA) with KBr pellet.
The samples were scanned from 500ꢀ4000 cmꢀ1. UVꢀVis spectra were acquired on a
Quawell Q5000 microvolume UVꢀVis Spectrophotometer. Fluorescence spectra were
achieved by a FS5 spectrofluorometer (Edinburgh Instruments, UK). The amount of FA
in CF7 was quantified spectrophotometrically by absorbance measurement at 280 nm by
using the calibration curve of standard solutions of FA in DMSO. The Cy7 conjugation
efficiencies were determined by using the calibration curve of standard solutions of
ALKꢀCy7 in DMSO through fluorescent measurements of Cy7 (λex = 633 nm).
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Preparation and Characterization of CF7Ns. CF7Ns was prepared through
selfꢀassembly as follows. CF7 (1 mg) was dissolved in DMSO (1mL), and the solution
was added into ddH2O drop by drop and stirred. Then the DMSO was removed by
dialysis. The excess solvent could be evaporated to obtain the appropriate concentration
of CF7Ns. C7 selfꢀassembled nanoparticles (C7Ns) and CF selfꢀassembled nanoparticles
(CFNs) were obtained according to the same method. Fluorescence measurements were
conducted with an excitation wavelength of 633 nm to scan the respective emission
profiles. The mean particle size, zeta potential, and polydispersity index (PDI) was
measured by the dynamic light scanning (DLS) using a Zetasizer Nano ZS Particle Size
and Zeta Potential Analyzer (Malvern Instruments Ltd., UK). The morphology of CF7Ns
was observed using atomic force microscopy (AFM) on a Multimode 8 AFM series
(Bruker, USA) in tapping/AC mode. The scanning electron microscopy (SEM)
micrographs were obtained by NOVA NANOSEM 230 (FEI, USA). The critical micelle
concentration (CMC) of CF7 is detected using FITC as a fluorescence probe. CF7
dissolved in DMSO were diluted in ddH2O to various concentrations, and then 1 ꢁg/mL
FITC in methanol was added. The fluorescence intensity at λem = 519 nm was monitored
using a Hitachi Fꢀ7000 fluorescence spectrometer with a quartz fluorescence cell at λex =
490 nm.
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Stability Study. The stability of CF7Ns was studied in DMEM or DMEM
o
containing 10% FBS for one week at 25 C. At certain time points (1, 3, 5, and 7 days),
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samples were collected to monitor the changes in particle size.
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In Vitro Drug Release of CF7Ns. In vitro release of Cy7 dye from CF7Ns was
performed using the dialysis bag diffusion technique with lysozyme (1.25
mg/mL)ꢀcontained 40% ethanolꢀPBS as dissolution medium. 1 mL of CF7Ns prepared
from 5 mg of CF7 was diluted with 1 mL dissolution medium. The suspension was then
put into a pretreated dialysis bag. The samples were incubated with 20 mL 40%
ethanolꢀlysozymeꢀPBS solution (pH = 5.5 or 7.4) at 37 oC in a lightꢀsealed condition. At
predetermined time intervals, samples were taken out and the concentration of the
released Cy7 dye was determined based on a calibration curve of standard solutions of
free ALKꢀCy7 in dissolution medium using the fluorescence spectra method as described
above. The release experiments were carried out in triplicates and the results were
expressed as mean ± standard deviation (SD).
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Cell Culture. Human cervical cancer HeLa cells and human liver cancer HepG2
cells were purchased from Cell Resource Center of Shanghai Institute for Biological
Sciences (Chinese Academy of Sciences, Shanghai, China). HeLa cells were cultured in
DMEM culture medium mixed with 10% fetal bovine serum (FBS) and 100 U/mL
penicillin and 100 µg mLꢀ1 streptomycin. HepG2 cells were grown in RPMI 1640
medium containing 10% FBS, 0.1mg/mL streptomycin and 100 U/mL penicillin. Cells
were cultured at 37 oC in a humidified incubator of 5% CO2.
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Cellular Uptake of CF7Ns. HeLa and HepG2 cells were seeded in a 6ꢀwell plate
with 1.5 mL medium and cultured for 24 h. Afterwards cells were incubated with C7Ns
and CF7Ns with or without free FA (4 mg/mL) for 2 h at 37 oC. Then cells were collected
and subjected to flow cytometry (Becton Dickinson FACSAriaIII cell sorter). 10,000
events were recorded for each sample. The data was processed by FlowJo software.
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Confocal Fluorescence Microscopy Studies. HeLa and HepG2 cells were seeded
in a 24ꢀwell plate and cultured for 24 h, followed by incubation with Cy7, C7Ns, and
CF7Ns at concentration equivalent to 1.4 ꢁg/mL of Cy7. After incubation for 2 h, the
cells were washed three times with PBS (pH = 7.4). Then DAPI (10 µg/mL) was added to
stain cell nucleus for 15 min. In the end, the cells were fixed with 4.0% (w/v)
paraformaldehyde for 15 min at room temperature in the dark. The confocal images were
taken by Leica confocal microscope (Leica TCS SP8, Germany). Negative controls were
used for each experiment to minimize the background color.
In Vitro Cytotoxicity Studies. The cytotoxicity of CF7Ns was determined by the
MTT assay. To evaluate the efficacy of PDT therapy with different power densities, cells
were exposed to a NIR irradiation with power densities of 1.0, 2.0, and 2.5 W/cm2 for 1,
5, and 10 min, respectively.
HeLa and HepG2 cells were separately seeded in a 96ꢀwell plate at a density of 8000
cells per well and cultured for 24 h. Then cells were treated with Cy7, C7Ns, or CF7Ns
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with different concentrations (0.07, 0.35, 0.7, 1.4, 2.8 ꢁg/mL of Cy7) with or without
exposition to a NIR irradiation by an infrared lamp with a power density of 2.0 W/cm2
for 5 min. Control cells were exposed to 0.1% DMSO. After incubation for 24 and 48 h,
cells were washed three times with PBS and then 150 µL of MTT solution was added.
The cells were incubated for 4 h at 37 oC. After the medium was removed, 150 µL of
DMSO was added to the well and incubated for 10 min. The absorbance of the solution
was measured at 570 nm by TECAN Infinite F200 Microplate Reader with six replicates
analyzed at each dose.
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Cell Cycle Analysis. HeLa cells were seeded on the 6ꢀwell plates and incubated
with Cy7, C7Ns, and CF7Ns at concentration equivalent to 1.4 ꢁg/mL of Cy7 with
exposition to a NIR irradiation (2.0 W/cm2, 5 min) at 37 oC for 48 h. Cells without
treatment were used as control. After incubation, cells were collected, washed twice with
PBS, fixed with 70% cold ethanol, and stored at 4 oC overnight. Cells were centrifuged
again and washed with cold PBS. The cellular DNA was stained with fluorescent solution
(1% Triton Xꢀ100, 0.01% RNase A, and 0.05% PI) for 30 min at 37 oC in darkness. The
DNA content was measured by flow cytometry, and the percentage of cells in each phase
of the cell cycle was evaluated using the ModFit software. 10,000 events were recorded
for each sample. All experiments were performed three times.
Cells Apoptosis Assay. Cells Apoptosis was detected by using an Annexin
VꢀFITC/PI Staining Kit. HeLa cells were placed into 6ꢀwell plates at a density of 5×105
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cells per well. Cells were incubated with Cy7, C7Ns, and CF7Ns at concentration
equivalent to 1.4 ꢁg/mL of Cy7 with or without exposition to a NIR irradiation
(2.0W/cm2, 5min) for 48 h. The cells were harvested, suspended with 500 ꢁL of binding
buffer, and stained with 5 µL of Annexin VꢀFITC and PI following the manufacturer’s
instructions. After reaction at room temperature in the dark for 15 min, all the samples
were immediately detected by using flow cytometry. 10,000 events were recorded for
each sample. All experiments were performed three times.
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Measurement of intracellular ROS. Intracellular ROS generation was detected by
reactive oxygen species assay kit (Beyotime Biotechnology, Haimen, China). HeLa cells
were seeded in 6ꢀwell plate and cultured for 24 h, followed by treatment with Cy7, C7Ns,
and CF7Ns at concentration equivalent to 1.4 ꢁg/mL of Cy7 for 24 h. Then cells were
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loaded with DCFHꢀDA dye (10 ꢁM) in serumꢀfree DMEM medium for 20 min at 37 C.
After washing three times with PBS, the cells were irradiated with NIR lamp for 5 min.
DCF fluorescence intensity was detected using the flow cytometry to quantify the levels
of ROS.
Western Blot Analysis. HeLa cells (pretreated with 20 ng/mL ILꢀ1β for 4 h) were
seeded on the 6ꢀwell plates and incubated with Cy7, C7Ns, and CF7Ns at concentration
equivalent to 1.4 ꢁg/mL of Cy7 with an exposure to NIR irradiation at 37 oC for 24 h.
Cells were collected and the cell lysates were prepared by treating RIPA lysis buffer.
Then equal amounts of protein were separated by SDSꢀPAGE and transferred to
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polyvinylidene difluoride (PVDF) membranes. The membranes were blocked by
blocking solution and incubated with antibodies against Survivin, p21 and GAPDH
overnight at 4 oC, followed by incubation with secondary antibodies for 2 h at room
temperature. The target proteins were detected by Super Signal West Pico
chemiluminescent substrate kit and ChemiDoc XRS system. The expression of proteins
of interest was analyzed with Image Lab analysis software.
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Statistical Analysis. All data were expressed as the mean ± SD for at least three
separate experiments. Statistical analysis was performed using the Student's tꢀtest. The
differences were considered significant for p value < 0.05 and p < 0.01was indicative of a
very significant difference.
RESULTS AND DISCUSSION
Synthesis and Characterization of ALK-F and ALK-Cy7. ALKꢀF and ALKꢀCy7
were synthesized using previously reported methods (Scheme 1)11, 26. The UVꢀvis spectra
of ALKꢀF and ALKꢀCy7 were recorded in DMSO (Figure 1A). ALKꢀF showed an
adsorption peak at 280 nm, whereas ALKꢀCy7 demonstrated no absorption signal
between 250 nm and 350 nm. The fluorescence emission spectrum showed that
ALKꢀCy7 had a characteristic emission at about 801 nm when excited at 633 nm (Figure
1C), but no fluorescent signal could be found in ALKꢀF. The UVꢀvis spectra of ALKꢀF
and fluorescence spectra of ALKꢀCy7 with different concentrations were shown with
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good linearity (Figure 1B, D). The calibration curve of ALKꢀF concentration against
absorption values at length of 280 nm was y = 36.889x+0.0209 (R2 = 0.992) and the
calibration curve of ALKꢀCy7 concentration against fluorescent intensity (λex/λem =
633/801 nm) was y = 1627.4x+184.16 (R2 = 0.991). The good linear relationship can be
used for subsequent quantitative determination of the grafting efficiencies of FA and Cy7
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onto the Cs backbone. The H NMR spectra of ALKꢀF and ALKꢀCy7 were shown in
Figure 2. They were consistent with the previously reported results29, 30
.
Synthesis and Characterization of CF7. In this study, FA and Cy7 were
conjugated to Cs by click reaction to afford CF7. Due to the insolubility of Cs in organic
solvents31, the substitution of Cs at a specific position was difficult, which greatly limited
the generation of structurally diverse Cs derivatives with different properties for various
applications. Several approaches have been reported using Nꢀsubstituted derivatives,
including imido32, acyl33, imino34 and phthaloyl derivatives as precursors to make
regioselective modification of Cs. Among them, Nꢀphthaloylꢀchitosan could be the best
choice because of its highly solubility in polar aprotic solvents which offering the
opportunity for the regioselective substitution at Cꢀ6 position. Apart from facilitating
regioselective modification of Cs, phthaloylꢀmodification might give some bioactivities
to the modified materials. Some reports demonstrated that anticancer drugs coordinated
with phthaloylꢀmodified amino acids could obtain promoted production of intracellular
ROS and enhanced anticancer activity35, 36. So we chose Nꢀphthaloylꢀchitosan as
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precursors for subsequent modification. The amine groups of Cs were firstly reacted with
phthalic anhydride to get Nꢀphthaloylꢀchitosan (2; Scheme 2a). Then bromide group was
introduce to the Cꢀ6 position of Cs with NBS and TPP to afford
6ꢀbromideꢀ6ꢀdeoxyꢀNꢀphthaloylꢀchitosan (3; Scheme 2b). Finally sodium azide was used
to react with the bromide derivatives to obtain 6ꢀazidoꢀ6ꢀdeoxyꢀNꢀphthaloylꢀchitosan
(CsꢀN3; 4; Scheme 2c). The click coupling of CsꢀN3, ALKꢀF and ALKꢀCy7 was carried
out in DMSO and water using in situ generated Cu (I) catalyst by CuSO4 and sodium
ascorbate (Scheme 2d) 30. The coupling reaction was carried out in mixed cosolvent
system of DMSO and water because CsꢀN3 was only soluble in organic solvents such as
DMF, DMSO, NMP, but CuSO4 and sodium ascorbate were soluble in water. Click
chemistry has been used to prepare various highly regioꢀselective Cs derivatives based on
Nꢀphthaloylꢀchitosan precursors37. Generally, the phthaloyl protecting group was
removed in the final step by aqueous hydrazine monohydrate under the temperature of
100 °C. Considering of the fragile structure of Cy7 and the bioactivity of the phthaloyl
group, the deprotection reaction was not carried out and the obtained CF7 was directly
used for subsequent studies.
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The successful conjugation of FA and Cy7 onto Cs was confirmed by the H NMR
(Figure 2 and Figure S1), FTIR (Figure 3A), UVꢀVis (Figure 3B), and fluorescence
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spectra (Figure 3C). Compared with the H NMR spectrum of CsꢀN3, the spectrum of
1
CF7 showed additional peaks at 1.0~2.0 ppm and 6.5~8.5 ppm. According to the H
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NMR spectra of ALKꢀF and ALKꢀCy7, the peaks at 1.0~2.0 ppm were the characteristic
peaks of ALKꢀCy7, and the peaks at 6.5~8.5 ppm were the characteristic peaks of both
ALKꢀF and ALKꢀCy7. The signal of formed triazole groups might appear at δ = 7.5~8.5
ppm. Because of the multiple peaks at 6.5~8.5 ppm, it is difficult to distinguish it from
them. FTIR spectra were carried out to detect the typical azido groups of CsꢀN3. The
FTIR spectra of CsꢀN3 showed the azido group absorption band at 2100 cmꢀ1, a band at
1715 cmꢀ1 due to –C=O stretching vibrations of the phthalic anhydride, and three
adsorption bands between 630 and 800 cmꢀ1 corresponding to orthoꢀsubstituted of
benzene. Compared with the FTIR spectrum of CsꢀN3, the disappearance of the peak at
2100 cmꢀ1 in FTIR spectrum of CF7 indicated the consumption of the azide groups,
suggesting the successful click cyclization (Figure 3A). The FTIR spectra of ALKꢀF and
ALKꢀCy7 were also determined and the ALKꢀF had characteristic peaks between 1500
and 1650 cmꢀ1. The appearance of the characteristic peak at about 1600 cmꢀ1 in FTIR
spectrum of CF7 indicated the successful conjugation of FA onto Cs. CF and C7 were
synthesized as well to be used as control in later experiments. The characteristic peaks
between 1500 and 1650 cmꢀ1 appeared in FTIR spectrum of CF, demonstrating the
successful conjugation of FA onto Cs.
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UVꢀVis and fluorescence spectroscopic characterization of CF7 were performed to
further confirm the conjugation of FA and Cy7 to Cs. CF7 and CF both showed an
adsorption peak at 280 nm (Figure 3B), whereas CsꢀN3 and ALKꢀCy7 showed no
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adsorption at 280 nm, indicating the introduction of FA groups in CF7 and CF.
Fluorescence spectra demonstrated that CF7 and C7 had characteristic emission peaks at
about 801 nm when excited at 633 nm, which is the fluorescence characteristics of
ALKꢀCy7, indicting the successfully grafting of ALKꢀCy7 to Cs (Figure 3C).
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In order to optimize the grafting efficiencies of FA and Cy7 linked onto the Cs,
several batches of parallel synthesis of CF7 using different reaction feed mass ratios of
ALKꢀF, ALKꢀCy7 and CsꢀN3 were performed under the same condition. In the parallel
synthesis, we found that the water solubility of CF7 gradually decreased with the
increasing feed mass ratios of ALKꢀF, which makes the Cs derivatives difficult to be
used further. When increasing the feed mass ratios of ALKꢀCy7, the fluorescene
intensities of C7 and CF7 increased to some extent followed by a sharp decline.
Fluorescence selfꢀquenching might happen when too many fluorescence functional
groups were assembled in one Cs backbone38. The optimized reaction feed mass ratio of
CsꢀN3∶ALKꢀF∶ALKꢀCy7 was 2∶1∶1. The solubility of CF7 obtained under this
condition was excellent. According to the calibration curve of ALKꢀF (Figure 1B), the
content of FA in CF7 and CF could be quantitatively detected. The FA conjugation
efficacies in CF and CF7 were 0.25 ± 0.02 mg/mg and 0.18 ± 0.01 mg/mg, respectively
(Table 1). According to the calibration curve of ALKꢀCy7 (Figure 1D), the content of
Cy7 in CF7 and C7 could be quantitatively calculated. The Cy7 grafting capacities in C7
and CF7 were 0.08 ± 0.01 mg/mg and 0.07 ± 0.02 mg/mg, respectively (Table 1).
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Preparation and Characterization of CF7Ns. A large variety of selfꢀassembled
nanoparticles based on amphiphilic chitosan derivatives have been reported as drug
delivery vehicles39. Cs modified with the hydrophobic moiety could result in amphiphile
to form selfꢀassembled nanoparticles which could encapsulate anticancer drugs and
deliver them to targeted tumor tissues via EPR effects. Similarly, CF7 in which Cs was
modified with hydrophobic FA and Cy7 moieties could selfꢀassembled into CF7Ns
(Figure 4A) and the selfꢀassembly could be driven by the hydrophobic interactions
between phthalic anhydride groups, FA, Cy7 groups, and the hydrogen bonding between
chitosan chains40. The CMC of CF7 is detected using FITC as a fluorescence probe. An
obvious turning point was observed in the curve of the fluorescence intensity of
CF7/FITC solution at λex/λem = 490 nm /519 nm versus CF7 concentration, which
corresponded to the CMC of CF7, indicating that CF7 could selfꢀassemble into
thermodynamically stable micelles at concentrations higher than 4 ꢁg/mL (Figure S2).
The formation of selfꢀassembled nanoparticles was first verified using DLS. The CF7Ns
have a narrow particle size distribution (Figure 4B) and an average diameter of about
296.1 nm (Table 1). The particle size of CF7Ns was larger than that of C7Ns and CFNs,
which could be due to the stronger intermolecular repulsion and the steric effects of FA
and Cy7 moieties inside the CF7Ns. All C7Ns, CFNs, and CF7Ns had a negative zeta
potential because the amino groups in Cs backbone were substituted by phthalic
anhydride. The change of particle sizes of CF7Ns was studied in two different media,
DMEM or DMEM + 10% FBS, for one week. The results showed that CF7Ns remained
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stable with no significant changes in size after 7 days (Figure 4C), indicating the potential
of CF7Ns for systemic application. The morphology of CF7Ns observed by a AFM
shows filamentous clusters (Figure 4D), indicating that the long chain polymer CF7 could
twist together to form nanoclusters. The size of CF7Ns measured by AFM was about 240
nm, which was slightly smaller than that measured by DLS. One possible reason for this
phenomenon is that the samples for obtaining AFM image was in airꢀdry condition, in
which the particles shrank and the size get smaller. As the optical properties of the
fluorescent materials could be affected by the solvents, the fluorescence spectra of CFNs,
C7Ns, and CF7Ns were measured in water as well. The SEM micrograph of CF7Ns was
shown in Figure 4E, which further proves that the long chain polymer CF7 could twist
together to form nanoparticles. C7Ns and CF7Ns had a characteristic emission at about
801 nm when excited at 633 nm (Figure 4F). The emission spectra of C7Ns and CF7Ns
were agreed with ALKꢀCy7, and no obvious spectral shifts were observed.
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For effective PDT, PSs should be delivered to the target site without aggregation
and degradation. Some available PSs such as porphyrins aggregate seriously in the
biological fluid environment during the delivery process, which causes fluorescence
quenching and consequently reduces the PDT effects6, 41, 42. Additionally, some PSs are
rapidly degraded due to the heat produced by NIR light irradiation which also lessens
the therapeutic effects of PDT43. TDDS possessed sustained drug release
characteristics could offer one effective solution to these problems. The PSs released
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from TDDS at the target site slowly could effectively avoid the occurrence of
aggregation during the delivery process and the degradation at the action site. To study
the release pattern of Cy7 dyes from CF7Ns, the in vitro release of Cy7 dye from CF7Ns
was performed in PBS at pH 7.4 to simulate normal physiological condition and at pH
5.5 to simulate the tumor microenvironment. Because the hydrolysis of Cs in PBS is very
slow, lysozyme, which is an enzyme abundant in biological fluids and human lymphatic
and kidney tissues44 was added to the dissolution medium to accelerate the hydrolysis of
Cs45. To enhance the solubility of the released Cy7 dye, 40% of ethanol was added to the
dissolution medium. The samples were taken out at certain time points and the released
Cy7 dye was quantified by fluorescence spectra. The release rate of Cy7 from CF7Ns
showed a sustained release profile in both buffers (Figure 4G). The sustainedꢀrelease
manner of CF7Ns could be beneficial to enhance the bioavailability and the PDT effects
of Cy7. From Figure 4G, it was also found that the release rate of Cy7 at pH 5.5 was
faster than that at pH 7.4. The cumulative release amount of Cy7 at pH 5.5 was almost
65% at 48 h, whereas the cumulative release amount of Cy7 was only 30% at pH 7.4. The
pHꢀdependant drug release characteristics of CF7Ns could be attributed to the
protonation of –OH groups at the Cꢀ4 positions of Cs, which led to the partial
dissociation of hydrogen bonding between Cs chains. It was reasonable to expect that the
sustained and pHꢀdependant drug release in tumor site could bring more opportunities for
onꢀdemand drug delivery and improved antiꢀtumor efficacy.
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Cellular Uptake and Intracellular Distribution. FRs were highly expressed in
several human tumor cells including cancers of the ovary, breast, liver, kidney, and lung46.
We chose FRꢀpositive tumor cells (HeLa cells)47 and FRꢀnegative tumor cells (HepG2
cells )48 to investigate the cellular uptake efficiencies and the intracellular distribution
behaviors of the CF7Ns. As shown in Figure 5A, Cy7, C7Ns and CF7Ns could enter the
cells. Compared with free Cy7 which is distributed throughout the cytoplasm and nuclei
of the entire cell, C7Ns and CF7Ns is mainly accumulated in the cytoplasm. The
fluorescence intensity of HeLa cells incubated with CF7Ns was stronger than that
incubated with C7Ns, suggesting that the internalization of CF7Ns is easier than that of
C7Ns. However, the fluorescence intensities did not show obvious difference in HepG2
cells incubated with CF7Ns and C7Ns. These results indicated that the internalization of
CF7Ns into HeLa cells could be FRꢀmediated. The FRꢀselective internalization could
insure the tumorꢀtargeted NIR imaging by CF7Ns49.
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To further quantify the results of FR mediated endocytosis, flow cytometry was
used to measure the cellular uptake efficiencies of C7Ns and CF7Ns in HeLa and HepG2
cells (Figure 5B, C). The results from flow cytometry were in accordance with that
acquired by confocal microscopy. There was no significant difference between the uptake
of C7Ns and CF7Ns by HepG2 cells. With the help of FR, HeLa cells could grab much
more CF7Ns than C7Ns. A free FA competition study was also carried out in HeLa and
HepG2 cells to verify the FRꢀmediated endocytosis of CF7Ns. Because of the
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competitive binding of FA to FR, the endocytosis of CF7Ns was distinctly blocked. The
results made clear that the uptake and internalization of CF7Ns into FRꢀpositive tumor
cells (HeLa cells) is FRꢀmediated endocytosis, indicating that CF7Ns could be a
promising carrier for NIR fluorescence probes for the FRꢀpositive tumorꢀtargeted NIR
imaging.
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Cytotoxicity Studies. MTT assay was used to test the cytotoxicity of the carrier
materials and the nanotheranostics. Low or neglected cytotoxicity is very important for a
biomaterial to be used for TDDS. To evaluate the safety of the carrier materials and
eliminate the effects of nonꢀactive substances on the cell viability, the viability of HeLa
and HepG2 cells treated with Cs, CsꢀN3 and CFNs were first evaluated. As shown in
Figure 6, no doseꢀdependent toxicity was found in Cs, CsꢀN3, and CFNs treated HeLa
and HepG2 cells. Nearly 90% of cells could survive after incubation with Cs in a wide
range of concentrations (from 20 µg/mL to 150 µg/mL) for 24 h and 48 h. CsꢀN3
displayed certain inhibitory effects on both cells, but the cell viabilities could increase to
80% when incubated with CFNs in HepG2 cells, suggesting that the inhibition effects of
CsꢀN3 were mainly from the azide groups50 in Cs and the inhibition could be reversed by
FA modification. In HeLa cells, CFNs demonstrated a bit stronger inhibition effects than
Cs and CsꢀN3 after 48 h incubation time, which could attribute to the interaction of the
FA moieties with FRs in HeLa cells, thus affecting the normal proliferation of cells51.
Nevertheless, the viability of HeLa cells was still about 75% after 48 h incubation with
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CFNs at 150 µg/mL. The above results indicated that Cs, CsꢀN3, and CFNs had no
obvious effects on cell viability and these nanomaterials could have good
biocompatibility for in vivo applications.
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Cy7 was reported to generate singlet oxygen upon excitation with NIR light, which
led to cell death. Therefore, PDT effects of CF7Ns were studied in both HeLa and HepG2
cells. In order to determine the optimal irradiation condition for PDT, the viability of
HeLa cells incubated with different concentrations of CF7Ns were exposed to different
power densities of NIR light for different irradiation times followed by incubation for 24
h. As shown in Figure 7AꢀC, the cell viability of HeLa cells was closely related with the
irradiation time and the intensity of NIR light. Cells exposed to NIR light with a power
density of 1.0 W/cm2 did not show any difference in cell viability with respect to those
cells without NIR irradiation for 1, 5, or 10 min. When cells were irradiated with NIR
light at 2.5 W/cm2, almost 80% growth inhibition was observed even in the control cells
without CF7Ns treatment, indicating the severe hurt to cells. When cells were exposed to
NIR light with a power density of 2.0 W/cm2, satisfactory PDT effects without obvious
sideꢀeffects could be obtained especially with the irradiation time of 5 min. Therefore,
NIR light with a power density of 2.0 W/cm2 and the irradiation time of 5 min was
selected as the irradiation condition for PDT.
To study the FRꢀtargeted PDT effects of CF7Ns, cell viabilities of HeLa and HepG2
cells exposed to different concentrations of CF7Ns for 24 and 48 h with and without NIR
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light exposure were compared with that of Cy7 and C7Ns (Figure 7DꢀG). Cy7, C7Ns and
CF7Ns all showed doseꢀdependent toxicity to two cells without NIR irradiation,
suggesting that Cy7 moieties have inhibitory effects on cell proliferation. As shown in
Figure 6, CFNs had little effect on cell proliferation at a concentration of 60 µg/mL,
indicating that the toxicity of C7Ns and CF7Ns is mainly attributed to the Cy7 moieties.
In HepG2 cells, CF7Ns showed comparable inhibition effects with C7Ns for 24 and 48 h
treatment (Figure 7F,G). In contrast, CF7Ns showed significant enhanced inhibition than
C7Ns in HeLa cells, indicating that CF7Ns could be taken up more easily by FRꢀpositive
cancer cells to exert antiꢀcancer effects (Figure 7D,E). The more cellular uptake of
CF7Ns in HeLa cells made CF7Ns have stronger PDT effects than that of C7Ns. The cell
viability of HeLa cells treated with 40 µg/mL of CF7Ns (equivalent to 2.8 ꢁg/mL of Cy7)
for 48 h with NIR irradiation could be as low as 14%. The more effective protection and
utilization of PSs by sustained and pHꢀdependent release of Cy7 from CF7Ns could be
another reason for the improved PDT efficacy.
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Cell Cycle Analysis and Cells Apoptosis Assay. To further determine how CF7Ns
affected the viability of HeLa cells under NIR irradiation, cell cycle distribution was
analyzed after 48 h treatment. The representative flow cytometric DNA contents of HeLa
cells after incubation with Cy7, C7Ns and CF7Ns with or without NIR irradiation were
shown in Figure 8A. C7Ns and CF7Ns could arrest the cell cycle in G0/G1 phase. The
NIR irradiation further deactivated the recognized cancer cells by arresting the cells at the
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G0/G1 phase and preventing them from going into the S phase and G2/M phase. More
induction of G0/G1 phase arrest was found in cells incubated with CF7Ns than in cells
treated C7Ns, which further indicated that FAꢀmodified nanoparticles have ability to
target FR positive cancer cells to exert inhibitory effects.
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The induction of apoptosis is one of the important mechanisms for antitumor activity.
Flow cytometric analysis of cell apoptosis was shown in Figure 8B. In correspondence
with the cytotoxicity results, the amount of apoptotic cells in Cy7 and C7Nsꢀtreating
groups increased from 8.08% to 36.8% and from 6.26% to 27.8% after NIR irradiation
(Figure 8B) respectively, indicating that Cy7 photosensitizer could induce cell apoptosis
under NIR irradiation. The results also showed that around 53.4% and 5.57% of cells
were in apoptotic stage after incubated with CF7Ns and C7Ns, suggesting that the CF7Ns
with FAꢀmodification could facilitate FRꢀmediated internalization and promote apoptosis
induction. As high as 70% of early and late stage apoptotic cells were detected in CF7Ns
group under NIR irradiation, suggesting that Cy7 under NIR irradiation could cause more
apoptosis to improve therapeutic efficacy. The antitumor effects of PDT could result
from the oxidative stressꢀinduced cellular response such as disruption of homeostasis,
changes in lipid metabolism, and ion transportation52, which lead to activation of the
protein kinase pathway, the expression of transcription factors and cytokines, and the
release of a number of mediators responsible for the process of cell death, thus resulting
in apoptosis and/or necrosis53.
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Measurement of intracellular ROS. Light, PS and oxygen are three key
components involved in a typical PDT process. Under light irradiation, a PS in ground
state is jumped into a highꢀenergy state, leading to energy transfer to neighboring oxygen
to generate of ROS54. The ROS was reported to be able to destroy tumors by directly
inducing cancer cell apoptosis55 and disrupt tumor vasculatures by exerting
antiꢀangiogenesis effect56. To investigate whether NIR irradiation of Cy7 could cause the
ROS production, HeLa cells after treatment were incubated with DCFHꢀDA. As shown in
Figure 9A, the quantity of ROS increased significantly after treated with Cy7, C7Ns, and
CF7Ns with NIR irradiation, suggesting that Cy7 with NIR irradiation could cause the
generation of ROS in HeLa cells. CF7Ns showed the strongest generation of ROS, which
is highly agreed with the results of cytotoxicity. The results forcefully proved that CF7Ns
with NIR irradiation induced HeLa cells apoptosis in relation with the generation of ROS.
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Effects of CF7Ns on the expression of apoptosis-related proteins. Previous
studies have demonstrated that Survivin is one of the strongest apoptosis inhibitory
factors and its expression is related to cell apoptosis and cell cycle regulation57. The
tumor suppressor protein p21 could act an inhibitor of cell cycle progression that arrests
cells in the G1 and G2 phases58,59. Therefore, two proteins Surviving and p21 were
chosen to study the effects of CF7Ns on the expression of apoptosisꢀrelated cytokines.
The western blotting analysis for Surviving and p21 in HeLa cells was shown in Figure
9B. Compared with control group, the expression of p21 significantly increased and the
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expression of Survivin decreased in C7Ns and CF7Ns with NIR irradiation. The results
further proved that CF7Ns under NIR irradiation could cause cells to undergo apoptosis
by affecting the expression of apoptosisꢀrelated proteins.
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CONCLUSIONS
FA and Cy7 modified Csꢀbased nanotheranostics were synthesized in this study. The
diameter of CF7Ns measured by DLS was about 291.6 nm and the morphology of CF7Ns
showed filamentous clusters of particles. The release of Cy7 from CF7Ns demonstrated a
sustained and pHꢀdependant drug release manner, which was important for improved
PDT efficacy. The tumor targeted imaging effect of CF7Ns was clearly demonstrated by
flow cytometry and confocal fluorescence microscopy in FRꢀpositive HeLa cells. The
cytotoxicity studies and cells apoptosis assay showed that CF7Ns under NIR irradiation
led more apoptotic in HeLa cells compared with C7Ns to improve the therapy efficacy.
These results indicated that CF7Ns could be a valuable nanotheranostics for targeted NIR
fluorescence imaging and PDT in different kinds of FRꢀpositive tumors.
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