Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression

SummaryThe present study investigated the effects of crotamine, a cell-penetrating peptide from rattlesnake venom, at different exposure times and concentrations, on both developmental competence and gene expression (ATP1A1, AQP3, GLUT1 and GLUT3) of in vitro fertilized (IVF) bovine embryos. In Experiment 1, presumptive zygotes were exposed to 0.1 μM crotamine for 6, 12 or 24 h and control groups (vehicle and IVF) were included. In Experiment 2, presumptive zygotes were exposed to 0 (vehicle), 0.1, 1 and 10 μM crotamine for 24 h. Additionally, to visualize crotamine uptake, embryos were exposed to rhodamine B-labelled crotamine and subjected to confocal microscopy. In Experiment 1, no difference (P > 0.05) was observed among different exposure times and control groups for cleavage and blastocyst rates and total cells number per blastocyst. Within each exposure time, mRNA levels were similar (P > 0.05) in embryos cultured with or without crotamine. In Experiment 2, concentrations as high as 10 μM crotamine did not affect (P > 0.05) the blastocyst rate. Crotamine at 0.1 and 10 μM did not alter mRNA levels when compared with the control (P > 0.05). Remarkably, only 1 μM crotamine decreased both ATP1A1 and AQP3 expression levels relative to the control group (P < 0.05). Also, it was possible to visualize the intracellular localization of crotamine. These results indicate that crotamine can translocate intact IVF bovine embryos and its application in the culture medium is possible at concentrations from 0.1–10 μM for 6–24 h.

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In Vitro and In Vivo Cell Uptake of a Cell-Penetrating Peptide Conjugated with Fluorescent Dyes Having Different Chemical Properties

Cancers ◽

10.3390/cancers13092245 ◽

2021 ◽

Vol 13 (9) ◽

pp. 2245

Author(s):

Hideo Takakura ◽

Honoka Sato ◽

Kohei Nakajima ◽

Motofumi Suzuki ◽

Mikako Ogawa

Keyword(s):

Fluorescent Dyes ◽

Chemical Properties ◽

Cyanine Dyes ◽

Cell Uptake ◽

Cell Penetrating Peptide ◽

Cell Penetrating ◽

A Cell ◽

Targeting Strategy

In molecular imaging, a targeting strategy with ligands is widely used because specificity can be significantly improved. In fluorescence imaging based on a targeting strategy, the fluorescent dyes conjugated with ligands may affect the targeting efficiency depending on the chemical properties. Herein, we used a cell-penetrating peptide (CPP) as a ligand with a variety of fluorescent cyanine dye. We investigated in vitro and in vivo cell uptake of the dye-CPP conjugates when cyanine dyes with differing charge and hydrophilicity/lipophilicity were used. The results showed that the conjugates with positively charged and lipophilic cyanine dyes accumulated in cancer cells in vitro, but there was almost no accumulation in tumors in vivo. On the other hand, the conjugates with negatively charged and hydrophilic cyanine dyes did not accumulate in cancer cells in vitro, but fluorescence was observed in tumors in vivo. These results show that there are some cases in which the cell uptake of the dye-peptide conjugates may differ significantly between in vitro and in vivo experiments due to the chemical properties of the fluorescent dyes. This suggests that attention should be paid to the chemical properties of fluorescent dyes in fluorescence imaging based on a targeting strategy.

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In vitro assessment of a novel polyrotaxane-based drug delivery system integrated with a cell-penetrating peptide

Journal of Controlled Release ◽

10.1016/j.jconrel.2007.08.029 ◽

2007 ◽

Vol 124 (1-2) ◽

pp. 43-50 ◽

Cited By ~ 69

Author(s):

Cheol Moon ◽

Young Min Kwon ◽

Won Kyu Lee ◽

Yoon Jeong Park ◽

Victor C. Yang

Keyword(s):

Drug Delivery ◽

Drug Delivery System ◽

Delivery System ◽

Cell Penetrating Peptide ◽

Cell Penetrating ◽

A Cell ◽

In Vitro Assessment

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Enzymatic Ligation of Antibody and Cell-Penetrating Peptide for Efficient and Cell-Specific siRNA Delivery

10.21203/rs.3.rs-567665/v1 ◽

2021 ◽

Author(s):

Yu Ando ◽

Hikaru Nakazawa ◽

Daisuke Miura ◽

Mitsuo Umetsu

Keyword(s):

Sirna Delivery ◽

Research Progress ◽

Cell Penetrating Peptide ◽

E Coli ◽

C Terminus ◽

Maximum Reaction ◽

Cell Penetrating ◽

A Cell ◽

Candidate Molecule

Abstract A fusion protein comprising an antibody and a cell-penetrating peptide is a candidate molecule capable of efficient and cell-specific delivery of siRNA into cells in order to reduce the side effects of nucleic acid drugs. However, their expression in bacterial hosts, required for their development, often fails, impeding research progress. In this study, we separately prepared anti-EGFR nanobodies with the K-tag sequence MRHKGS at the C-terminus and arginine-9 (R9) with the Q-tag sequence LLQGS at the N-terminus, and enzymatically ligated them in vitro by microbial transglutaminase to generate Nanobody-R9, which is not expressed as a fused protein in E. coli. Nanobody-R9 exhibited a maximum reaction efficiency of 85.1%, without changing the properties of the nanobody or R9. Nanobody-R9 successfully delivered siRNA into the cells, and the cellular influx of siRNA increased with increase in the ratio of Nanobody-R9 to siRNA. We further demonstrated that the Nanobody-R9–siRNA complex, at a 30:1 ratio, induced RNAi of target mRNA with approximately 52% efficiency compared to lipofectamine.

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