New polyphosphoramidate with a spermidine side chain as a gene carrier.
A new cationic polymer (PPA-SP), polyphosphoramidate bearing spermidine side chain, was prepared as a non-viral vector for gene delivery. PPA-SP was synthesized from poly(1,2-propylene H-phosphonate) by the Atherton-Todd reaction. The weight average molecular weight of PPA-SP was 3.44x10(4) with a number average degree of polymerization of 90, as determined by GPC/LS/RI method. The average net positive charge per polymer chain was 102. PPA-SP was able to condense plasmid DNA efficiently and formed complexes at an N/P ratio (free amino groups in polymer to phosphate groups in DNA) of 2 and above, as determined by agarose gel electrophoresis. This new gene carrier offered significant protection to DNA against nuclease degradation at N/P ratios above 2, and showed lower cytotoxicity than PLL and PEI in cell culture. The LD(50) of PPA-SP was 85 microg/ml in COS-7 cells, in contrast to 20 and 42 microg/ml for PLL and PEI, respectively. The complexes prepared in saline at N/P ratios of 5 approximately 10 had an average size of 250 nm and zeta-potential of 26 mV. PPA-SP mediated efficient gene transfection in a number of cell lines, and the transfection protocol was optimized in HEK293 cells using a luciferase plasmid as a marker gene. Gene expression mediated by PPA-SP was greatly enhanced when chloroquine was used in conjunction at a concentration of 100 microM. Under the optimized condition, PPA-SP/DNA complexes yield a luciferase expression level closed to PEI/DNA complexes or Transfast mediated transfection. In a non-invasive CNS gene delivery model, PPA-SP/DNA complexes yielded comparable bcl-2 expression as PEI/DNA complexes in mouse brain stem following injection of the complexes in the tongue.
Wang
Jun
J
Johns Hopkins Singapore Biomedical Centre, Tissue and Therapeutic Engineering Laboratory, 10 Medical Drive, Singapore 117597, Singapore.
Zhang
Peng-Chi
PC
Lu
Hong-Fang
HF
Ma
Nan
N
Wang
Shu
S
Mao
Hai-Quan
HQ
Leong
Kam W
KW
D000818:Animals; D019556:COS Cells / Q000187:drug effects / Q000378:metabolism; D002522:Chlorocebus aethiops; D004337:Drug Carriers / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011108:Polymers / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics; D013095:Spermidine / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D004337:Drug Carriers; D011108:Polymers; D013095:Spermidine