Evaluation of Rhenium and Technetium-99m Complexes Bearing Quinazoline Derivatives as Potential EGFR Agents.

Τhe Epidermal Growth Factor Receptor tyrosine kinase inhibitor (EGFR-TKI) 6-amino-4-[(3-bromophenyl) amino]quinazoline was derivatized with 6-bromohexanoyl-chloride and coupled with the tridentate chelating agents N-(2-pyridylmethyl) aminoethyl acetic acid (PAMA) and L(+)-cysteine bearing the donor atom set NNO and SNO, respectively. The rhenium precursors ReBr(CO)5 and fac-[NEt4]2[ReBr3(CO)3] were used for the preparation of the Re complexes fac-[Re(NNO)(CO)3] (5a) and fac-[Re(SNO)(CO)3] (7a) which were characterized by NMR and IR spectroscopies. Subsequently, the new potential EGFR inhibitors were labeled with the fac-[99mTc(CO)3]+ core in high yield and radiochemical purity (>90%) by ligand exchange reaction using the fac-[99mTc][Tc(OH2)3(CO)3]+ precursor. The radiolabeled complexes were characterized by comparative HPLC analysis with the analogous rhenium (Re) complexes as references. In vitro studies in the A431 cell lines showed that both ligands and Re complexes inhibit A431 cell growth. Complex 5a demonstrated the highest potency (IC50 = 8.85 ± 2.62 µM) and was further assessed for its capacity to inhibit EGFR autophosphorylation, presenting an IC50 value of 26.11 nM. Biodistribution studies of the 99mTc complexes in healthy mice showed high in vivo stability for both complexes and fast blood and soft tissue clearance with excretion occurring via the hepatobiliary system.

Synthesis and preclinical evaluation of rhenium and technetium-99m "4 + 1" mixed-ligand complexes bearing quinazoline derivatives as potential EGFR imaging agents.

Epidermal growth factor receptors (EGFR) of tyrosine kinase (TK) have shown high expression levels in most cancers and are considered a promising target for cancer diagnosis and therapy. Expanding the investigation for novel targeted radiopharmaceuticals, an EGFR inhibitor such as 4-aminoquinazoline derivatives along with a radionuclide such as technetium-99m (99mTc) could be ideal. Thus, we report herein the synthesis, characterization, and biological evaluation of new "4 + 1" mixed-ligand ReIII- and 99mTcIII-complexes of the general formula [99mTc][Tc(NS3)(CN-R)] bearing tris(2-mercaptoethyl)-amine (NS3) as the tetradentate tripodal ligand and a series of isocyanide derivatives (CN-R) of tyrosine kinase inhibitor (3-bromophenyl)quinazoline-4,6-diamine as the monodentate ligand. The quinazoline isocyanide derivatives 4a-d were prepared in two steps and reacted with the [Re(NS3)PMe2Ph] precursor leading to the final complexes 5a-d in high yield. All compounds were characterized by elemental analysis, IR, and NMR spectroscopies. In vitro studies, for their potency to inhibit the cell growth, using intact A431 cells indicate that the quinazoline derivatives 4a-d and the Re complexes 5a-d significantly inhibit the A431 cell growth. In addition, the EGFR autophosphorylation study of complex 5b shows an IC50 value in the nanomolar range. The corresponding "4 + 1" 99mTc-complexes 6a-d were prepared by employing the [99mTc]TcEDTA intermediate and the appropriate monodentate 4a-d in a two-step synthetic procedure with a radiochemical yield (RCY) from 63 to 77 % and a radiochemical purity (RCP) > 99 % after HPLC purification. Their structures have been established by HPLC comparative studies using the well-characterized Re-complexes 5a-d as reference. All 99mTc-complexes remain stable for at least 6 h, and their logD7.4 values confirmed their anticipated lipophilic character. Biodistribution studies in healthy Swiss albino mice of 99mTc-complexes showed hepatobiliary excretion and initial fast blood clearance. Complex 6b was also tested in Albino SCID mice bearing A431 tumors and showed rapid tumor uptake at 5 min (2.80 % ID/g) with a moderate tumor/muscle ratio (2.06) at 4 h p.i. The results encourage further investigation for this type of 99mTc-complexes as single-photon emission computed tomography (SPECT) radio agents for imaging tumors overexpressing EGFR.

Synthesis, characterization and evaluation of 68Ga labelled monomeric and dimeric quinazoline derivatives of the HBED-CC chelator targeting the epidermal growth factor receptor.

Tyrosine kinase (TK) receptors including epidermal growth factor receptors (EGFRs) are known to be overexpressed in a wide variety of solid tumors associated with poor prognosis. The HBED-CC chelator N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid 1 was coupled via one or both its propionic acid moieties with the quinazoline EGFR-TK inhibiting pharmacophore 4-amino-N-(4-((3-bromophenyl)amino)quinazolin-6-yl)butanamide 3 resulting in either a monomeric 4 or a dimeric 5 species. Ligands 4 and 5 reacted with Ga3+ generating the corresponding complexes Ga4 and Ga5. Both ligands and complexes were characterized with mass spectrometry and NMR spectroscopy and evaluated in vitro with MTT assays in A431 cells, where they showed IC50 values in the range 51.6 to 68.8 µM. Labeling of ligands 4 and 5 with the PET radionuclide 68Ga was quantitative and resulted in tracers [68Ga]Ga4 and [68Ga]Ga5 with radiochemical purities greater than 98%, which were also characterised by comparative RP-HPLC studies with Ga4 and Ga5 respectively. Radiotracers [68Ga]Ga4 and [68Ga]Ga5 were stable (intact tracer over 98%) in the reaction mixture (120 min) and in human serum (30 min). Both tracers were evaluated in vivo with biodistribution experiments in SCID mice bearing A431 tumors presenting tumor uptake of 1.34 for [68Ga]Ga4 and 1.01 %ID/g for [68Ga]Ga5 at 5 min, which was slightly decreased at 60 min p.i. and then remained stable until 120 min p.i. To the best of our knowledge, this is the first report of monomeric and dimeric quinazoline conjugates with the chelator HBED-CC, which can serve as a basis for further development of EGFR-TKI targeting tracers.

New labeled derivatives of the neuroprotective peptide colivelin: synthesis, characterization, and first in vitro and in vivo applications.

Colivelin (CL), first reported in 2005, is the most potent member of the humanin family of neuroprotective peptides with in vitro and in vivo rescuing action against insults associated with Alzheimer's disease (AD). The objective of the present work is the design, synthesis and characterization of specific CL derivatives that can be used as molecular probes in the investigation of the unknown mechanism of CL action. Within this framework, three CL derivatives bearing suitable tags, i.e., the fluorescent moiety FITC, the streptavidin-counterpart biotinyl-group, and the (99m)Tc-radiometal chelating unit dimethylGly-Ser-Cys, were developed and subsequently applied in biological evaluation experiments. Specifically, the FITC-labeled derivative of CL was used in confocal microscopy, where specific binding at the periphery of F11 cells was observed; the biotin-labeled derivative of CL was used in an in-house developed ELISA-type assay, where specific and concentration-dependent binding with the ß-amyloid peptide of AD was shown; finally, the (99m)Tc-radiolabeled derivative of CL was used in in vivo biodistribution studies in healthy Swiss Albino mice, where 0.58% of the radioactivity administered was measured in the mouse brain 2min after injection. The above first successful applications of the CL probes demonstrate their potential to contribute in the field of neuroprotective peptides.

Radiochemical and radiobiological assessment of a pyridyl-S-cysteine functionalized bombesin derivative labeled with the (99m)Tc(CO)3(+) core.

Bombesin is a neuropeptide widely studied due to its ability to target various types of cancers. Technetium-99m on the other hand is ideal for diagnostic tumor targeting. The aim of the present study is the investigation of the coupling of the ligand (S)-(2-(2'-pyridyl)ethyl)-d,l-cysteine with the BN-peptide Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met(CONH2) through the spacer aminohexanoic acidand the labeling of the resulting derivative MBN with the synthon [M(CO)3(H2O)3](+) (M=(99m)Tc, Re). The peptide was synthesized according to the SPPS method, purified and characterized by ESI-MS. The new (99m)Tc-labeled biomolecule was stable in vitro, showed high affinity for the human GRP receptor expressed in PC3 cells and the rate of internalization was found to be time-dependent tissue distribution of the radiopeptide was evaluated in normal mice and in prostate cancer experimental models and significant radioactivity uptake was observed in the pancreas of normal mice as well as in PC3 tumors. Dynamic studies of the radiopeptide showed satisfactory tumor images.

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.

Unsymmetrical, monocarboxyalkyl meso-arylporphyrins in the photokilling of breast cancer cells using permethyl-ß-cyclodextrin as sequestrant and cell uptake modulator.

In the search for photosensitizers with chemical handles to facilitate their integration into complex drug delivery nanosystems, new, unsymmetrically substituted, water insoluble meso-tetraphenylporphyrin and meso-tetra(m-hydroxyphenyl)porphyrin derivatives bearing one carboxyalkyl side chain were synthesized. Permethyl-ß-cyclodextrin (pMßCD) was their ideal monomerizing host and highly efficient shuttle to transfer them into water. New assembly modes of the extremely stable (Kbinding > 1012 M-2) 2:1 complexes were identified. The complexes are photostable and do not disassemble in FBS-containing cell culture media for 24 h. Incubation of breast cancer MCF-7 cells with the complexes results in intense intracellular fluorescence, strongly enhanced in the endoplasmic reticulum (ER), high photokilling efficiency (~90%) and low dark toxicity. pMßCD stands out as a very capable molecular isolator of mono-carboxyalkyl-arylporphyrins that increases uptake and modulates their localization in the cells. The most efficient porphyrins are envisaged as suitable photosensitizers that can be linked to biocompatible drug carriers for photo- and chemo-therapy applications.

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.

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.

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.

Spacer site modifications for the improvement of the in vitro and in vivo binding properties of (99m)Tc-N(3)S-X-bombesin[2-14] derivatives.

It has been shown that gastrin releasing peptide receptors (GRPRs) are overexpressed in various types of cancer cells. Bombesin is an analogue of the mammalian GRP that binds with high specificity and affinity to GRPRs. Significant research efforts have been lately devoted to the design of radiolabeled 8 or 14 aminoacid bombesin (BN) peptides for the detection (either with gamma or positron emitting radionuclides) and therapy (with beta(-) emitting radionuclides) of cancer. The specific aim of the present study was to further investigate the radiolabeled peptide structure and to determine whether the total absence of a linker or the use of a basic diverse amino acid linker could influence the biodistribution profile of the new compounds for specific targeting of human prostate cancer. Thus, two new derivatives with the structure Gly-Gly-Cys-X-BN[2-14], where linker X is either zero (I) or Orn-Orn-Orn (Orn: ornithine) (II) were designed and synthesized. The corresponding (99m)Tc-BN derivatives were obtained with high radiochemical yield (>98%) and had almost identical retention times in RP-HPLC with the (185/187)Re complexes, which were also characterized by ESI-MS. Metabolic stability was found to be high in human plasma, moderate in PC-3 cells, and rather low in mouse liver and kidney homogenates for both BN derivatives studied. The BN derivative without the spacer was less stable in cell culture and liver homogenates. A satisfactory binding affinity to GRPRs, in the nanomolar range, was obtained for both BN derivatives as well as for their Re complexes, with BN (II) demonstrating the highest one. In vitro internalization/externalization assays indicated that approximately 6% of BN (I) and approximately 25% of BN (II) were internalized into PC-3 cells. In vivo evaluation in normal Swiss mice and in tumor bearing SCID mice showed that BN (II) presented higher tumor and pancreas uptake than BN (I). Small animal SPECT dynamic imaging, carried out after an injection of BN (II) in mice bearing PC-3 tumors, resulted in PC-3 tumor delineation with low background activity. Overall, this study performed for two new N(3)S-X-BN[2-14] derivatives indicated that hydrophilicity and charge strongly affected the in vitro and in vivo binding properties and the biodistribution pattern. This finding is confirmed by SPECT imaging of BN (II), which is under further in vivo evaluation for detecting cancer-positive GRPRs.

Remarkable Brain Penetration of Cyclopentadienyl M(CO)3+ (M = 99mTc, Re) Derivatives of Benzothiazole and Benzimidazole Paves the Way for Their Application as Diagnostic, with Single-Photon-Emission Computed Tomography (SPECT), and Therapeutic Agents for Alzheimer's Disease.

The synthesis and evaluation of three novel 99mTc complexes (99mTc-1-3) and their corresponding Re complexes (Re-1-3), in which the phenyl ring of 2-phenylbenzothiazole or 2-phenylbenzimidazole is replaced by the cyclopentadienyl tricarbonyl [Cp99mTc(CO)3] core, are reported. Both 99mTc and Re complexes were prepared from the corresponding ferrocenyl derivatives, and the Re complexes were fully characterized by elemental analysis, spectroscopic methods, and X-ray crystallography. The complexes exhibit effective in vitro binding to ß-amyloid (Aß) plaques and fibrils, inhibit Aß fibril formation, and significantly reduce Aß-induced cytotoxicity and reactive oxygen species production in neuronal cell cultures. The brain uptake of the 99mTc complexes ranges between 7.94 and 3.99% ID/g at 2 min p.i., being the highest recorded for potential 99mTc Aß plaque imaging probes in mice. Powered by their high brain uptake, the complexes represent strong theranostic candidates against Alzheimer's disease combining single-photon-emission computed tomography diagnostic (99mTc complexes) and antiamyloid therapeutic (Re complexes) potential.

Development of a specific IgY-based ELISA for prothymosin alpha, a bioactive polypeptide with diagnostic and therapeutic potential.

Prothymosin alpha (ProTα) is a highly conserved polypeptide (109 amino acids in humans) with diagnostic and therapeutic potential; ProTα exerts intra- and extra-cellular biological functions associated with cell proliferation, apoptosis and immune regulation, while it has been suggested to act as a damage-associated molecular pattern (DAMP) or alarmin. In this work, chicken polyclonal anti-ProTα antibodies that had been developed several years ago were immunochemically evaluated and proven to retain immunoreactivity for ProTα, with remarkable thermal and pH stability. Moreover, the antibodies showed practically no cross-reactivity with a series of ProTα-fragments, eventually intracellularly produced -such as ProTα[1-28] (also known as Tα1) and ProTα[100-109], which exert per se biological activity and might be present in biological samples along with the intact molecule, being therefore highly specific for whole-length ProTα. Based on the above antibodies (IgYs-3e), a highly specific competitive ProTα-ELISA with well-studied analytical characteristics (intra- and inter-assay CVs: ≤5% and ≤12%, respectively, limit of detection: 2.1 ng/mL, recovery: 88-104%) was developed. The new ProTα-ELISA was applied to the analysis of supernatants of HeLa cells driven to necrosis; intact ProTα was measured in cell culture supernatants, at levels that seemed to depend on % cell necrosis.

Immunocytological and preliminary immunohistochemical studies of prothymosin alpha, a human cancer-associated polypeptide, with a well-characterized polyclonal antibody.

Prothymosin alpha (ProTalpha) is a nuclear polypeptide of great biological and, possibly clinical, importance, because its expression levels have been associated with early diagnosis/prognosis of human cancer. It is therefore interesting to raise easily available and cost-effective antibodies that would be applied to develop reliable ProTalpha immunodiagnostics. In this study, New Zealand white rabbits and laying hens were parallel immunized against intact ProTalpha or the synthetic fragments ProTalpha[1-28], ProTalpha[87-109], and ProTalpha[101-109], all conjugated to keyhole limpet hemocyanin (KLH). The corresponding antibodies G and Y were immunochemically evaluated in parallel with ELISA and Western blot systems and applied to fluorescence immunocytology experiments using various cancer cell lines and normal cells. The antibody G raised against ProTalpha[101-109]/KLH had excellent functional characteristics in the Western blot and immunocytology experiments, where the fluorescent signal was almost exclusively shown in the cell nucleus independently of the cells assayed. The above antibody has been applied to preliminary IHC staining of human cancer prostate tissues, leading to a high percentage of clearly and intensively stained nuclei in the adenocarcinoma tissue; this antibody can be further used in cancer tissue immunostaining and in research concerning the role of ProTalpha in tumorigenesis.

The multiple T-maze in vivo testing of the neuroprotective effect of humanin analogues.

Humanin (HN) and its analogues have been shown to protect cells against death induced by various Alzheimer's disease (AD) genes and amyloid-beta-peptides in vitro; the analogues [Gly(14)]-HN and colivelin have also been shown to be potent in reversing learning and memory impairment induced by scopolamine or quinuclidinyl benzilate (QNB) in mice or rats in vivo using the Y-maze or multiple T-maze tests. This paper describes the activity of new peptides of the HN family, after i.p. administration, on QNB-induced impairment of spatial memory in the multiple T-maze test in rats. The following peptides have been studied: HN analogues truncated either on the C- or N-terminus, or analogues having a tert-Leu in place of Leu in the central part of the molecule, the active HN core PAGASRLLLLTGEIDLP (RG-PAGA) and its analogues having three or five leucines instead of four, and finally the recently described hybrid peptide colivelin (i.e. a peptide having the activity-dependent neurotrophic factor SALLRSIPA attached to the N-terminus of the active RG-PAGA) and its des-Leu- and plus-Leu-analogues. While the truncated analogues and most of the tert-Leu containing analogues were devoid of activity, the analogues of the RG-PAGA were active, i.e. they reversed the impairment of spatial memory irrespective of the number of Leu present in their sequence. The highest activity was shown by colivelin and its des-Leu-analogue. These results demonstrate the potential of HN analogues in the modulation of the cholinergic system, which plays an important role in the cognitive deficits associated with AD and other neurodegenerative diseases.

A Novel Metal-Based Imaging Probe for Targeted Dual-Modality SPECT/MR Imaging of Angiogenesis.

Superparamagnetic iron oxide nanoparticles with well-integrated multimodality imaging properties have generated increasing research interest in the past decade, especially when it comes to the targeted imaging of tumors. Bevacizumab (BCZM) on the other hand is a well-known and widely applied monoclonal antibody recognizing VEGF-A, which is overexpressed in angiogenesis. The aim of this proof-of-concept study was to develop a dual-modality nanoplatform for in vivo targeted single photon computed emission tomography (SPECT) and magnetic resonance imaging (MRI) of tumor vascularization. Iron oxide nanoparticles (IONPs) have been coated with dimercaptosuccinic acid (DMSA), for consequent functionalization with the monoclonal antibody BCZM radiolabeled with 99mTc, via well-developed surface engineering. The IONPs were characterized based on their size distribution, hydrodynamic diameter and magnetic properties. In vitro cytotoxicity studies showed that our nanoconstruct does not cause toxic effects in normal and cancer cells. Fe3O4-DMSA-SMCC-BCZM-99mTc were successfully prepared at high radiochemical purity (>92%) and their stability in human serum and in PBS were demonstrated. In vitro cell binding studies showed the ability of the Fe3O4-DMSA-SMCC-BCZM-99mTc to bind to the VEGF-165 isoform overexpressed on M-165 tumor cells. The ex vivo biodistribution studies in M165 tumor-bearing SCID mice showed high uptake in liver, spleen, kidney and lungs. The Fe3O4-DMSA-SMCC-BCZM-99mTc demonstrated quick tumor accumulation starting at 8.9 ± 1.88%ID/g at 2 h p.i., slightly increasing at 4 h p.i. (16.21 ± 2.56%ID/g) and then decreasing at 24 h p.i. (6.01 ± 1.69%ID/g). The tumor-to-blood ratio reached a maximum at 24 h p.i. (~7), which is also the case for the tumor-to-muscle ratio (~18). Initial pilot imaging studies on an experimental gamma-camera and a clinical MR camera prove our hypothesis and demonstrate the potential of Fe3O4-DMSA-SMCC-BCZM-99mTc for targeted dual-modality imaging. Our findings indicate that Fe3O4-DMSA-SMCC-BCZM-99mTc IONPs could serve as an important diagnostic tool for biomedical imaging as well as a promising candidate for future theranostic applications in cancer.

Tumor Targeting via Sialic Acid: [68Ga]DOTA-en-pba as a New Tool for Molecular Imaging of Cancer with PET.

PURPOSE: The aim of this study was to demonstrate the potential of Ga-68-labeled macrocycle (DOTA-en-pba) conjugated with phenylboronic vector for tumor recognition by positron emission tomography (PET), based on targeting of the overexpressed sialic acid (Sia). PROCEDURES: The imaging reporter DOTA-en-pba was synthesized and labeled with Ga-68 at high efficiency. Cell binding assay on Mel-C and B16-F10 melanoma cells was used to evaluate melanin production and Sia overexpression to determine the best model for demonstrating the capability of [68Ga]DOTA-en-pba to recognize tumors. The in vivo PET imaging was done with B16-F10 tumor-bearing SCID mice injected with [68Ga]DOTA-en-pba intravenously. Tumor, blood, and urine metabolites were assessed to evaluate the presence of a targeting agent. RESULTS: The affinity of [68Ga]DOTA-en-pba to Sia was demonstrated on B16-F10 melanoma cells, after the production of melanin as well as Sia overexpression was proved to be up to four times higher in this cell line compared to that in Mel-C cells. Biodistribution studies in B16-F10 tumor-bearing SCID mice showed blood clearance at the time points studied, while uptake in the tumor peaked at 60 min post-injection (6.36 ± 2.41 % ID/g). The acquired PET images were in accordance with the ex vivo biodistribution results. Metabolite assessment on tumor, blood, and urine samples showed that [68Ga]DOTA-en-pba remains unmetabolized up to at least 60 min post-injection. CONCLUSIONS: Our work is the first attempt for in vivo imaging of cancer by targeting overexpression of sialic acid on cancer cells with a radiotracer in PET.

A Triphenylphosphonium-Functionalized Mitochondriotropic Nanocarrier for Efficient Co-Delivery of Doxorubicin and Chloroquine and Enhanced Antineoplastic Activity.

Drug delivery systems that target subcellular organelles and, in particular, mitochondria are considered to have great potential in treating disorders that are associated with mitochondrial dysfunction, including cancer or neurodegenerative diseases. To this end, a novel hyperbranched mitochondriotropic nanocarrier was developed for the efficient co-delivery of two different (both in chemical and pharmacological terms) bioactive compounds. The carrier is based on hyperbranched poly(ethyleneimine) functionalized with triphenylphosphonium groups that forms ~100 nm diameter nanoparticles in aqueous media and can encapsulate doxorubicin (DOX), a well-known anti-cancer drug, and chloroquine (CQ), a known chemosensitizer with arising potential in anticancer medication. The anticancer activity of this system against two aggressive DOX-resistant human prostate adenocarcinoma cell lines and in in vivo animal studies was assessed. The co-administration of encapsulated DOX and CQ leads to improved cell proliferation inhibition at extremely low DOX concentrations (0.25 µΜ). In vivo experiments against DU145 human prostate cancer cells grafted on immunodeficient mice resulted in tumor growth arrest during the three-week administration period and no pervasive side effects. The findings put forward the potential of such targeted low dose combination treatments as a therapeutic scheme with minimal adverse effects.

68Ga-radiolabeled AGuIX nanoparticles as dual-modality imaging agents for PET/MRI-guided radiation therapy.

AIM: The aim of this study was to develop a dual-modality positron emission tomography/magnetic resonance (PET/MR) imaging probe by radiolabeling gadolinium-containing AGuIX derivatives with the positron-emitter Gallium-68 (68Ga). MATERIALS & METHODS: AGuIX@NODAGA nanoparticles were labeled with 68Ga at high efficiency. Tumor accumulation in an appropriate disease model was assessed by ex vivo biodistribution and in vivo PET/MR imaging. RESULTS:  68Ga-AGuIX@NODAGA was proven to passively accumulate in U87MG human glioblastoma tumor xenografts. Metabolite assessment in serum, urine and tumor samples showed that 68Ga-AGuIX@NODAGA remains unmetabolized up to at least 60 min postinjection. CONCLUSION: This study demonstrates that 68Ga-AGuIX@NODAGA can be used as a dual-modality PET/MR imaging agent with passive accumulation in the diseased area, thus showing great potential for PET/MR image-guided radiation therapy.

Synthesis, characterization, and biological evaluation of M(I)(CO)3(NNO) complexes (M = Re, 99mTc) conjugated to 2-(4-aminophenyl)benzothiazole as potential breast cancer radiopharmaceuticals.

The synthesis and biological evaluation of new M(I)(CO)3(NNO) (M = Re, 99mTc) complexes attached to the antitumor agent 2-(4-aminophenyl)benzothiazole are reported. The fluorescent rhenium complex enters MCF-7 breast cancer cells but does not enter normal HFFF-2 and MRC-5 cells. The analogous radioactive 99mTc complex produces fast blood and soft tissue clearance when administered to healthy mice. These complexes are promising candidates for developing radiopharmaceuticals for imaging (99mTc) and targeted radiotherapy (186Re, 188Re) of breast cancer.