Added: Lashawnta Perlman - Date: 02.12.2021 21:01 - Views: 34044 - Clicks: 8377
Try out PMC Labs and tell us what you think. Learn More. These studies suggest that large cyclic peptides containing arginine and tryptophan can be used as a molecular transporter of specific compounds. The properties of the cell membrane create a barrier for the efficient cellular delivery of therapeutics. The cell membrane contains phospholipids, which are negatively charged and extremely hydrophobic. The phospholipids have been shown to obstruct the transportation of negatively charged or water-insoluble molecules. For example, phosphopeptides are cell-impermeable compounds that are used as probes for studying phosphoprotein—protein interactions.
Cell-penetrating peptides CPPs have been introduced as a drug delivery system for various applications. Other commonly used drug delivery systems include liposomes and micelles. Encapsulating the drug in a liposome has demonstrated to increase the efficiency of drug delivery and prevent damage to the normal tissue. However, a study has shown that liposomes still cause drug leakage, 7 especially for highly hydrophilic drugs such as doxorubicin Dox that is one of the most widely administered drugs for the treatment of cancer.
Furthermore, the high dose of the drug has been shown to cause irreversible damage to the cardiac muscle. There are many ways that the cellular uptake of CPPs may occur. The most common ways include direct penetration and endocytosis. Direct penetration may include inverted micelle formation and pore formation. The endocytotic pathway involves several pathways, such as macropinocytosis, phagocytosis, endocytosis-independent of clathrin, or endocytosis-dependent on the coat proteins clathrin or caveolin. Cem peptides review best strategy to overcome this limitation is to create a compound that will bypass the endosomal pathway or enhance the rate of escape from the endosome.
We have ly reported the application of cyclic peptide CPPs containing arginine R and tryptophan W residues for delivery of different cargo Cem peptides review. The data confirmed that cyclic peptides containing alternating tryptophan and arginine residues, [WR] 4 and [WR] 5improved the cellular uptake of compounds, such as anti-HIV drugs, Dox, phosphopeptides, and siRNA or generated peptide nanostructures. The cyclic nature of the peptides provided higher stability versus the linear counterparts and more efficient cell penetration. We also showed that the cellular uptake of [WR] 4 and [WR] 5 is independent of the endocytotic pathway.
In continuation of our efforts to develop structure—cellular uptake relationship for cyclic peptides containing W and R residues, herein we synthesized and evaluated amphiphilic cyclic CPPs composed of increasing s of W and R residues, compared their efficiency with [WR] 5 in improving the cellular uptake of cell-impermeable compounds, established structure—molecular transporter efficiency relationship, and evaluated the mechanism of cellular uptake. The studies allowed us to determine if there was any effect on the molecular transporter efficiency of the cyclic peptide by increasing the of hydrophobic and positively charged residues.
Cytotoxicity of the cyclic peptides was determined to find an appropriate noncytotoxic concentration for cellular uptake studies. Using leukemia cancer CCRF-CEM cells, the cellular uptake studies were evaluated to measure the uptake of fluorescence-labeled compounds [e. Structure—molecular transporter efficiency relationship of the compounds was determined. A fluorescence-labeled derivative of the most efficient peptide was evaluated in the presence and absence of different endocytosis inhibitors, and the uptake was evaluated using fluorescence microscopy.
The linear side-chain protected peptides were assembled on H-Trp Boc chlorotrityl resin first. As a representative example, the synthesis of [WR] 9 is depicted in Scheme 1. To synthesize the fluorescence-labeled cyclic peptide Scheme 2the peptide sequence was partially modified. Appropriately protected amino acids were assembled on H-Trp Boc chlorotrityl resin 0. The coupling reaction was completed in overnight. The cytotoxicity was gradually increased as the of tryptophan and arginine increased in the cyclic peptides. Further studies were required to find a noncytotoxic concentration for [WR] 9.
These studies showed that the toxicity of [WR] Cem peptides review precluded it from being used at a higher concentration and suggested that this compound could be used alternatively as an anticancer agent at higher concentrations. Thus, this concentration was used for performing cellular uptake studies.
Cells and fluorescence-labeled phosphopeptide alone were used as negative controls. On the basis of these data, [WR] 9 was found to be more effective when compared with [WR] 5 in improving the cellular uptake of the phosphopeptide, presumably because a higher of positive charges and the larger size of [WR] 9 provide adequate interaction and encapsulation of a large linear negatively charged phosphopeptide in comparison to [WR] 5. Thus, cyclic [WR] 9 was found to be the most effective peptide as the molecular transporter of fluorescence-labeled phosphopeptide among the synthesized peptides.
Cem peptides review, further studies were performed with [WR] 9. These data indicate that an appropriate ratio of 5 to 1 between [WR] 9 and phosphopeptide is required for more efficient uptake. The presence of [WR] 9 also showed enhanced cellular uptake of the anti-HIV drugs but not as ificant as the phosphopeptide and in comparison to [WR] 5. However, [WR] 9 showed to be ificantly more efficient in the delivery of a larger linear phosphopeptide in comparison to [WR] 5.
The cellular uptake of the peptide was also time-dependent and was at its highest after 1 h. Thus, the cellular uptake of the fluorescently labeled peptide was found to be concentration- and time-dependent. NT is no treatment. This suggests that the mechanism of uptake could be dependent on clathrin-mediated or caveolae-mediated endocytosis and phagocytosis.
The cellular uptake of the peptide decreased ificantly by 5. These data indicate that the peptide has a cell permeability property and could be used in transporting appropriate cargo molecules across the cellular membrane as described above.
However, the exact localization of the peptide needs further investigation. A of cyclic amphipathic peptide-containing arginine and tryptophan residues, namely [WR] 5[WR] 6[WR] 7[WR] 8and [WR] 9 were synthesized through Fmoc solid-phase chemistry and compared for their efficiency in improving the cellular uptake of cell-impermeable compounds and establish a structure—molecular transporter efficiency relationship.
However, [WR] 9 showed to be the most efficient in improving the uptake of the phosphopeptide. However, these inhibitors did not completely block the uptake. Therefore, we can conclude the uptake of this peptide is not completely dependent on endocytosis and other mechanisms of entry are possible.
These data suggest that increasing the of alternating positively charged arginine residues and hydrophobic tryptophan residues in a cyclic peptide was an optimal approach for generating compounds with efficient molecular transporter properties for large molecules, such as a negatively charged phosphopeptide.
The work advances the scientific knowledge in the area of cyclic peptide-based delivery systems for improving the cellular uptake of cell-impermeable compounds. Other chemicals and reagents were purchased from Sigma-Aldrich. The solvent was filtered. The Fmoc-protected amino acid was coupled to the N -terminal in the presence of 2- 1 H -benzotriazolyl -1,1,3,3-tetramethyluronium hexafluorophosphate 3 equiv Cem peptides review, DIPEA 6 equivand DMF mixing for 1 h under nitrogen gas.
The reaction solution was filtered after the coupling reaction completed. The resin was washed with 15 mL of DMF for 5 min twice. The followed amino acids were coupled using the same protocol to assemble the complete linear protected peptide on trityl resin. The crude material was obtained as a white solid.
MALDI analysis was used to confirm the molecular weight of the linear peptides after small cleavage of the peptide from the resin. The protected linear peptides 0. The cyclization reaction occurred overnight under inert condition using nitrogen. MALDI analysis was used to monitor the completion of cyclization by checking the molecular weight of the cyclized peptide. The solvents were removed under low pressure.
The cold diethyl ether was added to precipitate the crude peptide and was Cem peptides review to obtain solid peptide. MALDI analysis confirmed the molecular weight of the cyclic peptides without any protecting groups.
All the peptides were purified using reversed-phase HPLC and lyophilized. The coupling reaction was completed overnight. CRL to determine the viability of the peptides according to the ly reported procedure. LLC-PK1 cells were seeded at cells in 0. Cell viability was then determined by measuring the fluorescence intensity at nm using a SpectraMax M2 microplate spectrophotometer. The fluorescence intensity was measured in the presence and absence of the synthesized peptides.
The cells were then washed with PBS two times. All assays were performed in triplicates. FACS analysis was performed to measure the fluorescence intensity intracellularly and to determine whether the cyclic peptides were facilitating the cargos to cross the membrane. To induce ATP depletion, the cells were pre-incubated with 0. FACS was performed as ly described. After 3 h incubation, cells were centrifuged at Cem peptides review for 5 min. The media was then added to the cells. The cells were then placed on the cover slip and were detected by using a Keyence fluorescence microscope BZ-X Keyence Corp.
The authors would like to acknowledge financial support for this research from the Chapman University School of Pharmacy. National Center for Biotechnology InformationU. ACS Omega. Published online Nov Samara E. Author information Article notes Copyright and information Disclaimer. Corresponding author. Phone: Fax: R. Fax: K. Received Sep 29; Accepted Nov This is an open access article published under an ACS AuthorChoicewhich permits copying and redistribution of the article or any adaptations for non-commercial purposes.
This article has been cited by other articles in PMC. Introduction The properties of the cell membrane create a barrier for the efficient cellular delivery of therapeutics.Cem peptides review
email: [email protected] - phone:(892) 708-6223 x 9120
CEM Corp. Products