Niosomal virosome derived by vesicular stomatitis virus glycoprotein as a new gene carrier

https://doi.org/10.1016/j.bbrc.2020.10.054Get rights and content

Highlights

  • Virosomes as membranous vesicles with viral fusion protein in their membrane are versatile vehicles for cargo delivery.

  • VSV-G is one of the proteins used in the construction of virosomes.

  • Niosomes are composed of non-ionic surfactants which have amphipathic characteristic and overall neutral charge.

  • This pioneering study verified that niosome could be used as an bilayer instead of liposome in virosome preparation.

Abstract

Virosomes as membranous vesicles with viral fusion protein in their membrane are versatile vehicles for cargo delivery. The vesicular stomatitis virus glycoprotein (VSV-G) is a common fusogenic protein used in virosome preparation. This glycoprotein has been used in liposomal systems so far, but in this study, we have tried to use the niosomal form instead of liposome for. Niosomes are vesicular systems composed of non-ionic surfactants. Niosomes were constructed by the thin-film hydration method. VSV-G gene in pMD2.G plasmid was expressed in the HEK293T cell line and then was reconstituted in the niosome bilayer. The formation of niosomal virosomes was confirmed with different methods such as SDS-PAGE gel, western blotting, and transmission electron microscopy (TEM). The efficiency of niosomal virosome was investigated with the pmCherry reporter gene. SDS-PAGE and western blotting proved the expression and successful insertion of protein into the bilayer. The TEM images showed the spike projection of VSV-G on the surface of niosomes. The transfection results showed high efficiency of niosomal virosomes as a novel carrier. This report has verified that niosome could be used as an efficient bilayer instead of liposome to construct virosomes.

Introduction

Gene therapy has been approved to be effective for the treatment of certain hereditary diseases (e.g., congenital immune-dysfunction due to adenosine deaminase deficiency), and it also can be applied to other genetic disorders such as malignant tumors. Gene therapy is completely dependent on the efficiency of gene delivery systems [1]. These systems could be categorized into viral and non-viral forms [2,3]. Non-viral vectors are attractive for researchers to be used as a promising alternative to viruses. These vectors have some considerable properties such as ease of pharmacologic production, low toxicity, lack of pathogenicity, and immunogenicity [4] which prove their potential as beneficial vectors. However, low levels of gene transfer by these vectors as compared to viruses restrict their application in gene delivery. There are a variety of non-viral vectors for gene delivery like polymers, peptides, mesoporous nanoparticles, organic-inorganic hybrids, and micro-vesicles such as liposomes and niosomes [5].

Among non-viral vectors, the niosome has attracted a lot of attention due to its remarkable properties. Niosomes are vesicular systems composed of non-ionic surfactants which have amphipathic characteristic and overall neutral charge [6,7]. These non-ionic surfactants are safe and inexpensive which could be used in biomedicine e.g. as drug carriers for both hydrophilic and hydrophobic drugs. Niosomes are highly stable and their leakage is only a little more than liposomes [8]. Besides, they could be easily prepared and the presence of non-ionic surfactants makes them less toxic than liposomes [9]. Therefore, the niosome is an interesting choice for cargo delivery.

Several viruses have been considered to be used as foreign gene-transducing vectors [10]. But the main problem with these vectors is that none of them could be used as risk-free or long-lasting treatments because of their potential for malignant alternations and cell destroying activity against the recipient cells [11,12]. Virosomes are the reconstruction of lipid membranes and viral spike proteins but without the viral genetic materials [13,14]. Liposomal virosomes have been constructed so far. Studies have proved that virosomes not only could carry different biologically active components like proteins, nucleic acid, peptides, and DNA [[15], [16], [17]] but also they could be used to carry therapeutic agents for drug delivery purposes [18]. Viral coated proteins are assumed to act independently of the other virion components, therefore they could be utilized in other non-viral gene transfer systems for the targeted selection of a certain organ or tissue [19]. These proteins have a key role in the entrance of viruses into the host cells and are different from one virus to another. As an instance, surface glycoproteins of influenza are hemagglutinin and neuraminidase [20] or G-protein is the glycoprotein of vesicular stomatitis virus (VSV) [21] and S-glycoprotein is the glycoprotein of coronavirus [9].

As mentioned, VSV-G is one of the proteins used in the construction of virosomes. It has been reported that VSV-G can restructure into the lipid bilayer spontaneously, however the controlled condition in which this protein could conserve its fusogenic activity in such lipids is necessary [22,23]. VSV-G is made up of 511 amino acids which could be in three conformations including a) prefusion conformation that is found in high pH ranges, b) the active form that has some interaction with the membrane of the host cell and c) the postfusion conformation [24]. This glycoprotein has been inserted in liposomes or used solitary for gene delivery enhancement [25]. To the best of our knowledge, no research has been accomplished for the investigation of niosomal virosomes instead of liposomes. Therefore, this study aims to evaluate the possibility of the preparation of niosomal virosomes. For this purpose, the expression of VSV-G protein in the HEK293T cell line was carried out and the possibility of VSV-G entrance into the cell membrane was evaluated.

Section snippets

Materials

Cell lines were purchased from the Pasteur Institute of Iran (Tehran, Iran). Maxi-prep plasmid extraction kit was purchased from Yekta Tajhiz Azma, Iran. Hoechst dye, fetal bovine serum (FBS), trypsin, DMEM medium, and Opti-MEM medium were all provided from Thermo scientific (Carlsbad, CA). Cholesterol, Span 80, Span 60, Tween 80, Tween 60, and secondary antibody were obtained from Sigma (Steinheim, Germany). The primary antibody was purchased from Santa Cruz Company (USA). Valid commercial

Niosome formation

Light microscopic examination confirmed the formation of niosomes which was carried out successfully in both formulations by the thin-film hydration method. Saturated and unsaturated niosomes are presented in Fig. 1a and Fig. 1b. Due to the adequate formation of both saturated and unsaturated niosomes and considering the previous studies [8], unsaturated niosome was chosen for the next steps.

Niosome toxicity

For the investigation of niosome toxicity, HEK293T cells were cultured in a 96-well plate. As can be

Discussion

Due to the importance of VSV-G, some investigation about the expression and the different methods for its extraction and purification has been accomplished so far [27]. In this study, the expression and purification of VSV-G with different methods like the construction of cellular syncytium, and with a polyclonal antibody against VSV-G protein in HEK293T cells have been confirmed. There are also several studies about the development of VSV-G virosomes. There are two different methods for the

Conclusion

Although niosomes and liposomes are different in structure and composition, they have some essential characteristics in common. The results of this study have proved that due to the remarkable stability of niosomes compared to liposomes and the suitable compatibility of VSV-G in the niosome structure, niosomes are compatible alternatives for liposomes in the construction of virosomes. Moreover, considering the role of Span80 as a versatile assistant in gene delivery, this component is an

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study was supported by a grant from Kerman University of Medical Sciencesand the authors greatly appreciate financial support.

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