Transfecting pDNA to E. coli DH5α using bovine serum albumin nanoparticles as a delivery vehicle

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 isolation 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.

In vivo compaction dynamics of bacterial DNA: A fingerprint of DNA/RNA demixing?

“…This indicates that electrostatic forces play an important role in the compaction mechanism and that it is crucial that the non-neutralized electrostatic charges along the DNA not be screened too severely for compaction to occur. However, it may be felt that these results are not fully conclusive, since attractive interactions between DNA and BSA proteins and the formation of relatively weak BSA-DNA complexes and coacervates have been reported [50]. Therefore, it cannot be completely excluded that the fewer positively charged patches on the surface of BSA molecules are sufficient to let these proteins bind to DNA and cause conformational changes and compaction.…”

A segregative phase separation scenario of the formation of the bacterial nucleoid

“…It was indeed first shown that the addition of 5-10% (w/v) of bovine serum albumin (BSA) to the buffer compacts long DNA molecules to densities close to that of the nucleoid. [39,40] Since the surface of BSA proteins displays small positive patches and the formation of weak BSA-DNA coacervates has been reported, [41] it can admittedly not be completely excluded that the observed compaction of the DNA by BSA proteins corresponds actually to complex coacervation (associative phase separation) rather than segregative phase separation. Still, unambiguous confirmation of the efficiency of the segregative phase separation mechanism came shortly after from experiments performed with negatively charged silica nanoparticles with diameter in the range 20-135 nm, which showed that introduction of a few percents thereof in the buffer also leads to the gradual compaction of the DNA.…”

Albumin-Based Nanoparticles for the Delivery of Doxorubicin in Breast Cancer

“…For the preparation of ABNs, various techniques like desolvation, thermal gelation, emulsification, nano spray drying, and self-assembly can be used. However, desolvation is the most commonly used method, where ethanol is employed as a desolvating agent and glutaraldehyde as a cross-linker [ 39 , 41 ]. Unfortunately, using glutaraldehyde as a cross-linker has some disadvantages like toxicity, potential reaction with encapsulated drug, and longer reaction time to prepare albumin nanoparticles [ 17 , 42 ].…”