Study of the antimicrobial activity of cationic carbosilane dendrimers against clinical strains of multidrug-resistant bacteria and their biofilms.

Introduction: Antimicrobial Resistance is a serious public health problem, which is aggravated by the ability of the microorganisms to form biofilms. Therefore, new therapeutic strategies need to be found, one of them being the use of cationic dendritic systems (dendrimers and dendrons). Methods: The aim of this study is to analyze the in vitro antimicrobial efficacy of six cationic carbosilane (CBS) dendrimers and one dendron with peripheral ammonium groups against multidrug-resistant bacteria, some of them isolated hospital strains, and their biofilms. For this purpose, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC) and minimum eradication biofilm concentration (MBEC) studies were carried out. In addition, the cytotoxicity on Hela cells of those compounds that proved to be the most effective was analyzed. Results: All the tested compounds showed in vitro activity against the planktonic forms of methicillin-resistant Staphylococcus aureus and only the dendrimers BDSQ017, BDAC-001 and BDLS-001 and the dendron BDEF-130 against their biofilms. On the other hand, only the dendrimers BDAC 001, BDLS-001 and BDJS-049 and the dendron BDEF-130 were antibacterial in vitro against the planktonic forms of multidrug-resistant Pseudomonas aeruginosa, but they lacked activity against their preformed biofilms. In addition, the dendrimers BDAC-001, BDLS-001 and BDSQ-017 and the dendron BDEF-130 exhibited a good profile of cytotoxicity in vitro. Discussion: Our study demonstrates the possibility of using the four compounds mentioned above as possible topical antimicrobials against the clinical and reference strains of multidrug-resistant bacteria.

Dendritic systems for bacterial outer membrane disruption as a method of overcoming bacterial multidrug resistance.

The alarming rise of multi-drug resistant microorganisms has increased the need for new approaches through the development of innovative agents that are capable of attaching to the outer layers of bacteria and causing permanent damage by penetrating the bacterial outer membrane. The permeability (disruption) of the outer membrane of Gram-negative bacteria is now considered to be one of the main ways to overcome multidrug resistance in bacteria. Natural and synthetic permeabilizers such as AMPs and dendritic systems seem promising. However, due to their advantages in terms of biocompatibility, antimicrobial capacity, and wide possibilities for modification and synthesis, highly branched polymers and dendritic systems have gained much more interest in recent years. Various forms of arrangement, and structure of the skeleton, give dendritic systems versatile applications, especially the possibility of attaching other ligands to their surface. This review will focus on the mechanisms used by different types of dendritic polymers, and their complexes with macromolecules to enhance their antimicrobial effect, and to permeabilize the bacterial outer membrane. In addition, future challenges and potential prospects are illustrated in the hope of accelerating the advancement of nanomedicine in the fight against resistant pathogens.

Potential anti-adhesion activity of novel carbosilane zwitterionic dendrimers against eukaryotic and prokaryotic pathogenic microorganisms.

The development of biofilms on different surfaces continues to be a major public health problem. The antimicrobial resistance and the difficulty of finding drugs capable of combating these established biofilms generates the urgent need to find compounds that prevent cells from settling and establishing of these complex communities of microorganisms. Zwitterionic modification of nanomaterials allows the formation of a hydration layer, and this highly hydrophilic surface provides antifouling properties as well as a good biocompatibility by preventing non-specific interactions. Thus, they are appropriate candidates to prevent microbial adhesion to different surfaces and, in consequence, avoid biofilm formation. For this reason, we have incorporated zwitterionic moieties in multivalent systems, as are carbosilane dendrimers. Characterization of these systems was performed using nuclear magnetic resonance and mass spectrometry. It has been analysed if the new molecules have capacity to inhibit the biofilm formation in Candida albicans, Staphylococcus aureus and Pseudomonas aeruginosa. The results showed that they were more effective against S. aureus, observing a biofilm reduction of 81.5% treating with 32 mg/L of G2SiZWsf dendrimer and by 72.5% using 32 mg/L of the G3SiZWsf dendrimer. Finally, the absence of cytotoxicity was verified by haemolysis and cytotoxicity studies in human cells lines.

Mechanistic insight of lysozyme transport through the outer bacteria membrane with dendronized silver nanoparticles for peptidoglycan degradation.

Drug resistance has become a global problem, prompting the entire scientific world to seek alternative methods of dealing with resistant pathogens. Among the many alternatives to antibiotics, two appear to be the most promising: membrane permeabilizers and enzymes that destroy bacterial cell walls. Therefore, in this study, we provide insight into the mechanism of lysozyme transport strategies using two types of carbosilane dendronized silver nanoparticles (DendAgNPs), non-polyethylene glycol (PEG)-modified (DendAgNPs) and PEGylated (PEG-DendAgNPs), for outer membrane permeabilization and peptidoglycan degradation. Remarkably, studies have shown that DendAgNPs can build up on the surface of a bacterial cell, destroying the outer membrane, and thereby allowing lysozymes to penetrate inside the bacteria and destroy the cell wall. PEG-DendAgNPs, on the other hand, have a completely different mechanism of action. PEG chains containing a complex lysozyme resulted in bacterial aggregation and an increase in the local enzyme concentration near the bacterial membrane, thereby inhibiting bacterial growth. This is due to the accumulation of the enzyme in one place on the surface of the bacteria and penetration into it through slight damage of the membrane due to interactions of NPs with the membrane. The results of this study will help propel more effective antimicrobial protein nanocarriers.

Carbosilane dendritic nanostructures, highly versatile platforms for pharmaceutical applications.

Dendrimers are multifunctional molecules with well-defined size and structure due to the step-by-step synthetic procedures required in their preparation. Dendritic constructs based on carbosilane scaffolds present carbon-carbon and carbon-silicon bonds, which results in stable, lipophilic, inert, and flexible structures. These properties are highly appreciated in different areas, including the pharmaceutical field, as they can increase the interaction with cell membranes and improve the therapeutic action. This article summarizes the most recent advances in the pharmaceutical applications of carbosilane dendritic molecules, from therapeutics to diagnostics and prevention tools. Dendrimers decorated with cationic, anionic, or other moieties, including metallodendrimers; supramolecular assemblies; dendronized nanoparticles and surfaces; as well as dendritic networks like hydrogels are described. The collected examples confirm the potential of carbosilane dendrimers and dendritic materials as antiviral or antibacterial agents; in therapy against cancer, neurodegenerative disease, or oxidative stress; or many other biomedical applications. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Immobilization of Alcalase on Silica Supports Modified with Carbosilane and PAMAM Dendrimers.

Enzyme immobilization is a powerful strategy for enzyme stabilization and recyclability. Materials covered with multipoint molecules are very attractive for this goal, since the number of active moieties to attach the enzyme increases with respect to monofunctional linkers. This work evaluates different dendrimers supported on silica to immobilize a protease enzyme, Alcalase. Five different dendrimers were employed: two carbosilane (CBS) dendrimers of different generations (SiO2-G0Si-NH2 and SiO2-G1Si-NH2), a CBS dendrimer with a polyphenoxo core (SiO2-G1O3-NH2), and two commercial polyamidoamine (PAMAM) dendrimers of different generations (SiO2-G0PAMAM-NH2 and SiO2-G1PAMAM-NH2). The results were compared with a silica support modified with a monofunctional molecule (2-aminoethanethiol). The effect of the dendrimer generation, the immobilization conditions (immobilization time, Alcalase/SiO2 ratio, and presence of Ca2+ ions), and the digestion conditions (temperature, time, amount of support, and stirring speed) on Alcalase activity has been evaluated. Enzyme immobilization and its activity were highly affected by the kind of dendrimer and its generation, observing the most favorable behavior with SiO2-G0PAMAM-NH2. The enzyme immobilized on this support was used in two consecutive digestions and, unlike CBS supports, it did not retain peptides released in the digestion.

Bacteria capture with magnetic nanoparticles modified with cationic carbosilane dendritic systems.

Bacteria elimination from water sources is key to obtain drinkable water. Hence, the design of systems with ability to interact with bacteria and remove them from water is an attractive proposal. A diversity of polycationic macromolecules has shown bactericide properties, due to interactions with bacteria membranes. In this work, we have grafted cationic carbosilane (CBS) dendrons and dendrimers on the surface of iron oxide magnetic nanoparticles (MNP), leading to NP (ca. 10 nm) that interact with bacteria by covering bacteria membrane. Application of an external magnetic field removes MNP from solution sweeping bacteria attached to them. The interaction of the MNP with Gram-positive S. aureus bacteria is more sensible to the size of dendritic system covering the MNP, whereas interaction with Gram-negative E. coli bacteria is more sensible to the density of cationic groups. Over 500 ppm of NPM, MNP covered with dendrons captured over 90% of both type of bacteria, whereas MNP covered with dendrimers were only able to capture S. aureus bacteria (over 90%) but not E. coli bacteria. Modified MNP were characterized by transmission electron microscopy (TEM), thermogravimetric analysis (TGA), Fourier-transform infrared spectroscopy (FTIR), Z potential and dynamic light scattering (DLS). Interaction with bacteria was analyzed by UV, TEM and scanning electron microscopy (SEM). Moreover, the possibility to recycle cationic dendronized MNP was explored.

Triazine-Carbosilane Dendrimersomes Enhance Cellular Uptake and Phototoxic Activity of Rose Bengal in Basal Cell Skin Carcinoma Cells.

Background: The search for new formulations for photodynamic therapy is intended to improve the outcome of skin cancer treatment using significantly reduced doses of photosensitizer, thereby avoiding side effects. The incorporation of photosensitizers into nanoassemblies is a versatile way to increase the efficiency and specificity of drug delivery into target cells. Herein, we report the loading of rose bengal into vesicle-like constructs of amphiphilic triazine-carbosilane dendrons (dendrimersomes) as well as biophysical and in vitro characterization of this novel nanosystem. Methods: Using established protocol and analytical and spectroscopy techniques we were able to synthesized dendrons with strictly designed properties. Engaging biophysical methods (hydrodynamic diameter and zeta potential measurements, analysis of spectral properties, transmission electron microscopy) we confirmed assembling of our nanosystem. A set of in vitro techniques was used for determination ROS generation, (ABDA and H2DCFDA probes), cell viability (MTT assay) and cellular uptake (flow cytometry and confocal microscopy). Results: Encapsulation of rose bengal inside dendrimersomes enhances cellular uptake, intracellular ROS production and concequently, the phototoxicity of this photosensitizer. Conclusion: Triazine-carbosilane dendrimersomes show high capacity as drug carriers for anticancer photodynamic therapy.

The Antibacterial Effect of PEGylated Carbosilane Dendrimers on P. aeruginosa Alone and in Combination with Phage-Derived Endolysin.

The search for new microbicide compounds is of an urgent need, especially against difficult-to-eradicate biofilm-forming bacteria. One attractive option is the application of cationic multivalent dendrimers as antibacterials and also as carriers of active molecules. These compounds require an adequate hydrophilic/hydrophobic structural balance to maximize the effect. Herein, we evaluated the antimicrobial activity of cationic carbosilane (CBS) dendrimers unmodified or modified with polyethylene glycol (PEG) units, against planktonic and biofilm-forming P. aeruginosa culture. Our study revealed that the presence of PEG destabilized the hydrophilic/hydrophobic balance but reduced the antibacterial activity measured by microbiological cultivation methods, laser interferometry and fluorescence microscopy. On the other hand, the activity can be improved by the combination of the CBS dendrimers with endolysin, a bacteriophage-encoded peptidoglycan hydrolase. This enzyme applied in the absence of the cationic CBS dendrimers is ineffective against Gram-negative bacteria because of the protective outer membrane shield. However, the endolysin-CBS dendrimer mixture enables the penetration through the membrane and then deterioration of the peptidoglycan layer, providing a synergic antimicrobial effect.

PEGylation of Dendronized Gold Nanoparticles Affects Their Interaction with Thrombin and siRNA.

The use of nonviral carriers based on nanomaterials is a promising strategy for modern gene therapy aimed at protecting the genetic material against degradation and enabling its efficient cellular uptake. To improve the effectiveness of nanocarriers in vivo, they are often modified with poly(ethylene glycol) (PEG) to reduce their toxicity, limit nonspecific binding by proteins in the bloodstream, and extend blood half-life. Thus, the selection of an appropriate degree of surface PEGylation is crucial to preserve the interaction of nanoparticles with the genetic material and to ensure its efficient transport to the site of action. Our research focuses on the use of innovative gold nanoparticles (AuNPs) coated with cationic carbosilane dendrons as carriers of siRNA. In this study, using dynamic light scattering and zeta potential measurements, circular dichroism, and gel electrophoresis, we investigated dendronized AuNPs modified to varying degrees with PEG in terms of their interactions with siRNA and thrombin to select the most promising PEGylated carrier for further research.

Dendritic Nanotheranostic for the Delivery of Infliximab: A Potential Carrier in Rheumatoid Arthritis Therapy.

Antibodies are macromolecules that specifically recognize their target, making them good candidates to be employed in various therapies. The possibility of attaching a drug to an immunoglobulin makes it possible to release it specifically into the affected tissue as long as it overexpresses the target. However, chemical coupling could affect the functionality (specificity and affinity) of the antibody. It has been observed that the use of intermediaries, such as dendrimers, could resolve this issue. Because carbosilane dendrimers have aroused great interest in the field of biomedicine, this report describes the synthesis of an anionic carbosilane dendrimer with a fluorochrome on its surface that then forms a conjugate with an antibody. It has been used as immunoglobulin and infliximab, whose target is TNF-α, which is a cytokine that is overexpressed in the inflamed area or even in the blood of patients with autoimmune diseases, such as rheumatoid arthritis. In addition, the integrity and functionality of the antibody has been studied to see if they have been affected after the chemical coupling process.

Immobilization of thermolysin enzyme on dendronized silica supports. Evaluation of its feasibility on multiple protein hydrolysis cycles.

This work evaluates different dendrimer-silica supports for the immobilization of enzymes by multipoint covalent binding. Thermolysin was immobilized on two dendrimers (PAMAM and carbosilane) with two different generations (zero (G0) and first (G1)). Results were compared with a control, a silica support functionalized with a monofunctional molecule. Dendrimers increased the number of available sites to bind the enzyme. Despite the enzyme was immobilized on all supports, G0 dendrimers immobilized a 30% more enzyme than G1. Thermolysin immobilized on G0 dendrimer supports showed the highest activity and could be employed in three consecutive hydrolysis cycles. Optimal immobilization time was 1 h while optimal protein loading was 25 mg enzyme/100 mg support. Enzyme activity was promoted when using 5 mg of immobilized enzyme at 750 rpm, 60 °C, and 2 h of hydrolysis. Under these conditions, the activity of thermolysin increased up to the 78% of the free enzyme activity. Kinetics of the hydrolysis reaction using the immobilized thermolysin was also studied and compared with the obtained using the free thermolysin. The addition of ZnCl2 and NaCl during the immobilization procedure increased thermolysin activity in the second (22% more) and in the third (14% more) hydrolysis clycles.

Carbosilane Dendrimers Loaded with siRNA Targeting Nrf2 as a Tool to Overcome Cisplatin Chemoresistance in Bladder Cancer Cells.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is considered as the master regulator of antioxidant and cytoprotective gene expressions. Moreover, it plays a pivotal role in cancer progression. Nrf2 mediates the adaptive response which contributes to the resistance to chemotherapeutic pro-oxidant drugs, such as cisplatin (CDDP), in various tumors, including bladder cancers. For this reason, Nrf2 could be a promising target to overcome chemoresistance. There are several known Nrf2 pharmacological inhibitors; however, most of them are not specific. The use of a specific small interfering RNA (siRNA) targeting the Nrf2 gene (siNrf2) loaded into nanovehicles is an attractive alternative, since it can increase specificity. This study aimed to evaluate the biological activity of siNrf2 loaded on guanidine-terminated carbosilane dendrimers (GCDs) in overcoming CDDP resistance in bladder cancer cells with a high level of Nrf2. Parameters such as viability, proliferation, apoptosis, migration, and oxidative stress level were taken into account. Results demonstrated that siNrf2-GCD treatment sensitized CDDP-resistant cells to CDDP treatment. Moreover, data obtained by treating the non-cancerous human kidney HK-2 cell line strongly suggest a good safety profile of the carbosilane dendrimers loaded with siNrf2. In conclusion, we suggest that siNrf2-GCD is a promising drug delivery system for gene therapy to be used in vivo; and it may represent an important tool in the therapy of CDDP-resistant cancer.

In Vitro Activity of Carbosilane Cationic Dendritic Molecules on Prevention and Treatment of Candida Albicans Biofilms.

Candida spp. are one of the most common fungal pathogens. Biofilms formed by Candidaalbicans offer resistance mechanisms against most antifungal agents. Therefore, development of new molecules effective against these microorganisms, alone or in combination with antifungal drugs, is extremely necessary. In the present work, we carried out a screening process of different cationic carbosilane dendritic molecules against C. albicans. In vitro activity against biofilm formation and biofilms was tested in both Colección Española de Cultivos Tipo (CECT) 1002 and clinical C. albicans strains. Cytotoxicity was studied in human cell lines, and biofilm alterations were observed by scanning electron microscopy (SEM). Antifungal activity of the carbosilane dendritic molecules was assessed by monitoring cell viability using both established and novel cell viability assays. One out of 14 dendritic molecules tested, named BDSQ024, showed the highest activity with a minimum biofilm inhibitory concentration (MBIC) for biofilm formation and a minimum biofilm damaging concentration (MBDC) for existing biofilm of 16-32 and 16 mg/L, respectively. Synergy with amphotericin (AmB) and caspofungin (CSF) at non-cytotoxic concentrations was found. Therefore, dendritic compounds are exciting new antifungals effective at preventing Candida biofilm formation and represent a potential novel therapeutic agent for treatment of C. albicans infection in combination with existing clinical antifungals.

pH-Sensitive Dendrimersomes of Hybrid Triazine-Carbosilane Dendritic Amphiphiles-Smart Vehicles for Drug Delivery.

Supramolecular constructions of amphiphilic dendritic molecules are promising vehicles for anti-cancer drug delivery due to the flexibility of their architecture, high drug loading capacity and avoiding off-target effects of a drug. Herein, we report a new class of amphiphilic dendritic species-triazine-carbosilane dendrons readily self-assembling into pH-sensitive dendrimersomes. The dendrimersomes efficiently encapsulate anticancer drugs doxorubicin and methotrexate. Chemodrug-loaded dendrimersomes have dose-related cytotoxic activity against leukaemia cell lines 1301 and K562. Our findings suggest that triazine-carbosilane dendrimersomes are prospective drug carriers for anti-cancer therapy.

Silver Nanoparticles Surface-Modified with Carbosilane Dendrons as Carriers of Anticancer siRNA.

Gene therapy is a promising approach in cancer treatment; however, current methods have a number of limitations mainly due to the difficulty in delivering therapeutic nucleic acids to their sites of action. The application of non-viral carriers based on nanomaterials aims at protecting genetic material from degradation and enabling its effective intracellular transport. We proposed the use of silver nanoparticles (AgNPs) surface-modified with carbosilane dendrons as carriers of anticancer siRNA (siBcl-xl). Using gel electrophoresis, zeta potential and hydrodynamic diameter measurements, as well as transmission electron microscopy, we characterized AgNP:siRNA complexes and demonstrated the stability of nucleic acid in complexes in the presence of RNase. Hemolytic properties of free silver nanoparticles and complexes, their effect on lymphocyte proliferation and cytotoxic activity on HeLa cells were also examined. Confocal microscopy proved the effective cellular uptake of complexes, indicating the possible use of this type of silver nanoparticles as carriers of genetic material in gene therapy.

Functionalization of silica with amine and ammonium alkyl chains, dendrons and dendrimers: Synthesis and antibacterial properties.

Materials modified with ammonium groups on the surface have shown antibacterial activity. In this paper, alkyl chains, carbosilane (CBS) dendrimers and dendrons and poly(amidoamine) (PAMAM) dendrimers containing amine and ammonium groups have been grafted to silica surface and the influence of molecule structure on the stability and on antibacterial activity have been evaluated. These materials have been characterized by thermogravimetric analysis (TGA), zeta (Z) potential, scanning electron microscopy (SEM), infrared spectroscopy (IR) and nuclear magnetic resonance (13C CP MAS NMR). The degree of silica functionalization depends on type of outer groups, amine or ammonium, type and core of dendrimer, and length of chains. The Z potential measurements of these materials in water suspensions were used to test their stability in this medium. These measurements showed, for some of the modified silicas, the diminishing of Z potential from positive values toward zero, probably due to interaction of the functional groups with the silica surface. This variation was also dependent on ligand structure and peripheral functions. Finally, studies of inhibition of bacteria growth stand out again the relevance of ligand structure and number of functional groups on silica surface. The most active systems were those with more surface covered, those with cationic groups further away from silica surface and higher dendritic generation.

New synthetic procedure for the antiviral sulfonate carbosilane dendrimer G2-S16 and its fluorescein-labelled derivative for biological studies.

The anionic carbosilane (CBS) dendrimer with sulfonate groups G2-S16 is a promising compound for the preparation of a microbicide gel to prevent HIV infection. However, until now its synthesis required aggressive conditions. Hence, a reliable synthetic procedure is very important to face GMP conditions and clinical trials. In this study, G2-S16 has been prepared by a new approach that involves the addition of an amine-terminated dendrimer to ethenesulfonyl fluoride (C2H3SO3F, ESF) and then transformation to the sulfonate dendrimer by treatment with a base. This strategy also makes feasible the synthesis of a labelled sulfonate dendrimer (G2-S16-FITC) to be used as a molecular probe for in vivo experiments. Interestingly, G2-S16-FITC enters into human peripheral blood mononuclear cells (PBMCs).

Effect of PEGylation on the biological properties of cationic carbosilane dendronized gold nanoparticles.

Heterofunctionalized gold nanoparticles (AuNPs) were obtained in a one pot reaction of gold precursor with cationic carbosilane dendrons (first to third generations, 1-3G) and (polyethylene)glycol (PEG) ligands in the presence of a reducing agent. The final dendron/PEG proportion on AuNPs depends on the initial dendron/PEG ratio (3/1, 1/1, 1/3) and dendron generation. AuNPs were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), ultraviolet spectroscopy (UV-VIS), thermogravimetric analysis (TGA), nuclear magnetic resonance (1H NMR) and zeta potential (ZP). Several assays have been carried out to determine the relevance of PEG/dendron ratio and dendron generation in the biomedical properties of PEGylated AuNPs and the results have been compared with those obtained for non-PEGylated AuNPs. Finally, analyses of PEG recognition by anti-PEG antibodies were carried out. In general, haemolysis, platelet aggregation and toxicity were reduced after PEGylation of AuNPs, the effect being dependent on dendron generation and dendron/PEG ratio. Dendron generation determines the exposure of PEG ligand and the interaction of this ligand with AuNPs environment. On the other hand, increasing PEG proportion diminishes toxicity but also favors interaction with antibodies.

Feasibility of cationic carbosilane dendrimers for sustainable protein sample preparation.

Protein sample preparation is the bottleneck in the analysis of proteins. The aim of this work is to evaluate the feasibility of carbosilane dendrimers functionalized with cationic groups to make easier this step. Anionic carbosilane dendrimers (sulphonate- and carboxylate-terminated) have already demonstrated their interaction with proteins and their potential in protein sample preparation. In this work, interactions between positively charged carbosilane dendrimers and different model proteins were studied when working under different pH conditions, dendrimer concentrations, and dendrimer generations. Amino- and trimethylammonium-terminated carbosilane dendrimers presented, in some cases, weak interactions with proteins. Unlike them, carbosilane dendrimers with terminal dimethylamino groups could interact, in many cases, with proteins and these interactions were affected by the pH, the dendrimer concentration, and the dendrimer generation. Moreover, dendrimer precipitation was observed at all pHs, although just second and fourth generation (2 G and 4 G) dendrimers resulted in the formation of complexes with proteins. Under experimental conditions promoting dendrimer-protein interactions, 2 G dimethylamino-terminated dendrimers were proposed as an alternative to other methods used in analytical chemistry or analysis in which an organic solvent or a resin are required to enrich/purify proteins in a complex sample.