Beneficial Effects of Milk-Derived Extracellular Vesicles on Liver Fibrosis Progression by Inhibiting Hepatic Stellate Cell Activation.

Liver fibrosis is the consequence of various chronic liver diseases, resulting in accumulation of extracellular matrix, following the activation and proliferation of hepatic stellate cells (HSCs). Based on the milk-derived extracellular vesicles' (MDEs') characteristics and biological proprieties, we investigate whether MDEs may regulate fibrotic progression by inhibiting HSCs' activation via the MDEs' miRNA content. In order to study this question, we examined the effect of human and cow MDEs on HSCs isolated from murine livers, on activation, proliferation and their proteins' expression. We have shown that MDEs are able to enter into HSCs in vitro and into the livers in vivo. MDEs inhibited HSCs' proliferation following stimulation with PDGF. Moreover, in vivo treatment with MDEs resulted in an increase of in miRNA-148 and Let7a expression in HSCs. In contrast, treatment with MDEs reduced the expression of miR-21 in HSCs. In addition, MDEs regulate HSC activation, as was shown by downregulation of collagen I expression and alpha smooth muscle actin, and upregulation of PPARγ. MDEs carrying beneficial miRNAs can be a nontoxic natural target for treatment of liver cirrhosis.

Cow and Human Milk-Derived Exosomes Ameliorate Colitis in DSS Murine Model.

The aim of this study was to investigate the therapeutic effect of cow and human milk derived exosomes (MDEs) on colitis. We used gavage administration of fluorescent labeled MDEs to track their localization patterns in vivo and studied their therapeutic effect on colitis in a dextran sulfate sodium (DSS)-induced colitis model. MDEs attenuated the severity of colitis induced by DSS and statistically reduced the histopathological scoring grade and shortening of the colon. Likewise, treatment with MDEs reduced the expression of interleukin 6 and tumor necrosis factor-α. Moreover, miRNAs highly expressed in milk, such as miRNA-320, 375, and Let-7, were found to be more abundant in the colon of MDE-treated mice compared with untreated mice; contrastingly, the expression of their target genes, mainly DNA methyltransferase 1 (DNMT1) and DNMT3 were downregulated. Furthermore, the level of TGF-ß was upregulated in the colon of MDE-treated mice. We demonstrated that MDEs have a therapeutic and anti-inflammatory effect on colitis, involving several complementary pathways in its mechanism of action. The therapeutic effects of MDEs might have implications for the possible addition of MDEs as a nutrient in enteral nutrition formulas for patients with inflammatory bowel disease.

White matter lesions, cerebral inflammation and cognitive function in a mouse model of cerebral hypoperfusion.

Development of specific treatments for vascular dementia requires appropriate animal models. Bilateral carotid artery stenosis (BCAS) employs metal coils wrapped around both common carotid arteries to induce cerebral hypoperfusion, white matter lesions and memory impairment in mice. We focused on the relationship of memory impairment induced by BCAS to white matter lesions demonstrated by ex vivo magnetic resonance imaging (MRI). We found a significant effect of BCAS on perceptual learning in the novel object recognition test and on number of errors and latency to platform in the radial arm water maze. MRI analysis revealed a significant effect of BCAS on diffusion tensor imaging (DTI) parameters in white matter areas. After correction for multiple testing, significantly lower fractional anisotropy (FA) values were found in the corpus callosum and anterior commissure and significantly higher mean diffusivity values in the internal capsule. Focusing on the corpus callosum, we found that correlations between FA and number of errors on the RAWM test were significant after controlling for treatment. We further found that the effects of BCAS on cognition were partly mediated by its effects on white matter integrity. Immunofluorescence studies demonstrated significantly higher microglia cell density and soma size in the corpus callosum of BCAS mice compared to controls, and these parameters were correlated with the imaging data. The results of this study indicate that cognitive deficits induced by cerebral hypoperfusion due to BCAS result in part from microglia activation and disruption of white matter integrity, supporting the face and construct validity of this unique model of vascular dementia.

Intracerebroventricular Streptozotocin Induces Obesity and Dementia in Lewis Rats.

BACKGROUND: Animal models of dementia associated with metabolic abnormalities play an important role in understanding the bidirectional relationships between these pathologies. Rodent strains develop cognitive dysfunctions without alteration of peripheral metabolism following intracerebroventricular administration of streptozotocin (icv-STZ). OBJECTIVE: We aimed to estimate the effect of icv-STZ on cognitive functions and peripheral metabolism in Lewis rats, which are rarely used for the induction of cognitive abnormalities. METHODS: Inbred adult Lewis rats were treated with single icv-STZ (3 mg/kg). Cognitive functions were assessed using Morris water maze (MWM) test and locomotion by the Open Field test. Metabolic alterations were studied using histological and biochemical analysis of brain and peripheral tissues. RESULTS: The icv-STZ induced rapid weight decline during the first two weeks. Thereafter, the rats showed an accelerated weight gain. Three months after the icv-STZ treatment, the rats were severely obese and revealed fatty liver, pancreatic islet hypertrophy, significantly elevated levels of blood insulin, leptin, and adiponectin, but intact peripheral glucose homeostasis. The icv-STZ rats expressed amyloid-ß deposits in blood vessels of leptomeningeal area, microgliosis, astrogliosis, and spongiosis in fimbria-fornix area of hippocampus. Locomotor activities of icv-STZ treated and sham-operated rats were similar. In the MWM test, the icv-STZ treated rats demonstrated severely impaired spatial learning during both acquisition and reversal phases. CONCLUSIONS: Icv-STZ treated Lewis rats develop severe dementia associated with obesity and peripheral metabolic abnormalities. This animal model may be useful for exploring the pathophysiological relationship between obesity and dementia and provides a new tool for development of effective therapy.

Differentially Severe Cognitive Effects of Compromised Cerebral Blood Flow in Aged Mice: Association with Myelin Degradation and Microglia Activation.

Bilateral common carotid artery stenosis (BCAS) models the effects of compromised cerebral blood flow on brain structure and function in mice. We compared the effects of BCAS in aged (21 month) and young adult (3 month) female mice, anticipating a differentially more severe effect in the older mice. Four weeks after surgery there was a significant age by time by treatment interaction on the radial-arm water maze (RAWM; p = 0.014): on the first day of the test, latencies of old mice were longer compared to the latencies of young adult mice, independent of BCAS. However, on the second day of the test, latencies of old BCAS mice were significantly longer than old control mice (p = 0.049), while latencies of old controls were similar to those of the young adult mice, indicating more severe impairment of hippocampal dependent learning and working memory by BCAS in the older mice. Fluorescence staining of myelin basic protein (MBP) showed that old age and BCAS both induced a significant decrease in fluorescence intensity. Evaluation of the number oligodendrocyte precursor cells demonstrated augmented myelin replacement in old BCAS mice (p < 0.05) compared with young adult BCAS and old control mice. While microglia morphology was assessed as normal in young adult control and young adult BCAS mice, microglia of old BCAS mice exhibited striking activation in the area of degraded myelin compared to young adult BCAS (p < 0.01) and old control mice (p < 0.05). These findings show a differentially more severe effect of cerebral hypoperfusion on cognitive function, myelin integrity and inflammatory processes in aged mice. Hypoperfusion may exacerbate degradation initiated by aging, which may induce more severe neuronal and cognitive phenotypes.

Physicochemical parameters affecting liposomal bisphosphonates bioactivity for restenosis therapy: internalization, cell inhibition, activation of cytokines and complement, and mechanism of cell death.

Partial inactivation and transient depletion of monocytes/macrophages by liposomal bisphosphonates (LIP-BPs) is widely experimented in various inflammatory disorders including restenosis. Previous studies on activation of cytokines by LIP-BPs are limited to certain cell lines. Moreover, the correlation between in vitro and in vivo studies and complement (C) activation has not been reported. We report here a comprehensive study on the bioactivity of LIP-BPs on various cells' internalization and proliferation, mechanism of cell death, cytokines (in vitro and in vivo) and C activation (in the rat, rabbit and pig). The role of the following parameters has been determined i) drug type (clodronate/alendronate); ii) vesicles size (60-800nm); iii) charge (neutral/negative/ positive); and iv) cell culture type (various cell lines and primary cultures). It was found that monocyte/macrophage inhibition and cytokine activation depend on the cell type, with a limited correlation to the bioactivity obtained in the rat and rabbit models of restenosis. Negatively charged liposomes (85+/-20nm) effectively depleted rabbit's monocytes (67% depletion), with a minor activation of cytokines and no C activation. It is concluded that cell culture studies are insufficient for assessing cytokine activation, and that by controlling LIP-BP properties (size, charge and drug type) optimal bioactivity could be achieved.

Liposomal simvastatin attenuates neointimal hyperplasia in rats.

Monocytes, macrophages, and inflammation play a key role in the process of neointimal proliferation and restenosis. The present study evaluated whether systemic and transient depletion of monocytes could be obtained by a single intravenous (IV) injection of simvastatin liposomes, for the inhibition of neointima formation. Balloon-injured carotid artery rats (n = 30) were randomly assigned to treatment groups of free simvastatin, simvastatin in liposomes (3 mg/kg), and saline (control). Stenosis and neointima to media ratio (N/M) were determined 14 days following single IV injection at the time of injury by morphometric analysis. Depletion of circulating monocytes was determined by flow cytometry analyzes of blood specimens. Inhibition of RAW264.7, J774, and THP-1 proliferation by simvastatin-loaded liposomes and free simvastatin was determined by the 3-(4, 5-dimethylthiazolyl-2)-2, 5- diphenyltetrazolium bromide assay. Simvastatin liposomes were successfully formulated and were found to be 1.5-2 times more potent than the free drug in suppressing the proliferation of monocytes/macrophages in cell cultures of RAW 264.7, J774, and THP-1. IV injection of liposomal simvastatin to carotid-injured rats (3 mg/kg, n = 4) resulted in a transient depletion of circulating monocytes, significantly more prolonged than that observed following treatment with free simvastatin. Administration to balloon-injured rats suppressed neointimal growth. N/M at 14 days was 1.56 +/- 0.16 and 0.90 +/- 0.12, control and simvastatin liposomes, respectively. One single systemic administration of liposomal simvastatin at the time of injury significantly suppresses neointimal formation in the rat model of restenosis, mediated via a partial and transient depletion of circulating monocytes.

A new double emulsion solvent diffusion technique for encapsulating hydrophilic molecules in PLGA nanoparticles.

The commonly utilized techniques for encapsulating hydrophilic molecules in NP suffer from low encapsulation efficiency because of the drug rapid partitioning to the external aqueous phase. We hypothesized that combining the double emulsion system with a partially water-soluble organic solvent, could result in better encapsulation yield of hydrophilic molecules in nano-sized NP, and the utilization of both biocompatible surfactants and solvents. As a model drug we used alendronate, a hydrophilic low MW bisphosphonate. The new NP preparation technique, double emulsion solvent diffusion (DES-D), resulted in improved formulation characteristics including smaller size, lower size distribution, higher encapsulation yield, and more biocompatible ingredients in comparison to classical methods. The utilization of partially water-miscible organic solvent (ethyl acetate) enabled rapid diffusion through the aqueous phase forming smaller NP. In addition, the formulated alendronate NP exhibited profound inhibition of raw 264 macrophages, depletion of rabbit's circulating monocytes, and inhibition of restenosis in the rat model. It is concluded that the new technique is advantageous in terms of smaller size, lower size distribution, higher encapsulation yield, and more biocompatible ingredients, with unaltered bioactivity.

Single and double emulsion manufacturing techniques of an amphiphilic drug in PLGA nanoparticles: formulations of mithramycin and bioactivity.

Formulation of hydrophilic compounds in nanoparticles is problematic due to their escape to the external aqueous phase. The certain amphiphilic nature of mithramycin, utilized clinically in cancer, makes its incorporation into nanoparticles an interesting challenge, elucidating the formulation factors of amphiphilics in nanoparticles. We hypothesized that mithramycin nanoparticles could provide more effective therapy of restenosis due to its antiproliferating and potential monocyte inhibition properties. The nanoprecipitation technique (designed for lipophilic compounds) was found preferable, with better encapsulation efficiency, than the emulsification solvent diffusion (ESD) technique (79.3 +/- 3.1% and 40.8 +/- 1.1%, respectively). The double emulsion solvent diffusion (DESD) method, designed for hydrophilic compounds, yielded similar encapsulation efficiency (80%). Nanoparticles size was, 110 +/- 36, 130 +/- 30, and 160 +/- 31 nm, ESD, nanoprecipitation, and DESD techniques, respectively. Mithramycin solution and in nanoparticles significantly inhibited RAW264 macrophages and smooth muscle cells in a dose-dependent relationship, and reduced the number of circulating monocytes in rabbits. However, no inhibition of restenosis was obtained in the rat carotid model following i.v. administration of mithramycin nanoparticles. It can be concluded that PLGA-based polymeric nanoparticles of mithramycin can be formulated by techniques suitable for lipophilic/hydrophilic compounds. The ineffectiveness in the rat restenosis model is probably due to the short depletion period of circulating monocytes and lack of arterial targeting.

Preparation of alendronate liposomes for enhanced stability and bioactivity: in vitro and in vivo characterization.

Liposomes containing bisphosphonates have been shown to deplete circulating monocytes and reduce experimental restenosis. However, acceptable shelf life was not achieved, and the disruption extent and rate of the vesicles in the circulation has not been examined. Designing an optimal liposomal formulation in general, and for an anti-inflammatory effect in particular, requires careful consideration of the factors that contribute to their in vitro stability and integrity in the blood after injection. An improved liposomal alendronate formulation was prepared by a modified thin lipid film hydration technique followed by extrusion, resulting in relatively smaller size vesicles, narrow size distribution, and low drug to lipid ratio in comparison to the reverse phase evaporation method. In order to rule out premature leakage of the drug, the integrity of the vesicles was examined by means of size-exclusion chromatography in vitro and in vivo, with subsequent analysis of size, drug (fractions of encapsulated and free) and lipid concentrations. Vesicles were found to be stable in serum, with 15 +/- 3% leakage of the drug after 10 min in rabbit's circulation, and intact liposomes were detected in the circulation 24 h following administration. It is concluded that the new formulation results in increased stability (2.5 years) as determined by the insignificant changes in vesicle size, drug leakage, lipid and drug stability, in vitro bioactivity (macrophages inhibition), as well as in vivo in depleting circulating monocytes and inhibition of restenosis in rabbits. Our in vitro stability results regarding dilution in serum paralleled in vivo data. Thus, in vitro assessment may provide a valuable tool in assessing in vivo integrity of liposomal formulations.

Delivery of serotonin to the brain by monocytes following phagocytosis of liposomes.

Many drugs are not able to enter the brain due to the presence of the blood-brain barrier (BBB) and therefore cannot be used in the treatment of diseases of the brain. Since it is now known that the brain is under immunological surveillance, we hypothesized that phagocytic cells of the innate immune system, mainly neutrophils and monocytes, can be exploited as transporters of drugs to the brain. To target circulating mononuclear phagocytic cells, negatively-charged nano-sized liposomes were formulated encapsulating serotonin, a BBB impermeable neurological drug. Brain uptake, biodistribution, and the mechanism of brain transport were examined in vitro and in rats and rabbits by utilizing double-radiolabeled (3)H (in the membrane) and (14)C-serotonin (in the core), and liposomes with fluorescent markers (membrane and core). The brain uptake of liposomal serotonin was significantly higher (0.138%+/-0.034 and 0.097%+/-0.011, vs. 0.068%+/-0.02 and 0.057%+/-0.01, 4 h and 24 h after IV administration in rats, serotonin liposomes and in solution, respectively). The same brain uptake of both empty and serotonin liposomes, the co-localization in the brain of both markers, and the unchanged ratio of (3)H:(14)C suggest that intact liposomes entered the brain. Since treatment of animals by liposomal alendronate resulted with inhibition of monocytes but not of neutrophils, and with no brain delivery, it is suggested that monocytes are the main transporters of liposomes to the brain.

Additive-free albumin nanoparticles of alendronate for attenuating inflammation through monocyte inhibition.

AIMS: Particulated dosage forms of bisphosphonates, such as polymeric nanoparticles and liposomes, deplete circulating monocytes and attenuate inflammation. The aim of this work was to develop a novel formulation of albumin nanoparticles with no crosslinkers that encapsulate the bisphosphonate, alendronate and, further, to examine its bioactivity in vitro and in vivo. RESULTS: The novel formulation was prepared by desolvation of human serum albumin in acidic pH induced by alendronate, which enables an electrostatic interaction between albumin and the acidic drug. The mean particle size of the negatively charged nanoparticle was 250-300 nm and drug-entrapment efficiency was 49%. The formulation can be filter sterilized and lyophilized for increased stability. Alendronate nanoparticles exhibited significant inhibitory effects on RAW264 macrophage growth and a significant attenuation of stenosis in rats. CONCLUSION: It is concluded that bioactive nanoparticles of human albumin can be formulated without crosslinkers and potentially toxic additives.

Nanosuspensions of alendronate with gallium or gadolinium attenuate neointimal hyperplasia in rats.

Monocytes/macrophages play a pivotal role in the formation of neointinal hyperplasia following vascular injury. Transient depletion of circulating monocytes by particulate delivery systems containing bisphosphonates, such as alendronate, results in restenosis inhibition. We hypothesized that a self-suspendable nanoparticulate dosage form, with a minimum amount of expients, could be formulated by complexing the negatively charged alendronate with gallium or gadolinium. We further hypothesized that a synergistic biological effect could be obtained by nanosuspensions of alendronate with these counter ions. Nanosuspensions (150-250 nm) of alendronate-gallium and alendronate-gadolinium were successfully formulated with no additives except for the active agents and HCl for pH adjustment. Both nanosuspensions exhibited macrophage cell line growth inhibition in a dose-response relationship in comparison to the various agents in solution and in liposomes. A synergistic effect of the nanosuspensions was observed in the inhibition of raw264 macrophages, and in reducing IL-1beta and TNF-alpha secretion in cell culture. Single IV administration at the time of injury, of alendronate-gallium or alendronate-gadolinium nanosuspensions resulted in inhibition of neointimal hyperplasia and stenosis in the rat model of vascular injury. The results correlated with the significant reduction of circulating monocytes. The nanosuspensions possess the advantages of no additives for minimal provocation of side effects, and the potential of immunomodulating inflammatory disorders.