Folate receptor-mediated intracellular delivery of recombinant caspase-3 for inducing apoptosis.

Recombinant reversed caspase-3 (rev-caspase-3) is a pro-apoptotic gene capable of intracellular autocatalytic processing, which leads to programmed cell death. Folate receptor-specific intracellular delivery of the rev-caspase-3 gene into KB cells over-expressing folate receptors was explored by employing the folate-poly(ethylene glycol)-polyethylenimine (FOL-PEG-PEI) conjugate as a nonviral polymeric carrier. Using luciferase as a reporter gene, the conditions for formulation of DNA/polymer polyplexes were pre-optimized to attain the highest folate receptor-mediated gene transfection efficiency. FOL-PEG-PEI conjugate complexed with rev-caspase-3 plasmid in an optimized condition gave rise to a great increase in expression and activation of exogenous rev-caspase-3 in KB cells when pretreated with doxorubicin. The synthesized conjugate exhibited higher transfection efficiency than other commercially available transfection agents due to a unique mechanism of folate-receptor mediated endocytic gene transfer. The transfected cells showed a significant extent of apoptosis by rev-caspase-3. This study suggests the potential of using folate-receptor-mediated delivery of rev-caspase-3 gene for inducing tumor cell death in a target-specific manner.

Target-specific gene silencing by siRNA plasmid DNA complexed with folate-modified poly(ethylenimine).

A target-specific delivery system of green fluorescent protein (GFP) small interfering RNA (siRNA) plasmid DNA was developed by using folate-modified cationic polyethylenimine (PEI). A GFP siRNA plasmid vector (pSUPER-siGFP), which inhibits the synthesis of GFP, was constructed and used for suppressing GFP expression in folate receptor over-expressing cells (KB cells) in a target-specific manner. A PEI-poly(ethylene glycol)-folate (PEI-PEG-FOL) conjugate was synthesized as a pSUPER-siGFP plasmid gene carrier. KB cells expressing GFP were treated with various formulations of pSUPER-siGFP/PEI-PEG-FOL complexes to inhibit expression of GFP. The formulated complexes were characterized under various conditions. Their GFP gene inhibition and cellular uptake behaviors were explored by confocal microscopy and flow cytometry analysis. pSUPER-siGFP/PEI-PEG-FOL complexes inhibited GFP expression of KB cells more effectively than pSUPER-siGFP/PEI complexes with no folate moieties and showed far reduced extent of inhibition for folate receptor deficient cells (A549 cells). The results indicated that folate receptor-mediated endocytosis was a major pathway in the process of cellular uptake, suggesting that targeted delivery of siRNA vector could be achieved to a specific cell.

Folate receptor mediated intracellular protein delivery using PLL-PEG-FOL conjugate.

To develop a receptor-mediated intracellular delivery system that can transport therapeutic proteins or other bioactive macromolecules into a specific cell, a di-block copolymer conjugate, poly(L-lysine)-poly(ethylene glycol)-folate (PLL-PEG-FOL), was synthesized. The PLL-PEG-FOL conjugate was physically complexed with fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) in an aqueous phase by ionic interactions. Cellular uptake of PLL-PEG-FOL/FITC-BSA complexes was greatly enhanced against a folate receptor over-expressing cell line (KB cells) compared to a folate receptor deficient cell line (A549 cells). The presence of an excess amount of free folate (1 mM) in the medium inhibited the intracellular delivery of PLL-PEG-FOL/FITC-BSA complexes. This suggests that the enhanced cellular uptake of FITC-BSA by KB cells in a specific manner was attributed to folate receptor-mediated endocytosis of the complexes having folate moieties on the surface. The PLL-PEG-FOL di-block copolymer could be potentially applied for intracellular delivery of a wide range of other biological active agents that have negative charges on the surface.

Biodegradable nanoparticles containing protein-fatty acid complexes for oral delivery of salmon calcitonin.

Biodegradable nanoparticles containing salmon calcitonin (sCT) were formulated using protein-fatty acid complexes, and their in vitro transport against a Caco-2 cell monolayer and the extent of in vivo oral uptake were assessed. Positively charged sCT was hydrophobically ion paired to form physical complexes with fatty acid, phospholipid, and surfactant. Among the complexes, sodium oleate was used to form sCT-oleate complexes, which were characterized and formulated into biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles. Endocytosis of sCT nanoparticles by Caco-2 cells was studied by flow cytometry. Transcytosis of sCT across the Caco-2 monolayer was also quantitated by an ELISA method. The sCT nanoparticles were orally administered to Sprague-Dawley rats, and serum sCT was monitored. Biodegradable polymeric nanoparticles containing a loading amount of sCT as high as 2.7% (w/w) were prepared based on the complexation of sCT with sodium oleate. A greater amount of sCT nanoparticles could be delivered into Caco-2 cells compared with free sCT, and sCT could also be transported from the apical side to the basolateral side of the Caco-2 monolayer. In vivo experiments using a rat animal model showed the possibility of oral uptake of sCT. This study showed that physical complexation of sCT with amphiphilic molecules enabled the formation of sCT-loaded PLGA nanoparticles at a high loading efficiency and that sCT-PLGA nanoparticles were transported across the Caco-2 cell monolayer and were readily taken up in vivo following oral admistration.