مقالات پذیرفته شده در سومین کنگره بین المللی زیست پزشکی
Overexpression of Cdk9 by nanoparticle delivery system in C2C12 myoblast cells
Overexpression of Cdk9 by nanoparticle delivery system in C2C12 myoblast cells
Vahideh Tarhriz,1Leila Abkhooie,2,*Elham Mansouri,3MirMahdi Abolghasemi,4Mohammad Saeid Hejazi,5
1. Molecular Medicine Research Center, Biomedicine institute, Tabriz University of Medical Sciences, Tabriz, Iran 2. Department of Molecular Medicine, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran 3. Molecular Medicine Research Center, Biomedicine institute, Tabriz University of Medical Sciences, Tabriz, Iran 4. Molecular Medicine Research Center, Biomedicine institute, Tabriz University of Medical Sciences, Tabriz, Iran 5. Department of Molecular Medicine, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
Introduction: Gene delivery systems need crucial update in the issue of nanocarriers. Layered double hydroxides (LDHs) are known as biocompatible inorganic lamellar nanomaterials with versatile properties. Two-dimensional (2D) layered nanostructures provide preservative transferring for their cargos due to physicochemical properties with timely release. In the present study, we show that easy and cost-effective production of Zn/Al-LDH nanoparticles proposed them as potential alternatives for the traditional routs of gene delivery.
Methods: In the present study, Zn–Al layered double hydroxides were synthesized by a coprecipitation method and characterized by FTIR, XRD, SEM, TEM and Zeta potential tests. MTT assay was carried out to measure both the antibacterial activity and cytotoxicity of the nanomaterials. Cdk9 gene was cloned in plasmid pCEP4 for gene expression enhancement in C2C12 cells. Zn/Al-LDH/plasmid/gene (pCEP4/Cdk9) evaluated on C2C12 myoblast cells. For investigation of gene/plasmid delivery by Zn/Al-LDH. Agarose gel electrophoresis was used for determination of the appropriate pCEP4/Cdk9 gene loading into the interlayer of LDH. The Real-Time PCR and Western blotting tests were carried out in transfected and control cells.
Results: Scanning Electron Microscope (SEM) indicated that the structure of Zn/Al-LDH nanoparticles is uniform and homogeneous and showed the narrow size distribution of Zn/Al-LDH nanoparticles with a mean diameter of _74 nm. The particle size in the range of 60–80 nm and the layer morphology of the Zn/Al-LDH were indicated by the TEM micrograph. The XRD pattern of Zn/Al-LDH is shown in the range of 2θ=20–700 that the Zn/Al-LDH sample indicates characteristic diffractions of hydrotalcite. Moreover, the FT-IR spectra of Zn/Al-LDH, VP-Zn/Al-LDH and MD-Zn/Al-LDH nanoparticles in the 400–4000 cm-1 showed the exhibit basic characteristic peaks at ⁓451 cm-1 that were attributed to the presence of M–O (metal–oxygen group) bonds stretching vibration. Also, MTT assay showed that Zn/Al-LDH nanoparticles with a size of (200–300) nm have a less negative effect on C2C12 cells, and 94% of the cells were healthy compared to the control group. Moreover, the result of MTT assay for antibacterial activity demonstrated that bacterial proliferation approximately decreased by 30–40% in comparison to the control group.
Observation of agarose gel electrophoresis was shown significant changes in the concentration of pCEP4/Cdk9 indicated high capacity of LDH nanoparticles in the absorption of DNA.
The results of Real-Time and Western Blotting of Cdk9 gene confirmed the potent ability of Zn/Al-LDH to load and in the delivery of the gene.
Conclusion: In the present study, Zn/Al-LDH nanoparticles were provided with the lateral size of 5–300 nm and the optimal size for effective gene loading of Zn/Al-LDH nanoparticles is 30–100 nm. We used Zn/Al-LDH as Cdk9 gene carrier which was cloned in plasmid pCEP4 for gene expression enhancement in C2C12 cells. Zn/Al-LDH nanoparticles are able to bind to
the plasmid tightly and facilitate their uptake into C2C12 cells and the Zn/Al-LDH can be utilized as an effective carrier in gene delivery and gene therapy. The expression of Cdk9 increased in both RNA and protein level.
our results about appeared that the Zn/Al-LDH nanoparticles can work as valuable devices for drug delivery and cellular uptaking of qualities. Hence, since of easy and inexpensive generation of Zn/Al-LDH nanoparticles, they are proposed as appropriate and reliable choices to traditional and expensive strategies such as Lipofectamine for gene conveyance.