Tiziana Modena

Long-term release of clodronate from biodegradable microspheres.

This paper describes the formulation of a biodegradable microparticulate drug delivery system containing clodronate, a bisphosphonate intended for the treatment of bone diseases. Microspheres were prepared with several poly(D,L-lactide-co-glycolide) (PLGA) copolymers of various molecular weights and molar compositions and 1 poly(D,L-lactide) (PDLLA) homopolymer by a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation procedure. Critical process parameters and formulation variables (ie, addition of stabilizing agents) were evaluated for their effect on drug encapsulation efficiency and clodronate release rate from microparticles Well-formed clodronate-loaded microspheres were obtained for all polymers by selecting suitable process parameters (inner water/oil volume ratio 1∶16, temperature-raising rate in the solvent evaporation step 1°C/min, 2% wt/vol NaCl in the external aqueous phase). Good yields were obtained in all batches of clodronate microspheres (above 60%); drug encapsulation efficiencies ranged between 49% and 75% depending on the polymer used. Clodronate release from all copolymer microspheres was completed in about 48 hours, while those from PDLLA microspheres required about 20 days. The change of microsphere composition by adding a surfactant such as Span 20 or a viscosing agent such as carboxymethylcellulose extended the long-term release up to 3 months. Clodronate was successfully entrapped in PLGA and PDLLA microspheres, and drug release could be modulated from 48 hours up to 3 months by suitable selection of polymer, composition, additives, and manufacturing conditions.

Chitosan glutamate nanoparticles for protein delivery: Development and effect on prolidase stability

Purpose: To evaluate the feasibility of exploiting ultrasonication coupled with ionotropic gelation in order to prepare tripolyphosphate (TPP)-chitosan glutamate nanoparticles suitable for the delivery of the enzyme prolidase. Methods: All the parameters for the preparation of TPP-chitosan nanoparticles in terms of components weight ratio, ultrasonication conditions and time-saving nanoparticles recovery conditions were optimized. The best formulation was loaded with the prolidase. All the nanoparticles were characterized in terms of morphology, size, polydispersity, zeta-potential, yield of the process and encapsulation efficiency. The in-vitro activity of the prolidase was assessed by capillary electrophoresis (CE). Results and conclusions: A TPP to chitosan weight ratio of 0.2:1 combined with one ultrasonication cycle (4 min using the probe-type sonifier at 75% power) obtained well-formed nanoparticles of spherical shape, mean size of ∼ 365 nm (polydispersity index 0.3) and a + 17.94 mV zeta potential. A satisfactory prolidase encapsulation efficiency (43%) was obtained with a yield of the preparation process of ∼55%. In vitro study of activity of prolidase, as free enzyme or released from chitosan nanoparticles, highlighted the ability of chitosan to stabilize the enzyme during all the steps of the preparation process and to modulate the enzyme activity up to 48 h.

Evaluation of process parameters involved in chitosan microsphere preparation by the o/w/o multiple emulsion method

Chitosans are interesting biopolymers largely studied for applications in the medical and pharmaceutical fields. In this work, an o/w/o multiple emulsion technique was used for the preparation of hydrophobic drug loaded microspheres. Moreover, the influence of critical variables (concentration of acetic acid in the polymer solution and drug-polymer ratio) on microsphere morphology and drug content was evaluated. Two chitosans of different molecular weights and deacetylation degree were employed; ketoprofen, a non-steroidal anti- inflammatory drug, was chosen as the hydrophobic model drug. The multiple emulsion method produced well-formed microspheres with good yields. Acetic acid concentration in the polymeric solutions influenced particle size and drug content of the microspheres. The highest drug encapsulation efficiencies were obtained for the lowest theoretical drug/chitosan ratio.

Ex vivo evaluation of prolidase loaded chitosan nanoparticles for the enzyme replacement therapy

Prolidase loaded chitosan nanoparticles were set up in order to suggest an innovative therapeutic approach for Prolidase Deficiency (PD), a rare autosomal inherited disorder of the connective tissue. The satisfactory drug loading efficiency (42.6+/-2.1%) as well as the suitable physical characteristics (mean diameter of 365.5+/-35.1 nm and a positive zeta-potential of 17.94+/-0.12 mV) was achieved. In order to verify the compatibility of the chitosan nanoparticles with cells, the influence of the nanoparticles on the growth and the viability (MTT assay) of cultured skin fibroblasts were determined: the nanoparticles showed a good biocompatibility up to 5 microg of chitosan/10,000 fibroblasts. Uptake of chitosan nanoparticles by fibroblasts was verified by confocal microscopy using FITC-labelled chitosan nanoparticles. The ex vivo experiments were performed by incubating different amounts of prolidase loaded chitosan nanoparticles with skin human fibroblasts from PD patients for scheduled times. The restored prolidase activity was quantitatively monitored by a capillary electrophoretic method and confirmed by cells morphological observations. Standing from the nanoparticles internalization, the enzymatic activity was progressively restored reaching the best value (about 66%) after 5 days of co-incubation. Moreover, prolidase loaded chitosan nanoparticles permitted to restore prolidase activity in PD fibroblasts for a prolonged period of time (8 days).

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.

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.

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.

Investigation on Process Parameters Involved in Polylactide-Co-Glycolide Microspheres Preparation

AbstractIndomethacin loaded polylactide-co-glycolide (PLGA) microspheres were prepared by emulsification solvent evaporation. The preparation involves several process parameters that can affect the morphological characteristics, the “in vitro” and “in vivo” dissolution behaviour of microspheres.The evaluation of three process parameters, emulsification stirring rate, emulsifier concentration and dispersed phase to continuous phase ratio was carried out in order to correlate them to some microsphere properties.Results show that the variables evaluated affect mainly microspheres drug content and, at less extent, particle size.

Testing of “In Vitro” Dissolution Behaviour of Microparticulate Drug Delivery Systems

AbstractNo official dissolution method exists concerning microparticulate drug delivery systems. The purpose of this work is the evaluation of different dissolution methods commonly used to test the in vitro release behaviour of microparticulate drug delivery systems. The influence of different environmental conditions, as stirring speed, ionic strength and presence of surfactant, on drug release is also evaluated.Four dissolution methods, based on different equipments (USP dissolution test apparatus, rotating bottle apparatus, shaker incubator, recycling flow through cell), are performed on the same batch of indomethacin loaded poly-D, L-lactide (PDLLA) microspheres prepared by spray drying. The results obtained with the methods tested show the influence of in vitro dissolution method employed and of the environmental conditions on drug release profile.

In vitro evaluation of chondroitin sulphate-chitosan microspheres as carrier for the delivery of proteins.

A novel formulation based on chondroitin sulphate/chitosan microspheres (CS/CH) has been investigated for oral delivery of macromolecules using ovalbumin as the model protein (OVA). The microspheres were prepared by a new emulsion-complex coacervation method. Physico-chemical properties of the polymers constituting microparticulate matrix were investigated by IR, DSC, TGA and X-ray diffraction analyses. In vitro tests were performed to evaluate the drug delivery system degradation and the protein release under conditions simulating the intestinal fluids. The ability of colonic enzymes to degrade the microparticulate systems was simulated employing the chondroitinase ABC enzyme. Results showed that the different CS/CH compositions influenced both microparticles stability and the protein release rate. Only the microspheres composed by 1:1 chondroitin sulphate–chitosan ratio achieved an OVA release profile suitable to a possible colon targeting. These microspheres released ∼30% of ovalbumin encapsulated in 24 h in the different aqueous media tested, while they released 100% of protein in the presence of chondroitinase. The preliminary results demonstrated that chondroitin sulphate-chitosan microspheres can be a suitable delivery system for protein drug envisaged to oral administration.