Structural study of DNA condensation induced by novel phosphorylcholine-based copolymers for gene delivery and relevance to DNA protection.
Poly[2-(dimethylamino)ethyl methacrylate-b-2-methacryloyloxyethyl phosphorylcholine] (DMA-MPC) is currently under investigation as a new vector candidate for gene therapy. The DMA block has been previously demonstrated to condense DNA effectively. The MPC block contains a phosphorylcholine (PC) headgroup, which can be found naturally in the outside of the cell membrane. This PC-based polymer is extremely hydrophilic and acts as a biocompatible steric stabilizer. In this study, we assess in detail the morphologies of DNA complexes obtained using the diblock copolymer series DMA(x)MPC30 (where the mean degree of polymerization of the MPC block was fixed at 30 and the DMA block length was systematically varied) using transmission electron microscopy (TEM) and liquid atomic force microscopy (AFM). Both techniques indicate more compact complex morphologies (more efficient condensation) as the length of the cationic DMA block increases. However, the detailed morphologies of the DMA(x)MPC30-DNA complexes observed by TEM in vacuo and by AFM in aqueous medium are different. This phenomena is believed to be related to the highly hydrophilic nature of the MPC block. TEM studies revealed that the morphology of the complexes changes from loosely condensed structures to highly condensed rods, toroids, and oval-shaped particles as the DMA moiety increases. In contrast, morphological changes from plectonemic loops to flower-like and rectangular block-like structures, with an increase in highly condensed central regions, are observed by in situ AFM studies. The relative population of each structure is clearly dependent on the polymer molecular composition. Enzymatic degradation assays revealed that only the DMA homopolymer provided effective DNA protection against DNase I degradation, while other highly condensed copolymer complexes, as judged from TEM and gel electrophoresis, only partially protected the DNA. However, AFM images indicated that the same highly condensed complexes have less condensed regions, which we believe to be the initiation sites for enzymatic attack. This indicates that the open structures observed by AFM of the DNA complexation by the DMA(x)MPC30 copolymer series are closer to in vivo morphology when compared to TEM.
Chim
Y T A
YT
Laboratory of Biophysics and Surface Analysis, School of Pharmacy, The University of Nottingham, NG7 2RD, UK.
Lam
J K W
JK
Ma
Y
Y
Armes
S P
SP
Lewis
A L
AL
Roberts
C J
CJ
Stolnik
S
S
Tendler
S J B
SJ
Davies
M C
MC
D001704:Biopolymers / Q000737:chemistry / Q000494:pharmacology; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D004247:DNA / Q000008:administration & dosage* / Q000737:chemistry / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D008689:Methacrylates / Q000737:chemistry* / Q000494:pharmacology; D018625:Microscopy, Atomic Force; D046529:Microscopy, Electron, Transmission; D009690:Nucleic Acid Conformation; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*
D001704:Biopolymers; D002412:Cations; D008689:Methacrylates; C512256:poly(2-(dimethylamino)ethyl methacrylate-b-2-methacryloyloxyethyl phosphorylcholine); D010767:Phosphorylcholine; D004247:DNA; D003850:Deoxyribonuclease I