Ammonium trifluoroborate-modified poly(ß-aminoesters): A case study for PET-guided in vivo pharmacokinetic studies of a non-viral gene delivery system.

Nucleic acid-based therapies have become a game-changing player in our way of conceiving pharmacology. Nevertheless, the inherent lability of the phosphodiester bond of the genetic material with respect to the blood nucleases severely hampers its delivery in naked form, therefore making it necessary to use delivery vectors. Among the potential non-viral vectors, polymeric materials such as the poly(ß-aminoesters) (PBAEs) stand out as promising gene carriers thanks to their ability to condense nucleic acids in the form of nanometric polyplexes. To keep advancing these systems into their translational preclinical phases, it would be highly valuable to gain accurate insights of their in vivo pharmacokinetic profile. We envisaged that positron emission tomography (PET)-guided imaging could provide us with both, an accurate assessment of the biodistribution of PBAE-derived polyplexes, as well shed light on their clearance process. In this sense, taking advantage of the efficient [19F]-to-[18F]­fluorine isotopic exchange presented by the ammonium trifluoroborate (AMBF3) group, we have designed and synthesized a new 18F-PET radiotracer based on the chemical modification of a linear poly(ß-aminoester). As proof of concept, the incorporation of the newly developed 18F-PBAE into a model nanoformulation was shown to be fully compatible with the formation of the polyplexes, their biophysical characterization, and all their in vitro and in vivo functional features. With this tool in hand, we were able to readily obtain key clues about the pharmacokinetic behavior of a series of oligopeptide-modified PBAEs (OM-PBAEs). The observations described in this study allow us to continue supporting these polymers as an outstanding non-viral gene delivery vector for future applications.

Infection-specific PET imaging with 18F-fluorodeoxysorbitol and 2-[18F]F-ρ-aminobenzoic acid: An extended diagnostic tool for bacterial and fungal diseases.

Introduction: Suspected infectious diseases located in difficult-to-access sites can be challenging due to the need for invasive procedures to isolate the etiological agent. Positron emission tomography (PET) is a non-invasive imaging technology that can help locate the infection site. The most widely used radiotracer for PET imaging (2-deoxy-2[18F] fluoro-D-glucose: [18F]FDG) shows uptake in both infected and sterile inflammation. Therefore, there is a need to develop new radiotracers able to specifically detect microorganisms. Methods: We tested two specific radiotracers: 2-deoxy-2-[18F]-fluoro-D-sorbitol ([18F]FDS) and 2-[18F]F-ρ-aminobenzoic acid ([18F]FPABA), and also developed a simplified alternative of the latter for automated synthesis. Clinical and reference isolates of bacterial and yeast species (19 different strains in all) were tested in vitro and in an experimental mouse model of myositis infection. Results and discussion: Non-lactose fermenters (Pseudomonas aeruginosa and Stenotrophomonas maltophilia) were unable to take up [18F]FDG in vitro. [18F]FDS PET was able to visualize Enterobacterales myositis infection (i.e., Escherichia coli) and to differentiate between yeasts with differential assimilation of sorbitol (i.e., Candida albicans vs. Candida glabrata). All bacteria and yeasts tested were detected in vitro by [18F]FPABA. Furthermore, [18F]FPABA was able to distinguish between inflammation and infection in the myositis mouse model (E. coli and Staphylococcus aureus) and could be used as a probe for a wide variety of bacterial and fungal species.

In Vivo Evaluation of Multifunctional Gold Nanorods for Boron Neutron Capture and Photothermal Therapies.

The incidence and mortality of cancer demand more innovative approaches and combination therapies to increase treatment efficacy and decrease off-target side effects. We describe a boron-rich nanoparticle composite with potential applications in both boron neutron capture therapy (BNCT) and photothermal therapy (PTT). Our strategy is based on gold nanorods (AuNRs) stabilized with polyethylene glycol and functionalized with the water-soluble complex cobalt bis(dicarbollide) ([3,3'-Co(1,2-C2B9H11)2]-), commonly known as COSAN. Radiolabeling with the positron emitter copper-64 (64Cu) enabled in vivo tracking using positron emission tomography imaging. 64Cu-labeled multifunctionalized AuNRs proved to be radiochemically stable and capable of being accumulated in the tumor after intravenous administration in a mouse xenograft model of gastrointestinal cancer. The resulting multifunctional AuNRs showed high biocompatibility and the capacity to induce local heating under external stimulation and trigger cell death in heterogeneous cancer spheroids as well as the capacity to decrease cell viability under neutron irradiation in cancer cells. These results position our nanoconjugates as suitable candidates for combined BNCT/PTT therapies.

In vivo multimodal imaging of adenosine A1 receptors in neuroinflammation after experimental stroke.

Adenosine A1 receptors (A1ARs) are promising imaging biomarkers and targets for the treatment of stroke. Nevertheless, the role of A1ARs on ischemic damage and its subsequent neuroinflammatory response has been scarcely explored so far. Methods: In this study, the expression of A1ARs after transient middle cerebral artery occlusion (MCAO) was evaluated by positron emission tomography (PET) with [18F]CPFPX and immunohistochemistry (IHC). In addition, the role of A1ARs on stroke inflammation using pharmacological modulation was assessed with magnetic resonance imaging (MRI), PET imaging with [18F]DPA-714 (TSPO) and [18F]FLT (cellular proliferation), as well as IHC and neurofunctional studies. Results: In the ischemic territory, [18F]CPFPX signal and IHC showed the overexpression of A1ARs in microglia and infiltrated leukocytes after cerebral ischemia. Ischemic rats treated with the A1AR agonist ENBA showed a significant decrease in both [18F]DPA-714 and [18F]FLT signal intensities at day 7 after cerebral ischemia, a feature that was confirmed by IHC results. Besides, the activation of A1ARs promoted the reduction of the brain lesion, as measured with T2W-MRI, and the improvement of neurological outcome including motor, sensory and reflex responses. These results show for the first time the in vivo PET imaging of A1ARs expression after cerebral ischemia in rats and the application of [18F]FLT to evaluate glial proliferation in response to treatment. Conclusion: Notably, these data provide evidence for A1ARs playing a key role in the control of both the activation of resident glia and the de novo proliferation of microglia and macrophages after experimental stroke in rats.

Pre-targeting with ultra-small nanoparticles: boron carbon dots as drug candidates for boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is a promising cancer treatment exploiting the neutron capture capacity and subsequent fission reaction of boron-10. The emergence of nanotechnology has encouraged the development of nanocarriers capable of accumulating boron atoms preferentially in tumour cells. However, a long circulation time, required for high tumour accumulation, is usually accompanied by accumulation of the nanosystem in organs such as the liver and the spleen, which may cause off-target side effects. This could be overcome by using small-sized boron carriers via a pre-targeting strategy. Here, we report the preparation, characterisation and in vivo evaluation of tetrazine-functionalised boron-rich carbon dots, which show very fast clearance and low tumour uptake after intravenous administration in a mouse HER2 (human epidermal growth factor receptor 2)-positive tumour model. Enhanced tumour accumulation was achieved when using a pretargeting approach, which was accomplished by a highly selective biorthogonal reaction at the tumour site with trans-cyclooctene-functionalised Trastuzumab.

Radiochemistry: A Useful Tool in the Ophthalmic Drug Discovery.

Positron Emission Tomography (PET) and Single Photon Emission Computerized Tomography (SPECT) are ultra-sensitive, fully translational and minimally invasive nuclear imaging techniques capable of tracing the spatiotemporal distribution of positron (PET) or gamma (SPECT) emitter-labeled molecules after administration into a living organism. Besides their impact in the clinical diagnostic, PET and SPECT are playing an increasing role in the process of drug development, both during the evaluation of the pharmacokinetic properties of new chemical entities as well as in the proof of concept, proof of mechanism and proof of efficacy studies. However, they have been scarcely applied in the context of ophthalmic drugs. In this paper, the basics of nuclear imaging and radiochemistry are briefly discussed, and the few examples of the use of these imaging modalities in ophthalmic drug development reported in the literature are presented and discussed. Finally, in a purely theoretical exercise, some labeling strategies that could be applied to the preparation of selected ophthalmic drugs are proposed and potential applications of nuclear imaging in ophthalmology are projected.

Gold Nanoparticles as Boron Carriers for Boron Neutron Capture Therapy: Synthesis, Radiolabelling and In vivo Evaluation.

Background: Boron Neutron Capture Therapy (BNCT) is a binary approach to cancer therapy that requires accumulation of boron atoms preferentially in tumour cells. This can be achieved by using nanoparticles as boron carriers and taking advantage of the enhanced permeability and retention (EPR) effect. Here, we present the preparation and characterization of size and shape-tuned gold NPs (AuNPs) stabilised with polyethylene glycol (PEG) and functionalized with the boron-rich anion cobalt bis(dicarbollide), commonly known as COSAN. The resulting NPs were radiolabelled with 124I both at the core and the shell, and were evaluated in vivo in a mouse model of human fibrosarcoma (HT1080 cells) using positron emission tomography (PET). Methods: The thiolated COSAN derivatives for subsequent attachment to the gold surface were synthesized by reaction of COSAN with tetrahydropyran (THP) followed by ring opening using potassium thioacetate (KSAc). Iodination on one of the boron atoms of the cluster was also carried out to enable subsequent radiolabelling of the boron cage. AuNPs grafted with mPEG-SH (5 Kda) and thiolated COSAN were prepared by ligand displacement. Radiolabelling was carried out both at the shell (isotopic exchange) and at the core (anionic absorption) of the NPs using 124I to enable PET imaging. Results: Stable gold nanoparticles simultaneously functionalised with PEG and COSAN (PEG-AuNPs@[4]-) with hydrodynamic diameter of 37.8 ± 0.5 nm, core diameter of 19.2 ± 1.4 nm and ξ-potential of -18.0 ± 0.7 mV were obtained. The presence of the COSAN on the surface of the NPs was confirmed by Raman Spectroscopy and UV-Vis spectrophotometry. PEG-AuNPs@[4]- could be efficiently labelled with 124I both at the core and the shell. Biodistribution studies in a xenograft mouse model of human fibrosarcoma showed major accumulation in liver, lungs and spleen, and poor accumulation in the tumour. The dual labelling approach confirmed the in vivo stability of the PEG-AuNPs@[4]-. Conclusions: PEG stabilized, COSAN-functionalised AuNPs could be synthesized, radiolabelled and evaluated in vivo using PET. The low tumour accumulation in the animal model assayed points to the need of tuning the size and geometry of the gold core for future studies.

P2X4 receptor controls microglia activation and favors remyelination in autoimmune encephalitis.

Microglia survey the brain microenvironment for signals of injury or infection and are essential for the initiation and resolution of pathogen- or tissue damage-induced inflammation. Understanding the mechanism of microglia responses during pathology is hence vital to promote regenerative responses. Here, we analyzed the role of purinergic receptor P2X4 (P2X4R) in microglia/macrophages during autoimmune inflammation. Blockade of P2X4R signaling exacerbated clinical signs in the experimental autoimmune encephalomyelitis (EAE) model and also favored microglia activation to a pro-inflammatory phenotype and inhibited myelin phagocytosis. Moreover, P2X4R blockade in microglia halted oligodendrocyte differentiation in vitro and remyelination after lysolecithin-induced demyelination. Conversely, potentiation of P2X4R signaling by the allosteric modulator ivermectin (IVM) favored a switch in microglia to an anti-inflammatory phenotype, potentiated myelin phagocytosis, promoted the remyelination response, and ameliorated clinical signs of EAE Our results provide evidence that P2X4Rs modulate microglia/macrophage inflammatory responses and identify IVM as a potential candidate among currently used drugs to promote the repair of myelin damage.

In vivo imaging of Α7 nicotinic receptors as a novel method to monitor neuroinflammation after cerebral ischemia.

In vivo positron emission tomography (PET) imaging of nicotinic acetylcholine receptors (nAChRs) is a promising tool for the imaging evaluation of neurologic and neurodegenerative diseases. However, the role of α7 nAChRs after brain diseases such as cerebral ischemia and its involvement in inflammatory reaction is still largely unknown. In vivo and ex vivo evaluation of α7 nAChRs expression after transient middle cerebral artery occlusion (MCAO) was carried out using PET imaging with [11 C]NS14492 and immunohistochemistry (IHC). Pharmacological activation of α7 receptors was evaluated with magnetic resonance imaging (MRI), [18 F]DPA-714 PET, IHC, real time polymerase chain reaction (qPCR) and neurofunctional studies. In the ischemic territory, [11 C]NS14492 signal and IHC showed an expression increase of α7 receptors in microglia and astrocytes after cerebral ischemia. The role played by α7 receptors on neuroinflammation was supported by the decrease of [18 F]DPA-714 binding in ischemic rats treated with the α7 agonist PHA 568487 at day 7 after MCAO. Moreover, compared with non-treated MCAO rats, PHA-treated ischemic rats showed a significant reduction of the cerebral infarct volumes and an improvement of the neurologic outcome. PHA treatment significantly reduced the expression of leukocyte infiltration molecules in MCAO rats and in endothelial cells after in vitro ischemia. Despite that, the activation of α7 nAChR had no influence to the blood brain barrier (BBB) permeability measured by MRI. Taken together, these results suggest that the nicotinic α7 nAChRs play a key role in the inflammatory reaction and the leukocyte recruitment following cerebral ischemia in rats.

Evaluation of the novel TSPO radiotracer [18F] VUIIS1008 in a preclinical model of cerebral ischemia in rats.

BACKGROUND: In vivo positron-emission tomography (PET) imaging of transporter protein (TSPO) expression is an attractive and indispensable tool for the diagnosis and therapy evaluation of neuroinflammation after cerebral ischemia. Despite several radiotracers have shown an excellent capacity to image neuroinflammation, novel radiotracers such as [18F] VUIIS1008 have shown promising properties to visualize and quantify the in vivo expression of TSPO. METHODS: Longitudinal in vivo magnetic resonance (MRI) and PET imaging studies with the novel TSPO radiotracer 2-(5,7-diethyl-2-(4-(2-[18F] fluoroethoxy) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl)-N, N-diethylacetamide ([18F] VUIIS1008), and (N, N-diethyl-2-(2-[4-(2-fluoroethoxy)-phenyl]-5,7-dimethyl-pyrazolo [1,5-a] yrimidin-3-yl)-acetamide ([18F] DPA-714) were carried out before and at days 1, 3, 7, 14, 21, and 28 following the transient middle cerebral artery occlusion (MCAO) in rats. RESULTS: MRI images showed the extension and evolution of the brain infarction after ischemic stroke in rats. PET imaging with [18F] VUIIS1008 and [18F] DPA714 showed a progressive increase in the ischemic brain hemisphere during the first week, peaking at day 7 and followed by a decline from days 14 to 28 after cerebral ischemia. [18F] DPA714 uptake showed a mild uptake increase compared to [18F] VUIIS1008 in TSPO-rich ischemic brain regions. In vivo [18F] VUIIS1008 binding displacement with VUIIS1008 was more efficient than DPA714. Finally, immunohistochemistry confirmed a high expression of TSPO in microglial cells at day 7 after the MCAO in rats. CONCLUSIONS: Altogether, these results suggest that [18F] VUIIS1008 could become a valuable tool for the diagnosis and treatment evaluation of neuroinflammation following ischemic stroke.