Role of functionalized liposomes in drug delivery through the blood-brain barrier

The central nervous system (CNS), one of the most delicate microenvironments of the body, is protected by the blood-brain barrier (BBB). The protective properties of the BBB are conferred by the intricate architecture of its endothelium coupled with multiple specific transport systems expressed on the surface of endothelial cells in the brain vasculature. Although the BBB has a key role in regulation of neural biochemical environment, essential for maintaining neuronal integrity, it limits drug delivery to the CNS. In fact, less than 2% of all US Food and Drug Agency (FDA)-approved small-molecule drugs cross the intact BBB to varying degrees. Due to their flexible physicochemical and biophysical properties, the liposomes represent an attractive tool to deliver therapeutic molecules across the BBB. Moreover, their surface can be easily modified with ligands to improve their target and delivery. The peptide gH625, identified as a membrane-perturbing domain in glycoprotein H (gH) of Herpes Simplex virus 1, has been used extensively for vector-mediated strategies that enable passage of a large variety of small molecules as well as proteins across cell membranes in vitro. The goal of this Ph.D. project was to develop a new carrier system to deliver therapeutic molecules through the BBB with high efficacy and minimal toxicity. The project was divided in two parts. In the first part, in vitro and in vivo experiments were performed to investigate the capacity of gH625 peptide to enter and accumulate in neuron and astrocyte cell lines, and its ability to cross the blood-brain barrier in rats. In the second part of the project, the efficiency of liposomes functionalized with gH625 was evaluated on both in vitro model of rat BBB by using a neuroprotective peptide like PACAP (pituitary adenylate cyclase-activating polypeptide) and on in vivo mouse brain by using a hypothermic neuropeptide. The results show that gH625 peptide has a significant ability to penetrate brain cells. In fact, gH625 can be efficiently incorporated by human neuroblastoma (SH-SY5Y) and glioblastoma-astrocytoma cell lines (U-87 MG) without alteration of their cell viability. Furthermore, the in vivo experiments demonstrate that, despite the blood filtration action of the liver, carried out through the gH625 uptake of Kupffer cells, gH625 substantially reaches the brain BBB vessels. In particular, gH625 can be highly accumulated in endothelial cells of the BBB and taken up in some neurons. The results of second part of project show that the functionalization of liposomes with gH625 improves their passage through the endothelium of in vitro BBB model, thus resulting in an increased transport of PACAP and its less accumulation in the endothelial cells. Moreover, the toxicity studies reveal that the gH625-liposomes are nontoxic and do not affect tight junction organization in the BBB endothelium. Finally, further in vivo results demonstrated that the gH625 peptide may improve the efficiency of liposomes in mice. The results of this study suggest that gH625 peptide is a valuable tool to develop functionalized nanosystems for drug delivery to the brain. In particular, gH625-mediated liposomes represent a promising strategy to deliver therapeutic agents to CNS. Taken together, these data may have importance for the treatment of brain diseases and tracking of nanosystems in vivo

Catestatin and GABAAR related feeding habits rely on dopamine, ghrelin plus leptin neuroreceptor expression variations

Catestatin (CST), an endogenously small sympathoinhibitory peptide is capable of interfering with the major cerebral neuroreceptor-blocking site, i.e. γ-aminobutyric acidA receptor (GABAAR) system especially in limbic brain areas that are involved with feeding behaviors. The GABAARergic-related effects seem to derive from its interaction with other molecular neuroreceptors such as dopaminergic, ghrelin and leptinergic. In this context, the present study aimed to investigate probable feeding responses (eating and drinking) induced by treatment with CST and the GABAAR antagonist bicucullin (BIC) alone or simultaneously (CST+BIC) in the Syrian hibernating hamster (Mesocricetus auratus) model. Hamsters that received these compounds via intracerebroventricular infusions displayed notable variations of feeding and drinking bouts. In particular, an anorexigenic response was evident following treatment with CST while BIC evoked a significant increase of eating and drinking behaviors. Surprisingly when both agents were given simultaneously, a predominating anorexigenic response was detected as shown by evident CST-dependent reduction of feeding bouts. Contextually such behaviors, especially those following the combined treatment were tightly correlated with the significantly increased cerebral dopamine receptor 1 (D1) plus reduced ghrelin receptor (GhsR) and leptin receptor (LepR) transcript levels. Overall, the anorexigenic effect of CST deriving from its tight interaction with GABAARs activity plus D1 and GhsR transcripts tends to propose these neuronal elements as pivotal factors responsible for feeding disorders

Polystyrene nanoparticles internalization in human gastric adenocarcinoma cells

The increase in the use of nanoparticles, as a promising tool for drug delivery or as a food additive, raises questions about their interaction with biological systems, especially in terms of evoked responses. In this work, we evaluated the kinetics of uptake of 44 nm (NP44) and 100 nm (NP100) unmodified polystyrene nanoparticles (PS-NPs) in gastric adenocarcinoma (AGS) cells, as well as the endocytic mechanism involved, and the effect on cell viability and gene expression of genes involved in cell cycle regulation and inflammation processes. We showed that NP44 accumulate rapidly and more efficiently in the cytoplasm of AGS compared to NP100; both PS-NPs showed an energy dependent mechanism of internalization and a clathrin-mediated endocytosis pathway. Dose response treatments revealed a non-linear curve. PS-NPs also affected cell viability, inflammatory gene expression and cell morphology. NP44 strongly induced an up-regulation of IL-6 and IL-8 genes, two of the most important cytokines involved in gastric pathologies. Our study suggests that parameters such as time, size and concentration of NPs must be taken carefully into consideration during the development of drug delivery systems based on NPs and for the management of nanoparticles associated risk factors

Expression and potential role of the peptide orexin-A in prostate cancer

The peptides orexin-A and orexin-B and their G protein-coupled OX1 and OX2 receptors are involved in multiple physiological processes in the central nervous system and peripheral organs. Altered expression or signaling dysregulation of orexins and their receptors have been associated with a wide range of human diseases including narcolepsy, obesity, drug addiction, and cancer. Although orexin-A, its precursor molecule prepro-orexin and OX1 receptor have been detected in the human normal and hyperplastic prostate tissues, their expression and function in the prostate cancer (PCa) remains to be addressed. Here, we demonstrate for the first time the immunohistochemical localization of orexin-A in human PCa specimens, and the expression of prepro-orexin and OX1 receptor at both protein and mRNA levels in these tissues. Orexin-A administration to the human androgen-dependent prostate carcinoma cells LNCaP up-regulates OX1 receptor expression resulting in a decrease of cell survival. Noteworthy, nanomolar concentrations of the peptide counteract the testosterone-induced nuclear translocation of the androgen receptor in the cells: the orexin-A action is prevented by the addition of the OX1 receptor antagonist SB-408124 to the test system. These findings indicate that orexin-A/OX1 receptor interaction interferes with the activity of the androgen receptor which regulates PCa onset and progression, thus suggesting that orexin-A and its receptor might represent novel therapeutic targets to challenge this aggressive cancer

Triclosan and estradiol effects on human prostate cells

Xenoestrogens are estrogen-mimicking compounds that are commonly found in personal care products and pesticides. The activity of xenoestrogens in the human body involves interference with estrogen receptor binding. Triclosan (TCS), a lesserknown xenoestrogen, is a broad-spectrum antibacterial commonly used in cosmetics, toothpastes, soap and other consumer products. The widespread use of TCS and its detection in human breast milk, urine and serum have raised concerns regarding its association with various health outcomes. Recent evidence suggests that TCS may play a role in cancer development, perhaps through its estrogenicity. In the present work we have studied the effects of TCS and Estrogen (E2) on human prostate adenocarcinoma epithelial cells (LNCaP) in order to highlight estrogen and xenoestrogen influence on human prostate. Although androgens are the most important hormones in the normal development of the male reproductive system, more recently, it has been suggested a central role for estrogen in male reproductive system and it has been hypothesized that high level of estrogens may disturb the endocrine control of the male reproductive capability. We examined the effects of TCS and E2 on the proliferation of the LNCaP through MTT assay. They were both able to increase cell proliferation at concentration of 10-8 M after 24h of treatment. In order to study estrogen receptor (ER) involvement, we evaluated the cellular localization and expression of ERs with immunofluorescence and western blot techniques after treatment with TCS and E2. Finally, through Real Time PCR analysis we have investigated gene expression of several molecular targets of estrogen pathway. We have observed that treatment with TCS and E2 induced an upregulation of Ki-67, cyclin D1 and cyclin E. We have also observed an upregulation of proinflammatory cytochines Il-1β after TCS and E2 treatment. These results confirm the estrogenic activity of TCS and suggest that estrogen and xenoestrogens may interfere with molecular pathways of prostate physiolog

Valproic Acid Synergizes With Cisplatin and Cetuximab in vitro and in vivo in Head and Neck Cancer by Targeting the Mechanisms of Resistance

Recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) is a devastating malignancy with a poor prognosis. The combination of cisplatin (CDDP) plus cetuximab (CX) is one of the standard first-line treatments in this disease. However, this therapeutic regimen is often associated with high toxicity and resistance, suggesting that new combinatorial strategies are needed to improve its therapeutic index. In our study, we evaluated the antitumor effects of valproic acid (VPA), a well-known antiepileptic agent with histone deacetylase inhibitory activity, in combination with CDDP/CX doublet in head and neck squamous cell carcinoma (HNSCC) models. We demonstrated, in HNSCC cell lines, but not in normal human fibroblasts, that simultaneous exposure to equitoxic doses of VPA plus CDDP/CX resulted in a clear synergistic antiproliferative and pro-apoptotic effects. The synergistic antitumor effect was confirmed in four different 3D-self-assembled spheroid models, suggesting the ability of the combined approach to affect also the cancer stem cells compartment. Mechanistically, VPA enhanced DNA damage in combination treatment by reducing the mRNA expression of ERCC Excision Repair 1, a critical player in DNA repair, and by increasing CDDP intracellular concentration via upregulation at transcriptional level of CDDP influx channel copper transporter 1 and downregulation of the ATPAse ATP7B involved in CDDP-export. Valproic acid also induced a dose-dependent downregulation of epidermal growth factor receptor (EGFR) expression and of MAPK and AKT downstream signaling pathways and prevent CDDP- and/or CX-induced EGFR nuclear translocation, a well-known mechanism of resistance to chemotherapy. Indeed, VPA impaired the transcription of genes induced by non-canonical activity of nuclear EGFR, such as cyclin D1 and thymidylate synthase. Finally, we confirmed the synergistic antitumor effect also in vivo in both heterotopic and orthotopic models, demonstrating that the combined treatment completely blocked HNSCC xenograft tumors growth in nude mice. Overall, the introduction of a safe and generic drug such as VPA into the conventional treatment for R/M HNSCC represents an innovative and feasible antitumor strategy that warrants further clinical evaluation. A phase II clinical trial exploring the combination of VPA and CDDP/CX in R/M HNSCC patients is currently ongoing in our institute

A cell-penetrating peptide as a tool for delivery to blood brain barrier (BBB)

The high impermeability and selectivity of the BBB prevent the transport of many drugs into the brain, making them ineffective for the treatment of central nervous system diseases. The cell-penetrating peptides (CPPs) represent a new strategy to functionalize carriers in order to deliver therapeutic molecules to the brain. Several studies have demonstrated that gH625, a peptide derived from the glycoprotein H of herpes simplex virus 1, is able to cross the membrane bilayer and represents an ideal CPP for the delivery BBB, because of its ability to escape from the endocytic pathways1. In this study we evaluated the internalization of gH625 in human neuroblastoma (SH-SY5Y) and astrocytoma (U87-MG) cells and in rat brain. Fluorescence and spectrofluorimetric in vitro analyses show a good rate of uptake in both cell lines after 2h of treatment with gH625 labeled with 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (gH625-NBD) (Fig.1). The internalization is almost complete if higher concentration was used (5μM) and the signal is prevalently found within the cytoplasm. Immunofluorescence studies, using anti-GFAP and anti-BBB antibodies, were performed after intravenous administration (3h) of gH625-NBD (160μg/100 g bw) in rats. Five images for each experimental class were analyzed with ImageJ 1.48 software; the deconvolutionlab plugin was used to deconvolve image channels through the Tikhonov-Miller’s algorithm; the Co-localization Colormap plugin was then used to evaluate the degree of correlation between pair of pixels in the red and green channels, resulting in the distribution of the values of the normalized mean deviation product (nMDP) and the index of correlation as the fraction of positively correlated pixels in the image2. Co-localization studies produced a color scale map (from -1 to 1) where negative indexes (cold colors) represent no co-localization and indexes above 0 (hot colors) represent co-localization. The study reveals a high co-localization score with BBB and low co-localization score with GFAP protein of astrocytes; interestingly few neurons were labeled for gH625-NBD, indicating the passage through the BBB (Fig.2). The index of correlation shows poor positive correlation in gH625/anti-GFAP and high positive correlation in the gH625/anti-BBB. These data show that gH625 is up taken by neuronal cells and reaches the rat brain. Taken together, our results can be considered as a preliminary data to develop a liposome-based systems which involves the use of gH625 as an efficient drugs delivery through the BBB. 1Guarnieri D et al. 2013 Drug delivery: shuttle-mediated nanoparticle delivery to the BBB. Small 9(6): 806 2Jaskolski F et al. 2005 An automated method to quantify and visualize colocalized fluorescent signals. J of Neur Meth 146(1):42-4

Endocytic pathways involved in polystyrene nanoparticle uptake in human gastric adenocarcinoma cells

Nanoparticles (NPs) are promising tools in medical fields, both in diagnosis and therapy (Schlorf et al., 2012; Wickline and Lanza, 2008). Despite this high applicative potential, little is known about their interaction with biological systems, almost in terms of endocytic pathways and toxicity. The first step to develop a good drug delivery systems based on NPs is to well characterize these molecular aspects. Thus, in this work, with a quantitative and qualitative approach, we studied the uptake of two representative sizes of polystyrene nanoparticles (PS-NPs), 44 nm (NP44) and 100 nm (NP100), labeled with FITC and ROD, respectively, in human adenocarcinoma gastric cells (AGS). The experiments were performed after exposure with 10μg/mL NPs for different times of incubation and temperatures (37°C and 4°C), with or without well known endocytosis inhibitor drugs (dynasore for clathrin dependent pathways and EIPA for macropinocytosis/phagocytosis). Quantitative spectrofluorimetric assays reveal a time-dependent kinetics of internalization at 37°C, with maximum values after 30 min and a decrease after 1 h for both NPs sizes. Precisely, NP44 show a high rate of uptake and a quickly internalization compared to NP100 (Fig. 1). Fluorescent images demonstrate that NPs are able to accumulate in the cytoplasm after 1 and 4 h, without reaching cell nuclei. However, NP100 tend to form aggregate after long exposition times (Fig. 3), while NP44 present an uniform cytoplasmatic distribution at all times considered (Fig. 4). Endocytosis inhibition tests show a null internalization at 4° C and a strong reduction of the uptake rate after treatment with dynasore for both NPs; EIPA, instead, partially affects NPs uptake (Fig. 2). In conclusion, in this study, we demonstrated that PS-NPs are internalized by AGS cells in a size and time dependent manner; probably, as suggest by other authors, they undergo a release process (Iversen et al., 2011). Moreover, we show that this uptake occurs through an energy dependent mechanism and that clathrin mediated endocytosis seems to be the privileged endocytic pathway for PS-NPs. References: Iversen TG., SkotlandT. Sandvig K. (2011). Endocytosis and intracellular transport of nanoparticles: Present knowledge and need for future studies. Nonotoday 6:176-181. Schlorf T, Meincke M, Kossel E, Gluer CC, Jansen O, Mentlein R (2011). Biological properties of iron oxide nanoparticles for cellular and molecular magnetic resonance imaging. Int J Mol Sci. 12(1):12–23. Wickline S.A., Lanza G.M. (2003). Nanotechnology for molecular imaging and targeted therapy. Circulation 107: 1092–1095

PACAP-LOADED LIPOSOME DELIVERY ACROSS THE BBB: A LIGHT-SHEET MICROSCOPY STUDY

The blood-brain barrier (BBB) impermeability and selectivity prevent the transport of many therapeutic molecules into the brain, making ineffective their use for treatment of neurological diseases.1 Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuroprotective peptide proposed for treatment of central nervous system (CNS) diseases.2 However, its clinical use is limited by the efflux component of peptide transport system-6 (PTS-6), which reduces its brain uptake3, and also for its low stability in human plasma, rapid degradation and peripheral actions.4 Nanocarrier-mediated method is a non-invasive strategy to explore for brain drug delivery; among them, liposomes are attractive tools that can be easily modified to improve their delivery. 5 We developed liposomes loaded with PACAP and functionalized on the surface with gH625 peptide, a membrane-perturbing domain in glycoprotein H of Herpes simplex virus 1. gH625 can traverse the membrane bilayer and deliver several cargoes across cell membranes in vitro6 and crosses the BBB in vivo.7We evaluated the efficiency of gH625-liposomes to deliver PACAP to the brain in Swiss CD1 mice after intravenous administration using light sheet fluorescence microscopy. Our results show that gH625-liposomes ameliorate both PACAP reaching and crossing the BBB, increasing the number of neuronal cells labeled with PACAP. These data suggest that gH625-liposomes represent a promising strategy to deliver therapeutic agents to CNS for the treatment of neurological diseases but also to provide an effective imaging and/or diagnostic tool for the brain