Different Strategies for Formation of PEGylated EGF-Conjugated PEI/DNA Complexes for Targeted Gene Delivery

Blessing, Thomas; Kursa, Malgorzata; Holzhauser, Robert; Kircheis, Ralf; Wagner, Ernst

doi:10.1021/bc0001488

Cited by 235 publications

(175 citation statements)

References 52 publications

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“…(Peng et al, 2001;Wolschek et al, 2002;Ogris et al, 2003;Pereboeva et al, 2007). In our own work, we have most often used murine EGF, which, in contrast to human EGF, does not carry any lysines in its primary amino acid sequence (Blessing et al, 2001;Wolschek et al, 2002;Ogris et al, 2003). Hence, convenient coupling via the N-terminal primary amine to heterobifunctional cross-linkers, such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), can occur without interfering with the binding domain of EGF.…”

Section: Discussionmentioning

confidence: 99%

Disconnecting the Yin and Yang Relation of Epidermal Growth Factor Receptor (EGFR)-Mediated Delivery: A Fully Synthetic, EGFR-Targeted Gene Transfer System Avoiding Receptor Activation

et al. 2011

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The epidermal growth factor receptor (EGFR) is upregulated within a high percentage of solid tumors and hence is an attractive target for tumor-targeted therapies including gene therapy. The natural EGFR ligand epidermal growth factor (EGF) has been used for this purpose, despite the risk of mitogenic effects due to EGFR activation. We have developed a fully synthetic, EGFR-targeted gene delivery system based on PEGylated linear polyethylenimine (LPEI), allowing evaluation of different EGFR-binding peptides in terms of transfection efficiency and EGFR activation. Peptide sequences directly derived from the human EGF molecule enhanced transfection efficiency with concomitant EGFR activation. Only the EGFR-binding peptide GE11, which has been identified by phage display technique, showed specific enhancement of transfection on EGFR-overexpressing tumor cells including glioblastoma and hepatoma, but without EGFR activation. EGFR targeting led to high levels of cell association of fluorescently labeled polyplexes after only 30 min of incubation. EGF pretreatment of cells induced enhanced cellular internalization of all polyplex types tested, pointing at generally enhanced macropinocytosis. EGF polyplexes diminished cell surface expression of EGFR for up to 4 hr, whereas GE11 polyplexes did not. In a clinically relevant orthotopic prostate cancer model, intratumorally injected GE11 polyplexes were superior in inducing transgene expression when compared with untargeted polyplexes.

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Section: Discussionmentioning

confidence: 99%

Disconnecting the Yin and Yang Relation of Epidermal Growth Factor Receptor (EGFR)-Mediated Delivery: A Fully Synthetic, EGFR-Targeted Gene Transfer System Avoiding Receptor Activation

et al. 2011

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“…27 However, to date, most targeting sterically stabilized gene delivery vehicles have been studied in vitro. [3][4][5][6][7][8][9] As the ultimate goal of any potential gene therapy is delivery in vivo, a comparison study of TsPLP both in vitro and in vivo was undertaken to elucidate the influence of the PEGylation on gene delivery efficiency. PLP showed the lowest in vitro transfection activity (Figure 2), and this is likely due to the PEG molecule preventing association of the lipoplex with the target cell 2,12 and/or due to PEG inhibition of fusion between lipid and endosomal membrane, which is necessary for the intracellular release of exogenously delivered DNA.…”

Section: Discussionmentioning

confidence: 99%

“…In this study, postcoated TsPLP was prepared essentially according to a strategy described by Blessing et al 3 Briefly, DNA was mixed with liposomes composed of DOTAP and DOPE (1:1 molar ratio) to form a lipid/DNA complex (lipoplex). Subsequently, heterobifunctional PEG (NHS-PEG-MAL) was added to form a Mal-PEGlipoplex, in which a coupling reaction occurs between the NHS group of bifunctional PEG and the amine residue of DOPE (Figure 1).…”

Section: In Vitro Transfection Activitiesmentioning

confidence: 99%

“…2 The application of this modification (PEGylation) to nonviral gene delivery systems has drawn considerable interest. [3][4][5][6][7][8][9] Benefits of PEGylation have been described and include improvement of systemic circulation of lipoplexes or polyplexes; 1,10 improvement of the solubility of polyplexes thereby allowing the preparation of higher therapeutic concentrations without subsequent aggregation; 10,11 and reduction of in vivo toxicity of the polyplex. 10 However, disadvantages of PEGylation have also been observed.…”

Section: Introductionmentioning

confidence: 99%

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A sterically stabilized immunolipoplex for systemic administration of a therapeutic gene

Pirollo

Rait

et al. 2004

Gene Ther

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A sterically stabilized immunolipoplex (TsPLP), containing an antitransferrin receptor single-chain antibody fragment (TfRscFv)-PEG molecule, has been developed to specifically and efficiently deliver a therapeutic gene to tumor cells. A postcoating preparation strategy was employed in which a DNA/lipid complex (lipoplex) was formed first and then sequentially conjugated with PEG and TfRscFv. The complex prepared by this method was shown to be superior in ability to deliver genes to tumor cells than when prepared by a common precoating strategy, in which DNA is mixed with TfRscFv-PEG conjugated liposome. Using prostate cancer cell line DU145, a comparison was made between the in vitro and in vivo gene delivery efficiencies of four complexes, Lipoplex (LP), PEG-Lipoplex (PLP), TfRscFv-PEG-Lipoplex (TsPLP) and our standard TfRscFv-Lipoplex (TsLP). In vitro, the order of transfection efficiency was TsLP4LPETsPLP4PLP. However, in vivo the order of transfection efficiency, after systemic administration via the tail vein, was TsPLP4TsLP4LP or PLP with TsPLPmediated exogenous gene expression in tumor being twofold higher than when mediated by TsLP. This suggests that the in vitro transfection efficiency of TsPLP was not indicative of its in vivo efficiency. In addition, it was found that the level of exogenous gene expression in the tumor mediated by TsPLP was higher than that mediated by TsLP and did not decrease over the time. More importantly, high exogenous gene expression in tumor, but low expression in liver, was observed after an i.v. delivery of TsPLP carrying either the GFP reporter gene or the p53 gene, indicating that tumor preferential targeting was maintained by this complex in the presence of PEG. These findings show that incorporation of PEG into our targeted lipoplex results in a more efficient delivery of the complex to the tumor cells, possibly by inhibiting the first pass clearance observed with non-PEG containing liposomes. Therefore, these data demonstrate that TsPLP is a improvement over our previously established tumor targeted gene delivery complex for systemic gene therapy of cancer.

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“…Intravenous injection of a PEGylated polyplex would result in passive accumulation of the polyplex in a tumor. However, to advance the beneficial effects of gene therapies, researchers have begun to target polycations in a noninvasive but directed manner 7,8 by complexing polycations with antibodies, vitamins, glycolipids, and receptor ligands, like transferrin (Tf).…”

Section: Introductionmentioning

confidence: 99%

Optical imaging of transferrin targeted PEI/DNA complexes in living subjects

Hildebrandt¹,

Iyer²,

Wagner

et al. 2003

Gene Ther

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Noninvasive optical bioluminescence imaging systems are important tools for evaluating gene expression in vivo for study of individual and temporal variation in a living animal. In this report, we demonstrate that expression of the firefly luciferase reporter gene (fl) delivered by transferrin (Tf) targeted polyethylenimine (PEI) complexes with, or without, poly(ethylene glycol) (PEG) modifications can be imaged in living A/J mice bearing N2A tumors using a cooled charged coupled device (CCD) camera. Tf-PEI-PEG, Tf-PEI, and PEI (positive control) complexes were tail-vein injected and mice were imaged at 5, 24, 48, and 72 h after complex injection. After imaging, the organs were analyzed ex vivo for firefly luciferase protein (FL) activity. The Tf and PEG modified formulations show significantly (Po0.05) higher FL activity in vivo and ex vivo at the tumor as compared to other organs, including the lungs (a site of high expression with PEI, the positive control). Furthermore, the in vivo bioluminescent signal correlated well (R 2 ¼0.83) with ex vivo FL activity. These data support that noninvasive imaging of fl reporter expression can be used to monitor the specificity of Tf-PEI and Tf-PEI-PEG polyplex targeting of N2A tumors in A/J mice.

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Different Strategies for Formation of PEGylated EGF-Conjugated PEI/DNA Complexes for Targeted Gene Delivery

Cited by 235 publications

References 52 publications

Disconnecting the Yin and Yang Relation of Epidermal Growth Factor Receptor (EGFR)-Mediated Delivery: A Fully Synthetic, EGFR-Targeted Gene Transfer System Avoiding Receptor Activation

Disconnecting the Yin and Yang Relation of Epidermal Growth Factor Receptor (EGFR)-Mediated Delivery: A Fully Synthetic, EGFR-Targeted Gene Transfer System Avoiding Receptor Activation

A sterically stabilized immunolipoplex for systemic administration of a therapeutic gene

Optical imaging of transferrin targeted PEI/DNA complexes in living subjects

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