Rheological and Viscoelastic Analysis of Hybrid Formulations for Topical Application.

The rheological and viscoelastic properties of hybrid formulations composed of vehicles designed for cutaneous topical application and loaded with ultradeformable liposomes (UDL) were assessed. UDL were selected for their established ability to transport both lipophilic and hydrophilic compounds through the skin, and are applicable in pharmaceuticals and cosmetics. Formulations underwent flow analysis and were fitted to the Herschel-Bulkley model due to their prevalent non-Newtonian behavior in most cases. Linear viscoelastic regions (LVR) were identified, and G' and G″ moduli were determined via frequency sweep steps, considering the impact of temperature and aging. The formulations exhibited non-Newtonian behavior with pseudoplastic traits in most cases, with UDL incorporation inducing rheological changes. LVR and frequency sweep tests indicated predominantly elastic solid behavior, with G' higher than G″, at different temperatures and post-production times. Tan δ values also illustrated a predominant solid-like behavior over liquid. This study provides pivotal insights into the rheological and viscoelastic features of topical formulations, emphasizing the crucial role of meticulous vehicle and formulation selection when incorporating UDL or analogous liposomal drug delivery systems.

Rheological, Physical, and Spectroscopical Characterization of Gamma-Irradiated Albumin Nanoparticles Loaded with Anthocyanin.

Anthocyanins are the main active compounds in blueberry. However, they have poor oxidation stability. If anthocyanins are encapsulated in protein nanoparticles, their oxidation resistance could be increased as a result of the slowing down of the oxidation process. This work describes the advantages of using a γ-irradiated bovine serum albumin nanoparticle bound to anthocyanins. The interaction was characterized biophysically, mainly by rheology. By computational calculation and simulation based on model nanoparticles, we estimated the number of molecules forming the albumin nanoparticles, which allowed us to infer the ratio of anthocyanin/nanoparticles. Measurements by UV-vis spectroscopy, FTIR spectroscopy, fluorescence spectroscopy, dynamic light scattering (DLS), ζ potential, electron transmission microscopy, and rheology at room (25 °C) and physiological (37 °C) temperatures were performed. The spectroscopy measurements allowed identifying additional hydrophobic sites created during the irradiation process of the nanoparticle. On the basis of the rheological studies, it was observed that the BSA-NP trend is a Newtonian flow behavior type for all the temperatures selected, and there is a direct correlation between dynamic viscosity and temperature values. Furthermore, when anthocyanins are added, the system increases its resistance to the flow as reflected in the morphological changes observed by TEM, thus confirming the relationship between viscosity values and aggregate formation.

The Topical Nanodelivery of Vismodegib Enhances Its Skin Penetration and Performance In Vitro While Reducing Its Toxicity In Vivo.

Vismodegib is a first-in-class inhibitor for advanced basal cell carcinoma treatment. Its daily oral doses present a high distribution volume and several side effects. We evaluated its skin penetration loaded in diverse nanosystems as potential strategies to reduce side effects and drug quantities. Ultradeformable liposomes, ethosomes, colloidal liquid crystals, and dendrimers were able to transport Vismodegib to deep skin layers, while polymeric micelles failed at this. As lipidic systems were the most effective, we assessed the in vitro and in vivo toxicity of Vismodegib-loaded ultradeformable liposomes, apoptosis, and cellular uptake. Vismodegib emerges as a versatile drug that can be loaded in several delivery systems for topical application. These findings may be also useful for the consideration of topical delivery of other drugs with a low water solubility.

BSA-capped gold nanoclusters as potential theragnostic for skin diseases: Photoactivation, skin penetration, in vitro, and in vivo toxicity.

BSA-capped gold nanoclusters are promising theragnostic systems that can be excited to render both fluorescence emission and reactive oxygen species. Although their synthesis and photoluminescence properties are already well described, more accurate information about their use as photosensitizers is required in order to advance towards health applications. In this work, we have obtained BSA-capped gold nanoclusters and characterized their photophysics by different techniques. Singlet oxygen production was detected upon irradiation, which was enough to produce toxicity on two cell lines. Remarkably, an internal energy transfer, probably due to the presence of smaller nanoclusters and the contribution of oxidized residues of BSA in the system, caused fluorescence emission near 640 nm after excitation in the UV range. Additionally, the system was capable of penetrating human skin beyond the stratum corneum, which enhances the potential of these nanoclusters as bifunctional photodynamic therapy effectors and biomarkers with application in a diversity of skin diseases. In the absence of radiation, BSA-capped gold nanoclusters did not cause toxicity in vitro, while their toxic effect on an in vivo model as zebrafish was determined.

Comparative skin penetration profiles of formulations including ultradeformable liposomes as potential nanocosmeceutical carriers.

BACKGROUND: Ultradeformable liposomes are promising carriers for cosmeceutical actives as they can be loaded with molecules of different polarities, and they present unique penetration properties. AIMS: While those features have already been tested, we wanted to know whether their special penetration properties could be maintained after incorporation in diverse cosmetic vehicles, including commercial products already in the market. METHODS: Ultradeformable liposomes loaded with a lipophilic and a hydrophilic fluorescent probe were prepared by lipid film resuspension, followed by extrusion and incorporation to different vehicles and commercial products. Penetration was determined in human and pig skin by incubation, with the Saarbrücken penetration model, followed by the recovery of the probes or by fluorescence microscopy. RESULTS: The incorporation of ultradeformable liposomes to cosmetic vehicles did not alter their penetration in most of the cases for human skin explants. Pig skin penetration presented significant differences compared with human explants. CONCLUSIONS: Ultradeformable liposomes could be useful as versatile cosmeceutical carriers in final product formulations.

Nanoferulic: From a by-product of the beer industry toward the regeneration of the skin.

BACKGROUND: Brewers' spent grain (BSG) is one of the most abundant by-products of the beer industry and causes serious environmental problems. Ferulic acid (FA) is an antioxidant with potential cosmeceutical applications. FA was extracted from BSG, developing a method of high extraction performance in order to be encapsulated in ultradeformable liposomes (Nanoferulic, NF). AIMS: To obtain a product with high added value such as FA, from a residue currently underused, using simple and economical chemical methods. To load FA into a nanosystem designed for the topical route, its encapsulation has the purpose to take profit from its photoprotective, anti-inflammatory, and antioxidant properties in the deep layers of the skin. METHODS: Ferulic acid was obtained from dried BSG using acid and basic treatments in series. NF was prepared by lipid film resuspension of a solution containing FA obtained from BSG. Size and Z-potential were determined. Cytotoxicity was assessed in vitro. Skin penetration was assessed by NF determination at different skin depths and by confocal microscopy. RESULTS: The yield of the extraction process was 0.43% on a dry basis. Encapsulation rendered liposomes of around 140 nm with 92% of encapsulation efficiency. No toxicity was observed in all the tested concentrations. Successful results were obtained from the regeneration studies. CONCLUSIONS: It was possible to develop a nanosystem containing FA, generating a high-value commercial input for the pharmaceutical and cosmeceutical industry. The use of BSG generated in industrial scale would help to reduce the volume of highly polluting waste.

Nanoformulation for potential topical delivery of Vismodegib in skin cancer treatment.

Vismodegib (Erivedge®, Genentech) is a first-in-class inhibitor of the hedgehog signaling pathway for the treatment of basal cell carcinoma (BCC). The treatment currently consists of the oral administration of Erivedge® capsules. Although it has shown therapeutic efficacy in clinical trials, there are many side effects related to its systemic distribution. In this work, we have incorporated vismodegib to ultradeformable liposomes in order to obtain a nano-drug delivery system via topical route, which could be useful to reduce systemic distribution -and consequently side effects- while achieving a viable epidermis-specific target where neoplastic events of BCC develop. Vismodegib was loaded into liposomes composed of soy phosphatidylcholine and sodium cholate, and the obtained formulation was characterized by different techniques, both experimental and computational. Several analyses were performed,with a special focus on the interaction of the drug with the liposomal membrane. Additionally, the penetration of Vismodegib delivered by ultradeformable liposomes was assessed on human skin explants. This is one of the first works that propose the topical route for Vismodegib and the first, to our knowledge, in stabilizing this active into a nano-drug delivery system specifically designed for penetrating the stratum corneum impermeable barrier.

Comparative toxicity of PEG and folate-derived blue-emitting silicon nanoparticles: in vitro and in vivo studies.

AIM: Amino functionalization is a first step modification aiming to achieve biomedical applications of silicon nanoparticles, for example, for photodynamic therapy or radiotherapy. Nevertheless, toxicity and low quantum yields due to the positive charge of amino groups emerge as a problem that could be solved with subsequent derivatizations. MATERIALS & METHODS: Folic and PEG-conjugated nanoparticles were obtained from amino-functionalized silicon nanoparticle (NH2SiNP). Cytotoxicity was determined on a tumor cell line at low and high concentrations. Four end points of in vivo toxicity were evaluated on zebrafish (Danio rerio). RESULTS: Folic acid functionalization reduced the cytotoxicity in comparison to amino and PEG-functionalized nanoparticles. In zebrafish, folic functionalization lowered toxicity in general while PEG increased it. CONCLUSION: Functionalization of NH2SiNP with folic acid reduced the toxic effects in vitro and in vivo. This could be useful for therapeutic applications. PEG functionalization did not lower the toxicity.

Zebrafish (Danio rerio) model as an early stage screening tool to study the biodistribution and toxicity profile of doxorubicin-loaded mixed micelles.

Doxorubicin (DOX) hydrochloride is a powerful anthracycline antibiotic used for the treatment of various types of malignancies, particularly ovarian and metastatic breast cancer. However, DOX presents severe side effects, such as hepatotoxicity, nephrotoxicity, dose-limiting myelosuppression, brain damage and cardiotoxicity. A liposomal formulation, Doxil®, was approved by the FDA, which has managed to reduce the number of cardiac events in patients with metastatic breast cancer. However, in comparison to free DOX, Doxil® has not shown significant improvements regarding survival. We have previously designed DOX-loaded mixed micelles (MMDOX) composed of D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) and Tetronic® T1107. To assess the potential toxic effects of this novel formulation, in this work the zebrafish (Danio rerio) model was used to evaluate its in vivo toxicity and teratogenicity. This study evaluated and compared the effects of DOX exposure from different formulations (free DOX, MMDOX and Doxil®) on the swimming activity, morphological alterations, cardiac rhythm, lethality rate and DOX biodistribution. MMDOX showed lower lethal effects, morphological alterations and neurotoxic effects than the free drug. This study shows the potential of the MMDOX to be an effective DOX-delivery system because it could reduce the side effects.

Toward biomedical application of amino-functionalized silicon nanoparticles.

Silicon blue-emitting nanoparticles (NPs) are promising effectors for photodynamic therapy and radiotherapy, because of their production of reactive oxygen species (ROS) upon irradiation. RESULTS: Amino-functionalized silicon NPs (NH2SiNP) were intrinsically nontoxic below 100 µg/ml in vitro (on two tumor cell lines) and in vivo (zebrafish larvae and embryos). NH2SiNP showed a moderate effect as a photosensitizer for photodynamic therapy and reduced ROS generation in radiotherapy, which could be indicative of a ROS scavenging effect. Encapsulation of NH2SiNP into ultradeformable liposomes improved their skin penetration after topical application, reaching the viable epidermis where neoplastic events occur. CONCLUSION: Subsequent derivatizations after amino-functionalization and incorporation to nanodrug delivery systems could expand the spectrum of the biomedical application of these kind of silicon NPs.

Skin penetration and UV-damage prevention by nanoberries.

BACKGROUND: Ethanolic extract from blueberry (Vaccinium myrtillus) is rich in anthocyanins and thus exhibits antioxidant activity. On the other hand, ultradeformable liposomes are capable of penetrating to the impermeable barrier of skin. Nanoberries are ultradeformable liposomes carrying blueberry extract. OBJECTIVES: In this study, their capacity to penetrate the stratum corneum and photodamage prevention were tested, with the aim of developing a topical formulation for skin protection from environmental damage. METHODS: Nanoberries were prepared by lipid film resuspension with ethanolic extract from blueberry, followed by sonication and incorporation to a gel. Size, zeta potential, deformability, rheology, and viscoelasticity were determined. Toxicity was assessed in vivo in zebrafish model, while in vitro cytotoxicity assay was performed on HaCaT and HEK-293T cell lines. Skin penetration was evaluated with the Saarbrücken penetration model followed by tape stripping, cryosection, or optical sectioning. UV-damage protection and photoprotection were determined by ad hoc methods with UVA, UVB, and UVC radiation on HaCaT cells. Wound assay was performed on HaCaT cells. RESULTS: Nanoberries of about 100 nm, with differential elastic properties, did penetrate the stratum corneum, with low toxicity. When HaCaT cells were exposed to UV radiation in the presence of nanoberries, their viability was maintained. CONCLUSIONS: Nanoberries could be effective to protect the skin from sun photodamage.

Nano-formulation for topical treatment of precancerous lesions: skin penetration, in vitro, and in vivo toxicological evaluation.

With the aim of improving the topical delivery of the antineoplastic drug 5-fluorouracil (5FU), it was loaded into ultradeformable liposomes composed of soy phosphatidylcholine and sodium cholate (UDL-5FU). The liposome populations had a mean size of 70 nm without significant changes in 56 days, and the ultradeformable formulations were up to 324-fold more elastic than conventional liposomes. The interaction between 5FU and the liposomal membrane was studied by three methods, and also release profile was obtained. UDL-5FU did penetrate the stratum corneum of human skin. At in vitro experiments, the formulation was more toxic on a human melanoma-derived than on a human keratinocyte-derived cell line. Cells captured liposomes by metabolically active processes. In vivo toxicity experiments were carried out in zebrafish (Danio rerio) larvae by studying the swimming activity, morphological changes, and alterations in the heart rate after incubation. UDL-5FU was more toxic than free 5FU. Therefore, this nano-formulation could be useful for topical application in deep skin precancerous lesions with advantages over current treatments. This is the first work that assessed the induction of apoptosis, skin penetration in a Saarbrücken penetration model, and the toxicological effects in vivo of an ultradeformable 5FU-loaded formulation.

Actividad in vitro contra Leishmania y permeación en piel humana de liposomas ultradeformables de miltefosina / In vitro activity against Leishmania and human skin permeation of miltefosine ultradeformable liposomes

Introducción: los liposomas ultradeformables de miltefosina (LUD-MIL) constituyen una opción para el tratamiento tópico en leishmaniasis cutánea penetrando los estratos de la piel hasta la dermis, sitio donde habita el parásito. Objetivo: diseñar LUD-MIL y determinar su actividad contra L. (Viannia) panamensis y L. (V.) braziliensis y la permeación en piel humana. Métodos: los LUD-MIL, liposomas convencionales de fosfatidilcolina (LConv) y LUD-MIL-fluorescente (LUD-MIL-Fluo) fueron preparados por el método de rehidratación de película lipídica. Se caracterizaron fisicoquímicamente y se determinaron: la liberación en membrana semisintéticas, la retención en las capas de la piel y la permeación en piel humana. La citotoxicidad en THP-1 fue determinada por el ensayo colorimétrico de MTT y la actividad en promastigotes y amastigotes intracelulares por recuento microscópico. Resultados: el tamaño, índice de polidispersión, potencial Z y concentración de fosfolípidos de los LUD-MIL fue de 100,7 nm, 0,147, -12,0 mV y 53,24 mM respectivamente. El flujo de MIL a través de la membrana fue mayor con LUD-MIL que con MIL-libre. El tratamiento con LUD-MIL indujo menor acumulación de la MIL en el estrato corneo y mayor permeación que el tratamiento con MIL libre. Los LUD-MIL y los LConv-MIL mantuvieron la actividad de la MIL en los parásitos y células. Los LUD-MIL fueron más tóxicos para las células que los LConv y la MIL y más activos en amastigotes intracelulares de L. (V.) braziliensis. Conclusión: los LUD-MIL preparados conservaron la actividad anti-Leishmania de la MIL y permitieron la liberación del compuesto en membranas y piel humana. Ensayos en modelos experimentales de leishmaniasis cutánea para evaluar la actividad de estas formulaciones son urgentes de realizar(AU)

Introduction: miltefosine ultradeformable liposomes (MIL-LUD) are an option for the topical treatment of cutaneous leishmaniasis penetrating the skin layers to the dermis where the parasite inhabits. Objective: to design MIL-LUD and determine their in vitro activity against L. (Viannia) panamensis and L. (V.) braziliensis and to determine human skin permeation. Methods: MIL-LUD, phosphatidylcholine liposomes (MIL-LConv) and fluorescent MIL-LUD (MIL-LUD-Fluo) were prepared by lipid film rehydration method. They were physicochemically characterized to determine drug release in semisynthetic membrane, retention in skin layers and permeation on human skin membranes. Cytotoxicity in THP-1 was determined by the MTT colorimetric test and activity in promastigotes and intracellular amastigotes by microscopic counting. Results: the size, the polydispersion index, the Zeta potential and phospholipid content were 100.7 nm, 0.147, -12.0mV and 53.24mM, respectively for MIL-LUD. MIL flow through the semisynthetic membrane was greater with MIL-LUD than MIL-free treatment. MIL-LUD treatment induced lower MIL accumulation in the stratum corneum and increased permeation than MIL free treatment. The MIL-LUD and MIL-Conv maintained MIL activity in parasites and cells. The MIL-LUD was more toxic to cells than MIL-Conv and more active against intracellular amastigotes of L. (V.) braziliensis. Conclusion: prepared LUD -MIL retained the anti-leishmanial activity of the MIL and allowed the compound release in human skin and membranes. Testing of experimental cutaneous leishmaniasis models to evaluate the activity of these formulations are urgently needed(AU)

Nanoberries for topical delivery of antioxidants.

With the aim of improving the antioxidant activity of polyphenols from blueberries (Vaccinium myrtillus) on skin targets after topical application, ethanolic extracts from three blueberry varieties (named Millenia, O'Neal, and Blue Crisp) were loaded into ultradeformable liposomes. These nanocarriers are known to be capable of penetrating through the stratum corneum reaching its deeper layers and the viable epidermis. On the other hand, blueberries contain large amounts of polyphenols, whose antioxidant properties as tissue protectors against processes mediated by reactive oxygen species have been extensively proved. Blueberries are usually consumed as edible products, but their antioxidant compounds are poorly absorbed. The antioxidant properties of the extracts were screened before and after being loaded into ultradeformable liposomes made of soy phosphatidylcholine and sodium cholate, of nearly 100 nm in size at 0.223 extract/lipid w/w. The ethanolic extract-loaded ultradeformable liposomes (nanoberries) from Millenia variety retained an 85% of the antioxidant capacity of the free extract and showed low cytotoxicity on HaCaT cells (less than 20%) at active concentration against free radicals.

In vitro phototoxicity of ultradeformable liposomes containing chloroaluminum phthalocyanine against New World Leishmania species.

The use of photodynamic therapy (PDT) against cutaneous leishmaniasis (CL) based on chloroaluminum phthalocyanine (ClAlPc) is a promissory alternative therapy. The main purpose of this article was to assess the internalization and in vitro phototoxic activities of ClAlPc encapsulated in ultradeformable liposomes (UDL-ClAlPc) in Leishmania parasites and mammalian cells. Cell internalization was determined by fluorescence microscopy, cell and parasite damage by standard MTT or direct microscopic analysis and a phototoxic index (PI) was calculated as the compound activity (IC(50)) at 0 J/cm(2)/IC(50) at 17 J/cm(2). Liposomal and free ClAlPc were internalized by infected and non-infected THP-1 cells and co-localized in the mitochondria. Treatment of UDL-ClAlPc was almost 10 times more photoactive than free ClAlPc on THP-1 cells and promastigotes and intracellular amastigotes of Leishmania chagasi and Leishmania panamensis. Liposomal compounds were active on non-irradiated and irradiated cells however PI higher than 50 were calculated. PI for amphotericin B referential drug were lower than 1.2. Empty liposomes tested at the same lipid concentration of active ClPcAl-liposomes were non-toxic. Upon photodynamic treatment a nonselective-parasite activity against intracellular amastigotes were observed and loss of membrane integrity resulting in a release of parasites was detected. Further studies oriented to evaluate both the state of infection after PDT and the effectiveness of UDL as delivery vehicles of ClAlPc in CL experimental models are required.

Curcumin-loaded lipid and polymeric nanocapsules stabilized by nonionic surfactants: an in vitro and In vivo antitumor activity on B16-F10 melanoma and macrophage uptake comparative study.

Curcumin is a polyphenol obtained from the plant Curcuma longa (called turmeric) that displays several pharmacological activities, including anti-inflammatory, antioxidant, antimicrobial and antitumoral activity, but clinical use has been limited by its poor solubility in water and, consequently, minimal systemic bioavailability. We have therefore formulated the drug into nanocarrier systems in an attempt to improve its therapeutic properties. This study evaluates the effect of intraperitoneally administered nanocapsules containing curcumin on subcutaneous melanoma in mice inoculated with B16-F10 cells, and on the cytotoxicity activity against B16-F10 cells in vitro. Phagocytic uptake of formulations was also evaluated upon incubation with macrophage J774 cells by fluorescence microscopy. Lipid and polymeric nanocapsules were prepared by the phase inversion and nanoprecipitation methods, respectively. The uptake of the lipid nanocapsules prepared using Solutol HS15 was significantly reduced in J774 cells. Curcumin, as free drug or as drug-loaded nanocapsules, was administrated at a dose of 6 mg/kg twice a week for 21 days. Free drug and curcumin-loaded nanocapsules significantly reduced tumor volume (P < 0.05 vs. control), but no difference was found in the antitumor activity displayed by lipid and polymeric nanocapsules. This assumption was supported by the in vitro study, in which free curcumin as well as loaded into nanocapsules caused significant reduction of cell viability in a concentration- and time-dependent manner.

Sunlight triggered photodynamic ultradeformable liposomes against Leishmania braziliensis are also leishmanicidal in the dark.

Being independent of artificial power sources, self administered sunlight triggered photodynamic therapy could be a suitable alternative treatment for cutaneous leishmaniasis, that avoids the need for injectables and the toxic side effects of pentavalent antimonials. In this work we have determined the in vitro leishmanicidal activity of sunlight triggered photodynamic ultradeformable liposomes (UDL). ZnPc is a hydrophobic Zn phthalocyanine that showed 20% anti-promastigote activity (APA) and 20% anti-amastigote activity (AA) against Leishmania braziliensis (strain 2903) after 15min sunlight irradiation (15J/cm(2)). However, when loaded in UDL as UDL-ZnPc (1.25µM ZnPc-1mM phospholipids) it elicited 100% APA and 80% AA at the same light dose. In the absence of host cell toxicity, UDL and UDL-ZnPc also showed non-photodynamic leishmanicidal activity. Confocal laser scanning microscopy of cryosectioned human skin mounted in non-occlusive Saarbrücken Penetration Model, showed that upon transcutaneous administration ZnPc penetrated nearly 10 folds deeper as UDL-ZnPc than if loaded in conventional liposomes (L-ZnPc). Quantitative determination of ZnPc confirmed that UDL-ZnPc penetrated homogeneously in the stratum corneum, carrying 7 folds higher amount of ZnPc 8 folds deeper than L-ZnPc. It is envisioned that the multiple leishmanicidal effects of UDL-ZnPc could play a synergistic role in prophylaxis or therapeutic at early stages of the infection.

Etanidazole in pH-sensitive liposomes: design, characterization and in vitro/in vivo anti-Trypanosoma cruzi activity.

In this work, the hydrophilic, low molecular weight and trypanocidal drug etanidazole (ETZ) was loaded in pH-sensitive liposomes (L-ETZ). Liposomes were made of dioleoyl-phosphatidylethanolamine: cholesteryl hemisuccinate (DOPE:CHEMS, 6:4, mol:mol), of 380 nm size at 14% ETZ/total lipid (w/w) ratio. To follow their uptake and intracellular fate by fluorescence microscopy, pH-sensitive liposomes were loaded with the fluorophore/quencher pair HPTS/DPX. A fast and massive delivery of the liposomal aqueous content into the cytosol of murine J774 macrophages was observed. L-ETZ vesicles were phagocytosed by both uninfected and Trypanosoma cruzi-infected macrophages. A 72% of anti-amastigote activity (AA) was demonstrated on L-ETZ-treated J774 cells, whereas the same dose of free ETZ rendered 0% AA. Endovenous administration of L-ETZ at 14 microg/mouse dose provoked significant decrease in parasitemia levels of T. cruzi-infected mice. Conversely, inoculation of a 180-fold higher dose of free ETZ failed in reducing the number of bloodstream trypomastigotes. Hence, these results point to develop systems, such as L-ETZ, designed for selective delivery of drugs to the cytoplasm of phagocytic cells, thus enhancing the efficacy of molecules considered poorly active.