Preparation, cytotoxicity, and in vivo antitumor efficacy of 111In-labeled modular nanotransporters

Abstract

Purpose: Modular nanotransporters (MNTs) are a polyfunctional platform designed to achieve receptor-specific delivery of short-range therapeutics into the cell nucleus by receptor-mediated endocytosis, endosome escape, and targeted nuclear transport. This study evaluated the potential utility of the MNT platform in tandem with Auger electron emitting ¹¹¹In for cancer therapy.

Methods: Three MNTs developed to target either melanocortin receptor-1 (MC1R), folate receptor (FR), or epidermal growth factor receptor (EGFR) that are overexpressed on cancer cells were modified with p-SCN-Bn-NOTA and then labeled with ¹¹¹In in high specific activity. Cytotoxicity of the ¹¹¹In-labeled MNTs was evaluated on cancer cell lines bearing the appropriate receptor target (FR: HeLa, SK-OV-3; EGFR: A431, U87MG.wtEGFR; and MC1R: B16-F1). In vivo micro-single-photon emission computed tomography/computed tomography imaging and antitumor efficacy studies were performed with intratumoral injection of MC1R-targeted ¹¹¹In-labeled MNT in B16-F1 melanoma tumor-bearing mice.

Results: The three NOTA-MNT conjugates were labeled with a specific activity of 2.7 GBq/mg with nearly 100% yield, allowing use without subsequent purification. The cytotoxicity of ¹¹¹In delivered by these MNTs was greatly enhanced on receptor-expressing cancer cells compared with ¹¹¹In nontargeted control. In mice with B16-F1 tumors, prolonged retention of ¹¹¹In by serial imaging and significant tumor growth delay (82% growth inhibition) were found.

Conclusion: The specific in vitro cytotoxicity, prolonged tumor retention, and therapeutic efficacy of MC1R-targeted ¹¹¹In-NOTA-MNT suggest that this Auger electron emitting conjugate warrants further evaluation as a locally delivered radiotherapeutic, such as for ocular melanoma brachytherapy. Moreover, the high cytotoxicity observed with FR- and EGFR-targeted ¹¹¹In-NOTA-MNT suggests further applications of the MNT delivery strategy should be explored.

Keywords: Auger electrons; cancer; melanoma; nuclear delivery; radionuclide therapy.

Figure 1

Schematic presentation of MNTs used in the study. Notes: (A) Principal scheme of MNT structure and representation of the function of each module. (B) Schematic presentation of MNT transport from the target cell surface to its nucleus. (C) MNTs used in the current study. MNT is designed to recognize and bind through its ligand module to internalizable receptors expressed on the surface of target cells; following subsequent internalization by receptor-mediated endocytosis, the MNT escapes from the endosomes with the help of its endosomolytic module; finally, the MNT binds through its nuclear localization signal module to importins in the hyaloplasm and this complex is transported through the nuclear pore into the target cell nucleus. MSH targeted to melanocortin receptor-1, EGF targeted to EGF receptor, or FA targeted to folate receptors served as different ligand modules; tDTox served as the endosomolytic module, the optimized SV-40 large T-antigen NLS was responsible for importin binding and transport into the nucleus through nuclear pore, and Escherichia coli HMP was used as the carrier module. Abbreviations: EGF, epidermal growth factor; FA, folic acid; HMP, hemoglobin-like protein; MNTs, modular nanotransporters; MSH, α-melanocyte-stimulation hormone; NLS, nuclear localization sequence; p-SCN-Bn-NOTA, chelator S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid; tDTox, the diphtheria toxin translocation domain.

Figure 2

Labeling of NOTA–MNT-MSH with ¹¹¹In as a function of: (A) reaction time, (B) SDS concentration in the reaction mixture, (C) citrate concentration, and (D) reaction mixture pH. Abbreviations: CMC, critical micelle concentration; MNT, modular nanotransporter; MSH, α-melanocyte-stimulation hormone; SDS, sodium dodecyl sulfate.

Figure 3

(A) Autoradiography of SDS-PAGE gels of ¹¹¹In-NOTA–MNT-MSH (lane 1), ¹¹¹In-NOTA–MNT-FA (lane 2), ¹¹¹In-NOTA–MNT-EGF (lane 3), and control ¹¹¹In (lane 4). “Standard labels” indicate the bands from the ColorBurst™ Electrophoresis Markers. Prominent main band corresponds to the expected molecular weight for these constructs (70–76 kDa); negligible bands migrating directly ahead of bromophenol blue (compared to lane 4) correspond to free ¹¹¹In (3%–5%). The faint bands (1%–10%), corresponding to an M_w of 20–50 kDa, are MNT-NOTA protein degradation products that were eventually labeled. (B) Autoradiography of SDS-PAGE gels of ¹¹¹In-NOTA–MNT-MSH at different ¹¹¹In/MNT ratios in the reaction mixture: 0.2 GBq/mg (lane 1), 1.1 GBq/mg (lane 2), 2.0 GBq/mg (lane 3), 4.4 GBq/mg (lane 4), 7.3 GBq/mg (lane 5). Abbreviations: EGF, epidermal growth factor; FA, folic acid; MNT, modular nanotransporter; MSH, α-melanocyte-stimulation hormone; SDS, sodium dodecyl sulfate.

Figure 4

Cytotoxicity of ¹¹¹In-NOTA–MNT-EGF on A431 cells with either high (2.7 GBq/mg) or low (0.2 GBq/mg) SA, unlabeled NOTA–MNT-EGF, and ¹¹¹In control as a function of (A) radioactivity or (B) MNT concentration in the media. The solid lines represent a fit of the data to a mono-exponential (¹¹¹In, high SA ¹¹¹In-NOTA–MNT-EGF) or a bi-exponential model (¹¹¹In-low SA NOTA–MNT-EGF). Note: Dashed lines indicate 95% confidence intervals. Abbreviations: EGF, epidermal growth factor; MNT, modular nanotransporter; SA, specific acivity.

Figure 5

Cytotoxicity of: ¹¹¹In-NOTA–MNT-FA on FR-positive (A) HeLa and (B) SK-OV-3; (C) ¹¹¹In-NOTA–MNT-EGF on EGFR-positive U87MG.wtEGFR cells; and (D) ¹¹¹In-NOTA–MNT-MSH on MC1R-positive B16-F1 cells, all compared to ¹¹¹In control and also FA-blocked ¹¹¹In-NOTA–MNT-FA for HeLa cells. Note: The significance of the difference was calculated using Kruskal–Wallis test. Abbreviations: EGFR, epidermal growth factor receptor; FA, folic acid; FR, folate receptor; MC1R, melanocortin receptor-1; MNT, modular nanotransporter; MSH, α-melanocyte-stimulation hormone.

Figure 6

Serial SPECT/CT imaging of C57 black mice bearing B16-F1 melanoma tumors after intratumoral injection of ¹¹¹In-NOTA–MNT-MSH. Notes: (A) SPECT/CT (color/gray) images of representative animal at indicated times after injection; (B) decay-corrected clearance of ¹¹¹In activity from the tumor. Dashed lines indicate 95% confidence interval. The ring in the abdominal region presented on the CT scans of the mice is the breathing sensor. Abbreviations: MNT, modular nanotransporter; MSH, α-melanocyte-stimulation hormone; SPECT/CT, single-photon emission computed tomography/computed tomography.

Figure 7

In vivo therapy of B16-F1 melanoma-bearing mice with ¹¹¹In-NOTA–MNT-MSH (♦), using (A) 2.6, (B) 5.2, and (C) 10.4 MBq/mice, ¹¹¹In with the same activity (▲), NOTA–MNT-MSH with the same concentration (◊), or saline (■). Note: *P<0.05 compared to control groups, according to the one-way ANOVA test. Abbreviations: ANOVA, analysis of variance; MNT, modular nanotransporter; MSH, α-melanocyte-stimulation hormone.