Perfluorocarbon Nanodroplets as Potential Nanocarriers for Brain Delivery Assisted by Focused Ultrasound-Mediated Blood-Brain Barrier Disruption.

The management of brain diseases remains a challenge, particularly because of the difficulty for drugs to cross the blood-brain barrier. Among strategies developed to improve drug delivery, nano-sized emulsions (i.e., nanoemulsions), employed as nanocarriers, have been described. Moreover, focused ultrasound-mediated blood-brain barrier disruption using microbubbles is an attractive method to overcome this barrier, showing promising results in clinical trials. Therefore, nanoemulsions combined with this technology represent a real opportunity to bypass the constraints imposed by the blood-brain barrier and improve the treatment of brain diseases. In this work, a stable freeze-dried emulsion of perfluorooctyl bromide nanodroplets stabilized with home-made fluorinated surfactants able to carry hydrophobic agents is developed. This formulation is biocompatible and droplets composing the emulsion are internalized in multiple cell lines. After intravenous administration in mice, droplets are eliminated from the bloodstream in 24 h (blood half-life (t1/2) = 3.11 h) and no long-term toxicity is expected since they are completely excreted from mice' bodies after 72 h. In addition, intracerebral accumulation of tagged droplets is safely and significantly increased after focused ultrasound-mediated blood-brain barrier disruption. Thus, the proposed nanoemulsion appears as a promising nanocarrier for a successful focused ultrasound-mediated brain delivery of hydrophobic agents.

Hydrophilic Fluorescent Nanoprodrug of Paclitaxel for Glioblastoma Chemotherapy.

Highly water-soluble, nontoxic organic nanoparticles on which paclitaxel (PTX), a hydrophobic anticancer drug, has been covalently bound via an ester linkage (4.5% of total weight) have been prepared for the treatment of glioblastoma. These soft fluorescent organic nanoparticles (FONPs), obtained from citric acid and diethylenetriamine by microwave-assisted condensation, show suitable size (Ø = 17-30 nm), remarkable solubility in water, softness as well as strong blue fluorescence in an aqueous environment that are fully retained in cell culture medium. Moreover, these FONPs were demonstrated to show in vitro safety and preferential internalization in glioblastoma cells through caveolin/lipid raft-mediated endocytosis. The PTX-conjugated FONPs retain excellent solubility in water and remain stable in water (no leaching), while they showed anticancer activity against glioblastoma cells in two-dimensional and three-dimensional culture. PTX-specific effects on microtubules reveal that PTX is intracellularly released from the nanocarriers in its active form, in relation with an intracellular-promoted lysis of the ester linkage. As such, these hydrophilic prodrug formulations hold major promise as biocompatible nanotools for drug delivery.

Laser- synthesized TiN nanoparticles as promising plasmonic alternative for biomedical applications.

Exhibiting a red-shifted absorption/scattering feature compared to conventional plasmonic metals, titanium nitride nanoparticles (TiN NPs) look as very promising candidates for biomedical applications, but these applications are still underexplored despite the presence of extensive data for conventional plasmonic counterparts. Here, we report the fabrication of ultrapure, size-tunable TiN NPs by methods of femtosecond laser ablation in liquids and their biological testing. We show that TiN NPs demonstrate strong and broad plasmonic peak around 640-700 nm with a significant tail over 800 nm even for small NPs sizes (<7 nm). In vitro tests of laser-synthesized TiN NPs on cellular models evidence their low cytotoxicity and excellent cell uptake. We finally demonstrate a strong photothermal therapy effect on U87-MG cancer cell cultures using TiN NPs as sensitizers of local hyperthermia under near-infrared laser excitation. Based on absorption band in the region of relative tissue transparency and acceptable biocompatibility, laser-synthesized TiN NPs promise the advancement of biomedical modalities employing plasmonic effects, including absorption/scattering contrast imaging, photothermal therapy, photoacoustic imaging and SERS.

5-HT2A receptor SPECT imaging with [¹²³I]R91150 under P-gp inhibition with tariquidar: More is better?

INTRODUCTION: Pharmacological P-glycoprotein (P-gp) inhibition with tariquidar (TQD) is considered a promising strategy for the augmentation of radiotracer brain uptake. However, a region-dependent effect may compromise the robustness of quantitative studies. For this reason, we studied the effect of a TQD pretreatment on 5-HT2A imaging with [(123)I]R91150 and compared results with those obtained in Mdr1a knock-out (KO) rats. METHODS: Ex vivo autoradiography was performed in TQD (15 mg/kg) pretreated wild-type (WT-TQD), Mdr1a knock-out (KO) and untreated WT rats for Specific Binding Ratio (SBR) estimation. In vivo dynamic SPECT imaging with serial arterial blood sampling was performed in the former two groups of rats and kinetic analysis was performed with a one tissue-compartment (1TC) model and the Specific Uptake Ratio (SUR). Results were analyzed statistically using repeated measures ANOVA. RESULTS: SBR values differed between WT-TQD, Mdr1a KO and WT rats in a region-dependent manner (p<0.0001). In vivo brain uptake of radiotracer did not differ between groups. Similarly, kinetic analysis provided distribution volume (V(T)) values that did not differ significantly between groups. SUR binding potential (BPND) values from both groups highly correlated with corresponding V(T) (r=0.970, p<0.0001 and r=0.962, p<0.0001, respectively). However, SUR measured over averaged images between 100 and 120 min, using cerebellum as reference region, demonstrated values that were, by average, 2.99±0.53 times higher in the WT-TQD group, with the difference between groups being region-dependent (p<0.001). In addition, coefficient of variation of the SUR BPND values across brain regions was significantly higher in the WT-TQD rats (41.25%±9.63% versus 11.13%±5.59%, p<0.0001). CONCLUSION: P-gp inhibition with TQD leads to region-dependent effect in the rat brain, with probably sub-optimal effect in cerebellum. This warrants attention when it is used as a reference region for quantitative studies.

In Vivo Quantification of 5-HT2A Brain Receptors in Mdr1a KO Rats with 123I-R91150 Single-Photon Emission Computed Tomography.

Our goal was to identify suitable image quantification methods to image 5-hydroxytryptamine2A (5-HT2A) receptors in vivo in Mdr1a knockout (KO) rats (i.e., P-glycoprotein KO) using 123I-R91150 single-photon emission computed tomography (SPECT). The 123I-R91150 binding parameters estimated with different reference tissue models (simplified reference tissue model [SRTM], Logan reference tissue model, and tissue ratio [TR] method) were compared to the estimates obtained with a comprehensive three-tissue/seven-parameter (3T/7k)-based model. The SRTM and Logan reference tissue model estimates of 5-HT2A receptor (5-HT2AR) nondisplaceable binding potential (BPND) correlated well with the absolute receptor density measured with the 3T/7k gold standard (r > .89). Quantification of 5-HT2AR using the Logan reference tissue model required at least 90 minutes of scanning, whereas the SRTM required at least 110 minutes. The TR method estimates were also highly correlated to the 5-HT2AR density (r > .91) and only required a single 20-minute scan between 100 and 120 minutes postinjection. However, a systematic overestimation of the BPND values was observed. The Logan reference tissue method is more convenient than the SRTM for the quantification of 5-HT2AR in Mdr1a KO rats using 123I-R91150 SPECT. The TR method is an interesting and simple alternative, despite its bias, as it still provides a valid index of 5-HT2AR density.

SPECT imaging of glioma with radioiodinated CLINDE: evidence from a mouse GL26 glioma model.

BACKGROUND: Recent research has demonstrated the potential of 18-kDa translocator protein (TSPO) to serve as a target for nuclear imaging of gliomas. The aim of this study was to evaluate SPECT imaging of GL26 mouse glioma using radioiodinated CLINDE, a TSPO-specific tracer. METHODS: GL26 cells, previously transfected with an enhanced green fluorescent protein (EGFP)-expressing lentivirus, were stereotactically implanted in the striatum of C57/Bl6 mice. At 4 weeks post-injection, dynamic SPECT scans with [(123)I]CLINDE were performed. A displacement study assessed specificity of tracer binding. SPECT images were compared to results of autoradiography, fluorescence microscopy, in situ nucleic acid hybridization, histology, and immunohistochemistry. Western blotting was performed to verify TSPO production by the tumor. RESULTS: Specific uptake of tracer by the tumor is observed with a high signal-to-noise ratio. Tracer uptake by the tumor is indeed 3.26 ± 0.32 times higher than that of the contralateral striatum, and 78% of the activity is displaceable by unlabeled CLINDE. Finally, TSPO is abundantly expressed by the GL26 cells. CONCLUSIONS: The present study demonstrates the feasibility of [(123)I]CLINDE SPECT in translational studies and underlines its potential for clinical glioma SPECT imaging.

Design of a serotonin 4 receptor radiotracer with decreased lipophilicity for single photon emission computed tomography.

With the aim to develop a suitable radiotracer for the brain imaging of the serotonin 4 receptor subtype (5-HT4R) using single photon emission computed tomography (SPECT), we synthesized and evaluated a library of di- and triazaphenanthridines with lipophilicity values which were in the range expected to favour brain penetration, and which demonstrated specific binding to the target of interest. Adding additional nitrogen atoms to previously described phenanthridine ligands exhibiting a high unspecific binding, we were able to design a radioiodinated compound [(125)I]14. This compound exhibited a binding affinity value of 0.094 nM toward human 5-HT4R and a high selectivity over other serotonin receptor subtypes (5-HTR). In vivo SPECT imaging studies and competition experiments demonstrated that the decreased lipophilicity (in comparison with our previously reported compounds 4 and 5) allowed a more specific labelling of the 5-HT4R brain-containing regions.

A modified simplified reference tissue model for the quantification of dopamine D2/3 receptors with [18F]fallypride images.

Defluorination of [18F]fallypride and accumulation of 18F in skull and glands leads to the contamination of brain structures with spillover activity due to partial volume effects, leading to considerable errors in binding potential estimations. Here we propose a modification of the simplified reference tissue model (SRTM) to take into account the contribution of skull activity to the radioactivity kinetic pattern in cerebellum and target regions. It consists of the introduction of an additional parameter for each volume of interest (sT) and one for the cerebellum (sR), corresponding to the fraction of skull activity contaminating these structures. Using five rat positron emission tomography experiments, we applied the modified SRTM (SRTMc), which resulted in excellent fits. As a relative means of comparison of results, we applied factor analysis (FA) to decompose dynamic data into images corresponding to brain and skull activity. With the skull factor images, we estimated the "true" sT and sR values, ultimately permitting us to fix the sR value. Parameters obtained with the SRTMc were closely correlated with values obtained from FA-corrected data. In conclusion, we propose an efficient method for reliable quantification of dopamine D2/3 receptors with single-injection [18F]fallypride scans that is potentially applicable to human studies where 18F skull accumulation compromises binding parameter estimation.

Small-animal single-photon emission computed tomographic imaging of the brain serotoninergic systems in wild-type and mdr1a knockout rats.

The pharmacokinetic properties of radiotracers are crucial for successful in vivo single-photon emission computed tomographic (SPECT) imaging. Our goal was to determine if MDR1A-deficient animals could allow better SPECT imaging outcomes than wild-type (WT) animals for a selection of serotoninergic radioligands. Thus, we compared the performances of 123I-p-MPPI, 123I-R91150, 123I-SB207710, and 123I-ADAM radioligands, for imaging of their respective targets (5-hydroxytryptamine [5-HT]1A, 5-HT2A, 5-HT4, and serotonin transporter [SERT]), in WT and Mdr1a knockout (KO) rats. With 123I-SB207710, virtually no SPECT signal was recorded in the brain of WT or KO animals. For 123I-p-MPPI, low nondisplaceable binding potentials (BPND, mean ± SD) were observed in WT (0.49 ± 0.25) and KO (0.89 ± 0.52) animals. For 123I-ADAM, modest imaging contrast was observed in WT (1.27 ± 0.02) and KO (1.31 ± 0.09) animals. For 123I-R91150, the BPND were significantly higher in Mdr1a KO (3.98 ± 0.65) animals compared to WT animals (1.22 ± 0.26). The pharmacokinetics of 123I-SB207710 and 123I-p-MPPI do not make them ideal tracers for preclinical SPECT neuroimaging. 123I-ADAM showed adequate brain uptake regardless of Mdr1a expression and appeared suitable for preclinical SPECT neuroimaging in both animal strains. The use of Mdr1a KO animals significantly improved the brain penetration of 123I-R91150, making this animal strain an interesting option when considering SPECT neuroimaging of 5-HT2A receptors in rat.

Quantification of GABAA receptors in the rat brain with [(123)I]Iomazenil SPECT from factor analysis-denoised images.

PURPOSE: In vivo imaging of GABAA receptors is essential for the comprehension of psychiatric disorders in which the GABAergic system is implicated. Small animal SPECT provides a modality for in vivo imaging of the GABAergic system in rodents using [(123)I]Iomazenil, an antagonist of the GABAA receptor. The goal of this work is to describe and evaluate different quantitative reference tissue methods that enable reliable binding potential (BP) estimations in the rat brain to be obtained. METHODS: Five male Sprague-Dawley rats were used for [(123)I]Iomazenil brain SPECT scans. Binding parameters were obtained with a one-tissue compartment model (1TC), a constrained two-tissue compartment model (2TCc), the two-step Simplified Reference Tissue Model (SRTM2), Logan graphical analysis and analysis of delayed-activity images. In addition, we employed factor analysis (FA) to deal with noise in data. RESULTS: BPND obtained with SRTM2, Logan graphical analysis and delayed-activity analysis was highly correlated with BPF values obtained with 2TCc (r=0.954 and 0.945 respectively, p<0.0001). Equally significant correlations were found between values obtained with 2TCc and SRTM2 in raw and FA-denoised images (r=0.961 and 0.909 respectively, p<0.0001). Scans of at least 100min are required to obtain stable BPND values from raw images while scans of only 70min are sufficient from FA-denoised images. These images are also associated with significantly lower standard errors of 2TCc and SRTM2 BP values. CONCLUSION: Reference tissue methods such as SRTM2 and Logan graphical analysis can provide equally reliable BPND values from rat brain [(123)I]Iomazenil SPECT. Acquisitions, however, can be much less time-consuming either with analysis of delayed activity obtained from a 20-minute scan 50min after tracer injection or with FA-denoising of images.

Synthesis and structure-affinity relationships of selective high-affinity 5-HT(4) receptor antagonists: application to the design of new potential single photon emission computed tomography tracers.

The work described herein aims at finding new potential ligands for the brain imaging of 5-HT(4) receptors (5-HT(4)Rs) using single-photon emission computed tomography (SPECT). Starting from the nonsubstituted phenanthridine compound 4a, exhibiting a K(i) value of 51 nM on the 5-HT(4)R, we explored the structure-affinity in this series. We found that substitution in position 4 of the tricycle with a fluorine atom gave the best result. Introduction of an additional nitrogen atom inside the tricyclic framework led to an increase of both the affinity and selectivity for 5-HT(4)R, suggesting the design of the antagonist 4v, exhibiting a high affinity of 0.04 nM. Several iodinated analogues were then synthesized as potential SPECT tracers. The iodinated compound 11d was able to displace the reference radioiodinated 5-HT(4)R antagonist (1-butylpiperidin-4-yl)methyl-8-amino-7-iodo[(123)I]-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate {[(123)I]1, [(123)I]SB 207710} both in vitro and in vivo in brain. Compound 11d was radiolabeled with [(125)I]iodine, providing a potential SPECT candidate for brain imaging of 5-HT(4)R.

Quantification of dopamine D(2/3) receptors in rat brain using factor analysis corrected [18F]Fallypride images.

The goal of this work is to quantify the binding parameters of [(18)F]Fallypride in the striatal and extrastriatal regions of the rat brain using factor analysis (FA) to correct small animal PET kinetic imaging for spillover defluorination radioactivity. Eleven rats were employed for YAP-(S)PET acquisitions and metabolite studies. All kinetic parameters including B'(max) and K(d)V(R) were estimated with a three-tissue compartment seven-parameter model (3T-7k) on the basis of all the FA-corrected data from the multi-injection protocol. Binding potential (BP(ND)) was calculated with Logan's graphical analysis taking cerebellum as the reference region and using the first injection raw (BP(ND-RAW)) and FA-corrected (BP(ND-FA)) data. Three distinct factors corresponding to free+non-specific binding, specific binding and skull and gland accumulation were recovered from FA with their corresponding spatial distributions. The resulting reconstructed images without skull and gland accumulation were improved to provide a better contrast between specific and non-specific regions. Very bad fits were obtained when using time-activity curves (TACs) calculated from the raw [(18)F]Fallypride data, whereas all TACs were well fitted by the 3T-7k model after FA correction. FA-corrected data enables the cerebellar region to be used as reference for the Logan approach. The magnitude of the BP(ND-FA) values was increased from 21% to 108% across regions and the rank order of BP(ND-FA) values (Cx

Synthesis and characterization of a iodine-125-labeled pyrrolo[1,2-a]thieno[3,2-e]pyrazine and evaluation as a potential 5-HT4R SPECT tracer.

In the aim to find new radiotracers for the in vivo imaging of 5-HT(4) receptors by ultra-high resolution quantitative SPECT, we have developed the synthesis of a radioiodinated 5-HT(4) ligand using an iododestannylation procedure. The [(125)I]-ligand was obtained in a high radiochemical yield. Preliminary autoradiographic and ex vivo studies failed to show a specific labeling of 5-HT(4) receptors.