Agnese Miro

Influence of the co-encapsulation of different non-ionic surfactants on the properties of PLGA insulin-loaded microspheres

The aim of this work was to produce insulin-loaded microspheres allowing the preservation of peptide stability during both particle processing and insulin release. Our strategy was to combine the concepts of using surfactants to improve insulin stability while optimising overall microsphere characteristics such as size, morphology, peptide loading and release. Bovine insulin was encapsulated within poly(lactide-co-glycolide) (PLGA 50:50, Resomer RG504H) microspheres by the multiple emulsion-solvent evaporation technique. Microspheres were prepared by adding to the primary emulsion three non-ionic surfactants, poloxamer 188, polysorbate 20 and sorbitan monooleate 80, at different concentrations (1.5 and 3. 0% w/v). The presence of surfactants was found to decrease the mean diameter and to affect the morphology of the microspheres. Insulin encapsulation efficiency was reduced in the presence of surfactants and especially for sorbitan monooleate 80, in a concentration-dependent mode. The influence of the surfactants on the interactions between insulin and PLGA together with the primary emulsion stability were found to be the major determinants of insulin encapsulation. The release of insulin from microspheres was biphasic, showing an initial burst effect followed by a near zero-order release for all the batches prepared. The initial burst was related to the presence of insulin molecules located onto or near to the microsphere surface. In the presence of surfactants, a faster insulin release with respect to microspheres encapsulating insulin alone was observed. Insulin stability within microspheres after processing, storage and release was evaluated by reversed phase- and size-exclusion-HPLC. The analysis of microsphere content after processing and 6 months of storage showed that insulin did not undergo any chemical modification within microspheres. On the contrary, during the period of sustained release insulin was transformed in a high-molecular weight product, the amount of which was related to the surfactant used. In conclusion, polysorbate 20 at 3% w/v concentration was the most effective in giving regular shaped particles with both good insulin loading and slow release, and limiting insulin modification within microspheres.

Dry powders based on PLGA nanoparticles for pulmonary delivery of antibiotics: Modulation of encapsulation efficiency, release rate and lung deposition pattern by hydrophilic polymers

Although few experimental studies have been handled so far to exploit the potential of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) in the production of dry powders for antibiotic inhalation, there has been no comprehensive study on the role played by NP composition. In this work, we try to shed light on this aspect by designing and developing a pulmonary delivery system for antibiotics, such as tobramycin (Tb), based on PLGA NPs embedded in an inert microcarrier made of lactose, referred to as nano-embedded micro-particles (NEM). At nanosize level, helper hydrophilic polymers were used to impart the desired surface, bulk and release properties to PLGA NPs prepared by a modified emulsion-solvent diffusion technique. Results showed that poly(vinyl alcohol) (PVA) and chitosan (CS) are essential to optimise the size and modulate the surface properties of Tb-loaded PLGA NPs, whereas the use of alginate (Alg) allows efficient Tb entrapment within NPs and its release up to one month. Optimized formulations display good in vitro antimicrobial activity against P. aeruginosa planktonic cells. Furthermore, spray-drying of the NPs with lactose yielded NEM with peculiar but promising flow and aerosolization properties, while preserving the peculiar NP features. Nonetheless, in vivo biodistribution studies showed that PVA-modified Alg/PLGA NPs reached the deep lung, while CS-modified NPs were found in great amounts in the upper airways, lining lung epithelial surfaces. In conclusion, PLGA NP composition appears to play a crucial role in determining not only the technological features of NPs but, once processed in the form of NEM, also their in vitro/in vivo deposition pattern.

Insulin-loaded PLGA/cyclodextrin large porous particles with improved aerosolization properties: In vivo deposition and hypoglycaemic activity after delivery to rat lungs

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.

Spectrophotometric determination of polyethylenimine in the presence of an oligonucleotide for the characterization of controlled release formulations

Polyethylenimine (PEI) is a cationic polymer that can be associated to oligonuclotides to promote their transfection both in vitro and in vivo. The controlled release of oligonucleotide/polyethylenimine complexes from biodegradable systems can result in an increased cellular internalisation of the oligonucleotide and a reduced cytotoxicity of the complex. This effect strongly depends on the amount of PEI loaded in and released from the delivery system. In this work we describe a rapid, sensitive and reproducible spectrophotometric method for the quantitative analysis of PEI by itself or in the presence of an associated oligonucleotide. PEI does not possess chromophores, hence the determination by ordinary spectrophotometry is not possible. However, upon addition of copper (II) ions, PEI forms a dark blue cuprammonium complex that can be detected by UV-vis spectrophotometry. The optimum conditions in terms of optical parameters, copper (II) concentration required for a quantitative PEI complexation, and the most suitable medium for the reaction were ascertained. A linear relationship (r(2)=0.9997) between absorbance and amounts of PEI was found at lambda(max) of 285 nm over the concentration range 5.0-50.0 microg ml(-1). The detection limit (QOD) was 4.0 microg ml(-1). The method was validated for the quantitation of PEI in the presence of an oligonucleotide, which absorbs at 285 nm as well.

Engineered PLGA nano‐ and micro‐carriers for pulmonary delivery: challenges and promises

Objectives  The aim of this review is to summarize the current state‐of‐the‐art in poly(lactic‐co‐glycolic acid) (PLGA) carriers for inhalation. It presents the rational of use, the potential and the recent advances in developing PLGA microparticles and nanoparticles for pulmonary delivery. The most promising particle engineering strategies are discussed, highlighting the advantages along with the major challenges for researchers working in this field.

Modulation of drug release from hydrogels by using cyclodextrins: the case of nicardipine/β-cyclodextrin system in crosslinked polyethylenglycol

A simple approach is presented to modulate drug delivery from swellable systems by using complexants. The effect of complexants has been interpreted by means of simple mass balances on diffusing species and the involved relevant parameters have been individuated. The application of this strategy to the release of nicardipine (NIC) from swellable systems by using beta-cyclodextrin (CD) as complexant has evidenced the potential of the approach to tailor drug release. Crosslinked polyethyleneglycol has been synthesized, characterized and used as the swellable matrix. Swelling kinetics, NIC and CD diffusivities in the swollen matrix and NIC/CD phase solubility studies have been performed. The polymer matrix has been loaded with pure NIC or with NIC and CD at different ratios and release kinetics evaluated. Release profiles have shown that the presence of CD significantly affected drug delivery by decreasing the effective diffusivity of NIC. The higher the CD/NIC ratio the slower is the release. This effect has been interpreted on the basis of the proposed model and physically sound assumptions.

Alginate-hyaluronan composite hydrogels accelerate wound healing process.

In this paper we propose polysaccharide hydrogels combining alginate (ALG) and hyaluronan (HA) as biofunctional platform for dermal wound repair. Hydrogels produced by internal gelation were homogeneous and easy to handle. Rheological evaluation of gelation kinetics of ALG/HA mixtures at different ratios allowed understanding the HA effect on ALG cross-linking process. Disk-shaped hydrogels, at different ALG/HA ratio, were characterized for morphology, homogeneity and mechanical properties. Results suggest that, although the presence of HA does significantly slow down gelation kinetics, the concentration of cross-links reached at the end of gelation is scarcely affected. The in vitro activity of ALG/HA dressings was tested on adipose derived multipotent adult stem cells (Ad-MSC) and an immortalized keratinocyte cell line (HaCaT). Hydrogels did not interfere with cell viability in both cells lines, but significantly promoted gap closure in a scratch assay at early (1 day) and late (5 days) stages as compared to hydrogels made of ALG alone (p<0.01 and 0.001 for Ad-MSC and HaCaT, respectively). In vivo wound healing studies, conducted on a rat model of excised wound indicated that after 5 days ALG/HA hydrogels significantly promoted wound closure as compared to ALG ones (p<0.001). Overall results demonstrate that the integration of HA in a physically cross-linked ALG hydrogel can be a versatile strategy to promote wound healing that can be easily translated in a clinical setting.

Engineering gas-foamed large porous particles for efficient local delivery of macromolecules to the lung

Gas-foamed large porous particles (gfLPP) based on poly(lactic-co-glycolic) acid (PLGA) have been recently suggested as potential carriers for pulmonary drug delivery. In this work, we attempt to engineer gfLPP for efficient local delivery of macromolecules in the lungs. Particles were fabricated by the double emulsion-solvent evaporation technique using ammonium bicarbonate as porogen. To improve particle technological properties, two lipid aid excipients, namely dipalmitoylphosphatidylcholine (DPPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), were tested. Preliminary technological studies performed on unloaded gfLPP showed that the addition of an appropriate amount of NH(4)(HCO(3)), which spontaneously produces CO(2) and NH(3) during solvent evaporation, is essential to achieve a homogeneous population of highly porous particles with optimal aerodynamic properties. Then, the effect of the presence of DPPC or DOTAP upon the properties of gfLPP containing a model hydrophilic macromolecule, rhodamine B isothiocyanate-dextran (Rhod-dex), was assessed. We found that in the case of hydrophilic macromolecules unable to interact with PLGA end-groups, such as Rhod-dex, excipient addition is essential to increase the amount of drug entrapped within gfLPP, being as high as 80% only for DPPC- or DOTAP-engineered gfLPP. Also Rhod-dex release profile from gfLPP was strongly affected by excipient addition in the initial formulation, with lipid-engineered gfLPP allowing for a more prolonged release of Rhod-dex as compared to excipient-free gfLPP. A further modulation of Rhod-dex initial release rate could be achieved when DOTAP was used, likely due to the electrostatic interactions occurring between macromolecule and cationic phospholipid. Conceiving the developed gfLPP for drug inhalation, DPPC- and DOTAP-engineered gfLPP displayed optimal MMAD(exp) values falling within the range 6.1-7.6 microm and very low geometric standard deviations (GSD) varying between 1.2 and 1.3. In vivo deposition studies performed after intra-tracheal administration of gfLPP in rats confirmed the ability of the developed dry powders to deposit along bronchia and bronchioles. In perspective, lipid-engineered gfLPP represent a viable alternative to LPP developed so far to achieve local and prolonged release of hydrophilic macromolecules, such as nucleic acids, in the lungs.

Improving the efficacy of inhaled drugs in cystic fibrosis: Challenges and emerging drug delivery strategies

Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians associated with early death. Although the faulty gene is expressed in epithelia throughout the body, lung disease is still responsible for most of the morbidity and mortality of CF patients. As a local delivery route, pulmonary administration represents an ideal way to treat respiratory infections, excessive inflammation and other manifestations typical of CF lung disease. Nonetheless, important determinants of the clinical outcomes of inhaled drugs are the concentration/permanence at the lungs as well as the ability of the drug to overcome local extracellular and cellular barriers. This review focuses on emerging delivery strategies used for local treatment of CF pulmonary disease. After a brief description of the disease and formulation rules dictated by CF lung barriers, it describes current and future trends in inhaled drugs for CF. The most promising advanced formulations are discussed, highlighting the advantages along with the major challenges for researchers working in this field.

Chromatographic indexes on immobilized artificial membranes for the prediction of transdermal transport of drugs.

A set of 12 drugs, consisting of structurally unrelated neutral, basic, acidic and amphoteric compounds, was examined by high performance liquid chromatrography (HPLC) on a model of fluid membrane bilayers, the immobilized artificial membrane (IAM) column. The logarithms of chromatographic capacity factors extrapolated to 100% aqueous phase at pH 5.5 (log kw) were measured and compared to the n-octanol/water partition coefficients (log P). The scale derived from the IAM system was different from the lipophilicity scale expressed by the log P, due to the peculiar capability of phospholipids to well accommodate the ionized form of some molecules and show additive or repulsive extra-interactions when particular structural motifs on the molecule are present. The relationship between log P and log kw previously obtained for compounds interacting on IAM phase by a uniquely lipophilicity-based mechanism, allowed us to calculate, from log P, the values of log kw expected for the drugs considered. These values were subtracted from the log kw experimentally determined and the differences were assumed to quantify the amount of extra-interactions (hydrogen bond and electrostatic interactions) with phospholipids (delta log kw). The coefficients of permeability through the human skin (Kp) for the compounds considered did not correlate with either log kw or log P values. However, the Kp values correlated well with the delta log kw values indicating that the higher the ability of a molecule to cross the skin barrier, the lower its component of interaction with phospholipids not accounted for by lipophilicity-based interactions.