title: DNA amplification via polymerase chain reaction inside miniemulsion droplets with subsequent poly(n-butylcyanoacrylate) shell formation and delivery of polymeric capsules into mammalian cells.
abstract: There is a growing interest in the development of stable nanocapsules that could deliver the bioactive compounds within the living organism, and to release them without causing any toxic effects. Here the miniemulsion droplets were first used as "nanoreactors" for the amplification of single-molecule dsDNA template (476 and 790 base pairs) through PCR. Afterwards, each droplet was surrounded with a biodegradable PBCA shell by interfacial anionic polymerization, enabling therefore to deliver the PCR products into the cells. The size of the initial miniemulsion droplets and the final polymeric capsules was in the range of 250 and 320 nm, mainly depending on the type of the continuous phase and presence of dsDNA template molecules. The formation of PCR products was resolved with gel electrophoresis and detected with fluorescence spectroscopy in the presence of DNA specific dye (SYBRGreen). TEM studies were performed to prove the formation of the polymeric shell. The shell thickness was measured to be within 5-15 nm and the average molecular weight of the formed PBCA polymer was around 75000 g · mol(-1) . For the cell uptake experiments, the obtained nanocapsules were transferred from the organic phase into aqueous medium containing a water-soluble surfactant. The effect of the surfactant type (anionic, cationic or non-ionic) on the HeLa cell viability and nanocapsule uptake behavior was studied by CLSM and FACS. Confocal analysis demonstrated that nanocapsules stabilized with cationic (CTMA-Cl) and non-ionic (Lutensol AT50) surfactants show almost the same uptake, whereas capsules redispersed in anionic (SDS) surfactant possess a 30% higher uptake. The release of the encapsulated material within the cell was studied on the example of Cy5-labeled oligonucleotides showing the colocalization with mitochondria of MSCs cells.
authors.affiliation: Max Planck Institute for Polymer Research, Ackermannweg 10, Mainz, Germany.
authors.forename: Grit
authors.initials: G
authors.lastname: Baier
authors.forename: Anna
authors.initials: A
authors.lastname: Musyanovych
authors.forename: Katharina
authors.initials: K
authors.lastname: Landfester
authors.forename: Andreas
authors.initials: A
authors.lastname: Best
authors.forename: Steffen
authors.initials: S
authors.lastname: Lorenz
authors.forename: Volker
authors.initials: V
authors.lastname: Mailänder
mesh_terms: D002232:Carbocyanines / Q000032:analysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D016503:Drug Delivery Systems / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004655:Emulsions / Q000737:chemistry* / Q000378:metabolism; D004659:Enbucrilate / Q000138:chemical synthesis*; D005260:Female; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D008970:Molecular Weight; D053769:Nanocapsules / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009821:Oils / Q000737:chemistry; D016133:Polymerase Chain Reaction; D013501:Surface-Active Agents / Q000737:chemistry; D014280:Triglycerides / Q000737:chemistry
chemical_list: D002232:Carbocyanines; D004655:Emulsions; D053769:Nanocapsules; D009821:Oils; D013501:Surface-Active Agents; D014280:Triglycerides; C085321:cyanine dye 5; C022834:miglyol 812; D004247:DNA; D004659:Enbucrilate