Effect of nanocapsules containing docosahexaenoic acid in mice with chronic inflammation.

BACKGROUND: Omega 3 fatty acids, such as docosahexaenoic acid (DHA) have been widely consumed as supplements to control chronic inflammation. Nanocapsules containing DHA (MLNC-DHA-a1) were developed and showed excellent stability. Thus, our objective was to evaluate the effect of MLNC-DHA-a1 nanocapsules on biomarkers of chronic inflammation. METHODS: Cells viability was determined by flow cytometry. The uptake of MLNC-DHA-a1 nanocapsules by macrophages and their polarization were determined. In vivo, LDLr(-,-) mice were fed a Western diet to promote chronic inflammation and were treated with MLNC-DHA-a1 nanocapsules, intravenously injected via the caudal vein once a week for 8 weeks. RESULTS: MLNC-DHA-a1 nanocapsules decreased the concentration of TNFα (p = 0.02) in RAW 264.7 cells compared to the non-treated group (NT), with no changes in IL-10 (p = 0.29). The nanocapsules also exhibited an increase in the M2 (F4/80+ CD206) phenotype (p < 0.01) in BMDM cells. In vivo, no difference in body weight was observed among the groups, suggesting that the intervention was well tolerated. However, compared to the CONT group, MLNC-DHA-a1 nanocapsules led to an increase in IL-6 (90.45 ×13.31 pg/mL), IL-1ß (2.76 ×1.34 pg/mL) and IL-10 (149.88 ×2.51 pg/mL) levels in plasma. CONCLUSION: MLNC-DHA-a1 nanocapsules showed the potential to promote in vitro macrophage polarization and were well-tolerated in vivo. However, they also increased systemic pro-inflammatory cytokines. Therefore, considering that this immune response presents a limitation for clinical trials, further studies are needed to identify the specific compound in MLNC-DHA-a1 that triggered the immune response. Addressing this issue is essential, as MLNC-DHA-a1 tissue target nanocapsules could contribute to reducing chronic inflammation.

The Anti-Arthritic Activity of Diclofenac Lipid-Core Nanocapsules: Stereological Analysis Showing More Protection of Deep Joint Components.

Diclofenac is the most prescribed nonsteroidal anti-inflammatory drug worldwide and is used to relieve pain and inflammation in inflammatory arthritis. Diclofenac is associated with serious adverse effects, even in regular-dose regimens. Drug delivery systems can overcome this issue by reducing adverse effects and optimizing their efficacy. This study evaluated the activity of lipid-core nanocapsules loaded with diclofenac (DIC-LNCs) in an experimental model of adjuvant-induced arthritis. The diclofenac nanoformulation was obtained via self-assembly. A stereological analysis approach was applied for the morphological quantification of the volume, density, and cellular profile count of the metatarsophalangeal joints of rats. Proinflammatory cytokines and biochemical profiles were also obtained. Our results showed that the diclofenac nanocapsule DIC-LNCs were able to reduce arthritis compared with the control group and the DIC group. DIC-LNCs efficiently reduced proinflammatory cytokines, C-reactive protein, and xanthine oxidase levels. Additionally, DIC-LNCs reduced the loss of synoviocytes and chondrocytes compared with the DIC (p < 0.05) and control groups (p < 0.05). These data suggest that DIC-LNCs have anti-arthritic activity and preserve joint components, making them promising for clinical use.

Folic Acid-Doxorubicin-Double-Functionalized-Lipid-Core Nanocapsules: Synthesis, Chemical Structure Elucidation, and Cytotoxicity Evaluation on Ovarian (OVCAR-3) and Bladder (T24) Cancer Cell Lines.

PURPOSE: Folic acid-doxorubicin-double-functionalized-lipid-core nanocapsules (LNC-CS-L-Zn+2-DOX-FA) were prepared, characterized, and evaluated in vitro against ovarian and bladder cancer cell lines (OVCAR-3 and T24). METHODS: LNC-CS-L-Zn+2-DOX-FA was prepared by self-assembly and interfacial reactions, and characterized using liquid chromatography, particle sizing, transmission electron microscopy, and infrared spectroscopy. Cell viability and cellular uptake were studied using MTT assay and confocal microscopy. RESULTS: The presence of lecithin allows the formation of nanocapsules with a lower tendency of agglomeration, narrower size distributions, and smaller diameters due to an increase in hydrogen bonds at the surface. LNC-L-CS-Zn+2-DOX-FA, containing 98.00 ± 2.34 µg mL-1 of DOX and 105.00 ± 2.05 µg mL-1 of FA, had a mean diameter of 123 ± 4 nm and zeta potential of +12.0 ± 1.3 mV. After treatment with LNC-L-CS-Zn+2-DOX-FA (15 µmol L-1 of DOX), T24 cells had inhibition rates above 80% (24 h) and 90% (48 h), whereas OVCAR-3 cells showed inhibition rates of 68% (24 h) and 93% (48 h), showing higher cytotoxicity than DOX.HCl. The fluorescent-labeled formulation showed a higher capacity of internalization in OVCAR-3 compared to T24 cancer cells. CONCLUSION: Lecithin favored the increase of hydrogen bonds at the surface, leading to a lower tendency of agglomeration for nanocapsules. LNC-CS-L-Zn+2-DOX-FA is a promising therapeutic agent against tumor-overexpressing folate receptors.

Dermatopharmacokinetic and pharmacodynamic evaluation of a novel nanostructured formulation containing capsaicinoids for treating neuropathic pain.

The in vivo skin penetration by dermal microdialysis and the pharmacological efficacy of a chitosan hydrogel containing capsaicinoids-loaded nanocapsules (CHNCCaps) was evaluated in this study. Such gel has previously been proven to control capsaicinoids release and decrease the drugs side effects in humans. The nanocapsules containing capsaicinoids had an average size around 150 nm, with a low polydispersity index, positive zeta potential, and high encapsulation efficiency of the drugs. The CHNCCaps showed intact nanocapsules, a slightly acid pH value, and a pseudoplastic behavior suitable for topical application. Microdialysis experiments showed a 1.6-fold increase in the concentration of capsaicinoids in the dermis (after 12 h of its application) when CHNCCaps was administered compared to a chitosan hydrogel containing capsaicinoids in hydroethanolic solution (CHETCaps) and the commercial cream. The CHNCCaps showed antiallodynic and antihyperalgesic effects from 6 h to 96 h after treatment initiation, whereas CHETCaps and the commercial cream showed antiallodynic and antihyperalgesic effects only at 48 h and 96 h after treatment initiation, respectively. CHNCCaps and the commercial cream maintained antihyperalgesic activity for 6 days after treatment interruption. For mechanical allodynia, the antinociceptive effect was maintained for 48 h after treatment interruption only with CHNCCaps. In conclusion, CHNCCaps is a promising formulation for treating peripheral neuropathic pain.

Docosahexaenoic acid nanoencapsulated with anti-PECAM-1 as co-therapy for atherosclerosis regression.

Atherosclerosis is a non-resolving inflammatory condition that underlies major cardiovascular diseases.Recent clinical trial using an anti-inflammatory drug has showna reduction of cardiovascular mortality, but increased the susceptibility to infections. For this reason, tissue target anti-inflammatory therapies can represent a better option to regress atherosclerotic plaques. Docosahexaenoic acid (DHA) is a natural omega 3 fatty acidcomponentof algae oil and acts asaprecursor of several anti-inflammatory compounds, such the specialized proresolving lipid mediators(SPMs). During the atherosclerosis process, the inflammatory condition of the endothelium leads to the higher expression of adhesion molecules, such as Endothelial Cell Adhesion Molecule Plate 1 (PECAM-1 or CD31), as part of the innate immune response. Thus, the objective of this study was to develop lipid-core nanocapsules with DHA constituting the nucleus and anti-PECAM-1 on their surface and drive this structure to the inflamed endothelium. Nanocapsules were prepared by interfacial deposition of pre-formed polymer method. Zinc-II was added to bind anti-PECAM-1 to the nanocapsule surface by forming an organometallic complex. Swelling experiment showed that the algae oil act as non-solvent for the polymer (weight constant weight for 60 days, p > 0.428) indicating an adequate material to produce kinetically stable lipid-core nanocapsules (LNC). Five formulations were synthesized: Lipid-core nanocapsules containing DHA (LNC-DHA) or containing Medium-chain triglycerides (LNC-MCT), multi-wall nanocapsules containing DHA (MLNC-DHA) or containing MCT (MLNC-MCT) and the surface-functionalized (anti-PECAM-1) metal-complex multi-wall nanocapsules containing DHA (MCMN-DHA-a1). All formulations showed homogeneous macroscopic aspects without aggregation. The mean size of the nanocapsules measured by laser diffraction did not show difference among the samples (p = 0.241). Multi-wall nanocapsules (MLNC) showed a slight increase in the mean diameter and polydispersity index (PDI) measured by DLS, lower pH and an inversion in the zeta-potential (ξP) compared to LNCs. Conjugation test for anti-PECAM-1 showed 94.80% of efficiency. The mean diameter of the formulation had slightly increased from 160 nm (LCN-DHA) and 162 nm (MLNC-DHA) to 164 nm (MCMN-DHA-a1) indicating that the surface functionalization did not induce aggregation of the nanocapsules. Biological assays showed that the MCMN-DHA-a1 were uptaken by the HUVEC cells and did not decrease their viability. The surface-functionalized (anti- PECAM-1) metal-complex multi-wall nanocapsules containing DHA (MCMN-DHA-a1) can be considered adequate for pharmaceutical approaches.

Pequi (Caryocar brasiliense Cambess)-Loaded Nanoemulsion, Orally Delivered, Modulates Inflammation in LPS-Induced Acute Lung Injury in Mice.

Pequi is a Brazilian fruit used in folk medicine for pulmonary diseases treatment, but its oil presents bioavailability limitations. The use of nanocarriers can overcome this limitation. We developed nanoemulsions containing pequi oil (pequi-NE) and evaluated their effects in a lipopolysaccharide (LPS)-induced lung injury model. Free pequi oil or pequi-NE (20 mg/kg) was orally administered to A/J mice 16 and 4 h prior to intranasal LPS exposure, and the analyses were performed 24 h after LPS provocation. The physicochemical results revealed that pequi-NE comprised particles with mean diameter of 174-223 nm, low polydispersity index (0.11 ± 0.01), zeta potential of -7.13 ± 0.08 mV, and pH of 5.83 ± 0.12. In vivo evaluation showed that free pequi oil pretreatment reduced the influx of inflammatory cells into bronchoalveolar fluid (BALF), while pequi-NE completely abolished leukocyte accumulation. Moreover, pequi-NE, but not free pequi oil, reduced myeloperoxidase (MPO), TNF-α, IL-1ß, IL-6, MCP-1, and KC levels. Similar anti-inflammatory effects were observed when LPS-exposed animals were pre-treated with the nanoemulsion containing pequi or oleic acid. These results suggest that the use of nanoemulsions as carriers enhances the anti-inflammatory properties of oleic acid-containing pequi oil. Moreover, pequi's beneficial effect is likely due its high levels of oleic acid.

(-)-linalool-Loaded Polymeric Nanocapsules Are a Potential Candidate to Fibromyalgia Treatment.

Fibromyalgia (FM) is a chronic disease that has as main characteristic generalized musculoskeletal pain, which can cause physical and emotional problems to patients. However, pharmacological therapies show side effects that hamper the adhesion to treatment. Given this, (-)-linalool (LIN), a monoterpene with several therapeutic properties already reported in scientific literature as anti-depressive, antinociceptive, anti-inflammatory, and antihyperalgesic also demonstrated therapeutic potential in the treatment of FM. Nevertheless, physicochemical limitations as high volatilization and poor water-solubility make its use difficult. In this perspective, this present research had performed the incorporation of LIN into polymeric nanocapsules (LIN-NC). Size, morphology, encapsulation efficiency, cytotoxicity, and drug release were performed. The antihyperalgesic effect of LIN-NC was evaluated by a chronic non-inflammatory muscle pain model. The results demonstrated that the polymeric nanocapsules showed particle size of 199.1 ± 0.7 nm with a PDI measurement of 0.13 ± 0.01. The drug content and encapsulation efficiency were 13.78 ± 0.05 mg/mL and 80.98 ± 0.003%, respectively. The formulation did not show cytotoxicity on J774 macrophages. The oral treatment with LIN-NC and free-LIN increased the mechanical withdrawal threshold on all days of treatment in comparison with the control group. In conclusion, LIN-NC is a promising proposal in the development of phytotherapy-based nanoformulations for future clinical applications.

Healing of dermal wounds property of Caryocar brasiliense oil loaded polymeric lipid-core nanocapsules: formulation and in vivo evaluation.

Cutaneous lesions lead to complications in patients, since they may be recurrent and also represent risk of progression to infection and/or amputation. Therefore, effective, protective, and topical treatments of easy application and removal need to be developed to provide effective alternatives to patients. The Caryocar brasiliense Cambess (CBC) presents important pharmacological activities and proved in the healing process. This paper reports the improvement of the CBC nanostructured (LNCCBC and LNCCBC+) activity in dermal wounds in vivo. The oil was physico-chemically characterized and used in the development of lipid-core nanocapsules (LNCs), coated (LNCCBC+) or without chitosan (LNCCBC), in concentration of 1.0 mg mL-1. Hydrogel (HG) was tested in vivo on lesions in the back of male Wistar rats for 14 days. The oil presented appropriate physico-chemical characteristics for its use, such as moisture 0.76 %, acidity 0.85 % and oleic acid 25.90 %. The LNCs showed nanometric size (around 200 nm), monomodal distribution, slight acid pH and zeta potential of + 22.1 mV in accordance with the composition. The nanostructured oil induced dermal healing in vivo showing significantly better improvement than free oil. LNCCBC+ showed best results showing the higher increase of the production of type 1 collagen, an important protein to the healing repair. These results suggest that development of formulations LNCCBC and LNCCBC+ are promising and important alternative for the treatment of dermal wounds, avoiding complications related to cutaneous lesions.

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.

High doses of lipid-core nanocapsules do not affect bovine embryonic development in vitro.

The improvement of in vitro embryo production by culture media supplementation has been a potential tool to increase blastocyst quality and development. Recently, lipid-core nanocapsules (LNC), which were developed for biomedical applications as a drug-delivery system, have demonstrated beneficial effects on in vitro embryo production studies. LNCs have a core composed of sorbitan monostearate dispersed in capric/caprylic triglyceride. Based on that, we firstly investigated if LNCs supplemented during in vitro oocyte maturation had affinity to the mineral oil placed over the top of the IVM media. Also, the effects of LNC supplementation in different concentrations (0; 0.94; 4.71; 23.56; 117.80 and 589.00µg/mL) during the in vitro maturation protocol were evaluated in oocytes and blastocysts by in vitro tests. LNCs seemed not to migrate to the mineral oil overlay during the in vitro oocyte maturation. Interestingly, LNCs did not show toxic effects in the oocyte in vitro maturation rate, cumulus cells expansion and oocyte viability. The highest LNCs concentration tested (589µg/mL) generated the lowest ROS and GSH levels, and reduced apoptosis rate when compared to the control. Additionally, toxic effects in embryo development and quality were not observed. The LNC supramolecular structure demonstrated to be a promising nanocarrier to deliver molecules in oocytes and embryos, aiming the improvement of the embryo in vitro development.

Ciprofloxacin-loaded lipid-core nanocapsules as mucus penetrating drug delivery system intended for the treatment of bacterial infections in cystic fibrosis.

Treatment of bacterial airway infections is essential for cystic fibrosis therapy. However, effectiveness of antibacterial treatment is limited as bacteria inside the mucus are protected from antibiotics and immune response. To overcome this biological barrier, ciprofloxacin was loaded into lipid-core nanocapsules (LNC) for high mucus permeability, sustained release and antibacterial activity. Ciprofloxacin-loaded LNC with a mean size of 180nm showed a by 50% increased drug permeation through mucus. In bacterial growth assays, the drug in the LNC had similar minimum inhibitory concentrations as the free drug in P. aeruginosa and S. aureus. Interestingly, formation of biofilm-like aggregates, which were observed for S. aureus treated with free ciprofloxacin, was avoided by exposure to LNC. With the combined advantages over the non-encapsulated drug, ciprofloxacin-loaded LNC represent a promising drug delivery system with the prospect of an improved antibiotic therapy in cystic fibrosis.

Bromelain-Functionalized Multiple-Wall Lipid-Core Nanocapsules: Formulation, Chemical Structure and Antiproliferative Effect Against Human Breast Cancer Cells (MCF-7).

PURPOSE: This study was conducted a promising approach to surface functionalization developed for lipid-core nanocapsules and the merit to pursue new strategies to treat solid tumors. METHODS: Bromelain-functionalized multiple-wall lipid-core nanocapsules (Bro-MLNC-Zn) were produced by self-assembling following three steps of interfacial reactions. Physicochemical and structural characteristics, in vitro proteolytic activity (casein substrate) and antiproliferative activity (breast cancer cells, MCF-7) were determined. RESULTS: Bro-MLNC-Zn had z-average diameter of 135 nm and zeta potential of +23 mV. The complex is formed by a Zn-N chemical bond and a chelate with hydroxyl and carboxyl groups. Bromelain complexed at the nanocapsule surface maintained its proteolytic activity and showed anti-proliferative effect against human breast cancer cells (MCF-7) (72.6 ± 1.2% at 1.250 µg mL-1 and 65.5 ± 5.5% at 0.625 µg mL-1). Comparing Bro-MLNC-Zn and bromelain solution, the former needed a dose 160-folds lower than the latter for a similar effect. Tripan blue dye assay corroborated the results. CONCLUSIONS: The surface functionalization approach produced an innovative formulation having a much higher anti-proliferative effect than the bromelain solution, even though both in vitro proteolytic activity were similar, opening up a great opportunity for further studies in nanomedicine.

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.

Nanoencapsulation of Rose-Hip Oil Prevents Oil Oxidation and Allows Obtainment of Gel and Film Topical Formulations.

The rose-hip oil holds skin regenerating properties with applications in the dermatological and cosmetic area. Its nanoencapsulation might favor the oil stability and its incorporation into hydrophilic formulations, besides increasing the contact with the skin and prolonging its effect. The aim of the present investigation was to develop suitable rose-hip-oil-loaded nanocapsules, to verify the nanocapsule effect on the UV-induced oxidation of the oil and to obtain topical formulations by the incorporation of the nanocapsules into chitosan gel and film. The rose-hip oil (500 or 600 µL), polymer (Eudragit RS100®, 100 or 200 mg), and acetone (50 or 100 mL) contents were separately varied aiming to obtain an adequate size distribution. The results led to a combination of the factors acetone and oil. The developed formulation showed average diameter of 158 ± 6 nm with low polydispersity, pH of 5.8 ± 0.9, zeta potential of +9.8 ± 1.5 mV, rose-hip oil content of 54 ± 1 µL/mL and tendency to reversible creaming. No differences were observed in the nanocapsules properties after storage. The nanoencapsulation of rose-hip oil decreased the UVA and UVC oxidation of the oil. The chitosan gel and film containing rose-hip-oil-loaded nanocapsules showed suitable properties for cutaneous use. In conclusion, it was possible to successfully obtain rose-hip-oil-loaded nanocapsules and to confirm the nanocapsules effect in protecting the oil from the UV rays. The chitosan gel and film were considered interesting alternatives for incorporating the nanoencapsulated rose-hip oil, combining the advantages of the nanoparticles to the advantages of chitosan.

Pharmacological Improvement and Preclinical Evaluation of Methotrexate-Loaded Lipid-Core Nanocapsules in a Glioblastoma Model.

Glioblastoma multiforme is a devastating cerebral tumor with an exceedingly poor prognosis. Methotrexate (MTX) is a folic acid analogue that inhibits DNA synthesis by binding to dihydrofolate reductase. Biodegradable nanoparticles are emerging as a promising system for drug delivery to specific tissues. The aims of the current study were pharmacological improvement and preclinical evaluation of MTX-loaded lipid-core nanocapsules (MTX-LNCs) in a glioblastoma model. Cell viability was assessed using the MTT assay, and the cell cycle was characterized by flow cytometry analysis of propidium iodide staining. Apoptosis was measured using an AnnexinV kit and by examining active caspase-3 immunocontent. In vivo glioma implantation was performed in rats, followed by measurement of the tumor size and tumoral apoptosis, BCL-2 immunohistochemistry and analyses of toxicological parameters. MTX-LNCs with increased encapsulation efficiency were successfully prepared. Our in vitro results showed a decrease in glioma cell viability after MTX-LNC treatment that was preceded by cell cycle arrest, leading the cells to undergo apoptotic death, as indicated by AnnexinV staining and increased active caspase-3 protein levels. In the in vivo glioma model, we observed a decrease in the tumor size and an increase in apoptosis in the tumor microenvironment (based on the AnnexinV assay and BCL-2 measurement). MTX-LNC treatment decreased the leukocyte number but altered neither toxicological tissue marker expression nor metabolic parameters. The present results reveal that MTX-LNCs represented an efficient formulation in a preclinical model of glioma and are a potential candidate for clinical trials.

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.

A novel approach to arthritis treatment based on resveratrol and curcumin co-encapsulated in lipid-core nanocapsules: In vivo studies.

Resveratrol and curcumin are two natural polyphenols extensively used due to their remarkable anti-inflammatory activity. The present work presents an inedited study of the in vivo antioedematogenic activity of these polyphenols co-encapsulated in lipid-core nanocapsules on Complete Freund's adjuvant (CFA)-induced arthritis in rats. Lipid-core nanocapsules were prepared by interfacial deposition of preformed polymer. Animals received a single subplantar injection of CFA in the right paw. Fourteen days after arthritis induction, they were treated with resveratrol, curcumin, or both in solution or loaded in lipid-core nanocapsules (1.75 mg/kg/twice daily, i.p.), for 8 days. At the doses used, the polyphenols in solution were not able to decrease paw oedema. However, nanoencapsulation improved the antioedematogenic activity of polyphenols at the same doses. In addition, the treatment with co-encapsulated polyphenols showed the most pronounced effects, where an inhibition of 37-55% was observed between day 16 and 22 after arthritis induction. This treatment minimized most of the histological changes observed, like fibrosis in synovial tissue, cartilage and bone loss. In addition, unlike conventionally arthritis treatment, resveratrol and curcumin co-encapsulated in lipid-core nanocapsules did not alter important hepatic biochemical markers (ALP, AST, and ALT). In conclusion, the strategy of co-encapsulating resveratrol and curcumin in lipid-core nanocapsules improves their efficacy as oedematogenic agents, with no evidence of hepatotoxic effects. This is a promising strategy for the development of new schemes for treatment of chronic inflammation diseases, like arthritis.

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.

New strategy to surface functionalization of polymeric nanoparticles: one-pot synthesis of scFv anti-LDL(-)-functionalized nanocapsules.

PURPOSE: In general, the surface functionalization of polymeric nanoparticles is carried out by covalently bounding ligands to the nanoparticle surface. This process can cause a lack or decrease of the ligand specificity to its target receptor, besides the need of purification steps. We proposed a ligand-metal-chitosan-lecithin complex as a new strategy to functionalize the surface of biodegradable nanoparticles. METHODS: One pot synthesis of scFv anti-LDL(-)-functionalized nanocapsules was carried out by self-assembly and interfacial reactions. Particle sizing techniques, lipid peroxidation and molecular recognition by enzyme linked immuno sorbent assays were carried out. RESULTS: The selected formulation had unimodal size distribution with mean diameter of about 130 nm. The metals in the complex did not enhance the oxidative stress, and the scFv anti-LDL(-)-functionalized nanocapsules recognized LDL(-) and did not react with native LDL indicating the maintenance of the active site of the fragment. CONCLUSIONS: The one pot synthesis, using the ligand-metal-chitosan-lecithin complex to functionalize the surface of the biodegradable nanocapsules, maintained the active site of the antibody fragment making the device interesting for applications in nanomedicine.

Prednisolone-loaded nanocapsules as ocular drug delivery system: development, in vitro drug release and eye toxicity.

OBJECTIVE: To develop non-toxic aqueous ocular drug delivery systems containing prednisolone by means of its nanoencapsulation. MATERIALS AND METHODS: Nanocapsules were prepared by interfacial deposition of preformed polymer [poly(ε-caprolactone) or Eudragit® RS100]. Particle size distribution was determined by laser diffractometry, photon correlation spectroscopy and nanoparticle tracking analysis. Ocular irritation and cytotoxicity were evaluated in vitro on the chorioallantoic membrane (CAM) and rabbit corneal epithelial cell line, respectively. RESULTS AND DISCUSSION: Nanocapsules showed mean particle sizes between 100 and 300 nm and prednisolone encapsulation efficiency of around 50%. Controlled release of prednisolone occurred for 5 h for both formulations according to the biexponential model. Both formulations were found to be non-irritant in the CAM test and non-cytotoxic toward rabbit corneal epithelial cells. CONCLUSIONS: Encapsulation of prednisolone in nanocapsules was reported for the first time, being suitable for producing eye drops for the treatment of ocular inflammatory and no eye toxicity was indicated.