Construction and application of elastin like polypeptide containing IL-4 receptor targeting peptide

Abstract

Various human solid tumors highly express IL-4 receptors which amplify the expression of some of anti-apoptotic proteins, preventing drug-induced cancer cell death. Thus, IL-4 receptor targeted drug delivery can possibly increase the therapeutic efficacy in cancer treatment. Macromolecular carriers with multivalent targeting moieties offered great advantages in cancer therapy as they not only increase the plasma half-life of the drug but also allow delivery of therapeutic drugs to the cancer cells with higher specificity, minimizing the deleterious effects of the drug on normal cells. In this study we designed a library of elastin like polypeptide (ELP) polymers containing tumor targeting AP1 peptide using recursive directional ligation method. AP1 was previously discovered as an atherosclerotic plaque and breast tumor tissue homing peptide using phage display screening method, and it can selectively bind to the interleukin 4 receptor (IL-4R). The fluorescently labeled [AP1-V12]6, an ELP polymer containing six AP1 enhanced tumor-specific targeting ability and uptake efficiency in H226 and MDA-MB-231 cancer cell lines in vitro. Surface plasmon resonance analysis showed that multivalent presentation of the targeting ligand in the ELP polymer increased the binding affinity towards IL-4 receptor compared to free peptide. The binding of [AP1-V12]6 to cancer cells was remarkably reduced when IL-4 receptors were blocked by antibody against IL-4 receptor further confirmed its binding. Importantly, the Cy5.5-labeled [AP1-V12]6 demonstrated excellent homing and longer retention in tumor tissues in MDA-MB-231 xenograft mouse model. Immunohistological studies of tumor tissues further validated the targeting efficiency of [AP1-V12]6 to tumor tissue. These results indicate that designed [AP1-V12]6 can serve as a novel carrier for selective delivery of therapeutic drugs to tumors.

Figure 1. Monomer genes of V₁₄ and AP1-V₁₂.

(A) [VGVPG]₁₄ and (B) VGRKRLDRNG[VGVPG]₁₂ monomeric genes and their corresponding polypeptide sequences.

Figure 2. Protein expression and thermal characterization.

(A) Polypeptide sequences of (a) [V₁₄]₆ and (b) [AP1-V₁₂]₆, expressed proteins were visualized by Coomassie Blue staining after SDS-PAGE analysis. Left lane: protein molecular weight marker (in kDa). Expected sizes of [V₁₄]₆ (∼35 kDa) and [AP1-V₁₂]₆ (∼37 kDa) are indicated on the right. (B) The turbidity profiles of [V₁₄]₆ and [AP1-V₁₂]₆ protein verses OD₃₅₀ as a function of temperature (increased at a rate of 1°C min⁻¹) were obtained.

Figure 3. In vitro binding assays of [V₁₄]₆ and [AP1-V₁₂]₆ polymer.

(A, B) H226, (C, D) MDA-MB-231 and (E, F) H460 cells were incubated with 10 µM of [V₁₄]₆, [AP1-V₁₂]₆ and AP1 for 1 h at 4°C. Cell binding was determined using flow cytometry. Histograms are representative of three independent experiments. Graphical bars (on right) represent the percent of Alexa 488 labeled polymer bound to cells as mean ±SD of data obtained from three separate experiments performed in triplicates. ***P<0.0001, **P<0.001, and *P<0.05, one-way ANOVA; n = 3. (G, H) H226 cells (3×10⁵ cells) were pre- incubated with different concentrations (1, 5 and 10 µg/mL) of anti-IL-4 receptor antibody followed by 1 h incubation with 10 µM Alexa-labeled [AP1-V₁₂]₆ at 4°C. The cells were further suspended in 300 µL of PBS after washing and analyzed using flow cytometry. Histograms are representative of three independent experiments. Graphical bars represent the percent of Alexa 488 labeled polymer bound to cells as mean ±SD of data obtained from three separate experiments performed in triplicates. ***P<0.0001, One way ANOVA; n = 3.

Figure 4. Analysis of cellular localization of [AP1-V₁₂]₆ polymer.

Confocal laser scanning microscopic images of H226 cancer cells treated with 10 µM of [AP1-V₁₂]₆, AP1, or [V₁₄]₆ at (A) 4°C and (B) 37°C. Representative confocal images of three experiments (scale bar 20 µm). Right panels: Examination of [AP1-V₁₂]₆, AP1 and [V₁₄]₆ cellular location by Z-section scanning of confocal microscopic images. Representative confocal images of three experiments (scale bar 10 µm).

Figure 5. In vitro cell viability and proliferation assay.

(A, B) MDA-MB-231 cells were plated in 96 well plates in serum (10% FBS) containing media and further treated with different concentration of [AP1-V₁₂]₆ and [V₁₄]₆ (1, 5, 10, 20 µM) for various time intervals (12, 24, 48, and 72 h). Cell viability was accessed by measuring WST-8 absorbance at 450 nm (n = 5) samples at different time intervals. The graph represents the percentage of cell viability in treated cells compared to control (untreated) cells. The result shown here is the representative data of 3 independent experiments. (C) For proliferation assay, MDA-MB-231 (2×10³) cells were serum starved (1% FBS) for 16 h with or without different concentrations of IL-4. To check the effect of [AP1-V₁₂]₆ and [V₁₄]₆ on IL-4 stimulation, the cells were grown in low serum (1% FBS) media containing different concentration of IL-4 (10, 50, 100 ng/ml) and [AP1-V₁₂]₆ and [V₁₄]₆ (10 µM) for 24, 48, and 72 h. Cell proliferation was analyzed by measuring the WST-8 absorbance at 450 nm (n = 5 samples). The graph represents the percentage of cell viability in treated cells compared to control (untreated) cells. The result shown here is the representative data of 3 independent experiments. A t-test was performed to determine the significance of various groups after IL-4 and polymer treatments. Control versus IL-4 (100 ng/ml), *  = P<0.05 and **  = P<0.001, IL-4 (100 ng/ml) versus [AP1-V₁₂]₆ + IL-4 (100 ng/ml), +  = P<0.05 and ++  = P<0.001.

Figure 6. IL-4 R binding affinity of [AP1-V₁₂]₆ polymer.

Determination of binding kinetics. Normalized (A) [AP1-V₁₂]₆ protein (125 nM - 1 µM), (B) AP1 peptide (3.125 - 50 µM) and (C) [V₁₄]₆ (1 µM) binding curves (black line) with fits (red line) obtained using Scrubber 2. Histograms are representative of three independent experiments.

Figure 7. In vivo, ex vivo imaging and biodistribution of [AP1-V₁₂]₆ polymers.

(A) Cy5.5-labeled [V₁₄]₆ and [AP1-V₁₂]₆ (8 mg/kg) were intravenously injected into MDA-MB-231 tumor xenografted nude mice. Biodistribution was determined by collecting in vivo fluorescence images at different time points. Scale bar indicates normalized fluorescence intensity (NC). Representative optical images of three experiments. (B) Quantitation of fluorescence intensities in tumor sites at respective time points (n = 3). (C) Analysis of fluorescence intensities for tumors and organs from ex vivo images (n = 3). (D) Histological analysis of [AP1-V₁₂]₆ polymer (red) localization in tumors. Nuclei were stained with DAPI (blue), and IL-4R expression on cells was visualized by anti-IL-4 receptor antibody staining (green). Representative confocal images of three experiments (scale bar 50 µm).