ß-Glucan Grafted Microcapsule, a Tool for Studying the Immunomodulatory Effect of Microbial Cell Wall Polysaccharides.

ß-(1,3)-Glucan is one of the antigenic components of the bacterial as well as fungal cell wall. We designed microcapsules (MCs) ligated with ß-(1,3)-glucan, to study its immunomodulatory effect. The MCs were obtained by interfacial polycondensation between diacyl chloride (sebacoyl chloride and terephtaloyl chloride) and diethylenetriamine in organic and aqueous phases, respectively. Planar films were first designed to optimize monomer compositions and to examine the kinetics of film formation. MCs with aqueous fluorescent core were then obtained upon controlled emulsification-polycondensation reactions using optimized monomer compositions and adding fluorescein into the aqueous phase. The selected MC-formulation was grafted with Curdlan, a linear ß-(1,3)-glucan from  Agrobacterium species or branched ß-(1,3)-glucan isolated from the cell wall of Aspergillus fumigatus. These ß-(1,3)-glucan grafted MCs were phagocytosed by human monocyte-derived macrophages, and stimulated cytokine secretion. Moreover, the blocking of dectin-1, a ß-(1,3)-glucan recognizing receptor, did not completely inhibit the phagocytosis of these ß-(1,3)-glucan grafted MCs, suggesting the involvement of other receptors in the recognition and uptake of ß-(1,3)-glucan. Overall, grafted MCs are a useful tool for the study of the mechanism of phagocytosis and immunomodulatory effect of the microbial polysaccharides.

Strategies for Prevention and Treatment of Trichomonas vaginalis Infections.

The last estimated annual incidence of Trichomonas vaginalis worldwide exceeds that of chlamydia and gonorrhea combined. This critical review updates the state of the art on advances in T. vaginalis diagnostics and strategies for treatment and prevention of trichomoniasis. In particular, new data on treatment outcomes for topical administration of formulations are reviewed and discussed.

Phase solubility studies and anti-Trichomonas vaginalis activity evaluations of metronidazole and methylated ß-cyclodextrin complexes: Comparison of CRYSMEB and RAMEB.

Metronidazole (MTZ) is a 5-nitroimidazole drug used for the treatment of Trichomonas vaginalis parasitic infection. Aqueous formulations containing MTZ are restricted because apparent solubility in water of this drug is low. In this context, two methylated-ß-cyclodextrins (CRYSMEB and RAMEB) were used as a tool to increase apparent solubility of MTZ in water. CRYSMEB was limited by its own solubility in water (15% w/w, 12.59 mM), while RAMEB at a concentration of 40% w/w (300.44 mM) allowed a maximal increase of apparent solubility of MTZ (3.426% w/w, 200.19 mM). From our knowledge, this corresponds to the highest enhancement of MTZ apparent aqueous solubility ever reported in the literature using methylated cyclodextrins. In vitro evaluations showed that anti-T. vaginalis activity of MTZ formulated with CRYSMEB and RAMEB was preserved.

Supramolecular Chitosan Micro-Platelets Synergistically Enhance Anti-Candida albicans Activity of Amphotericin B Using an Immunocompetent Murine Model.

PURPOSE: The aim of this work is to design new chitosan conjugates able to self-organize in aqueous solution in the form of micrometer-size platelets. When mixed with amphotericin B deoxycholate (AmB-DOC), micro-platelets act as a drug booster allowing further improvement in AmB-DOC anti-Candida albicans activity. METHODS: Micro-platelets were obtained by mixing oleoyl chitosan and α-cyclodextrin in water. The formulation is specifically-engineered for mucosal application by dispersing chitosan micro-platelets into thermosensitive pluronic® F127 20 wt% hydrogel. RESULTS: The formulation completely cured C. albicans vaginal infection in mice and had a superior activity in comparison with AmB-DOC without addition of chitosan micro-platelets. In vitro studies showed that the platelets significantly enhance AmB-DOC antifungal activity since the IC50 and the MIC90 decrease 4.5 and 4.8-times. Calculation of fractional inhibitory concentration index (FICI = 0.198) showed that chitosan micro-platelets act in a synergistic way with AmB-DOC against C. albicans. No synergy is found between spherical nanoparticles composed poly(isobutylcyanoacrylate)/chitosan and AmB-DOC. CONCLUSION: These results demonstrate for the first time the ability of flattened chitosan micro-platelets to have synergistic activity with AmB-DOC against C. albicans candidiasis and highlight the importance of rheological and mucoadhesive behaviors of hydrogels in the efficacy of the treatment.

Thermosensitive and mucoadhesive pluronic-hydroxypropylmethylcellulose hydrogel containing the mini-CD4 M48U1 is a promising efficient barrier against HIV diffusion through macaque cervicovaginal mucus.

To be efficient, vaginal microbicide hydrogels should form a barrier against viral infections and prevent virus spreading through mucus. Multiple particle tracking was used to quantify the mobility of 170-nm fluorescently labeled COOH-modified polystyrene particles (COOH-PS) into thermosensitive hydrogels composed of amphiphilic triblock copolymers with block compositions EOn-POm-EOn (where EO refers to ethylene oxide and PO to propylene oxide) containing mucoadhesive hydroxypropylmethylcellulose (HPMC). COOH-PS were used to mimic the size and the surface charge of HIV-1. Analysis of COOH-PS trajectories showed that particle mobility was decreased by Pluronic hydrogels in comparison with cynomolgus macaque cervicovaginal mucus and hydroxyethylcellulose hydrogel (HEC; 1.5% by weight [wt%]) used as negative controls. Formulation of the peptide mini-CD4 M48U1 used as an anti-HIV-1 molecule into a mixture of Pluronic F127 (20 wt%) and HPMC (1 wt%) did not affect its anti-HIV-1 activity in comparison with HEC hydrogel. The 50% inhibitory concentration (IC50) was 0.53 µg/ml (0.17 µM) for M48U1-HEC and 0.58 µg/ml (0.19 µM) for M48U1-F127-HPMC. The present work suggests that hydrogels composed of F127-HPMC (20/1 wt%, respectively) can be used to create an efficient barrier against particle diffusion in comparison to conventional HEC hydrogels.

The unexpected increase of clotrimazole apparent solubility using randomly methylated ß-cyclodextrin.

Clotrimazole (CTZ) and cyclodextrin (CD) inclusion complexes having improved apparent water solubility were obtained from phase solubility diagrams. ß-CD (1.5% w/w) and hydroxypropyl-ß-CD (40% w/w) offered poor CTZ solubility enhancements (12 and 384 times, respectively). Unexpectedly, the apparent solubility of CTZ was 9980 times increased from 0.4 µg.mL(-1) (1.42 µM) without CD to 4.89 mg.mL(-1) (14.9 mM) using randomly-methylated ß-CD (Me-ß-CD) (40% w/w). This is the highest apparent CTZ solubility improvement ever reported in the literature using conventional CDs. Quantitative nuclear magnetic resonance ((1) H-NMR) coupled with two-dimensional nuclear Overhauser effect (NOESY) experiments and molecular docking calculations showed that the highest interactions with Me-ß-CD were reported for CTZ two phenyl groups. A lower interaction was reported for chlorophenyl, while imidazole had the weakest interaction with Me-ß-CD.

Drug-free chitosan coated poly(isobutylcyanoacrylate) nanoparticles are active against trichomonas vaginalis and non-toxic towards pig vaginal mucosa.

PURPOSE: The present work reports a non-conventional therapeutic strategy based on the use of vaginally-applied formulations for the treatment of trichomoniasis due to Trichomonas vaginalis without adding a drug. METHODS: The formulations were based on a thermosensitive pluronic® F127 hydrogel containing mucoadhesive poly(isobutylcyanoacrylate) nanoparticles coated with a mixture of chitosan and thiolated chitosan (75/25 wt%). The nanoparticles were obtained by anionic emulsion polymerization of isobutylcyanoacrylate. The anti-T. vaginalis activity of the formulations was evaluated in vitro. RESULTS: Chitosan-coated nanoparticles showed a strong anti-T. vaginalis activity at 100 µg/mL independently on the proportion of thiolated chitosan. No anti-T. vaginalis activity was reported neither with chitosan-uncoated poly(isobutylcyanoacrylate) nanoparticles nor with chitosan used as a solution. These results suggest that the anti-T. vaginalis activity was related to poly(isobutylcyanoacrylate) nanoparticles but only when they are coated with chitosan. Histological analysis of ex vivo pig vaginal mucosa in contact with pluronic® F127 hydrogel containing poly(isobutylcyanoacrylate) nanoparticles coated with the mixture chitosan/thiolated chitosan (75/25 wt%) did not reveal any toxicity. CONCLUSION: This study demonstrated that poly(isobutylcyanoacrylate) nanoparticles coated with chitosan were active against T. vaginalis without adding a drug. Besides their anti-T. vaginalis activity, the formulations are non-toxic towards pig vaginal mucosa.

MiniCD4 microbicide prevents HIV infection of human mucosal explants and vaginal transmission of SHIV(162P3) in cynomolgus macaques.

In complement to an effective vaccine, development of potent anti-HIV microbicides remains an important priority. We have previously shown that the miniCD4 M48U1, a functional mimetic of sCD4 presented on a 27 amino-acid stable scaffold, inhibits a broad range of HIV-1 isolates at sub-nanomolar concentrations in cellular models. Here, we report that M48U1 inhibits efficiently HIV-1(Ba-L) in human mucosal explants of cervical and colorectal tissues. In vivo efficacy of M48U1 was evaluated in nonhuman primate (NHP) model of mucosal challenge with SHIV(162P3) after assessing pharmacokinetics and pharmacodynamics of a miniCD4 gel formulation in sexually matured female cynomolgus macaques. Among 12 females, half were treated with hydroxyethylcellulose-based gel (control), the other half received the same gel containing 3 mg/g of M48U1, one hour before vaginal route challenge with 10 AID(50) of SHIV(162P3). All control animals were infected with a peak plasma viral load of 10(5)-10(6) viral RNA (vRNA) copies per mL. In animals treated with miniCD4, 5 out of 6 were fully protected from acquisition of infection, as assessed by qRT-PCR for vRNA detection in plasma, qPCR for viral DNA detection in PBMC and lymph node cells. The only infected animal in this group had a delayed peak of viremia of one week. These results demonstrate that M48U1 miniCD4 acts in vivo as a potent entry inhibitor, which may be considered in microbicide developments.

Cyclodextrin-based supramolecular nanogels decorated with mannose for short peptide encapsulation.

Nanogels are aqueous dispersions of hydrogel particles formed by physically or chemically cross-linked polymer networks of nanoscale size. Herein, we devised a straightforward technique to fabricate a novel class of physically cross-linked nanogels via a self-assembly process in water involving α-cyclodextrin and a mannose molecule that was hydrophobically modified using an alkyl chain. The alkyl chain-modified mannose was synthesized in five steps, starting with D-mannose. Subsequently, nanogels were formed by subjecting α-cyclodextrin and the hydrophobically modified mannose to magnetic stirring in water. By adjusting the mole ratio between the hydrophobically modified mannose and α-cyclodextrin, nanogels with an average 100-150 nm diameter were obtained. Physicochemical and structural analyses by 1H NMR and X-ray diffraction unveiled a supramolecular and hierarchical mechanism underlying the creation of these nanogels. The proposed mechanism of nanogel formation involves two distinct steps: initial interaction of hydrophobically modified mannose with α-cyclodextrin resulting in the formation of inclusion complexes, followed by supramolecular interactions among these complexes, ultimately leading to nanogel formation after 72 h of stirring. We demonstrated the nanogels' ability to encapsulate a short peptide ([p-tBuF2, R5]SHf) as a water-soluble drug model. This discovery holds promise for potentially utilizing these nanogels in drug delivery applications.

Hyaluronan nanoplatelets exert an intrinsic anti-inflammatory activity in a rat model of bladder painful syndrome/interstitial cystitis.

Glycosaminoglycan (GAG) replenishment therapy consists of the instillation of GAG solutions directly in the bladder to alleviate Bladder Painful Syndrome/Interstitial Cystitis (BPS/IC). However, several issues were reported with this strategy because the GAG solutions are rapidly eliminated from the bladder by spontaneous voiding, and GAG have low bioadhesive behaviors. Herein, GAG nanomaterials with typical flattened morphology were obtained by a self-assembly process. The formation mechanism of those nanomaterials, denoted as nanoplatelets, involves the interaction of α-cyclodextrin cavity and alkyl chains covalently grafted on the GAG. Three GAG were used in this investigation, hyaluronan (HA), chondroitin sulfate (CS), and heparin (HEP). HA NP showed the best anti-inflammatory activity in an LPS-induced in vitro inflammation model of macrophages. They also exhibited the best therapeutic efficacy in a BPS/IC rat inflammation model. Histological examinations of the bladders revealed that HA NP significantly reduced bladder inflammation and regenerated the bladder mucosa. This investigation could open new perspectives to alleviate BPS/IC through GAG replenishment therapy.

Mucosal application of the broadly neutralizing antibody 10-1074 protects macaques from cell-associated SHIV vaginal exposure.

Passive immunization using broadly neutralizing antibodies (bNAbs) is investigated in clinical settings to inhibit HIV-1 acquisition due to the lack of a preventive vaccine. However, bNAbs efficacy against highly infectious cell-associated virus transmission has been overlooked. HIV-1 transmission mediated by infected cells present in body fluids likely dominates infection and aids the virus in evading antibody-based immunity. Here, we show that the anti-N-glycans/V3 loop HIV-1 bNAb 10-1074 formulated for topical vaginal application in a microbicide gel provides significant protection against repeated cell-associated SHIV162P3 vaginal challenge in non-human primates. The treated group has a significantly lower infection rate than the control group, with 5 out of 6 animals fully protected from the acquisition of infection. The findings suggest that mucosal delivery of potent bnAbs may be a promising approach for preventing transmission mediated by infected cells and support the use of anti-HIV-antibody-based strategies as potential microbicides in human clinical trials.

Polyamide nanocapsules and nano-emulsions containing Parsol® MCX and Parsol® 1789: in vitro release, ex vivo skin penetration and photo-stability studies.

PURPOSE: To prepare polyamide nanocapsules for skin photo-protection, encapsulating α-tocopherol, Parsol®MCX (ethylhexyl methoxycinnamate) and/or Parsol®1789 (butyl methoxydibenzoylmethane). METHODS: Nanocapsules were obtained by combining spontaneous emulsification and interfacial polycondensation reaction between sebacoyl chloride and diethylenetriamine. Nano-emulsions used as control were obtained by the same process without monomers. The influence of carrier on release rate was studied in vitro with a membrane-free model. Epidermal penetration of encapsulated sunscreens was ex vivo evaluated using Franz diffusion cells. Ability of encapsulated sunscreens to improve photo-stability was verified by comparing percentage of degradation after UV radiation exposure. RESULTS: Sunscreen-containing nanocapsules (260-400 nm) were successfully prepared; yield of encapsulation was >98%. Parsol®MCX and Parsol®1789 encapsulation led to decreased release rate by up to 60% in comparison with nano-emulsion and allowed minimum penetration through pig ear epidermis. Presence of polyamide shell protected encapsulated sunscreen filters from photo-degradation without affecting their activity. CONCLUSIONS: Encapsulation of Parsol®MCX and Parsol®1789 into oil-core of polyamide nanocapsules allowed protection from photo-degradation, controlled release from nanocapsules, and limited penetration through pig ear epidermis.

The counterbalanced effect of size and surface properties of chitosan-coated poly(isobutylcyanoacrylate) nanoparticles on mucoadhesion due to pluronic F68 addition.

PURPOSE: To evaluate of the effect of size and surface characteristics of poly(isobutylcyanoacrylate) nanoparticles coated with pluronic F68 and thiolated chitosan on mucoadhesion. METHODS: Nanoparticles were obtained by radical emulsion polymerization in presence of different amounts of F68 (0-4%w/v). Mucoadhesion was ex vivo evaluated by applying nanoparticle suspension on rat intestinal mucosa and quantifying the amount of attached nanoparticles after incubation. RESULTS: F68 unimers added in the polymerization medium allowed decreasing nanoparticle size from 251 to 83 nm, but resulted in nanoparticle surface modification. The amount of thiolated chitosan onto nanoparticle surface was decreased resulting in lower thiol groups and zeta potential. Consequently, the decrease of nanoparticle hydrodynamic diameter resulted in eight-fold-increase of the number of nanoparticles attached to the mucosa but a significant decrease of the weight of attached nanoparticles was observed. This unexpected result was due to a decrease of the amount of chitosan and thiolated chitosan available to interact with mucus upon addition of F68 in the polymerization medium. CONCLUSIONS: Addition of F68 should not be recommended to improve the amount of mucoadherent nanoparticles. Further studies could allow understanding if the low amount of small size nanoparticles could be able to improve oral bioavailability.

Effect of micro- and nanoparticle shape on biological processes.

In the drug delivery field, there is beyond doubt that the shape of micro- and nanoparticles (M&NPs) critically affects their biological fate. Herein, following an introduction describing recent technological advances for designing nonspherical M&NPs, we highlight the role of particle shape in cell capture, subcellular distribution, intracellular drug delivery, and cytotoxicity. Then, we discuss theoretical approaches for understanding the effect of particle shape on internalization by the cell membrane. Subsequently, recent advances on shape-dependent behaviors of M&NPs in the systemic circulation are detailed. In particular, the interaction of M&NPs with blood proteins, biodistribution, and circulation under flow conditions are analyzed. Finally, the hurdles and future directions for developing nonspherical M&NPs are underscored.

Advances in the treatment of inflammatory bowel disease: Focus on polysaccharide nanoparticulate drug delivery systems.

The complex pathogenesis of inflammatory bowel disease (IBD) explains the several hurdles for finding an efficient approach to cure it. Nowadays, therapeutic protocols aim to reduce inflammation during the hot phase or maintain remission during the cold phase. Nonetheless, these drugs suffer from severe side effects or poor efficacy due to low bioavailability in the inflamed region of the intestinal tract. New protocols based on antibodies that target proinflammatory cytokines are clinically relevant. However, besides being expensive, their use is associated with a primary nonresponse or a loss of response following a long administration period. Accordingly, many researchers exploited the physiological changes of the mucosal barrier for designing nanoparticulate drug delivery systems to target inflamed tissues. Others exploited biocompatibility and relative affordability of polysaccharides to test their intrinsic anti-inflammatory and healing properties in IBD models. This critical review updates state of the art on advances in IBD treatment. Data on using polysaccharide nanoparticulate drug delivery systems for IBD treatment are reviewed and discussed.

Shape stability of ellipsoidal nanomaterials prepared by physical deformation.

The nonspherical shape of nanomaterials (NMs) represents a key attribute for controlling their biological behaviors. Analyzing shape stability over time represents a significant concern because nonspherical NMs are likely to rearrange into a thermodynamically more stable spherical shape. In this investigation, ellipsoidal NMs were designed by physical deformation of core/shell nanospheres composed of poly(isobutylcyanoacrylate) and chitosan or a mixture of chitosan and thiolated chitosan. After optimizing the process parameters for designing ellipsoidal NMs, the shape stability during storage was investigated for 6 months at different temperatures (4 °C, 20 °C and 40 °C). The NM shape was examined by analyzing the aspect ratio from images obtained by electron microscopy techniques. The results demonstrated the feasibility of designing shape-persistent ellipsoidal NMs by physical deformation of spherical particles.

Real-time visualization of morphology-dependent self-motion of hyaluronic acid nanomaterials in water.

Drug delivery to target sites is often limited by inefficient particle transport through biological media. Herein, motion behaviors of spherical and nonspherical nanomaterials composed of hyaluronic acid were studied in water using real-time multiple particle tracking technology. The two types of nanomaterials have comparable surface compositions and surface potentials, and they have equivalent diameters. The analysis of nanomaterial trajectories revealed that particles with flattened morphology and a high aspect ratio, designated nanoplatelets, exhibited more linear trajectories and faster diffusion in water than nanospheres. Fitting the plots of mean square displacement vs. time scale suggests that nanoplatelets exhibited hyperdiffusive behavior, which is similar to the motion of living microorganisms. Furthermore, at 37 °C, the surface explored by a nanoplatelet was up to 33-fold higher than that explored by a nanosphere. This investigation on morphology-dependent self-motion of nanomaterials could have a significant impact on drug delivery applications by increasing particle transport through biological media.

Role of the interactions of soft hyaluronan nanomaterials with CD44 and supported bilayer membranes in the cellular uptake.

Increasing valence by acting on nanomaterial morphology can enhance the ability of a ligand to specifically bind to targeted cells. Herein, we investigated cell internalization of soft hyaluronic acid (HA) nanoplatelets (NPs) that exhibit a typical hexagonal shape, flat surfaces and high aspect ratio (Γ≈12 to 20), as characterized by atomic force microscopy in hydrated conditions. Fluorescence imaging revealed that internalization of HA-NPs by a T24 tumor cell line and by macrophages was higher than native polysaccharide in a dose-dependent and time-dependent manners. The ability of HA-NPs to efficiently compete with native HA assessed using Bio-layer interferometry showed that NPs had a stronger interaction with recombinant CD44 receptor compared to native HA. The results were discussed regarding physical properties of the NPs and the implication of multivalent interactions in HA binding to CD44. Experiments conducted on supported bilayer membranes with different compositions showed that non-specific interactions of NPs with lipid membranes were negligible. Our findings provide insights into intracellular drug delivery using soft HA-NPs through receptor-mediated multivalent interactions.

What can isothermal titration microcalorimetry experiments tell us about the self-organization of surfactants into micelles?

The aim of the present review is to give a concise analysis of the thermodynamic parameters obtained from isothermal titration microcalorimetry (ITC) experiments for the characterization of the self-organization of surfactants into micelles. This review is also focused on works describing some methods allowing to overcome ITC limitation and to extract accurate thermodynamic values from ITC data.

Trichomonas vaginalis Motility Is Blocked by Drug-Free Thermosensitive Hydrogel.

Trichomonas vaginalis motility in biological fluids plays a prominent, but understudied, role in parasite infectivity. In this study, the ability of a thermosensitive hydrogel (pluronic F127) to physically immobilize T. vaginalis was investigated. Blocking parasite motility could prevent its attachment to the mucosa, thus reducing the acquisition of the infection. The trajectory of individual parasites was monitored by multiple particle tracking. Mean square displacement, diffusivity, and velocity were calculated from x, y coordinates during time. Major results are that T. vaginalis exhibited different types of trajectories in a diluted solution composed of lactate buffer similar to "run-and-tumble" motion reported for flagellated bacteria. The fastest T. vaginalis specimen moves with a velocity of 19 µm/s. Observation of T. vaginalis movements showed that the cell body remains rigid during swimming and that the propulsive forces necessary to generate the movement are the result of flagellar beating. Parasite motility was partially slowed down using hydroxyethylcellulose hydrogel, used as a reference for the development of vaginal microbicides, while 100% of T. vaginalis were immobile in F127 hydrogel. Once completed by biological investigations on mice, this report suggests using drug-free formulation composed of F127 as a new strategy to prevent T. vaginalis attachment to the mucosa. The concept will be extended to other flagellated organisms where the motility is driven by cilia and flagella.