177Lu-Labeled Iron Oxide Nanoparticles Functionalized with Doxorubicin and Bevacizumab as Nanobrachytherapy Agents against Breast Cancer.

The use of conventional methods for the treatment of cancer, such as chemotherapy or radiotherapy, and approaches such as brachytherapy in conjunction with the unique properties of nanoparticles could enable the development of novel theranostic agents. The aim of our current study was to evaluate the potential of iron oxide nanoparticles, coated with alginic acid and polyethylene glycol, functionalized with the chemotherapeutic agent doxorubicin and the monoclonal antibody bevacizumab, to serve as a nanoradiopharmaceutical agent against breast cancer. Direct radiolabeling with the therapeutic isotope Lutetium-177 (177Lu) resulted in an additional therapeutic effect. Functionalization was accomplished at high percentages and radiolabeling was robust. The high cytotoxic effect of our radiolabeled and non-radiolabeled nanostructures was proven in vitro against five different breast cancer cell lines. The ex vivo biodistribution in tumor-bearing mice was investigated with three different ways of administration. The intratumoral administration of our functionalized radionanoconjugates showed high tumor accumulation and retention at the tumor site. Finally, our therapeutic efficacy study performed over a 50-day period against an aggressive triple-negative breast cancer cell line (4T1) demonstrated enhanced tumor growth retention, thus identifying the developed nanoparticles as a promising nanobrachytherapy agent against breast cancer.

Improvement of the Effectiveness of HER2+ Cancer Therapy by Use of Doxorubicin and Trastuzumab Modified Radioactive Gold Nanoparticles.

In the present article, we describe a multimodal radiobioconjugate that contains a chemotherapeutic agent (doxorubicin, DOX), a ß-emitter (198Au), and a guiding vector (trastuzumab, Tmab) for targeted therapy of cancers overexpressing HER2 receptors. To achieve this goal, radioactive gold nanoparticles (198AuNPs) with a mean diameter of 30 nm were synthesized and coated with a poly(ethylene glycol) (PEG) linker conjugated to DOX and monoclonal antibody (Tmab) via peptide bond formation. In vitro experiments demonstrated a high affinity of the radiobioconjugate to HER2 receptors and cell internalization. Cytotoxicity experiments performed using the MTS assay showed a significant decrease in the viability of SKOV-3 cells. A synergistic cytotoxic effect due to the simultaneous presence of DOX and 198Au was revealed after 48 h of treatment with 2.5 MBq/mL. Flow cytometry analysis indicated that DOX-198AuNPs-Tmab mainly induced cell cycle arrest in the G2/M phase and late apoptosis. Dose-dependent additive and synergistic effects of the radiobioconjugate were also shown in spheroid models. Ex vivo biodistribution experiments were performed in SKOV-3 tumor-bearing mice, investigating different distributions of the 198AuNPs-DOX and DOX-198AuNPs-Tmab after intravenous (i.v.) and intratumoral (i.t.) administration. Finally, in vivo therapeutic efficacy studies on the same animal model demonstrated very promising results, as they showed a significant tumor growth arrest up to 28 days following a single intratumoral injection of 10 MBq. Therefore, the proposed multimodal radiobioconjugate shows great potential for the local treatment of HER2+ cancers.

Old Drug, New Delivery Strategy: MMAE Repackaged.

Targeting therapy is a concept that has gained significant importance in recent years, especially in oncology. The severe dose-limiting side effects of chemotherapy necessitate the development of novel, efficient and tolerable therapy approaches. In this regard, the prostate specific membrane antigene (PSMA) has been well established as a molecular target for diagnosis of, as well as therapy for, prostate cancer. Although most PSMA-targeting ligands are radiopharmaceuticals used in imaging or radioligand therapy, this article evaluates a PSMA-targeting small molecule-drug conjugate, and, thus, addresses a hitherto little-explored field. PSMA binding affinity and cytotoxicity were determined in vitro using cell-based assays. Enzyme-specific cleavage of the active drug was quantified via an enzyme-based assay. Efficacy and tolerability in vivo were assessed using an LNCaP xenograft model. Histopathological characterization of the tumor in terms of apoptotic status and proliferation rate was carried out using caspase-3 and Ki67 staining. The binding affinity of the Monomethyl auristatin E (MMAE) conjugate was moderate, compared to the drug-free PSMA ligand. Cytotoxicity in vitro was in the nanomolar range. Both binding and cytotoxicity were found to be PSMA-specific. Additionally, complete MMAE release could be reached after incubation with cathepsin B. In vivo, the MMAE conjugate displayed good tolerability and dose-dependent inhibition of tumor growth. Immunohistochemical and histological studies revealed the antitumor effect of MMAE.VC.SA.617, resulting in the inhibition of proliferation and the enhancement of apoptosis. The developed MMAE conjugate showed good properties in vitro, as well as in vivo, and should, therefore, be considered a promising candidate for a translational approach.

Preliminary Evaluation of Iron Oxide Nanoparticles Radiolabeled with 68Ga and 177Lu as Potential Theranostic Agents.

Theranostic radioisotope pairs such as Gallium-68 (68Ga) for Positron Emission Tomography (PET) and Lutetium-177 (177Lu) for radioisotopic therapy, in conjunction with nanoparticles (NPs), are an emerging field in the treatment of cancer. The present work aims to demonstrate the ability of condensed colloidal nanocrystal clusters (co-CNCs) comprised of iron oxide nanoparticles, coated with alginic acid (MA) and stabilized by a layer of polyethylene glycol (MAPEG) to be directly radiolabeled with 68Ga and its therapeutic analog 177Lu. 68Ga/177Lu- MA and MAPEG were investigated for their in vitro stability. The biocompatibility of the non-radiolabeled nanoparticles, as well as the cytotoxicity of MA, MAPEG, and [177Lu]Lu-MAPEG were assessed on 4T1 cells. Finally, the ex vivo biodistribution of the 68Ga-labeled NPs as well as [177Lu]Lu-MAPEG was investigated in normal mice. Radiolabeling with both radioisotopes took place via a simple and direct labelling method without further purification. Hemocompatibility was verified for both NPs, while MTT studies demonstrated the non-cytotoxic profile of the nanocarriers and the dose-dependent toxicity for [177Lu]Lu-MAPEG. The radiolabeled nanoparticles mainly accumulated in RES organs. Based on our preliminary results, we conclude that MAPEG could be further investigated as a theranostic agent for PET diagnosis and therapy of cancer.

Synthesis and in vitro proof-of-concept studies on bispecific iron oxide magnetic nanoparticles targeting PSMA and GRP receptors for PET/MR imaging of prostate cancer.

Prostate cancer (PCa) is the most common malignancy worldwide in men. This is a proof-of-concept study describing the development of 68Ga-magnetic iron oxide nanoparticles (mNP) targeting prostate specific membrane antigen (PSMA) and gastrin releasing peptide (GRPR) receptors as potential tools for diagnosis of PCa with PET/MRI. Two pharmacophores targeting PSMA, 1, and GRPR, 2, were coupled to mNPs carrying -SH (mNP-S1/2) or -NH2 (mNP-N1/2) groups. The mNP-S1/2 and mNP-N1/2 were characterized for their size, zeta potential, structure, and efficiency of functionalization using dynamic light scattering (DLS), FT-IR and RP-HPLC. A direct 68Ga-labelling procedure was followed, where 68Ga-mNP-N1/2 proved superior to 68Ga-mNP-S1/2 regarding radiolabelling efficiency, and thus were further evaluated in vitro. Toxicity studies in PCa cells (LNCaP, PC-3) showed low toxicity, and minimal hemolysis of red blood cells. In vitro assays in cells expressing PSMA (LNCaP), and GRPR (PC-3), showed specific time-dependent binding (40 min to plateau), high avidity (PC-3: Kd = 28.27 nM, LNCaP: Kd = 11.49 nM) and high internalization rates for 68Ga-mNP-N1/2 in both cell lines.

Biodistribution of Mesoporous Carbon Nanoparticles via Technetium-99m Radiolabelling after Oral Administration to Mice.

The use of ordered mesoporous matrices, and in particular carbon-based mesoporous nanoparticles has shown great potential towards enhancing the bioavailability of orally administered drugs. Nevertheless, elucidation of the in vivo absorption, distribution, and excretion of such carriers is essential for understanding their behaviour, and radiolabelling provides a very useful way to track their occurrence inside the body. In this work, uniform spherical CMK-1-type ordered mesoporous carbon nanoparticles have been radiolabelled with Technetium-99m (99mTc) and traced after oral administration to mice. Ex vivo biodistribution studies showed that the radiolabelled nanoparticles accumulated almost exclusively in the gastrointestinal tract; complete elimination of the radiotracer was observed within 24 h after administration, with practically no uptake into other main organs. These findings along with the results from in vitro stability studies indicate that the spherical carbon nanoparticles examined could be safely used as drug carriers with minimal side effects, but also support the great value of radiolabelling methods for monitoring the particles' behaviour in vivo.

Chelator-Free/Chelator-Mediated Radiolabeling of Colloidally Stabilized Iron Oxide Nanoparticles for Biomedical Imaging.

The aim of this study was to develop a bioimaging probe based on magnetic iron oxide nanoparticles (MIONs) surface functionalized with the copolymer (p(MAA-g-EGMA)), which were radiolabeled with the positron emitter Gallium-68. The synthesis of the hybrid MIONs was realized by hydrolytic condensation of a single ferrous precursor in the presence of the copolymer. The synthesized MagP MIONs displayed an average Dh of 87 nm, suitable for passive targeting of cancerous tissues through the enhanced permeation and retention (EPR) effect after intravenous administration, while their particularly high magnetic content ascribes strong magnetic properties to the colloids. Two different approaches were explored to develop MIONs radiolabeled with 68Ga: the chelator-mediated approach, where the chelating agent NODAGA-NHS was conjugated onto the MIONs (MagP-NODAGA) to form a chelate complex with 68Ga, and the chelator-free approach, where 68Ga was directly incorporated onto the MIONs (MagP). Both groups of NPs showed highly efficient radiolabeling with 68Ga, forming constructs which were stable with time, and in the presence of PBS and human serum. Ex vivo biodistribution studies of [68Ga]Ga- MIONs showed high accumulation in the mononuclear phagocyte system (MPS) organs and satisfactory blood retention with time. In vivo PET imaging with [68Ga]Ga-MagP MIONs was in accordance with the ex vivo biodistribution results. Finally, the MIONs showed low toxicity against 4T1 breast cancer cells. These detailed studies established that [68Ga]Ga- MIONs exhibit potential for application as tracers for early cancer detection.

Synthesis and In Vitro Evaluation of Gold Nanoparticles Functionalized with Thiol Ligands for Robust Radiolabeling with 99mTc.

Radiolabeled gold nanoparticles (AuNPs) have been widely used for cancer diagnosis and therapy over recent decades. In this study, we focused on the development and in vitro evaluation of four new Au nanoconjugates radiolabeled with technetium-99m (99mTc) via thiol-bearing ligands attached to the NP surface. More specifically, AuNPs of two different sizes (2 nm and 20 nm, referred to as Au(2) and Au(20), respectively) were functionalized with two bifunctional thiol ligands (referred to as L1H and L2H). The shape, size, and morphology of both bare and ligand-bearing AuNPs were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques. In vitro cytotoxicity was assessed in 4T1 murine mammary cancer cells. The AuNPs were successfully radiolabeled with 99mTc-carbonyls at high radiochemical purity (>95%) and showed excellent in vitro stability in competition studies with cysteine and histidine. Moreover, lipophilicity studies were performed in order to determine the lipophilicity of the radiolabeled conjugates, while a hemolysis assay was performed to investigate the biocompatibility of the bare and functionalized AuNPs. We have shown that the functionalized AuNPs developed in this study lead to stable radiolabeled nanoconstructs with the potential to be applied in multimodality imaging or for in vivo tracking of drug-carrying AuNPs.

A Proof-of-Concept Study on the Therapeutic Potential of Au Nanoparticles Radiolabeled with the Alpha-Emitter Actinium-225.

Actinium-225 (225Ac) is receiving increased attention for its application in targeted radionuclide therapy, due to the short range of its emitted alpha particles in conjunction with their high linear energy transfer, which lead to the eradication of tumor cells while sparing neighboring healthy tissue. The objective of our study was the evaluation of a gold nanoparticle radiolabeled with 225Ac as an injectable radiopharmaceutical form of brachytherapy for local radiation treatment of cancer. Au@TADOTAGA was radiolabeled with 225Ac at pH 5.6 (30 min at 70 °C), and in vitro stability was evaluated. In vitro cytotoxicity was assessed in U-87 MG cancer cells, and in vivo biodistribution was performed by intravenous and intratumoral administration of [225Ac]225Ac-Au@TADOTAGA in U-87 MG tumor-bearing mice. A preliminary study to assess therapeutic efficacy of the intratumorally-injected radio-nanomedicine was performed over a period of 22 days, while the necrotic effect on tumors was evaluated by a histopathology study. We have shown that [225Ac]225Ac-Au@TADOTAGA resulted in the retardation of tumor growth after its intratumoral injection in U87MG tumor-bearing mice, even though very low activities were injected per mouse. This gold nanoparticle radiopharmaceutical could be applied as an unconventional brachytherapy in injectable form for local radiation treatment of cancer.