Selenosugars targeting the infective stage of Trypanosoma brucei with high selectivity.

Earlier evidences showed that diglycosyl diselenides are active against the infective stage of African trypanosomes (top hits IC50 0.5 and 1.5 µM) but poorly selective (selectivity index <10). Here we extended the study to 33 new seleno-glycoconjugates with the aim to improve potency and selectivity. Three selenoglycosides and three glycosyl selenenylsulfides displayed IC50 against bloodstream Trypanosoma brucei in the sub-µM range (IC50 0.35-0.77 µM) and four of them showed an improved selectivity (selectivity index >38-folds vs. murine and human macrohages). For the glycosyl selenylsulfides, the anti-trypanosomal activity was not significantly influenced by the nature of the moiety attached to the sulfur atom. Except for a quinoline-, and to a minor extent a nitro-derivative, the most selective hits induced a rapid (within 60 min) and marked perturbation of the LMWT-redox homeostasis. The formation of selenenylsulfide glycoconjugates with free thiols has been identified as a potential mechanism involved in this process.

Applications of the ROS-Responsive Thioketal Linker for the Production of Smart Nanomedicines.

Reactive oxygen species (ROS)-sensitive drug delivery systems (DDS) specifically responding to altered levels of ROS in the pathological microenvironment have emerged as an effective means to enhance the pharmaceutical efficacy of conventional nanomedicines, while simultaneously reducing side effects. In particular, the use of the biocompatible, biodegradable, and non-toxic ROS-responsive thioketal (TK) functional group in the design of smart DDS has grown exponentially in recent years. In the design of TK-based DDS, different technological uses of TK have been proposed to overcome the major limitations of conventional DDS counterparts including uncontrolled drug release and off-target effects. This review will focus on the different technological uses of TK-based biomaterials in smart nanomedicines by using it as a linker to connect a drug on the surface of nanoparticles, form prodrugs, as a core component of the DDS to directly control its structure, to control the opening of drug-releasing gates or to change the conformation of the nano-systems. A comprehensive view of the various uses of TK may allow researchers to exploit this reactive linker more consciously while designing nanomedicines to be more effective with improved disease-targeting ability, providing novel therapeutic opportunities in the treatment of many diseases.

99mTechnetium- or Cy7-Labeled Fab(Tocilizumab) as Potential Multiple Myeloma Imaging Agents.

BACKGROUND: Multiple Myeloma (MM) is a malignant hematologic disorder and the second most common blood cancer. Interleukin-6 (IL-6) has been identified as a crucial factor for the proliferation and survival of MM cells and the overexpression of IL-6 receptor is being studied as a molecular target for therapeutic and diagnostic use in myelomas and other comorbidities. Tocilizumab is a humanized monoclonal antibody that binds IL-6R. OBJECTIVE: We aim to label and evaluate Fab(Tocilizumab) with 99mTechnetium or Cy7 as potential MM imaging agents. METHODS: IL-6R distribution was analyzed by Laser Confocal Microscopy (LCM) in MM cell lines. Fab(Tocilizumab) was produced by the digestion of Tocilizumab with papain for 24h at 37°C, derivatized with NHS-HYNIC-Tfa and radiolabeled with 99mTc. Radiochemical stability and in vitro cell assays were evaluated. Biodistribution and SPECT/CT were performed. Also, Fab(Tocilizumab) was labeled with Cy7 for in vivo fluorescence imaging up to 72h. RESULTS: LCM analysis demonstrates IL-6R distribution on MM cell lines. Incubation with papain resulted in complete digestion of Tocilizumab and exhibited a good purity and homogeneity. Radiolabeling with 99mTc via NHS-HYNIC-Tfa was found to be fast, easy, reproducible and stable, revealing high radiochemical purity and without interfering with IL-6R recognition. Biodistribution and SPECT/CT studies showed a quick blood clearance and significant kidney and MM engrafted tumor uptake. Cy7-Fab(Tocilizumab) fluorescent imaging allowed MM1S tumor identification up to 72h p.i. CONCLUSION: These new molecular imaging agents could potentially be used in the clinical setting for staging and follow-up of MM through radioactive whole-body IL-6R expression visualization in vivo. The fluorescent version could be used for tissue sample evaluation and to guide surgical excision, if necessary.

Synthesis, Characterization, and In Vitro Studies of an Reactive Oxygen Species (ROS)-Responsive Methoxy Polyethylene Glycol-Thioketal-Melphalan Prodrug for Glioblastoma Treatment.

Glioblastoma (GBM) is the most frequent and aggressive primary tumor of the brain and averages a life expectancy in diagnosed patients of only 15 months. Hence, more effective therapies against this malignancy are urgently needed. Several diseases, including cancer, are featured by high levels of reactive oxygen species (ROS), which are possible GBM hallmarks to target or benefit from. Therefore, the covalent linkage of drugs to ROS-responsive molecules can be exploited aiming for a selective drug release within relevant pathological environments. In this work, we designed a new ROS-responsive prodrug by using Melphalan (MPH) covalently coupled with methoxy polyethylene glycol (mPEG) through a ROS-cleavable group thioketal (TK), demonstrating the capacity to self-assembly into nanosized micelles. Full chemical-physical characterization was conducted on the polymeric-prodrug and proper controls, along with in vitro cytotoxicity assayed on different GBM cell lines and "healthy" astrocyte cells confirming the absence of any cytotoxicity of the prodrug on healthy cells (i.e. astrocytes). These results were compared with the non-ROS responsive counterpart, underlining the anti-tumoral activity of ROS-responsive compared to the non-ROS-responsive prodrug on GBM cells expressing high levels of ROS. On the other hand, the combination treatment with this ROS-responsive prodrug and X-ray irradiation on human GBM cells resulted in an increase of the antitumoral effect, and this might be connected to radiotherapy. Hence, these results represent a starting point for a rationale design of innovative and tailored ROS-responsive prodrugs to be used in GBM therapy and in combination with radiotherapy.

Nanomedicine Against Aß Aggregation by ß-Sheet Breaker Peptide Delivery: In Vitro Evidence.

The accumulation of amyloid ß (Aß) triggers a cascade of toxic events in Alzheimer's disease (AD). The KLVFF peptide can interfere with Aß aggregation. However, the peptide suffers from poor bioavailability and the inability to cross the blood-brain barrier. In this work, we study the possibility of adopting nanomedicine to overcome KLVFF limits in biodistribution. We produced new engineered polymeric nanoparticles (NPs), and we evaluated the cellular toxicity of these NPs and validated that KVLFF peptides released by NPs show the same promising effects on AD pathology. Our results revealed the successful generation of KVLFF loaded NPs that, without significant effects on cell heath, are even more potent in reversing Aß-induced pathologies compared to the free peptide. Therefore, NPs will significantly advance KVLFF treatment as a therapeutic option for AD.

ROS-responsive "smart" polymeric conjugate: Synthesis, characterization and proof-of-concept study.

New approaches integrating stimuli-responsive linkers into prodrugs are currently emerging. These "smart" prodrugs can enhance the effectivity of conventional prodrugs with promising clinical applicability. Oxidative stress is central to several diseases, including cancer. Therefore, the design of prodrugs that respond to ROS stimulus, allowing a selective drug release in this condition, is fairly encouraging. Aiming to investigate the ROS-responsiveness of prodrugs containing the ROS-cleavable moiety, Thioketal (TK), we performed proof-of-concept studies by synthesizing ROS-responsive conjugate, namely mPEG-TK-Cy5, through exploiting Cy5 fluorescent dye. We demonstrated that, differently to non-ROS-responsive control conjugate (mPEG-Cy5), mPEG-TK-Cy5 shows a selective release of Cy5 in response to ROS in both, ROS-simulated conditions and in vitro on glioblastoma cells. Our results confirm the applicability of TK-technology in the design of ROS-responsive prodrugs, which constitutes a promising approach in cancer treatment. The translatability of this technology for other diseases treatment makes this a highly relevant and promising approach.

Evaluation of chromosomal aberrations induced by 188Re-dendrimer nanosystem on B16f1 melanoma cells.

PURPOSE: To study the rhenium-188 labeling of polyamidoamine (PAMAM) generation 4 (G4) dendrimer and its evaluation on biodistribution and chromosomal aberrations in melanoma cells induced by ionizing radiation as potential treatment agent. MATERIALS AND METHODS: Dendrimers were first conjugated with Suc-HYNIC (succinimidyl 6-hydrazinopyridine-3-carboxylic acid hydrochloride). Dendrimer-HYNIC was then incubated with 188ReO4-. Biodistribution was performed administrating 188Re-dendrimer to normal (NM) or melanoma-bearing mice (MBM). Chromosome aberration test was conducted in order to measure treatment capacity of 188Re-dendrimer in melanoma cells. RESULTS: Radiolabeling yield of dendrimer was approx. 70%. Biodistribution studies in NM showed blood clearance with hepatic and renal depuration. MBM showed a similar pattern of biodistribution with tumor uptake of 6% of injected dose. Aberrant metaphases quantified in control cells were 7%, increasing to 29.5% in cells treated with 15µCi (0.555 MBq) of 188Re-dendrimer for 24 h. CONCLUSIONS: 188Re-dendrimer can produce double-stranded breaks in DNA induced by ionizing radiation in melanoma cells in vitro.

Tocilizumab Labeling with 99mTechnetium via HYNIC as a Molecular Diagnostic Agent for Multiple Myeloma.

BACKGROUND: Multiple myeloma is the second most common hematological malignancy. Interleukin-6 (IL-6) is one of the key molecules related to growth, survival and proliferation of myeloma cells. Tocilizumab is a humanized monoclonal antibody directed against receptor of IL-6. OBJECTIVE: To radiolabel Tocilizumab with 99mTechnetium as a potential imaging agents for MM. METHODS: IL-6R expression was studied by laser confocal microscopy in MM cell lines (U266, NCI-H929 and MM1S). Tocilizumab was derivatized with NHS-HYNIC-Tfa and radiolabeling with 99mTc. Radiochemical stability was determined. In-vitro binding and immunoreactive fraction assays were performed. Biodistribution and SPECT/CT imaging were evaluated in healthy BALB/c and MM-bearing BALB/c nude mice. RESULTS: LCM studies allowed us to demonstrate that U266, NCI-H929 and MM1S cells present high expression of IL-6R in cell membrane. Radiolabeling was carried out in a fast, reproducible, easy and stable way having high radiochemical purity and did not interfere with epitope recognition. The immunoreactive fraction of 99mTc- HYNIC-Tocilizumab was 86.35%. Biodistribution showed a high uptake in liver, spleen, gastrointestinal tract and kidneys. SPECT/CT imaging of MM-bearing BALB/c nude mice showed liver uptake and a high tumor selective uptake at 24 hours. CONCLUSIONS: Our results support the potential role of 99mTc-HYNIC-Tocilizumb as a novel MM radiotracer for targeting IL-6 expression in-vivo. We describe the development of a formulation kit to radiolabeling monoclonal antibodies in a clinical setting. We hope that these novel molecular imaging agents will open the path to new diagnostic and therapeutic strategies for MM disease.

Technetium-99m- or Cy7-Labeled Rituximab as an Imaging Agent for Non-Hodgkin Lymphoma.

INTRODUCTION: Rituximab was the first monoclonal antibody approved for the treatment of B-cell non-Hodgkin lymphoma (NHL) expressing CD20 antigen. This antibody has also the potential to be used as a specific fluorescent and radiolabel agent for targeting NHL. OBJECTIVE: To radiolabel rituximab with technetium-99m (99mTc) or Cy7 and evaluate both probes as potential imaging agents for NHL. METHODS: Rituximab was derivatized with the trifluoroacetyl hydrazino protected form of succinimidyl ester of HYNIC and radiolabeled with 99mTc. Radiochemical stability and in vitro cell assays were evaluated. Biodistribution and single-photon emission computed tomography/computed tomography (SPECT/CT) were performed. Raji cells were transfected with luciferase for bioluminescent NHL imaging up to 21 days. Rituximab was labeled with Cy7 for in vivo noninvasive fluorescence imaging up to 96 h. RESULTS: Radiolabeling was carried out in a fast, reproducible, easy, and stable way with high radiochemical purity and did not interfere with epitope recognition. Biodistribution and SPECT/CT studies showed high liver and discrete tumor uptake. Bioluminescence and fluorescence studies helped us evaluate rituximab-Cy7 in Raji subcutaneous engraftment in BALB/c nude mice. CONCLUSIONS: Our results support the potential use of rituximab labeled either with 99mTc or Cy7 as a molecular imaging tool for staging, restaging, and guiding surgical excision of tumors, which merits further evaluation.

The Effect of A Hexanoic Acid Linker Insertion on the Pharmacokinetics and Tumor Targeting Properties of the Melanoma Imaging Agent 99mTc-HYNIC-cycMSH.

BACKGROUND: Lactam cyclized alpha-melanocyte stimulating hormone (α-MSH) analogues exhibit high stability and affinity for the MC1-R receptors over expressed in melanoma cells. Recently, we reported a novel 99mTc-HYNIC-cycMSH4-13 analogue with the HYNIC chelator directly attached to the lactam cyclized ring. OBJECTIVE: In this study we proposed the introduction of a 6-aminohexanoic acid (Ahx) linker between the HYNIC chelator and lactam cyclized peptide cycMSH4-13 to reduce steric hindrance and improve the melanoma targeting and imaging proprieties of the radiolabeled peptide. METHOD: HYNIC-Ahx-cycMSH4-13 peptide was synthesized on an automated peptide synthesizer and displayed an IC50 of 0.3 nM using B16/F1 cells. The 99mTc/tricine radiolabeled peptide was examined for radiochemical purity, stability and cell binding. In vivo, biodistribution and planar gamma imaging studies were performed in B16/F1 melanoma tumor bearing C57BK mice. RESULTS: 99mTc-HYNIC-Ahx-cycMSH4-13 was obtained with a radiochemical purity > 95%, was stable up to 24 h at room temperature and exhibited high binding and rapid internalization in B16/F1 cells. In vivo biodistribution studies showed a tumor uptake of 4.92 ± 0.92 % ID/g and 2.78 ± 1.48 % ID/g at 2 h and 4 h post injection, respectively. Whole-body clearance was rapid through urinary excretion. The melanoma tumors were clearly visualized by planar gamma imaging. CONCLUSION: 99mTc-HYNIC-Ahx-cycMSH4-13 was shown radiochemically stability and exhibited rapid and selective uptake in melanoma cells and tumors. Imaging studies yielded promising preclinical results, warranting further evaluation of 99mTc-HYNIC-cycMSH analogs as melanoma specific imaging agents.

In vitro and in vivo uptake studies of PAMAM G4.5 dendrimers in breast cancer.

BACKGROUND: Breast cancer is the second leading cause of cancer death worldwide. Nanotechnology approaches can overcome the side effects of chemotherapy as well as improve the efficacy of drugs. Dendrimers are nanometric size polymers which are suitable as drug delivery systems. To the best of our knowledge, studies on the application of PAMAM G4.5 (polyamidoamine half generation 4) dendrimers as potential drug delivery systems in breast cancer have not been reported. In this work we developed a PAMAM G4.5 dendrimer containing FITC (fluorescein isothiocyanate) dye to study their uptake by murine breast cancer cells and BALB/c mice breast tumors. RESULTS: We performed a reaction between FITC and PAMAM G4.5 dendrimers which were previously derivatized with piperazine (linker molecule), characterized them by (1)H NMR (proton nuclear magnetic resonance) spectroscopy and MALDI-TOF (matrix-assisted laser desorption/ionization- time-of-flight) mass spectrometry. The experimental data indicated that 2 FITC molecules could be bound covalently at the PAMAM G4.5 dendrimer surface, with 17 FITC molecules probably occluded in PAMAM dendrimers cavity. PAMAM-FITC dendrimer (PAMAM G4.5-piperazinyl-FITC dendrimer) size distribution was evaluated by DLS (dynamic light scattering) and TEM (transmission electron microscopy). The nanoparticle hydrodynamic size was 96.3 ± 1.4 nm with a PdI (polydispersion index) of 0.0296 ± 0.0171, and the size distribution measured by TEM was 44.2 ± 9.2 nm. PAMAM-FITC dendrimers were neither cytotoxic in 4T1 cells nor hemolytic up to 24 h of incubation. In addition, they were uptaken in vitro by 4T1 cells and in vivo by BALB/c mice breast tumors. PAMAM G4.5-piperazinyl-FITC dendrimer intracellular distribution was observed through histologic analysis of the tumor by laser confocal microscopy. CONCLUSION: These results indicate that PAMAM G4.5 dendrimers enter tumor tissue cells, being good candidates to be used as antitumor drug delivery systems for breast cancer treatment and diagnosis.

Superparamagnetic iron-oxide nanoparticles mPEG350- and mPEG2000-coated: cell uptake and biocompatibility evaluation.

Superparamagnetic iron oxide nanoparticles (SPIONS) were synthesized by thermal decomposition of an organometallic precursor at high temperature and coated with a bi-layer composed of oleic acid and methoxy-polyethylene glycol-phospholipid. The formulations were named SPION-PEG350 and SPION-PEG2000. Transmission electron microscopy, X-ray diffraction and magnetic measurements show that the SPIONs are near-spherical, well-crystalline, and have high saturation magnetization and susceptibility. FTIR spectroscopy identifies the presence of oleic acid and of the conjugates mPEG for each sample. In vitro biocompatibility of SPIONS was investigated using three cell lines; up to 100µg/ml SPION-PEG350 showed non-toxicity, while SPION-PEG2000 showed no signal of toxicity even up to 200µg/ml. The uptake of SPIONS was detected using magnetization measurement, confocal and atomic force microscopy. SPION-PEG2000 presented the highest internalization capacity, which should be correlated with the mPEG chain size. The in vivo results suggested that SPION-PEG2000 administration in mice triggered liver and kidney injury. FROM THE CLINICAL EDITOR: The potential use of superparamagnetic iron oxide nanoparticles (SPIONS) in the clinical setting have been studied by many researchers. The authors synthesized two types of SPIONS here and investigated the physical properties and biological compatibility. The findings should provide more data on the design of SPIONS for clinical application in the future.

Effect of resveratrol on behavioral performance of streptozotocin-induced diabetic mice in anxiety tests.

The aim of this study was to evaluate with anxiety tests the effect of resveratrol (RSV) on streptozotocin (STZ)-induced diabetic mouse behavioral performance at the second and fourth week of treatment. Confirmed diabetic mice (>250 mg/dl of glucose in blood after STZ injection) were treated with RSV (RDM, n=12) or control treated (DM, n=12) for 4 weeks. DM and RDM were tested in the Open Field Test (OFT) and Elevated Plus Maze (EPM). In the second week of RSV treatment, a higher grooming frequency (P<0.05) and a lower defecation and rearing frequency (P<0.05) were detected in the OFT in the RDM group compared with the DM. There was a higher grooming frequency (P<0.05) and higher percentage of entries in open arms (P<0.05) in the RDM group than in the DM group in the EPM. However, in the fourth week of RSV treatment, the only effect observed was a higher grooming frequency in the RDM group than in the DM group (P<0.05) in the EPM. In conclusion, RSV treatment in diabetic mice provoked anxiolytic-like effects in both tests (OFT and EPM), and these effects were observed in a short time window (2 weeks). It is suggested that RSV may help diabetic animals to adapt to new stressing and anxiety situations and thus to improve their welfare.

Diabetes increases stiffness of live cardiomyocytes measured by atomic force microscopy nanoindentation.

Stiffness of live cardiomyocytes isolated from control and diabetic mice was measured using the atomic force microscopy nanoindentation method. Type 1 diabetes was induced in mice by streptozotocin administration. Histological images of myocardium from mice that were diabetic for 3 mo showed disorderly lineup of myocardial cells, irregularly sized cell nuclei, and fragmented and disordered myocardial fibers with interstitial collagen accumulation. Phalloidin-stained cardiomyocytes isolated from diabetic mice showed altered (i.e., more irregular and diffuse) actin filament organization compared with cardiomyocytes from control mice. Sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a) pump expression was reduced in homogenates obtained from the left ventricle of diabetic animals compared with age-matched controls. The apparent elastic modulus (AEM) for live control or diabetic isolated cardiomyocytes was measured using the atomic force microscopy nanoindentation method in Tyrode buffer solution containing 1.8 mM Ca(2+) and 5.4 mM KCl (physiological condition), 100 nM Ca(2+) and 5.4 mM KCl (low extracellular Ca(2+) condition), or 1.8 mM Ca(2+) and 140 mM KCl (contraction condition). In the physiological condition, the mean AEM was 112% higher for live diabetic than control isolated cardiomyocytes (91 ± 14 vs. 43 ± 7 kPa). The AEM was also significantly higher in diabetic than control cardiomyocytes in the low extracellular Ca(2+) and contraction conditions. These findings suggest that the material properties of live cardiomyocytes were affected by diabetes, resulting in stiffer cells, which very likely contribute to high diastolic LV stiffness, which has been observed in vivo in some diabetes mellitus patients.