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Intracellular delivery of fluorescent protein into viable wheat microspores using cationic peptides.

ABSTRACT: Microspores are specialized generative cells with haploid genome that demonstrate the amenability toward embryogenesis under certain conditions. The induced microspore culture technique is largely exploited by the breeding programs of wheat and other crops due to its high efficiency for generation of the large number of haploid plants in the relatively short period of time. The ability to produce mature double haploid plant from a single cell has also attracted attention of the plant biotechnologists in the past few years. More importantly, the possibility to deliver proteins for improvement of embryogenesis and the genome modification purposes holds great potential for transgene-free wheat biotechnology. In the present study, we examined the ability of cationic and amphipathic cell penetrating peptides (CPPs) to convey a covalently-linked mCherry protein inside the viable microspores. We demonstrate that the affinity of CPPs to the microspore cells dependents on their charge with the highest efficiency of CPP-mCherry binding to the cells achieved by cationic CPPs (penetratin and R9). Additionally, due to overall negative charge of the microspore cell wall, the successful uptake of the protein cargo by live microspore cells is attained by utilization of a reversible disulfide bond between the R9 CPP and mCherry protein. Overall, the approach proposed herein can be applied by the other biotechnology groups for the fast and efficient screening of the different CPP candidates for their ability to deliver proteins inside the viable plant cells.

SUBMITTER: Bilichak A

PROVIDER: S-EPMC4552043 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Intracellular delivery of fluorescent protein into viable wheat microspores using cationic peptides.

Bilichak Andriy A Luu Justin J Eudes François F

Frontiers in plant science 20150828

Microspores are specialized generative cells with haploid genome that demonstrate the amenability toward embryogenesis under certain conditions. The induced microspore culture technique is largely exploited by the breeding programs of wheat and other crops due to its high efficiency for generation of the large number of haploid plants in the relatively short period of time. The ability to produce mature double haploid plant from a single cell has also attracted attention of the plant biotechnolo ...[more]

PMID: 26379691

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Project description:Rapid, facile, and noncovalent cell membrane modification with alkyl-grafted anionic polymers was sought as an approach to enhance intracellular delivery and bioactivity of cationic peptides. We synthesized a library of acrylic acid-based copolymers containing varying amounts of an amine-reactive pentafluorophenyl acrylate monomer followed by postpolymerization modification with a series of alkyl amines to afford precise control over the length and density of aliphatic alkyl side chains. This synthetic strategy enabled systematic investigation of the effect of the polymer structure on membrane binding, potentiation of peptide cell uptake, pH-dependent disruption of lipid bilayers for endosome escape, and intracellular bioavailability. A subset of these polymers exhibited pKa of ∼6.8, which facilitated stable membrane association at physiological pH and rapid, pH-dependent endosomal disruption upon endocytosis as quantified in Galectin-8-YFP reporter cells. Cationic cell penetrating peptide (CPP) uptake was enhanced up to 15-fold in vascular smooth muscle cells in vitro when peptide treatment was preceded by a 30-min pretreatment with lead candidate polymers. We also designed and implemented a new and highly sensitive assay for measuring the intracellular bioavailability of CPPs based on the NanoLuciferase (NanoLuc) technology previously developed for measuring intracellular protein-protein interactions. Using this split luciferase class of assay, polymer pretreatment enhanced intracellular delivery of the CPP-modified HiBiT peptide up to 30-fold relative to CPP-HiBiT without polymer pretreatment (p < 0.05). The overall structural analyses show that polymers containing 50:50 or 70:30 molar ratios of carboxyl groups to alkyl side chains of 6-8 carbons maximized peptide uptake, pH-dependent membrane disruption, and intracellular bioavailability and that this potentiation effect was maximized by pairing with CPPs with high cationic charge density. These results demonstrate a rapid, mild method for polymer modification of cell surfaces to potentiate intracellular delivery, endosome escape, and bioactivity of cationic peptides.

Project description:Negatively charged tissues are ubiquitous in the human body and are associated with a number of common diseases yet remain an outstanding challenge for targeted drug delivery. While the anionic proteoglycans are critical for tissue structure and function, they make tissue matrix dense, conferring a high negative fixed charge density (FCD) that makes drug penetration through the tissue deep zones and drug delivery to resident cells extremely challenging. The high negative FCD of these tissues is now being utilized by taking advantage of electrostatic interactions to create positively charged multi-stage delivery methods that can sequentially penetrate through the full thickness of tissues, create a drug depot and target cells. After decades of work on attempting delivery using strong binding interactions, significant advances have recently been made using weak and reversible electrostatic interactions, a characteristic now considered essential to drug penetration and retention in negatively charged tissues. Here we discuss these advances using examples of negatively charged tissues (cartilage, meniscus, tendons and ligaments, nucleus pulposus, vitreous of eye, mucin, skin), and delve into how each of their structures, tissue matrix compositions and high negative FCDs create barriers to drug entry and explore how charge interactions are being used to overcome these barriers. We review work on tissue targeting cationic peptide and protein-based drug delivery, compare and contrast drug delivery designs, and also present examples of technologies that are entering clinical trials. We also present strategies on further enhancing drug retention within diseased tissues of lower FCD by using synergistic effects of short-range binding interactions like hydrophobic and H-bonds that stabilize long-range charge interactions. As electrostatic interactions are incorporated into design of drug delivery materials and used as a strategy to create properties that are reversible, tunable and dynamic, bio-electroceuticals are becoming an exciting new direction of research and clinical work.

Dataset Information

Intracellular delivery of fluorescent protein into viable wheat microspores using cationic peptides.

Publications

Intracellular delivery of fluorescent protein into viable wheat microspores using cationic peptides.

Similar Datasets

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