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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 attractionstrategy. This HAmodification 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 fluorescentlylabeled mesoporous carbon nanoparticles, with or without HA functionalization, demonstrated that HA-UMCS are able to specifically 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 figures may appear in colour only in the online journal) large specific surface area (orpore volume) andtunable 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 recentdecades, a variety of nanocarriers have been proposed and studied for the delivery of nanomedicines [7, 8, 22]. Compared with conventional polymer-based or organic lipidnanoparticles, inorganic matrix nanoparticles exhibit some particular properties that make thempotential 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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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
efflux.
immunogenicity and biodegradability [6, 15, 16, 25, 26]. Simutaneously, HA can specifically bind to CD44 receptors which are overexpressed in many typesof 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 itsalready excellent propertiesthrough the synergistic beneficial combination of UMCS and HA. The advantagesof HA capping UMCS include: (1) HA improves the physiological dispersion stability of UMCS necessary for intravenous administration [2]; (2) itslarge molecular size hinders drug leakage into the blood circulation; (3) itshydrophilic nature prolongs the blood circulation time of nanoparticles like PEG [3, 20]; (4) itsspecific 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 acceleratingdrug release. In addition, doxorubicin (DOX) and verapamil (VER) were encapsulated inthe 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 usingP-gp glycoprotein.
targeted drug delivery [11].Mesoporous carbon nanocarriers can alsoprotect the encapsulated agents from degradation during delivery, as well as improve their membranepermeability [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 thatpumpout 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 thatcankill cancer cells usingsynergistic 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 forencapsulatingdrug 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 ofPBS (pH 7.4) buffer solution, ultrasonicated and stirred for 24 h in darkness at room temperature. The DOX-loaded NH2-UMCSwerecollected 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 theNH2UMCSby solvent evaporation. The previous product (DOX/ NH2-UMCS) was added into10 ml of ethanol solution of verapamil (verapamil:NH2-UMCS=1:2, wt). Then, the mixture was stirred for 24 h to ensure thatverapamil adsorbed homogeneously onto the outer pore channels of theNH2UMCS.The ethanol in samples was thenremoved 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 thesame as the method above. To evaluate the DOX loading efficiency, the unbound DOX was measuredusing ultraviolet (UV) spectroscopy (UV-2000, Unico, USA) at 490 nm relative to a calibration curve performed underidentical 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 andtetraethyl orthosilicatewere purchased from Sigma-Aldrich (StLouis, MO, USA). Fluorescent probes containing Hoechst 33342, rhodamine-phalloidin were bought from Molecular Probes Inc. (Eugene, OR, USA). Bovine serum albuminwas purchased from Amreso (USA). Cell culture Dulbecco’s Modified EagleMedium (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
UMCSof 350 nm diameter were preparedusing aspherical nanosilica matrix (SNM) as a template and furfuryl alcohol as acarbon precursor, as previously described [28]. AnATS AH100D homogenizer (ATS Engineer Inc., China) was used to increase the homogeneity of the SNM. After procedural carbonization, aSi–C composite was added to 10% hydrofluoric acid to remove the silica template, and UMCSwere obtained. Then, acarboxyl group was introduced to the surface of theUMCSby 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 noxideas the coupling agent, by sonication for 4 h to prepare NH2-UMCS [29]. Finally, NH2-UMCS (1 mg) wereadded 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 theVER/ DOX/HA-UMCS nanoparticles was performed in triplicate at apH of 7.4 (the physiological blood circulation pH) and apH 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 ofPBS solution with apH of 5.0 or 7.4. The sample tubes were transferred into an orbital shaker water bath and shakenat 100 rpm and37 °C. Atdesignated 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 usinga Nanosizer Nano-ZS90 (Malvern Instruments Ltd, Malvern, UK). Fourier transform infrared (FT-IR) spectra wereobtained usingan FT-IR spectrometer (Bruker IFS 55, Fallanden, Switzerland) withthe KBr pellet technique, and thespectral 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 DMEMsupplemented 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 adensity 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 theaddition of nanoparticles. After incubation, the cells were rinsed with cold PBSthree times, trypsinized with 0.25% pancreatin and resuspended in PBS. The numberof nanoparticles ingested by cells was determined by a flow cytometer (Becton Dickinson FACS Calibur, Mountain View, USA). The events measured in theFL-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 adose of 5 mg DOX kg−1 and 5 mg VERkg−1, respectively. After 4 h, themice 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 μgml−1) and VER (0.01, 0.1, 1.0, 5.0, and 10.0 μgml−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 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, iMark, America). Cell growth was expressed as a percentage, defined as the ratio of the absorbance in the treated well compared with the control wellmultiplied 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