Javier de la Mata

Highly Efficient Transfection of Rat Cortical Neurons Using Carbosilane Dendrimers Unveils a Neuroprotective Role for HIF-1α in Early Chemical Hypoxia-Mediated Neurotoxicity

PurposeTo study the effect of a non-viral vector (carbosilane dendrimer) to efficiently deliver small interfering RNA to postmitotic neurons to study the function of hypoxia-inducible factor-1α (HIF1-α) during chemical hypoxia-mediated neurotoxicity.MethodsChemical hypoxia was induced in primary rat cortical neurons by exposure to CoCl2. HIF1-α levels were determined by Western Blot and toxicity was evaluated by both MTT and LDH assays. Neurons were incubated with dendriplexes containing anti-HIF1-α siRNA and both uptake and HIF1-α knockdown efficiency were evaluated.ResultsWe report that a non-viral vector (carbosilane dendrimer) can deliver specific siRNA to neurons and selectively block HIF1-α synthesis with similar efficiency to that achieved by viral vectors. Using this method, we have found that this transcription factor plays a neuroprotective role during the early phase of chemical hypoxia-mediated neurotoxicity.ConclusionThis work represents a proof-of-concept for the use of carbosilane dendrimers to deliver specific siRNA to postmitotic neurons to block selected protein synthesis. This indicates that this type of vector is a good alternative to viral vectors to achieve very high transfection levels in neurons. This also suggests that carbosilane dendrimers might be very useful for gene therapy.

In vivo delivery of siRNA to the brain by carbosilane dendrimer.

Nanotechnology offers a new platform for therapeutic delivery of antiretrovirals to the central nervous system (CNS). Nanoformulated antiretroviral drugs offer multifunctionality, that is, the ability to package multiple diagnostic and therapeutic agents in the same nanocompose, along with the added provisions of site-directed delivery, delivery across the blood-brain-barrier (BBB), and controlled release of therapeutics. We studied the viability of dendrimers and dendriplexes in human primary astrocytes, as well as their uptake by these astrocytes. Functional validation was performed by using specific siRNA against HIV-1 Nef to interfere to HIV-1 infectivity. A high efficiency in Nef silencing, reducing HIV-1 infectivity was observed in astrocytes treated with dendriplexes compared with control or siRandom treated astrocytes. More interestingly, we studied the biodistribution of the second generation of carbosilane dendrimer loaded with FITC (2G-(SNMe3I)11-FITC) in vivo, in BALB/c mice. Dendriplexes were inoculated into BALB/c mice by the retro-orbital venous plexus, and their localization was determined after 1 and 24h post-injection. Dendriplexes were detected inside the brain by a sensitive imaging system of fluorescent imaging in vivo (IVIS Lumina), and by confocal microscopy analysis of sections of OCT-embedded tissues. The 2G-(SNMe3I)11-FITC dendrimer transported efficiently siRNA into the brain, crossing the BBB. Moreover, this dendrimer successfully delivered and transfected siRNA to HIV-infected human primary astrocytes and achieved gene silencing without causing cytotoxicity. These results highlight the potential of this nanoformulation in the treatment of neurological disorders.

Monocyclopentadienyl alkyl alkylidene niobium(V) and tantalum(V) complexes. X-ray crystal structure of Ta(η5-Cp′)(CH2SiMe3)2(CHSiMe3)

Abstract Reactions of monocyclopentadienyl complexes M(η5-Cp′)Cl4 with trimethylsilylmethyl lithium leads to the formation of alkylidene derivatives M(η5-Cp′)(CH2SiMe3)2(CHSiMe3), where M = Nb (1); Ta (2); Cp′= C5Me5 and M = Ta; Cp′ Me3Si(C5H4) (3), 1,3-(Me3Si)2(C5H3) (4). The new complexes were characterized by 1H and 13C NMR spectroscopy and the molecular structure of 2 was studied by X-ray diffraction methods.

Half-sandwich isocyanide, phosphine, methyl and phosphanido pentamethylcyclopentadienylniobium (V) complexes

Abstract [NbCp★Cl4] Cp★ = ν5-C5Me5) reacts with one molar equivalent of isocyanides and phosphines to give pseudo-octahedral adducts [NbCp★Cl4·L], ( L = 2,4,6- Me 3 C 6 H 2 NC , 1 ; PMe 3 , 2 ; PMe 2 Ph , 3 ; PMePh 2 , 4) and the dinuclear complex [∗[NbCp★Cl4∗]2(μ-dmpe)], 5. Reactions of [NbCp★Cl4] with appropriate amounts of MgClMe yield the correspodning dimethyl [NbCp★Cl2Me2], 6, and tetramethyl [NbCp★Me4], 7 derivatives. Similarly, when [∗[NbCp★Cl3∗2(μ-O)] an and 3 are treated with the alkylating magnesium reagent, [∗[NbCp★Me3∗]2(μ-O)], 8, and [NbCp★Cl3Me(PMe2Ph)], 9, are obtained in high yields. A new phosphanido-niobium(V) complex [NbCp★Cl3(PPh2)] 10, is formed in the reaction of the starting tetrachloro complex with one equivalent of LiPPh2. All the complexes were characterized by IR and NMR spectroscopy, and most of them were studied by mass spectrometry.

Interaction of cationic carbosilane dendrimers and their complexes with siRNA with erythrocytes and red blood cell ghosts.

Abstract We have investigated the interactions between cationic NN16 and BDBR0011 carbosilane dendrimers with red blood cells or their cell membranes. The carbosilane dendrimers used possess 16 cationic functional groups. Both the dendrimers are made of water-stable carbon–silicon bonds, but NN16 possesses some oxygen–silicon bonds that are unstable in water. The nucleic acid used in the experiments was targeted against GAG-1 gene from the human immunodeficiency virus, HIV-1. By binding to the outer leaflet of the membrane, carbosilane dendrimers decreased the fluidity of the hydrophilic part of the membrane but increased the fluidity of the hydrophobic interior. They induced hemolysis, but did not change the morphology of the cells. Increasing concentrations of dendrimers induced erythrocyte aggregation. Binding of short interfering ribonucleic acid (siRNA) to a dendrimer molecule decreased the availability of cationic groups and diminished their cytotoxicity. siRNA–dendrimer complexes changed neither the fluidity of biological membranes nor caused cell hemolysis. Addition of dendriplexes to red blood cell suspension induced echinocyte formation.

Dendronized PLGA nanoparticles with anionic carbosilane dendrons as antiviral agents against HIV infection

The development of novel strategies to prevent HIV-1 infection is of outstanding relevance. PLGA nanoparticles have become some of the most used nanosized materials for biomedical applications. Polyanionic carbosilane dendrimers and dendrons have shown potent and broad-spectrum anti-HIV-1 activity. Therefore, PLGA nanoparticles functionalized with carbosilane anionic dendrons through a carbodiimide-mediated reaction have been prepared with the aim of them becoming novel antiviral agents against HIV infection. The biocompatibility of the new dendronized nanoparticles has been explored in PBMC and HEC-1A cells. Both inhibition experiments showed differences between dendronized nanoparticles, free dendrons and PLGA nanoparticles. The results obtained with the dendronized nanoparticles indicate that these systems are powerful anti-HIV agents, compared to dendrons or dendrimers alone or PLGA NPs, suggesting an enhancement of their interaction with viral or cell receptors. The topology and size of these new systems could be responsible for the higher activity observed.

Polycationic carbosilane dendrimer decreases angiogenesis and tumor-associated macrophages in tumor-bearing mice

Therapies against cancer have been improved and progressed during recent decades, initially were using chemotherapeutic drugs, which directly affect tumor cells, but nowadays are focused in cellular therapies aimed at treating the tumor stroma, because tumor and stromal cells jointly control development and tumor progression. Immunotherapy is of great relevance because it could modify the tumor stroma, controlling tumor growth. Tumor-associated macrophages have been proposed as target cells owing to the positive correlation between the high content of macrophages and the adverse prognosis of tumor development. 2G-03NN24 dendrimer had previously shown immunomodulatory effects by reducing the functional capabilities of human anti-inflammatory macrophages, leading them to a pro-inflammatory state, and thereby helping to control tumor development. New dendrimer capabilities against tumor mass are described and presented in in vivo studies using tumor-bearing mice. MC38 cells were used to induce tumors in C57BL/6 mice. Tumor growth was evaluated during 21 days and tumors were stained with hematoxylin/eosin to analyze the histopathology features. Tumor histopathology studies show that 2G-03NN24 dendrimer decreases the tumor size and the number of intratumoral blood vessels. Furthermore, cellular populations on tumor mass were analyzed by an immunofluorescence assay. Evaluation of tumor-associated macrophages indicates that 2G-03NN24 dendrimer reduces the amount of tumor-associated macrophages, creating a more favorable microenvironment within tumors. Data defines 2G-03NN24 as a candidate for finding a new antitumor compound based on cellular therapies.