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Hyaluronic acid modified mesoporous carbon nanoparticles for targeted drug delivery to
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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.
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
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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.
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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.
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Nanotechnology 27 (2016) 135102 L Wan et al
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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).
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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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