A novel method for DNA delivery into bacteria using cationic copolymers

Abstract

Amphiphilic copolymers have a wide variety of medical and biotechnological applications, including DNA transfection in eukaryotic cells. Still, no polymer-primed transfection of prokaryotic cells has been described. The reversible additionfragmentation chain transfer (RAFT) polymer synthesis technique and the reversible deactivation radical polymerization variants allow the design of polymers with well-controlled molar mass, morphology, and hydrophilicity/hydrophobicity ratios. RAFT was used to synthesize two amphiphilic copolymers containing different ratios of the amphiphilic poly [2-(dimethyl-amino) ethyl methacrylate] and the hydrophobic poly [methyl methacrylate]. These copolymers bound to pUC-19 DNA and successfully transfected non-competent Escherichia coli DH5a, with transformation efficiency in the range of 10 3 colony-forming units per mg of plasmid DNA. These results demonstrate prokaryote transformation using polymers with controlled amphiphilic/hydrophobic ratios.

Cationic Polymers Enable Internalization of Negatively Charged Chemical Probe into Bacteria

“…To our knowledge, the only example of related work was reported by de Souza and coworkers where they showcased the ability of cationic copolymers to promote the transformation of pDNA into a non-competent E. coli DH5α strain. 43 Transformation with these polymers yielded CFU in the range of 10 3 /μg of DNA, whereas a normal transformation ranges in 10 6 – 10 7 CFU/μg of DNA. 43, 61…”

“…To our knowledge, the only example of related work was reported by de Souza and coworkers where they showcased the ability of cationic copolymers to promote the transformation of pDNA into a non-competent E. coli DH5α strain. 43 Transformation with these polymers yielded CFU in the range of 10 3 /µg of DNA, whereas a normal transformation ranges in 10 6 -10 7 CFU/µg of DNA. 43,61 Finally, our data indicate that the investigated polymers may provide a generalizable strategy for bacterial permeabilization as they also promoted probe entry into the Gram-positive organism, B. subtilis.…”

“…43 Transformation with these polymers yielded CFU in the range of 10 3 /µg of DNA, whereas a normal transformation ranges in 10 6 -10 7 CFU/µg of DNA. 43,61 Finally, our data indicate that the investigated polymers may provide a generalizable strategy for bacterial permeabilization as they also promoted probe entry into the Gram-positive organism, B. subtilis. These cells remained more viable and showed less damage than those treated with lysozyme, an enzyme used to degrade peptidoglycan in the cell wall.…”

“…One key discovery has been the importance of complex architectures such as micelles (termed micelleplex when complexed with genetic material) for better delivery. [38][39][40][41][42] The cationic nature of these materials suggests that they may also interact favorably with bacterial cells 2,20,28,[43][44] . In addition, the ease of synthesis of these types of molecules could make them a promising new class of bacterial delivery reagents.…”

“…The cationic nature of these materials suggests that they may also interact favorably with bacterial cells 2, 20, 28, 43–44 In addition, the ease of synthesis of these types of molecules could make them a promising new class of bacterial delivery reagents. While eukaryotic membranes and prokaryotic cell envelopes differ, the ability of these polymers to complex with negatively charged genetic materials makes them promising candidates for the delivery of other negatively charged molecules such as the adenosine triphosphate (ATP)-based probe, BODIPY-FL-ATPγS.…”