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Hyaluronic acid modified mesoporous carbon nanoparticles for targeted drug delivery to

CD44-overexpressing cancer cells

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2016 Nanotechnology 27 135102

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Nanotechnology

Nanotechnology 27 (2016) 135102 (11pp) doi:10.1088/0957-4484/27/13/135102

Hyaluronic acid modied mesoporous


carbon nanoparticles for targeted drug
delivery to CD44-overexpressing
cancer cells
Long Wan1,2,3, Jian Jiao1, Yu Cui1, Jingwen Guo4, Ning Han1, Donghua Di1,
Di Chang1, Pu Wang4, Tongying Jiang1 and Siling Wang1
1
School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016,
Peoples Republic of China
2
Department of Pharmacy, The First Afliated Hospital of China Medical University, Nanajingbei Street
155, Shenyang, Peoples Republic of China
3
College of Pharmaceutical Science, China Medical University, Puhe Road 77, Shenyang 110122, Peoples
Republic of China
4
College of Life Science and Health, Northeastern University, Wenhuadong Road 89, Shenyang 110015,
Peoples Republic of China

E-mail: silingwang@syphu.edu.cn

Received 22 November 2015, revised 20 January 2016


Accepted for publication 26 January 2016
Published 22 February 2016

Abstract
In this paper, hyaluronic acid (HA) functionalized uniform mesoporous carbon spheres (UMCS)
were synthesized for targeted enzyme responsive drug delivery using a facile electrostatic
attractionstrategy. This HAmodication ensuredstable drug encapsulation in mesoporous
carbon nanoparticles in anextracellular environment while increasingcolloidal stability,
biocompatibility, cell-targeting ability, and controlled cargo release. The cellular uptake
experiments of uorescentlylabeled mesoporous carbon nanoparticles, with or without HA
functionalization, demonstrated that HA-UMCS are able to specically target cancer cells
overexpressing CD44 receptors. Moreover, thecargoloadeddoxorubicin (DOX) and verapamil
(VER) exhibiteda dual pH and hyaluronidase-1 responsive release in the tumor
microenvironment. In addition,VER/DOX/HA-UMCS exhibited a superior therapeutic effect
on anin vivo HCT-116 tumor in BALB/c nude mice. In summary, it is expected that HA-UMCS
will offer a new methodfor targeted co-delivery of drugs to tumors overexpressing CD44
receptors.

Keywords: mesoporous carbon nanoparticles, hyaluronic acid, controlled release, co-delivery


(Some gures may appear in colour only in the online journal)

1. Introduction large specic surface area (orpore volume) andtunable


size(or morphology or pore structure) allow these inor-
In recentdecades, a variety of nanocarriers have been ganic mesoporous materials to selectively encapsulate dif-
proposed and studied for the delivery of nanomedicines ferent drugs of interest and to be tuned to optimize cellular
[7, 8, 22]. Compared with conventional polymer-based or uptake [9, 17, 31]. In particular, mesoporous carbon nano-
organic lipidnanoparticles, inorganic matrix nanoparticles particles with a strong adsorption capacity [10, 23],
exhibit some particular properties that make thempotential stacking interactions [12, 14] and easily functionalized
therapeutic and diagnostic agents [13, 21, 24, 32]. Their surface [19] have attracted great interest with regard to

0957-4484/16/135102+11$33.00 1 2016 IOP Publishing Ltd Printed in the UK


Nanotechnology 27 (2016) 135102 L Wan et al

Figure 1. Schematic illustration of VER/DOX-loaded HA-UMCS for pH/redox-triggered drug release and inhibition of P-gpmediated drug
efux.

targeted drug delivery [11].Mesoporous carbon nano- immunogenicity and biodegradability [6, 15, 16, 25, 26].
carriers can alsoprotect the encapsulated agents from Simutaneously, HA can specically bind to CD44 receptors
degradation during delivery, as well as improve their which are overexpressed in many typesof cancer cells [4, 18].
membranepermeability [32]. In addition, HA-functionalized nanomedicine delivery systems
Multidrug resistance (MDR) is one of the main obstacles to can penetrate cancer cells more efciently through HA receptor-
successful chemotherapy treatment of cancer. MDR is often mediated endocytosis. The biocompatibility and specic afnity
caused by the overexpression of a 170 kDa glycoprotein within of HA for CD44 receptors ensures its great application potential
the plasma membrane known as P-glycoprotein (P-gp). In for treatments targeting cancer.
addition, a wide range of anticancer drugs are substrates of P-gp In this research, as shown in gure 1, a uniform meso-
thatpumpout the drug molecules from the inside of cancer porous carbon spheres (UMCS)-based inorganic/organic drug
cells, reducing intracellular drug accumulation. Therefore, if the delivery system has been developed for the rst time to
activity of P-gp could be restrained, we would obtain double the improve itsalready excellent propertiesthrough the synergistic
anticancer effects with half the dose. Therefore, combination benecial combination of UMCS and HA. The advantagesof
therapy has been used in chemotherapy treatment thatcankill HA capping UMCS include: (1) HA improves the physiolo-
cancer cells usingsynergistic effects before the cancer adapts, gical dispersion stability of UMCS necessary for intravenous
reducing the probability of cancer cell mutation. Howe- administration [2]; (2) itslarge molecular size hinders drug
ver,widespread application of combination therapy is limited leakage into the blood circulation; (3) itshydrophilic nature
since the different membrane transport properties and biodis- prolongs the blood circulation time of nanoparticles like PEG
tribution of many drugs results in suboptimal drug combina- [3, 20]; (4) itsspecic interaction with CD44 receptors
tions in the tumor region. Due to their high drug-loading improves the selective targeting of nanoparticles; (5) HA
capacity and their extensive applicability forencapsulatingdrug macromolecules are rapidly degraded and broken down into
molecules with different natures, mesoporous carbon nano- fragments by the hyaluronidases abundant in malignant tumors
particles make the simultaneous delivery of multiple agents for [1, 5, 25] thereby acceleratingdrug release. In addition, dox-
combination therapy possible. orubicin (DOX) and verapamil (VER) were encapsulated inthe
A perfect targeted drug delivery system should transport mesopores of the core UMCS for combination therapy at high
toxic agents to the specic target site without any drug leakage drug loading (25% for each drug). For the rst time, human
into the blood circulation, while allowing the drug to be colon cancer HCT-116 cells were inoculated into BALB/c
released quickly at the target site based on an environmental nude mice to obtain in vivo antitumor effects. The results we
change. Hyaluronic acid (HA), a natural anionic glycosami- obtained have conrmed the specic selectivity of HA-UMCS
noglycan, is one of the major components of the extracellular and an enhanced efciency in treating CD44 receptors over-
matrix in normal tissues like epithelial, connective, and expressed cancer cells, while effectively inhibiting the DOX
neural tissues, and it exhibits good biocompatibility, non- efux usingP-gp glycoprotein.

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Nanotechnology 27 (2016) 135102 L Wan et al

2. Materials and methods 2.4. Preparation of DOX and VER loaded nanoparticles

Firstly, DOX hydrochloride (40 mg) and NH2-


2.1. Materials
UMCS(10 mg) were added into 10 ml ofPBS (pH 7.4)
Hexadecylamine andtetraethyl orthosilicatewere purchased buffer solution, ultrasonicated and stirred for 24 h in darkness
from Sigma-Aldrich (StLouis, MO, USA). Fluorescent at room temperature. The DOX-loaded NH2-UMCSwer-
probes containing Hoechst 33342, rhodamine-phalloidin were ecollected by centrifugation at 13 000 rpm for 10 min and
bought from Molecular Probes Inc. (Eugene, OR, USA). repeated washing with PBS (pH 7.4). Secondly, verapamil
Bovine serum albuminwas purchased from Amreso (USA). was encapsulated into the outer mesopores of theNH2-
Cell culture Dulbeccos Modied EagleMedium (DMEM), UMCSby solvent evaporation. The previous product (DOX/
penicillin-streptomycin, fetal bovine serum (FBS) were pur- NH2-UMCS) was added into10 ml of ethanol solution of
verapamil (verapamil:NH2-UMCS=1:2, wt). Then, the
chased from GIBCO, Invitrogen Co. (Carlsbad, USA).
mixture was stirred for 24 h to ensure thatverapamil adsorbed
Trypsin and phosphate buffer solution (PBS) were obtained
homogeneously onto the outer pore channels of theNH2-
from Mcgene Co (Beijing, China). Sodium hyaluronate
UMCS.The ethanol in samples was thenremoved by con-
(MW100 KDa) was a product of Shanghai Alading Bio-
stant stirring at 40 C. Thirdly, HA macromolecules were
chem Co. Ltd (China). All the other reagents in the experi- grafted onto the surface of the VER/DOX/NH2-UMCS by
ments were of analytical grade and used without additional electrostatic attraction. The drug-loaded process of COOH-
purication. UMCS was thesame as the method above.
To evaluate the DOX loading efciency, the unbound
DOX was measuredusing ultraviolet (UV) spectroscopy
2.2. Preparation of nanoparticles (UV-2000, Unico, USA) at 490 nm relative to a calibration
UMCSof 350 nm diameter were preparedusing aspherical curve performed underidentical conditions. The loading
efciency of DOX could be calculated according to the ori-
nanosilica matrix (SNM) as a template and furfuryl alcohol as
ginal and residual DOX concentrations and volumes. The
acarbon precursor, as previously described [28]. AnATS
calculation equation of DOX follows below. The loading
AH100D homogenizer (ATS Engineer Inc., China) was used
amount of VER was almost the same as the initial addition
to increase the homogeneity of the SNM. After procedural
amount.
carbonization, aSiC composite was added to 10% hydro-
uoric acid to remove the silica template, and UMCSwere
2.5. In vitro release experiment
obtained. Then, acarboxyl group was introduced to the sur-
face of theUMCSby wet oxidation. Then,200 mg of the The in vitro release prole of DOX and VER from theVER/
COOH-UMCS was dispersed in 100 ml of ethylenediamine, DOX/HA-UMCS nanoparticles was performed in triplicate at
using N-[(dimethylamino)-1H-1,2,3-triazolo [46] pyridin-1- apH of 7.4 (the physiological blood circulation pH) and
ylmethylene]-N-methylmethanaminium hexauorophosphate apH of 5.0 (the endosomal pH of cancer cells). Briey, 1 mg
noxideas the coupling agent, by sonication for 4 h to prepare of DOX was introduced into a centrifuge tube and dispersed
NH2-UMCS [29]. Finally, NH2-UMCS (1 mg) wereadded in 5 ml ofPBS solution with apH of 5.0 or 7.4. The sample
into 10 ml of HA (0.1 mg ml1) solution, sonicated for 10 min tubes were transferred into an orbital shaker water bath and
and centrifuged at 13 000 rpm. for 10 min to remove extra shakenat 100 rpm and37 C. Atdesignated time intervals, a
volume of 3 ml release medium was taken after the tubes were
non-adsorbed HA, so that HA-UMCS were obtained.
centrifuged at 12 000 rpm for 5 min. Another 3 ml of fresh
release medium was added into the tubes and the precipitate
was resuspended by vortex for continuous measurement. The
2.3. Characterization of nanoparticles
amount of DOX or VER in the supernatant buffer solution
An SEM (ZEISS, SUPRA 35, Germany) and a TEM (Tecnai was quantied with the UVvisible absorption spectra due to
G2 F30, FEI, The Netherlands, operated at 200 kV) were used the respective calibration curves at 490 nm and 278 nm.
to study the overall shape and pore characteristics of the
nanoparticles. A surface area instrument (V-Sorb 2800P, 2.6. Cell culture and nanoparticle uptake
Beijing, China) was used to analyze the surface area and pore
HCT-116 cells and 3T3 cells were respectively maintained in
size distribution of nanoparticles according to the Brunauer DMEMsupplemented with 10% (v:v) FBS, penicillin (1%, v:
EmmettTeller (BET) and BarrettJoynerHalenda (BJH) v) and streptomycin (1%, v:v) in 5% CO2 at 37 C. For
methods from the adsorption isotherms of N2. -potentials and confocal microscope analysis, HCT-116 cells or 3T3 cells
particle sizes were measured usinga Nanosizer Nano-ZS90 were cultured on coverslips in 24-well plates.
(Malvern Instruments Ltd, Malvern, UK). Fourier transform A quantitative study of the cellular uptake of nano-
infrared (FT-IR) spectra wereobtained usingan FT-IR particles by FACS analysis was performed as follows. HCT-
spectrometer (Bruker IFS 55, Fallanden, Switzerland) with- 116 cells or 3T3 cells were seeded in 12-well plates at
the KBr pellet technique, and thespectral region ranged from adensity of 5105 cells per well 12 h prior to study. Then
400 to 4000 cm1. the medium was removed, and the fresh cell culture medium

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Nanotechnology 27 (2016) 135102 L Wan et al

containing DOX/HA-UMCS nanoparticles were added into via the tail vein,DOX, DOX+VER, DOX/COOH-UMCS,
wells and incubated for another 2 h at 37 C. The con- VER/DOX/COOH-UMCS, DOX/HA-UMCS and VER/
centration of DOX was 1 g ml1. In the receptor competitive DOX/HA-UMCS at adose of 5 mg DOX kg1 and 5 mg
experiment, cells were pretreated with free HA macro- VERkg1, respectively. After 4 h, themice were sacriced
molecules for 1 h at 37 C prior to theaddition of nano- and tissues were excised and imaged using Carestream
particles. After incubation, the cells were rinsed with cold Molecular Imaging In Vivo MS FX PRO system at the opti-
PBSthree times, trypsinized with 0.25% pancreatin and mal wavelength (ex: 470 nm, em: 600 nm).
resuspended in PBS. The numberof nanoparticles ingested
by cells was determined by a ow cytometer (Becton Dick- 2.10. In vivo antitumor efficacy
inson FACS Calibur, Mountain View, USA). The events
measured in theFL-2 channel were tens of thousands and the Mice bearing HCT-116 tumors were randomly divided into
gated viable cells were quantied for the mean uorescence six groups (n=6) and administrated intravenously with
intensity (MFI). saline (control), DOX, DOX+VER, DOX/COOH-UMCS,
VER/DOX/COOH-UMCS, DOX/HA-UMCS and VER/
DOX/HA-UMCS via tail vein injection at days 1, 5, 9 and 13
2.7. Cytotoxicity assay
(5 mg DOX kg1 and 5 mg VER kg1). Body weight and
The in vitro cytotoxicity of DOX-Sol, VER/DOX-Sol, tumor volume were monitored and recorded every three days.
DOX/HA-UMCS nanoparticles, VER/DOX/HA-UMCS After 21 days, the mice were sacriced and the tumors were
nanoparticles and blank nanoparticles was evaluated by SRB excised, weighed and photographed. The antitumor efcacy
assay [27]. Briey, HCT-116, 3T3, MCF-7 and MCF-7/Adr was evaluated using the following formula: inhibition rate
cells were seeded in 96-well plates at a density of 1104 (%)=1We/Wc100%, where We represents the tumor
cells/well (180 l/well) overnight prior to the addition of weight in the experimental group and Wc is the tumor weight
preparations. Then, 20 l of the fresh cell culture medium in the control group.
containing serial concentrations of DOX (0.01, 0.1, 1.0, 5.0,
and 10.0 gml1) and VER (0.01, 0.1, 1.0, 5.0, and 2.11. Statistical analysis
10.0 gml1) was added into the wells cultured with the cells
for another 48 h. Simultaneously, serial concentrations of The results are expressed as the meanSD. Statistical ana-
blank HA-UMCS nanoparticles were added to study their lysis was performed using the students t-test. P<0.05 was
cytotoxicity. considered signicantly different.
After 48 h ofincubation, the cells were stained with 0.4%
SRB as previously described [28]. The absorbance was
measured at 490 nm using a microplate reader (Bio-rad, 3. Results and discussion
iMark, America). Cell growth was expressed as a percentage,
dened as the ratio of the absorbance in the treated well 3.1. Synthesis and characterization of HA-UMCS
compared with the control wellmultiplied by 100. IC50, the nanoparticles
drug concentration at which inhibition of 50% cell growth From the typical SEM image shown in gure 2(A), it can be
occurs, was calculated using SPSS 17.0. seen that UMCS nanoparticles are uniformly monodispersed
spheres with a diameter of about 350 nm, consistent with the
2.8. Animals and tumor model results of adynamic light scatteringanalysis (384 nm,
Male BALB/c nude mice aged 5 weeks (1620 g) were PDI=0.026), the uniform particle size distribution of which
purchased from the Experimental Animal Center of Shenyang helps reduce the effect of differences in size on cellular uptake
Pharmaceutical University and kept under a 12 h light/dark [30]. The representative TEM image (gure 2(B)) shows that
cycle at the Animal Care Facility. Animal experiments were the emanative slit-shaped pores of UMCS help stable drug
carried out in accordance with the Guidelines for Animal loading.
Experimentation of Shenyang Pharmaceutical University, and The nitrogen adsorptiondesorption plots of the UMCS,
the protocol was approved by the Animal Ethics Committee COOH-UMCS, NH2-UMCS and HA-UMCS nanoparticles
of this institution. The tumor model was generated by injec- (gure 3(A)) show the typical type IV isotherm and H4
tion of 1107 HCT-116 cells into the right axilla of nude hysteresis loop, corresponding to typical mesoporous mate-
mice. The tumor volume was monitored using vernier calipers rials with a narrow slit-like pore size distribution, which is
every day and calculated as V=(W2L)/2, where W and L consistent with the TEM observations. As seen from table 1,
are theshortest and longest axes of the tumor in mm, after the introduction of carboxyl groups onto the UMCS, the
respectively. The tumors were allowed to grow up to specic surface area (SBET) and pore volume (Vt) decreased to
100 mm3 before experiment. 537 m2 g1 and 0.351 cm3 g1, respectively,but the pore
diameter (WBJH) increased to 3.8 nmas a result of the
destruction on the micro-pore-channel of thenanoparticles
2.9. In vivo distribution of HA-UMCS nanoparticles
byacidic ammonium persulfate solution. In addition,the
To evaluate the in vivo tumor targeting effect of HA-UMCS micro-pore-channel was almost blocked after the functiona-
nanoparticles, the HCT-116 tumor-bearing mice were given, lization of ethanediamine (gure 3(B))resulted in a certain

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Nanotechnology 27 (2016) 135102 L Wan et al

Figure 2. SEM image (A) and TEM image (B) of UMCS nanoparticles.

Table 1. N2 Adsorptiondesorption parameters of different


functionalized UMCS nanoparticles.
Sample SBET (m2 g1) Vt (cm3 g1) Dp (nm)
UMCS 1113 0.753 3.6
COOH-UMCS 537 0.351 3.8
NH2-UMCS 215 0.235 3.8
HA-UMCS 166 0.185 3.8

Figure 4. -potential analysis of COOH-UMCS, NH2-UMCS and


HA-UMCS nanoparticles in PBS solution.

the successful modication of HA onto the nanoparticles. The


unchanged type IV isotherm (gure 3(A)) and pore diameter
(gure 3(B)) conrmed that the HA was grafted onto the
surface of NH2-UMCS, rather than in the pore channels.
The surface modication of the UMCS nanoparticles was
investigated using a -potential instrument. As can be seen
Figure 3. N2adsorption isotherms (A) and the pore size distribution from gure 4(a), the -potential of COOH-UMCS, NH2-
curves (B). UMCS and HA-UMCS is 25.4, +24.9 and 20.8 mV,
respectively, conrming the successful modication of the
amine groups (NH2-UMCS) on the negatively charged car-
decrease in SBET and Vt. However, the effect of destruction of boxylated UMCS nanoparticles surface (COOH-UMCS), and
micro-pore-channels on thedrug loading of nanoparticles is subsequent capping of HA on the positively charged NH2-
not signicant, asthe micro-pore-channel is too small to UMCS. The negatively charged surface of HA-UMCS
encapsulate drug molecules. HA-functionalized NH2-UMCS nanoparticles maintainslong-term stability in the blood cir-
showed a sharp reductionin SBET and Vt (table 1), suggesting culation system. The results of the transmittance spectrometry

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Nanotechnology 27 (2016) 135102 L Wan et al

Figure 5. FT-IR spectra of (a) pristine uniform mesoporous carbon Figure 6. In vitro release proles of VER and DOX from HA-UMCS
spheres, (b) COOH-UMCS, (c) NH2-UMCS, (d) HA-UMCS. nanoparticles in the presence of Hyal-1 and without Hyal-1, in
pH 7.4 and 5.0 PBS buffer. Data represent the meanSD, n=3.

analysis conrmed the good dispersion stability of the HA-


between DOX and HA. After the introduction of Hayl-1,
UMCS nanoparticles, and less than4% variation in trans-
approximately 75% of DOX was released within 24 h fol-
mittance was obtained in 72 h (data not shown).
lowing incubation at pH 5.0, about 7 times higher than the
The FT-IR spectrum was also used to monitor the action
group without Hayl-1, which indicated that the gate keeper
of UMCS nanoparticle medication. As shown in gure 5, a
HA was broken down into fragments by Hayl-1, and could no
new intense band at 1724 cm1 emerged after carboxylated
longer stop the drug release. Furthermore, VER showed a
modication of the UMCS nanoparticles, the characteristic
faster release rate than DOX, becauseVER was loaded in the
peak of COOH groups. With subsequent ethylenediamine
outer pores of theUMCS and was very water soluble at a
conjugation, the COOH peak disappeared and overlapped
pH of 5.0. The faster release of VER guarantees the efcient
stretching vibrations of CONH and NH groups were seen
suppression of P-gp producing a high DOX accumulation in
at 1580 cm1. In addition, the presence of a new wide band at
cells. This specic pH and enzyme-triggered drug release
1299 cm1 corresponded to CN bond stretching. After the
from HA-UMCS is highly desirable for cancer-targeted drug
capping of HA, two new peaks at 1110 and 1166 cm1
delivery since it would signicantly minimizepremature drug
appeared, indicating the CO stretching vibrations from C
release during circulation in the blood, yet ensure a sufcient
OC of HA. A weak band at 1454 cm1 was due to the
level of drug in the tumor tissues with the combined passive
bending vibration of CH3 and the carboxyl peak was
and active targeting of nanoparticles.
detected again, but masked by the strong CONH stretch
vibration bands. The FT-IR results conrmed that HA mac-
romolecules were successfully attached to the UMCS nano-
3.3. Cellular uptake of DOX/HA-UMCS nanoparticles
particles, and this was consistent with the results of the -
potential analysis. To study the selective cellular uptake of the HA-UMCS
nanoparticles, encapsulated DOX was used as a uorescence
probe and the targeting effect was visualized by confocal
3.2. In vitro release of VER and DOX from functionalized
microscopy. As shown in gure 7(B), stronger red uorescent
nanoparticles
signals of DOX were apparent inside the HCT-116 cells,
A UVvis analysis showed that the drug-loading ratio of demonstrating the HA-UMCS nanoparticles are taken up by
VER:DOX:NH2-UMCS was 1:1:2. The in vitro release pro- the HA receptors overexpressing cancer cells. Bycontrast, in
le of DOX and VER from VER/DOX/HA-UMCS nano- the 3T3 cell group (gure 7(A)), a weaker red uorescent
particles was performed in phosphate buffer solution at a signal was observed, suggesting that the cells without over-
pH of 7.4 (the physiological pH) and a pH of 5.0 (the expression of HA receptors were not subjected to the invasion
endosomal pH) at a temperature of 37 C. Hyaluronidase-1 of nanoparticles. Furthermore, when the HCT-116 cells were
(150 U ml1) was used in vitro to mimic the in vivo specic pre-cultured with free HA, the uptake of the HA-UMCS
enzyme-triggered controlled release in cancer regions. As nanoparticles was markedly reduced because of the blocking
shown in gure 6, without Hayl-1, only about 4% and 11% of of CD44 receptors (gure 8).
DOX release was obtained in pH 7.4 and pH 5.0 PBS buffer To achieve a quantitative comparison, the endocytosis of
solution, respectively. The acidic solution of pH 5.0 improved DOX/HA-UMCS nanoparticles by HCT-116 cell lines or
the cumulative DOX release, due to the increased solubility of 3T3 cells was further analyzed by ow cytometry. As shown
DOX at the lower pH and dissociation of electrostatic binding in gure 7(C), the MFIvalues show that HCT-116 cells took

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Nanotechnology 27 (2016) 135102 L Wan et al

signal intensity showed a sharp decline in a concentration-


dependent manner, indicating that the interaction between
HA-UMCS nanoparticles and CD44 receptors was weakened
by the competition of free HA macromoleculesand there-
duction in the subsequent HA receptor-mediated endocytosis
of nanoparticles. We also carried out experiments at +4 C to
study the ATP-dependent internalization by high-afnity HA
receptors, which are inactive at this low temperature. The
uptake of HA-UMCS nanoparticles was obviously inhibited
(gure 8). The above results proved that HA-UMCS nano-
particles can specically target CD44-overexpressing cancer
cells through HA-receptor-mediated endocytosis and there is
a reduction in nanoparticle invasion in the cells without
overexpression of CD44 receptors.

3.4. In vitro cytotoxicity

The in vitro cytotoxicity of various DOX preparations and


blank HA-UMCS nanoparticles was established in HCT-116,
3T3, MCF-7 and MCF-7/Adr cells using the SRB assay. The
results in gure 9 show that HA-UMCS are almost com-
pletely non-toxic to all four cell lines, indicating that HA-
UMCS are excellent biocompatible nanocarriers within the
Figure 7. Specic endocytosis of DOX/HA-UMCS nanoparticles by test concentration range. A dose-dependent cytotoxicity to all
(A) 3T3 cells and(B) HCT-116 cells. The MFIof DOX-labeled
nanoparticles in cells was measured using ow cytometry, and the
the test cells was observed for free DOX and DOX-loaded
values were normalized to the values of the HCT-116 cells (C). NPs. As shown in gures 9(A) and (B), the formulation of
DOX/HA-UMCS was more cytotoxic to HCT-116 cells than
to 3T3 cells, consistent with the IC50 results (0.263 g
ml1for HCT-116 cells and 24.501 g ml1 for 3T3 cells).
The increased cytotoxicity could be attributed to the increased
cellular uptake of HA-UMCS by CD44 receptor-over-
expressing cancer cells through receptor mediated endocy-
tosis. Simultaneously, the HA surface modication decreased
the interaction between NPs and normal cells aslike charges
repel, so that HA-UMCS do not only improve the anticancer
effects of DOX on CD44 receptors overexpressing cancer
cells, but also reduce the side effects on normal cells.
Also, as can be seen from gures 9(C) and (D), the co-
delivery of VER with DOX was more cytotoxic to the studied
cells compared with DOX alone, especially to the MCF-7/
Adr cell line. Due to the overexpression of P-gp, the IC50
value of DOX for MCF-7/Adr cells was as high as 32.633 g
ml1 (table 2), which was about 76 times more resistant
to DOX compared with the original MCF-7 cells
(IC50=0.43 g ml1). After the addition of VER, the cyto-
toxicity of DOX was increased as a result of the increased
DOX intracellular accumulation. In addition, the group of
Figure 8. The intracellular DOX uorescence of functionalized DOX+VER exhibited the strongest anticancer effects, the
nanoparticles was analyzed using ow cytometry. Alternatively, free reason for which may be the fact that diffusion of free drug
HA was added or the cells were maintained at +4 C. molecules into cells ensures faster and greater cytotoxic
effects in comparison with drug-loaded nanoparticles thatare
up ve times more DOX/HA-UMCS nanoparticles than 3T3 initially internalized into cells, with subsequent intracellular
cells, as a result of the apparent difference in the number of release and production of cell death.
CD44 receptors. To further conrm the specic interaction of However, no-one can ignore the side effects produced by
HA-UMCS nanoparticles with CD44 receptors of HCT-116 free DOX and VER in clinical situations. Therefore, a tar-
cells, free HA macromolecules were added to the culture geted drug delivery system is essential for the successful
medium prior to the addition of DOX/HA-UMCS nano- treatment of cancer. In addition, the co-delivery of DOX and
particles. As can be seen from gure 8, the mean uorescent VER via HA-UMCS resulted in markedly increased

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Nanotechnology 27 (2016) 135102 L Wan et al

Figure 9. Cytotoxicity of blank nanoparticles and different DOX formulations in 3T3 cells (A), HCT-116 cells (B), MCF-7 cells (C) and
MCF-7/Adr cells. Results are expressed as the meanSD, n=6. The concentration of blank HA-UMCS was consistent with the drug
loading (two times the concentration of DOX).

chemotherapeutic agents can signicantly increase the antic-


Table 2. IC50 values of free DOX, free VER/DOX, DOX/HA-
ancer effects.
UMCS and VER/DOX/HA-UMCS nano-formulations on cancer
cells.
IC50 (g ml1) 3.5. In vivo distribution of HA-UMCS nanoparticles
MCF-
Samples 3T3 HCT-116 MCF-7 7/Adr The in vivo distribution of different DOX preparations was
observed using the FX Pro in vivo imaging system. As shown
DOX 2.861 1.700 0.430 32.633 in gure 10, DOX, even although administered with VER,
VER/DOX 2.903 0.978 0.242 1.195 exhibited a low accumulation in tumors, indicating that the
DOX/HA-UMCS 24.501 0.263 6.283 18.448 combination of DOX with VER did not improve the tumor-
VER/DOX/ 27.955 0.323 5.360 3.572
specic uptake of DOX. Simultaneously, the relatively high
HA-UMCS
concentration of DOX in normal tissues would produce
severe systemic toxicity. By contrast, when DOX was loaded
in the HA-UMCS nanoparticles, the uorescence signal
intensities in the tumor were markedly increased, suggesting
cytotoxicity to MCF-7/Adr cells compared with the free that HA-modied UMCS nanoparticles can specically deli-
DOX or DOX/HA-UMCS, indicating a successful intracel- ver DOX to CD44-overexpressing cancer cells in vivo. Co-
lular release of VER that inhibits the efux of P-gp and delivery with VER increased the content of DOX in tumors
increases the intracellular accumulation of DOX. Taken producing the successful suppression of P-gp by VER.
together, the multifunctional targeted drug delivery system Although a certain numberof non-targeted nanoparticles
HA-UMCS for the combination of MDR modulators and COOH-UMCS permeate into the tumor sites with the blood

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Nanotechnology 27 (2016) 135102 L Wan et al

Figure 10. Fluorescence images of tissues and tumor at 4 h after intravenous administration of different DOX preparations.

Figure 11. Antitumor effects of different DOX preparations on HCT-116 tumor-bearing nude mice. (A) Changes in the tumor volume and (B)
body weight after administration. (C) Representative images of excised HCT-116 tumors. (D) The tumor weight of the excised tumors. The
different groups of (C) and (D) are(a) saline, (b) DOX, (c) DOX+VER, (d) DOX/COOH-UMCS, (e) VER/DOX/COOH-UMCS (f)
DOX/HA-UMCS and (g) VER/DOX/HA-UMCS groups. Data are expressed as the meanSD, n=6. Statistical signicance: *P<0.05
and ***P<0.0005.

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Nanotechnology 27 (2016) 135102 L Wan et al

circulation, there was a signicant difference in DOX content body weight did not change much, conrming the absence of
between the targeted and non-targeted groups.The reason for any serious systemic toxicity. The high antitumor activity and
this may be that the passive target efciency is much weaker low systemic toxicity make HA-UMCS an ideal nanodrug
than that of the active target and the uncontrolled-release delivery system for the co-delivery of chemotherapeutic drugs
delivery system COOH-UMCS could sustain most of the and P-gp inhibitors into tumors for more effective cancer
DOX released in the target tissues. The uncontrolled-release treatment.
COOH-UMCS leads to the leakage of DOX during circula-
tion in the blood, increasing the accumulation of DOX in
normal tissues. Therefore, the targeted drug delivery system 4. Conclusion
ofHA-UMCS nanoparticles examined in our study does not
only improve the antitumor effect of DOX, but also reduces In summary, HA-UMCS exhibit a variety of attractive proper-
the damage to normal tissues. ties in terms of targeted anticancer co-delivery, such asstable
drug encapsulation in mesopores of carbon nanoparticles,
3.6. In vivo antitumor efficacy
improved targeted cellular internalization, and stimulated release
within cancer cells with inactivation of P-gp increasing drug
To investigate whether the increased cytotoxicity and targeted accumulation. Therefore, HA-UMCS loaded with DOX and
biodistribution would bring about improved therapeutic VER can signicantly increase the cell cytotoxicity and induce
effects, the antitumor efcacy was examined in mice with cell apoptosis. Importantly, VER/DOX/HA-UMCS exhibited
HCT-116 tumors. As can be seen from gure 11, free DOX high cancer accumulation and anticancer effects with low sys-
displayed a signicant in vivo tumor inhibition effect com- temic toxicity. This versatile co-delivery system based on HA-
pared with the saline group. The tumor growth rate was conjugated carbon nanoparticles is a promising targeted delivery
reduced in a way that produced a nal tumor volume, and nanovehicle for efcient combination cancer therapy.
weight of the free DOX group was 62.09% and 62.32% of
saline group, respectively. The DOX/COOH-UMCS group
exhibited a similar inhibitory effect as the free DOX group, Acknowledgments
with a 39.86% and 41.39% suppression of the tumor volume
and tumor weight, respectively. In addition, the addition of This work was supported by the National Basic Research
VER did not produce any obvious improvement in antitumor Program of China (973 Program) (No. 2015CB932100),
efcacy, either in combination with free DOX or encapsulated National Natural Science Foundation of China (No.
in DOX/COOH-UMCS where the VER was released before 81273449).
the nanoparticles arrived at the tumor site, indicating that free
VER would not increase the antitumor efcacy of DOX at a
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