Highly Sensitive "Off/On" EPR Probes to Monitor Enzymatic Activity.

The assessment of unregulated level of enzyme activity is a crucial parameter for early diagnoses in a wide range of pathologies. In this study, we propose the use of electron paramagnetic resonance (EPR) as an easy method to probe carboxylesterase (CE) enzymatic activity in vitro. For this application, were synthesized two amphiphilic, nitroxide containing esters, namely Tempo-C12 (T-C12) and Tempo-2-C12 (T-2-C12). They exhibit low solubility in water and form stable micelles in which the radicals are EPR almost silent, but the hydrolysis of the ester bond yields narrows and intense EPR signals. The intensity of the EPR signals is proportional to the enzymatic activity. CEs1, CEs2 and esterase from porcine liver (PLE) were investigated. The obtained results show that T-C12 and T-2-C12-containing systems display a much higher selectivity toward the CEs2, with a Limit of Detection of the same order of those ones obtained with optical methods.

Biotinylation of a MRI/Gd BNCT theranostic agent to access a novel tumour-targeted delivery system.

A new biotin based BNCT (Boron Neutron Capture Therapy)-MRI theranostic is here reported (Gd-AL01) in order to exploit the high tumour specificity of biotin and the selectivity of BNCT in a synergistic manner. The key is the preparation of an intermediate where an o-carborane is linked to two amino groups orthogonally protected via the exploitation of two consecutive Mitsunobu reactions. The aim is its functionalisation in two different steps with biotin as the biological vector and Gd-DOTA as the MRI probe and GdNCT agent. Cell uptake was evaluated on HeLa tumour cells overexpressing biotin receptors. The internalised boron is proportional to the concentration of the theranostic agent incubated in the presence of cells. A maximum value of 77 ppm is reached and a well detectable signal intensity increase in the T1 weighted image of HeLa cells was observed, differently from clinically used GdHPDO3A, where no contrast is detected. These excellent results indicate that Gd-AL01 can be applied as a theranostic probe in BNCT studies.

LDL mediated delivery of Paclitaxel and MRI imaging probes for personalized medicine applications.

BACKGROUND: The combination of imaging and therapeutic agents in the same smart nanoparticle is a promising option to perform a minimally invasive imaging guided therapy. In this study, Low density lipoproteins (LDL), one of the most attractive biodegradable and biocompatible nanoparticles, were used for the simultaneous delivery of Paclitaxel (PTX), a hydrophobic antitumour drug and an amphiphilic contrast agent, Gd-AAZTA-C17, in B16-F10 melanoma cell line. These cells overexpress LDL receptors, as assessed by flow cytometry analysis. RESULTS: PTX and Gd-AAZTA-C17 loaded LDLs (LDL-PTX-Gd) have been prepared, characterized and their stability was assessed under 72 h incubation at 37 °C and compared to LDL loaded with Gd-AAZTA-C17 (LDL-Gd) and LDL-PTX. The cytotoxic effect of LDL-PTX-Gd was evaluated by MTT assay. The anti-tumour drug loaded into LDLs showed a significantly higher toxicity on B16-F10 cells with respect to the commercially available formulation Paclitaxel kabi (PTX Kabi) used in clinical applications. Tumour cells uptake was initially assessed by ICP-MS and MRI on B16-F10 cell line. By the analysis of the image signal intensity, it was possible to extrapolate the amount of internalized PTX indirectly by the decrease of relaxation times caused by Gd, proportional to its concentration. Finally, the treatment with PTX loaded LDL on B16-F10 tumour bearing mice resulted in a marked reduction of tumour growth compared to the administration of PTX Kabi alone. CONCLUSIONS: LDLs are selectively taken-up by tumour cells and can be successfully exploited for the selective delivery of Paclitaxel and imaging agents. For the first time the anon invasive "in vivo" determination of the amount of PTX accumulated in the tumour was possible, thanks to the use of theranostic agents of natural origin.

Towards Enhanced MRI Performance of Tumor-Specific Dimeric Phenylboronic Contrast Agents.

It is known that phenylboronic acid (PBA) can target tumor tissues by binding to sialic acid, a substrate overexpressed by cancer cells. This capability has previously been explored in the design of targeting diagnostic probes such as Gd- and 68Ga-DOTA-EN-PBA, two contrast agents for magnetic resonance imaging (MRI) and positron emission tomography (PET), respectively, whose potential has already been demonstrated through in vivo experiments. In addition to its high resolution, the intrinsic low sensitivity of MRI stimulates the search for more effective contrast agents, which, in the case of small-molecular probes, basically narrows down to either increased tumbling time of the entire molecule or elevated local concentration of the paramagnetic ions, both strategies resulting in enhanced relaxivity, and consequently, a higher MRI contrast. The latter strategy can be achieved by the design of multimeric GdIII complexes. Based on the monomeric PBA-containing probes described recently, herein, we report the synthesis and characterization of the dimeric analogues (GdIII-DOTA-EN)2-PBA and (GdIII-DOTA-EN)2F2PBA. The presence of two Gd ions in one molecule clearly contributes to the improved biological performance, as demonstrated by the relaxometric study and cell-binding investigations.

Solid-phase synthesis and evaluation of tumour-targeting phenylboronate-based MRI contrast agents.

Paramagnetic macrocycles functionalized with phenylboronic moieties have proven to be interesting for MRI applications based on their ability to recognize cancer cells and generate local contrast. However, full use of the potential of this class of compounds is hampered by laborious and inefficient synthetic and, especially, purification procedures. The amphiphilic character of water-soluble phenylboronates renders them difficult compounds to be prepared through conventional solution synthesis due to the tendency to aggregate and form adducts with other nucleophiles. The new strategy described herein exploits the advantage of solid-phase synthesis with the application of DEAM-PS resin for anchorage and the subsequent simplified derivatization of boronates. GdDOTA-EN-PBA and its fluorinated analogue GdDOTA-EN-F2PBA were synthesized in a much easier, faster and economically convenient way to achieve good yields and purity. Furthermore, the effect of electron-withdrawing fluorine atoms on the aromatic ring of the latter compound was investigated by comparing the physico-chemical properties of both compounds as well as their binding affinity towards melanoma cancer cells.

Design, synthesis and preliminary in-vitro studies of novel boronated monocarbonyl analogues of Curcumin (BMAC) for antitumor and ß-amiloyd disaggregation activity.

Curcumin is currently being investigated for its capacity to treat many types of cancer and to prevent the neuron damage that is observed in Alzheimer's disease (AD). However, its clinical use is limited by its low stability and solubility in aqueous solutions. In this study, we propose a completely new class of boronated monocarbonyl analogues of Curcumin (BMAC, 6a-c), in which a carbonyl group replaces the Curcumin ß-diketone functionality, and an ortho-carborane, an icosahedral boron cluster, substitutes one of the two phenolic rings. BMAC antitumor activity against MCF7 and OVCAR-3 cell lines was assessed in vitro and compared to that of Curcumin and the corresponding MAC derivative. BMAC 6a-c showed efficiencies that are comparable to that of MAC and superior to that of Curcumin in both the cell lines. Moreover, the inhibition of the formation of ß-amyloid aggregates by BMAC 6a-c was evaluated and it was shown that compound 6c, which contains two OH moieties, has a better efficiency than Curcumin. The presence of a second -OH group can enhance the compound's binding efficacy with ß-amyloid aggregates. For the future, the presence of at least one carborane group means that the BMAC antitumor effect can be coupled with Boron Neutron Capture Therapy.

Combining magnetic nanoparticles and icosahedral boron clusters in biocompatible inorganic nanohybrids for cancer therapy.

The potential biomedical applications of the MNPs nanohybrids coated with m-carboranylphosphinate (1-MNPs) as a theranostic biomaterial for cancer therapy were tested. The cellular uptake and toxicity profile of 1-MNPs from culture media by human brain endothelial cells (hCMEC/D3) and glioblastoma multiform A172 cell line were demonstrated. Prior to testing 1-MNPs' in vitro toxicity, studies of colloidal stability of the 1-MNPs' suspension in different culture media and temperatures were carried out. TEM images and chemical titration confirmed that 1-MNPs penetrate into cells. Additionally, to explore 1-MNPs' potential use in Boron Neutron Capture Therapy (BNCT) for treating cancer locally, the presence of the m-carboranyl coordinated with the MNPs core after uptake was proven by XPS and EELS. Importantly, thermal neutrons irradiation in BNCT reduced by 2.5 the number of cultured glioblastoma cells after 1-MNP treatment, and the systemic administration of 1-MNPs in mice was well tolerated with no major signs of toxicity.

MRI visualization of neuroinflammation using VCAM-1 targeted paramagnetic micelles.

The detection of neuroinflammatory processes using innovative and non-invasive imaging techniques is of great help to deeply investigate the onset and progression of neurodegenerative diseases. Since Vascular Cell Adhesion Molecule (VCAM-1) is over expressed at the blood brain barrier in the event of neuroinflammation, the goal of this work was the testing of MRI detectable micelles targeted towards VCAM-1 to visualize inflamed regions in a mouse model of acute neuroinflammation. The developed probe allowed for the early detection of the disease, with higher T1 signal enhancement and more precise localization in comparison to untargeted micelles or to the clinically approved contrast agent MultiHance. Moreover, the relatively long blood half-life of the nanosystem (ca. 6.3 h) guaranteed a good accumulation in the inflamed regions, paving the way to future diagnostic/theranostic applications, implying the loading of neuroprotective or even anti-cancer drugs inside the core of the micelles.

The hydroboration reaction as a key for a straightforward synthesis of new MRI-NCT agents.

In this study the hydroboration reaction has been exploited to produce in only four steps a new lipophilic GdBNCT/MRI agent (PB01). As a matter of fact, the formation of a new B­C bond to link the decaborane with the lipophilic moiety greatly simplifies the synthesis of PB01 with respect to the previously reported dual agents. The complexes obtained (PB01a and PB01b) have been fully characterised from the relaxometric point of view and, after disaggregation with HPßCD, both isomers display high affinity for low density lipoproteins (LDLs) that can be exploited as specific carriers of these therapeutic and diagnostic agents for tumour cells. The LDL loading capacity is different for the two isomers. In fact, LDL can be loaded with 75 and 300 units of PB01a and PB01b, respectively, and for this reason, the isomer PB01b results to be the best candidate to perform MRI guided BNCT.

A theranostic approach based on the use of a dual boron/Gd agent to improve the efficacy of Boron Neutron Capture Therapy in the lung cancer treatment.

This study aims at developing an innovative theranostic approach for lung tumor and metastases treatment, based on Boron Neutron Capture Therapy (BNCT). It relies on to the use of low density lipoproteins (LDL) as carriers able to maximize the selective uptake of boron atoms in tumor cells and, at the same time, to quantify the in vivo boron distribution by magnetic resonance imaging (MRI). Tumor cells uptake was initially assessed by ICP-MS and MRI on four types of tumor (TUBO, B16-F10, MCF-7, A549) and one healthy (N-MUG) cell lines. Lung metastases were generated by intravenous injection of a Her2+ breast cancer cell line (i.e. TUBO) in BALB/c mice and transgenic EML4-ALK mice were used as primary tumor model. After neutron irradiation, tumor growth was followed for 30-40 days by MRI. Tumor masses of boron treated mice increased markedly slowly than the control group. From the clinical editor: In this article, the authors described an improvement to existing boron neutron capture therapy. The dual MRI/BNCT agent, carried by LDLs, was able to maximize the selective uptake of boron in tumor cells, and, at the same time, quantify boron distribution in tumor and in other tissues using MRI. Subsequent in vitro and in vivo experiments showed tumor cell killing after neutron irradiation.

Design of PLGA based nanoparticles for imaging guided applications.

An amphiphilic Gd(III) complex has been efficiently loaded in polylactic-co-glycolic acid nanoparticles (PLGA-NPs) to yield a novel, high sensitive magnetic resonance imaging (MRI) contrast agent for imaging guided drug delivery applications. As the Gd(III) complex is soluble in organic solvents, the nanoparticles were prepared as oil/water emulsions. PLGA-NPs were stable, in buffer, for more than 1 week without any release of the incorporated agents. The millimolar relaxivity of the Gd(III) complex incorporated in the particles (140 nm diameter) was of 21.7 mM(-1) s(-1) at 21.5 MHz, a value that is about 5 times higher than that observed with the commercially available contrast agents used in clinic. The relaxometric efficiency of these particles resulted inversely proportional to the particle size measured by dynamic light scattering. The high stability and sensitivity of PLGA-NPs allowed their accumulation in vivo in murine melanoma xenograft as shown in the corresponding MR images. Once loaded with drug and contrast agents, PLGA nanoparticles can be proposed as efficient theranostic MRI agents.

Synthesis of a carborane-containing cholesterol derivative and evaluation as a potential dual agent for MRI/BNCT applications.

In this study the synthesis and characterization of a new dual, imaging and therapeutic, agent is proposed with the aim of improving the efficacy of Boron Neutron Capture Therapy (BNCT) in cancer treatment. The agent (Gd-B-AC01) consists of a carborane unit (ten boron atoms) bearing a cholesterol unit on one side (to pursue the incorporation into the liposome bi-layer) and a Gd(iii)/1,4,7,10-tetraazacyclododecane monoamide complex on the other side (as a MRI reporter to attain the quantification of the B/Gd concentration). In order to endow the BNCT agent with specific delivery properties, the liposome embedded with the MRI/BNCT dual probes has been functionalized with a pegylated phospholipid containing a folic acid residue at the end of the PEG chain. The vector allows the binding of the liposome to folate receptors that are overexpressed in many tumor types, and in particular, in human ovarian cancer cells (IGROV-1). An in vitro test on IGROV-1 cells demonstrated that Gd-B-AC01 loaded liposomes are efficient carriers for the delivery of the MRI/BNCT probes to the tumor cells. Finally, the BNCT treatment of IGROV-1 cells showed that the number of surviving cells was markedly smaller when the cells were irradiated after internalization of the folate-targeted GdB10-AC01/liposomes.

A quantitative relaxometric version of the ELISA test for the measurement of cell surface biomarkers.

Quantitative measurement of marker expression in diseased cells is still a topic of considerable interest and different methodologies are currently under intense scrutiny. This work aims at developing an in vitro diagnostic method based on the release of paramagnetic species from relaxometrically "silent" liposomes operated by the action of a phospholipase A2 (PLA2) previously targeted to the epitope of interest. The released paramagnetic species causes an increase of the longitudinal water proton relaxation rate proportional to the number of PLA2 bound to the cell outer surface. The sensitivity of the herein proposed method, named R-ELISA, was attempted in the detection of folate receptor expression on human ovarian cancer cells by functionalizing PLA2 with folic acid. Receptor/cell number of 8.3×10(5) has been measured on IGROV-1 cells. The R-ELISA assay can detect nanomolar cell suspension receptor concentrations and has been validated by well-established spectrofluorimetric procedures.

Combining BNCT with carbonic anhydrase inhibition for mesothelioma treatment: Synthesis, in vitro, in vivo studies of ureidosulfamido carboranes.

Mesothelioma is a malignant neoplasm of mesothelial cells caused by exposure to asbestos. The average survival time after diagnosis is usually nine/twelve months. A multi-therapeutic approach is therefore required to treat and prevent recurrence. Boronated derivatives containing a carborane cage, a sulfamido group and an ureido functionality (CA-USF) have been designed, synthesised and tested, in order to couple Boron Neutron Capture Therapy (BNCT) and the inhibition of Carbonic Anhydrases (CAs), which are overexpressed in many tumours. In vitro studies showed greater inhibition than the reference drug acetazolamide (AZ). To increase solubility in aqueous media, CA-USFs were used as inclusion complexes of hydroxypropyl ß-cyclodextrin (HP-ß-CD) in all the inhibition and cell experiments. BNCT experiments carried out on AB22 (murine mesothelioma) cell lines showed a marked inhibition of cell proliferation by CA-USFs, and in one case a complete inhibition of proliferation twenty days after neutron irradiation. Finally, in vivo neutron irradiation experiments on a mouse model of mesothelioma demonstrated the efficiency of combining CA IX inhibition and BNCT treatment. Indeed, a greater reduction in tumour mass was observed in treated mice compared to untreated mice, with a significant higher effect when combined with BNCT. For in vivo experiments CA-USFs were administered as inclusion complexes of higher molecular weight ß-CD polymers thus increasing the selective extravasation into tumour tissue and reducing clearance. In this way, boron uptake was maximised and CA-USFs demonstrated to be in vivo well tolerated at a therapeutic dose. The therapeutic strategy herein described could be expanded to other cancers with increased CA IX activity, such as melanoma, glioma, and breast cancer.

A radical containing micellar probe for assessing esterase enzymatic activity with ultra-low field Overhauser-enhanced magnetic resonance imaging.

The ability to track altered enzyme activity using a non-invasive imaging protocol is crucial for the early diagnosis of many diseases but is often challenging. Herein, we show that Overhauser magnetic resonance imaging (OMRI) can be used to monitor enzymatic conversion at an ultra-low field (206 µT) using a highly sensitive "off/on" probe with a nitroxide stable radical containing ester, named T2C12-T80. This TEMPO derivative containing probe forms stable electron paramagnetic resonance (EPR) silent micelles in water that are hydrolysed by esterases, thus yielding narrow EPR signals whose intensities correlate directly with specific enzymatic activity. The responsiveness of the probe to tumours, facilitated by increased esterase activity, was initially determined by comparing EPR signals measured upon incubation with 3T3 (healthy fibroblasts used as control), HepG2 (human hepatoma) and Hs766T (human pancreatic cancer cells) cell lysates and then with Hs766T and 3T3 living cells. Next, Overhauser MR images were detected on a phantom containing the probe and the esterases to show that the approach is well suited for being translated to the in vivo detection at the earth's magnetic field. Regarding detection sensitivity, ultra-low field OMRI (ULF-OMRI) is beneficial over OMRI at higher fields (e.g. 0.2 T) since Overhauser enhancements are significantly higher and the technique is safe in terms of the specific absorption rate.

A carborane-derivative "click" reaction under heterogeneous conditions for the synthesis of a promising lipophilic MRI/GdBNCT agent.

In this study, the Huisgen reaction has been used to functionalise a carborane cage with a lipophilic moiety and a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) ligand to obtain a new Gd boron neutron-capture therapy (BNCT)/magnetic resonance imaging (MRI) agent. The introduction of the triazole units has been accomplished under both heterogeneous conditions, by the use of a Cu-supported ionic-liquid catalyst, and homogeneous conditions. The ability of the Gd complex of the synthesised ligand to form stable adducts with low-density lipoproteins (LDLs) has been evaluated and then MRI has been performed on tumour melanoma cells incubated in the presence of a Gd-complex/LDL imaging probe. It has been concluded that the high amount of intracellular boron necessary to perform BNCT can be reached even in the presence of a relatively low-boron-containing LDL concentration.

4-Amino-TEMPO loaded liposomes as sensitive EPR and OMRI probes for the detection of phospholipase A2 activity.

This work aims at developing a diagnostic method based on Electron Paramagnetic Resonance (EPR) measurements of stable nitroxide radicals released from "EPR silent" liposomes. The liposome destabilisation and consequent radical release is enzymatically triggered by the action of phospholipase A2 (PLA2) present in the biological sample of interest. PLA2 are involved in a broad range of processes, and changes in their activity may be considered as a unique valuable biomarker for early diagnoses. The minimum amount of PLA2 measured "in vitro" was 0.09 U/mL. Moreover, the liposomes were successfully used to perform Overhauser-enhanced Magnetic Resonance Imaging (OMRI) in vitro at 0.2 T. The amount of radicals released by PLA2 driven liposome destabilization was sufficient to generate a well detectable contrast enhancement in the corresponding OMRI image.

A novel pH sensitive theranostic PLGA nanoparticle for boron neutron capture therapy in mesothelioma treatment.

This study aims to develop poly lactic-co-glycolic acid (PLGA) nanoparticles with an innovative imaging-guided approach based on Boron Neutron Capture Therapy for the treatment of mesothelioma. The herein-reported results demonstrate that PLGA nanoparticles incorporating oligo-histidine chains and the dual Gd/B theranostic agent AT101 can successfully be exploited to deliver a therapeutic dose of boron to mesothelioma cells, significantly higher than in healthy mesothelial cells as assessed by ICP-MS and MRI. The selective release is pH responsive taking advantage of the slightly acidic pH of the tumour extracellular environment and triggered by the protonation of imidazole groups of histidine. After irradiation with thermal neutrons, tumoral and healthy cells survival and clonogenic ability were evaluated. Obtained results appear very promising, providing patients affected by this rare disease with an improved therapeutic option, exploiting PLGA nanoparticles.

15N magnetic resonance hyperpolarization via the reaction of parahydrogen with 15N-propargylcholine.

(15)N-Propargylcholine has been synthesized and hydrogenated with para-H(2). Through the application of a field cycling procedure, parahydrogen spin order is transferred to the (15)N resonance. Among the different isomers formed upon hydrogenation of (15)N-propargylcholine, only the nontransposed derivative contributes to the observed N-15 enhanced emission signal. The parahydrogen-induced polarization factor is about 3000. The precise identification of the isomer responsible for the observed (15)N enhancement has been attained through a retro-INEPT ((15)N-(1)H) experiment. T(1) of the hyperpolarized (15)N resonance has been estimated to be ca. 150 s, i.e., similar to that reported for the parent propargylcholine (144 s). Experimental results are accompanied by theoretical calculations that stress the role of scalar coupling constants (J(HN) and J(HH)) and of the field dependence in the formation of the observed (15)N polarized signal. Insights into the good cellular uptake of the compound have been gained.

MRI-guided neutron capture therapy by use of a dual gadolinium/boron agent targeted at tumour cells through upregulated low-density lipoprotein transporters.

The upregulation of low-density lipoprotein (LDL) transporters in tumour cells has been exploited to deliver a sufficient amount of gadolinium/boron/ligand (Gd/B/L) probes for neutron capture therapy, a binary chemio-radiotherapy for cancer treatment. The Gd/B/L probe consists of a carborane unit (ten B atoms) bearing an aliphatic chain on one side (to bind LDL particles), and a Gd(III)/1,4,7,10-tetraazacyclododecane monoamide complex on the other (for detection by magnetic resonance imaging (MRI)). Up to 190 Gd/B/L probes were loaded per LDL particle. The uptake from tumour cells was initially assessed on cell cultures of human hepatoma (HepG2), murine melanoma (B16), and human glioblastoma (U87). The MRI assessment of the amount of Gd/B/L taken up by tumour cells was validated by inductively coupled plasma-mass-spectrometric measurements of the Gd and B content. Measurements were undertaken in vivo on mice bearing tumours in which B16 tumour cells were inoculated at the base of the neck. From the acquisition of magnetic resonance images, it was established that after 4-6 hours from the administration of the Gd/B/L-LDL particles (0.1 and 1 mmol kg(-1) of Gd and (10)B, respectively) the amount of boron taken up in the tumour region is above the threshold required for successful NCT treatment. After neutron irradiation, tumour growth was followed for 20 days by MRI. The group of treated mice showed markedly lower tumour growth with respect to the control group.