an anti-ros/hepatic fibrosis drug delivery system based on ... · an anti-ros/hepatic fibrosis...

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An anti-ROS/hepatic brosis drug delivery system based on salvianolic acid B loaded mesoporous silica nanoparticles Qianjun He a , Jiamin Zhang b , Feng Chen a , Limin Guo a , Ziyan Zhu b , Jianlin Shi a, * a State Key Laboratory of High Performance Ceramics and Superne Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Ding-Xi Road, Shanghai 200050, China b Shanghai (Red Cross) Blood Center, Shanghai Institute of Blood Transfusion, Shanghai, 200051, China article info Article history: Received 13 April 2010 Accepted 1 July 2010 Available online 31 July 2010 Keywords: Mesoporous silica Nanoparticle Drug release Anti-hepatic brosis Blood compatibility Salvianolic acid abstract The rhodamine B (RhB) covalently grafted SBA-15-structured mesoporous silica nanoparticles (MSNs- RhB) of high surface area (750 m 2 g 1 ), large pore volume (0.7 cm 3 g 1 ), uniform particle size (about 400 nm) and positively charged surface (29.6 5.0 mV), has been developed as a drug delivery system (SAB@MSNs-RhB) for anti-ROS (reactive oxygen species)/hepatic brosis by loading a negatively charged drug salvianolic acid B (SAB). The dosage formulation SAB@MSNs-RhB effectively protected the loaded drug SAB from decomposition. The multi-release experimental results showed that SAB@MSNs-RhB exhibited an outstanding SAB sustained-release property, and relatively high SAB release rates and concentrations in a long term after the consumption of previously released SAB as compared to SAB loaded MSNs (SAB@MSNs) of negatively charged surface (31.1 2.6 mV). The inuences of the drug concentration, incubation time, drug formula and drug carrier on the ROS level, proliferative activity and cytotoxicity of LX-2 cells were evaluated. The results showed that the inhibiting effect of SAB@MSNs-RhB on the ROS level and proliferative activity of LX-2 cells was more remarkable than free SAB in a long term (72 h), and became more intensive with the increase of the sample concentration and the incubation time. SAB@MSNs-RhB enhanced the cellular drug uptake, the drug bioaccessability and efcacy for anti- ROS/hepatic brosis via the nanoparticles-mediated endocytosis and the sustained release of the drug. There was no visible cytotoxicity of free SAB, MSNs-RhB and SAB@MSNs-RhB against LX-2 cells in a broad concentration range (0.5e100 mM) and incubation time periods up to 72 h. The blood compatibility of the carrier MSNs-RhB was evaluated by investigating the hemolysis and coagulation behaviors in a broad concentration range (50e500 mg mL 1 ) under in vitro conditions. The results suggested that MSNs-RhB possessed good blood compatibility. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction There are over ve hundred million patients bearing various hepatitis, including hepatitis A, B, C and E, currently in the world, and the disease incidence rates of fatty liver, alcoholic liver and drug hepatitis are also increasing. Various chronic liver diseases are very much prone to hepatic brosis which is the stage which must be passed through during pathological changes into hepatocir- rhosis and even into hepatic carcinoma, therefore anti-hepatic brosis is one of the most important approaches to prevent or even reverse the malignant transformation [1]. Millions of patients need the treatment of hepatic brosis per year. For the past few years, a large amount of plant-derived drugs has been exploited to reduce the probability of the malignant transformation into hepatocir- rhosis [1,2]. It was previously reported that some antioxidants derived from Chinese medicines, such as Salvia miltiorrhiza, gly- cyrrhizin, tetrandrine, oxymatrine, curcumin, and so on, could suppress peroxidation and eliminate reactive oxygen species (ROS) in hepatic stellate cells (HSCs), and consequently, reduce or even reverse hepatic brosis in treated animals [2]. Thereinto, Salvia is one of the most commonly used agents for treating brosis [3e5]. Salvianolic acid B (SAB) is a major water-soluble polyphenolic acid extracted from Radix Salviae miltiorrhizae (Sm), which is a common herbal medicine that has been clinically used in China for more than one thousand of years as a blood-circulation accelerating agent and antioxidant. SAB has been proved capable of inhibiting hepatic brosis and reversing chronic hepatitis B by double-blind double-dummy clinical follow-up observations, and is a very promising clinic drug for anti-hepatic brosis [5]. However, SAB is unstable and subject to degradation and oxidation, which leads to * Corresponding author. Tel.: þ86 21 52412714; fax: þ86 21 52413122. E-mail address: [email protected] (J. Shi). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.07.008 Biomaterials 31 (2010) 7785e7796

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Page 1: An anti-ROS/hepatic fibrosis drug delivery system based on ... · An anti-ROS/hepatic fibrosis drug delivery system based on salvianolic acid B loaded mesoporous silica nanoparticles

lable at ScienceDirect

Biomaterials 31 (2010) 7785e7796

Contents lists avai

Biomaterials

journal homepage: www.elsevier .com/locate/biomater ia ls

An anti-ROS/hepatic fibrosis drug delivery system based on salvianolic acid Bloaded mesoporous silica nanoparticles

Qianjun He a, Jiamin Zhang b, Feng Chen a, Limin Guo a, Ziyan Zhu b, Jianlin Shi a,*a State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Ding-Xi Road,Shanghai 200050, Chinab Shanghai (Red Cross) Blood Center, Shanghai Institute of Blood Transfusion, Shanghai, 200051, China

a r t i c l e i n f o

Article history:Received 13 April 2010Accepted 1 July 2010Available online 31 July 2010

Keywords:Mesoporous silicaNanoparticleDrug releaseAnti-hepatic fibrosisBlood compatibilitySalvianolic acid

* Corresponding author. Tel.: þ86 21 52412714; faxE-mail address: [email protected] (J. Shi).

0142-9612/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.biomaterials.2010.07.008

a b s t r a c t

The rhodamine B (RhB) covalently grafted SBA-15-structured mesoporous silica nanoparticles (MSNs-RhB) of high surface area (750 m2 g�1), large pore volume (0.7 cm3 g�1), uniform particle size (about400 nm) and positively charged surface (29.6 � 5.0 mV), has been developed as a drug delivery system(SAB@MSNs-RhB) for anti-ROS (reactive oxygen species)/hepatic fibrosis by loading a negatively chargeddrug salvianolic acid B (SAB). The dosage formulation SAB@MSNs-RhB effectively protected the loadeddrug SAB from decomposition. The multi-release experimental results showed that SAB@MSNs-RhBexhibited an outstanding SAB sustained-release property, and relatively high SAB release rates andconcentrations in a long term after the consumption of previously released SAB as compared to SABloaded MSNs (SAB@MSNs) of negatively charged surface (�31.1 � 2.6 mV). The influences of the drugconcentration, incubation time, drug formula and drug carrier on the ROS level, proliferative activity andcytotoxicity of LX-2 cells were evaluated. The results showed that the inhibiting effect of SAB@MSNs-RhBon the ROS level and proliferative activity of LX-2 cells was more remarkable than free SAB in a long term(72 h), and became more intensive with the increase of the sample concentration and the incubationtime. SAB@MSNs-RhB enhanced the cellular drug uptake, the drug bioaccessability and efficacy for anti-ROS/hepatic fibrosis via the nanoparticles-mediated endocytosis and the sustained release of the drug.There was no visible cytotoxicity of free SAB, MSNs-RhB and SAB@MSNs-RhB against LX-2 cells in a broadconcentration range (0.5e100 mM) and incubation time periods up to 72 h. The blood compatibility of thecarrier MSNs-RhB was evaluated by investigating the hemolysis and coagulation behaviors in a broadconcentration range (50e500 mg mL�1) under in vitro conditions. The results suggested that MSNs-RhBpossessed good blood compatibility.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

There are over five hundred million patients bearing varioushepatitis, including hepatitis A, B, C and E, currently in the world,and the disease incidence rates of fatty liver, alcoholic liver anddrug hepatitis are also increasing. Various chronic liver diseases arevery much prone to hepatic fibrosis which is the stage which mustbe passed through during pathological changes into hepatocir-rhosis and even into hepatic carcinoma, therefore anti-hepaticfibrosis is one of the most important approaches to prevent or evenreverse the malignant transformation [1]. Millions of patients needthe treatment of hepatic fibrosis per year. For the past few years,a large amount of plant-derived drugs has been exploited to reduce

: þ86 21 52413122.

All rights reserved.

the probability of the malignant transformation into hepatocir-rhosis [1,2]. It was previously reported that some antioxidantsderived from Chinese medicines, such as Salvia miltiorrhiza, gly-cyrrhizin, tetrandrine, oxymatrine, curcumin, and so on, couldsuppress peroxidation and eliminate reactive oxygen species (ROS)in hepatic stellate cells (HSCs), and consequently, reduce or evenreverse hepatic fibrosis in treated animals [2]. Thereinto, Salvia isone of the most commonly used agents for treating fibrosis [3e5].Salvianolic acid B (SAB) is a major water-soluble polyphenolic acidextracted from Radix Salviae miltiorrhizae (Sm), which is a commonherbal medicine that has been clinically used in China for morethan one thousand of years as a blood-circulation acceleratingagent and antioxidant. SAB has been proved capable of inhibitinghepatic fibrosis and reversing chronic hepatitis B by double-blinddouble-dummy clinical follow-up observations, and is a verypromising clinic drug for anti-hepatic fibrosis [5]. However, SAB isunstable and subject to degradation and oxidation, which leads to

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Q. He et al. / Biomaterials 31 (2010) 7785e77967786

the reduced clinical effectiveness and industrialization bottlenecks.One of the solutions to the problems is to develop the sustained-release dosage forms of SAB [6,7].

It is well known that mesoporous silica nanoparticles (MSNs)possess some excellent properties such as facile multi-functional-ization, excellent biocompatibility and biodegradability, highspecific surface area and pore volume, tunable pore structures andexcellent physicochemical stability [8e11]. Thus the applicationstudies of MSNs in the biomedical field have attracted greatattention [12e25]. MSNs have been intensively suggested aseffective carriers for controllable drug delivery [12e19], bio-signalprobing [19,20], gene transport and expression [19e22], biomark-ing [23] and many other important bioapplications [24]. Especially,the drug sustained-release features of MSNs owing to their uniquepore structures and facile surface modification are drawingincreasing attention in the biomedical field. MSNs often need to bemodified for the special charge property of the surface and have theappropriate pore properties such as pore size, mesostructureordering and porosity in order to regulate the drug sustained-release profiles [26e29]. As for the present drug SAB, which iseasily ionized into negatively charged ions in polar medium and hasa relatively large molecular size of w1.1 nm � 1.6 nm, positivelycharged SBA-15-type MSNs of larger pore size than MCM-41-typeMSNs are thought to be a suitable carrier for the desired sustained-release features.

In our present work, we introduced a co-condensation methodto covalently graft rhodamine B (RhB) groups within the meso-porous channels of SBA-15-type MSNs (MSNs-RhB) for the positivecharging on the surface of MSNs as well as the red fluorescentemission. SAB molecules (ions) were loaded into MSNs-RhB bya vacuum nano-casting route [30] to construct a drug (SAB) deliverysystem (SAB@MSNs-RhB), and were expected to release chronicallyand steadily due to the electrostatic attraction between the posi-tively charged MSNs-RhB and negative SAB ions. Furthermore, theinhibiting effect of SAB@MSNs-RhB on the ROS level and prolifer-ation activity of LX-2 cells (a kind of HSCs) was investigated, sinceHSCs are mainly activated by ROS to induce fibrosis and are thepathological key of the fibrosis formation [31e33]. In addition, theblood compatibility of the drug carrier MSNs-RhB was also inves-tigated for bio-safety.

In addition, the key motives of using RhB is to positively chargethe surface of MSNs and label MSNs for fluorescence simulta-neously, thus other positively charged dye groups also could beused to be homogeneously grafted within the mesoporous chan-nels of MSNs by the co-condensation method, and similar sus-tained-release behaviors might be received by virtue of theelectrostatic attraction between the positively charged MSNs andnegative SAB ions. In our works, a high grafting amount and gooddispersivity of RhB groups grafted within the mesoporous channelsof MSNs could be obtained, owing to the unique virtues of the co-condensation method as previously reported [34]. Therefore, themuch sustained release of negative drugs fromMSNs-RhB could beexpected.

2. Materials and methods

2.1. Synthesis and characterization of SBA-15-type MSNs-RhB

Based on a newly developed bottom-up tailoring methodology and a facile co-condensation approach as described in our previous reports [34,35], subsphaeroidalSBA-15-type MSNs-RhB with RhB groups monodispersed and covalently bondedwithin the pore channels were synthesized. Firstly, 0.40 g of ZrOCl2$8H2O and 1.25 gof Pluronic P123 (EO20PO70EO20, Aldrich, Mn ¼ 5800) were dissolved in turn in100 mL of 2 M HCl aqueous solution at 35 �C under vigorously stirring. After thesolution became clear, a 2.8 mL mixed solution of tetraethoxysilane (TEOS) anda synthetic silane coupling agent of side-chained RhB (RhB-APTES) in amolar ratio of10000 to 1 was added. The vigorous stirring was continued for 24 h under a light-

sealed condition, and then the red as-synthesized materials were collected bycentrifugation. The as-synthesized materials were extracted several times witha mixed solution of ethanol (150 mL) and hydrochloric acid (36e38%, 2 mL) tocompletely remove surfactants. The final products MSNs-RhB were dried overnightat 80 �C in vacuum, and then stored under a cool, dry and light-sealed condition. Inorder to compare the drug release behaviors between SBA-15-type MSNs-RhB andMSNs without the RhB modification, pure SBA-15-type MSNs was also synthesizedby adding TEOS as the single silicon source in accordancewith the above-mentionedprocedures for the synthesis of MSNs-RhB. Above-mentioned RhB-APTES wasprepared by the dehydration reaction between RhB and APTES using 1-[3-(dime-thylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC$HCl) as both dehydrantand catalyst under a light-sealed and dry condition. Excessive EDC$HCl was used andlong enough reaction time was given in order to complete the above dehydrationreaction [34].

The morphology and mesostructure of MSNs-RhB and MSNs were observed viatransmission electron microscopy (TEM) and scanning electron microscopy (SEM),which were performed on a JEMe2010 electron microscope with an acceleratingvoltage of 200 kV and a JSM-6700F electron microscope, respectively. The meso-structure ordering of MSNs-RhB and MSNs was characterized by small-angle X-raydiffraction (SAXRD), and the SAXRD data were recorded on Rigaku D/Max-2550 Vdiffractometer using Cu Ka radiation (40 kV and 40 mA) at a scanning rate of0.4�/min over the range of 0.7e6.0� with a step width of 0.002� . The porosities ofMSNs-RhB and MSNs were measured by nitrogen adsorptionedesorption isothermexperiments, which were carried out on a Micromeritics Tristar 3000 analyzer at77 K under a continuous adsorption condition, with all samples were dried for 12 hat 100 �C under nitrogen before measurements. Average pore diameter was calcu-lated from desorption branches of the nitrogen adsorptionedesorption isotherms bythe BarretteJoynereHalenda (BJH) method, and specific surface area and porevolume were calculated by BrunauereEmmetteTeller (BET) and BJH methods,respectively. The mean zeta potentials of MSNs-RhB and MSNs in PBS weremeasured via a zeta potential analyzer (ZetaPlus, Brookhaven Instrument Corp.,Holtsville, NY, USA).

2.2. Stability measurements of free SAB and SAB in SAB@MSNs-RhB

To measure the stability of SAB, seven equal portions of dry SAB (5 mg) andSAB@MSNs-RhB (15 mg) containing equal amount of SAB were exposed undernatural illumination in air at 60 �C for the accelerated decomposition to distinguishtheir stabilities more evidently. At an interval of 1 day, one portion of SAB orSAB@MSNs-RhBwas completely dissolved into 30mL aqueous solution of HF (0.5 M).The absorbance of the obtained clear solutions at 284 nmwas immediately recordedon a Shimadzu UV-3101PC UV-vis absorption spectrophotometer for the measure-ment of SAB concentrations. A calibration curvewas drawn by using SAB solutions ofgiven concentrations according to the Lambert and Beer’s law. The decompositionpercentages of free SAB and SAB within SAB@MSNs-RhB at the different time pointswere calculated by comparing with the blank control at the zero exposure time.

2.3. In vitro drug loading and release

Two drug delivery systems (DDSs), SAB@MSNs-RhB and SAB@MSNs, wereconstructed by a previously reported vacuum nano-casting route [30] by taking SAB(SigmaeAldrich) as the model drug, and positively charged MSNs-RhB and nega-tively chargedMSNs as the drug carriers, respectively. Initially, MSNs-RhB andMSNswere dried 12 h at 120 �C in vacuum, and then further kept in vacuum at roomtemperature for 3 h. Then 10 mg SAB was completely dissolved into 5 mL dryethanol, and then refrigerated at �20 �C for 30 min. The ethanol solution of SAB(2e3 drops) was added dropwise into dry MSNs-RhB or MSNs until full soakage.Then the soaked SAB@MSNs-RhB and SAB@MSNs were vacuumized slowly at roomtemperature for 30 min. The above-mentioned soakageevacuum procedures wererepeated until 5 mL of the ethanol solution of SABwas used up. Finally, the preparedSAB@MSNs-RhB and SAB@MSNs were sealed in brown bottles and stored at �20 �C.

We introduced a multi-release method to effectively distinguish the differentdrug release behaviors of SAB@MSNs and SAB@MSNs-RhB. Sterilized dialysis bagswith dialyzer molecular-weight cut-off 3500 Daltonwere used to carry out the drugrelease experiments. These dialysis bags were pretreated prior to use as follows.These dialysis bags were fully immersed into 50% aqueous solution of ethanol andboiled 1 h, and thenwashed with 50% ethanol, 10 mmol L�1 NaHCO3 and 1mmol L�1

EDTA in turn. Phosphate buffered saline (PBS, 0.15 M, pH 7.4) which was prepared bydissolving 8 g of NaCl, 0.2 g of KCl, 1.15 g of Na2HPO4 and 0.2 g of KH2PO4 in 1 L ofdeionized water, was used as the drug release medium.

SAB@MSNs-RhB and SAB@MSNs (10 mg) were respectively dispersed into 2 mLPBS, and then the solutions were put into pretreated dialysis bags with 1/3 air gapwhich sealed with dialysis bag holders. The sealed dialysis bags were put into brownbottles and then 18 mL PBS was added. These bottles were shaken at a speed of100 rpm at 37 �C under a light-sealed condition. At certain time intervals, 3 mL of therelease medium was taken out for measuring the released drug concentration byvirtue of UV-vis absorption technique, and then was returned to the original releasemedium. After 2 days, the release systems reached the drug release equilibrium.Then 18 mL release medium was replaced with the equivalent-volume fresh PBS.

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Fig. 1. SAXRD patterns of samples MSNs and MSNs-RhB.

Q. He et al. / Biomaterials 31 (2010) 7785e7796 7787

At intervals of 2 days, the same procedures were repeated. For the measurement ofreleased SAB concentrations, the absorbance of the release medium at 284 nm wasrecorded on a Shimadzu UV-3101PC UV-vis absorption spectrophotometer. A cali-bration curve of SAB-released concentrations was plotted by using SAB solutions ofgiven concentrations according to the Lambert and Beer’s law.

2.4. Measurements of intracellular ROS levels

LX-2 cell lines were cultured in DMEM medium containing 10% fetal bovineserum (FBS). LX-2 Cells were maintained at 37 �C in a humidified and 5% CO2

incubator. For all experiments, LX-2 cells were harvested by using PBS solution andre-suspended in fresh DMEM medium before plating.

ROS levels in LX-2 cells respectively treated with free SAB, MSNs-RhB andSAB@MSNs-RhB were measured by the 20 ,70-dichlorofluorescein diacetate (DCF-DA,Beyotime Biotechnology) assay. Non-fluorescent DCF-DA would transform intogreen fluorescent 20 ,70-dichlorofluorescein (DCF) under the oxidation of intracellularROS [36]. LX-2 cells were seeded in 96-well plates at a density of 103 cells per well.After incubation for 24 h at 37 �C in 100 mL DMEM medium containing 10% FBS,culture mediumwas discarded and then cells were respectively treated with 100 mLpH 7.4 PBS solutions of MSNs-RhB, free SAB and SAB@MSNs at the same concen-trations (0.5 mM, 5 mM, 50 mM and 100 mM). After incubation for 72 h, culture mediawere discarded, and then cells were rinsed three times with PBS. 100 mL of 10 mMDCF-DA solution of FBS-free DMEM medium was then added. After incubation for20 min, cells were rinsed three times with PBS again to remove the residual DCF-DA,and then were directly observed on a Leica fluorescence microscopy (Leica DM ILLED) for the fluorescent analysis of ROS levels in LX-2 cells.

2.5. Cell viability and proliferation activity assay with LX-2 cells

In vitro cytotoxicity of free SAB, MSNs-RhB and SAB@MSNs-RhB against LX-2cells and the proliferation activity of LX-2 cells were assessed by the CCK-8 (CellCounting Kit-8, Beyotime Biotechnology) assay. The statistical evaluation of datawasperformed using a two-tailed unpaired Student’s t-test. A p-value of less than 0.05was considered statistically significant. Each data point is represented asmean � standard deviation (SD) of six independent experiments (n ¼ 6, n indicatesthe number of wells in a plate for each experimental condition). The time and dosedependences of the cytotoxicity and the cell proliferation activity were investigatedat different time points of incubation (24 h, 48 h and 72 h) at various molarconcentrations of 0.5 mM, 5 mM, 50 mM and 100 mM. Free SAB solutions containing thesame SAB concentrations with SAB@MSNs-RhB solutions, and the carrier MSNs-RhBsolutions containing the same silica concentrations with SAB@MSNs-RhB solutions,were used for comparison.

For the cytotoxicity assay, LX-2 cells were seeded in 96-well plates at a density of104 cells per well. After incubation for 24 h at 37 �C in 100 mL DMEM mediumcontaining 10% FBS, culture mediumwas discarded and then cells were treated with100 mL of free SAB, MSNs-RhB and SAB@MSNs-RhB solutions of different molarconcentrations (0.5 mM, 5 mM, 50 mM and 100 mM) for 24 h, 48 h and 72 h at 37 �C ina humidified and 5% CO2 incubator. Then a 10 mL CCK-8 solutionwas added and thencells were incubated for another 1 h. The absorbance was monitored at 450 nm onamicro-plate reader (Bio-Tek ELx800). A culturemediumwithout the addition of theDDS nanoparticles was used as the blank control. The cytotoxicity was expressed asthe percentage of the cell viability as compared with the blank control.

For the cell proliferation activity assay, LX-2 cells were seeded in 96-well platesat a density of 103 cells per well with 100 mL of DMEM medium containing 10% FBS.Cells were respectively incubated with 100 mL of free SAB, MSNs-RhB andSAB@MSNs-RhB solutions of different molar concentrations (0.5 mM, 5 mM, 50 mM and100 mM) for 24 h, 48 h and 72 h at 37 �C in a humidified and 5% CO2 incubator. Then10 mL CCK-8 solution was added and cells were incubated for another 1 h. Theabsorbance was monitored at 450 nm on a micro-plate reader (Bio-Tek ELx800).A culture medium without the addition of the DDS nanoparticles was used as theblank control. Cell numbers were measured by the CCK-8 assay, and the cellproliferation activitywas expressed by the relative cell number after incubationwithnanoparticles as compared with the blank control.

2.6. Blood compatibility analysis

The blood compatibility of SAB@MSNs-RhB was evaluated by measuringhemolysis and coagulation behaviors under in vitro conditions. The levels ofprothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen(Fib) were measured to evaluate coagulation behaviors of SAB@MSNs-RhB. Freshhuman blood anticoagulated with citric-acid-dextrose solution and Fresh FrozenPlasma (FFP) were obtained from the Shanghai (Red Cross) Blood Center. The plasmawas removed from fresh human blood by centrifugation for 10 min at 3000 rpm,then the upper solution was sucked out, and residual HRBCs at the bottom ofcentrifugal tubes were washed three times with sterile isotonic PBS solution, andthen were diluted to 1/10 of their volume with sterile isotonic PBS solution for thehemolysis assay.

For the hemolysis determination, three batches of diluted HRBCs suspensions(300 mL) were added into: (a) 1200 mL of nanoparticle PBS suspension at the

concentration of 500 mg mL�1; (b) 1200 mL of superpure water as a positive control;(c) 1200 mL of sterile isotonic PBS solution as a negative control, respectively. Themixtures were gently shaken up, and then kept still for 2 h at room temperature.Finally, themixtureswere centrifuged for 2min at 4000 r/min and the absorbance ofthe upper clear solution at 541 nm was recorded in a Shimadzu UV-3101PC UV-visabsorption spectrophotometer. The hemolysis percentages of MSNs-RhB andSAB@MSNs-RhB samples were calculated by dividing the absorbance differencebetween the sample and the negative control by the absorbance difference betweenpositive and negative controls, and multiplying the resulting ratio by 100.

For the coagulation determination, FFP was thawed at 37 �C in a water bath, andthen 50 mL PBS solutions of MSNs-RhB and SAB@MSNs-RhB samples at differentconcentrations were respectively mixed with 450 mL thawed plasma. After staticincubation of 5 min at 37 �C, the mixed solutions were centrifuged, and then theupper clear solutions were sucked out and were used to measure PT, APTT and Fibvalues on a ACL� 7000 fully-automatic blood coagulation analyzer by using theHemosIL� kit (Instrumentation Laboratory Company, Lexington, MA 02421-3125,USA). Blank control experiments were done by adding identical volumes of PBS.

Each experiment was repeated three times. The statistical evaluation of datawasperformed using a two-tailed unpaired Student’s t-test. A p-value of less than 0.05was considered statistically significant.

3. Results and discussion

3.1. The morphology and mesostructure of SBA-15-type MSNs andMSNs-RhB

From the SAXRD patterns of MSNs and MSNs-RhB shown inFig. 1, three distinct diffraction peaks at 2q ¼ 0.94�, 1.45� and 1.66�

indexed to (100), (110) and (200) planes, respectively, reveal thatboth MSNs and MSNs-RhB have a highly ordered SBA-15-type 2Dhexagonal (P6mm) symmetry. Furthermore, from the TEM imagesof MSNs and MSNs-RhB respectively shown in Fig. 2c and d, bothMSNs and MSNs-RhB exhibit highly ordered mesoporous channelsin the [001] direction in accordance with the above-mentionedSAXRD results. SEM and TEM analyses indicated that both MSNsand MSNs-RhB had a subsphaeroidal morphology, a high dis-persivity and uniform particle size of about 400 nm (Fig. 2).

As shown in Fig. 3, both MSNs and MSNs-RhB have the classicaltype-IV nitrogen adsorptionedesorption isotherms with H1-typehystereses and well-defined steps at relative pressures (P/P0) of0.6e0.8. This suggests that bothMSNs andMSNs-RhB have uniformmesoporous channels and relatively narrow pore size distribution,in accordance with the results obtained from TEM imaging and

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Fig. 2. SEM and TEM images of samples MSNs ((a) and (c), respectively) and MSNs-RhB ((b) and (d), respectively). The scale bars of a, b, c and d correspond to 1 mm, 1 mm, 100 nmand 100 nm, respectively.

Q. He et al. / Biomaterials 31 (2010) 7785e77967788

SAXRD data. Moreover, MSNs and MSNs-RhB have relatively largepore sizes (5.0 nm and 4.9 nm, respectively), high specific surfaceareas (780 m2 g�1 and 750 m2 g�1, respectively) and large porevolumes (0.8 cm3 g�1 and 0.7 cm3 g�1, respectively). Therefore, it

Fig. 3. Nitrogen adsorptionedesorption isotherms of samples MSNs and MSNs-RhB.

should be feasible for them to hold SAB molecules (molecular sizew1.1 nm � 1.6 nm) with high loading capacities.

Furthermore, the zeta potentials of MSNs and MSNs-RhB weremeasured to be �31.1 � 2.6 mV and 29.6 � 5.0 mV, respectively.This suggests that MSNs-RhB has been positively charged toa great extent by RhB groups covalently grafted within the mes-oporous channels. This positive charging property of MSNs-RhB isexpected useful to pull negatively charged SAB in polar medium(such as ethanol and water) into their mesoporous channels byvirtue of electrostatic attraction and/or hydrogen bonding, asschematically illustrated in Fig. 4. In addition, SAB molecules(ions) were expected to release in a more sustained way fromthe constructed SAB@MSNs-RhB, due to such an electrostaticattraction, than from SAB@MSNs constructed using negativelycharged MSNs.

3.2. The stability of SAB within SAB@MSNs-RhB

In order to confirm the protection effect of SAB from degrada-tion and oxidation by MSNs-RhB, decomposition acceleratingexperiments were carried out under natural illumination in air at60 �C for both free SAB and SAB within SAB@MSNs-RhB. As shownin Fig. 5, the decomposition rates of free SAB are much higher thanthose of SAB within SAB@MSNs-RhB. After 7-day decomposition,about 31% of free SAB and only 1% of SAB within SAB@MSNs-RhBare decomposed. Therefore, MSNs-RhB as a carrier plays animportant role in the effective protection of the loaded drug SABfrom decompostion. It is thought that chemically inert features and

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Fig. 4. Construction of the drug delivery system SAB@MSNs-RhB as schematicallyillustrated by the cutaway diagram of mesoporous structure. There is an ionizationbalance between SAB molecules and negative ions in ethanol. Both SAB molecules andnegative ions can be loaded within the mesoporous channels and bind to the meso-porous walls of SAB@MSNs-RhB mainly via hydrogen bonding between silanol groupson the SAB@MSNs-RhB and phenolic hydroxyls and carboxyls of SAB, and electrostaticattraction between quarter ammonium head groups on the SAB@MSNs-RhB andionized phenolic hydroxyls and carboxyls of SAB.

Q. He et al. / Biomaterials 31 (2010) 7785e7796 7789

highly porous structure of MSNs-RhB could prevent oxygen fromcontacting and oxidizing SAB within SAB@MSNs-RhB, and preventthe direct irradiation of natural light which would induce andaccelerate the oxidation and degradation of SAB [37].

3.3. In vitro drug release behaviors of SAB@MSNs and SAB@MSNs-RhB

In order to conveniently distinguish the difference in the sus-tained drug release behaviors between SAB@MSNs-RhB andSAB@MSN, the multi-release properties at an interval of 48 h wereinvestigated, as shown in Fig. 6a and b. The drug release profiles of

Fig. 5. The time dependence of the decomposition percentage of free SAB (B) and SABwithin SAB@MSNs-RhB (6).

SAB@MSNs-RhB and SAB@MSNs in Fig. 6a can be linearly fitted(Fig. 6c) with a kind of hyperbolic function as follows: t/Qt ¼ a þ btfor the first release, (t � 48)/Qt ¼ a þ b(t � 48) for the secondrelease, and (t � 96)/Qt ¼ a þ b(t � 96) for the third release.Thereinto, Qt is the drug-released percentage at time t, and a andb are adjustable parameters with phenomenological meanings: 1/aand 1/b express the initial release rate (slop of the release curve attime t ¼ 0) and the maximum release, respectively [38]. Theselinear fittings had high correlation coefficients of over 0.999, asshown in Fig. 6c. Combining with these linear fitting results (a andb values), the time dependence of the drug release rates ofSAB@MSNs-RhB and SAB@MSNs in the three release stages wasobtained according to the equation: y ¼ dQt/dt ¼ a/(a þ bt)2.To further compare the SAB release rates between SAB@MSNs-RhBand SAB@MSNs, the time dependence of the ratio (R) between thedrug release rates of SAB@MSNs-RhB and SAB@MSNs in the threerelease stages was calculated, as shown in Fig. 6d.

From Fig. 6a, a portion of SAB was released fast in the initialstage from SAB@MSNs and SAB@MSNs-RhB under the same releaseconditions. This could be resulted from the relatively fast desorp-tion of SAB adsorbed on the outside surface of both SAB@MSNs andSAB@MSNs-RhB. To enhance the bioavailability of SAB, this portionof SAB adsorbed on the outside surface of SAB@MSNs andSAB@MSNs-RhB should not be washed down because they do nothave cytotoxicity in a broad concentration range of 0.5e100 mMagainst LX-2 cells, which will be indicated and discussed therein-after in the Section 3.5. From Fig. 6d, it is apparent that, comparedwith SAB@MSNs-RhB, SAB@MSNs have remarkably higher SABrelease rates in the first release stage (0e48 h), and the differencebetween their SAB release rates became more and more apparentalong with the release process. Such a difference in the first releasestage could be attributed to the electrostatic attraction betweenpositively charged MSNs-RhB and negatively ionized SAB mole-cules with six phenolic hydroxyls and two carboxyls in eachmolecule in pH 7.4 PBS. After 48 h of release, a balance betweenrelease and adsorption was reached, and the SAB-releasedconcentrations and percentages from SAB@MSNs and SAB@MSNs-RhB reached 121.0 mg mL�1 and 91.2 mg mL�1 (Fig. 6a), 80.7% and60.8% (Fig. 6b), respectively. Therefore, SAB@MSNs-RhB showedmuch more sustained SAB release than SAB@MSNs in the firstrelease period of 48 h.

During the second release (48e96 h), the ratios between theSAB release rates of SAB@MSNs-RhB and SAB@MSNs kept almostconstant at 0.8. At the end of this release stage, the released SABconcentrations and accumulated percentages from SAB@MSNs andSAB@MSNs-RhB reached 35.2 mg mL�1 and 28.2 mg mL�1 (Fig. 6a),96.1% and 73.5% (Fig. 6b), respectively. Therefore, SAB@MSNs stillkept higher SAB release rates in the second release stage thanSAB@MSNs-RhB, and absolute majority (96.1%) of SAB had releasedout from SAB@MSNs, however there still was 26.5% of SAB left inthe SAB@MSNs-RhB DDS at the end of the second release.

It is noticeable that during the third release (96e144 h), veryinterestingly, the SAB release rate of SAB@MSNs-RhB becamesignificantly higher than that of SAB@MSNs in opposition to thecase of the foregoing release stages, and the difference between theSAB release rates of two DDSs became more and more apparentalong with the release process in this stage (Fig. 6d). This could bedue to the remarkably more amount of SAB remaining inSAB@MSNs-RhB than in SAB@MSNs. Correspondingly, the releasedSAB concentrations from SAB@MSNs-RhB became ever higher thanthose from SAB@MSNs in this release stage (Fig. 6a). At the end ofthe third release stage (144 h), the SAB-released concentrations andaccumulated percentages of SAB@MSNs and SAB@MSNs-RhBreached 5.5 mg mL�1 and 8.0 mg mL�1 (Fig. 6a), 97.4% and 77.3%(Fig. 6b), respectively. It could be found that there was still

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Fig. 6. Drug release profiles of samples SAB@MSNs-RhB and SAB@MSNs in pH 7.4 PBS at 37 �C: (a) SAB-released concentration vs time plots, (b) SAB-released percentage vs timeplots, (c) fitting lines, and (d) the time dependence of the ratios of SAB release rates between SAB@MSNs-RhB and SAB@MSNs in the three release stages (0e48 h, 48e96 h and96e144 h).

Q. He et al. / Biomaterials 31 (2010) 7785e77967790

a considerable SAB amount of 23.7% not released from SAB@MSNs-RhB, and consequently relatively high SAB release rate andconcentration from SAB@MSNs-RhB would be kept in the releasemedium even beyond 144 h of release. Therefore, compared withSAB@MSNs, SAB@MSNs-RhB had apparently more outstanding SABsustained-release capability, and more importantly, after theconsumption of previously released SAB in 96 h, SAB@MSNs-RhBstill exhibited relatively high SAB release rates and concentrationsin the release medium. Such drug release features of SAB@MSNs-RhB are expected useful to enhance the long-term efficacy of anti-hepatic fibrosis.

3.4. Suppression of ROS levels in LX-2 cells by SAB@MSNs-RhB

Oxidative stress has been reported closely associated withchronic liver diseases of various etiologies, and ROS acts assignalingmolecules in the pathways or networks of hepatic fibrosis.There were several reports showing that the increase of oxidativestress or the ROS signaling cascade will activate the proliferation ofHSCs and induce hepatic fibrosis [32,33]. It was previously reportedthat SAB as one of available antioxidants could scavenge ROS inHSCs and consequently reduce or even reverse hepatic fibrosis intreated animals [39]. In our present work, we constructed

SAB@MSNs-RhB to delivery SAB into LX-2 cells by virtue of anenhanced nanoparticles-mediated endocytosis, consequentlyenhance the efficacy of scavenging ROS.

DCF-DA was introduced to evaluate ROS levels in LX-2 cellstreated with free SAB, MSNs-RhB and SAB@MSNs-RhB at differentconcentrations of 0.5 mM, 5 mM, 50 mM and 100 mM, as shown in Fig. 7.Green fluorescent intensity and coverage area in LX-2 cells reflectROS levels. By comparing green fluorescent (Fig. 7a) and bright field(Fig. 7b) images of blank control without adding any drug, it can befound that a great number of ROS has bestrewed all LX-2 cells. FromFig. 7kem, plenty of MSNs-RhB nanoparticles of red fluorescencehave been uptaken by LX-2 cells, however the ROS level keepsalmost identically high with that in blank control (Fig. 7a).However, ROS levels in LX-2 cells treated with free SAB largelydecreased with the increase of SAB concentrations, as shown inFig. 7cej. Comparatively, as SAB@MSNs-RhB could be easilyuptaken by LX-2 cells, the ROS levels decreased correspondinglywith the increase of SAB@MSNs-RhB concentrations, as shown inFig. 7ney. Importantly, compared with free SAB, ROS levels in LX-2cells treated with SAB@MSNs-RhB at the same SAB concentrationswere remarkably lower. Therefore, we believe that the SAB@MSNs-RhB DDS has mediated, and consequently also enhanced, thecellular uptake of SAB and the bioavailability of SAB for scavenging

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Fig. 7. Suppression on ROS levels of LX-2 cells by the free drug SAB, the carrier MSNs-RhB and the drug delivery system SAB@MSNs-RhB at different concentrations (0.5 mM, 5 mM,50 mM and 100 mM). Thereinto, green fluorescence intensity reflects the ROS levels, and the red fluorescence stands for MSNs-RhB in LX-2 cells. Figs. (a)/(b) are green fluorescent/bright field images of blank control, respectively; Figs. (c)/(d), (e)/(f), (g)/(h) and (i)/(j) are green fluorescent/bright field images of LX-2 cells treated with free SAB at concentrationsof 0.5 mM, 5 mM, 50 mM and 100 mM, respectively; Figs. (k)/(l)/(m) are green fluorescent/bright field/red fluorescent images of LX-2 cells treated with MSNs-RhB at the concentration of100 mM, respectively; Figs. (n)/(o)/(p), (q)/(r)/(s), (t)/(u)/(v) and (w)/(x)/(y) are green fluorescent/bright field/red fluorescent images of LX-2 cells treated with SAB@MSNs-RhB atdifferent concentrations 0.5 mM, 5 mM, 50 mM and 100 mM, respectively.

Q. He et al. / Biomaterials 31 (2010) 7785e7796 7791

ROS via the nanoparticles-mediated endocytosis, most probablyowing to the small nano-size of MSNs as previously reported [40].Thereby, the sustained-release features and the nanoparticles-mediated endocytosis are expected useful in inhibiting the prolif-eration of LX-2 cells.

3.5. Inhibition of the proliferation activity of LX-2 cells bySAB@MSNs-RhB

The inhibiting effect on the proliferation of LX-2 cells bySAB@MSNs-RhB, free SAB and MSNs-RhB was investigatedcomparatively, and the influence of the sample concentration andthe incubation time on the proliferation activity of LX-2 cells wasalso presented, and the results are shown in Fig. 8. The cell prolif-eration was neither hindered nor accelerated when LX-2 cells wereexposed to MSNs-RhB for 24 h (Fig. 8a), 48 h (Fig. 8b) and 72 h(Fig. 8c) in a broad concentration range of 0.5e100 mM.

As for free SAB, the cell proliferation activity became remarkablylower with the increase of the SAB concentration, and the 24-h cellproliferation activity decreased by about 8% and 41% at SABconcentrations of 0.5 mM and 100 mM, respectively (Fig. 8a). Inaddition, the cell proliferation activity also decreased with theincrease of incubation time, and the inhibiting effect of free SAB onthe cell proliferation activity became continuously weaker, and the48-h and 72-h cell proliferation activity decreased by only about46% and 49% at the SAB concentration of 100 mM, respectively(Fig. 8b and c). This is probably due to the long-term extracellularstay and consequent oxidative deactivation of free SAB.

As for SAB@MSNs-RhB, the cell proliferation activity alsobecame remarkably lower with the increase of the SAB concen-tration, and the 24-h cell proliferation activity decreased by about4% and 32% at SAB@MSNs-RhB concentrations of 0.5 mM and 100 mM,respectively (Fig. 8a). Compared with free SAB of the sameconcentrations and incubation time, the inhibiting effect ofSAB@MSNs-RhB on the cell proliferation activity seemed weaker in

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Fig. 8. Inhibiting effect on the LX-2 cell proliferation activity by the free drug SAB, the carrier MSNs-RhB and the drug delivery system SAB@MSNs-RhB at different concentrations(0.5 mM, 5 mM, 50 mM and 100 mM) in 24 h (a), 48 h (b) and 72 h (c) of incubation, respectively.

Q. He et al. / Biomaterials 31 (2010) 7785e77967792

a short release period of 24 h, probably because large numbers ofSAB molecules and/or ions were still adsorbed within the meso-porous channels, and thus the concentration of the released SABwas lower than that of free SAB. However, with the increase ofincubation time, the inhibiting effect of SAB@MSNs-RhB on the cellproliferation activity became more and more remarkable, and the48-h and 72-h cell proliferation activity decreased by about 59%and 69%, respectively, at SAB@MSNs-RhB concentrations of 100 mM(Fig. 8b and c). The difference in the inhibiting effect between freeSAB and SAB@MSNs-RhB became even more distinct at the pro-longed incubation, most probably owing to the sustained SABrelease and well-maintained drug efficacy by the nanoparticles-mediated endocytosis as indicated in Fig. 7. SAB molecules (ions)within SAB@MSNs-RhB were released slowly and mainly in LX-2cells, whichmostly prevented the loss and oxidative deactivation ofSAB outside LX-2 cells and therefore kept its inhibiting effect on thecell proliferation activity. In conclusion, SAB@MSNs-RhB had anapparent sustained-release behavior, therefore exhibited anevident longer term inhibiting effect on the cell proliferationactivity as compared with free SAB.

3.6. In vitro cytotoxicity of SAB@MSNs-RhB against LX-2 cells

The bio-safety of the drug carrier MSNs-RhB also must beconsidered. Herein, we investigated the in vitro cytotoxicity of freeSAB, MSNs-RhB and SAB@MSNs-RhB in a broad concentrationrange (0.5e100 mM) and in different incubation time intervals (24 h,48 h and 72 h) against LX-2 cells. From Fig. 9, it can be found that allthree samples, free SAB, MSNs-RhB and SAB@MSNs-RhB, do notshow visible cytotoxicity against LX-2 cells in spite of sampleconcentrations and incubation time. Therefore, SAB@MSNs-RhBshould be a safe DDS, and thus after SAB is loaded intoMSNs-RhB, itis unnecessary to rinse away SAB molecules adsorbed on theoutside surface of SAB@MSNs-RhB for the high drug bioavailability.

3.7. Blood compatibility of MSNs-RhB

As one of drug carriers, the blood compatibility of MSNs-RhBand MSNs is attracting great and special attention [41]. RecentlySlowing and co-workers [42] have found that the hemolytic activityof MCM-41-type MSNs is highly dependent on MSNs

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Fig. 9. Cytotoxicity of the free drug SAB, the carrier MSNs-RhB and the drug delivery system SAB@MSNs-RhB at different concentrations (0.5 mM, 5 mM, 50 mM and 100 mM) againstLX-2 cells in 24 h (a), 48 h (b) and 72 h (c) of incubation, respectively.

Q. He et al. / Biomaterials 31 (2010) 7785e7796 7793

concentrations and surface negative potentials. We have demon-strated the mostly inhibited hemolytic activity of MCM-41-typeMSNs by the surface PEGylation [43]. Very recently, Lin [44] hasindicated the great impact of the pore structure of MCM-41-typeMSNs on their hemolytic behaviors. However, hemolysis andcoagulation behaviors of SBA-15-typeMSNs, which have a differentmesoporous structure from MCM-41-type MSNs, has not beenreported so far as far as we know. In this work, we evaluated theblood compatibility of SBA-15-type MSNs and MSNs-RhB withnegative and positive surface potentials respectively by investi-gating their hemolysis and coagulation behaviors. As to theircoagulation behaviors, PT was used to evaluate the extrinsic andcommon coagulation pathways, APTT was used to evaluate theintrinsic and common coagulation pathways, and Fib was used toevaluate the abnormality of coagulation factor I.

The hemolytic phenomena of SBA-15-type MSNs and MSNs-RhB are almost invisible by direct observation, as suggested by theinset in Fig. 10a. From curves in Fig. 10a, it is found that thehemolysis percentages of both SBA-15-type MSNs and MSNs-RhBincrease slightlywith the increase of nanoparticle concentration in

the range of 50e500 mgmL�1. At the concentration of 500 mgmL�1,the hemolysis percentage of SBA-15-type MSNs is rather low atabout 1.4%, which is tenfold lower than that (14.2%) of MCM-41-type MSNs at the same concentration as previously reported by us[43]. It is believed that the distinct difference in the hemolyticactivities between SBA-15-type and MCM-41-type MSNs could berelated to their mesopore structures. Furthermore, compared withSBA-15-type MSNs, the hemolysis percentages of SBA-15-typeMSNs-RhB are remarkably lower than SBA-15-type MSNs at thesame concentrations in the range of 50e500 mg mL�1. The hemo-lysis percentage of SBA-15-type MSNs-RhB at the concentration of500 mg mL�1 is as low as 0.3% (Fig. 10a). This should be due to thepositive surface potential of SBA-15-typeMSNs-RhB co-condensedwith quarter ammonium groups which could surpress the damageto RBCs membrane linked with the trimethyl-ammonium headgroups, because the negative surface redicals of MSNswas thoughtto cause irreversible damage to RBCs membrane by electrostaticattractions as previously reported by Slowing [42]. Therefore, SBA-15-type MSNs-RhB have negligible hemolysis activity in the broadconcentration rang of 50e500 mg mL�1.

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Fig. 10. Hemolysis percentages (a) and coagulation properties of MSNs and MSNs-RhB at various concentrations of 50e500 mg mL�1: (b) APTT, (c) PT, and (d) Fib. The inset in(a) shows the hemolysis of the negative PBS control, samples MSNs and MSNs-RhB of different concentrations in mg mL�1 as labelled on the tubes, and the positive water control.

Q. He et al. / Biomaterials 31 (2010) 7785e77967794

Owing to the extensive mesoporosities, high surface areas andhigh absorption capacities, zeolite and mesoporous bioactive glasscan be used for fast hemostasis [45e48]. Recently, C.S. Liu reporteda kind of Ag and Ca incorporated MCM-41-type mesoporous silicapowder used for antibacteria and hemostasis, and experimentalresults showed that its hemostasis was mainly derived from twofactors: (1) the inducement of hematoblast conglomeration andfibrous intension enhancement owing to high water absorptioncapacity, (2) the activation of the intrinsic pathway by releasedcalcium ions [49]. However, MSNs-induced coagulation should becompletely prevented in order to avoid any thrombosis when theyare administrated by vein injection.

It can clearly be found from Fig. 10b, c and d that neither ofthe APTT, PT and Fib values of SBA-15-type MSNs and MSNs-RhBexceeds their normal ranges of 31.5e43.5 s, 0.82e1.15 of PTR(PT ratios between MSNs samples and the reference sample) and2e4 g L�1, respectively, and all three parameters are very closeto blank control values and almost independent of concentrationin a broad range of 50e500 mg mL�1. In addition, there is notstatistically significant difference in APTT, PT and Fib valuesbetween MSNs and MSNs-RhB at the same concentrations of

50e500 mg mL�1. This suggests that both SBA-15-type MSNs andMSNs-RhB in a broad concentration range of 50e500 mg mL�1

have not activated the intrinsic, extrinsic and common coagula-tion pathways. This is utterly different from the dry mesoporoussilica powder previously reported by C.S. Liu [49], because allhydrophilic mesoporous channels of MSNs and MSNs-RhB havebeen fully filled with PBS during experimental operation in thepresent study, and no space is left for further water absorptionwhen mixed with plasma. Thus both MSNs and MSNs-RhB hadnot effected the normal coagulation/anti-coagulation functions ofplasma, i.e., the blood compatibility of SBA-15-type MSNs-RhB issatisfactory.

4. Conclusions

In summary, a drug delivery system SAB@MSNs-RhB for anti-ROS/hepatic fibrosis has been constructed by loading a negativelycharged drug SAB into the positively charged fluorescent SBA-15-type MSNs-RhB, which is of high surface area (750 m2 g�1), largepore volume (0.7 cm3 g�1) and uniform particle size (about400 nm). The dosage formulation SAB@MSNs-RhB effectively

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Q. He et al. / Biomaterials 31 (2010) 7785e7796 7795

protected the loaded drug SAB from decomposition. The multi-release experiments showed that SAB@MSNs-RhB possesseda much more significant SAB sustained-release capability thanSAB@MSNs, and higher SAB release rates and concentrations in theprolonged release process beyond 96 h after the consumption ofpreviously released SAB. Compared with free SAB, SAB@MSNs-RhBexhibited more remarkable inhibiting effect on ROS levels and onthe proliferation activity of LX-2 cells in a considerably long term(72 h). SAB@MSNs-RhB showed the enhanced cellular drug uptake,bioaccessability and efficacy for ROS level suppression and anti-hepatic fibrosis by the nanoparticles-mediated endocytosis andthe sustained SAB release. There was no visible cytotoxicity ofSAB@MSNs-RhB against LX-2 cells in a broad concentration range(0.5e100 mM) and incubation time periods up to 72 h. MSNs-RhBshowed the negligible hemolysis activity and had not effected thenormal coagulation/anti-coagulation functions of human plasma ina broad concentration range (50e500 mg mL�1).

Acknowledgements

We greatly acknowledge financial supports from the NationalNature Science Foundation of China (Grant Nos. 20633090,50823007 and 50972154), National 863 High-Tech Program(Grant No. 2007AA03Z317), Shanghai Rising-Star Program (GrantNo. 07QA14061), Shanghai Nano-Science Project (Grant No.0852nm03900) and CASKJCX Projects (Grant Nos. KJCX2-YW-M02and KJCX2-YW-210).

Appendix

Figures with essential color discrimination. Figs. 4, 6, 8e10 inthis article are difficult to interpret in black andwhite. The full colorimages can be found in the online version, at doi:10.1016/j.biomaterials.2010.07.008.

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