Elsevier

Aquaculture

Volumes 358–359, 15 August 2012, Pages 14-22
Aquaculture

Synthesis and characterization of CS/TPP nanoparticles for oral delivery of gene in fish

https://doi.org/10.1016/j.aquaculture.2012.06.012Get rights and content

Abstract

This work examines the potential use of chitosan tripolyphosphate (CS/TPP) nanoparticles for gene delivery in different tissues of fish through oral route. The porin gene of Vibrio anguillarum was used to construct a DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP. The CS/TPP nanoparticles were synthesized by ionic gelation process and these nanoparticles particles were characterized. The morphology and particle size measurements of the nanoparticles were studied by field emission scanning electron microscopy (FE-SEM) and FTIR (Fourier Transform Infrared Spectra). The pVAOMP was encapsulated by CS/TPP nanoparticles by mixing of equal volume of heated CS/TPP and pVAOMP DNA solutions. The encapsulation efficiency of CS/TPP nanoparticles was found to be 79.9% of DNA binding with CS/TPP nanoparticles. The stability of plasmid DNA was also determined after encapsulation using DNase I and chitosanase. The cytotoxicity of CS/TPP nanoparticles was evaluated by MTT assay using fish cell line. The expression of gene was confirmed by immunohistochemistry, ELISA and real-time PCR analyses. The results indicate that DNA can be easily delivered into fish by feeding with CS/TPP nanoparticles.

Highlights

► CS/TPP nanoparticle ► Non viral gene delivery ► Encapsulate pVAOMP DNA ► In vitro and in vivo study

Introduction

Vibriosis is one of the important fish bacterial diseases caused by Vibrio anguillarum and is responsible for severe economic loss in aquaculture industry worldwide (Anderson and Norton, 1991, Austin and Austin, 1993). Asian sea bass (Lates calcarifer) is an important cultivable fish for aquaculture in India. V. anguillarum is a major problem in sea bass farming in India and worldwide. Although different vaccine formulations have been used to prevent vibriosis and no effective vaccine is available against this important pathogen (Boesen et al., 1997, Joosten et al., 1997). Current vaccine research is oriented towards replacement of conventional vaccines with more effective and safer approaches, such as DNA vaccines. Immunization with antigen encoding plasmid DNA can elicit very strong and long-lasting humoral and cellular immune responses. This new approach offers economic, environmental and safety advantages, which are particularly attractive for the aquaculture industry (Heppell and Davis, 2000, Kwang, 2000). Intramuscular injection was used by different workers for DNA vaccination in fish (Anderson et al., 1996, Hansen et al., 1991, Lorenzen et al., 1998, Rajesh Kumar et al., 2007, Sulaiman et al., 2000). This method is obviously impractical and not feasible for field application. Hence, it is necessary to develop a simple and cost effective delivery system to deliver DNA vaccine for mass vaccination in fish farms. Recently, a number of new techniques have been developed to introduce a foreign DNA into cells. One approach is a non-viral delivery system.

Viral gene delivery systems have been found to have high transfection efficiency for a wide range of cell types but they have some disadvantages such as virtually induced inflammatory responses and oncogenic effects (Simon et al., 1993). To overcome these problems, non-viral gene delivery systems using biopolymers have been developed to deliver genes with minimal host immune responses (Bozkir and Saka, 2004a, MacLaughlin et al., 1998, Mao et al., 2001, Rajesh Kumar et al., 2008b, Ramos et al., 2005, Romoren et al., 2002). Cationic polymers were found to be promising delivery systems among the non-viral delivery systems (Garnett, 1999). Among the polymers used to encapsulate pDNA are poly (d,l-lactic-co-glycolic) acid (PLGA) (Capan et al., 1999), gelatine (Leong et al., 1998) and chitosan (Bozkir and Saka, 2004a, Hirosue et al., 2001, Lee et al., 2001). Chitosan (CS) is a natural biodegradable, biocompatible and non-toxic biopolymer extracted from the shells of crustaceans. Mumper et al. (1995) have used the chitosan for the first time to deliver the plasmid in the cellular system. Chitosan and its derivatives have been examined extensively for medical and pharmaceutical applications especially in artificial organs, targeted drug delivery, drug transport, protein delivery gene transfer and so on (Lavertu et al., 2006, Pan et al., 2002, Sakai et al., 2001, Wei and Zhang, 2007). Due to the presence of amino groups, CS possess positive charge and can effectively bind negatively charged DNA resulting in nanoparticles of various sizes (Bozkir and Saka, 2004b, Ishii et al., 2001) and also protect DNA from nuclease degradation (MacLaughlin et al., 1998, Mao et al., 2001, Richardson et al., 1999). CS has favorable biocompatibility characteristics as well as the ability to increase membrane permeability, both in vitro and in vivo and can be degraded by lysozyme in serum (Aspden et al., 1996, Takeuchi et al., 2001). Based on the above characteristics, chitosan has been considered as a promising carrier for gene delivery. Chitosan has been used to deliver various reporter genes orally in different animals (MacLaughlin et al., 1998, Roy et al., 1999, Takeuchi et al., 2001).

Gene transfer for transient expression in fish is very important for the application of DNA vaccines to prevent viral and bacterial diseases of economically important fish species (Anderson et al., 1996). Ramos et al. (2005) have reported the oral delivery of a construct expressing the β-galactosidase reporter gene into fish by encapsulating the DNA in chitosan and incorporating it into fish feeds and observed the expression of the gene in stomach, spleen and gill tissue. Romoren et al. (2002) have investigated the use of chitosan–DNA formulations to deliver the gene in fish by immersion method. The CS/TPP nanoparticles have been used as an alternative to chitosan to encapsulate peptides, proteins, pDNA and siRNA by various workers (Calvo et al., 1997a, Calvo et al., 1997b, Csaba et al., 2009, Cuna et al., 2002, Fernandez Urrusuno et al., 1999, Gan et al., 2005, Katas and Alpar, 2006, Vila et al., 2004, Wang et al., 2009, Yang et al., 2009). Calvo et al (1997b) have developed a technique for developing chitosan nanogels by adding a cross-linking agent, i.e. tripolyphosphate (TPP), into the aqueous phase containing chitosan. CS/TPP nanoparticles with entrapped siRNA have been found to be better vector for delivering the siRNA compared to chitosan siRNA complexes (Katas and Alpar, 2006). Csaba et al. (2009) have adapted ionic gelation technique for the encapsulation of different nucleic acids (plasmid DNA and short oligonucleotides) into chitosan–TPP nanoparticles and evaluated their potential as gene delivery nanocarriers. In addition, chitosan–TPP can penetrate deep into tissues through fine capillaries and this allows efficient delivery of proteins, drug and plasmid DNA in the body (Gan et al., 2005). In the present study, an attempt was made to make use of CS/TPP nanoparticles as an alternative to chitosan to deliver porin gene of V. anguillarum to fish through oral route.

Section snippets

Materials

Low molecular weight chitosan (CS) and tripolyphosphate (TPP) were purchased from Sigma Aldrich (St. Louis, Missouri, USA). Chitosanase (Streptomyces griseus) and DNase I were purchased from Promega (Madison, USA). Acetic acid and ethanol were purchased from Hi-media (Mumbai, India). Plasmid (pVAOMP) DNA was constructed and produced in large scale in our lab. The plasmid was prepared from Escherichia coli DH5α transformants using alkaline lysis protocols as described by Sambrook et al. (1989).

FTIR and SEM characterization of CS/TPP nanoparticles

The results of FTIR spectra of CS and CS/TPP nanoparticles are shown in Fig. 1. To investigate CS‐TPP nanoparticle formation, FTIR spectra of CS and CS-TPP nanoparticles were recorded. The main IR bands of pure CS and CS–TPP were reported in Table 1 which showed the presence of the Pdouble bondO and Psingle bondO groups at the frequency of 1203 cm- 1 and 1240 cm- 1, respectively and the band shifts (from 1647 cm- 1 and 1588 cm- 1, corresponding to Csingle bondO and Nsingle bondH stretching, respectively in pure CS, to 1738 cm- 1 and 1643 cm- 1 for

Discussion

In the present study, CS/TPP nanoparticles were used to deliver the gene in fish through oral route. Oral delivery of plasmid-VAOMP encapsulated in CS/TPP nanoparticles through feeding with fish feeds resulted in expression of OMP gene of V. anguillarum in gill, heart, muscle, liver and intestine of these fish but not in controls. The ionic gelation method was followed to prepare CS/TPP nanoparticles using low molecular weight chitosan. The size of the nanoparticles ranged from 30 to 60 nm.

Conclusions

Ionic gelation method was used to prepare CS/TPP nanoparticles. CS/TPP naanoparticles are an interesting alternative to other chitosan based delivery systems for oral plasmid DNA delivery. CS/TPP nanoparticles are suitable for the simultaneous encapsulation and sustained release of DNA molecules. The results of the present study suggest that CS/TPP nanoparticles can be used to deliver pVAOMP DNA in fish through oral route. CS/TPP nanoparticles are simple to prepare, safe and exhibit a

Acknowledgments

The authors thank the management of C. Abdul Hakeem College for providing the facilities to carry out this work. This work was funded by Department of Science and Technology, Government of India, New Delhi, India.

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