Cellulose Nanocrystal-Based Emulsion of Thyme Essential Oil: Preparation and Characterisation as Sustainable Crop Protection Tool.

Essential oil-based pesticides, which contain antimicrobial and antioxidant molecules, have potential for use in sustainable agriculture. However, these compounds have limitations such as volatility, poor water solubility, and phytotoxicity. Nanoencapsulation, through processes like micro- and nanoemulsions, can enhance the stability and bioactivity of essential oils. In this study, thyme essential oil from supercritical carbon dioxide extraction was selected as a sustainable antimicrobial tool and nanoencapsulated in an oil-in-water emulsion system. The investigated protocol provided high-speed homogenisation in the presence of cellulose nanocrystals as stabilisers and calcium chloride as an ionic crosslinking agent. Thyme essential oil was characterised via GC-MS and UV-vis analysis, indicating rich content in phenols. The cellulose nanocrystal/essential oil ratio and calcium chloride concentration were varied to tune the nanoemulsions' physical-chemical stability, which was investigated via UV-vis, direct observation, dynamic light scattering, and Turbiscan analysis. Transmission electron microscopy confirmed the nanosized droplet formation. The nanoemulsion resulting from the addition of crosslinked nanocrystals was very stable over time at room temperature. It was evaluated for the first time on Pseudomonas savastanoi pv. savastanoi, the causal agent of olive knot disease. In vitro tests showed a synergistic effect of the formulation components, and in vivo tests on olive seedlings demonstrated reduced bacterial colonies without any phytotoxic effect. These findings suggest that crosslinked cellulose nanocrystal emulsions can enhance the stability and bioactivity of thyme essential oil, providing a new tool for crop protection.

Synthesis of a light-responsive platinum curcumin complex, chemical and biological investigations and delivery to tumor cells by means of polymeric nanoparticles.

Platinum-based anticancer drugs are common in chemotherapy, but problems such as systemic toxicity and acquired resistance of some tumors hamper their clinical applications and therapeutic efficacy. It is necessary to synthesize Pt-based drugs and explore strategies to reduce side effects and improve pharmacokinetic profiles. Photo-responsive chemotherapeutics have emerged as an alternative strategy against several cancers, as photoactivation offers spatial selectivity and fewer side effects. Here, we combine chemical synthesis and nanotechnology to create a multifunctional platinum drug delivery system based on the novel metal complex [Pt(ppy)(curc)] (ppy = deprotonated 2-phenylpyridine, curc = deprotonated curcumin)] embodying the naturally occurring bioactive molecule, curcumin. The ultrasonication method coupled with the layer-by-layer technology was employed to produce nanocolloids, which demonstrated a good biocompatibility, higher solubility in aqueous solution, stability, large drug loading, and good biological activity in comparison with the free drug. In vitro release experiments revealed that the polymeric nanoformulation is relatively stable under physiological conditions (pH = 7.4 and 37 °C) but sensitive to acidic environments (pH = 5.6 and 37 °C) which would trigger the release of the loaded drug. Our approach modifies the bioavailability of this Pt-based drug increasing its therapeutic action in terms of both cytotoxic and anti-metastasis effects.

Evaluation of the Antitumor Effects of Platinum-Based [Pt(η1-C2H4-OR)(DMSO)(phen)]+ (R = Me, Et) Cationic Organometallic Complexes on Chemoresistant Pancreatic Cancer Cell Lines.

Pancreatic cancer is one of the most lethal malignancies with an increasing incidence and a high mortality rate, due to its rapid progression, invasiveness, and resistance to anticancer therapies. In this work, we evaluated the antiproliferative and antimigratory activities of the two organometallic compounds, [Pt(η1-C2H4-OMe)(DMSO)(phen)]Cl (1) and [Pt(η1-C2H4-OEt)(DMSO)(phen)]Cl (2), on three human pancreatic ductal adenocarcinoma cell lines with different sensitivity to cisplatin (Mia PaCa-2, PANC-1, and YAPC). The two cationic analogues showed superimposable antiproliferative effects on the tested cells, without significant differences depending on alkyl chain length (Me or Et). On the other hand, they demonstrated to be more effective than cisplatin, especially on YAPC cancer cells. For the interesting cytotoxic activity observed on YAPC, further biological assays were performed, on this cancer cell line, to evaluate the apoptotic and antimetastatic properties of the considered platinum compounds (1 and 2). The cytotoxicity of 1 and 2 compounds appeared to be related to their intracellular accumulation, which was much faster than that of cisplatin. Both 1 and 2 compounds significantly induced apoptosis and cell cycle arrest, with a high accumulation of sub-G1 phase cells, compared to cisplatin. Moreover, phenanthroline-containing complexes caused a rapid loss of mitochondria membrane potential, ΔΨM, if compared to cisplatin, probably due to their cationic and lipophilic properties. On 3D tumor spheroids, 1 and 2 significantly reduced migrated area more than cisplatin, confirming an antimetastatic ability.

Low-Intensity Light-Responsive Anticancer Activity of Platinum(II) Complex Nanocolloids on 2D and 3D In Vitro Cancer Cell Model.

This study aimed to evaluate the therapeutic efficacy of low-intensity visible light responsive nanocolloids of a Pt-based drug using a 2D and three-dimensional (3D) in vitro cancer cell model. Biocompatible and biodegradable polymeric nanocolloids, obtained using the ultrasonication method coupled with Layer by Layer technology, were characterized in terms of size (100 ± 20 nm), physical stability, drug loading (78%), and photoactivation through spectroscopy studies. The in vitro biological effects were assessed in terms of efficacy, apoptosis induction, and DNA-Pt adducts formation. Biological experiments were performed both in dark and under visible light irradiation conditions, exploiting the complex photochemical properties. The light-stimuli responsive nanoformulation gave a significant enhancement in drug bioactivity. This allowed us to achieve satisfying results by using nanomolar drug concentration (50 nM), which was ineffective in darkness condition. Furthermore, our nanocolloids were validated in 3D in vitro spheroids using confocal microscopy and cytofluorimetric assay to compare their behavior on culture in 2D monolayers. The obtained results confirmed that these nanocolloids are promising tools for delivering Pt-based drugs.

Enhanced Bioactivity of Pomegranate Peel Extract following Controlled Release from CaCO3 Nanocrystals.

Pomegranate peel extract is rich of interesting bioactive chemicals, principally phenolic compounds, which have shown antimicrobial, anticancer, and antioxidative properties. The aim of this work was to improve extract' bioactivity through the adsorption on calcium carbonate nanocrystals. Nanocrystals revealed as efficient tools for extract adsorption reaching 50% of loading efficiency. Controlled release of the contained metabolites under acidic pH has been found, as it was confirmed by quantitative assay and qualitative study through NMR analysis. Specific functionality of inorganic nanocarriers could be also tuned by biopolymeric coating. The resulting coated nanoformulations showed a great antimicrobial activity against B. cinerea fungus preventing strawberries disease better than a commercial fungicide. Furthermore, nanoformulations demonstrated a good antiproliferative activity in neuroblastoma and breast cancer cells carrying out a higher cytotoxic effect respect to free extract, confirming a crucial role of nanocarriers. Finally, pomegranate peel extract showed a very high radical scavenging ability, equal to ascorbic acid. Antioxidant activity, measured also in intracellular environment, highlighted a protective action of extract-adsorbed nanocrystals twice than free extract, providing a possible application for new nutraceutical formulations.

Visible Light-Activated Water-Soluble Platicur Nanocolloids: Photocytotoxicity and Metabolomics Studies in Cancer Cells.

Nanoparticle-based drug delivery systems for cancer therapy offer a great promising opportunity as they specifically target cancer cells, also increasing the bioavailability of anticancer drugs characterized by low water solubility. Platicur, [Pt(cur) (NH3)2](NO3), is a cis-diamine-platinum(II) complex linked to curcumin. In this work, an ultrasonication method, coupled with layer by layer technology, allows us to obtain highly aqueous stable Platicur nanocolloids of about 100 nm. The visible light-activated Platicur nanocolloids showed an increased drug release and antitumor activity on HeLa cells, with respect to Platicur nanocolloids in darkness. This occurrence could give very interesting insight into selective activation of the nanodelivered Pt(II) complex and possible side-effect lowering. For the first time, the metabolic effects of Platicur nanocolloid photoactivation, in the HeLa cell line, have been investigated using an NMR-based metabolomics approach coupled with statistical multivariate data analysis. The reported results highlight specific metabolic differences between photoactivated and non-photoactivated Platicur NC-treated HeLa cancer cells.

CaCO3 as an Environmentally Friendly Renewable Material for Drug Delivery Systems: Uptake of HSA-CaCO3 Nanocrystals Conjugates in Cancer Cell Lines.

Chemical and biochemical functionalization of nanoparticles (NPs) can lead to an active cellular uptake enhancing their efficacy thanks to the targeted localization in tumors. In the present study calcium carbonate nano-crystals (CCNs), stabilized by an alcohol dehydration method, were successfully modified by grafting human serum albumin (HSA) on the surface to obtain a pure protein corona. Two types of CCNs were used: naked CaCO3 and the (3-aminopropyl)triethoxysilane (APTES) modified CaCO3-NH2. The HSA conjugation with naked CCN and amino-functionalized CCN (CCN-NH2) was established through the investigation of modification in size, zeta potential, and morphology by Transmission Electron Microscopy (TEM). The amount of HSA coating on the CCNs surface was assessed by spectrophotometry. Thermogravimetric analysis (TGA) and Differential scanning calorimetry (DSC) confirmed the grafting of APTES to the surface and successive adsorption of HSA. Furthermore, to evaluate the effect of protein complexation of CCNs on cellular behavior, bioavailability, and biological responses, three human model cancer cell lines, breast cancer (MCF7), cervical cancer (HeLa), and colon carcinoma (Caco-2) were selected to characterize the internalization kinetics, localization, and bio-interaction of the protein-enclosed CCNs. To monitor internalization of the various conjugates, chemical modification with fluorescein-isothiocyanate (FITC) was performed, and their stability over time was measured. Confocal microscopy was used to probe the uptake and confirm localization in the perinuclear region of the cancer cells. Flow cytometry assays confirmed that the bio-functionalization influence cellular uptake and the CCNs behavior depends on both cell line and surface features.

Cell-Penetrating CaCO3 Nanocrystals for Improved Transport of NVP-BEZ235 across Membrane Barrier in T-Cell Lymphoma.

Owing to their nano-sized porous structure, CaCO3 nanocrystals (CaCO3NCs) hold the promise to be utilized as desired materials for encapsulating molecules which demonstrate wide promise in drug delivery. We evaluate the possibility to encapsulate and release NVP-BEZ235, a novel and potent dual PI3K/mTOR inhibitor that is currently in phase I/II clinical trials for advanced solid tumors, from the CaCO3NCs. Its chemical nature shows some intrinsic limitations which induce to administer high doses leading to toxicity; to overcome these problems, here we proposed a strategy to enhance its intracellular penetration and its biological activity. Pristine CaCO3 NCs biocompatibility, cell interactions and internalization in in vitro experiments on T-cell lymphoma line, were studied. Confocal microscopy was used to monitor NCs-cell interactions and cellular uptake. We have further investigated the interaction nature and release mechanism of drug loaded/released within/from the NCs using an alternative approach based on liquid chromatography coupled to mass spectrometry. Our approach provides a good loading efficiency, therefore this drug delivery system was validated for biological activity in T-cell lymphoma: the anti-proliferative test and western blot results are very interesting because the proposed nano-formulation has an efficiency higher than free drug at the same nominal concentration.

Nanostructured polysaccharidic microcapsules for intracellular release of cisplatin.

Carbohydrate polimeric microcapsules were assembled using a LbL approach onto a CaCO3 core. The microcapsules were used to delivery the anticancer drug cisplatin into HeLa and MCF-7 cancer cell lines. Drug encapsulation, measured by ICP spectroscopy, was around 50% of the charging solution. Fluorimetric measurements showed an efficient cellular uptake of polysacchardic microcapsules in both cell lines. The drug-loaded capsules demonstrated a better efficiency against cell viability than the free drug. Specifically, the amount of platinum reaching genomic DNA was measured, showing that encapsulation improves the nuclear delivery of the drug for both cell lines.

Glucose capped silver nanoparticles induce cell cycle arrest in HeLa cells.

This study aims to determine the interaction (uptake and biological effects on cell viability and cell cycle progression) of glucose capped silver nanoparticles (AgNPs-G) on human epithelioid cervix carcinoma (HeLa) cells, in relation to amount, 2×103 or 2×104 NPs/cell, and exposure time, up to 48h. The spherical and well dispersed AgNPs (30±5nm) were obtained by using glucose as reducing agent in a green synthesis method that ensures to stabilize AgNPs avoiding cytotoxic soluble silver ions Ag+ release. HeLa cells take up abundantly and rapidly AgNPs-G resulting toxic to cells in amount and incubation time dependent manner. HeLa cells were arrested at S and G2/M phases of the cell cycle and subG1 population increased when incubated with 2×104 AgNPs-G/cell. Mitotic index decreased accordingly. The dissolution experiments demonstrated that the observed effects were due only to AgNPs-G since glucose capping prevents Ag+ release. The AgNPs-G influence on HeLa cells viability and cell cycle progression suggest that AgNPs-G, alone or in combination with chemotherapeutics, may be exploited for the development of novel antiproliferative treatment in cancer therapy. However, the possible influence of the cell cycle on cellular uptake of AgNPs-G and the mechanism of AgNPs entry in cells need further investigation.

Surface reactivity and in vitro toxicity on human bronchial epithelial cells (BEAS-2B) of nanomaterials intermediates of the production of titania-based composites.

Titanium dioxide (TiO2) nanoparticles (NPs) are manufactured worldwide in large quantities for use in a wide range of applications. Evaluating the hazards associated with TiO2 NPs is crucial as it enables risk assessment related to human and environmental exposure. In this study the in vitro human toxicity of a set of TiO2 NPs modified with acetic, oleic and boric acids were studied in order to assess the hazard in view of a future scale-up of the synthesis. The surface reactivity of the powders under simulated solar illumination and in the dark has been evaluated by means of EPR spectroscopy. Human bronchial epithelial cells (BEAS-2B) have been chosen as a model for lung epithelium. Cytotoxicity has been assessed by measuring the cells membrane integrity by lactate dehydrogenase (LDH) assay, and the inflammatory response evaluated as nitric oxide (NO) and TNF-α production, and oxidative stress measured as intracellular reduced glutathione (GSH) levels, and induced lipoperoxidation. Aeroxide P25 was used for comparison. The results demonstrated a low photoreactivity and toxic effects lower than Aeroxide P25 of the nano-TiO2 powders, probably as a consequence of the presence of acidic moieties at the surface.

Photodynamic activity of thiophene-derived lysosome-specific dyes.

The photodynamic activity occurring through the lysosome photo-damage is effective in terms of triggered synergic effects which can avoid chemo-resistance pathways. The potential photodynamic activity of two fluorescent lysosome-specific probes was studied providing their interaction with human serum albumin, demonstrating their in vitro generation of singlet oxygen and investigating the resulted photo-toxic effect in human cancer cells.

Halloysite clay nanotubes for resveratrol delivery to cancer cells.

Halloysite is natural aluminosilicate clay with hollow tubular structure which allows loading with low soluble drugs using their saturated solutions in organic solvents. Resveratrol, a polyphenol known for having antioxidant and antineoplastic properties, is loaded inside these clay nanotubes lumens. Release time of 48 h is demonstrated. Spectroscopic and ζ-potential measurements are used to study the drug loading/release and for monitoring the nanotube layer-by-layer (LbL) coating with polyelectrolytes for further release control. Resveratrol-loaded clay nanotubes are added to breast cell cultures for toxicity tests. Halloysite functionalization with LbL polyelectrolyte multilayers remarkably decrease nanotube self-toxicity. MTT measurements performed with a neoplastic cell lines model system (MCF-7) as function of the resveratrol-loaded nanotubes concentration and incubation time indicate that drug-loaded halloysite strongly increase of cytotoxicity leading to cell apoptosis.

TGF-beta inihibitor-loaded polyelectrolyte multilayers capsules for sustained targeting of hepatocarcinoma cells.

In this review we will report on recent advanced in polyelectrolyte capsules for targeted drug delivery (eg of growth factor inhibitor) against epatocarcinoma. Degradable polyelectrolyte multilayers capsules (PMCs) are of particular interest for cancer therapy since under physiological conditions they can be enzymatically degraded upon cell interaction. Small bioactive molecules such as TGF-Beta inhibitors can be incorporated inside them. Nano-to-microscale delivery systems can enhance efficacy at single cell level for targeted therapy. Layer-by-layer (LbL) self-assembled capsules are novel carriers maximizing drug administration and improving antimetastatic activity of TGF-Beta inhibitors in Hepatocellular Carcinoma (HCC).

Polyelectrolyte capsules as carriers for growth factor inhibitor delivery to hepatocellular carcinoma.

The efficient internalization of TGF-beta inhibitor-loaded polyelectrolyte capsules and particles is studied in two HCC cell lines. Two polyelectrolyte pairs (biocompatible but not degradable and biodegradable crosslinked with gluteraldehyde) are employed for coating. The capsules are characterized by SEM. LY is successfully loaded inside the core and embedded between polymer layers. MS is used to quantify the loading efficiency by comparing post-loading and core-loading methods, since both coated templates and hollow shells are used as carriers. CLSM confirms dissolution of the pre-formed multilayer upon enzymatic degradation as the method of release, and migration assays demonstrate a higher inhibition efficiency of TGF-beta in tailored biodegradable capsules compared to free LY administration.

Resveratrol downregulates Akt/GSK and ERK signalling pathways in OVCAR-3 ovarian cancer cells.

Phytochemicals constitute a heterogeneous group of substances with an evident role in human health. Their properties on cancer initiation, promotion and progression are well documented. Particular attention is now devoted to better understand the molecular basis of their anticancer action. In the present work, we studied the effect of resveratrol on the ovarian cancer cell line OVCAR-3 by a proteomic approach. Our findings demonstrate that resveratrol down-regulates the protein cyclin D1 and, in a concentration dependent manner, the phosphorylation levels of protein kinase B (Akt) and glycogen synthase kinase-3ß (GSK-3ß). The dephosphorylation of these kinases could be responsible for the decreased cyclin D1 levels observed after treatment. We also showed that resveratrol reduces phosphorylation levels of the extracellular signal-regulated kinase (ERK) 1/2. Chemical inhibitors of phosphatidylinositol 3-kinase (PI3K) and ERK both increased the in vitro therapeutic efficacy of resveratrol. Moreover, resveratrol had an inhibitory effect on the AKT phosphorylation in cultured cells derived from the ascites of ovarian cancer patients and in a panel of human cancer cell lines. Thus, resveratrol shows antitumor activity in human ovarian cancer cell lines targeting signalling pathway involved in cell proliferation and drug-resistance.

Lapatinib/Paclitaxel polyelectrolyte nanocapsules for overcoming multidrug resistance in ovarian cancer.

The sonication-assisted layer-by-layer (SLBL) technology was developed to combine necessary factors for an efficient drug-delivery system: (i) control of nanocolloid size within 100 - 300 nm, (ii) high drug content (70% wt), (iii) shell biocompatibility and biodegradability, (iv) sustained controlled release, and (v) multidrug-loaded system. Stable nanocolloids of Paclitaxel (PTX) and lapatinib were prepared by the SLBL method. In a multidrug-resistant (MDR) ovarian cancer cell line, OVCAR-3, lapatinib/PTX nanocolloids mediated an enhanced cell growth inhibition in comparison with the PTX-only treatment. A series of in vitro cell assays were used to test the efficacy of these formulations. The small size and functional versatility of these nanoparticles, combined with their ability to incorporate various drugs, indicates that lapatinib/PTX nanocolloids may have in vivo therapeutic applications.

Drug-loaded polyelectrolyte microcapsules for sustained targeting of cancer cells.

In this review we will overview novel nanotechnological nanocarrier systems for cancer therapy focusing on recent development in polyelectrolyte capsules for targeted delivery of antineoplastic drugs against cancer cells. Biodegradable polyelectrolyte microcapsules (PMCs) are supramolecular assemblies of particular interest for therapeutic purposes, as they can be enzymatically degraded into viable cells, under physiological conditions. Incorporation of small bioactive molecules into nano-to-microscale delivery systems may increase drug's bioavailability and therapeutic efficacy at single cell level giving desirable targeted therapy. Layer-by-layer (LbL) self-assembled PMCs are efficient microcarriers that maximize drug's exposure enhancing antitumor activity of neoplastic drug in cancer cells. They can be envisaged as novel multifunctional carriers for resistant or relapsed patients or for reducing dose escalation in clinical settings.

Plant oil bodies: novel carriers to deliver lipophilic molecules.

Oil bodies (OBs) are specialised organelles ubiquitously detected in plant oil seeds, which serve as lipid storage compartments. OBs consist of a hydrophobic core of triacylglycerol (TAGs), surrounded by a monolayer of phospholipids (PLs) embedded with some specific proteins with a size ranging from 0.5 to 2 µm. In this work, we report an easy method to reconstitute OBs starting from their constituents and to encapsulate lipophilic molecules, i.e. the fluorescent fluorescein isothiocyanate (FITC) and carboxyfluorescein (CF), into reconstituted OBs. This methods allowed us to produce OBs 4- to 10-fold smaller (50-200 nm) than the native one and to obtain a good recovery (about 40%) of both the fluorescent compounds used in the present work. The properties of reconstituted OBs were investigated by a combination of Brewster angle microscopy, scanning force microscopy, ζ-potential techniques. OBs were stable and formed ordered monolayers when patterned on hydrophobic substrates whereas they showed a higher tendency to aggregate into larger, coalescing OBs when were deposited onto hydrophilic substrates or at the air/water interface. Furthermore, we verified the uptake of FITC-loaded OBs by the MCF-7 breast cancer cell line. Our results indicated that OBs could be envisaged as novel carriers to deliver hydrophobic bioactive compounds.