Elita Montanari

Hyaluronic acid nanohydrogels as a useful tool for BSAO immobilization in the treatment of melanoma cancer cells

An alternative anticancer therapy based on the use of bovine serum amine oxidase (BSAO), an enzyme that converts polyamines over-expressed in malignant cells, into hydrogen peroxide and aldehyde(s), thus inducing high cytotoxicity in cancer cells, was recently proposed. With the aim of improving the system efficacy by exploiting a nanotechnology approach, BSAO is covalently immobilized onto injectable nanohydrogels (NHs) based on cholesterol-graft-hyaluronic acid (HA-CH), a biocompatible conjugate that spontaneously leads to self-assembled structures in aqueous solutions. In this study, the physicochemical properties of the HA-CH-based NHs and the NHs cytocompatibility are reported. The properties of the NHs-BSAO system are also studied in terms of protein residual activity, both in vitro and on a model melanoma cell line.

Chasing bacteria within the cells using levofloxacin-loaded hyaluronic acid nanohydrogels.

In the present work, an innovative approach based on the delivery of levofloxacin (LVF) from polysaccharide nanohydrogels for the treatment of bacterial intracellular infections is described. The nanohydrogels (NHs) were obtained by self-assembling of the hyaluronic acid-cholesterol amphiphilic chains in aqueous environment. LVF, a fluoroquinolone antibiotic scarcely efficient in intracellular infections, was entrapped within such NHs by nanoprecipitation, thus forming a drug delivery system (LVF-NHs) that was tested for its activity on different bacteria strains. The MIC values of levofloxacin-loaded nanohydrogels were determined for Staphylococcus aureus and Pseudomonas aeruginosa strains and compared to those obtained using free LVF. The intracellular antimicrobial activity of LVF-NHs and free LVF was compared on HeLa epithelial cell line infected by the above mentioned bacteria, and the increase in antibacterial efficacy of LVF-NHs with respect to that of free LVF was evidenced. The obtained results allow to conclude that this new approach can be considered as really promising method for intracellular infection treatments.

One-step formation and sterilization of gellan and hyaluronan nanohydrogels using autoclave

The sterilization of nanoparticles for biomedical applications is one of the challenges that must be faced in the development of nanoparticulate systems. Usually, autoclave sterilization cannot be applied because of stability concerns when polymeric nanoparticles are involved. This paper describes an innovative method which allows to obtain, using a single step autoclave procedure, the preparation and, at the same time, the sterilization of self-assembling nanohydrogels (NHs) obtained with cholesterol-derivatized gellan and hyaluronic acid. Moreover, by using this approach, NHs, while formed in the autoclave, can be easily loaded with drugs. The obtained NHs dispersion can be lyophilized in the presence of a cryoprotectant, leading to the original NHs after re-dispersion in water.

Highly versatile nanohydrogel platform based on riboflavin-polysaccharide derivatives useful in the development of intrinsically fluorescent and cytocompatible drug carriers

In this work we describe a new nanohydrogel platform, based on polysaccharides modified with the hydrophobic and fluorescent molecule riboflavin tetrabutyrate, which leads to innovative structures useful for drug delivery applications. Hyaluronic acid and pullulan were chosen as representative of anionic and neutral polysaccharides, respectively, and the bromohexyl derivative of riboflavin tetrabutyrate was chemically linked to these polymer chains. Because of such derivatization, polymer chains were able to self-assemble in aqueous environment thus forming nanohydrogels, with mean diameters of about 312 and 210 nm, for hyaluronan and pullulan, respectively. These new nanohydrogels showed low polydispersity index, and negative ζ-potential. Moreover, the nanohydrogels, which can be easily loaded with model drugs, showed long-term stability in water and physiological conditions and excellent cytocompatibility. All these properties allow to consider these intrinsically fluorescent nanohydrogels suitable for the formulation of innovative drug dosage forms.

Hyaluronan-cholesterol nanohydrogels: Characterisation and effectiveness in carrying alginate lyase

Although in recent years several methods have been studied and developed to obtain different types of nanosized drug delivery systems, the set up of suitable procedures and materials remains highly expensive, their preparation is time consuming and often not feasible for a scale-up process. Furthermore, the sterilisation and storage of nanocarrier formulations represents a complicated but mandatory step for their effective use. In our previous work we assessed the use of an autoclaving process to achieve, in one simple step, sterile self-assembled hyaluronan-cholesterol (HA-CH) and hyaluronan-riboflavin (HA-Rfv) nanohydrogels (NHs). In the present work, the effect of the high temperature on HA-CH has been studied in detail. HA-CH suspensions were characterised in terms of size and polydispersity by Dynamic Light Scattering at different temperatures and conditions; the HA-CH chemical structure and its molecular weight were assessed via FT-IR and GPC analysis after the sterilising cycle in an autoclave at 121°C for 20min. The obtained NHs were then observed with TEM and AFM microscopy, in both dry and liquid conditions. The Youngs modulus of the NHs was determined, evidencing the soft nature of these nanosystems; the critical aggregation concentration (c.a.c) of the nanosuspension was also assessed. Thereafter, alginate lyase (AL) was conjugated to NHs, with the aim of developing a useful system for therapies against bacterial infections producing alginate biofilms. The conjugation efficiency and the enzymatic activity of AL were determined after immobilisation. The AL-NHs system showed the ability to depolymerise alginate, offering an opportunity to be a useful nanosystem for the treatment of biofilm-associated infections.

Design of Hybrid Gels Based on Gellan-Cholesterol Derivative and P90G Liposomes for Drug Depot Applications

Gels are extensively studied in the drug delivery field because of their potential benefits in therapeutics. Depot gel systems fall in this area, and the interest in their development has been focused on long-lasting, biocompatible, and resorbable delivery devices. The present work describes a new class of hybrid gels that stem from the interaction between liposomes based on P90G phospholipid and the cholesterol derivative of the polysaccharide gellan. The mechanical properties of these gels and the delivery profiles of the anti-inflammatory model drug diclofenac embedded in such systems confirmed the suitability of these hybrid gels as a good candidate for drug depot applications.

Pursuing Intracellular Pathogens with Hyaluronan. From a ‘Pro-Infection’ Polymer to a Biomaterial for ‘Trojan Horse’ Systems

Hyaluronan (HA) is among the most important bioactive polymers in mammals, playing a key role in a number of biological functions. In the last decades, it has been increasingly studied as a biomaterial for drug delivery systems, thanks to its physico-chemical features and ability to target and enter certain cells. The most important receptor of HA is ‘Cluster of Differentiation 44’ (CD44), a cell surface glycoprotein over-expressed by a number of cancers and heavily involved in HA endocytosis. Moreover, CD44 is highly expressed by keratinocytes, activated macrophages and fibroblasts, all of which can act as ‘reservoirs’ for intracellular pathogens. Interestingly, both CD44 and HA appear to play a key role for the invasion and persistence of such microorganisms within the cells. As such, HA is increasingly recognised as a potential target for nano-carriers development, to pursuit and target intracellular pathogens, acting as a ‘Trojan Horse’. This review describes the biological relationship between HA, CD44 and the entry and survival of a number of pathogens within the cells and the subsequent development of HA-based nano-carriers for enhancing the intracellular activity of antimicrobials.

Hyaluronan‐Based Nanohydrogels for Targeting Intracellular S. Aureus in Human Keratinocytes

Staphylococcus aureus is one of the most significant human pathogens that is frequently isolated in a wide range of superficial and systemic infections. The ability of S. aureus to invade and survive within host cells such as keratinocytes and host immune cells has been increasingly recognized as a potential factor in persistent infections and treatment failures. The incorporation of antibiotics into hyaluronan-cholesterol nanohydrogels represents a novel paradigm in the delivery of therapeutic agents against intracellular bacteria. The work presented herein shows that NHs quickly enter human keratinocytes and accumulate into lysosomes. When used for targeting intracellular S. aureus the antimicrobial activity of loaded levofloxacin is enhanced, possibly changing the antibiotic intracellular fate from cytosol to lysosome. Indeed, gentamicin, an antibiotic that predominantly accumulates in lysosomes, shows significant and equal antibacterial activity when entrapped into NHs. These results strongly suggest that lysosomal formulations may display preferential activity toward intracellular S. aureus, opening new avenues for the use of HA-based NHs for treatment of such skin infections.

Behind Resveratrol Stabilization by Carboxymethylated (1,3/1,6)-β-d-Glucan: Does the Polyphenol Play a Role in Polymer Structural Organization?

Resveratrol stability in solution can be improved by combining the polyphenol with carboxymethylated (1,3/1,6)-β-d-glucan (CM-glucan), a carbohydrate polymer widely used in the food and pharmaceutical industries. The present work was undertaken to elucidate the mechanism behind this stabilizing effect. The supramolecular structural, physico-chemical and morphological features of the CM-glucan/resveratrol complex have been studied under different physical and chemical stimuli by means of spectroscopic techniques, microscopy and physical methods such as UV-Visible spectroscopy (UV-Vis), spectrofluorimetry, Circular Dichroism (CD), Infrared spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC), Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM). Our experimental data indicate that CM-glucan conformational organized architecture in aqueous solution is enhanced in the presence of resveratrol, suggesting that the polyphenol is able to confer a high degree of order to the polymer by a probable cooperative structural organization that results in a long term stabilization for the polyphenol.