Phase-Separated Liposomes Hijack Endogenous Lipoprotein Transport and Metabolism Pathways to Target Subsets of Endothelial Cells In Vivo.

Plasma lipid transport and metabolism are essential to ensure correct cellular function throughout the body. Dynamically regulated in time and space, the well-characterized mechanisms underpinning plasma lipid transport and metabolism offers an enticing, but as yet underexplored, rationale to design synthetic lipid nanoparticles with inherent cell/tissue selectivity. Herein, a systemically administered liposome formulation, composed of just two lipids, that is capable of hijacking a triglyceride lipase-mediated lipid transport pathway resulting in liposome recognition and uptake within specific endothelial cell subsets is described. In the absence of targeting ligands, liposome-lipase interactions are mediated by a unique, phase-separated ("parachute") liposome morphology. Within the embryonic zebrafish, selective liposome accumulation is observed at the developing blood-brain barrier. In mice, extensive liposome accumulation within the liver and spleen - which is reduced, but not eliminated, following small molecule lipase inhibition - supports a role for endothelial lipase but highlights these liposomes are also subject to significant "off-target" by reticuloendothelial system organs. Overall, these compositionally simplistic liposomes offer new insights into the discovery and design of lipid-based nanoparticles that can exploit endogenous lipid transport and metabolism pathways to achieve cell selective targeting in vivo.

In Vivo Tracking of the Degradation of Mesoporous Silica through 89 Zr Radio-Labeled Core-Shell Nanoparticles.

While mesoporous silica nanoparticles (MSNs) are extensively studied as high-potential drug delivery platforms, the successful clinical translation of these nanocarriers strongly depends on their biodistribution, biodegradation, and elimination patterns in vivo. Here, a novel method is reported to follow the in vivo degradation of MSNs by tracking a radioactive label embedded in the silica structure. Core-shell silica nanoparticles (NPs) with a dense core and a mesoporous shell are labeled with low quantities of the positron emitter 89 Zr, either in the dense core or in the mesoporous shell. In vivo positron emission tomography imaging and ex vivo organ measurements reveal a remarkable difference in the 89 Zr biodistribution between the shell-labeled and the core-labeled NPs. Release of the radiotracer from shell-labeled NPs is used as a probe of the extent of silica dissolution, and a prompt release of the radioisotope is observed, with partial excretion already in the first 2 h post injection, and a slower accumulation in bones over time. On the other hand, when 89 Zr is embedded in the nanoparticle core, the biodistribution remains largely unchanged during the first 6 h. These findings indicate that MSNs have fast, hour-scale, degradation kinetics in vivo.

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.

Oral Treatment with Iododiflunisal Delays Hippocampal Amyloid-ß Formation in a Transgenic Mouse Model of Alzheimer's Disease: A Longitudinal in vivo Molecular Imaging Study1.

BACKGROUND: Transthyretin (TTR) is a tetrameric, amyloid-ß (Aß)-binding protein, which reduces Aß toxicity. The TTR/Aß interaction can be enhanced by a series of small molecules that stabilize its tetrameric form. Hence, TTR stabilizers might act as disease-modifying drugs in Alzheimer's disease. OBJECTIVE: We monitored the therapeutic efficacy of two TTR stabilizers, iododiflunisal (IDIF), which acts as small-molecule chaperone of the TTR/Aß interaction, and tolcapone, which does not behave as a small-molecule chaperone, in an animal model of Alzheimer's disease using positron emission tomography (PET). METHODS: Female mice (AßPPswe/PS1A246E/TTR+/-) were divided into 3 groups (n = 7 per group): IDIF-treated, tolcapone-treated, and non-treated. The oral treatment (100 mg/Kg/day) was started at 5 months of age. Treatment efficacy assessment was based on changes in longitudinal deposition of Aß in the hippocampus (HIP) and the cortex (CTX) and determined using PET-[18F]florbetaben. Immunohistochemical analysis was performed at age = 14 months. RESULTS: Standard uptake values relative to the cerebellum (SUVr) of [18F]florbetaben in CTX and HIP of non-treated animals progressively increased from age = 5 to 11 months and stabilized afterwards. In contrast, [18F]florbetaben uptake in HIP of IDIF-treated animals remained constant between ages = 5 and 11 months and significantly increased at 14 months. In the tolcapone-treated group, SUVr progressively increased with time, but at lower rate than in the non-treated group. No significant treatment effect was observed in CTX. Results from immunohistochemistry matched the in vivo data at age = 14 months. CONCLUSION: Our work provides encouraging preliminary results on the ability of small-molecule chaperones to ameliorate Aß deposition in certain brain regions.

Radiochemical examination of transthyretin (TTR) brain penetration assisted by iododiflunisal, a TTR tetramer stabilizer and a new candidate drug for AD.

It is well settled that the amyloidogenic properties of the plasma protein transporter transthyretin (TTR) can be modulated by compounds that stabilize its native tetrameric conformation. TTR is also present in cerebrospinal fluid where it can bind to Aß-peptides and prevent Aß aggregation. We have previously shown that treatment of Alzheimer's Disease (AD) model mice with iododiflunisal (IDIF), a TTR tetramer stabilizing compound, prevents AD pathologies. This evidence positioned IDIF as a new lead drug for AD. In dissecting the mechanism of action of IDIF, we disclose here different labeling strategies for the preparation of 131I-labeled IDIF and 131I- and 124I-labeled TTR, which have been further used for the preparation of IDIF-TTR complexes labeled either on the compound or the protein. The biodistribution of all labeled species after intravenous administration has been investigated in mice using ex vivo and in vivo techniques. Our results confirm the capacity of TTR to cross the blood brain barrier (BBB) and suggest that the formation of TTR-IDIF complexes enhances BBB permeability of both IDIF and TTR. The increased TTR and IDIF brain concentrations may result in higher Aß-peptide sequestration capacity with the subsequent inhibition of AD symptoms as we have previously observed in mice.

Immuno-PET Imaging and Pharmacokinetics of an Anti-CEA scFv-based Trimerbody and Its Monomeric Counterpart in Human Gastric Carcinoma-Bearing Mice.

Monoclonal antibodies (mAbs) are currently used as therapeutic agents in different types of cancer. However, mAbs and antibody fragments developed so far show suboptimal properties in terms of circulation time and tumor penetration/retention. Here, we report the radiolabeling, pharmacokinetic evaluation, and determination of tumor targeting capacity of the previously validated anti-CEA MFE23-scFv-based N-terminal trimerbody (MFE23N-trimerbody), and the results are compared to those obtained for the monomeric MFE23-scFv. Dissection and gamma-counting studies performed with the 131I-labeled protein scaffolds in normal mice showed slower blood clearance for the trimerbody, and accumulation in the kidneys, the spleen, and the liver for both species. These, together with a progressive uptake in the small intestine, confirm a combined elimination scheme with hepatobiliary and urinary excretion. Positron emission tomography studies performed in a xenograft mouse model of human gastric adenocarcinoma, generated by subcutaneous administration of CEA-positive human MKN45 cells, showed higher tumor accumulation and tumor-to-muscle (T/M) ratios for 124I-labeled MFE23N-trimerbody than for MFE23-scFv. Specific uptake was not detected with PET imaging in CEA negative xenografts as indicated by low T/M ratios. Our data suggest that engineered intermediate-sized trivalent antibody fragments could be promising candidates for targeted therapy and imaging of CEA-positive tumors.

Application of polymersomes engineered to target p32 protein for detection of small breast tumors in mice.

Triple negative breast cancer (TNBC) is the deadliest form of breast cancer and its successful treatment critically depends on early diagnosis and therapy. The multi-compartment protein p32 is overexpressed and present at cell surfaces in a variety of tumors, including TNBC, specifically in the malignant cells and endothelial cells, and in macrophages localized in hypoxic areas of the tumor. Herein we used polyethylene glycol-polycaprolactone polymersomes that were affinity targeted with the p32-binding tumor penetrating peptide LinTT1 (AKRGARSTA) for imaging of TNBC lesions. A tyrosine residue was added to the peptide to allow for 124I labeling and PET imaging. In a TNBC model in mice, systemic LinTT1-targeted polymersomes accumulated in early tumor lesions more than twice as efficiently as untargeted polymersomes with up to 20% ID/cc at 24 h after administration. The PET-imaging was very sensitive, allowing detection of tumors as small as ∼20 mm3. Confocal imaging of tumor tissue sections revealed a high degree of vascular exit and stromal penetration of LinTT1-targeted polymersomes and co-localization with tumor-associated macrophages. Our studies show that systemic LinTT1-targeted polymersomes can be potentially used for precision-guided tumor imaging and treatment of TNBC.