Optimization of factors influencing the transfection efficiency of folate–PEG–folate-graft-polyethylenimine
Introduction
An improvement in the design of polymeric gene carriers is needed to overcome the several obstacles encountered, from the site of administration to cellular uptake and nuclear entry, before plasmid DNA can function as a gene. Although viruses are known to be extremely efficient in the delivery of foreign genes into cells and tissues [1], there is a growing need to develop biocompatible polymeric gene carriers, which would not elicit immune responses and toxic side-effects.
The incorporation of the hydrophilic polymer polyethylene glycol (PEG) into polymeric gene carriers has been shown to improve the biocompatibility of self-assembling polymer/DNA complexes. PEGylated polymers have been shown to stabilize cationic polymer/DNA complexes under physiological conditions, increase water solubility, and reduce toxicity [2], [3].
Certain extracellular membrane receptor proteins can be utilized for site-specific gene delivery. Cancerous cells divide rapidly and need folic acid for DNA synthesis. This morphological phenomenon is best noted by up-regulation of membrane folate binding protein expression for a subsequent increase in folate internalization [4], [5], [6]. Folic acid has been linked to drugs, proteins, liposomes, and cationic polymers for enhanced uptake by cancer cells [7], [8], [9], [10], [11].
Polyethylenimine (PEI) is a water-soluble polymer with primary (25%), secondary (50%), and tertiary (25%) amine groups [12]. When solutions containing PEI become more acidic, the primary, secondary, and tertiary amine groups begin to protonate [11], [13], [14]. PEI serves as a DNA condensing and endosomal disruption agent due to its positive charge at neutral pH and further positive charge generation as the pH decreases [15], [16]. PEI has been modified to be multifunctional for enhanced gene transfer efficiency and reduced cytotoxicity [11], [17], [18]. Previous reports show that, when there is no receptor-mediated internalization of polymer/pLuc complexes, PEGylation decreased the transfection efficiency of PEI [18].
A combination of extended circulation, water solubility, receptor targeting, endosomal disruption, and reduced toxicity are needed for enhanced gene delivery [11], [19], [20], [21], [22]. In the present study, we aimed at optimizing the different variables which have a direct influence on the gene transfer efficiencies of polymeric gene carriers. We synthesized and characterized folate–PEG–folate-graft-PEIs of various grafting ratios to optimize the factors influencing transfection efficiency in vitro.
Section snippets
Materials
Folic acid, ethidium bromide, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide (MTT), dicyclohexylcarbodiimide (DCC), polyethylenimine ∼25,000 Mw (PEI), and Sephadex G-15 were all purchased from Sigma (St. Louis, MO, USA). Polyethyleneglycol-bis-amine ∼3400 Mw (PEG) was purchased from Shearwater (Huntsville, AL, USA). Pyridine, dimethyl sulfoxide (DMSO), and glucose were purchased from Aldrich (Milwaukee, WI, USA).
Synthesis of polymers for modified gene carrier
A synthesis scheme for PEI conjugates was reported in a previous publication [11]. Qualitative ninhydrin assays indicated the formation of PEG-bis-folate after the first reaction step. This was supported by MALDI-TOF mass spectroscopy, and absorbance at 363 nm, where each FPF was determined to have two folate molecules, giving an average molecular weight of ∼4282. The absorbance profile ranging from 210 to 500 nm was identical for folic acid, FPF, FPF-2.3g-PEI, FPF-5.2g-PEI, FPF-9.2g-PEI, and
Discussion
Up-regulation of folate binding proteins on malignant tissues has been shown to allow enhanced gene delivery and expression via folate–lipid and folate–polymer gene carrier systems [4], [5], [7], [8], [9], [10], [11]. Cultured CT-26 colon adenocarcinoma and KB oral epidermoid cells should express the folate receptor and normal smooth muscle cells (SMC) should not [6]. In this study, CT-26 and KB cells were maintained in folate-free media, while the normal smooth muscle cells were maintained in
Acknowledgements
Our special thanks to Alex Zlotnikov for technical assistance and to Expression Genetics, Inc. for financial support.
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