title: Enhanced gene expression through temperature profile-induced variations in molecular architecture of thermoresponsive polymer vectors.
abstract: BACKGROUND
  Successful non-viral gene targeting requires vectors to meet two conflicting needs-strong binding to protect the genetic material during transit and weak binding at the target site to enable release. Responsive polymers could fulfil such requirements through the switching of states, e.g. the chain-extended coil to chain-collapsed globule phase transition that occurs at a lower critical solution temperature (LCST), in order to transport nucleic acid in one polymer state and release it in another.
  
  
  METHODS
  The ability of new synthetic polycations based on poly(ethyleneimine) (PEI) with grafted neutral responsive poly(N-isopropylacrylamide) (PNIPAm) chains to condense DNA into particles with architectures varying according to graft polymer LCST was assessed using a combination of fluorescence spectroscopy, dynamic light scattering (DLS), zeta sizing, gel retardation and atomic force microscopy studies. Transfection assays were conducted under experimental conditions wherein the polymer components were able to cycle across their LCST.
  
  
  RESULTS
  Two PEI-PNIPAm conjugate polymers with different LCSTs displayed coil-globule transitions when complexed to plasmid DNA, leading to variations in molecular architecture as shown by changes in emission maxima of an environment-sensitive fluorophore attached to the PNIPAm chains. Gel retardation assays demonstrated differences in electrophoretic mobilities of polymer-DNA complexes with temperatures below and above polymer LCSTs. Atomic force micrographs showed changes in the structures of polymer-DNA complexes for a polymer undergoing a phase transition around body temperature but not for the polymer with LCST outside this range. Transfection experiments in C2C12 and COS-7 cells demonstrated that the highest expression of transgene occurred in an assay that involved a 'cold-shock' below polymer LCST during transfection.
  
  
  CONCLUSIONS
  Designed changes in thermoresponsive polycation vector configuration via temperature-induced phase transitions enhanced transgene expression. The results indicate that changes in molecular architecture induced by a carefully chosen stimulus during intracellular trafficking can be used to enhance gene delivery.
authors.affiliation: School of Pharmacy and Biomedical Sciences, and Institute of Biomedical and Biomolecular Science, University of Portsmouth, St. Michael's Building, White Swan Road, Portsmouth PO1 2DT, UK.
authors.forename: Matthieu D
authors.initials: MD
authors.lastname: Lavigne
authors.forename: Sivanand S
authors.initials: SS
authors.lastname: Pennadam
authors.forename: James
authors.initials: J
authors.lastname: Ellis
authors.forename: Laura L
authors.initials: LL
authors.lastname: Yates
authors.forename: Cameron
authors.initials: C
authors.lastname: Alexander
authors.forename: Dariusz C
authors.initials: DC
authors.lastname: Górecki
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000737:chemistry*; D051379:Mice; D011094:Polyethyleneimine / Q000737:chemistry; D011108:Polymers / Q000737:chemistry*; D013696:Temperature; D014162:Transfection
chemical_list: D000180:Acrylic Resins; D011108:Polymers; C052970:poly-N-isopropylacrylamide; D011094:Polyethyleneimine