Ionogels Derived from Fluorinated Ionic Liquids to Enhance Aqueous Drug Solubility for Local Drug Administration.

Gelatin is a popular biopolymer for biomedical applications due to its harmless impact with a negligible inflammatory response in the host organism. Gelatin interacts with soluble molecules in aqueous media as ionic counterparts such as ionic liquids (ILs) to be used as cosolvents to generate the so-called Ionogels. The perfluorinated IL (FIL), 1-ethyl-3-methylpyridinium perfluorobutanesulfonate, has been selected as co-hydrosolvent for fish gelatin due to its low cytotoxicity and hydrophobicity aprotic polar structure to improve the drug aqueous solubility. A series of FIL/water emulsions with different FIL content and their corresponding shark gelatin/FIL Ionogel has been designed to enhance the drug solubility whilst retaining the mechanical structure and their nanostructure was probed by simultaneous SAXS/WAXS, FTIR and Raman spectroscopy, DSC and rheological experiments. Likewise, the FIL assisted the solubility of the antitumoural Doxorubicin whilst retaining the performing mechanical properties of the drug delivery system network for the drug storage as well as the local administration by a syringe. In addition, the different controlled release mechanisms of two different antitumoral such as Doxorubicin and Mithramycin from two different Ionogels formulations were compared to previous gelatin hydrogels which proved the key structure correlation required to attain specific therapeutic dosages.

Multifunctional PLA/Gelatin Bionanocomposites for Tailored Drug Delivery Systems.

A series of bionanocomposites composed of shark gelatin hydrogels and PLA nanoparticles featuring different nanostructures were designed to generate multifunctional drug delivery systems with tailored release rates required for personalized treatment approaches. The global conception of the systems was considered from the desired customization of the drug release while featuring the viscoelastic properties needed for their ease of storage and posterior local administration as well as their biocompatibility and cell growth capability for the successful administration at the biomolecular level. The hydrogel matrix offers the support to develop a direct thermal method to convert the typical kinetic trapped nanostructures afforded by the formulation method whilst avoiding the detrimental nanoparticle agglomeration that diminishes their therapeutic effect. The nanoparticles generated were successfully formulated with two different antitumoral compounds (doxorubicin and dasatinib) possessing different structures to prove the loading versatility of the drug delivery system. The bionanocomposites were characterized by several techniques (SEM, DLS, RAMAN, DSC, SAXS/WAXS and rheology) as well as their reversible sol-gel transition upon thermal treatment that occurs during the drug delivery system preparation and the thermal annealing step. In addition, the local applicability of the drug delivery system was assessed by the so-called "syringe test" to validate both the storage capability and its flow properties at simulated physiological conditions. Finally, the drug release profiles of the doxorubicin from both the PLA nanoparticles or the bionanocomposites were analyzed and correlated to the nanostructure of the drug delivery system.

Characterization of Tuna Gelatin-Based Hydrogels as a Matrix for Drug Delivery.

The skin of yellowfin tuna is one of the fishery industry solid residues with the greatest potential to add extra value to its circular economy that remains yet unexploited. Particularly, the high collagen content of fish skin allows generating gelatin by hydrolysis, which is ideal for forming hydrogels due to its biocompatibility and gelling capability. Hydrogels have been used as drug carriers for local administration due to their mechanical properties and drug loading capacity. Herein, novel tuna gelatin hydrogels were designed as drug vehicles with two structurally different antitumoral model compounds such as Doxorubicin and Crocin to be administrated locally in tissues with complex human anatomies after surgical resection. The characterization by gel permeation chromatography (GPC) of purified gelatin confirmed their heterogeneity composition, exhibiting three major bands that correspond to the ß and α chains along with high molecular weight species. In addition, the Fourier Transform Infrared (FT-IR) spectra of gelatin probed the secondary structure of the gelatin showing the simultaneous existence of α helix, ß sheet, and random coil structures. Morphological studies at different length scales were performed by a multi-technique approach using SAXS/WAXS, AFM and cryo-SEM that revealed the porous network formed by the interaction of gelatin planar aggregates. In addition, the sol-gel transition, as well as the gelation point and the hydrogel strength, were studied using dynamic rheology and differential scanning calorimetry. Likewise, the loading and release profiles followed by UV-visible spectroscopy indicated that the novel gelatin hydrogels improve the drug release of Doxorubicin and Crocin in a sustained fashion, indicating the structure-function importance in the material composition.

Numerical modelling of acoustic streaming during the ultrasonic melt treatment of direct-chill (DC) casting.

Acoustic streaming and its attendant effects in the sump of a direct-chill (DC) casting process are successfully predicted under ultrasonic treatment for the first time. The proposed numerical model couples acoustic cavitation, fluid flow, heat and species transfer, and solidification to predict the flow pattern, acoustic pressure, and temperature fields in the sump. The model is numerically stable with time steps of the order of 0.01 s and therefore computationally attractive for optimization studies necessitating simulation times of the order of a minute. The sump profile is altered by acoustic streaming, with the slurry region depressed along the centreline of the billet by a strong central jet. The temperature gradient in the transition zone is increased, potentially interfering with grain refinement. The cooling rate in the sump is also altered, thereby modifying the dendrite arm spacing of the as-cast billet. The relative position of the sonotrode affects the sump profile, with the sump depth decreased by around 5 mm when the sonotrode is moved above the graphite ring level by 100 mm. The acoustic streaming jet penetrates into the slurry zone and, as a result, the growth direction of dendritic grains in the off-centre position is altered.

A targeted functional RNA interference screen uncovers glypican 5 as an entry factor for hepatitis B and D viruses.

UNLABELLED: Chronic hepatitis B and D infections are major causes of liver disease and hepatocellular carcinoma worldwide. Efficient therapeutic approaches for cure are absent. Sharing the same envelope proteins, hepatitis B virus and hepatitis delta virus use the sodium/taurocholate cotransporting polypeptide (a bile acid transporter) as a receptor to enter hepatocytes. However, the detailed mechanisms of the viral entry process are still poorly understood. Here, we established a high-throughput infectious cell culture model enabling functional genomics of hepatitis delta virus entry and infection. Using a targeted RNA interference entry screen, we identified glypican 5 as a common host cell entry factor for hepatitis B and delta viruses. CONCLUSION: These findings advance our understanding of virus cell entry and open new avenues for curative therapies. As glypicans have been shown to play a role in the control of cell division and growth regulation, virus-glypican 5 interactions may also play a role in the pathogenesis of virus-induced liver disease and cancer.

Modification of the hepatitis B virus envelope protein glycosylation pattern interferes with secretion of viral particles, infectivity, and susceptibility to neutralizing antibodies.

UNLABELLED: The envelope proteins of hepatitis B virus (HBV) bear an N-linked glycosylation site at N146 within the immunodominant a-determinant in the antigenic loop (AGL) region. This glycosylation site is never fully functional, leading to a nearly 1/1 ratio of glycosylated/nonglycosylated isoforms in the viral envelope. Here we investigated the requirement for a precise positioning of N-linked glycan at amino acid 146 and the functions associated with the glycosylated and nonglycosylated isoforms. We observed that the removal of the N146 glycosylation site by mutagenesis was permissive to envelope protein synthesis and stability and to secretion of subviral particles (SVPs) and hepatitis delta virus (HDV) virions, but it was detrimental to HBV virion production. Several positions in the AGL could substitute for position 146 as the glycosylation acceptor site. At position 146, neither a glycan chain nor asparagine was absolutely required for infectivity, but there was a preference for a polar residue. Envelope proteins bearing 5 AGL glycosylation sites became hyperglycosylated, leading to an increased capacity for SVP secretion at the expense of HBV and HDV virion secretion. Infectivity-compatible N-glycosylation sites could be inserted at 3 positions (positions 115, 129, and 136), but when all three positions were glycosylated, the hyperglycosylated mutant was substantially attenuated at viral entry, while it acquired resistance to neutralizing antibodies. Taken together, these findings suggest that the nonglycosylated N146 is essential for infectivity, while the glycosylated form, in addition to its importance for HBV virion secretion, is instrumental in shielding the a-determinant from neutralizing antibodies. IMPORTANCE: At the surface of HBV particles, the immunodominant a-determinant is the main target of neutralizing antibodies and an essential determinant of infectivity. It contains an N-glycosylation site at position 146, which is functional on only half of the envelope proteins. Our data suggest that the coexistence of nonglycosylated and glycosylated N146 at the surface of HBV reflects the dual function of this determinant in infectivity and immune escape. Hence, a modification of the HBV glycosylation pattern affects not only virion assembly and infectivity but also immune escape.

I223R mutation in influenza A(H1N1)pdm09 neuraminidase confers reduced susceptibility to oseltamivir and zanamivir and enhanced resistance with H275Y.

BACKGROUND: Resistance of pandemic A(H1N1)2009 (H1N1pdm09) virus to neuraminidase inhibitors (NAIs) has remained limited. A new mutation I223R in the neuraminidase (NA) of H1N1pdm09 virus has been reported along with H275Y in immunocompromised patients. The aim of this study was to determine the impact of I223R on oseltamivir and zanamivir susceptibility. METHODS: The NA enzymatic characteristics and susceptibility to NAIs of viruses harbouring the mutations I223R and H275Y alone or in combination were analyzed on viruses produced by reverse genetics and on clinical isolates collected from an immunocompromised patient with sustained influenza H1N1pdm09 virus shedding and treated by oseltamivir (days 0-15) and zanamivir (days 15-25 and 70-80). RESULTS: Compared with the wild type, the NA of recombinant viruses and clinical isolates with H275Y or I223R mutations had about two-fold reduced affinity for the substrate. The H275Y and I223R isolates showed decreased susceptibility to oseltamivir (246-fold) and oseltamivir and zanamivir (8.9- and 4.9-fold), respectively. Reverse genetics assays confirmed these results and further showed that the double mutation H275Y and I223R conferred enhanced levels of resistance to oseltamivir and zanamivir (6195- and 15.2-fold). In the patient, six days after initiation of oseltamivir therapy, the mutation H275Y conferring oseltamivir resistance and the I223R mutation were detected in the NA. Mutations were detected concomitantly from day 6-69 but molecular cloning did not show any variant harbouring both mutations. Despite cessation of NAI treatment, the mutation I223R persisted along with additional mutations in the NA and the hemagglutinin. CONCLUSIONS: Reduced susceptibility to both oseltamivir and zanamivir was conferred by the I223R mutation which potentiated resistance to both NAIs when associated with the H275Y mutation in the NA. Concomitant emergence of the I223R and H275Y mutations under oseltamivir treatment underlines the importance of close monitoring of treated patients especially those immunocompromised.

The nucleocapsid region of HIV-1 Gag cooperates with the PTAP and LYPXnL late domains to recruit the cellular machinery necessary for viral budding.

HIV-1 release is mediated through two motifs in the p6 region of Gag, PTAP and LYPX(n)L, which recruit cellular proteins Tsg101 and Alix, respectively. The Nucleocapsid region of Gag (NC), which binds the Bro1 domain of Alix, also plays an important role in HIV-1 release, but the underlying mechanism remains unclear. Here we show that the first 202 residues of the Bro1 domain (Bro(i)) are sufficient to bind Gag. Bro(i) interferes with HIV-1 release in an NC-dependent manner and arrests viral budding at the plasma membrane. Similar interrupted budding structures are seen following over-expression of a fragment containing Bro1 with the adjacent V domain (Bro1-V). Although only Bro1-V contains binding determinants for CHMP4, both Bro(i) and Bro1-V inhibited release via both the PTAP/Tsg101 and the LYPX(n)L/Alix pathways, suggesting that they interfere with a key step in HIV-1 release. Remarkably, we found that over-expression of Bro1 rescued the release of HIV-1 lacking both L domains. This rescue required the N-terminal region of the NC domain in Gag and the CHMP4 binding site in Bro1. Interestingly, release defects due to mutations in NC that prevented Bro1 mediated rescue of virus egress were rescued by providing a link to the ESCRT machinery via Nedd4.2s over-expression. Our data support a model in which NC cooperates with PTAP in the recruitment of cellular proteins necessary for its L domain activity and binds the Bro1-CHMP4 complex required for LYPX(n)L-mediated budding.

Entry of hepatitis delta virus requires the conserved cysteine residues of the hepatitis B virus envelope protein antigenic loop and is blocked by inhibitors of thiol-disulfide exchange.

Hepatitis delta virus (HDV) particles are coated with the envelope proteins (large, middle, and small) of the hepatitis B virus (HBV). The large protein bears an infectivity determinant in its pre-S1 domain, whereas a second determinant has been proposed to map to the cysteine-rich antigenic loop (AGL) within the S domain of all three envelope proteins (G. Abou Jaoudé and C. Sureau, J. Virol. 79:10460-10466, 2006). In this study, the AGL cysteines were substituted by serine or alanine, and the mutants were evaluated for their function at viral entry using HDV particles and susceptible HepaRG cells. Mutations of cysteines 121 to 149 were tolerant of the production of HDV virions. The mutations altered the structure and antigenicity of the conserved "a" determinant of the AGL, as measured by conformation-sensitive antibodies, and they created a block to infectivity. Substitution of Cys-90 or Cys-221, located outside of the AGL, had no impact on the "a" determinant or viral entry. Furthermore, infectivity was maintained when the AGL CxxC motif at position 121 to 124 was modified by single-amino-acid deletion or insertion, suggesting that cysteines 121 and 124 are not catalyzers of thiol/disulfide exchange. However, membrane-impermeable inhibitors of thiol/disulfide isomerazation demonstrated a dose-dependent inhibition of infection in an in vitro assay when applied to the virus prior to inoculation or during the virus-cell interaction period. Overall, the results demonstrate the essential role of the AGL cysteines at viral entry, and they establish a correlation between the cysteine disulfide network, the conformation of the "a" determinant, and infectivity.

Myristoylation signal transfer from the large to the middle or the small HBV envelope protein leads to a loss of HDV particles infectivity.

A myristate linked to the N-terminus of the large hepatitis B virus (HBV) envelope protein was found to be required for infectivity of the hepatitis delta virus (HDV). Myristoylation of the large HBV envelope protein being known as indispensable for HBV infectivity, this result further demonstrates the similarities between the HBV and HDV entry pathways. In addition, the transfer of the N-myristoylation signal from the large to the middle or the small HBV envelope protein led in both cases to a loss of HDV infectivity. Hence, it is suggested that viral entry could depend on a physical link, or a spatial association, between the N-terminal receptor-binding polypeptide of the large protein and the myristoyl anchor linked to glycine-2.