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Hyaluronic acid modified mesoporous carbon nanoparticles for targeted drug delivery to CD44-overexpressing cancer cells

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Nanotechnology Nanotechnology 27 (2016) 135102 (11pp)

doi:10.1088/0957-4484/27/13/135102

Hyaluronic acid modified 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, People’s Republic of China 2 Department of Pharmacy, The First Affiliated Hospital of China Medical University, Nanajingbei Street 155, Shenyang, People’s Republic of China 3 College of Pharmaceutical Science, China Medical University, Puhe Road 77, Shenyang 110122, People’s Republic of China 4 College of Life Science and Health, Northeastern University, Wenhuadong Road 89, Shenyang 110015, People’s Republic of China E-mail: [email protected] 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 attractionstrategy. This HAmodification ensuredstable drug encapsulation in mesoporous carbon nanoparticles in anextracellular environment while increasingcolloidal stability, biocompatibility, cell-targeting ability, and controlled cargo release. The cellular uptake experiments of fluorescentlylabeled mesoporous carbon nanoparticles, with or without HA functionalization, demonstrated that HA-UMCS are able to specifically target cancer cells overexpressing CD44 receptors. Moreover, thecargoloadeddoxorubicin (DOX) and verapamil (VER) exhibiteda dual pH and hyaluronidase-1 responsive release in the tumor microenvironment. In addition,VER/DOX/HA-UMCS exhibited a superior therapeutic effect on anin vivo HCT-116 tumor in BALB/c nude mice. In summary, it is expected that HA-UMCS will offer a new methodfor targeted co-delivery of drugs to tumors overexpressing CD44 receptors. Keywords: mesoporous carbon nanoparticles, hyaluronic acid, controlled release, co-delivery (Some figures may appear in colour only in the online journal) large specific surface area (orpore volume) andtunable size(or morphology or pore structure) allow these inorganic mesoporous materials to selectively encapsulate different drugs of interest and to be tuned to optimize cellular uptake [9, 17, 31]. In particular, mesoporous carbon nanoparticles with a strong adsorption capacity [10, 23],π–π stacking interactions [12, 14] and easily functionalized surface [19] have attracted great interest with regard to

1. Introduction In recentdecades, a variety of nanocarriers have been proposed and studied for the delivery of nanomedicines [7, 8, 22]. Compared with conventional polymer-based or organic lipidnanoparticles, inorganic matrix nanoparticles exhibit some particular properties that make thempotential therapeutic and diagnostic agents [13, 21, 24, 32]. Their 0957-4484/16/135102+11$33.00

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© 2016 IOP Publishing Ltd Printed in the UK

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

efflux.

immunogenicity and biodegradability [6, 15, 16, 25, 26]. Simutaneously, HA can specifically bind to CD44 receptors which are overexpressed in many typesof cancer cells [4, 18]. In addition, HA-functionalized nanomedicine delivery systems can penetrate cancer cells more efficiently through HA receptormediated endocytosis. The biocompatibility and specific affinity of HA for CD44 receptors ensures its great application potential for treatments targeting cancer. In this research, as shown in figure 1, a uniform mesoporous carbon spheres (UMCS)-based inorganic/organic drug delivery system has been developed for the first time to improve itsalready excellent propertiesthrough the synergistic beneficial combination of UMCS and HA. The advantagesof HA capping UMCS include: (1) HA improves the physiological dispersion stability of UMCS necessary for intravenous administration [2]; (2) itslarge molecular size hinders drug leakage into the blood circulation; (3) itshydrophilic nature prolongs the blood circulation time of nanoparticles like PEG [3, 20]; (4) itsspecific interaction with CD44 receptors improves the selective targeting of nanoparticles; (5) HA macromolecules are rapidly degraded and broken down into fragments by the hyaluronidases abundant in malignant tumors [1, 5, 25] thereby acceleratingdrug release. In addition, doxorubicin (DOX) and verapamil (VER) were encapsulated inthe mesopores of the core UMCS for combination therapy at high drug loading (25% for each drug). For the first time, human colon cancer HCT-116 cells were inoculated into BALB/c nude mice to obtain in vivo antitumor effects. The results we obtained have confirmed the specific selectivity of HA-UMCS and an enhanced efficiency in treating CD44 receptors overexpressed cancer cells, while effectively inhibiting the DOX efflux usingP-gp glycoprotein.

targeted drug delivery [11].Mesoporous carbon nanocarriers can alsoprotect the encapsulated agents from degradation during delivery, as well as improve their membranepermeability [32]. Multidrug resistance (MDR) is one of the main obstacles to successful chemotherapy treatment of cancer. MDR is often caused by the overexpression of a 170 kDa glycoprotein within the plasma membrane known as P-glycoprotein (P-gp). In addition, a wide range of anticancer drugs are substrates of P-gp thatpumpout the drug molecules from the inside of cancer cells, reducing intracellular drug accumulation. Therefore, if the activity of P-gp could be restrained, we would obtain double the anticancer effects with half the dose. Therefore, combination therapy has been used in chemotherapy treatment thatcankill cancer cells usingsynergistic effects before the cancer adapts, reducing the probability of cancer cell mutation. However,widespread application of combination therapy is limited since the different membrane transport properties and biodistribution of many drugs results in suboptimal drug combinations in the tumor region. Due to their high drug-loading capacity and their extensive applicability forencapsulatingdrug molecules with different natures, mesoporous carbon nanoparticles make the simultaneous delivery of multiple agents for combination therapy possible. A perfect targeted drug delivery system should transport toxic agents to the specific target site without any drug leakage into the blood circulation, while allowing the drug to be released quickly at the target site based on an environmental change. Hyaluronic acid (HA), a natural anionic glycosaminoglycan, is one of the major components of the extracellular matrix in normal tissues like epithelial, connective, and neural tissues, and it exhibits good biocompatibility, non-

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2. Materials and methods

2.4. Preparation of DOX and VER loaded nanoparticles

Firstly, DOX hydrochloride (40 mg) and NH2UMCS(10 mg) were added into 10 ml ofPBS (pH 7.4) buffer solution, ultrasonicated and stirred for 24 h in darkness at room temperature. The DOX-loaded NH2-UMCSwerecollected by centrifugation at 13 000 rpm for 10 min and repeated washing with PBS (pH 7.4). Secondly, verapamil was encapsulated into the outer mesopores of theNH2UMCSby solvent evaporation. The previous product (DOX/ NH2-UMCS) was added into10 ml of ethanol solution of verapamil (verapamil:NH2-UMCS=1:2, wt). Then, the mixture was stirred for 24 h to ensure thatverapamil adsorbed homogeneously onto the outer pore channels of theNH2UMCS.The ethanol in samples was thenremoved by constant stirring at 40 °C. Thirdly, HA macromolecules were grafted onto the surface of the VER/DOX/NH2-UMCS by electrostatic attraction. The drug-loaded process of COOHUMCS was thesame as the method above. To evaluate the DOX loading efficiency, the unbound DOX was measuredusing ultraviolet (UV) spectroscopy (UV-2000, Unico, USA) at 490 nm relative to a calibration curve performed underidentical conditions. The loading efficiency of DOX could be calculated according to the original and residual DOX concentrations and volumes. The calculation equation of DOX follows below. The loading amount of VER was almost the same as the initial addition amount.

2.1. Materials

Hexadecylamine andtetraethyl orthosilicatewere purchased from Sigma-Aldrich (StLouis, MO, USA). Fluorescent probes containing Hoechst 33342, rhodamine-phalloidin were bought from Molecular Probes Inc. (Eugene, OR, USA). Bovine serum albuminwas purchased from Amreso (USA). Cell culture Dulbecco’s Modified EagleMedium (DMEM), penicillin-streptomycin, fetal bovine serum (FBS) were purchased from GIBCO, Invitrogen Co. (Carlsbad, USA). Trypsin and phosphate buffer solution (PBS) were obtained from Mcgene Co (Beijing, China). Sodium hyaluronate (MW≈100 KDa) was a product of Shanghai Alading Biochem Co. Ltd (China). All the other reagents in the experiments were of analytical grade and used without additional purification.

2.2. Preparation of nanoparticles

UMCSof 350 nm diameter were preparedusing aspherical nanosilica matrix (SNM) as a template and furfuryl alcohol as acarbon precursor, as previously described [28]. AnATS AH100D homogenizer (ATS Engineer Inc., China) was used to increase the homogeneity of the SNM. After procedural carbonization, aSi–C composite was added to 10% hydrofluoric acid to remove the silica template, and UMCSwere obtained. Then, acarboxyl group was introduced to the surface of theUMCSby wet oxidation. Then,200 mg of the COOH-UMCS was dispersed in 100 ml of ethylenediamine, using N-[(dimethylamino)-1H-1,2,3-triazolo [4–6] pyridin-1ylmethylene]-N-methylmethanaminium hexafluorophosphate noxideas the coupling agent, by sonication for 4 h to prepare NH2-UMCS [29]. Finally, NH2-UMCS (1 mg) wereadded into 10 ml of HA (0.1 mg ml−1) solution, sonicated for 10 min and centrifuged at 13 000 rpm. for 10 min to remove extra non-adsorbed HA, so that HA-UMCS were obtained.

2.5. In vitro release experiment

The in vitro release profile of DOX and VER from theVER/ DOX/HA-UMCS nanoparticles was performed in triplicate at apH of 7.4 (the physiological blood circulation pH) and apH of 5.0 (the endosomal pH of cancer cells). Briefly, 1 mg of DOX was introduced into a centrifuge tube and dispersed in 5 ml ofPBS solution with apH of 5.0 or 7.4. The sample tubes were transferred into an orbital shaker water bath and shakenat 100 rpm and37 °C. Atdesignated time intervals, a volume of 3 ml release medium was taken after the tubes were 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 amount of DOX or VER in the supernatant buffer solution was quantified with the UV–visible absorption spectra due to the respective calibration curves at 490 nm and 278 nm.

2.3. Characterization of nanoparticles

An SEM (ZEISS, SUPRA 35, Germany) and a TEM (Tecnai G2 F30, FEI, The Netherlands, operated at 200 kV) were used to study the overall shape and pore characteristics of the nanoparticles. A surface area instrument (V-Sorb 2800P, Beijing, China) was used to analyze the surface area and pore size distribution of nanoparticles according to the Brunauer– Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH) methods from the adsorption isotherms of N2. ζ-potentials and particle sizes were measured usinga Nanosizer Nano-ZS90 (Malvern Instruments Ltd, Malvern, UK). Fourier transform infrared (FT-IR) spectra wereobtained usingan FT-IR spectrometer (Bruker IFS 55, Fallanden, Switzerland) withthe KBr pellet technique, and thespectral region ranged from 400 to 4000 cm−1.

2.6. Cell culture and nanoparticle uptake

HCT-116 cells and 3T3 cells were respectively maintained in DMEMsupplemented with 10% (v:v) FBS, penicillin (1%, v: v) and streptomycin (1%, v:v) in 5% CO2 at 37 °C. For confocal microscope analysis, HCT-116 cells or 3T3 cells were cultured on coverslips in 24-well plates. A quantitative study of the cellular uptake of nanoparticles by FACS analysis was performed as follows. HCT116 cells or 3T3 cells were seeded in 12-well plates at adensity of 5×105 cells per well 12 h prior to study. Then the medium was removed, and the fresh cell culture medium 3

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containing DOX/HA-UMCS nanoparticles were added into wells and incubated for another 2 h at 37 °C. The concentration of DOX was 1 μg ml−1. In the receptor competitive experiment, cells were pretreated with free HA macromolecules for 1 h at 37 °C prior to theaddition of nanoparticles. After incubation, the cells were rinsed with cold PBSthree times, trypsinized with 0.25% pancreatin and resuspended in PBS. The numberof nanoparticles ingested by cells was determined by a flow cytometer (Becton Dickinson FACS Calibur, Mountain View, USA). The events measured in theFL-2 channel were tens of thousands and the gated viable cells were quantified for the mean fluorescence intensity (MFI).

via the tail vein,DOX, DOX+VER, DOX/COOH-UMCS, VER/DOX/COOH-UMCS, DOX/HA-UMCS and VER/ DOX/HA-UMCS at adose of 5 mg DOX kg−1 and 5 mg VERkg−1, respectively. After 4 h, themice were sacrificed and tissues were excised and imaged using Carestream Molecular Imaging In Vivo MS FX PRO system at the optimal wavelength (λex: 470 nm, λem: 600 nm). 2.10. In vivo antitumor efficacy

Mice bearing HCT-116 tumors were randomly divided into six groups (n=6) and administrated intravenously with 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 (5 mg DOX kg−1 and 5 mg VER kg−1). Body weight and tumor volume were monitored and recorded every three days. After 21 days, the mice were sacrificed and the tumors were excised, weighed and photographed. The antitumor efficacy was evaluated using the following formula: inhibition rate (%)=1−We/Wc×100%, where We represents the tumor weight in the experimental group and Wc is the tumor weight in the control group.

2.7. Cytotoxicity assay

The in vitro cytotoxicity of DOX-Sol, VER/DOX-Sol, DOX/HA-UMCS nanoparticles, VER/DOX/HA-UMCS nanoparticles and blank nanoparticles was evaluated by SRB assay [27]. Briefly, HCT-116, 3T3, MCF-7 and MCF-7/Adr cells were seeded in 96-well plates at a density of 1×104 cells/well (180 μl/well) overnight prior to the addition of preparations. Then, 20 μl of the fresh cell culture medium containing serial concentrations of DOX (0.01, 0.1, 1.0, 5.0, and 10.0 μgml−1) and VER (0.01, 0.1, 1.0, 5.0, and 10.0 μgml−1) was added into the wells cultured with the cells for another 48 h. Simultaneously, serial concentrations of blank HA-UMCS nanoparticles were added to study their cytotoxicity. After 48 h ofincubation, 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, iMark, America). Cell growth was expressed as a percentage, defined as the ratio of the absorbance in the treated well compared with the control wellmultiplied by 100. IC50, the drug concentration at which inhibition of 50% cell growth occurs, was calculated using SPSS 17.0.

2.11. Statistical analysis

The results are expressed as the mean±SD. Statistical analysis was performed using the student’s t-test. P

Hyaluronic acid modified mesoporous carbon nanoparticles for targeted drug delivery to CD44-overexpressing cancer cells.

In this paper, hyaluronic acid (HA) functionalized uniform mesoporous carbon spheres (UMCS) were synthesized for targeted enzyme responsive drug deliv...
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