F.J. de la Mata

In Vitro Studies of Water-Stable Cationic Carbosilane Dendrimers As Delivery Vehicles for Gene Therapy Against HIV and Hepatocarcinoma

Here we present a synthetic procedure for water-stable carbosilane dendrimers containing ammonium groups at the periphery of type Gn-{[Si(CH2)3N+(Me)(Et)CH2CH2N+Me3]x (CF3SO3 -)y} which have been used as non-viral vectors for transfecting different types of nucleic acids against two different medical problems, HIV and hepatocarcinoma. These systems have shown to be non-toxic in both PBMC and HepG2 cell lines under the experimental conditions and are able to form nanoconjugates with nucleic acids perfectly stable over time and in a wide range of pH values, which leads to the conclusion that the interaction between dendrimer and nucleic acid is very strong. In addition, a high degree of transfection using these nanoconjugates has been observed, ranging from 70-90% depending on the generation and in the particular case of PBMC transfection with anti-HIV oligonucleotides. However, besides of the good properties shown by the dendrimers here prepared as transfecting agents, only moderate effect was observed in functional experiments for hepatocarcinoma, as a result of the strong interaction between dendrimer and nucleic acid. Nevertheless, it is important to mention that an IRS-4 knock-down of 40% in HepG2 achieves an analogous degree of cell sensitization to cancer treatment, which may represent a major advance in the hepatocarcinoma treatment when appropriate dendrimers as transfection agents are used.

In vitro comparative assessment of different viability assays in Acanthamoeba castellanii and Acanthamoeba polyphaga trophozoites

The species of the genus Acanthamoeba are opportunistic protozoan parasites that cause different diseases in humans, such as amoebic keratitis and granulomatous encephalitis. The rise in the rate of Acanthamoeba keratitis, mainly due to the increase in contact lens wearers, turns the development of viability assays using a multi-well plate reader as a tool for screening new antiamoebic agents in vitro into an important goal. In our study, the viability assays PrestoBlue®, resazurin sodium salt, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and CellTiter96® were tested for their suitability as time-saving alternatives to the classical manual or direct-counting method, assessing the effect of the antiamoebic agent chlorhexidine digluconate and temperature on Acanthamoeba castellanii (ATCC® 30234™) and Acanthamoeba polyphaga 2961. Although resazurin and MTT have already been previously used in amoeba viability assays to test the activities of antiamoebic agents in vitro, it is the first time that PrestoBlue® and CellTiter96® are used for this purpose. Results indicated that the viability assays were strain-dependent leading in some cases to an overestimation of the real situation of viable cells. This implies that each viability assay ought to be set up for each amoeba strain studied.

A study of ortho- and para-siloxyanilines for the synthesis of mono-, bi-, and tetra-nuclear early transition metal–imido complexes

Abstract The siloxyanilines o -Me 3 SiOC 6 H 4 NH 2 ( 1 ) and p -RMe 2 SiOC 6 H 4 NH 2 (R=H ( 2 ); R=Me ( 3 )), and their N-silylated derivatives p -Me 3 SiOC 6 H 4 NHSiMe 3 ( 4 ) and p -Me 3 SiOC 6 H 4 N(SiMe 3 ) 2 ( 5 ) have been prepared from ortho - or para -aminophenol and used in the synthesis of imido complexes. Thus, binuclear [{Ti(η 5 -C 5 H 5 )Cl}{μ-NC 6 H 4 ( p -OSiMe 3 )}] 2 ( 6 ) and mononuclear [TiCl 2 {NC 6 H 4 ( p -OSiMe 3 )}(py) 3 ] ( 7 ) imido complexes have been obtained from the reaction of 3 and [Ti(η 5 -C 5 H 5 )Cl 3 ] or [TiCl 2 (N t Bu)(py) 3 ], respectively. In contrast, the reaction of 1 with TiCl 4 and t Bupy affords the titanocycle [TiCl 2 {OC 6 H 4 ( o -NH)N,O}( t Bupy) 2 ] ( 8 ). Compound 5 has also been used to prepare the niobium imide complex [NbCl 3 {NC 6 H 4 ( p -OSiMe 3 )}(MeCN) 2 ] ( 9 ), by its reaction with NbCl 5 in CH 3 CN. These findings have been applied to the synthesis of polynuclear systems. Thus, chlorocarbosilane Si[CH 2 CH 2 CH 2 Si(Me) 2 Cl] 4 (CS–Cl) has been functionalized with the ortho - and para -aminophenoxy groups to give 10 and 11 , respectively. The use of 11 has allowed the formation of the tetranuclear compound 12 . Attempts to synthesize terminal imido titanium complexes from 10 and TiCl 4 in the presence of t Bupy and Et 3 N, give complex 8 and carbosilane CS–Cl.

In vitro anti-Acanthamoeba synergistic effect of chlorhexidine and cationic carbosilane dendrimers against both trophozoite and cyst forms

Acanthamoeba sp. are the causative agents of severe illnesses in humans such as Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). Medical therapy is not yet well established. Treatments of AK last for several months and generate toxicity, resistances appear due to the cysts stage and recurrences can occur. In this study has been demonstrated that the combination of chlorhexidine digluconate (CLX) and carbosilane dendrimers containing ammonium or guanidine moieties has in vitro synergistic effect against Acanthamoeba polyphaga. This synergy provokes an important reduction in the minimal trophozoite amoebicidal concentration (MTAC) of CLX, which means a reduction of their toxic effects on human cells. Moreover, some CLX/dendrimer combinations show important activity against the cyst resistance stage.

In vitro evaluation of the effectiveness of new water-stable cationic carbosilane dendrimers against Acanthamoeba castellanii UAH-T17c3 trophozoites.

Acanthamoeba is one of the most common free-living amoebas which is widespread in the environment and can infect humans, causing diseases such as keratitis and encephalitis. In this paper we examine for the first time the amebicidal activity of the family of cationic dendrimers nG-[Si{(CH2)3N+(Me)(Et)(CH2)2NMe3+}2I−]x (where n denotes the generations: zero (n = 0, x = 1), first (n = 1, x = 4), and second (n = 2, x = 8); for simplicity, they were named as 0G-CNN2, 1G-CNN8, and 2G-CNN16, respectively) against Acanthamoeba castellanii UAH-T17c3 trophozoites. In order to test the amebicidal activity, we cultured the strain A. castellanii UAH-T17c3 in PYG-Bactocasitone medium and later, we treated it with different concentrations of these dendrimers and monitored the effects and damage by optical count, flow cytometry, and scanning electron microscopy. The results showed that all the nanosystems assayed had a strong amebicidal activity. The dendrimer 1G-CNN8 was the most effective against the amoeba. In the morphology of treated throphozoites of A. castellanii UAH-T17c3 analyzed by light and scanning electron microscopy techniques, morphological changes were evident in amoeba cells, such as loss of pseudopodia, ectoplasm increase, roundness, and cellular lysis. Furthermore, flow cytometry results showed alterations in cell granularity, which was dose–time dependent. In conclusion, this family of cationic carbosilane dendrimers has a strong amebicidal activity against the trophozoites of A. castellanii UAH-T17c3 in vitro. They could potentially become new agents significant to the development of new amebicidal compounds for prevention and therapy of Acanthamoeba infections.

Characterization of carboxylate-terminated carbosilane dendrimers and their evaluation as nanoadditives in capillary electrophoresis for vegetable protein profiling.

Protein profiles are becoming an important tool to differentiate and classify varieties of several cultivars and to obtain a specific fingerprint for them. The use of protein profiles for these purposes needs to achieve high separation efficiencies to obtain a high number of well resolved peaks. In this work, carbosilane dendrimers with interior carbon-silicon bonds and negatively charged in the dendrimer surface with carboxylic acid as functional groups were employed as nanoadditives to separate soybean and olive seeds proteins. First, these dendrimers were characterized using CE to evaluate their possible impurities. A potentiometric titration was later carried out to determine their pK(a) values. Afterwards, the characterized dendrimers were used to improve the protein profiles obtained by EKC for vegetable proteins. Different dendrimer generations (G1, G2, and G3) and concentrations (0.01-1% m/v) were tested. The highest dendrimer generation G3 at 0.1% (m/v) allowed observing the best protein profiles for soybean and olive seeds. These results demonstrate that carboxylate-terminated carbosilane dendrimers are attractive nanoadditives in EKC for the effective separation of vegetable proteins.

Biophysical Characterization of Glycodendrimers As Nano-carriers for HIV Peptides

This paper examines the formation and stability of nano-complexes that could provide a new therapeutic approach against HIV-1 infection. Poly(propylene imine) glycodendrimers decorated with 2(nd) generation cationic carbosilane dendrons were generated and their use in polyplex formation checked. Owing to their positively-charged terminal amino groups the hybrid glycodendrimers can bind anionic peptides. It was shown that they form nano-complexes with the HIV-derived peptides P24, Gp160 and Nef. Complexes 130-190 nm in size were formed in molar ratios (dendrimer/ peptide) of (3-4):1. These were sufficiently stable over time and at different pHs. The results obtained suggest that the hybrid dendrimers studied can be considered as alternative carriers for delivering HIV peptides to dendritic cells.

Carbosilane dendrimers as carriers of siRNA

Despite the enormous possibilities of RNAi, there still exist many problems that need to be addressed. Obstacles in delivery, target cell transfection, stability/degradation, transient activity, secondary effects, toxicity caused by the delivery vector, and resistance all hinder the path of carrying out in vivo experiments with RNAi and further developing RNAi as a new therapy for clinical use. Notwithstanding, the majority of research that uses RNAi depends on a delivery vector of some kind. This review offers a brief overview of the current status of carbosilane dendrimers as siRNA delivery vectors.

Prevention of vaginal and rectal herpes simplex virus type 2 transmission in mice: mechanism of antiviral action

Topical microbicides to stop sexually transmitted diseases, such as herpes simplex virus type 2 (HSV-2), are urgently needed. The emerging field of nanotechnology offers novel suitable tools for addressing this challenge. Our objective was to study, in vitro and in vivo, antiherpetic effect and antiviral mechanisms of several polyanionic carbosilane dendrimers with anti-HIV-1 activity to establish new potential microbicide candidates against sexually transmitted diseases. Plaque reduction assay on Vero cells proved that G2-S16, G1-S4, and G3-S16 are the dendrimers with the highest inhibitory response against HSV-2 infection. We also demonstrated that our dendrimers inhibit viral infection at the first steps of HSV-2 lifecycle: binding/entry-mediated events. G1-S4 and G3-S16 bind directly on the HSV-2, inactivating it, whereas G2-S16 adheres to host cell-surface proteins. Molecular modeling showed that G1-S4 binds better at binding sites on gB surface than G2-S16. Significantly better binding properties of G1-S4 than G2-S16 were found in an important position for affecting transition of gB trimer from G1-S4 prefusion to final postfusion state and in several positions where G1-S4 could interfere with gB/gH–gL interaction. We demonstrated that these polyanionic carbosilan dendrimers have a synergistic activity with acyclovir and tenofovir against HSV-2, in vitro. Topical vaginal or rectal administration of G1-S4 or G2-S16 prevents HSV-2 transmission in BALB/c mice in values close to 100%. This research represents the first demonstration that transmission of HSV-2 can be blocked by vaginal/rectal application of G1-S4 or G2-S16, providing a step forward to prevent HSV-2 transmission in humans.

Synthesis of mono- and dinuclear cyclopentadienyl–aryloxy titanium(IV) complexes

Abstract This paper reports the reactivity of monocyclopentadienyl titanium complexes [Ti(C5R5)Cl3] (R=H or Me) with phenol and hydroquinones to afford mono- and dinuclear aryloxy derivatives, respectively. The reaction of hydroquinones HO(C6H2XY)OH (X=Y=H, Me; X=H, Y=Me) with [Ti(C5H5)Cl3] gave red–orange microcrystalline solids of the corresponding dinuclear complexes [{Ti(C5H5)Cl2}2{μ-O(C6H2XY)O}] (X=Y=H (1); X=H, Y=Me (2); X=Y=Me (3)) in high yields. However, their dilithium salts Li2[O(C6H2XY)O] must be used in the treatment with [Ti(C5Me5)Cl3] to produce the expected dinuclear derivatives [{Ti(C5Me5)Cl2}2{μ-O(C6H2XY)O}] (X=Y=H (4); X=H, Y=Me (5); X=Y=Me (6)) as red–orange microcrystals in high yields too. Reaction of [Ti(C5R5)Cl3] with 4-allyl-2-methoxyphenol (commonly called eugenol) [C6H3(OH)(OMe)(C3H5)] or its lithium salt led to the monoaryloxy complexes [Ti(C5R5)Cl2{OC6H3(OMe)(C3H5)}] (R=H (7), Me (8)).