Inhibition of endocytosis using specific inhibitors.

<p>Effects of inhibitors (sucrose: clathrin-mediated endocytosis inhibitor; amiloride: macropinocytosis inhibitor; filipin: caveolae-mediated endocytosis inhibitor) on the internalization of FITC-BSA complexes with Pro-DeliverIN, Xfect, and TurboFect were evaluated using Huh-7 monolayer cultured cells (A) and MCTS (B). Error bars in the graph represent the standard deviation, n = 3 (A) or 4 (B). *<i>p</i><0.05 and **<i>p</i><0.01. The results obtained from monolayer cultured cells (A) and MCTS (B) were similar. Pro-DeliverIN appeared to be internalized into Huh-7 cells by clathrin-mediated endocytosis and caveolae-mediated endocytosis. The uptake route of Xfect was mainly by macropinocytosis, but also slightly by caveolae-mediated endocytosis. TurboFect was preferentially internalized into Huh-7 cells through macropinocytosis. </p

CLSM observations (40X objective) of each FITC-BSA complex using MCTS.

<p>A: Schematic illustration of MCTS observed in these experiments. The full size of MCTS was observed from the top view with the Z-stack mode from the top to middle positions. The optical thickness and the distance of the confocal section from the top of MCTS were 0.60 μm and 20 μm, respectively. B: The intra-MCTS distribution of FITC-BSA (green) complexes with Pro-DeliverIN, Xfect, and TurboFect was observed by CLSM using the 40X objective with staining nuclei (blue) shown in A. A certain amount of FITC-BSA complexes with Pro-DeliverIN and Xfect was observed in the inner layered cells. On the other hand, TurboFect showed FITC-BSA distribution only in the surface-first layered cells. The scale bars represent 50 μm.</p

Cellular uptake of each protein transfection reagent/FITC-BSA complex.

<p>FITC-BSA complexes with Pro-DeliverIN, Xfect, and TurboFect were applied to Huh-7 monolayer cultured cells (A) and MCTS (B). Error bars in the graph represent the standard deviation, n = 4. *<i>p</i><0.05 and **<i>p</i><0.01. In the case of monolayer cultured cells (A), TurboFect and Xfect achieved higher cellular uptake efficiency than that of Pro-DeliverIN. MCTS experiments revealed the higher uptake of TurboFect than of Pro-DeliverIN and Xfect. The uptake of protein transfection reagents was markedly higher by monolayer cultured cells than by MCTS.</p

CLSM observations (100X objective) of each FITC-BSA complex using MCTS.

<p>A: Schematic illustration of MCTS observed in these experiments. About one quater size of MCTS was observed from the top view. The optical thickness and the distance of the confocal section from the top of MCTS were 0.38 μm and 20 μm, respectively. B: The intra-MCTS distribution of FITC-BSA (green) complexes with Pro-DeliverIN, Xfect, and TurboFect was observed in detail by CLSM using the 100X objective with staining nuclei (blue). A small amount of FITC-BSA was observed in the nuclei, especially in TurboFect. The scale bars represent 20 μm.</p

Cell-Penetrating Peptides Using Cyclic α,α-Disubstituted α‑Amino Acids with Basic Functional Groups

In the delivery of cell-impermeable molecules, cell-penetrating peptides (CPPs) have been attracting increasing attention as intracellular delivery tools. In the present study, we designed four types of cyclic α,α-disubstituted α-amino acids (dAAs) with basic functional groups on their five-membered rings and different chiralities at the α-position and introduced them into arginine (Arg)-rich peptides. The evaluation of cell-penetrating abilities indicated that these peptides exhibited better cell permeabilities than an Arg nonapeptide. Furthermore, peptides containing dAAs delivered plasmid DNA (pDNA) better than a commercially available transfection reagent with a longer incubation time. These results demonstrate that the introduction of cyclic dAAs with basic functional groups into Arg-rich peptides is an effective strategy for the design of CPPs as a pDNA delivery tool

Cell-Penetrating Helical Peptides Having l‑Arginines and Five-Membered Ring α,α-Disubstituted α‑Amino Acids

Cell-penetrating peptides are powerful tools in the delivery of drugs, proteins, and nucleic acids into cells; therefore, focus has recently been placed on their development. In this study, we synthesized seven types of peptides possessing three l-arginines (l-Arg) and six l-leucines (l-Leu) and/or 1-aminocyclopentane-1-carboxylic acids (Ac₅c), and investigated their secondary structures and cell-penetrating abilities. The peptide composed of an equal number of l-Arg, l-Leu, and Ac₅c formed 3₁₀/α-helical structures in TFE solution and exhibited the highest cell-penetrating ability of all the peptides examined. Additional cellular uptake studies revealed that the incorporation of Ac₅c into peptides led to improved tolerability against serum. The results of the present study will help in the design of novel cell-penetrating peptides

Enhanced and Prolonged Cell-Penetrating Abilities of Arginine-Rich Peptides by Introducing Cyclic α,α-Disubstituted α‑Amino Acids with Stapling

Cell-penetrating peptides are receiving increasing attention as drug delivery tools, and the search for peptides with high cell-penetrating ability and negligible cytotoxicity has become a critical research topic. Herein, cyclic α,α-disubstituted α-amino acids were introduced into arginine-rich peptides and an additional staple was provided in the side chain. The peptides designed in the present study showed more enhanced and prolonged cell-penetrating abilities than an arginine nonapeptide due to high resistance to protease and conformationally stable helical structures

Twisted Structure of a Cyclic Hexapeptide Containing a Combination of Alternating l‑Leu‑d‑Leu-Aib Segments

We designed and synthesized a <i>C</i>₂-symmetric cyclic hexapeptide, <i>cyclo</i>(l-Leu-d-Leu-Aib)₂ (<b>2</b>), which contains l- and d-amino acids and achiral Aib residues. The conformation of <b>2</b> was analyzed in the crystalline state and in solution, which was a unique figure-eight-shaped conformation

Helical-Peptide-Catalyzed Enantioselective Michael Addition Reactions and Their Mechanistic Insights

Helical peptide foldamer catalyzed Michael addition reactions of nitroalkane or dialkyl malonate to α,β-unsaturated ketones are reported along with the mechanistic considerations of the enantio-induction. A wide variety of α,β-unsaturated ketones, including β-aryl, β-alkyl enones, and cyclic enones, were found to be catalyzed by the helical peptide to give Michael adducts with high enantioselectivities (up to 99%). On the basis of X-ray crystallographic analysis and depsipeptide study, the amide protons, N(2)–H and N(3)–H, at the N terminus in the α-helical peptide catalyst were crucial for activating Michael donors, while the N-terminal primary amine activated Michael acceptors through the formation of iminium ion intermediates

Twisted Structure of a Cyclic Hexapeptide Containing a Combination of Alternating l‑Leu‑d‑Leu-Aib Segments

We designed and synthesized a <i>C</i>₂-symmetric cyclic hexapeptide, <i>cyclo</i>(l-Leu-d-Leu-Aib)₂ (<b>2</b>), which contains l- and d-amino acids and achiral Aib residues. The conformation of <b>2</b> was analyzed in the crystalline state and in solution, which was a unique figure-eight-shaped conformation