Efficient Delivery of Cell Impermeable Phosphopeptides by a Cyclic Peptide Amphiphile Containing Tryptophan and Arginine

Phosphopeptides are valuable reagent probes for studying protein–protein and protein–ligand interactions. The cellular delivery of phosphopeptides is challenging because of the presence of the negatively charged phosphate group. The cellular uptake of a number of fluorescent-labeled phosphopeptides, including F′-GpYLPQTV, F′-NEpYTARQ, F′-AEEEIYGEFEAKKKK, F′-PEpYLGLD, F′-pYVNVQN-NH2, and F′-GpYEEI (F′ = fluorescein), was evaluated in the presence or absence of a [WR]4, a cyclic peptide containing alternative arginine (R) and tryptophan (W) residues, in human leukemia cells (CCRF-CEM) after 2 h incubation using flow cytometry. [WR]4 improved significantly the cellular uptake of all phosphopeptides. PEpYLGLD is a sequence that mimics the pTyr1246 of ErbB2 that is responsible for binding to the Chk SH2 domain. The cellular uptake of F′-PEpYLGLD was enhanced dramatically by 27-fold in the presence of [WR]4 and was found to be time-dependent. Confocal microscopy of a mixture of F′-PEpYLGLD and [WR]4 in live cells exhibited intracellular localization and significantly higher cellular uptake compared to that of F′-PEpYLGLD alone. Transmission electron microscopy (TEM) and isothermal calorimetry (ITC) were used to study the interaction of PEpYLGLD and [WR]4. TEM results showed that the mixture of PEpYLGLD and [WR]4 formed noncircular nanosized structures with width and height of 125 and 60 nm, respectively. ITC binding studies confirmed the interaction between [WR]4 and PEpYLGLD. The binding isotherm curves, derived from sequential binding models, showed an exothermic interaction driven by entropy. These studies suggest that amphiphilic peptide [WR]4 can be used as a cellular delivery tool of cell-impermeable negatively charged phosphopeptides

Surface Decorated Gold Nanoparticles by Linear and Cyclic Peptides as Molecular Transporters

Gold nanoparticles (AuNPs) were synthesized in situ in a green and rapid method from the reaction of reducing linear and cyclic peptides containing tryptophan and lysine residues, (KW)5 and cyclic [KW]5, with an aqueous solution of HAuCl4 and were evaluated as cellular nanodrug delivery systems. The cyclic or linear nature of the peptide was found to determine the morphology and size of the formed peptide-AuNPs and their in vitro molecular transporting efficiency. While cyclic [KW]5-AuNPs formed sponge-like agglomerates, linear (KW)5-AuNPs demonstrated ball-shaped structures. A comparative flow cytometry study showed that the cellular uptake of fluorescence-labeled anti-HIV drugs (emtricitabine (FTC) and lamivudine (3TC)) in human leukemia (CCRF-CEM) cells, and a negatively charged cell-impermeable phosphopeptide (GpYEEI) in human ovarian adecarcinoma (SK-OV-3) cells was significantly higher in the presence of cyclic [KW]5-AuNPs than that of linear (KW)5-AuNPs, parent cyclic [KW]5, and linear (KW)5 peptides. For example, the cellular uptake of F′-GpYEEI was enhanced 12.8-fold by c[KW]5-AuNPs. Confocal microscopy revealed the localization of fluorescence-labeled-3TC in the presence of c[KW]5-AuNPs mostly in nucleus in SK-OV-3 cells after 1 h. On the other hand, l(KW)5-AuNPs delivered fluorescence-labeled-3TC in cytoplasm. These data suggest that noncell penetrating peptides can be converted to efficient molecular transporters through peptide-capped AuNPs formation

Peptide-Amphiphile Containing Arginine and Fatty Acyl Chains as Molecular Transporters

Peptide amphiphiles (PAs) are promising tools for the intracellular delivery of numerous drugs. PAs are known to be biodegradable systems. Here, four PA derivatives containing arginine and lysine conjugated with fatty acyl groups with different chain length, namely PA1: R-K(C14)-R, PA2: R-K(C16)-R, PA3: K(C14)-R-K(C14), and PA4: K(C16)-R-K(C16), where C16 = palmitic acid and C14 = myristic acid, were synthesized through Fmoc chemistry. Flow cytometry studies showed that among all synthesized PAs, only K(C16)-R-K(C16), PA4 was able to enhance the cellular uptake of a fluorescence-labeled anti-HIV drug 2\u27,3\u27-dideoxy-3\u27-thiacythidine (F′-3TC, F′ = fluorescein) and a biologically important phosphopeptide (F′-PEpYLGLD) in human leukemia cells (CCRF-CEM) after 2 h incubation. For example, the cellular uptake of F′-3TC and F′-PEpYLGLD was enhanced approximately 7.1- and 12.6-fold in the presence of the PA4 compared to those of the drugs alone. Confocal microscopy of F′-3TC and F′-PEpYLGLD loaded PA4 in live cells showed significantly higher intracellular localization than the drug alone in human ovarian cells (SK-OV-3) after 2 h incubation. The HPLC results showed that loading of Dox by the peptide amphiphile was 56% after 24 h. The loaded Dox was released (34%) within 48 h intracellularly. The CD results exhibited that the secondary structure of the peptide was changed upon interactions with Dox. Mechanistic studies revealed that endocytosis is the major pathway of the internalization. These studies suggest that PAs containing appropriate sequence of amino acids, chain length, charge, and hydrophobicity can be used as cellular delivery tools for transporting drugs and biomolecules

Self-Assembled Surfactant Cyclic Peptide Nanostructures as Stabilizing Agents

A number of cyclic peptides, including [FR]4, [FK]4, [WR]4, [CR]4, [AK]4, and [WK]n (n = 3-5) containing L-amino acids were synthesized using solid-phase peptide synthesis. We hypothesized that an optimal balance of hydrophobicity and charge could generate self-assembled nanostructures in aqueous solution by intramolecular and/or intermolecular interactions. Among all the designed peptides, [WR]n (n = 3-5) generated self-assembled vesicle-like nanostructures at room temperature as shown by Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), and/or Dynamic light scattering (DLS). This class of peptides represents the first report of surfactant-like cyclic peptides that self-assemble into nanostructures. A plausible mechanistic insight of self-assembly of [WR]5 was investigated by molecular modeling studies. Modified [WR]5 analogues, such as [WMeR]5. [WR(Me)2]5, [WMeR(Me)2]5,and [WdR]5 exhibited different morphologies than [WR]5 as shown by TEM observations. [WR]5 exhibited significant stabilizing effect for generated silver nanoparticles and glyceraldehyde-3-phosphate dehydrogenase activity. These studies established a new class of surfactant-like cyclic peptides that self-assembled into nanostructures and could have potential applications for stabilizing of silver nanoparticles and protein biomolecules

Synthesis and Biological Evaluation of 5′-O-Dicarboxylic Fatty Acyl Monoester Derivatives of Anti-HIV Nucleoside Reverse Transcriptase Inhibitors

A number of 5′-O-dicarboxylic fatty acyl monoester derivatives of 3′-azido-3′-deoxythymidine (zidovudine, AZT), 2′,3′-didehydro-2′,3′-dideoxythymidine (stavudine, d4T), and 3′-fluoro-3′-deoxythymidine (alovudine, FLT) were synthesized to improve the lipophilicity and potentially the cellular delivery of parent polar 2′,3′-dideoxynucleoside (ddN) analogs. The compounds were evaluated for their anti-HIV activity. Three different fatty acids with varying chain length of suberic acid (octanedioic acid), sebacic acid (decanedioic acid), and dodecanedioic acid were used for the conjugation with the nucleosides. The compounds were evaluated for anti-HIV activity and cytotoxicity. All dicarboxylic ester conjugates of nucleosides exhibited significantly higher anti-HIV activity than that of the corresponding parent nucleoside analogs. Among all the tested conjugates, 5′-O-suberate derivative of AZT (EC50 = 0.10 nM) was found to be the most potent compound and showed 80-fold higher anti-HIV activity than AZT without any significant toxicity (TC50 \u3e500 nM)

Ga-doped Pt-Ni Octahedral Nanoparticles as a Highly Active and Durable Electrocatalyst for Oxygen Reduction Reaction

Bimetallic PtNi nanoparticles have been considered as a promising electrocatalyst for oxygen reduction reaction (ORR) in polymer electrolyte membrane fuel cells (PEMFCs) owing to their high catalytic activity. However, under typical fuel cell operating conditions, Ni atoms easily dissolve into the electrolyte, resulting in degradation of the catalyst and the membrane-electrode assembly (MEA). Here, we report gallium-doped PtNi octahedral nanoparticles on a carbon support (Ga-PtNi/C). The Ga-PtNi/C shows high ORR activity, marking an 11.7-fold improvement in the mass activity (1.24 A mgPt-1) and a 17.3-fold improvement in the specific activity (2.53 mA cm-2) compare to the commercial Pt/C (0.106 A mgPt-1 and 0.146 mA cm-2). Density functional theory calculations demonstrate that addition of Ga to octahedral PtNi can cause an increase in the oxygen intermediate binding energy, leading to the enhanced catalytic activity toward ORR. In a voltage-cycling test, the Ga-PtNi/C exhibits superior stability to PtNi/C and the commercial Pt/C, maintaining the initial Ni concentration and octahedral shape of the nanoparticles. Single cell using the Ga-PtNi/C exhibits higher initial performance and durability than those using the PtNi/C and the commercial Pt/C. The majority of the Ga-PtNi nanoparticles well maintain the octahedral shape without agglomeration after the single cell durability test (30,000 cycles). This work demonstrates that the octahedral Ga-PtNi/C can be utilized as a highly active and durable ORR catalyst in practical fuel cell applications

Design, Synthesis, Antiviral Activity, and Pre-Formulation Development of Poly-L-Arginine-Fatty Acyl Derivatives of Nucleoside Reverse Transcriptase Inhibitors

The objective of this work was to design conjugates of anti-HIV nucleosides conjugated with fatty acids and cell-penetrating poly-L-arginine (polyArg) peptides. Three conjugates of polyArg cell-penetrating peptides with fatty acyl derivatives of alovudine (FLT), lamivudine (3TC), and emtricitabine (FTC) were synthesized. In general, the compounds exhibited anti-HIV activity against X4 and R5 cell-free virus with EC50 values of 1.5–16.6 μM. FLT-CO-(CH2)12-CO-(Arg)7 exhibited EC50 values of 2.9 μM and 3.1 μM against X4 and R5 cell-free virus, respectively. The FLT conjugate was selected for further preformulation studies by determination of solution state degradation and lipid solubility. The compound was found to be stable in neutral and oxidative conditions and moderately stable in heated conditions. [Refer to PDF for graph