Cell-impermeable and negatively charged compounds' cellular uptake across the cell membranes remains challenging. Herein, the synthesis of four linear [(WWRR) 2 , (WWRR) 3 , (WWRR) 4 , and (WWRR) 5 ] and four cyclic ([WWRR] 2 , [WWRR] 3 , [WWRR] 4 , and [WWRR] 5 ) peptides containing alternate two tryptophan (WW) and two arginine (RR) residues and their biological evaluation as molecular transporters are reported. The peptides did not show any significant cytotoxicity in different cell lines (MDA-MB-23, SK-OV-3, and HEK 293) at a concentration of 5 μM and after 3 h of incubation time. The uptake of fluorescence-labeled cargo molecules (F′-GpYEEI, F′-siRNA, and F′-3TC) in the presence of the peptides was monitored in different cell lines (SK-OV-3 and MDA-MB-231) with fluorescence-activated cell sorting. Among all the peptides, [WWRR] 5 (C4) showed the highest cellular uptake of cargo molecules, indicating it can act as effective molecular transporter. Confocal microscopy in MDA-MB-231 cells showed the cellular uptake of F′-GpYEEI in the presence of C4 and the intracellular localization of fluorescence-labeled C4 (F′-C4) in the cytosol. The F′-C4 cellular uptake was found to be concentration-and time-dependent, as shown by flow cytometry in MDA-MB-231 cells. Confocal microscopy and flow cytometry of F′-C4 in MDA-MB-231 cells were examined alone and in the presence of different endocytosis inhibitors (chlorpromazine, methyl-β-cyclodextrin, chloroquine, and nystatin). The data showed that the cellular uptake of F′-C4 in the presence of chlorpromazine, chloroquine, and methyl-β-cyclodextrin was reduced but not completely eliminated, indicating that both energy-independent and energy-dependent pathways contributed to the cellular uptake of F′-C4. Similar results were obtained using the confocal microscopy of C4 and F′-GpYEEI in the presence of endocytosis inhibitors (chlorpromazine, methyl-β-cyclodextrin, chloroquine, and nystatin). These data indicate that C4 has the potential to be used as a cell-penetrating peptide and cargo transporter.
Recent approvals of siRNA-based products motivated the scientific community to explore siRNA as a treatment option for several intractable ailments, especially cancer. The success of approved siRNA therapy requires a suitable and safer drug delivery agent. Herein, we report a series of oleyl conjugated histidine–arginine peptides as a promising nonviral siRNA delivery tool. The conjugated peptides were found to bind with the siRNA at N/P ratio ≥ 2 and demonstrated complete protection for the siRNA from early enzymatic degradation at N/P ratio ≥ 20. Oleyl-conjugated peptide -siRNA complexes were found to be noncytotoxic in breast cancer cells (MCF-7 and MDA-MB-231) and normal breast epithelial cells (MCF 10A) at N/P ratio of ~40. The oleyl-R3-(HR)4 and oleyl-R4-(HR)4 showed ~80-fold increased cellular uptake in MDA-MB-231 cells at N/P 40. Moreover, the conjugated peptides-siRNA complexes form nanocomplexes (~115 nm in size) and have an appropriate surface charge to interact with the cell membrane and cause cellular internalization. Furthermore, this study provides a proof-of-concept that oleyl-R5-(HR)4 can efficiently silence STAT-3 gene (~80% inhibition) in MDA-MB-231 cells with similar effectiveness to Lipofectamine. Further exploration of this approach holds a great promise in discovering a successful in vivo siRNA delivery agent with a favorable pharmacokinetic profile.
The entry of proteins through the cell membrane is challenging, thus limiting their use as potential therapeutics. Seven cell-penetrating peptides, designed in our laboratory, were evaluated for the delivery of proteins. Fmoc solid-phase peptide synthesis was utilized for the synthesis of seven cyclic or hybrid cyclic–linear amphiphilic peptides composed of hydrophobic (tryptophan (W) or 3,3-diphenylalanine (Dip) and positively-charged arginine (R) residues, such as [WR]4, [WR]9, [WWRR]4, [WWRR]5, [(RW)5K](RW)5, [R5K]W7, and [DipR]5. Confocal microscopy was used to screen the peptides as a protein delivery system of model cargo proteins, green and red fluorescein proteins (GFP and RFP). Based on the confocal microscopy results, [WR]9 and [DipR]5 were found to be more efficient among all the peptides and were selected for further studies. [WR]9 (1–10 µM) + protein (GFP and RFP) physical mixture did not show high cytotoxicity (>90% viability) in triple-negative breast cancer cells (MDA-MB-231) after 24 h, while [DipR]5 (1–10 µM) physical mixture with GFP exhibited more than 81% cell viability. Confocal microscopy images revealed internalization of GFP and RFP in MDA-MB-231 cells using [WR]9 (2–10 μM) and [DipR]5 (1–10 µM). Fluorescence-activated cell sorting (FACS) analysis indicated that the cellular uptake of GFP was concentration-dependent in the presence of [WR]9 in MDA-MB-231 cells after 3 h of incubation at 37 °C. The concentration-dependent uptake of GFP and RFP was also observed in the presence of [DipR5] in SK-OV-3 and MDA-MB-231 cells after 3 h of incubation at 37 °C. FACS analysis indicated that the cellular uptake of GFP in the presence of [WR]9 was partially decreased by methyl-β-cyclodextrin and nystatin as endocytosis inhibitors after 3 h of incubation in MDA-MB-231 cells, whereas nystatin and chlorpromazine as endocytosis inhibitors slightly reduced the uptake of GFP in the presence of [DipR]5 after 3 h of incubation in MDA-MB-231. [WR]9 was able to deliver therapeutically relevant proteins (Histone H2A) at different concentrations. These results provide insight into the use of amphiphilic cyclic peptides in the delivery of protein-related therapeutics.
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