Tumor detection and elimination by a targeted gallium corrole

Abstract

Sulfonated gallium(III) corroles are intensely fluorescent macrocyclic compounds that spontaneously assemble with carrier proteins to undergo cell entry. We report in vivo imaging and therapeutic efficacy of a tumor-targeted corrole noncovalently assembled with a heregulin-modified protein directed at the human epidermal growth factor receptor (HER). Systemic delivery of this protein-corrole complex results in tumor accumulation, which can be visualized in vivo owing to intensely red corrole fluorescence. Targeted delivery in vivo leads to tumor cell death while normal tissue is spared. These findings contrast with the effects of doxorubicin, which can elicit cardiac damage during therapy and required direct intratumoral injection to yield similar levels of tumor shrinkage compared with the systemically delivered corrole. The targeted complex ablated tumors at >5 times a lower dose than untargeted systemic doxorubicin, and the corrole did not damage heart tissue. Complexes remained intact in serum and the carrier protein elicited no detectable immunogenicity. The sulfonated gallium(III) corrole functions both for tumor detection and intervention with safety and targeting advantages over standard chemotherapeutic agents.

Fig. 1.

Detection of HerGa in vivo targeting and intracellular trafficking. Live animal imaging of corrole fluorescence after IV delivery of either free corrole or targeted complex. (A) Nude mice bearing human HER2⁺ tumors (≈300 cubic mm) received a single IV injection of either S2Ga or HerGa (15 nmoles with respect to corrole dose) and were imaged at 2.5 hr postinjection using a noninvasive small animal fluorescence imaging system (35). Schematic to the left indicates the whole body and tumor orientation of the mice in the fluorescent images. (B) Images capturing time course of corrole circulation in mice after receiving HerGa as described in A. Arrows in both A and B point to tumors. Corrole fluorescence is indicated by blue-red pseudocoloring with fluorescence intensity represented according to the color bar on the right. (C–E) Intracellular trafficking in live cells. MDA-MB-435 cells were treated with HerGa at 1 μM final corrole concentration and live cells imaged by fluorescence microscopy at the indicated time points after treatment. (C and D) High-resolution spinning disk micrographs (Perkin-Elmer/Improvision) showing apparent vesicles and corrole fluorescence (pseudocolored green) distribution in cytoplasm at 15 min (C) and 2 hr (D). (E) Micrograph of corrole fluorescence (red) distribution in cytoplasm as captured by inverted fluorescence microscopy at up to 24 hr after uptake. In C–E, fluorescence images were overlaid on brightfield images. n, nucleus. (Scale bars, 10 μm.)

Fig. 2.

Targeted cytotoxicity in a mixed culture of HER2⁺ and HER2⁻ human cancer cells. Cell cultures containing 1:1 cell ratio of MDA-MB-435 (ErbB2⁺,GFP⁻):MDA-MB-231 (ErbB2⁻,GFP⁺) (Top) were treated with HerPBK10 alone (A), HerPBK10-S2Ga (B), S2Ga (C), or doxorubicin (Dox) (D) at 0.5 μM final drug (corrole or Dox) concentration. HerPBK10 alone was added at the equivalent concentration to the same protein in the complex. Cells were treated daily for 1 week in complete (serum-containing) medium and cells assessed at the indicated time points for GFP fluorescence (to determine relative MDA-MB-231 number) and crystal violet staining (to determine total cell number). Cell survival was determined by calculating the relative doubling time (DT) of experimental (exp) cells normalized by control (con) cells based on the crystal violet stains (total cells) and GFP fluorescence (MDA-MB-231 cells). The DT of MDA-MB-435 was determined by subtracting the DT of MDA-MB-231 from the total cell DT.

Fig. 3.

Targeted tumor growth intervention by HerGa. Effect of systemically-delivered HerGa or individual components on tumor growth. Nude mice bearing HER2⁺ tumors received daily IV injections for 1 week of the indicated reagents when the tumors reached 250–300 cubic mm (tumor inoculation and treatment schedule depicted in A; SC, subcutaneous). Graph in B presents tumor volumes measured before, during, and after treatment. Mice were euthanized at 25 days after the last day of treatment, and tissues harvested for histological assessment. n = 5–9 tumors per treatment group. (C) Comparative doses of Dox delivered IV or intratumorally (IT) on tumor growth. Treatment schedule is the same as in B. (D) Effect on myocardia of hearts harvested from treated mice at the end of the experiments in B and C (“Dox” hearts were harvested from mice receiving Dox IT). Paraffin-fixed specimens were processed for immunofluorescence against myosin and imaged at 60× magnification.

Fig. 4.

Neutralizing antibody induction and serum stability. (A) Immunocompetent (C57BL/6) mice received an initial s.c. inoculation of HerPBK10 protein (0.05 or 0.5 mg/kg) or Ad5-GFP (1.2 × 10⁹ pfu per mouse) followed by blood collection scheduled every 7 days up to day 35 after the initial inoculation as summarized by the time chart (Upper). On day 21, respective mice received a second inoculation of each corresponding reagent. Sera isolated from bleeds were assessed by ELISA for relative antibody titer produced against HerPBK10 (Lower). Arrows denote days of antigen inoculation. n = 4 mice per treatment group dose. (B) Effect of immune sera on target cell binding. Ligand-receptor binding was tested by measuring the level of cell attachment to HerPBK10-coated plates in immune or preimmune serum collected from mice in A. Cells suspended in either preimmune serum, immune serum from Ad5 or HerPBK10-inoculated mice, or complete media containing 10% bovine serum but no mouse serum were incubated on HerPBK10-coated wells for 1 hr at 37 °C, followed by removal of free cells and measurement of attached cells by crystal violet assay. The level of receptor-specific binding was assessed on separate cells preincubated with competitive inhibitor (Her). Un, untreated mice. *, P <0.01 compared with cells attached in preimmune serum, as determined by the 2-tailed unpaired t test. (C) Relative corrole retention by HerGa during incubation in human serum. Human serum (Omega Scientific, Inc.) was immobilized by overnight incubation at 4 °C in 96-well plates. The next day, the wells were washed with PBS, and HerGa or S2Ga alone (50 μM final) were added to each well. After incubation at 37 °C for the indicated time points, samples were removed from the wells and measured for corrole fluorescence. Corrole retention or loss by the complex was assessed by comparing the fluorescence of preserum and postserum incubation samples (RFU 424 nm excitation wavelength/620 nm emission wavelength of postincubation sample/input). Fluorescence values are shown as a percentage of input fluorescence (of fluorescence of complex before serum incubation). *, P <0.03 compared with incubation without serum. Significances were determined by 2-tailed paired t tests.