The Effect of Natural-Based Formulation (NBF) on the Response of RAW264.7 Macrophages to LPS as an In Vitro Model of Inflammation.

Macrophages are some of the most important immune cells in the organism and are responsible for creating an inflammatory immune response in order to inhibit the passage of microscopic foreign bodies into the blood stream. Sometimes, their activation can be responsible for chronic inflammatory diseases such as asthma, tuberculosis, hepatitis, sinusitis, inflammatory bowel disease, and viral infections. Prolonged inflammation can damage the organs or may lead to death in serious conditions. In the present study, RAW264.7 macrophages were exposed to lipopolysaccharide (LPS; 20 ng/mL) and simultaneously treated with 20 µg/mL of natural-based formulation (NBF), mushroom-cannabidiol extract). Pro-inflammatory cytokines, chemokines, and other inflammatory markers were analyzed. The elevations in the presence of interleukin-6 (IL-6), cycloxygenase-2 (COX-2), C-C motif ligand-5 (CCL5), and nitrite response, following exposure to LPS, were completely inhibited by NBF administration. IL-1ß and tumor necrosis factor alpha (TNF-α) release were inhibited by 3.9-fold and 1.5-fold, respectively. No toxic effect of NBF, as assessed by lactate dehydrogenase (LDH) release, was observed. Treatment of the cells with NBF significantly increased the mRNA levels of TLR2, and TLR4, but not NF-κB. Thus, it appears that the NBF possesses anti-inflammatory and immunomodulatory effects which can attenuate the release of pro-inflammatory markers. NBF may be a candidate for the treatment of acute and chronic inflammatory diseases and deserves further investigation.

The TSPO Ligands MGV-1 and 2-Cl-MGV-1 Differentially Inhibit the Cigarette Smoke-Induced Cytotoxicity to H1299 Lung Cancer Cells.

TSPO is involved in cigarette smoke (CS)-induced cellular toxicity, which may result in oral and pulmonary diseases and lung cancer. H1299 lung cancer cells were exposed directly to CS. The H1299 cells were pretreated with our TSPO ligands MGV-1 and 2-Cl-MGV-1 (Ki = 825 nM for both) at a concentration of 25 µM 24 h prior to CS exposure. Cell death and apoptotic markers were measured, in addition to TSPO expression levels, ATP synthase activity, generation of reactive oxygen species (ROS), depolarization of mitochondrial membrane potential (ΔΨm), cAMP and LDH levels. Pretreatment with MGV-1 and 2-Cl-MGV-1 (25 µM), 24 h prior to CS exposure, differentially attenuated the CS-induced cellular insult as well as cell death in H1299 lung cancer cells. These protective effects included prevention of ATP synthase reversal, ROS generation, depolarization of the mitochondrial membrane and elevation in LDH. The preventive efficacy of 2-Cl-MGV-1 was superior to that achieved by MGV-1. Both ligands did not prevent the elevation in cAMP. These findings may indicate a mild protective effect of these TSPO ligands in CS-related pulmonary and keratinocyte cellular pathology.

Liposome-based targeting of dopamine to the brain: a novel approach for the treatment of Parkinson's disease.

Delivery of drugs into the brain is poor due to the blood brain barrier (BBB). This study describes the development of a novel liposome-based brain-targeting drug delivery system. The liposomes incorporate a diacylglycerol moiety coupled through a linker to a peptide of 5 amino acids selected from amyloid precursor protein (APP), which is recognized by specific transporter(s)/receptor(s) in the BBB. This liposomal system enables the delivery of drugs across the BBB into the brain. The brain-directed liposomal system was used in a mouse model of Parkinson's disease (PD). Intra-peritoneal (IP) administration of liposomes loaded with dopamine (DA) demonstrated a good correlation between liposomal DA dose and the behavioral effects in hemiparkinsonian amphetamine-treated mice, with an optimal DA dose of 60 µg/kg. This is significantly lower dose than commonly used doses of the DA precursor levodopa (in the mg/kg range). IP injection of the APP-targeted liposomes loaded with a DA dose of 800 µg/kg, resulted in a significant increase in striatal DA within 5 min (6.9-fold, p < 0.05), in amphetamine-treated mice. The increase in striatal DA content persisted for at least 3 h after administration, which indicates a slow DA release from the delivery system. No elevation in DA content was detected in the heart or the liver. Similar increases in striatal DA were observed also in rats and mini-pigs. The liposomal delivery system enables penetration of compounds through the BBB and may be a candidate for the treatment of PD and other brain diseases.

Liposomal Carrier Conjugated to APP-Derived Peptide for Brain Cancer Treatment.

Brain tumors are hard to treat with the currently available therapy. The major obstacle in the treatment of brain tumors is the lack of therapeutic strategies capable to penetrate the blood-brain barrier (BBB). The BBB is an endothelial interface that separates the brain from the circulatory blood system and prevents the exposure of the central nervous system (CNS) to circulating toxins and potentially harmful compounds. Unfortunately, the BBB prevents also the penetration of therapeutic compounds into the brain. We present here a drug-delivery liposomal carrier, conjugated to a peptide inserted in the liposomal membrane, which is putatively recognized by BBB transporters. The peptide is a short sequence of 5 amino acids (RERMS) present in the amyloid precursor protein (APP). This APP-targeted liposomal system was designed specifically for transporting compounds with anti-cancer activity via the BBB into the brain in an effective manner. This drug-delivery liposomal carrier loaded with the anti-cancer compounds temozolomide (TMZ), curcumin, and doxorubicin crossed the BBB in an in vitro model as well as in vivo (mice model). In the in vitro model, the targeted liposomes crossed the BBB model fourfold higher than the non-targeted liposomes. Labeled targeted liposomes penetrated the brain in vivo 35% more than non-targeted liposomes. Treatment of mice that underwent intracranial injection of human U87 glioblastoma, with the targeted liposomes loaded with the three tested anti-cancer agents, delayed the tumor growth and prolonged the mice survival in a range of 45% -70%. It appears that the targeted liposomal drug-delivery system enables better therapeutic efficacy in a SCID mouse model of glioblastoma compared to the corresponding non-targeted liposomes and the free compounds.

Lung-Derived Microscaffolds Facilitate Diabetes Reversal after Mouse and Human Intraperitoneal Islet Transplantation.

There is a need to develop three-dimensional structures that mimic the natural islet tissue microenvironment. Endocrine micro-pancreata (EMPs) made up of acellular organ-derived micro-scaffolds seeded with human islets have been shown to express high levels of key beta-cell specific genes and secrete quantities of insulin per cell similar to freshly isolated human islets in a glucose-regulated manner for more than three months in vitro. The aim of this study was to investigate the capacity of EMPs to restore euglycemia in vivo after transplantation of mouse or human islets in chemically diabetic mice. We proposed that the organ-derived EMPs would restore the extracellular components of the islet microenvironment, generating favorable conditions for islet function and survival. EMPs seeded with 500 mouse islets were implanted intraperitoneally into streptozotocin-induced diabetic mice and reverted diabetes in 67% of mice compared to 13% of controls (p = 0.018, n = 9 per group). Histological analysis of the explanted grafts 60 days post-transplantation stained positive for insulin and exhibited increased vascular density in a collagen-rich background. EMPs were also seeded with human islets and transplanted into the peritoneal cavity of immune-deficient diabetic mice at 250 islet equivalents (IEQ), 500 IEQ and 1000 IEQ. Escalating islet dose increased rates of normoglycemia (50% of the 500 IEQ group and 75% of the 1000 IEQ group, n = 3 per group). Human c-peptide levels were detected 90 days post-transplantation in a dose-response relationship. Herein, we report reversal of diabetes in mice by intraperitoneal transplantation of human islet seeded on EMPs with a human islet dose as low as 500 IEQ.

Beta Cells Secrete Significant and Regulated Levels of Insulin for Long Periods when Seeded onto Acellular Micro-Scaffolds.

The aim of this work is to obtain significant and regulated insulin secretion from human beta cells ex vivo. Long-term culture of human pancreatic islets and attempts at expanding human islet cells normally result in loss of beta-cell phenotype. We propose that to obtain proper ex vivo beta cell function, there is a need to develop three-dimensional structures that mimic the natural islet tissue microenvironment. We here describe the preparation of endocrine micro-pancreata (EMPs) that are made up of acellular organ-derived micro-scaffolds seeded with human intact or enzymatically dissociated islets. We show that EMPs constructed by seeding whole islets, freshly enzymatically-dissociated islets or even dissociated islets grown first in standard monolayer cultures express high levels of key beta-cell specific genes and secrete quantities of insulin per cell similar to freshly isolated human islets in a glucose-regulated manner for more than 3 months in vitro.

Kidney-specific microscaffolds and kidney-derived serum-free conditioned media support in vitro expansion, differentiation, and organization of human embryonic stem cells.

We report a novel method for culturing human embryonic stem cells (HES) using 300-µm-thick acellular kidney-derived microscaffolds (KMSs) that allow cells to obtain nutrients and gasses simply by diffusion, enabling the KMSs to be used readily ex vivo under defined culture conditions, without the need for vascularization/perfusion or transplantation. Standard histology and scanning electron microscopy show that HES grow and expand according to the complex structure dictated by the scaffold. We further show that the expression levels of NPHS-1, REN, AQP-1, SLC2A2, and ANPEP were 7.6-, 5.1-, 128-, 4.3-, and 3.9-fold higher, respectively, when the HES were grown on KMS compared with the HES grown on collagen. Similarly, the levels of these genes were 10-, 30-, 4.6-, 7.5-, and 3-fold higher, respectively, when the HES were grown on KMS compared with the HES grown on Matrigel. We have also shown that culturing HES in 5% kidney-derived serum-free conditioned media can lead to the upregulation of NPHS-1, REN, and EPO by 3-, 18-, and 15-fold, respectively. This article demonstrates a novel way of growing HES in vitro whereby the beneficial biophysical as well as biochemical surroundings of the seeded cells provide a more in vivo-like environment, thereby assisting in differentiation of the HES toward a renal lineage. The approach presented here may provide a powerful tool for in vitro study of HES differentiation toward kidney-specific cell lineages under controlled conditions.

Kidney derived micro-scaffolds enable HK-2 cells to develop more in-vivo like properties.

Many cell lines, despite the fact that they are easy to culture, tend to lose some of their in vivo characteristics in vitro, we therefore decided to investigate whether culturing HK-2 cells on kidney derived micro-scaffolds (KMS) could improve proximal tubule functionality to these cells. Kidney derived micro-scaffolds (KMS) have been prepared that, due to the fact that they are only 300 µm in depth, allow for transfer of gases and nutrients via diffusion whilst maintaining the kidney's intricate microstructure. Culturing HK-2 on KMS shows significant increase in expression of AQP-1, ATP1B1, SLC23A1 and SLC5A2 after 1, 2 and 3 weeks compared with HK-2 grown under standard tissue culture conditions. Additionally, very high levels of expression of CCL-2 (15-30 fold increase) and LRP-2 (25-200 fold increase) were observed when the HK-2 were grown on KMS compared with HK-2 grown under standard tissue culture conditions. Furthermore, HK-2 cells grown under standard conditions released higher levels of Il-6 and Il-8 compared with primary tubule cells (Asterand AS-9-2) and secreted no MCP-1 or RANTES as opposed to primary cells that released MCP-1 and RANTES following stimulation. However, HK-2 grown on KMS showed both a marked decrease in Il-6/Il-8 secretion in line with the primary cells and secreted MCP-1 as well. These results show that the micro-environment of the KMS assists in restoring in vivo like properties to the HK-2 cells.