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.

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.

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.

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.

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.

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.

Hierarchically built hyaluronan nano-platelets have symmetrical hexagonal shape, flattened surfaces and controlled size.

Most of nanomaterials composed of hyaluronic acid (HA) used for drug targeting are spherical. In this investigation, we suggest that the morphology of HA nanomaterials could be considered as a new parameter to control their interactions with cells. However, designing nanomaterials with elongated morphology and controlled size is still challenging. The aim of this study was to design and to characterize non-spherical HA nanomaterials with flat surfaces and to highlight main parameters controlling the size. Nanoparticles were formed by mixing HA hydrophobically-modified with palmitoyl groups (PA-HA) and α-cyclodextrin in water. These particles, called nano-platelets, had symmetrical hexagonal shape, flattened surfaces and were 9-fold larger than thick. Small nano-platelets with well-defined shape were obtained with low PA-HA degree of substitution, by adding 5 wt% of α-cyclodextrin solution for a fixed concentration of PA-HA (1 wt%) (569 nm) and for long stirring periods (735-538 nm for 72-168 h). PA-HA was successfully conjugated to a near-infrared fluorescent probe suitable for in vitro and in vivo experiments without nano-platelet size and surface charge modification. This is the first report showing the design of non-spherical and flattened HA nano-platelets that could be used to study the impact of nanomaterial shape on molecular interactions with cells.

Topically Applied Chitosan-Coated Poly(isobutylcyanoacrylate) Nanoparticles Are Active Against Cutaneous Leishmaniasis by Accelerating Lesion Healing and Reducing the Parasitic Load.

Parenteral administration of amphotericin B-deoxycholate (AmB-DOC) or pentavalent antimonials to cure cutaneous leishmaniasis (CL) results in severe adverse reactions, while topically applied antileishmanial drugs are ineffective despite their good tolerance. This work is aimed to investigate whether poly(isobutylcyanoacrylate) nanoparticles coated with chitosan (Cs-NPs) could provide intrinsic antileishmanial activity after topical application. In vitro evaluations revealed that nanoparticles were active against the promastigote, axenic amastigote, and intramacrophage forms of Leishmania major. In vivo evaluations after repetitive topical applications on the skin of mice infected with L. major showed that Cs-NPs combined or not with AmB-DOC allowed partial healing of the lesion characterized by histological analyses. The parasitic load of skin specimens collected from mice was significantly reduced compared with that from nontreated mice, as analyzed by quantitative polymerase chain reaction (q-PCR). Ultrastructure characterizations by electron microscopy of L. major promastigotes after incubation with Cs-NPs showed morphological alterations, including aberrant shape and swelling of mitochondria and parasitic vacuoles.

Surface-dependent endocytosis of poly(isobutylcyanoacrylate) nanoparticles by Trichomonas vaginalis.

Previous data from our research group showed that chitosan-coated poly(isobutylcyanoacrylate) nanoparticles (NPs) (denoted PIBCA/Chito20) exhibited intrinsic anti-Trichomonas vaginalis activity, while PIBCA/pluronic® F68 without chitosan (PIBCA/F68) were inactive. However, the mechanism of anti-T. vaginalis activity of chitosan-coated PIBCA NPs is still unknown. Our hypothesis is that chitosan-coated NPs are internalized by the parasite, contrarily to PIBCA/F68. In this investigation, the impact of NP surface on their internalization by the protozoan was studied using flow cytometry and parasite morphological changes after different incubation times with PIBCA/Chito20 NPs were monitored by electron microscopy. Flow-cytometry revealed that PIBCA/Chito20 NPs were uptaken by T. vaginalis as early as 10-min-incubation. Drastic cell morphological transformations were observed from scanning electron microscopy and transmission electron microscopy after incubation with PIBCA/Chito20 NPs. Numerous pits were seen on cell membrane since 10 min. Gradual increase in contact time increased NP endocytosis and induced proportional damages to T. vaginalis membrane. Then, investigation of whether PIBCA/Chito20 NPs can improve MTZ anti-T. vaginalis activity was studied using checkerboard experiment. Calculation of fractional inhibitory concentration index (FICI = 3.53) showed an additive effect between NPs and MTZ.

Hierarchical supramolecular platelets from hydrophobically-modified polysaccharides and α-cyclodextrin: Effect of hydrophobization and α-cyclodextrin concentration on platelet formation.

Micro- and nano-platelets are a group of particles with typical flat surfaces and hexagonal shape. They are obtained by hierarchical self-assembly in water of α-cyclodextrin and polysaccharides hydrophobically-modified with alkyl chains. It is expected that the formation of well structured and cohesive platelets is driven by the interaction between alkyl chains grafted on polysaccharides and α-cyclodextrin. The objective of this investigation is to tune platelet formation by modifying these two parameters, independently on polysaccharide composition. A systematic study was conducted by varying polysaccharide type (dextran, pullulan, amylopectin), degree of substitution (DS:0.1-5.6%) and α-cyclodextrin concentration (0-10 wt%) for a fixed concentration of polysaccharide esterified with palmitoyl groups (1 wt%). Characterizations include ATR-FTIR, elemental analysis, solid state 13C NMR and transmission electron microscopy. Abundant and well-organized hexagonal platelets were obtained with high DS (4.2-5.6%) and a concentration of α-cyclodextrin higher than 2.5 wt%. Isothermal titration calorimetry revealed a sequential binding with a stoichiometry of 2 α-cyclodextrin molecules for 1 palmitoyl group grafted on dextran. This is the first report showing the possibility to control platelet formation by modifying DS and α-cyclodextrin concentration, independently on polysaccharide composition.

New insights on the structure of hexagonally faceted platelets from hydrophobically modified chitosan and α-cyclodextrin.

A new class of non-spherical particles was recently designed in our research group by mixing a polysaccharide grafted with fatty acids and α-cyclodextrin in water. Because their flat surfaces, and according to their size, particles are called micro- or nano-platelets. Here, we varied the composition of fatty acids grafted on chitosan (oleic acid, palmitic acid or stearic acid) and characterized platelet morphology. Transmission electron microscopy (TEM), cryogenic TEM, scanning electron microscopy, atomic force microscopy (AFM) and confocal laser scanning microscopy experiments showed that the platelets have a preferentially hexagonal shape with sharp edges, independently on alkyl chain grafted on chitosan. Furthermore, AFM topographic analysis of platelet surface showed parallel thin terraces with 12-14-nm height, suggesting a multi-layered structure alternating chitosan and fatty-acid/α-cyclodextrin inclusion complexes. We also revealed for the first time that a simple magnetic mixing of fatty acids with α-cyclodextrin in water results from solid inclusion complexes with a crystalline structural organization characterized by powder X-ray diffraction. Our results demonstrate that fatty acid/α-cyclodextrin interaction is the driving force for platelet formation.

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.

Autoassemblies of α-Cyclodextrin and Grafted Polysaccharides: Crystal Structure and Specific Properties of the Platelets.

Cyclodextrins (CDs) are a family of oligosaccharides with a toroid shape, which exhibit a remarkable ability to include guest molecules in their internal cavity, providing a hydrophobic environment for poorly soluble molecules. Recently, new types of inclusions of α CDs with alkyl grafted polysaccharide chains (pullulan, chitosan, dextran, amylopectin, chondroitin sulfate...) have been prepared which are autoassembled into micro- and nanoplatelets. We report in this paper an extensive investigation of platelets with different compositions, including their reversible hydration (thermogravimetric analysis), crystalline structure (powder X-ray diffraction), dimensions and shapes (scanning electron microscopy-field emission gun), thermal properties, solubility, and melting (micro-differential scanning calorimetry). The crystalline platelets exhibit layered structures intercalating the polysaccharide backbones and CD complexes hosting the grafted alkyl chains. The monoclinic symmetry of columnar-type crystals suggests a head-to-tail arrangement of the CDs. The platelets have a preferentially hexagonal shape with sharp edges, variable sizes, and thicknesses and sometimes show incomplete layers (terraces). The crystal parameters change upon dehydration. Melting temperatures of platelets in aqueous solutions exceed 100 °C. Finally, we discuss the potential relation between the platelet structure and applications for mucoadhesive devices.

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.

M48U1 and Tenofovir combination synergistically inhibits HIV infection in activated PBMCs and human cervicovaginal histocultures.

Microbicides are considered a promising strategy for preventing human immunodeficiency virus (HIV-1) transmission and disease. In this report, we first analyzed the antiviral activity of the miniCD4 M48U1 peptide formulated in hydroxyethylcellulose (HEC) hydrogel in activated peripheral blood mononuclear cells (PBMCs) infected with R5- and X4-tropic HIV-1 strains. The results demonstrate that M48U1 prevented infection by several HIV-1 strains including laboratory strains, and HIV-1 subtype B and C strains isolated from the activated PBMCs of patients. M48U1 also inhibited infection by two HIV-1 transmitted/founder infectious molecular clones (pREJO.c/2864 and pTHRO.c/2626). In addition, M48U1 was administered in association with tenofovir, and these two antiretroviral drugs synergistically inhibited HIV-1 infection. In the next series of experiments, we tested M48U1 alone or in combination with tenofovir in HEC hydrogel with an organ-like structure mimicking human cervicovaginal tissue. We demonstrated a strong antiviral effect in absence of significant tissue toxicity. Together, these results indicate that co-treatment with M48U1 plus tenofovir is an effective antiviral strategy that may be used as a new topical microbicide to prevent HIV-1 transmission.

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.