Enhanced cellular uptake and metabolic stability of lipo‐oligoarginine peptides

Abstract

Developing efficient cellular delivery vectors is crucial for designing novel therapeutic agents to enhance their plasma membrane permeability and metabolic stability in cells. Previously, we engineered cell penetrating peptide vectors named as “lipo-oligoarginine peptides” (LOAPs) by conjugating a proper combination of fatty acid and oligoarginine that translocated into cell easily without adverse effect on cell viability. In the present study, we report a systemic evaluation of cellular uptake and metabolic stability of LOAPs in Jurkat cells by introducing different combination of D-Arg residues in the peptide backbone. The cellular uptake and intracellular fate, cell viability, and metabolic stability and proteolytic degradation patterns of various LOAPs consisted of different combination of L- and D-Arg sequences were confirmed by flow cytometry, cytotoxicity assay, and analytical RP-HPLC with MALDI-TOF mass. All investigated LOAPs penetrated the cell efficiently with low cellular toxicity. The LOAPs having D-Arg residues at their N-termini seemed to have better metabolic stability than the LOAPs having C-terminal D-Arg residues. The metabolic degradation patterns were similar among all investigated LOAPs. The major hydrolytic site was between lauroyl group and β-Ala residue. Without the lipid chain, the oligoarginine peptide was pumped out of cells easily. The results presented in this study suggest that structurally modified LOAPs could be used as a novel CPP design toward improved therapeutic application.

Macromolecular Uptake of Alkyl‐Chain‐Modified Guanidinoglycoside Molecular Transporters

“…The dependency of cellular internalization on the alkyl chain length has also been reported for acylated CPP analogs [19–24] and amphiphilic aminoglycosides. [25–26] Additionally, the impact of hydrophobicity on uptake has been documented for systems including polymers [27] , amphiphiles [28] , and oligoarginine carriers with different counterions.…”

Enhancing the Cellular Delivery of Nanoparticles Using Lipo‐Oligoarginine Peptides

“…[30–32] ALP and MLP were purified from preparative HPLC, and their molecular weights were characterized by electrospray ionization (ESI; Supporting Information, Figure S1). To obtain optimal incorporation of LOAP incorporation, various concentrations of ALP or MLP were allowed to react with AuNPs.…”

“…It is well known that an improved cellular uptake can be achieved if appropriate hydrophobic lipid chains (C8 to C16) are anchored on peptides, suggesting the critical contribution of fatty acids in cellular trafficking activity. [28–32] Although the fluorescence intensity of fAuNPs was lower than it of free LOAPs, considerable cellular uptake of fAuNPs was achieved. Bare AuNPs did not exhibit any noticeable fluorescence signal due to the absence of FITC dyes.…”

“…[28–34] Previously, we developed lipo-oligoarginine peptides (LOAPs) by conjugating a proper combination of hydrophobic fatty acids and positively charged oligoarginines, which enhanced the cellular uptake of cargo into cells. [30–32] One particular LOAP, myristoylated hendeca-D-arginine peptide (MLP, Figure 1), exhibited the most reliable results for elevated cellular uptake rate, intracellular localization, low cellular toxicity, and enhanced metabolic stability. [31] Therefore, we prepared functionalized AuNPs (fAuNPs) by incorporating MLP on their surfaces to improve cellular uptake rate and intracellular trafficking with minimal cellular toxicity.…”

Strategies to Stabilize Cell Penetrating Peptides forIn VivoApplications

“…Conjugation of oligoarginines with fatty acid chains through a β-Ala residue at the N-terminal edge, enhanced their metabolic stability and cell penetration efficiency[161]. These lipo-oligoarginine peptides were further optimized by introducing different combination of D-Arg residues in the peptide backbone[162]. Coupling of a four octanoic acid (C8) tail at the N-terminal lysine (using both α and ϵamines) of Tat induced the formation of well-defined nanofibers able to encapsulate small hydrophobic drugs, such as paclitaxel, and to efficiently transport it into cells[163].…”