Efficient spatially targeted gene editing using a near-infrared activatable protein-conjugated nanoparticle for brain applications.

Spatial control of gene expression is critical to modulate cellular functions and deconstruct the function of individual genes in biological processes. Light-responsive gene-editing formulations have been recently developed; however, they have shown limited applicability in vivo due to poor tissue penetration, limited cellular transfection and the difficulty in evaluating the activity of the edited cells. Here, we report a formulation composed of upconversion nanoparticles conjugated with Cre recombinase enzyme through a photocleavable linker, and a lysosomotropic agent that facilitates endolysosomal escape. This formulation allows in vitro spatial control in gene editing after activation with near-infrared light. We further demonstrate the potential of this formulation in vivo through three different paradigms: (i) gene editing in neurogenic niches, (ii) gene editing in the ventral tegmental area to facilitate monitoring of edited cells by precise optogenetic control of reward and reinforcement, and (iii) gene editing in a localized brain region via a noninvasive administration route (i.e., intranasal).

Mitochondrial and metabolic remodelling in human skin fibroblasts in response to glucose availability.

Cell culture conditions highly influence cell metabolism in vitro. This is relevant for preclinical assays, for which fibroblasts are an interesting cell model, with applications in regenerative medicine, diagnostics and therapeutic development for personalized medicine, and the validation of ingredients for cosmetics. Given these cells' short lifespan in culture, we aimed to identify the best cell culture conditions and promising markers to study mitochondrial health and stress in normal human dermal fibroblasts (NHDF). We tested the effect of reducing glucose concentration in the cell medium from high glucose (HGm) to a more physiological level [low glucose medium (LGm)], or its complete removal and replacement by galactose [medium that forces oxidative phosphorylation (OXPHOSm)], always in the presence of glutamine and pyruvate. We have demonstrated that only with OXPHOSm was it possible to observe the selective inhibition of mitochondrial adenosine triphosphate (ATP) production. This reliance on mitochondrial ATP was accompanied by changes in oxygen consumption rate and extracellular acidification rate, oxidation of citric acid cycle substrates, fatty acids, lactate, and other substrates, increased mitochondrial network extension and polarization, the increased protein content of voltage-dependent anion channel (VDAC) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha and changes in several key transcripts related to energy metabolism. LGm did not promote significant metabolic changes in NHDF, although mitochondrial network extension and VDAC protein content were increased compared to HGm-cultured cells. Our results indicate that short-term adaptation to OXPHOSm is ideal for studying mitochondrial health and stress in NHDF.

Refinement of a differentiation protocol using neuroblastoma SH-SY5Y cells for use in neurotoxicology research.

Since most models used to study neuronal dysfunction display disadvantages and ethical concerns, a fast and reproducible in vitro model to study mitochondria-related neurodegeneration is required. Here, we optimized and characterized a 3-day retinoic acid-based protocol to differentiate the SH-SY5Y cell line into a neuronal-like phenotype and investigated alterations in mitochondrial physiology and distribution. Differentiation was associated with p21-linked cell cycle arrest and an increase in cell mass and area, possibly associated with the development of neurite-like extensions. Notably, increased expression of mature neuronal markers (neuronal-specific nuclear protein, microtubule-associated protein 2, ßIII tubulin and enolase 2) was observed in differentiated cells. Moreover, increased mitochondrial content and maximal area per cell suggests mitochondrial remodeling. To demonstrate that this model is appropriate to study mitochondrial dysfunction, cells were treated for 6 h with mitochondrial toxicants (rotenone, antimycin A, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) and 6-hydroxydopamine (6-OHDA)). Differentiated cells were more susceptible to increasing concentrations of FCCP, antimycin A, and rotenone, while 6-OHDA showed a distinct dose-dependent neurotoxicity pattern. Even though differentiated cells did not exhibit a fully mature/differentiated neuronal phenotype, the protocol developed can be used to study neurotoxicity processes, mitochondrial dynamics, and bioenergetic impairment, representing an alternative to study mitochondrial impairment-related pathologies in vitro.

Gd- and Eu-Loaded Iron Oxide@Silica Core-Shell Nanocomposites as Trimodal Contrast Agents for Magnetic Resonance Imaging and Optical Imaging.

Superparamagnetic maghemite core-porous silica shell nanoparticles, γ-Fe2O3@SiO2 (FS), with 50 nm diameter and a 10 nm core, impregnated with paramagnetic complexes b-Ln ([Ln(btfa)3(H2O)2]) (where btfa = 4,4,4-trifluoro-l-phenyl-1,3-butanedione and Ln = Gd, Eu, and Gd/Eu), performing as promising trimodal T1-T2 MRI and optical imaging contrast agents, are reported. These nanosystems exhibit a high dispersion stability in water and no observable cytotoxic effects, witnessed by intracellular ATP levels. The structure and superparamagnetic properties of the maghemite core nanocrystals are preserved upon imbedding the b-Ln complexes in the shell. Hela cells efficiently and swiftly internalize the NPs into the cytosol, with no observable cytotoxicity below a concentration of 62.5 µg mL-1. These nanosystems perform better than the free b-Gd complex as T1 (positive) contrast agents in cellular pellets, while their performance as T2 (negative) contrast agents is similar to the FS. Embedding of the b-Eu complex in the silica pores endows the nanoparticles with strong luminescence properties. The impregnation of gadolinium and europium complexes in a 1:1 ratio afforded a trimodal nanoplatform performing as a luminescent probe and a double T1 and T2 MRI contrast agent even more efficient than b-Gd used on its own, as observed in cell-labeled imaging experiments and MRI cell pellets.

(Gd,Yb,Tb)PO4 up-conversion nanocrystals for bimodal luminescence-MR imaging.

Up-conversion (Gd,Yb,Tb)PO(4) materials and their potential for bimodal imaging have received little attention in the literature. Herein, we report the first study on the up-conversion emission of (Gd,Yb,Tb)PO(4) nanocrystals synthesized via a hydrothermal method at 150 °C. These materials exhibit ultraviolet, blue and green up-conversion emissions upon excitation with a 980 nm continuous wave laser diode. The intensity of the blue-emission band at 479 nm, ascribed to the cooperative up-conversion emission of a pair of excited Yb(3+) ions, depends on the Yb(3+)/Tb(3+) concentration ratio, calcination temperature and particle size. Strong green up-conversion emission of Tb(3+) is observed at 543 nm for the (5)D(4)→(7)F(5) transition. Relaxometry measurements reveal that the nanocrystals are efficient T(2)-weighted (negative) contrast agents which, combined with visible-light emission generated by infrared excitation, affords them considerable potential for being used in bimodal, photoluminescence-magnetic resonance, imaging.

Lanthanide-DTPA grafted silica nanoparticles as bimodal-imaging contrast agents.

The design and synthesis of a combined MRI-optical probe for bio-imaging are reported. The materials studied join the properties of lanthanide (Ln(3+)) complexes and nanoparticles (NPs), offering an excellent solution for bimodal imaging. The hybrid SiO(2)@APS/DTPA:Gd:Ln (Ln = Eu(3+) or Tb(3+)) (APS: 3-aminopropyltriethoxysilane, DTPA: diethylenetriamine pentaacetic acid) system increases the payload of the active magnetic centre (Gd(3+)) and introduces a Ln(3+) long-life excited state (Eu(3+): 0.35 ± 0.02 ms, Tb(3+): 1.87 ± 0.02 ms), with resistance to photobleaching and sharp emission bands. The Eu(3+) ions reside in a single low-symmetry site. Although the photoluminescence emission is not influenced by the simultaneous presence of Gd(3+) and Eu(3+), a moderate r(1) increase and a larger enhancement of r(2) are observed, particularly at high fields, due to susceptibility effects on r(2). The presence of Tb(3+) instead of Eu(3+) further raises r(1) but decreases r(2). These values are constant over a wide (5-13) pH range, indicating the paramagnetic NPs stability and absence of leaching. The uptake of NPs by living cells is fast and results in an intensity increase in the T(1)-weighted MRI images. The optical properties of the NPs in cellular pellets are also studied, confirming their potential as bimodal imaging agents.

Fine tuning of the relaxometry of γ-Fe2O3@SiO2 nanoparticles by tweaking the silica coating thickness.

We report the fine-tuning of the relaxometry of gamma-Fe2O3@SiO2 core-shell nanoparticles by adjusting the thickness of the coated silica layer. It is clear that the coating thickness of Fe2O3@SiO2 nanoparticles has a significant impact on the r(1) (at low B0 fields), r(2), and r(2)* relaxivities of their aqueous suspensions. These studies clearly indicate that the silica layer is heterogeneous and has regions that are porous to water and others-that are not. It is also shown, that the viability and the mitochondrial dehydrogenase expression of the microglial cells do not appear to be sensitive to the vesicular load with these core-shell nanoparticles. The adequate silica-shell thickness can therefore be tuned to allow for both a sufficiently high response as contrast agent, and-adequate grafting of targeted biomolecules.