Spray-dried raloxifene submicron particles for pulmonary delivery: Development and in vivo pharmacokinetic evaluation in rats.

Raloxifene hydrochloride (RH) is a selective oestrogen receptor modulator used for the treatment of osteoporosis. Even though 60% of an oral dose is quickly absorbed via the gastrointestinal tract, the absolute bioavailability of RH is only 2-3% in humans due to extensive first-pass metabolism. Various approaches to improve RH bioavailability have been reported over the past few years; however, none have focused on the development of products for pulmonary administration. Therefore, in this study, submicron particles containing RH were produced for pulmonary administration with the aim to limit first-pass metabolism. Powders were produced by vibrational atomisation spray drying with a high process yield (>80%). The drug content was between 440 and 890 mg·g-1, and powders had a high encapsulation efficiency (>95%), mean particle size of 400-700 nm, low residual moisture (<2%) and spherical shape. These powders showed an improved drug dissolution rate compared to the raw RH material. Moreover, they presented high dose uniformity (95-100%), a high in vitro respirable fraction (>55%) and adequate mass median aerodynamic diameter for pulmonary delivery (<5 µm). The pharmacokinetic study in male Wistar rats demonstrated an absolute bioavailability of 47.20% after pulmonary administration of the particles. Therefore, these submicron-sized powders are promising for pulmonary RH delivery as a dry powder medicine.

Hydrogel containing silibinin nanocapsules presents effective anti-inflammatory action in a model of irritant contact dermatitis in mice.

The current study developed an innovative Pemulen® TR2 hydrogel containing silibinin-loaded pomegranate oil-based nanocapsules (HP-NC SB) intending cutaneous application. The formulation anti-inflammatory activity in an in vivo model and biometric studies on the skin of healthy volunteers were also performed. The nanocapsules were prepared using the interfacial deposition of preformed polymer technique and the hydrogels were obtained by thickening of nanocapsules suspension with Pemulen® TR2. Formulations with free compound, vehicle and blank nanocapsules were also produced. The hydrogels were evaluated concerning pH, silibinin content, particle size, spreadability profile, rheology, in vitro drug release, cutaneous permeation, bioadhesive potential and cutaneous biometry evaluation. Furthermore, a model of contact dermatitis croton oil-induced in mice was performed to evaluate the hydrogels anti-inflammatory potential. The formulations presented adequate characteristics for skin administration: particle within nanometric size, pH values in the acid range, silibinin content close theoretical values (1 mg/g) and non-Newtonian pseudoplastic behavior. Nano-based hydrogels showed high bioadhesive properties, increased silibinin in vitro release profile and its retention in the stratum corneum. The best anti-inflammatory effect was exhibited by HP-NC SB, which reduced both ear edema and inflammatory cells infiltration in comparison to the induced group. Furthermore, cutaneous biometric evaluation showed that formulations containing free or nanoencapsulated silibinin caused no modification in normal skin conditions (pH, tissue hydration, transepidermal water loss and erythema). In summary, the results demonstrated that the Pemulen® TR2 hydrogel containing NC SB was successfully developed, indicating its potential as an alternative treatment for irritant contact dermatitis.

Co-Nanoencapsulation of Vitamin D3 and Curcumin Regulates Inflammation and Purine Metabolism in a Model of Arthritis.

We analyzed the effects of a nanoencapsulated association of curcumin and vitamin D3 on purine metabolism enzymes in neutrophils, lymphocytes, and platelets in a model of adjuvant-induced arthritis (AIA) in rats. Following AIA induction, the animals were treated for 15 days with free and nanoencapsulated curcumin (4 mg/kg), nanocapsules of vitamin D3 (VD3) (15.84 IU/day), a nanoencapsulated combination of curcumin and VD3, vehicle, or blank nanocapsules. The animals were euthanized, and blood was collected to evaluate the activities of E-NTPDase, adenosine deaminase (ADA), and myeloperoxidase (MPO), as well as reactive oxygen species (ROS) levels and biochemical parameters. Also, the liver and kidney were collected for histological analysis. The changes in the activities of purinergic enzymes indicated that inflammation was significantly reverted by vitamin D3 and curcumin co-nanoencapsulation treatments in the arthritic rats. The reduction of inflammation was confirmed by the reduction in the signs and symptoms of AIA, as well as in MPO activity by all formulations. The treatments with nanocapsules reverted histological alterations in the kidney. Serum parameters were not altered by the induction or treatments. Our results suggest that co-nanoencapsulation of vitamin D3 and curcumin is an efficient alternative adjuvant treatment for rheumatoid arthritis as it reverts the changes in the purine metabolism and reduces arthritis-associated inflammation.

Effects of Fish and Grape Seed Oils as Core of Haloperidol-Loaded Nanocapsules on Oral Dyskinesia in Rats.

Haloperidol is a widely used antipsychotic, despite the severe motor side effects associated with its chronic use. This study was carried out to compare oral dyskinesia induced by different formulations of haloperidol-loaded nanocapsules containing caprylic/capric triglycerides, fish oil or grape seed oil (GSO) as core, as well as free haloperidol. Haloperidol-loaded lipid-core nanocapsules formulations were prepared, physicochemical characterized and administered (0.5 mg kg-1-ip) to rats for 28 days. Oral dyskinesia was evaluated acutely and subchronically and after that cell viability and free radical generation in cortex and substantia nigra. All formulations presented satisfactory physicochemical parameters. Acutely, all formulations were able to prevent oral dyskinesia development in comparison to free haloperidol, except haloperidol-loaded nanocapsules containing GSO, whose effect was only partial. After subchronic treatment, all haloperidol-loaded nanocapsules formulations prevented oral dyskinesia in relation to free drug. Also, haloperidol-loaded nanocapsules containing fish oil and GSO were more effective than caprylic/capric triglycerides nanocapsules and free haloperidol in cell viability preservation and control of free radical generation. Our findings showed that fish oil formulation may be considered as the best formulation of haloperidol-loaded lipid-core nanocapsules, being able to prevent motor side effects associated with chronic use of antipsychotic drugs, as haloperidol.

Effects of Two Types of Melatonin-Loaded Nanocapsules with Distinct Supramolecular Structures: Polymeric (NC) and Lipid-Core Nanocapsules (LNC) on Bovine Embryo Culture Model.

Melatonin has been used as a supplement in culture medium to improve the efficiency of in vitro produced mammalian embryos. Through its ability to scavenge toxic oxygen derivatives and regulate cellular mRNA levels for antioxidant enzymes, this molecule has been shown to play a protective role against damage by free radicals, to which in vitro cultured embryos are exposed during early development. In vivo and in vitro studies have been performed showing that the use of nanocapsules as active substances carriers increases stability, bioavailability and biodistribution of drugs, such as melatonin, to the cells and tissues, improving their antioxidant properties. These properties can be modulated through the manipulation of formula composition, especially in relation to the supramolecular structures of the nanocapsule core and the surface area that greatly influences drug release mechanisms in biological environments. This study aimed to evaluate the effects of two types of melatonin-loaded nanocapsules with distinct supramolecular structures, polymeric (NC) and lipid-core (LNC) nanocapsules, on in vitro cultured bovine embryos. Embryonic development, apoptosis, reactive oxygen species (ROS) production, and mRNA levels of genes involved in cell apoptosis, ROS and cell pluripotency were evaluated after supplementation of culture medium with non-encapsulated melatonin (Mel), melatonin-loaded polymeric nanocapsules (Mel-NC) and melatonin-loaded lipid-core nanocapsules (Mel-LNC) at 10-6, 10-9, and 10-12 M drug concentrations. The highest hatching rate was observed in embryos treated with 10-9 M Mel-LNC. When compared to Mel and Mel-NC treatments at the same concentration (10-9 M), Mel-LNC increased embryo cell number, decreased cell apoptosis and ROS levels, down-regulated mRNA levels of BAX, CASP3, and SHC1 genes, and up-regulated mRNA levels of CAT and SOD2 genes. These findings indicate that nanoencapsulation with LNC increases the protective effects of melatonin against oxidative stress and cell apoptosis during in vitro embryo culture in bovine species.

Pomegranate seed oil nanoemulsions improve the photostability and in vivo antinociceptive effect of a non-steroidal anti-inflammatory drug.

The combination of pomegranate seed oil and ketoprofen in nanoemulsions aiming to improve the antinociceptive effect was evaluated according to the writhing test and Complete Freud's Adjuvant induced paw inflammation in mice. The formulations showed adequate characteristics and improved ketoprofen's photostability against UVC radiation exposure. The dialysis bag technique showed that 100% of the drug was released from the nanoemulsions after 3h and the oil amount had no influence on the releasing. Furthermore, time- and dose-response curves were obtained to determine the antinociceptive effect of the formulations. In the post-test, the nanoemulsion containing ketoprofen significantly reduced abdominal constrictions in time-response curve, showing effect up to 12h while the free ketoprofen showed effect up to 3h. In addition, the blank nanoemulsion presented a reduction of abdominal constriction up to 1h of pre-treatment. Regarding the dose-response curve, the free ketoprofen presents effect at 0.5mg/Kg dose and nanoemulsion at 1.0mg/Kg dose. Time- and dose-response curves were performed to determine the antinociceptive effect in inflammatory pain. After the evaluation of mechanical allodynia testing at the Von Frey Hair, the free ketoprofen showed effect up to 6h while nanoemulsions presented effect up to 10h. Moreover, acute toxicity was performed with ALT and AST activity evaluations and urea levels. After 7 days of treatment, no toxic effects for nanoemulsions were found. In conclusion, ketoprofen-loaded pomegranate seed oil nanoemulsions presented adequate characteristics and a high antinociceptive activity in the animal models tested.

Free and nanoencapsulated vitamin D3 : effects on E-NTPDase and E-ADA activities in an animal model with induced arthritis.

UNLABELLED: The effect of vitamin D3 in oral solution (VD3 ) and vitamin D3 -loaded nanocapsules (NC-VD3 ) was analysed in animals with complete Freund's adjuvant (CFA) induced arthritis (AR). For this purpose, we evaluated scores for arthritis, thermal hyperalgesia and paw oedema, as well as histological analyses and measurements of the activity of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) and ecto-adenosine deaminase (E-ADA) enzymes in rat lymphocytes. Haematological and biochemical parameters were also determined. The doses administered were 120 UI/day of VD3 and 15.84 UI/day of NC-VD3 . Fifteen days after the induction of AR, the groups were treated for 15 days with vitamin D3 . The results demonstrated that VD3 was able to reduce arthritis scores, thermal hyperalgesia and paw oedema in rats with CFA-induced arthritis. However, treatment with NC-VD3 did not reduce arthritis scores. The histological analyses showed that both formulations were able to reduce the inflammatory changes induced by CFA. The activity of E-NTPDase in rat lymphocytes was higher in the AR compared with the control group, while the activity of E-ADA was lower. This effect was reversed after the 15-day treatment. Data from this study indicates that both forms of vitamin D3 seem to contribute to decreasing the inflammatory process induced by CFA, possibly altering the activities of ectoenzymes. Copyright © 2016 John Wiley & Sons, Ltd. SIGNIFICANCE OF THE STUDY: The effects promoted by both formulations of vitamin D3 , either in oral solution or nanoencapsulated form, strongly suggests the softening of the inflammatory process induced by complete Freund's adjuvant (CFA), possibly altering the E-NTPDase and E-ADA activities. However, it is known that vitamin D has a beneficial effect on the modulation of the immune system components responsible for the inflammatory process. Moreover, the establishment of responses to treatment with vitamin D3 may provide an alternative for inhibiting the proinflammatory response, assisting in our understanding of the immunopathology of this disease and possibly improving the signs and symptoms that hinder the quality of life of patients with rheumatoid arthritis. HIGHLIGHTS: Evaluation of the effects on the E-NTPDase and E-ADA activities in an animal model of induced arthritis. Two formulations of vitamin D3 were used: form oral solution and nanoencapsulated. Vitamin D3 seems to contribute to the inflammatory process induced by CFA. Vitamin D3 possibly alters the E-NTPDase and E-ADA activities. Vitamin D3 may be an alternative supplementary treatment for chronic arthritis.

Tretinoin-loaded lipid-core nanocapsules decrease reactive oxygen species levels and improve bovine embryonic development during in vitro oocyte maturation.

In vitro oocyte maturation (IVM) protocols can be improved by adding chemical supplements to the culture media. Tretinoin is considered an important retinoid in embryonic development and its association with lipid-core nanocapsules (TTN-LNC) represents an innovative way of improving its solubility, and chemical stability, and reducing its toxicity. The effects of supplementing IVM medium with TTN-LNC was evaluated by analyzing production of reactive oxygen species (ROS), S36-phosphorilated-p66Shc levels and caspase activity in early embryonic development, and expression of apoptosis and pluripotency genes in blastocysts. The lowest concentration tested (0.25µM) of TTN-LNC generated higher blastocyst rate, lower ROS production and S36-p66Shc amount. Additionally, expression of BAX and SHC1 were lower in both non-encapsulated tretinoin (TTN) and TTN-LNC-treated groups. Nanoencapsulation allowed the use of smaller concentrations of tretinoin to supplement IVM medium thus reducing toxic effects related with its use, decreasing ROS levels and apoptose frequency, and improving the blastocyst rates.

Nanoencapsulation of rice bran oil increases its protective effects against UVB radiation-induced skin injury in mice.

Excessive UV-B radiation by sunlight produces inflammatory and oxidative damage of skin, which can lead to sunburn, photoaging, and cancer. This study evaluated whether nanoencapsulation improves the protective effects of rice bran oil against UVB radiation-induced skin damage in mice. Lipid-core nanocapsules containing rice bran oil were prepared, and had mean size around 200 nm, negative zeta potential (∼-9 mV), and low polydispersity index (<0.20). In order to allow application on the skin, a hydrogel containing the nanoencapsulated rice bran oil was prepared. This formulation was able to prevent ear edema induced by UVB irradiation by 60 ± 9%, when compared with a hydrogel containing LNC prepared with a mixture of medium chain triglycerides instead of rice bran oil. Protein carbonylation levels (biomarker of oxidative stress) and NF-κB nuclear translocation (biomarker of pro-inflammatory and carcinogenesis response) were reduced (81% and 87%, respectively) in animals treated with the hydrogel containing the nanoencapsulated rice bran oil. These in vivo results demonstrate the beneficial effects of nanoencapsulation to improve the protective properties of rice bran oil on skin damage caused by UVB exposure.

Redispersible liposomal-N-acetylcysteine powder for pulmonary administration: development, in vitro characterization and antioxidant activity.

Liposomal dry powders of N-acetylcysteine (SD-NAC-Lip) were developed for pulmonary administration. Liposomes were prepared by reverse phase evaporation and spray dried using lactose (10%, w/w) as drying adjuvant. The powders were characterized according to process yield, drug content, residual water content, particle size distribution, morphology and redispersion behavior. In vitro aerosol performance was evaluated using an eight-stage Andersen Cascade Impactor. Moreover, in vitro antioxidant activity was determined by measuring thiobarbituric acid reactive species (TBARS) present in the lungs of healthy Wistar rats after induction of oxidation by iron/EDTA. The spray-drying process had a high yield (71%±2), drug content (mg/g) according to the expected value, moisture content below 9%, geometric mean diameter under 3µm with span value lower than 1. Spherical particles were observed by scanning electron microscopy. Liposomal dry-powders were able to recover the nanometric size of the original dispersion after their redispersion in aqueous medium, as shown by laser diffraction and transmission electron microscopy. Furthermore, the powders presented aerodynamic diameter of about 7µm and respirable fraction above 30%, indicating suitable properties for pulmonary use. The encapsulation of N-acetylcysteine in liposomes was essential to maintain its in vitro antioxidant activity after the drying process. In addition, the powder containing the encapsulated drug had better in vitro antioxidant activity than the liquid and solid formulations containing the non-encapsulated drug, which makes it a good candidate for the treatment of pulmonary diseases associated with oxidative stress.

Influence of the type of vegetable oil on the drug release profile from lipid-core nanocapsules and in vivo genotoxicity study.

The use of rice bran (RB), soybean (SB) or sunflower seed (SF) oils to prepare lipid-core nanocapsules (LNCs) as controlled drug delivery systems was investigated. LNCs were prepared by interfacial deposition using the preformed polymer method. All formulations showed negative zeta potential and adequate nanotechnological characteristics (particle size 220-230 nm, polydispersity index < 0.20). The environmental safety was evaluated through an in vivo protocol (Allium cepa test) and LNCs containing RB, SB or SF oils did not present genotoxic potential. Clobetasol propionate (CP) was selected as a model drug to evaluate the influence of the type of vegetable oil on the control of the drug release from LNCs. Biphasic drug release profiles were observed for all formulations. After 168 h, the concentration of drug released from the formulation containing SF oil was lower (0.36 mg/mL) than from formulations containing SB (0.40 mg/mL) or RB oil (0.45 mg/mL). Good correlations between the consistency indices for the LNC cores and the burst and sustained drug release rate constants were obtained. Therefore, the type of the vegetal oil was shown as an important factor governing the control of drug release from LNCs.

Curcumin-loaded lipid-core nanocapsules as a strategy to improve pharmacological efficacy of curcumin in glioma treatment.

In this study, we developed curcumin-loaded lipid-core nanocapsules (C-LNCs) in an attempt to improve the antiglioma activity of this polyphenol. C-LNC showed nanotechnological properties such as nanometric mean size (196 nm), 100% encapsulation efficiency, polydispersity index below 0.1, and negative zeta potential. The in vitro release assays demonstrated a controlled release of curcumin from lipid-core nanocapsules. In C6 and U251MG gliomas, C-LNC promoted a biphasic delivery of curcumin: the first peak occurred early in the treatment (1-3h), whereas the onset of the second phase occurred after 48 h. In C6 cells, the cytotoxicity of C-LNC was comparable to non-encapsulated curcumin only after 96 h, whereas C-LNCs were more cytotoxic than non-encapsulated curcumin after 24h of incubation in U251MG. Induction of G2/M arrest and autophagy were observed in C-LNC as well as in free-curcumin treatments. In rats bearing C6 gliomas, C-LNC (1.5mg/kg/day, i.p.) decreased the tumor size and malignance and prolonged animal survival when compared to same dose of non-encapsulated drug. In addition, serum markers of tissue toxicity and histological parameters were not altered. Considered overall, the data suggest that the nanoencapsulation of curcumin in LNC is an important strategy to improve its pharmacological efficacy in the treatment of gliomas.

Improved photostability and reduced skin permeation of tretinoin: development of a semisolid nanomedicine.

The aims of this work were to increase the photostability and to reduce the skin permeation of tretinoin through nanoencapsulation. Tretinoin is widely used in the topical treatment of various dermatological diseases such as acne, psoriasis, skin cancer, and photoaging. Tretinoin-loaded lipid-core polymeric nanocapsules were prepared by interfacial deposition of a preformed polymer. Carbopol hydrogels containing nanoencapsulated tretinoin presented a pH value of 6.08±0.14, a drug content of 0.52±0.01 mg g(-1), pseudoplastic rheological behavior, and higher spreadability than a marketed formulation. Hydrogels containing nanoencapsulated tretinoin demonstrated a lower photodegradation (24.17±3.49%) than the formulation containing the non-encapsulated drug (68.64±2.92%) after 8h of ultraviolet A irradiation. The half-life of the former was seven times higher than the latter. There was a decrease in the skin permeability coefficient of the drug by nanoencapsulation, independently of the dosage form. The liquid suspension and the semisolid form provided K(p)=0.31±0.15 and K(p)=0.33±0.01 cm s(-1), respectively (p≤0.05), while the samples containing non-encapsulated tretinoin showed K(p)=1.80±0.27 and K(p)=0.73±0.12 cm s(-1) for tretinoin solution and hydrogel, respectively. Lag time was increased two times by nanoencapsulation, meaning that the drug is retained for a longer time on the skin surface.