Ruthenium atoms located in the surfaces of carbosilane dendrimers markedly increase their anti-tu... more Ruthenium atoms located in the surfaces of carbosilane dendrimers markedly increase their anti-tumor properties. Carbosilane dendrimers have been widely studied as carriers of drugs and genes owing to such characteristic features as monodispersity, stability, and multivalence. The presence of ruthenium in the dendrimer structure enhances their successful use in anti-cancer therapy. In this paper, the activity of dendrimers of generation 1 and 2 against 1301 cells was evaluated using Transmission Electron Microscopy, comet assay and Real Time PCR techniques. Additionally, the level of reactive oxygen species (ROS) and changes of mitochondrial potential values were assessed. The results of the present study show that ruthenium dendrimers significantly decrease the viability of leukemia cells (1301) but show low toxicity to non-cancer cells (peripheral blood mononuclear cells—PBMCs). The in vitro test results indicate that the dendrimers injure the 1301 leukemia cells via the apoptosis...
Candida albicans is a human pathogen of significant clinical relevance. This pathogen is resistan... more Candida albicans is a human pathogen of significant clinical relevance. This pathogen is resistant to different drugs, and most clinical antifungals are not effective against the prevention and treatment of C. albicans infections. As with other microorganisms, it can produce biofilms that serve as a barrier against antifungal agents and other substances, contributing to infection in humans and environmental tolerance of this microorganism. Thus, resistances and biofilm formation make treatment difficult. In addition, the complete eradication of biofilms in implants, catheters and other medical devices, is challenging and necessary to prevent relapses of candidemia. Therefore, it is a priority to find new molecules or combinations of compounds with anti-Candida biofilm activity. Due to the difficulty of treating and removing biofilms, the aim of this study was to evaluate the in vitro ability of different generation of cationic carbosilane dendrons derived from 4-phenylbutyric acid, ...
Background: There is a need for equations with which to calculate the intrinsic rate constants th... more Background: There is a need for equations with which to calculate the intrinsic rate constants that can further characterise enzyme catalysed reactions despite what seems to be conventional differences in methodology in the literature. Methods: Theoretical, experimental (Bernfeld method), and computational methods. Objectives: 1) To derive an alternative intrinsic rate constant equations consistent with their dimension, 2) derive electrostatic intermolecular potential energy equation, (e), 3) calculate the intrinsic rate constants for forward (k1) and reverse (k2) reactions, and 4) define the dependence or otherwise of kinetic constants on diffusion and deduce the catalytic efficiency. Results and Discussion: The ultimate quantitative results were ~ 64.69 0.49 exp (+3)/ min (k2) (and kd () = ~ 60.66 exp (+3)/ min), ~ 1594.48 11.99 exp (+3) exp (+3) L/mol.min (k1) (and ka () = ~1482.47 exp (+3) L/mol.min), ~ 58.00 10.83 exp (+3) /min, the apparent rate constant for reverse reaction (kb), and ~ 75.83 10.83 exp (+3) /min, the rate constant for product formation (k3). The catalytic efficiency was: 3.025 exp (+ 9) L / mol. Conclusion: The relevant equations were derived. Based on the derived equations the intrinsic rate constants can be calculated. Since k3 is > kb, then k3 is diffusion controlled and it appears that the enzyme has reached kinetic perfection. The evaluation of rate constants either from the perspective of diffusion dependency or independency cannot be valid without Avogadro number.
PLGA nanoparticles functionalized with carbosilane anionic dendrons have been prepared. The bioco... more PLGA nanoparticles functionalized with carbosilane anionic dendrons have been prepared. The biocompatibility and HIV activity have been explored in PBMC and HEC-1A cells. The results indicate that these systems are powerful anti-HIV agents.
Taylor & Francis makes every effort to ensure the accuracy of all the information (the "Content")... more Taylor & Francis makes every effort to ensure the accuracy of all the information (the "Content") contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
P.S. It is a published abstract. Background: Short oligodeoxynucleotides (ODNs) are a new class o... more P.S. It is a published abstract. Background: Short oligodeoxynucleotides (ODNs) are a new class of drugs for an antisense therapy of cancer and infectious diseases. Water soluble carbosilane dendrimers have been recently described by our groups as ODNs carriers. Our aim was to study the interaction between dendriplexes and serum albumin. Materials and methods: The interactions between bovine serum albumin (BSA), carbosilane dendrimers and ODNs used in anti HIV1 therapy were examined using fluorescence and gel-electrophoresis. Results: The interaction between ODNs and dendrimers depended on the type of the ODN and dendrimer. The interactions between carbosilane dendrimers and BSA were weak and occurred preferentially at protein surface. BSA was able to destroy partially some types of ODN/dendrimer complexes. Conclusions: Carbosilane dendrimers can effectively bind ODNs. The stability of complex depends on electrostatic interactions between dendrimer and ODN. Formation of dendriplexes...
ABSTRACT In the last few years many anti-viral drugs have been developed to fight against H1V-1 i... more ABSTRACT In the last few years many anti-viral drugs have been developed to fight against H1V-1 infection and have been proved to control infection. Highly active antiretroviral therapy (HAART) has made an immense impact on fighting the HIV/AIDS pandemic, however, it still possesses major drawbacks such as the need of keeping greater than 95% adherence to be effective and it is a life-long therapy. The additional problems of availability and drug cost, side effects of the treatment, the emergence of drug resistance, the wide array of viral isolates, high mutability of the virus, and coinfections with other diseases such as tuberculosis or hepatitis, among many other issues, demand the research into addressing HIV infection to be continued and further expanded with the objective of making new discoveries and improving current therapies. Intensification of successful anti retroviral therapy has been proposed as a strategy that could control residual replication, diminish the HIV latent reservoirs, and be useful for eradication purposes. However, recently reported studies have failed to show any significant impact of treatment intensification on residual HIV viremia in patients with chronic infection treated with HAART. As a consequence, intensification is close to be rejected for eradication purposes. Therefore, the HIV/AIDS pandemic will not be eradicated without substantial new developments and innovation. A new approach should focus on improving the efficacy and safety of short nucleic acids (oligonucleotides and/or RNA interference (RNAi)-based antiviral drugs) and how this technology is being developed as a new therapeutic tool to fight against HIV infection. The mechanism of RNAi involves short double-stranded segments of RNA termed short interfering RNA (siRNA). The introduction of these oligonucleotides into the cytoplasm of the cell results in degradation of mRNA complementary to the sequence contained in the siRNA. RNAi is a technique that has been used to successfully target HIV replication. However, there still exist some problems that need to be addressed such as obstacles in delivery, target cell transfection, stability/degradation, transient activity, secondary effects, toxicity caused by 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. Furthermore, new advances of these issues can be addressed by using the correct delivery vector. Therefore, a great deal of research is being put into finding a suitable partner and chauffeur for these valuable small nucleic acid (SNA) molecules. The ideal delivery agent should protect the oligonucleotide (ODN) from binding unspecifically to serum proteins and should protect the siRNA from degradation. Also, it should transport ODN or siRNA to the target cells or tissues and facilitate their transfection into the cytoplasm of the cells. The ideal delivery should remain fairly innocuous all the time and should cause little or no adverse effects. To overcome these drawbacks and to make gene therapy (GT) a real alternative, non-viral vehicles such as cationic liposomes, conventional polymers, or dendrimers, have been developed. Once in contact with negatively charged DNA [oligodcoxynuclcotidcs (ODNs) or siRNA], cationic systems form electrostatic complexes with the nucleic acids, namely lipoplexes, polyplexes, or dendriplexes respectively. The use of liposomes for transfection purposes was first described in 1987/ Cationic lipids prepared for this purpose are commercially available (i.e. Cytofcctin™ or Lipofcctin™). However, they have side-effects such us lung inflammatory reactions, and a possible failure of transfection in the presence of serum. The main drawback of the use of conventional degradable polymers as delivery agents is their thermodynamic instability, that results in a short in vivo life-time of the active species, besides their polydispersity. Dendrimers represent an alternative approach for the transfection of SNAs in a wide range of cells. Dendrimers are nanoscopic monodisperse polymers with highly branched three dimensional structures that have shown a wide potential for biological applications. The major advantage of dendrimers over the rest of the vehicles is based on their uniform structure and versatile modification of their skeletons and surfaces, which gives the basis for a precise characterization of the complex [vector/nucleic acid or vector/drug] and a more accurate and systematic research of the delivery process. These systems have shown stability and non-immunogenic properties for their potential use in a high variety of therapeutic applications and have been successfully used as carriers for delivery of nucleic acids and drugs.
Advanced Delivery and Therapeutic Applications of RNAi, 2013
ABSTRACT This chapter offers an overview of the current status of dendrimers as synthetic carrier... more ABSTRACT This chapter offers an overview of the current status of dendrimers as synthetic carriers for siRNA delivery, with an emphasis on how structural alterations to dendrimers affect their transfection efficiencies and cytotoxicities.
ABSTRACT Cationic carbosilane dendrimers with two ammonium groups per branch [GnO3(SiONN)m]2m+ (n... more ABSTRACT 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.
New amphiphilic hyperbranched polymers possessing hydrophobic skeletons and hydrophilic terminal ... more New amphiphilic hyperbranched polymers possessing hydrophobic skeletons and hydrophilic terminal groups have been prepared and characterized. The synthetic strategy involved the formation of a new stable matrix with aromatic units within a carbosilane backbone, as well as the use of a classical polycarbosilane matrix. Both of them with allyl groups on the surface have narrow polydispersity values. Molecular weight and polydispersity of the hyperbranched polymers were obtained using gel permeation chromatography with multi-angle light scattering, and determination of the average number of functional groups present on the surface was achieved using 1 H NMR spectroscopy. The introduction of ionic groups was carried out via thiol-ene reactions with various thiol derivatives. The thermal properties of the polymers were also analysed using differential scanning calorimetry and zeta potential measurements.
The use of dendrimers for biomedical applications has emerged with promising results. 2G-NN16 is ... more The use of dendrimers for biomedical applications has emerged with promising results. 2G-NN16 is a carbosilane dendrimer with sixteen positive charges per molecule tested to be capable to bind and release antisense oligonucleotides (ODNs) and small interference RNA (siRNA) in vitro. In spite of low cytotoxicity observed for these dendrimers, little is known about cellular changes they produce in cells in general and in immune cells in particular. Genomic technologies allow us to identify global gene expression profile changes in macrophages exposed to a non-toxic concentration (5 microM) of 2G-NN16, alone or complexed with a random siRNA (dendriplex). Results were confirmed by quantitative real-time RT-PCR. Exposing macrophages to this dendrimer or dendriplex causes multiple gene expression changes, but no specific action of random siRNA was detected. Pathway analysis of differentially expressed genes shows the altered functions to be immune response, proliferation and transcription regulation. Interleukin 17F (IL17F) was the most regulated gene. Global gene expression profiles are a highly sensitive method to measure the toxicity degree of a gene delivery vehicle. The strong repression of IL17F, IL23R and IL23A, all of which are involved in autoimmune disease, by this particular dendrimer suggests a potential pharmacological application.
To study the effect of a non-viral vector (carbosilane dendrimer) to efficiently deliver small in... more To 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-1alpha (HIF1-alpha) during chemical hypoxia-mediated neurotoxicity. Chemical hypoxia was induced in primary rat cortical neurons by exposure to CoCl(2). HIF1-alpha levels were determined by Western Blot and toxicity was evaluated by both MTT and LDH assays. Neurons were incubated with dendriplexes containing anti-HIF1-alpha siRNA and both uptake and HIF1-alpha knockdown efficiency were evaluated. We report that a non-viral vector (carbosilane dendrimer) can deliver specific siRNA to neurons and selectively block HIF1-alpha 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. This 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.
& The "Materials and Methods" section stated, "Control and dendrimer probes were labeled, respect... more & The "Materials and Methods" section stated, "Control and dendrimer probes were labeled, respectively, with Alexa Fluor 647 reactive dye decapack and Alexa Fluor 555 reactive dye decapack." This section should state, "Control and dendrimer probes were labeled, respectively, with Alexa Fluor 555 reactive dye decapack and Alexa Fluor 647 reactive dye decapack." & There was a mistake in the microarray quantification files, and a new analysis has been done. The new microarray data have been corrected in Gene Expression Omnibus (Series accession number GSE12405). As a consequence of this mistake, Figure 3, Figure 4, Table I, Table II and Supplemental Table have been changed as follows. In spite of this error, conclusions remain valid. CHANGES AND IMPLICATIONS Change: The number of genes over-expressed or underrepressed more than twice is 2,706 and 5,560 genes for Dendrimer (DM) and Dendriplex (DX), respectively. Implication: This change has no effect on discussion, and it is not relevant because the analysis is performed on a selection of these genes. Change: The number of genes differentially expressed, applying filtering by p-value (<0.05), average signal (>32) and z-score (>1.8 or <-1.8), are 141 and 215 for DM and DX exposure, respectively. Implication: A number of selected genes by dendrimer or dendriplex exposure is now more homogeneous.
Novel amine- and ammonium-terminated carbosilane dendrimers of type G(n)-[Si{CH(2)O-(C(6)H(4))-3-... more 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.
Treatment of dendriplexes formed between water-soluble carbosilane dendrimers and phosphorothioat... more 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.
... 5) is better prepared by method d by treatment of the dilithium salt Li 2 [1,4-O(2,3-C 6 H 2 ... more ... 5) is better prepared by method d by treatment of the dilithium salt Li 2 [1,4-O(2,3-C 6 H 2 Me 2 )O---] with [Ti(C 5 H 5 ) 2 Cl 2 ] whereas the trimetallic compound [{Ti(C 5 H 5 ) 2 Cl} 3 (μ 3 -1,3,5-C 6 H 3 O 3 ---)] 9) can be prepared directly from 1,3,5-trihidroxybenzene in the ...
Dendrimers have been proposed as new carriers for drug delivery. They have distinctive characteri... more 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.
Skip to Main Content. Due to scheduled maintenance access to the Wiley Online Library may be disr... more Skip to Main Content. Due to scheduled maintenance access to the Wiley Online Library may be disrupted as follows: Monday, 6 September - New York 0400 EDT to 0500 EDT; London 0900 BST to 1000 BST; Singapore 1600 to 1700. ...
A new mechanism of gene expression inhibition has been discovered as RNA interference, in which t... more 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 zetapotential, 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.
Ruthenium atoms located in the surfaces of carbosilane dendrimers markedly increase their anti-tu... more Ruthenium atoms located in the surfaces of carbosilane dendrimers markedly increase their anti-tumor properties. Carbosilane dendrimers have been widely studied as carriers of drugs and genes owing to such characteristic features as monodispersity, stability, and multivalence. The presence of ruthenium in the dendrimer structure enhances their successful use in anti-cancer therapy. In this paper, the activity of dendrimers of generation 1 and 2 against 1301 cells was evaluated using Transmission Electron Microscopy, comet assay and Real Time PCR techniques. Additionally, the level of reactive oxygen species (ROS) and changes of mitochondrial potential values were assessed. The results of the present study show that ruthenium dendrimers significantly decrease the viability of leukemia cells (1301) but show low toxicity to non-cancer cells (peripheral blood mononuclear cells—PBMCs). The in vitro test results indicate that the dendrimers injure the 1301 leukemia cells via the apoptosis...
Candida albicans is a human pathogen of significant clinical relevance. This pathogen is resistan... more Candida albicans is a human pathogen of significant clinical relevance. This pathogen is resistant to different drugs, and most clinical antifungals are not effective against the prevention and treatment of C. albicans infections. As with other microorganisms, it can produce biofilms that serve as a barrier against antifungal agents and other substances, contributing to infection in humans and environmental tolerance of this microorganism. Thus, resistances and biofilm formation make treatment difficult. In addition, the complete eradication of biofilms in implants, catheters and other medical devices, is challenging and necessary to prevent relapses of candidemia. Therefore, it is a priority to find new molecules or combinations of compounds with anti-Candida biofilm activity. Due to the difficulty of treating and removing biofilms, the aim of this study was to evaluate the in vitro ability of different generation of cationic carbosilane dendrons derived from 4-phenylbutyric acid, ...
Background: There is a need for equations with which to calculate the intrinsic rate constants th... more Background: There is a need for equations with which to calculate the intrinsic rate constants that can further characterise enzyme catalysed reactions despite what seems to be conventional differences in methodology in the literature. Methods: Theoretical, experimental (Bernfeld method), and computational methods. Objectives: 1) To derive an alternative intrinsic rate constant equations consistent with their dimension, 2) derive electrostatic intermolecular potential energy equation, (e), 3) calculate the intrinsic rate constants for forward (k1) and reverse (k2) reactions, and 4) define the dependence or otherwise of kinetic constants on diffusion and deduce the catalytic efficiency. Results and Discussion: The ultimate quantitative results were ~ 64.69 0.49 exp (+3)/ min (k2) (and kd () = ~ 60.66 exp (+3)/ min), ~ 1594.48 11.99 exp (+3) exp (+3) L/mol.min (k1) (and ka () = ~1482.47 exp (+3) L/mol.min), ~ 58.00 10.83 exp (+3) /min, the apparent rate constant for reverse reaction (kb), and ~ 75.83 10.83 exp (+3) /min, the rate constant for product formation (k3). The catalytic efficiency was: 3.025 exp (+ 9) L / mol. Conclusion: The relevant equations were derived. Based on the derived equations the intrinsic rate constants can be calculated. Since k3 is > kb, then k3 is diffusion controlled and it appears that the enzyme has reached kinetic perfection. The evaluation of rate constants either from the perspective of diffusion dependency or independency cannot be valid without Avogadro number.
PLGA nanoparticles functionalized with carbosilane anionic dendrons have been prepared. The bioco... more PLGA nanoparticles functionalized with carbosilane anionic dendrons have been prepared. The biocompatibility and HIV activity have been explored in PBMC and HEC-1A cells. The results indicate that these systems are powerful anti-HIV agents.
Taylor & Francis makes every effort to ensure the accuracy of all the information (the "Content")... more Taylor & Francis makes every effort to ensure the accuracy of all the information (the "Content") contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
P.S. It is a published abstract. Background: Short oligodeoxynucleotides (ODNs) are a new class o... more P.S. It is a published abstract. Background: Short oligodeoxynucleotides (ODNs) are a new class of drugs for an antisense therapy of cancer and infectious diseases. Water soluble carbosilane dendrimers have been recently described by our groups as ODNs carriers. Our aim was to study the interaction between dendriplexes and serum albumin. Materials and methods: The interactions between bovine serum albumin (BSA), carbosilane dendrimers and ODNs used in anti HIV1 therapy were examined using fluorescence and gel-electrophoresis. Results: The interaction between ODNs and dendrimers depended on the type of the ODN and dendrimer. The interactions between carbosilane dendrimers and BSA were weak and occurred preferentially at protein surface. BSA was able to destroy partially some types of ODN/dendrimer complexes. Conclusions: Carbosilane dendrimers can effectively bind ODNs. The stability of complex depends on electrostatic interactions between dendrimer and ODN. Formation of dendriplexes...
ABSTRACT In the last few years many anti-viral drugs have been developed to fight against H1V-1 i... more ABSTRACT In the last few years many anti-viral drugs have been developed to fight against H1V-1 infection and have been proved to control infection. Highly active antiretroviral therapy (HAART) has made an immense impact on fighting the HIV/AIDS pandemic, however, it still possesses major drawbacks such as the need of keeping greater than 95% adherence to be effective and it is a life-long therapy. The additional problems of availability and drug cost, side effects of the treatment, the emergence of drug resistance, the wide array of viral isolates, high mutability of the virus, and coinfections with other diseases such as tuberculosis or hepatitis, among many other issues, demand the research into addressing HIV infection to be continued and further expanded with the objective of making new discoveries and improving current therapies. Intensification of successful anti retroviral therapy has been proposed as a strategy that could control residual replication, diminish the HIV latent reservoirs, and be useful for eradication purposes. However, recently reported studies have failed to show any significant impact of treatment intensification on residual HIV viremia in patients with chronic infection treated with HAART. As a consequence, intensification is close to be rejected for eradication purposes. Therefore, the HIV/AIDS pandemic will not be eradicated without substantial new developments and innovation. A new approach should focus on improving the efficacy and safety of short nucleic acids (oligonucleotides and/or RNA interference (RNAi)-based antiviral drugs) and how this technology is being developed as a new therapeutic tool to fight against HIV infection. The mechanism of RNAi involves short double-stranded segments of RNA termed short interfering RNA (siRNA). The introduction of these oligonucleotides into the cytoplasm of the cell results in degradation of mRNA complementary to the sequence contained in the siRNA. RNAi is a technique that has been used to successfully target HIV replication. However, there still exist some problems that need to be addressed such as obstacles in delivery, target cell transfection, stability/degradation, transient activity, secondary effects, toxicity caused by 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. Furthermore, new advances of these issues can be addressed by using the correct delivery vector. Therefore, a great deal of research is being put into finding a suitable partner and chauffeur for these valuable small nucleic acid (SNA) molecules. The ideal delivery agent should protect the oligonucleotide (ODN) from binding unspecifically to serum proteins and should protect the siRNA from degradation. Also, it should transport ODN or siRNA to the target cells or tissues and facilitate their transfection into the cytoplasm of the cells. The ideal delivery should remain fairly innocuous all the time and should cause little or no adverse effects. To overcome these drawbacks and to make gene therapy (GT) a real alternative, non-viral vehicles such as cationic liposomes, conventional polymers, or dendrimers, have been developed. Once in contact with negatively charged DNA [oligodcoxynuclcotidcs (ODNs) or siRNA], cationic systems form electrostatic complexes with the nucleic acids, namely lipoplexes, polyplexes, or dendriplexes respectively. The use of liposomes for transfection purposes was first described in 1987/ Cationic lipids prepared for this purpose are commercially available (i.e. Cytofcctin™ or Lipofcctin™). However, they have side-effects such us lung inflammatory reactions, and a possible failure of transfection in the presence of serum. The main drawback of the use of conventional degradable polymers as delivery agents is their thermodynamic instability, that results in a short in vivo life-time of the active species, besides their polydispersity. Dendrimers represent an alternative approach for the transfection of SNAs in a wide range of cells. Dendrimers are nanoscopic monodisperse polymers with highly branched three dimensional structures that have shown a wide potential for biological applications. The major advantage of dendrimers over the rest of the vehicles is based on their uniform structure and versatile modification of their skeletons and surfaces, which gives the basis for a precise characterization of the complex [vector/nucleic acid or vector/drug] and a more accurate and systematic research of the delivery process. These systems have shown stability and non-immunogenic properties for their potential use in a high variety of therapeutic applications and have been successfully used as carriers for delivery of nucleic acids and drugs.
Advanced Delivery and Therapeutic Applications of RNAi, 2013
ABSTRACT This chapter offers an overview of the current status of dendrimers as synthetic carrier... more ABSTRACT This chapter offers an overview of the current status of dendrimers as synthetic carriers for siRNA delivery, with an emphasis on how structural alterations to dendrimers affect their transfection efficiencies and cytotoxicities.
ABSTRACT Cationic carbosilane dendrimers with two ammonium groups per branch [GnO3(SiONN)m]2m+ (n... more ABSTRACT 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.
New amphiphilic hyperbranched polymers possessing hydrophobic skeletons and hydrophilic terminal ... more New amphiphilic hyperbranched polymers possessing hydrophobic skeletons and hydrophilic terminal groups have been prepared and characterized. The synthetic strategy involved the formation of a new stable matrix with aromatic units within a carbosilane backbone, as well as the use of a classical polycarbosilane matrix. Both of them with allyl groups on the surface have narrow polydispersity values. Molecular weight and polydispersity of the hyperbranched polymers were obtained using gel permeation chromatography with multi-angle light scattering, and determination of the average number of functional groups present on the surface was achieved using 1 H NMR spectroscopy. The introduction of ionic groups was carried out via thiol-ene reactions with various thiol derivatives. The thermal properties of the polymers were also analysed using differential scanning calorimetry and zeta potential measurements.
The use of dendrimers for biomedical applications has emerged with promising results. 2G-NN16 is ... more The use of dendrimers for biomedical applications has emerged with promising results. 2G-NN16 is a carbosilane dendrimer with sixteen positive charges per molecule tested to be capable to bind and release antisense oligonucleotides (ODNs) and small interference RNA (siRNA) in vitro. In spite of low cytotoxicity observed for these dendrimers, little is known about cellular changes they produce in cells in general and in immune cells in particular. Genomic technologies allow us to identify global gene expression profile changes in macrophages exposed to a non-toxic concentration (5 microM) of 2G-NN16, alone or complexed with a random siRNA (dendriplex). Results were confirmed by quantitative real-time RT-PCR. Exposing macrophages to this dendrimer or dendriplex causes multiple gene expression changes, but no specific action of random siRNA was detected. Pathway analysis of differentially expressed genes shows the altered functions to be immune response, proliferation and transcription regulation. Interleukin 17F (IL17F) was the most regulated gene. Global gene expression profiles are a highly sensitive method to measure the toxicity degree of a gene delivery vehicle. The strong repression of IL17F, IL23R and IL23A, all of which are involved in autoimmune disease, by this particular dendrimer suggests a potential pharmacological application.
To study the effect of a non-viral vector (carbosilane dendrimer) to efficiently deliver small in... more To 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-1alpha (HIF1-alpha) during chemical hypoxia-mediated neurotoxicity. Chemical hypoxia was induced in primary rat cortical neurons by exposure to CoCl(2). HIF1-alpha levels were determined by Western Blot and toxicity was evaluated by both MTT and LDH assays. Neurons were incubated with dendriplexes containing anti-HIF1-alpha siRNA and both uptake and HIF1-alpha knockdown efficiency were evaluated. We report that a non-viral vector (carbosilane dendrimer) can deliver specific siRNA to neurons and selectively block HIF1-alpha 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. This 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.
& The "Materials and Methods" section stated, "Control and dendrimer probes were labeled, respect... more & The "Materials and Methods" section stated, "Control and dendrimer probes were labeled, respectively, with Alexa Fluor 647 reactive dye decapack and Alexa Fluor 555 reactive dye decapack." This section should state, "Control and dendrimer probes were labeled, respectively, with Alexa Fluor 555 reactive dye decapack and Alexa Fluor 647 reactive dye decapack." & There was a mistake in the microarray quantification files, and a new analysis has been done. The new microarray data have been corrected in Gene Expression Omnibus (Series accession number GSE12405). As a consequence of this mistake, Figure 3, Figure 4, Table I, Table II and Supplemental Table have been changed as follows. In spite of this error, conclusions remain valid. CHANGES AND IMPLICATIONS Change: The number of genes over-expressed or underrepressed more than twice is 2,706 and 5,560 genes for Dendrimer (DM) and Dendriplex (DX), respectively. Implication: This change has no effect on discussion, and it is not relevant because the analysis is performed on a selection of these genes. Change: The number of genes differentially expressed, applying filtering by p-value (<0.05), average signal (>32) and z-score (>1.8 or <-1.8), are 141 and 215 for DM and DX exposure, respectively. Implication: A number of selected genes by dendrimer or dendriplex exposure is now more homogeneous.
Novel amine- and ammonium-terminated carbosilane dendrimers of type G(n)-[Si{CH(2)O-(C(6)H(4))-3-... more 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.
Treatment of dendriplexes formed between water-soluble carbosilane dendrimers and phosphorothioat... more 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.
... 5) is better prepared by method d by treatment of the dilithium salt Li 2 [1,4-O(2,3-C 6 H 2 ... more ... 5) is better prepared by method d by treatment of the dilithium salt Li 2 [1,4-O(2,3-C 6 H 2 Me 2 )O---] with [Ti(C 5 H 5 ) 2 Cl 2 ] whereas the trimetallic compound [{Ti(C 5 H 5 ) 2 Cl} 3 (μ 3 -1,3,5-C 6 H 3 O 3 ---)] 9) can be prepared directly from 1,3,5-trihidroxybenzene in the ...
Dendrimers have been proposed as new carriers for drug delivery. They have distinctive characteri... more 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.
Skip to Main Content. Due to scheduled maintenance access to the Wiley Online Library may be disr... more Skip to Main Content. Due to scheduled maintenance access to the Wiley Online Library may be disrupted as follows: Monday, 6 September - New York 0400 EDT to 0500 EDT; London 0900 BST to 1000 BST; Singapore 1600 to 1700. ...
A new mechanism of gene expression inhibition has been discovered as RNA interference, in which t... more 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 zetapotential, 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.
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Papers by Paula Ortega