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Multifunctional cationic polymer decorated and drug intercalated layered silicate (NLS) for early gastric cancer prevention Xue Jin a, b , Xiurong Hu a , Qiwen Wang a , Kai Wang a , Qi Yao a , Guping Tang a, b, ** , Paul K. Chu b, * a Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Hangzhou 310028, PR China b Department of Physics & Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China article info Article history: Received 28 November 2013 Accepted 14 December 2013 Available online 16 January 2014 Keywords: Montmorillonite Attapulgite Cationic polymer Drug intercalate Gastric cancer abstract A multifunctional compound that can prevent early gastric cancer is produced by intercalating 3.20% and 1.64% of 5-FU into the interlayer of montmorillonite (MMT) and attapulgite (At), respectively. A low molecular weight cationic polymer, polyethylenimine (PEI1200), is incorporated into the surface of the 5- FU-MMT and 5-FU-At to form the multifunctional layered silicate (NLS). The chemical structure and surface morphology of the NLS are characterized and the model drug of 5-FU is intercalated into the MMT and At. The cell viability determined by the MTT assay on the BGC-823 cell lines show that over 80% of the cells are live under the experimental conditions. The PEI-5-FU-MMT and PEI-5-FU-At can carry the report gene to the BGC-823 and COS-7 cell lines efciently. Western blotting assay shows that the pTrail protein of the BGC-823 cell lines treated with PEI-5-FU-MMT/pTrail and PEI-5-FU-At/pTrail is up- regulated, whereas the cFLIP protein is down-regulated at 48 h compared to free 5-FU, PEI1200, MMT, and At, providing evidence that the NLS can increase the sensitivity of pTrail gene and improve the effects of pTrail gene therapy. Moreover, the Helicobacter pylori (HP) bacteria are adsorbed and immo- bilized efciently on the surface of the NLS according to the LIVE/DEAD Ò BacLightÔ Bacterial Viability Kit in the confocal uorescence analysis. The histochemical analyses provide evidence that NLS/pTrail can prevent early gastric mucosa effectively. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Natural layered silicates (NLS) have unique properties [1e4] and in particular, montmorillonite (MMT) and attapulgite (At) are compatible with polymers after suitable surface modication. These two NLS are phyllosilicate clay minerals composed of crys- talline hydrated octahedral layered magnesium aluminum silicate with exchangeable cations. MMT has a 2:1 layered structure and octahedral silica sheet, whereas At has a discontinuous octahedral structure comprising alternating 2:1 aluminosilicate modules and hydrated channels [5e7]. As medical clays, MMT and At can absorb dietary toxins, bacterial toxins associated with gastrointestinal disturbance, as well as hydrogen ions in acidosis and metabolic toxins [8e10]. Recently, polymer/layered medical clay composites have attracted much attention because of the favorable properties rendered by the combination of polymer with layered silicate. Moreover, cationic-polymer decorated NLS composites offer the advantage of intercalated drugs and carried genes producing therapeutic effects [11e 13]. Gastric cancer is the second leading cause of cancer-related deaths in the world. Early gastric cancer progresses through a se- ries of histological steps initiated by the serial transition from normal mucosa to chronic supercial gastritis, atrophic gastritis, intestinal metaplasia, and nally dysplasia and early gastric cancer [14e16]. Helicobacter pylori infection is associated with gastritis, peptic ulcer disease, or gastric cancer. It colonizes in the human gastrointestinal tract, shows signicant genetic diversity, and is reected in sequence variations within otherwise well-conserved gene and by the presence of non-conserved genes, mobile genetic elements, and chromosomal rearrangements [17e19]. Infection by H. pylori is one of the key processes in inducing early gastric cancer and hence, it is important to control the process of chronic super- cial gastritis and atrophic gastritis. Polyethylenimine (PEI) has been one of the extensively studied polycations and considered the gold standard both in vitro and * Corresponding author. Tel.: þ852 34427724; fax: þ852 34420542. ** Corresponding author. Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Hangzhou 310028, PR China. Tel./fax: þ86 571 88273284. E-mail addresses: [email protected] (G. Tang), [email protected] (P.K. Chu). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.12.040 Biomaterials 35 (2014) 3298e3308

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Page 1: Multifunctional cationic polymer decorated and drug ... · Multifunctional cationic polymer decorated and drug intercalated layered silicate (NLS) for early gastric cancer prevention

Multifunctional cationic polymer decorated and drug intercalatedlayered silicate (NLS) for early gastric cancer prevention

Xue Jin a,b, Xiurong Hu a, Qiwen Wang a, Kai Wang a, Qi Yao a, Guping Tang a,b,**,Paul K. Chu b,*

a Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Hangzhou 310028, PR ChinabDepartment of Physics & Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China

a r t i c l e i n f o

Article history:Received 28 November 2013Accepted 14 December 2013Available online 16 January 2014

Keywords:MontmorilloniteAttapulgiteCationic polymerDrug intercalateGastric cancer

a b s t r a c t

A multifunctional compound that can prevent early gastric cancer is produced by intercalating 3.20% and1.64% of 5-FU into the interlayer of montmorillonite (MMT) and attapulgite (At), respectively. A lowmolecular weight cationic polymer, polyethylenimine (PEI1200), is incorporated into the surface of the 5-FU-MMT and 5-FU-At to form the multifunctional layered silicate (NLS). The chemical structure andsurface morphology of the NLS are characterized and the model drug of 5-FU is intercalated into theMMT and At. The cell viability determined by the MTT assay on the BGC-823 cell lines show that over 80%of the cells are live under the experimental conditions. The PEI-5-FU-MMT and PEI-5-FU-At can carry thereport gene to the BGC-823 and COS-7 cell lines efficiently. Western blotting assay shows that the pTrailprotein of the BGC-823 cell lines treated with PEI-5-FU-MMT/pTrail and PEI-5-FU-At/pTrail is up-regulated, whereas the cFLIP protein is down-regulated at 48 h compared to free 5-FU, PEI1200, MMT,and At, providing evidence that the NLS can increase the sensitivity of pTrail gene and improve theeffects of pTrail gene therapy. Moreover, the Helicobacter pylori (HP) bacteria are adsorbed and immo-bilized efficiently on the surface of the NLS according to the LIVE/DEAD� BacLight� Bacterial Viability Kitin the confocal fluorescence analysis. The histochemical analyses provide evidence that NLS/pTrail canprevent early gastric mucosa effectively.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Natural layered silicates (NLS) have unique properties [1e4] andin particular, montmorillonite (MMT) and attapulgite (At) arecompatible with polymers after suitable surface modification.These two NLS are phyllosilicate clay minerals composed of crys-talline hydrated octahedral layered magnesium aluminum silicatewith exchangeable cations. MMT has a 2:1 layered structure andoctahedral silica sheet, whereas At has a discontinuous octahedralstructure comprising alternating 2:1 aluminosilicate modules andhydrated channels [5e7]. As medical clays, MMT and At can absorbdietary toxins, bacterial toxins associated with gastrointestinaldisturbance, as well as hydrogen ions in acidosis and metabolictoxins [8e10]. Recently, polymer/layered medical clay composites

have attracted much attention because of the favorable propertiesrendered by the combination of polymer with layered silicate.Moreover, cationic-polymer decorated NLS composites offer theadvantage of intercalated drugs and carried genes producingtherapeutic effects [11e13].

Gastric cancer is the second leading cause of cancer-relateddeaths in the world. Early gastric cancer progresses through a se-ries of histological steps initiated by the serial transition fromnormal mucosa to chronic superficial gastritis, atrophic gastritis,intestinal metaplasia, and finally dysplasia and early gastric cancer[14e16]. Helicobacter pylori infection is associated with gastritis,peptic ulcer disease, or gastric cancer. It colonizes in the humangastrointestinal tract, shows significant genetic diversity, and isreflected in sequence variations within otherwise well-conservedgene and by the presence of non-conserved genes, mobile geneticelements, and chromosomal rearrangements [17e19]. Infection byH. pylori is one of the key processes in inducing early gastric cancerand hence, it is important to control the process of chronic super-ficial gastritis and atrophic gastritis.

Polyethylenimine (PEI) has been one of the extensively studiedpolycations and considered the gold standard both in vitro and

* Corresponding author. Tel.: þ852 34427724; fax: þ852 34420542.** Corresponding author. Institute of Chemical Biology and PharmaceuticalChemistry, Zhejiang University, Hangzhou 310028, PR China. Tel./fax: þ86 57188273284.

E-mail addresses: [email protected] (G. Tang), [email protected](P.K. Chu).

Contents lists available at ScienceDirect

Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

0142-9612/$ e see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biomaterials.2013.12.040

Biomaterials 35 (2014) 3298e3308

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in vivo. PEI has repeated basic units with a backbone of two carbonatoms followed by one nitrogen atom and contains primary, sec-ondary, and, in the case of branched PEI, tertiary amino groups,each of which has the potential to be protonated. The positively-charged amino groups may interact with negatively-chargedphosphate groups of DNA molecules to form the polymer DNAcomplexes. High molecular weight (HMW) PEI shows high trans-gene expression but significant cytotoxicity, whereas low molecu-lar weight (LMW, molecular weight less than 2000 Da) PEIcontrarily displays lower toxicity but poor transfection activity as aresult of the poor ability to condense DNA [20,21].

The tumor necrosis factor (TNF) is a ligand-type cytokinemolecule and the TNF-related apoptosis-inducing ligand (pTrail) isa type II transmembrane molecule, in which the carboxyl-terminusof the receptor-binding domain protrudes extracellularly. Recom-binant soluble human pTrail has been employed in clinical inves-tigation of cancer therapy because it has been shown to induceapoptosis in various types of human cancer. It functions by trig-gering the apoptotic signal cascade through binding cognate re-ceptors on the cell surface. 5-FU is an anticancer drug widely usedclinically for early gastric cancer [22,23]. The mechanism is toinhibit thymidylate synthase or act as the false bases in DNA andRNA, thereby killing tumor cells in the S-phase of the cell cycle.However, a major obstacle in the successful treatment of gastriccancer is the resistance of gastric cancer cells to current chemo-therapy and cell toxicity.

It has been shown that pTrail sensitivity in cell lines can bemodulated by concomitant treatment with an array of genotoxicagents and this phenomenon occurs in a pTrail-dependent manner.In fact, pTrail/5-FU can be sensitized by combined treatment withpTrail and 5-FU in tumor cell lines [24,25]. In the study, a syner-gistic platform combining the functions of chemotherapeutic drugand therapeutic gene is described for early gastric cancer preven-tion. 5-FU as a model drug is intercalated into the MMTand At. Lowmolecular weight polyethylenimine (PEI, 1200Da) is coated on thesurface of the NLS to carry the therapeutic gene pTrail to form themultifunctional compound. The adhesion of bacteria on the NLS ismeasured and synergistic release of the drug and gene is investi-gated systematically both in vitro and in vivo.

2. Materials and methods

2.1. Materials

The Na-montmorillonite (Na-MMT) was purified at Ningcheng, Neimengguprovince, China and Na-Attapulgite (Na-At) was purified at Xuyi, Jiangsu province,China. 5-fluorouracil (5-FU) and polyethyleneimine (PEI 1200Da) were purchasedfrom Sigma, pEGFPwas purchased from KeyGen Biotech, and l was supplied by YrbioBiological Technology Co. The BGC-823 cells were maintained in the RPMI1640medium supplemented with 10% FCS (fetal calf serum). COS-7 cells were cultured inDulbecco’s Modified Eagle’s Medium (DMEM) with 10% FCS.

2.2. Synthesis of PEI-5-FU-NLS

The 5-FU-MMT and 5-FU-At were prepared by an ion-exchange reaction. The 5-FU solution (0.075 mol/L or 0.025 mol/L) was adjusted to a pH of 4 by hydrochloricacid and added to the montmorillonite or attapulgite suspension (5 mg/mL) in equalvolume. The suspension was stirred at room temperature overnight, washed threetimes with deionized water, and centrifuged at 3000 rpm. The obtained 5-FU-MMTand 5-FU-At was freeze-dried for 72 h. PEI1200 (0.02 mmol) and 0.5 g of 5-FU-MMTor 5-FU-At were fixed in 10 mL of distilled water for 2 h followed by washing andfreeze drying.

2.3. Characterization of PEI-5-FU-NLS

The X-ray powder diffraction (XRD) spectra were collected on the Rigaku D/max2550 PC X-ray diffractometer for 0.5 s/step in the range 0.5� and 30� . The step sizewas 0.02� . The Fourier transform infrared spectroscopy (FT-IR) spectra were ac-quired on the Bruker Veefor22) at ambient temperature between 4000 and480 cm�1. The drug content was determined by thermogravimetric analysis (SDTQ600 Thermogravimetric Analyzer, USA). The sample (10mg) was heated from roomtemperature to 800 �C at a rate of 10 �C/min under air flow.

Gel electrophoresis was performed at room temperature in the TAE buffer with1% (w/w) agarose gel at 80 V for 40 min. The DNA was visualized by UV (254 nm)illumination. The particle size and zeta-potential measurements were performed ona Zetasizer Nano ZS (Malvern Instruments, Southborough, MA) at 25 �C by dynamiclight scattering (DLS) using the Zetasizer 3000 (Malvern Instruments, Worcester-shire, UK).

2.4. Surface morphology of PEI-5-FU-NLS

The morphology of NLS was studied by transmission electron microscopy (TEM)on the Philips CM100 electron microscope operated at 100 kV and equipped with aLaB6 gun and Nikon Digital camera) and atomic force microscopy (AFM) on theDIINNOVA (Veeco, USA). Prior to TEM, 10 mL of the complex containing 0.05 mg ofpDNA at the weight ratio of 20 was dropped onto carbon-coated copper grids. Afterdrying for 5 min at room temperature, the images were recorded by electron mi-croscope. Before conducting AFM, the complex solution was dropped onto the sili-con surface and then the solution was removed with a piece of tissue paper. Theimage analysis was performed using the nanoscope software.

Scheme 1. Schematic diagram of modification NLS.

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2.5. Cell behavior

2.5.1. Cell viability assayThe cytotoxicity of NLS was evaluated by the MTT assay. The BGC-823 cell lines

were seeded at 8 � 103 cells/well in 200 mL serum-medium on 96-well plates. After24 h, the medium was replaced with 200 mL of serum-free culture media con-taining serial dilutions of complex solutions at a series of weight ratios for 4 h. The

culture medium was replaced by 100 mL of the medium with 10% FBS and 0.5 mg/mL filtered MTT solution for another 4 h. Finally, each well was replaced with100 mL DMSO and measured spectrophotometrically on an ELISA plate reader(Model 550, Bio-Rad) at a wavelength 570 nm. The relative cell growth (%) relatedto the control cells cultured in the media without the polymer was calculated by[A]test/[A]control � 100.

Fig. 1. A Transmission electron microscopy (TEM) images of NLSs: (a) MMT, (b) PEI/MMT, (c) PEI-5-FU/MMT, (d) At, (e) PEI-At and (f) PEI-5-FU/At. B Atomic force microscopy (AFM)(up) and Scanning electron microscopy (SEM) (down) images of NLSs: (a)(e) MMT, (b)(f) PEI-5-FU-MMT, (c)(g) At, (d)(h) PEI-5-FU-At.

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2.5.2. In vitro transfection efficiency assayThe luciferase transfection efficiency mediated by PEI-5-FU/NLS was evaluated

using the BGC-823 cell lines. The cells were cultured on 6 well plate at 1.5 � 106 perwell for 24 h. The medium was changed with 10 mg of plasmid and 200 mg of PEI-5-FU/NLS complexes in the serum-free RPMI1640 medium (0.8 ml) and the cells wereincubated for 4 h. The complex medium was replaced again by the 10% serumRPMI1640 medium and incubated for 48 h post-transfection. The luciferase activitywas measured according to standard protocols of luciferase assay system (Promega).The total protein was measured according to a BCA protein assay kit (Pierce) andluciferase activity was reported in terms of RLU/mg cellular protein. The expressionof pEGFP was analyzed by confocal fluorescence microscopy (Leica SPE).

2.5.3. Antiproliferative activity assayThe Annexin V-FITC Apoptosis Detection Kit (Keygen, Jiangsu, China) was

employed in the measurement of the apoptosis rate induced by pTrail. The cells

were detached with the EDTA-free trypsin solution and washed. 5 mL of FITC-conjugated annexin V and 5 mL of propidium iodide (PI) were added to 500 mL ofthe cell suspension and incubated for 15 min at room temperature in darknessprior to flow cytometric analysis. The proteins of the cells were extracted after 48 htransfection for western blot. The cell lysates were electrophoresed, transferred toblots, and probed by reacting with antibodies as reported previously. The anti-bodies used were b-actin antibody (Keygen), cFLIP-antibody (Biosynthesis, Beijing,China), Trail-antibody (Boster, Wuhan, China), and Goat anti-rabbit IgG-HRP-anti-body. The immunoreactivity was detected by enhanced chemiluminescence (ECL;Keygen) and the blots were visualized and quantified using the Gel-Pro32 software.

2.6. Bacteria adhesion studies

The antibacterial activity was assessed by scanning electron microscopy andconfocal fluorescence microscopy. The bacterial solution was first adjusted to1.5 � 106 CFU/mL by a phosphate buffer solution (PBS) and 50 mL of particles with

Fig. 2. A Powder X-ray Diffraction (XRD) curve of NLSs. 5-FU, PEI-5-FU/MMT and MMT (left). 5-FU,PEI-5-FU/At and At (right). B Fourier transform infrared spectroscopy (FT-IR)spectra of 5-FU, MMT, 5-FU/MMT and PEI-5-FU/MMT (left). 5-FU, At and PEI-5-FU/At (right). C Thermogravimetric analysis (TGA) curve of NLSs. MMT, 5-FU/MMT and PEI-5-FU/MMT(up). At, 5-FU/At and PEI-5-FU/At (down).

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different concentrations (0.01 and 0.05 g/ml) were added to 1 mL of the bacteria so-lution. Itwas incubated at 37 �C and agitated at a speed of 230 rpm for 1 h. The solutionwas stained by LIVE/DEAD� BacLight� Bacterial Viability Kit for confocal fluorescenceanalysis. Prior to microscopic examination, the mediumwas placed in glutaraldehyde(2.5%) for 3 h and then distilled water. The bacteria and particle solution was centri-fuged and after washing, the sample was prepared on a silicon slice.

2.7. In vivo prevention

The preventive effects of PEI-5-FU-NLS were assessed using ethanol-inducedacute gastritis. Mice (BALB/C mice, female) 6 weeks old were randomly dividedinto three groups, including the PBS control group, PEI-5-FU-MMT/pTrail model

Fig. 2. (continued).

Table 1Average size and zeta potential of NLSs.

Compounds Size/nm z-Potential/mV

MMT 1285 � 483 0.43 � 0.295-FU-MMT 321 � 62 �1.77 � 0.71PEI1200-5-FU-MMT 623 � 308 þ60.03 � 8.31AT 3127 � 1157 �9.67 � 0.295-FU-AT 354 � 56 �3.27 � 0.48PEI1200-5-FU-At 602.80 � 13.86 þ32.6 � 5.09

Fig 3. The agarose gel electrophoresis assay of PEI-5-FU/MMT and PEI-5-FU/At atdifferent weight ratio.

Fig. 4. The drug release behavior of 5-FU-MMT and 5-FU-At in artificial gastric juice atroom temperature (25 �C). The drug-loaded particles are immersed in 10 ml of aliquotsin the simulated human gastric media (PBS, HCl adjust to pH 1.2) and incubated in aconstant temperature shaking bed at 37 �C and 80 rpm. The drug concentrations aredetermined by UV/vis spectrophotometry (Hitachi, UV-3400) at 265 nm.

Fig. 5. Cell viability of MMT, PEI-5-FU-MMT (a), At, PEI-5-FU-At (b) in BGC-823 celllines for concentrations between 0.005 and 0.07 mg/mL.

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Fig. 6. A Transfection efficiency of PEI-5-FU/At/luc and PEI-5-FU/MMT/luc complexes in COS-7 (a) and BGC-823 (b) cell lines at 24 and 48 h. B Transfection efficiency of PEI-5-FU/At/pEGFP (a) and PEI-5-FU/MMT/pEGFP (b) complexes in BGC-823 cell lines at 48 h.

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group, and PEI-5-FU-At/pTrail model group. After ethanol (75%) administration for aweek, the therapy groups of PEI-5-FU-MMT/pTrail and PEI-5-FU-At/pTrail were fedthree times every 3 days. The control group received the same amount of normalsaline instead of the complexes. The animals were killed by cervical dislocation afteradministration of the particle solution at day 3 and day 21.

2.8. H&E staining assay

The animal experiments were performed in accordance with the CAPN (ChinaAnimal Protection Law) and the protocols were approved by Zhejiang UniversityAnimal Care and Use Committee. Athymic female mice (BALB/c strain) (4e6 weeks,18e22 g weight) were purchased from the University of Zhejiang Animal CareCenter and maintained in a pathogen-free environment at a controlled temperature(24 �C).

After opening the stomach along the greater curvature, the specimens werewashed by 0.1 mol/L PBS three times, fixed in 4% formaldehyde, dehydrated withgradient ethanol, and embedded in paraffin. The tissue sections (5 mm) weredewaxed and rehydrated according to a standard protocol and stained with hema-toxylin and eosin (H&E) for microscopic observation.

2.9. Statistical analysis

The data shown in this paper are presented as means � standard deviation.Statistical significance (p< 0.05) was evaluated by using the Student t-testwhen onlytwo groups were compared. If more than two groups were compared, evaluation ofsignificance was performed using one-way analysis of variance (ANOVA) followedby Bonferroni’s post hoc test. In all the tests, the statistical significance was set atp < 0.05.

3. Results and discussion

3.1. Characterization of PEI-5-FU-NLS

3.1.1. Surface morphology of PEI-5-FU-NLSThe PEI-5-FU/MMT and PEI-5-FU/At are fabricated by inter-

calating drugs into the NLS using a stirring technique as shown inScheme 1 [11,26]. After purification by centrifugation and filtration,the morphology of the NLS is examined by transmission electronmicroscopy and according to Fig. 1A (b and c), the clay platelets ofMMT are randomly distributed in a face-to-face stacked configu-ration. The 5-FU is intercalated in the space between the clay layersfinely but the MMTwithout 5-FU shows agglomeration (Fig. 1Aea).On the PEI-5-FU/At, the PEI is grafted onto the surface of At and 5-FU is intercalated in the network structure, resulting in slightlylarger diameters of the single rods (Fig. 1Aed,e,f). Compared to theMMT and At matrices, the different octahedral layered structureresults in different intercalation configurations which affect therelease rate of 5-FU from NLS in vitro and in vivo. The AFM and SEMimages confirm the size and surface morphology of the PEI-5-FU-NLS (Fig. 1B aeh).

3.1.2. Structure of PEI-5-FU-NLSThe structure is consistent with that shown by XRD (Fig. 2A). 5-

FU is first adsorbed on the free surface of the NLS and replaces Naþ

in the interlayer, followed by 5-FU replacing the eOH groups toform ionic bonds with Al3þ andMg2þ in the NLS. The FTIR spectra of5-FU, MMT, PEI1200-5-FU-MMT, At and PEI1200-5-FU-At areshown in Fig. 2B. The band at 1037 cm�1 arises from the stretchingvibration of the SieOH band from MMT and At. After PEI1200coating onto the surface of NLS, the CeH band at 2968 cm�1 fromPEI appears. According to the TGA profile of pure NLS, 5-FU-NLS,and PEI-5-FU-NLS (Fig. 2C), the intercalated concentrations of 5-FUin MMT and At are 3.20% and 1.64%, respectively. The importantproperties of the drug-intercalated NLS are summarized in Table 1.The particle size of 5-FU/MMTand 5-FU/At is about 300 nm and thesurface charges are negative, �1.77 and �3.27 mV, respectively.After polyethylenimine incorporation into the drug-intercalatedNLS, the particle size increases to 600 nm and the surface chargeschange to 60.1 and 36.2 mV, respectively, indicating that thecationic polymer encapsulated NLS can take genes to the cells. The

agarose gel electrophoresis assay confirms that at weight ratio of 2and 3, the PEI1200-5-FU-MMT and PEI1200-5-FU-At can condenseDNA (Fig. 3). Owing to the neutral or negative charge nature of thesurface for MMT and At, these assembled PEI1200 serves as a“positive shell” of the NLS to interact with negatively charged DNAand as a result, renders even stronger complexing capability.

3.1.3. In vitro drug releaseThe 5-FU release from NLS is monitored in artificial gastric juice

at room temperature (25 �C) (Fig. 4). 5-FU is gradually released after1 day from PEI-MMT and PEI-At. Comparing the two NLS, therelease from PEI-At is swifter than that from PEI-MMT. This stemsfrom the discontinuous octahedral structure of At and the weakforce with 5-FU thereby enabling easy drug release from PEI-Atin vitro. After 7 days, three times of 5-FU escape from PEI-Atcompared to PEI-MMT.

3.2. Cell behavior

3.2.1. Cell viability and gene transfection efficiencyThe cytological behavior of the drug-intercalated NLS is studied

using the BGC-823 cell line (human gastric carcinoma). As shown inFig. 5, lower cytotoxicity is observed fromMMT, PEI-5-FU-MMT, At,

Fig. 7. Protein levels of pTrail and cFLIP in BGC-823 cell lines treated with 5-FU,PEI1200, At, MMT, PEI-5-FU/At/pTrail, and PEI-5-FU/MMT/pTrail and PBS (Control). Forthe protein analysis, the proteins were extracted after 48 h.

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and PEI-5-FU-At at concentrations between 0.005 and 0.07 mg/mL,and the cell viability is over 80% under the experimental conditions.

The gene transfection efficiency is determined using the BGC-823 and COS-7 cell lines (African green monkey Cercopithecusaethiops fibroblast-like kidney cells) at 24 h and 48 h mediated byPEI-5-FU/MMT (Fig. 6-A). The results are influenced by the celllines, weight ratios, and time. For the COS-7 cell lines, the luciferaseexpression mediated by PEI-5-FU/MMT is 2 times higher at 48 hthan 24 h for a weight ratio of 10, whereas for the BGC-823 celllines, it is 5 times higher at 48 h for the same weight ratio. Tofurther verify the gene delivery capability of PEI-5-FU/MMT/pDNAand PEI-5-FU/At/pDNA, the gene expression of pEGFP is visualizedby fluorescence microscopy. Using the optimal transfection w/wratio of 20, the images demonstrate that PEI-5FU/MMT and PEI-5-FU/At can carry the pEGFP report gene to the BGC-823 cell linesat 48 h and stronger fluorescence signals are observed (Fig. 6-B).

3.2.2. Antiproliferative activity assayThe cFLIP protein interferes with the initial proteolytic step of

apoptosis induced by pTrail (tumor necrosis factor-relatedapoptosis inducing ligand) and is a potent suppressor of the celldeath pathway [27,28]. The aberrant up-regulation of the cFLIPprotein which would be present in cancers and the small inter-fering RNA-mediated down-regulation are found to be sufficient tosensitize the pTrail-resistant tumor cell lines to apoptosis inducedby pTrail. Consequently, the identification drugs which sensitize

cancer cells to pTrail by cFLIP down-regulation result in the iden-tification of DNA-damaging drugs as potent down-regulators ofcFLIP and sensitizers to pTrail-induced apoptosis. 5-FU, the DNA-damaging agent, can trigger down-regulation of cFLIP. In the pre-sent study, pTrail and cFLIP proteins are analyzed by westernblotting in the BGC-823 cell lines after treatment withMMT, At, PEI,PEI-5-FU-MMT, and PEI-5-FU-At for 48 h and b-actin is used as theprotein level reference. The pTrail protein of the cells treated withPEI-5-FU-MMT/pTrail and PEI-5-FU-At/pTrail is up-regulated butthe cFLIP protein is down-regulated at 48 h in comparisonwith free5-FU, PEI1200, MMT, and At. It indicates that the 5-FU intercalatedNLS system can suppress cFLIP protein efficiently. Furthermore, theNLS can express pTrail gene, up-regulate pTrail protein, and inducetumor cell apoptosis (Fig. 7). The apoptosis data obtained from theBGC-823 cell lines treated by PEI-5-FU-MMT, PEI-5-FU-At, andcontrol are shown in Fig. 8 and Table 2. The attractive force between5-FU and At is weaker and so more 5-FU is released from At than

Fig. 8. Induction of apoptosis on the BGC-823 cell lines by PEI-5FU/MMT/pTrail and PEI-5-FU/At/pTrail and control for 48 h.

Table 2Cell apoptosis analysis by flow cytometry.

Sample Cell apoptosis

Early and late apoptotic (%) Cell death (%)

PEI-5-FU-MMT/pTrail 7.03 8.92PEI-5-FU-At/pTrail 4.47 16.35Control 0.14 0.13

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MMT. The results show that PEI-5-FU-At can suppress the expres-sion of cFLIP protein, increase the sensitivity of pTrail gene, andimprove the effects of pTrail gene therapy.

3.3. Bacteria adhesion and viability

Fig. 9 shows the morphological changes of H. pylori (HP) incu-bated with NLS (a and b designating PEI-5-FU-MMT/pTrail and cand d for PEI-5-FU-At/pTrail at 24 h and 48 h, respectively). After

PEI is conjugated onto 5-FU-MMT and 5-FU-At, the zeta potentialschange from �1.77 � 0.71 mV to 60.03 � 8.31, and �9.67 � 0.29 to32.6 � 5.09 mV, respectively. The strong adsorption and immobi-lization capacity leads to adhesion of more HP bacteria on thesurface of NLS from 24 h to 48 h. To obtain more evidence, in situlife-dead staining of bacterial organisms attached to the NLS sur-face is conducted to determine the viability and ratio of dead bac-teria to total number of bacteria attached [29e31]. Fig. 10 displaysthe epifluorescence microscopy images of the HP bacteria and the

Fig. 10. Viability of Helicobacter pylori attached to PEI-5-FU-MTT/pTrail and PEI-5-FU-At/pTrail as determined by a fluorescent vital stain at 24 h and 48 h. The living bacterial cellsexhibit green fluorescence (Syto 9 dye) while the dead microorganisms are characterized by a red fluorescence (propidium iodide). (For interpretation of the references to color inthis figure legend, the reader is referred to the web version of this article.)

Fig. 9. Morphology of Helicobacter pylori after incubation with PEI-5-FU/MMT/pTrail (a, b) and PEI-5-FU/At/pTrail (c, d) at 24 h and 48 h.

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majority of the HP is observed to be viable after attachment toMMTand At at 24 h as characterized by 90e100% of the HP showinggreen fluorescence, meaning that MMT and At can adsorb bacteriaeffectively. After treatment with PEI-5-FU-MMT/pTrail and PEI-5-FU-At/pTrail, 30%e20% of the attached bacteria are found to bedead and display significant red fluorescence compared to theMMTand At groups. The images of PEI-5-FU-MMT/pTrail and PEI-5-FU-At/pTrail reveal extensive red fluorescence on the surface at 48 h.For both NLS samples, more than 70e80% of the H. pylori areobserved to be dead. Reduction of HP viability is related to drug andgene release, which is interacted and carried in the NLS, and isfound to be significantly different compared to MMT and At (10e20% red fluorescence). Notably, co-delivery of drug and gene affectsthe viability of the attached bacteria. As shown by the drug andprotein measurement, shielding of the cationic polymer and drugdelivery is the reason for the enhanced antibacterial efficacy of thistype of NLS.

3.4. In vivo prevention

Fig. 11 shows the histochemical analysis of the gastric mucosatreated by NLS/pTrail at days 3 and 21 after ethanol (75%) admin-istration [32]. According to Fig. 11 (a and b), in the NLS/pTrailtherapy groups (n ¼ 10 and concentration ¼ 0.3 mg/ml), the gastricmucosal injury is obviously slighter than that of the control group(Fig.11-c, n¼ 3) at day 3. The area of erosion and ulcers aremild anda few of the inflammatory cells infiltrate. After treatment for 21days, the gastric mucosa is smooth (Fig. 11 d and e, n ¼ 10 andconcentration ¼ 0.3 mg/ml) and the layers have clear boundaries.There are no significant atrophy parietal cells and chief cells. Thecurative effect of the PEI-5-FU-MMT/pTrail group is better than thatof the PEI-5-FU-At/pTrail group. The surface positive charge on theNLS is an important factor for bacteria adhesion and protection ofthe gastric mucosa. In contrast, the gastric mucosa surface of theuntreated group (n¼ 3, Fig. 11f) is unevenwith ulcers and bleeding.It is congested and has edema. With telangiectasia, the mucuslayers are damaged and the submucosal gastric glands areincomplete.

4. Conclusion

Montmorillonite (MMT) and attapulgite (At) are phyllosilicateclay minerals having the structure of crystalline hydrated

octahedral layered magnesium aluminum silicate. Comparing to At,MMT is a 2:1 layered structure and has an octahedral silica sheet,whereas At has a discontinuous octahedron structure consisting ofalternating 2:1 aluminosilicate modules and hydrated channels.Hence, more 5-FU is intercalated into the MMT (weight ratio of3.20%) than At (1.64%). These twomaterials can be conjugated withthe cationic-polymer, polyethylenimine (PEI1200) to efficientlyabsorb H. pylori (HP) bacteria in vitro and in vivo. The modifiednatural layered silicates (NLS) are multifunctional compoundswhich can take 5-FU and pTrail gene to prevent early gastric cancersynergistically.

Acknowledgments

This work was jointly supported by the National High Tech-nology Development Program of China (863 Program2007AA03Z355, 2009AA02Z416), National Natural Science Foun-dation of China (grant # 30970711 and 21074111), Major Scientificand Technological Innovation Project of Hangzhou (20122511A43),Hong Kong Research Grants Council (RGC) General Research Funds(GRF) Nos. 112510 and 112212, and City University of Hong KongApplied Research Grant Nos. 9667066 and 9667069.

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