Cellular uptake and anti-tumor activity of gemcitabine conjugated with new amphiphilic cell penetrating peptides

Abstract

Gemcitabine (Gem) is used as a single agent or in combination with other anticancer agents to treat many types of solid tumors. However, it has many limitations such as a short plasma half-life, dose-limiting toxicities and drug resistance. Cell-penetrating peptides (CPPs) are short peptides which may deliver a large variety of cargo molecules into the cancerous cells. The current study was designed to evaluate the antiproliferative activity of gemcitabine chemically conjugated to CPPs. The peptides were synthesized using solid phase synthesis procedure. The uptake efficiency of CPPs into cells was examined by flow cytometry and fluorescent microscopy. The synthesized peptides were chemically conjugated to Gem and the in vitro cytotoxicity of conjugates was tested by MTT assay on A594 cell line. According to the obtained results, cellular uptake was increased with increasing the concentration of CPPs. On the other hand the coupling of Gem with peptides containing block sequence of arginine (R5W3R4) and some alternating sequences (i.e. [RW]6 and [RW]3) exhibited improved antitumor activity of the drug. The findings in this study support the advantages of using cell-penetrating peptides for improving intracellular delivery of Gem into tumor as well as its activity.

Keywords: cell penetrating peptide; drug delivery; gemcitabine; toxicity.

Table 1. Peptide sequences

Figure 1. Schematic representing synthesis of R5W3R4 peptide

Figure 2. Schematic representing preparing of gemcitabine-R5W3R4 conjugate

Figure 3. Cytotoxicity of peptides on A549 cells after 72 h incubation

Figure 4. Fluorescence microscopy, visualization of FITC-labeled, R5W3R4 (A, B), [RW]6 (C, D), [RW]5 (E, F), [RW]4 (G, H), and [RW]3 (I, J) in A459 cells. The top photos show fluorescence microscopy and the bottom bright field of A459 cells. Live cells were treated with 25 µm of peptides for 1.5 h at 37 °C. Control cells were incubated in RPMI medium without the peptides.

Figure 5. Cellular uptake of FITC-labeled peptides in live A549 cells after incubation. The uptake was measured as the relative fraction of positive cells (%) from flow cytometry analysis of all living cells positive for the fluorophore.

Figure 6. Toxicity of Gem-R5W3R4 (A), Gem-[RW]6 (B), Gem-[RW5] (C), Gem-[RW]4 (D), Gem-[RW]3 (E) and Gem to A549 cells. The cells were incubated for 3 days in 10 % FBS with or without peptides and analyzed for proliferation by MTT assay.