Iron oxide nanoparticles for magnetically-guided and magnetically-responsive drug delivery.

In this review, we discuss the recent advances in and problems with the use of magnetically-guided and magnetically-responsive nanoparticles in drug delivery and magnetofection. In magnetically-guided nanoparticles, a constant external magnetic field is used to transport magnetic nanoparticles loaded with drugs to a specific site within the body or to increase the transfection capacity. Magnetofection is the delivery of nucleic acids under the influence of a magnetic field acting on nucleic acid vectors that are associated with magnetic nanoparticles. In magnetically-responsive nanoparticles, magnetic nanoparticles are encapsulated or embedded in a larger colloidal structure that carries a drug. In this last case, an alternating magnetic field can modify the structure of the colloid, thereby providing spatial and temporal control over drug release.

Pharmacokinetics of resveratrol metabolic profile in healthy humans after moderate consumption of red wine and grape extract tablets.

A pharmacokinetic study of the metabolic profile of resveratrol has been performed in healthy men after moderate red wine (RW) consumption. The bioavailability of resveratrol is highly influenced by several factors such as the food matrix and, therefore, this study has been compared with a pilot study in which men ingested grape extract (GE) tablets as a nutraceutical, containing similar total amounts of resveratrol than RW. Blood and urine samples were taken before and at several time points after intervention and then analyzed by SPE and LC-ESI-MS/MS. Up to 17 resveratrol and piceid derivatives were identified, including those formed by the intestinal microbiota. Resveratrol glucosides were found in plasma as intact forms and reached the lowest maximum concentrations 1h after both interventions. Higher plasma concentrations and longer times (t(max)) were observed for resveratrol glucuronides due to phase II metabolism and even higher values for conjugates derived from microbiota, such as dihydroresveratrol-glucuronides. The same trend was observed for total excreted amounts in urine samples. When both treatments were compared, statistically significant differences for some metabolites were obtained, which may be due to the different composition of resveratrol and piceid in both sources. However, GE formulation seems to delay resveratrol absorption, staying longer in the gut where could be metabolized to a greater degree, since 2.1-3.6-fold higher urinary concentrations of microbial metabolites were observed after GE intervention at 12-24h urinary fraction. Therefore, supplement intake could be also a way to bring resveratrol benefits to human health.

Analgesic and antiallodynic effects of antidepressants after infiltration into the rat.

Tricyclic antidepressants (TCA) have potent local anesthetic properties and may produce a long-lasting pain blockade that could be of interest in relieving chronic pain states such as neuropathic pain, but there are only few data comparing their dose-response curves of analgesic activity under the same experimental conditions. This study examines the time course of pain-relieving properties of 7 TCA in heat-induced paw withdrawal after subcutaneous administration. Mixed inhibitors of norepinephrine and serotonin uptake (amitriptyline, nortriptyline, imipramine, desipramine, doxepin) and selective inhibitors of serotonin uptake (fluoxetine and fluvoxamine) were assayed. The TCA with the longest analgesic activity were selected to test its antiallodynic effect in the neuropathic pain model of chronic sciatic nerve constriction injury. Bupivacaine was used as a reference drug in both experiments. A dose versus time of maximal analgesic effect curve was constructed for each drug. The longest analgesic effect was obtained for doxepin and imipramine. Although low doses of amitriptyline showed the same activity than doxepin, higher doses failed to reach the same effect. Selective inhibitors of serotonin showed no action at all doses tested. In the chronic sciatic nerve constriction injury model, doxepin and, to a smaller degree, amitriptyline and imipramine protected from allodynia; bupivacaine was ineffective. The antiallodynic effect always lasted less long than the analgesic effect. These observations provide support for the potential use of TCA as durable analgesics. Doxepin overall showed the most outstanding results in pain relief.

Absorption and pharmacokinetics of green tea catechins in beagles.

The present study evaluates for the first time in dogs, the kinetics of green tea catechins and their metabolic forms in plasma and urine. Ten beagles were administered 173 mg (12.35 mg/kg body weight) of catechins as a green tea extract, in capsules. Blood samples were collected during 24 h after intake and urine samples were collected during the following periods of time: 0-2, 2-6, 6-8 and 8-24 h. Two catechins with a galloyl moiety and three conjugated metabolites were detected in plasma. Most of the detected forms in plasma reached their maximum plasma concentration (Cmax) at around 1 h. Median Cmax for ( - )-epigallocatechin-3-gallate (EGCG), ( - )-epicatechin-3-gallate (ECG), ( - )-epigallocatechin glucuronide (EGC-glucuronide), ( - )-epicatechin glucuronide (EC-glucuronide), ( - )-epicatechin sulphate (EC-sulphate) were 0.3 (range 0.1-1.9), 0.1 (range 0-0.4), 0.8 (range 0.2-3.9), 0.2 (range 0.1-1.7) and 1 (range 0.3-3.4) micromol/l, respectively. The areas under the plasma concentration v. time curves (AUC0 --> 24) were 427 (range 102-1185) micromol/l x min for EGC-glucuronide, 112 (range 53-919) micromol/l x min for EC-sulphate, 71 (range 26-306) micromol/l x min for EGCG, 40 (range 12-258) micromol/l x min for EC-glucuronide and 14 (range 0.1-124) micromol/l x min for ECG. The values of mean residence time (MRT0 --> 24) were 5 (range 2-16), 2 (range 1-11), 10 (range 2-13), 3 (range 2-16) and 2.4 (range 1-18) h for EGCG, ECG, EGC-glucuronide, EC-glucuronide and EC-sulphate, respectively. In urine, catechins were present as conjugated forms, suggesting bile excretion of EGCG and ECG. Green tea catechins are absorbed following an oral administration and EGC-glucuronide is the metabolic form that remains in the organism for a longer period of time, suggesting that this compound could suffer an enterohepatic cycle.

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.

Thymus essential oil extraction, characterization and incorporation in phospholipid vesicles for the antioxidant/antibacterial treatment of oral cavity diseases.

The aim of the work was to extract, characterize, and formulate Thymus capitatus (Tymbra capitata) essential oil in phospholipid vesicles: liposomes, glycerosomes and Penetration Enhancer-containing Vesicles (PEVs). The steam-distilled essential oil was mainly composed of carvacrol. The oil was mixed with lecithin and water to produce liposomes, or different ratios of water/glycerol or water/propylene glycol (PG) to produce glycerosomes and PG-PEVs, respectively. Cryo-TEM showed the formation of unilamellar, spherical vesicles, and light scattering disclosed that their size increased in the presence of glycerol or PG, which improved long-term stability. The formulations were highly biocompatible, and capable of counteracting oxidative stress and favouring wound repair in keratinocytes, thanks to enhanced uptake. The antibacterial activity of the oil was demonstrated against cariogenic Streptococcus mutans, Lactobacillus acidophilus, and commensal Streptococcus sanguinis. The combination of antioxidant and antibacterial activities of Thymus essential oil formulations may be useful for the treatment of oral cavity diseases.

Chitosan and hyaluronan coated liposomes for pulmonary administration of curcumin.

Aiming at improving the nebulization performances and lung antioxidant protection of curcumin, chitosan or hyaluronan-coated liposomes were prepared and their characteristics and performances were compared with that of uncoated liposomes. Curcumin loaded liposomes displayed a diameter lower than 100nm, the coating with both polymers led to a small increase of vesicle size around 130nm and the zeta potential turned to positive values using chitosan while remained negative using hyaluronan. Chitosan allowed the formation of more lamellar and stiffer vesicles with a higher bilayer thickness (dB∼59Ǻ) with respect to the uncoated liposomes, whereas hyaluronan allowed the interdigitation of the bilayers (dB∼47Ǻ) due to the polymer intercalation between phospholipid head groups resulting in vesicles mainly organized in uncorrelated bilayers. Both polymer coatings, especially hyaluronan, greatly improved the stability of the vesicles, especially during the nebulization process, promoting the deposition of the phytodrug in the furthest stages of the impactor in high amount (≥50%). Polymer coated vesicles were biocompatible and improved the curcumin ability to protect A549 cells from the oxidative stress induced by hydrogen peroxide, restoring healthy conditions (cell relative metabolic activity 100%). In particular, a synergic effect of curcumin and hyaluronan was observed resulting in a proliferative effect and a subsequent further enhancement of cell relative metabolic activity up to 120%.

Oil-in-water nanoemulsions are suitable for carrying hydrophobic compounds: Indomethacin as a model of anti-inflammatory drug.

Oil-in-water nanoemulsions are increasingly being used as delivery systems for encapsulating lipophilic components in functional food, personal care and pharmaceutical products. In the current study, we developed a multimodal platform to carry hydrophobic indomethacin or magnetic nanoparticles, or both. As a consequence, this platform has great potential for therapeutic or imaging purposes. By optimizing the system composition and homogenization conditions, a nanoemulsion with a mean droplet diameter of about 200nm and a low polydispersity index (<0.2) was formed. The plain nanoemulsion was shown to be innocuous in cellular studies and did not present acute toxicity (observed in a rat model). More interesting was the finding that nanoemulsions loaded with indomethacin presented a significantly different anti-inflammatory than the free drug.

Combination of argan oil and phospholipids for the development of an effective liposome-like formulation able to improve skin hydration and allantoin dermal delivery.

Allantoin is traditionally employed in the treatment of skin ulcers and hypertrophic scars. In the present work, to improve its local deposition in the skin and deeper tissues, allantoin was incorporated in conventional liposomes and in new argan oil enriched liposomes. In both cases, obtained vesicles were unilamellar, as confirmed by cryo-TEM observation, but the addition of argan oil allowed a slight increase of the mean diameter (∼130nm versus ∼85nm). The formulations, especially those containing argan oil, favoured the allantoin accumulation in the skin, in particular in the dermis (∼8.7µg/cm(2)), and its permeation through the skin (∼33µg/cm(2)). The performances of vesicles as skin delivery systems were compared with those obtained by water dispersion of allantoin and the commercial gel, Sameplast(®). Moreover, in this work, for the first time, the elastic and viscous moduli of the skin were measured, underlining the different hydrating/moisturizing effects of the formulations. The application of ARG liposomes seems to provide a softening and relaxing effect on the skin, thus facilitating the drug accumulation and passage into and trough it.

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.

The tomato sauce making process affects the bioaccessibility and bioavailability of tomato phenolics: a pharmacokinetic study.

Tomato sauce is the most commonly consumed processed tomato product worldwide, but very little is known about how the manufacturing process may affect the phenolic composition and bioavailability after consumption. In a prospective randomised, cross-over intervention study, we analysed the plasma and urinary levels of tomato phenolic compounds and their metabolites after acute consumption of raw tomatoes and tomato sauce, enriched or not with refined olive oil during production. Respectively, eleven and four phenolic metabolites were found in urine and plasma samples. The plasma concentration and urinary excretion of naringenin glucuronide were both significantly higher after the consumption of tomato sauce than raw tomatoes. The results suggest that the mechanical and thermal treatments during tomato sauce manufacture may help to deliver these potentially bioactive phenolics from the food matrix more effectively than the addition of an oil component, thus increasing their bioavailability.

Identification and nanoentrapment of polyphenolic phytocomplex from Fraxinus angustifolia: in vitro and in vivo wound healing potential.

The aim of the present study was to elucidate the polyphenolic composition of Fraxinus angustifolia leaf and bark extracts, and to evaluate their efficacy in wound healing. Quercetin, catechin, rutin and tannic acid were identified as the main components of the extracts. In order to improve their skin bioavailability, the polyphenolic phytocomplexes were incorporated in different nanovesicles, namely ethosomes and phospholipid vesicles containing Transcutol(®) P (Trc) or ethylene glycol (EG). The latter had never been used before as a component of phospholipid vesicles, and it was found to play a key role in improving extract efficacy in wound healing. Results of cryogenic transmission electron microscopy (cryo-TEM), Photon Correlation Spectroscopy (PCS) and Small-Angle X-ray Scattering (SAXS) showed that ethosomes and EG-PEVs were small, monodispersed, unilamellar vesicles, while Trc-PEVs were larger, less homogeneously dispersed and multilamellar, with a large bilayer thickness. Free extracts did not show relevant ability to protect in vitro human keratinocytes from H2O2 damages, while when entrapped in nanovesicles, they significantly inhibited H2O2 stress damages, probably related to a higher level of cell uptake. On the other hand, in vivo results showed that the highest antioxidant and anti-inflammatory effects were provided by the phytocomplexes in EG-PEVs, which favoured wound healing. Moreover, non-entrapped F. angustifolia extracts showed a marginal effect, comparable to that of free quercetin dispersion (control). In conclusion, our results depict that these extracts may find potential applications in biomedicine.

Influence of olive oil on carotenoid absorption from tomato juice and effects on postprandial lipemia.

The potential benefits of tomato-rich diets for the cardiovascular system have been related to plasma concentrations of carotenoids. In addition, the bioavailability of carotenoids from foods depends on their chemical structure, processing and the food matrix. Our aim was to evaluate the effect of adding oil to tomato juice (not treated with heat) on the bioavailability of plasma carotenoids and postprandial lipid response. In a randomized, controlled, crossover feeding trial, eleven healthy volunteers were assigned to receive a single ingestion of 750g of tomato juice (TJ) containing 10% of refined olive oil/70kg body weight (BW) and 750g of TJ without oil/70kg BW on two different days. All lycopene isomers increased significantly in subjects consuming TJ with oil, reaching the maximum concentration at 24h. LDL cholesterol and total cholesterol decreased significantly 6h after the consumption of TJ with oil, which significantly correlated with an increase of trans-lycopene and 5-cis-lycopene, respectively.

Thermal hyperalgesia and light touch allodynia after intradermal Mycobacterium butyricum administration in rat.

We examined the time course (7 weeks) of thermal hyperalgesia and light touch allodynia in rats after intradermal administration of Mycobacterium butyricum. Nociceptive thresholds to heat and light touch were assessed. Paw edema and temperature, motor function, body weight, and propioception were also tested. Some rats developed arthritis (named AA rats) but others did not (named non-AA rats). Both groups were compared with healthy animals. Persistent hyperalgesia was found in both groups; in AA rats it appeared before clinical evidence of arthritis. Transient allodynia ocurred only after edema development and fell when edema decreased. Motor function was impaired only in AA rats. The results of this study demonstrate that hyperalgesia, but not allodynia, appeared after Mycobacterium butyricum in both groups, suggesting that changes in sensitivity were not merely the result of local hypersensitivity of the inflamed tissue, but may also be due to alterations in nociception in the central nervous system.

Assessment of diclofenac permeation with different formulations: anti-inflammatory study of a selected formula.

The aim of this study was to improve the transdermal permeation of sodium diclofenac. Permeation studies were carried out in vitro using human skin (0.4 mm thick) from plastic surgery as a membrane. Four liquid formulations of 1% (w/w) sodium diclofenac were assayed: three ternary solvent systems (M4, M5, M6) and one microemulsion (M3). A 1% (w/w) solution of sodium diclofenac and a commercially available semisolid preparation were tested as reference formulations. The following permeation parameters for diclofenac were assessed: permeability coefficient, flux and drug permeated and retained in the skin at 24 h. The highest values of these parameters were obtained with formula M4, which contains transcutol 59.2%, oleic acid 14.9% and d-limonene 5% (w/w) as permeation enhancers. The anti-inflammatory activity of this formula was compared with that of the semisolid preparation on carrageenan-induced paw edema in rats. As expected from in vitro results, the M4 diclofenac delivery system showed higher activity than the semisolid preparation, both when applied locally (to the inflammation area) and when applied systemically (to the back). Neither treatment irritated the skin when tested on rabbits in a 72-h trial. These results suggest that topical delivery of sodium diclofenac with an absorption enhancer such as a mixture of oleic acid and d-limonene (M4) may be an effective medication for both dermal and subdermal injuries.

Improvement of quercetin protective effect against oxidative stress skin damages by incorporation in nanovesicles.

Quercetin was incorporated in glycerosomes, new phospholipid-glycerol vesicles, and their protective effect against oxidative stress skin damages was extensively evaluated. In particular, the concentration-dependent effect of glycerol (from 10 to 50%) on vesicle suitability as cutaneous carriers of quercetin was carefully assessed. All vesicles were unilamellar and small in size (∼80-110 nm), as confirmed by cryo-TEM observation, with a drug incorporation efficiency ranging between 81 and 91%. SAXS studies, performed to investigate the bilayer arrangement, indicated a strong, dose-dependent interaction of glycerol with the polar portions of the phospholipid molecules, while quercetin did not significantly change the bilayer packing. In vitro studies on newborn pig skin underlined the concentration-dependent ability of glycerosomes to promote quercetin accumulation in the different layers, also confirmed by confocal microscopic observation of skin treated with fluorescent vesicles. Quercetin incorporated into liposomal and glycerosomal nanoformulations showed a strong ability to scavenge free radicals (DPPH test) and protect human keratinocytes in vitro against hydrogen peroxide damage. Moreover, quercetin-loaded vesicles were avidly taken up by keratinocytes in vitro. Overall, results indicate 40 and 50% glycerosomes as promising nanosystems for the improvement of cutaneous quercetin delivery and keratinocyte protection against oxidative stress damage.

External magnetic field-induced selective biodistribution of magnetoliposomes in mice.

This study looked at the effect of an external magnet on the biodistribution of magnetoliposomes intravenously administrated in mice (8 mg iron/kg) with and without induced acute inflammation. Our results showed that due to enhanced vascular permeability, magnetoliposomes accumulated at the site of inflammation in the absence of an external magnetic field, but the amount of iron present increased under the effect of a magnet located at the inflammation zone. This increase was dependent on the time (20 or 60 min) of exposure of the external magnetic field. It was also observed that the presence of the magnet was associated with lower amounts of iron in the liver, spleen, and plasma than was found in mice in which a magnet had not been applied. The results of this study confirm that it is possible to target drugs encapsulated in magnetic particles by means of an external magnet.

Single-pass intestinal perfusion to establish the intestinal permeability of model drugs in mouse.

The aim of the present work was to study the intestinal permeabilities (P(eff)) of five model drugs: furosemide, piroxicam, naproxen, ranitidine and amoxicillin in the in situ intestinal perfusion technique in mice and compare them with corresponding rat and human in vivo P(eff) values. The main experimental conditions were: mice CD1 30-35 g, test drug concentrations in perfusion experiments (the highest dose strength dissolved in 250 mL of PBS pH 6.2) and flow rate of 0.2 mL/min. The test compounds were assayed following a validated HPLC method. The effective permeability coefficients at steady-state were calculated after correcting the outlet concentration following the gravimetric correction method proposed by Sutton et al. (2001). The permeability coefficient values ranged from 0.1751±0.0756×10(-4) cm/s for ranitidine to 17.19±4.16×10(-4) cm/s for naproxen. The mouse method correctly assigned the BCS permeability classification of a given drug and a correlation between mouse permeability data and the fraction of an oral dose absorbed in humans was achieved (FA=1-exp(-34,745·P(eff(mouse))); R=0.9631). Based on the results obtained, we conclude that mouse can be considered a valuable tool in the evaluation of intestinal permeability in order to predict the extent of human gastrointestinal absorption following oral administration of a drug.

Effect of magnet implant on iron biodistribution of Fe@C nanoparticles in the mouse.

The in vivo biodistribution of Fe@C nanoparticles (NP) was tested in mice bearing an inflammatory focus induced by injecting carrageenan into an air pouch previously formed on their back. The animals were intravenously injected NP with a high (60 mg/kg) or a low iron dose (6 mg/kg) and sacrificed 2 h later. Blood and organ samples (liver, spleen, lung, and kidney) were obtained; washed exudates were also collected. Iron concentration in plasma, blood cells, organs, and exudates was determined by flameless atomic-absorption-spectroscopy after digestion of organic material. Pouch exudate volume increased in all groups of mice with experimental inflammation. After i.v. administration of the high and low dose of NP, iron in exudate increased by 83.3% and 92.2%, respectively. A similar increase in hepatic iron appeared after the high dose (78%), but no increase appeared after the low dose. When the magnet was present, a 157% and 119% increase of iron in exudate appeared after both doses of NPs, but only the high dose of NP increased iron liver (60%). The presence of a magnetic field in the pouch favored selective biodistribution of NP in the inflammatory focus. These results indicate that mice with an inflammatory compartment are suitable for primary screening of different NP types. They also show that selective biodistribution is greater when a low dose of NP was used and that distribution in the target organ was increased by the magnetic field.

Magnetoliposomes prepared by reverse-phase followed by sequential extrusion: characterization and possibilities in the treatment of inflammation.

Anionic ferrofluid was encapsulated in 200nm-diameter liposomes. The process involved phase-reverse evaporation followed by sequential extrusion. Magnetoliposomes were characterized by transmission electron microscopy, Doppler laser electrophoresis, SQUID magnetometry, dynamic light scattering and iron content by atomic absorption spectrophotometry. The absence of hysteresis of the magnetic power of particles at room temperature is characteristic of a material with superparamagnetic properties. The encapsulation efficiency was determined for several iron/phospholipid ratios, and this parameter ranged from 0.016 to 0.024mg iron per mmole of phospholipids, depending on the initial magnetite concentration. In comparison with magnetoliposomes that were obtained solely by extrusion, this method afforded significantly better encapsulation (P=0.0002). Magnetic particles were intravenously administered to healthy or inflammation-induced mice. After 1h, the content of iron was determined in exudates, liver, spleen and plasma. Magnetoliposomes accumulated in the exudates collected from the inflammation site, which suggests that these particles could be loaded with the drugs needed to treat some inflammatory processes.