Folate-PEG-folate-graft-polyethylenimine-based gene delivery.
Folate-polyethylene glycol-folate-grafted-polyethylenimine (FPF-g-PEI) was synthesized by linking folic acid to both ends of a mono-functional PEG and then grafting to PEI. The graft ratio was determined using Beer's law by measuring the UV absorbance at 363 nm. The pH profile, diameter and shape of the carriers were determined. Transfection efficiencies were optimized in normal smooth muscle cells (SMC) and CT-26 colon adenocarcinoma cells using plasmid DNA encoding luciferase reporter gene. Free folic acid was shown to inhibit transfection with FPF-2.3 g-PEI at neutral charge ratio. Relative toxicity between PEI and the modified carrier was measured using MTT colorimetric assay. Therapeutic potential of pmIFN-gamma complexed with these polymeric carriers in terms of gene expression was determined at protein and mRNA levels using ELISA and RT-PCR. FPF-g-PEI was determined to have 2.3 folate-PEG-folate (FPF) linear polymers grafted to each PEI molecule. The average molecular weight was measured to be approximately 33,500 Mw and the pH profile was characteristic of endosomal disruption capacity. Atomic Force Microscopy (AFM) and Dynamic Laser Light Scattering (DLLS) indicated FPF-2.3 g-PEI and PEI (at 2 w/w ratio) efficiently condensed plasmid DNA resulting in oblique spheroid polyplexes with a mean diameter of approximately 150 nm. FPF-2.3 g-PEI was superior to PEI in terms of cytotoxicity and transfection efficiency in cancer cells. Smooth muscle cells showed no specificity for folate tethered complexes, where PEI/pLuc complexes yielded higher efficiencies.
Benns
J M
JM
Center for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, 30 South 2000 East, RM 201, Salt Lake City, UT 84112-5820, USA.
Maheshwari
A
A
Furgeson
D Y
DY
Mahato
R I
RI
Kim
S W
SW
D002352:Carrier Proteins / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D016503:Drug Delivery Systems; D011992:Endosomes / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000737:chemistry*; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007371:Interferon-gamma / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D018625:Microscopy, Atomic Force; D010316:Particle Size; D010957:Plasmids / Q000235:genetics; D011092:Polyethylene Glycols / Q000737:chemistry*; D011094:Polyethyleneimine / Q000737:chemistry*; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D007371:Interferon-gamma; D011094:Polyethyleneimine; D005492:Folic Acid; D008156:Luciferases