The purpose was to design a cancer-specific short peptide for the efficient delivery of siRNA into target (cancer) cells.
- ◉ A549 (human lung adenocarcinoma cell line)
- ◉ MRC5 (human normal cell line)
- ◉ Gene CYP1A1-targeting gene
- ◉ Three types of peptides used in this research: buforin IIB (BIIB), MV1, and MV2
Buforin IIb has been known to target cancer cells by its positive charge. In this research, we design a modified version of Buforin IIB peptide sequence (MV1 and MV2) to find the optimized peptide sequence for low cytotoxicity and enhanced siRNA delivery into targeted cancer cells.
We discovered that buforin IIb had RLLR repeats at the C-terminal end. We hypothesized that deleting the RLLR sequence may lower cell cytotoxicity and increase the siRNA delivery because the shorter peptide together with a positive charge has the advantage of entering through the cell membrane.
- ◉ We planned to perform a cell cytotoxicity test using MTT assay with various concentrations of different versions of peptides
- ◉ We planned to perform an siRNA transfection test and measure the target gene expression level using agarose gel-based RT-PCR.
- ◉ The MTT assay
It is used to measure cellular metabolic activity as an indicator of cell viability, proliferation, and cytotoxicity. This colorimetric assay is based on the reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) to purple formazan crystals by metabolically active cells. The viable cells contain NAD(P)H-dependent oxidoreductase enzymes which reduce the MTT to formazan. The insoluble formazan crystals are dissolved using a solubilization solution and the resulting colored solution is quantified by measuring absorbance at 570 nanometers using a multi-well spectrophotometer. The darker the solution, the greater the number of viable, metabolically active cells.
- ◉ Agarose gel-based RT-PCR
Reverse transcription converts the RNA to cDNA. Then PCR amplification amplifies the template cDNA. Agarose gel electrophoresis allows detection and quantification of amplified DNA. PCR amplification can be broken down into three phases. The exponential phase occurs when the reaction components are in excess, thereby theoretically allowing for a doubling of the PCR product per cycle. In the next phase, the amplification of the PCR product occurs linearly rather than exponentially as the reagents are partially depleted. Then, the plateau phase represents the point in which the amplifications have further slowed or have completely stopped. The end product of PCR is analyzed in the agarose gel. The band intensity of the amplified gene detected on the agarose gel is correlated with the target RNA copy number at the linear phase.
- ◉ MTT: absolute units
It is measured by the spectrophotometer – absorbance of 570 nm.
- ◉ RT-PCR: relative units
The target gene expression must be normalized by GAPDH (housekeeping gene).
Both MV1 and MV2 have low cytotoxicity compared to buforin IIB.
siRNA-CYP1A1 (20 µM) showed the lowest CYP1A1 expression level, meaning that MV1 successfully delivers siRNA targeting CYP1A1 only in lung cancer cells (A549). The cytotoxicity and RT-PCR results indicate that MV1 was the best peptide to achieve both low cytotoxicity and delivery of siRNA into lung cancer cells.