Optimization of factors influencing the transfection efficiency of folate–PEG–folate-graft-polyethylenimine

doi:10.1016/S0168-3659(01)00513-2

Journal of Controlled Release

Volume 79, Issues 1–3, 19 February 2002, Pages 255-269

https://doi.org/10.1016/S0168-3659(01)00513-2 Get rights and content

Abstract

Folate–poly(ethylene glycol)–folate-grafted-polyethylenimine (FPF-g-PEI) was synthesized over a range of grafting ratios of folate–poly(ethylene glycol)–folate (FPF) to polyethylenimine (PEI). The conjugation was determined using the absorbance at 363 nm for each polymer. FPF-g-PEIs were determined to have 2.3, 5.2, 9.3 and 20 FPF linear polymers grafted to each PEI. The average molecular weight was calculated to be ∼34,848, 47,266, 64,823 and 110,640 Da, respectively. The pH profiles of FPF-g-PEIs suggest that the polymers have endosomal disruption capacity, and the gel electrophoretic band retardation showed efficient condensation of DNA. The transfection efficiency, determined using plasmid encoding luciferase, was dependent on the cell type and was different for CT-26 colon adenocarcinoma, KB oral epidermoid, and normal smooth muscle cells (SMC). The relative toxicity of polymer/plasmid complexes was determined using the MTT colorimetric assay. At neutral charge ratio, FPF-g-PEI/pLuc complexes were less toxic to cells and showed higher transfection in cancer cells compared to PEI/pLuc complexes. Smooth muscle cells showed no specificity for FPF-g-PEI/pLuc complexes, whereas PEI/pLuc complexes showed a higher transfection efficiency. The transfection efficiency increased when neutral polymer/DNA complex concentrations increased, but decreased when positively charged polymer/DNA complex concentrations increased. There was little increase in toxicity when FPF-5.2g-PEI/pLuc complex concentrations increased.

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 M_w (PEI), and Sephadex G-15 were all purchased from Sigma (St. Louis, MO, USA). Polyethyleneglycol-bis-amine ∼3400 M_w (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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Optimization of factors influencing the transfection efficiency of folate–PEG–folate-graft-polyethylenimine

Abstract

Introduction

Section snippets

Materials

Synthesis of polymers for modified gene carrier

Discussion

Acknowledgements

Biochim. Biophys. Acta

J. Controlled Release

Arch. Biochem. Biophys.

J. Biol. Chem.

Mol. Ther.

Viral vectors in gene therapy

Annu. Rev. Microbiol.

Folate-binding protein is a marker for ovarian cancer

Cancer Res.

Folate deficiency reduces the GPI-anchored folate-binding protein in rat renal tubules

Am. J. Physiol. Cell. Physiol.

Transfection of folate–polylysine DNA complexes: evidence for lysosomal delivery

Bioconjug. Chem.

Folate-mediated targeting of therapeutic and imaging agents to cancers

Crit. Rev. Ther. Drug Carrier Syst.

Folate copolymer-mediated transfection of cultured cells

Bioconjug. Chem.

A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity

Pharm. Res.