Metabolomics reveals differential mechanisms of toxicity of hyperbranched poly(ethyleneimine)-derived nanoparticles to the soil-borne fungus Verticillium dahliae Kleb.

There is a consensus on the urge for the discovery and assessment of alternative, improved sources of bioactivity that could be developed as plant protection products (PPPs), in order to combat issues that the agrochemical sector is facing. Based on the recent advances in nanotechnology, nanoparticles seem to have a great potential towards the development of the next generation nano-PPPs used as active ingredients (a.i.) per se or as nanocarriers in their formulation. Nonetheless, information on their mode(s)-of-action (MoA) and mechanisms of toxicity is yet largely unknown, representing a bottleneck in their further assessment and development. Therefore, we have undertaken the task to assess the fungitoxicity of hyperbranched poly(ethyleneimine) (HPEI), quaternized hyperbranched poly(ethyleneimine) (QPEI), and guanidinylated hyperbranched poly(ethyleneimine) (GPEI) nanoparticles to the soil-born plant pathogenic fungus Verticillium dahliae Kleb, and dissect their effects on its metabolism applying GC/EI/MS metabolomics. Results revealed that functionalization of HPEI nanoparticles with guanidinium end groups (GPEI) increases their toxicity to V. dahliae, while functionalization with quaternary ammonium end groups (QPEI) decreases it. The treatments with the nanoparticles affected the chemical homeostasis of the fungus, altering substantially its amino acid pool, energy production, and fatty acid content, causing additionally oxidative and osmotic stresses. To the best of our knowledge, this is the first report on the comparative toxicity of HPEI, QPEI, and GPEI to filamentous fungi applying metabolomics. The findings could be exploited in the study of the quantitative structure-activity relationship (QSAR) of HPEI-derived nanoparticles and their further development as nano-PPPs.

Functionalized Hyperbranched Polyethylenimines as Thermosensitive Drug Delivery Nanocarriers with Controlled Transition Temperatures.

The low critical solution temperature phase transition (Tc) that is exhibited by thermosensitive polymers is strongly dependent on polymer concentration, pH, ionic strength, as well as the presence of specific molecules or ions in solution. Therefore, polymers with Tc values above 37 °C that are useful for hyperthermia therapy are not readily available. In the present study, temperature-sensitive hyperbranched polyethylenimine derivatives were developed through stepwise functionalization with isobutylamide groups. Although factors such as the concentration of polymer, sodium chloride, phosphate ions, and pH considerably affect the transition temperature, it was possible to obtain a hyperbranched derivative having the required Tc (38-39 °C) for the given aqueous medium required in cell experiments through careful selection of the degree of substitution. This thermosensitive derivative can encapsulate doxorubicin (DOX), a well-known anticancer agent, and was further studied as a temperature-triggered drug delivery system. Although the polymeric carrier showed no notable toxicity at temperatures either below or above the transition temperature, the thermoresponsive drug-loaded formulation exhibited increased DOX cellular uptake and improved in vitro cytotoxicity at 40 °C.

Male infertility-linked point mutation reveals a vital binding role for the C2 domain of sperm PLCζ.

Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus that evokes intracellular calcium (Ca2+) oscillations that are essential for the initiation of egg activation during mammalian fertilisation. A recent genetic study reported a male infertility case that was directly associated with a point mutation in the PLCζ C2 domain, where an isoleucine residue had been substituted with a phenylalanine (I489F). Here, we have analysed the effect of this mutation on the in vivo Ca2+ oscillation-inducing activity and the in vitro biochemical properties of human PLCζ. Microinjection of cRNA or recombinant protein corresponding to PLCζI489F mutant at physiological concentrations completely failed to cause Ca2+ oscillations and trigger development. However, this infertile phenotype could be effectively rescued by microinjection of relatively high (non-physiological) amounts of recombinant mutant PLCζI489F protein, leading to Ca2+ oscillations and egg activation. Our in vitro biochemical analysis suggested that the PLCζI489F mutant displayed similar enzymatic properties, but dramatically reduced binding to PI(3)P and PI(5)P-containing liposomes compared with wild-type PLCζ. Our findings highlight the importance of PLCζ at fertilisation and the vital role of the C2 domain in PLCζ function, possibly due to its novel binding characteristics.

Drug Delivery Systems Based on Hydroxyethyl Starch.

The advantageous biological properties of hydroxyethyl starch (HES) triggered research interest toward the design and synthesis of drug delivery systems (DDSs) based on this polysaccharide. Convenient reaction schemes, including one-step reactions, led to the synthesis of HES conjugates with selected anticancer molecules or therapeutic proteins. Nanocapsules and hydrogels based on HES were also prepared and studied as prospective drug delivery systems. Formulations originating from these drug conjugates and also from nanocapsules and hydrogels loaded with drugs were characterized, highlighting the extension of their half-life in plasma, which is a critical property as far as their efficacy is concerned. Results obtained in vitro and in vivo proved promising, justifying the undertaking of additional experiments with such systems, including their multifunctionalization. The promising formulations that are discussed in this Topical Review is expected to further increase interest in applying HES for molecular constructing novel DDSs with enhanced efficacy, which may, in the future, find clinical applications.

An in vitro and in vivo study of peptide-functionalized nanoparticles for brain targeting: The importance of selective blood-brain barrier uptake.

Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood-brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial barriers shows great potential as a therapeutic strategy in a wide variety of diseases. Functionalizing nanoparticles with peptides allows for more efficient targeting to specific organs. We have evaluated the hemocompatibilty, cytotoxicity, endothelial uptake, efficacy of delivery and safety of liposome, hyperbranched polyester, poly(glycidol) and acrylamide-based nanoparticles functionalized with peptides targeting brain endothelial receptors, in vitro and in vivo. We used an ELISA-based method for the detection of nanoparticles in biological fluids, investigating the blood clearance rate and in vivo biodistribution of labeled nanoparticles in the brain after intravenous injection in Wistar rats. Herein, we provide a detailed report of in vitro and in vivo observations.

Essential Role of the EF-hand Domain in Targeting Sperm Phospholipase Cζ to Membrane Phosphatidylinositol 4,5-Bisphosphate (PIP2).

Sperm-specific phospholipase C-ζ (PLCζ) is widely considered to be the physiological stimulus that triggers intracellular Ca(2+) oscillations and egg activation during mammalian fertilization. Although PLCζ is structurally similar to PLCδ1, it lacks a pleckstrin homology domain, and it remains unclear how PLCζ targets its phosphatidylinositol 4,5-bisphosphate (PIP2) membrane substrate. Recently, the PLCδ1 EF-hand domain was shown to bind to anionic phospholipids through a number of cationic residues, suggesting a potential mechanism for how PLCs might interact with their target membranes. Those critical cationic EF-hand residues in PLCδ1 are notably conserved in PLCζ. We investigated the potential role of these conserved cationic residues in PLCζ by generating a series of mutants that sequentially neutralized three positively charged residues (Lys-49, Lys-53, and Arg-57) within the mouse PLCζ EF-hand domain. Microinjection of the PLCζ EF-hand mutants into mouse eggs enabled their Ca(2+) oscillation inducing activities to be compared with wild-type PLCζ. Furthermore, the mutant proteins were purified, and the in vitro PIP2 hydrolysis and binding properties were monitored. Our analysis suggests that PLCζ binds significantly to PIP2, but not to phosphatidic acid or phosphatidylserine, and that sequential reduction of the net positive charge within the first EF-hand domain of PLCζ significantly alters in vivo Ca(2+) oscillation inducing activity and in vitro interaction with PIP2 without affecting its Ca(2+) sensitivity. Our findings are consistent with theoretical predictions provided by a mathematical model that links oocyte Ca(2+) frequency and the binding ability of different PLCζ mutants to PIP2. Moreover, a PLCζ mutant with mutations in the cationic residues within the first EF-hand domain and the XY linker region dramatically reduces the binding of PLCζ to PIP2, leading to complete abolishment of its Ca(2+) oscillation inducing activity.

Triphenylphosphonium Decorated Liposomes and Dendritic Polymers: Prospective Second Generation Drug Delivery Systems for Targeting Mitochondria.

Targeting specific intracellular organelles has been a biological process of significant interest. Specifically, for mitochondrial targeting, conventional liposomal and dendritic polymer nanoparticles were selected to be presented in this miniperspective. Both types of nanoparticles were decorated on their external surface with triphenylphosphonium cation (TPP), a well-known and effective mitochondrial targeting moiety. Due to their advantageous specificity toward mitochondria, these nanoparticles may be considered as prospective second generation drug delivery systems (DDSs). Functionalized liposomal and dendritic nanoparticles are conveniently prepared, and although they encounter several hurdles on their route from the extracellular environment to the interior of mitochondria, they manage to be accumulated inside them in experiments in vitro. Therefore, the TPP-functionalized nanoparticles presented in this miniperspective can prove effective DDSs and efforts should be continued to obtain results that will trigger further studies including clinical studies, hopefully leading to effective drugs for mitochondrial diseases. In fact, since these DDSs enter and act at the site where the dysfunction exists, a new medicine subspecialty is emerging, the so-called mitochondrial medicine.

Intercalation study of low-molecular-weight hyperbranched polyethyleneimine into graphite oxide.

We report for the first time the intercalation of low-molecular-weight hyperbranched polyethyleneimine (PEI) into graphite oxide (GO) for the facile, bulk synthesis of novel graphene-based hybrid (GO-PEI) materials exhibiting tailored interlayer galleries. The size of the intercalant as well as the loading in GO were systematically investigated to determine their contribution to the basal spacing of the resulting materials. Powder X-ray diffraction measurements demonstrated the generation of constrained hybrid systems along the c axis that exhibit considerably increased interlayer distances compared with the starting, pristine GO. The results of X-ray photoelectron and FTIR studies are consistent with a "grafting-to" process of the intercalated PEI with the oxygen functional groups present along the GO framework. Furthermore, it was found that a great number of the nitrogen-containing groups in PEI still remain available within the newly formed, confined micro-environment of intercalated GO galleries. The increased surface area of the GO-PEI hybrids in conjunction with the remaining available active groups of intercalated PEI render the synthesised hybrids very attractive candidates as nanostructured adsorbents.

Carbon Nanodisks Decorated with Guanidinylated Hyperbranched Polyethyleneimine Derivatives as Efficient Antibacterial Agents.

Non-toxic carbon-based hybrid nanomaterials based on carbon nanodisks were synthesized and assessed as novel antibacterial agents. Specifically, acid-treated carbon nanodisks (oxCNDs), as a safe alternative material to graphene oxide, interacted through covalent and non-covalent bonding with guanidinylated hyperbranched polyethyleneimine derivatives (GPEI5K and GPEI25K), affording the oxCNDs@GPEI5K and oxCNDs@GPEI25K hybrids. Their physico-chemical characterization confirmed the successful and homogenous attachment of GPEIs on the surface of oxCNDs, which, due to the presence of guanidinium groups, offered them improved aqueous stability. Moreover, the antibacterial activity of oxCNDs@GPEIs was evaluated against Gram-negative E. coli and Gram-positive S. aureus bacteria. It was found that both hybrids exhibited enhanced antibacterial activity, with oxCNDs@GPEI5K being more active than oxCNDs@GPEI25K. Their MIC and MBC values were found to be much lower than those of oxCNDs, revealing that the GPEI attachment endowed the hybrids with enhanced antibacterial properties. These improved properties were attributed to the polycationic character of the oxCNDs@GPEIs, which enables effective interaction with the bacterial cytoplasmic membrane and cell walls, leading to cell envelope damage, and eventually cell lysis. Finally, oxCNDs@GPEIs showed minimal cytotoxicity on mammalian cells, indicating that these hybrid nanomaterials have great potential to be used as safe and efficient antibacterial agents.

Chimeras of sperm PLCζ reveal disparate protein domain functions in the generation of intracellular Ca2+ oscillations in mammalian eggs at fertilization.

Phospholipase C-zeta (PLCζ) is a sperm-specific protein believed to cause Ca(2+) oscillations and egg activation during mammalian fertilization. PLCζ is very similar to the somatic PLCδ1 isoform but is far more potent in mobilizing Ca(2+) in eggs. To investigate how discrete protein domains contribute to Ca(2+) release, we assessed the function of a series of PLCζ/PLCδ1 chimeras. We examined their ability to cause Ca(2+) oscillations in mouse eggs, enzymatic properties using in vitro phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and their binding to PIP2 and PI(3)P with a liposome interaction assay. Most chimeras hydrolyzed PIP2 with no major differences in Ca(2+) sensitivity and enzyme kinetics. Insertion of a PH domain or replacement of the PLCζ EF hands domain had no deleterious effect on Ca(2+) oscillations. In contrast, replacement of either XY-linker or C2 domain of PLCζ completely abolished Ca(2+) releasing activity. Notably, chimeras containing the PLCζ XY-linker bound to PIP2-containing liposomes, while chimeras containing the PLCζ C2 domain exhibited PI(3)P binding. Our data suggest that the EF hands are not solely responsible for the nanomolar Ca(2+) sensitivity of PLCζ and that membrane PIP2 binding involves the C2 domain and XY-linker of PLCζ. To investigate the relationship between PLC enzymatic properties and Ca(2+) oscillations in eggs, we have developed a mathematical model that incorporates Ca(2+)-dependent InsP3 generation by the PLC chimeras and their levels of intracellular expression. These numerical simulations can for the first time predict the empirical variability in onset and frequency of Ca(2+) oscillatory activity associated with specific PLC variants.

Mitochondrial delivery of doxorubicin by triphenylphosphonium-functionalized hyperbranched nanocarriers results in rapid and severe cytotoxicity.

PURPOSE: To develop a novel hyperbranched polymer-based nanocarrier for efficient drug delivery to cell mitochondria. Also to study for the first time the cytotoxic effect of doxorubicin via mitochondria-specific delivery system. METHODS: We introduced alkyltriphenylphosphonium groups (TPP) to a poly(ethylene imine) hyperbranched polymer (PEI). We harnessed the hydrophobic assembly of these alkylTPP functionalized PEI molecules into ~100 nm diameter nanoparticles (PEI-TPP) and further encapsulated the chemotherapy agent doxorubicin (DOX), to produce the mitotropic nanoparticles PEI-TPP-DOX. RESULTS: By administering PEI-TPP-DOX to human prostate carcinoma cells DU145, we found that: (i) PEI-TPP-DOX specifically localized at cell mitochondria as revealed by the inherent DOX fluorescence; (ii) in contrast to the slow apoptotic cell death incurred by DOX over the period of days at micromolar concentrations, PEI-TPP-DOX triggered rapid and severe cytotoxicity within few hours of incubation and at submicromolar incubation concentrations. This cytotoxicity was mainly found to be of a necrotic nature, not precluding autophagy related death pathways to a smaller extent. CONCLUSIONS: We have elaborated a versatile mitotropic nanocarrier; furthermore, using this platform, we have developed a mitochondrial-doxorubicin formulation with exceptional cytocidal properties, even in nanomolar concentrations.

Carbon Dots-Biomembrane Interactions and Their Implications for Cellular Drug Delivery.

The effect of carbon dots (CDs) on a model blayer membrane was studied as a means of comprehending their ability to affect cell membranes. Initially, the interaction of N-doped carbon dots with a biophysical liposomal cell membrane model was investigated by dynamic light scattering, z-potential, temperature-modulated differential scanning calorimetry, and membrane permeability. CDs with a slightly positive charge interacted with the surface of the negative-charged liposomes and evidence indicated that the association of CDs with the membrane affects the structural and thermodynamic properties of the bilayer; most importantly, it enhances the bilayer's permeability against doxorubicin, a well-known anticancer drug. The results, like those of similar studies that surveyed the interaction of proteins with lipid membranes, suggest that carbon dots are partially embedded in the bilayer. In vitro experiments employing breast cancer cell lines and human healthy dermal cells corroborated the findings, as it was shown that the presence of CDs in the culture medium selectively enhanced cell internalization of doxorubicin and, subsequently, increased its cytotoxicity, acting as a drug sensitizer.

A Triphenylphosphonium-Functionalized Delivery System for an ATM Kinase Inhibitor That Ameliorates Doxorubicin Resistance in Breast Carcinoma Mammospheres.

The enzyme ataxia-telangiectasia mutated (ATM) kinase is a pluripotent signaling mediator which activates cellular responses to genotoxic and metabolic stress. It has been shown that ATM enables the growth of mammalian adenocarcinoma stem cells, and therefore the potential benefits in cancer chemotherapy of a number of ATM inhibitors, such as KU-55933 (KU), are currently being investigated. We assayed the effects of utilizing a triphenylphosphonium-functionalized nanocarrier delivery system for KU on breast cancer cells grown either as a monolayer or in three-dimensional mammospheres. We observed that the encapsulated KU was effective against chemotherapy-resistant mammospheres of breast cancer cells, while having comparably lower cytotoxicity against adherent cells grown as monolayers. We also noted that the encapsulated KU sensitized the mammospheres to the anthracycline drug doxorubicin significantly, while having only a weak effect on adherent breast cancer cells. Our results suggest that triphenylphosphonium-functionalized drug delivery systems that contain encapsulated KU, or compounds with a similar impact, are a useful addition to chemotherapeutic treatment schemes that target proliferating cancers.

Co-assembled MoS2-TiO2 Inverse Opal Photocatalysts for Visible Light-Activated Pharmaceutical Photodegradation.

Heterostructured photocatalytic materials in the form of photonic crystals have been attracting attention for their unique light harvesting ability that can be ideally combined with judicious compositional modifications toward the development of visible light-activated (VLA) photonic catalysts, though practical environmental applications, such as the degradation of pharmaceutical emerging contaminants, have been rarely reported. Herein, heterostructured MoS2-TiO2 inverse opal films are introduced as highly active immobilized photocatalysts for the VLA degradation of tetracycline and ciprofloxacin broad-spectrum antibiotics as well as salicylic acid. A single-step co-assembly method was implemented for the challenging incorporation of MoS2 nanosheets into the nanocrystalline inverse opal walls. Compositional tuning and photonic band gap engineering of the MoS2-TiO2 photonic films showed that integration of low amounts of MoS2 nanosheets in the inverse opal framework maintains intact the periodic macropore structure and enhances the available surface area, resulting in efficient VLA antibiotic degradation far beyond the performance of benchmark TiO2 films. The combination of broadband MoS2 visible light absorption and photonic-assisted light trapping together with the enhanced charge separation that enables the generation of reactive oxygen species via firm interfacial coupling between MoS2 nanosheets and TiO2 nanoparticles is concluded as a competent approach for pharmaceutical abatement in water bodies.

Preparation of multicompartment lipid-based systems based on vesicle interactions.

Various strategies for constructing artificial multicompartment vesicular systems, which primitively mimic the structure of eukaryotic cells, are presented. These model systems are appropriate for addressing several issues such as the understanding of cell processes, the development of nanoreactors and novel multicompartment delivery systems for specific drug applications, the transport through bilayer membranes, and also hypothesizing on the evolution of eukaryotic cells as originating from the symbiotic association of prokaryotes.

Antibiotic-Loaded Hyperbranched Polyester Embedded into Peptide-Enriched Silk Fibroin for the Treatment of Orthopedic or Dental Infections.

The development of innovative osteoconductive matrices, which are enriched with antibiotic delivery nanosystems, has the invaluable potential to achieve both local contaminant eradication and the osseointegration of implanted devices. With the aim of producing safe, bioactive materials that have osteoconductive and antibacterial properties, novel, antibiotic-loaded, functionalized nanoparticles (AFN)-based on carboxylic acid functionalized hyperbranched aliphatic polyester (CHAP) that can be integrated into peptide-enriched silk fibroin (PSF) matrices with osteoconductive properties-were successfully synthesized. The obtained AFNPSF sponges were first physico-chemically characterized and then tested in vitro against eukaryotic cells and bacteria involved in orthopedic or oral infections. The biocompatibility and microbiological tests confirmed the promising characteristics of the AFN-PSF products for both orthopedic and dental applications. These preliminary results encourage the establishment of AFN-PSF-based preventative strategies in the fight against implant-related infections.

Vancomycin-nanofunctionalized peptide-enriched silk fibroin to prevent methicillin-resistant Staphylococcus epidermidis-induced femoral nonunions in rats.

Introduction: Implant-related infections and infected fractures are significant burdens in orthopedics. Staphylococcus epidermidis is one of the main causes of bone infections related to biofilm formation upon implants. Current antibiotic prophylaxis/therapy is often inadequate to prevent biofilm formation and results in antibiotic resistance. The development of bioactive materials combining antimicrobial and osteoconductive properties offers great potential for the eradication of microorganisms and for the enhancement of bone deposition in the presence of infections. The purpose of this study is to prevent the development of methicillin-resistant S. epidermidis (MRSE)-infected nonunion in a rat model. Methods: To this end, a recently developed in our laboratories bioactive material consisting of antibiotic-loaded nanoparticles based on carboxylic acid functionalized hyperbranched aliphatic polyester (CHAP) that are integrated into peptide-enriched silk fibroin sponges with osteoconductive properties (AFN-PSF) was employed, whose biocompatibility and microbiological tests provided proof of its potential for the treatment of both orthopedic and dental infections. In particular, non-critical femoral fractures fixed with plates and screws were performed in Wistar rats, which were then randomly divided into three groups: 1) the sham control (no infection, no treatment); 2) the control group, infected with MRSE and treated with peptide-enriched silk fibroin sponges incorporating non-drug-loaded functionalized nanoparticles (PSF); 3) the treated group, infected with MRSE and treated with peptide-enriched silk fibroin sponges incorporating vancomycin-loaded functionalized nanoparticles (AFN-PSF). After 8 weeks, bone healing and osteomyelitis were clinically assessed and evaluated by micro-CT, microbiological and histological analyses. Results: The sham group showed no signs of infection and complete bone healing. The PSF group failed to repair the infected fracture, displaying 75% of altered bone healing and severe signs of osteomyelitis. The AFN-PSF treated group reached 70% of fracture healing in the absence of signs of osteomyelitis, such as abscesses in the cortical and intraosseous compartments and bone necrosis with sequestra. Discussion: AFN-PSF sponges have proven effective in preventing the development of infected nonunion in vivo. The proposed nanotechnology for local administration of antibiotics can have a significant impact on patient health in the case of orthopedic infections.

Interaction of vesicles: adhesion, fusion and multicompartment systems formation.

In this review, interactions of selected vesicles, induced either by molecular recognition or by electrostatic interactions, for the simulation of cell-cell and cell-drug interaction processes are discussed. In order of increasing complexity, examples of vesicles adhesion are presented at first, followed by recognition experiments in which fusion takes place. This differentiation in behavior was primarily attributed to the structural features of interacting vesicular pairs primarily affected by concentration and lateral phase separation of the anchored recognizable groups. In certain cases, fusion is accompanied by multicompartmentalization of the obtained aggregates. In connection with the formation of these multicompartment systems, it was proposed that an analogous mechanism could be operating in the evolution of eukaryotes during the symbiosis of prokaryotes.

Engineering Mitochondriotropic Carbon Dots for Targeting Cancer Cells.

Aiming to understand and enhance the capacity of carbon dots (CDs) to transport through cell membranes and target subcellular organelles-in particular, mitochondria-a series of nitrogen-doped CDs were prepared by the one-step microwave-assisted pyrolysis of citric acid and ethylenediamine. Following optimization of the reaction conditions for maximum fluorescence, functionalization at various degrees with alkylated triphenylphosphonium functional groups of two different alkyl chain lengths afforded a series of functionalized CDs that exhibited either lysosome or mitochondria subcellular localization. Further functionalization with rhodamine B enabled enhanced fluorescence imaging capabilities in the visible spectrum and allowed the use of low quantities of CDs in relevant experiments. It was thus possible, by the appropriate selection of the alkyl chain length and degree of functionalization, to attain successful mitochondrial targeting, while preserving non-toxicity and biocompatibility. In vitro cell experiments performed on normal as well as cancer cell lines proved their non-cytotoxic character and imaging potential, even at very low concentrations, by fluorescence microscopy. Precise targeting of mitochondria is feasible with carefully designed CDs that, furthermore, are specifically internalized in cells and cell mitochondria of high transmembrane potential and thus exhibit selective uptake in malignant cells compared to normal cells.