Blood-brain barrier dysfunction in hemorrhagic transformation: a therapeutic opportunity for nanoparticles and melatonin.

Stroke is the second leading cause of death worldwide, estimated that one-sixth of the world population will suffer it once in their life. The most common type of this medical condition is the ischemic stroke (IS), produced by a thrombotic or embolic occlusion of a major cerebral artery or its branches, leading to the formation of a complex infarct region caused by oxidative stress, excitotoxicity, and endothelial dysfunction. Nowadays, the immediate treatment for IS involves thrombolytic agents or mechanical thrombectomy, depending on the integrity of the blood-brain barrier (BBB). A common stroke complication is the hemorrhagic transformation (HT), which consists of bleeding into the ischemic brain area. Currently, better treatments for IS are urgently needed. As such, the neurohormone melatonin has been proposed as a good candidate due to its antioxidant, anti-inflammatory, and neuroprotective effects, particularly against lipid peroxidation and oxidative stress during brain ischemia. Here, we proposed to develop intravenous or intranasal melatonin nanoformulation to specifically target the brain in patients with stroke. Nowadays, the challenge is to find a formulation able to cross the barriers and reach the target organ in an effective dose to generate the pharmacological effect. In this review, we discuss the current literature about stroke pathophysiology, melatonin properties, and its potential use in nanoformulations as a novel therapeutic approach for ischemic stroke.

Versatile Methodology to Encapsulate Gold Nanoparticles in PLGA Nanoparticles Obtained by Nano-Emulsion Templating.

PURPOSE: Gold nanoparticles have been proved useful for many biomedical applications, specifically, for their use as advanced imaging systems. However, they usually present problems related with stability and toxicity. METHODS: In the present work, gold-nanoparticles have been encapsulated in polymeric nanoparticles using a novel methodology based on nano-emulsion templating. Firstly, gold nanoparticles have been transferred from water to ethyl acetate, a solvent classified as class III by the NIH guidelines (low toxic potential). Next, the formation of nano-emulsions loaded with gold nanoparticles has been performed using a low-energy, the phase inversion composition (PIC) emulsification method, followed by solvent evaporation giving rise to polymeric nanoparticles. RESULTS: Using this methodology, high concentrations of gold nanoparticles (>100 pM) have been encapsulated. Increasing gold nanoparticle concentration, nano-emulsion and nanoparticle sizes increase, resulting in a decrease on the stability. It is noteworthy that the designed nanoparticles did not produce cytotoxicity neither hemolysis at the required concentration. CONCLUSIONS: Therefore, it can be concluded that a novel and very versatile methodology has been developed for the production of polymeric nanoparticles loaded with gold nanoparticles. Graphical Abstract Schematic representation of AuNP-loaded polymeric nanoparticles preparation from nano-emulsion templating.

Ferulic acid-loaded polymeric nanoparticles prepared from nano-emulsion templates facilitate internalisation across the blood-brain barrier in model membranes.

A hydroxycinnamic acid derivative, namely ferulic acid (FA) has been successfully encapsulated in polymeric nanoparticles (NPs) based on poly(lactic-co-glycolic acid) (PLGA). FA-loaded polymeric NPs were prepared from O/W nano-emulsion templates using the phase inversion composition (PIC) low-energy emulsification method. The obtained PLGA NPs exhibited high colloidal stability, good drug-loading capacity, and particle hydrodynamic diameters in the range of 74 to 117 nm, depending on the FA concentration used. In vitro drug release studies confirmed a diffusion-controlled mechanism through which the amount of released FA reached a plateau at 60% after 6 hours-incubation. Five kinetic models were used to fit the FA release data as a function of time. The Weibull distribution and Korsmeyer-Peppas equation models provided the best fit to our experimental data and suggested quasi-Fickian diffusion behaviour. Moderate dose-response antioxidant and radical scavenging activities of FA-loaded PLGA NPs were demonstrated using the DPPH˙ assay achieving inhibition activities close to 60 and 40%, respectively. Cell culture studies confirmed that FA-loaded NPs were not toxic according to the MTT colorimetric assay, were able to internalise efficiently SH-SY5Y neuronal cells and supressed the intracellular ROS-level induced by H2O2 leading to 52% and 24.7% of cellular viability at 0.082 and 0.041 mg mL-1, respectively. The permeability of the NPs through the blood brain barrier was tested with an in vitro organ-on-a-chip model to evaluate the ability of the FA-loaded PLGA and non-loaded PLGA NPs to penetrate to the brain. NPs were able to penetrate the barrier, but permeability decreased when FA was loaded. These results are promising for the use of loaded PLGA NPs for the management of neurological diseases.

Formation of Pegylated polyurethane and Lysine-coated polyurea nanoparticles obtained from O/W nano-emulsions.

The present work describes the formation of Pegylated polyurethane and Lysine-coated polyurea nanoparticles obtained from O/W nano-emulsions via an interfacial polycondensation process in the aqueous solution/polysorbate 80/diisocyanate/medium chain triglyceride systems. The initial nano-emulsions were prepared using the phase inversion composition (PIC) method. Dynamic light scattering studies revealed the changes in the particle size occurring during the process of nanoparticle formation. Well-defined polymeric nanoparticles with a small particle diameter (below 80 nm) and low polydispersity index were obtained using a highly hydrophilic component (polyethylene glycol or lysine) and an aliphatic diisocyante monomer. FT-IR and AFM studies showed that the polymeric matrix of nanoparticles was built by copolymers derived from reaction between the diisocyanate and the hydroxyl groups of both nonionic surfactant and the highly hydrophilic component. Pegylated-polyurethane and lysine-coated polyurea nanoparticles designed in this study are promising tools for future applications in biomedical sciences.

Drug delivery properties of macroporous polystyrene solid foams.

PURPOSE: Polymeric porous foams have been evaluated as possible new pharmaceutical dosage forms. METHODS: These materials were obtained by polymerization in the continuous phase of highly concentrated emulsions prepared by the phase inversion temperature method. Their porosity, specific surface and surface topography were characterized, and the incorporation and release of active principles was studied using ketoprofen as model lipophilic molecule. RESULTS: Solid foams with very high pore volume, mainly inside macropores, were obtained by this method. The pore morphology of the materials was characterized, and very rough topography was observed, which contributed to their nearly superhydrophobic properties. These solid foams could be used as delivery systems for active principles with pharmaceutical interest, and in the present work ketoprofen was used as a model lipophilic molecule. CONCLUSIONS: Drug incorporation and release was studied from solid foam disks, using different concentrations of the loading solutions, achieving a delayed release with short lag-time.

Rosmarinic Acid-Loaded Polymeric Nanoparticles Prepared by Low-Energy Nano-Emulsion Templating: Formulation, Biophysical Characterization, and In Vitro Studies.

Rosmarinic acid (RA), a caffeic acid derivative, has been loaded in polymeric nanoparticles made up of poly(lactic-co-glycolic acid) (PLGA) through a nano-emulsion templating process using the phase-inversion composition (PIC) method at room temperature. The obtained RA-loaded nanoparticles (NPs) were colloidally stable exhibiting average diameters in the range of 70-100 nm. RA was entrapped within the PLGA polymeric network with high encapsulation efficiencies and nanoparticles were able to release RA in a rate-controlled manner. A first-order equation model fitted our experimental data and confirmed the prevalence of diffusion mechanisms. Protein corona formation on the surface of NPs was assessed upon incubation with serum proteins. Protein adsorption induced an increase in the hydrodynamic diameter and a slight shift towards more negative surface charges of the NPs. The radical scavenging activity of RA-loaded NPs was also studied using the DPPH·assay and showed a dose-response relationship between the NPs concentration and DPPH inhibition. Finally, RA-loaded NPs did not affect the cellular proliferation of the human neuroblastoma SH-SY5Y cell line and promoted efficient cellular uptake. These results are promising for expanding the use of O/W nano-emulsions in biomedical applications.

Complexation Nanoarchitectonics of Carbon Dots with Doxorubicin toward Photodynamic Anti-Cancer Therapy.

Carbon dots (Cdots) are known as photosensitizers in which the nitrogen doping is able to improve the oxygen-photosensitization performance and singlet-oxygen generation. Herein, the characteristics of nanoconjugates of nitrogen-doped Cdots and doxorubicin were compared with the property of nitrogen-doped Cdots alone. The investigation was performed for the evaluation of pH-dependent zeta potential, quantum yield, photosensitization efficiency and singlet-oxygen generation, besides spectroscopy (UV-visible absorption and fluorescence spectra) and cytotoxicity on cancer model (HeLa cells). Encapsulation efficiency, drug loading, and drug release without and with light irradiation were also carried out. These investigations were always pursued under the comparison among different nitrogen amounts (ethylenediamine/citric acid = 1-5) in Cdots, and some characteristics strongly depended on nitrogen amounts in Cdots. For instance, surface charge, UV-visible absorbance, emission intensity, quantum yield, photosensitization efficiency and singlet-oxygen generation were most effective at ethylenediamine/citric acid = 4. Moreover, strong conjugation of DOX to Cdots via π-π stacking and electrostatic interactions resulted in a high carrier efficiency and an effective drug loading and release. The results suggested that nitrogen-doped Cdots can be considered promising candidates to be used in a combination therapy involving photodynamic and anticancer strategies under the mutual effect with DOX.

A nanocellulose-based platform towards targeted chemo-photodynamic/photothermal cancer therapy.

Cellulose nanocrystals (CNCs) have advantages as drug delivery carriers because of their biocompatibility and the presence of hydroxyl groups which favor chemical modification and drug binding. The present study describes the development of novel multifunctional rod-like CNCs-based carriers as therapeutic platforms: CNCs were hybridized with folic acid for actively targeting tumor cells, carbon dots (Cdots) for both imaging and photodynamic/photothermal treatments and doxorubicin (DOX) as an anticancer drug. Hybridized carriers displayed excellent drug-loading capacity. Moreover, Cdots-containing hybrids showed fluorescence and photosensitized singlet oxygen generation and photothermal behavior. Carriers exhibited pH-sensitive drug release because of changing interactions with DOX, and this release proved to be effective against in vitro cervical cancer cells, as evidenced by dose-dependent reduced cellular viabilities. Additionally, DOX release was promoted by light irradiation and the photodynamic behavior by reactive oxygen species was confirmed. These results demonstrate the potential of multifunctional CNCs-based carriers as platforms for multimodal photodynamic/photothermal-chemotherapy.

Ethylcellulose nanoparticles prepared from nano-emulsion templates as new folate binding haemocompatible platforms.

Ethylcellulose is a biocompatible polymer attracting increasing interest for biomedical applications. In the present work, the formation of folate-ethylcellulose nanoparticle complexes from nano-emulsion templates prepared by a low-energy approach, using aqueous components suitable for biomedical applications has been investigated. The composition of the aqueous component is shown to be crucial for the formation of stable nano-emulsions and influences the zeta potential values. The ethylcellulose nanoparticles with mean sizes around 100 nm were obtained from the nano-emulsions by solvent evaporation and showed positive zeta potential values above +20 mV due to the presence of the cationic surfactant. The nanoparticles were successfully complexed with folate, as evidenced by both particle size and zeta potential measurements. The complexes prepared with HEPES buffered glucose solution showed excellent haemocompatibility, which make them promising for parenteral therapeutic applications and also for those in which easy access to systemic circulation may occur, like in lungs.

Short and ultrashort antimicrobial peptides anchored onto soft commercial contact lenses inhibit bacterial adhesion.

Commercial soft contact lenses were chemically modified to incorporate antibacterial properties. Contact lenses and especially soft contact lenses present a risk of eye microbial infection that eventually may lead to vision loss. This is a significant health issue given the large population of contact lenses wearers worldwide. In order to introduce bactericidal activity in hydrogel contact lenses, one short and one ultrashort antimicrobial peptides, LKKLLKLLKKLLKL (LK) and IRIRIRIR (IR), were selected. These peptides were anchored on the surface of contact lenses using a linker (1,4-butanediol diglycidyl ether) under mild conditions (room temperature, pH = 7.4). Physical and chemical properties of peptide-functionalized contact lenses were investigated through several analytical techniques including wettability, Raman confocal microscopy, fluorescence studies, refractometry and spectrophotometry. These studies demonstrated that contact lens modification occurred at the nanolevel (ng/lens). Bacterial cultures showed that peptide-functionalized contact lenses can drastically reduce bacterial adhesion and viability when exposed to Pseudomonas aeruginosa and Staphylococcus aureus. These systems offer the potential to minimise corneal bacterial infection and represent a suitable platform for future ophthalmic devices.

Functionalized PLGA nanoparticles prepared by nano-emulsion templating interact selectively with proteins involved in the transport through the blood-brain barrier.

During the last few decades, extensive efforts has been made to design nanocarriers to transport drugs into the central nervous system (CNS). However, its efficacy is limited due to the presence of the Blood-Brain Barrier (BBB) which greatly reduces drug penetration making Drug Delivery Systems (DDS) necessary. Polymeric nanoparticles (NPs) have been reported to be appropriate for this purpose and in particular, poly(lactic-co-glycolic acid) (PLGA) has been used for its ability to entrap small molecule drugs with great efficiency and the ease with which it functionalizes NPs. Despite the fact that their synthetic identity has been studied in depth, the biological identity of such manufactured polymers still remains unknown as does their biodistribution and in vivo fate. This biological identity is a result of their interaction with blood proteins, the so-called "protein corona" which tends to alter the behavior of polymeric nanoparticles in the body. The aim of the present research is to identify the proteins bounded to polymeric nanoparticles designed to selectively interact with the BBB. For this purpose, four different PLGA NPs were prepared and analyzed: (i) "PLGA@Drug," in which a model drug was encapsulated in its core; (ii) "8D3-PLGA" NPs where the PLGA surface was functionalized with a monoclonal anti-transferrin receptor antibody (8D3 mAb) in order to specifically target the BBB; (iii) "8D3-PLGA@Drug" in which the PLGA@Drug surface was functionalized using the same antibody described above and (iv) bare PLGA NPs which were used as a control. Once the anticipated protein corona NPs were obtained, proteins decorating both bare and functionalized PLGA NPs were isolated and analyzed. Apart from the indistinct interaction with PLGA NPs with the most abundant serum proteins, specific proteins could also be identified in the case of functionalized PLGA NPs. These findings may provide valuable insight into designing novel vehicles based on PLGA NPs for crossing the BBB.

Cubic liquid crystalline structures in diluted, concentrated and highly concentrated emulsions for topical application: Influence on drug release and human skin permeation.

Novel emulsions with a nanostructured continuous phase have been proposed as controlled drug delivery systems to enhance topical delivery of active ingredients avoiding systemic effects. In this study, oil-in-water (O/W) emulsions with two surfactant/water (S/W) weight ratios of 40:60 and 35:65, and oil concentrations of 10 wt% (diluted emulsion), 40 wt% (concentrated emulsion) and 85 wt% (highly concentrated emulsion) have been investigated to identify the presence of liquid crystalline structures and their influence on drug release and skin permeation. The emulsions have been characterized in terms of visual appearance, rheology and drug release. The presence of cubic liquid crystalline structures in emulsions with S/W 40:60 was confirmed by small angle X-ray scattering (SAXS). Rheology results showed a markedly different behaviour in emulsions with S/W 40:60 compared with nonstructured emulsions. A model drug, diclofenac sodium (DS) was successfully incorporated in the emulsions. DS release was studied with hydrophilic and lipophilic membranes, and the amount of DS in the receptor solution was significantly lower in the formulations containing cubic liquid structures. An in vitro skin permeation study with dermatomed human skin showed that emulsions with a nanostructured continuous phase are suitable formulations for topical delivery with DS retention in skin layers. The results indicate that the amount of drug retained in skin structures may be tuned by modification of liquid crystal concentration and emulsion structure.

NLCs as a potential carrier system for transdermal delivery of forskolin.

Nanostructured lipid carriers (NLC) composed of the substances generally recognized as safe (GRAS) were obtained by using a hot high-pressure homogenization technique (HPH). The influence of the number of homogenization cycles and concentration of a decyl glucoside surfactant on the NLC properties were studied. The system's stability was assessed by macroscopic observation, light backscattering and zeta potential measurements. NLC particle size was measured using dynamic light scattering (DLS). The kinetically stable formulations were loaded with forskolin and selected for in vitro drug permeation study using the Franz cell method. Concentration of forskolin in the receptor solution (i.e. ethanol/PBS mixture) was analyzed with high performance liquid chromatography (HPLC) with UV detection. The obtained results have shown that NLC formulations could be used as effective carriers for forskolin permeation through the skin.

Personalized Nanomedicine: A Revolution at the Nanoscale.

Nanomedicine is an interdisciplinary research field that results from the application of nanotechnology to medicine and has the potential to significantly improve some current treatments. Specifically, in the field of personalized medicine, it is expected to have a great impact in the near future due to its multiple advantages, namely its versatility to adapt a drug to a cohort of patients. In the present review, the properties and requirements of pharmaceutical dosage forms at the nanoscale, so-called nanomedicines, are been highlighted. An overview of the main current nanomedicines in pre-clinical and clinical development is presented, detailing the challenges to the personalization of these therapies. Next, the process of development of novel nanomedicines is described, from their design in research labs to their arrival on the market, including considerations for the design of nanomedicines adapted to the requirements of the market to achieve safe, effective, and quality products. Finally, attention is given to the point of view of the pharmaceutical industry, including regulation issues applied to the specific case of personalized medicine. The authors expect this review to be a useful overview of the current state of the art of nanomedicine research and industrial production, and the future opportunities of personalized medicine in the upcoming years. The authors encourage the development and marketing of novel personalized nanomedicines.

Nano-emulsions as vehicles for topical delivery of forskolin.

Two O/W forskolin-loaded nano-emulsions (0.075% wt.) based on medium chain triglycerides (MCT) and stabilized by a nonionic surfactant (Polysorbate 80 or Polysorbate 40) were studied as forskolin delivery systems. The nano-emulsions were prepared by the PIC method. The mean droplet size of the nano-emulsions with Polysorbate 80 and Polysorbate 40 with oil/surfactant (O/S) ratios of 20/80 and 80% water concentration, measured by Dynamic Light Scattering (DLS), was of 118 nm and 111 nm, respectively. Stability of the formulations, as assessed by light backscattering for 24 h, showed that both nano-emulsions were stable at 25°C. Studies of forskolin in vitro skin permeation from the nano-emulsions and from a triglyceride solution were carried out at 32°C, using Franz-type diffusion cells. A mixture of PBS/ethanol (60/40 v/v) was used as a receptor solution. The highest flux and permeability coefficient was obtained for the system stabilized with Polysorbate 80 (6.91±0.75 µg · cm-2·h-1 and 9.21 · 10-3±1.00 · 10-3 cm · h-1, respectively) but no significant differences were observed with the flux and permeability coefficient value of forskolin dissolved in oil. The obtained results showed that the nano-emulsions developed in this study could be used as effective carriers for topical administration of forskolin.

Studies on the formation of O/W nano-emulsions, by low-energy emulsification methods, suitable for pharmaceutical applications.

The formation of O/W nano-emulsions suitable for pharmaceutical application and the solubilisation of a practically non-water-soluble drug, lidocaine, have been studied in water/non-ionic surfactant/oil systems. Nano-emulsions were prepared by using low-energy emulsification methods, changing the composition at constant temperature. Kinetic stability was assessed by measuring droplet diameter as a function of time. Lidocaine solubilisation was studied in nano-emulsions with high water content. In the water/Cremophor EL/Miglyol 812 system the lowest droplet sizes, from 14 to 39nm at 10/90 and 40/60 oil/surfactant ratios, respectively, and 90% of water content, were obtained with an emulsification method consisting of stepwise addition of water to oil/surfactant mixtures at 70 degrees C. Nano-emulsions of this system showed high kinetic stability. Droplet diameters did not exceed 67nm after a period of at least 7 months. The maximum lidocaine concentration solubilised in nano-emulsions of the water/Cremophor EL/Miglyol 812 system with 90 and 95% of water content was 3.5 and 2.1%, respectively. These values are within the therapeutic range of lidocaine.

Transdermal delivery of forskolin from emulsions differing in droplet size.

The skin permeation of forskolin, a diterpene isolated from Coleus forsholii, was studied using oil in water (O/W) emulsions as delivery formulations and also an oil solution for comparative purposes. Two forskolin-loaded emulsions of water/Brij 72:Symperonic A7/Miglyol 812:Isohexadecane, at 0.075 wt% forskolin concentration were prepared with the same composition and only differing in droplet size (0.38 µm and 10 µm). The emulsions showed high kinetic stability at 25 °C. In vitro study of forskolin penetration through human skin was carried out using the MicroettePlus(®) system. The concentration of the active in the receptor solution (i.e. ethanol/phosphate buffer 40/60, v/v) was analyzed by high performance liquid chromatography with UV detection. The obtained results showed that forskolin permeation from the emulsions and the oil solution, through human skin, was very high (up to 72.10%), and no effect of droplet size was observed.

Polyurethane and polyurea nanoparticles based on polyoxyethylene castor oil derivative surfactant suitable for endovascular applications.

The design of new, safe and effective nanotherapeutic systems is an important challenge for the researchers in the nanotechnology area. This study describes the formation of biocompatible polyurethane and polyurea nanoparticles based on polyoxyethylene castor oil derivative surfactant formed from O/W nano-emulsions by polymerization at the droplet interfaces in systems composed by aqueous solution/Kolliphor(®) ELP/medium chain triglyceride suitable for intravenous administration. Initial nano-emulsions incorporating highly hydrophilic materials were prepared by the phase inversion composition (PIC) method. After polymerization, nanoparticles with a small particle diameter (25-55 nm) and low polydispersity index were obtained. Parameters such as concentration of monomer, O/S weight ratio as well as the polymerization temperature were crucial to achieve a correct formation of these nanoparticles. Moreover, FT-IR studies showed the full conversion of the monomer to polyurethane and polyurea polymers. Likewise the involvement of the surfactant in the polymerization process through their nucleophilic groups to form the polymeric matrix was demonstrated. This could mean a first step in the development of biocompatible systems formulated with polyoxyethylene castor oil derivative surfactants. In addition, haemolysis and cell viability assays evidenced the good biocompatibility of KELP polyurethane and polyurea nanoparticles thus indicating the potential of these nanosystems as promising drug carriers.

Quantifying the bioadhesive properties of surface-modified polyurethane-urea nanoparticles in the vascular network.

Nanomedicine research is currently requiring new standard methods to quantify the biocompatibility and bioadhesivity of emerging biomaterials designed to be used in contact with blood or soft tissues. In this study, we used biotinylated polyurethane-urea nanoparticles as a model to examine the applicabitility of an adapted hemagglutination assay to quantify the bioadhesive potential of these nanoparticles to red blood cells and, in turn, to extrapolate this data to vascular endothelial cells. We demonstrated that biotinylated nanoparticles adsorb to human erythrocytes and preferentially gather in erythrocyte contact areas. Moreover, these nanoparticles promoted a higher percentage of pig and human erythrocyte agglutination than naked polyurethane-urea nanoparticles in a biotin concentration-dependent manner. Conversely, pegylated nanoparticles were used as a negative control of the technique thus showing decreasing hemagglutination values as compared to naked nanoparticles until a minimum threshold. Furthermore, hemagglutination assay demonstrated an excellent positive correlation with bioadhesion quantification in human endothelial cells and the endothelial layer of pig aorta thus validating the hemagglutination assay described here as a useful method for predicting nanoparticle bioadhesivity to vascular endothelium. Therefore, the methodology described here is a versatile and straightforward method that allows evaluating the bioadhesive features of surface-modified polyurethane-urea nanoparticles in contact with blood and the vascular network and appears as a powerful tool to better design any drug delivery systems or implantable devices for biomedical applications.

Formation of polymeric nano-emulsions by a low-energy method and their use for nanoparticle preparation.

Formation of polymeric O/W nano-emulsions has been studied in the water/polyoxyethylene 4 sorbitan monolaurate/ethylcellulose solution system by the phase inversion composition (PIC) method. These nano-emulsions were used for the preparation of nanoparticles by solvent evaporation. Composition variables such as O/S ratio or final water content as well as emulsification path have been found to play a key role in the formation of stable, nanometer sized emulsions. Nano-emulsions with a constant water content of 90 wt.% and O/S ratios from 50/50 to 70/30 showed an average droplet size of about 200 nm as assessed by dynamic light scattering. Mean nanoparticle diameters, as determined by transmission electron microscopy image analysis, were of the order of 50 nm and showed a slight increase as well as a broader size distribution at increasing O/S ratios. The findings verify that the low-energy emulsification methods are not only valid for aliphatic and semipolar oils, but also for a highly polar solvent such as ethylacetate containing a preformed polymer.