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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 bDepartment 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 2013 Accepted 14 December 2013 Available online 16 January 2014

    Keywords: Montmorillonite Attapulgite Cationic polymer Drug intercalate Gastric cancer

    a b s t r a c t

    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 efficiently. 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 efficiently on the surface of the NLS according to the LIVE/DEAD� BacLight� Bacterial Viability Kit in the confocal fluorescence analysis. The histochemical analyses provide evidence that NLS/pTrail can prevent early gastric mucosa effectively.

    � 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 modification. 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 [11e13].

    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 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 human gastrointestinal tract, shows significant genetic diversity, and is reflected 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- ficial 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: (G. Tang), (P.K. Chu).

    Contents lists available at ScienceDirect


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

    0142-9612/$ e see front matter � 2013 Elsevier Ltd. All rights reserved.

    Biomaterials 35 (2014) 3298e3308

  • in vivo. PEI has repeated basic units with a backbone of two carbon atoms 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-charged phosphate groups of DNA molecules to form the polymer DNA complexes. 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) PEI contrarily displays lower toxicity but poor transfection activity as a result of the poor ability to condense DNA [20,21].

    The tumor necrosis factor (TNF) is a ligand-type cytokine molecule and the TNF-related apoptosis-inducing ligand (pTrail) is a type II transmembrane molecule, in which the carboxyl-terminus of 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 induce apoptosis 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 used clinically for early gastric cancer [22,23]. The mechanism is to inhibit thymidylate synthase or act as the false bases in DNA and RNA, thereby killing tumor cells in the S-phase of the cell cycle. However, a major obstacle in the successful treatment of gastric cancer 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 be modulated by concomitant treatment with an array of genotoxic agents and this phenomenon occurs in a pTrail-dependent manner. In fact, pTrail/5-FU can be sensitized by combined treatment with pTrail and 5-FU in tumor cell lines [24,25]. In the study, a syner- gistic platform combining the functions of chemotherapeutic drug and therapeutic gene is described for early gastric cancer preven- tion. 5-FU as a model drug is intercalated into the MMTand At. Low molecular weight polyethylenimine (PEI, 1200Da) is coated on the surface of the NLS to carry the therapeutic gene pTrail to form the multifunctional compound. The adhesion of bacteria on the NLS is measured 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, Neimenggu province, China and Na-Attapulgite (Na-At) was purified at Xuyi, Jiangsu province, China. 5-fluorouracil (5-FU) and polyethyleneimine (PEI 1200Da) were purchased from Sigma, pEGFPwas purchased from KeyGen Biotech, and l was supplied by Yrbio Biological Technology Co. The BGC-823 cells were maintained in the RPMI1640 medium supplemented with 10% FCS (fetal calf serum). COS-7 cells were cultured in Dulbecco’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 hydrochloric acid and added to the montmorillonite or attapulgite suspension (5 mg/mL) in equal volume. The suspension was stirred at room temperature overnight, washed three times with deionized water, and centrifuged at 3000 rpm. The obtained 5-FU-MMT and 5-FU-At was freeze-dried for 72 h. PEI1200 (0.02 mmol) and 0.5 g of 5-FU-MMT or 5-FU-At were fixed in 10 mL of distilled water for 2 h followed by washing and freeze drying.

    2.3. Characterization of PEI-5-FU-NLS

    The X-ray powder diffraction (XRD) spectra were collected on the Rigaku D/max 2550 PC X-ray diffractometer for 0.5 s/step in the range 0.5� and 30� . The step size was 0.02� . The Fourier transform infrared spectroscopy (FT-IR) spectra were ac- quired on the Bruker Veefor22) at ambient temperature between 4000 and 480 cm�1. The drug content was determined by thermogravimetric analysis (SDT Q600 Thermogravimetric Analyzer, USA). The sample (10mg) was heated from room temperature to 800 �C at a rate of 10 �C/min under air flow.

    Gel electrophoresis was performed at room temperature in the TAE buffer with 1% (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 on a Zetasizer Nano ZS (Malvern Instruments, Southborough, MA) at 25 �C by dynamic light 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 wi