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Therapeutics, Targets, and Chemical Biology

Polymalic Acid–Based Nanobiopolymer Provides Efficient Systemic Breast Cancer Treatment by Inhibiting both HER2/neu Receptor Synthesis and Activity

Satoshi Inoue, Hui Ding, Jose Portilla-Arias, Jinwei Hu, Bindu Konda, Manabu Fujita, Andres Espinoza, Sonal Suhane, Marisa Riley, Marcus Gates, Rameshwar Patil, Manuel L. Penichet, Alexander V. Ljubimov, Keith L. Black, Eggehard Holler and Julia Y. Ljubimova
Satoshi Inoue
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Hui Ding
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Jose Portilla-Arias
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Jinwei Hu
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Bindu Konda
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Manabu Fujita
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Andres Espinoza
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Sonal Suhane
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Marisa Riley
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Marcus Gates
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Rameshwar Patil
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Manuel L. Penichet
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Alexander V. Ljubimov
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Keith L. Black
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Eggehard Holler
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Julia Y. Ljubimova
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DOI: 10.1158/0008-5472.CAN-10-3093 Published February 2011
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    Figure 1.

    The nanobiopolymer schematic. The nanobiopolymeric conjugate was designed to inhibit HER2/neu expression by AON and to attenuate HER2/neu-mediated cell signaling by Herceptin. The modules are HER2/neu morpholino AON (1) conjugated to the PMLA scaffold by disulfide bonds (S-S) that are cleaved by cytoplasmic glutathione to release the free drugs; targeting and/or effector antibodies comprising anti-TfR either alone or combination of mAbs to mouse TfR (2a), human TfR (2b), and Herceptin (2c) for tumor endothelial and cancer cell targeting, receptor-mediated endocytosis, and antitumor effect, PEG for drug protection (3), stretches of conjugated LOEt for endosomal escape of the drug (4), and optional fluorescent reporter dye (Alexa Fluor 680) for imaging (5). The nanopolymer also contained free, unsubstituted, pendant carboxyl groups for enhancing solubility and nonfunctional disulfide originating from chemical masking of excess sulfhydryls.

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    Figure 2.

    In vitro cell viability assay. HER2/neu overexpressing breast cancer cells (BT-474 and SKBR-3; Fig. 3A) were treated with various drugs (top row). After 72 hours, cell viability was determined using trypan blue exclusion assay. Percentages of cell growth were calculated as the average of cell counts for each group and expressed relative to parallel samples treated with PBS (control) set to 100%. Growth of tumor cells treated with P/mPEG/LOEt/AON/Herceptin/TfR(M) was significantly inhibited compared with other treatments in both cell lines. In cell lines expressing low amounts of HER2/neu (Fig. 3A), the only drug active in cell growth inhibition was the lead compound (bottom row). *, P < 0.05; **, P < 0.01; ***, P < 0.003 compared with PBS. The lead drug also showed significant differences at P < 0.005 when compared with all treatment groups in top row, and at P < 0.02 when compared with Herceptin in bottom row.

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    Figure 3.

    Changes of HER2/neu expression, Akt phosphorylation, and apoptosis upon various treatments of breast cancer cells in vitro. A, HER2/neu and TfR expression in cell lines. B, expression analysis of various markers. HER2/neu overexpressing breast cancer cells (a) were treated with various drugs. Western blot analysis showed decreased HER2/neu and phosphorylated Akt in Herceptin, P/mPEG/LOEt/Herceptin, AON or P/mPEG/LOEt/AON/TfR(H/M)-treated tumor cells but not in PBS or Endoporter-treated cells. P/mPEG/LOEt/AON/Herceptin/TfR(M) further reduced both HER2/neu and p-Akt. Generation of cleaved PARP as a measure of apoptosis was best seen in P/mPEG/LOEt/AON/Herceptin/TfR(M)-treated cells. GAPDH was used as an internal loading control. Breast cancer cell lines used in this study expressed high levels of TfR (A).

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    Figure 4.

    Distribution of various drugs labeled with Alexa Fluor 680 in live mice with BT-474 breast tumors and in isolated organs. Twenty-four hours after intravenous injection (left), control conjugate with IgG instead of targeting antibodies (top row) had little BT-474 tumor accumulation, confirmed after major organs analysis (right). Most of this control polymer accumulated in drug clearing organs, liver and kidneys. Polymer with Herceptin alone had a moderate tumor accumulation (middle row). The highest accumulation in breast tumor was observed with the lead drug. Arrows mark tumor implantation site.

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    Figure 5.

    Distribution of various drugs in BT-474 breast tumor cells. Animals treated intravenously with different drugs (Fig. 4) were sacrificed 24 hours after drug injection, their tumors were excised, and sections analyzed by confocal microscopy. Control conjugate P/mPEG/LOEt/IgG with attached Alexa Fluor 680 tracking dye (red) showed little if any tumor cell accumulation (top row). P/mPEG/LOEt/Herceptin displayed considerable accumulation in tumor cells, whereas the highest accumulation was observed for the lead drug P/mPEG/LOEt/AON/Herceptin/TfR(M), in complete accordance with live animal imaging (Fig. 4). Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm.

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    Figure 6.

    Mouse tumor inhibition, pathology, signaling, and apoptosis marker expression. A, 2 representative animals for each group and histopathologic analysis of respective tumors (H&E staining) are shown after treatment with different drug variants. Variable amounts of dead tissue are present in all nanobiopolymer-treated groups. In accordance with tumor size reduction data, the lead drug P/mPEG/LOEt/AON/Herceptin/TfR(M) caused pronounced disappearance of tumor cells with mostly necrotic areas present. B, tumor growth inhibition in mice. Animals treated with Herceptin, P/mPEG/LOEt/Herceptin, or P/mPEG/LOEt/AON/TfR(H/M) showed significant inhibition compared with PBS control (P < 0.03). P/mPEG/LOEt/AON/Herceptin/TfR(M) treatment produced the highest inhibition of tumor growth resulting in 80% to 95% tumor regression during the follow-up period compared with other treatment groups (P < 0.02 vs. Herceptin and other drugs; P < 0.001 vs. PBS). Error bars denote SEM. C, Expression of select markers after treatment of HER2/neu-positive tumors in vivo. Western blot analysis revealed the decrease in HER2/neu and p-Akt (but not total Akt) expression in Herceptin-, P/mPEG/LOEt/Herceptin-, or P/mPEG/LOEt/AON/TfR(H/M)-treated mice compared with PBS-treated ones. P/mPEG/LOEt/AON/Herceptin/TfR(M) further inhibited HER2/neu expression, with near disappearance of p-Akt band. PARP cleavage as a measure of apoptosis was also the most pronounced in P/mPEG/LOEt/AON/Herceptin/TfR(M)-treated mice compared with other groups. GAPDH was an internal control to normalize gel loading.

Tables

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  • Table 1.

    Nanobiopolymer versions, their sizes, and ζ potentials

    Nanobiopolymer variantVersionSize, nmζ potential, mV
    P/mPEG/LOEt/AON/Herceptin/TfR(M)Lead version with AON, Herceptin, and anti-TfR(M)22.1 ± 2.3−5.2 ± 0.4
    P/mPEG/LOEt/AON/TfR(H/M)With AON and anti-TfR(H/M)20.1 ± 2.4−5.7 ± 0.6
    P/mPEG/LOEt/HerceptinWith Herceptin alone15.1 ± 1.2−4.1 ± 0.4
    P/mPEG/LOEt/IgGControl version for imaging study with IgGN/AN/A

    N/A, Not applicable.

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    Cancer Research: 71 (4)
    February 2011
    Volume 71, Issue 4
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    Polymalic Acid–Based Nanobiopolymer Provides Efficient Systemic Breast Cancer Treatment by Inhibiting both HER2/neu Receptor Synthesis and Activity
    Satoshi Inoue, Hui Ding, Jose Portilla-Arias, Jinwei Hu, Bindu Konda, Manabu Fujita, Andres Espinoza, Sonal Suhane, Marisa Riley, Marcus Gates, Rameshwar Patil, Manuel L. Penichet, Alexander V. Ljubimov, Keith L. Black, Eggehard Holler and Julia Y. Ljubimova
    Cancer Res February 15 2011 (71) (4) 1454-1464; DOI: 10.1158/0008-5472.CAN-10-3093

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    Polymalic Acid–Based Nanobiopolymer Provides Efficient Systemic Breast Cancer Treatment by Inhibiting both HER2/neu Receptor Synthesis and Activity
    Satoshi Inoue, Hui Ding, Jose Portilla-Arias, Jinwei Hu, Bindu Konda, Manabu Fujita, Andres Espinoza, Sonal Suhane, Marisa Riley, Marcus Gates, Rameshwar Patil, Manuel L. Penichet, Alexander V. Ljubimov, Keith L. Black, Eggehard Holler and Julia Y. Ljubimova
    Cancer Res February 15 2011 (71) (4) 1454-1464; DOI: 10.1158/0008-5472.CAN-10-3093
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