In Vitro and in Vivo Suppression of He Pa to Cellular

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EUROPEAN JOURNAL OF CANCER 4 3 ( 2 0 0 7 ) 1 8 4 –1 9 3 available at www.sciencedirect.com journal homepage: www.ejconline.com In vitro and in vivo suppression of hepatocellular carcinoma growth by chitosan nanoparticles Lifeng Qia,c,*, Zirong Xua, Minli Chenb a Zhejiang University, Nano-biology Lab of Animal Science College, Hangzhou 310029, PR China Centre of Lab Animal Research, Zhejiang College of Traditional Chinese Medicine, Hangzhou 310053, PR China c Orthopaedic Research Lab, Depart
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  In vitro and in vivo suppression of hepatocellularcarcinoma growth by chitosan nanoparticles Lifeng Qi a,c, *, Zirong Xu a , Minli Chen b a Zhejiang University, Nano-biology Lab of Animal Science College, Hangzhou 310029, PR China b Centre of Lab Animal Research, Zhejiang College of Traditional Chinese Medicine, Hangzhou 310053, PR China c Orthopaedic Research Lab, Department of Orthopaedics, School of Medicine, West Virginia University, Morgantown 26506-9196, USA A R T I C L E I N F O Article history: Received 3 April 2006Received in revised form10 August 2006Accepted 31 August 2006 Available online 17 October 2006 Keywords: Hepatocellular carcinomaChitosan nanoparticlesAntitumourA B S T R A C TChitosan nanoparticles (CNP), a kind of widely used drug carrier, have shown potent cyto-toxic effects on various tumour cell lines in vitro and in vivo . This study sought to evaluatethe antitumour effect of CNP on growth of human hepatocellular carcinoma (BEL7402) andthepossiblemechanismsinvolved.Cellsweregrownintheabsenceandpresenceofvariousconcentrations of CNP with mean particle size of about 40 nm. Cell viability, ultrastructuralchanges, surface charge, mitochondrial membrane potential, reactive oxygen species (ROS)generation, lipid peroxidation, DNA fragmentation and fatty acid composition were ana-lysed by MTTassay, electron microscopy, zetasizer analysis, flow cytometry, spectrophoto-metric thiobarbituric (TBA) assays, DNA agarose gel electrophoresis and GC/MSrespectively. For in vivo experiments, male BABL/c nude mice were implanted with BEL7402cells subcutaneously to establish human hepatoma model. Chitosan, saline, and CNP withdifferent mean particle size (40, 70 and 100 nm) were administrated by oral administration(1 mg/kg body weight). Tumour and body weight were measured, morphologic changes of tumourandlivertissueswerestudiedunderelectronmicroscope. Invitro ,CNPexhibitedhighantitumouractivitieswithanIC 50 valueof15.01 l g/ml,6.19 l g/mland0.94 l g/mlaftertreat-mentfor24 h,48 hand72 hrespectively.CNPcouldinducecellnecrosisobservedbyelectronmicroscopeand DNAfragmentation. The antitumour mechanismwasmediated by neutral-isation of cell surface charge, decrease of mitochondrial membranepotential and inductionoflipidperoxidation.ThetumourgrowthinhibitoryratesonBEL7402cellsinnudemicetrea-tedwithchitosanandCNPwithdifferentmeanparticlesize(40,70and100 nm)were24.07%,61.69%, 58.98% and 34.91% respectively. Typical necrotic morphological changes of tumourtissues and no liver abnormalities were found under electron microscope. In this paper,results show a strong antitumour effect of CNP on human hepatoma cell line BEL7402 invitro and invivo .ThesefindingssuggestthatCNPcouldbeakindofpromisingagentforfur-ther evaluations in the treatment of hepatocellular carcinoma. Ó 2006 Elsevier Ltd. All rights reserved. 1. Introduction Hepatocellular carcinoma (HCC) is one of the most commonmalignancies worldwide. 1 Chemotherapy is one of the mostimportant treatments currently available for cancer diseases.Treatment of patients with HCC remains a clinical challengedue to the disappointing effects of most chemotherapies. 2 The efficacy of chemotherapy is limited and patients have 0959-8049/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.ejca.2006.08.029* Corresponding author: Tel.: +1 304 293 1065; fax: +1 304 293 7070.E-mail address:lifengqi01@hotmail.com(L. Qi). E U R O P E A N J O U R N A L O F C A N C E R 43 (2007) 184  –  193 available at www.sciencedirect.comjournal homepage: www.ejconline.com  to suffer from serious side effects, some of which are life-threatening. 3 Therefore, progresses in developing variouscontrolled and targeted drug delivery systems, especially onbiodegradable polymer nanoparticles, have attracted moreattention. Nanoparticles can provide a controlled and tar-geted way to deliver the encapsulated anticancer drugs andthus result in high efficacy with low side effects. 4 Doxorubi-cin-loaded nanoparticles were reported to show increasedcytotoxicity against hepatocellular carcinoma cells in vitro and in vivo . 5 Chitosan, the deacetylated derivative of chitin, is one of the abundant, renewable, nontoxic and biodegradable carbo-hydrate polymers. Chitosan has been applied broadly as afunctional biopolymer in food and pharmaceutics. Chitosanis known to have various biological activities including antit-umour activities, immuno-enhancing effects, antifungal andantimicrobial activities. 6,7 Chitosan nanoparticles (CNP) havebeen synthesised as drug carriers as reported in previousstudies. 8–10 The uniquecharacterof nanoparticles due to theirsmall size and quantum size effect could make CNP exhibitbiological activities. 11 CNP with small particle size and en-hanced zeta potential have been prepared and characterisedin our previous reports, 11,12 and their in vitro and in vivo cyto-toxic effects against various tumour cell lines were also stud-ied. It showed that CNP with small particle size and positivesurface charge could exhibit higher antitumour activity thanother chitosan derivatives, and the physiochemical propertiesof nanoparticles such as particle size and zeta potential couldmake a significant effect on their antitumour activity. 13 Theantitumour mechanism of CNP was related to its mem-brane-disrupting and apoptosis-inducing activities. 14 CNPalso showed significant dose- and size-dependent antitumouractivity against Sarcoma À 180 and hepatoma H22 in mice. 15 This study was undertaken to probe into the antitumourmechanism of CNP by investigating the effect on cell viability,cell morphology, DNA fragmentation, surface charge, mito-chondrial membrane potential, lipid peroxidation, fatty acidcomposition and the in vivo effect of CNP on human hepa-toma BEL7402 cells in nude mice. 2. Materials and methods 2.1. Drugs and chemicals CNP with mean particle size ranging from 40 nm to 100 nmand positive surface charge about 50 mV were preparedaccording to the method reported in our previous studies. 11,12 CNP were formed by coacervation between positively chargedchitosan (0.5%, w/v) and negatively charged sodium tripoly-phosphate (0.25%, w/v). Nanoparticles with different meansize were obtained by adjusting the volume ratio of chitosanto tripolyphosphate solution. Nanoparticles were purified bycentrifugation at 9000 · g for 30 min. Supernatants were dis-carded and chitosan nanoparticles were extensively rinsedwith distilled water to remove any NaOH residues, and freezedried before further use or analysis. Chitosan nanoparticleswith mean particle size about 40 nm were used for in vitro experiments. CNP were filtered by membrane with diameter0.45 l m and autoclaved to remove any contaminant beforeuse in cell culture. The obtained nanoparticles were stableunder the autoclaving conditions. 11 Trypsin and rhodamine123 were purchased from Sigma Chemicals (St. Louis, MO,USA). 2.2. Cell culture conditions BEL7402 cellswere obtained from the Cell Bank of the ChineseAcademy of Science, Shanghai, China. The cell line was cul-tured in RPMI À 1640 (Gibco, Life Technologies, Vienna, Aus-tria) supplemented with 10% heat-inactivated foetal bovineserum (Gibco) and 100 U/ml penicillin+0.1 mg/ml streptomy-cin in 75 cm 2 tissue plastic flasks (Corning, USA). Cells weremaintained at 37 ° C in a humidified 5% CO 2 atmosphere andmaintained in a log-phase-growth at about 3–6 · 10 5 cells/ml. 2.3. Cell viability assay Before use, BEL7402 cells were digested by 0.25% trypsin, col-lected and the cell numbercounted, then dilutedinto cell sus-pension at a density of 1 · 10 5  /ml in complete medium, andseeded into 96-well plates at 200 l l/well. After being culturedfor 24 h, the cells were immediately treated with variousdoses (25, 50, 75, 100 l g/ml) of CNP for another 24, 48, and72 h. The effect of different treatments on cell viability wasassessed by the tetrazolium dye (MTT) assay. 16 Control cellswere also cultured at the same time. Cell proliferation andinhibition curves were drawn, and the inhibitory concentra-tion against 50% cells (IC 50 ) was determined. 2.4. Ultrastructural cell morphology BEL7402 cells grown on glass coverslips were incubated with25 l g/ml CNP for 4 h. The appropriate solvent was added tothe control. CNP-treated and untreated cellswere fixed in glu-taraldehyde/paraformaldehyde solution and prepared forscanning electron microscopy (SEM) by the triple-fixationGTGO methods. 14 The surface morphology of cells was exam-ined by a XL30-ESEM scanning electron microscope.BEL7402 cells grown on glass coverslips were incubatedwith 25 l g/ml CNP for 24 h. 0.1 M PBS was added to the con-trols. CNP-treated and untreated cells were fixed in glutaral-dehyde/paraformaldehyde solution and prepared fortransmission electron microscopy (TEM) as previously de-scribed. 14 Observations and micrographs were made under a JEM-1200EX transmission electron microscope. 2.5. DNA fragmentation After treatment with 25 l g/ml CNP for 1 h and 4 h, BEL7402cells were collected and DNAwas extracted according to pre-vious methods. 14 The DNA samples thus obtained were runon a 1.5% agarose gel at 50 V and visualised by ethidium bro-mide staining under UV light. 2.6. Changes in surface potential of cells Zeta potential is defined as the difference in electrical poten-tial between the surface of the cells and the bulk surround-ing medium. 17 It is a measure of the net distribution of electrical charge on the surface of the cells. In this paper, E U R O P E A N J O U R N A L O F C A N C E R 43 (2007) 184  –  193 185  the changes in zeta potential of BEL7402 cells treated withCNP for different time were determined as followed.BEL7402 cells grown on glass coverslips were incubated with25 l g/ml CNP at intervals from 30 min to 4 h. The cells weredetached by adding 0.25% trypsin solution to prepare cellsuspension. Then the surface potential of cell suspensionwas determined using Zetasizer Nano-ZS90 (Malvern Instru-ments). The analysis was performed at a scattering angle of 90 ° at 25 ° C using samples diluted to different concentrationswith de-ionised water. 2.7. Determination of mitochondrial membrane potential To study mitochondrial membrane potential (MMP), the cellswere treated with 25 l g/ml CNP at different intervals from30 min to 4 h respectively, and then stained with 10 l g/mlrhodamine 123 which is easily sequestered by the mitochon-drial membrane. 18 Once the mitochondrial membrane poten-tial is lost, rhodamine 123 is subsequently washed out of thecells. The mitochondrial membrane potential was deter-mined using FACSCalibur flow cytometer (Becton Dickinson,San Jose, CA) and analysed by a Cell Quest software program(BD PharMingen, FRANKLIN Lakes, USA). 2.8. Determination of reactive oxygen species and lipid peroxidation The cells treated with various doses (25, 50, 75, 100 l g/ml) of CNP for 24 h were harvested, washed twice and resuspendedin Hank’s buffered saline solution (HBSS). Reactive oxygenspecies (ROS) production was studied by measuring the fluo-rescence intensity of dichlorofluorescein (DCF) as describedby Buyukavci and colleagues. 19 Non-fluorescent 2,7-dichloro-fluorescin-diacetate (DCFH-DA) diffuses into the cell throughthe plasma membrane and is hydrolysed within the cell toDCFH. Intracellular oxidation converts DCFH into the fluores-cent form,DCF. DCFH-DA (MolecularProbes, Eugene,OR, USA)was stored under liquidnitrogenvapouras a 1 mMstock solu-tion in ethanol. Cells were washed with PBS and incubated in1 ml of DMEM with 5 l M DCFH-DA (1:200 dilution) for 60 min.Samples were analysed using FACSCalibur flow cytometer(Becton Dickinson, San Jose, CA). Fluorescence intensity wascalculated as percent increase of fluorescence intensity whencompared with the control sample [(fluorescence intensity of cells in treated sample/fluorescence intensity of cells in thecontrol sample) · 100].Lipid peroxidation products were measured by the spec-trophotometric thiobarbituric (TBA, Sigma, MO, USA) assaysas described by Hofmanova and colleagues. 20 Briefly: 0.75 mlof cell suspension treated with various doses of CNP for24 h was added to the TBA reagent (0.5 ml of 15% trichloroace-tic acid; 0.5 ml of 0.25 N hydrochloric acid; 0.5 ml of 0.6% TBA).This mixture was incubated at 90 ° C for 45 min, then cooled,extracted with 2.25 ml of N-butanol, and centrifuged (5 min,1500 g). The absorbance of the upper phase was measuredat 532 nm. The concentration of thiobarbituric acid reactivesubstances (TBARs) was calculated from a standard calibra-tion curve generated with known amounts of 1,1,3,3-tetraeth-oxypropane (Sigma, MO, USA). 2.9. Fatty acid analysis The fatty acid composition of BEL7402 cell membrane wasdetermined after the cells were treated with 25 l g/ml CNPfor 4 h. The total lipids were extracted from the cells by using the procedure of Bligh and Dyer. 21 The lipid extract was drieddown under nitrogen and was saponified by incubation at65 ° C for 30 min to release the free fatty acids. Following acid-ification with hydrochloric acid, the free fatty acids were ta-ken up into benzene. Fatty acid methyl esters (FAME) wereprepared by using the method of Morrison and Smith. 22 Un-wanted salts were taken up and removed with distilled water,the FAME were dried down and dissolved in trimehylpentane,and then analysed by GC/MS (HP6890GC/5973MsM, USA). 2.10. Antitumour activity in vivo Male athymic BALB/c nude mice, 5-weeks-old weighing 15–25 g at the start of the study, were used. Human hepatomacells (BEL7402, about 5 · 10 6 per 0.2 ml) were implanted sub-cutaneously into right flank of the nude mice. Four days afterinoculation, when the tumour volumes reached2 · 2 · 2 mm 3 , animals were randomly assigned to five treat-ment groups ( n = 10 pergroup).Eachtreatmentgroup receivedrespective 1 mg/kg once daily chitosan or CNP with differentparticle size (40, 70, 100 nm) dissolved in physiological salineby oral administration (p.o.), while 0.9% saline was providedfor the control group. Positive control group of cisplatin(cDDP) was administered intravenously (0.75 mg/kg bodyweight). Mice were allowed food and water ad libitum for upto 4 weeks. The animals were sacrificed, and the tumourswere dissected and weighed. Inhibition ratio was calculatedby following formula: inhibition ratio (%) = [(A–B)/B] · 100,where A is average tumour weight of the negative controlgroup, and B is that of the treatment group. All experimentswere carried out according to the guidelines of the local EthicsCommittee for Animal Use.Tumour and liver specimenswere prefixed in 25 g/L glutar-aldehyde, then in 10 g/L OsO4, dehydrated in ethanol series,and replaced in propene oxide. 23 The sampleswere examinedwith a JEM-1200EX transmission electron microscope. 2.11. Statistical analyses Results are presented as mean±SD. The 2-way ANOVA andStudent’s t test were used to compare data from differenttreatment groups. When P was less than 0.05, differenceswere considered significant. 3. Results 3.1. Inhibitory effect on proliferation of BEL7402 cells Fig. 1shows the cell viability curve at various concentrationsof CNP. The inhibition of cell viability of BEL7402 cells by CNPwas clearly observed in a dose- and time-dependent manner.The median lethal concentration of CNP was 15.01 l g/ml,6.19 l g/ml and 0.94 l g/ml for BEL7402 at 24 h, 48 h and 72 hrespectively. 186 E U R O P E A N J O U R N A L O F C A N C E R 43 (2007) 184  –  193  3.2. Necrotic cell morphology The ultrastructural alterations of BEL7402 cells treated withCNP were observed under scanning electron microscope andtransmission electron microscope. Similar results as our pre-vious report were obtained. The control cell surface showedthe presence of numerous randomly distributed microvilli.After 4 h treatment with CNP, the cells broke into honeycombshape-like pieces, necrotic cell death as evidenced by an earlyloss of membrane integrity, pore forming surface morphologywas observed, and examined by SEM observations (Fig. 2). Thecompletely disruption of cell membrane,dispersedchromatinfragments, vacuolated cytoplasm and organelleswere also re-vealed under TEM (Fig. 3). The necrotic cell morphology indi-cated the unique penetrating mode of CNP against BEL7402cells. 3.3. DNA fragmentation DNA was extracted from cultured BEL7402 cells treated with25 l g/ml CNP for 1 h and 4 h, the occurrence of necrosis wasdetected by agarose gel electrophoresis. Specific DNA degra-dative smearing typical of necrotic degeneration 24 was prom-inent in cells incubated with CNP for 1 h, and the fragmentedDNA increased greatly in cells treated for 4 h (Fig. 4). Itshowed that CNP mainly induce the necrotic cell death result-ing from the disruption of cell membrane at early treatment. 3.3.1. Alterations of surface potential and mitochondrialmembrane potential (MMP) In this study, CNP caused a time-dependent decrease of neg-ative surface potential and MMP in BEL7402 cells treated with25 l g/ml CNP at different intervals from 30 min to 4 h (Fig. 5).After 4 h of treatment, the zeta potential of cells fell to À 0.92 mV from the À 9.39 mV of non-treated cells. It showedthat CNP could neutralise the negative surface charge of BEL7402 cells so as to damage the cell membrane.The percentage of cells with the loss of mitochondrialmembrane potential increased significantly with the increaseof treatment time, and reached 39% when treated for 4 h 0204060801000 Concentrations ( g/mL)   r   t  n  o  c   f  o  s   l   l  e  c  e   l   b  a   i  v   %  o   l Chitosan-72hCNP-24hCNP-48hCNP-72h 2550 75100 Fig. 1 – Effect of CNP on the viability of hepatoma BEL7402cells incubated in the presence of increasing concentrationsof CNP for 24 h, 48 h and 72 h in vitro .Fig. 2 – SEM photographs (the bar is 5 l m) of BEL7402 cells before (A) and after (B) 25 l g/ml CNP treatment for 4 h.Fig. 3 – TEM photographs of BEL7402 cells before (A) and after (B) 25 l g/ml CNP treatment for 24 h (  · 5000). E U R O P E A N J O U R N A L O F C A N C E R 43 (2007) 184  –  193 187
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