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.

The Latin American Federation of Biophysical Societies (LAFeBS).

The Latin American Federation of Biophysical Societies (LAFeBS) was constituted in 2007 in Montevideo, Uruguay, as a collaborative effort among the Biophysical Societies of Argentina, Brazil, and Uruguay. This visionary collaboration foresees the future of Biophysics in Latin America. In this commentary, we will briefly review the history of LAFeBS, the remarkable path undertaken since its foundation 16 years ago, and its key initiative, the Latin American Postgraduate Program in Biophysics (POSLATAM).

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.

Lipopolymers and lipids from lung surfactants in association with N-acetyl-l-cysteine: Characterization and cytotoxicity.

In the present work, we obtained polymeric diacetylene liposomes that can associate N-Acetyl-l-Cysteine (NAC), a broad spectrum mucolytic. The reason for studying these formulations is that they could be applied in the future as NAC delivery systems, with a possible dose reduction but maintaining its effect. Liposomes used herein are obtained by a photopolymerization reaction, thus gaining stability and rigidity. Lipids belonging to lung surfactant were added in different ratios to the formulations in order to maximize its possible interaction with the lung tissue. Because of lipopolymer stability, the oral or nasal route could be appropriated. This formulation could efficiently transport NAC to exert its mucolytic activity and help in diseases such as cystic fibrosis, which has abnormal mucus production. Also, this type of treatment could be useful in other types of diseases, interacting with the mucus layer and making the lung tissue more permeable to other therapies. Formulations so obtained presented high levels of polymerization. Also, they present small hollow fibers structures with a high number of polymeric units. These types of arrangements could present advantages in the field of drug delivery, giving the possibility of a controlled release. Lipopolymers with lipids from lung surfactant associated with NAC are promising complexes in order to treat not only respiratory illnesses. The stability of the formulation would allow its inoculation through other routes such as the oral one, helping the reposition of NAC as an antioxidant drug. Finally, these formulations are non-toxic and easy to produce.

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.

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.

Folic acid magnetic nanotheranostics for delivering doxorubicin: Toxicological and biocompatibility studies on Zebrafish embryo and larvae.

Doxorubicin (DOXO) is a chemotherapeutic agent widely used for the treatment of solid tumors and hematologic malignancies in both adults and children. However, DOXO causes short- and long-term cardiotoxicity and others undesirable side effects, such as nephrotoxicity and neurotoxicity. Magnetic nanoparticles (MNPs) allow the delivery of drugs specifically to target place, employing an external magnet. Moreover, they may act as contrast agents in MRI providing information on the diagnostic of diverse pathologies. In this way, two functions may be combined in a unique nanosystem known as theranostic. Also, the MNPs can be modified with folic acid (MNPs@FA) to increase the uptake by cancer cells that overexpress the FA receptors. In previous works, our collaborators obtained and characterized MNPs, MNPs@FA, and MNPs@FA@DOXO. It is essential to study the biosafety of nanotheranostic, and there is no published study of Fe3O4 nanoparticles developmental toxicity. Because of that, this work aimed to study the in vivo toxicity and biocompatibility of DOXO, MNPs@FA, and MNPs@FA@DOXO using zebrafish embryo and larvae as an animal model. Viability, developmental toxicity, changes in spontaneous movement (neurotoxicity), changes in cardiac rhythm (cardiotoxicity), and efficiency of DOXO-uptake were studied. While the 48-h treatment with 50 µg/mL of DOXO resulted in a 30% larvae death and the development of significant morphological abnormalities, the treatment with MNPs@FA@DOXO and MNPs@FA did not reduce the viability and did not cause developmental abnormalities. Besides, the MNPs@FA@DOXO reduced the cardiotoxicity and promoted a more rapid and significant uptake of DOXO by zebrafish larvae.

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.

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.

Nanotoxicological and teratogenic effects: A linkage between dendrimer surface charge and zebrafish developmental stages.

This article reports novel results about nanotoxicological and teratogenic effects of the PAMAM dendrimers DG4 and DG4.5 in zebrafish (Danio rerio). Zebrafish embryos and larvae were used as a rapid, high-throughput, cost-effective whole-animal model. The objective was to provide a more comprehensive and predictive developmental toxicity screening of DG4 and DG4.5 and test the influence of their surface charge. Nanotoxicological and teratogenic effects were assessed at developmental, morphological, cardiac, neurological and hepatic level. The effect of surface charge was determined in both larvae and embryos. DG4 with positive surface charge was more toxic than DG4.5 with negative surface charge. DG4 and DG4.5 induced teratogenic effects in larvae, whereas DG4 also induced lethal effects in both zebrafish embryos and larvae. However, larvae were less sensitive than embryos to the lethal effects of DG4. The platform of assays proposed and data obtained may contribute to the characterization of hazards and differential effects of these nanoparticles.

Diacetylenic lipids in the design of stable lipopolymers able to complex and protect plasmid DNA.

Different viral and non-viral vectors have been designed to allow the delivery of nucleic acids in gene therapy. In general, non-viral vectors have been associated with increased safety for in vivo use; however, issues regarding their efficacy, toxicity and stability continue to drive further research. Thus, the aim of this study was to evaluate the potential use of the polymerizable diacetylenic lipid 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) as a strategy to formulate stable cationic lipopolymers in the delivery and protection of plasmid DNA. Cationic lipopolymers were prepared following two different methodologies by using DC8,9PC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the cationic lipids (CL) 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), stearylamine (SA), and myristoylcholine chloride (MCL), in a molar ratio of 1:1:0.2 (DMPC:DC8,9PC:CL). The copolymerization methodology allowed obtaining cationic lipopolymers which were smaller in size than those obtained by the cationic addition methodology although both techniques presented high size stability over a 166-day incubation period at 4°C. Cationic lipopolymers containing DOTAP or MCL were more efficient in complexing DNA than those containing SA. Moreover, lipopolymers containing DOTAP were found to form highly stable complexes with DNA, able to resist serum DNAses degradation. Furthermore, neither of the cationic lipopolymers (with or without DNA) induced red blood cell hemolysis, although metabolic activity determined on the L-929 and Vero cell lines was found to be dependent on the cell line, the formulation and the presence of DNA. The high stability and DNA protection capacity as well as the reduced toxicity determined for the cationic lipopolymer containing DOTAP highlight the potential advantage of using lipopolymers when designing novel non-viral carrier systems for use in in vivo gene therapy. Thus, this work represents the first steps toward developing a cationic lipopolymer-based gene delivery system using polymerizable and cationic lipids.

Association with Amino Acids Does Not Enhance Efficacy of Polymerized Liposomes As a System for Lung Gene Delivery.

Development of improved drug and gene delivery systems directly into the lungs is highly desirable given the important burden of respiratory diseases. We aimed to evaluate the safety and efficacy of liposomes composed of photopolymerized lipids [1,2-bis-(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine] associated with amino acids as vectors for gene delivery into the lungs of healthy animals. Lipopolymer vesicles, in particular, are more stable than other types of liposomes. In this study, lipopolymers were associated with l-arginine, l-tryptophan, or l-cysteine. We hypothesized that the addition of these amino acids would enhance the efficacy of gene delivery to the lungs by the lipopolymers. l-Arginine showed the highest association efficiency due to its positive charge and better surface interactions. None of the formulations caused inflammation or altered lung mechanics, suggesting that these lipopolymers can be safely administered as aerosols. All formulations were able to induce eGFP mRNA expression in lung tissue, but the addition of amino acids reduced delivery efficacy when compared with the simple lipopolymer particle. These results indicate that this system could be further explored for gene or drug delivery targeting lung diseases.

Optimization and in vitro toxicity evaluation of G4 PAMAM dendrimer-risperidone complexes.

Risperidone is an approved antipsychotic drug belonging to the chemical class of benzisoxazole. This drug has low solubility in aqueous medium and poor bioavailability due to extensive first-pass metabolism and high protein binding (>90%). As new strategies to improve treatments efficiency are needed, we have studied cationic G4 PAMAM dendrimers' performance to act as efficient nanocarriers for this therapeutic drug. In this respect, we explored dendrimer-risperidone complexation dependence on solvent, temperature, pH and salt concentration, as well as in vitro cytotoxicity measured on L929 cell line and human red blood cells. The best dendrimer-risperidone incorporation was achieved when a mixture of 70:30 and 90:10 v/v chloroform:methanol was used, obtaining 17 and 32 risperidone molecules per dendrimer, respectively. No cytotoxicity on L929 cells was found when dendrimer concentration was below 3 × 10(-2) µM and risperidone concentration below 5.1 µM. Also, no significant hemolysis or morphological changes were observed on human red blood cells. Finally, attempting to obtain an efficient drug delivery system for risperidone, incorporation in G4 PAMAM dendrimers was optimized, improving drug solubility with low cytotoxicity.

Biodistribution of liposome/DNA systems after subcutaneous and intraperitoneal inoculation.

In this work, we analyzed protein interaction, cell toxicity, and biodistribution of liposome formulation for further possible applications as DNA vehicles in gene-therapy protocols. In relation to protein interaction, cationic liposomes showed the lowest protein interaction, but this parameter was incremented with DNA association. On the other hand, noncharged liposomes presented high protein interaction, but DNA association decreased this parameter. Protein interaction of polymeric liposomes did not change with DNA association. Cell toxicity of these three liposome formulations was low, cell death became present at concentrations higher than 0.5 mg/mL, and these concentrations were higher than those usually used in transfection assays. In the case of noncharged and polymeric liposomes, toxicity increased upon interaction with serum proteins. DNA/liposome-mediated tissue distribution was analyzed in Balb-c female mice. Results indicated that noncharged liposomes were able to deliver DNA to liver after intraperitoneal (i.p.) inoculation, while polymeric liposomes were able to deliver DNA to kidney by using the same inoculation route. Cationic liposomes were able to deliver DNA to a wide range of tissues by the i.p. route (e.g., liver, intestine, kidney, and blood). After subcutaneous inoculation, only cationic liposomes were able to deliver DNA to blood, but not the other two formulations within the detection limits of the method.

Alternative site of implantation affects tumor malignancy and metastatic potential in mice: its comparison to the flank model.

MC-C fibrosarcoma and B16F0 melanoma tumors were implanted intradermally in the dorsal region of the foot of mice. Tumor progression was compared to standard implantation in the flank. Although foot tumors only reached 13% (MC-C) and 25% (B16F0) of the mean volume of flank tumors, a more malignant phenotype in terms of histology and survival rate was observed in this type of tumors. Moreover, lung metastases were only detected in hosts bearing foot tumors, in contrast to MC-C and B16F0 populations with tumors growing in the flank. In addition, cellular influx and local immune reaction were higher in the dorsal region of the foot. According to our results, the dermis of the flank allows excessive tumor growth due to its low reactivity. Thus, differences in innate and adaptive immune effectors between the evaluated tumor microenvironments would account for the differences in tumor malignancy. Due to its striking differences with the standard flank inoculation, the tumor implantation model herein introduced could be a valuable tool to study the metastatic potential of different cell lines and the microenvironment components affecting tumor growth.

Brucella abortus MFP: a trimeric coiled-coil protein with membrane fusogenic activity.

The bacterial genus Brucella consists of a group of facultative intracellular pathogens which produces abortion and infertility in animals and a chronic debilitating febrile illness in humans. BMFP is a basic protein of Brucella abortus that belongs to a highly conserved group of homologue proteins of unknown structure and function in proteobacteria (COG2960). In this study, we report the structural and biochemical characterization of this protein. We found that BMFP has two structural domains: a carboxyl-terminal coiled-coil domain through which the protein self-associates as a trimer and a natively disordered amino-terminal domain which has propensity to adopt an amphipathic alpha-helical structure. This natively unfolded domain undergoes a structural rearrangement from unfolded to alpha-helix in the presence of high ionic strength, acidic pH, detergents, and phospholipid vesicles. Moreover, we demonstrated that the interaction of BMFP with phospholipid vesicles promotes in vitro membrane fusion. These results contribute to the elucidation of the structural and functional properties of this protein and its homologues present in most proteobacteria.