Biodegradable composite porous poly(dl-lactide-co-glycolide) scaffold supports mesenchymal stem cell differentiation and calcium phosphate deposition.

In recent decades, tissue engineering strategies have been proposed for the treatment of musculoskeletal diseases and bone fractures to overcome the limitations of the traditional surgical approaches based on allografts and autografts. In this work we report the development of a composite porous poly(dl-lactide-co-glycolide) scaffold suitable for bone regeneration. Scaffolds were produced by thermal sintering of porous microparticles. Next, in order to improve cell adhesion to the scaffold and subsequent proliferation, the scaffolds were coated with the osteoconductive biopolymers chitosan and sodium alginate, in a process that exploited electrostatic interactions between the positively charged biopolymers and the negatively charged PLGA scaffold. The resulting scaffolds were characterized in terms of porosity, degradation rate, mechanical properties, biocompatibility and suitability for bone regeneration. They were found to have an overall porosity of ∼85% and a degradation half time of ∼2 weeks, considered suitable to support de novo bone matrix deposition from mesenchymal stem cells. Histology confirmed the ability of the scaffold to sustain adipose-derived mesenchymal stem cell adhesion, infiltration, proliferation and osteo-differentiation. Histological staining of calcium and microanalysis confirmed the presence of calcium phosphate in the scaffold sections.

Reshaping antibiotics through hydrophobic drug-bile acid ionic complexation enhances activity against Staphylococcus aureus biofilms.

The antibiotic era is on the verge of a profound change and facing a ground shaking crisis. The frequent failures of antibiotic treatments are often associated with biofilm formation, which is responsible for chronic infections, exacerbation as well as reinfection. So far, albeit the large number of valuable strategies employed to combat biofilm formation, little success has been recorded. In this work, we propose a simple approach, based on hydrophobic ionic complexation with the bile acids, deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA), to enhance anti-biofilm activity of well-known antibiotics, namely kanamycin (K), amikacin (A) and vancomycin (V). Activity was evaluated against Staphylococcus aureus ATCC 29213 and six methicillin-resistant clinical isolates. The formation of a 1:4 ADCA and KDCA and 1:1 VUDCA complexes was confirmed by 1HNMR, in silico molecular dynamics simulations, as well as thermal, spectrophotometric and HPLC analyses. The complexes showed higher inhibition of S. aureus growth compared to parent drugs and a concentration-independent biofilm inhibition and dispersion capacity in the order KDCA > ADCA >>VUDCA, even at concentrations ten-fold below the MIC. S. aureus growth inhibition evaluated upon treatment with bile acid-drug sequential addition and the complexes as well as the measured complex stability in solution suggest a bile acid carrier role. The complexes showed in vivo toxicity only at 10×MIC concentration on the chicken embryo chorioallantoic membrane model in the order KDCA < ADCA < VUDCA. KDCA was safe at all concentrations. Although several aspects to be addressed, this approach is promising due to its simplicity, the proved in vitro anti-biofilm activity enhancement and tolerability. A potential pulmonary drug delivery application is envisaged.

Dynamic behavior of a spring-powered micronozzle needle-free injector.

Conventional injection is still the leading method to deliver macromolecular therapeutics. Needle injection is considered a low compliance administration strategy, principally due to pain and needle phobia. This has fostered the research on the development of alternative strategies to circumvent the skin barrier. Among needle-free drug delivery methods, jet injection is an old strategy with great potential not yet completely disclosed. Here, the design, engineering and dynamic behavior of a novel spring-powered micronozzle needle-free injector is presented. Fluid mechanics was first studied in air to calculate jet force and speed as well as injection duration in different conditions. Polyacrylamide gel was used to simulate a soft tissue and to investigate the jet evolution over time of different injected doses. Finally, ex vivo characterization was carried out on pig skin. Results evidenced a direct dependence of the force, velocity, and duration with the injection volume. The model material allowed individuating the different steps of jet penetration and to attempt a mechanistic explanation. A different behavior has been recorded in the skin with interesting findings for subcutaneous and/or dermal delivery. Peculiar features with respect to existing jet injectors confers to this device good potentiality for a future clinical application.

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.

A pool of peptides extracted from wheat bud chromatin inhibits tumor cell growth by causing defective DNA synthesis.

BACKGROUND: We previously reported that a pool of low molecular weight peptides can be extracted by alkali treatment of DNA preparations obtained from prokaryotic and eukaryotic cells after intensive deproteinization. This class of peptides, isolated from wheat bud chromatin, induces growth inhibition, DNA damage, G2 checkpoint activation and apoptosis in HeLa cells. In this work we studied their mechanism of action by investigating their ability to interfere with DNA synthesis. METHODS: BrdUrd comet assays were used to detect DNA replication defects during S phase. DNA synthesis, cell proliferation, cell cycle progression and DNA damage response pathway activation were assessed using 3H-thymidine incorporation, DNA flow cytometry and Western blotting, respectively. RESULTS: BrdUrd labelling close to DNA strand discontinuities (comet tails) detects the number of active replicons. This number was significantly higher in treated cells (compared to controls) from entry until mid S phase, but markedly lower in late S phase, indicating the occurrence of defective DNA synthesis. In mid S phase the treated cells showed less 3H-thymidine incorporation with respect to the controls, which supports an early arrest of DNA synthesis. DNA damage response activation was also shown in both p53-defective HeLa cells and p53-proficient U2OS cells by the detection of the phosphorylated form of H2AX after peptide treatment. These events were accompanied in both cell lines by an increase in p21 levels and, in U2OS cells, of phospho-p53 (Ser15) levels. At 24 h of recovery after peptide treatment the cell cycle phase distribution was similar to that seen in controls and CDK1 kinase accumulation was not detected. CONCLUSION: The data reported here show that the antiproliferative effect exhibited by these chromatin peptides results from their ability to induce genomic stress during DNA synthesis. This effect seems to be S-phase specific since surviving cells are able to progress through their normal cell cycle when the peptide fraction is removed from the culture medium. It is likely that the subsequent apoptosis is a consequence of the failed attempt of the tumour cells to repair the DNA damage induced by the peptides.

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.