Intracellular Delivery of Poorly Soluble Polyphenols: Elucidating the Interplay of Self-Assembling Nanocarriers and Human Chondrocytes.

Increased molecular understanding of multifactorial diseases paves the way for novel therapeutic approaches requiring sophisticated carriers for intracellular delivery of actives. We designed and characterized self-assembling lipid-core nanocapsules for coencapsulation of two poorly soluble natural polyphenols curcumin and resveratrol. The polyphenols were identified as high-potential therapeutic candidates intervening in the intracellular inflammation cascade of chondrocytes during the progress of osteoarthritis. To elucidate the interplay between chondrocytes and nanocapsules and their therapeutic effect, we pursued a complementary analytical approach combining label-free visualization with biological assays. Primary human chondrocytes did not show any adverse effects upon nanocapsule application and coherent anti-Stokes Raman scattering images visualized their intracellular uptake. Further, by systematically blocking different uptake mechanisms, an energy independent uptake into the cells could be identified. Additionally, we tested the therapeutic effect of the polyphenol-loaded carriers on inflamed chondrocytes. Treatment with nanocapsules resulted in a major reduction of nitric oxide levels, a well-known apoptosis trigger during the course of osteoarthritis. For a more profound examination of this protective effect on joint cells, we pursued studies with atomic force microscopy investigations. Significant changes in the cell cytoskeleton as well as prominent dents in the cell membrane upon induced apoptosis were revealed. Interestingly, these effects could not be detected for chondrocytes which were pretreated with the nanocapsules. Overall, besides presenting a sophisticated carrier system for joint application, these results highlight the necessity of establishing combinatorial analytical approaches to elucidate cellular uptake, the interplay of codelivered drugs and their therapeutic effect on the subcellular level.

Lipid-Core Nanocapsules Improved Antiedematogenic Activity of Tacrolimus in Adjuvant-Induced Arthritis Model.

Despite significant technological advances, rheumatoid arthritis remains an incurable disease with great impact on the life quality of patients. We studied the encapsulation of tacrolimus in lipidcore nanocapsules (TAC-LNC) as a strategy to enhance its systemic anti-arthritic properties. TAC-LNC presented unimodal distribution of particles with z-average diameter of 212 +/- 11, drug content close to the theoretical value (0.80 mg mL(-1)), and 99.43% of encapsulation efficiency. An in vitro sustained release was determined for TAC-LNC with anomalous transport mechanism (n = 0.61). In vivo studies using an arthritis model induced by Complete Freund's Adjuvant demonstrated that the animals treated with TAC-LNC presented a significantly greater inhibition of paw oedema after intraperitoneal administration. Furthermore, the encapsulation of TAC in lipid-core nanocapsules was potentially able to prevent hyperglycemia in the animals. In conclusion, TAC-LNC was prepared with 100% yield of nanoscopic particles having satisfactory characteristics for systemic use. This formulation represents a promising strategy to the treatment of rheumatoid arthritis in the near future.

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.

Free and nanoencapsulated curcumin suppress ß-amyloid-induced cognitive impairments in rats: involvement of BDNF and Akt/GSK-3ß signaling pathway.

Alzheimer's disease (AD), a neurodegenerative disorder exhibiting progressive loss of memory and cognitive functions, is characterized by the presence of neuritic plaques composed of neurofibrillary tangles and ß-amyloid (Aß) peptide. Drug delivery to the brain still remains highly challenging for the treatment of AD. Several studies have been shown that curcumin is associated with anti-amyloidogenic properties, but therapeutic application of its beneficial effects is limited. Here we investigated possible mechanisms involved in curcumin protection against Aß(1-42)-induced cognitive impairment and, due to its poor bioavailability, we developed curcumin-loaded lipid-core nanocapsules in an attempt to improve the neuroprotective effect of this polyphenol. Animals received a single intracerebroventricular injection of Aß(1-42) and they were administered either free curcumin or curcumin-loaded lipid-core nanocapsules (Cur-LNC) intraperitoneally for 10days. Aß(1-42)-infused animals showed a significant impairment on learning-memory ability, which was paralleled by a significant decrease in hippocampal synaptophysin levels. Furthermore, animals exhibited activated astrocytes and microglial cells, as well as disturbance in BDNF expression and Akt/GSK-3ß signaling pathway, beyond tau hyperphosphorylation. Our findings demonstrate that administration of curcumin was effective in preventing behavioral impairments, neuroinflammation, tau hyperphosphorylation as well as cell signaling disturbances triggered by Aß in vivo. Of high interest, Cur-LNC in a dose 20-fold lower presented similar neuroprotective results compared to the effective dose of free curcumin. Considered overall, the data suggest that curcumin is a potential therapeutic agent for neurocognition and nanoencapsulation of curcumin in LNC might constitute a promising therapeutic alternative in the treatment of neurodegenerative diseases such as AD.

Free and nanoencapsulated curcumin prevents cigarette smoke-induced cognitive impairment and redox imbalance.

Cigarette smoke-exposure promotes neurobiological changes associated with neurocognitive abnormalities. Curcumin, a natural polyphenol, have shown to be able to prevent cigarette smoke-induced cognitive impairment. Here, we investigated possible mechanisms involved in curcumin protection against cigarette smoke-induced cognitive impairment and, due to its poor bioavailability, we investigated the potential of using curcumin-loaded lipid-core nanocapsules (C-LNC) suspension. Rats were treated with curcumin and cigarette smoke, once a day, 5 days each week, for 30 days. Animals were divided into ten groups: I, control (vehicle/corn oil); II, curcumin 12.5mg/kg; III, curcumin 25mg/kg; IV, curcumin 50mg/kg; V, C-LNC 4 mg/kg; VI, tobacco exposed; VII, curcumin 12.5mg/kg along with tobacco exposure; VIII, curcumin 25mg/kg along with tobacco exposure; IX, curcumin 50mg/kg along with tobacco exposure; X, C-LNC 4 mg/kg along with tobacco exposure. Cigarette smoke-exposure impaired object recognition memory (P<0.001), indicated by the low recognition index, increased biomarkers of oxidative/nitrosative stress such as TBARS (P<0.05) and NOx (P<0.01), decreased antioxidant defenses such as NPSH content (P<0.01) and SOD activity (P<0.01) and inhibited the activities of enzymes involved in ion homeostasis such as Na(+),K(+)-ATPase and Ca(2+)-ATPase. Both curcumin formulations (free and nanoencapsulated) prevented the memory impairment, the redox imbalance and the alterations observed in the ATPases activities. Maintenance of ion homeostasis and redox balance is involved in the protective mechanism of curcumin against tobacco-induced cognitive impairment. Our results suggest that curcumin is a potential therapeutic agent for neurocognition and that C-LNC may be an alternative to its poor bioavailability.

Chitosan hydrogels containing nanoencapsulated phenytoin for cutaneous use: Skin permeation/penetration and efficacy in wound healing.

Although phenytoin is an antiepileptic drug used in the oral treatment of epilepsy, its off-label use as a cutaneous healing agent has been studied in recent years due to the frequent reports of gingival hyperplasia after oral administration. However, the cutaneous topical application of phenytoin should prevent percutaneous skin permeation. Therefore, the aim of this study was to evaluate the in vitro skin permeation/retention and in vivo effects of nanocapsules and nanoemulsions loaded with phenytoin and formulated as chitosan hydrogels on the healing process of cutaneous wounds in rats. The hydrogels had adequate pH values (4.9-5.6) for skin application, drug content of 0.025% (w/w), and non-Newtonian pseudoplastic rheological behaviour. Hydrogels containing nanocapsules and nanoemulsions enabled improved controlled release of phenytoin and adhesion to skin, compared with hydrogels containing non-encapsulated phenytoin. In vitro skin permeation studies showed that phenytoin permeation to the receptor compartment, and consequently the risk of systemic absorption, may be reduced by nanoencapsulation without any change in the in vivo performance of phenytoin in the wound healing process in rats.

Reconstituted spray-dried phenytoin-loaded nanocapsules improve the in vivo phenytoin anticonvulsant effect and the survival time in mice.

This study evaluated the in vivo anticonvulsant effect of a spray-dried powder for reconstitution containing phenytoin-loaded lipid-core nanocapsules. The effect of chitosan coating on redispersibility, gastrointestinal stability, and drug release from nanoparticles was evaluated during the development of the powders. Maltodextrin was used as adjuvant in the spray-drying process. Chitosan coating played an important role in redispersibility, and large particles (>100 µm) were obtained using the highest concentration of solids in the feed. However, after aqueous redispersion, volume-based particle size was reduced to about 1 µm. The release of nanoparticles from the surface of the spherical microagglomerates (roundness index = 0.75) was confirmed by SEM analysis. Powders reconstituted in water recovered partially the nanometric properties of the original suspensions and were stable for 24 h. Phenytoin-loaded chitosan-coated nanocapsules and their redispersed powders have good gastrointestinal stability, and are able to control drug release in simulated gastric and intestinal fluids. Besides that, the reconstituted powder containing chitosan-coated nanocapsules exhibited improved anticonvulsant activity against seizures induced by pilocarpine in mice, compared to the non-encapsulated drug, representing an important approach in anticonvulsant treatments for children and adults.

Tretinoin-loaded lipid-core nanocapsules overcome the triple-negative breast cancer cell resistance to tretinoin and show synergistic effect on cytotoxicity induced by doxorubicin and 5-fluororacil.

Nanostructured drug delivery systems have been extensively studied, mainly for applications in cancer therapy. The advantages of these materials include protection against drug degradation and improvement in both the relative solubility of poorly water soluble drugs as in targeting of therapy, due to the enhanced permeability and retention effect on tumor sites. In this work, we evaluate the antitumor activity of tretinoin-loaded lipid core nanocapsules (TT-LNC) in a tretinoin-resistant breast cancer cell-line, MDA-MB- 231, as well as the synergistic effect of combination of this treatment with 5-FU or DOXO. The inhibition of cell growth was assayed by MTT reduction. Live/Dead assay and DAPI staining evaluated cytotoxicity. Apoptosis was evaluated by Annexin V-PE/7AAD and the effect of chronic exposure was evaluated by colony formation assay. TT-LNC reduced the cell viability even at lower concentrations (1µM) and displayed synergistic effect with 5-FU or DOXO on cytotoxicity and colony formation inhibition. Our work shows a possibility of using nanocapsules to improve the antitumoral activity of TT for its use either alone or in combination with other chemotherapeutic drugs, especially considering the chronic effect.

Drug-loaded nanoemulsion as positive control is an alternative to DMSO solutions for in vitro evaluation of curcumin delivery to MCF-7 cells.

BACKGROUND: In vitro evaluation of toxicity and/or efficacy of nanostructured drug delivery systems involves the uses of different controls, including positive and negative controls, as well as a solution or dispersion of the drug in water. One of the most frequently solvent used to dilute poorly water soluble drugs to in vitro tests are dimethylsulfoxide (DMSO). However, its different specific surface area and different diffusion coefficients could make the comparative effects difficult. We proposed that a solvent-free dispersions having similar specific surface area could be a better control than drug in solution against cell lines. METHODS: We evaluate the effect of curcumin-loaded lipid-core nanocapsules, curcumin-loaded nanoemulsion and curcumin DMSO-water solution on viability and colony forming efficiency of human breast cancer cell line, MCF7. RESULTS: The cytotoxic effect of nanocapsules at 24-72h was similar to nanoemulsion and lower than drug solution. However, the nanocapsules had a superior anticancer activity when long periods (10days) were evaluated, which highlight the sustained drug release by nanocapsules. CONCLUSIONS: Our results showed a superior anticancer activity of curcumin-loaded lipid-core nanocapsules compared to curcumin-loaded nanoemulsion and curcumin dissolved in DMSO in long exposition time assay, wihch is not observed in short exposition time assays like MTT. When a poorly water-soluble drug is under investigation, the nanoemulsion prepared with the same compounds of the nanocapsules, except the polymer, could be a better control than DMSO-solution of drug.

Skin penetration behavior of lipid-core nanocapsules for simultaneous delivery of resveratrol and curcumin.

Polyphenols, which are secondary plant metabolites, gain increasing research interest due to their therapeutic potential. Among them, resveratrol and curcumin are two agents showing antioxidant, anti-inflammatory, antimicrobial as well as anticarcinogenic effects. In addition to their individual therapeutic effect, increased activity was reported upon co-delivery of the two compounds. However, due to the poor water solubility of resveratrol and curcumin, their clinical application is currently limited. In this context, lipid-core nanocapsules (LNC) composed of an oily core surrounded by a polymeric shell were introduced as drug carrier systems with the potential to overcome this obstacle. Furthermore, the encapsulation of polyphenols into LNC can increase their photostability. As the attributes of the polyphenols make them excellent candidates for skin treatment, the aim of this study was to investigate the effect of co-delivery of resveratrol and curcumin by LNC upon topical application on excised human skin. In contrast to the formulation with one polyphenol, resveratrol penetrated into deeper skin layers when the co-formulation was applied. Based on vibrational spectroscopy analysis, these effects are most likely due to interactions of curcumin and the stratum corneum, facilitating the skin absorption of the co-administered resveratrol. Furthermore, the interaction of LNC with primary human skin cells was analyzed encountering a cellular uptake within 24h potentially leading to intracellular effects of the polyphenols. Thus, the simultaneous delivery of resveratrol and curcumin by LNC provides an intelligent way for immediate and sustained polyphenol delivery for skin disease treatment.

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.

Poly(ϵ-caprolactone) microcapsules and nanocapsules in drug delivery.

INTRODUCTION: Poly(ϵ-caprolactone) (PCL), a biodegradable and biocompatible polymer, is useful to encapsulate a wide range of drugs making it an interesting material for the preparation of carriers with potential applications in therapeutics. AREAS COVERED: The design and development of those carriers to modulate drug release, to improve the drug stability or apparent solubility in aqueous media, as well as to target tissues and organs are discussed. EXPERT OPINION: Microencapsulation is a well-established process in pharmaceutical industry to protect drugs from chemical degradation and to control drug release. In this context, PCL is a useful polymer to prepare microcapsules. Nanoencapsulation, a more recent approach, offers new possibilities in drug delivery. PCL can be used as polymer to prepare different types of nanocapsules presenting diverse flexibility according to the chemical nature of the core. Those nanocapsules are capable of controlling drug release and improving photochemical stability. In addition, they can modulate cutaneous drug penetration/permeation and act as physical sunscreen due to their capability of light scattering. Considering the pharmaceutical point of view, PCL nanocapsules are versatile formulations, once they can be used in the liquid form, as well as incorporated into semi-solid or solid dosage forms.

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.