Claudia Colonna

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.

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).

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.

5-methyl-pyrrolidinone chitosan films as carriers for buccal administration of proteins.

The purpose of this research was to investigate 5-methyl-pyrrolidinone chitosan (MPC) films as carriers for buccal delivery of protein drugs. Placebo and protein-loaded MPC films were prepared by casting and were then cross-linked with tripolyphosphate at different pH conditions. Myoglobin (MHb) was chosen as the model protein because its molecular weight is under the permeability limit of the buccal mucosa. The observed characteristics like bioadhesiveness, swelling behavior, and in vitro release of MHb from loaded films furnish information on the functional behavior of these films. The results obtained show that the modulation of Mhb release was achieved only through chitosan cross-linking; the best results in release rate control were obtained by cross-linking performed at pH 6.5. Good bioadhesion properties were maintained even with high cross-linking degrees; the swelling index of MHb-loaded films at different cross-linking degrees evaluated at pH 7.4 and pH 6.4 were comparable to those of placebo films. By setting suitable tripolyphosphate cross-linking conditions for MPC films, one can control protein release without affecting bioadhesion.

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.

γ-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 60Co 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.

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.

Evaluation of bioadhesive performance of chitosan derivatives as films for buccal application

The purpose of this work is to evaluate the buccal bioadhesive performance of film.s made by four different chitosan derivatives: chitosan chloride (CL., 13 ), chitosan glutamate (G., 13 ), 5-methyl-pyrrolidinone chitosan (MP) and a partially reacetylated chitosan (RC). Films were prepared by casting and ionically cross-linked by tripolyphosphate (TPP) using increasing TPP:chitosan molar ratio (20:1-200:1). A rapid and simple in vitro test based on use of buccal cell suspension was modified to assess bioadhesion of solid dosage forms and it proved able to discriminate between bioadhesion properties of films made by different chitosans and with different TPP:chitosan molar ratios. All films, with the exception of the MP films, have biphasic bioadhesion profile, showing a bioadhesiveness drop over a fixed amount of TPP which depends on the type of chitosan. This comparative study has enabled G 213 and MP to be chosen as the most promising chitosan derivatives aimed at optimizing the designing of controlled drug delivery via buccal bioadhesive films.

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.