In vitro and in vivo toxicity evaluation of plant virus nanocarriers.

The use of biological self-assembling materials, plant virus nanoparticles in particular, appears very intriguing as it allows a great choice of symmetries and dimensions, easy chemical and biological engineering of both surface and/or internal cavity as well as safe and rapid production in plants. In this perspective, we present an initial evaluation of the safety profile of two structurally different plant viruses produced in Nicotiana benthamiana L. plants: the filamentous Potato virus X and the icosahedral Tomato bushy stunt virus. In vitro haemolysis assay was used to test the cytotoxic effects, which could arise by pVNPs interaction with cellular membranes, while early embryo assay was used to evaluate toxicity and teratogenicity in vivo. Data indicates that these structurally robust particles, still able to infect plants after incubation in serum up to 24h, have neither toxic nor teratogenic effects in vitro and in vivo. This work represents the first safety-focused characterization of pVNPs in view of their possible use as drug delivery carriers.

Lipid nanoparticles for brain targeting III. Long-term stability and in vivo toxicity.

PURPOSE: The aim of the work was to assess the long-term stability and the safety of lipid nanoparticles intended for brain drug delivery. METHODS: Lipid nanoparticles, prepared by high pressure homogenization, were stored at room temperature and 4°C and monitored for their mean hydrodynamic diameter and Gaussian distribution width over time. Cetylpalmitate and polysorbate(®) 80 chemical integrity were investigated by nuclear magnetic resonance on diagnostic signals. Nanoparticle toxicity was assessed in chicken embryos by chorioallantoic membrane assay and in rodents by brain histological evaluation. RESULTS: Data showed nanoparticle stability at 4°C over a period of time of 4 years with only a limited particle size increase while at room temperature destabilization was observed after 9 months. Nuclear magnetic resonance investigation confirmed the absence (<5%) of chemical degradation of the lipid matrix and the surfactant after 4 years of storage at 4°C. Chorioallantoic membrane assay and rat brain histology showed the absence of acute toxicity corroborating previously published data. CONCLUSIONS: Cetylpalmitate nanoparticle long-term physical and chemical stability, together with the in vivo safety, corroborate the existing evidences of the high value of colloidal lipids as parenteral formulations and carriers for brain drug delivery.

Capreomycin supergenerics for pulmonary tuberculosis treatment: preparation, in vitro, and in vivo characterization.

The pulmonary route is one of the main strategies investigated to improve tuberculosis therapy. The aim of this study was to develop a simple and scalable method to produce capreomycin inhalable powders to use as supergeneric. In vitro antimycobacterial activity and in vivo acute toxicity were assessed using agar proportion susceptibility test on Mycobacterium tuberculosis and chicken chorioallantoic membrane assay, respectively. Capreomycin and three different hydrophobic counterions, namely oleate, linoleate, and linolenate, were combined in solution to obtain hydrophobic ion-pairs that were successively spray-dried. Ion-pairing efficiency was influenced by the spray-dryer employed to produce the powder. In the case of capreomycin oleate, both instruments, mini and nano spray-dryer, were suitable to maintain a high ion-paired content, while for capreomycin linoleate and linolenate, mini spray-dryer was the most appropriate instrument. The three formulations showed morphology and particle sizes potentially suitable for inhalation. Capreomycin oleate and linoleate showed the same efficacy of capreomycin sulfate against M. tuberculosis, while capreomycin linolenate showed a reduced efficacy, even though strain growth was inhibited at 10(-4) mycobacterial inoculum. In vivo acute toxicity studies evidenced the lowest toxic potential for capreomycin oleate when compared to the single components or the other two salts. Overall, capreomycin oleate seems to possess the most promising characteristics to be used as supergenerics in pulmonary tuberculosis treatment.

The Wavy Erythropoiesis of Developing Chick Embryos. Isolation of Each Wave by a Differential Lysis and Identification of the Constituent Erythroid Types: (chick embryos/carbonic anhydrase activity/erythropoietic organs/ wave of erythropoiesis).

Erythroid carbonic anhydrase (CA) activity of chick embryos from the third day of incubation to the egg hatching has been determined. Five minor activity peaks with maxima at 3, 6, 9, 15 and 17 days of development and a major one with maximum at 19 days have been found. The correlation between the peak distribution and the timing of release into the blood stream of waves of newly produced erythroid cells has been demonstrated on the basis of the following observations: 1) a linear correlation exists between red cell maturation and increase of CA activity; 2) chick red cells undergo lysis in the "Ørskov" medium when their CA activity exceeds a threshold value (23±3 Units/109 red cells); and 3) the lysis kinetics of red cells in the Ørskov medium is proportional to their CA content. We have thus been able to distinguish the immature erythroid forms from the mature ones on the basis of their behaviour in the Ørskov medium. In the blood of developing chick embryos, we have found waves of newly produced red cells at about 2, 4, 7, 10, 16 and 18 days of development. The same experimental criteria allowed us to detect the waves of red cell production in the erythropoietic organs. One wave has been detected in the blood islands at about 2 days; four waves in the yolk sac at about 5, 6, 11 and 15 days; two waves in the spleen at about 18 and 20 days; two waves in the bone marrow at about 19 days of incubation and 1 day after hatching. Primitive erythroid cells are produced in the first two waves: that of blood islands at 2 days and that of yolk sac at 5 days. Definitive red cells are produced in the other waves with the exception of the second wave of spleen and of the second wave of bone marrow, which are constituted by red cells of adult type.