Developing Actively Targeted Nanoparticles to Fight Cancer: Focus on Italian Research.

Active targeting is a valuable and promising approach with which to enhance the therapeutic efficacy of nanodelivery systems, and the development of tumor-targeted nanoparticles has therefore attracted much research attention. In this field, the research carried out in Italian Pharmaceutical Technology academic groups has been focused on the development of actively targeted nanosystems using a multidisciplinary approach. To highlight these efforts, this review reports a thorough description of the last 10 years of Italian research results on the development of actively targeted nanoparticles to direct drugs towards different receptors that are overexpressed on cancer cells or in the tumor microenvironment. In particular, the review discusses polymeric nanocarriers, liposomes, lipoplexes, niosomes, solid lipid nanoparticles, squalene nanoassemblies and nanobubbles. For each nanocarrier, the main ligands, conjugation strategies and target receptors are described. The literature indicates that polymeric nanoparticles and liposomes stand out as key tools for improving specific drug delivery to the site of action. In addition, solid lipid nanoparticles, squalene nanoparticles and nanobubbles have also been successfully proposed. Taken together, these strategies all offer many platforms for the design of nanocarriers that are suitable for future clinical translation.

Nanosystems in Cosmetic Products: A Brief Overview of Functional, Market, Regulatory and Safety Concerns.

Nanosystems exhibit various innovative physico-chemical properties as well as a range of cosmetic functions, including increased skin retention for loaded compounds. The worldwide nano-market has therefore been consistently extensive in recent decades. This review summarizes the most important properties of nanosystems that are employed in cosmetics, including composition, functions and interactions with skin, with particular attention being paid to marketed products. Moreover, the worldwide regulatory landscape of nanomaterials used as cosmetic ingredients is considered, and the main safety concerns are indicated. In general, advanced physico-chemical characterization is preliminarily needed to assess the safety of nanomaterials for human health and the environment. However, there is currently a shortfall in global legislation as a universally accepted and unambiguous definition of a nanomaterial is still lacking. Therefore, each country follows its own regulations. Anyhow, the main safety concerns arise from the European context, which is the most restrictive. Accordingly, the poor dermal permeation of nanomaterials generally limits their potential toxic effects, which should be mainly ascribed to unwanted or accidental exposure routes.

Effect of Methyl-ß-Cyclodextrin and Trehalose on the Freeze-Drying and Spray-Drying of Sericin for Cosmetic Purposes.

Sericin is a protein extracted from Bombyx mori silk cocoons. Over the last decade, this wastewater product of the textile industry has shown many interesting biological properties. This protein is widely used in the cosmetic and biomedical fields. In this study, sericin has been obtained via a High-Temperature High-Pressure degumming process, and was dried using the freeze-drying (fd) and spray-drying (sd) techniques. Proteins tend to collapse during drying, hence, sericin has been dried in the presence of two selected carrier agents: methyl-ß-cyclodextrin and trehalose. The obtained powders have been analyzed using thermal investigation, microscopy (optical, SEM), and granulometric and spectroscopic analyses. Moreover, the percentage yield of the spray-drying process has been calculated. Both the agents were able to significantly improve the drying process, without altering the physico-chemical properties of the protein. In particular, the co-spray-drying of sericin with methyl-ß-cyclodextrin and trehalose gave good process yields and furnished a powder with low moisture content and handling properties that are better than those of the other studied dried products. These characteristics seem to be appropriate and fruitful for the manufacturing of cosmetic raw materials.

Ozonated Oils as Antimicrobial Systems in Topical Applications. Their Characterization, Current Applications, and Advances in Improved Delivery Techniques.

The search for a wide spectrum of antimicrobial agents that can avoid resistance while maintaining reasonable side effects has led to ozonated oils experiencing an increase in scientific interest and clinical applications. The treatment of vegetable oils with ozone leads to the creation of a reservoir of ozone that slowly releases into the skin thanks to the fact that ozone can be held as ozonides of unsaturated fatty acids. Interest in the use of ozonated oils has meant that several ozonated-vegetable-oil-containing products have been commercialized as cosmetic and pharmaceutical agents, and in innovative textile products with antibacterial activity. New approaches to the delivery of ozonated oils have very recently appeared in an attempt to improve their characteristics and reduce drawbacks, such as an unpleasant odor, high viscosity and undesired effects on skin, including irritation and rashes. The present review focuses on the current status of delivery agents that use ozonated oils as antimicrobial agents in topical (dermal, skin, and soft tissues) treatments. Challenges and future opportunities for these delivery systems will also be discussed.

Mesoporous silica nanoparticles as a promising skin delivery system for methotrexate.

The systemic administration of methotrexate (MTX), a commonly used, antineoplastic drug which is also used in cutaneous disorders, is primarily associated with prolonged retention in the body and consequently with side effects. Innovative drug delivery techniques and alternative administration routes would therefore contribute to its safe and effective use. The general objective of this study is thus the development of MTX-based preparations for the topical treatment of skin disorders. MCM-41-like nanoparticles (MSN), are herein proposed as carriers which can improve the cutaneous absorption and hence the bioavailability and efficacy of MTX. The MTX/MSN complex, prepared via the impregnation procedure, has been physico-chemically characterized, while its cell cultures have had their biocompatibility and bioactivity tested. Furthermore, a series of stable MTX-based dermal formulations has been developed, some containing shea butter, a natural fat. Ex-vivo porcine skin absorption and the transepidermal permeation of MTX have also been monitored in a variety of media using Franz diffusion cells. Interestingly, the epidermal accumulation of the active molecule was increased by its inclusion into MSN, regardless of the surrounding medium. Furthermore, the presence of shea butter enhanced the skin uptake of the drug both in the free and in the loaded form.

Thermoresponsive mesoporous silica nanoparticles as a carrier for skin delivery of quercetin.

Recently, mesoporous silica nanoparticles (MSNs) have emerged as promising drug delivery systems able to preserve the integrity of the carried substance and/or to selectively reach a target site; however, they have rarely been explored for skin application. In this study, thermoresponsive MSNs, designed to work at physiologic cutaneous temperature, are proposed as innovative topical carriers for quercetin (Q), a well-known antioxidant. The thermosensitive nanoparticles were prepared by functionalizing two different types of matrices, with pore size of 3.5nm (MSNsmall) and 5.0nm (MSNbig), carrying out a free radical copolymerization of N-isopropylacrylamide (NIPAM) and 3-(methacryloxypropyl)trimethoxysilane (MPS) inside the mesopores. The obtained copolymer-grafted MSNs (copoly-MSNs) were physico-chemically characterized and their biocompatibility was attested on a human keratinocyte cell line (HaCaT). The release profiles were assessed and the functional activity of Q, free or loaded, was evaluated in terms of antiradical and metal chelating activities. Ex vivo accumulation and permeation through porcine skin were also investigated. The characterization confirmed the copolymer functionalization of the MSNs. In addition, both the bare and functionalized silica matrices were found to be biocompatible. Among the copolymer-grafted complexes, Q/copoly-MSNbig exhibited more evident thermoresponsive behavior proving the potential of these thermosensitive systems for advanced dermal delivery.

Mesoporous silica as topical nanocarriers for quercetin: characterization and in vitro studies.

The flavonoid quercetin is extensively studied for its antioxidant and chemopreventive properties. However the poor water-solubility, low stability and short half-life could restrict its use in skin care products and therapy. The present study was aimed to evaluate the potential of aminopropyl functionalized mesoporous silica nanoparticles (NH2-MSN) as topical carrier system for quercetin delivery. Thermo gravimetric analysis, X-ray diffraction, high resolution transmission electron microscopy, nitrogen adsorption isotherms, FT-IR spectroscopy, zeta potential measurements and differential scanning calorimetry allowed analyzing with great detail the organic-inorganic molecular interaction. The protective effect of this vehicle on UV-induced degradation of the flavonoid was investigated revealing a certain positive influence of the inclusion on the photostability over time. Epidermal accumulation and transdermal permeation of this molecule were ex vivo evaluated using porcine skin mounted on Franz diffusion cells. The inclusion complexation with the inorganic nanoparticles increased the penetration of quercetin into the skin after 24h post-application without transdermal delivery. The effect of quercetin alone or given as complex with NH2-MSN on proliferation of JR8 human melanoma cells was evaluated by sulforhodamine B colorimetric proliferation assay. At a concentration 60 µM the complex with NH2-MSN was more effective than quercetin alone, causing about 50% inhibition of cell proliferation.

The protective effect of the mesoporous host on the photo oxidation of fluorescent guests: a UV-Vis spectroscopy study.

The inclusion of fluorescent molecules within the pores of an inorganic host system is one of the most common ways used for the preparation of highly fluorescent nanosystems. The confinement of labile molecules usually leads to better performances mainly due to their protection against environmental parameters which can concur with the deactivation of the fluorescent ones. In this work the protective effect of the siliceous mesoporous host on photo oxidation of fluorescent guest molecules has been investigated under different experimental conditions, highlighting an outstanding photostability if compared to the performances exerted by the fluorescent dye in solution.

MCM-41 as a useful vector for rutin topical formulations: synthesis, characterization and testing.

Rutin, the glycoside of quercetin, could be used in topical preparations because of its antioxidant and radical scavenging properties, but its employ in cosmetic and pharmaceutical products is limited by poor physico-chemical stability. These issues were addressed by preparing, characterizing and testing rutin inclusion complexes with MCM-41 mesoporous silica. The effect of surface functionalization with aminopropyl groups (NH2-MCM-41) on the molecules properties was studied. The organic/inorganic interaction was confirmed by many techniques. In particular, the high inclusion of rutin in the pores of NH2-MCM-41 was assessed by XRD, TGA, gas-volumetric analysis (BET), while FTIR spectroscopy allowed to analyse with great detail the molecular interaction with the inorganic surface. Rutin was stabilized against UV degradation, mostly by its inclusion in NH2-MCM-41. Ex vivo studies showed a greater accumulation in porcine skin in the case of rutin complexed with NH2-MCM-41. Not only antioxidant properties of rutin were maintained after immobilization but, with aminopropyl silica, the metal-chelating activity increased noticeably. The immobilization of rutin in aminopropyl silica resulted in better performance in terms of activity and photostability, suggesting the importance of functionalization in stabilizing organic molecules within silica pores.

Stabilization of quercetin flavonoid in MCM-41 mesoporous silica: positive effect of surface functionalization.

Antioxidants can prevent UV-induced skin damage mainly by neutralizing free radicals. For this purpose, quercetin (Q) is one of the most employed flavonoids even if the potential usefulness is limited by its unfavorable physicochemical properties. In this context, mesoporous silica (MCM-41) is herein proposed as a novel vehicle able to improve the stability and performance of this phenolic substrate in topical products. Complexes of Q with plain or octyl-functionalized MCM-41 were successfully prepared with different weight ratios by a kneading method, and then, they were characterized by XRD, gas-volumetric (BET), TGA, DSC, and FTIR analyses. The performances of the different complexes were evaluated in vitro in terms of membrane diffusion profiles, storage and photostability, antiradical and chelating activities. The physicochemical characterization confirmed an important host/guest interaction due to the formation of Si-OH/quercetin hydrogen-bonded adducts further strengthened by octyl functionalization through van der Waals forces. The immobilization of Q, particularly on octyl-functionalized silica, increased the stability without undermining the antioxidant efficacy opening the way for an innovative employment of mesoporous composite materials in the skincare field.

Specific effects of single antioxidants in the lipid peroxidation caused by nano-titania used in sunscreen lotions.

The effect of some additives, phenylalanine, ascorbyl palmitate and sodium ascorbyl phosphate on the oxidation of linoleic acid and porcine ear skin induced by UV irradiation was investigated, in the absence and in the presence of variously uncoated and coated titania powders. Such additives have, on the one hand, a scavenging activity toward the oxidizing species photogenerated by TiO(2), and on the other one an inhibitory effect toward UVB-induced peroxidation. Sodium ascorbyl phosphate and ascorbyl palmitate displayed a stronger antioxidant effect than phenylalanine toward linoleic acid peroxidation. On porcine skin all the three molecules exhibited both antiradical and antioxidant activity. Their protective effect against peroxidation was higher with porcine skin lipids than with linoleic acid, referable to the chemical differences in the two lipid substrates.

Role of particle coating in controlling skin damage photoinduced by titania nanoparticles.

TiO(2) nanoparticles hazard is associated to their photocatalytic activity causing release of DNA damaging ROS (Reactive Oxygen Species), lipid peroxidation and skin damage. Various coatings have been proposed to minimize photocatalysis, while keeping the potential to block UV radiations. Uncoated and variously coated commercial nano-titania have been classified on the basis of UVB-induced lipoperoxidation of linoleic acid. A selection of the most and the least protective specimens was then examined by ESR (Electron Spin Resonance) to evidence the presence of surface paramagnetic centres and the release of ROS in aqueous suspensions (spin trapping). Paramagnetic centres and ROS were correlated with the extent of lipid peroxidation. When tested on porcine skin (mimicking the human one), titania acted as on linoleic acid. The combined use of lipid peroxidation of simple fatty acids with ESR analysis is here proposed as a possible screening tool for the evaluation of the potential toxicity of nano-titania in sunscreen preparations.

Photodegradation of retinol and anti-aging effectiveness of two commercial emulsions.

Two commercial anti-aging products, RETI C and RETI C concentrate emulsions, containing retinol and vitamin C, were studied. The concentration of vitamin A was determined over time, subjecting the creams to an accelerated stability test. Both emulsions, when stored at 25 degrees C, showed a moderate decrease over time in retinol concentration, while after storage at 40 degrees C the percentage of retinol degraded increased over time. Under UVA irradiation, the retinol degraded to a greater extent than under UVB irradiation, both in RETI C and RETI C concentrate emulsions. In order to verify the anti-aging effectiveness of the emulsions, an in vivo test on some female volunteers was carried out, evaluating the visible results of the application of the creams on the skin surface. The creams were rather unstable after storage at 40 degrees C, but they were effective in treating the signs of aging and in reducing facial wrinkles.

Cholesteryl butyrate solid lipid nanoparticles as a butyric acid pro-drug: effects on cell proliferation, cell-cycle distribution and c-myc expression in human leukemic cells.

Cholesteryl butyrate solid lipid nanoparticles (chol-but SLN) have been proposed as a pro-drug to deliver butyric acid. We compared the effects on cell growth, cell-cycle distribution and c-myc expression of chol-but SLN and sodium butyrate (Na-but) in the human leukemic cell lines Jurkat, U937 and HL-60. In all the cell lines 0.5 and 1.0 mM chol-but SLN provoked a complete block of cell growth. Cell-cycle analysis demonstrated in Jurkat cells that 0.25 mM chol-but SLN caused a pronounced increase of G2/M cells and a decrease of G0/G1 cells, whereas in U937 and HL-60 cells chol-but SLN led to a dose-dependent increase of G0/G1 cells, with a decrease of G2/M cells. In Jurkat and HL-60 cells 0.5 mM chol-but SLN induced a significant increase of sub-G0/G1 apoptotic cells. Cell growth and cell-cycle distribution were unaffected by the same concentrations of Na-but. A concentration of 0.25 mM chol-but SLN was able to cause a rapid and transient down-regulation of c-myc expression in all the cell lines, whereas 1 mM Na-but caused a slight reduction of c-myc expression only in U937 cells. The results show how chol-but SLN affects the proliferation pattern of both myeloid and lymphoid cells to an extent greater than the natural butyrate.

Influence of ion pairing on topical delivery of retinoic acid from microemulsions.

The purpose of the present study was to determinate the significance of ion pairing on the topical permeation of retinoic acid (R.A) using microemulsions as delivery vehicles. Phenylalanine methyl ester, phenylalanine ethylester, histidine methyl ester, tryptophan methyl ester and valine methyl ester were used as counter ions. Results of diffusion studies through polydimethylsiloxane membrane (PDMS) indicate that retinoic acid permeation from ethanol-pH 6.4 buffer mixture significantly increased in the presence of counter ions. A linear relationship was found between apparent partition coefficients and permeation coefficients. The highest values were with valine methyl ester and phenylalanine ethyl ester. In order to develop alternative formulations for topical administration of R.A, microemulsions were evaluated as delivery vehicles. Oil-in-water (O/W) and water-in-oil (W/O) microemulsion formulations were prepared using water, isopropyl myristate, lecithin, caprylyl-capryl glucoside and ethanol or 1,2 hexanediol. Experiments with PDMS membranes showed decreasing permeabilities of R.A from microemulsions in the presence of counter ions. This was related to the increased lipophilicity and different vehicle membrane affinity of the ion pairs The ability of the systems to deliver R.A through the skin was evaluated in vitro using pig-skin. R.A permeabilities were much lower with microemulsions than with solution, while a large increase in R.A skin deposition was observed only from O/W microemulsions in the presence of counter ions. The depth of skin accumulation was below 100 microm after 24 h application. The results suggest that O/W microemulsions containing a counter ion can be used to optimise drug targeting without a concomitant increase in systemic absorption.

Incorporation of cyclosporin A in solid lipid nanoparticles (SLN).

The cyclic undecapeptide cyclosporin A (CyA) a potent immunosuppressive drug used in many therapies, is extremely hydrophobic. Commercial products employ solubilising agents to improve gastrointestinal absorption. In the present study CyA solid lipid nanoparticles (SLN) are prepared from warm o/w microemulsion, dispersed in cold water. The matrix chiefly consists of stearic acid, phosphatidylcholine and taurocholate; up to 13% of CyA can be incorporated. The average diameter of CyA-loaded SLNs is below 300 nm and transmission electron microscopy (TEM) analysis shows them to be spherical. In vitro release of CyA from SLNs is low. CyA-loaded SLNs can be proposed for most administration routes, in particular for the duodenal route.