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Revision as of 18:32, 9 October 2023

BP100-(KH)₉

Cell-Penetrating Peptides (CPP) are short peptides that can penetrate the cell membrane and deliver a wide range of cargoes into the cell. This part is the coding sequence of our chimeric CPP formed with BP100 and the polycationic peptide (KH)9. It is proven to deliver proteins and nucleic acids into the plant and prokaryotic cells. The polycationic peptide is an efficient nucleic acid carrier and has the ability to promote buffering effect on the pre-lysosomal vesicle. The cargo being negatively charged interacts with the polycationic end of the peptide through ionic interactions, whereas the BP100 region mediates cellular entry through carpet-mechanism.

Sequence and Features

CHARACTERIZATION

To determine the cytotoxicity of the peptide produced by BBa_K3224003 (Cell Penetrating Peptide - BP100(KH)9) in HeLa (immortalized human cervical cancer) and C2C12 (immortalized mouse myoblast) cell.

The cells were treated with different concentrations of the CPP encoded by the biobrick (4,8,16,32,64,120 μM/ml) of BP-100-(KH)9 and were incubated for different time intervals 24 h and 48 h.

MTT ASSAY OF CPP IN HELA CELLS (24 HRS)

FIGURE 1: MTT Assay of CPP in HeLa Cells (24 h)

When HeLa cells were treated with lower concentrations of CPP (4-16 µM) for 48 hours, ~15% cytotoxicity was observed. Higher concentrations of CPP (32-120 µM) showed ~25% cytotoxicity on the cells.

MTT ASSAY OF CPP IN HELA CELLS (48 HRS)

FIGURE 2: MTT Assay of CPP in HeLa cells (48 h)

When HeLa cells were treated with CPP for a time period of 48 hours, the cytotoxicity levels lie below 50% for lower concentrations of the peptide (4 and 8µM). For the concentrations from 16 to 120µM the cytotoxicity shoots up to ~100%.

MTT ASSAY OF CPP IN C2C12 CELLS (24 HRS)

FIGURE 3: MTT Assay of CPP in C2C12 Cells (24 h)

The action of lower concentrations of CPP (4 and 8µM) on C2C12 cells showed ~35% cytotoxicity. At higher concentrations of CPP (16 to120µM) ~135% cytotoxicity was observed.

MTT ASSAY OF CPP IN C2C12 CELLS (48 HRS)

FIGURE 4: MTT Assay of CPP in C2C12 Cells (48 h)

When C2C12 cell lines were treated with lower concentrations of CPP (4-32µM) for 48 hours, CPP showed ~100% cytotoxicity. At higher concentrations (64 and 120µM) the cytotoxicity goes beyond 150%.

OPTIMIZATION OF N:P RATIO:

The Cell Penetrating Peptide BP100-(KH)9 (BBa_K3224003) was synthesized in-vitro and purified by High-Performance Liquid Chromatography (HPLC). The in-vitro synthesized shRNA-like siRNA with a fluorophore and a quencher (BBa-K3224002) was mixed at 100 µM concentration, with varying concentrations of the chimeric CPP BP100- (KH)9 to make up different N/P ratios: 0.5, 1 and 2. Also, only siRNA without the CPP was added to the cells as delivery control. The fluorescence emitted was measured in Arbitrary Fluorescence Units (AFU). The other nucleotide construct, survivin shRNA (BBa_K3224001), was not used for this experiment as it is devoid of a fluorophore.

The intensity of the fluorescence emitted is proportional to the amount of cargo successfully delivered into the cells, and hence an indicator of the optimum N/P ratio in order to achieve improved transfection efficiency. From the graph, we observe that the N/P ratio of 0.5 gives higher intensity of fluorescence which can be attributed to the presence of higher amount of CPP for a given amount of siRNA to be transfected. Thus the efficient delivery of the nucleic acid has taken place by the CPP.

As the amount of protein used to make up the N:P ratios of 1.0 and 2.0 for the fixed concentration of nucleotide (100 µL) decreases, lesser number of nucleotides get delivered into the cells, which in turn accounts for the decrease in fluorescence emitted from the cells in these two experimental conditions. In addition to optimizing the N:P ratio, the current experiment also confirms that the two submitted parts work: CPP in terms of delivering the cargo, and shRNA-like siRNA in terms of emitting fluorescence.

Figure 5: Optimization of the N/P ratio of CPP-siRNA complexes

Contribution: New data add by SZU-China 2023