Correction: Mechanisms Underlying the Anti-Tumoral Effects of Citrus bergamia Juice.

[This corrects the article DOI: 10.1371/journal.pone.0061484.].

NF-κB mediates the antiproliferative and proapoptotic effects of bergamot juice in HepG2 cells.

AIMS: Among cancers, hepatocellular carcinoma is one of the commonest worldwide, and its incidence is increasing around the world. A lot of evidence underlines that natural substances usually consumed in the diet can have an important role in the prevention of cancer. In this study we investigated the molecular mechanisms underlying the antiproliferative activity of Citrus bergamia (bergamot) juice (BJ) in human hepatocellular carcinoma HepG2 cells. MAIN METHODS: HepG2 cells were exposed to BJ and then cell proliferation, cell cycle progression, apoptosis and NF-κB nuclear translocation were evaluated. KEY FINDINGS: Here we present results demonstrating that BJ reduced the growth rate of human hepatocellular carcinoma HepG2 cells in a time- and concentration-dependent manner, by a mechanism involving the activation of apoptotic machinery via both intrinsic and extrinsic pathways. Moreover, BJ increased expression of P53 and P21 proteins that may be responsible for the HepG2 cell cycle arrest in G2 phase. In addition, BJ reduced NF-κB nuclear translocation. SIGNIFICANCE: Our data demonstrate the ability of BJ in reducing the growth of HepG2 cells, revealing its mechanism of action and suggesting a promising role as anticancer drugs.

Effects of bergamot essential oil and its extractive fractions on SH-SY5Y human neuroblastoma cell growth.

OBJECTIVES: The goals were to investigate the mechanisms underlying the antiproliferative effects of bergamot essential oil (BEO) and to identify the compounds mainly responsible for its SH-SY5Y cells growth rate inhibition. METHODS: Five BEO extractive fractions (BEOs) differing in their chemical composition were used. Cell proliferation was determined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and cell count assays. Trypan blue exclusion test and Annexin V/PI staining were performed to assess their cytotoxic activity. Genotoxicity was detected by comet assay. The cell cycle was checked cytofluorimetrically. Reactive oxygen species (ROS) and Δψm were measured fluorimetrically. Western blotting analyses for some apoptosis-related proteins were carried out. KEY FINDINGS: Treatment of SH-SY5Y cells with some types of BEOs decreased cell growth rate by a mechanism correlated to both apoptotic and necrotic cell death. Coloured BEOs act by increasing ROS generation, responsible for the drop in Δψm, and modulate p38 and extracellular signal-regulated kinases (ERK ½) mitogen-activated protein kinases, p53, Bcl-2 and Bax signalling pathways. Finally, we identify bergamottin and 5-geranyloxy-7-methoxycoumarin as the bioactive molecules that could play a pivotal role in the antiproliferative effects exerted by coloured BEOs. CONCLUSIONS: Our study provides novel insights into the field of the antiproliferative effects of BEO, which could be exploited in the context of a multitarget pharmacological strategy.

Colloidal supramolecular aggregates for therapeutic application in neuromedicine.

Neuromedicine has recently been emerging on the research scene and presents interesting challenges in therapeutics. The range of therapies generally used to treat neurological disorders are limited in their efficacy and degree of patient compliance because of the necessity of multiple drug dosages, low drug concentration in the central nervous system and side effects. Moreover, therapeutics require standard drug dosages which cannot be personalized. The limiting obstacle in neuromedicine is still the blood-brain barrier, which prevents the accumulation of endogenous and exogenous compounds inside the brain. Various transporters located on the blood-brain barrier modulate the crossing of endogenous compounds. It has been discovered that these transporters can be used as pathways for the transport of therapeutic agents and macromolecules that pass the blood-brain barrier allowing the uptake of bioactive compounds into the central nervous system. Several attempts have recently been made to develop forms of nanomedicine capable of overcoming the limitations of conventional therapy, above all the crossing of the blood-brain barrier. An outstandingly promising option could be the use of colloidal supramolecular aggregates. These nanodrugs are safe, biodegradable, and biocompatible and can combine biomaterials useful for diagnostic and therapeutical applications. They can be modified using monoclonal antibodies, proteins, peptides and macromolecules, thus providing personalized neuromedicine, which can be used in the treatment of various neurological disorders. In this review, recent advancements of supramolecular colloidal devices as neuromedicines are discussed, with particular focus on the latest developments.

Anticancer activity of liposomal bergamot essential oil (BEO) on human neuroblastoma cells.

Citrus extracts, particularly bergamot essential oil (BEO) and its fractions, have been found to exhibit anticancer efficacy. However, the poor water solubility, low stability and limited bioavailability have prevented the use of BEO in cancer therapy. To overcome such drawbacks, we formulated BEO liposomes that improved the water solubility of the phytocomponents and increased their anticancer activity in vitro against human SH-SY5Y neuroblastoma cells. The results warrant further investigation of BEO liposomes for in vivo applications.

Mechanisms underlying the anti-tumoral effects of Citrus Bergamia juice.

Based on the growing deal of data concerning the biological activity of flavonoid-rich natural products, the aim of the present study was to explore in vitro the potential anti-tumoral activity of Citrus Bergamia (bergamot) juice (BJ), determining its molecular interaction with cancer cells. Here we show that BJ reduced growth rate of different cancer cell lines, with the maximal growth inhibition observed in neuroblastoma cells (SH-SY5Y) after 72 hs of exposure to 5% BJ. The SH-SY5Y antiproliferative effect elicited by BJ was not due to a cytotoxic action and it did not induce apoptosis. Instead, BJ stimulated the arrest in the G1 phase of cell cycle and determined a modification in cellular morphology, causing a marked increase of detached cells. The inhibition of adhesive capacity on different physiologic substrates and on endothelial cells monolayer were correlated with an impairment of actin filaments, a reduction in the expression of the active form of focal adhesion kinase (FAK) that in turn caused inhibition of cell migration. In parallel, BJ seemed to hinder the association between the neural cell adhesion molecule (NCAM) and FAK. Our data suggest a mechanisms through which BJ can inhibit important molecular pathways related to cancer-associated aggressive phenotype and offer new suggestions for further studies on the role of BJ in cancer treatment.

Gemcitabine-loaded liposomes: rationale, potentialities and future perspectives.

This review describes the strategies used in recent years to improve the biopharmaceutical properties of gemcitabine, a nucleoside analog deoxycytidine antimetabolite characterized by activity against many kinds of tumors, by means of liposomal devices. The main limitation of using this active compound is the rapid inactivation of deoxycytidine deaminase following administration in vivo. Consequently, different strategies based on its encapsulation/complexation in innovative vesicular colloidal carriers have been investigated, with interesting results in terms of increased pharmacological activity, plasma half-life, and tumor localization, in addition to decreased side effects. This review focuses on the specific approaches used, based on the encapsulation of gemcitabine in liposomes, with particular attention to the results obtained during the last 5 years. These approaches represent a valid starting point in the attempt to obtain a novel, commercializable drug formulation as already achieved for liposomal doxorubicin (Doxil(®), Caelyx(®)).

Anti-inflammatory activity of novel ammonium glycyrrhizinate/niosomes delivery system: human and murine models.

Today there is a very great deal of interest among members of the global natural products community in investigating new plant constituents. Recent studies demonstrate that liquorice extracts are useful in the treatment of dermatitis, eczema, and psoriasis, with an efficacy comparable to that of corticosteroids. In this work, niosomes made up of surfactants (Tween 85 and Span 20) and cholesterol at various concentrations were prepared to investigate the potential application of niosomes for the delivery of ammonium glycyrrhizinate (AG), useful for the treatment of various inflammatory based diseases. Vesicles were characterized evaluating dimensions, ζ potential, anisotropy, drug entrapment efficiency, stability, cytotoxicity evaluation and skin tolerability. Release profiles of ammonium glycyrrhizinate/niosomes were evaluated in vitro using cellulose membranes. The best formulation was used to evaluate the in vitro/in vivo efficacy of the ammonium glycyrrhizinate/niosomes in murine and human models of inflammation. The AG-loaded non-ionic surfactant vesicles showed no toxicity, good skin tolerability and were able to improve the drug anti-inflammatory activity in mice. Furthermore, an improvement of the anti-inflammatory activity of the niosome delivered drug was observed on chemically induced skin erythema in humans.

Improved in vitro and in vivo collagen biosynthesis by asiaticoside-loaded ultradeformable vesicles.

The potentiality of ultradeformable vesicles as a possible topical delivery system for asiaticoside, a natural compound obtained from Centella asiatica was evaluated, because this compound exhibits collagen biosynthesis promoting activity. Ultradeformable vesicles were prepared by the extrusion technique; these vesicles were composed of Phospholipon 100 and different molar fractions of sodium cholate as the edge activator. The physicochemical properties of the ultradeformable vesicles were investigated through differential scanning calorimetry and light scattering techniques. The potential cyctotoxicity and biological activity of asiaticoside-loaded ultradeformable vesicles were evaluated on primary human dermal fibroblast cells by determining the extracellular lactic dehydrogenase activity, the cellular viability and the biosynthetic production of collagen. In vitro permeation experiments through human stratum corneum and epidermis membranes were also carried out. Ultradeformable vesicles having sodium cholate molar fraction of 0.2 proved to be the most suitable topical carriers for asiaticoside. A sodium cholate content of >0.2 was observed to be cytotoxic probably due to its co-existence with other lipid aggregates, an example being mixed micelles. Asiaticoside-loaded ultradeformable vesicles with a sodium cholate molar fraction of 0.2 elicited the greatest degree of collagen biosynthesis in human fibroblasts. Ultradeformable vesicles provided the greatest in vitro skin permeation of asiaticoside showing a 10-fold increase with respect to the free drug solution and favoured an increase in in vivo collagen biosynthesis. Ultradeformable vesicles are therefore suitable carriers for the pharmaceutical and cosmetic application of the natural agent asiaticoside.

Paclitaxel-loaded ethosomes®: potential treatment of squamous cell carcinoma, a malignant transformation of actinic keratoses.

Topical application of anticancer drugs for the treatment of malignancies represents a new challenge in dermatology, potentially being an alternative therapeutic approach for the efficacious treatment of non-melanoma skin cancer, that is, actinic keratoses, and malignant lesions of the skin caused by ultraviolet radiation. Anti-proliferative and antimitotic drugs, including many of the taxanes, are currently under investigation for the treatment of cutaneous malignant transformation of actinic keratoses, particularly the squamous cell carcinoma. Paclitaxel-loaded ethosomes® are proposed as topical drug delivery systems for the treatment of this pathology due to their suitable physicochemical characteristics and enhanced skin penetration ability for deep dermal delivery. Our in vitro data show that the skin application of paclitaxel-loaded ethosomes® improved the permeation of paclitaxel in a stratum corneum-epidermis membrane model and increased its anti-proliferative activity in a squamous cell carcinoma model as compared to the free drug. The results obtained encouraged the use of the paclitaxel-loaded ethosomes® as the formulation for the potential treatment of squamous cell carcinoma, a malignant transformation of actinic keratoses.

Ethosomes® and transfersomes® containing linoleic acid: physicochemical and technological features of topical drug delivery carriers for the potential treatment of melasma disorders.

Two vesicular colloidal carriers, ethosomes® and transfersomes® were proposed for the topical delivery of linoleic acid, an active compound used in the therapeutic treatment of hyperpigmentation disorders, i.e. melasma, which is characterized by an increase of the melanin production in the epidermis. Dynamic light scattering was used for the physicochemical characterization of vesicles and mean size, size distribution and zeta potential were evaluated. The stability of formulations was also evaluated using the Turbiscan Lab® Expert based on the analysis of sample transmittance and photon backscattering. Ethosomes® and transfersomes® were prepared using Phospholipon 100 G®, as the lecithin component, and ethanol and sodium cholate, as edge activator agents, respectively. Linoleic acid at 0.05% and 0.1% (w/v) was used as the active ingredient and entrapped in colloidal vesicles. Technological parameters, i.e. entrapment efficacy, drug release and permeation profiles, were also investigated. Experimental findings showed that physicochemical and technological features of ethosomes® and transfersomes® were influenced by the lipid composition of the carriers. The percutaneous permeation experiments of linoleic acid-loaded ethosomes® and transfersomes® through human stratum corneum-epidermidis membranes showed that both carriers are accumulated in the skin membrane model as a function of their lipid compositions. The findings reported in this investigation showed that both vesicular carriers could represent a potential system for the topical treatment of hyperpigmentation disorders.

Gemcitabine-loaded PEGylated unilamellar liposomes vs GEMZAR: biodistribution, pharmacokinetic features and in vivo antitumor activity.

The systemic efficacy of the chemotherapeutic agents presently used to treat solid tumors is limited by their low therapeutic index. Previously, our research group improved the in vitro antitumoral activity of gemcitabine, an anticancer agent rapidly deaminated to the inactive metabolite 2',2'-difluorodeoxyuridine, entrapping it into unilamellar pegylated liposomes made up of 1,2-dipalmitoyl-snglycero-3-phosphocholine monohydrate/cholesterol/N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (6:3:1 molar ratio). In this work, we investigated the in vivo efficiency of the gemcitabine liposomal formulation (5mg/kg) with respect to the antitumoral commercial product GEMZAR (50mg/kg) on an anaplastic thyroid carcinoma xenograft model obtaining similar effects in terms of inhibition of tumor mass proliferation after 4weeks of treatment. The investigation of the carrier biodistribution and the drug pharmacokinetic profile furnished the rationalization of the efficacy of the vesicular system containing the active compound 10-fold less concentrated; in fact, liposomes promoted the concentration of the drug inside the tumor and they increased its plasmatic half-life. In addition, no signs of blood toxicity were observed when vesicular devices of effective doses of the drug were used.

Turbiscan lab expert analysis of the stability of ethosomes and ultradeformable liposomes containing a bilayer fluidizing agent.

The stability of vesicular drug carriers containing linoleic acid, as a model of bilayer fluidizing agent, was evaluated using a Turbiscan optical analyzer, an innovative analytical instrument able to determine the long-time stability of colloidal systems. Ethosomes and ultradeformable liposomes were prepared using Phospholipon 100G as the lecithin component, while ethanol and sodium cholate were used for the specific preparation of ethosomes and ultradeformable liposomes, respectively. The advantages of the Turbiscan optical analyzer are: (i) its ability to measure reversible (creaming and sedimentation) and irreversible (coalescence and segregation) destabilization phenomena directly in the sample without any dilution and (ii) to detect these phenomena much earlier and easier than other apparatuses. Turbiscan data showed that both colloidal vesicles demonstrate a good stability during the 3h of the experiment. No modification of Turbiscan backscattering profiles of colloidal suspensions occurred when different amounts of linoleic acid were used to prepare ethosomes and ultradeformable liposomes. No coalescence, sedimentation, flocculation or clarification occurred. The results were very encouraging and confirmed the fact that the Turbiscan optical analyzer can be used to study the stability of colloidal formulations even in the presence of deformable agents.

Innovative bola-surfactant niosomes as topical delivery systems of 5-fluorouracil for the treatment of skin cancer.

An innovative niosomal system made up of alpha,omega-hexadecyl-bis-(1-aza-18-crown-6) (Bola), Span 80 and cholesterol (2:5:2 molar ratio) was proposed as a topical delivery system for 5-fluorouracil (5-FU), largely used in the treatment of different forms of skin cancers. Bola-niosomes showed a mean size of approximately 400 nm, which were reduced to approximately 200 nm by a sonication procedure with a polydispersion index value of 0.1. Bola-niosomes showed a loading capacity of approximately 40% with respect to the amount of 5-FU added during the preparation. 5-FU-loaded bola-niosomes were tested on SKMEL-28 (human melanoma) and HaCaT (non-melanoma skin cancer with a specific mutations in the p53 tumor suppressor gene) to assess the cytotoxic activity with respect to the free drug. 5-FU-loaded bola-niosomes showed an improvement of the cytotoxic effect with respect to the free drug. Confocal laser scanning microscopy studies were carried out to evaluate both the extent and the time-dependent bola-niosome-cell interaction. The percutaneous permeation of 5-FU-loaded niosomes was evaluated by using human stratum corneum and epidermis membranes. Bola-niosomes provided an increase of the drug penetration of 8- and 4-folds with respect to a drug aqueous solution and to a mixture of empty bola-niosomes with a drug aqueous solution.