Nanobiopolymer for direct targeting and inhibition of EGFR expression in triple negative breast cancer

Abstract

Treatment options for triple negative breast cancer (TNBC) are generally limited to cytotoxic chemotherapy. Recently, anti-epidermal growth factor receptor (EGFR) therapy has been introduced for TNBC patients. We engineered a novel nanobioconjugate based on a poly(β-L-malic acid) (PMLA) nanoplatform for TNBC treatment. The nanobioconjugate carries anti-tumor nucleosome-specific monoclonal antibody (mAb) 2C5 to target breast cancer cells, anti-mouse transferrin receptor (TfR) antibody for drug delivery through the host endothelial system, and Morpholino antisense oligonucleotide (AON) to inhibit EGFR synthesis. The nanobioconjugates variants were: (1) P (BioPolymer) with AON, 2C5 and anti-TfR for tumor endothelial and cancer cell targeting, and EGFR suppression (P/AON/2C5/TfR), and (2) P with AON and 2C5 (P/AON/2C5). Controls included (3) P with 2C5 but without AON (P/2C5), (4) PBS, and (5) P with PEG and leucine ester (LOEt) for endosomal escape (P/mPEG/LOEt). Drugs were injected intravenously to MDA-MB-468 TNBC bearing mice. Tissue accumulation of injected nanobioconjugates labeled with Alexa Fluor 680 was examined by Xenogen IVIS 200 (live imaging) and confocal microscopy of tissue sections. Levels of EGFR, phosphorylated and total Akt in tumor samples were detected by western blotting. In vitro western blot showed that the leading nanobioconjugate P/AON/2C5/TfR inhibited EGFR synthesis significantly better than naked AON. In vivo imaging revealed that 2C5 increased drug-tumor accumulation. Significant tumor growth inhibition was observed in mice treated with the lead nanobioconjugate (1) [P = 0.03 vs. controls; P<0.05 vs. nanobioconjugate variant (2)]. Lead nanobioconjugate (1) also showed stronger inhibition of EGFR expression and Akt phosphorylation than other treatments. Treatment of TNBC with the new nanobioconjugate results in tumor growth arrest by inhibiting EGFR and its downstream signaling intermediate, phosphorylated Akt. The nanobioconjugate represents a new generation of nanodrugs for treatment of TNBC.

Figure 1. Nanobioconjugate schematic.

The new version of nanobioconjugate was designed to inhibit EGFR expression by Morpholino AON in vitro and in vivo. The components are Morpholino AON to EGFR (1) conjugated to the scaffold by disulfide bonds that are cleaved by glutathione in the cytoplasm to release the free AONs; targeting antibodies 2C5 and TfR either alone or as a combination of mAbs to tumor cells (2a), mouse TfR (2b) for tumor endothelial and cancer cell targeting, and receptor-mediated endocytosis; PEG for drug protection (3); L-leucine ethyl ester together with polymer-COOH (4) for endosomal escape of the drug, and optional fluorescent reporter dye (fluorescein or Alexa Fluor 680) for imaging (5).

Figure 2. Expression levels of 2C5 and EGFR (after treatment).

(2a) Expression level of 2C5 antigen and transferrin receptor in breast cancer cells. 2C5 antigen and TfR expression was studied by western blot analysis. All cell lines expressed TfR and 2C5 antigen. Consistent expression of TfR was seen in all cell lines. 2C5 antigen was also expressed by all cell lines, with the highest expression in MDA-MB-231 (not shown). β-Actin was an internal control to normalize gel loading. (2b) Inhibition of EGFR expression in vitro by P/AON/2C5. EGFR overexpressing breast cancer cells MDA-MB-468 (TNBC) and SKBR-3 were treated with either PBS (control), Endoporter (5 µM), two different concentrations of EGFR AON (5 µM or 10 µM) with Endoporter, or three different concentrations of nanobioconjugate (P/AON/2C5) (1.25 µM, 2.5 µM or 5 µM). Two different sequences of EGFR AONs were used for this study (shown in Materials and Methods, 1. Reagents section). Since EGFR AON version 2 inhibited EGFR expression better than version 1 in vitro, version 2 (shown here) was chosen for the entire study. 72 hour after treatment, total cell protein was harvested and subjected to Western blot analysis as described in Materials and Methods. Decreased EGFR expression was observed in AON, or P/AON/2C5-treated tumor cells but not in PBS or Endoporter-treated cells. In both cell lines, 10 µM of AON was required to inhibit EGFR. On the other hand, low concentrations (1.25 µM for SKBR-3 and 2.5 µM for MDA-MB-468) of the nanobioconjugate significantly inhibited EGFR expression. GAPDH was an internal control to normalize gel loading.

Figure 3. Significant accumulation of P/2C5 in MDA-MB-468 breast tumor in vivo.

(3a), An MDA-MB-468 subcutaneous tumor-bearing mouse was administered with P/2C5 intravenously. 24 hours later, the animal showed drug distribution mostly in the tumor, as well as in kidney and liver (drug clearing organs). (3b), Other than in kidney and liver, the drug was seen exclusively in the tumor.

Figure 4. Tumor-specific active targeting effect of the 2C5 antibody in vivo.

MDA-MB-468 subcutaneous tumor-bearing mice were injected with Alexa Fluor 680-labeled nanobioconjugates (P/IgG, control; P/2C5 or P/2C5/TfR) through their tail vein. The images were taken 24 hours after the drug injection using Xenogen IVIS 200 system. Although the P/IgG barely accumulated in the tumor by EPR effect, the P/2C5 accumulated in the tumor area significantly more than control (Fig. 4a and 4b, **p = 0.001). The highest drug accumulation was seen in P/2C5/TfR treated group where two different tumor targeting antibodies were combined (Fig. 4a and 4b: p = 0.002 vs P/2C5; ***p = 0.0001 vs P/IgG). Confocal microscopy confirmed drug delivery efficiency of the nanopolymer with dual targeting antibodies, P/2C5/TfR (Fig. 4c).

Figure 5. Tumor inhibition in mouse model, and effects on EGFR expression and Akt phosphorylation.

(5a) Tumor growth inhibition in mice. MDA-MB-468 subcutaneous breast tumors treated systemically with P/AON/2C5, or P/AON/2C5/TfR were significantly inhibited compared with PBS (control), P(polymer only), or P/2C5 (without anti-tumor component) (P<0.03). The highest inhibition of tumor growth was observed in mice treated with P/AON/2C5/TfR (p<0.03 vs. controls; p<0.05 vs. P/AON/2C5). Error bars denote SEM. (5b) Expression of EGFR and phosphorylated Akt (p-Akt) after treatment of EGFR-positive tumors in vivo. Western blot analysis showed the decrease in EGFR and p-Akt expression in P/AON/2C5-, or P/AON/2C5/TfR -treated mice compared to controls. The highest inhibition of EGFR and p-Akt was seen upon treatment with P/AON/2C5/TfR where 2 targeting antibodies were combined. GAPDH was an internal control to normalize gel loading. (5c) Histopathological analysis of tumors. Hematoxylin and eosin staining of tumors treated with PBS, P, P/2C5, P/AON/2C5 or the leading drug P/AON/2C5/TfR. Consistent with tumor size reduction data, the leading drug P/AON/2C5/TfR treated tumor showed significant reduction in the number of viable tumor cells as compared to other treatments.