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In the treatment of lung cancer, there is an urgent need of innovative medicines to optimize pharmacological responses of conventional chemotherapeutics while attenuating side effects. Here, we have exploited some relatively unexplored subtle differences in reduction potential, associated with cancer cell microenvironments in addition to the well-known changes in intracellular redox environment. We report the synthesis and application of novel redox-responsive PLGA (poly(lactic-co-glycolic acid)) -PEG (polyethylene glycol) nanoparticles (RR-NPs) programmed to change surface properties when entering tumor microenvironments, thus enhancing cell internalization of the particles and their drug cargo. The new co-polymers, in which PEG and PLGA were linked by 'anchiomeric effector' dithiylethanoate esters, were synthesized by a combination of ring-opening polymerization and Michael addition reactions and employed to prepare NPs. Non redox-responsive nanoparticles (nRR-NPs) based on related PLGA-PEG copolymers were also prepared as comparators. Spherical NPs of around 120 nm diameter with a low polydispersity index and negative zeta potential as well as good drug loading of docetaxel were obtained. The NPs showed prolonged stability in relevant simulated biological fluids and a high ability to penetrate an artificial mucus layer due to the presence of the external PEG coating. Stability, FRET and drug release studies in conditions simulating intracellular reductive environments demonstrated a fast disassembly of the external shell of the NPs, thus triggering on-demand drug release. FACS measurements and confocal microscopy showed increased and faster uptake of RR-NPs in both 2D- and 3D- cell culture models of lung cancer compared to nRR-NPs. In particular, the 'designed-in' reductive instability of RR-NPs in conditioned cell media, the fast PEG release in the extracellular compartment, as well as a diminution of uptake rate in control experiments where extracellular thiols were neutralized, suggested a partial extracellular release of the PEG fringe that promoted rapid internalization of the residual NPs into cells. Taken together, these results provide further evidence of the effectiveness of PEGylated reducible nanocarriers to permeate mucus layer barriers, and establish a new means to enhance cancer cell uptake of drug carriers by extra-and intra-cellular cleavage of protein- and cell-shielding hydrophilic blocks., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Flucytosine (5-fluorocytosine, 5-FC) is a fluorinated analogue of cytosine currently approved for the systemic treatment of fungal infections, which has recently demonstrated a very promising antivirulence activity against the bacterial pathogen Pseudomonas aeruginosa. In this work, we propose novel inhalable hyaluronic acid (HA)/mannitol composite dry powders for repositioning 5-FC in the local treatment of lung infections, including those affecting cystic fibrosis (CF) patients. Different dry powders were produced in one-step by spray-drying. Powder composition and process conditions were selected after in depth formulation studies aimed at selecting the 5-FC/HA/mannitol formulation with convenient aerosolization properties and drug release profile in simulated lung fluids. The optimized 5-FC/HA/mannitol powder for inhalation (HyaMan_FC#3) was effectively delivered from different breath-activated dry powder inhalers (DPI) already available to CF patients. Nevertheless, the aerodynamic assessment of fine particles suggested that the developed formulation well fit with a low-resistance DPI. HyaMan_FC#3 inhibited the growth of the fungus Candida albicans and the production of the virulence factor pyoverdine by P. aeruginosa at 5-FC concentrations that did not affect the viability of both wild type (16HBE14o-) and CF (CFBE41o-) human bronchial epithelial cells. Finally, pharmacokinetics of HyaMan_FC#3 inhalation powder and 5-FC solution after intratracheal administration in rats were compared. In vivo results clearly demonstrated that, when formulated as dry powder, 5-FC levels in both bronchoalveolar lavage fluid and lung tissue were significantly higher and sustained over time as compared to those obtained with the 5-FC solution. Of note, when the same 5-FC amount was administered intravenously, no significant drug amount was found in the lung at each time point from the injection. To realize a 5-FC lung concentration similar to that obtained by using HyaMan_FC#3, a 6-fold higher dose of 5-FC should be administered intravenously. Taken together, our data demonstrate the feasibility to deliver 5-FC by the pulmonary route likely avoiding/reducing the well-known side effects associated to the high systemic 5-FC doses currently used in humans. Furthermore, our results highlight that an appropriate formulation design can improve the persistence of the drug at lungs, where microorganisms causing severe infections are located., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Combination therapies for cancer aim to exploit either additive or synergistic effects arising from the action of two species with the final goal to maximize the therapeutic efficacy. In this work, we develop multifunctional nanoparticles (NPs) for co-delivery of the conventional anticancer drug docetaxel (DTX) and the second generation photosensitizer zinc-phthalocyanine (ZnPc) as potential dual carrier system for the combination of chemotherapy and photodynamic therapy (PDT). Biodegradable and amphiphilic block copolymers based on poly(ε-caprolactone) (PCL=B) and poly(ethylene oxide) (PEO=A), with AB and ABA architectures, were assembled in "core-shell" NPs and loaded with both DTX and ZnPc employing the melting/sonication method. Hydrodynamic diameters within the range 60-100nm and low polydispersity indexes were obtained. Zeta potential was negative for all the formulations and unaffected by drug encapsulation. Concerning drug loading ability of NPs, the entrapment efficiency was related to initial ZnPc/DTX ratio. Steady-stationary and time-resolved emission fluorescence measurements pointed out the embedding of monomeric ZnPc in the NPs, excluding the presence of ZnPc self-supramolecular oligomers. The release of DTX was biphasic whereas ZnPc remained mainly associated with NPs. Singlet oxygen generation was observed when ZnPc-loaded NPs were irradiated at 610nm within a 45min time range, despite that ZnPc was not released in the medium. Stability of NPs in the presence of serum proteins and plasma was excellent and no toxicity toward red blood cells was found. NPs cytotoxicity was evaluated in HeLa cells irradiated for 30min with a halogen lamp. After 72h, viability of cells treated with ZnPc/DTX-loaded NPs strongly decreased as compared to NPs loaded only with DTX, thus showing a combined effect of both DTX and ZnPc. Superior antitumor activity of ZnPc/DTX-loaded NPs as compared to DTX-loaded NPs was confirmed in an animal model of orthotopic amelanotic melanoma, thus pointing to the application of PEO-PCL NPs in the combined chemo-photodynamic therapy of cancer., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In this paper we have investigated the behavior of core-shell poly(ethylene oxide)-poly(epsilon-caprolactone) (PEO-PCL) micelles derived from copolymers with linear triblock (TR) and 4-arm star-diblock (ST) architectures for the delivery of docetaxel (DTX). DTX was loaded inside micelles (DTX-TR(m) and DTX-ST(m)) with high efficiency and released slowly for more than two weeks. DTX-loaded micelles based on both copolymers had very similar properties in terms of mean size, zeta potential, loading ability and release rate in buffered saline. However, the stability of DTX-ST(m) was very poor in aqueous media with proteins resulting in a strong and progressive aggregation. We studied the effect of increasing concentrations of free DTX or DTX-loaded micelles on growth inhibition of human breast MCF-7 and MDA-MB468 and prostate PC3 and DU145 adenocarcinoma cell lines. DTX-loaded TR micelles induced cell growth inhibition similarly to free DTX whereas DTX-ST(m) showed lower cytotoxicity. On the other hand, by normalizing IC(50) values for the actual amount of DTX released from micelles in the medium, DTX-loaded ST micelles became more active than free DTX in all cell lines tested. Both free DTX and DTX-loaded TR micelles displayed a significantly lower cytotoxic activity in G(2)/M phase synchronized cells, whereas cytotoxicity of DTX-loaded ST micelles did not change. Cytotoxicity was related to micelle stability, uptake and release rate in cell culture media. Our results suggest that for a correct interpretation of cytotoxicity of nanocarriers, the evaluation of their behavior in biologically relevant conditions is of utmost importance to select proper systems for further in vivo testing., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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.

A promising approach to control the time and space distribution of signalling molecules inside tissue engineering scaffolds consists in entrapping biodegradable microspheres releasing the protein locally for long time frames. However, a rational design of microsphere-integrated scaffolds requires the knowledge of protein release profiles directly within the polymeric template. In this work, PLGA microspheres encapsulating rhodamine-labelled bovine serum albumin (BSA-Rhod) as a model protein were produced in different formulation conditions and tested for their release features in solution and in collagen and collagen/hyaluronic acid (HA) scaffolds. BSA-Rhod release profiles from single microspheres in solution and within the scaffold were assessed by using a confocal laser scanning microscopy (CLSM)-assisted method. Results suggest that the same diffusion-erosion process controls BSA-Rhod release from microspheres in solution and collagen. Nonetheless, two main factors contribute protein release within the scaffold, that is water activity in the release environment and transport properties of the protein in the gel. While microsphere formulation mainly controls the induction time necessary to activate protein release, polymer scaffold composition governs the release rate. Thus, the fine regulation of a tissue engineering construct may be obtained by an appropriate combination of microspheres and scaffolds, providing a spatial and temporal control over signalling molecule delivery.

The aim of this work was to study the influence of cyclodextrin (CD) incorporation on the properties of protein-loaded poly(lactide-co-glycolide) (PLGA) microspheres, with particular regards to protein release kinetics. To this purpose, insulin-loaded microspheres were prepared by spray-drying emulsion or solution formulations, with or without hydroxypropyl-beta-cyclodextrin (HPbetaCD), and fully characterized for encapsulation efficiency and release kinetics of both insulin and cyclodextrin. Homogeneous populations of spherical microparticles entrapping both insulin and HPbetaCD were obtained. In order to get an insight into insulin/HPbetaCD interactions occurring inside microspheres, Fourier transform infrared (FTIR) analysis in the Amide I region was performed. FTIR spectra of dried microspheres containing HPbetaCD showed a change in insulin secondary structure, attributed to the presence of insulin/HPbetaCD complexes within microspheres. Insulin release was affected by the presence of HPbetaCD depending on the initial formulation conditions. In the case of microspheres prepared from emulsion, cyclodextrin reduced only insulin burst, whereas in the case of microspheres obtained from solution, the overall insulin release rate was slowed down. Combining the release kinetics of HPbetaCD with the FTIR results on hydrated microspheres, it was concluded that the formation of insulin/HPbetaCD complexes inside microspheres is critical to decrease protein diffusivity in the polymer matrix and achieve an effective modulation of protein release rate.

The potential of spray-drying technique for the encapsulation in poly(lactide-co-glycolide) (PLGA) microspheres of bovine insulin, a poorly stable peptide, has been investigated. Insulin-loaded microspheres were prepared by spray-drying different feeding liquids containing insulin and PLGA, that is a S/O dispersion, a W/O emulsion or an acetic acid solution. In the case of the emulsion, insulin was also co-encapsulated with either non-ionic surfactants such as polysorbate 20 and poloxamer 188, or complexing agents such as HPbetaCD. In the microspheres prepared from the acetic acid solution of insulin and PLGA, HPbetaCD was tested. Microspheres containing surfactants were aggregated, whereas good quality particles displaying a mean diameter in the range 12.1-27.9 microm were produced in the other cases. Insulin was efficiently loaded inside microspheres except for S/O formulation (only 22% of total insulin content was entrapped). The impact of the microencapsulation process on insulin chemical and conformational stability was assessed by HPLC, circular dichroism and turbidimetry studies. Under the adopted manufacture conditions, insulin was encapsulated in the native state and its chemical and conformational stability was preserved along the fabrication process. The formulations containing only insulin displayed low burst effects (6-11%), whereas the addition of surfactants resulted in much higher burst effects (49-54%) and faster release rate. The co-encapsulation of HPbetaCD slowed down the overall release rate and, in the case of microspheres prepared from the emulsion, allowed a constant insulin release up to 45 days. The study of insulin stability along the release phase showed that insulin was released in the intact form and un-released insulin was stable inside all the microsphere formulations. We conclude that insulin can be effectively encapsulated in PLGA microspheres by the spray-drying technique. Additives with complexing properties such as HPbetaCD have demonstrated a potential in optimizing the release rate of insulin when used in microspheres prepared from W/O emulsions.

Segmented poly(ether-ester-amide)s (PEEAs) derived from poly(epsilon-caprolactone) oligomers, sebacoyl chloride, hydrophilic diamide-diamines based on short sequences of ethylenoxy groups and containing amino acids, were used to produce matrix systems intended for the delivery of metronidazole in the periodontal pocket. PEEAs are soluble in chloroform and insoluble in water and show M(n) values in the range 8.5-18.6 kDa. The melting temperatures (53-59 degrees C) are close to that of poly(epsilon-caprolactone) (PCL) with a similar M(n). The water absorption of PEEAs is improved if compared with that of pure PCL and depends on both the length of oxyethylene sequences and the amino acid number, as well as on copolymer composition. Loaded-films containing 20% (w/w) of metronidazole were prepared by compression-molding. The release rate was diffusive in the first stage, whereas also other mechanisms, probably polymer degradation, contributed to the slower second phase. The rate of medium penetration within the film depended on PEEA hydrophilicity and crystallinity and was the main determinant governing the drug release rate. The opportunity to control effectively drug release rates by modulating the composition, and in turn the properties, of PEEAs is an attracting feature for their use in a number of drug delivery systems.

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.

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.