A biodistribution study of PEGylated PCL-based nanoparticles in C57BL/6 mice bearing B16/F10 melanoma.

One of the major drawbacks that limits the clinical application of nanoparticles is the lack of preliminary investigations related to their biocompatibility, biodegradability and biodistribution. In this work, biodegradable PEGylated polymer nanoparticles (NPs) have been synthesized by using macromonomers based on poly(ε-caprolaconte) oligomers. More in detail, NPs have been produced by adopting a surfactant-free semibatch emulsion polymerization process using PEG chains as a stabilizing agent. The NPs were also labeled with rhodamine B covalently bound to the NPs to quantitatively study their biodistribution in vivo. NPs were investigated in both in vitro and in vivo preclinical systems to study their biodistribution in mice bearing B16/F10 melanoma, as well as their biocompatibility and biodegradability. The NP concentration was evaluated in different tissues at several times after intravenous injection. The disappearance of the NPs from the plasma was biphasic, with distribution and elimination half-lives of 30 min and 15 h, respectively. NPs were retained in tumors and in filter organs for a long time, were still detectable after 7 d and maintained a steady concentration in the tumor for 120 h. 48 h after injection, 70 ± 15% of the inoculated NPs were excreted in the feces. The favorable tumor uptake, fast excretion and absence of cytotoxicity foster the further development of produced NPs as drug delivery carriers.

Intracerebroventricular administration of human umbilical cord blood cells delays disease progression in two murine models of motor neuron degeneration.

The lack of effective drug therapies for motor neuron diseases (MND), and in general for all the neurodegenerative disorders, has increased the interest toward the potential use of stem cells. Among the cell therapy approaches so far tested in MND animal models, systemic injection of human cord blood mononuclear cells (HuCB-MNCs) has proven to reproducibly increase, although modestly, the life span of SOD1G93A mice, a model of familial amyotrophic lateral sclerosis (ALS), even if only few transplanted cells were found in the damaged areas. In attempt to improve the potential efficacy of these cells in the central nervous system, we examined the effect and distribution of Hoechst 33258-labeled HuCB-MNCs after a single bilateral intracerberoventricular injection in two models of motor neuron degeneration, the transgenic SOD1G93A and wobbler mice. HuCB-MNCs significantly ameliorated symptoms progression in both mouse models and prolonged survival in SOD1G93A mice. They were localized in the lateral ventricles, even 4 months after administration. However, HuCB-MNCs were not found in the spinal cord ventral horns. This evidence strengthens the hypothesis that the beneficial role of transplanted cells is not due to cell replacement but is rather associated with the production and release of circulating protective factors that may act both at the central and/or peripheral levels. In particular, we show that HuCB-MNCs release a series of cytokines and chemokines with antiinflammatory properties that could be responsible of the functional improvement of mouse models of motor neuron degenerative disorders.

Mitochondrial oxidative metabolism in motor neuron degeneration (mnd) mouse central nervous system.

The mnd mouse spontaneously develops slowly evolving motoneuron pathology leading to progressive motor impairment. There is strong evidence that a complex interplay between oxidative stress, mitochondria abnormalities and alteration of glutamate neurotransmission plays an important role in the pathogenesis of motor neuron diseases. Therefore, we investigated the presence of mitochondrial dysfunction in frontal, central (comprising the motor area) and occipital regions of the cerebral cortex and in the spinal cord of 35-week-old mnd mice. Lipid peroxide derivatives reacting with thiobarbituric acid (TBARS) were measured in the cervical, thoracic and lumbar spinal cord. In addition biochemical and behavioural analyses were carried out in mnd mice chronically treated with l-carnitine from the 11th to the 34th week of life (mndT mice). Slight but significant alterations of mitochondrial enzyme activities were seen in the mnd cortical regions. The central area was the most affected and both complex I, IV and citrate synthase were decreased with respect to controls. The rate of oxygen consumption (QO2) was markedly decreased in both the upper (cervical + upper portion of the thoracic region) and lower (lumbar + lower portion of the thoracic region) mnd spinal cord. The level of TBARS showed a rostro-caudal trend to increase, being 30% higher in the lumbar tract of mnd mice in comparison with controls. L-carnitine treatment increased the mitochondrial enzyme activities in cortical regions towards control value and was effective in enhancing QO2 and decreasing TBARS levels in the spinal cord of mndT. Behavioural testing showed that L-carnitine significantly delayed the onset of motor behaviour impairment. This beneficial effect was declining at 35 week of age, when the biochemical measurements were performed.

Acetyl-L-carnitine shows neuroprotective and neurotrophic activity in primary culture of rat embryo motoneurons.

We evaluated the role of acetyl-L-carnitine (ALCAR) in protecting primary motoneuron cultures exposed to excitotoxic agents or serum-brain derived neurotrophic factor (BDNF) deprived. To exclude that ALCAR works as a metabolic source, we compared its effects with those of L-carnitine (L-CAR), that seems to have no neurotrophic effect. A concentration of 10 mM ALCAR, but not L-CAR, significantly reduced the toxic effect of 50 microM N-methyl-D-aspartate (NMDA, % viability: NMDA 45.4+/-2.80, NMDA+ALCAR 90.8+/-11.8; P<0.01) and of 5 microM kainate in cultured motoneurons (% viability: kainate 40.66+/-10.73; kainate+ALCAR 63.80+/-13.88; P<0.05). The effect was due to a shift to the right of the dose-response curve for kainate (EC50 for kainate 5.99+/-1.012 microM; kainate+ALCAR 8.62+/-1.13 microM; P<0.05). ALCAR, but not L-CAR, significantly protected against BDNF and serum-deprivation reducing the apoptotic cell death (% viability respect to control: without BDNF/serum 61.8+/-13.3: without BDNF/serum+ALCAR 111.8+/-13.9; P<0.01). Immunocytochemistry showed an increase in choline acethyltransferase and tyrosine kinaseB receptors in motoneurons treated with ALCAR but not with L-CAR. These results suggest that ALCAR treatment improves the motoneurons activity, acting as a neurotrophic factor.

Glutamate transporters in the spinal cord of the wobbler mouse.

We studied the role of glutamate excitotoxicity in motor neuron degeneration in the wobbler mouse (wr/wr), a model of amyotrophic lateral sclerosis and spinal muscular atrophies. Choline acetyltransferase (ChAT) activity was decreased in the cervical spinal cord and in the muscles innervated by nerves originating in this region of wobbler mice, but no differences were found in the lumbar spinal cord and in the hindleg muscles. Glial fibrillar acid protein (GFAP), a marker of reactive gliosis, was significantly higher in the cervical spinal cord of wobbler mice aged 4 weeks than in controls and the differences were more marked at 12 weeks; no differences were found in the lumbar spinal cord. In spite of this selective degeneration of motor neurons (resulting in strong decrease in the neuronal glutamate transporter EAAC1) and reactive gliosis in the cervical spinal cord, the levels of the glial glutamate transporter proteins GLT-1 and GLAST were similar in wobbler and control mice. Plasma concentrations of excitatory amino acids were no different at any time examined. Our results exclude the involvement of decrease in glutamate GLT 1 transporter in the motor neuron degeneration in wobbler mice.

Properties of Ca2+-activated K+ channels in erythrocytes from patients with myotonic muscular dystrophy.

Using the single-channel patch-clamp technique, Ca2+-activated K+ channels of erythrocytes from patients with myotonic muscular dystrophy (MyD) were studied. Elementary single-channel properties--conductance, rectification, kinetics, voltage- and calcium-dependence--measured in inside-out patches of MyD erythrocytes, did not differ significantly from those of control cells. The activity of the channels, studied in patches attached to red cells from MyD patients, exhibited mean patch currents which were significantly higher than the controls. The increased mean patch current was due to a higher opening frequency, associated with a reduced mean channel closed time. These results indicate that Ca2+-activated K+ channels of erythrocytes from patients either detect a higher intracellular calcium concentration and/or express an augmented calcium-sensitivity. Since these channels are targets for phosphorylation, our findings make it possible to identify defective kinase mechanisms, in minimally disturbed cells of the patient, at a molecular level of resolution.