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Yang Liu,1 Zhengyuan Zhou,2 Yutian Feng,2 Xiao-Guang Zhao,2 Ganesan Vaidyanathan,2 Michael R Zalutsky,1,2 Tuan Vo-Dinh1,3,4 1Department of Biomedical Engineering, Duke University, Durham, NC, 27708, USA; 2Department of Radiology, Duke University Medical Center, Durham, NC, 27710, USA; 3Department of Chemistry, Duke University, Durham, NC, 27708, USA; 4Fitzpatrick Institute for Photonics, Duke University, Durham, NC, 27708, USACorrespondence: Michael R ZalutskyDepartment of Radiology, Duke University Medical Center, 311 Research Drive, 161H Bryan Research Building, Durham, NC, 27710, USAEmail michael.zalutsky@duke.eduTuan Vo-DinhDepartment of Biomedical Engineering, Duke University, 1427 CIEMAS, Box 90281, Durham, NC, 27708, USATel +1 919 660-8520Email tuan.vodinh@duke.eduAim: To develop an innovative 211At nanoplatform with high radiolabeling efficiency and low in vivo deastatination for future targeted alpha-particle therapy (TAT) to treat cancer.Methods: Star-shaped gold nanoparticles, gold nanostars (GNS), were used as the platform for 211At radiolabeling. Radiolabeling efficiency under different reaction conditions was tested. Uptake in the thyroid and stomach after systemic administration was used to evaluate the in vivo stability of 211At-labeled GNS. A subcutaneous U87MG human glioma xenograft murine model was used to preliminarily evaluate the therapeutic efficacy of 211At-labeled GNS after intratumoral administration.Results: The efficiency of labeling GNS with 211At was almost 100% using a simple and rapid synthesis process that was completed in only 1 min. In vitro stability test in serum showed that more than 99% of the 211At activity remained on the GNS after 24 h incubation at 37°C. In vivo biodistribution results showed low uptake in the thyroid (0.44– 0.64%ID) and stomach (0.21– 0.49%ID) between 0.5 and 21 h after intravenous injection, thus indicating excellent in vivo stability of 211At-labeled GNS. The preliminary therapeutic efficacy study demonstrated that 211At labeled GNS substantially reduced tumor growth (P < 0.001; two-way ANOVA) after intratumoral administration.Conclusion: The new 211At radiolabeling strategy based on GNS has the advantages of a simple process, high labeling efficiency, and minimal in vivo dissociation, making it an attractive potential platform for developing TAT agents that warrants further evaluation in future preclinical studies directed to evaluating prospects for clinical translation.Keywords: astatine-211, 211At, gold nanostars, GNS, cancer therapy, targeted alpha-particle therapy, TAT

Tatiana A Slastnikova,1,* Andrey A Rosenkranz,1,2,* Natalia B Morozova,3 Maria S Vorontsova,3 Vasiliy M Petriev,4,5 Tatiana N Lupanova,1 Alexey V Ulasov,1 Michael R Zalutsky,6 Raisa I Yakubovskaya,3 Alexander S Sobolev1,2 1Laboratory of Molecular Genetics of Intracellular Transport, Institute of Gene Biology, Russian Academy of Sciences, 2Department of Biophysics, Biological Faculty, Lomonosov Moscow State University, 3Department of Anticancer Therapy Modifiers and Protectors, Moscow Hertsen Research Institute of Oncology, Russian Ministry of Health Care, Moscow, 4National Medical Research Radiological Center, Russian Ministry of Health Care, Obninsk, Moscow Region, 5Department of Nuclear Medicine, National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), Moscow, Russia; 6Department of Radiology, Duke University Medical Center, Durham, NC, USA *These authors contributed equally to this work 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: nuclear delivery, cancer, melanoma, radionuclide therapy, Auger electrons

Tatiana A Slastnikova1,2, Andrey A Rosenkranz1,2, Pavel V Gulak1, Raymond M Schiffelers3, Tatiana N Lupanova1,4, Yuri V Khramtsov1, Michael R Zalutsky5, Alexander S Sobolev1,21Laboratory of Molecular Genetics of Intracellular Transport, Institute of Gene Biology, Moscow, Russia; 2Department of Biophysics, Biological Faculty, Moscow State University, Vorobyevy Gory, Moscow, Russia; 3Laboratory for Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, the Netherlands; 4Department of Bioengineering, Biological Faculty, Moscow State University, Vorobyevy Gory, Moscow, Russia; 5Department of Radiology, Duke University Medical Center, Durham, NC, USABackground: Modular nanotransporters (MNT) are recombinant multifunctional polypeptides created to exploit a cascade of cellular processes, initiated with membrane receptor recognition to deliver selective short-range and highly cytotoxic therapeutics to the cell nucleus. This research was designed for in vivo concept testing for this drug delivery platform using two modular nanotransporters, one targeted to the α-melanocyte-stimulating hormone (αMSH) receptor overexpressed on melanoma cells and the other to the epidermal growth factor (EGF) receptor overexpressed on several cancers, including glioblastoma, and head-and-neck and breast carcinoma cells.Methods: In vivo targeting of the modular nanotransporter was determined by immunofluorescence confocal laser scanning microscopy and by accumulation of 125I-labeled modular nanotransporters. The in vivo therapeutic effects of the modular nanotransporters were assessed by photodynamic therapy studies, given that the cytotoxicity of photosensitizers is critically dependent on their delivery to the cell nucleus.Results: Immunohistochemical analyses of tumor and neighboring normal tissues of mice injected with multifunctional nanotransporters demonstrated preferential uptake in tumor tissue, particularly in cell nuclei. With 125I-labeled MNT{αMSH}, optimal tumor:muscle and tumor:skin ratios of 8:1 and 9.8:1, respectively, were observed 3 hours after injection in B16-F1 melanoma-bearing mice. Treatment with bacteriochlorin p-MNT{αMSH} yielded 89%–98% tumor growth inhibition and a two-fold increase in survival for mice with B16-F1 and Cloudman S91 melanomas. Likewise, treatment of A431 human epidermoid carcinoma-bearing mice with chlorin e6- MNT{EGF} resulted in 94% tumor growth inhibition compared with free chlorin e6, with 75% of animals surviving at 3 months compared with 0% and 20% for untreated and free chlorin e6-treated groups, respectively.Conclusion: The multifunctional nanotransporter approach provides a new in vivo functional platform for drug development that could, in principle, be applicable to any combination of cell surface receptor and agent (photosensitizers, oligonucleotides, radionuclides) requiring nuclear delivery to achieve maximum effectiveness.Keywords: drug delivery, nanobiotechnology, nanomedicine, cancer therapy, photosensitizers, multifunctional nanotransporter

Aim: To develop an innovative 211 At nanoplatform with high radiolabeling efficiency and low in vivo deastatination for future targeted alpha-particle therapy (TAT) to treat cancer., Methods: Star-shaped gold nanoparticles, gold nanostars (GNS), were used as the platform for 211 At radiolabeling. Radiolabeling efficiency under different reaction conditions was tested. Uptake in the thyroid and stomach after systemic administration was used to evaluate the in vivo stability of 211 At-labeled GNS. A subcutaneous U87MG human glioma xenograft murine model was used to preliminarily evaluate the therapeutic efficacy of 211 At-labeled GNS after intratumoral administration., Results: The efficiency of labeling GNS with 211 At was almost 100% using a simple and rapid synthesis process that was completed in only 1 min. In vitro stability test in serum showed that more than 99% of the 211 At activity remained on the GNS after 24 h incubation at 37°C. In vivo biodistribution results showed low uptake in the thyroid (0.44-0.64%ID) and stomach (0.21-0.49%ID) between 0.5 and 21 h after intravenous injection, thus indicating excellent in vivo stability of 211 At-labeled GNS. The preliminary therapeutic efficacy study demonstrated that 211 At labeled GNS substantially reduced tumor growth (P < 0.001; two-way ANOVA) after intratumoral administration., Conclusion: The new 211 At radiolabeling strategy based on GNS has the advantages of a simple process, high labeling efficiency, and minimal in vivo dissociation, making it an attractive potential platform for developing TAT agents that warrants further evaluation in future preclinical studies directed to evaluating prospects for clinical translation., Competing Interests: The authors report no conflicts of interest in this work., (© 2021 Liu et al.)

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 111 In 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 111 In in high specific activity. Cytotoxicity of the 111 In-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 111 In-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 111 In delivered by these MNTs was greatly enhanced on receptor-expressing cancer cells compared with 111 In nontargeted control. In mice with B16-F1 tumors, prolonged retention of 111 In 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 111 In-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 111 In-NOTA-MNT suggests further applications of the MNT delivery strategy should be explored., Competing Interests: The authors report no conflicts of interest in this work.