Selective sodium iodide symporter (NIS) genetherapy of glioblastoma mediatedby EGFR-targeted lipopolyplexes

Abstract

Lipo-oligomers, post-functionalized with ligands to enhance targeting, represent promising new vehicles for the tumor-specific delivery of therapeutic genes such as the sodium iodide symporter (NIS). Due to its iodide trapping activity, NIS is a powerful theranostic tool for diagnostic imaging and the application of therapeutic radionuclides. ¹²⁴I PET imaging allows non-invasive monitoring of the in vivo biodistribution of functional NIS expression, and application of ¹³¹I enables cytoreduction. In our experimental design, we used epidermal growth factor receptor (EGFR)-targeted polyplexes (GE11) initially characterized in vitro using ¹²⁵I uptake assays. Mice bearing an orthotopic glioblastoma were treated subsequently with mono-dibenzocyclooctyne (DBCO)-PEG₂₄-GE11/NIS or bisDBCO-PEG₂₄-GE11/NIS, and 24-48 h later, ¹²⁴I uptake was assessed by positron emission tomography (PET) imaging. The best-performing polyplex in the imaging studies was then selected for ¹³¹I therapy studies. The in vitro studies showed EGFR-dependent and NIS-specific transfection efficiency of the polyplexes. The injection of monoDBCO-PEG₂₄-GE11/NIS polyplexes 48 h before ¹²⁴I application was characterized to be the optimal regime in the imaging studies and was therefore used for an ¹³¹I therapy study, showing a significant decrease in tumor growth and a significant extension of survival in the therapy group. These studies demonstrate the potential of EGFR-targeted polyplex-mediated NIS gene therapy as a new strategy for the therapy of glioblastoma.

Keywords: DNA nanoparticle; EGFR-targeting; GBM; NIS; gene therapy; glioblastoma; polyplexes; radioiodine; sodium iodide symporter.

Graphical abstract

Figure 1

Formulation of functionalized polyplexes The cationic lipo-OAA containing an N-terminal azido group was mixed with pDNA at N/P 12 and incubated for 30 min at room temperature (A). Following the addition of a DBCO agent with 0.25 equivalents, another incubation for 4 h at room temperature was performed (B). Schematic depictions are shown of the sequence-defined lipo-OAA with the compound ID 1252 (K, lysine; C, cysteine; Y, tyrosine; H, histidine; Stp, succinoyltetraethylene-pentamine) (C) and the structure of PEGylation agents for post-functionalization containing monovalent (D) or bivalent (E) DBCO with ligand peptide GE11 for targeting of EGFR or without GE11 as negative non-targeting control. DLS measurements of formed polyplexes revealed a size of 120–140 nm with a uniform size distribution (PDI of <0.2) (F) and a zeta potential below 20 mV, whereas non-targeted PEGylated polyplexes showed more efficient surface shielding than polyplexes with targeted ligands (G) (∗p ≤ 0.05; n/s, not significant). Results are reported as mean ± SEM (n = 3). RT, room temperature.

Figure 2

EGFR-targeted NIS gene transfer in vitro Cell-surface expression of EGFR was measured by flow cytometry. A specific antibody detected the expression levels of human EGFR on GBM14, MCF-7, FTC-133, U87, and LN229 compared with isotype controls (A). ¹²⁵I transfection studies with GBM14, MCF-7, FTC-133, U87, and LN229 (n = 3) indicate a correlation between the receptor expression levels and the transfection efficiency of targeted polyplexes (bisDBCO-PEG₂₄-GE11/NIS) (B). Receptor specificity was shown by transfecting cells with untargeted polyplexes (monoDBCO-PEG₂₄/NIS n = 6 and bisDBCO-PEG₂₄/NIS n = 3), resulting in a significantly lower iodide uptake. Background radiation levels after control transfection with LUC-coding polyplexes (monoDBCO-PEG₂₄-GE11/LUC n = 6 and bisDBCO-PEG₂₄-GE11/LUC n = 3) or the addition of NIS-specific inhibitor perchlorate prove NIS dependency of iodide uptake (B and C) (∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001). Treatment with the selective EGFR inhibitor cetuximab resulted in a dose-dependent inhibition of the transfection of U87 cells using monoDBCO-PEG₂₄-GE11/NIS polyplexes, demonstrating the EGFR dependency of transfection with GE11 polyplexes (D) (∗∗p ≤ 0.01). Cell viability of U87 was affected neither by monoDBCO- nor by bisDBCO-polyplex treatment (E). Results are reported as mean ± SEM.

Figure 3

Polyplex-mediated NIS gene transfer in vivo U87 GBM (A) showed high membranous EGFR expression in the receptor staining compared with no EGFR expression in normal brain tissue (B). Original magnifications of 8× (scale bar: 300 μm), 15× (scale bar: 100 μm), and 40× (scale bar: 30 μm) were chosen. Tumoral iodide uptake in ¹²⁴I PET studies was significantly higher in mice treated with monoDBCO-PEG24-GE11/NIS (n = 5) (C) compared with non-targeting monoDBCO-PEG24/NIS (n = 4) (E). No tumoral iodide uptake above background was measured in mice that received LUC-coding monoDBCO-PEG24-GE11/LUC polyplexes (n = 3) (F), comparable to mice that did not bear a tumor (J). An interval of 48 h between systemic polyplex injection and iodide administration resulted in a higher iodide uptake than an interval of 24 h (n = 5) (C, D). Analogous outcomes and reproduction of the advantageous targeting effect of GE11 polyplexes were seen in studies with bisDBCO-PEG24-GE11/NIS (n = 5) (G) for targeted, bisDBCO-PEG24/NIS (n = 3) (H) for non-targeted, and bisDBCO-PEG24-GE11/LUC (n = 2) (I) for LUC-coding polyplexes. One representative image is shown for each group. Tumoral iodide uptake was measured by serial scannings over 5 h and quantified as the percentage of the injected dose per milliliter tumor (K) (∗p ≤ 0.05). Results are reported as mean ± SEM (S, snout, nasal secretion; T, thyroid; SG, salivary glands; St, stomach; B, bladder).

Figure 4

Analysis of NIS protein expression in U87 tumors ex vivo Immunohistochemical staining of NIS protein in GBM xenografts embedded in paraffin revealed a higher NIS expression (red) in mice treated with targeted polyplexes (monoDBCO-PEG₂₄-GE11/NIS [A] and bisDBCO-PEG₂₄-GE11/NIS [F]) 48 h before sacrifice compared with the 24h time point (B). No positive NIS staining in tumors of mice that received control polyplexes (C, D, G, and H) or untreated (E) mice was observed. Liver (I), lung (J), kidney (K), and spleen (L) did not show any NIS expression. One representative image with 20× original magnification is shown for each group (scale bar: 100 μm). A 40× original magnification was chosen for the close up (scale bar: 50 μm).

Figure 5

¹³¹I therapy studies in vivo GBM-bearing mice, confirmed by MRI on day 0, were treated with three cycles of i.v. injection of polyplexes on days 1, 5, and 9 followed by i.p. injection of 55.5 MBq ¹³¹I 48 h later, on days 3, 7, and 11. Tumor sizes were monitored twice a week by MRI. Exemplary MRI images of tumor sizes on day 18 of the therapy trial from a monoDBCO-PEG₂₄-GE11/NIS + ¹³¹I (A), a monoDBCO-PEG₂₄/NIS + ¹³¹I (B), a monoDBCO-PEG₂₄-GE11/NIS + NaCl (C), and a NaCl + NaCl (D)-treated mouse are shown. Tumors are highlighted by green lines. Injection of monoDBCO-PEG₂₄-GE11/NIS + ¹³¹I led to a decrease in tumor growth in the therapy group (n = 5) compared with control groups monoDBCO-PEG₂₄/NIS + ¹³¹I (n = 5; mean ± SEM; ∗p < 0.05 on day 15), monoDBCO-PEG₂₄-GE11/NIS + NaCl (n = 5; mean ± SEM), and NaCl + NaCl (n = 5; mean ± SEM; ∗p < 0.05 on days 18 and 21) (E). Therapy mice treated with monoDBCO-PEG₂₄-GE11/NIS + ¹³¹I (n = 5) showed a significant extension of survival compared with control groups monoDBCO-PEG₂₄/NIS + ¹³¹I (n = 5; ∗p < 0.05), monoDBCO-PEG₂₄-GE11/NIS + NaCl (n = 5; ∗∗p < 0.01), and NaCl + NaCl (n = 5; ∗∗p < 0.01) (F).

Figure 6

Analysis of proliferation index and blood vessel density of therapy tumors ex vivo Frozen tissue sections from GBM dissected after the therapy study were stained for Ki67 (green) for proliferation index and CD31 (red) for blood vessel density. Nuclei are stained with Hoechst (blue). The therapy group that received monoDBCO-PEG₂₄-GE11/NIS followed by ¹³¹I (A) showed fewer Ki67-positive cells (E) and a significantly smaller CD31-positive area (F) (∗p < 0.05, ∗∗∗p < 0.001) compared with the control groups treated with monoDBCO-PEG₂₄/NIS plus ¹³¹I (B), monoDBCO-PEG₂₄-GE11/NIS plus NaCl (C), or NaCl only (D). One representative picture of each group is shown at 20× original magnification (scale bar: 100 μm). Results are reported as mean ± SEM (for each group n = 4).