Active targeting of RGD-conjugated bioreducible polymer for delivery of oncolytic adenovirus expressing shRNA against IL-8 mRNA - PubMed (original) (raw)

Active targeting of RGD-conjugated bioreducible polymer for delivery of oncolytic adenovirus expressing shRNA against IL-8 mRNA

Jaesung Kim et al. Biomaterials. 2011 Aug.

Abstract

Even though oncolytic adenovirus (Ad) has been highlighted in the field of cancer gene therapy, transductional targeting and immune privilege still remain difficult challenges. The recent reports have noted the increasing tendency of adenoviral surface shielding with polymer to overcome the limits of its practical application. We previously reported the potential of the biodegradable polymer, poly(CBA-DAH) (CD) as a promising candidate for efficient gene delivery. To endow the selective-targeting moiety of tumor vasculature to CD, cRGDfC well-known as a ligand for cell-surface integrins on tumor endothelium was conjugated to CD using hetero-bifunctional cross-linker SM (PEG)(n). The cytopathic effects of oncolytic Ad coated with the polymers were much more enhanced dose-dependently when compared with that of naked Ad in cancer cells selectively. Above all, the most potent oncolytic effect was assessed with the treatment of Ad/CD-PEG(500)-RGD in all cancer cells. The enhanced cytopathic effect of Ad/RGD-conjugated polymer was specifically inhibited by blocking antibodies to integrins, but not by blocking antibody to CAR. HT1080 cells treated with Ad/CD-PEG(500)-RGD showed strong induction of apoptosis and suppression of IL-8 and VEGF expression as well. These results suggest that RGD-conjugated bioreducible polymer might be used to deliver oncolytic Ad safely and efficiently for tumor therapy.

Copyright © 2011 Elsevier Ltd. All rights reserved.

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Figures

Fig. 1

Fig. 1

The expression of CAR or integrins (αvβ3 and αvβ5) on the surface of human cancer cells (A549, HT1080 or MCF7). The status of CAR or integrins on each cell type was determined using flow cytometry. Cells were stained with ab9891 (CAR), MAB1976 (αvβ3) or MAB1961 (αvβ5) Ab followed by treatment of secondary Ab, goat anti-mouse IgG Alexa 647. (A) Flow cytometry of CAR expression level on the cell types. The solid line histograms represent anti CAR labeling. (B) Flow cytometry of integrins (αvβ3 and αvβ5) expression levels on the cells. The broken line histograms represent anti-αvβ3 integrin labeling and the solid line histograms represent anti-αvβ5 integrin labeling. Each filled histogram represents only secondary Ab labeling as a negative control.

Fig. 2

Fig. 2

Synthetic scheme of RGD-conjugated bioreducible polymer. Cyclic RGDfC (cRGDfC) was conjugated to poly(CBA-DAH) (CD) using hetero-bifunctional cross linker, NHS-PEGm-Maleimide. The PEGm represents PEG500 or PEG2000 with different size.

Fig. 3

Fig. 3

The cytopathic effect of naked Ad, Ad coated with CD (Ad/CD), CD-PEG500-RGD (Ad/CD-PEG500-RGD) or CD-PEG2000-RGD (Ad/CD-PEG2000-RGD) in human cancer (A549, HT1080 or MCF7) (A) or normal (HDF or BJ) (B) cells. Each cell type was treated with naked Ad, Ad/CD, Ad/CD-PEG500-RGD or Ad/CD-PEG2000-RGD at an MOI of 20 (A549), 50 (HDF or BJ), 100 (HT1080) or 200 (MCF7) with corresponding polymer molecules (3.1, 6.3, 12.5, 25, 50 or 100 × 104 polymer molecules per Ad particle). Cell viability was measured by MTT assay.

Fig. 4

Fig. 4

Competition assay using CAR Ab or integrin (αvβ3 and αvβ5) Abs. After HT1080 cells were incubated with CAR Ab or integrin Abs at 4°C for 1 h, Naked Ad, Ad/CD, Ad/CD-PEG500-RGD or Ad/CD-PEG2000-RGD was added to the medium and incubated at 37°C for 1 h. Each cytopathic effect was measured by MTT assay.

Fig. 5

Fig. 5

Exclusive killing effect of oncolytic Ad coated with CD-PEG500-RGD depending on RGD peptides by competition assay. HT1080 cells expressing high levels of integrins were treated with Ad/CD, Ad/CD-PEG500 or Ad/CD-PEG500-RGD at an MOI of 100 (2.5 × 105 polymer molecules per Ad particle). After incubation for 2 days, cell viability was measured by MTT assay.

Fig. 6

Fig. 6

Induction of apoptosis by Ad/polymers in HT1080 cells. The cells treated with naked Ad, Ad/CD, Ad/CD-PEG500-RGD or Ad/CD-PEG2000-RGD at an MOI of 50 (2.5 × 105 polymer molecules per Ad particle) were analyzed by flow cytometric analysis of Annexin V-FITC binding and PI uptake. The cells treated with 1 mM of CoCl2 for 18 hrs were served as an experimental positive control.

Fig. 7

Fig. 7

Down-regulation of IL-8 or VEGF expression by oncolytic Ad coated with each polymer in human cancer or normal cells. Human cancer (HT1080 or MCF7) or normal (HDF) cell type was treated with naked Ad, Ad/CD, Ad/CD-PEG500-RGD or Ad/CD-PEG2000-RGD at an MOI of 100, 200 or 50 (2.5 × 105 polymer molecules per Ad particle), respectively. After incubation for 2 (HT1080 and MCF7) or 4 (HDF) days, each conditioned medium was measured the expression of IL-8 (A) or VEGF (B) by human IL-8 or VEGF ELISA assay kit, respectively. Only polymer-treated cells were utilized as negative experimental groups to check the effect of each polymer itself on the expression levels of IL-8 or VEGF.

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