Reduction-sensitive amphiphilic dextran derivatives as theranostic nanocarriers for chemotherapy and MR imaging

Article abstract of DOI:10.1039/C6RA22373G

Reduction-sensitive, amphiphilic dextran derivatives were developed from disulfide-linked dextran-g-poly-(N-ε-carbobenzyloxy-l-lysine) graft polymer (Dex-g-SS-PZLL), and used as theranostic nanocarriers for chemotherapy and MR imaging. Dex-g-SS-PZLLs were

Full text of DOI:10.1039/C6RA22373G

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Reduction-sensitive amphiphilic dextran
derivatives as theranostic nanocarriers for
chemotherapy and MR imaging
Cite this: RSC Adv., 2016, 6, 114519
Hui-Kang Yang,†a Meng Qi,†a Lei Mo,a Rui-Meng Yang,a Xiang-Dong Xu,a
a
a
b
c
a
*
*
Jun-Fang Bao, Wen-Jie Tang, Jian-Tao Lin, Li-Ming Zhang and Xin-Qing Jiang
Reduction-sensitive, amphiphilic dextran derivatives were developed from disulfide-linked dextran-g-
poly-(N-3-carbobenzyloxy-L-lysine) graft polymer (Dex-g-SS-PZLL), and used as theranostic
nanocarriers for chemotherapy and MR imaging. Dex-g-SS-PZLLs were synthesized by click conjugation
between azidized dextran (Dex-N3, 40 kD) and a-alkyne-SS-PZLL (degree of polymerization ¼ 10, 15
and 25). The chemical structures of dextran derivatives were characterized by Fourier transform infrared
spectroscopy and nuclear magnetic resonance analyses. Owing to their amphiphilic nature, these
copolymers can self-assemble into spherical nanosized micelles in an aqueous medium, as confirmed
by fluorometry, transmission electron microscopy and dynamic light scattering. Interestingly, the
hydrodynamic radii of the micelles (65–100 nm in diameter) were dependent on the block length
of PZLL, and their critical micelle concentrations were in the range of 0.020–0.007 mg mLꢀ1, which
decreased as the length of PZLL increased. The anticancer drug doxorubicin (DOX) and
superparamagnetic iron oxide (SPIO) nanoparticles (NPs), as the magnetic resonance imaging (MRI)
contrast agent, were simultaneously encapsulated in the hydrophobic core of the micelles by the
dialysis method. The release profiles of encapsulated DOX from SPIO/DOX-loaded micelles were shown
to be rapid in the presence of 10 mM glutathione (GSH) within 24 h, whereas less than 30% DOX was
released from reduction insensitive Dex-g-PZLL micelles in 48 h. Only about 35% DOX was released
from Dex-g-SS-PZLL micelles in the same timeframe. According to the in vitro cytotoxicity test, it was
found that reduction-sensitive micelles showed higher toxicity to HepG2 cancer cells than the
reduction-insensitive micelles incubated with equivalent DOX concentration. Flow cytometry and
fluorescence microscopy analyses further demonstrated that the reduction-sensitive micelles exhibited
faster drug release behavior than reduction-insensitive micelles, which also led to higher cytotoxicity.
The SPIO/DOX-loaded micelles demonstrated excellent MRI contrast enhancement, and the r2 relaxivity
Received 7th September 2016
Accepted 14th November 2016
values of the SPIO/DOX-loaded micelles were up to 261.3 Fe mMꢀ1 ꢀ1. Consequently, these reduction-
s
DOI: 10.1039/c6ra22373g
sensitive amphiphilic dextran derivatives are promising theranostic nanocarriers for MR imaging and
chemotherapy.
www.rsc.org/advances
undesired side effects, effective tumor penetration and accu-
mulation via enhanced permeability and retention (EPR) and
endothelial leakiness (nanoEL) effects.1–7 The therapeutic and
1. Introduction
Polymeric nanocarriers play an important role in cancer therapy
and diagnosis, due to their improved water solubility, provision
of better bioavailability of hydrophobic drugs and diagnostic
agents, prolonged circulation time in the bloodstream, reduced
diagnostic agents are formulated in nanocarriers as a single
theranostic platform, which can then be further conjugated to
biological ligands for targeting. Aer loading the therapeutic
and diagnostic agents, the theranostic nanocarriers can achieve
systemic circulation, evade host defenses and deliver the drug
and diagnostic agents at the targeted site to diagnose and treat
the disease at the cellular and molecular level.8 As such, there
has been increasing interest in developing multifunctional
theranostic nanocarriers that incorporate diagnostic agents,
together with drugs, into polymeric micelles. Diagnostic agents
such as visible and NIR dyes, quantum dots, gold nanoparticles,
or superparamagnetic iron oxide (SPIO) and anticancer drugs
aDepartment of Radiology, Guangzhou First People's Hospital, Guangzhou Medical
University, Guangzhou 510180, China. E-mail: gzcmmcjxq@163.com; 123086302@
qq.com;
2986555610@qq.com;
2379828219@qq.com;
32656547@qq.com;
xxd2190@163.com; 605036656@qq.com; 770159633@qq.com
bDongguan Scientic Research Center, Guangdong Medical University, Dongguan
523808, China. E-mail: linjt326@163.com
cSchool of Materials Science and Engineering, Sun Yat-sen University, Guangzhou
510275, China. E-mail: ceszhlm@mail.sysu.edu.cn
† These authors contributed equally to this work.
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could be loaded into the nanocarriers and accumulated in cells efficiently with an applied external magnetic eld, and they
tumor tissues through EPR or “nanoEL” effects to predict the released the encapsulated drugs rapidly under the reduction
therapeutic efficacy.9–13 Feng et al. reported a PLA-TPGS-based environment.28
theranostic platform loading with both docetaxel and
Recent advances in nanomedical technology have focused
quantum dots.14 PLA-TPGS nanoparticles act as multimodal on developing a safe, biocompatible and biodegradable mate-
imaging systems when co-loaded with superparamagnetic iron rial. Biodegradable polymers are used for nanoparticle prepa-
oxide nanoparticles for MRI and with quantum dots for uo- ration to provide biological compatibility with less cytotoxicity.
rescence imaging.15 Gong et al. reported stable and tumor- Dextran and its derivatives are attractive candidates for bioma-
targeting, multifunctional worm-like polymer vesicles loaded terials in various elds, such as tissue engineering, imaging,
with SPIO and DOX simultaneously, which can deliver payloads gene and drug delivery, due to their hydrophilicity, non-toxicity
to the targeted tumor tissues, thus making the combination of and biodegradability.29–32 For example, hydrophobic dextran
chemotherapy and ultrasensitive MRI possible.16
derivatives, such as disulde bond-linked dextran–PCL and
Multidrug resistance (MDR) of cancer cells can cause dextran–PBLG diblock copolymer, were developed and applied
90% chemotherapeutic failure in patients with metastatic for triggered anticancer drug release.31,33 Commercially available
tumors.17,18 In order to overcome the MDR of cancer cells, highly MRI contrast agents such as Feridex and Resovist with a dextran-
elevated drug doses are usually used, which will cause and carboxydextran-coating, which stably disperse SPIO in
high toxicity and severe toxic side effects. It is desirable to aqueous media, have been already approved for use in clinical
develop polymeric micelles that can release their payloads MRI to assist in accurate cancer imaging.34–36
rapidly at the sites of tumors. For this purpose, stimuli-sensitive
Herein, we report the synthesis of reduction-sensitive
polymeric micelles that release payloads in response to an amphiphilic dextran derivatives as SPIO and DOX nano-
internal or external environmental stimulus, such as pH, carriers for controlled drug delivery and efficient MRI contrast
temperature, light, oxidation, or enzymatic degradation, have enhancement. Even though there are some studies on disulde
been developed for drug delivery.9,19–22 Particularly, reduction- bond linked micelles, reduction-responsive micelles based on
sensitive micelles, self-assembled from amphiphilic copoly- amphiphilic polysaccharides and polypeptides that are con-
mers, have attracted extensive attention for the active intracel- structed via click chemistry and act as theranostic platforms
lular delivery of anticancer drugs.23,24 It is reported that have rarely been reported.37 Amphiphilic dextran derivatives,
glutathione (GSH), a thiol-containing tripeptide capable of composed of dextran (Dex) and poly-(N-3-carbobenzyloxy-L-
cleaving disulde bonds by a redox reaction, is abundant in the lysine) (PZLL) were prepared via click conjugation between
cytoplasm of the cell (1–10 mM), whereas it is rarely present in azidized dextran and a-alkyne PZLL. The self-assembly behav-
blood plasma (2 mM).25,26 Hence, reduction-sensitive nano- iors of amphiphilic dextran derivatives in water were investi-
carriers have been developed to achieve the rapid release of gated using dynamic light scattering (DLS), transmission
encapsulated drugs inside the cells or under reductive condi- electron microscopy (TEM), and uorescence spectroscopy. As
tions mimicking that of the intracellular compartments. Yu shown in Scheme 1, the introduction of disulde bonds was
et al. designed a reduction and pH co-triggered magnetic expected to adjust the release of DOX, due to the reduction
nanogel based on sodium alginate (SA) to deliver DOX and SPIN responsive disassembling behaviors of the micelles. Their
simultaneously. They found that the nanogel could effectively intracellular drug delivery and growth inhibition to HepG2 cells
inhibit cell growth, while the nanogel exclusive of DOX was were investigated via MTT, ow cytometry assay and uores-
nontoxic.27 Zhang et al. synthesized a reduction-responsive, cence microscopy. Furthermore, the magnetic properties of the
multifunctional, theranostic nanosystem based on SPIO&DOX- micelles that contain SPIO were characterized to understand
PPLVs. These nanocarriers were internalized into the tumor their efficacy as MRI probes.
Scheme 1 Illustration of the reduction-responsive polymeric micelles for cancer chemotherapy and imaging.
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2.2 Synthesis of amphiphilic dextran derivatives containing
the disulde bond (Dex-g-SS-PZLL)
2. Experimental section
2.1 Materials
2.2.1 Preparation of disulde-containing a-alkyne-poly-(N-
3-carbobenzyloxy-L-lysine). Disulde-containing a-alkyne-poly-
(N-3-carbobenzyloxy-L-lysine) (a-alkyne-SS-PZLL), with different
degrees of polymerization, was prepared through the ring open
polymerization (ROP) of 3-carbobenzyloxy-L-lysine N-carbox-
yanhydride (Lys (Z)-NCA) initiated by (propargyl carbamate) ethyl
dithio ethylamine (PPA-Cyst), as shown in Scheme 2(A). In
a typical experiment, 5.0 g of N-3-carbobenzyloxy-L-lysine (17.8
mmol) was reacted with 3 g triphosgene (10.1 mmol) by using
anhydrous tetrahydrofuran (THF) as solvent. The Schlenk ask
Dextran (Mw ¼ 40 kDa) was purchased from Sinopharm Chem-
ical Reagents, Shanghai Co. Ltd., (China) and used as received.
1-Azido-2,3-epoxypropane was synthesized by two-step reactions
in our lab, according to the method reported previously
by Pahimanolis et al.38 Copper sulfate (CuSO4$5H2O) and sodium
ascorbate (99%) were purchased from Alfa Aesar. Doxorubicin
hydrochloride, N-3-carbobenzyloxy-L-lysine, copper sulfate, tri-
phosgene, iron(III) acetylacetonate (99%), benzyl alcohol,
N,N0-carbonyldiimidazole (CDI) and glutathione (GSH) were
purchased from Aladdin Chemical Company. Sodium
azide (99%), cystamine dihydrochloride, 2,2-dimethoxy-2-
phenylacetophenone (DMPA, 99%), and 3-aminopropyl-
triethoxysilane (APTS) were purchased from Aldrich. Dime-
thylformamide (DMF, Shanghai Sinopharm Chemical Reagent
Corp., 99.5%) was distilled from calcium hydride under reduced
pressure and stored over molecular sieves. (Propargyl carbamate)
ethyl dithio ethylamine was prepared according to previously
described procedures.39 Dulbecco's Modied Eagle's Medium
ꢁ
was placed in an oil bath regulated at 50 C for 1 hour under
static nitrogen pressure. Aer that, the organic solvent, THF, was
removed in vacuo; the obtained oil-like residue was rst dissolved
in ethyl acetate and then washed with cold 5% NaHCO3 solution
(3 ꢂ 150 mL). The ethyl acetate layer was collected and dried over
anhydrous MgSO4 and evaporated to give the white solid 3-car-
bobenzyloxy-L-lysine N-carboxyanhydride (Lys (Z)-NCA) with
a yield of 77%. ZLL-NCA (0.306 g, 1 mmol) was weighed in
a glovebox under pure argon, introduced into a ame-dried
Schlenk ask, and dissolved with 10 mL of anhydrous DMF.
The solution was stirred for 10 min, and then PPA-Cyst (0.024 g,
0.1 mmol) was added with a nitrogen-purged syringe. The solu-
(DMEM),
trypsin–ethylenediaminetetraaceticacid
(trypsin–
EDTA), and fetal bovine serum (FBS) were purchased from Gibco-
BRL (Canada). 40,6-Diamidino-2-phenylindole (DAPI) was
purchased from Beyotime Institute of Biotechnology (China). 3-
ꢁ
tion was stirred for 3 days at 25 C. Aer reaction, a-alkyne-SS-
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
PZLL was precipitated in methanol and a white powder deposit
was obtained. Aer the solvent was removed under reduced
pressure a white solid with a yield of 90% was obtained. The
degree of polymerization of the a-alkyne-SS-PZLL was obtained
from the relative integration of characteristic protons of the
chain ends to protons of polymer main chains. For example, the
relative integration of 4.13 ppm compared to that characteristic
of protons of the PZLL main chain, e.g., methylenic protons due
to the benzylic groups at 5.08 ppm, gave the degree of
(MTT) was purchased from Invitrogen Corporation (Washington,
USA). All other reagents were of analytical grade and used
without further purication. The human hepatocellular liver
carcinoma (HepG2) cell line was obtained from the Institute of
Biochemistry and Cell Biology, the Chinese Academy of Sciences
(Shanghai, China). Human umbilical vein endothelial (HUVEC)
cell line was purchased from the Animal Center of the Sun Yat-
sen University (Guangzhou, China).
Scheme 2 The synthesis route to amphiphilic dextran derivatives containing the disulfide bond (Dex-g-SS-PZLL).
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polymerization of a-alkyne-SS-PZLL. The a-alkyne-SS-PZLL has
three different degrees of polymerization; i.e., 10, 15 and 25, and
they were synthesized via the ROP by varying the ZLL-NCA/PPA-
Cyst molar ratio. The chemical structures of a-alkyne-SS-PZLLs
2.3 Synthesis of hydrophobic SPIO NPs
In many publications, oleic acid and oleylamine were used to
prevent SPIO particles from aggregation. This kind of SPIO NP
could be dispersed in hexane, THF and chloroform very well,
but the magnetic nanoparticles lead to systematic aggregation
in DMSO or DMF.40,41 As is known, Dex-g-SS-PZLL could only
be dissolved in DMSO; therefore, we needed to synthesize
hydrophobic SPIO NPs that could be dispersed in DMSO.
Hydrophobic SPIO NPs were prepared following a procedure
reported by Brougham et al.42 Iron(III) acetylacetonate (1 g) and
benzyl alcohol (15 mL) were placed in ask and the mixture
heated to reuxing (200 ꢁC) for 7 h under a ow of argon. Then,
the black-colored mixture was cooled to room temperature. The
black solution was precipitated with ethanol and separated
via centrifugation. Aer the Fe3O4 nanoparticles were redis-
persed in chloroform, APTS (50 mL) was added and the organic
solvent was tumbled for one hour. The nanoparticles were
precipitated in methanol (three times) to remove excess APTES,
and the obtained amino functionalized magnetic nanoparticles
(MNP-APTS) were redispersed in chloroform. To 200 mg of Lys
(Z)-NCA in a Schlenk tube, a 5 mL suspension of amino func-
1
were conrmed by H NMR (500 MHz, CDCl3/CF3CO2D, v/v ¼
85/15): 1.24 (m, 2H, –CH2CH2CH2–), 1.39 (m, 2H, –CHCH2CH2–),
1.7 (t, 2H, –CHCH2CH2–), 3.09 (2H, –NHCH2CH2–), 3.95 (2H,
–CCH2NH–), 4.13 (s, 2H, CH^CCH2O–), 4.41 (s, 1H, –NHCH–),
5.08 (s, 2H, –OCH2Ph), 7.30 (s, 5H, –Ph), 8.27 (s, 1H, –CONH–).
FT-IR (KBr, cmꢀ1): 3293 (n N–H); 2944 (n C–H), 1734 (n CO–O),
1652 (n C]O), 1546, 1386 (n CO–NH).
2.2.2 Preparation of azidated dextran. The introduction of
azide groups onto the backbone of dextran was carried out
according to a published method (Scheme 2(B)). In a typical
experiment, to 50 mL of 0.1 mol Lꢀ1 NaOH solution, 240 mg
dextran (1.5 mmol) and 180 mL (1.5 mmol) of freshly prepared
solution of 1-azido-2,3-epoxypropane were added successively
under magnetic stirring. The obtained clear reaction mixture
ꢁ
solution was stirred for 24 h at 30 C in a closed vial. Aer the
reaction, the solution was neutralized with acetic acid and
dialyzed in distilled water for 3 d (MWCO ¼ 14 000) and
lyophilized to obtain the azidized dextran with a yield of 82%.
FTIR (PerkinElmer Paragon 1000 spectrometer) and 1H NMR
(Bruker DPX-300 NMR spectrometer) analyses were used to
conrm the formation of azidized dextran. FTIR (KBr, cmꢀ1):
3460 cmꢀ1 (nO–H, pyranose), 2106 cmꢀ1 (azido groups), and
1022 cmꢀ1 (nC–O, pyranose). 1H NMR (500 MHz, D2O): d (ppm) ¼
5.60–4.50 (protons on anhydroglucose unit), 3.78 (–CH2N3), 3.61
(–CH2O–). 13C NMR (D2O, ppm) ¼ 97.96, 73.72, 71.60, 70.47,
69.82, 65.66 (dextran C–O), 72.21, 69.92 (C–O), 53.42 (C–N3).
Based on 13C NMR analysis, the degree of substitution (DS) of
azide groups in dextran was about 0.10.
tionalized magnetic nanoparticles (MNP-APTS) (15 mg mLꢀ1
)
in anhydrous chloroform was added under argon atmosphere.
The reaction mixture was stirred for 72 hours at room
temperature. PZLL graed magnetic nanoparticles were
precipitated in excess diethyl ether and separated as the
dry product. The dry magnetic particles were suspended in
anhydrous DMSO (5 mL) and stored at room temperature for
further use.
2.4 Characterization
2.2.3 Preparation of dextran derivatives containing the FTIR spectra of all samples were obtained in transmission mode
disulde bond. In order to obtain dextran derivatives contain- on a Perkin-Elmer Paragon 1000 spectrometer under ambient
ing disulde bonds (Dex-g-SS-PZLL), a-alkyne-SS-PZLLs were conditions. Samples were ground with KBr and then
conjugated with azidized dextran by a copper-catalyzed azide compressed into pellets. The spectra were obtained in the range
alkyne cyclization reaction, as shown in Scheme 2(C). The click of 500 to 4000 cmꢀ1
.
1H NMR spectral measurements were
reaction was carried out as follows: a-alkyne-SS-PZLL10 (0.12 g) carried out on a Bruker 500 NMR spectrometer by using
and a-azido dextran (0.10 g) were rstly co-dissolved in 10 mL of deuterated chloroform (CDCl3-d) or deuterated dimethyl sulf-
DMSO with N2-bubbling. Aer that, 30 mg of CuSO4$5H2O were oxide (DMSO-d6) as solvent, and tetramethylsilane (TMS) as the
introduced into the ask and the mixture was further bubbled internal standard. The size and size distribution of the micellar
with nitrogen for about 10 min until 60 mg of sodium ascorbate aggregates were determined via dynamic light scattering (DLS)
(NaAsc) was added. The Schlenk ask was placed in an oil bath using a BI-200SM Goniometer particle size analyzer (Broo-
regulated at 50 ꢁC for 3 days under static nitrogen pressure. The khaven Instruments Corporation, USA). Each analysis lasted for
excess a-alkyne-SS-PZLL10 was excluded by being dialyzed 300 s and the data were collected on an auto-correlator at 25 ꢁC
against DMSO for 1 day, with the dialysis tubing (MWCO ¼ 8–14 with a detection angle of scattered light at 90ꢁ. For each sample,
kDa), aer which the reaction mixture was dialyzed against the data from three measurements were averaged to obtain the
deionized water for 2 days. Aer the dialysis and lyophilization, mean ꢃ standard deviation (SD). The transmission electron
Dex-g-SS-PZLL10 was obtained as a white powder (0.19 g, 90.0%). microscopy (TEM) observation was performed using a JEOL
Dex-g-SS-PZLL20 and Dex-g-SS-PZLL25 were prepared using the JEM-2100F transmission electron microscope (JEM-2100F,
1
same method, with yields of 86.8 and 84.2%, respectively. H JEOL, Tokyo, Japan) operated at an accelerating voltage of 200
NMR (500 MHz, DMSO-d6): 3.1–4.0 ppm (m, dextran glucosidic keV. The samples were prepared by dropping 10 mL of 0.5 mg
protons), 4.7 ppm (s, dextran anomeric proton), 4.5, 4.9, and mLꢀ1 micelle suspension on the copper grid. A small drop of
5.1 ppm (s, dextran hydroxyl protons), 2.19 ppm (m, 2H, 1 wt% phosphotungstic acid solution (2 wt% in water) was
–CHCH2CH2–), 2.50 ppm (t, 2H, –CHCH2CH2–), 5.05 ppm (s, 2H, applied to the copper grid for staining the sample, and then
–OCH2–Ph), 7.25 ppm (s, 5H, –Ph), 7.86 ppm (s, 1H, –CONH–) blotted off with lter paper aer 1 min. The grid was nally
and 8.05 ppm (s, 1H, C]CHN–).
dried overnight before TEM observation.
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dissolution of the SPIO NPs. Fe concentration was determined
at the specic Fe absorption wavelength (248.3 nm) based on
a previously established calibration curve. The SPIO loading
content was calculated as the ratio of SPIO to the total weight of
the SPIO loaded micelles.
2.5 Preparation of micelles, and critical micelle
concentration (CMC)
Dex-g-SS-PZLL (10 mg) was dissolved in warm DMSO (2 mL),
then 4.0 mL of phosphate buffer (PB, 50 mM, pH 7.4) were
added into the DMSO slowly under stirring at room tempera-
ture, following which the mixed solution was dialyzed against
PB for 24 h at 25 ꢁC using a cellulose membrane bag (molecular
weight cutoff, MWCO 3500–4000).
SLC (%) ¼ w3/w4 ꢂ 100
(2)
where w3 is the weight of loaded SPIO, w4 is the weight of SPIO
The critical micelle concentration (CMC) was determined
using pyrene as a uorescence probe. The concentration of
Dex-g-SS-PZLL was varied from 1.0 to 4.88 ꢂ 10ꢀ4 mg mLꢀ1 and
the nal concentration of pyrene was xed at 6.0 ꢂ 10ꢀ7 mol
Lꢀ1. Before the measurement, the mixed solution of pyrene and
Dex-g-SS-PZLL was kept on a shaker at 37 ꢁC for 24 h to reach the
solubilization equilibrium in the dark. The uorescence spectra
were recorded using a uorescence spectrometer (RF-5301PC
Shimadzu); the excitation spectra of Dex-g-SS-PZLL/pyrene
solutions were scanned from 300 to 350 nm at room tempera-
ture, with an emission wavelength of 373 nm and a bandwidth
of 5 nm. The intensity ratios of I337 to I335 were plotted as
a function of the logarithm of Dex-g-SS-PZLL concentrations.
The CMC value was taken from the intersection of the tangent
to the curve at the inection with the horizontal tangent
through the points at low concentrations.
loaded micelles.
2.8 In vitro release of DOX
For the in vitro release of loaded DOX, its release proles from
Dex-g-SS-PZLL micelles were investigated at 37 ꢁC in two
different media; i.e., the DOX/SPIO loaded micelles were sus-
pended in 3 mL of PBS with 0 or 10 mM GSH, and the micelle
solution was immediately transferred into a dialysis bag
(MWCO 3500 Da). The release experiment was initiated by
placing the end-sealed dialysis bag in 27 mL of PBS containing
ꢁ
0 or 10 mM GSH with gentle shaking, in a 37 C water bath at
100 rpm. At selected time intervals, the buffered solution (3 mL)
outside the dialysis bag was removed and replaced with fresh
buffered solution of the same volume. The amount of released
DOX was determined using a UV-Vis spectrometer (UV-3150,
Shimadzu, Japan) at 480 nm.
2.6 Loading of DOX and SPIO into micelles
nꢀ1
X
Ve
Ci þ V0Cn
The DOX/SPIO-loaded micelles were prepared via a dialysis
method. In brief, 10 mg Dex-g-SS-PZLL, DOX$HCl (2 mg), an
equimolar amount of triethylamine (TEA) and 1.5 mg SPIO were
dissolved in 2 mL DMSO. Next, 5 mL of deionized water were
slowly added into the above mixed solution under stirring. The
mixed solution was dialyzed against deionized water for 2 days
to allow the formation of DOX/SPIO-loaded micelles and to
remove organic solvents and unencapsulated DOX dissolved in
aqueous solution (Mw cut-off: 14 000 Da). The DOX/SPIO-loaded
micelles were obtained by ltering the aqueous solution
through a 0.45 mm lter.
i¼1
mDOX
Er ð%Þ ¼
ꢂ 100%
(3)
where mDOX represents the amount of doxorubicin in the
micelles, V0 is the whole volume of the release media (V0 ¼ 30
mL), Ve is the volume of the replacement media (Ve ¼ 3 mL), and
Ci represents the concentration of DOX in the ith sample.
2.9 Cell viability assays
The relative cytotoxicities of Dex-g-SS-PZLL micelles against
human hepatocellular liver carcinoma (HepG2) cells and
human umbilical vein endothelial (HUVEC) cells were evaluated
in vitro by a MTT assay. Five multiple holes were set for every
2.7 Determination of the DOX and SPIO loading contents
For the determination of DOX loading amount, the DOX/SPIO- sample. The cells were seeded in a 96-well plate at a density of
loaded Dex-g-SS-PZLL micelles were broken in a mixed DMSO/ 1 ꢂ 104 cells per well in 100 mL of DMEM and incubated at 37 ꢁC
PBS solvent (v/v, 70/30), then the sample was ltered to in a 5% CO2 atmosphere for 24 h. Aer removing the culture
remove the insoluble SPIO and subjected to UV analysis medium, the Dex-g-SS-PZLL micelles solution was added to
(UV-3150, Shimadzu, Japan) at the wavelength of 480 nm. The the wells at a specic concentration (0–500 mg mLꢀ1). Aer co-
loading amount of DOX was determined from the calibration incubation with cells for 48 h, 20 mL of MTT solution in PBS
curve of DOX. Drug loading content (DLC) was calculated (5 mg mLꢀ1) was added to each well and the plate was incubated
according to the following equation:
for another 4 h at 37 ꢁC. Aer that, the medium containing
MTT was removed, and 150 mL of DMSO was added to each well
to dissolve the MTT formazan crystals. Finally, the plates were
shaken for 10 min, and the absorbance of formazan product
was measured at 490 nm by a microplate reader.
DLC (%) ¼ w1/w2 ꢂ 100
(1)
where w1 is the weight of loaded drug, w2 is the weight of drug
loaded micelles.
Atomic absorption spectroscopy (AAS) was used to measure
the loading amount of SPIO NPs inside the polymeric micelles.
Cell viability (%) ¼ [A490(sample)/A490(control)] ꢂ 100
(4)
In brief, the DOX/SPIO-loaded micelles were added to 1 M HCl where A490(sample) and A490(control) represent the absorbances
solution to allow the disaggregation of micelles and complete of the sample and control wells, respectively.
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The in vitro cytotoxicity assay of DOX/SPIO-loaded Dex-g-SS- 13.8/27.6/41.4/55.2/69.0 ms, ip angle: 180ꢁ, slice thickness:
PZLL micelles was performed against HepG2 cells by MTT 3.0 mm, matrix 444 ꢂ 448) was used to measure the transverse
assay. Five multiple holes were set for every sample. Briey, relaxation time. Relaxation times were obtained by tting
HepG2 cells were respectively cultured onto 96-well plates at the multi-echo data to a monoexponential decay curve using
a density of 1 ꢂ 104 cells per well and incubated in a humidied linearized least-squares optimization. Relaxivity values were
atmosphere of 5% CO2 at 37 ꢁC for 24 h. Aer that, the growth calculated via linear least-squares tting of 1/relaxation time
medium was replaced by 100 mL complete DMEM containing (sꢀ1) versus the iron concentration (mM Fe).
different DOX concentrations (0–10 mg mLꢀ1 DOX) and further
incubated for 24 h. Aer co-incubation with cells for 24 h, 20 mL
of MTT solution in PBS (5 mg mLꢀ1) was added to each well
3. Results and discussion
3.1 Preparation and characterization of amphiphilic dextran
and the plate was incubated for another 4 h at 37 ꢁC. Aer
that, the medium containing MTT was removed, and 150 mL of
DMSO was added to each well to dissolve the MTT formazan
crystals. Finally, the plates were shaken for 10 min, and the
absorbance of formazan product was measured at 490 nm by
a microplate reader. Cell viability (%) was calculated based on
the above method.
derivatives
Amphiphilic dextran derivatives containing disulde bonds
were prepared via the click conjugation reaction between azi-
dated dextran with a-alkyne-SS-PZLL (Scheme 2). The click
conjugation reaction between Dex-N3 and a-alkyne-SS-PZLL was
performed in DMSO using an alkyne/azido molar ratio of 1.3/
1. The resulting Dex-g-SS-PZLL-graed copolymer was puried
by dialyzing with DMSO and deionized water to remove excess
2.10 Cellular uptake studies
The cellular uptake and distribution of micelles were performed
using ow cytometry (Guava easyCyte 5HT-2L, Merck Millipore,
Germany) and inverted uorescence microscope (Axio Observ-
er.Z1, Carl Zeiss, Germany). For ow cytometry, the HepG2 cells
were seeded in 6-well plates at a density of 1 ꢂ 106 cells per well,
in 2 mL of complete DMEM and cultured at 37 ꢁC in the 5% CO2
atmosphere overnight. Cells were then washed with PBS and
incubated with free DOX and DOX/SPIO-loaded micelles (DOX
concentration: 10 mg mLꢀ1) at 37 ꢁC for an additional 4 h. Cells
without pretreatment were used for comparison. Aer the
culture medium was removed, the cells were washed with PBS
thrice and treated with trypsin. Then, 2 mL of PBS was added to
each culture well, and the cells were collected by centrifuging
the solutions for 5 min at 3000 rpm. Aer removal of the
supernatant, the cells were resuspended in 0.3 mL of PBS. A
minimum of 2 ꢂ 104 cells were analyzed from each sample.
For inverted uorescence microscope studies, HepG2 cells
were seeded in a 6-well plate (1 ꢂ 106 cells per well) in 2 mL of
DMEM and cultured at 37 ꢁC in the 5% CO2 atmosphere for
24 h. In order to observe the cells internalization, the culture
medium was replaced with 2 mL of DMEM containing free DOX
and DOX/SPIO loaded micelles (DOX concentration: 10 mg
mLꢀ1). Aer 4 h, the culture medium was removed and the cells
were washed with PBS then xed with 4% formaldehyde for
30 min at room temperature, and the slides were rinsed with
PBS three times. The cell nuclei were dyed with 4,6-diamidino-2-
phenylindole (DAPI) for 30 min and washed with PBS three
times. Finally, the samples were observed by inverted uores-
cence microscopy, and the photos were obtained with a 20ꢂ
objective. The samples were excited using a Mercury HBO power
supply. The excitation wavelengths of DOX and DAPI were 546 ꢃ
12 nm and 365 nm, respectively.
polypeptide and organic solvent.
Fig. 1 gives the FTIR spectra of azidated dextran, a-alkyne-SS-
PZLL and Dex-g-SS-PZLL. As shown in the FTIR spectra, the
characteristic absorption bands of azidated dextran appeared
at 3500 cmꢀ1 (nO–H, glucopyranose), 2922 cmꢀ1 (nC–O, gluco-
pyranose) and 2110 cmꢀ1 (azido group). The characteristic
absorption bands attributed to a-alkyne-SS-PZLL appeared at
3294 cmꢀ1 (nN–H), 2934 cmꢀ1 and 2841 cmꢀ1 (nC–H), 1742 cmꢀ1
(nC]O), as well as 1548 cmꢀ1 (nCO–NH). Aer the click conjuga-
tion, the spectrum of azidated dextran did not show the char-
acteristic absorption band of the azido group at 2110 cmꢀ1, but
exhibited the main characteristic absorption bands of azidated
dextran and a-alkyne-SS-PZLL, which conrmed the click
conjugation reaction. The FTIR results of our experiment are
similar to the previous results.44 The main difference between
alkyne-SS-PZLL and Dex-g-SS-PZLL in FTIR results was the band
2.11 Relaxivity measurement
T2 MR imaging was carried out using a 3.0 T MRI scanner (Verio,
Siemens, Erlangen, Germany) at room temperature, according
Fig. 1 FTIR spectra of azidated dextran (a), a-alkyne-SS-PZLL (b) and
to a reported protocol.43 T2 mapping sequence (TR: 1000 ms, TE: Dex-g-SS-PZLL (c).
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from 3000 to 3750 cmꢀ1. The chemical structure of Dex-g-SS-
PZLL was conrmed by H NMR. As shown in Fig. 2, the char-
3.2 Micelles formation and their properties
1
The dextran derivatives were prepared by graing hydrophilic
dextran with hydrophobic PZLL. It is known that amphiphilic
polymers can self-assemble into micelles in selected solvents. In
this study, the self-assembly behavior in aqueous medium was
investigated by uorometry in the presence of pyrene as a uo-
rescent probe, according to the literature.45 The variation in the
intensity ratio (I337/I335) of the vibronic peak at 337 nm to the
vibronic peak at 335 nm is quite sensitive to the polarity of
the microenvironment where pyrene is located. Fig. 3(a) gives
the change of I337/I335 with the concentration of Dex-g-SS-
PZLL15. We nd that at lower Dex-g-SS-PZLL15 micelles
concentrations, the I337/I335 values remain nearly unchanged. As
the micelles concentrations increase, the intensity ratio starts to
increase, implying the formation of the micellar self-aggregates
with hydrophobic domains. The critical micelle concentration
(CMC) could be determined by the crossover point of two
straight lines, as indicated in Fig. 3(a). As shown in Table 1, The
CMC values were found to decrease with the increase in
hydrophobic block length from PZLL10 to PZLL25, which may be
attributed to enhanced hydrophobicity of dextran derivatives.
For instance, the calculated CMC values of Dex-g-SS-PZLL10,
acteristic signals of the protons in the dextran segment were at
3.16–4.77 ppm and the proton resonance peak at 1.25 (14),
1.31 (13), 1.50–1.60 (12), 2.92 (15), 3.62 (10), 4.93 (16) and
7.31 ppm, which were the characteristic signals of PZLL.
Furthermore, the proton resonance signal at 8.05 ppm in the
spectrum indicated the formation of the triazole ring.38 These
results support the successful synthesis of the Dex-g-SS-PZLL-
graed copolymer. For comparison, a Dex-g-PZLL graed
copolymer without disulde bonds was also prepared.
Dex-g-SS-PZLL15, and Dex-g-SS-PZLL25 were 0.02 mg mLꢀ1
,
0.012 mg mLꢀ1 and 0.007 mg Lꢀ1, respectively.
The hydrodynamic diameter of the resulting micelles was
obtained by DLS analyses. As shown in Fig. 3(b), the Dex-g-SS-
PZLL15 micellar aggregates were found to have the mean
diameter (MD) of 75.3 ꢃ 2.0 nm and a narrow size distribution
with the polydispersity index (PDI) of 0.09 ꢃ 0.019. As shown
in Table 1, the mean diameters of the micelles were dependent
Fig. 2 1H NMR spectrum of Dex-g-SS-PZLL.
Fig. 3 (a) The change in I337/I335 with the concentration of Dex-g-SS-PZLL15; (b) the size and size distribution of Dex-g-SS-PZLL15 micelles; (c)
the change in the decay frequency, G, as a function of the square of the scattering vector q2 for aqueous systems of Dex-g-SS-PZLL15 micelles at
the test angles ranging from 50 to 130ꢁ; (d) the TEM photograph for the aqueous system of Dex-g-SS-PZLL15 micelles.
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Table 1 Characteristics of blank and DOX/SPIO-loaded micellesa
Blank micelles
DOX/SPIO-loaded micelles
Sample code
Diameterb (nm)
PDIb
CMCc (mg mLꢀ1
)
Diameterb (nm)
PDIb
DLC (%)
SLC (%)
Dex-g-SS-PZLL10
Dex-g-SS-PZLL15
Dex-g-SS-PZLL25
68.5 ꢃ 2.7
75.3 ꢃ 2.0
96.5 ꢃ 1.7
0.07 ꢃ 0.01
0.10 ꢃ 0.02
0.15 ꢃ 0.02
0.020
0.012
0.007
94.6 ꢃ 1.7
117.3 ꢃ 3.6
135.8 ꢃ 2.3
0.10 ꢃ 0.01
0.12 ꢃ 0.04
0.10 ꢃ 0.04
13.2
12.5
14.6
11.3
12.4
11.7
All aggregate solutions had a nal polymer concentration of 0.50 mg mLꢀ1
.
Measured by dynamic light scattering (DLS). c Determined by pyrene-
a
b
based uorescence spectrometry.
on the hydrophobic block length. The dextran derivatives with
The mean diameter and the morphology of DOX/SPIO-
the longer PZLL block formed larger micelles, indicating loaded micelles were determined by DLS and TEM measure-
a larger hydrophobic inner core. ments. Dynamic light scattering (DLS) measurements (Fig. 5(a))
In Fig. 3(c), the dependence of the decay frequency, G, as showed that the mean diameter was 117.3 nm for DOX/SPIO-
a function of the square of the scattering vector q2 (q is dened loaded Dex-g-SS-PZLL15 micelles. The TEM image of DOX/
as q ¼ 4pn sin(q/2)/l, where n is the refractive index of the SPIO-loaded Dex-g-SS-PZLL15 micelles (Fig. 5(b)) shows that
solution, q is the scattering angle, and l ¼ 632.8 nm is the they are uniform in shape and size distribution. Compared
wavelength of the incident laser light) was plotted. The linear to the blank micelles, the SPIO and DOX co-loaded micelles
variation of the relaxation frequency versus the squared scat- showed a signicant increase in size. The increase in micelle
tering vector q2 passing through the origin is the hallmark of sizes might be due to the hydrophobic DOX and SPIO
a translational diffusive process, conrming the presence of being encapsulated into the hydrophobic core of the Dex-g-SS-
spherical objects with a pure diffusive mode.46
PZLL15 micelles.49
TEM observations could reveal the morphology of the
micellar aggregates. To conrm the DLS results, TEM observa-
tion was carried out for aqueous systems of Dex-g-SS-PZLL15
micelles. Fig. 3(d) clearly shows that the spherical-like aggre-
gates were formed. The average diameter of the spherical-like
aggregates, according to the TEM images, was about 35 nm.
The average diameters calculated from TEM images were
smaller than those obtained by DLS results. This may be due to
the fact that DLS analysis reveals the hydrodynamic diameter
upon swelling in aqueous solution, whereas TEM reveals the
dimensions of aggregates in the dry state.
In order to assess the potential application of dextran
derivatives as theranostic nanocarriers for MR imaging and
chemotherapy, Dex-g-SS-PZLL15 was chosen as the reduction
sensitive graed polymer and Dex-g-PZLL was used as the
reduction-insensitive control to observe the effect of the disul-
de bond on drug release and cytotoxicity tests. Hydrophobic
SPIO and DOX were co-loaded into the micelles. The loading
contents of SPIO and DOX in the micelles are summarized in
Table 1. As a hydrophobic drug, DOX could be well encapsu-
lated into the hydrophobic inner cores of Dex-g-SS-PZLL
micelles, driven by the hydrophobic interactions between the
drug and the hydrophobic segments of conjugated polymer.
Fluorescence spectra revealed the interaction between DOX and
micelles. As shown in Fig. 4 the uorescence of the DOX loaded
micelles was nearly completely quenched when DOX was
encapsulated into the micelles, conrming the p–p stacking
interaction between DOX and phenyl groups of PZLL chains.47
DOX-loaded Dex-g-SS-PZLL15 micelles were treated with GSH
and ltered through a 220 nm lter membrane. In the uores-
cence spectrometry of the ltered solution, an emission peak
for DOX was observed again. These results suggest that the
release of DOX from disulde bonds containing micelles, trig-
gered by GSH, generates a uorescent signal.48
3.3 In vitro stimuli-responsive drug release
According to previous studies, GSH is abundant in the cyto-
plasm of the cell (1–10 mM), whereas it is rarely present in blood
plasma (ꢄ2 mM).25 The release behavior of DOX from the Dex-g-
SS-PZLL micelles was evaluated in the presence and absence
of 10.0 mM GSH in PBS at pH 7.4. As shown in Fig. 6, in the
absence of GSH, the DOX/SPIO-loaded micelles showed a sus-
tained and slow release. Even aer 48 h, only about 35% of DOX
could be released from these micelles. However, in the presence
of 10 mM GSH, mimicking the intracellular environment, DOX/
SPIO-loaded Dex-g-SS-PZLL micelles showed a burst release
Fig. 4 The fluorescence spectra of the Dex-g-SS-PZLL15 micelles,
DOX solution, Dex-g-SS-PZLL/DOX micelles and Dex-g-SS-PZLL/
DOX micelles treated with GSH (lexcitation ¼ 330 nm).
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Fig. 5 (a) Typical particle size distribution and (b) TEM image of the DOX/SPIO-loaded Dex-g-SS-PZLL15 micelles.
behavior, in which 77.2% of DOX was released in the initial 6 h HepG2 cells were treated with free DOX and DOX/SPIO-loaded
and a complete release of loaded DOX was observed within 12 h. micelles with various equivalent DOX concentrations from
As for the Dex-g-PZLL micelles, without the reduction insensi- 0.1 to 10 mg mLꢀ1. Aer incubation for 48 h, the cell viability was
tive disulde bonds, the release behavior of DOX was not evaluated by MTT assay. Fig. 8 shows the MTT assay results that
affected by GSH. These results suggest that the rapid release the free DOX has the highest cytotoxicity, regardless of the
of DOX was attributed to the cleavage of disulde linkages, DOX concentration. The viability of HepG2 cells was mainly
leading to disintegration of the micellar structure, triggered by dependent on the DOX concentrations. As the DOX concentra-
GSH.23,31 Since tumor cells show higher GSH content, this tions increased, the viability of HepG2 cells incubated with
observation demonstrated that the Dex-g-SS-PZLL micelles both DOX/SPIO-loaded micelles was decreased. However, when
might have potential applications as a tumor triggered drug the DOX concentration was over 0.5 mg mLꢀ1, the cytotoxicity
delivery platform.
of DOX/SPIO-loaded Dex-g-SS-PZLL15 micelles was higher than
that of Dex-g-PZLL, which can be attributed to the rapid release
of DOX from reduction sensitive micelles by cleavage of
the disulde bond in an intracellular environment.23 Although
free DOX can kill cancer cells more efficiently than DOX-loaded
micelles, it usually suffers from challenges including rapid drug
clearance, poor biodistribution, non-ideal physicochemical
properties of drugs (e.g., poor solubility), rapid drug degrada-
tion, and systemic toxicity.50 The use of micelles in general oen
achieves favorable biodistribution, prolonged release proles,
higher therapeutic effects and reduced side effects of the drug.
DOX interacts with DNA and the uorescence signal of DOX
allows us to follow their internalization and intracellular
distribution. Fluorescence microscopy and ow cytometry were
3.4 In vitro cell cytotoxicity and cellular uptake
MTT assay was used to evaluate the in vitro cytotoxicity of
the blank Dex-g-SS-PZLL15 micelles toward HUVEC and HepG2
cells. The cells were incubated with Dex-g-SS-PZLL15 at different
concentrations for 48 h. Fig. 7 shows that the viabilities of
HUVEC and HepG2 cells were over 85%, even with the Dex-g-SS-
PZLL15 concentrations up to 500 mg mLꢀ1. According to the
MTT results, Dex-g-SS-PZLL15 had low cytotoxicity and good
biocompatibility.
The DOX/SPIO-loaded Dex-g-SS-PZLL15 micelles were further
investigated to evaluate their potential therapeutic efficacy.
Fig. 6 The in vitro release behavior of loaded DOX from Dex-g-SS-
PZLL micelles in the absence and presence of GSH (37 ꢁC, pH 7.4). The Fig. 7 In vitro cytotoxicity of blank micelles against Hepg2 and HUVEC
DOX-loaded Dex-g-PZLL micelles were used as the control.
cells at different concentrations.
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higher cellular uptake level of free DOX than DOX-loaded
micelles could be attributed to different cellular uptake mech-
anisms. Free DOX was transported into cells by a passive
diffusion mechanism, while DOX-loaded micelles were inter-
nalized in the cells by an endocytosis mechanism.51,52 The
intense DOX accumulation in the nucleus for free DOX occurred
because the free DOX molecules that had diffused into the
cytosol were rapidly transported into the nucleus and avidly
bound to the chromosomal DNA.16
As shown in Fig. 9(b), ow cytometry analysis clearly
demonstrated that the highest uorescence intensity was
observed in the HepG2 cells incubated with free DOX,
compared to the cells with DOX-loaded micelles for 4 h, which
was consistent with the cellular uptake behavior observed by
uorescence microscopy and was attributed to different cellular
uptake mechanisms. Moreover, by comparison with the
reduction-sensitive Dex-g-SS-PZLL15, the reduction-insensitive
Dex-g-PZLL micelles showed the weakest uorescence inten-
sity in the HepG2 cells incubated with equivalent DOX
concentration. Therefore, based on the uorescence micro-
scope observation and ow cytometric analysis, it is conrmed
that the fast intracellular drug release from Dex-g-SS-PZLL
micelles under a reductive condition could be attributed to
the cleavage of the disulde bonds in the polymers.53
Fig. 8 Cytotoxicity of free DOX, Dex-g-SS-PZLL15 and Dex-g-PZLL
DOX/SPIO loaded micelles against HepG2 cells at different DOX
concentrations (incubation time 48 h).
used to evaluate the cellular uptake behavior of the DOX/SPIO-
loaded Dex-g-SS-PZLL micelles against HepG2 cells. For the
uorescence microscopy analysis, the cellular uptake of DOX
was assayed by incubating HepG2 cells with DOX/SPIO-loaded
micelles for 4 h at a DOX concentration of 10 mg mLꢀ1. As
shown in Fig. 9(a), aer 4 h of incubation of HepG2 cells with
DOX and DOX/SPIO-loaded micelles, strong DOX uorescence
appeared, possibly due to the fast internalization of micelles
and rapid release of DOX inside cells. It should be noted that
higher intracellular DOX uorescence intensity was observed
in the HepG2 cells incubated with free DOX than with DOX
loaded micelles. According to the uorescence microscope
images, we also found that free DOX mainly accumulated in the
cell nucleus; the DOX delivered by Dex-g-SS-PZLL15 micelles was
transported into the cell nucleus, and as for the Dex-g-PZLL
micelles, much stronger DOX uorescence appeared in the
cytoplasm with a weaker uorescence in the nucleus. The
3.5 Relaxivity measurement
SPIO nanoparticles are capable of shortening the transverse
relaxation time of water protons and thus could act as a T2
contrast agent in MRI.3 The in vitro MRI experiment was carried
out in a 3.0T Siemens MRI scanner at room temperature.
Fig. 10(a) shows the measurement of r2 relaxivity for the DOX/
SPIO-loaded Dex-g-SS-PZLL15 micelles. Relaxivity can be calcu-
lated through the linear least squares tting of 1/relaxation time
(sꢀ1) versus the iron concentration (mM Fe). According to our
measurements, the r2 for DOX/SPIO-loaded Dex-g-SS-PZLL15
Fig. 9 (a) Fluorescence microscope images of HepG2 cells after 4 h incubation with free DOX (A), DOX/SPIO-loaded Dex-g-SS-PZLL micelles
(B) and DOX/SPIO-loaded Dex-g-PZLL micelles (C). (b) DOX fluorescence intensity of HepG2 cells incubated with free DOX, DOX/SPIO-loaded
Dex-g-SS-PZLL15 micelles and DOX/SPIO-loaded Dex-g-PZLL micelles at 37 ꢁC for 4 h (DOX concentration ¼ 10 mg mLꢀ1), measured by flow
cytometry.
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the Natural Science Foundation of Guangdong Province
(S2013010012549, 2014A030313647), the Science and Technology
Plan Foundation of Guangzhou (201510010086, 201607010038).
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Acknowledgements
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