Incorporation of reversibly cross-linked polyplexes into LPDII vectors for gene delivery.
LPDII vectors are synthetic vehicles for gene delivery composed of polycation-condensed DNA complexed with anionic liposomes. In this study, we evaluated the stability and transfection properties of polyethylenimine (PEI, 25 kDa)/DNA polyplexes before and after covalent cross-linking with dithiobis(succinimidylpropionate) (DSP) or dimethyl x 3,3'-dithiobispropionimidate x 2HCl (DTBP), either alone or as a component of LPDII vectors. We found that cross-linking PEI/DNA polyplexes at molar ratios > or =10:1 (DSP or DTBP:PEI) stabilized these complexes against polyanion disruption, and that this effect was reversible by reduction with 20 mM dithioerythritol (DTE). Transfection studies with polyplexes cross-linked at molar ratios of 10:1-100:1 in KB cells, a folate receptor-positive oral carcinoma cell line, showed decreasing luciferase gene expression with increasing cross-linking ratio. Subsequently, polyplexes, cross-linked with DSP at a molar ratio of 10:1, were combined with anionic liposomes composed of diolein/cholesteryl hemisuccinate (CHEMS) (6:4 mol/mol), diolein/CHEMS/poly(ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE) (6:4:0.05 mol/mol), or diolein/CHEMS/folate-PEG-cholesterol (folate-PEG-Chol) (6:4:0.05 mol/mol) for LPDII formation. Transfection studies in KB cells showed that LPDII vectors containing cross-linked polyplexes mediated approximately 2-15-fold lower gene expression than LPDII prepared with un-cross-linked polyplexes, depending on the lipid:DNA ratio. Inclusion of PEG-DSPE at 0.5 mol % appeared to further decrease transfection levels approximately 2-5-fold. Compared with LPDII formulated with PEG-DSPE, LPDII incorporating 0.5 mol % folate-PEG-Chol exhibited higher luciferase activities at all lipid:DNA ratios tested, achieving an approximately 10-fold increase at a lipid:DNA ratio of 5. Compared with cross-linked LPDII vectors without PEG-DSPE, inclusion of folate-PEG-Chol increased luciferase activities 3-4-fold between lipid:DNA ratios of 1 and 5. Interestingly, inclusion of 1 mM free folate in the growth media during transfection increased transfection activity approximately 3-4-fold for cross-linked LPDII vectors and LPDII containing folate-PEG-Chol, but had no effect on the transfection activity of LPDII formulated with PEG-DSPE. However, in the presence of 5 mM free folate, the luciferase activity mediated by LPDII vectors containing folate-PEG-Chol was reduced approximately 6-fold. Transmission electron micrographs were also obtained to provide evidence of LPDII complex formation. Results showed that cross-linked LPDII vectors appear as roughly spherical aggregated complexes with a rather broad size distribution ranging between 300 and 800 nm.
Gosselin
Michael A
MA
Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, USA.
Guo
Wenjin
W
Lee
Robert J
RJ
D002352:Carrier Proteins / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004075:Diglycerides; D004355:Drug Stability; D058973:Folate Receptors, GPI-Anchored; D006801:Humans; D007624:KB Cells; D008024:Ligands; D008081:Liposomes; D010316:Particle Size; D010957:Plasmids / Q000008:administration & dosage / Q000493:pharmacokinetics; D011073:Polyamines / Q000138:chemical synthesis / Q000493:pharmacokinetics*; D000071228:Polyelectrolytes; D011094:Polyethyleneimine / Q000737:chemistry; D011956:Receptors, Cell Surface; D014162:Transfection / Q000379:methods* / Q000592:standards; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D004075:Diglycerides; D058973:Folate Receptors, GPI-Anchored; D008024:Ligands; D008081:Liposomes; D011073:Polyamines; D000071228:Polyelectrolytes; D011094:Polyethyleneimine; D011956:Receptors, Cell Surface; C013965:diolein