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Identification of novel carrier peptides for the specific delivery of therapeutics into cancer cells ¹

Institute for Cancer Research, Department of Immunology, Molecular Medicine Group, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway

²Correspondence: Institute for Cancer Research, Department of Immunology, Molecular Medicine Group, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway. E-mail: mosioud{at}ulrik.uio.no

SPECIFIC AIMS

Selective delivery of nucleic acids into pathogenic cells would improve their efficacy and minimize potential adverse side effects. In contrast to larger molecules (e.g., antibodies), small peptides would represent more appropriate delivery vehicles because of their excellent tissue penetration, easy synthesis and as well as conjugation to drugs and short oligonucleotides. We established a cell selection method in which peptides that are internalized by breast cancer cell line SKBR3 are selected after incubation with random peptide phage libraries. We explored the potential of the selected peptides to specifically deliver antisense oligonucleotides to cancer cells.

PRINCIPAL FINDINGS

1. Selection of cancer cell-specific binding peptides
The display of peptides on the surface of a bacteriophage provides an opportunity to identify peptides with desired binding specificity. The breast cancer cell line SKBR3 was used as an affinity matrix to isolate phages from 7-mer and 12-mer random peptide phage libraries. To eliminate phages with binding capacity to common receptors, the libraries were preabsorbed on human mammary epithelial and peripheral blood mononuclear cells prior to biopanning on subconfluent SKBR3 cells. To enrich for internalized phages, membrane-bound phages were eliminated by acid and trypsin treatments. After five or six rounds of selection, internalized phages were rescued, amplified, and random phage clones were analyzed for binding to SKBR3 by flow cytometry. The peptide sequences displayed by the positive phages were determined (Fig. 1 A, B). The sequences define a major core motif (LTVXPWY), which was not found in negative phages. None of the selected peptides bound to human primary cells from different tissue origin (e.g., epithelial, endothelial, hematopoietic).

View larger version (30K): [in this window] [in a new window]   Figure 1. Identification of cell-specific targeting peptides. Amino acid sequences of the peptides displayed by positive phages selected from the first (A) and second (B) sets of biopanning. Conserved residues are indicated by gray letters. C) Analysis of the phage uptake with an epifluorescence microscope (upper panel). Cell nuclei were counterstained with Hoechst (lover panel). D) Fluorescence images of SKBR3 cells incubated with fluorescein-labeled P7–2 peptide or fluorescein-labeled random peptide.

2. Internalization of the selected peptides by SKBR3
A phage displaying the LTVSPWY peptide sequence was chosen for further analysis. The binding of this phage to SKBR3 cells was abrogated by pretreatment with the synthetic peptide, indicating that binding of the phage is mediated by its displayed peptide. Uptake of the phage displaying the LTVSPWY peptide sequence (Ph7–2) by SKBR3 cells was evaluated by immunofluorescence. Figure 1C (upper panel) shows immunofluorescence images of SKBR3 cells after incubation with the Ph7–2 phage or with nonbinder phage (Ph-34). To visualize cell nuclei, cells were counterstained with Hoechst 33342 (lower panel). In contrast to the Ph-34 phage, the Ph7–2 phage showed strong cell staining. To further substantiate that the binding is directed by the displayed peptide, we analyzed the binding of 6-iodoacetamidofluorescein (6-IAF)-conjugated LTVSPWY peptide. Strong binding and cellular internalization were obtained (Fig. 1D ). No significant binding was observed when SKBR3 cells were incubated with a 6-IAF-conjugated random peptide. Taken together, the data suggest that these experimental conditions are favorable for enrichment of internalized peptides.

3. Peptide-mediated antisense uptake by SKBR3 cells
To determine whether the cancer cell binding peptide LTVSPWY could be used to enhance the delivery of antisense oligonucleotides, a fluorescein-conjugated antisense oligonucleotide against the ErbB2 receptor was designed and coupled via a disulfide bridge to the LTVSPWY peptide. SKBR3 cells incubated with the antisense-peptide conjugates for 4 h at 37°C showed strong intracellular staining, whereas no significant staining was obtained with the antisense alone (Fig. 2 A, B). Both molecules were tested at 0.5 µM final concentration.

View larger version (40K): [in this window] [in a new window]   Figure 2. The LTVSPWY peptide mediated antisense internalization. SKBR3 cells growing on chamber slides at 37°C were incubated with either fluorescein antisense or fluorescein antisense peptide conjugates for 4 h at 37°C, then visualized with an epifluorescence microscope (A, B). ErbB2 and ß-actin protein expression in SKBR3 cells treated with various test molecules (0.5 µM) for 72 h (C, D). SP = scrambled sequence-peptide conjugates; AP7–2 = antisense-peptide conjugates, P7–2 = LTVSPWY peptide, A = antisense alone, C = untreated and AMP7–2 = 2' O-methyl antisense peptide conjugates.

4. Inhibition of ErbB2 gene expression by the antisense-peptide conjugates
To assess the effects of the antisense-peptide conjugates on ErbB2 gene expression, SKBR3 cells were treated with test molecules (0.5 µM) for 72 h, then ErbB2 protein levels were determined. Addition of antisense peptide (AP7–2) conjugates to the cells resulted in 60% inhibition (Fig. 2C ). In contrast, neither the peptide (P7–2) nor the antisense alone (A) exhibited a significant inhibitory effect. A scrambled control sequence peptide conjugate (SP) was ineffective. Western blot analysis of the ErbB2 gene expression revealed no major difference between control and cells treated with 2' O-methyl-modified antisense-peptide conjugates (AMP7–2), thus indicating that the antisense-peptide conjugates function in living cells mainly via the activation of the endogenous RNase H. The observed inhibition effect is specific since ß-actin protein levels were not affected by the treatment.

CONCLUSION

Peptides identified by screening the phage libraries on breast cancer cell line SKBR3 have proved useful for delivering antisense oligonucleotides to cancer cells. Inhibition of the ErbB2 gene expression by the antisense-peptide conjugate indicates that the complex does function in cells. The selected peptides bind mainly to breast cancer cells and can potentially be used to target these cells.

View larger version (34K): [in this window] [in a new window]   Figure 3. Schematic representation of the experimental design and findings.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0280fje; to cite this article, use FASEB J. (December 17, 2002) 10.1096/fj.02-0280fje

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