Synthesis and biophysical evaluation of carbosilane dendrimers as therapeutic siRNA carriers.

Gene therapy presents an innovative approach to the treatment of previously incurable diseases. The advancement of research in the field of nanotechnology has the potential to overcome the current limitations and challenges of conventional therapy methods, and therefore to unlocking the full potential of dendrimers for use in the gene therapy of neurodegenerative disorders. The blood-brain barrier (BBB) poses a significant challenge when delivering therapeutic agents to the central nervous system. In this study, we investigated the biophysical properties of dendrimers and their complexes with siRNA directed against the apolipoprotein E (APOE) gene to identify an appropriate nanocarrier capable of safely delivering the cargo across the BBB. Our study yielded valuable insights into the complexation process, stability over time, the mechanisms of interaction, the influence of dendrimers on the oligonucleotide's spatial structure, and the potential cytotoxic effects on human cerebral microvascular endothelium cells. Based on our findings, we identified that the dendrimer G3Si PEG6000 was an optimal candidate for further research, potentially serving as a nanocarrier capable of safely delivering therapeutic agents across the BBB for the treatment of neurodegenerative disorders.

Magnetic nanoparticles coated with carboxylate-terminated carbosilane dendrons as a reusable and green approach to extract/purify proteins.

Extraction/purification of proteins, at both analytical and industrial levels, is a limiting step that usually requires the use of organic solvents and involves tedious work and a high cost. This work proposes a more sustainable alternative based on the use of magnetic nanoparticles (MNPs) coated with carboxylate-terminated carbosilane dendrons. MNPs coated with first- and second-generation carbosilane dendrons and bare MNPs were employed for the extraction of proteins with different molecular weights and charges. Interaction of proteins with MNPs significantly varied with the pH, the protein, and the dendron generation (different sizes and number of charges in the periphery). Optimal dendron:protein molar ratios and suitable conditions for disrupting interactions after protein extraction were also researched. Second-generation dendron-coated MNPs showed 100% retention capability for all proteins when using acidic conditions. They were reused without losing magnetism or interaction capacity after a disruption of protein-dendron interactions with 0.2% SDS at 100 °C for 10 min. The capacity of dendron-coated MNPs was successfully applied to the recovery/purification of proteins from two food by-products, olive seeds and cheese whey.

Sulfonate-ended carbosilane dendrimers with a flexible scaffold cause inactivation of HIV-1 virions and gp120 shedding.

Infection with human immunodeficiency virus type 1 (HIV-1) continues to be a global public health issue, especially in low-resource countries. Sexual transmission is responsible for the majority of HIV-1 infections worldwide. Women are more susceptible to HIV-1 acquisition than men and represent nearly 50% of the HIV-infected population. Topical vaginal microbicides that act at the earlier stages of infection offer a prevention strategy to reduce the acquisition of HIV-1. Dendrimers are nano-sized, radially symmetric molecules with a well-defined and monodisperse structure consisting of tree-like arms or branches. We perform a TZM.bl cell line-based screening of two families of carbosilane dendrimers (6 nanocompounds: G1-S12P, G2-S24P, G3-S48P, G1-C12P, G2-C24P and G3-C48P) that we have previously synthesized, containing 12, 24 or 48 sulfonate (or carboxylate) end-groups and a polyphenolic core. This work shows that second- and third-generation sulfonate-ended carbosilane dendrimers with a polyphenolic core (G2-S24P and G3-S48P, respectively) display low cytotoxicity (CC50 > 300 µM) with virucidal anti-R5-HIV-1 activity (EC50 < 50 nM; therapeutic index >6000) causing irreversible HIV-1 inactivation (80-90%) by loss of HIV-1 RNA (40%), gp120 shedding (70-80%) and p24 capsid protein release (45-60%). Herein, we demonstrate that sulfonate end-groups and a flexible scaffold from carbosilane dendrimers strongly influence their properties acting as potent virucides.

Efficacy of carbosilane dendrimers with an antiretroviral combination against HIV-1 in the presence of semen-derived enhancer of viral infection.

Amyloid fibrils, which are present in semen, were considered to be a cause of topical vaginal gel ineffectiveness in vivo after microbicides failed as HIV-1 prophylaxis. Therefore, it was necessary to determine whether a dendrimer was suitable for further evaluation in an in vitro model of semen-enhanced viral infection (SEVI). We demonstrated that SEVI in TZM.bl cell cultures increased the infectivity of R5-HIV-1NL(AD8), pTHRO.c and pCH058.c isolates, causing higher IC50 values for two polyanionic carbosilane dendrimers, G2-STE16 and G3-S16. However, both dendrimers maintained protection rates of 90% at non-toxic concentrations. When dendrimers were combined with Tenofovir/Maraviroc (TDF/MVC), the anti-HIV-1 effect remained at a minimum IC50 increase between 1- and 7-fold in the presence of amyloid fibrils. In peripheral blood mononuclear cells (PBMC), IC50 values were slightly influenced by the presence of semen. In brief, dendrimers combined with antiretrovirals showed a synergistic effect. This result plays a crucial role in new microbicide formulations, as it overcomes the negative effects of amyloid fibrils.

Antiviral mechanism of polyanionic carbosilane dendrimers against HIV-1.

Nanotechnology-derived platforms, such as dendrimers, are very attractive in several biological applications. In the case of human immunodeficiency virus (HIV) infection, polyanionic carbosilane dendrimers have shown great potential as antiviral agents in the development of novel microbicides to prevent the sexual transmission of HIV-1. In this work, we studied the mechanism of two sulfated and naphthylsulfonated functionalized carbosilane dendrimers, G3-S16 and G2-NF16. They are able to inhibit viral infection at fusion and thus at the entry step. Both compounds impede the binding of viral particles to target cell surface and membrane fusion through the blockage of gp120-CD4 interaction. In addition, and for the first time, we demonstrate that dendrimers can inhibit cell-to-cell HIV transmission and difficult infectious synapse formation. Thus, carbosilane dendrimers' mode of action is a multifactorial process targeting several proteins from viral envelope and from host cells that could block HIV infection at different stages during the first step of infection.

Dendrimers as nonviral vectors in dendritic cell-based immunotherapies against human immunodeficiency virus: steps toward their clinical evaluation.

Although the antiretroviral therapy has led to a long-term control of HIV-1, it does not cure the disease. Therefore, several strategies are being explored to develop an effective HIV vaccine, such as the use of dendritic cells (DCs). DC-based immunotherapies bear different limitations, but one of the most critical point is the antigen loading into DCs. Nanotechnology offers new tools to overcome these constraints. Dendrimers have been proposed as carriers for targeted delivery of HIV antigens in DCs. These nanosystems can release the antigens in a controlled manner leading to a more potent specific immune response. This review focuses on the first steps for clinical development of dendrimers to assess their safety and potential use in DC-based immunotherapies against HIV.

Enhanced activity of carbosilane dendrimers against HIV when combined with reverse transcriptase inhibitor drugs: searching for more potent microbicides.

Self-administered topical microbicides or oral preexposure prophylaxis could be very helpful tools for all risk groups to decrease the human immunodeficiency virus (HIV)-1 infection rates. Up until now, antiretrovirals (ARVs) have been the most advanced microbicide candidates. Nevertheless, the majority of clinical trials has failed in HIV-1 patients. Nanotechnology offers suitable approaches to develop novel antiviral agents. Thereby, new nanosystems, such as carbosilane dendrimers, have been shown to be safe and effective compounds against HIV with great potential as topical microbicides. In addition, because most of the attempts to develop effective topical microbicides were unsuccessful, combinatorial strategies could be a valid approach when designing new microbicides. We evaluated various combinations of anionic carbosilane dendrimers with sulfated (G3-S16) and naphthyl sulfonated (G2-NF16) ended groups with different ARVs against HIV-1 infection. The G3-S16 and G2-NF16 dendrimers showed a synergistic or additive activity profile with zidovudine, efavirenz, and tenofovir in the majority of the combinations tested against the X4 and R5 tropic HIV-1 in cell lines, as well as in human primary cells. Therefore, the combination of ARVs and polyanionic carbosilane dendrimers enhances the antiviral potency of the individual compounds, and our findings support further clinical research on combinational approaches as potential microbicides to block the sexual transmission of HIV-1.

Synergistic activity profile of carbosilane dendrimer G2-STE16 in combination with other dendrimers and antiretrovirals as topical anti-HIV-1 microbicide.

Polyanionic carbosilane dendrimers represent opportunities to develop new anti-HIV microbicides. Dendrimers and antiretrovirals (ARVs) acting at different stages of HIV replication have been proposed as compounds to decrease new HIV infections. Thus, we determined the potential use of our G2-STE16 carbosilane dendrimer in combination with other carbosilane dendrimers and ARVs for the use as topical microbicide against HIV-1. We showed that these combinations obtained 100% inhibition and displayed a synergistic profile against different HIV-1 isolates in our model of TZM.bl cells. Our results also showed their potent activity in the presence of an acidic vaginal or seminal fluid environment and did not activate an inflammatory response. This study is the first step toward exploring the use of different anionic carbosilane dendrimers in combination and toward making a safe microbicide. Therefore, our results support further studies on dendrimer/dendrimer or dendrimer/ARV combinations as topical anti-HIV-1 microbicide. FROM THE CLINICAL EDITOR: This paper describes the first steps toward the use of anionic carbosilane dendrimers in combination with antivirals to address HIV-1, paving the way to further studies on dendrimer/dendrimer or dendrimer/ARV combinations as topical anti-HIV-1 microbicides.

Development of sulphated and naphthylsulphonated carbosilane dendrimers as topical microbicides to prevent HIV-1 sexual transmission.

OBJECTIVES: For the last 20 years, the idea of alternative prevention strategies based on the use of topical vaginally products to inhibit HIV-1 infection in women has been established. The concept of a 'microbicide' product has been born out of the unavailability of a vaccine against HIV-1 and the problems of women in negotiating the use of preventive prophylaxis by their partners, especially in developing countries. DESIGN: We have developed and evaluated polyanionic carbosilane dendrimers G3-S16 and G2-NF16 with sulphated and naphthylsulphonated end groups as nonspecific microbicides. METHODS: Cellular in-vitro or in-vivo models were used to evaluate the safety, biocompatibility and anti-HIV ability of two polyanionic carbosilane dendrimers. RESULTS: Both dendrimers showed high biosafety in human epithelial cell lines derived from uterus and vagina and in primary blood human cells (PBMC). These dendrimers not only have a partial capacity to block the entry of different X4 and R5 HIV-1 isolates inside epithelial cells but protect the epithelial monolayer from cell disruption and also reduce HIV-1 infection of activated PBMC. Additionally, treatment of epithelial cells with G3-S16 or G2-NF16 dendrimers did not produce changes in proinflammatory cytokines profile, in proliferation of PBMC, on microbiota or sperm survival. Finally, no irritation or vaginal lesions were detected in female CD1(ICR) mice after dendrimers vaginal administration. CONCLUSION: These interesting results suggest that G3-S16 or G2-NF16 could be effective to inhibit HIV infection and transmission within genital mucosa as well as the spread of HIV transmission to human PBMC.

Synergistic activity of carbosilane dendrimers in combination with maraviroc against HIV in vitro.

OBJECTIVE: Dendrimers have been shown as effective and safe anti-HIV compounds with great potential as topical microbicides. Currently, the most advanced microbicides are based on antiretrovirals. However, nowadays none of them has shown any success in clinical trials. DESIGN: Antiviral activity and combinatorial profile of different combinations between CCR5 co-receptor antagonist, maraviroc (MRV), and polyanionic carbosilane dendrimers against HIV-1 strains were evaluated. METHODS: Cellular in-vitro models were used to evaluate the antiviral action of combinations of carbosilane dendrimers/MRV against CCR5 and dual tropic viral strains in TZM.bl cells and peripheral blood mononuclear cells. Combinatorial analysis was performed using Calcusyn software. RESULTS: In the majority of combinations tested, dendrimers showed synergistic profile with MRV against CCR5 and dual tropic HIV-1. CONCLUSION: The evaluated two-drug combinations increase their antiviral potency supporting further clinical investigations to develop combinatorial formulated topical microbicides to fight against the worldwide HIV spread.

Phenotype and functional analysis of human monocytes-derived dendritic cells loaded with a carbosilane dendrimer.

Dendritic cells (DCs) play a major role in development of cell-mediated immunotherapy due to their unique role in linking innate and adaptive immunities. In spite of improvement in this area, strategies employing ex vivo generated DCs have shown limited efficacy in clinical trials. Dendrimers have been proposed as new carriers for drug delivery in aim to ameliorate DCs antigen loading that is a pivotal point in DCs approaches. In this study, we have investigated the phenotypic and functional characteristics of human monocytes-derived dendritic cells after HIV-derived peptides uptake in vitro. We have found that iDCs and mDCs were able to capture efficiently water soluble carbosilane (CBS) dendrimer 2 G-NN16 and did not induce changes in maturation markers levels at the DCs surface. Therefore, CBS 2 G-NN16-loaded mDCs migrated as efficiently as unloaded DCs towards CCL19 or CCL21. Furthermore, DCs viability, activation of allogenic naïve CD4 + T cells by mDCs and secretion of cytokines were not significantly changed by 2 G-NN16 loading. Summing up, our data indicate that CBS 2 G-NN16 has no negative effects on the pivotal properties of DCs in vitro. It should therefore be feasible to further develop this antigen loading strategy for clinical use in immunotherapy against viral infections.