Phospholipid-stabilized mesoporous carbon nanospheres as versatile carriers for systemic delivery of amphiphobic SNX-2112 (a Hsp90 inhibitor) with enhanced antitumor effect

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Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

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Research Paper

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Phospholipid-stabilized mesoporous carbon nanospheres as versatile carriers for systemic delivery of amphiphobic SNX-2112 (a Hsp90 inhibitor) with enhanced antitumor effect

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Xingwang Zhang 1, Tianpeng Zhang 1, Yanghuan Ye, Huaqing Chen, Hua Sun, Xiaotong Zhou, Zhiguo Ma, Baojian Wu ⇑

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Division of Pharmaceutics, College of Pharmacy, Jinan University, PR China

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a r t i c l e

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Article history: Received 31 December 2014 Revised 21 April 2015 Accepted in revised form 22 April 2015 Available online xxxx Keywords: SNX-2112 Mesoporous carbon Amphiphobic Breast cancer Antitumor effect

a b s t r a c t Systemic delivery of amphiphobic drugs (insoluble in both water and oil) represents a formidable challenge in drug delivery. This work aimed to engineer a functional mesoporous carbon material to efficiently load SNX-2112, an amphiphobic anticancer agent, and to evaluate its performance in tumor-targeting delivery. Hydrothermal reaction combined with high-temperature activation was used to fabricate glucose-based mesoporous carbon nanospheres (MCNs). SNX-2112-loaded MCNs stabilized by phospholipid (SN-PMCNs) were prepared by the absorption/solvent diffusion/high-pressure homogenization method. The obtained SN-PMCNs were 180 nm around in particle size, showing a high drug load (42.7%) and acceptable physical stability. SN-PMCNs demonstrated an enhanced in vitro antitumor effect and increased uptake into cancer cells in comparison with the formulation of SNX-2112 solution (SN-Sol). The in vivo antitumor effect and biodistribution in 4T1 xenograft tumor mice, a breast cancer model, were also significantly improved through SN-PMCNs. It was shown that specific clathrin-dependent and nonspecific caveolae-dependent endocytosis were involved in the cellular trafficking of SN-PMCNs. Glucose transporter-mediated transport, prolonged body residence time and improved biodistribution via EPR effect were the main mechanisms of enhanced antitumor effect. SN-PMCNs have presented excellent tumor targeting properties and should be a promising carrier to address the systemic delivery of SNX-2112. Ó 2015 Published by Elsevier B.V.

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1. Introduction

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Advances in chemistry, drug design and bioactivity screening have significantly accelerated the drug discovery, resulting in numerous highly active entities. However, the majority of drug candidates are plagued with poor solubility and/or permeability.

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Abbreviations: CNs, carbon nanospheres; MCNs, mesoporous carbon nanospheres; SN-PMCNs, SNX-2112-loaded MCNs stabilized by phospholipid; SN-Sol, SNX-2112 solution; EPR effect, enhanced permeability and retention effect; BCS, biopharmaceutics classification system; BCA, bicinchoninic acid; SEM, scanning electron microscope; TEM, transmission electron microscope; DL, drug load; MWCO, molecular weight cut-off; PBS, phosphate buffered saline; FTIR, fourier transform infrared spectroscopy; PDI, polydispersity index; IC50, half maximal inhibitory concentration; RES, reticuloendothelial system. ⇑ Corresponding author at: Division of Pharmaceutics, College of Pharmacy, Jinan University, 601 West Huangpu Avenue, Guangzhou 510632, PR China. Tel./fax: +86 20 85220482. E-mail address: [email protected] (B. Wu). 1 These authors contributed equally to this work.

In fact, over 70% of drugs on the market or under development are identified as BCS II or IV ones [1], many of which are simultaneously insoluble in water and oil (i.e. amphiphobic). Typical amphiphobic drugs include anticancer agents, such as raloxifene [2], paclitaxel [3], and Z-GP-Dox (a doxorubicin derivative) [4]. Systemic delivery of amphiphobic drugs is of high challenge due to poor solubility and incompatibility with excipients. To achieve systemic delivery of insoluble drugs, a variety of nanoparticle-based formulations have been explored, including liposomes [5], polymeric micelles [6], polymeric nanoparticles [7], lipid nanocarriers [8], and nanocrystals [9]. Although these systems have demonstrated potential in solubilization and targeted delivery of poorly water-soluble drugs, they are less effective in drug loading in the case of amphiphobic drugs due to significant drug precipitation from the carriers. Hence, it is imperative to develop novel nanocarriers for an improved drug load and more effective drug delivery.

http://dx.doi.org/10.1016/j.ejpb.2015.04.023 0939-6411/Ó 2015 Published by Elsevier B.V.

Please cite this article in press as: X. Zhang et al., Phospholipid-stabilized mesoporous carbon nanospheres as versatile carriers for systemic delivery of amphiphobic SNX-2112 (a Hsp90 inhibitor) with enhanced antitumor effect, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.04.023

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In recent years, mesoporous materials have received growing interests in interfacial catalysis, energy reserve, and drug delivery [10]. Mesoporous nanomaterials as drug carriers possess several superior characteristics, such as tunable drug release, modifiable targetability and high drug load [11]. Owing to large pore volume and high specific surface area, mesoporous materials can achieve a high entrapment rate toward amphiphobic drugs by adsorption. Saha et al. synthesized mesoporous carbons using a soft-template technique to orally deliver three model drugs (captopril, furosemide and ranitidine), and showed that the material had excellent potential in the release control and drug loading [12]. In another study, they demonstrated the newer biomaterial of low toxicity and good biocompatibility by the cytotoxicity test and protein adsorption experiment [13]. Hydrophilic mesoporous carbon nanoparticles as carriers of camptothecin have also been reported. The carrier exhibited a good water dispersibility and sustained drug release, and could be internalized into cells to effectively inhibit their growth [14]. Although carbon-based materials are readily available and low-toxic, their merits in drug delivery of amphiphobic drugs have not been fully established. Mesoporous carbon nanospheres (MCNs) can be readily obtained by hydrothermal synthesis [15]. Various substances can be used as carbon sources to fabricate MCNs, such as phenolic resol, sucrose and glucose. It was shown that glucose uptake and glycolytic metabolism were highly active in cancer cells than normal cells [16]. Overexpression of glucose transporter has been verified in a variety of cancer cells, including breast cancer cell [17]. MCNs that use glucose as carbon source are incompletely carbonized, on which glucose residues are retained. Thus, MCNs based on glucose may be a promising carrier for targeted delivery of amphiphobic drug. SNX-2112 is a heat-shock protein 90 (Hsp90) inhibitor that can degrade Hsp90 client proteins to induce cell cycle arrest and apoptosis, thereby killing cancer cells [18]. However, SNX-2112 is almost insoluble in water and oil, and also poorly soluble in other lipophilic excipients. The solubility is just 7.55 lg/mL in water and 90.1 lg/mL in soybean oil. The Log P and pKa are determined to be 1.89 and 11.67, respectively. The insoluble nature significantly limits development of the compound toward clinical stages. Although there are hydrophilic hydroxyl and carboxyl groups on the surfaces of MCNs, MCNs are hydrodynamically unstable as injectable carriers. In this work, phospholipid-stabilized MCNs (PMCNs) were developed for systemic and specific delivery of SNX-2112 (Scheme 1). Multiple techniques such as loading drug by adsorption, stabilizing carriers by phospholipid coating, and targeting delivery by glucose transporter are integrated into the engineering of SNX-2112-loaded PMCNs (SN-PMCNs). The suitability and antitumor effects of SN-PMCNs were evaluated by a series of in vitro experiments and a breast cancer xenograft mouse model.

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2. Materials and methods

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2.1. Materials

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SNX-2112 and AT-533 (internal standard, an analogue of SNX-2112) with a purity of >98.0% were kindly provided by Prof. Yifei Wang (Biomedicine Research and Development Center, Jinan University, Guangzhou, China). Soybean lecithin (S100) was supplied by Lipoid (Ludwigshafen, Germany). Chlorpromazine, simvastatin, Filipin, trypsin and EDTA were obtained from Sigma–Aldrich (Shanghai, China). Glucose, 5-aminofluorescein and sucrose were purchased from Aladdin (Shanghai, China). RPMI 1640, DMEM, fetal bovine serum (FBS) and penicillin– streptomycin were purchased from Gibco BRL (Gaithersburg, MD, USA). BCA protein assay kit, RIPA lysis buffer and phenylmethanesulfonyl fluoride (PMSF) were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Deionized water was prepared by

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a water purifier (Chengdu, China). All other chemicals and reagents used were of analytical grade.

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2.2. Synthesis and characterization of MCNs

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Carbon nanospheres (CNs) were synthesized by hydrothermal reaction using glucose as carbon source [19]. Typically, 6 g of glucose was dissolved in 60 mL of deionized water to form a clear solution, and then the solution was placed in a Teflon-lined autoclave and maintained at 200 °C. To obtain suitable CNs, the hydrodynamic size was monitored using a particle size analyzer (Zetasizer Nano ZS, Malvern, Worcestershire, UK) in real time. The reaction was terminated by cooling the system to room temperature. The products were filtrated and washed alternately with ethanol and water. The purified CNs were dried at 70 °C for 8 h followed by impregnation with ZnCl2 solution (0.8 M) for 12 h. The impregnated CNs were harvested by filtration and proceeded to dry at 80 °C for 12 h. An activation procedure was performed on the material at 400 °C under N2 atmosphere for 3 h. After cooling down, the material was subjected to HCl (0.5 M) disintegration to create porosity. Finally, MCNs were obtained after washing and drying. MCNs were characterized by BET nitrogen adsorption, scanning and transmission electron microscopies. The surface area and pore size of MCNs were calculated based on the N2 adsorption/desorption isotherm obtained from a surface area and porosity analyzer (Micrometritics TriStar, Norcross, USA). MCNs were first outgassed at 250 °C for 3 h under vacuum to a final pressure of 0.25 Pa and then the isotherm was measured at 77 K over the relative pressure (P/Po). The specific surface area and adsorbed volume of MCNs were determined using the Brunauer–Emmett–Teller (BET) equation. The surface area was calculated from the BET model. The pore volume versus diameter distribution was calculated by analyzing the adsorption and desorption branches of the isotherm using the Barrett–Joyner–Halenda (BJH) method [20]. To inspect the surface morphology, MCNs were immobilized to supporters by drying them under a lamp. Fixed particles were then coated with platinum/palladium and photographed using a Zeiss XL-30E SEM (Oberkochen, Germany). For insight into the interior morphology, the sample was prepared as above without surface coating and observed with a Philips Tecnai 10 TEM (Amsterdam, Netherlands).

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2.3. Adsorption isotherm of MCNs versus SNX-2112

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Adsorption isotherm was plotted by accumulative absorption of SNX-2112 against time at 25 °C. In detail, 50 mg of MCNs was added into 50 mL of SNX-2112 solution (dissolved in 80% ethanol, 15 mg/mL) and agitated with a magnetic stirrer. At predetermined intervals, the sample (200 lL) was withdrawn and centrifuged at 6000g for 10 min to separate SNX-2112-loaded MCNs from the system. The concentration of SNX-2112 in supernatant (free drug) was determined by HPLC as described below. The accumulative adsorption quantity of SNX-2112 relative to MCNs at various time points (qt, mg/g) was calculated by the equation: qt = (C0  Ct)  V/M, where C0 and Ct (mg/mL) respectively represent the initial and real-time concentration of SNX-2112, V denotes the volume of system, and M is the weight of MCNs used.

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2.4. Preparation of SN-PMCNs

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SN-PMCNs were prepared by an absorption/solvent-diffusion/ high pressure homogenization technique. Briefly, 30 mg of SNX-2112 was dissolved in 1 mL 80% ethanol, into which 30 mg of MCNs was introduced. After absorption for 6 h, the solvent was removed by evaporation at 45 °C under reduced pressure until

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Scheme 1. Construction of targeted delivery system of amphiphobic SNX-2112 using glucose-based mesoporous carbon nanospheres (MCNs). SNX-2112 was incorporated into MCNs via adsorption which was then stabilized by phospholipid. The engineered system could enhance the antitumor effect of SNX-2112 by EPR effect and glucose transporter-mediated transport.

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no solvent was residual. Afterward, 1 mL of ethanol solution containing 15 mg soybean lecithin was added and stirred for 30 min to completely disperse SNX-2112-absorbed MCNs. The suspension was then rapidly injected into 10 mL water with a syringe and SNPMCNs formed upon the solvent diffusion into the aqueous phase. Subsequently, the resulting nanosuspensions were homogenized through a Microfluidizer (Nano DeBee, Massachusetts, USA) for 8 cycles at 20,000 psi.

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2.5. Characterization of SN-PMCNs

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The particle size and f potential of SN-PMCNs were measured by Zetasizer Nano ZS. The sample was appropriately diluted with water and subjected to measurement for particle size and f potential analyses at 25 °C. The morphology of SN-PMCNs was observed by TEM. TEM sample was prepared following the same procedure of MCNs. Drug load (DL) was determined after separating SNX-2112 from SN-PMCNs by centrifugation. Separation was performed on a centrifugal filter device (AmiconÒ Ultra-0.5, MWCO 50 K, Millipore, USA) at 8,000 g. The concentration of SNX-2112 in the filtrate denoting free SNX-2112 (Mfre) was quantified by HPLC. The DL was calculated according to the equation: DL (%) = (Mtot – Mfre)/ Mtot  100%, where Mtot denoted the total amount of SNX-2112 used in the formulation. For quantification of SNX-2112, samples were determined by HPLC using Dionex Ultimate 3000 HPLC system (Thermo Scientific, MA, USA). SNX-2112 was separated against a C18 column (Syncronis C18, 5 lm, 4.6  250 mm) guarded with a precolumn at 40 °C and monitored at 251 nm with an injection

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volume of 20 lL. The mobile phase consisted of 70% methanol and 30% water pumped at a flow rate of 1.0 mL/min.

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2.6. In vitro drug release of SN-PMCNs

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Reverse bulk equilibrium dialysis technique was used to study the release of SNX-2112 from SN-PMCNs. The release medium was composed of PBS (pH 7.4) and 1% Tween 80. The end-ligated dialysis bags containing 0.5 mL of blank medium and a ceramic ball were immersed into 1000 mL release medium. After equilibrium with the bulk phase, 10 mL of SN-PMCNs (2.5 mg/mL) suspensions was introduced and reversely dialyzed at 37 °C under agitation. At specific time points, dialysis bags were withdrawn and the concentration of SNX-2112 in dialysates was determined by HPLC. The percentage of drug release was calculated.

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2.7. In vitro cytotoxicity assay

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The in vitro anticancer activity of SN-PMCNs was evaluated on three cancer cell lines (4T1, HepG-2 and MCF-7). Tumor cell lines of 4T1, HepG-2 and MCF-7 were purchased from Shanghai Center of Cell Source (Shanghai, China). The cells were cultured in RPMI 1640 or DMEM medium containing 10% FBS, 100 IU/mL penicillin and 100 lg/mL streptomycin at 37 °C in an atmosphere of 90% relative humidity and 5% CO2. Briefly, cells were seeded in 96-well plates at a density of 5  103 cells/well and cultured for 24 h. After that, the cells were incubated with various concentrations of SNX-2112 solution (SN-Sol, solubilized in 50% propylene glycol, v/v) or SN-PMCNs for another 24 h. Cell viability was measured by MTT assay according to a reported procedure [21].

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2.8. Cellular uptake and internalization

a Concentrator Plus (Eppendorf, NY, USA). The residuals were reconstituted in 100 lL 60% methanol for HPLC analysis.

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Cellular uptake of SN-PMCNs was investigated on 4T1 cells that were cultured as above. The cells were washed with 37 °C PBS (pH 7.4). The effect of drug concentration on cellular uptake was investigated by incubating cells with different concentrations of treatment media (SN-PMCNs or SN-Sol, diluted in RPMI 1640 medium). To examine the effect of incubation time on cellular uptake, treatment media equivalent to 25 lg/mL SNX-2112 were added and incubated with cells for 0.5, 1, 2, and 4 h. Finally, the media were removed and cells were washed triply with ice-cold PBS. The cells were lysed with RIPA Lysis Buffer (0.1% PMSF) followed by centrifugation at 12,000g for 15 min (4 °C). The supernatant was collected and its protein content was determined using BCA Protein Assay Kit. For quantification of SNX-2112, cell lysates were collected and SNX-2112 were extracted with 50% methanol (800 lL/sample). The extraction liquid was centrifuged at 12,000g for 10 min. The concentration of SNX-2112 in the supernatants was analyzed by HPLC. To observe cellular internalization of SN-PMCNs, 5-aminofluorescein-labeled MCNs were prepared by one-pot synthesis of glucose and 5-aminofluorescein at a ratio of 100/1. Then, fluorescence-labeled SN-PMCNs were prepared according to the abovementioned method. The well-cultured 4T1 cells incubated with fluorescence-labeled SN-PMCNs for 2 h. The incubation medium was removed, and the cells were rinsed with PBS twice. The cells were subsequently fixed in 4% paraformaldehyde and examined by confocal laser scanning microscopy (CLSM) (Zeiss LSM510 Meta, Oberkochen, Germany).

2.11. In vivo antitumor effect of SN-PMCNs

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The antitumor effect of SN-PMCNs was evaluated on BALB/c mice bearing breast tumor. Female BALB/c mice were inoculated subcutaneously with 0.1 mL of 4T1 tumor cell suspensions (1  108 cells/mL). Tumor volume (V) was measured daily by a caliper and calculated using the formula: V = (L  W2)/2 (mm3). When the tumor volume grew up to 80–120 mm3, mice were randomly divided into three groups: saline, SN-Sol and SN-PMCNs. They were intravenously administered of two preparations (5 mg/kg) or saline via the tail vein every other day. The tumor diameter and body weight of each mouse were measured every two days. At the end of experiments, the animals were put to death to harvest tumor tissues. The tumor mass was weighted and photographed.

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2.12. Histological examination

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Hematoxylin-eosin (HE) staining was adopted to examine the histological alterations of the liver and spleen tissues following the antitumor experiment. Briefly, tissue samples treated with 4% paraformaldehyde were dehydrated using Carnoy’s fluid and then prepared into paraffin-embedded sections. After being stained by HE, the histomorphology of tissue was inspected and photographed using an Olympus imaging system (Olympus IX70, Tokyo, Japan).

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2.13. Biodistribution in 4T1 xenograft mice

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2.10. Pharmacokinetic studies

The induction of tumor was followed the same procedure as described above. When the tumor volume reached to 250–300 mm3, mice were randomly divided into 2 groups (24 per group, n = 6  4) and administrated with SN-Sol and SN-PMCNs (9 mg/kg) via the tail vein, respectively. At 1, 3, 6 and 12 h, bloods were sampled from the eyeball of mice and plasmas were collected as described above. After that, the animals were killed by cervical dislocation and the organs of the heart, liver, lung, kindly, lung, intestine, and tumor were harvested. The tissues were then washed with ice-cold saline and dried with filter paper. The tissues were homogenized with saline at a weight ratio of 1/2 before extraction. The extraction of SNX-2112 from tissue samples was the same as that of the blood samples. The tissue SNX-2112 was quantified by an UPLC-QTOF/MS system. Quantitation was performed based on the full scan analysis and extracted positive ion chromatograms using MassLynx version 4.1. The instrument configuration and parameter settings referred to the literature published previously [22]. Student’s t-test was used to analyze and compare the results.

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4T1 cells were used to investigate the cellular trafficking pathway of SN-PMCNs, which were seeded in 6-well plates (5  105 cells/well) and grew for 24 h. To explore the cellular trafficking pathway, glucose (110 mM), hypertonic sucrose (450 mM), chlorpromazine (0.03 mM), simvastatin (0.05 mM), filipin (1.5 lM) and sodium azide (15 mM) were separately added into the wells. After pre-incubation for 0.5 h, the cells were treated with SN-PMCNs (25 lg/mL) for 4 h at 37 °C. Subsequently, the cells were washed with PBS and lysed with RIPA Lysis Buffer (0.1% PMSF) followed by centrifugation at 12,000g for 15 min (4 °C). To study the effect of temperature on cellular uptake of SN-PMCNs, the cells were pre-treated at 4 °C for 0.5 h, and then incubated with the SN-PMCNs solutions (25 lg/mL) for 4 h at 4 °C. Quantifications of protein and SNX-2112 accorded with the established methods above in the cellular uptake.

3. Results and discussion

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3.1. Synthesis and characterization of MCNS

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CNs could be readily synthesized by the hydrothermal reaction. The resultant CNs reacting for 3 h were 150 nm in particle size as determined by Nano ZS analyzer. ZnCl2 used as dehydrant can facilitate the formation of porous carbon structure [23]. Under the action of high temperature, ZnCl2 is converted into ZnO during the process of activation where N2 prevents excess carbonization. Mesopores are formed when ZnO in CNs is disintegrated by HCl. MCNs from glucose were incompletely carbonized and possessed abundant hydroxyl and carboxyl groups (3400 cm1 and 1550 cm1) as revealed by FTIR (Fig. 2A). It was also observed that

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Male Sprague–Dawley rats (230 ± 20 g) were randomly divided into two groups (n = 5). The rats were fasted overnight prior to administration but allowed free access to water. All animal experiments were conducted according to the Guidelines on the Care and Use of Animals for Scientific Purposes (2004, Singapore) and approved by the Experimental Animal Ethical Committee of Jinan University (Guangzhou, China). Rats were administered with SN-PMCNs and SN-Sol by intrajugular injection at a dose of 5 mg/kg. Blood (0.30 mL) was collected from the jugular vein at pre-determined time intervals and centrifuged at 5,000 g for 5 min to collect plasma with a centrifuge (Eppendorf, NY, USA). The plasma SNX-2112 was recovered by a deproteinization procedure using 500 lL of acetonitrile. The samples were supplemented with 20 lL of 100 lg/mL AT-533 as internal standard and vortexed for 5 min. After centrifugation at 12,000g for 10 min, the supernatants were transferred to tubes followed by evaporation using

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overly carbonized CNs was difficultly dispersed with water due to the formation of aggregates. The BET plot of N2 adsorption onto MCNs is given in Fig. S1. The BET surface area of MCNs was calculated based on the BET model to be 2864 m2/g, which was considerably large and thus competent to adsorb enough drug molecules. Fig. S2 exhibits the N2 adsorption/desorption isotherms of MCNs with or without treatment of ZnCl2. The BET surface area of ZnCl2-treated MCNs was just 214 m2/g, indicating that ZnCl2 played a positive role in the formation of porosity. As shown in Fig. 2B, the pore diameter of synthetic MCNs ranged from 2 nm to 10 nm, demonstrating the mesoporous nature of MCNs. They were spherical and porous in structure from the morphology observed with SEM (Fig. 2C). Meanwhile, the porosity of MCNs can be confirmed by TEM photograph where a translucent corona surrounding the sphere core, an inkling of hollow structure, was visualized clearly (Fig. 2D). These results indicated that MCNs have been successfully fabricated.

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3.2. Adsorption performance for SNX-2112

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Fig. 3 depicts the adsorption of MCNs toward SNX-2112 as a function of time. The adsorption capacity of MCNs toward SNX-2112 was assessed on the removal of SNX-2112 from the aqueous solution. The adsorption arrived to an equilibrium after stirring for 6 h, approximately. The maximal adsorption payload at 12 h was up to 1.90 mg/mg, almost two times the weight of MCNs. The result indicated that SNX-2112 can be strongly adsorbed by mesoporous material via multiplying Van der Waals force. Mesoporous materials are substances structurally containing pores of 2–50 nm diameter according to IUPAC [24]. Our prepared MCNs were approximately 150 nm in particle size with the inner pores between the mesoporous range, which provided an opportunity to load SNX-2112 by an adsorption mechanism.

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3.3. Preparation and characterization of SN-PMCNs

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To achieve high drug load via adsorption and obtain hydrodynamically stable SN-PMCNs, a combined method of absorption/ solvent-diffusion/high pressure homogenization was used. The obtained SN-PMCNs possessed a mean size of 180 nm (PDI < 0.3) with a zeta potential of 34.4 mv (Fig. 4A). It was a little larger than that of MCNs, which might be ascribable to the coating of phospholipid. SN-PMCNs were near-spherical in morphology as revealed by TEM (Fig. 4B). The rate of DL for SN-PMCNs was 42.7%, suggesting a good load capacity. When kept in ambient conditions for one month, there was no significant change in particle size of SN-PMCNs, showing a suitable physical stability. Interestingly, SN-PMCNs were positively charged that may be beneficial to cellular uptake due to the negative nature of cell membranes. In addition to providing stable surface, the highly positive potential of SN-PMCNs would enhance the response of targeting to slightly acidic tumorous tissues [25].

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3.4. In vitro drug release

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The in vitro release profile of SNX-2112 from the preparation versus time is shown in Fig. 5. It can be seen that SNX-2112 was able to be released slowly from PMCNs. The accumulative release was less than 20% at 1 h, and could be up to 85% at 12 h. The release was well described by Weibull model with a high correlation coefficient of 0.9961. According to the fitted Weibull function (Log (ln(1  y) = 0.919 Log x  0.3663), the time needed for releasing 50% of SNX-2112 (t50) was calculated to be 3.10 h. The result revealed that SNX-2112 could be released from PMCNs in a manner of tardiness via desorption or adsorbates interchange. The results from differential scanning calorimetry (Fig. S3) showed

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Fig. 1. Chemical structure of SNX-2112.

that SNX-2112 distributing onto the surface of particles was amorphous, indicating that the drug molecules were loaded in the pores of carbon nanospheres by adsorption rather than deposition. The characteristic dispersion and release of drug was ascribable to the high affinity of SNX-2112 to MCNs. Although the release data obtained by dialysis method did not fully represent the real release behavior of preparation in the blood stream, it was useful for characterization of drug release from the formulation.

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3.5. In vitro antitumor activity

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The antitumor activities of SN-PMCNs against 4T1, MCF-7 and HepG2 cell lines with SN-Sol as control are shown in Fig. 6. The blank carriers showed less toxicity relative to the culture medium with cell survivals over 93% on three cell lines. Both SN-PMCNs and SN-Sol presented a concentration-dependent cytotoxic effect, but the cytotoxicity of SN-PMCNs was more notable compared to SN-Sol. The calculated IC50 values of two formulations at 24 h treatment are summarized in Table 1. It could be seen that the IC50 values of SN-PMCNs increased following the order of 4T1, MCF-7 and HepG2, of which 4T1 cell was most sensitive to the formulation. The IC50 values of SN-PMCNs decreased twofold roughly on MCF-7 and HepG-2 cell lines in comparison with the control, whereas it was almost fivefold decrease in 4T1 cell lines. It was indicative of more SNX-2112 molecules entering into the cells that induced the death of cells. The in vitro cytotoxicity studies demonstrated that PMCNs can facilitate the transport of SNX-2112 into the tumor cells through a certain ligand-mediated mechanism. It might be attributed to glucose transporter-mediated endocytosis by the glucose residues that are preserved on the surface of MCNs [26].

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3.6. Cellular uptake and internalization

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To investigate the effect of PMCNs on cellular uptake of SNX-2112, the cellular uptake of SN-PMCNs was examined on 4T1 cell line with SN-Sol as reference. The cellular uptake of two SNX-2112 preparations against concentration and time is shown in Fig. 7. It was a concentration-dependent uptake below middle concentration (25 lg/mL) for two formulations (Fig. 7A). Reversely, at high concentration, the amount of cellular uptake reduced relative to the group of middle concentration. It could be explained by that high-level of SNX-2112 immediately induced apoptosis of lots of cells that resulted in reduced accumulative uptake. However, the cellular uptakes of 4T1 cells treated with SN-PMCNs were all higher than those of SN-Sol-treated groups at

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Fig. 2. Characteristics of MCNs prepared by a hydrothermal method at 200 °C: (A) FTIR spectrum; (B) nitrogen adsorption/desorption isotherm and pore diameter distribution (inset); (C) SEM micrograph, and (D) TEM micrograph.

Fig. 3. Kinetic adsorption curve of SNX-2112 over MCNs at 25 °C.

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various levels. The cellular uptake was quantified based on the total protein level of cells. Hence, the result released a clear signal that PMCNs can enhance the cellular uptake of SNX-2112. Generally, the process of cellular uptake is associated with the time of exposure. As shown in Fig. 7B, the cellular uptake of SNX-2112 by 4T1 cell increased over time in the cases of SN-PMCNs and SN-Sol. Although the cellular uptake of solution group increased with the time, the uptrend of uptake was rather limited, indicating a possible SNX-2112 efflux [22]. However, there was a significant time-dependent uptake of SNX-2112 in the case

of SN-PMCNs, which illustrated that SNX-2112 can be continually taken up into the cells in the form of SN-PMCNs. The coating of phospholipid and/or incorporation of SNX-2112 molecules into the inner cavities of PMCNs shielded the unfavorable characteristics of drug that increased the cellular uptake of SNX-2112 via glucose transporter-mediated influx and attenuated drug efflux. Fig. 8 exhibits the co-localization of nucleus stained by Hoechst 33258 and 5-aminofluorescein-labeled SN-PMCNs within 4T1 cells. The CLSM micrographs provided direct evidence for entrance of SN-PMCNs into the cytoplasm of 4T1 cells. In addition, from the fluorescence intensity and distribution, it could be seen that SN-PMCNs were internalized into the 4T1 cells in large quantity. The easy internalization of SN-PMCNs offers the feasibility that SN-PMCNs carry SNX-2112 across the cell membrane and release the payload within the cytoplasm. The size of MCNs is far larger than the cytomembrane pores and hence passive diffusion into the cytoplasm is incredible. Accordingly, an active transport such as carrier-mediated one is substantially involved in the process of PMCNs internalization. We assume that it can be ascribed to a glucose transporter-mediated endocytosis.

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The internalization of nanoparticles is generally categorized into macropinocytosis, clathrin-dependent, caveolae-dependent and other receptors-mediated endocytosis [27,28]. To study the mechanism responsible for the cellular uptake of SN-PMCNs, 4T1 cells were treated with four typical pharmacological inhibitors, i.e. hypertonic sucrose (nonspecific), chlorpromazine hydrochloride (specific), simvastatin (nonspecific) and Filipin (specific). They are known as inhibitors of clathrin-dependent endocytosis

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Fig. 4. Particle size distribution (A) and TEM micrograph (B) of SN-PMCNs.

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and inhibitors of caveolae-dependent endocytosis, respectively [29]. The relative uptake of SN-PMCNs in the presence of inhibitors is displayed in Fig. 9. When the cells co-cultured with hypertonic sucrose, chlorpromazine hydrochloride, simvastatin and Filipin, the uptake rate of SNX-2112 on 4T1 cell was reduced by 4%, 49.8%, 97.2% and 11.3% compared with the control, respectively. These results strongly suggested that specific clathrin-mediated endocytosis and nonspecific caveolae-dependent endocytosis play key roles in the cellular uptake of SN-PMCNs.

Fig. 5. The release curve of SNX-2112 from SN-PMCNs performed based on reverse bulk equilibrium dialysis with SN-Sol as reference (n = 3, mean ± SD).

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Glucose transporters are overexpressed in the breast cancer cells [17]. The uptake rate was significantly reduced (>50%) in the presence of high concentration of glucose when 4T1 cells were incubated with SN-PMCNs. It showed that there was a competition between free glucose and SN-PMCNs in binding onto the cell surface, revealing that glucose transporter-mediated transport was the reason for enhanced uptake of SNX-2112. We also investigated the effect of energy on the cellular uptake. As shown in Fig. 9, the cellular uptake had a significant suppression (Paired t-test, P < 0.05) compared with the control at 4 °C. It was indicative that the process of endocytosis might be energy-dependent. At low temperature, the ATP output is largely limited due to the decline of energy-productive enzymes activity, resulting in reduction of cellular uptake. In order to further elucidate the event, 4T1 cells were incubated with sodium azide (SA), an inhibitor of mitochondrial energy synthesis [30]. As a consequence, the uptake of SNPMCNs on 4T1 cells was reduced by 25% around. The facts above proved that the cellular uptake of SN-PMCNs on 4T1 cells is required of energy. The carrier-mediated endocytosis and glucose transporter-mediated transport are generally active processes [27]. Therefore, it can be concluded that specific clathrin-dependent and nonspecific caveolae-dependent endocytosis are primarily involved in the uptake of SN-PMCNs where the energy is simultaneously requisite.

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3.8. Pharmacokinetics

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The pharmacokinetic profiles of SN-PMCNs and SN-Sol after i.v. administration to SD rats are shown in Fig. 10. There was significant difference in pharmacokinetics for two preparations characterized by rapid elimination for solution and obvious retention for SN-PMCNs. Main pharmacokinetic parameters calculated based on non-compartmental model are given in Table 2. The AUC0?1 of SN-PMCNs was 1.815 lgh/mL, which was 1.73 times the value of SN-Sol. In addition, the half-life (T1/2) and MRT for the group of SN-PMCNs were 4.57-fold and 8.13-fold as high as those of SN-Sol. Likewise, it was significantly different in the clearance (CL) between two preparations. The group of SN-Sol exhibited a higher peak concentration (Cmax) than SN-PMCNs. It might be attributed to the distinction of SNX-2112 release from two formulations into the blood. SN-Sol can immediately perfuse the circulatory system as injected into the blood and be facilely recovered from the plasma. However, the entrapment of SNX-2112 into the MCNs was through an adsorption force, making it unable to promptly release into the blood. The release kinetics is closely related to the adsorption force between the drug and the carrier materials, and further related with the drug nature. SNX-2112 possesses more electronegative atoms as shown in Fig. 1, including three F, four O and four N atoms, rendering the molecule more polarity. This brings about an increased Van der Waals force for adsorption, and thus retards the release. The highly insoluble nature of SNX-2112 is another reason for lack of burst release. Besides, the portion of SNX-2112 loaded in PMCNs cannot be retrieved from the blood, resulting in low SNX-2112 exposure to blood. Altogether, SN-PMCNs possessed a slower plasma elimination and longer systemic circulation time. The pharmacokinetic results turned out that PMCNs are endued with long circulating nature that is advantageous to enhance the antitumor effect of SNX-2112.

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3.9. In vivo antitumor effect of SN-PMCNs

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The changes in body weight, average tumor weight and volume, and typical tumor masses of 4T1 xenograft mice after treatment with saline or SNX-2112 preparations for 16 days are presented in Fig. 11. There was no significant body weight loss in mice receiving saline, SN-Sol and SN-PMCNs (Fig. 11A), indicating that the

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Fig. 6. In vitro antitumor activity of SN-PMCNs at various levels assessed on 4T1, HepG-2 and MCF-7 cell lines. The cells were treated for 24 h with SN-PMCNs using SN-Sol as a control. The gray column signifies the relative cell viability of blank carriers at a carbon concentration of 250 lg/mL.

Table 1 The half maximal inhibitory concentration (IC50) values of SN-PMCNs and the reference on three tumor cell lines. Cell line

4T1 MCF-7 HepG-2

IC50 (lg/mL) SN-PMCNs

SN-Sol

7.193** 12.01** 24.05**

37.84 32.13 72.14

Paired-t test, significantly different from the solution formulation. ** p < 0.01.

Fig. 7. Cellular uptake of SN-PMCNs and SN-Sol on 4T1 cells at 25 lg/mL of SNX2112: (A) the effect of drug concentration on uptake; (B) the effect of incubation time on uptake.

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mice had a fitting tolerance to the given dose of SNX-2112. However, the average tumor weight of mice treated with SN-PMCNs was merely 52.23% and 67.88% as heavy as that of SN-Sol and saline group (Fig. 11B), respectively. The tumors of

mice treated with saline or SN-Sol expanded rapidly during the experiment. In contrast, the tumor growth was significantly slowed down when the mice were administrated with SN-PMCNs (Fig. 11C). SN-PMCNs demonstrated a more forceful antitumor effect than SN-Sol, which was consistent with the results of the in vitro cell experiments. The typical tumor masses excised from 4T1 xenograft model after treatment are presented in Fig. 11D. Neither complete tumor growth regression nor toxicity-induced death of mouse was observed in any experimental groups. Anyhow, SN-PMCNs had significant advantages over SN-Sol in inhibiting the growth of 4T1 solid tumor. The merits of SNPMCNs might be accounted for by the passive targeting due to EPR effect that increased the release of SNX-2112 within the tumor tissue [31], and by the active transport via glucose ligand-mediated internalization that enhanced the cytoplasmic concentration of SNX-2112 [32]. For further insight into the pathology of vital organs, the histologies of liver and spleen mostly exposed to nanoparticles were checked. There were no significant acute injuries of liver and spleen from either SN-PMCNs or SN-Sol treatment (Fig. 12). However, some pathological changes appeared in the liver of the saline group, showing hepatocyte swelling and eosinophilic staining. This might result from the inflammation of the liver or metastasis of cancer cells to the liver. It has been reported that 4T1 cells (breast cancer cells) could spontaneously produce a highly metastatic tumor by infiltration into the liver, lymph node and brain [33,34]. However, there were no signals of organic lesions in the liver of mice treated with SN-PMCNs or SN-Sol, demonstrating excellent anti-tumor effects of them. In addition, there were apparent MCNs-associated black dots (indicated as green arrow) being in the liver and spleen tissue sections. The preferential distribution of SN-PMCNs into the liver, spleen and lung potentially prevented the development of cancerometastasis. Taken together, SN-PMCNs exhibited enhanced in vivo antitumor activity on the xenograft tumor model and inhibited the progression of tumor. It was noted that SN-Sol showed an inferior antitumor effect contrast with SN-PMCNs on the xenograft tumor mice and possessed a large injection toxicity due to the use of organic solvent. The mice took place blood vessel ulceration at the injection site after two weeks of SN-Sol injection that caused a have-to termination of experiments on the sixteenth day.

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3.10. Biodistribution in the tumor-bearing mice

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Biodistribution study is helpful to understand the therapeutic action and delivery mechanism of formulated drug. SNX-2112 levels in the blood and organs of heart, liver, lung, kidney, spleen, and tumor after i.v. administration of SN-Sol and SN-PMCNs are

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Fig. 8. CLSM observation of the internalization of SN-PMCNs. Blue represents the nucleus stained by Hoechst 33258 (Hoe) and green denotes 5-aminofluorescein-labeled SNPMCNs (5-AF-SN-PMCNs). Fluorescence-labeled nanoparticles are indicated by red arrows. (For the interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 9. Cellular trafficking pathway of SN-PMCNs characterized by various inhibitors and limited conditions. Endocytosis inhibitors used include hypertonic sucrose (non-specific clathrin-dependent), chlorpromazine (specific clathrin-dependent), simvastatin (non-specific caveolin-dependent) and Filipin (specific caveolin-dependent). The dependence on energy of cellular uptake was evaluated through a low temperature (4 °C) or the presence of sodium azide (SA).

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showed in Fig. 13. Like the pharmacokinetics, SN-Sol presented a high blood distribution and fast diffusion into the periphery with the time relative to SN-PMCNs. Despite low level in the blood, SN-PMCNs possessed a persistence in distribution, indicating a sustained release effect of SNX-2112. In the heart, the distribution of SN-PMCNs was higher than that of SN-Sol after 3 h, but the concentration of SNX-2112 decreased over the time. It was shown that those SN-PMCNs distributed into the heart could be eluted by abundant blood flow. However, significantly high distribution into the liver, spleen and lung was provided with SN-PMCNs. It is the intrinsic characteristics of particulate injection that particles larger than 100 nm are readily sequestrated into the RES of the liver, spleen, lung and kidney [35]. Almost all RES-like organs had high SNX-2112 distribution in terms of SN-PMCNs, which was of notable advantage to treat primary liver or lung cancer. Reversely, there was high intestinal concentration of SNX-2112 in the group of SN-Sol. The distribution of SN-PMCNs in the intestine was lower

Fig. 10. Plasma concentration–time curves of SN-Sol and SN-PMCNs after i.v. administration to SD rats at the dose of 5 mg/kg SNX-2112. Data expressed as mean ± SD (n = 6).

Table 2 Pharmacokinetic comparison between SN-PMCNs and SN-Sol after i.v. administration to rats at a dose of 1.0 mg/kg (n = 6, mean ± SD). Parameters

SN-PMCNs

SN-Sol

T1/2 (h) Tmax (h) Cmax (lg/mL) MRT (h) CL (L/h) AUC0?1 (lg h/mL)

36.74 ± 2.699** 0.200 ± 0.007* 0.055 ± 0.007** 54.42 ± 7.982** 0.557 ± 0.067* 1.815 ± 0.194**

8.036 ± 1.125 0.167 ± 0.006 0.434 ± 0.049 6.691 ± 1.957 0.959 ± 0.100 1.052 ± 0.121

AUC: area under plasma SNX-2112 concentration versus time curve; One-way ANOVA, compared with SN-Sol. * p < 0.05. ** p < 0.01.

than that of SN-Sol at various time points. It might be ascribable to the easy perfusion of free SNX-2112 over entrapped SNX-2112 at this tissue. Both SN-Sol and SN-PMCNs showed high SNX-2112 distribution in the tumor tissue. Interestingly, the concentrations of SNX-2112 in the tumor for SN-Sol were higher than those of SN-PMCNs in the first instance; however they apparently reduced with the duration of distribution. At 12 h, the drug concentration in

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Fig. 11. In vivo antitumor effects of SNX-2112 formulations in 4T1 tumor xenograft model: (A) changes in the body weight of subjects as a function of time in tumor-bearing female BALB/c mice; (B) the average weight of tumor mass of each group at the time of sacrifice (ANOVA, ⁄p < 0.05, compared with control or SN-Sol); (C) the tumor growth curves of control and therapeutic groups; (D) representative tumor photographs from mice subjected to treatment for 16 days.

Fig. 12. Histological features of liver and spleen sections of mice after treatment with saline or SNX-2112 formulations for 16 days that are stained with hematoxylin-eosin.

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the tumor for SN-Sol was significantly lower than that of SNPMCNs, demonstrating a difference in distribution between two formulations. SN-PMCNs have higher SNX-2112 distribution in the liver, spleen, lung and tumor compared to SN-Sol. The tissue

concentration of SNX-2112 for SN-Sol appeared rapid recession in all tissues. In contrast, SN-PMCNs produced an excellent maintenance of high drug concentration, especially in the tumor. It was clear that SN-PMCNs showed great potential in the improvement of biodistribution and therapeutic efficacy.

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Fig. 13. Biodistribution of SNX-2112 in 4T1 tumor xenograft mice after injection of SN-PMCNs and SN-Sol for 1, 3, 6 and 12 h.

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4. Conclusions

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In this work, it demonstrates the suitability of mesoporous carbon material as novel nanocarriers for systemic delivery of amphiphobic anticancer drug. Glucose-based MCNs possessed a strong adsorption toward SNX-2112 and could enhance the in vitro and in vivo delivery efficiency. It was documented that glucose transporter-mediated endocytosis might be responsible for enhanced in vitro cellular uptake of SNX-2112 in terms of SNPMCNs, and the internalization of SN-PMCNs was closely associated with specific clathrin-dependent and nonspecific caveolae-dependent endocytosis. The in vivo antitumor effect of SNX-2112 was also significantly enhanced as formulated into PMCN. Prolonged body residence time and improved tumor targeting were proposed as causes for enhanced antitumor activity of SN-PMCNs. Overall, PMCNs developed here represent a promising carrier for systemic delivery of amphiphobic antitumor agents.

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Acknowledgments

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This work was co-supported by the National Natural Science Foundation of China (81402855) and the China Postdoctoral Science Foundation (2014M562253).

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Appendix A. Supplementary material

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejpb.2015.04.023.

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Please cite this article in press as: X. Zhang et al., Phospholipid-stabilized mesoporous carbon nanospheres as versatile carriers for systemic delivery of amphiphobic SNX-2112 (a Hsp90 inhibitor) with enhanced antitumor effect, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.04.023