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Paolo Rainone,1– 3 Antonella De Palma,4 Francesco Sudati,5 Valentina Roffia,4 Valentina Rigamonti,6 Lucia Salvioni,6 Miriam Colombo,6 Marilena Ripamonti,2 Antonello Enrico Spinelli,7 Davide Mazza,7 Pierluigi Mauri,4 Rosa Maria Moresco,1,2,7 Davide Prosperi,6 Sara Belloli2,7 1Department of Medicine and Surgery, University of Milano-Bicocca, Monza, 20900, Italy; 2Institute of Molecular Bioimaging and Physiology of CNR, Segrate, 20090, Italy; 3Doctorate School of Molecular and Translational Medicine, University of Milan, Milan, Italy; 4Institute of Biomedical Technologies of CNR, Segrate, 20090, Italy; 5PET and Nuclear Medicine Unit, San Raffaele Scientific Institute, Milan, 20132, Italy; 6NanoBioLab, Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, 20126, Italy; 7Experimental Imaging Center, San Raffaele Scientific Institute, Milan, 20132, ItalyCorrespondence: Sara BelloliInstitute of Molecular Bioimaging and Physiology of CNR, Via Fratelli Cervi 93, Segrate, 20090, ItalyTel +39 02 26433640Fax +39 02 26432717Email belloli.sara@hsr.itIntroduction: The overexpression of Human Epidermal Growth Factor Receptor 2 (HER2) is usually associated with aggressive and infiltrating breast cancer (BC) phenotype, and metastases. Functionalized silica-based nanocarriers (SiNPs) can be labeled for in vivo imaging applications and loaded with chemotherapy drugs, making possible the simultaneous noninvasive diagnosis and treatment (theranostic) for HER2-positive BC.Methods: Firstly, FITC-filled SiNPs, were engineered with two different amounts of Hc-TZ (trastuzumab half-chain) per single nanoparticle (1:2 and 1:8, SiNPs to Hc-TZ ratio), which was 99mTc-radiolabeled at histidine residues for ex vivo and in vivo biodistribution evaluations. Secondly, nanoparticles were loaded with DOX and their in vitro and ex vivo/in vivo delivery was assessed, in comparison with liposomal Doxorubicin (Caelyx). Finally, the treatment efficacy of DOX-SiNPs-TZ (1

Paolo Rainone,1– 3 Antonella De Palma,4 Francesco Sudati,5 Valentina Roffia,4 Valentina Rigamonti,6 Lucia Salvioni,6 Miriam Colombo,6 Marilena Ripamonti,2 Antonello Enrico Spinelli,7 Davide Mazza,7 Pierluigi Mauri,4 Rosa Maria Moresco,1,2,7 Davide Prosperi,6 Sara Belloli2,7 1Department of Medicine and Surgery, University of Milano-Bicocca, Monza, 20900, Italy; 2Institute of Molecular Bioimaging and Physiology of CNR, Segrate, 20090, Italy; 3Doctorate School of Molecular and Translational Medicine, University of Milan, Milan, Italy; 4Institute of Biomedical Technologies of CNR, Segrate, 20090, Italy; 5PET and Nuclear Medicine Unit, San Raffaele Scientific Institute, Milan, 20132, Italy; 6NanoBioLab, Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, 20126, Italy; 7Experimental Imaging Center, San Raffaele Scientific Institute, Milan, 20132, ItalyCorrespondence: Sara BelloliInstitute of Molecular Bioimaging and Physiology of CNR, Via Fratelli Cervi 93, Segrate, 20090, ItalyTel +39 02 26433640Fax +39 02 26432717Email belloli.sara@hsr.itIntroduction: The overexpression of Human Epidermal Growth Factor Receptor 2 (HER2) is usually associated with aggressive and infiltrating breast cancer (BC) phenotype, and metastases. Functionalized silica-based nanocarriers (SiNPs) can be labeled for in vivo imaging applications and loaded with chemotherapy drugs, making possible the simultaneous noninvasive diagnosis and treatment (theranostic) for HER2-positive BC.Methods: Firstly, FITC-filled SiNPs, were engineered with two different amounts of Hc-TZ (trastuzumab half-chain) per single nanoparticle (1:2 and 1:8, SiNPs to Hc-TZ ratio), which was 99mTc-radiolabeled at histidine residues for ex vivo and in vivo biodistribution evaluations. Secondly, nanoparticles were loaded with DOX and their in vitro and ex vivo/in vivo delivery was assessed, in comparison with liposomal Doxorubicin (Caelyx). Finally, the treatment efficacy of DOX-SiNPs-TZ (1

TLQP-21 is a neuropeptide that is involved in the control of several physiological functions, including energy homeostasis. Since TLQP-21 could oppose the early phase of diet-induced obesity, it has raised a huge interest, but very little is known about its mechanisms of action on peripheral tissues. Our aim was to investigate TLQP-21 distribution in brain and peripheral tissues after systemic administration using positron emission tomography. We report here the radiolabeling of NODA-methyl phenylacetic acid (MPAA) functionalized JMV5763, a short analog of TLQP-21, with [F-18]aluminum fluoride. Labeling of JMV5763 was initially performed manually, on a small scale, and then optimized and implemented on a fully automated radiosynthesis system. In the first experiment, mice were injected in the tail vein with [F-18]JMV5763, and central and peripheral tissues were collected 13, 30, and 60 min after injection. Significant uptake of [F-18]JMV5763 was found in stomach, intestine, kidney, liver, and adrenal gland. In the CNS, very low uptake values were measured in all tested areas, suggesting that the tracer does not efficiently cross the blood-brain barrier. Pretreatment with nonradioactive JMV5763 caused a significant reduction of tracer uptake only in stomach and intestine. In the second experiment, PET analysis was performed in vivo 10-120 min after i.v. [F-18]JMV5763 administration. Results were consistent with those of the ex vivo determinations. PET images showed a progressive increase of [F-18]JMV5763 uptake in intestine and stomach reaching a peak at 30 min, and decreasing at 120 min. Our results demonstrate that F-18-labeling of TLQP-21 analogs is a suitable method to study its distribution in the body.

TLQP-21 is a neuropeptide that is involved in the control of several physiological functions, including energy homeostasis. Since TLQP-21 could oppose the early phase of diet-induced obesity, it has raised a huge interest, but very little is known about its mechanisms of action on peripheral tissues. Our aim was to investigate TLQP-21 distribution in brain and peripheral tissues after systemic administration using positron emission tomography. We report here the radiolabeling of NODA-methyl phenylacetic acid (MPAA) functionalized JMV5763, a short analog of TLQP-21, with [F-18]aluminum fluoride. Labeling of JMV5763 was initially performed manually, on a small scale, and then optimized and implemented on a fully automated radiosynthesis system. In the first experiment, mice were injected in the tail vein with [F-18]JMV5763, and central and peripheral tissues were collected 13, 30, and 60 min after injection. Significant uptake of [F-18]JMV5763 was found in stomach, intestine, kidney, liver, and adrenal gland. In the CNS, very low uptake values were measured in all tested areas, suggesting that the tracer does not efficiently cross the blood-brain barrier. Pretreatment with nonradioactive JMV5763 caused a significant reduction of tracer uptake only in stomach and intestine. In the second experiment, PET analysis was performed in vivo 10-120 min after i.v. [F-18]JMV5763 administration. Results were consistent with those of the ex vivo determinations. PET images showed a progressive increase of [F-18]JMV5763 uptake in intestine and stomach reaching a peak at 30 min, and decreasing at 120 min. Our results demonstrate that F-18-labeling of TLQP-21 analogs is a suitable method to study its distribution in the body.

The human epidermal growth factor receptor 2 (HER2) is normally associated with a highly aggressive and infiltrating phenotype in breast cancer lesions with propensity to spread into metastases. In clinic, the detection of HER2 in primary tumors and in their metastases is currently based on invasive methods. Recently, nuclear molecular imaging techniques, including positron emission tomography and single photon emission computed tomography (SPECT), allowed the detection of HER2 lesions in vivo. We have developed a99mTc-radiolabeled nanosilica system, functionalized with a trastuzumab half-chain, able to act as drug carrier and SPECT radiotracer for the identification of HER2-positive breast cancer cells. To this aim, nanoparticles functionalized or not with trastuzumab half-chain, were radiolabeled using the99mTc-tricarbonyl approach and evaluated in HER2 positive and negative breast cancer models. Cell uptake experiments, combined with flow cytometry and fluorescence imaging, suggested that active targeting provides higher efficiency and selectivity in tumor detection compared to passive diffusion, indicating that our radiolabeling strategy did not affect the nanoconjugate binding efficiency. Ex vivo biodistribution of99mTc-nanosilica in a SK-BR-3 (HER2+) tumor xenograft at 4 h postinjection was higher in targeted compared to nontargeted nanosilica, confirming the in vitro data. In addition, viability and toxicity tests provided evidence on nanoparticle safety in cell cultures. Our results encourage further assessment of silica99mTc-nanoconjugates to validate a safe and versatile nanoreporter system for both diagnosis and treatment of aggressive breast cancer.

Paolo Rainone,1,2,* Benedetta Riva,3,* Sara Belloli,1 Francesco Sudati,4 Marilena Ripamonti,1 Paolo Verderio,3 Miriam Colombo,3 Barbara Colzani,3 Maria Carla Gilardi,1 Rosa Maria Moresco,5 Davide Prosperi3 1Institute of Molecular Bioimaging and Physiology, CNR, Segrate (MI), 2Doctorate School of Molecular and Translational Medicine, University of Milan, Milan, 3NanoBioLab, Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milano, 4PET and Nuclear Medicine Unit, San Raffaele Scientific Institute, Milan, 5Department of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy *These authors contributed equally to this work Abstract: The human epidermal growth factor receptor 2 (HER2) is normally associated with a highly aggressive and infiltrating phenotype in breast cancer lesions with propensity to spread into metastases. In clinic, the detection of HER2 in primary tumors and in their metastases is currently based on invasive methods. Recently, nuclear molecular imaging techniques, including positron emission tomography and single photon emission computed tomography (SPECT), allowed the detection of HER2 lesions in vivo. We have developed a 99mTc-radiolabeled nanosilica system, functionalized with a trastuzumab half-chain, able to act as drug carrier and SPECT radiotracer for the identification of HER2-positive breast cancer cells. To this aim, nanoparticles functionalized or not with trastuzumab half-chain, were radiolabeled using the 99mTc-tricarbonyl approach and evaluated in HER2 positive and negative breast cancer models. Cell uptake experiments, combined with flow cytometry and fluorescence imaging, suggested that active targeting provides higher efficiency and selectivity in tumor detection compared to passive diffusion, indicating that our radiolabeling strategy did not affect the nanoconjugate binding efficiency. Ex vivo biodistribution of 99mTc-nanosilica in a SK-BR-3 (HER2+) tumor xenograft at 4 h postinjection wa

Paolo Rainone,1,2,* Benedetta Riva,3,* Sara Belloli,1 Francesco Sudati,4 Marilena Ripamonti,1 Paolo Verderio,3 Miriam Colombo,3 Barbara Colzani,3 Maria Carla Gilardi,1 Rosa Maria Moresco,5 Davide Prosperi3 1Institute of Molecular Bioimaging and Physiology, CNR, Segrate (MI), 2Doctorate School of Molecular and Translational Medicine, University of Milan, Milan, 3NanoBioLab, Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milano, 4PET and Nuclear Medicine Unit, San Raffaele Scientific Institute, Milan, 5Department of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy *These authors contributed equally to this work Abstract: The human epidermal growth factor receptor 2 (HER2) is normally associated with a highly aggressive and infiltrating phenotype in breast cancer lesions with propensity to spread into metastases. In clinic, the detection of HER2 in primary tumors and in their metastases is currently based on invasive methods. Recently, nuclear molecular imaging techniques, including positron emission tomography and single photon emission computed tomography (SPECT), allowed the detection of HER2 lesions in vivo. We have developed a 99mTc-radiolabeled nanosilica system, functionalized with a trastuzumab half-chain, able to act as drug carrier and SPECT radiotracer for the identification of HER2-positive breast cancer cells. To this aim, nanoparticles functionalized or not with trastuzumab half-chain, were radiolabeled using the 99mTc-tricarbonyl approach and evaluated in HER2 positive and negative breast cancer models. Cell uptake experiments, combined with flow cytometry and fluorescence imaging, suggested that active targeting provides higher efficiency and selectivity in tumor detection compared to passive diffusion, indicating that our radiolabeling strategy did not affect the nanoconjugate binding efficiency. Ex vivo biodistribution of 99mTc-nanosilica in a SK-BR-3 (HER2+) tumor xenograft at 4 h postinjection wa

The human epidermal growth factor receptor 2 (HER2) is normally associated with a highly aggressive and infiltrating phenotype in breast cancer lesions with propensity to spread into metastases. In clinic, the detection of HER2 in primary tumors and in their metastases is currently based on invasive methods. Recently, nuclear molecular imaging techniques, including positron emission tomography and single photon emission computed tomography (SPECT), allowed the detection of HER2 lesions in vivo. We have developed a99mTc-radiolabeled nanosilica system, functionalized with a trastuzumab half-chain, able to act as drug carrier and SPECT radiotracer for the identification of HER2-positive breast cancer cells. To this aim, nanoparticles functionalized or not with trastuzumab half-chain, were radiolabeled using the99mTc-tricarbonyl approach and evaluated in HER2 positive and negative breast cancer models. Cell uptake experiments, combined with flow cytometry and fluorescence imaging, suggested that active targeting provides higher efficiency and selectivity in tumor detection compared to passive diffusion, indicating that our radiolabeling strategy did not affect the nanoconjugate binding efficiency. Ex vivo biodistribution of99mTc-nanosilica in a SK-BR-3 (HER2+) tumor xenograft at 4 h postinjection was higher in targeted compared to nontargeted nanosilica, confirming the in vitro data. In addition, viability and toxicity tests provided evidence on nanoparticle safety in cell cultures. Our results encourage further assessment of silica99mTc-nanoconjugates to validate a safe and versatile nanoreporter system for both diagnosis and treatment of aggressive breast cancer.