Pharmaceutical Nanotechnology
Preparation and evaluation of chitosan–DNA–FAP-B nanoparticles as a novel non-viral vector for gene delivery to the lung epithelial cells

https://doi.org/10.1016/j.ijpharm.2011.02.043Get rights and content

Abstract

Gene delivery using cationic polymers such as chitosan shows good biocompatibility, but reveals low transfection efficiency. Fibronectin Attachment Protein of Mycobacterium bovis (FAP-B) which is responsible for the attachment of many Mycobacteria on the Fibronectin molecule of epithelial cell membrane can be considered as a new targeting ligand and can improve transfection rates in epithelial cells. In this study, chitosan–DNA nanoparticles were prepared using coacervation process. The effect of stirring speed and charge ratio (N/P) on the size and zeta potential of nanoparticles were evaluated. FAP-B ligand was added to nanoparticles at the specific condition to form chitosan–DNA–FAP-B nanoparticles via electrostatic attraction. Transfection efficiency of the final nanoparticles was investigated in A549 (alveolar epithelial cells). Cell viability was investigated using MTT assay. The optimum speed of stirring which was yielded the smallest chitosan–DNA nanoparticles with a narrow distribution (227 ± 43 nm), was 500 rpm with the corresponding N/P ratio of 20. Chitosan–DNA–FAP-B nanoparticles presented the size of 279 ± 27 nm with transfection efficiency about 10-fold higher than chitosan–DNA nanoparticles and resulted in 97.3% cell viability compared to 71.7% using Turbofect controls.

Chitosan–DNA–FAP-B nanoparticles showed good transfection efficiency without cell toxicity. They have small particle size around 279 nm which make them a promising candidate as a novel non-viral gene vector for gene delivery to lung epithelial cells.

Graphical abstract

In vitro transfection efficiency. (A) Naked DNA, (B) chitosan–DNA nanoparticles, (C) chitosan–DNA–FAP-B nanoparticles (chitosan/FAP-B = 1/0.5), (D) chitosan–DNA–FAP-B nanoparticles (chitosan/FAP-B = 1/1), (E) chitosan–DNA–FAP-B nanoparticles (chitosan/FAP-B = 1/2), (F) Turbofect. The relative transfection efficiency was calculated as relative light units (RLU) luminescence during 10 s at constant number of cells per well. The data are given as mean ± SD (n = 3).

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Introduction

The respiratory system is an excellent target for noninvasive localized drug delivery due to its large surface area and its accessibility. The airway and the alveolar epithelium are the sites where genetic disorders such as cystic fibrosis or inherited surfactant protein B deficiency and lung cancer have their major fatal impacts. For these reasons, the lung has been considered as an attractive target for gene therapy interventions (Vadolas et al., 2002, Okamoto et al., 2003, Mao et al., 2001).

The success in gene therapeutic strategies depends on an efficient system for the delivery of nucleic acid into the target cells. Both viral and non-viral gene delivery systems have been used in clinical trials to treat maladies such as cystic fibrosis and several forms of cancer (Vadolas et al., 2002, Okamoto et al., 2003). Non-viral delivery systems have been increasingly proposed as safer alternatives to viral vectors as they could be synthesized with higher purity and quality degree and less immunogenic response, are targetable, stable in storage, and easy to produce in large quantities. These advantages have provided the impetus to continue their development (Mao et al., 2001).

Cationic polymers have been shown as promising carriers among the non-viral gene delivery systems. Many cationic polymers, such as chitosan, polylysine, polyethyleneimine, dendrimers, poly (a-(4-aminobutyl)-L-glycolic acid) as well as cationic liposomes have been investigated for gene delivery. Nevertheless, an ideal polymeric gene carrier with high efficacy of gene transfer, targeting ability, good biocompatibility and stability has yet to emerge (Mao et al., 2001, Desmedt et al., 2000, Liu and Yao, 2002).

In recent years, the potential of chitosan as a polycationic gene carrier has been explored (Roy et al., 1999, Leong et al., 1998, MacLaughlin et al., 1998, Mao et al., 1996, Richardson et al., 1999). Chitosan is a polysaccharide copolymer of N-acetyl-D-glucosamine and D-glucosamine. Its cationic polyelectrolyte nature provides a strong electrostatic interaction with negatively charged DNA and protects it from nuclease degradation (Mao et al., 2001, Liu and Yao, 2002, Borchard, 2001). Chitosan has good biocompatibility and toxicity profile and has been widely used in pharmaceutical research (Dodane and Vilivalam, 1998) however; its application in vivo has been limited due to its low transfection efficiency.

Several cell-specific ligands, such as galactose, folic acid, transferrin or epidermal growth factor have been shown to improve transfection efficiency by receptor-specific interaction and endocytosis into target cells (Liu and Yao, 2002, Chan et al., 2007, Ogris et al., 2003, Mansouria et al., 2006). FAP-B (Fibronectin Attachment Protein of BCG) is one of the most expressed surface proteins of Mycobacterium bovis (BCG) and is reported to be responsible for the attachment of mycobacteria on the Fibronectin molecule of epithelial cells membranes. It is known that Fibronectin (FAP-B receptor) is expressed on lung, intestinal and bladder human epithelial cells membranes. The molecular weight of FAP-B is about 45–47 kDa and its structure is full of Proline and Alanine. By this reason, it is called Apa (Alanin–Prolin-rich antigen) (Laqueyrerie et al., 1995, Romain et al., 1993, Zhao et al., 2000). Abolhassani et al., 2006 have shown that BCG cannot be translocated through the epithelial cell line cultures from apical side to the basolateral compartment without FAP-B interactions. FAP-B is not detectable in HK-BCG (Heat-killed BCG) extracts and, as expected, HK-BCG was not able to translocate through epithelial cells. In addition, a mutant strain of BCG deleted from the FAP-B gene (BCG Apa) could not translocate the intestinal or lung epithelial cells. On the other hand, the inert latex beads coated with the FAP-B translocated through this monolayer cells whereas those coated with ovalbumin protein or non-coated beads failed to do so (Abolhassani et al., 2006). Therefore, it was decided to investigate the novel concept of using FAP-B in gene delivery and evaluate its potential in improving the transfection efficiency of complex DNA nanoparticles.

In this study, a non-viral gene vector with improved transfection efficiency to epithelial cells was prepared via interaction of chitosan–DNA nanoparticles with FAP-B ligand. Its transfection ability and cell viability were evaluated in alveolar epithelial cells (A549). Based on our findings, it seems chitosan–DNA–FAP-B nanoparticles may be a promising carrier for targeted gene delivery to Fibronectin molecules (FAP-B receptors) of epithelial cell membrane.

Section snippets

Materials

Chitosan Chitoclear (Mw = 126,000 Da/mol, deacetylation degree 98%) was purchased from Primex (Iceland). The plasmid pGL3-control vector encoding firefly lucifrase driven by an SV40 promoter was purchased from Bioneer (South Korea). FAP-B (Fibronectin Attachment Protein of BCG) (Mw = 45 kDa) was kindly provided by Prof G. Marchal (Institut Pasteur, Paris, France) (Romain et al., 1993). Turbofect reagent was obtained from Fermentas (Thermo Fisher Scientific, Canada). Cell culture media, Dulbecco's

Preparation and characterization of chitosan–DNA nanoparticles

The chitosan–DNA nanoparticles were formed as a result of complex coacervation between chitosan and DNA (Mao et al., 2001, Elfinger et al., 2009, Schmitz et al., 2007, Issa et al., 2006). The cationic characteristic of chitosan is a crucial parameter for the complex formation with DNA bearing negative charges. The size of nanoparticles is important for cellular uptake and can be affected by the speed of stirring during the formation of coacervates. To investigate such an effect, nanoparticles

Conclusions

Chitosan–DNA nanoparticles were successfully prepared by complex coacervation process under defined conditions. The optimum speed of stirring during coacervation was found to be 500 rpm with an ideal N/P ratio of 20 which resulted in the smallest nanoparticles. FAP-B was added to the chitosan–DNA nanoparticles as a ligand for attachment to its specific receptors present at the surface of epithelial cells. Chitosan–DNA–FAP-B nanoparticles had similar and small particle size as chitosan–DNA

Acknowledgements

This work would not have been possible without the invaluable excellent technical assistant of following colleagues: Dr. Zare, Dr. Khanahmad, Mr. Rahimi, and Miss Sohrabi. This work has been supported by Pasteur Institute of Iran and Tehran University of Medical Sciences (grant 57-6858).

References (35)

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