Fabrication of trail – s layer/ graphene quantum dot complexes for induction of apoptosis in colon cancer cells

Shima Lotfollahzadeh,1 Elahe sadat hosseini,2 Maryam nikkhah,3,*

1. Department of Nanobiothechnology, Faculty of Biological Sciences, Tarbiat Modares University
2. Department of Nanobiothechnology, Faculty of Biological Sciences, Tarbiat Modares University
3. Department of Nanobiothechnology, Faculty of Biological Sciences, Tarbiat Modares University

Abstract

Introduction

Tumor necrosis factor (tnf) related apoptosis inducing ligand (trail), known as a cytokine of the tnf superfamily, is considered as a promising anti-tumor agent due to its ability to induce apoptosis selectively in a wide variety of tumor cells while sparing vital normal cells. however, its therapeutic efficacy has been hampered by its extremely short half-life and weak pharmacokinetic profile. to overcome these obstacles trail-based nano therapies, aiming to significantly improve its circulating time in vivo have been employed. crystalline bacterial cell surface layers (s-layers) are two-dimensional proteinaceous arrays that have been identified in a great number of different species of bacteria and represent an almost universal feature of archaea .one of the most fascinating properties of isolated native s-layer proteins and s-layer fusion proteins is being capable to self-assemble into monomolecular crystalline arrays in suspension and recrystallize at liquid-surface interfaces, lipid structures and on solid supports (e.g. polymers, metals, silicon wafers) . in this context we have fabricated trail – s-layer fusion protein conjugates with graphene quantum dots (gqds) to benefit self-assembly properties of s-layer protein to improve the systemic circulation and elevate antitumor activity of trail as well as tracking and real time monitoring of the complex via fluorescent gqds.

Methods

Expression and purification of trail – s-layer fusion protein: trail – s-layer fusion protein cdna was cloned into prokaryotic expression vector pet28a and was expressed in iptg induced e.coli bl21(de3) cells and purified using immobilized metal-ion affinity chromatography. purified proteins was analyzed by sds-page. synthesis and characterization of gqd : non-toxic blue-luminescent graphene quantum dots (gqds) were synthesized by hydrothermal method and characterized by uv-vis, photoluminescence (pl), fourier transform infrared (ftir) and raman spectroscopies, atomic force microscopy (afm) and transmission electron microscopy (tem) conjugation of fusion protein and gqds : conjugation was achieved by mixing of fusion protein aqueous solution (0.16 mg/ml) and gqds (0.05mg/ml) followed by incubation at room temperature for 2h in dark. trail – s-layer /gqd complexes were characterized by uv-vis and photoluminescence (pl) spectroscopies, gel retardation assay, size and surface charge(ζ-potential) obtained by dynamic light scattering (dls) in the presence or absence of 〖cacl〗_2 (bivalent cations help to recrystallization) , atomic force microscopy (afm) and transmission electron microscopy (tem) cytotoxicity assay, cell imaging and apoptosis assay : ht29 cell line was cultured for all cellular assays .the viability of the cells was determined by mtt assay. doxorubicin (dox), treatment of the cells was done to sensitize ht29 cells to trail. fluorescence microscopy was used for cell imaging and the percentage of apoptotic cells were measured by flow cytometry assay.

Results

Trail – s-layer fusion protein expression was analyzed by sds-page 12.5% and purified protein as a single band was obtained. gqds characterization was performed by all mentioned method and non-toxic highly blue-luminescent graphene quantum dots with a narrow size distribution was achieved. investigation of the trail–s-layer /gqd complex formation was performed at first by gel retardation assay. results showed that free fusion protein and gqd travel a longer distance in the gel compared to the complex. the change in the ζ-potential and size of the complex reveals that electrostatic interactions have been established . formation of gqd-protein complex were further characterized by uv−vis and fluorescence spectroscopies. in the uv−vis spectra, two absorption peaks appear at ∼280 and ∼337 nm, characteristic of protein and gqd, respectively, indicating successful conjugation of the protein and gqds . furthermore , the fluorescence of gqds were partially quenched indicating the strong interaction between protein and gqd . the morphology and size distribution of both gqds and complexe were determined by afm. the cytotoxicity of different concentrations of, trail – s-layer fusion protein was investigated by mtt assay .the results showed that fusion protein had a well cytotoxic effect on ht29 cells. tracking of the complex analyzed by fluorescence microscopy technique .

Conclusion

In this present study we applied a novel trail – s-layer fusion protein conjugated gqds to improve physicochemical, pharmacokinetic and antitumor properties of trail as well as real time monitoring of delivery. according to the results, our designed nano-complex could be considered as a promising therapeutic potential agent for antitumor biotherapy and an important step forward successful clinical applications .

Keywords

key words: trail – s-layer fusion protein, self-assembly, graphene quantum dot