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Anti-(ds)-DNA antibodies are the serological hallmark of Systemic Lupus Erythematosus (SLE). They assemble in the bloodstream with (ds)-DNA, forming immunocomplexes, which spread all over the body causing, among the other symptoms, lupic glomerulonephritis. Pathological manifestations of the disease may be reduced by destabilizing or inhibiting the formation of the immunocomplexes. In this respect, glycodendrimers showed peculiar interacting abilities towards this kind of biomolecule. Various generations of open-shell maltose-decorated poly(amidoamine) (PAMAM) and poly(propyleneimine) (PPI) dendrimers and two oligopeptides with different polyethylene glycol units were synthesized and characterized, and then tested for their anti-SLE activity. The activity of glycodendrimers and oligopeptides was evaluated in human plasma from patients with SLE, compared to healthy plasma, by means of an enzyme-linked immunosorbent assay (ELISA), and electron paramagnetic resonance (EPR) characterization using spin-label and spin-probe techniques. Different strategies for the immunocomplex formation were tested. The results show that both kinds of glycodendrimers and oligopeptides inhibited the formation of immunocomplexes. Also, a partial breakdown of preformed immunocomplexes was observed. Both ELISA and EPR analyses indicated a better activity of glycodendrimers compared to oligopeptides, the 3 rd generation PPI dendrimer being the most promising against SLE. This study highlights the possibility to develop a new class of dendritic therapeutics for the treatment of Lupus in pre-clinical studies.

The activity and interacting ability of a polyamidoamine (PAMAM) dendrimer modified with 4- N -methylpiperazine-1,8-naphthalimide units (termed D) and complexed by Cu(ii) ions, towards healthy and cancer cells were studied. Comparative electron paramagnetic resonance (EPR) studies of the Cu(ii)-D complex are presented: coordination mode, chemical structure, flexibility and stability of these complexes, in the absence and presence of myeloid cancer cells and peripheral blood mononuclear cells (PBMC). The interactions of Cu(ii) ions in the biological media at different equilibrium times were studied, highlighting different stability and interacting conditions with the cells. Furthermore, flow cytometry and confocal analysis, trace the peculiar properties of the dendrimers in PBMC and U937 cells. Indeed, a new probe (Fly) was used as a potential fluorescent tool for biological imaging of Cu(ii). The study highlights that dendrimer and, mainly, the Cu(ii) metallodendrimer are cytotoxic agents for the cells, specifically for U937 tumor cells, inducing mitochondrial dysfunction, ROS increase and lysosome involvement. The metallodendrimer shows antitumor selectivity, fewer affecting healthy PBMC, inducing a massive apoptotic cell death on U937 cells, in line with the high stability of this complex, as verified by EPR studies. The results underline the potentiality of this metallodendrimer to be used as anticancer drug., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

Iminopyridine-decorated carbosilane metallodendrimers have recently emerged as a promising strategy in the treatment of cancer diseases. Their unique features such as the nanometric size, the multivalent nature and the structural perfection offer an extraordinary platform to explore structure-to-property relationships. Herein, we showcase the outstanding impact on the antitumor activity of a parameter not explored before: the iminopyridine substituents in meta position. New Cu(II) carbosilane metallodendrimers, bearing methyl or methoxy substituents in the pyridine ring, were synthesized and thoroughly characterized. Electron Paramagnetic Resonance (EPR) was exploited to unveil the properties of the metallodendrimers. This study confirmed the presence of different coordination modes of the Cu(II) ion (Cu-N 2 O 2 , Cu-N 4 and Cu-O 4 ), whose ratios were determined by the structural features of the dendritic molecules. These metallodendrimers exhibited IC 50 values in the low micromolar range (<6 μM) in tumor cell lines such as HeLa and MCF-7. The subsequent in vitro assays on both healthy (PBMC) and tumor (U937) myeloid cells revealed two key facts which improved the cytotoxicity and selectivity of the metallodrug: First, maximizing the Cu-N 2 O 2 coordination mode; second, adequately selecting the pair ring-substituent/metal-counterion. The most promising candidates, G 1 (-CH 3 )Cl (8) and G 1 (-OCH 3 )NO 3 (17), exhibited a substantial increase in the antitumor activity in U937 tumor cells, compared to the non-substituted counterparts, probably through two different ROS-production pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Copper(II) carbosilane metallodendrimers are promising nanosized anticancer metallodrugs. The precise control on their design enables an accurate structure-to-activity study. We hypothesized that different structural features, such as the dendrimer generation and metal counterion, modulate the interaction with tumor cells, and subsequently, the effectivity and selectivity of the therapy. A computer-aided analysis of the electron paramagnetic resonance (EPR) spectra allowed us to obtain dynamical and structural details on the interactions over time between the dendrimers and the cells, the myeloid U937 tumor cells and peripheral blood mononuclear cells (PBMC). The intracellular fate of the metallodendrimers was studied through a complete in vitro evaluation, including cytotoxicity, cytostaticity, and sublethal effects regarding mitochondria function, lysosomal compartments, and autophagic organelle involvement. EPR results confirmed a higher membrane stabilization for chloride dendrimers and low generation complexes, which ultimately influence the metallodrug uptake and intracellular fate. The in vitro evaluation revealed that Cu(II) metallodendrimers are cytostatic and moderate cytotoxic agents for U937 tumor cells, inducing death processes through the mitochondria-lysosome axis as well as autophagic vacuole formation, while barely affecting healthy monocytes. The study provided valuable insight into the mechanism of action of these nanosized metallodrugs and relevant structural parameters affecting the activity.

Dendrimers exhibit unique interactions with cell membranes, arising from their nanometric size and high surface area. To a great extent, these interactions define their biological activity and can be reported in situ by spin-labelling techniques. Schiff-base carbosilane ruthenium (II) metallodendrimers are promising antitumor agents with a mechanism of action yet to explore. In order to study their in situ interactions with model cell membranes occurring at a molecular level, namely cetyltrimethylammonium bromide micelles (CTAB) and lecithin liposomes (LEC), electron paramagnetic resonance (EPR) was selected. Both a spin probe, 4-( N , N -dimethyl- N -dodecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl bromide (CAT12), able to enter the model membranes, and a spin label, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) covalently attached at newly synthesized heterofunctional dendrimers, were used to provide complementary information on the dendrimer-membrane interactions. The computer-aided EPR analysis demonstrated a good agreement between the results obtained for the spin probe and spin label experiments. Both points of view suggested the partial insertion of the dendrimer surface groups into the surfactant aggregates, mainly CTAB micelles, and the occurrence of both polar and hydrophobic interactions, while dendrimer-LEC interactions involved more polar interactions between surface groups. We found out that subtle changes in the dendrimer structure greatly modified their interacting abilities and, subsequently, their anticancer activity.

Current cancer therapies present serious drawbacks including severe side-effects and development of drug resistance. Strategies based on nanosized metallodrugs combine the structural diversity and non-classical modes of action of metal complexes with the selectivity arising from the unique interaction of nanoparticles with biological membranes. A new family of water-soluble copper(ii) carbosilane metallodendrimers was synthesized and characterized as a nanotechnological alternative to current therapies. The interactions occurring over time between the dendrimers, at different generations (G 0 to G 2 ) and with different Cu(ii) counter-ions (nitrate vs. chloride), and cell-membrane models (cethyl-trimethylammonium bromide (CTAB) micelles and lecithin liposomes) were investigated using a computer-aided analysis of the electron paramagnetic resonance (EPR) spectra. The EPR analysis provided structural and dynamical information on the systems indicating that the increase in generation and the change of the Cu(ii) contra-ion - from nitrate to chloride - produce an increased relative amount and strength of interaction of the dendrimer with the model membranes. Interestingly, the stabilization effect produced a lower toxicity towards cancer cells. The cytotoxic effect of Cu(ii) metallodendrimers was verified by an in vitro screening in a selection of tumor cell lines, revealing the impact of multivalency on the effectivity and selectivity of the metallodrugs. As a proof-of-concept, first-generation dendrimer G 1 -Cu(ONO 2 ) 2 was selected for in-depth in vitro and in vivo antitumor evaluation towards resistant prostate cancer. The Cu(ii)-metallodendrimers produced a significant tumor size reduction with no signs of toxicity during the experiment, confirming their promising potential as anticancer metallodrugs.

A series of new organometallic carbosilane dendrimers functionalized with Copper(II) complex on the surface were synthesized and characterized as potential anticancer agents. These metallodendrimers were synthesized through the reaction of dendritic ligands containing N,N- and N,O- donor atoms able to act as chelating agents with CuCl 2 as metallic ion precursor. The structural characterization of these complexes was addressed through the use of different analytical and spectroscopical techniques. Particularly, an electron paramagnetic resonance study was performed to corroborate the coordination properties of these dendritic ligands. A preliminary study was carried out to establish the cytotoxicity of the new synthesized compounds in human prostate (PC3) and human cervical (HeLa) cancer cell lines in order to evaluate their potential as anticancer agents and compare their activity with other copper or analogous ruthenium metallodendrimers., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Poly(propyleneimine) glycodendrimers fully modified with maltose units were administered to different cancer cell lines and their effect on cell viability was evaluated by using MTS assay and flow cytometry. The mechanism of dendrimer-cell interactions was investigated by the electron paramagnetic resonance (EPR) technique by using a new nitroxide-conjugated glycodendrimer. The nitroxide groups did not modify both the biological properties (cell viability and apoptosis degree) of the dendrimers in the presence of the cells and the dendrimer-cell interactions. Since this class of dendrimers is already known to be biocompatible for human healthy cells, noncancer cells such as human peripheral blood mononuclear cells (PBMCs) and macrophages were also treated with the glycodendrimer, and EPR spectra of the nitroxide-conjugated glycodendrimer were compared for cancer and noncancer cells. It was found that this dendrimer selectively affects the cell viability of tumor cells, while, surprisingly, PBMC proliferation is induced. Moreover, H-bond-active glycodendrimer-cell interactions were different for the different cancer cell lines and noncancer cells. The nitroxide-conjugated glycodendrimer was able to interact with the cell membrane and eventually cross it, getting in contact with cytosol antioxidants. This study helps to clarify the potential anticancer effect of this class of dendrimers opening to future applications of these macromolecules as new antitumor agents.

Anionic carbosilane dendrons decorated with sulfonate functions and one thiol moiety at the focal point have been used to synthesize water-soluble gold nanoparticles (AuNPs) through the direct reaction of dendrons, gold precursor, and reducing agent in water, and also through a place-exchange reaction. These nanoparticles have been characterized by NMR spectroscopy, TEM, thermogravimetric analysis, X-ray photoelectron spectroscopy (XPS), UV/Vis spectroscopy, elemental analysis, and zeta-potential measurements. The interacting ability of the anionic sulfonate functions was investigated by EPR spectroscopy with copper(II) as a probe. Different structures and conformations of the AuNPs modulate the availability of sulfonate and thiol groups for complexation by copper(II). Toxicity assays of AuNPs showed that those produced through direct reaction were less toxic than those obtained by ligand exchange. Inhibition of HIV-1 infection was higher in the case of dendronized AuNPs than in dendrons., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A synthetic protocol has been designed to incorporate the DO3A ligand to the focal point of cationic or anionic carbosilane dendrons, affording a set of bifunctional chelating agents (BFCAs) useful for potential biomedical applications. The complexation behavior study of ionic BFCAs has been accomplished by UV-vis and electron paramagnetic resonance spectroscopy as well as potentiometric titrations. The presence of the dendron branches modifies the complexation capacity of the macrocyclic ring with respect to that of the 1,4,7,10-tetraazacyclodocecane-N,N',N″,N‴-tetraacetic acid (DOTA) ligand. Also, a different behavior has been observed in the carboxylate-terminated dendrons against analogous sulfonate- or amine-terminated dendrons in the contribution of the branches and peripheral groups to the coordination modes. The presence or not of Cu-S2O2 coordination sites and the generation can be important factors to take into account for considering a particular biomedical application.

Poly-L-lysine is a biocompatible polymer used for drug or gene delivery, for transport through cellular membranes, and as nanosized magnetic resonance imaging contrast agents. Cu(II)-poly-L-lysine complexes are of particular interest for their role in biocatalysis. In this study, poly-L-lysine dendrigrafts (DGLs) at different generations (G2, G3, and G4) are synthesized and characterized in absence and presence of Cu(II) by means of electron paramagnetic resonance (EPR), UV-Vis, potentiometric titration and circular dichroism (CD). The analysis is performed as a function of the [Cu(II)]/[Lys] (=R) molar ratio, pH and generation by identifying differently flexible complexes in different dendrimer regions. The amine sites in the lateral chains become increasingly involved with the increase of pH. The good agreement and complementarity of the results from the different techniques provide an integrate view of the structural and dynamic properties of Cu(II)-DGL complexes implementing their use as biocatalysts., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Understanding the early onset of neurodegeneration is crucial to deploy specific treatments for patients before the process becomes irreversible. Copper has been proposed as a biomarker for many neurodegenerative disorders, being the ion released by pathologically unfolded proteins involved in many biochemical pathways. Dendrimers are macromolecules that bind metal ions with a large ion/ligand ratio, thus, allowing a massive collection of copper. This work provides structural information, obtained via molecular modeling and EPR, for the binding sites of copper in polypropyleneimine (PPI) dendrimers, especially in the maltose decorated form that has potential applications in diagnosis and therapies for various types of neurodegenerations. The analysis of the EPR spectra showed that, at the lowest Cu concentrations, the results are well supported by the calculations. Moreover, EPR analysis at increasing Cu(II) concentration allowed us to follow the saturation behavior of the interacting sites identified by the modeling study.

The structure of copper(II) complexes formed with triethanolamine (TEA) core poly(amidoamine) (PAMAM) dendrimers from generation 0 (G0) to 4 (G4) were investigated by the electron paramagnetic resonance (EPR) technique and molecular simulations. Different square planar coordination modes were detected as a function of copper(II) concentration, whose dynamic evolution relates to the high structural flexibility peculiar to this dendrimer family. Modulated by generation and solvation effects, copper(II) complexation begins at the dendrimer core and progresses to the dendrimer periphery. Differently from the ethylenediamine (EDA) core PAMAM dendrimers, the copper complexes involving the TEA core showed high mobility and saturation of the internal sites above the 1 : 1 molar ratio between the dendrimers and the ions. Therefore, by combining EPR and molecular simulations for the first time, ultimately we obtained unique information on structure, dynamics and copper interacting ability of these dendrimers which could be successfully exploited in biomedical applications.

Herein we describe the synthesis and characterization of new sulfonated and carboxylated poly(propyleneimino) (PPI) dendrimers with the ethylenediamino (EDA) core, at generations 1, 2 and 3. By means of UV-Vis and EPR spectroscopy, using Cu(2+) as a probe, we concluded that these dendrimers show a specific pattern in the coordination of metal ions. In agreement with the UV-Vis studies, EPR spectra of carboxylated compounds are constituted by 3 different signals which appear and then disappear with increasing copper concentration, corresponding to the saturation of different copper complexation sites. At the lowest copper concentration up to a 1:1 molar ratio between Cu(II) and the dendrimer, the spectrum is characteristic of a CuN2O2 coordination at the core of the dendrimer. The spectrum appearing at higher Cu(II) concentrations indicates a peripheral location of the ions coordinating one nitrogen and 3 oxygen atoms in a square planar geometry in restricted mobility conditions. For the highest concentrations tested, copper ions are confined at the external dendrimer surface with CuO4 coordination. For sulfonate systems, the EPR results are in line with a weaker interaction of Cu(II) with the nitrogen sites and a stronger interaction with the oxygen (SO3(-)) groups with respect to the interactions measured by EPR for carboxylate systems.

A complex system, based on a dendrimer solubilized in the aqueous core of water-in-oil microemulsion, may combine the advantages of both dendrimers and microemulsions to provide better control of drug release. We report for the first time the use of EPR technique to determine the effect of solubilized dendrimer on the structure of the microemulsion. The solubilized poly(propyleneimine) (PPI-G2) interacts with sodium bis(2-ethylhexyl) sulfosuccinate (AOT). EPR analysis provided information on polarity, microviscosity, and molecular order of the systems. Polarity and microviscosity increased from unloaded water-in-oil microemulsion to the system loaded with 0.2 wt % PPI-G2, but remained unchanged with higher PPI-G2 loads. The degree of order also increased with 0.2 wt % PPI-G2 with only minor additional increase with larger quantities (25 wt %) of PPI-G2. Variations in pH only slightly affected the structure of microemulsion in the absence and presence of the loaded dendrimers. Aliphatic oils with longer lipophilic chains enhanced the structural order of the microemulsion. On increasing water content, polarity and degree of order increased. PPI-G2 dendrimer in small loads is attracted by the negatively charged AOT and thus intercalates in the interface of the droplets. Yet, at higher PPI-G2 loads, the excess molecules are solubilized in the water core.

Micro- and macrostructural behaviors of three different lyotropic liquid crystals (LLCs) loaded with a dendrimer, namely second generation poly(propylene imine) (PPI-G2), were studied by means of rheology and electron paramagnetic resonance (EPR). The three mesophases were L(α), Q(224), and H(II) composed of glycerol monooleate (GMO) and water-PPI-G2 solution (and d-α-tocopherol (vitamin E) in the case of H(II)). We characterized the impact of PPI-G2 interactions with the components of the host mesophases on their structural characteristics on different length scales. The incorporation of PPI-G2 within the L(α) and H(II) systems induced the formation of more elastic hexagonal systems with a "solidlike" behavior, while in the Q(224) system a different trend with a "liquidlike" behavior was observed. As a result, the dendrimer induced a remarkable change in both the structural and viscoelastic properties of the systems. Hence, the microenvironment in the interface region within the systems was monitored by computer-aided EPR using 5-doxylstearic acid (5-DSA) as a pH-dependent probe. The microviscosity (τ) and order (S) of systems were found to be sensitive to the PPI-G2 presence: when PPI-G2 concentration increased, τ and S increased in both the L(α) and Q(224) systems. In the H(II) systems two trends were observed, reflecting a decrease in τ and S up to 10 wt % PPI-G2 and subsequently their increase at higher dendrimer concentrations. It was assessed that PPI-G2 interacted strongly with the GMO hydroxyl groups in the L(α) phase, with the water molecules in the Q(224) systems. In the H(II) mesophase strong interactions with both the water and GMO hydroxyl molecules were detected.

The aggregation behavior of the amyloid peptide Aβ(1-28) and the prion peptide PrP(185-208) - both responsible for neurodegenerative disorders - was analyzed in the absence and in the presence of poly(propylene imine) (PPI) dendrimers at generation 5 (G5) with a dense shell of maltose and maltotriose units. Thioflavin T (ThT) fluorescence assay and circular dichroism (CD) experiments indicated that fibril formation is enhanced at low dendrimer concentration, while it is prevented at relatively high dendrimer concentrations. Computer aided EPR analysis by means of the selected spin probe 4-octyl-dimethylammonium,2,2,6,6-tetramethyl-piperidine-1-oxyl bromide (CAT8) further demonstrated this behavior, but also provided detailed information on the mechanism of fibril formation and on the different behavior of the differently decorated dendrimers. The CAT8 radicals were progressively trapped at the peptide interphase when peptide aggregates were formed, also monitoring pre-fibrillar structures. At later time, a phase separation of the CAT8 radicals monitors the formation of further supramolecular structures where the probes become squeezed among fibrillar aggregates. The addition of small amounts of dendrimers promotes the formation of peptide fibrils breaking them and providing a larger amount of ends that serve as sites of replications. Conversely, a high amount of dendrimers allows the peptides to well separate from each other such preventing their aggregation. EPR results also indicate that the perturbation played by PPI(G5)-Maltose are more effective onto PrP(185-208) than onto Aβ(1-28), while PPI(G5)-Maltotriose is less effective towards PrP(185-208) in both promoting aggregation and preventing it by changing the dendrimer concentration. These results provide useful information about the mechanism and interactions which regulate the ability of macromolecules like the dendrimers to favor, prevent or cure neurodegenerative diseases.

We describe here the use of anionic carbosilane dendrimers to obtain new copper complexes. UV-Vis and a computer aided analysis of the EPR spectra provided information about the coordination modes of copper depending on the nature of the dendrimer and about the geometry and structure of the complexes in solution. Some of these metallo-dendrimers have been tested "in vitro" as antiviral compounds in the inhibition of HIV infection in pre and post-infection treatment.

A key pathological event of prion and Alzheimer diseases is the formation of prion and amyloid plaques generated by peptide aggregation in the form of fibrils. Dendrimers have revealed their ability to prevent fibril formation and therefore cure neurodegenerative diseases. To provide information about the kinetics and the mechanism of peptide fibril formation and about the ability of the dendrimers to prevent peptide aggregation, we performed a computer-aided EPR analysis of the selected nitroxide spin probe 4-octyl-dimethylammonium,2,2,6,6-tetramethyl-piperidine-1-oxyl bromide (CAT8) in water solutions of the β-amyloid peptide Aβ 1-28 and the prion peptide PrP 185-208, which contain the fibril nucleation sites, in the absence and in the presence of phosphorus dendrimers. After a careful selection of the experimental conditions that allow aggregation to occur and to be monitored by EPR analysis over time, it was found that the Aβ 1-28 fibrils formed in 220 min at 0.5 mM peptide, 0.05 mM CAT8, 0.04 mg/mL heparin, and pH = 5. As a consequence, the interacting sites available for cooperative interactions with CAT8 were engaged in the peptide-peptide interactions and a fraction of the probe was extracted in the fluid fibril/water interphase, while another fraction was trapped at the peptide/peptide interphase, showing a decrease in mobility. Conversely, in the presence of the dendrimer (at the selected, after several trials, peptide/dendrimer molar ratio = 50), due to dipole-dipole interactions with peptide monomers, the probe remained at the dendrimer/peptide interphase and the spectral parameters negligibly changed over time. A fraction of probes inserted in PrP 185-208 low-packed aggregates and monitored their fast formation after 90 min. However, the binding organization of the prion peptide negligibly changed upon aggregation in comparison to Aβ 1-28. It is proposed that dendrimers mainly interfere in the lag (nucleation) phase of the prion peptide.

Dendrimers are a new class of nanotechnological polymers suitable for drug targeting, microarray systems or detoxication. The present study is devoted to a detailed analysis of binding between PAMAM dendrimers and bovine serum albumin (fatty acid free or loaded with oleic, linoleic, oleic+linoleic or oleic+linoleic+arachodonic acids) by measuring zeta-potential, fluorescence quenching, fluorescence anisotropy and electron paramagnetic resonance. Addition of PAMAM G2 and G6 dendrimers to protein solutions resulted in attachment to the protein molecule. The PAMAM dendrimers also competed with BSA for fatty acids if two or three fatty acids were loaded per protein. This can lead to the extraction of fatty acids from BSA to the PAMAM dendrimer.

Spin-probe and spin-label techniques were used to study the interactions of the Abeta 1-28 peptide involved in Alzheimer disease and the PrP 106-126 peptide suspected to be preferentially involved in spongiform encephalopathies with three different types of dendrimers. A computer-aided EPR analysis of a positively charged and a neutral spin probe was performed by comparing the pure dendrimer and peptide systems with the dendrimer-peptide ones. Also spin-labeled PAMAM dendrimers were used to test the interactions. The results show the interactions between dendrimer and peptide monomer to be stronger for Abeta 1-28 than for PrP 106-126. PAMAM dendrimers perturb the aggregation of the peptides more than PPI dendrimers do.