Covalent Conjugation of Amphotericin B to Hyaluronic Acid: An Injectable Water-Soluble Conjugate with Reduced Toxicity and Anti-Leishmanial Potential.

Amphotericin B (AmB) is a highly hydrophobic drug with significant leishmanicidal activity whose use is limited by its poor water solubility and adverse effects. Polymer-drug conjugates are proposed as a delivery system designed to overcome those limitations while improving drug bioavailability, safety, and activity. Here, AmB was covalently linked to periodate-oxidized hyaluronic acid (HA) (oxidation degree of 30.1 ± 5.6%) via a Schiff base (HA-AmB imine). The conjugate presents high water solubility and self-assembles into particles with a mean size of 88.2 ± 17.6 nm, a negative charge (-28.3 ± 0.9 mV), and a drug content of 17.8 ± 1.4%. Spectroscopic studies revealed the presence of AmB in aggregate and super-aggregated forms in the conjugate, which could explain the significant reduction of the in vitro cytotoxicity and hemolytic activity. The formulation showed not only in vitro anti-leishmanial activity against L. infantum-infected macrophages (IC50 = 0.023 µM) but also against an in vivo infected mouse model, promoting a 1.32- and a 4.98-log10 suppression of the L. infantum burden in the spleens and liver, respectively, without toxic effects. In summary, this study describes the safe and effective use of water-soluble HA-AmB imine conjugates for leishmaniasis treatment.

Ultrathin, Strong, and Highly Flexible Ti3C2Tx MXene/Bacterial Cellulose Composite Films for High-Performance Electromagnetic Interference Shielding.

The fabrication of ultrathin films that are electrically conductive and mechanically strong for electromagnetic interference (EMI) shielding applications is challenging. Herein, ultrathin, strong, and highly flexible Ti3C2Tx MXene/bacterial cellulose (BC) composite films are fabricated by a scalable in situ biosynthesis method. The Ti3C2Tx MXene nanosheets are uniformly dispersed in the three-dimensional BC network to form a mechanically entangled structure that endows the MXene/BC composite films with excellent mechanical properties (tensile strength of 297.5 MPa at 25.7 wt % Ti3C2Tx) and flexibility. Importantly, a 4 µm thick Ti3C2Tx/BC composite film with 76.9 wt % Ti3C2Tx content demonstrates a specific EMI shielding efficiency of 29141 dB cm2 g-1, which surpasses those of most previously reported MXene-based polymer composites with similar MXene contents and carbon-based polymer composites. Our findings show that the facile, environmentally friendly, and scalable fabrication method is a promising strategy for producing ultrathin, strong, and highly flexible EMI shielding materials such as the freestanding Ti3C2Tx/BC composite films for efficient EMI shielding to address EMI problems of a fast-developing modern society.

SLN and NLC for topical, dermal, and transdermal drug delivery.

Introduction: From a biopharmaceutical standpoint, the skin is recognized as an interesting route for drug delivery. In general, small molecules are able to penetrate the stratum corneum, the outermost layer of the skin. In contrast, the delivery of larger molecules, such as peptides and proteins, remains a challenge. Nanoparticles have been exploited not only to enhance skin penetration of drugs but also to expand the range of molecules to be clinically used.Areas covered: This review focus on Solid lipid nanoparticles (SLN) and Nanostructured lipid carriers (NLC) for skin administration. We discuss the selection criteria for lipids, surfactants, and surface modifiers commonly in use in SLN/NLC, their production techniques, and the range of drugs loaded in these lipid nanoparticles for the treatment of skin disorders.Expert opinion: Depending on the lipid and surfactant composition, different nanoparticle morphologies can be generated. Both SLN and NLC are composed of lipids that resemble those of the skin and sebum, which contribute to their enhanced biocompatibility, with limited toxicological risk. SLN and NLC can be loaded with very chemically different drugs, may provide a tunable release profile, can be produced in a sterilized environment, and be scaled-up without the need for organic solvents.

Tailoring Bacteria Response by Piezoelectric Stimulation.

Bacteria are simple organisms with a remarkable capacity for survival by adapting to different environments, which is a result of their long evolutionary history. Taking into consideration these adapting mechanisms, this work now investigates the effect of electrically active microenvironments on bacteria and on how this stimulation may trigger bacteria growth inhibition or proliferation. Electrical microenvironments are generated via stimulation of a piezoelectric polymer with a mechanical cue, thus developing an electrical response and a variation on the surface charge of the polymeric material. Specifically, Gram-positive Staphylococcus epidermidis and Gram-negative Escherichia coli were grown overnight under static and dynamic conditions on piezoelectric poly(vinylidene) fluoride (PVDF) films to further study bacteria behavior under: (i) the effect of the material surface charge in static conditions, (ii) the mechanical effect, and (iii) the piezoelectric effect, the last two performed under dynamic conditions. Bacteria viability in planktonic and biofilm forms was measured, and the microorganism morphology was characterized. Whereas E. coli responds little to any of the stimuli application, S. epidermidis growth can be regulated through the material surface charge and by the applied frequency. Positively charged PVDF induces bacterial growth inhibition in planktonic and adhered cells in static conditions, whereas antifouling properties are obtained when a mechanical or piezoelectric effect at 4 Hz stimuli is applied. By increasing the stimuli to 40 Hz, however, the adhesion of bacteria is promoted. In conclusion, the behavior of certain bacteria species is tailored through the application of piezoelectric materials, which provide sufficient mechanoelectrical stimuli for growth or inhibition of bacteria, allowing for the design of suitable anti- and promicrobial strategies. Such strategies are only found in studies related to mammalian cells, whereas in bacterial cells this type of stimuli are still unknown. Thus, this work provides one of the first insights on the effect of piezoelectric stimuli on bacterial cells.

Tuning Myoblast and Preosteoblast Cell Adhesion Site, Orientation, and Elongation through Electroactive Micropatterned Scaffolds.

Electroactive polymers are being increasingly used in tissue engineering applications. Together with the electromechanical clues, morphological ones have been demonstrated to determine cell proliferation and differentiation. This work reports on the micropatterning of poly(vinylidene fluoride-co-trifluoroethylene), P(VDF-TrFE) scaffolds, and their interaction with myoblast and preosteoblasts cell lines, selected based on their different functional morphology. The scaffolds were obtained by soft lithography and obtained in the form of arrays of lines, intermittent lines, hexagons, linear zigzags, and curved zigzags with dimensions of 25, 75, and 150 µm. Moreover, the scaffolds were tested in cell adhesion assays of myoblasts and preosteoblasts cell lines. The results show that more linear surface topographies and dense morphology have a large potential in the regeneration of musculoskeletal tissue, while nonpatterned scaffolds or more anisotropic surface microstructures present largest potential to promote the growth and regeneration of bone tissue. In this way, cell adhesion site, orientation, and elongation can be controlled by choosing properly the topography and morphology of the scaffolds, indicating their suitability and potential for further proliferation and differentiation assays.

In Vivo Imaging of Glycol Chitosan-Based Nanogel Biodistribution.

The preclinical development of nanomedicines raises several challenges and requires a comprehensive characterization. Among them is the evaluation of the biodistribution following systemic administration. In previous work, the biocompatibility and in vitro targeting ability of a glycol chitosan (GC) based nanogel have been validated. In the present study, its biodistribution in the mice is assessed, using near-infrared (NIR) fluorescence imaging as a tool to track the nanogel over time, after intravenous administration. Rapid whole body biodistribution of both Cy5.5 labeled GC nanogel and free polymer is found at early times. It remains widespreadly distributed in the body at least up to 6 h postinjection and its concentration then decreases drastically after 24 h. Nanogel blood circulation half-life lies around 2 h with the free linear GC polymer presenting lower blood clearance rate. After 24 h, the blood NIR fluorescence intensity associated with both samples decreases to insignificant values. NIR imaging of the organs shows that the nanogel had a body clearance time of ≈48 h, because at this time point a weak signal of NIR fluorescence is observed only in the kidneys. Hereupon it can be concluded that the engineered GC nanogel has a fairly long blood circulation time, suitable for biomedical applications, namely, drug delivery, simultaneously allowing efficient and quick body clearance.

Acetylated bacterial cellulose coated with urinary bladder matrix as a substrate for retinal pigment epithelium.

This work evaluated the effect of acetylated bacterial cellulose (ABC) substrates coated with urinary bladder matrix (UBM) on the behavior of retinal pigment epithelium (RPE), as assessed by cell adhesion, proliferation and development of cell polarity exhibiting transepithelial resistance and polygonal shaped-cells with microvilli. Acetylation of bacterial cellulose (BC) generated a moderate hydrophobic surface (around 65°) while the adsorption of UBM onto these acetylated substrates did not affect significantly the surface hydrophobicity. The ABS substrates coated with UBM enabled the development of a cell phenotype closer to that of native RPE cells. These cells were able to express proteins essential for their cytoskeletal organization and metabolic function (ZO-1 and RPE65), while showing a polygonal shaped morphology with microvilli and a monolayer configuration. The coated ABC substrates were also characterized, exhibiting low swelling effect (between 1.5-2.0 swelling/mm(3)), high mechanical strength (2048MPa) and non-pyrogenicity (2.12EU/L). Therefore, the ABC substrates coated with UBM exhibit interesting features as potential cell carriers in RPE transplantation that ought to be further explored.

Mechanical fatigue performance of PCL-chondroprogenitor constructs after cell culture under bioreactor mechanical stimulus.

In tissue engineering of cartilage, polymeric scaffolds are implanted in the damaged tissue and subjected to repeated compression loading cycles. The possibility of failure due to mechanical fatigue has not been properly addressed in these scaffolds. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. This is related to inherent discontinuities in the material due to the micropore structure of the macro-pore walls that act as stress concentration points. In this work, chondrogenic precursor cells have been seeded in poly-ε-caprolactone (PCL) scaffolds with fibrin and some were submitted to free swelling culture and others to cyclic loading in a bioreactor. After cell culture, all the samples were analyzed for fatigue behavior under repeated loading-unloading cycles. Moreover, some components of the extracellular matrix (ECM) were identified. No differences were observed between samples undergoing free swelling or bioreactor loading conditions, neither respect to matrix components nor to mechanical performance to fatigue. The ECM did not achieve the desired preponderance of collagen type II over collagen type I which is considered the main characteristic of hyaline cartilage ECM. However, prediction in PCL with ECM constructs was possible up to 600 cycles, an enhanced performance when compared to previous works. PCL after cell culture presents an improved fatigue resistance, despite the fact that the measured elastic modulus at the first cycle was similar to PCL with poly(vinyl alcohol) samples. This finding suggests that fatigue analysis in tissue engineering constructs can provide additional information missed with traditional mechanical measurements.

siRNA Inhibition of Endocytic Pathways to Characterize the Cellular Uptake Mechanisms of Folate-Functionalized Glycol Chitosan Nanogels.

Glycol chitosan nanogels have been widely used in gene, drug, and contrast agent delivery in an effort to improve disease diagnosis and treatment. Herein, we evaluate the internalization mechanisms and intracellular fate of previously described glycol chitosan nanogels decorated with folate to target the folate receptor. Uptake of the folate-decorated nanogel was impaired by free folate, suggesting competitive inhibition and shared internalization mechanisms via the folate receptor. Nanogel uptake was shown to occur mainly through flotillin-1 and Cdc42-dependent endocytosis. This was determined by inhibition of uptake reduction observed upon siRNA depletion of these two proteins and the pathways that they regulate. The data also suggest the involvement of the actin cytoskeleton in nanogel uptake via macropinocytosis. After 7 h of incubation with HeLa cells, approximately half of the nanogel population was localized in endolysosomal compartments, whereas the remaining 50% of the material was in undefined regions of the cytoplasm. Glycol chitosan nanogels may thus have potential as drug delivery vectors for targeting different intracellular compartments.

Recombinant CBM-fusion technology - Applications overview.

Carbohydrate-binding modules (CBMs) are small components of several enzymes, which present an independent fold and function, and specific carbohydrate-binding activity. Their major function is to bind the enzyme to the substrate enhancing its catalytic activity, especially in the case of insoluble substrates. The immense diversity of CBMs, together with their unique properties, has long raised their attention for many biotechnological applications. Recombinant DNA technology has been used for cloning and characterizing new CBMs. In addition, it has been employed to improve the purity and availability of many CBMs, but mainly, to construct bi-functional CBM-fused proteins for specific applications. This review presents a comprehensive summary of the uses of CBMs recombinantly produced from heterologous organisms, or by the original host, along with the latest advances. Emphasis is given particularly to the applications of recombinant CBM-fusions in: (a) modification of fibers, (b) production, purification and immobilization of recombinant proteins, (c) functionalization of biomaterials and (d) development of microarrays and probes.

Biocompatibility of a self-assembled glycol chitosan nanogel.

The research of chitosan-based nanogel for biomedical applications has grown exponentially in the last years; however, its biocompatibility is still insufficiently reported. Hence, the present work provides a thorough study of the biocompatibility of a glycol chitosan (GC) nanogel. The obtained results showed that GC nanogel induced slight decrease on metabolic activity of RAW, 3T3 and HMEC cell cultures, although no effect on cell membrane integrity was verified. The nanogel does not promote cell death by apoptosis and/or necrosis, exception made for the HMEC cell line challenged with the higher GC nanogel concentration. Cell cycle arrest on G1 phase was observed only in the case of RAW cells. Remarkably, the nanogel is poorly internalized by bone marrow derived macrophages and does not trigger the activation of the complement system. GC nanogel blood compatibility was confirmed through haemolysis and whole blood clotting time assays. Overall, the results demonstrated the safety of the use of the GC nanogel as drug delivery system.

Structural analysis of dextrins and characterization of dextrin-based biomedical hydrogels.

The characterization of several commercial dextrins and the analysis of the potential of dextrin derived hydrogels for biomedical applications were performed in this work. The structural characterization of dextrins allowed the determination of the polymerization and branching degrees, which ranged from 6 to 17 glucose residues and 2 to 13%, respectively. Tackidex, a medical grade dextrin was choosen for further characterization. The combination of hydrogel with a dextrin nanogel and urinary bladder matrix was achieved without compromising the mechanical properties or microstructure. The encapsulation of cells, preserving its viability, confirms the biocompatibility of the injectable hydrogels, which have therefore great potential for biomedical applications.

Glycol chitosan-based nanogel as a potential targetable carrier for siRNA.

A self-assembled glycol chitosan nanogel (GC) is synthesized by chemically grafting hydrophobic chains onto a polysaccharide, which is comprehensively characterized. The obtained macromolecular micelle is decorated with folate-conjugated poly(ethylene glycol) (PEG) (GCFA). An average size distribution of 250 and 200 nm is observed, respectively for the GC and GCFA nanogels. Differential cell localization is observed on incubating the materials with HeLa cells. Whereas the GC nanogel is detected on the cell surface, GCFA is localized in the cytoplasm. The cell viability is not compromised by the nanogels. Interestingly, GC nanogel is poorly internalized by bone marrow derived macrophages (BMDMs), and GCFA is not phagocytosed. Given its ability to complex siRNA, the targetable GC nanogel can be a promising vehicle for siRNA delivery.

Biocompatibility of poly(lactic acid) with incorporated graphene-based materials.

The incorporation of graphene-based materials has been shown to improve mechanical properties of poly(lactic acid) (PLA). In this work, PLA films and composite PLA films incorporating two graphene-based materials - graphene oxide (GO) and graphene nanoplatelets (GNP) - were prepared and characterized regarding not only biocompatibility, but also surface topography, chemistry and wettability. The presence of both fillers changed the films surface topography, increasing the roughness, and modified the wettability - the polar component of surface free energy increased 59% with GO and decreased 56% with GNP. Mouse embryo fibroblasts incubated with both fillers exceeded the IC(50) in both cases with a concentration of 10 µg mL(-1). No variations in cell proliferation at the surface of the composite films were observed, except for those containing GO after 24 h incubation, which presented higher cell proliferation than pristine PLA films. Platelet adhesion to PLA and PLA/GNP films was lower in the presence of plasma proteins than when no proteins were present. Furthermore, incorporation of GNP into PLA reduced platelet activation in the presence of plasma proteins. The results indicated that low concentrations of GO and GNP may be incorporated safely in PLA to improve aspects relevant for biomedical applications, such as mechanical properties.

Polymeric nanogels as vaccine delivery systems.

Polymeric nanogels find a relevant field of application in the formulation of a new generation of therapeutic and preventive vaccines, aiming at the fine-tuned modulation of the immune response. Intrinsic properties of polymeric nanogels, such as material chemistry, size and shape, surface charge, and hydrophobicity or hydrophilicity, may be determining factors in shaping the induced immune response. These materials can thus work as synthetic adjuvants, which can also be conjugated with immunostimulants. Polymeric nanogels protect vaccine antigens from degradation in vivo and, surface-conjugated with antibodies or specific ligands, could increase active targeting specificity. This review covers the recent published data concerning the modulation of innate and adaptive immune responses by engineered polymeric nanogels and their potential application as delivery systems in vaccination. FROM THE CLINICAL EDITOR: In this review, the utility of polymeric nanogels is discussed as adjuvants and protective agents for enhanced vaccination with more robust immune response and a more uniform outcome.

Unraveling the uptake mechanisms of mannan nanogel in bone-marrow-derived macrophages.

The mechanisms associated with the cellular internalization of nanomedicines must be carefully considered when designing drug- and vaccine-delivery systems. The cellular fate and effects of nanomedicines depend to a large extent on the cell uptake routes. A self-assembled mannan nanogel is developed as a vaccination platform for antigen and adjuvant delivery. The mannan nanogel uptake by murine bone-marrow-derived macrophages is found to be time-, concentration-, and energy-dependent, involving mannose-receptor-mediated phagocytosis and clathrin-mediated endocytosis. The nanogel is also visualized in the cytosol suggesting endolysosomal escape. These results indicate that mannan nanogel is a promising versatile carrier for intracellular delivery of vaccines or therapeutic agents.

Self-assembled mannan nanogel: cytocompatibility and cell localization.

Amphiphilic mannan, produced by the Michael addition of hydrophobic 1-hexadecanethiol to vinyl methacrylated mannan, self-assembles in aqueous medium through hydrophobic interactions among alkyl chains. Resultant nanogel is stable, spherical, polydisperse, with 50-140 nm mean hydrodynamic diameter depending on the polymer degree of substitution, and nearly neutral negative surface charge. No cytotoxicity of mannan nanogel is detected up to about 0.4 mg/mL in mouse embryo fibroblast cell line 3T3 and mouse bone marrow-derived macrophages (BMDM) using cell proliferation, lactate dehydrogenase and Live/Dead assays. Comet assay, under the tested conditions, reveals no DNA damage in fibroblasts but possible in BMDM. BMDM internalize the mannan nanogel, which is observed in vesicles in the cytoplasm by confocal laser scanning microscopy. Confocal colocalization image analysis denotes that the entrance and exit of nanogel and FM 4-64 might occur by the same processes--endocytosis and exocytosis--in BMDM. Physicochemical characteristics, in vitro cytocompatibility and uptake of self-assembled mannan nanogel by mouse BMDM are great signals of the potential applicability of this nanosystem for macrophages targeted delivery of vaccines or drugs, acting as potential nanomedicines, always with the key goal of preventing and/or treating diseases.

Biocompatibility of mannan nanogel--safe interaction with plasma proteins.

BACKGROUND: Self-assembled mannan nanogels are designed to provide a therapeutic or vaccine delivery platform based on the bioactive properties of mannan to target mannose receptor expressed on the surface of antigen-presenting cells, combined with the performance of nanogels as carriers of biologically active agents. METHODS: Proteins in the corona around mannan nanogel formed in human plasma were identified by mass spectrometry after size exclusion chromatography or centrifugation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Structural changes and time dependent binding of human apolipoprotein A-I (apoA-I) and human serum albumin (HSA) to mannan nanogel were studied using intrinsic tryptophan fluorescence and circular dichroism spectroscopy. The mannan nanogel effect on blood coagulation and fibrillation of Alzheimer's disease-associated amyloid ß peptide and hemodialysis-associated amyloidosis ß2 microglobulin was evaluated using thrombin generation assay or thioflavin T fluorescence assay, respectively. RESULTS: The protein corona around mannan nanogel is formed through a slow process, is quite specific comprising apolipoproteins B-100, A-I and E and HSA, evolves over time, and the equilibrium is reached after hours to days. Structural changes and time dependent binding of apoA-I and HSA to mannan nanogel are minor. The mannan nanogel does not affect blood coagulation and retards the fibril formation. CONCLUSIONS: Mannan nanogel has a high biosafety and biocompatibility, which is mandatory for nanomaterials to be used in biomedical applications. GENERAL SIGNIFICANCE: Our research provides a molecular approach to evaluate the safety aspects of nanomaterials, which is of general concern in society and science.

Development of a hybrid dextrin hydrogel encapsulating dextrin nanogel as protein delivery system.

Dextrin, a glucose polymer with low molecular weight, was used to develop a fully resorbable hydrogel, without using chemical initiators. Dextrin was first oxidized (oDex) with sodium periodate and then cross-linked with adipic acid dihidrazide, a nontoxic cross-linking molecule. Furthermore, a new bidimensional composite hydrogel, made of oxidized dextrin incorporating dextrin nanogels (oDex-nanogel), was also developed. The oDex hydrogels showed good mechanical properties and biocompatibility, allowing the proliferation of mouse embryo fibroblasts 3T3 cultured on top of the gel. The gelation time may be controlled selecting the concentrations of the polymer and reticulating agent. Both the oDex and oDex-nanogel hydrogels are biodegradable and present a 3-D network with a continuous porous structure. The obtained hybrid hydrogel enables the release of the dextrin nanogel over an extended period of time, paralleling the mass loss curve due to the degradation of the material. The dextrin nanogel allowed the efficient incorporation of interleukin-10 and insulin in the oDex hydrogel, providing a sophisticated system of controlled release. The new hydrogels present promising properties as an injectable carrier of bioactive molecules. Both proteins and poorly water-soluble low-molecular-weight drugs are efficiently encapsulated in the nanogel, which performs as a controlled release system entrapped in the hydrogel matrix.

Supramolecular assembled nanogel made of mannan.

The supramolecular assembly of amphiphilic mannan, synthesized by the Michael addition of hydrophobic 1-hexadecanethiol to vinyl methacrylated mannan, originates in aqueous medium the formation of a nanogel, stabilized by hydrophobic interactions among alkyl chains. The critical aggregation concentration, calculated by fluorescence spectroscopy ranged between 0.002 and 0.01 mg/mL, depending on the polymer degree of substitution. The cryo-field emission scanning electron microscopy showed spherical macromolecular micelles with diameters between 100 and 500 nm. The dynamic light scattering analysis revealed a polydisperse colloidal system, with mean hydrodynamic diameter between 50 and 140 nm, depending on the polymer degree of substitution. The nanogel is negatively charged, stable over a 6 months storage period, and stable at pH 3-8, salt or urea solutions. Bovine serum albumin and curcumin were spontaneously incorporated in the nanogel, being stabilized by the hydrophobic domains, opening the possibility for future applications as potential delivery systems for therapeutic molecules. In vitro assays were carried out to characterize the biocompatibility of the nanogel. A toxic effect of mannan-C(16) was observed, specific to mouse macrophage-like cell line J774, not affecting mouse embryo fibroblast cell line 3T3 viability.