A series of cyclic peptides, [(DipR)(WR)₄], [(DipR)₂(WR)₃], [(DipR)₃(WR)₂], [(DipR)₄(WR)], and [DipR]₅, and their linear counterparts containing arginine (R) as positively charged residues and tryptophan (W) or diphenylalanine (Dip) as hydrophobic residues, were synthesized and evaluated for their molecular transporter efficiency. The in vitro cytotoxicity of the synthesized peptides was determined in human epithelial ovary adenocarcinoma cells (SK-OV-3), human lymphoblast peripheral blood cells (CCRF-CEM), human embryonic epithelial kidney healthy cells (HEK-293), human epithelial mammary gland adenocarcinoma cells (MDA-MB-468), pig epithelial kidney normal cells (LLC-PK1), and human epithelial fibroblast uterine sarcoma cells (MES-SA). A concentration of 5–10 µM and 3 h incubation were selected in uptake studies. The cellular uptake of a fluorescent-labeled phosphopeptide, stavudine, lamivudine, emtricitabine, and siRNA was determined in the presence of peptides via flow cytometry. Among the peptides, [DipR]₅ (10 µM) was found to be the most efficient transporter and significantly improved the uptake of F’-GpYEEI, i.e., by approximately 130-fold after 3 h incubation in CCRF-CEM cells. Confocal microscopy further confirmed the improved delivery of fluorescent-labeled [DipR]₅ (F’-[K(DipR)₅]) alone and F’-GpYEEI in the presence of [DipR]₅ in MDA-MB-231 cells. The uptake of fluorescent-labeled siRNA (F’-siRNA) in the presence of [DipR]₅ with N/P ratios of 10 and 20 was found to be 30- and 50-fold higher, respectively, compared with the cells exposed to F’-siRNA alone. The presence of endocytosis inhibitors, i.e., nystatin, chlorpromazine, chloroquine, and methyl β-cyclodextrin, did not completely inhibit the cellular uptake of F’-[K(DipR)₅] alone or F’-GpYEEI in the presence of [DipR]₅, suggesting that a combination of mechanisms contributes to uptake. Circular dichroism was utilized to determine the secondary structure, while transmission electron microscopy was used to evaluate the particle sizes and morphology of the peptides. The data suggest the remarkable membrane transporter property of [DipR]₅ for improving the delivery of various small molecules and cell-impermeable negatively charged molecules (e.g., siRNA and phosphopeptide).

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.

Background. We have previously reported peptides composed of sequential arginine (R) residues paired with tryptophan (W) or 3,3-diphenyl-L-alanine residues (Dip), such as cyclic peptides [R₄W₄] and [R₄(Dip)₃], as antibacterial agents. Results. Herein, we report antibacterial and antifungal activities of five linear peptides, namely ((DipR)₄(WR)), ((DipR)₃(WR)₂), ((DipR)₂(WR)₃), ((DipR)(WR)₄), and (DipR)₄R, and five cyclic peptides [(DipR)₄(WR)], [(DipR)₃(WR)₂], [(DipR)₂(WR)₃], [(DipR)(WR)₄], and [DipR]₅, containing alternate positively charged R and hydrophobic W and Dip residues against fungal, Gram-positive, and Gram-negative bacterial pathogens. The minimum inhibitory concentrations (MICs) of all peptides were determined by the micro-broth dilution method against Methicillin-Resistant Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Streptococcus pneumoniae, and Bacillus subtilis. Fungal organisms were Candida albicans, Candida parapsilosis, and Aspergillus fumigatus. [DipR]₅ and ((DipR)₂(WR)₃) showed MIC values of 0.39–25 µM and 0.78–12.5 µM against Gram-positive and Gram-negative bacteria strains, respectively. The highest activity was observed against S. pneumoniae with MIC values of 0.39–0.78 µM among tested compounds. [DipR]₅ demonstrated MIC values of 6.6 µM against C. parapsilosis and 1.6 µM against A. fumigatus, whereas fluconazole showed MIC values of 3.3 µM and >209 µM, respectively. Conclusions. These findings highlight the potential of these peptides as broad-spectrum antimicrobial agents.

Melanoma is the most fatal type of skin cancer and is notoriously resistant to chemotherapies. The response of melanoma to current treatments is difficult to predict. To combat these challenges, in this study, we utilize a small peptide to increase drug delivery to melanoma cells. A peptide library array was designed and screened using a peptide array-whole cell binding assay, which identified KK-11 as a novel human melanoma-targeting peptide. The peptide and its D-amino acid substituted analogue (VPWxEPAYQrFL or D-aa KK-11) were synthesized via a solid-phase strategy. Further studies using FITC-labeled KK-11 demonstrated dose-dependent uptake in human melanoma cells. D-aa KK-11 significantly increased the stability of the peptide, with 45.3% remaining detectable after 24 h with human serum incubation. Co-treatment of KK-11 with doxorubicin was found to significantly enhance the cytotoxicity of doxorubicin compared to doxorubicin alone, or sequential KK-11 and doxorubicin treatment. In vivo and ex vivo imaging revealed that D-aa KK-11 distributed to xenografted A375 melanoma tumors as early as 5 min and persisted up to 24 h post tail vein injection. When co-administered, D-aa KK-11 significantly enhanced the anti-tumor activity of a novel nNOS inhibitor (MAC-3-190) in an A375 human melanoma xenograft mouse model compared to MAC-3-190 treatment alone. No apparent systemic toxicities were observed. Taken together, these results suggest that KK-11 may be a promising human melanoma-targeted delivery vector for anti-melanoma cargo.

We have previously reported peptides containing subsequent arginine (R) residues followed by tryptophan (W) or 3,3-diphenyl-L-alanine residues (Dip), such as cyclic peptides [R4W4] and [R4(Dip)3], as antibacterial agents. Herein, we report antibacterial and antifungal activities of ten linear peptides, ((DipR)4(WR)), ((DipR)3(WR)2), ((DipR)2(WR)3), ((DipR)(WR)4), (DipR)4R, and cy-clic peptides [(DipR)4(WR)], [(DipR)3(WR)2], [(DipR)2(WR)3], [(DipR)(WR)4], and [DipR]5 con-taining alternate positively charged R and hydrophobic W and Dip residues against fungal, Gram-positive and Gram-negative bacterial pathogens. The minimum inhibitory concentrations (MIC) of all peptides were determined by the micro-broth dilution method against Methicil-lin-Resistant Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Streptococcus pneumoniae, and Bacillus subtilis. Fungal organisms were Candida albicans, Candida parapsilosis, and Aspergillus fumigatus. [DipR]5 and ((DipR)2(WR)3) showed MIC values of 0.39-6.3 µM and 3.1-25 µM against Gram-positive and Gram-negative bacteria strains, respectively. The highest activity was ob-served against S. pneumoniae with MIC values of 0.39-0.78 µM among tested compounds. [DipR]5 demonstrated MIC values of 6.6 µM against C. parapsilosis and 1.6 µM against A. fumigatus, whereas fluconazole showed MIC values of 3.3 µM and >209 µM, respectively.