Effective in Vivo Targeting of Influenza Virus through a Cell-Penetrating/Fusion Inhibitor Tandem Peptide Anchored to the Plasma Membrane.

The impact of influenza virus infection is felt each year on a global scale when approximately 5-10% of adults and 20-30% of children globally are infected. While vaccination is the primary strategy for influenza prevention, there are a number of likely scenarios for which vaccination is inadequate, making the development of effective antiviral agents of utmost importance. Anti-influenza treatments with innovative mechanisms of action are critical in the face of emerging viral resistance to the existing drugs. These new antiviral agents are urgently needed to address future epidemic (or pandemic) influenza and are critical for the immune-compromised cohort who cannot be vaccinated. We have previously shown that lipid tagged peptides derived from the C-terminal region of influenza hemagglutinin (HA) were effective influenza fusion inhibitors. In this study, we modified the influenza fusion inhibitors by adding a cell penetrating peptide sequence to promote intracellular targeting. These fusion-inhibiting peptides self-assemble into ∼15-30 nm nanoparticles (NPs), target relevant infectious tissues in vivo, and reduce viral infectivity upon interaction with the cell membrane. Overall, our data show that the CPP and the lipid moiety are both required for efficient biodistribution, fusion inhibition, and efficacy in vivo.

In Vivo Efficacy of Measles Virus Fusion Protein-Derived Peptides Is Modulated by the Properties of Self-Assembly and Membrane Residence.

Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV hemagglutinin (H) and fusion (F) envelope glycoproteins; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad repeat (HR) regions of F can inhibit MV infection at the entry stage. In previous work, we have generated potent MV fusion inhibitors by dimerizing the F-derived peptides and conjugating them to cholesterol. We have shown that prophylactic intranasal administration of our lead fusion inhibitor efficiently protects from MV infection in vivo We show here that peptides tagged with lipophilic moieties self-assemble into nanoparticles until they reach the target cells, where they are integrated into cell membranes. The self-assembly feature enhances biodistribution and the half-life of the peptides, while integration into the target cell membrane increases fusion inhibitor potency. These factors together modulate in vivo efficacy. The results suggest a new framework for developing effective fusion inhibitory peptides. IMPORTANCE: Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. We show here that specific biophysical properties regulate the in vivo efficacy of MV F-derived peptides.

Efeito da dexametasona e melatonina exógenas sobre parâmetros sanguíneos, progesterona, carboidratos totais e histomorfometria de órgãos em ratas prenhes / The effect of dexamethasone and exogenous melatonin on blood parameters, progesterone, total carbo hydrates and histomorphometry of organs in pregnant rats

A dexametasona é utilizada em casos de gestação com risco de prematuridade; porém, doses suprafisiológicas podem afetar a embriogênese. A melatonina tem demonstrado prevenir efeitos deletérios dos glicocorticoides. Assim, avaliamos a influência da melatonina sobre efeitos da dexametasona em ratas prenhes através dos seguintes parâmetros: 1. Hemograma e perfil glicídico; 2. Níveis de progesterona; e 3. Histomorfometria e histoquímica. Foram utilizadas 20 ratas divididas nos grupos: I - ratas prenhes que receberam placebo (Controle); II - ratas prenhes tratadas com dexametasona (0,8mg/kg); III - ratas prenhes tratadas com melatonina (0,5mg/kg); IV - ratas prenhes tratadas com dexametasona e melatonina. Todos os tratamentos foram iniciados 10 dias após confirmação do acasalamento até o final da prenhez. O sangue foi coletado no 7º, 14º e 21º dia de prenhez. As dosagens de carboidratos e progesterona foram realizadas pelo método antrona e ELISA, respectivamente. O fígado, rins e adrenais foram analisados histoquímica e morfometricamente. No 7º dia de prenhez, não houve alteração significativa nos parâmetros analisados. Porém, no 14º dia de prenhez, houve aumento significativo do volume de hematócrito, redução do número total de hemácias e leucócitos, neutrofilia, linfopenia, eosinopenia e redução do diâmetro das hemácias nas matrizes tratadas com dexametasona. Esses efeitos permaneceram no 21º dia de prenhez, exceto para o hematócrito, o qual reduziu. Verificou-se ainda redução significativa dos níveis de glicose (21º dia de prenhez) e progesterona (14º e 21º dia de prenhez). Não houve alteração nos parâmetros morfométricos e histoquímico no fígado, rins e adrenais. A dexametasona na dosagem de 0,8mg/kg, administrada a partir do terço médio da prenhez, produz alterações hematológicas, bioquímicas e hormonais em ratas, sendo prevenidas pela melatonina; porém não afeta o fígado, rins e adrenais quanto aos parâmetros morfométricos e histoquímicos.

Dexamethasone is used in cases of pregnancy with prematurity risk. However, it may affect the embryogenesis when used in supraphysiological doses. Melatonin has been shown to prevent the deleterious effects of glucocorticoids. Therefore, the influence of melatonin on the effects of dexamethasone on pregnant rats was evaluated through the following parameters: 1. Hemogram and glucose profile; 2. Progesterone levels; 3. Histomorphometry and histochemistry analyses. Twenty female rats were divided into the following groups: I - rats that received placebo (Control); II - rats treated with dexamethasone (0.8mg/kg); III - rats treated with melatonin (0.5mg/kg); IV - rats treated with dexamethasone and melatonin. All treatments started 10 days after confirmation of mating and lasted until the end of the pregnancy. Blood samples were collected on the 7th, 14th, and 21st day. Carbohydrate and progesterone levels were determined with the antrona and ELISA method, respectively. The liver, kidneys, and adrenal glands were analyzed morphometrically and histochemically. On the 7th day of pregnancy there were no significant changes in the parameters analyzed. However, at 14 days of pregnancy there was a significant increase of hematocrit, reducing the total number of erythrocytes and leukocytes, neutrophilia, lymphopenia, eosinopenia and reduced diameter of red blood cells in rats treated with dexamethasone. These effects remained on the 21st of day of pregnancy, except for the hematocrit, which was reduced. There was also a significant reduction in glucose levels (21st day) and progesterone (14th and 21st days). There was no change in the histochemical and morphometric parameters in the liver, kidneys and adrenals. Dexamethasone at a dosage of 0.8mg/kg administered from the middle third of pregnancy produces hematological, biochemical and hormonal changes in rats, being prevented by melatonin, but does not affect the liver...

A focus on glucose-mediated drug delivery to the central nervous system.

Drug delivery to the central nervous system (CNS) is a timely and challenging issue: 95 percent; of the pharmacological drugs cannot be delivered to the brain. This is mainly due to the blood-brain barrier (BBB), a highly selective boundary that hampers the passage of most compounds into the CNS. To overcome this problem, several approaches exist to deliver a therapeutic drug to the brain that takes into account not only the chemical properties of the drug but also the type of transport used at the BBB. One of those strategies is the glucose-mediated drug delivery which will be the focus of the present review. Glucose-mediated drug delivery requires the attachment of glycosyl moieties to a drug and the use of endogenous glucose transporters as a way to circumvent the blood-brain barrier. Glycosylated drugs display improved cell penetrability, enhanced biodistribution, stability and low toxicity. Examples such as glycosylation of ibuprofen and different opioids result in an enhanced central effect and will be discussed.

Inhibition of nociceptive responses after systemic administration of amidated kyotorphin.

BACKGROUND AND PURPOSE: Kyotorphin (KTP; L-Tyr-L-Arg), an endogenous neuropeptide, is potently analgesic when delivered directly to the central nervous system. Its weak analgesic effects after systemic administration have been explained by inability to cross the blood-brain barrier (BBB) and detract from the possible clinical use of KTP as an analgesic. In this study, we aimed to increase the lipophilicity of KTP by amidation and to evaluate the analgesic efficacy of a new KTP derivative (KTP-amide - KTP-NH(2) ). EXPERIMENTAL APPROACH: We synthesized KTP-NH(2) . This peptide was given systemically to assess its ability to cross the BBB. A wide range of pain models, including acute, sustained and chronic inflammatory and neuropathic pain, were used to characterize analgesic efficacies of KTP-NH(2) . Binding to opioid receptors and toxicity were also measured. KEY RESULTS: KTP-NH(2) , unlike its precursor KTP, was lipophilic and highly analgesic following systemic administration in several acute and chronic pain models, without inducing toxic effects or affecting motor responses and blood pressure. Binding to opioid receptors was minimal. KTP-NH(2) inhibited nociceptive responses of spinal neurons. Its analgesic effects were prevented by intrathecal or i.p. administration of naloxone. CONCLUSIONS AND IMPLICATIONS: Amidation allowed KTP to show good analgesic ability after systemic delivery in acute and chronic pain models. The indirect opioid-mediated actions of KTP-NH(2) may explain why this compound retained its analgesic effects although the usual side effects of opioids were absent, which is a desired feature in next-generation pain medications.

In vitro blood-brain barrier models--latest advances and therapeutic applications in a chronological perspective.

The first generation of in vitro models providing successful isolation of viable brain endothelial cells from different species, which could be maintained in cell culture, have emerged around thirty years ago. However, the time consuming and the difficulty of working with primary culture cells led to the development of simpler models employing cell lines with blood-brain barrier properties. The creation, in late nineties, of a transgenic mouse harboring the temperature sensitive simian virus 40 large T-antigen as a source of conditionally immortalized brain endothelial cell lines circumvented the problems of in vitro transfection of tumour inducing gene in primary cells. These different ways to obtain cultures of brain endothelial cells have profited from the discovery of different cellular factors that allow the growth of differentiated cells on plastic filters. Although cell preparations and culture conditions of brain endothelial cells are based on the same principle, there are two main models for studying the blood-brain barrier: the static and the more recently described dynamic model. Dynamic models were created in order to replicate the physiological in vivo environment of the blood-brain barrier. The large pool of in vitro models is being enlarged since each laboratory improves its model adding small differences adapted to the research interests. The great impact of blood-brain barrier studies in the development of therapies related to the central nervous system supports the interests of this review about in vitro models.

Hepatitis C virus core protein binding to lipid membranes: the role of domains 1 and 2.

We have analysed and identified different membrane-active regions of the Hepatitis C virus (HCV) core protein by observing the effect of 18-mer core-derived peptide libraries from two HCV strains on the integrity of different membrane model systems. In addition, we have studied the secondary structure of specific membrane-interacting peptides from the HCV core protein, both in aqueous solution and in the presence of model membrane systems. Our results show that the HCV core protein region comprising the C-terminus of domain 1 and the N-terminus of domain 2 seems to be the most active in membrane interaction, although a role in protein-protein interaction cannot be excluded. Significantly, the secondary structure of nearly all the assayed peptides changes in the presence of model membranes. These sequences most probably play a relevant part in the biological action of HCV in lipid interaction. Furthermore, these membranotropic regions could be envisaged as new possible targets, as inhibition of its interaction with the membrane could potentially lead to new vaccine strategies.

Does aliphatic chain length influence carbocyanines' orientation in supported lipid multilayers?

UV-Vis linear dichroism was used to study the orientation of 3,3'-dihexyloxacarbocyanine (DiOC6(3)) and 3,3'-dihexadecyloxacarbocyanine (DiOC16(3)) in supported multilayers of dipalmitoylphosphatidylcholine (DPPC) and dilauroylphosphatidylcholine (DLPC). Orientational probability density functions were similar for the two carbocyanines in both lipids. Multimodal distributions were found in all cases. The main peak is at 9 degrees-11 degrees relative to the bilayer normal axis, except for DiOC16(3) in DLPC multilayers (main peaks at 13 degrees and 90 degrees). Quenching studies revealed that the two carbocyanines are localized at the interface region of the membrane regardless of the lipid matrix they are inserted in. Combining these data with linear dichroism results lead to the conclusion that both the aliphatic chain length of carbocyanines and the lipid phase have little influence in the structural organization of these probes in lipidic bilayers. The partition constants of DiOC6 (3), K(p), were determined from fluorescence anisotropy measurements; the values obtained were K(p) (DPPC) = (2.39 +/- 0.05) x 10(3) and K(p) (DLPC) = (5.01 +/- 1.15) x 10(3).

Joint determination by Brownian dynamics and fluorescence quenching of the in-depth location profile of biomolecules in membranes.

The in-depth molar distribution function of fluorophores is revealed by a new methodology for fluorescence quenching data analysis in membranes. Brownian dynamics simulation was used to study the in-depth location profile of quenchers. A Lorentzian profile was reached. Since the Stern-Volmer equation is valid at every depth in the membrane for low quencher concentrations, the molar distribution of the fluorophore (also regarded as a Lorentzian) can be achieved. The average location and the broadness of the fluorophore distribution can be calculated. The importance of the knowledge of the location width is demonstrated and discussed, since this parameter reveals important conclusions on structural features of the interaction of membranes with probes and biomolecules (e.g., conformational freedom in proteins), as well as photophysical properties (e.g., differential fluorophore quantum yields). Subsequent use of this methodology by the reader does not, necessarily, involve the performance of simulations and is not limited to the use of Lorentzian function distributions.

A photophysical study of the polyene antibiotic filipin. Self-aggregation and filipin--ergosterol interaction.

Filipin, a macrolide polyene antibiotic, is known to interact selectively with ergosterol, a constituent of fungi membranes. In this work, the fluorescence resonance energy transfer (FRET) between a fluorescent analog of ergosterol, dehydroergosterol (DHE), and filipin was measured in small unilamellar vesicles of dipalmitoylphosphatidylcholine at 25 degrees C. The time-resolved FRET results were rationalized in the framework of the mean concentration model, and were complemented with steady-state fluorescence intensity, anisotropy and absorption measurements. The results point to the formation of both DHE--filipin aggregates (evidence from static quenching of DHE fluorescence by filipin) and filipin--filipin aggregates (evidence from: (i) the FRET acceptor concentration distributions; (ii) spectral changes of filipin absorption in the vesicles, the excitonic interaction suggesting a stack arrangement; (iii) filipin fluorescence self-quenching), even in presence of DHE and low antibiotic mole fractions (<1 mol%). These results point out that apparently contradictory biochemical models for the action of filipin (some based on the presence of sterols, others not) can be equally valid. Moreover, since results (ii) and (iii) are also observed when a sterol is present, both models of action can actually coexist in membranes with a low sterol content.

Conformation and dynamic properties of a saturated hydrocarbon chain confined in a model membrane: a Brownian dynamics simulation.

A Brownian dynamics simulation of a saturated hydrocarbon chain with simple mean-field potentials, namely anchorage, orientation and enclosing, reproducing a biological membrane environment is presented. The simulation was performed for a time equivalent to 1.4 micros thanks to the simplicity of our model. The results are compared with those obtained for a hydrocarbon chain simulated in the absence of the membrane potentials but with confinement. With the appropriate choice of parameters, equilibrium properties, such as deuterium order parameter, chain length, tilt angle and geometry, and dynamic properties, such as dihedral angle transition rate, rotational and translational diffusion, recovered from our simulations, correctly reproduced, are consistent with hydrocarbon-derived molecule experimental results and simulation results obtained from other more complex studies.

The pentaene macrolide antibiotic filipin prefers more rigid DPPC bilayers: a fluorescence pressure dependence study.

Filipin is a pentaene macrolide antibiotic which was previously shown to incorporate more extensively into DPPC bilayers below the main phase transition temperature than above this temperature. This result was extremely unusual because drugs tend to be expelled from ordered gel phases. However, such results could not be safely attributed to the phase change of the bilayer itself because the temperature was changing concomitantly. In this work we changed the bilayer phase isothermally (53 degrees C) by hydrostatic pressure variation and discovered that filipin has a slightly more extensive incorporation in the pure DPPC gel phase (P>ca. 54.4 MPa): Kp,lc approximately 3x10(3) vs. Kp,gel approximately 6x10(3). The presence of sterols (45% molar ergosterol or cholesterol) caused an increase in the partition coefficients, regardless of pressure, ergosterol having a more pronounced effect (Kp approximately 2x10(4)-6x10(4)). Kp was pressure dependent in both cases, but mainly with cholesterol (Kp approximately 2x10(3)-2x10(4)). At variance with cholesterol, when ergosterol was used, no phase transition was detected. This difference cannot be due to a more extended uptake of filipin by cholesterol-containing membranes, and so must be due to specific interactions with cholesterol. In agreement with this finding, we discovered that filipin is more tightly packed (lower partial molar volume) in the cholesterol-rich phase than in the ergosterol-rich phase. Our results also point to a 2:1 DPPC:cholesterol stoichiometry in the cholesterol-rich phase (17% molar cholesterol). All partition coefficients were calculated from steady-state fluorescence anisotropy measurements.

Simulation of the distribution and diffusion of a rigid amphipathic particle embedded in a model membrane.

We simulate, by Brownian dynamics, the distribution, orientation and diffusion of a rigid molecule, represented as a dumbbell, with amphipathic nature, embedded in a model membrane. The significant features of a biological membrane are reproduced by means of a Maier-Saupe orienting potential, an enclosing potential and a lipophobic potential. We also evaluate the equilibrium quantities, such as order parameter, and dynamic features, such as rotational or translational diffusivity, of the embedded molecule in terms of the system parameters and compare the obtained results with those obtained from model independent theory.

Filipin-induced lesions in planar phospholipid bilayers imaged by atomic force microscopy.

Filipin is a macrolide polyene with antifungal activity belonging to the same family of antibiotics as amphotericin B and nystatin. Despite the spectroscopy and electron microscopy studies of its interaction with natural membranes and membrane model systems, several aspects of its biochemical action, such as the role of membrane sterols, remain to be completely understood. We have used atomic force microscopy (AFM) to study the effect of filipin on dipalmitoylphosphatidylethanolamine bilayers in the presence and absence of cholesterol. The bilayers were prepared by Langmuir-Blodgett deposition over mica and imaged under water. It was shown that filipin-induced lesions could only be found in membranes with cholesterol. In close agreement with electron microscopy results, we have reported the presence of densely packed circular protrusions in the membrane with a mean diameter of 19 nm (corrected for convolution with AFM tip) and 0.4 nm height. Larger circular protrusions (90 nm diameter and 2.5 nm height) and doughnut-shaped lesions were also detected. These results demonstrate that filipin-induced lesions in membranes previously observed by electron microscopy are not biased by artifacts resulting from sample preparation. Filipin aggregates in aqueous solution could also be imaged for the first time. These polydisperse spherical structures were observed in samples with and without cholesterol.

Fluorescence quenching data interpretation in biological systems. The use of microscopic models for data analysis and interpretation of complex systems.

In micro-heterogeneous media (e.g. membranes, micelles and colloidal systems), the fluorescence decay in the absence of quencher is usually intrinsically complex, e.g. due to the existence of several sub-populations with different micro-environments. In this case it is impossible to analyze data in detail (accounting for transient effects) and simpler formalisms are needed. The objective of the present work is to present and discuss such simpler formalisms. The goal is to achieve simple data analysis and meaningful, clear data interpretation in complex systems using microscopic models that consider several sub-populations of chromophores. Two points are dealt with in detail. (i) It is shown that the approximation of the transient effects by the quenching sphere-of-action model is not always possible. The quenching sphere-of-action concept can be regarded as a valuable tool, although crude, only in a limited range of experimental conditions, namely time resolution. (ii) The Stern-Volmer equation usually used for data analysis is only valid for a limited range of small and moderate equilibrium association constants, Ka, although this is frequently overlooked in the literature. Self-consistency criteria are presented for the proposed methods. The well-known downward curvature due to a fraction of fluorophores which is not accessible to the quencher is only a limiting case from a set of possible situations which result in deviations to linearity. A systematic classification of the different types of quenching is presented.

Continuous particle size distribution analysis with dynamic light scattering. MAXAMPER: a regularization method using the maximum amplitude for the average error and the Lagrange's multipliers method.

This work deals with a new method to evaluate the scattered light intensity distributions with particle size by means of photon correlation spectroscopy. Basically, the data analysis consists in: 1) To find the least squares solution (L2 norm), and 2) To find the solution that is not significantly different from the least squares solution (within a certain significance level) that simultaneously minimizes the sum of the modulus of the residuals (L1 norm). A simple procedure is achieved by using the Lagrange's multipliers method. The two aspects that debilitate CONTIN (a rather empirical regularization based on the continuity of the distributions and the non-suitable use of the Fisher's F test) are prevented in this work, granting more meaningful results. The method was applied to simulated curves, in several typical situations (mono-modal and bi-modal, narrow and broad distributions). The quality of the results is better for narrower mono-modal distributions and bi-modal distributions having well separated peaks, as expected. Latex beads (nanospheres) having 114 nm in diameter were used to test the method in experimental conditions. MAXAMPER better reproduces monodispersed distribution profiles than CONTIN and the first moments of the distributions are in agreement with the expected value in most of the analysis.

Interaction of the major epitope region of HIV protein gp41 with membrane model systems. A fluorescence spectroscopy study.

Fluorescence spectroscopy (both steady-state and time-resolved) was used to study the fragment 579-601 of gp41 ectodomain (HIV-1), a highly conserved sequence and major epitope, regarding (1) structural information, (2) interaction with membrane model systems, and (3) location in the phospholipid bilayer. The peptide was characterized both in its monomeric (after reduction of the disulfide bond between cysteine residues) and in the dimeric forms. The change of the fluorescence anisotropy between monomer and dimer was rationalized on the basis of energy migration, and a distance between the two tryptophan (Trp) residues of approximately 6 A was obtained. Using different fluorescence spectroscopy approaches, it was demonstrated that, despite the fact that monomeric gp41 fragment incorporates in the membrane model systems studied, the dimeric form does not interact with these vesicles. A methodology based on the increase of the mean fluorescence lifetime averaged by the preexponentials was derived, to obtain the partition coefficient of the peptide in the different lipid systems. Fluorescence quenching using lipophilic probes and red edge excitation shift (REES) were used to study the location of the gp41 fragment in the membrane. It was concluded that the Trp residue is located in a shallow position, near the interface. The REES results show an uncommonly large wavelength shift (18 nm) for the gp41 fragment incorporated in the membrane. Our results are consistent with a "two steps" model for the gp41 fusion mechanism similar to the one proposed for influenza virus hemagglutinin.

Teaching light scattering spectroscopy: the dimension and shape of tobacco mosaic virus.

The tobacco mosaic virus is used as a model molecular assembly to illustrate the basic potentialities of light scattering techniques (both static and dynamic) to undergraduates. The work has two objectives: a pedagogic one (introducing light scattering to undergraduate students) and a scientific one (stabilization of the virus molecular assembly structure by the nucleic acid). Students are first challenged to confirm the stabilization of the cylindrical shape of the virus by the nucleic acid, at pH and ionic strength conditions where the coat proteins alone do not self-assemble. The experimental intramolecular scattering factor is compared with the theoretical ones for several model geometries. The data clearly suggest that the geometry is, in fact, a rod. Comparing the experimental values of gyration radius and hydrodynamic radius with the theoretical expectations further confirms this conclusion. Moreover, the rod structure is maintained over a wider range of pH and ionic strength than that valid for the coat proteins alone. The experimental values of the diffusion coefficient and radius of gyration are compared with the theoretical expectations assuming the dimensions detected by electron microscopy techniques. In fact, both values are in agreement (length approximately 300 nm, radius approximately 20 nm).

The transverse location of the fluorescent probe trans-parinaric acid in lipid bilayers.

The transverse location of trans-parinaric acid in spherical vesicles made up from dipalmitoylphosphatidylcholine has been investigated by the differential quenching of the probe fluorescence by 5- and 16-doxylstearic acid derivatives. The quenching data are interpreted in terms of a local fluorophore concentration factor. In this way it was found that the polyene of t-PnA is located within the inner part of the bilayer (presumably aligned with the bilayer lipids), both in the gel and in the liquid crystalline phases.