Michela Mori

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.

Platelet lysate formulations based on mucoadhesive polymers for the treatment of corneal lesions

Objectives  Growth factors contained in platelet α‐granules initiate and modulate tissue repair and are proposed for the treatment of soft and hard‐tissue surgical conditions and in the management of non‐healing wounds. Platelet lysate is a hemoderivative obtained from platelet‐rich plasma and is capable of releasing a pool of growth factors. Many medical and surgical techniques have been proposed for the treatment of corneal lesions; management of these conditions remains problematic and healing with standard protocols is unattainable. The aim of this study was to develop formulations suitable for prolonging the contact of platelet lysate with the damaged cornea for the time necessary to exert a therapeutic effect.

Ionic polymeric micelles based on chitosan and fatty acids and intended for wound healing. Comparison of linoleic and oleic acid

Chitosan is well known for its positive properties in wound healing. Also unsaturated fatty acids are described as able to accelerate tissue repairing mechanisms. In this work hydrophobically modified chitosan was obtained by ionic interaction with either oleic or linoleic acid. In aqueous environment self-assembling into nanoparticles occurred. The presence of hydrophobic domains, similar to those present in polymeric micelles, was demonstrated by changes in pyrene spectra. Both oleate and linoleate derivatives showed mucoadhesion behaviour. Cytotoxicity tests on human dermal fibroblasts demonstrated good biocompatibility of especially oleate derivatives. Clarithromycin, a poorly soluble model drug proposed for use in infected wounds was successfully encapsulated in both oleic and linoleic based polymeric micelles. The ionic structure of the carriers is responsible for their loosening at neutral pH and in the presence of salts. This behaviour should impair parenteral administration of the systems, but can be useful for topical delivery where the micelle components, chitosan and fatty acid, can play a positive role in dermal regeneration and tissue repairing.

Thermosensitive eyedrops containing platelet lysate for the treatment of corneal ulcers

Corneal lesions cause significant pain and visual impairment and, in many cases, are unresponsive to conventional treatments. Platelet lysate (PL) is an haemoderivative rich in growth factors (GFs) that are released by platelets after freeze-thawing destruction of platelet rich plasma (PRP). The aim of the present work was to develop thermosensitive and mucoadhesive eyedrops to maintain and prolong the contact of platelet lysate (PL) with cornea ulcers. A sterile vehicle based on chondroitin sulphate sodium (CS) and hydroxypropylmethyl cellulose (HPMC) was developed. An extemporaneous loading of the vehicle with PL was performed and the obtained formulation was able to quickly thermogelify at about 32 °C and was characterized by good mucoadhesive properties. ELISA evidenced that the growth factor PDGF AB was compatible with the vehicle and stable in the formulation up to 15 days of storage at 2-8 °C. In vitro wound healing and proliferation test (performed using rabbit corneal epithelial cells (RCE)) showed that the formulation enhanced cell growth and put in evidence a synergistic effect of CS and PL in stimulating cell proliferation. The overall results indicate that PL loaded in thermosensitive and mucoadhesive eyedrops can be profitably employed to treat corneal lesions.

Calcium alginate particles for the combined delivery of platelet lysate and vancomycin hydrochloride in chronic skin ulcers.

The aim of the present work was the development of a powder formulation for the combined delivery of platelet lysate and of a model antibiotic drug, vancomycin hydrochloride (VCM), in chronic skin ulcers. Calcium alginate particles were prepared by freeze-drying beads obtained by ionic gelation method. The experimental conditions adopted permitted the complete loading of VCM and of PDGF AB, the growth factor chosen as representative of those contained in PL. Such particles where able to absorb PBS (mimicking wound exudate), to form a gel and to modulate the release of VCM and of PDGF AB. They are characterized by enhancement properties of human fibroblast proliferation due to PL presence. In particular, PL, when loaded in alginate particles, was able not only to increase the number of viable cells, but also the number of cells in proliferative phase. Such properties were comparable to those of fresh PL indicating the capability of calcium alginate particles to load PL bioactive substances without altering their activity. The formulation developed is characterized by an easier and a less painful administration with respect to traditional gauzes and semisolid preparations and permits the loading in the same dosage form of active substances of different nature avoiding eventual incompatibility problems.

In vitro assessment of biopolymer-modified porous silicon microparticles for wound healing applications.

The wound healing stands as very complex and dynamic process, aiming the re-establishment of the damaged tissues integrity and functionality. Thus, there is an emerging need for developing biopolymer-based composites capable of actively promoting cellular proliferation and reconstituting the extracellular matrix. The aims of the present work were to prepare and characterize biopolymer-functionalized porous silicon (PSi) microparticles, resulting in the development of drug delivery microsystems for future applications in wound healing. Thermally hydrocarbonized PSi (THCPSi) microparticles were coated with both chitosan and a mixture of chondroitin sulfate/hyaluronic acid, and subsequently loaded with two antibacterial model drugs, vancomycin and resveratrol. The biopolymer coating, drug loading degree and drug release behavior of the modified PSi microparticles were evaluated in vitro. The results showed that both the biopolymer coating and drug loading of the THCPSi microparticles were successfully achieved. In addition, a sustained release was observed for both the drugs tested. The viability and proliferation profiles of a fibroblast cell line exposed to the modified THCPSi microparticles and the subsequent reactive oxygen species (ROS) production were also evaluated. The cytotoxicity and proliferation results demonstrated less toxicity for the biopolymer-coated THCPSi microparticles at different concentrations and time points comparatively to the uncoated counterparts. The ROS production by the fibroblasts exposed to both uncoated and biopolymer-coated PSi microparticles showed that the modified PSi microparticles did not induce significant ROS production at the concentrations tested. Overall, the biopolymer-based PSi microparticles developed in this study are promising platforms for wound healing applications.

Preparation and characterization of polysaccharide-based nanoparticles with anticoagulant activity

The aim of this study was to produce and characterize nanoparticles (NPs), combining chondroitin sulfate (CS) and fucoidan (FC) with chitosan for therapeutic purposes. These NPs were characterized by dynamic light scattering, zeta potential determination, and transmission electronic microscopy. The anticoagulant activity was determined for FC NPs and compared with FC solution at the same concentration. FC NPs showed regular shapes and better anticoagulant activity than free polysaccharide solution. FC solution did not affect coagulation compared to FC NPs, which increased up to two-fold, even at a lower concentration. Cytotoxicity and permeability tests were conducted using Caco-2 cell monolayer, exhibiting no toxic effect in this cell line and higher permeability for NP2 samples than FC solution at the same concentration.

The role of chitosan as a mucoadhesive agent in mucosal drug delivery

Mucoadhesive properties of chitosan are supported by an exceedingly high number of papers and have been studied using a number of different methods. Also, the myriad of chitosan derivatives that have been synthesized and proposed for pharmaceutical use have been studied for mucoadhesion, looking at the changes in mucoadhesivity induced by structure modifications. The review will briefly recall the possible mechanisms involved in chitosan mucoadhesivity, among which ionic interactions with mucin chains and hydration state are the prevailing ones. Furthermore, adhesion mechanism inevitably depends on the environmental conditions as well as on the type of physical and pharmaceutical form considered. Therefore, the testing methods used for mucoadhesivity should be carefully chosen. The present review will report on the experience acquired by the authors in the field. The focus will be on some examples of liquid, semisolid, solid and nanoparticulate formulations based on either chitosan or chitosan derivatives and on how it is possible to choose the most suitable testing method depending on the envisaged mucoadhesion mechanism and on the physical/pharmaceutical form under examination.

New therapeutic platforms for the treatment of epithelial and cutaneous lesions.

There are still so-called unmet needs in the treatment of epithelial and cutaneous lesions. Mucositis, ocular lesions, chronic skin wounds represent typical examples. These pathologies do not yet afford a satisfactory treatment. In particular chronic wounds represent a major health care burden, likely to increase as the population ages. Healing of epithelial and cutaneous lesions progresses through a complex cascade of events starting with the secretion in the local environment of a pool of growth factors, cytokines and proteins from the serum and degranulating platelets. Recently platelet lysate (PL), a hemoderivative obtained by platelet destruction by freeze-thawing of a platelet rich plasma (PRP) sample in the presence of an anticoagulant agent, has proved capable of promoting the healing of buccal and corneal lesions. Since the efficacy of growth factors (GFs) critically depends on the way they are made available to the injured tissue, the development of suitable therapeutic vehicles is of paramount importance to release GFs according to the repairing requirements. The present work focuses on the development and testing of few such formulations, in particular a mucoadhesive gel and an in situ gelling buccal spray for the treatment of oral mucositis, and a thermosensitive eye drop solution for corneal lesions. Besides technological characterization, the formulations have been preliminarily screened with an ELISA assay, a cell proliferation test and an in vitro wound healing test. Some formulations have been used in early clinical trials.

Sponge-Like Dressings Based on the Association of Chitosan and Sericin for the Treatment of Chronic Skin Ulcers. I. Design of Experiments–Assisted Development

The aim of the present work was the development of sponge-like dressings based on chitosan glutamate (high molecular weight) and sericin for the treatment of chronic skin ulcers. Dressings were prepared by freeze-drying and glycine was added as cryoprotectant agent. Dressing development was assisted by design of experiments, using a simplex centroid design. Mechanical resistance, hydration propensity, viscous, and viscoelasticity properties of dressings were considered as response variables. The superimposition of the contour plots, calculated by the best fit model for each response variable, permitted to individuate a region of the factor space where the dressing of optimized quantitative composition was chosen. Such a dressing was able to absorb high amount of phosphate-buffered saline forming a gel characterized by rheological properties enabling both a lubricant and a protective effect. The optimized formulation was characterized by optimal mechanical properties and by cell proliferation and antioxidant activity on human fibroblast cell line.