Paula Ortega

Characterization of carbosilane dendrimers as effective carriers of siRNA to HIV-infected lymphocytes.

One of the primary limitations of RNA interference as a technique for gene regulation is effective delivery of siRNA into the target cells. Dendrimers are nanoparticles that are increasingly being used as oligonucleotide and drug delivery vehicles. We have developed amino-terminated carbosilane dendrimers (CBS) as a means to protect and transport siRNA. Initially, stability studies showed that CBS bind siRNA via electrostatic interactions. Dendrimer-bound siRNA was found to be resistant to degradation by RNase. Cytotoxicity assays of CBS/siRNA dendriplexes with peripheral blood mononuclear cells (PBMC) and the lymphocytic cell line SupT1 revealed a maximum safe dendrimer concentration of 25 microg/ml. Next, utilizing flow cytometry and confocal microscopy, lymphocytes were seen to be successfully transfected by fluorochrome-labeled siRNA either naked or complexed with CBS. Dendriplexes with +/- charge ratio of 2 were determined to have the highest transfection efficiency while maintaining a low level of toxicity in these systems including hard-to-transfect HIV-infected PBMC. Finally, CBS/siRNA dendriplexes were shown to silence GAPDH expression and reduce HIV replication in SupT1 and PBMC. These results point to the possibility of utilizing dendrimers such as CBS to deliver and transfect siRNA into lymphocytes thus allowing the use of RNA interference as a potential alternative therapy for HIV infection.

Carbosilane Dendrimers to Transfect Human Astrocytes with Small Interfering RNA Targeting Human Immunodeficiency Virus

BackgroundHIV infection of the CNS is the principle cause of HIV-associated dementia in adults and encephalopathy in children. Gene therapy techniques such as small interfering RNA (siRNA) possess great potential in drug development, but first they must overcome the key obstacle of reaching the interior of the affected cells. A successful delivery vector for anti-HIV drugs that is capable of crossing the blood-brain barrier (BBB) could provide a way of addressing this issue. Non-viral vectors such as dendrimers offer a means for effectively delivering and transfecting siRNA to the target cells.ObjectiveTo evaluate the application of gene therapy for reducing HIV replication in human astrocytes.MethodsWe used the 2G-NN16 amino-terminated carbosilane dendrimer as a method for delivering siRNA to HIV-infected human astrocytes. We tested the cytotoxicity in human astrocytoma cells caused by 2G-NN16 and dendriplexes formed with siRNA (siRNA/2G-NN16) by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium-bromide (MTT) and lactate dehydrogenase assays. The ability to transfect human astrocytes with siRNA/2G-NN16 dendriplexes was tested by flow cytometry and immunofluorescence microscopy. To assess the potential capability of siRNA/2G-NN16 dendriplexes for crossing the BBB, we used an in vitro transcytosis assay with bovine brain microvascular endothelial cells. HIV-1 inhibition assays using 2G-NN16 and siRNA/2G-NN16 dendriplexes were determined by quantification of the viral load from culture supernatants of the astrocytes.ResultsA gradual time-controlled degradation of the 2G-NN16 dendrimer and liberation of its siRNA cargo between 12 and 24 hours was observed via gel electrophoresis. There was no cytotoxicity in HIV-infected or non-infected human astrocytoma cells when treated with up to 24 μg/mL of 2G-NN16 dendrimer or siRNA/2G-NN16 dendriplexes, and siRNA/2G-NN16 dendriplexes were seen to successfully transfect human astrocytes even after crossing an in vitro BBB model. More interestingly, transfected siRNA was observed to exert a biologic effect, as dendriplexes were shown to down-regulate the housekeeping gene GAPDH and to reduce replication of HIV-1 strains X4-HIV NL4-3 and R5-HIV BaL in human astrocytes.ConclusionsThe 2G-NN16 dendrimer successfully delivers and transfects siRNA to HIV-infected human astrocytes and achieves gene silencing without causing cytotoxicity.Both authors have contributed equally to this paper.

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.

Amine and ammonium functionalization of chloromethylsilane-ended dendrimers. Antimicrobial activity studies

Novel amine- and ammonium-terminated carbosilane dendrimers of type G(n)-[Si{CH(2)O-(C(6)H(4))-3-NMe(2)}](x) or G(n)-[Si{CH(2)O-(C(6)H(4))-3-NMe(3)(+)I(-)}](x) have been synthesized and characterized up to second generation by phenolysis of (chloromethyl)silyl-terminated dendrimers with 3-dimethylamine phenol and subsequent quaternization with methyl iodide. Quaternized carbosilane dendrimers are stable in protic solvents and can be solubilised in water after the addition of less than 1% of dimethyl sulfoxide. A study of the antimicrobial activity of these cationic dendrimers of first and second generation against both gram-positive and gram-negative bacteria is also described. The results obtained demonstrate that the new ammonium-terminated carbosilane dendrimers can be considered as multivalent biocides.

Water-soluble carbosilane dendrimers protect phosphorothioate oligonucleotides from binding to serum proteins

Treatment of dendriplexes formed between water-soluble carbosilane dendrimers and phosphorothioate oligodeoxynucleotides (ODN) with the anionic detergent sodium dodecyl sulfate disrupted the complexes indicating that the nature of the union in such dendriplexes is merely electrostatic. However, dendriplexes were not dissociated by serum proteins like bovine or human serum albumins, as assessed by gel electrophoresis and fluorescence experiments. This would imply a dendrimer-mediated protective effect able to prevent ODN interactions with serum proteins and additionally could translate into a reduction of the ODN doses needed to achieve the biological effects. The employment of carbosilane dendrimers as carriers may solve the problem of ODN kidnapping by plasmatic proteins as a key drawback for therapeutics involving ODNs. As examples, transfection processes on normal primary peripheral blood cells and diagnosis of HIV infection in the presence of serum have been assayed.

Gene Therapy in HIV‐Infected Cells to Decrease Viral Impact by Using an Alternative Delivery Method

The ability of dendrimer 2G‐[Si{O(CH2)2N(Me)2+(CH2)2NMe3+(I−)2}]8 (NN16) to transfect a wide range of cell types, as well as the possible biomedical application in direct or indirect inhibition of HIV replication, was investigated. Cells implicated in HIV infection such as primary peripheral blood mononuclear cells (PBMC) and immortalized suspension cells (lymphocytes), primary macrophages and dendritic cells, and immortalized adherent cells (astrocytes and trophoblasts) were analyzed. Dendrimer toxicity was evaluated by mitochondrial activity, cell membrane rupture, release of lactate dehydrogenase, erythrocyte hemolysis, and the effect on global gene expression profiles using whole‐genome human microarrays. Cellular uptake of genetic material was determined using flow cytometry and confocal microscopy. Transfection efficiency and gene knockdown was investigated using dendrimer‐delivered antisense oligonucleotides and small interfering RNA (siRNA). Very little cytotoxicity was detected in a variety of cells relevant to HIV infection and erythrocytes after NN16 dendrimer treatment. Imaging of cellular uptake showed high transfection efficiency of genetic material in all cells tested. Interestingly, NN16 further enhanced the reduction of HIV protein 24 antigen release by antisense oligonucleotides due to improved transfection efficiency. Finally, the dendrimer complexed with siRNA exhibited therapeutic potential by specifically inhibiting cyclooxygenase‐2 gene expression in HIV‐infected nervous system cells. NN16 dendrimers demonstrated the ability to transfect genetic material into a vast array of cells relevant to HIV pathology, combining high efficacy with low toxicity. These results suggest that NN16 dendrimers have the potential to be used as a versatile non‐viral vector for gene therapy against HIV infection.

Carbosilane dendrimers NN8 and NN16 form a stable complex with siGAG1.

A new mechanism of gene expression inhibition has been discovered as RNA interference, in which the ability of double-stranded RNA to stimulate specific degradation of an mRNA target with a complementary sequence to one of the double-stranded RNA strands. Water-soluble carbosilane dendrimers containing ammonium or amine groups at their periphery are biocompatible molecules that may be good candidates as non-viral carriers of small interfering RNA. In studying the formation of complex between anti-HIV siRNA siGAG1 and carbosilane dendrimers NN8 and NN16 by circular dichroism, fluorescence, and zeta-potential, the size of nanoparticles formed has been estimated by dynamic light scattering. At a charge ratio of 1:3-4 (siGAG1:dendrimer), the dendriplexes formed were in the size range of 250-350 nm.

Binding Properties of Water-Soluble Carbosilane Dendrimers

Dendrimers have been proposed as new carriers for drug delivery. They have distinctive characteristics, such as uniform and controlled size, monodispersity and modifiable surface group functionality, which make them extremely useful for biomedical applications. In this study, the binding capacity of water-soluble carbosilane dendrimers was examined. A double fluorimetric titration method with 1-anilinonaphthalene-8-sulphonic acid (ANS) was used to estimate the binding constant and the number of binding centers per dendrimer molecule. The data obtained suggest that ANS interacts non-covalently with the dendrimers. Second generation dendrimers have an open, asymmetric structure that allows them to encapsulate ANS. The ability of the polymers to interact with DNA was assessed by an ethidium bromide (EB) displacement assay. All the dendrimers studied bound to DNA in competition with EB, though the strength of binding varied. Dendrimer interactions with a protein (BSA) were tested using fluorescence quenchers. The dendrimers caused no conformation change in the protein, indicating that interactions between carbosilane dendrimers and BSA are weak and occur preferentially at the protein surface.

Synthesis of polymetallic Group 4 complexes bridged by benzenediolate and triolate ligands. X-ray crystal structure of [{Ti(C5Me5)Cl2}2{μ-1,4-O(2,3-C6H2Me2)O-}]

Abstract This paper reports the synthesis of polymetallic complexes in which two or three Group 4 metals are linked to a benzene core through oxo groups. Four methods have been evaluated for the synthesis of such derivatives: from the appropriated alcohol with (a) methyl complexes via methane elimination, (b) chloride compounds in the presence of a Lewis base, or (c) a zirconium hydride, and (d) from the lithium salt of the alcohol and chloride complexes. Method a has been used for the synthesis of bimetallic and trimetallic (pentamethylcyclopentadienyl)titanium(IV) complexes [{Ti(C5Me5)Cl2}2{μ-1,4-O(C6H2XY)O}] (X=Y=H (1); X=H, Y=Me (2); X=Y=Me (3)), [{Ti(C5Me5)Me2}2{μ-1,4-O(C6H2Me2)O}] (4), and [{Ti(C5Me5)X2}3(μ3-1,3,5-C6H3O3)] (X=Cl (7), Me (8)) from the corresponding hydroquinones 1,4-HO(2,3-C6H2XY)OH (X=Y=H, Me; X=H, Y=Me) or 1,3,5-trihydroxibenzene and [Ti(C5Me5)Cl2Me] or [Ti(C5Me5)Me3], respectively. Bis(cyclopentadienyl)titanium bimetallic complex [{Ti(C5H5)2Cl}2{μ-1,4-O(C6H2Me2)O}] (5) is better prepared by method d by treatment of the dilithium salt Li2[1,4-O(2,3-C6H2Me2)O] with [Ti(C5H5)2Cl2] whereas the trimetallic compound [{Ti(C5H5)2Cl}3(μ3-1,3,5-C6H3O3)] (9) can be prepared directly from 1,3,5-trihidroxybenzene in the presence of NEt3 (method b). Finally, bis(cyclopentadienyl)zirconium complexes [{Zr(C5H5)2Cl}2{μ-1,4-O(C6H2Me2)O}] (6) and [{Zr(C5H5)2Cl}3(μ3-1,3,5-C6H3O3)] (10) are obtained from [Zr(C5H5)2ClH] (method c). The structure of complex 3 has been determined by X-ray diffraction methods.

Study of cationic carbosilane dendrimers as potential activating stimuli in macrophages

Cationic carbosilane dendrimers with two ammonium groups per branch [GnO3(SiONN)m]2m+ (n = 1, m = 6; n = 2, m = 12; n = 3, m = 24), derived from the 1,3,5-trihydroxybenzene (1,3,5-C6H3(OH)3) core, have been synthesized from reactions of dendrimers functionalized with Si–Cl bonds and the alcohol amine 2-{[2-(dimethylamino)ethyl]methylamino}ethanol (HO(CH2)2N(Me)(CH2)2NMe2) and further addition of MeI. The cationic dendrimers obtained were soluble in water, although they decomposed slowly in this medium. The toxicity and inflammatory activity of these dendrimers were evaluated in M1 macrophages and the results compared with the known carbosilane dendrimer 2G-NN16, derived from a Si atom core. The data showed good toxicity profiles for dendrimers [GnO3(SiONN)m]2m+ of first and second generation and 2G-NN16 and also that these dendrimers are not sensed as activating stimuli for macrophages and, as consequence, they do not cause an inflammatory response.