gH625-liposomes as tool for pituitary adenylate cyclase-activating polypeptide brain delivery.

The blood-brain barrier (BBB) regulates the traffic of molecules into the central nervous system (CNS) and also limits the drug delivery. Due to their flexible properties, liposomes are an attractive tool to deliver drugs across the BBB. We previously characterized gH625, a peptide derived from Herpes simplex virus 1. The present study investigates the efficiency of liposomes functionalized on their surface with gH625 to promote the brain uptake of neuroprotective peptide PACAP (pituitary adenylate cyclase-activating polypeptide). Using a rat in vitro BBB model, we showed that the liposomes preparations were non-toxic for the endothelial cells, as assessed by analysis of tight junction protein ZO1 organization and barrier integrity. Next, we found that gH625 improves the transfer of liposomes across endothelial cell monolayers, resulting in both low cellular uptake and increased transport of PACAP. Finally, in vivo results demonstrated that gH625 ameliorates the efficiency of liposomes to deliver PACAP to the mouse brain after intravenous administration. gH625-liposomes improve both PACAP reaching and crossing the BBB, as showed by the higher number of brain cells labelled with PACAP. gH625-liposomes represent a promising strategy to deliver therapeutic agents to CNS and to provide an effective imaging and diagnostic tool for the brain.

Surface decoration with gH625-membranotropic peptides as a method to escape the endo-lysosomal compartment and reduce nanoparticle toxicity.

The membranotropic peptide gH625 is able to transport different cargos (i.e., liposomes, quantum dots, polymeric nanoparticles) within and across cells in a very efficient manner. However, a clear understanding of the detailed uptake mechanism remains elusive. In this work, we investigate the journey of gH625-functionalized polystyrene nanoparticles in mouse-brain endothelial cells from their interaction with the cell membrane to their intracellular final destination. The aim is to elucidate how gH625 affects the behavior of the nanoparticles and their cytotoxic effect. The results indicate that the mechanism of translocation of gH625 dictates the fate of the nanoparticles, with a relevant impact on the nanotoxicological profile of positively charged nanoparticles.

Enhanced intracellular translocation and biodistribution of gold nanoparticles functionalized with a cell-penetrating peptide (VG-21) from vesicular stomatitis virus.

Reduced toxicity and ease of modification make gold nanoparticles (GNPs) suitable for targeted delivery, bioimaging and theranostics by conjugating cell-penetrating peptides (CPPs). This study presents the biodistribution and enhanced intracellular uptake of GNPs functionalized with VG-21, a CPP derived from vesicular stomatitis virus glycoprotein (G). Cell penetrating efficiency of VG-21 was demonstrated using CellPPD web server, conjugated to GNPs and were characterized using, UV-visible and FTIR spectroscopy, transmission electron microscopy, dynamic light scattering and zeta potential. Uptake of VG-21 functionalized GNPs (fGNPs) was tested in eukaryotic cell lines, HEp-2, HeLa, Vero and Cos-7, using flow cytometry, fluorescence and transmission electron microscopy (TEM), and inductively coupled plasmon optical emission spectroscopy (ICP-OES). The effects of nanoparticles on stress and toxicity related genes were studied in HEp-2 cells. Cytokine response to fGNPs was studied in vitro and in vivo. Biodistribution of nanoparticles was studied in BALB/c mice using TEM and ICP-OES. VG-21, GNPs and fGNPs had little to no effect on cell viability. Upon exposure to fGNPs, HEp-2 cells revealed minimal down regulation of stress response genes. fGNPs displayed higher uptake than GNPs in all cell lines with highest internalization by HEp-2, HeLa and Cos-7 cells, in endocytotic vesicles and nuclei. Cytokine ELISA showed that mouse J774 cells exposed to fGNPs produced less IL-6 than did GNP-treated macrophage cells, whereas TNF-α levels were low in both treatment groups. Biodistribution studies in BALB/c mice revealed higher accumulation of fGNPs than GNPs in the liver and spleen. Histopathological analyses showed that fGNP-treated mice accumulated 35 ng/mg tissue and 20 ng/mg tissue gold in spleen and liver respectively, without any adverse effects. Likewise, serum cytokines were low in both GNP- and fGNP-treated mice. Thus, VG-21-conjugated GNPs have enhanced cellular internalization and are suitable for various biomedical applications as nano-conjugates.

Distribution characteristics of DNA vaccine encoded with glycoprotein C from Anatid herpesvirus 1 with chitosan and liposome as deliver carrier in ducks.

BACKGROUND: A eukaryotic expression plasmid encoding glycoprotein C (gC) of Anatid herpesvirus 1 (AnHV-1) (pcDNA3.1-gC) was constructed and validated. The tissue distribution of chitosan/DNA complexes, liposome/DNA complexes and pcDNA3.1-gC alone were evaluated using a quantitative real-time PCR based TaqMan™ probe following intramuscular administration in ducklings. RESULTS: Compared with pcDNA3.1-gC alone, liposomes universally increased the plasmid DNA copy number at the injection sites, liver, spleen, heart, brain, bursa of Fabricius, and especially in the enteron (esophagus, duodenum, rectum, and cecum). Chitosan also universally increased the plasmid DNA copy number at the injection sites, liver, spleen, heart, brain and esophagus. Compared with lipoplex-gC, higher chitosan-gC plasmid DNA copy numbers were detected at the injection sites, liver, spleen, heart, brain and esophagus. In contrast, compared with lipoplex-gC, lower copy numbers of chitosan-gC plasmid DNA were detected in the duodenum, rectum and cecum. CONCLUSIONS: The results of this study demonstrated that chitosan and liposomes mediated rapid and extensive plasmid distribution in duck tissues, with low levels maintained from 1 d after DNA vaccination.

In vitro investigation of efficient photodynamic therapy using a nonviral vector; hemagglutinating virus of Japan envelope.

Photodynamic therapy (PDT) is a photochemical modality approved for cancer treatment. PDT has demonstrated efficacy in early stage lung cancer and esophageal cancer. The accumulation of photosensitizers in cancer cells is necessary to enhance the therapeutic benefits of PDT; however, photosensitizers have low uptake efficiency. To overcome this limitation, a drug delivery system, such as the hemagglutinating virus of Japan envelope (HVJ-E) vector, is required. In this study, the combination of PDT and HVJ-E was investigated for enhancing the efficacy of PDT. The photosensitizers that were evaluated included 5-aminolaevulinic acid (5-ALA), protoporphyrin IX (PPIX), and HVJ-PPIX. The uptake of the photosensitizers as increased twenty-fold with the addition of HVJ-E. The cytotoxicity of conventional 5-ALA was enhanced by the addition of HVJ-E vector. In conclusion, HVJ-E vector improved the uptake of photosensitizers and the PDT effect.

Mucosal or systemic administration of rE2 glycoprotein antigen loaded PLGA microspheres.

We have evaluated the ability of recombinant E2 antigen, as a surfactant free formulation of poly (D,L-lactide-co-glycolide) (PLGA) microspheres, to elicit a systemic immune response after administration by mucosal routes (oral and nasal) in comparison to intramuscular route. The sequence encoding a truncated E2 glycoprotein of the classical swine fever virus (CSFV) was expressed in insect cells following infection with recombinant baculovirus, as a His-tagged recombinant antigen. The recombinant E2 glycoprotein (rE2) antigen was co-encapsulated with rabbit serum albumin (RSA) as a protein stabilizer. rE2/RSA loaded PLGA microspheres, with a mean diameter of 4 microm were obtained by a water in oil in water solvent extraction method (w/o/w). Rabbits were immunized with 10 microg of rE2 formulated in PLGA microspheres administrated by three different routes (oral, nasal and intramuscular). After 60 days, each rabbit in all three groups was challenge with 5 microg of rE2 glycoprotein solution by intradermal administration. Blood samples were collected weekly for 90 days and specific rE2 antigen antibodies measured. This work showed that rE2 antigen loaded microspheres was able to initiate an immune response. The intradermal challenge after nasal and oral administration had a clear boost effect on the systemic immune response. Moreover, the response after nasal administration was more intense and less variable than oral route. In conclusion, these data demonstrate a high potential of rE2 loaded PLGA microspheres for their use as a mucosal subunit vaccine.

Expression of squamous cell carcinoma antigen-1 in liver enhances the uptake of hepatitis B virus envelope-derived bio-nanocapsules in transgenic rats.

We previously developed the bio-nanocapsule, which consists of hepatitis B virus envelope L proteins. The bio-nanocapsule can be used to deliver genes and drugs specifically to the human liver-derived tissues in xenograft models, presumably by utilizing the human liver-specific mechanism of hepatitis B virus infection. The hepatitis B virus tropism is highly restricted to humans and higher primates. Thus, to evaluate the in vivo therapeutic effects of forthcoming bio-nanocapsule-based medicines, it will be crucial to develop an animal model whose liver is susceptible to both bio-nanocapsule and hepatitis B virus. In the present study, we aimed to establish a bio-nanocapsule-susceptible animal model using transgenic rats expressing squamous cell carcinoma antigen-1 (SCCA1), which has been proposed to be a receptor for hepatitis B virus, interacting with the hepatitis B virus envelope protein and enhancing the cellular uptake of hepatitis B virus. We show that the recombinant SCCA1 protein interacts directly with bio-nanocapsule and inhibits its attachment to the cultured human liver-derived cells. Furthermore, we have established a transgenic rat that specifically expresses SCCA1 in the liver and also demonstrate that the amount of bio-nanocapsule accumulated in the liver is significantly increased by the SCCA1 expression. Histological analysis suggests that bio-nanocapsule is preferentially incorporated into the SCCA1-expressing hepatocytes but not into macrophages, such as Küppfer cells, nor into endothelial cells. Therefore, this animal model is expected to be useful for the development of bio-nanocapsule-based medicines.

Métodos de determinación del tropismo viral: tests genotípicos y fenotípicos / Methods for determining viral tropism: genotype and phenotype tests

La reciente aprobación del primer antagonista de CCR5,maraviroc (MVC, Celsentri®), con una actividad antiviralespecífica frente a variantes virales R5-trópicas hagenerado la necesidad de disponer de ensayos para ladeterminación del tropismo viral en todos los pacientescandidatos a iniciar tratamiento con este nuevo fármaco.Aunque los métodos genotípicos parecen ser laherramienta más útil por su rapidez y sencillez, en el casodel tropismo viral, las técnicas fenotípicas son actualmenteconsideradas las más fiables. En los últimos años, se handesarrollado diferentes ensayos fenotípicos para ladeterminación del uso del correceptor. Sin embargo, elensayo fenotípico de TrofileTM es el ensayo utilizadoactualmente para la determinación de tropismo, ya que esel único que cuenta con validación clínica. Dado que lapresencia de variantes X4-trópicas en la población viral seha asociado con fracaso virológico a MVC, el principal retode las herramientas tanto genotípicas como fenotípicas esoptimizar su sensibilidad para la detección de variantesX4-trópicas presentes de forma minoritaria en la poblaciónviral. Al mismo tiempo, se está evaluando la correlaciónentre métodos genotípicos y fenotípicos para determinarsi las herramientas genotípicas pueden utilizarse paratomar decisiones terapéuticas(AU)

The recent approval of the first CCR5 antagonist,Maraviroc (MVC, Celsentri®), with specific antiviral activityagainst R5-tropic virus variants has generated the need forstudies to determine the viral tropism in all those patientcandidates for starting treatment with this new drug.Although genotyping methods appear to be the mostuseful tool due to its speed and simplicity, in the case ofviral tropism, phenotyping techniques are currentlyconsidered the most reliable. In the last few years, different phenotyping assays have been developed todetermine the use of the co-receptor. However, the TrofileTMphenotype assay is currently the one most used for thedetermination of tropism, since it is the only one that hasbeen clinically validated. Given that the presence of X4-tropic variants in the viral population has been associatedwith virological failure to MVC, the main challenge of bothgenotyping and phenotyping tools is to optimise theirsensitivity for detecting X4-tropic variants present in aminority of the viral population. At the same time, thecorrelation between genotyping/phenotyping methods isbeing evaluated to determine whether genotyping tools canbe useful to make therapeutic decisions(AU)

Magnetically targeted viral envelopes: a PET investigation of initial biodistribution.

Gene and drug therapy for organ-specific diseases in part depends on the efficient delivery to a particular region of the body. We examined the biodistribution of a viral envelope commonly used as a nanoscale gene delivery vehicle using positron emission tomography (PET) and investigated the magnetic alteration of its biodistribution. Iron oxide nanoparticles and (18)F-fluoride were encapsulated by hemagglutinating virus of Japan envelopes (HVJ-Es). HVJ-Es were then injected intravenously in the rat and imaged dynamically using high-resolution PET. Control subjects received injections of encapsulated materials alone. For magnetic targeting, permanent magnets were fixed on the head during the scan. Based on the quantitative analysis of PET images, HVJ-Es accumulated in the liver and spleen and activity remained higher than control subjects for 2 h. Histological sections of the liver confirmed imaging findings. Pixel-wise activity patterns on coregistered PET images of the head showed a significantly different pattern for the subjects receiving magnetic targeting as compared to all control groups. Imaging demonstrated the initial biodistribution of a viral envelope within the rodent by providing quantitative behavior over time and in specific anatomical regions. Magnetic force altered the biodistribution of the viral envelope to a target structure, and could enable region-specific delivery of therapeutic vehicles noninvasively.

In vivo protein delivery to human liver-derived cells using hepatitis B virus envelope pre-S region.

Human hepatocyte-specific delivery of green fluorescent protein was succeeded in the mouse xenograft model by fusion with hepatitis B virus surface antigen pre-S regions (pre-S(1+2)), not with each pre-S region. The entire pre-S region would be useful for human liver-specific delivery of therapeutic proteins and bio-imaging fluoroproteins in biomedical field.

Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein.

360 million people are chronically infected with the human hepatitis B virus (HBV) and are consequently prone to develop liver cirrhosis and hepatocellular carcinoma. As approved therapeutic regimens-which modulate patients' antiviral defenses or inhibit the viral reverse transcriptase-are generally noncurative, strategies interfering with other HBV replication steps are required. Expanding on our demonstration that acylated peptides derived from the large HBV envelope protein block virus entry in vitro, we show their applicability to prevent HBV or woolly monkey hepatitis B virus infection in vivo, using immunodeficient urokinase-type plasminogen activator (uPA) mice repopulated with primary human or Tupaia belangeri hepatocytes. Accumulation of the peptides in the liver, their extraordinary inhibitory potency and specific mode of action permit subcutaneous delivery at very low doses. Inhibition of hepadnavirus entry thus constitutes a therapeutic approach to prevent primary HBV infection, such as after liver transplantation, and might also restrain virus spread in chronically infected patients.

Use of a peptide derived from foot-and-mouth disease virus for the noninvasive imaging of human cancer: generation and evaluation of 4-[18F]fluorobenzoyl A20FMDV2 for in vivo imaging of integrin alphavbeta6 expression with positron emission tomography.

Expression of the epithelial-specific integrin alphavbeta6 is low or undetectable in most adult tissues but may be increased during wound healing and inflammation and is up-regulated dramatically by many different carcinomas, making alphavbeta6 a promising target for the in vivo detection of cancer using noninvasive imaging. In addition, alphavbeta6 is recognized as promoting invasion and correlates with aggressive behavior of human cancers and thus agents that recognize alphavbeta6 specifically in vivo will be an essential tool for the future management of alphavbeta6-positive cancers. Recently, we identified the peptide NAVPNLRGDLQVLAQKVART (A20FMDV2), derived from foot-and-mouth disease virus, as a potent inhibitor of alphavbeta6. Using flow cytometry and ELISA, we show that this peptide is highly selective, inhibiting alphavbeta6-ligand binding with a IC50 of 3 nmol/L, an activity 1,000-fold more selective for alphavbeta6 than for other RGD-directed integrins (alphavbeta3, alphavbeta5, and alpha5beta1). A20FMDV2 was radiolabeled on solid-phase using 4-[18F]fluorobenzoic acid, injected into mice bearing both alphavbeta6-negative and alphavbeta6-positive (DX3puro/DX3purobeta6 cell lines) xenografts and imaged using a small animal positron emission tomography (PET) scanner. Rapid uptake (<30 min) and selective retention (>5 h) of radioactivity in the alphavbeta6-positive versus the alphavbeta6-negative tumor, together with fast renal elimination of nonspecifically bound activity, resulted in specific imaging of the alphavbeta6-positive neoplasm. These data suggest that PET imaging of alphavbeta6-positive tumors is feasible and will provide an important new tool for early detection and improved management of many types of cancers.

Nanoparticles for the delivery of genes and drugs to human hepatocytes.

Hepatitis B virus envelope L particles form hollow nanoparticles displaying a peptide that is indispensable for liver-specific infection by hepatitis B virus in humans. Here we demonstrate the use of L particles for the efficient and specific transfer of a gene or drug into human hepatocytes both in culture and in a mouse xenograft model. In this model, intravenous injection of L particles carrying the gene for green fluorescent protein (GFP) or a fluorescent dye resulted in observable fluorescence only in human hepatocellular carcinomas but not in other human carcinomas or in mouse tissues. When the gene encoding human clotting factor IX was transferred into the xenograft model using L particles, factor IX was produced at levels relevant to the treatment of hemophilia B. The yeast-derived L particle is free of viral genomes, highly specific to human liver cells and able to accommodate drugs as well as genes. These advantages should facilitate targeted delivery of genes and drugs to the human liver.

Retargeting of viral glycoproteins into a non-exporting compartment during the myogenic differentiation of rat L6 cells.

We have analysed protein trafficking during the differentiation of rat L6 myoblasts into myotubes. Different proteins were found to lose different amounts of their processing by the Golgi apparatus during the myogenic differentiation, indicating that they were transported to this organelle with differing efficiencies. In order to investigate the destination of the nonprocessed glycoproteins we analysed the behaviour of vesicular stomatitis virus (VSV) and Semliki Forest virus glycoproteins in the presence of Brefeldin A, which returns the enzymes of the Golgi apparatus to the ER. Such experiments indicated that during myogenesis a fraction of both glycoproteins was shunted into a compartment that did not participate recycling with the Golgi apparatus. Immunofluorescence studies with the mutant VSV tsO45 G protein suggested that this compartment was diffusively distributed. We investigated whether the cytoplasmic tail had a role in the myogenic transport modulation by analysing the behaviour of recombinant VSV G proteins. Exchanging the cytoplasmic tail or the tail plus the membrane anchor had no effect, suggesting that the luminal portion was responsible for the diverted transport. Taken together, the results suggest that during the myogenesis of L6 myoblasts, varying fractions of different viral glycoproteins were sorted from the ER into a specific compartment that did not recycle with the Golgi apparatus.

Distribution of adjuvant MF59 and antigen gD2 after intramuscular injection in mice.

MF59, which is an adjuvant approved for human use, typically elicits higher antibody titers than alum when used in combination with a variety of recombinant and natural subunit antigens. The mechanisms responsible for the adjuvant action of MF59 are not fully understood. In particular, little is known about the in vivo distribution of MF59 and of antigen after intramuscular (i.m.) injection. The goal of the present study was to determine the distribution of MF59 injected with soluble antigen gD2 from type 2 herpes simplex virus (HSV) and to compare the distribution of gD2 injected with or without MF59. At 4 h, 36% of the injected dose of labeled MF59 was in the quadriceps muscle and about 50% was in the inguinal fat surrounding the muscle. Half of the initial amount of labeled MF59 in muscle was detected 42 h after injection. The amount of labeled MF59 in the draining lymph nodes was maximal 2 d after injection, which represented 0.1-0.3% of the injected dose. At 4 h, 12% of the injected dose of labeled gD2 was found in the muscle. The presence of MF59 did not significantly modify the distribution of gD2. The results indicate that MF59 and gD2 distribute and are cleared independently after i.m. injection. Importantly, MF59 is unlikely to have a repository effect, whereby it slowly releases the antigen.

Gbetagamma-mediated regulation of Golgi organization is through the direct activation of protein kinase D.

We have shown previously that the betagamma subunits of the heterotrimeric G proteins regulate the organization of the pericentriolarly localized Golgi stacks. In this report, evidence is presented that the downstream target of Gbetagamma is protein kinase D (PKD), an isoform of protein kinase C. PKD, unlike other members of this class of serine/threonine kinases, contains a pleckstrin homology (PH) domain. Our results demonstrate that Gbetagamma directly activates PKD by interacting with its PH domain. Inhibition of PKD activity through the use of pharmacological agents, synthetic peptide substrates, and, more specifically, the PH domain of PKD prevents Gbetagamma-mediated Golgi breakdown. Our findings suggest a possible mechanism by which the direct interaction of Gbetagamma with PKD regulates the dynamics of Golgi membranes and protein secretion.

Clofibrate inhibits membrane trafficking to the Golgi complex and induces its retrograde movement to the endoplasmic reticulum.

Insights into the function of the Golgi complex have been provided by experiments performed with various inhibitors of membrane trafficking, such as the macrocyclic lactone brefeldin A (BFA), a compound that inhibits constitutive secretion, prevents the formation of coatomer-coated transport vesicles, and stimulates the retrograde movement of Golgi resident enzymes back to the ER. We show here that the structurally unrelated compound clofibrate, a peroxisome proliferator (PP) and hypolipidemic agent, also reversibly disrupts the morphological and functional integrity of the Golgi complex in a manner similar to BFA. In the presence of clofibrate, the forward transport of newly synthesized secretory proteins from the ER to the Golgi is dramatically inhibited. Moreover, clofibrate causes Golgi membranes to travel rapidly in a microtubule-dependent manner back to the ER, forming a hybrid ER-Golgi tubulovesicular membrane network. These affects appear to be independent of clofibrate's ability to stimulate the PP-activated receptor (PPAR) alpha pathway because other PPAR stimulators (DEHP, WY-14643) did not alter the Golgi complex or induce retrograde trafficking. These data suggest that PPAR alpha-independent, clofibrate-sensitive proteins participate in regulating Golgi-to-ER retrograde membrane transport, and, equally importantly, that clofibrate may be used as a pharmacological tool for investigating Golgi membrane dynamics.

Cholesterol is required for surface transport of influenza virus hemagglutinin.

Transport from the TGN to the basolateral surface involves a rab/N-ethylmaleimide-sensitive fusion protein (NSF)/soluble NSF attachment protein (SNAP)/SNAP receptor (SNARE) mechanism. Apical transport instead is thought to be mediated by detergent-insoluble sphingolipid-cholesterol rafts. By reducing the cholesterol level of living cells by 60-70% with lovastatin and methyl-beta-cyclodextrin, we show that the TGN-to-surface transport of the apical marker protein influenza virus hemagglutinin was slowed down, whereas the transport of the basolateral marker vesicular stomatitis virus glycoprotein as well as the ER-to-Golgi transport of both membrane proteins was not affected. Reduction of transport of hemagglutinin was accompanied by increased solubility in the detergent Triton X-100 and by significant missorting of hemagglutinin to the basolateral membrane. In addition, depletion of cellular cholesterol by lovastatin and methyl-beta-cyclodextrin led to missorting of the apical secretory glycoprotein gp-80, suggesting that gp-80 uses a raft-dependent mechanism for apical sorting. Our data provide for the first time direct evidence for the functional significance of cholesterol in the sorting of apical membrane proteins as well as of apically secreted glycoproteins.

Transport of envelope proteins of Sendai virus, HN and F0, is blocked at different steps by thapsigargin and other perturbants to intracellular Ca2+.

The effects of thapsigargin (Tg), a specific inhibitor of Ca(2+)-ATPase of the endoplasmic reticulum (ER), on replication of Sendai virus (HVJ) in BALB3T3 cells were examined. In the presence of Tg, the cells infected with HVJ did not release viral particles to the culture medium. Tg inhibited almost completely the expression of viral envelope proteins, HN and F0, on the cell surface, although it did not affect the synthesis of viral proteins. Two other inhibitors of Ca(2+)-ATPase of the ER, 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) and cyclopiazonic acid (CPA), as well as Ca(2+)-ionophores such as A23187 and ionomycin, also inhibited the expression of HN protein on the cell surface. Tg seemed to inhibit the intracellular transport or maturation of the viral membrane proteins by perturbing intracellular distribution of Ca2+ ions. In the presence of Tg, HN protein remained sensitive to endoglycosidaseH (endoH) for 3 h after its synthesis. On the other hand, F0 protein became resistant to endoH and sensitive to neuraminidase even in the presence of Tg. These results indicate that the transport of HN protein is blocked at the ER or the cis-Golgi region, while that of F0 protein is blocked at the post-Golgi stage in the presence of Tg.