Wound Healing Activity of Nanoclay/Spring Water Hydrogels

Background: hydrogels prepared with natural inorganic excipients and spring waters are commonly used in medical hydrology. Design of these clay-based formulations continues to be a field scarcely addressed. Safety and wound healing properties of different fibrous nanoclay/spring water hydrogels were addressed. Methods: in vitro biocompatibility, by means of MTT assay, and wound healing properties were studied. Confocal Laser Scanning Microscopy was used to study the morphology of fibroblasts during the wound healing process. Results: all the ingredients demonstrated to be biocompatible towards fibroblasts. Particularly, the formulation of nanoclays as hydrogels improved biocompatibility with respect to powder samples at the same concentration. Spring waters and hydrogels were even able to promote in vitro fibroblasts motility and, therefore, accelerate wound healing with respect to the control. Conclusion: fibrous nanoclay/spring water hydrogels proved to be skin-biocompatible and to possess a high potential as wound healing formulations. Moreover, these results open new prospects for these ingredients to be used in new therapeutic or cosmetic formulations.This research was funded by Ministerio de Ciencia e Innovación, CGL2016–80833-R; Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía, P18-RT-3786 and Ministerio de Educación, Cultura y Deporte, FPU15/01577.Peer reviewe

Ciprofloxacin carrier systems based on hectorite/halloysite hybrid hydrogels for potential wound healing applications

The design of multifunctional nanomaterials which can help the healing processes of skin, preventing the bacterial infections, is crucial for the development of suitable therapy for the treatment of chronic lesions. The use of clay minerals in wound healing applications is well documented since the prehistoric period and offers several advantages due to their intrinsic properties. Herein, we report the development of ciprofloxacin carrier systems based on hectorite/halloysite (Ht/Hal) hybrid hydrogels for potential wound healing applications. To achieve this objective firstly the ciprofloxacin molecules were loaded onto Hal by a supramolecular and covalent approach. The so obtained fillers were thoroughly investigated by several techniques and at molecular level by means of quantum mechanics calculations along with empirical interatomic potentials. Afterwards the modified Hal were used as filler for Ht hydrogels. The introduction of modified Hal, in hectorite hydrogel, helps the gel formation with an improvement of the rheological properties. The in vitro kinetic release from both the fillers and from the hybrid hydrogels was studied both at skin's pH (5.4) and under neutral conditions (pH 7.4); in addition, the factors controlling the ciprofloxacin release process were determined and discussed. Finally, the in vitro biocompatibility of the Hal fillers was evaluated by means of cytotoxic assays and laser scanning confocal microscopy on normal human dermal fibroblasts.The work was carried out in the frame of the PON “AIM: Attrazione e Mobilità Internazionale” No. 1808223-1 project. Authors are thankful to H.A. Duarte for providing atomic coordinates of halloysite, to the CSIC Computational Center and the University of Granada Computation Center for computation facilities, and the Andalusian project grants RNM-1897 and P18-RT-3786 , and the Spanish MINECO projects , PCIN-2017-098 , FIS2016-77692-C2-2-P and CGL2016-80833-R , for the financial support of this research

Mycelium-based biomaterials as smart devices for skin wound healing

Introduction: Recently, mycelia of Ganoderma lucidum and Pleurotus ostreatus, edible fungi, have been characterized in vitro as self-growing biomaterials for tissue engineering since they are constituted of interconnected fibrous networks resembling the dermal collagen structure.Aim: This work aims to investigate the biopharmaceutical properties of G. lucidum and P. ostreatus mycelia to prove their safety and effectiveness in tissue engineering as dermal substitutes.Methods: The mycelial materials were characterized using a multidisciplinary approach, including physicochemical properties (morphology, thermal behavior, surface charge, and isoelectric point). Moreover, preclinical properties such as gene expression and in vitro wound healing assay have been evaluated using fibroblasts. Finally, these naturally-grown substrates were applied in vivo using a murine burn/excisional wound model.Conclusions: Both G. lucidum and P. ostreatus mycelia are biocompatible and able to safely and effectively enhance tissue repair in vivo in our preclinical model

NANOPARTICLES AND NANOFIBERS AS DRUG DELIVERY SYSTEMS OF POORLY SOLUBLE DRUGS

La via sistemica rappresenta la scelta più comune per la somministrazione di attivi e per lottenimento delleffetto terapeutico. Ad essa sono associate una serie di limitazioni tra cui la distribuzione aspecifica del farmaco responsabile della scarsa efficacia terapeutica e degli effetti collaterali che si tramutano in effetti tossici su siti differenti dal bersaglio terapeutico. Per questo motivo, vie di somministrazione a rilascio locale hanno di recente attratto la ricerca scientifica nel formulare sistemi in grado di veicolare lagente terapeutico su cellule o tessuti specifici; tale operazione è nota come drug targeting. In questo contesto, trovano largo interessa la nanomedicina e lingegneria tissutale, campi che in maniera sinergistica collaborano nellideazione di sistemi innovativi volti alla somministrazione del farmaco in situ. La strategia si basa sulla realizzazione di impianti intelligenti, capaci di risolvere limitazioni legate ai convenzionali sistemi di somministrazione. Numerose ricerche hanno messo in evidenza come sistemi nanoparticellari e sistemi nanofibrosi, ottenuti tramite limpiego di biopolimeri naturali e semisintetici, siano particolarmente efficaci nella veicolazione di principi attivi poco solubili o scarsamente biodisponibili. Quando opportunamente funzionalizzati e somministrati in prossimità del sito bersaglio, entrambi i sistemi favoriscono il rilascio sito-specifico del farmaco, riducendo lesposizione sistemica dellattivo, minimizzando la tossicità e migliorandone lefficacia. Lefficienza di tali sistemi è sia legata alla scelta dei biomateriali, ma soprattutto allelevata area superficiale data dalle dimensioni nanometriche. Biocompatibilità, biodegradabilità e capacità di essere facilmente suscettibili a modifiche chimiche sono aspetti ricercati nella formulazione di sistemi di rilascio di questo tipo. Il presente progetto di dottorato si inserisce in questo ambito ed è, specificatamente volto allottenimento di nanosistemi (nanoparticelle, nanofibre e/o sistemi ibridi) in grado di migliorare la biostabilità e la biodisponibilità di farmaci poco solubili e garantire un rilascio sito-specificoThe systemic route is the most common choice to administrate active molecules and to reach the therapeutic effect. Several limitations, including non-specific drug distribution, uncontrolled side effects and poor therapeutic efficacy that turn into toxic effects on sites different from the therapeutic target, mostly affect this route. For this reason, locally-released administration routes have recently attracted scientific research and novel systems aimed to carry therapeutic agents directly to specific cells or tissues are nowadays developed. In this context, nanomedicine and tissue engineering are broadly involved. These fields can work together in designing innovative and smart systems for in situ drug administration, solving all limitations linked to conventional drug delivery systems. Numerous studies have shown that nanoparticle and nanofibrous systems, obtained from natural and semi-synthetic biopolymers, are particularly effective in conveying poorly soluble or poorly bioavailable active ingredients. When properly functionalized or administered near the target site, both systems favor site-specific drug release, reducing systemic drug exposure, minimizing toxicity and improving its efficacy. The efficiency of these systems is linked either to the biomaterials employed, but especially to the high surface area given by the nanometric dimensions. Biocompatibility, biodegradability, and ability to be easily chemically modified are sought aspects during the formulation of this kind of nanosystems. This doctoral project is specifically aimed at developing novel polymeric nano-systems (nanoparticles, nanofibres and / or hybrid systems) able to improve biostability and bioavailability of poorly soluble drugs and guaranteeing a site-specific release

Chitosan Oleate Salt as an Amphiphilic Polymer for the Surface Modification of Poly-Lactic-Glycolic Acid (PLGA) Nanoparticles. Preliminary Studies of Mucoadhesion and Cell Interaction Properties

Most of the methods of poly-lactic-glycolic acid (PLGA) preparation involve the passage through the emulsification of a PLGA organic solution in water followed by solvent evaporation or extraction. The choice of the droplet stabilizer during the emulsion step is critical for the dimensions and the surface characteristics of the nanoparticles (NPs). In the present work, a recently described ionic amphiphilic chitosan derivative, chitosan oleate salt (CS-OA), was proposed for the first time to prepare PLGA NPs. A full factorial design was used to understand the effect of some formulation and preparation parameters on the NP dimensions and on encapsulation efficiency (EE%) of Nile red, used as a tracer. On the basis of the DoE study, curcumin loaded NPs were prepared, having 329 ± 42 nm dimensions and 68.75% EE%. The presence of a chitosan coating at the surface was confirmed by positive zeta potential and resulted in mucoadhesion behavior. The expected improvement of the interaction of the chitosan surface modified nanoparticles with cell membrane surface was confirmed in Caco-2 cell culture by the internalization of the loaded curcumin

Innovative Strategies in Tendon Tissue Engineering

The tendon is a highly aligned connective tissue that transmits force from muscle to bone. Each year, more than 32 million tendon injuries have been reported, in fact, tendinopathies represent at least 50% of all sports injuries, and their incidence rates have increased in recent decades due to the aging population. Current clinical grafts used in tendon treatment are subject to several restrictions and there is a significant demand for alternative engineered tissue. For this reason, innovative strategies need to be explored. Tendon replacement and regeneration are complex since scaffolds need to guarantee an adequate hierarchical structured morphology and mechanical properties to stand the load. Moreover, to guide cell proliferation and growth, scaffolds should provide a fibrous network that mimics the collagen arrangement of the extracellular matrix in the tendons. This review focuses on tendon repair and regeneration. Particular attention has been devoted to the innovative approaches in tissue engineering. Advanced manufacturing techniques, such as electrospinning, soft lithography, and three-dimensional (3D) printing, have been described. Furthermore, biological augmentation has been considered, as an emerging strategy with great therapeutic potential

Development of Berberine-Loaded Nanoparticles for Astrocytoma Cells Administration and Photodynamic Therapy Stimulation

Berberine (BBR) is known for its antitumor activity and photosensitizer properties in anti-cancer photodynamic therapy (PDT), and it has previously been favorably assayed against glioblastoma multiforme (GBM)-derived cells. In this work, two BBR hydrophobic salts, dodecyl sulfate (S) and laurate (L), have been encapsulated in PLGA-based nanoparticles (NPs), chitosan-coated by the addition of chitosan oleate in the preparation. NPs were also further functionalized with folic acid. All the BBR-loaded NPs were efficiently internalized into T98G GBM established cells, and internalization increased in the presence of folic acid. However, the highest mitochondrial co-localization percentages were obtained with BBR-S NPs without folic acid content. In the T98G cells, BBR-S NPs appeared to be the most efficient in inducing cytotoxicity events and were therefore selected to assess the effect of photodynamic stimulation (PDT). As a result, PDT potentiated the viability reduction for the BBR-S NPs at all the studied concentrations, and a roughly 50% reduction of viability was obtained. No significant cytotoxic effect on normal rat primary astrocytes was observed. In GBM cells, a significant increase in early and late apoptotic events was scored by BBR NPs, with a further increase following the PDT scheme. Furthermore, a significantly increased depolarization of mitochondria was highlighted following BBR-S NPs’ internalization and mostly after PDT stimulation, compared to untreated and PDT-only treated cells. In conclusion, these results highlighted the efficacy of the BBR-NPs-based strategy coupled with photoactivation approaches to induce favorable cytotoxic effects in GBM cells

Development of alginate-spermidine micro/nanogels as potential antioxidant and anti-inflammatory tool in peripheral nerve injuries. Formulation studies and physico-chemical characterization

The development of a successful strategy to ensure a full recovery in patients affected by peripheral nerve injury (PNI), one of the most debilitating pathologies, is, still today, a major clinical challenge. Herein, spermidine (SP), an endogenous polyamine, is employed with a dual role: as cross-linking agent for alginate (ALG) and as antioxidant and anti-inflammatory compound. In particular, micro/nanogels based on the ionic interaction between ALG and SP were obtained via ionotropic gelation. Different ALG concentrations and viscosity grades and different SP concentrations were considered. The influence of such variables on micro/nanogels size was investigated by means of a Design of Experiments (DoE) approach (full factorial design). The formation of micro/ nanogels was proved by Scanning Electron Microscope (SEM) analysis and by rheological and profilometry measurements. Fourier Transform Infrared (FTIR) measurements performed on nanogels of optimal composition confirmed SP-ALG interaction. The addition of trehalose as cryoprotectant agent to nanogel dispersion was considered in view of the employment of freeze-drying process to obtain a stable product. Moreover, in vitro studies on Schwann cells proved the ability of SP of expressing antioxidant and antiinflammatory properties, even if involved in the formation of nanogels

Chitosan Oleate Coated Poly Lactic-Glycolic Acid (PLGA) Nanoparticles versus Chitosan Oleate Self-Assembled Polymeric Micelles, Loaded with Resveratrol

Chitosan oleate (CS-OA), a chitosan salt with amphiphilic properties, has demonstrated the ability to self-assemble in aqueous environment to give polymeric micelles useful to load poorly soluble drugs. More recently, CS-OA was proposed to stabilize nanoemulsions during the preparation by emulsification and solvent evaporation of poly lactic-glycolic acid (PLGA) nanoparticles (NPs) loaded with curcumin. Positive mucoadhesive behavior and internalization properties were demonstrated for these NPs attributable to the presence of positive charge at the NP surface. In the present paper, two CS-OA-based nanosystems, micelles and PLGA NPs, were compared with the aim of elucidating their physico-chemical characteristics, and especially their interaction with cell substrates. The two systems were loaded with resveratrol (RSV), a hydrophobic polyphenol endowed with anti-cancerogenic, anti-inflammatory, and heart/brain protective effects, but with low bioavailability mainly due to poor aqueous solubility. Calorimetric analysis and X-ray spectra demonstrated amorphization of RSV, confirming its affinity for hydrophobic domains of polymeric micelles and PLGA core of NPs. TGA decomposition patterns suggest higher stability of PLGA-NPs compared with polymeric micelles, that anyway resulted more stable than expected, considering the RSV release profiles, and the cell line interaction results