Functionalization and Characterization of Magnetic Nanoparticles for the Detection of Ferritin Accumulation in Alzheimer's Disease

Early diagnosis in Alzheimer's disease (AD), prior to the appearance of marked clinical symptoms, is critical to prevent irreversible neuronal damage and neural malfunction that lead to dementia and death. Therefore, there is an urgent need to generate new contrast agents which reveal by a noninvasive method the presence of some of the pathological signs of AD. In the present study, we demonstrate for the first time a new nanoconjugate composed of magnetic nanoparticles bound to an antiferritin antibody, which has been developed based on the existence of iron deposits and high levels of the ferritin protein present in areas with a high accumulation of amyloid plaques (particularly the subiculum in the hippocampal area) in the brain of a transgenic mouse model with five familial AD mutations. Both in vitro and after intravenous injection, functionalized magnetic nanoparticles were able to recognize and bind specifically to the ferritin protein accumulated in the subiculum area of the AD transgenic mice.Fil: Fernández Cabada, Tamara. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Politécnica de Madrid; EspañaFil: Martínez Serrano, Alberto. Consejo Superior de Investigaciones Científicas; España. Universidad Autónoma de Madrid; EspañaFil: Cussó, Lorena. Instituto de Investigacion Sanitaria Gregorio Marañón; España. Universidad Carlos III de Madrid; España. Centro de Investigación Biomédica en Red de Salud Mental; EspañaFil: Desco, Manuel. Instituto de Investigacion Sanitaria Gregorio Marañón; España. Centro de Investigación Biomédica en Red de Salud Mental; España. Universidad Carlos III de Madrid; EspañaFil: Ramos Gómez, Milagros. Universidad Politécnica de Madrid; Españ

Combination of Single-Photon Emission Computed Tomography and Magnetic Resonance Imaging to Track ¹¹¹In-Oxine-Labeled Human Mesenchymal Stem Cells in Neuroblastoma-Bearing Mice

Homing is an inherent, complex, multistep process performed by cells such as human bone marrow mesenchymal stem cells (hMSCs) to travel from a distant location to inflamed or damaged tissue and tumors. This ability of hMSCs has been exploited as a tumor-targeting strategy in cell-based cancer therapy. The purpose of this study was to investigate the applicability of ¹¹¹In-oxine for tracking hMSCs in vivo by combining single-photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI). ¹¹¹In-labeled hMSCs (10⁶ cells) were infused intraperitoneally in neuroblastoma-bearing mice, whereas a control group received a dose of free ¹¹¹In-oxine. SPECT and MRI studies were performed 24 and 48 hours afterwards. Initially, the images showed similar activity in the abdomen in both controls and hMSC-injected animals. In general, abdominal activity decreases at 48 hours. hMSC-injected animals showed increased uptake in the tumor area at 48 hours, whereas the control group showed a low level of activity at 24 hours, which decreased at 48 hours. In conclusion, tracking ¹¹¹In-labeled hMSCs combining SPECT and MRI is feasible and may be transferable to clinical research. The multimodal combination is essential to ensure appropriate interpretation of the images.This work was funded in part by grants from Ministerio de Economía y Competitividad (PLE2009-0115), Red Tematica de Investigación Cooperativa en Cancer (RTICC/ISCIII; RD12/0036/0027), the Madrid Regional Government (S-BIO-0204-2006–MesenCAM and P2010/BMD-2420-CellCAM), and the Ministerio de Ciencia e Innovación (CEN-20101014 and TEC-2010-21619-C04-01).Publicad

Development and Validation of Non-Integrative, Self-Limited, and Replicating Minicircles for Safe Reporter Gene Imaging of Cell-Based Therapies

Reporter gene (RG) imaging of cell-based therapies provides a direct readout of therapeutic efficacy by assessing the fate of implanted cells. To permit long-term cellular imaging, RGs are traditionally required to be integrated into the cellular genome. This poses a potential safety risk and regulatory bottleneck for clinical translation as integration can lead to cellular transformation. To address this issue, we have developed non-integrative, replicating minicircles (MCs) as an alternative platform for safer monitoring of cells in living subjects. We developed both plasmids and minicircles containing the scaffold/matrix attachment regions (S/MAR) of the human interferon-beta gene, driven by the CMV promoter, and expressing the bioluminescence RG firefly luciferase. Constructs were transfected into breast cancer cells, and expanded S/MAR minicircle clones showed luciferase signal for greater than 3 months in culture and minicircles remained as episomes. Importantly, luciferase activity in clonal populations was slowly lost over time and this corresponded to a loss of episome, providing a way to reversibly label cells. To monitor cell proliferation in vivo, 1.5×10(6) cells carrying the S/MAR minicircle were implanted subcutaneously into mice (n = 5) and as tumors developed significantly more bioluminescence signal was noted at day 35 and 43 compared to day 7 post-implant (p<0.05). To our knowledge, this is the first work examining the use of episomal, self-limited, replicating minicircles to track the proliferation of cells using non-invasive imaging in living subjects. Continued development of S/MAR minicircles will provide a broadly applicable vector platform amenable with any of the numerous RG technologies available to allow therapeutic cell fate to be assessed in individual patients, and to achieve this without the need to manipulate the cell's genome so that safety concerns are minimized. This will lead to safe tools to assess treatment response at earlier time points and improve the precision of cell-based therapies.The authors would like to acknowledge the imaging support provided by the Stanford Small Animal Imaging FacilityPublicad

Dual-labeled nanoparticles based on small extracellular vesicles for tumor detection.

Small extracellular vesicles (sEVs) are emerging natural nanoplatforms in cancer diagnosis and therapy, through the incorporation of signal components or drugs in their structure. However, for their translation into the clinical field, there is still a lack of tools that enable a deeper understanding of their in vivo pharmacokinetics or their interactions with the cells of the tumor microenvironment. In this study, we have designed a dual-sEV probe based on radioactive and fluorescent labeling of goat milk sEVs. The imaging nanoprobe was tested in vitro and in vivo in a model of glioblastoma. In vitro assessment of the uptake of the dual probe in different cell populations (RAW 264.7, U87, and HeLa) by optical and nuclear techniques (gamma counter, confocal imaging, and flow cytometry) revealed the highest uptake in inflammatory cells (RAW 264.7), followed by glioblastoma U87 cells. In vivo evaluation of the pharmacokinetic properties of nanoparticles confirmed a blood circulation time of ~ 8 h and primarily hepatobiliary elimination. The diagnostic capability of the dual nanoprobe was confirmed in vivo in a glioblastoma xenograft model, which showed intense in vivo uptake of the SEV-based probe in tumor tissue. Histological assessment by confocal imaging enabled quantification of tumor populations and confirmed uptake in tumor cells and tumor-associated macrophages, followed by cancer-associated fibroblasts and endothelial cells. We have developed a chemical approach for dual radioactive and fluorescent labeling of sEVs. This methodology enables in vivo and in vitro study of these vesicles after exogenous administration. The dual nanoprobe would be a promising technology for cancer diagnosis and a powerful tool for studying the biological behavior of these nanosystems for use in drug delivery.This study was supported by Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III, project “PI20/01632” and “PT20/00044”, co-funded by the European Regional Development Fund (ERDF), “A way of making Europe”, by Comunidad de Madrid, project “Y2018/NMT-4949 (NanoLiver-CM)” and “S2017/ BMD-3867 (RENIM-CM)”, co-funded by the European Structural and Invest‑ ment Fund, and by Agencia Estatal de Investigación “PID2019‐110369RB‐I00/ AEI/https://doi.org/10.13039/501100011033” grant. This work was also sup‑ ported by “Diagnosis and treatment follow‐up of severe Staphylococcal infec‑ tions with anti‐Staphylococcal antibodies and immune‐PET ‐ Grant Fundación BBVA a Equipos de Investigación Científca 2018 and Ramon Areces Grant “Imagen molecular de la infección por Clostridiodes difcile”. Grant EQC2019006674-P funded by MCIN/AEI /https://doi.org/10.13039/501100011033 and by “ERDF A way of making Europe”. A. Santos-Coquillat is grateful for fnancial support from Consejería de Educación e Investigación Comunidad de Madrid, co-fnanced by European Social Fund (ESF) grant PEJD-2018-POST/BMD-9592 and the Sara Borrell Fellowship from Ministerio de Ciencia e Innovación, Insti‑ tuto de Salud Carlos III grant CD19/00136. M.I. González is funded by Instituto de Investigación Sanitaria Gregorio Marañón, Intramural Programme for the Promotion of R&D&I 2021, Sub-programme "Predoctoral training contract".S

Structural and Functional Brain Abnormalities in Mouse Models of Lafora Disease.

Mutations in the EPM2A and EPM2B genes, encoding laforin and malin proteins respectively, are responsible for Lafora disease, a fatal form of progressive myoclonus epilepsy with autosomal recessive inheritance. Neuroimaging studies of patients with Lafora disease have shown different degrees of brain atrophy, decreased glucose brain uptake and alterations on different brain metabolites mainly in the frontal cortex, basal ganglia and cerebellum. Mice deficient for laforin and malin present many features similar to those observed in patients, including cognitive, motor, histological and epileptic hallmarks. We describe the neuroimaging features found in two mouse models of Lafora disease. We found altered volumetric values in the cerebral cortex, hippocampus, basal ganglia and cerebellum using magnetic resonance imaging (MRI). Positron emission tomography (PET) of the cerebral cortex, hippocampus and cerebellum of Epm2a-/- mice revealed abnormal glucose uptake, although no alterations in Epm2b-/- mice were observed. Magnetic resonance spectroscopy (MRS) revealed significant changes in the concentration of several brain metabolites, including N-acetylaspartate (NAA), in agreement with previously described findings in patients. These data may provide new insights into disease mechanisms that may be of value for developing new biomarkers for diagnosis, prevention and treatment of Lafora disease using animal models

Oregano essential oil: an effective and non-toxic approach for prevent or treat resistant candida species

Vulvovaginal candidiasis (VVC) is one of the most prevalent vaginal infectious diseases, and the emergence of drug-resistant Candida strains has presented a growing challenge in its treatment. This highlights the urgent need to develop effective and non-toxic alternative treatments. In this context, essential oils (EOs) have emerged as a promising alternative considering low toxicity and high antimicrobial activity. This work is divided into two parts, the first consists of evaluating the effect of the vapor phase of oregano EO (VP-OEO) on biofilms of antifungal-resistant Candida species (Candida albicans and Candida glabrata) quantified by colony forming units enumeration and determine their mode of action by flow cytometry. Interestingly, the VP-EOs has shown to be more effective against Candida growth than their liquid form. Indeed, the results revealed high antifungal activity of VP-OEO against these drug-resistant strains, significantly reducing biofilm formation and mature biofilms, with impact on membrane integrity and metabolic activity of the fungal cells. The second part consists of the design and evaluation of nanoencapsulated OEO (KNP-OEO) as another alternative application of OEO for VVC treatment. These nanoparticles provided stability to OEO and controlled release of the EO. The results demonstrated complete inhibition of C. albicans growth. Moreover, in in vivo assay with BALB/C female mice, a single intravaginal application of KNP-OEO reduced C. albicans growth and preserved a healthy vaginal microbiota, including Lactobacillus species. In conclusion, these studies highlight the promising efficacy of OEO as an alternative for VVC treatment. Both approaches, VP-OEO and OEO-KNP, showed effective antifungal activity against drug-resistant strains while preserving vaginal health. These therapeutic options not only combat antifungal resistance, but also potentially propose a safer option for women's health due to their natural characteristics. However, further research is needed to confirm these promising results and advance the development of these alternative VVC therapies.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and grant ref 2020.05720.BD for Liliana Fernandes. Also, this study was supported by LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020 and Maria Elisa Rodrigues thanks FCT for funding through program DL 57/2016—Norma transitória. Sofia Costa de Oliveira acknowledges national funds through FCT, I.P., within the scope of the project "RISE - LA/P/0053/2020. Nuno Pereira Mira acknowledges support from FCT through its funding of research focused on Candida-lactobacilii interactions through LactoCan project (contract number: PTDC/BIA-MIC/31515/2017), iBB (contract: UIDB/04565/2020) and i4HB funding (contract: LA/P/0140/2020). Lorena Cussó acknowledges support by Agencia Estatal de Investigación (PRE2020-095268 MCIN / AEI /10.13039/501100011033 and by "ESF Investing in your future"), by Instituto de Salud Carlos III (PT20/00044) and co-funded by European Union (ERDF, "A way to make Europe")info:eu-repo/semantics/publishedVersio

Cardiac Extracellular Matrix Hydrogel Enriched with Polyethylene Glycol Presents Improved Gelation Time and Increased On-Target Site Retention of Extracellular Vesicles

Stem-cell-derived extracellular vesicles (EVs) have demonstrated multiple beneficial effects in preclinical models of cardiac diseases. However, poor retention at the target site may limit their therapeutic efficacy. Cardiac extracellular matrix hydrogels (cECMH) seem promising as drug-delivery materials and could improve the retention of EVs, but may be limited by their long gelation time and soft mechanical properties. Our objective was to develop and characterize an optimized product combining cECMH, polyethylene glycol (PEG), and EVs (EVs–PEG–cECMH) in an attempt to overcome their individual limitations: long gelation time of the cECMH and poor retention of the EVs. The new combined product presented improved physicochemical properties (60% reduction in half gelation time, p < 0.001, and threefold increase in storage modulus, p < 0.01, vs. cECMH alone), while preserving injectability and biodegradability. It also maintained in vitro bioactivity of its individual components (55% reduction in cellular senescence vs. serum-free medium, p < 0.001, similar to EVs and cECMH alone) and increased on-site retention in vivo (fourfold increase vs. EVs alone, p < 0.05). In conclusion, the combination of EVs–PEG–cECMH is a potential multipronged product with improved gelation time and mechanical properties, increased on-site retention, and maintained bioactivity that, all together, may translate into boosted therapeutic efficacy

A clinically compatible drug-screening platform based on organotypic cultures identifies vulnerabilities to prevent and treat brain metastasis

We report a medium‐throughput drug‐screening platform (METPlatform) based on organotypic cultures that allows to evaluate inhibitors against metastases growing in situ. By applying this approach to the unmet clinical need of brain metastasis, we identified several vulnerabilities. Among them, a blood–brain barrier permeable HSP90 inhibitor showed high potency against mouse and human brain metastases at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis applied to metastases treated with the chaperone inhibitor uncovered a novel molecular program in brain metastasis, which includes biomarkers of poor prognosis and actionable mechanisms of resistance. Our work validates METPlatform as a potent resource for metastasis research integrating drug‐screening and unbiased omic approaches that is compatible with human samples. Thus, this clinically relevant strategy is aimed to personalize the management of metastatic disease in the brain and elsewhere

Suppression of 18F-FDG signal in the bladder on small animal PET-CT.

INTRODUCTION:Retention of 2-deoxy-2-[18F]fluoro-D-glucose 18F-FDG in the bladder causes more problems in small animal research than in human research owing to the smaller size of the subject. Catheterization has been proposed to reduce bladder spillover both in human studies and in small animal research. Noninvasive alternatives such as hydration plus furosemide also seem to be a promising pre-imaging strategy for decreasing bladder spillover. Our main goal was to measure the effects of the combination of furosemide and hydration for reducing bladder signal directly on mouse bowel 18F-FDG-PET images. METHODS:Nine mice were divided into two groups: the control group (C, n = 4) and the treatment group (n = 5). The clearance protocol combines hyperhydration and a single furosemide dose during the 18F-FDG uptake period. Two images were acquired on different days in treated mice to evaluate two different furosemide doses (low dose, LD, 3.5 mg/kg; and high dose, HD, 7 mg/kg). A region of interest was drawn on each computed tomography image (bladder, kidneys, liver, muscle, and bone marrow). To quantify bladder spillover, two different areas of the colon were selected. RESULTS:A remarkable reduction in bladder spillover was achieved on 18F-FDG -PET in both groups. Our imaging findings were quantified, and both furosemide doses induced a decrease in mean standard uptake values (SUVmean) compared with the controls (LD 1.46 ± 0.54 and HD 1.05 ± 0.29; controls: 8.90 ± 3.4 [p-value < 0.05]). CONCLUSION:We validated a non-invasive, easy, and harmless pre-imaging alternative for decreasing 18F-FDG bladder spillover. Our study shows the effect of furosemide on bladder spillover directly on 18F-FDG-PET images by measuring SUVmean in the bladder, colon, liver, muscle, and bone marrow

Assessment of the anti-biofilm effect of micafungin in an animal model of catheter-related candidemia.

In cases where catheter-related candidemia (CRC) must be managed without catheter withdrawal, antifungal lock therapy using highly active anti-biofilm (HAAB) agents is combined with systemic treatment. However, the activity of HAAB agents has never been studied in in vivo models using bioluminescence. We assessed the efficacy of micafungin using a bioluminescent Candida albicans SKCA23-ACTgLuc strain in an animal model of CRC. We divided 33 female Wistar rats into five groups: sham (A), infected nontreated (B), treated with lock therapy (0.16 mg/ml) (C), systemically treated only (1 mg/kg) (D), and systemically treated+lock (E). Catheters were colonized 24 h before insertion into the femoral vein (day 0). Treatment started on day 1 and lasted 7 days, followed by 7 days of surveillance. Bioluminescence assays were carried out on days 1, 3, 5, and 14, together with daily monitoring of clinical variables. Postmortem microbiological cultures from the catheter and several tissue samples were also obtained. Overall, 28 rats (84.8%) completed the study. Group B animals showed significant weight loss at days 2, 4, and 5 compared with groups C and D (P < .05). In group B, no animals survived after day 7, 75% had CRC, and bioluminescence remained constant 5 days after catheter implantation. Positive catheter culture rates in groups C, D, and E were, respectively, 83.3%, 62.5%, and 25.0% (P = .15). Micafungin proved to be a HAAB agent when administered both systemically and in lock therapy in an animal model of CRC, although the bioluminescence signal persists after treatment. This persistence should be further analyzed.S