Transfecting pDNA to E. coli DH5α using bovine serum albumin nanoparticles as a delivery vehicle Jitendra Wagh, b Kuldeep J. Patel, c Parth Soni, c Krutika Desai, c Pratik Upadhyay d and Hemant P. Soni a * ABSTRACT: We describe the formulation of bovine serum albumin nanoparticles (BSA-NPs) by the coacervation method using surfactants. Plasmids (pUC18, pUC18egfp and pBBR1MCS-2) isolated from E. coli were incorporated into the BSA matrix by incubating in albumin solution prior to formulation of NPs. Plasmid incorporation was calculated by % yield, entrapment efficiency, DNA loading capacity and release of entrapped DNA by comparing with blank NPs. BSA-DNA binding studies were carried out by using fluorescence spectroscopy and Fourier Transform Infra Red Spectroscopy (FT-IR). The surface charge distribution of the NPs loaded with plasmid was calculated using zeta potential. The photoluminescence of BSA-NPs was quenched when loaded with pDNA, confirming the interaction of DNA with BSA. Altogether, these results provide evidences for the excellent DNA carrying efficiency of BSA-NPs without loss of plasmid’s integrity. The NPs were used to transfect E. coli DH5α strain lacking ampicillin resistance. They, however, showed ampicillin resistance subsequent to transfection with plasmid encoding ampicillin resistance gene. Effect of transfection was confirmed by confocal microscopy and by the isola- tion of the plasmid by agarose gel electrophoresis from the transfected bacterial culture. This study clearly demonstrates the efficacy of BSA-NPs as delivery vehicle for pDNA transfection. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: BSA nanoparticles; Gene delivery; Enhanced green fluorescence protein Introduction Transfection of foreign plasmid DNA or RNA into host cell nucleus to modify, change, or silence expression of gene is a challenging task. Availability of strategy in this context can ensure the produc- tion of specific protein that can help to mitigate conditions like Parkinson’s disease (1) and cystic fibrosis (2) or even provide relief to painful chemotherapy treatments in cancer (3). The cellular nu- clease enzymes constitute the major obstacle in this task as they can degrade naked plasmids in the cytoplasm before entry into the nucleus leading to low transfection efficiency (4–6). Various strategies have been devised to overcome this problem like trap- ping such pDNA in a specialized carrier of both natural and syn- thetic origin (7). Such a carrier should not only bind with pDNA but also with receptors on the cell membrane for successful entry into cells. After entering cytoplasm, it should neither interact with other organelles nor interfere with any biological process (8). Ideally, it should be degraded once it has entered the cytoplasm and release the cargo without causing any harm to the cell. There are carriers that can directly traverse the cell membrane and enter the cytoplasm and viruses are the naturally evolved machinery ideal for such a process (9,10). However, there are serious drawbacks with such systems like with immunogenicity (11,12), oncogenicity (13) and recombination efficiency (14), which restrict the successful application of them. Many chemical agents such as cationic lipids like 2,3-di-oleoyloxytrimethyl ammonium propane (15), fullerenes and their derivatives (16,17), biodegradable poly- mers and dendrimer based materials e.g. PLL-b-PEG (18–20), PAGA (21), PAMAM type dendrimers etc.; carbohydrate based polymeric cages e.g. β-cyclodextrins (22,23), chitosan (24,25) etc. have been considered as nonviral gene-delivery vectors. But they also have their own drawbacks, e.g. high concentrations of fullerene-based materials can induce inflammation promoting the development of cancer (26). Recent reviews have focused on different types of gene-delivery systems, barriers encountered during nonviral gene delivery and the techniques to overcome them (27,28). Considering these, there is a need for development of efficient and specific delivery vehicles either natural or synthetic. Protein, being a macromolecule, can very well satisfy all such requirements to act as a gene-delivery vector (29). They are capable of self- assembly into various shapes and morphologies with well defined * Correspondence to: Dr. Hemant P Soni. Department of Chemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara-390 002, Gujarat, India. E-mail: drhpsoni@yahoo.co.in a Department of Chemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara-390 002, Gujarat, India b Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara-390 002, Gujarat, India c Department of Biotechnology, Ashok & Rita Patel Institute of Integrated Study & Research in Biotechnology and Allied Sciences (ARIBAS), Sardar Patel University, V V Nagar, Gujarat, India d LJ Institute of Pharmacy, Sarkhej, Ahmedabad, Gujarat, India Abbreviations: AAV, adeno-associated virus; BSA, bovine serum albumin; BSA-NPs, bovine serum albumin nanoparticles; DLS, Dynamic Light Scatter- ing; ESGT, European Society of Gene Therapy; FTIR, Fourier transform infra red; LAFU, laminar air flow unit; LB, Luria Broth. Luminescence 2015; 30: 583–591 Copyright © 2014 John Wiley & Sons, Ltd. Research article Received: 5 June 2014, Revised: 13 August 2014, Accepted: 4 September 2014 Published online in Wiley Online Library: 24 October 2014 (wileyonlinelibrary.com) DOI 10.1002/bio.2789 583