Dynamics of nucleic acid/cationic polymer complexation and disassembly under biologically simulated conditions using in situ atomic force microscopy.
Elucidating dynamic morphological changes of gene-carrying vectors and their nucleic acid release under varying intracellular conditions has been a technical challenge. Atomic force microscopy (AFM) was used to observe nucleic acid/polymer polyplexes under endosomal and reducible cytosolic conditions. Both ketalized (acid-degradable) and unmodified (nondegradable) polyethylenimine (PEI) in linear and branched forms were used to prepare plasmid DNA- or siRNA-complexing polyplexes. Then, the polyplexes' complexation and disassembly were observed by in situ AFM in various differentially changing buffers that represent intracellular conditions. Results demonstrated obvious morphological destruction of DNA/ketalized linear PEI (KL-PEI) polyplexes under mildly acidic endosomal conditions, whereas no morphological changes were observed by DNA/ketalized branched PEI (KB-PEI) under the same conditions. In addition, siRNA was more efficiently dissociated from KL-PEI than KB-PEI under the same conditions. Nondegradable PEI did not show any evidence that DNA or siRNA was released. Anionic biomacromolecules (e.g., heparan sulfate), which was hypothesized to dissociate nucleic acids from cationic polymers, did not successfully disassemble polyplexes but appeared to be adsorbed on cationic polymers. The in situ AFM results combined with in vitro cellular transfection and gene silencing indicated that efficient endosomal escape of plasmid DNA in a compact polyplex form is required for efficient gene expression. On the contrary, rapid dissociation of siRNA from its cationic carrier is crucial for efficient gene silencing. The findings of this study may provide new insightful information for designing stimuli-responsive nonviral gene vectors as well as expanding tools for investigating nonviral vectors in nanoscales under biologically inspired conditions.
Shim
Min Suk
MS
Department of Chemical Engineering and Materials Science, University of California, Irvine, California 92697, USA.
Wang
Xi
X
Ragan
Regina
R
Kwon
Young Jik
YJ
D000818:Animals; D051379:Mice; D018625:Microscopy, Atomic Force; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics; D011094:Polyethyleneimine / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000235:genetics; D014162:Transfection / Q000295:instrumentation*
D011108:Polymers; D034741:RNA, Small Interfering; D011094:Polyethyleneimine
Micron. 2002;33(4):385-97
11814877
J Control Release. 2002 May 17;81(1-2):201-17
11992692
Mol Ther. 2005 Mar;11(3):336-47
15727930
J Gene Med. 2007 Jun;9(6):479-87
17410614
J Virol. 2003 Jun;77(11):6332-40
12743290
Proc Natl Acad Sci U S A. 2007 Oct 9;104(41):16050-5
17913877
Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7297-301
7638184
Science. 2003 Nov 7;302(5647):1002-5
14605360
J Control Release. 2009 Feb 10;133(3):206-13
18992289
Biotechnol Bioeng. 2000 Mar 5;67(5):598-606
10649234
Nucleic Acids Res. 2001 Sep 15;29(18):3882-91
11557821
Biomacromolecules. 2008 Feb;9(2):444-55
18186606
J Pharm Sci. 2003 Feb;92(2):203-17
12532370
Bioconjug Chem. 2009 Jan;20(1):5-14
19053311
Nucleic Acids Res. 1997 Aug 1;25(15):3095-101
9224610
J Biol Chem. 2003 Aug 29;278(35):32578-86
12807905
Gene Ther. 1997 Aug;4(8):823-32
9338011
Biotech Histochem. 2006 Mar-Jun;81(2-3):87-97
16908433
Gene Ther. 2005 Dec;12(24):1734-51
16079885
Curr Opin Biotechnol. 1996 Feb 1;7(1):78-84
8791307
Biotechnol Bioeng. 2001 Feb 5;72(3):331-8
11135203
Langmuir. 2005 Apr 12;21(8):3591-8
15807606
Mol Ther. 2000 Oct;2(4):302-17
11020345
Bioconjug Chem. 2006 Jan-Feb;17(1):101-8
16417257
J Biol Chem. 1998 Mar 27;273(13):7507-11
9516451
J Gene Med. 2004 Jan;6(1):76-84
14716679
Bioconjug Chem. 2009 Mar 18;20(3):488-99
19199781
Adv Drug Deliv Rev. 2001 Dec 31;53(3):341-58
11744176
Nucleic Acids Res. 2005 May 24;33(9):e86
15914665
Bioconjug Chem. 1999 Nov-Dec;10(6):993-1004
10563768
Biomacromolecules. 2004 Jan-Feb;5(1):32-9
14715005