Low-Generation Cationic Phosphorus Dendrimers: Novel Approach to Tackle Drug-Resistant S. aureus In Vitro and In Vivo.

The incessant, global increase in antimicrobial resistance (AMR) is a very big challenge for healthcare systems. AMR is predicted to grow at an alarming pace, with a dramatic increase in morbidity, mortality, and a 100 trillion US$ loss to the global economy by 2050. The mortality rate caused by methicillin-resistant S. aureus (MRSA) is much higher as compared to infections caused by drug-susceptible S. aureus. Additionally, there is a big paucity of therapeutics available for treatment of serious infections caused by MRSA. Thus, the discovery and development of novel therapies is an urgent, unmet medical need. In this context, we synthesized AE4G0, a low-generation cationic-phosphorus dendrimer expressing potent antimicrobial activity against S. aureus and Enterococcus sp., and demonstrating a broad selectivity index against eukaryotic cells. AE4G0 exhibits concentration-dependent, bactericidal activity and synergizes with gentamicin, especially against gentamicin-resistant MRSA NRS119. Fluorescence and scanning electron microscopy demonstrate that treatment with AE4G0 led to the utter destruction of S. aureus ATCC 29213 without inducing resistance, despite repeated exposure. When tested in vivo, AE4G0 demonstrates significant efficacy against S. aureus ATCC 29213, alone and in combination with gentamicin against gentamicin-resistant S. aureus NRS119 in the murine skin model of infection. Taken together, AE4G0 demonstrates the potential to be translated as a novel therapeutic option for the treatment of topical, drug-resistant S. aureus infections.

CD133-Functionalized Gold Nanoparticles as a Carrier Platform for Telaglenastat (CB-839) against Tumor Stem Cells.

The failure of a long-lasting curative therapeutic benefit of currently applied chemotherapies against malignant cancers is suggested to be caused by the ineffectiveness of such interventions on cancer stem cells (CSCs). CD133/AC133 is a cell surface protein previously shown to have potential to identify CSCs in various tumors, including brain tumors. Moreover, an increase in the rate of cellular metabolism of glutamine and glucose are contributors to the fast cellular proliferation of some high-grade malignancies. Inhibition of glutaminolysis by utilizing pharmacological inhibitors of the enzyme glutaminase 1 (GLS1) can be an effective anti-CSC strategy. In this study, the clinical-stage GLS1 inhibitor Telaglenastat (CB-839) was loaded into PEGylated gold nanoparticles equipped with the covalently conjugated CD133 aptamer (Au-PEG-CD133-CB-839) and exposed to a collection of CD133-positive brain tumor models in vitro. Our results show that Au-PEG-CD133-CB-839 significantly decreased the viability of CD133-postive cancer cells in a dose-dependent manner, which was higher as compared to the effects of treatment of the cells with the individual components of the assembled nanodrug. Interestingly, the treatment effect was observed in glioblastoma stem cells modeling different transcriptomic subtypes of the disease. The presented platform is the fundament for subsequent target specificity characterization and in vivo application.

In Vitro Validation of the Therapeutic Potential of Dendrimer-Based Nanoformulations against Tumor Stem Cells.

Tumor cells with stem cell properties are considered to play major roles in promoting the development and malignant behavior of aggressive cancers. Therapeutic strategies that efficiently eradicate such tumor stem cells are of highest clinical need. Herein, we performed the validation of the polycationic phosphorus dendrimer-based approach for small interfering RNAs delivery in in vitro stem-like cells as models. As a therapeutic target, we chose Lyn, a member of the Src family kinases as an example of a prominent enzyme class widely discussed as a potent anti-cancer intervention point. Our selection is guided by our discovery that Lyn mRNA expression level in glioma, a class of brain tumors, possesses significant negative clinical predictive value, promoting its potential as a therapeutic target for future molecular-targeted treatments. We then showed that anti-Lyn siRNA, delivered into Lyn-expressing glioma cell model reduces the cell viability, a fact that was not observed in a cell model that lacks Lyn-expression. Furthermore, we have found that the dendrimer itself influences various parameters of the cells such as the expression of surface markers PD-L1, TIM-3 and CD47, targets for immune recognition and other biological processes suggested to be regulating glioblastoma cell invasion. Our findings prove the potential of dendrimer-based platforms for therapeutic applications, which might help to eradicate the population of cancer cells with augmented chemotherapy resistance. Moreover, the results further promote our functional stem cell technology as suitable component in early stage drug development.

Supramolecular Self-Associations of Amphiphilic Dendrons and Their Properties.

This review presents precisely defined amphiphilic dendrons, their self-association properties, and their different uses. Dendrons, also named dendritic wedges, are composed of a core having two different types of functions, of which one type is used for growing or grafting branched arms, generally multiplied by 2 at each layer by using 1→2 branching motifs. A large diversity of structures has been already synthesized. In practically all cases, their synthesis is based on the synthesis of known dendrimers, such as poly(aryl ether), poly(amidoamine) (in particular PAMAM), poly(amide) (in particular poly(L-lysine)), 1→3 branching motifs (instead of 1→2), poly(alkyl ether) (poly(glycerol) and poly(ethylene glycol)), poly(ester), and those containing main group elements (poly(carbosilane) and poly(phosphorhydrazone)). In most cases, the hydrophilic functions are on the surface of the dendrons, whereas one or two hydrophobic tails are linked to the core. Depending on the structure of the dendrons, and on the experimental conditions used, the amphiphilic dendrons can self-associate at the air-water interface, or form micelles (eventually tubular, but most generally spherical), or form vesicles. These associated dendrons are suitable for the encapsulation of low-molecular or macromolecular bioactive entities to be delivered in cells. This review is organized depending on the nature of the internal structure of the amphiphilic dendrons (aryl ether, amidoamine, amide, quaternary carbon atom, alkyl ether, ester, main group element). The properties issued from their self-associations are described all along the review.

Non-Covalent Associates of siRNAs and AuNPs Enveloped with Lipid Layer and Doped with Amphiphilic Peptide for Efficient siRNA Delivery.

Elaboration of non-viral vehicles for delivery of therapeutic nucleic acids, in particular siRNA, into a cell is an actively growing field. Gold nanoparticles (AuNPs) occupy a noticeable place in these studies, and various nanoconstructions containing AuNPs are reported. We aimed our work to the rational design of AuNPs-based siRNA delivery vehicle with enhanced transfection efficiency. We optimized the obtaining of non-covalent siRNAs-AuNPs cores: ionic strength, temperature and reaction time were determined. Formation of cores was confirmed using gel electrophoresis. Stable associates were prepared, and then enveloped into a lipid layer composed of phosphatidylcholine, phosphatidylethanolamine and novel pH-sensitive lipidoid. The constructions were modified with [Str-(RL)4G-NH2] peptide (the resulting construction). All intermediate and resulting nanoconstructions were analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM) to control their physico-chemical properties. To examine the biological effect of the delivery vehicle, green fluorescent protein (GFP)-expressing human embryonic kidney (HEK) Phoenix cells were incubated with the resulting construction containing anti-GFP siRNA, with the siRNA effect being studied by flow cytometry and confocal microscopy. Transfection of the cells with the resulting construction reduced the GFP fluorescence as efficiently as Lipofectamin 3000. Thus, siRNA vehicle based on non-covalently bound siRNA-AuNP core and enveloped into a lipid layer provides efficient delivery of siRNA into a cell followed by specific gene silencing.

Amphiphilic carbosilane dendrons as a novel synthetic platform toward micelle formation.

A novel family of amphiphilic ionic carbosilane dendrons containing fatty acids at the focal point were synthesized and characterized. They spontaneously self-assembled in aqueous solution into micelles both in the absence and presence of salt, as confirmed by surface tension, conductivity, and DLS measurements. Dendron based micelles have spherical shapes and increase in size on decreasing dendron generation. These dendritic micelles have been demonstrated to be able to form complexes with therapeutic macromolecules such as siRNA and show a high loading capacity for drugs such as procaine, suggesting their potential use as nanocarriers for therapeutics.

Recent Advances in Nucleic Acid Targeting Probes and Supramolecular Constructs Based on Pyrene-Modified Oligonucleotides.

In this review, we summarize the recent advances in the use of pyrene-modified oligonucleotides as a platform for functional nucleic acid-based constructs. Pyrene is of special interest for the development of nucleic acid-based tools due to its unique fluorescent properties (sensitivity of fluorescence to the microenvironment, ability to form excimers and exciplexes, long fluorescence lifetime, high quantum yield), ability to intercalate into the nucleic acid duplex, to act as a π-π-stacking (including anchoring) moiety, and others. These properties of pyrene have been used to construct novel sensitive fluorescent probes for the sequence-specific detection of nucleic acids and the discrimination of single nucleotide polymorphisms (SNPs), aptamer-based biosensors, agents for binding of double-stranded DNAs, and building blocks for supramolecular complexes. Special attention is paid to the influence of the design of pyrene-modified oligonucleotides on their properties, i.e., the structure-function relationships. The perspectives for the applications of pyrene-modified oligonucleotides in biomolecular studies, diagnostics, and nanotechnology are discussed.

Polycationic phosphorous dendrimer potentiates multiple antibiotics against drug-resistant mycobacterial pathogens.

Mycobacterium tuberculosis (Mtb), causative agent of tuberculosis (TB) and non-tubercular mycobacterial (NTM) pathogens such as Mycobacterium abscessus are one of the most critical concerns worldwide due to increased drug-resistance resulting in increased morbidity and mortality. Therefore, focusing on developing novel therapeutics to minimize the treatment period and reducing the burden of drug-resistant Mtb and NTM infections are an urgent and pressing need. In our previous study, we identified anti-mycobacterial activity of orally bioavailable, non-cytotoxic, polycationic phosphorus dendrimer 2G0 against Mtb. In this study, we report ability of 2G0 to potentiate activity of multiple classes of antibiotics against drug-resistant mycobacterial strains. The observed synergy was confirmed using time-kill kinetics and revealed significantly potent activity of the combinations as compared to individual drugs alone. More importantly, no re-growth was observed in any tested combination. The identified combinations were further confirmed in intra-cellular killing assay as well as murine model of NTM infection, where 2G0 potentiated the activity of all tested antibiotics significantly better than individual drugs. Taken together, this nanoparticle with intrinsic antimycobacterial properties has the potential to represents an alternate drug candidate and/or a novel delivery agent for antibiotics of choice for enhancing the treatment of drug-resistant mycobacterial pathogens.

Dendrimers for Drug Delivery: Where Do We Stand in 2023?

Dendrimers are highly symmetric, hyperbranched macromolecules consisting of repeating structural units [...].

Dendrimer-Mediated Delivery of DNA and RNA Vaccines.

DNA and RNA vaccines (nucleic acid-based vaccines) are a promising platform for vaccine development. The first mRNA vaccines (Moderna and Pfizer/BioNTech) were approved in 2020, and a DNA vaccine (Zydus Cadila, India), in 2021. They display unique benefits in the current COVID-19 pandemic. Nucleic acid-based vaccines have a number of advantages, such as safety, efficacy, and low cost. They are potentially faster to develop, cheaper to produce, and easier to store and transport. A crucial step in the technology of DNA or RNA vaccines is choosing an efficient delivery method. Nucleic acid delivery using liposomes is the most popular approach today, but this method has certain disadvantages. Therefore, studies are actively underway to develop various alternative delivery methods, among which synthetic cationic polymers such as dendrimers are very attractive. Dendrimers are three-dimensional nanostructures with a high degree of molecular homogeneity, adjustable size, multivalence, high surface functionality, and high aqueous solubility. The biosafety of some dendrimers has been evaluated in several clinical trials presented in this review. Due to these important and attractive properties, dendrimers are already being used to deliver a number of drugs and are being explored as promising carriers for nucleic acid-based vaccines. This review summarizes the literature data on the development of dendrimer-based delivery systems for DNA and mRNA vaccines.

Effects of Dendrimer-microRNA Nanoformulations against Glioblastoma Stem Cells.

Glioblastoma is a rapidly progressing tumor quite resistant to conventional treatment. These features are currently assigned to a self-sustaining population of glioblastoma stem cells. Anti-tumor stem cell therapy calls for a new means of treatment. In particular, microRNA-based treatment is a solution, which in turn requires specific carriers for intracellular delivery of functional oligonucleotides. Herein, we report a preclinical in vitro validation of antitumor activity of nanoformulations containing antitumor microRNA miR-34a and microRNA-21 synthetic inhibitor and polycationic phosphorus and carbosilane dendrimers. The testing was carried out in a panel of glioblastoma and glioma cell lines, glioblastoma stem-like cells and induced pluripotent stem cells. We have shown dendrimer-microRNA nanoformulations to induce cell death in a controllable manner, with cytotoxic effects being more pronounced in tumor cells than in non-tumor stem cells. Furthermore, nanoformulations affected the expression of proteins responsible for interactions between the tumor and its immune microenvironment: surface markers (PD-L1, TIM3, CD47) and IL-10. Our findings evidence the potential of dendrimer-based therapeutic constructions for the anti-tumor stem cell therapy worth further investigation.

Controlling differentiation of stem cells via bioactive disordered cues.

Ideal bone tissue engineering is to induce bone regeneration through the synergistic integration of biomaterial scaffolds, bone progenitor cells, and bone-forming factors. Biomimetic scaffolds imitate the native extracellular matrix (ECM) and are often utilized in vitro as analogues of the natural ECM to facilitate investigations of cell-ECM interactions and processes. In vivo, the cellular microenvironment has a crucial impact on regulating cell behavior and functions. A PET surface was activated and then functionalized with mimetic peptides to promote human mesenchymal stem cell (hMSC) adhesion and differentiation into an osteogenic lineage. Spray technology was used to randomly micropattern peptides (RGD and BMP-2 mimetic peptides) on the PET surface. The distribution of the peptides grafted on the surface, the roughness of the surfaces and the chemistry of the surfaces in each step of the treatment were ascertained by atomic force microscopy, fluorescence microscopy, time-of-flight secondary ion mass spectrometry, Toluidine Blue O assay, and X-ray photoelectron spectroscopy. Subsequently, cell lineage differentiation was evaluated by quantifying the expression of immunofluorescence markers: osteoblast markers (Runx-2, OPN) and osteocyte markers (E11, DMP1, and SOST). In this article, we hypothesized that a unique combination of bioactive micro/nanopatterns on a polymer surface improves the rate of morphology change and enhances hMSC differentiation. In DMEM, after 14 days, disordered micropatterned surfaces with RGD and BMP-2 led to a higher osteoblast marker expression than surfaces with a homogeneous dual peptide conjugation. Finally, hMSCs cultured in osteogenic differentiation medium (ODM) showed accelerated cell differentiation. In ODM, our results highlighted the expression of osteocyte markers when hMSCs were seeded on PET surfaces with random micropatterns.

Amphiphilic Triazine-Phosphorus Metallodendrons Possessing Anti-Cancer Stem Cell Activity.

Dendritic molecules bearing metal complexes in their structure (metallodendrimers and metallodendrons) are considered prospective therapeutic entities. In particular, metallodendrons raise interest as antitumor agents for the treatment of poorly curable or drug-resistant tumors. Herein, we have synthesized amphiphilic triazine-phosphorus dendrons bearing multiple copper (II) or gold (III) complexes on the periphery and a branched hydrophobic fragment at the focal point. Due to their amphiphilic nature, metallodendrons formed single micelles (mean diameter ~9 nm) or multi-micellar aggregates (mean diameter ~60 nm) in a water solution. We have tested the antitumor activity of amphiphilic metallodendrons towards glioblastoma, a malignant brain tumor with a notoriously high level of therapy resistance, as a model disease. The metallodendrons exhibit higher cytotoxic activity towards glioblastoma stem cells (BTSC233, JHH520, NCH644, and SF188 cell lines) and U87 glioblastoma cells (IC50 was 3-6 µM for copper-containing dendron and 11-15 µM for gold-containing dendron) in comparison with temozolomide (IC50 >100 µM)-the clinical standard of care for glioblastoma. Our findings show the potential of metallodendron-based nanoformulations as antitumor entities.

Effects of Cationic Dendrimers and Their Complexes with microRNAs on Immunocompetent Cells.

Short regulatory oligonucleotides are considered prospective tools for immunotherapy. However, they require an adequate carrier to deliver potential therapeutics into immune cells. Herein, we explore the potential of polycationic dendrimers as carriers for microRNAs in peripheral blood mononuclear cells of healthy donors. As an oligonucleotide cargo, we use a synthetic mimic and an inhibitor of miR-155, an important factor in the development and functioning of immunocompetent cells. Dendrimers bind microRNAs into low-cytotoxic polyelectrolyte complexes that are efficiently uptaken by immunocompetent cells. We have shown these complexes to affect the number of T-regulatory cells, CD14+ and CD19+ cell subpopulations in non-activated mononuclear cells. The treatment affected the expression of HLA-DR on T-cells and PD-1 expression on T- and B-lymphocytes. It also affected the production of IL-4 and IL-10, but not the perforin and granzyme B production. Our findings suggest the potential of dendrimer-mediated microRNA-155 treatment for immunotherapy, though the activity of microRNA-dendrimer constructions on distinct immune cell subsets can be further improved.

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.

Dendriplex-Impregnated Hydrogels With Programmed Release Rate.

Hydrogels are biocompatible matrices for local delivery of nucleic acids; however, functional dopants are required to provide efficient delivery into cells. In particular, dendrimers, known as robust nucleic acid carriers, can be used as dopants. Herein, we report the first example of impregnating neutral hydrogels with siRNA-dendrimer complexes. The surface chemistry of dendrimers allows adjusting the release rate of siRNA-containing complexes. This methodology can bring new materials for biomedical applications.

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.

Cationic Polymers for the Delivery of the Ebola DNA Vaccine Encoding Artificial T-Cell Immunogen.

BACKGROUND: According to current data, an effective Ebola virus vaccine should induce both humoral and T-cell immunity. In this work, we focused our efforts on methods for delivering artificial T-cell immunogen in the form of a DNA vaccine, using generation 4 polyamidoamine dendrimers (PAMAM G4) and a polyglucin:spermidine conjugate (PG). METHODS: Optimal conditions were selected for obtaining complexes of previously developed DNA vaccines with cationic polymers. The sizes, mobility and surface charge of the complexes with PG and PAMAM 4G have been determined. The immunogenicity of the obtained vaccine constructs was investigated in BALB/c mice. RESULTS: It was shown that packaging of DNA vaccine constructs both in the PG envelope and the PAMAM 4G envelope results in an increase in their immunogenicity as compared with the group of mice immunized with the of vector plasmid pcDNA3.1 (a negative control). The highest T-cell responses were shown in mice immunized with complexes of DNA vaccines with PG and these responses significantly exceeded those in the groups of animals immunized with both the combination of naked DNAs and the combination DNAs coated with PAMAM 4G. In the group of animals immunized with complexes of the DNA vaccines with PAMAM 4G, no statistical differences were found in the ability to induce T-cell responses, as compared with the group of mice immunized with the combination of naked DNAs. CONCLUSIONS: The PG conjugate can be considered as a promising and safe means to deliver DNA-based vaccines. The use of PAMAM requires further optimization.

Topological Aspects of the Design of Nanocarriers for Therapeutic Peptides and Proteins.

Supramolecular chemistry holds great potential for the design of versatile and safe carriers for therapeutic proteins and peptides. Nanocarriers can be designed to meet specific criteria for given application (exact drug, administration route, target tissue, etc.). However, alterations in the topology of formulation components can drastically change their activity. This is why the supramolecular topology of therapeutic nanoconstructions has to be considered. Herein, we discuss several topological groups used for the design of nanoformulations for peptide and protein delivery: modification of polypeptide chains by host-guest interactions; packaging of proteins and peptides into liposomes; complexation and conjugation with dendrimers. Each topological type has its own advantages and disadvantages, so careful design of nanoformulations is needed. Ideally, each case where nanomedicine is needed requires a therapeutic construction specially created for that taking into account features of the administration route, target tissue, or organ, properties of a drug, its bioavailability, etc. The wide number of studies in the field of protein delivery by supramolecular and nanocarriers for proteins and peptides evidence their increasing potential for different aspects of the innovative medicine. Although significant progress has been achieved in the field, there are several remaining challenges to be overcome in future.

Interaction of Hydrophobic Tungsten Cluster Complexes with a Phospholipid Bilayer.

Nanoconstructions composed of lipid vesicles and inorganic units (nanoparticles, metal complexes) arouse much interest across materials science and nanotechnology as hybrid materials combining useful functionalities from both parts. Ideally, these units are to be embedded into the bilayer to keep the biophysical performance of lipid vesicles having inorganic moieties screened from the environment. This can be achieved by doping a lipid bilayer with cluster complexes of transition metals. In this work, we report the preparation of nanoparticles from trinuclear W3S4 cluster complexes and egg phosphatidylcholine. A systematic study of their properties was performed by the differential scanning calorimetry, NMR spectroscopy, dynamic light scattering, and transmission electron microscopy. Phospholipids and clusters have been found to spontaneously self-assemble into novel cluster-lipid hybrid materials. The behavior of clusters in the hydrophobic lipid environment is determined by the structure of the ligands and cluster-to-lipid ratio. Intact cluster complexes bearing compact hydrophobic ligands are embedded into the hydrophobic midplane of a lipid bilayer, whereas cluster complexes bearing larger ligands drive the aggregation of lipids and cluster complexes. Considering these differences, it could be possible to obtain different self-assembled associates such as cluster-doped liposomes or lipid-covered crystals. These cluster-lipid hybrids can be a platform for the design of new materials for nanotechnology.