Formulative Study and Characterization of Novel Biomaterials Based on Chitosan/Hydrolyzed Collagen Films.

To date, the need for biomaterials capable of improving the treatment of chronic skin wounds remains a clinical challenge. The aim of the present work is to formulate and characterize chitosan (Cs)/hydrolyzed collagen (HC) films as potential biomaterials with improved mechanical and hydration performances compared to single component formulations. Films were made by the solvent casting method, with or without glycerin and/or PEG1500 as plasticizers, resulting in a total of eight formulations. All films were characterized by their physico-chemical characteristics and their mechanical and hydration features. A full factorial design was also used to statistically assess the effect of HC concentration, type and concentration of plasticizers and their possible interactions on mechanical and swelling behaviors. Solid state characterization confirmed the hybrid nature of the films, with suggested electrostatic interactions between Cs and HC. Mechanical and swelling properties, along with the analysis of the experimental design, allowed the identification of formulations containing high HC concentration (2% w/v) and glycerin or glycerin/PEG1500 as more suitable candidates for skin wound treatment. Finally, viability assay of immortalized human keratinocytes (HaCaT) showed no statistical differences in cell survival compared to the complete culture medium, suggesting their potential as a promising tool for biomedical applications.

Natural and Synthetic Clay Minerals in the Pharmaceutical and Biomedical Fields.

Clay minerals are historically among the most used materials with a wide variety of applications. In pharmaceutical and biomedical fields, their healing properties have always been known and used in pelotherapy and therefore attractive for their potential. In recent decades, the research has therefore focused on the systematic investigation of these properties. This review aims to describe the most relevant and recent uses of clays in the pharmaceutical and biomedical field, especially for drug delivery and tissue engineering purposes. Clay minerals, which are biocompatible and non-toxic materials, can act as carriers for active ingredients while controlling their release and increasing their bioavailability. Moreover, the combination of clays and polymers is useful as it can improve the mechanical and thermal properties of polymers, as well as induce cell adhesion and proliferation. Different types of clays, both of natural (such as montmorillonite and halloysite) and synthetic origin (layered double hydroxides and zeolites), were considered in order to compare them and to assess their advantages and different uses.

Characterization and Molecular Modelling of Non-Antibiotic Nanohybrids for Wound Healing Purposes.

The healing process of chronic wounds continues to be a current clinical challenge, worsened by the risk of microbial infections and bacterial resistance to the most frequent antibiotics. In this work, non-antibiotic nanohybrids based on chlorhexidine dihydrochloride and clay minerals have been developed in order to design advanced therapeutic systems aimed to enhance wound healing in chronic lesions. To prepare the nanohybrids, two methodologies have been compared: the intercalation solution procedure and the spray-drying technique, the latter as a one-step process able to reduce preparation times. Nanohybrids were then fully studied by solid state characterization techniques. Computational calculations were also performed to assess the interactions between the drug and the clays at the molecular level. In vitro human fibroblast biocompatibility and antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa were assessed to check biocompatibility and potential microbicidal effects of the obtained nanomaterials. The results demonstrated the effective organic/inorganic character of the nanohybrids with homogeneous drug distribution into the clayey structures, which had been confirmed by classical mechanics calculations. Good biocompatibility and microbicidal effects were also observed, especially for the spray-dried nanohybrids. It was suggested that it could be due to a greater contact area with target cells and bacterial suspensions.

Norfloxacin-Loaded Electrospun Scaffolds: Montmorillonite Nanocomposite vs. Free Drug.

Infections in nonhealing wounds remain one of the major challenges. Recently, nanomedicine approach seems a valid option to overcome the antibiotic resistance mechanisms. The aim of this study was the development of three types of polysaccharide-based scaffolds (chitosan-based (CH), chitosan/chondroitin sulfate-based (CH/CS), chitosan/hyaluronic acid-based (CH/HA)), as dermal substitutes, to be loaded with norfloxacin, intended for the treatment of infected wounds. The scaffolds have been loaded with norfloxacin as a free drug (N scaffolds) or in montmorillonite nanocomposite (H-hybrid-scaffolds). Chitosan/glycosaminoglycan (chondroitin sulfate or hyaluronic acid) scaffolds were prepared by means of electrospinning with a simple, one-step process. The scaffolds were characterized by 500 nm diameter fibers with homogeneous structures when norfloxacin was loaded as a free drug. On the contrary, the presence of nanocomposite caused a certain degree of surface roughness, with fibers having 1000 nm diameters. The presence of norfloxacin-montmorillonite nanocomposite (1%) caused higher deformability (90%-120%) and lower elasticity (5-10 mN/cm2), decreasing the mechanical resistance of the systems. All the scaffolds were proven to be degraded via lysozyme (this should ensure scaffold resorption) and this sustained the drug release (from 50% to 100% in 3 days, depending on system composition), especially when the drug was loaded in the scaffolds as a nanocomposite. Moreover, the scaffolds were able to decrease the bioburden at least 100-fold, proving that drug loading in the scaffolds did not impair the antimicrobial activity of norfloxacin. Chondroitin sulfate and montmorillonite in the scaffolds are proven to possess a synergic performance, enhancing the fibroblast proliferation without impairing norfloxacin's antimicrobial properties. The scaffold based on chondroitin sulfate, containing 1% norfloxacin in the nanocomposite, demonstrated adequate stiffness to sustain fibroblast proliferation and the capability to sustain antimicrobial properties to prevent/treat nonhealing wound infection during the healing process.

Halloysite- and Montmorillonite-Loaded Scaffolds as Enhancers of Chronic Wound Healing.

The increase in life expectancy and the increasing prevalence of diabetic disease and venous insufficiency lead to the increase of chronic wounds. The prevalence of ulcers ranges from 1% in the adult population to 3-5% in the over 65 years population, with 3-5.5% of the total healthcare expenditure, as recently estimated. The aim of this work was the design and the development of electrospun scaffolds, entirely based on biopolymers, loaded with montmorillonite (MMT) or halloysite (HNT) and intended for skin reparation and regeneration, as a 3D substrate mimicking the dermal ECM. The scaffolds were manufactured by means of electrospinning and were characterized for their chemico-physical and preclinical properties. The scaffolds proved to possess the capability to enhance fibroblast cells attachment and proliferation with negligible proinflammatory activity. The capability to facilitate the cell adhesion is probably due to their unique 3D structure which are assisting cell homing and would facilitate wound healing in vivo.

Montmorillonite-norfloxacin nanocomposite intended for healing of infected wounds.

Background: Chronic cutaneous wounds represent a major issue in medical care and are often prone to infections. Purpose: The aim of this study was the design of a clay mineral-drug nanocomposite based on montmorillonite and norfloxacin (NF, antimicrobial drug) as a powder for cutaneous application, to enhance wound healing in infected skin lesions. Methods: The nanocomposite has been prepared by means of an intercalation solution procedure. Adsorption isotherm, solid-state characterization of the nanocomposite, drug loading capacity and its release have been performed. Moreover, cytocompatibility, in vitro fibroblast proliferation and antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus were assessed. Results: The clay drug adsorption isotherm demonstrates that the mechanism of NF intercalation into montmorillonite galleries is the adsorption as one single process, due to the charge-charge interaction between protonated NF and negatively charged montmorillonite edges in the interlayer space. Nanocomposite is biocompatible and it is characterized by antimicrobial activity greater than the free drug: this is due to its nanostructure and controlled drug release properties. Conclusion: Considering the results obtained, NF-montmorillonite nanocomposite seems a promising tool to treat infected skin lesions or skin wounds prone to infection, as chronic ulcers (diabetic foot, venous leg ulcers) and burns.

Halloysite nanotubes as tools to improve the actual challenge of fixed doses combinations in tuberculosis treatment.

Halloysite nanotubes (HLNTs) were used as nanocarriers of the tuberculostatic agent isoniazid (INH), a BCS (Biopharmaceutics Classification System) class III drug. Self-assembling nanohybrids (INH-loaded HLNTs) with an average outer diameter of 90 nm and polydispersity index of 0.7 approximately, were obtained by spontaneous adsorption of INH molecules to HLNTs powder in aqueous medium. The nanohybrids were aimed to improve oral drug bioavailability and reduce physicochemical incompatibility of INH with other concomitantly administered tuberculostatic agents. In vitro drug release from INH-loaded HLNTs was successfully fitted to a diffusive kinetic law founded on the adsorption-desorption equilibrium between drug molecules in solution and solid inorganic excipients. INH-loaded HLNTs showed good in vitro biocompatibility toward Caco-2 cells at the concentrations studied (up to 1233 µg/mL), with improved cell proliferation. Permeability tests showed that INH transport across Caco-2 cellular membranes was greatly enhanced and fluorescent microscopy confirmed that the drug encapsulated into nanohybrid was effectively internalized by the cells. INH-loaded HLNTs enhanced stability of the drug in presence of other tuberculostatic agents, both in binary and quaternary combinations. It has been demonstrated that simple interaction between INH with HLNTs leads to drug permeability and stability improvements that could greatly facilitate the design of multiple drug dosage forms, an actual challenge in oral treatment of tuberculosis. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2019.

Conformational polymorphic changes in the crystal structure of the chiral antiparasitic drug praziquantel and interactions with calcium carbonate.

Praziquantel is an antiparasitic drug used for decades. Currently, the praziquantel commercial preparation is a racemic mixture, in which only the levo-enantiomer possesses anthelmintic activity. The knowledge of its properties in the solid state and other chemical-physical properties is necessary for improving its efficacy and applications. Drug solid dispersions were prepared with calcium carbonate at 1:5 drug to excipient weight ratio by solvent evaporation method. Then, the modification of the crystal structure of the racemic polymorph of praziquantel in presence of calcium carbonate has been studied by means of several analytical techniques (DSC, TGA, XRD, SEM, FTIR, Raman spectroscopy and chiral liquid chromatography). This study has been completed with atomistic calculations based on empirical interatomic force fields and quantum mechanics methods applied to the crystal structure of praziquantel and of intermolecular interactions. The results evidenced that calcium carbonate provoked a conformational change in the praziquantel molecule yielding the formation of different polymorphs of praziquantel crystal. These alterations were not observed replacing calcium carbonate with colloidal silica as excipient in the solid dispersion.

Halloysite and chitosan oligosaccharide nanocomposite for wound healing.

Halloysite is a natural nanotubular clay mineral (HNTs, Halloysite Nano Tubes) chemically identical to kaolinite and, due to its good biocompatibility, is an attractive nanomaterial for a vast range of biological applications. Chitosan oligosaccharides are homo- or heterooligomers of N-acetylglucosamine and D-glucosamine, that accelerate wound healing by enhancing the functions of inflammatory and repairing cells. The aim of the work was the development of a nanocomposite based on HNTs and chitosan oligosaccharides, to be used as pour powder to enhance healing in the treatment of chronic wounds. A 1:0.05 wt ratio HTNs/chitosan oligosaccharide nanocomposite was obtained by simply stirring the HTNs powder in a 1% w/w aqueous chitosan oligosaccharide solution and was formed by spontaneous ionic interaction resulting in 98.6% w/w HTNs and 1.4% w/w chitosan oligosaccharide composition. Advanced electron microscopy techniques were considered to confirm the structure of the hybrid nanotubes. Both HTNs and HTNs/chitosan oligosaccharide nanocomposite showed good in vitro biocompatibility with normal human dermal fibroblasts up to 300µg/ml concentration and enhanced in vitro fibroblast motility, promoting both proliferation and migration. The HTNs/chitosan oligosaccharide nanocomposite and the two components separately were tested for healing capacity in a murine (rat) model. HTNs/chitosan oligosaccharide allowed better skin reepithelization and reorganization than HNTs or chitosan oligosaccharide separately. The results suggest to develop the nanocomposite as a medical device for wound healing. STATEMENT OF SIGNIFICANCE: The present work is focused on the development of halloysite and chitosan oligosaccharide nanocomposite for wound healing. It considers a therapeutic option for difficult to heal skin lesions and burns. The significance of the research considers two fundamental aspects: the first one is related to the development of a self-assembled nanocomposite, formed by spontaneous ionic interaction, while the second one is related to the possibility to find an effective treatment for cutaneous non healing lesions. The characterization of this hybrid system involves a multidisciplinary approach considering integrated techniques of solid state investigation and advanced electron microscopies, and in vitro/in vivo models to understand biocompatibility and proliferation properties (enhancement of in vitro fibroblast motility, proliferation and migration, and of in vivo burn healing), to understand safety and effectiveness of the developed nanocomposite.

Hybrid systems based on "drug - in cyclodextrin - in nanoclays" for improving oxaprozin dissolution properties.

A combined approach based on drug complexation with cyclodextrins, and complex entrapment in nanoclays has been investigated, to join in a single delivery system the benefits of these carriers and potentiate their ability to improve the dissolution properties of oxaprozin (OXA), a poorly water-soluble anti-inflammatory drug. Based on previous studies, randomly methylated ß-cyclodextrin (RAMEB) was chosen as the most effective cyclodextrin for OXA complexation. Adsorption equilibrium studies performed on three different clays (sepiolite, attapulgite, bentonite) allowed selection of sepiolite (SV) for its greater adsorption power towards OXA. DSC and XRPD studies indicated drug amorphization in both binary OXA-RAMEB coground and OXA-SV cofused products, due to its complexation or very fine dispersion in the clay structure, respectively. The drug amorphous state was maintained also in the ternary OXA-RAMEB-SV cofused system. Dissolution studies evidenced a clear synergistic effect of RAMEB complexation and clay nanoencapsulation in improving the OXA dissolution properties, with an almost 100% increase in percent dissolved and dissolution efficiency compared to the OXA-RAMEB coground system. Therefore, the proposed combined approach represents an interesting tool for improving the therapeutic effectiveness of poorly soluble drugs, and reducing the CD amount necessary for obtaining the desired drug solubility and dissolution rate increase.

Folk pharmaceutical formulations in western Mediterranean: identification and safety of clays used in pelotherapy.

ETHNOPHARMACOLOGICAL RELEVANCE: Clays are naturally occurring ingredients of many natural health products, being included in most of ancient Mediterranean/European medical texts and currently used to prepare therapeutic hot-muds (peloids) in several thermal stations of the Mediterranean region. Clays are included in the formulation of peloids as vehicles of the mineral-medicinal water, to obtain inorganic gels with rheological and thermal properties suitable to be topically applied. Knowledge about formulations and preparation procedures of these traditional medicines has been orally transmitted since ancient times. Increasing recognition of the therapeutic utility of these traditional and natural health care substances make necessary a full ethnopharmaceutic research to ascertain those compositional characters that allow to establish quality attributes and corresponding requirements for these materials and products, including identity, purity, richness and safety. MATERIALS AND METHODS: Five clay samples (A, B, C, D and E) currently used in various spa centers of southern European/Mediterranean countries were studied. X-Ray diffraction (XRD) and X-ray fluorescence (XRF) data were used to asses sample identity and richness. Elemental impurities and microbiological contaminants were also determined and compared to normative limits. Particle size distribution was related to their safety as powder materials. RESULTS: Samples A, C, D and E were identified as "high purity clay", while sample B was identified as a mix of clay minerals and carbonates. The presence of carbonates in this sample could compromise its suitability for pelotherapy. The studied clays meet the main normative limits for metals impurities, with the exception of arsenic in sample A and nickel in sample B. The samples comply with the microbiological limits proposed by European legislation for medicinal products. According to the particle size of the studied samples, prevention and control of dust exposure must be considered. CONCLUSIONS: Despite their demonstrated longevity, the use of clays in traditional medicine formulations as peloids greatly requires comprehension of their identity and safety attributes. Continuity of these mineral substances as recognized health care ingredients oblige to conduct interdisciplinary research to know the features that sustain their traditional use in the preparation of medicines (ethnopharmaceutics).

Montmorillonite-chitosan-silver sulfadiazine nanocomposites for topical treatment of chronic skin lesions: in vitro biocompatibility, antibacterial efficacy and gap closure cell motility properties.

Silver compounds and especially silver sulfadiazine (AgSD) are reported as effective antimicrobial agents against almost all known bacteria, fungi and some viruses. However, AgSD has been shown to be cytotoxic toward fibroblasts and keratinocytes in vitro and consequently to retard wound healing in vivo. The aim of the present work was to evaluate the in vitro biocompatibility (cytotoxicity and proliferation), antimicrobial efficacy and cell motility gap closure (assay of wound closure) of MT/CS nanocomposites loaded with silver sulfadiazine (AgSD). It is envisioned to be administered as a powder or a dressing for cutaneous application in the treatment of skin ulcers. The loading of AgSD in MT/CS nanocomposites aimed at preventing the delay in wound healing, by decreasing the cytotoxicity of AgSD and maintaining its antimicrobial properties. Nanocomposites were prepared by using different amounts of MT (100-2000 mg) and 40 ml of a 1% (w/w) chitosan glutamate aqueous solution. The relative amounts of AgSD and chitosan in the systems were assessed by suitable analytic methods. The nanocomposite prepared using 100mg of MT was characterized for in vitro biocompatibility and proliferation and for wound healing using normal human dermal fibroblasts (NHDF). Antimicrobial properties were evaluated against four reference bacterial strains: Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, and Pseudomonas aeruginosa. AgSD loaded in the 100 MT/CS nanocomposite showed good in vitro biocompatibility and gap closure properties (fibroblasts) and maintained AgSD antimicrobial properties, especially against P. aeruginosa, that often complicates skin lesions.

Solid state characterisation of silver sulfadiazine loaded on montmorillonite/chitosan nanocomposite for wound healing.

Biopolymer chitosan/montmorillonite nanocomposites loaded with silver sulfadiazine for wound healing purposes were prepared via intercalation solution technique. Structure and morphology of loaded nanocomposites were studied and compared with pure components and unloaded nanocomposites. X-ray diffraction, Fourier transformed infrared spectroscopy, high resolution transmission electron microscopy coupled with energy-dispersion X-ray analysis, thermal and elemental analysis were employed for the characterisation. The results confirmed that the drug was effectively loaded in the three-dimensional nanocomposite structures, in which chitosan chains were adsorbed in monolayers into the clay mineral interlayer spaces.

Intestinal permeability of oxytetracycline from chitosan-montmorillonite nanocomposites.

A nanocomposite based on chitosan and montmorillonite was developed as carrier to improve oral bioavailability of oxytetracycline. The nanocomposite was prepared by simple solid-liquid interaction and loaded with the drug. The loaded nanocomposite was characterized by X-ray powder diffraction, thermal analysis, FTIR spectroscopy and zeta potential. Caco-2 cell cultures were used to evaluate in vitro cytotoxicity and drug permeation. Confocal laser scanning microscopy was also performed to evaluate the eventual entrapment of drug into the Caco-2 cells. Results showed that the nanocomposite was internalized into the cells and effectively enhanced drug permeation, being also biocompatible towards Caco-2 cells.

Networking and rheology of concentrated clay suspensions "matured" in mineral medicinal water.

This work studied the influence of "maturation" conditions (time and agitation) on aggregation states, gel structure and rheological behaviour of a special kind of pharmaceutical semisolid products made of concentrated clay suspensions in mineral medicinal water. Maturation of the samples was carried out in distilled and sulphated mineral medicinal water, both in static conditions (without agitation) and with manual stirring once a week, during a maximum period of three months. At the measured pH interval (7.5-8.0), three-dimensional band-type networks resulting from face/face contacts were predominant in the laminar (disc-like) clay suspensions, whereas the fibrous (rod-like) particles formed micro-aggregates by van der Waals attractions. The high concentration of solids in the studied systems greatly determined their behaviour. Rod-like sepiolite particles tend to align the major axis in aggregates promoted by low shearing maturation, whereas aggregates of disc-like smectite particles did not have a preferential orientation and their complete swelling required long maturation time, being independent of stirring. Maturation of both kinds of suspensions resulted in improved rheological properties. Laminar clay suspensions became more structured with time, independently from static or dynamic maturation conditions, whereas for fibrous clay periodic agitation was also required. Rheological properties of the studied systems have been related to aggregation states and networking mechanisms, depending on the type of clay minerals constituents. Physical stability of the suspensions was not impaired by the specific composition of the Graena medicinal water.

Supramolecular structure of 5-aminosalycilic acid/halloysite composites.

This paper assesses the supramolecular structure of nanocomposites prepared by including the anti-inflammatory drug 5-aminosalycilic acid in halloysite nanotubes. Halloysite tubes have sub-micron individual lengths with outer diameters ∼0.1 µm, as observed by FESEM. The mercury intrusion plots showed bimodal profiles with pore dimensions ∼10 and 0.06 µm. X-ray diffraction and thermogravimetric results revealed changes in the hydration form of the clay after the interaction. The groups associated to the interaction were studied by FTIR. The location of the drug in the composites was determined after uranium staining of its amino groups by X-EDS microanalysis coupled with HREM. The drug was located both inside and on the surface of the halloysite nanotubes. These results confirm the occurrence of two concomitant interaction mechanisms: rapid adsorption of 5-ASA at the external halloysite surface followed by slow adsorption of the drug inside the tubes.

Penetration and distribution of thiocolchicoside through human skin: comparison between a commercial foam (Miotens) and a drug solution.

Penetration and distribution of thiocolchicoside from a commercially available foam (Miotens 0.25%, w/v) through human excised full-thickness skin were evaluated using two different in vitro apparatus: a Franz diffusion cell and a Saarbruecken penetration model-based cell. In order to evaluate the intrinsic capability of the drug to penetrate into the skin, a simple drug aqueous solution prepared at the same drug concentration as Miotens was also tested. Results showed that both apparatus were suitable to study thiocolchicoside penetration into human skin. Penetrated drug amounts were comparable using the two apparatus, probably because skin acts as "sink" for the drug. Miotens was found to significantly promote thiocolchicoside accumulation into full human skin thickness in comparison with the simple drug solution. The mixture of propylene glycol and propylene glycol diperlargonate contained into Miotens foam has been proven to be effective to promote penetration of thiocolchicoside into human skin.

Influence of complex solubility on formulations based on lambda carrageenan and basic drugs.

The purpose of the present work was to compare the behavior of some drug/carrageenan complexes having different solubility in water, in a controlled release formulation. Diltiazem HCl, bupropion HCl, metoprolol tartrate, and tramadol HCl were used as model drugs. The complexes were characterized by means of solubility measurements, release test at constant surface area, and water uptake measurements, and the results were related to their performance in controlled release formulations. For the more soluble complexes (involving metoprolol and tramadol) the occurrence of gelation after hydration was observed, while diltiazem complex apparently did not gellify; bupropion behavior was intermediate. A correspondence was found between the observed differences in complex solubility and hydration-gelation behavior and the drug release profiles. For all the drugs considered, the release was completed in about 10 to 12 hours, but different kinetics were observed depending on the solubility of the complexes. All the considered complexes seem suitable for controlled release purposes, although the data obtained show the relevance of the complex solubility to drug release profiles.