Study on hydrophilicity and degradability of chitosan/polylactide-co-polycaprolactone nanofibre blend electrospun membrane.

Electrospinning is an interesting technique to produce polymer membranes made of entangled nanofibres. The technique is raising interest in pharmaceutical and biomedical areas. Either electrospun membranes are studied for tissue regeneration purposes, or incorporation of nanoparticles in electrospun membranes can be an opportunity to control the delivery of drug or to obtain dual drug delivery system. In this work suspensions of hydrochloride chitosan salt in copolymer polylactide-co-polycaprolactone (PLA-PCL) solution were electrospun in order to assess an advanced study for developing polymer nanofibre blend membrane loaded with chitosan polymer. The aim of the work was to investigate the properties and stability of chitosan/PLA-PCL electrospun membranes considering their application for tissue regeneration and drug delivery. The electrospun membranes were characterized for their physico-chemical (FT-IR) morphology (SEM) and in vitro biological properties (cytocompatibility and cells engraftment). Results show that homogeneous electrospun PLA-PCL/chitosan blend nanofibres in the range size 800 nm were obtained. Chitosan was loaded inside the nanofibres up to 27.2% (w/w) without modifying nanofibre shape, and only 6% of the loaded chitosan resulted to be on the nanofibre surface. The presence of chitosan in the nanofibres has shown to accelerate the electrospun membranes degradation in vitro.

Emerging and re-emerging infectious disease in otorhinolaryngology.

SUMMARY: Emerging and re-emerging infectious disease in otorhinolaryngology (ENT) are an area of growing epidemiological and clinical interest. The aim of this section is to comprehensively report on the epidemiology of key infectious disease in otorhinolaryngology, reporting on their burden at the national and international level, expanding of the need of promoting and implementing preventive interventions, and the rationale of applying evidence-based, effective and cost- effective diagnostic, curative and preventive approaches. In particular, we focus on i) ENT viral infections (HIV, Epstein-Barr virus, Human Papilloma virus), retrieving the available evidence on their oncogenic potential; ii) typical and atypical mycobacteria infections; iii) non-specific granulomatous lymphadenopathy; iv) emerging paediatric ENT infectious diseases and the prevention of their complications; v) the growing burden of antimicrobial resistance in ENT and the strategies for its control in different clinical settings. We conclude by outlining knowledge gaps and action needed in ENT infectious diseases research and clinical practice and we make references to economic analysis in the field of ENT infectious diseases prevention and care.

Multivariate analysis for the optimization of microfluidics-assisted nanoprecipitation method intended for the loading of small hydrophilic drugs into PLGA nanoparticles.

Design of Experiment-assisted evaluation of critical process (total flow rate, TFR, flow rate ratio, FRR) and formulation (polymer concentration and structure, drug:polymer ratio) variables in a novel microfluidics-based device, a staggered herringbone micromixer (SHM), for poly(lactic-co-glycolic acid) copolymer (PLGA) nanoparticles (NPs) manufacturing was performed in order to systematically evaluate and mathematically describe their effects on NPs sizes and drug encapsulation; a small hydrophilic moiety, N-acetylcysteine, was chosen as challenging model drug. SHM-assisted nanoprecipitation method consistently yielded NPs with tailor made sizes (in the range of 100-900 nm) and polydispersity index range from 0.061 to 0.286. Significant effects on NPs sizes were highlighted for TFR and FRR: increasing TFR (from 5 to 15 mL/min) and decreasing FRR (from 1:1 to 1:5 v/v, acetonitrile: buffer) NPs with mean diameter <200 nm were obtained. SHM technique allowed for flexible, application-specific tuning of PLGA NPs size using organic solvents with relatively low toxicity (acetone, acetonitrile), varying aqueous phase composition (Tris buffer vs PVA aqueous solution) and PLGA characteristics (Mw ranging from 25-90 kDa, capped or un-capped PLGA, different lactide:glycolide molar ratio). A very satisfactory N-Ac encapsulation efficiency (more than 67%) and a prolonged release (by 168 h) were achieved.

Design of 3D scaffolds for tissue engineering testing a tough polylactide-based graft copolymer.

The aim of this research was to investigate a tough polymer to develop 3D scaffolds and 2D films for tissue engineering applications, in particular to repair urethral strictures or defects. The polymer tested was a graft copolymer of polylactic acid (PLA) synthesized with the rationale to improve the toughness of the related PLA homopolymer. The LMP-3055 graft copolymer (in bulk) demonstrated to have negligible cytotoxicity (bioavailability >85%, MTT test). Moreover, the LMP-3055 sterilized through gamma rays resulted to be cytocompatible and non-toxic, and it has a positive effect on cell biofunctionality, promoting the cell growth. 3D scaffolds and 2D film were prepared using different LMP-3055 polymer concentrations (7.5, 10, 12.5 and 15%, w/v), and the effect of polymer concentration on pore size, porosity and interconnectivity of the 3D scaffolds and 2D film was investigated. 3D scaffolds got better results for fulfilling structural and biofunctional requirements: porosity, pore size and interconnectivity, cell attachment and proliferation. 3D scaffolds obtained with 10 and 12.5% polymer solutions (3D-2 and 3D-3, respectively) were identified as the most suitable construct for the cell attachment and proliferation presenting pore size ranged between 100 and 400µm, high porosity (77-78%) and well interconnected pores. In vitro cell studies demonstrated that all the selected scaffolds were able to support the cell proliferation, the cell attachment and growth resulting to their dependency on the polymer concentration and structural features. The degradation test revealed that the degradation of polymer matrix (ΔMw) and water uptake of 3D scaffolds exceed those of 2D film and raw polymer (used as control reference), while the mass loss of samples (3D scaffold and 2D film) resulted to be controlled, they showed good stability and capacity to maintain the physical integrity during the incubation time.

Sub-unit vaccine against S. aureus-mediated infections: set-up of nano-sized polymeric adjuvant.

The aim of this work was the design of a novel adjuvanted system for vaccination against S. aureus-mediated infections: in particular, poly-lactide-co-glycolide (PLGA) nanoparticles were developed in order to efficiently load and boost a sub-unit model vaccine, namely a purified recombinant collagen binding bacterial adhesin fragment (CNA19). At first, the assessment of the actual immunogenicity of free CNA19 via subcutaneous administration was evaluated, in order to consider it as subunit antigen model. Secondly, for the development of CNA19 loaded PLGA nanoparticles, a preliminary study was focused on the production of well-formed nanoparticles by w/o/w double emulsion method exploiting ultrasonication cycles under mild conditions, then the optimization of the freeze-drying conditions and different CNA19 loading methods were considered (encapsulation, adsorption of on blank or CNA19 encapsulated nanoparticles). The set-up preparation method (process yield of about 83%) permitted to obtain CNA19 loaded nanoparticles with spherical shape, narrow size distribution (187.41 ± 51.2 nm), a slightly negative zeta-potential (-2.91 ± 0.64 mV) and to elicit satisfactory protein encapsulation efficiency (75.91 ± 4.22%) and loading capacity (8.59 ± 0.33 µg CNA19/nanoparticles mg). Then, CNA19 loaded PLGA nanoparticles were characterized by (i) an in vitro release test performed at different temperatures, namely 4°C, 25°C and 37°C, testing the antigen integrity (SDS-PAGE) and activity (ELISA); (ii) an in vitro stability study in terms of dimension and surface charge performed in a 21 days period of time. At 37°C there was evidence of a sustained release of the antigen, in active form, for almost 240 h with a burst release of about 20% in the first 2h. At 4°C stability tests and activity assays allowed to identify storage conditions useful to maintain CNA19 activity and easily NP re-suspendability with intact physical characteristics. Furthermore the evaluation of CNA19 loaded nanoparticles cytotoxicity (up to 10.652 mg PLGA/ml) by MTT assay and the study of cellular up-take assessed on human fibroblasts confirmed the feasibility to formulate a dosage form useful for vaccination against S. aureus-mediated infections.

Diaminobenzidine photoconversion is a suitable tool for tracking the intracellular location of fluorescently labelled nanoparticles at transmission electron microscopy.

Chitosan-based nanoparticles (NPs) deserve particular attention as suitable drug carriers in the field of pharmaceutics, since they are able to protect the encapsulated drugs and/or improve their efficacy by making them able to cross biological barriers (such as the blood-brain barrier) and reach their intracellular target sites. Understanding the intracellular location of NPs is crucial for designing drug delivery strategies. In this study, fluorescently-labelled chitosan NPs were administered in vitro to a neuronal cell line, and diaminobenzidine (DAB) photoconversion was applied to correlate fluorescence and transmission electron microscopy to precisely describe the NPs intracellular fate. This technique allowed to demonstrate that chitosan NPs easily enter neuronal cells, predominantly by endocytosis; they were found both inside membrane-bounded vesicles and free in the cytosol, and were observed to accumulate around the cell nucleus.

Long-term effect of gamma irradiation on the functional properties and cytocompatibility of multiblock co-polymer films.

The purpose of this work was to investigate the long-term effect of gamma-irradiation treatment on the functional properties of PEG-PDLLA and PEG-PLGA films and to evaluate the cytocompatibility of sterilized samples. Chemical and thermal properties, and cytocompatibility of sterilized films were detected for samples at time zero and after storage at 5 ± 3°C for 60 days. An in vitro degradation study was carried out on polymer samples to examine the effect of sterilization on the degradation performances of co-polymer films. Incubated samples were characterized in terms of film surface structure (SEM), chemical (GPC) and thermal (DSC) properties. The study performed on films upon gamma sterilization showed no significant changes of the PEG-PDLLA and PEG-PLGA film structure, while GPC analysis highlighted that the effect of gamma irradiation was dependent on the Mw and composition of polymers. DSC traces suggested more pronounced gamma-ray effects on the PEG-PLGA multiblock co-polymer. During the stability study important changes in terms of structure surface, thermal properties and cytocompatibility were observed and investigated. Data collected during the in vitro degradation study emphasized the need to know and investigate the degradation performances and behaviour of polymer or polymer systems (as DDS, scaffolds and bandage) treated with gamma rays.

Induction of an in vitro reversible hypometabolism through chitosan-based nanoparticles.

OBJECTIVE: Chitosan-based nanoparticles (NPs) were prepared to promote intracellular sustained delivery of the synthetic delta opioid D-Ala(2)-D-Leu(5)-enkephalin (DADLE), prolonging peptide activity and inducing a safe and reversible hypometabolic state. MATERIALS AND METHODS: NPs were prepared by combining ionotropic gelation and ultrasonication treatment. NP uptake studies and the effects of encapsulated DADLE on HeLa cells proliferation were tested by transmission electron microscopy (TEM) analysis, by immuno-fluorescence and immuno-cytochemistry. RESULTS: DADLE-loaded NPs are produced with suitable characteristics, a satisfactory process yield (55.4% ± 2.4%) and encapsulation efficiency (64.6% ± 2.1%). NPs are effective in inducing a hypometabolic stasis at a 10(-4) M DADLE concentration. Moreover, as seen from the immunofluorescence study, the effect persists through the recovery period (72 h). Indeed, NPs labelled by anti-enkephalin antibody inside cell nucleus reassert that the in vivo release of the peptide can be prolonged with respect to the case of free peptide supply. CONCLUSION: The nanoparticulate drug delivery system described seems to be effective in inducing and prolonging a sort of hibernation-like state in the cells.

gamma-irradiation of PEGd,lPLA and PEG-PLGA multiblock copolymers: II. effect of oxygen and EPR investigation.

The purpose of this research was to evaluate how the presence of oxygen can affect irradiation-induced degradation reactions of PEGd,lPLA and PEG-PLGA multiblock copolymers submitted to gamma irradiation and to investigate the radiolytic behavior of the polymers. PEGd,lPLA, PEG-PLGA, PLA, and PLGA were irradiated by using a (60)Co irradiation source in air and under vacuum at 25 kGy total dose. Mw and Mn were evaluated by gel permeation chromatography. The stability study was carried out on three samples sets: (a) polymer samples irradiated and stored in air, (b) polymer samples irradiated and stored under vacuum, and (c) polymer samples irradiated under vacuum and stored in air. The thermal and radiolytic behavior was investigated by differential scanning calorimetry and electron paramagnetic resonance (EPR), respectively. Samples irradiated in air showed remarkable Mw and Mn reduction and Tg value reduction due to radiation-induced chain scission reactions. Higher stability was observed for samples irradiated and stored under vacuum. EPR spectra showed that the presence of PEG units in multiblock copolymer chains leads to: (a) decrease of the radiolytic yield of radicals and (b) decrease of the radical trapping efficiency and faster radical decay rates. It can be concluded that the presence of oxygen during the irradiation process and the storage phase significantly increases the entity of irradiation-induced damage.

Non-viral dried powders for respiratory gene delivery prepared by cationic and chitosan loaded liposomes.

The aim of this work was to investigate lipid-based dried powders as transfection competent carriers capable of promoting the expression of therapeutic genes. The lipid-based vectors were prepared by combining different cationic lipids 1,2-dioleoyl-3-trimethylammoniumpropane chloride (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 3beta(N(N',N-dimethylaminoethane) carbamoyl) cholesterol hydrochloride (DC-Chol) or by mixing of anionic lipids (1,2-dimyristoyl-sn-glycero-3-phospocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt (DMPG) and chitosan salts. Spray drying of the formulations was performed using carbohydrates as thermoprotectant excipients and some amino acids as aerosolisation enhancers. Both the lipidic vectors and the dried powders were characterized for morphology, size, zeta potential (Z-potential) and a yield of the process. Agarose gel electrophoresis was used to examine the structural integrity of dehydrated plasmid DNA (pDNA). The biological functionality of the powders was quantified using the in vitro cell transfection. Among the several lipids and lipid-polymer mixtures tested, the best-selected formulations had spherical shape, narrow size distribution (mean diameter<220 nm, P.I.<0.250), a positive zeta-potential (>25 mV) with a good yield of the process (>65%). The set-up spray drying parameters allowed to obtain good yield of the process (>50%) and spherically shaped particles with the volume-weighted mean diameter (d[4,3])<6 microm in the respirable range. The set-up conditions for the preparation of the lipid dried powders did not adversely affect the structural integrity of the encapsulated pDNA. The powders kept a good transfection efficiency as compared to the fresh colloidal formulations. Lipid-based spray dried powders allowed the development of stable and viable formulations for respiratory gene delivery. In vitro dispersibility and deposition studies are in progress to determine the aerosolisation properties of the powders.

gamma-Irradiation of PEGd,lPLA and PEG-PLGA multiblock copolymers. I. Effect of irradiation doses.

To evaluate the effects of different gamma irradiation doses on PEGd,lPLA and PEG-PLGA multiblock copolymers. The behaviour of the multiblock copolymers to irradiation was compared to that of PLA, PLGA polymers. PEGd,lPLA, PEG-PLGA, PLA and PLGA polymers were irradiated by using a (60)Co irradiation source at 5, 15, 25 and 50 kGy total dose. Characterization was performed on all samples before and after irradiation, by nuclear magnetic resonance (NMR), infrared absorption spectrophotometry (FTIR) and gel permeation chromatography (GPC). The effect of gamma irradiation on polymer stability was also evaluated. Results of NMR and FTIR suggest an increase in -OH and -COOH groups, attributed to scission reactions induced by irradiation treatment. Data of GPC analysis showed that the weight average molecular weight (Mw) of polymer samples decreased with increasing irradiation dose. The extent of Mw degradation expressed as percentage of Mw reduction was more prominent for polymers with high molecular weight as PEGd,lPLA and PLA. The dominant effect of gamma-irradiation on both polymer samples was chain scission. The multiblock copolymer PEGd,lPLA presented higher sensitivity to irradiation treatment with respect to PLA, likely due to the presence of PEG in the matrix. The effect of gamma irradiation continues over a much longer period of time after gamma irradiation has been performed. It is suggested that the material reacts with oxygen to form peroxyl free radicals, which may further undergo degradation reactions during storage after irradiation.

Poly(D,L-lactide) nanoencapsulation to reduce photoinactivation of a sunscreen agent.

The use of sunscreens is the 'gold standard' for protecting the skin from ultraviolet light. Octyl methoxycinnamate (OMC) is one of the most widely used UVB filter but it can act as a sensitizer or photoallergen. When exposed to sunlight, OMC can change from the primary trans-form to cis-form and the isomerization, not reversible, conducts to a reduction of the UVB filtering efficiency because the trans-form has a higher extinction coefficient. Photostability is the most important characteristic of effective sunscreens and it can be influenced by formulation ingredients and by applying technological strategies. In this work, photostability experiments, performed on emulsion-gels containing different percentages of OMC free or loaded in poly(D,L-lactide) nanoparticles, were carried out. The presence of a polymeric envelop may act to protect the active ingredient. In this study, the influence of poly(D,L-lactide) matrices on the photochemical stability of the sunscreen agent was investigated. As highlighted in this study, free OMC in different formulations has different photoisomerization degree. Moreover, a dissimilar behaviour was observed by studying different sunscreen concentrations in the same cosmetic formulation. Photostability results show a significant reduction in photoisomerization degree for formulations containing sunscreen loaded in nanoparticles, highlighting that the encapsulation is a suitable strategy to improve OMC photostability. Moreover, sun protection factor (SPF) results show that the UVB filter protective power is also maintained after encapsulation.

Site-directed PEGylation as successful approach to improve the enzyme replacement in the case of prolidase.

The first aim of this work was to perform site-directed PEGylation of the enzyme prolidase at sulphydril groups by methoxy-polyethylene glycol-maleimide (Mal-PEG, Mw 5000 Da) in order to obtain a safe conjugation product more stable than the native enzyme. Prolidase is a cytosolic aminoacyl-l-proline hydrolase whose deficiency causes the onset of rare autosomal recessive disorder called prolidase deficiency (PD). The second purpose of this work was to investigate whether biodegradable chitosan nanoparticles loaded with PEGylated prolidase could be effective in releasing active enzyme inside fibroblasts as a possible therapeutic approach for PD. The SDS-PAGE analysis and the ESI-MS spectra confirmed the presence of the PEGylated prolidase: in particular the main conjugation product (m/z=about 65,000 Da) corresponded to the enzyme with two residues of Mal-PEG. In this study it was demonstrated the lack of toxicity (MTT assay) and the prolonged activity (40.6+/-2.6% after 48h of incubation at 37 degrees C) of the PEGylated enzyme. The PEGylated prolidase loaded chitosan nanoparticles had spherical shape, narrow size distribution (271.6+/-45.5 nm), a positive zeta-potential (15.93+/-0.26 mV) with a good preparation yield (54.6+/-3.6%) and protein encapsulation efficiency (44.8+/-4.6%). The ex vivo evaluation of prolidase activity on PD fibroblasts individuated a good level of prolidase activity replaced (about 72% after only 2 days of incubation) up to 10 days with improved morphological cell features.

Polyethylenglycol-co-poly-D,L-lactide copolymer based microspheres: preparation, characterization and delivery of a model protein.

PURPOSE: To prepare and characterize polyethylenglycol-co-poly-D,L-lactide (PEG-D,L-PLA) multiblock copolymer microspheres containing ovalbumin. Microsphere batches made of Poly-D,L-lactide (PLA) homopolymers were prepared in order to evaluate how the presence of PEG segments into PEG-D,L-PLA copolymer could affect the behaviour of microspheres as carrier of protein drugs. METHODS: The PEG-D,L-PLA and PLA microspheres, loaded with the model protein ovalbumin, were prepared using double emulsion solvent evaporation method. The effect of PEG segments in the microparticles matrix, on the morphology, size distribution, encapsulation efficiency and release behaviour was studied. RESULTS: According to the results, PEG-D,L-PLA microspheres were more hydrophilic than PLA microparticles and with lower glass transition temperature. The surface of PEG-D,L-PLA microspheres was not as smooth as that of PLA microparticles, the mean diameter of PEG-D,L-PLA microparticles was bigger than that of PLA microspheres. Protein release from the microspheres was affected by the morphological structure of PEG-D,L-PLA microspheres and properties of PEG-D,L-PLA copolymer. This study suggests that PEG-D,L-PLA multiblock copolymer may be used as carrier in protein delivery systems for different purposes.

Efficacy of oleuropein against UVB irradiation: preliminary evaluation.

Oleuropein, a phenolic compound derived from olive leaves and oil, is known to possess several biological properties, many of which may be attributed to its antioxidant and free radical-scavenging activities. Nevertheless, up to now, the cosmetic activity of this molecule has not been extensively investigated. The aim of this work was to evaluate the cosmetic properties of oleuropein against UVB-induced erythema. To this end, an emulsion and an emulgel containing oleuropein were prepared, applied and evaluated on healthy volunteers who had undergone UVB irradiation to investigate its protective and/or lenitive activity. Protective effect was assayed by application of topical preparations before irradiation and lenitive effect was evaluated after erythema induction. Vitamin E was used as the reference compound. Our study was carried out by using noninvasive techniques to assess specific skin parameters: barrier function, skin colour and microcirculation. Results clearly showed that oleuropein formulations highlighted lenitive efficacy by reducing erythema, transepidermal water loss and blood flow of about 22%, 35% and 30% respectively. The study allowed us to point out the lenitive property of oleuropein, opening the way to further trials to deepen our specific knowledge about this natural molecule, which could be used in association with other active ingredients in cosmetics to repair UV damages.

Preparation and in vitro evaluation of thiolated chitosan microparticles.

The objective of this study was to prepare a microparticulate drug delivery system being based on a new thiomer, namely a chitosan 2-iminothiolane conjugate (chitosan-TBA conjugate). Due to thiol groups being immobilized on chitosan, chitosan-TBA conjugate exhibits improved mucoadhesive and permeation enhancing properties. Because of these features microparticulate drug delivery systems based on chitosan-TBA conjugate might be a promising tool for the non-invasive administration of hydrophilic macromolecular drugs. Chitosan-TBA conjugate microspheres were prepared by the emulsification/solvent evaporation method. Fluorescein-isothiocyanate labelled dextran (FITC-dextran) was chosen as a model hydrophilic drug. Microspheres have been characterized by morphological analysis, thiol group content, swelling behaviour, polymer degradation drug load determination, dissolution test and mucoadhesion studies. Results reported in this work demonstrated the possibility to obtain stable microspheres without cross-linking agents. Thiolated chitosan microspheres seem to be more stable in aqueous media with respect to unmodified chitosan. The degradability by lysozyme appears quite similar for both polymers, showing that chemical modification does not influence the biodegradable properties of chitosan. Microspheres were able to control the drug release for at least 1 h, exhibiting comparatively strong mucoadhesive properties. The chitosan-TBA conjugate microparticles remain on the mucosa in a 2.5-fold higher concentration with respect to unmodified chitosan microparticles. These data suggest that chitosan-TBA conjugate microspheres have the potential to be used as a mucoadhesive drug delivery system.

Biodegradable microspheres for prolidase delivery to human cultured fibroblasts.

Prolidase deficiency (PD) is a rare autosomal recessive disorder caused by inadequate levels of the cytosolic exopeptidase prolidase (E.C. 3.4.13.9), for which there is not, as yet, a resolutive cure. We have investigated whether biodegradable microspheres loaded with prolidase could release active enzyme inside cells, to consider this system as a possible therapeutic approach for prolidase deficiency. Poly(lactide-co-glycolide) microspheres were prepared, modifying the classical double emulsion solvent evaporation method to mitigate the burst effect of the enzyme from the microspheres. Ex-vivo experiments were performed, by incubating microencapsulated prolidase with cultured fibroblasts from PD patients and from controls, to determine the amount of active enzyme delivered to the cells. The microparticulate drug delivery system described carried small amounts of active prolidase inside fibroblasts, ensuring a response to the intracellular accumulation of X-Pro dipeptides, the mechanism that is supposed to be responsible for the development of clinical manifestations of this disorder in man. A positive result of the presence of active enzyme inside cells was an improvement in fibroblast shape.

Poly(lactide-co-glycolide) microspheres containing bupivacaine: comparison between gamma and beta irradiation effects.

The beta- and gamma-irradiation effects on stability of microspheres made of poly(lactide-co-glycolide) 50:50 copolymer (PLGA) containing bupivacaine (BU) were studied. Microspheres containing 10, 25, and 40% w/w, respectively, of BU were prepared by spray drying and irradiated in air with beta- and gamma-irradiation at a dose of 25 kGy. Morphology (atomic force microscopy, particle-size analysis), physico-chemical characteristics (DSC and FT-IR spectroscopy), drug content and in vitro dissolution profile of microspheres were all determined; the stability of irradiated microspheres was evaluated over a 9-month period. The decrease of BU content in gamma-irradiated microspheres was almost always constant independent of the amount of BU per sample, therefore it was in inverse proportion to drug loading (range between 5 and 15%). BU release rate increased immediately after irradiation and increased slightly until 90 days of storage. As far as beta-irradiated microspheres are concerned, BU content decreased in a significant way (approximately 3%) only in microspheres containing 10% w/w of BU. Immediately after irradiation, drug release rate in beta-irradiated microspheres increased less than in the corresponding gamma-irradiated microspheres, and it did not change further over the following storage period. BU-loaded microspheres have been shown to be more stable against beta- than gamma-irradiation. AFM revealed that the surface roughness of the irradiated microspheres increases depending on irradiation. As such, if a parameter is quantifiable, it is proposed as a marker of degradation due to ionizing radiation.

PLGA microspheres for oral osteopenia treatment: preliminary "in vitro"/"in vivo" evaluation.

The aim of this work was to prepare and to evaluate "in vitro"/"in vivo" microspheres based on poly(D,L-lactide-co-glycolide) copolymers containing ipriflavone, for the local treatment of oral bone loss. The first objective was the preparation and "in vitro" characterization of ipriflavone loaded microspheres, by emulsion/solvent evaporation method. Process parameters such as drug:polymer weight ratio, and molecular weight of copolymers, were also investigated. The second objective was to elaborate a suitable animal model of mandibular osteoporosis, to evaluate the efficacy of these microparticulate drug delivery systems. "In vivo" experiments were carried out on female rats, in which oral osteopenia was induced by gonadectomy and molar avulsion. Morphometric analysis of mandibular segment were carried out to quantify the development of oral osteopenia and the efficacy of drug loaded microspheres. Results showed that ipriflavone loaded PLGA microspheres can be successfully obtained with good "in vitro" characteristics, utilizing the emulsification/solvent evaporation method. "In vivo" experiments revealed that local administration of microspheres produced only mild inflammation on the injection site. Morphometric analyses showed, at the level of the third molar, a slight increase in spongy and total bone mass on rat jaw treated with microspheres with respect to control. Control animals exhibited a scarce degree of osteopenia demonstrating that this animal model is not suitable for this purpose.

Periodontal delivery of ipriflavone: new chitosan/PLGA film delivery system for a lipophilic drug.

The aim of the present work was to design a film dosage form for sustained delivery of ipriflavone into the periodontal pocket. For this purpose, monolayer composite systems made of ipriflavone loaded poly(D,L-lactide-co-glycolide) (PLGA) micromatrices in a chitosan film form, were obtained by emulsification/casting/evaporation technique. Multilayer films, made of three layers of polymers (chitosan/PLGA/chitosan), were also prepared and compared to monolayer films for their "in vitro" characteristics. Morphology and physico-chemical properties of the different systems were evaluated. The influence of pH, ionic strength and enzymatic activity on film degradation, was also investigated. Significant differences in swelling, degradation and drug release were highlighted, depending on film structure and composition. In vitro experiments demonstrated that the composite micromatricial films represent a suitable dosage form to prolong ipriflavone release for 20 days.