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.

Imaging of nano-hydroxyapatite/chitosan scaffolds using a cone beam computed tomography device on rat calvarial defects with histological verification.

OBJECTIVES: Τhis study aims at determining the ability of cone beam computed tomography (CBCT) to visualize critical-size defects (CSD) created at rat calvaria and filled with 75/25 w/w nano-hydroxyapatite/chitosan (nHAp/CS) scaffolds, prior to their histological investigation. MATERIALS AND METHODS: Thirty adult Sprague Dawley rats, 15 males and 15 females, were used. Two CSD, 5 mm in diameter, were bilaterally trephined in the parietal bone. The right CSD was filled with nHAp/CS scaffold, while the left CSD remained empty, as the control group. Two female rats died post-operatively. Rats were euthanized at 2, 4, and 8 weeks post-surgery. Twenty-eight specimens (15 × 2 × 10 mm) were resected-containing both CSDs-and then scanned using a NewTom VGi CBCT imaging unit (Verona, Italy). The manufacturer's software trace region profile tool (NNT v6.2, Verona, Italy) was used in selected axial slices. The greyscale value (in VGiHU) and the traced/selected region of interest (ROI, in mm2) of those areas were automatically calculated. Subsequently, all specimens were histologically examined. RESULTS: An increased VGiHU (P = 0.000), was observed in the experimental group relative to the control group. The ROI of CSD (in mm2) was significantly reduced (P = 0.001) from the fourth to the eighth week in both groups. No statistically significant difference between male and female rats (P = 0.188) was observed with respect to VGiHU. CONCLUSIONS: The nHAp/CS scaffolds are easily visualized using a particular high-resolution CBCT device. CLINICAL RELEVANCE: Both the CBCT measurements and also the histological results suggest that the nHAp/CS scaffold presence contributes to new bone formation in rat calvarial CSD.

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.

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.

Collagen from the Marine Sponges Axinella cannabina and Suberites carnosus: Isolation and Morphological, Biochemical, and Biophysical Characterization.

In search of alternative and safer sources of collagen for biomedical applications, the marine demosponges Axinella cannabina and Suberites carnosus, collected from the Aegean and the Ionian Seas, respectively, were comparatively studied for their insoluble collagen, intercellular collagen, and spongin-like collagen content. The isolated collagenous materials were morphologically, physicochemically, and biophysically characterized. Using scanning electron microscopy and transmission electron microscopy the fibrous morphology of the isolated collagens was confirmed, whereas the amino acid analysis, in conjunction with infrared spectroscopy studies, verified the characteristic for the collagen amino acid profile and its secondary structure. Furthermore, the isoelectric point and thermal behavior were determined by titration and differential scanning calorimetry, in combination with circular dichroism spectroscopic studies, respectively.

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.

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.

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.

The effect of nano-hydroxyapatite/chitosan scaffolds on rat calvarial defects for bone regeneration.

BACKGROUND: This study aims at determining the biological effect of 75/25 w/w nano-hydroxyapatite/chitosan (nHAp/CS) scaffolds on bone regeneration, in terms of fraction of bone regeneration (FBR), total number of osteocytes (Ost), and osteocyte cell density (CD), as well as its biodegradability. METHODS: Two critical-size defects (CSDs) were bilaterally trephined in the parietal bone of 36 adult Sprague-Dawley rats (18 males and 18 females); the left remained empty (group A), while the right CSD was filled with nHAp/CS scaffold (group B). Two female rats died postoperatively. Twelve, 11, and 11 rats were euthanized at 2, 4, and 8 weeks post-surgery, respectively. Subsequently, 34 specimens were resected containing both CSDs. Histological and histomorphometric analyses were performed to determine the FBR, calculated as [the sum of areas of newly formed bone in lateral and central regions of interest (ROIs)]/area of the original defect, as well as the Ost and the CD (Ost/mm2) in each ROI of both groups (A and B). Moreover, biodegradability of the nHAp/CS scaffolds was estimated via the surface area of the biomaterial (BmA) in the 2nd, 4th, and 8th week post-surgery. RESULTS: The FBR of group B increased significantly from 2nd to 8th week compared to group A (P = 0.009). Both the mean CD and the mean Ost values of group B increased compared to group A (P = 0.004 and P < 0.05 respectively). Moreover, the mean value of BmA decreased from 2nd to 8th week (P = 0.001). CONCLUSIONS: Based on histological and histomorphometric results, we support that 75/25 w/w nHAp/CS scaffolds provide an effective space for new bone formation.

Synthesis and characterization of multifunctional hyperbranched polyesters as prospective contrast agents for targeted MRI.

Based on a commercially available hyperbranched aliphatic polyester, novel multifunctional gadolinium complexes were prepared bearing protective PEG chains, a folate targeting ligand and EDTA or DTPA chelate moieties. Their relatively high water relaxivity values coupled with biodegradability of the hyperbranched scaffold, folate receptor specificity render these non-toxic dendritic polymers promising candidates for MRI applications.

Targeting breast cancer stem-like cells using chloroquine encapsulated by a triphenylphosphonium-functionalized hyperbranched polymer.

Cancer stem cells (CSCs) have garnered increasing attention over the past decade, as they are believed to play a crucial role in tumor progression and drug resistance. Accumulating evidence provides insight into the function of autophagy in maintenance and survival of CSCs. Here, we studied the impact of a mitochondriotropic triphenylphosphonium-functionalized dendrimeric nanocarrier on cultured breast cancer cell lines, grown either as adherent cells or as mammospheres that mimic a stem-like phenotype. The nanocarrier manifested a substantial cytotoxicity both alone as well as after encapsulation of chloroquine, a well-known autophagy inhibitor. The cytotoxic effects of the nanocarrier could be ascribed to interference with mitochondrial function. Importantly, mammospheres were selectively sensitive to encapsulated chloroquine and this depends on the expression of the gene encoding ATM kinase. Ataxia-telangiectasia mutated (ATM) kinase is an enzyme that functions as an essential signaling mediator that enables growth of cancer stem cells through the regulation of autophagy. We noted that this ATM-dependent sensitivity of mammospheres to encapsulated chloroquine was independent of the status of the tumor suppressor gene p53. Our study suggests that breast cancer stem cells, as they are modeled by mammospheres, are sensitive to encapsulated chloroquine, depending on the expression of the ATM kinase, which is thereby characterized as a potential biomarker for sensitivity to this type of treatment.

A combination drug delivery system employing thermosensitive liposomes for enhanced cell penetration and improved in vitro efficacy.

Drug-loaded thermosensitive liposomes are investigated as drug delivery systems in combination with local mild hyperthermia therapy due to their capacity to release their cargo at a specific temperature range (40-42 °C). Additional benefit can be achieved by the development of such systems that combine two different anticancer drugs, have cell penetration properties and, when heated, release their drug payload in a controlled fashion. To this end, liposomes were developed incorporating at low concentration (5 mol%) a number of monoalkylether phosphatidylcholine lipids, encompassing the platelet activating factor, PAF, and its analogues that induce thermoresponsiveness and have anticancer biological activity. These thermoresponsive liposomes were efficiently (>90%) loaded with doxorubicin (DOX), and their thermal properties, stability and drug release were investigated both at 37 ◦C and at elevated temperatures. In vitro studies of the most advantageous liposomal formulation containing the methylated PAF derivative (methyl-PAF, edelfosine), an established antitumor agent, were performed on human prostate cancer cell lines. This system exhibits controlled release of DOX at 40-42 °C, enhanced cell uptake due to the presence of methyl-PAF, and improved cell viability inhibition due to the combined action of both medications.

Structural features of interacting complementary liposomes promoting formation of multicompartment structures.

The structural features of complementary liposomes and factors favoring formation of multicompartment systems are investigated. Specifically, liposomal formulations consisting of PEGylated unilamellar liposomes with guanidinium moieties located at the distal end of polyethylene glycol (PEG) chains interact with complementary multilamellar liposomes bearing phosphate moieties. Furthermore, the number of PEG chains attached to the unilamellar interface of the liposomes is enhanced by incorporating PEGylated cholesterol in their bilayer. While molecular recognition of the liposomes is the driving force for initiating multicompartmentalization, it is the enhanced PEGylation at the liposomal interface that synergistically promotes fusion resulting in large and well-formed multicompartment systems. A mechanism is proposed according to which initial adhesion of the liposomes, followed by reorganization of their membrane lipids, leads to giant bilayer aggregates incorporating large liposomes.