Encapsulation of Large-Size Plasmids in PLGA Nanoparticles for Gene Editing: Comparison of Three Different Synthesis Methods

The development of new gene-editing technologies has fostered the need for efficient and safe vectors capable of encapsulating large nucleic acids. In this work we evaluate the synthesis of large-size plasmid-loaded PLGA nanoparticles by double emulsion (considering batch ultrasound and microfluidics-assisted methodologies) and magnetic stirring-based nanoprecipitation synthesis methods. For this purpose, we characterized the nanoparticles and compared the results between the different synthesis processes in terms of encapsulation efficiency, morphology, particle size, polydispersity, zeta potential and structural integrity of loaded pDNA. Our results demonstrate particular sensibility of large pDNA for shear and mechanical stress degradation during double emulsion, the nanoprecipitation method being the only one that preserved plasmid integrity. However, plasmid-loaded PLGA nanoparticles synthesized by nanoprecipitation did not show cell expression in vitro, possibly due to the slow release profile observed in our experimental conditions. Strong electrostatic interactions between the large plasmid and the cationic PLGA used for this synthesis may underlie this release kinetics. Overall, none of the methods evaluated satisfied all the requirements for an efficient non-viral vector when applied to large-size plasmid encapsulation. Further optimization or alternative synthesis methods are thus in current need to adapt PLGA nanoparticles as delivery vectors for gene editing therapeutic technologies

In-situ preparation of ultra-small Pt nanoparticles within rod-shaped mesoporous silica particles: 3-D tomography and catalytic oxidation of n-hexane

The shape and porous configuration of supports are key parameters to design outstanding catalysts. However, the selection of a proper mesoporous support, such as SBA-15, by itself does not guarantee accessibility to catalytic sites. The distribution of the active phase and its stability are strongly related to the procedure used to deposit it on the catalytic substrate. Herein, we have prepared rod-shaped SBA-15 silica supports functionalized with amine groups to facilitate the electrostatic attraction and a good distribution of the resulting Pt-based catalytic nanoparticles along the pore walls. Additionally, the use of tetrakis-(hydroxymethyl)-phosphonium chloride (THPC) as both reductant and stabilizer is presented as a novel alternative for Pt nanoparticle synthesis. The behaviour of this catalyst in the total oxidation of n-hexane demonstrates high activity and excellent stability after 70 h on reaction stream. STEM-HAADF and 3-D tomography were used to confirm the presence of the metallic nanoparticles within the mesochannels and to corroborate their reduced sintering after reaction

In Cellulo Bioorthogonal Catalysis by Encapsulated AuPd Nanoalloys. Overcoming Intracellular Deactivation

Bioorthogonal metallocatalysis has opened up a xenobiotic route to perform nonenzymatic catalytic transformations in living settings. Despite their promising features, most metals are deactivated inside cells by a myriad of reactive biomolecules, including biogenic thiols, thereby limiting the catalytic functioning of these abiotic reagents. Here we report the development of cytocompatible alloyed AuPd nanoparticles with the capacity to elicit bioorthogonal depropargylations with high efficiency in biological media. We also show that the intracellular catalytic performance of these nanoalloys is significantly enhanced by protecting them following two different encapsulation methods. Encapsulation in mesoporous silica nanorods resulted in augmented catalyst reactivity, whereas the use of a biodegradable PLGA matrix increased nanoalloy delivery across the cell membrane. The functional potential of encapsulated AuPd was demonstrated by releasing the potent chemotherapy drug paclitaxel inside cancer cells. Nanoalloy encapsulation provides a novel methodology to develop nanoreactors capable of mediating new-to-life reactions in cell

Radon Mitigation Applications at the Laboratorio Subterraneo de Canfranc (LSC)

The Laboratorio Subterraneo de Canfranc (LSC) is the Spanish national hub for low radioactivity techniques and the associated scientific and technological applications. The concentration of the airborne radon is a major component of the radioactive budget in the neighborhood of the detectors. The LSC hosts a Radon Abatement System, which delivers a radon suppressed air with 1.1 & PLUSMN;0.2 mBq/m(3) of Rn-222. The radon content in the air is continuously monitored with an Electrostatic Radon Monitor. Measurements with the double beta decay demonstrators NEXT-NEW and CROSS and the gamma HPGe detectors show the important reduction of the radioactive background due to the purified air in the vicinity of the detectors. We also discuss the use of this facility in the LSC current program which includes NEXT-100, low background biology experiments and radiopure copper electroformation equipment placed in the radon-free clean room

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

Continuous microfluidic synthesis and functionalization of gold nanorods

AbstractA microfluidic platform with multiple reactant additions has been used for the continuous, unseeded synthesis of gold nanorods. This system allows separate control of the seed forming and nanorod growing stages, increasing reproducibility and reducing undesired effects related to a poor control of the seed aging stage. Also, on-stream surface functionalization with poly(ethylene glycol)-methyl ether thiol (SH-PEG) has been performed, allowing a 100-fold reduction in the consumption of this reactant compared to the amounts used in a conventional batch reactor. The system also facilitates tuning the concentration of halide anions (Cl− and Br−) that are used to modify the aspect ratio of the resulting rods

Continuous synthesis of drug-loaded nanoparticles using microchannel emulsification and numerical modeling: effect of passive mixing

By using interdigital microfluidic reactors, monodisperse poly(d,l lactic-co-glycolic acid) nanoparticles (NPs) can be produced in a continuous manner and at a large scale (~10 g/h). An optimized synthesis protocol was obtained by selecting the appropriated passive mixer and fluid flow conditions to produce monodisperse NPs. A reduced NP polydispersity was obtained when using the microfluidic platform compared with the one obtained with NPs produced in a conventional discontinuous batch reactor. Cyclosporin, an immunosuppressant drug, was used as a model to validate the efficiency of the microfluidic platform to produce drug-loaded monodisperse poly(d,l lactic-co-glycolic acid) NPs. The influence of the mixer geometries and temperatures were analyzed, and the experimental results were corroborated by using computational fluid dynamic three-dimensional simulations. Flow patterns, mixing times, and mixing efficiencies were calculated, and the model supported with experimental results. The progress of mixing in the interdigital mixer was quantified by using the volume fractions of the organic and aqueous phases used during the emulsification–evaporation process. The developed model and methods were applied to determine the required time for achieving a complete mixing in each microreactor at different fluid flow conditions, temperatures, and mixing rates

Optimization of superfluorinated PLGA nanoparticles for enhanced cell labelling and detection by 19F-MRI

Fluorine-19 (19F) Magnetic Resonance Imaging (MRI) is an emergent imaging technique for molecular imaging and cell tracking. Lack of intrinsic 19F signals in tissues allows unambiguous in vivo detection of exogenous fluorinated probes, complementary to the anatomical and multiparametric information obtained by standard 1H-MRI. However, the intrinsic low sensitivity of MRI technique requires the need of designing increasingly effective fluorinated tracers. PERFECTA, with its 36 magnetically equivalent 19F atoms and a designed branched molecular structure, represents an excellent superfluorinated tracer. In this paper, we report the development of PERFECTA loaded PLGA NPs stabilized by different coatings as promising 19F-MRI probes. The results clearly show the optimal cellular uptake of the produced colloidally stable PERFECTA loaded PLGA NPs without impact on cells viability. Importantly, NPs stabilization with the anionic surfactant sodium cholate (NaC) clearly enhances NPs internalization within cells with respect to PVA-coated NPs. Moreover, the optimized NPs are characterized by shorter T1 relaxation times with respect to other PERFECTA formulations that would allow the increase of 19F-MRI sensitivity with fast imaging acquisitions.F.B.B. and P.M. are thankful to the project NiFTy funded by MUR (PRIN2017, no. 2017MYBTXC) and to the NEWMED project, ID: 1175999 (funded by Regione Lombardia POR FESR 2014 2020). F.B.B. and C.C. also acknowledge financial support from P2RY12 project, ID: GR-2016- 02361325 (funded by the Italian Ministry of Health). We would like to thank Dr Marina Cretich from “Istituto di Scienze e Tecnologie Chimiche” (SCITEC-CNR) in Milan for the use of NTA Instrument. Experimental Imaging Center at IRCCS Ospedale San Raffaele is acknowledged for access to the preclinical MRI scanner. M S-A thanks the Spanish Government for an FPU PhD Research fellowship and AIRC- Fundation for cancer research for a Postdoctoral fellowship in Italy. CIBER-BBN (initiative funded by the VI National R&D&i Plan 2008– 2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund) and Nanbiosis ICTS are gratefully acknowledged. J.S. acknowledges the European Research Council (ERC Advanced Grant CADENCE number 742684). The electron microscopy studies were conducted at the Laboratorio de Microscopias Avanzadas-ICTS ELECMI, Universidad de Zaragoza, Spain.Peer reviewe