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.

Development of Nutraceutical Emulsions as Risperidone Delivery Systems: Characterization and Toxicological Studies.

Emulsions are gaining increasing interest to be applied as drug delivery systems. The main goal of this work was the formulation of an oil/water nutraceutical emulsion (NE) for oral administration, enriched in omega 3 (ω3) and omega 6 (ω6), and able to encapsulate risperidone (RISP), an antipsychotic drug widely used in the treatment of autism spectrum disorders (ASD). RISP has low solubility in aqueous medium and poor bioavailability because of its metabolism and high protein binding. Coadministration of ω3, ω3, and vitamin E complexed with RISP might increase its bioavailability and induce a synergistic effect on the treatment of ASD. Here, we developed an easy and quick method to obtain NEs and then optimized them. The best formulation was chosen after characterization by particle size, defects of the oil-in-water interface, zeta potential (ZP), and in vitro drug release. The formulation selected was stable over time, with a particle size of around 3 µm, a ZP lower than -20 mV and controlled drug release. To better understand the biochemical properties of the formulation obtained, we studied in vitro toxicity in the Caco-2 cell line. After 4 h of treatment, an increase in cellular metabolism was observed for all RISP concentrations, but emulsions did not change their metabolic rate, except at the highest concentration without drug (25 µg/mL), which showed a significant reduction in metabolism respect to the control. Additionally, locomotor activity and heart rate in zebrafish were measured as parameters of in vivo toxicity. Only the highest concentration (0.625 µg/mL) showed a cardiotoxic effect, which corresponds to the decrease in spontaneous movement observed previously. As all the materials contained in the formulations were US FDA approved, the NE selected would be good candidate for clinical trials.

Optimization and in vivo toxicity evaluation of G4.5 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%). Since new strategies to improve efficient treatments are needed, we studied the efficiency of anionic G4.5 PAMAM dendrimers as nanocarriers for this therapeutic drug. To this end, we explored dendrimer-risperidone complexation dependence on solvent concentration, pH and molar relationship. The best dendrimer-risperidone incorporation (46 risperidone molecules per dendrimer) was achieved with a mixture of chloroform:methanol 50∶50 v/v solution pH 3. In addition, to explore the possible effects of this complex, in vivo studies were carried out in the zebrafish model. Changes in the development of dopaminergic neurons and motoneurons were studied using tyrosine hydroxylase and calretinin, respectively. Physiological changes were studied through histological sections stained with hematoxylin-eosin to observe possible morphological brain changes. The most significant changes were observed when larvae were treated with free risperidone, and no changes were observed when larvae were treated with the complex.

Relationship between the adjuvant and cytotoxic effects of the positive charges and polymerization in liposomes.

Vaccine development today encounters a main obstacle, which is the need for effective adjuvants suitable for clinical trials. Aluminum salts, discovered 70 years ago and, very recently, MF59, are the only types of adjuvants currently used in vaccines licensed by the U.S. Food and Drug Administration. Liposomes represent an alternative approach to vaccine adjuvants. In this article, we describe the inflammatory response and biological effect of polymerization and the addition of positive charges in liposome formulations. Nonpolymerized cationic (NP(+)) liposomes significantly reduce metabolism in Vero cells after 24 hours. Correspondingly, both NP(+) and polymerized cationic (P(+)) liposomes reduce cell viability following a 48-hour incubation. Similar results were obtained with cells from the peritoneal cavities of mice. Paradoxically, those liposomes that presented clearly cytostatic or cytotoxic effects in vitro stimulated metabolism and had a mitogenic effect in vivo. Finally, the adjuvant effect was tested by immunization in BALB/c mice. The major effect was obtained with NP(+) liposomes. Accordingly, we also demonstrated that NP(+) liposomes injected into the dermis produced an outstanding inflammatory reaction, showing the histopathological characteristics of an inoculation granuloma. Thus, positive charge would play an important role in the immunoadjuvant effect of liposomes by conferring them cytotoxic capacity.

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.