Research Article: The use of tail-anchored protein chimeras to enhance liposomal cargo delivery

Date Published: February 22, 2019

Publisher: Public Library of Science

Author(s): Abbi Abdelrehim, Lior Shaltiel, Ling Zhang, Yechezkel Barenholz, Stephen High, Lynda K. Harris, Gantumur Battogtokh.

http://doi.org/10.1371/journal.pone.0212701

Abstract

Liposomes are employed as drug delivery vehicles offering a beneficial pharmacokinetic/distribution mechanism for in vivo therapeutics. Therapeutic liposomes can be designed to target specific cell types through the display of epitope-specific targeting peptides on their surface. The majority of peptides are currently attached by chemical modification of lipid constituents. Here we investigate an alternative and novel method of decorating liposomes with targeting ligand, using remotely and spontaneously inserting chimeric tail-anchored membrane (TA) proteins to drug loaded liposomes.

An artificial TA protein chimera containing the transmembrane domain from the spontaneously inserting TA protein cytochrome b5 (Cytb5) provided a robust membrane tether for the incorporation of three different targeting moieties into preformed liposomes. The moieties investigated were the transactivator of transcription (TAT) peptide, the EGF-receptor binding sequence GE11 and the placental and tumour homing ligand CCGKRK. In all cases, TA protein insertion neither significantly altered the size of the liposomes nor reduced drug loading. The efficacy of this novel targeted delivery system was investigated using two human cell lines, HeLa M and BeWo. Short term incubation with one ligand-modified TA chimera, incorporating the TAT peptide, significantly enhanced liposomal delivery of the encapsulated carboxyfluorescein reporter.

The Cytb5 TA was successfully employed as a membrane anchor for the incorporation of the desired peptide ligands into a liposomal drug delivery system, with minimal loss of cargo during insertion. This approach therefore provides a viable alternative to chemical conjugation and its potential to accommodate a wider range of targeting ligands may provide an opportunity for enhancing drug delivery.

Partial Text

Encapsulation of cytotoxic chemotherapeutics in liposomes improves their bioavailability and reduces their toxicity to non-cancerous tissue [1,2]. Liposomes are a versatile delivery system and modification of the liposomal surface with cell-binding ligands has been shown to facilitate active targeting, enhancing delivery and uptake in specific tissues [2–5]. Antibodies and peptides are frequently employed as targeting ligands and are commonly attached to liposomes through various chemical conjugation reactions [2,5–7], However, chemical modification of the liposomal surface with ligands can add complexity to the synthesis and increase the cost when scaling up the process [8].

In the present study, an alternative method for the display of ligands at the surface of liposomes without the need for chemical coupling was developed. This method exploits the TA of Cytb5 as an anchoring platform for N-terminally fused ligands. The ability for spontaneous membrane incorporation appears to be solely retained within the Cytb5 TA, since it has been shown to be capable of incorporating non-native N-terminal domains into lipid bilayers [17,18]. Here, we use the Syb2 N-terminal domain as a linker between the Cytb5 TA and three different ligands that have been demonstrated to significantly enhance the uptake of liposomes by their cellular targets [24,27,31].

The spontaneously inserting TA region of Cytb5 is capable of anchoring N-terminal domains incorporating cell binding ligands into liposomes. Modification with specific TA protein chimeras variably enhanced the uptake of liposomes containing fluorescent and/or cytotoxic cargoes in human cell culture models. We conclude that the Cytb5 TA presents an exciting opportunity to anchor a range of polypeptides onto the surface of synthetic lipid bilayers, and that with further optimisation this approach may be suitable for enhanced drug delivery.

 

Source:

http://doi.org/10.1371/journal.pone.0212701

 

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