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NF-kB blockade by decoy oligonucleotides (ODNs) able to interfere with NF-kB transcriptional activity may be of great help in limiting the progression of CF lung chronic inflammation. Although data proving the validity of this approach are just emerging, early studies on CF airway epithelial cell lines are encouraging. Major concerns arise from the need of appropriate systems for ODN inhalation, that are able to efficiently target the desired region of the lungs, protect the ODN in the biological environment and release it in a sustained manner. The technological approaches currently adopted for lung delivery of ODNs reliy on the use of lipidic nanoparticles or cationic liposomes, providing new formulation options for dispersed liquid droplet dosage forms (i.e., bulky, expensive and more time-consuming nebulizers) with poor control over ODN release rate. Prompted by this, we are currently developing respirable dry powders, for prolonged pulmonary delivery of a decoy ODN to NF-kB (dec-ODN), consisting of biodegradable large porous particles (LPP) based on poly(lactic-co-glycolic) acid (PLGA). The effects of dec-ODN LPP on NF-kB/DNA binding activity as well as IL-6 and IL-8 mRNA levels were studied on DF508 CFTR-mutated human bronchial epithelial cells stimulated with LPS from P. aeruginosa for 24 and 72 h. Finally, the effects of intratracheally administered naked dec-ODN or dec-ODN LPP on neutrophil lung recruitment induced by LPS from P. aeruginosa at 6, 24 and 72 h were investigated in a rat model of airway inflammation.

Lo sviluppo di Drug Delivery Systems sempre più sofisticati rappresenta oggi la ricerca di frontiera nel settore tecnologico-farmaceutico. In tale ambito, il lavoro di ricerca oggetto della tesi di dottorato ha avuto come obiettivo la progettazione, la realizzazione e l’applicazione di sistemi microparticellari per il rilascio modificato di biofarmaci al polmone. Tali sistemi sono stati progettati per essere somministrati per via inalatoria direttamente in forma di polvere secca. A tal fine, sono state sviluppate particelle porose di grandi dimensioni o Large porous particles (LPP) che rappresentano una valida strategia formulativa per l’ottenimento di polveri respirabili. L’impiego, poi, di materiali a base di polimeri d’uso consolidato, quali l’acido poli(lattico-co-glicolico), può essere di grande aiuto nella produzione di polveri che combinino l’abilità di raggiungere il sito bersaglio ad una attività terapeutica a lungo termine. In particolare, tali sistemi possono migliorare la stabilità in vivo del farmaco veicolato, diminuirne la tossicità, prolungarne il tempo di permanenza in situ e permettere la combinazione con altri principi attivi o eccipienti di supporto (e.g., promotori d’assorbimento). Il lavoro di tesi si è articolato in due fasi successive. Entrambe le fasi di ricerca hanno previsto uno studio approfondito di tipo tecnologico-formulativo confortato, poi, da studi farmacologici condotti in collaborazione con il Dipartimento di Farmacologia Sperimentale di questa Università. Nel corso della prima fase sono state studiate le potenzialità applicative di LPP a base di PLGA per il rilascio al polmone di insulina. Sulla base dell’esperienza maturata, nell’ambito di un progetto finanziato dalla Fondazione Italiana per la Fibrosi Cistica (FFC#5/2007), sono state sviluppate LPP a base di PLGA per il rilascio controllato al polmone di un decoy ODN diretto contro NF-B per il trattamento della fibrosi cistica. I dati raccolti dimostrano che le LPP di PLGA progettate rappresentano un carrier sicuro ed efficace per il rilascio controllato di proteine ed oligonucleotidi al polmone. Le polveri contenenti insulina raggiungono gli alveoli, rilasciano la proteina che, assorbita in forma attiva, mostra un’efficacia terapeutica dose-dipendente. Modificando il progetto formulativo, è stato possibile ottenere LPP in grado di rilasciare lentamente un dec-ODN a livello locale, inibire l’attivazione di NF-B e ridurre i livelli di IL-6 ed IL-8. Pertanto, le LPP sviluppate rappresentano anche un ottimo carrier per ridurre l’infiammazione cronica polmonare nei pazienti affetti da fibrosi cistica.

Background Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Neutrophil-dominated inflammation and chronic bacterial infection are still considered the primary cause of bronchioectasis, respiratory failure and consequent death in CF patients. Activation of nuclear factor (NF)-κB is responsible for overproduction of cytokines, such as interleukin (IL)-6 and IL-8, in airways of CF patients. Thus, decoy oligodeoxynucleotides against NF-κB (dec-ODN) may limit lung inflammation in CF. In the present study, we studied the effects of dec-ODN delivered through biodegradable and respirable poly(D,L-lactide-co-glycolide) large porous particles (LPP) on IL-6 and IL-8 mRNA expression as well as NF-κB/DNA binding activity in cystic fibrosis cells stimulated with lipopolysaccharide (LPS) from Pseudomonas aeruginosa. Methods dec-ODN LPP were prepared by a modified double emulsion technique and characterized in terms of size, morphology, tapped density and dec-ODN loading. Human epithelial bronchial IB3-1 (CFTR-mutated) as well as S9 (CFTR-corrected) were stimulated with LPS from P. aeruginosa for 24 and 72 h in the absence or presence of naked dec-ODN or dec-ODN LPP. Results Stimulation of cells with LPS from P. aeruginosa caused an increase of IL-6 and IL-8 mRNA levels, which were significantly inhibited by dec-ODN LPP at 24 and 72 h, whereas naked dec-ODN inhibited those only at 24 h. Similar effects were exhibited by dec-ODN LPP or naked dec-ODN on NF-κB/DNA binding activity. Conclusions Our observations indicate that respirable biodegradable dec-ODN LPP may represent a promising strategy for inhibiting NF-κB transcriptional activity and related gene expression and, thus, reduce lung chronic inflammation in CF patients. Copyright © 2011 John Wiley & Sons, Ltd.

The development of a novel generation of drug-eluting stent (DES) relies upon the idea to obtain very flexible platforms able to overcome some issues associated to available devices and widen their field of application, especially to the currently emerging biotech therapeutics. Here, we propose a new concept of DES named microsphere-integrated drug-eluting stent (MIDES) composed of drug eluting biodegradable poly(D,L-lactide-co-glycolide) microspheres--encapsulating an hydrophilic model molecule (dextran)--fully integrated in a poly(2-hydroxy-ethyl-methacrylate) coating. By implementing a modified spray-coating technique, we have been able to achieve a thin (10 μm), smooth, and homogeneous hydrogel surface embedding underneath a population of two different microparticles formulations--Dex502H and Dex506. The amount of drug can be tailored, resulting in a dextran loading as high as 1.4 μg/cm, by simply reiteration of coating layer deposition making the MIDES a custom-made device where the release kinetics can be further modified by opportunely choosing microsphere properties. DES use is nowadays restricted to delivery of hydrophobic pharmaceuticals; release of hydrophilic therapeutics from MIDES can, however, be finely controlled by specifically engineering biodegradable microspheres. By varying polymer resomer, we obtained a tunable release rate in the first month of delivery. Depending on the microspheres properties release profile changes drastically moving from a biphasic release, in the case of Dex502H, with a burst of about 20% in the first day to a more sustained release for Dex506 particles. As proof of principle, we also demonstrated that MIDES approach can allows the delivery of two different agents opening up the way to a multitherapy in restenosis treatment.

The aim of the present work is to develop large porous particles (LPP) of poly (lactide-co-glycolide) (PLGA) containing insulin with optimal aerodynamic properties and to test their in vivo potential, in pulmonary delivery. Insulin-loaded LPP were fabricated by a double emulsion method by aid of hydroxypropyl-beta-cyclodextrin (HPbetaCD). Conceiving this system for the controlled release of insulin to the lungs, the aerosolization properties and the release features in simulated lung fluids of PLGA/HPbetaCD/insulin LPP were investigated in depth. The technological results show that the combination of appropriate amounts of insulin and HPbetaCD plays a crucial role to achieve PLGA/HPbetaCD/insulin LPP with the desired bulk and aerodynamic properties, that is a highly porous structure, a very low density (0.1 g/ml), an experimental mass mean aerodynamic diameter (MMAD(exp)) ranging from 4.01 to 7.00 and a fine particle fraction (FPF) estimated to be 26.9-89.6% at the different airflow rates tested (i.e. 30-90 l/min). Confocal microscopy studies, performed after administration of labeled PLGA/HPbetaCD/insulin LPP to the rat lung by means of a low-scale dry powder inhaler (DPI), suggest that particles reach alveoli and remain in situ after delivery. The pharmacological effect of PLGA/HPbetaCD/insulin LPP was confirmed by dose-response studies performed on both normoglycaemic and streptozotocin-induced diabetic rats. While insulin solutions administered via pulmonary route are unable to cause a significant hypoglycaemic effect, insulin delivered through PLGA/HPbetaCD/insulin LPP at the same doses (0.5-4.0 IU/kg) significantly reduces blood glucose level as a function of the administered dose in both animal models. The developed LPP, tested in hyperglycaemic rats at evident pathological conditions, exerts a very significant and longer hypoglycaemic effect even at insulin doses as low as 0.5 IU/kg (about 0.5 mg of PLGA/HPbetaCD/insulin LPP per rat) as compared to a insulin solution. Taken together, our results support the viability of a dry powder formulation based on biodegradable LPP for the controlled release of insulin to the lungs. In vivo data show that PLGA/HPbetaCD/insulin LPP are able to reach alveoli, release insulin, which is absorbed in its bioactive form.