Barium ferrite nanoparticles (BaFeNPs) were investigated as vehicles for 223Ra radionuclide in targeted α-therapy. BaFe nanoparticles were labeled using a hydrothermal Ba2+ cations replacement by 223Ra with yield reaching 61.3 ± 1.8%. Radiolabeled nanoparticles were functionalized with 3-phosphonopropionic acid (CEPA) linker followed by covalent conjugation to trastuzumab (Herceptin®). Thermogravimetric analysis and radiometric method with the use of [131I]-labeled trastuzumab revealed that on average 19–21 molecules of trastuzumab are attached to the surface of one BaFe–CEPA nanoparticle. The hydrodynamic diameter of BaFe–CEPA–trastuzumab conjugate is 99.9 ± 3.0 nm in water and increases to 218.3 ± 3.7 nm in PBS buffer, and the zeta potential varies from +27.2 ± 0.7 mV in water to −8.8 ± 0.7 in PBS buffer. The [223Ra]BaFe–CEPA–trastuzumab radiobioconjugate almost quantitatively retained 223Ra (>98%) and about 96% of 211Bi and 94% of 211Pb over 30 days. The obtained radiobioconjugate exhibited high affinity, cell internalization and cytotoxicity towards the human ovarian adenocarcinoma SKOV-3 cells overexpressing HER2 receptor. Confocal studies indicated that [223Ra]BaFe–CEPA–trastuzumab was located in peri-nuclear space. High cytotoxicity of the [223Ra]BaFe–CEPA–trastuzumab bioconjugate was confirmed by radiotoxicity studies on SKOV-3 cell monolayers and 3D-spheroids. In addition, the magnetic properties of the radiobioconjugate should allow for its use in guide drug delivery driven by magnetic field gradient.

It has been proven and confirmed in numerous repeated tests, that the use of a combination of several therapeutic methods gives much better treatment results than in the case of separate therapies. Particularly promising is the combination of ionizing radiation and magnetic hyperthermia in one drug. To achieve this objective, magnetite nanoparticles have been modified in their core with α emitter 225Ac, in an amount affecting only slightly their magnetic properties. By 3-phosphonopropionic acid (CEPA) linker nanoparticles were conjugated covalently with trastuzumab (Herceptin®), a monoclonal antibody that recognizes ovarian and breast cancer cells overexpressing the HER2 receptors. The synthesized bioconjugates were characterized by transmission electron microscopy (TEM), Dynamic Light Scattering (DLS) measurement, thermogravimetric analysis (TGA) and application of 131I-labeled trastuzumab for quantification of the bound biomolecule. The obtained results show that one 225Ac@Fe3O4-CEPA-trastuzumab bioconjugate contains an average of 8–11 molecules of trastuzumab. The labeled nanoparticles almost quantitatively retain 225Ac (>98%) in phosphate-buffered saline (PBS) and physiological salt, and more than 90% of 221Fr and 213Bi over 10 days. In human serum after 10 days, the fraction of 225Ac released from 225Ac@Fe3O4 was still less than 2%, but the retention of 221Fr and 213Bi decreased to 70%. The synthesized 225Ac@Fe3O4-CEPA-trastuzumab bioconjugates have shown a high cytotoxic effect toward SKOV-3 ovarian cancer cells expressing HER2 receptor in-vitro. The in-vivo studies indicate that this bioconjugate exhibits properties suitable for the treatment of cancer cells by intratumoral or post-resection injection. The intravenous injection of the 225Ac@Fe3O4-CEPA-trastuzumab radiobioconjugate is excluded due to its high accumulation in the liver, lungs and spleen. Additionally, the high value of a specific absorption rate (SAR) allows its use in a new very perspective combination of α radionuclide therapy with magnetic hyperthermia.

Nanobodies (Nbs) are the smallest antibody-derived fragments with beneficial pharmacokinetic properties for molecular imaging and radionuclide therapy. Human Epidermal Growth Factor Receptor type 2 (HER2) is overexpressed in numerous carcinomas and portends a poor prognosis. Therefore, HER2-targeting nanobodies are very attractive vectors for TRT, especially when labeled with α-particle emitters. The aim of this study was to evaluate the therapeutic potential of the anti-HER2 Nb 2Rs15d labeled with 225Ac. Anti-HER2 Nb 2Rs15d was coupled with the bifunctional chelate p-SCN-Bn-DOTA and further was labeled with 225Ac. Its binding affinity and specificity for HER2, together with immunoreactive fraction (IF), were evaluated on SKOV-3 (HER2+) and MDA-MB-231 (HER2-) cells. Its in vitro cytotoxicity was assessed using MTS and clonogenic assays. In vivo, 225Ac-DOTA-Nb 2Rs15d and a non-targeting control 225Ac-DOTA-Nb R3b23 were evaluated in female athymic nude mice subcutaneously xenografted with SKOV-3 and MDAMB-231 tumors, both alone and with molar excess of unlabeled 2Rs15d. After determination of maximum tolerated dose (MTD), the therapeutic efficacy of 225Ac-DOTA-Nb 2Rs15d was investigated in mice bearing intraperitoneal SKOV-3.IP1/luciferase+ xenografted metastases against several controls, a trastuzumab regimen and a combination of both 225Ac-DOTA-Nb 2Rs15d and trastuzumab. The yield of DOTA-Nb 2Rs15d labeling was high (>90%), with radiochemical purity ≥95%. 225Ac-DOTA-Nb 2Rs15d bound specifically to HER2+ cells with ∼75% IF, a KD of 3.50±0.17nM and lack of competition with trastuzumab or pertuzumab in vitro. Cytotoxicity studies demonstrated that 225Ac-DOTA-Nb 2Rs15d significantly reduced SKOV-3 cell viability in a dose-dependent and HER2-mediated manner, compared to 225Ac-DOTA or 225Ac-DOTA-Nb R3b23 as controls. Tumor uptake in SKOV-3 xenografted mice was high and specific (∼8%), whereas in MDA-MB-231 was <0.5% already 1h pi. Its accumulation in kidneys was reduced almost 3-fold by coinjection 225Ac-DOTA-Nb 2Rs15d with 150 mg/kg Gelofusine. Therapy studies indicated that 225Ac-DOTA-Nb 2Rs15d increased Median Survival significantly, which measured 83 days compared to about 49 days for animals treated with controls PBS and 225Ac-DOTA-Nb R3b23, and to 72 days in case of trastuzumab regimen. The most extensive therapeutic effect (MD∼97 days) was observed for the combination of both 225Ac-DOTA-Nb 2Rs15d and trastuzumab. 225Ac-DOTA-Nb 2Rs15d efficiently targets HER2+ cells both in vitro and in vivo. Strong signs of therapeutic potential were observed in vitro, which were confirmed also at in vivo setting in mice bearing SKOV-3 xenografts. This study underlines the strong potential of 225Ac-DOTA-Nb 2Rs15d as a new radioconjugate for TAT and supports its further development towards the clinic.

The 225Ac radioisotope exhibits very attractive nuclear properties for application in radionuclide therapy. Unfortunately, the major challenge for radioconjugates labelled with 225Ac is that traditional chelating moieties are unable to sequester the radioactive daughters in the bioconjugate which is critical to minimize toxicity to healthy, non-targeted tissues. In the present work, we propose to apply TiO2 nanoparticles (NPs) as carrier for 225Ac and its decay products. The surface of TiO2 nanoparticles with 25 nm diameter was modified with Substance P (5-11), a peptide fragment which targets NK1 receptors on the glioma cells, through the silan-PEG-NHS linker. Nanoparticles functionalized with Substance P (5-11) were synthesized with high yield in a two-step procedure, and the products were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and thermogravimetric analysis (TGA). The obtained results show that one TiO2-bioconjugate nanoparticle contains in average 80 peptide molecules on its surface. The synthesized TiO2-PEG-SP(5-11) conjugates were labelled with 225Ac by ion-exchange reaction on hydroxyl (OH) functional groups on the TiO2 surface. The labelled bioconjugates almost quantitatively retain 225Ac in phosphate-buffered saline (PBS), physiological salt and cerebrospinal fluid (CSF) for up to 10 days. The leaching of 221Fr, a first decay daughter of 225Ac, in an amount of 30% was observed only in CSF after 10 days. The synthesized 225Ac-TiO2-PEG-SP(5-11) has shown high cytotoxic effect in vitro in T98G glioma cells; therefore, it is a promising new radioconjugate for targeted radionuclide therapy of brain tumours.

Introduction: Alpha particle emitting isotopes are of considerable interest for radionuclide therapy because of their high cytotoxicity and short path length. Among the many alpha emitters, Ra-223 exhibits very attractive nuclear properties for application in radionuclide therapy. The decay of this radioisotope and its daughters is accompanied by the emission of four alpha-particles, releasing 27.9 MeV of cumulative energy. Unfortunately the lack of an appropriate bifunctional ligand for radium has so far been a main obstacle for the application of Ra-223 in receptor targeted alpha therapy. In our studies we investigated the use of nanozeolite-Substance P bioconjugates as vehicles for Ra-223 radionuclides for targeted alpha therapy.Methods: The sodium form of an A-type of nanozeolite (NaA) was synthesized using the template method. Next, the nanozeolite particles were conjugated to the Substance P (5-11) peptide fragment, which targets NK-1 receptors on glioma cells. The obtained bioconjugate was characterized by transmission emission spectroscopy, thermogravimetric analysis and dynamic light scattering analysis. The NaA-silane-PEG-SP(5-11) bioconjugates were labeled with Ra-223 by exchange of the Na+ cation and the stability, receptor affinity and cytotoxicity of the obtained radiobioconjugates were tested.Results: The Ra-223-labeled nanozeolite bioconjugate almost quantitatively retains Ra-223 in vitro after 6 days, while the retention of decay products varies from 90 to 95%. The synthesized (223)RaA-silane-PEG-SP(5-11) showed high receptor affinity toward NK-1 receptor expressing glioma cells and exhibited a high cytotoxic effect in vitro.Conclusions: Substance P functionalized nanozeolite-A represents a viable solution for the use of the Ra-223 in vivo generator as a therapeutic construct for targeting glioma cells. (C) 2016 Elsevier Inc. All rights reserved.

Targeted radiotherapies maximize cytotoxicity to cancer cells. In this work, we describe the synthesis, characterization, and biodistribution of antibody conjugated gold-coated lanthanide phosphate nanoparticles containing 177Lu. [177Lu]Lu0.5Gd0.5(PO4)@Au@PEG800@Ab nanoparticles combine the radiation resistance of crystalline lanthanide phosphate for stability, the magnetic properties of gadolinium for facile separations, and a gold coating that can be readily functionalized for the attachment of targeting moieties. In contrast to current targeted radiotherapeutic pharmaceuticals, the nanoparticle-antibody conjugate can target and deliver multiple beta radiations to a single biologically relevant receptor. Up to 95% of the injected dose was delivered to the lungs using the monoclonal antibody mAb-201b to target the nanoparticles to thrombomodulin receptors. The 208 keV gamma ray from 177Lu decay (11%) can be used for SPECT imaging of the radiotherapeutic agent, while the moderate energy beta emitted in the decay can be highly effective in treating metastatic disease.

The At-211 radioisotope exhibits very attractive nuclear properties for application in radionuclide therapy. Unfortunately use of At-211 is limited, because astatine as the heaviest halogen forms weak bond with carbon atoms in the biomolecules which makes At-211 bioconjugates unstable in physiological conditions. In our work we propose a new solution for binding of At-211 which consists of using nanoparticles of titanium dioxide modified with silver atoms as carriers for At-211. Ag+ cations have been absorbed on the nanometer-sized TiO2 particles (15 and 32 nm) through ion exchange process and were reduced in Tollens' reaction. The obtained TiO2-Ag nanoparticles were labeled with At-211. It was found that labeling yields were almost quantitative under reducing conditions, while under oxidizing conditions they dropped to about 80%. The labeled nanoparticles exhibited very high stability in physiological salt, PBS buffer, solutions of peptides (0.001 M cysteine, 0.001 M glutathione) and in human blood serum. To make TiO2/Ag nanoparticles well dispersed in water and biocompatible their surface was modified with a silane coupling agent containing poly(ethyleneglycol) molecules. The developed functionalization approach will allow us to attach biomolecules to the TiO2/Ag surface.