Search

This contribution reports the design, synthesis, photochemical properties and drug inclusion capability of two novel β-cyclodextrin (βCD) conjugates, βCD-NBFNO1 and βCD-NBFNO2, covalently integrating an N-nitroso amino-nitro-benzofurazan in the primary and secondary hydroxyl rims of the βCD scaffold, respectively through flexible spacers of different length. Both βCD conjugates are water-soluble and release nitric oxide (NO) under the input of either blue or green light, with quantum yields ΦNO (blue) = 0.13, 0.31 and ΦNO (green) = 0.007, 0.013 respectively, the former representing the largest values ever reported for nonmetal-containing NO donors activatable by visible light. The good contrast between the fluorescence green emission of the chromogenic moiety after and before the NO release permits the easy and in real-time quantification of the amount of NO generated, without the addition of external fluorescent agents. Despite the presence of the appendages, these βCD derivatives are also able to complex betaxolol, a β-blocker drug widely used for the reduction of the intraocular pressure, with binding constants Kb = 500 ± 50 and 1100 ± 100 M−1, respectively, without affecting the photochemical performances. In view of the well-known vasodilator properties of NO, the present βCD derivatives represent intriguing candidates for biopharmaceutical research studies addressed to combined therapeutic ocular applications.

Core-shell polymeric nanoparticles (NPs) made of either di-block or tri-block poly-e-caprolactone and polyethylene glycol copolymers, covalently integrating Rhodamine B in the core or the shell have been prepared and a green fluorescent NO photodonor entrapped therein. One- and two-photon fluorescence experiments demonstrate that effective Forster Resonance Energy Transfer (FRET) occurs exclusively in the di-block NPs having the Rhodamine in the core, accounting for a localization of the NO photoreleaser in the inner part of the polymeric nanocarrier. These di-block NPs are stable in the presence of human serum albumin and their cargo release NO under exclusive excitation with visible light. Two-photon imaging experiments carried out using 900 nm NIR light, demonstrate that the release of the NO photodonor can be monitored in biological tissue, herein human skin, and provide insights into the integrity and penetration depth of the NPs. Toxicity experiments performed on NCTC keratinocyte cell lines in the dark and upon visible light irradiation show good biocompatibility of the polymeric system that therefore has a great potential in light of the multifaceted therapeutic role of NO.

With the aim to develop alternative therapeutic tools for the treatment of resistant cancers, here we propose targeted Pluronic(®) P123/F127 mixed micelles (PMM) delivering niclosamide (NCL) as a repositioning strategy to treat multidrug resistant non-small lung cancer cell lines. To build multifunctional PMM for targeting and imaging, Pluronic(®) F127 was conjugated with biotin, while Pluronic(®) P123 was fluorescently tagged with rhodamine B, in both cases at one of the two hydroxyl end groups. This design intended to avoid any interference of rhodamine B on biotin exposition on PMM surface, which is a key fundamental for cell trafficking studies. Biotin-decorated PMM were internalized more efficiently than non-targeted PMM in A549 lung cancer cells, while very low internalization was found in NHI3T3 normal fibroblasts. Biotin-decorated PMM entrapped NCL with good efficiency, displayed sustained drug release in protein-rich media and improved cytotoxicity in A549 cells as compared to free NCL (P

Nowadays, the downregulation of genes involved in the pathogenesis of severe lung diseases through local siRNA delivery appears an interesting therapeutic approach. In this study, we propose novel hybrid lipid-polymer nanoparticles (hNPs) consisting of poly(lactic-co-glycolic) acid (PLGA) and dipalmitoyl phosphatidylcholine (DPPC) as siRNA inhalation system. Background: Nowadays, the downregulation of genes involved in the pathogenesis of severe lung diseases through local siRNA delivery appears an interesting therapeutic approach. In this study, we propose novel hybrid lipid-polymer nanoparticles (hNPs) consisting of poly(lactic-co-glycolic) acid (PLGA) and dipalmitoyl phosphatidylcholine (DPPC) as siRNA inhalation system. Methods: A panel of DPPC/PLGA hNPs was prepared by emulsion/solvent diffusion and fully characterized. A combination of model siRNAs against the sodium transepithelial channel (ENaC) was entrapped in optimized hNPs comprising or not poly(ethylenimine) (PEI) as third component. siRNA-loaded hNPs were characterized for encapsulation efficiency, release kinetics, aerodynamic properties, and stability in artificial mucus (AM). The fate and cytotoxicity of hNPs upon aerosolization on a triple cell co-culture model (TCCC) mimicking human epithelial airway barrier were assessed. Finally, the effect of siRNA-loaded hNPs on ENaC protein expression at 72 hours was evaluated in A549 cells. Results: Optimized muco-inert hNPs encapsulating model siRNA with high efficiency were produced. The developed hNPs displayed a hydrodynamic diameter of approximate to 150nm, a low polydispersity index, a negative potential close to -25mV, and a peculiar triphasic siRNA release lasting for 5 days, which slowed down in the presence of PEI. siRNA formulations showed optimal in vitro aerosol performance after delivery with a vibrating mesh nebulizer. Furthermore, small-angle X-ray scattering analyses highlighted an excellent stability upon incubation with AM, confirming the potential of hNPs for direct aerosolization on mucus-lined airways. Studies in TCCC confirmed that fluorescent hNPs are internalized inside airway epithelial cells and do not exert any cytotoxic or acute proinflammatory effect. Finally, a prolonged inhibition of ENaC protein expression was observed in A549 cells upon treatment with siRNA-loaded hNPs. Conclusions: Results demonstrate the great potential of hNPs as carriers for pulmonary delivery of siRNA, prompting toward investigation of their therapeutic effectiveness in severe lung diseases.

The management of wounds in patients on anticoagulant therapy who require oral surgical procedures is problematic and often results in a nonsatisfactory healing process. Here, we report a method to prepare an advanced dressing able to avoid uncontrolled bleeding by occluding the postextractive alveolar wounds, and simultaneously, capable of a fast release of tranexamic acid (TA). Composite alginate/hyaluronan (ALG/HA) sponge dressings loaded with TA were prepared by a straightforward internal gelation method followed by a freeze-drying step. Both blank and drug-loaded sponges were soft, flexible, and elegant in appearance and nonbrittle in nature. Scanning electron microscopy analysis confirmed the porous nature of these dressings. The integration of HA influenced the microstructure, reducing the porosity, modifying the water uptake kinetic, and increasing the resistance to compression. TA release from ALG/HA sponges showed a controlled release up to 3 h, and it was faster in the presence of HA. Finally, an invitro clotting test performed on human whole blood confirmed that the TA-loaded sponges significantly reduce the blood clotting index by 30% compared with ALG/HA20 sponges. These results suggest that, if placed in a socket cavity, these dressings could give a relevant help to the blood hemostasis after dental extractions, especially in patients with coagulation disorders.

In the present work, we propose silk fibroin/alginate (SF/Alg) beads embedding simvastatin-loaded biodegradable microparticles as a versatile platform capable of tuning SVA release and in so doing osteogenic effects. In a first part of the study, microparticles of poly(lactic-co-glycolic) acid incorporating simvastatin either as lactone (SVL) or as hydroxyacid form (SVA) were prepared by spray-drying. While SVA-loaded microparticles released the drug in three days, long-term release of SVA could be obtained from SVL-loaded microparticles. In this latter case, SVL was promptly transformed to the osteogenic active SVA during release. When tested on mesenchymal stem cells, a time- and dose-dependent effect of SVL-loaded microparticles on cell proliferation and alkaline phosphatase (ALP) activity was found. Thereafter, SVL-loaded microparticles were embedded in SF/Alg beads to limit the initial simvastatin burst and to achieve easier implantation as well. Microparticle-embedded beads showed no cytotoxicity while ALP activity increased. If correctly exploited, the developed system may be suitable as osteogenic polymer scaffolds releasing correct amount of the drug locally for long time-frames.

The inflammatory process plays a crucial role in the onset and progression of several lung pathologies, including cystic fibrosis (CF),andthe involvement of NF-κBis widely recognized.Thespecific inhibition of NF-κB by decoy oligonucleotides delivered within the lung may be beneficial, although rationally designed systems are needed to optimize their pharmacological response. Prompted by this need, we have developed and tested in vivo an inhalable dry powder for the prolonged delivery of a decoy oligodeoxynucleotide to NF-κB (dec-ODN), consisting of large porous particles (LPPs) based on poly(lactic-co-glycolic) acid. First, LPPs containing dec- ODN (dec-ODN LPPs) were engineered to meet the aerodynamic criteria crucial for pulmonary delivery, to gain an effective loading of dec-ODN, to sustain its release, and to preserve its structural integrity in lung lining fluids. We then investigated the effects of dec- ODN LPPs in a rat model of lung inflammation induced by the intratracheal aerosolization of LPS from Pseudomonas aeruginosa. Theresults show that a single intratracheal insufflation of dec-ODN LPPs reduced the bronchoalveolar neutrophil infiltration induced by LPS for up to 72 hours, whereas naked dec-ODN was able to inhibit it only at 6 hours. The persistent inhibition of neutrophil infiltrate was associated with reduced NF-κB/DNA binding activity, as well as reduced IL-6, IL-8, and mucin-2 mRNA expression in lung homogenates. We consider it noteworthy that the developed LPPs, preventing the accumulation of neutrophils and NF-κB-related gene expression, may provide a new therapeutic option for the local treatment of inflammation associated with lung disease. Copyright © 2013 by the American Thoracic Society.

Effective delivery of tumor antigens to APCs is one of the key steps for eliciting a strong and durable immune response to tumors. Several cancer vaccines have been evaluated in clinical trials, based on soluble peptides, but results have not been fully satisfactory. To improve immunogenicity particles provide a valid strategy to display and/or incorporate epitopes which can be efficiently targeted to APCs for effective induction of adaptive immunity. In the present review, we report some leading technologies for developing particulate vaccines employed in cancer immunotherapy, highlighting the key parameters for a rational design to elicit both humoral and cellular responses.

In oral surgery, the management of patients on anticoagulant therapy is still challenging because of the risks of uncontrolled bleeding and, if the therapy is discontinued, the possibility of undesired thromboembolic complications. The use of local hemostatic agents may be a viable alternative to overcome these issues. Along these lines, the aim of this work was to evaluate the use of newly developed swelling matrices loaded with tranexanic acid in preventing postextractive bleeding in patients with no modification of oral anticoagulant therapy. The matrices are made up of cellulose and are able to fit the tridimensional postextractive alveolar cavity, thus assuring also a mechanical contribution to homeostasis. The potential of this new therapeutic approach in reducing hospitalization, removing the risk of infections, and lowering the number of hemorrhagic complications was demonstrated.

The molecular foundations of the use of (2-hydroxypropyl)-?- cyclodextrin (HP?CD) as solubility promoter of triamcinolone acetonide (TrA), a corticosteroid with very low aqueous solubility, was investigated by a multidisciplinary spectroscopic and computational approach. Aqueous solutions of TrA and HP?CD were investigated by UV and NMR spectroscopies. The association constant was determined by phase solubility diagrams and by the Foster-Fyfe method whereas the nature of the drug/cyclodextrin aggregates was probed by using the NMR DOSY technique. ROE measurements in solution led to stereochemical information regarding the nature of inclusion processes. TrA/HP?CD powders were prepared and investigated by Raman spectroscopy supported by computational methods. A molecular interaction of the hydroxyacyl chain with cyclodextrin, not identified in solution, was detected. Raman imaging experiments confirmed the attainment of a molecularly homogeneous system when the TrA/HP?CD molar ratio was 1:7 whereas TrA crystallized for mixtures richer in TrA (1:3.5) forming domains with size in the range of 10-15 ?m. We demonstrate that the combined use of several spectroscopical techniques with specific responsivities allows a detailed depiction of drug/cyclodextrin interaction useful in the development of novel pharmaceutical formulation. © 2012 Elsevier Ltd.

Nanotechnology development provides new strategies to treat cancer by integration of different treatment modalities in a single multifunctional nanoparticle. In this scenario, we applied the multifunctional Pluronic P123/F127 mixed micelles for Verteporfin-mediated photodynamic therapy in PC3 and MCF-7 cancer cells. Micelles functionalization aimed the targeted delivery by the insertion of biotin moiety on micelle surface and fluorescence image-based through rhodamine-B dye conjugation in the polymer chains. Multifunctional Pluronics formed spherical nanoparticulated micelles that efficiently encapsulated the photosensitizer Verteporfin maintaining its favorable photophysical properties. Lyophilized formulations were stable at least for 6months and readily reconstituted in aqueous media. The multifunctional micelles were stable in protein-rich media due to the dual Pluronic mixed micelles characteristic: high drug loading capacity provided by its micellar core and high kinetic stability due its biocompatible shell. Biotin surface functionalized micelles showed higher internalization rates due biotin-mediated endocytosis, as demonstrated by competitive cellular uptake studies. Rhodamine B-tagged micelles allowed monitoring cellular uptake and intracellular distribution of the formulations. Confocal microscopy studies demonstrated a larger intracellular distribution of the formulation and photosensitizer, which could drive Verteporfin to act on multiple cell sites. Formulations were not toxic in the dark condition, but showed high Verteporfin-induced phototoxicity against both cancer cell lines at low drug and light doses. These results point Verteporfin-loaded multifunctional micelles as a promising tool to further developments in photodynamic therapy of cancer.

The combination of conventional anticancer therapy with other treatment modalities such as photodynamic therapy (PDT) is paving the way to novel more effective treatment of solid tumors via light exposure. With this idea in mind, in this paper, nanoparticles (NPs) based on Heptakis (2-oligo(ethyleneoxide)-6-hexadecylthio-)-beta-CD (SC160H) for dual delivery of Zinc-Phthalocyanine (ZnPc) and Docetaxel (DTX) were developed pointing to their potential application as nanomedicine for the combined photodynamic and chemo-therapy of solid tumors. NPs prepared by the emulsion -solvent evaporation technique displayed a hydrodynamic diameter of congruent to 200 nm, a negative zeta potential (cAt 27 mV) and a satisfactory entrapment efficiency of both drugs at a specific mass ratio. On these bases, NPs containing DTX and ZnPc with theoretical loading of 5% and 0.2% respectively (ZnPc/DTX5-NPs) were selected for further investigations. The allocation of ZnPc and DTX into the colloid was investigated by complementary spectroscopic techniques. In particular, fluorescence emission studies showed the entrapment of ZnPc as a monomer in the carrier, with a low tendency to self -aggregate and consequently a fairly high propensity to photogenerate singlet oxygen. The interaction of SC160H with DTX, co-entrapped with ZnPc, was elucidated by 1H NMR and 2D ROESY, which suggested the presence of the chemotherapeutic in the hydrophobic portion of SC160H. ZnPc/DTX5-NPs were fairly stable in different biological relevant media within 24h. Finally, in vitro potential of the nanoassembly was evaluated in HeLa cancer cells by cell viability exploring both effects of DTX and ZnPc. Overall, results suggest the suitability of NPs based on SC16OH for delivering a combination of DTX with ZnPc to cancer cells, thus inducing photodynamic and antimitotic effects. (C) 2016 Elsevier B.V. All rights reserved.

In this study we attempted to develop Pluronic micelles delivering the photodynamic therapy photosensitizers benzoporphyrin derivatives (BPD). The BPD A-ring (BPDMA or Verteporfin ® , the active drug of FDA/USA approved Visudyne ® ), its regioisomer ring-B (BPDMB, not used in Visudyne ® formulation due its poor solubility) and a BPDMA/BPDMB mixture (BPD-Mixt) were formulated in Pluronic P123 or F127 as well as P123/F127 mixed micelles at two different mass ratios. P123/F127 presented the lowest critical micelle concentration showing high stability due synergistic aggregation of P123 and F127. Mixed micelles allowed the encapsulation of BPD as monomers enhancing their photophysical properties and stability during time even under diluted conditions. High loading was attributed to the strong hydrophobic affinity of BPD for micelle core especially in the binary system due synergistic aggregation of P123 and F127 demonstrating the high potential of these micelles to encapsulate hydrophobic drugs. The in vitro assays showed a photo-activity of BPD-Mixt comparable to that of BPDMA against HeLa and A549 cancer cells under red light. The use of BPD-mixed formulations avoids the complex separation steps of these regioisomers and implies in cost reduction. The proposed system allies costs reduction and photodynamic efficiency, which stimulates further development on this nanosystem and may be of clinical interest for cancer PDT.

We have recently demonstrated that the specific inhibition of nuclear factor-κB by a decoy oligonucleotide (dec-ODN) delivered through inhalable large porous particles (LPP) made of poly(lactic-co-glycolic acid) (PLGA) may be highly beneficial for long-term treatment of lung inflammation. Nevertheless, besides chronic inflammation, multifunctional systems aimed to control also infection are required in chronic lung diseases, such as cystic fibrosis (CF). In this work, we tested the hypothesis that engineering PLGA-based LPP with branched poly(ethylenimine) (PEI) may improve LPP properties for pulmonary delivery of dec-ODN, with particular regard to the treatment of Pseudomonas aeruginosa lung infections. After getting insight into the role of PEI on the technological properties of PLGA-based LPP for delivery of dec-ODN, the putative synergistic effect of PEI free or PEI released from LPP on in vitro antimicrobial activity of tobramycin (Tb) and aztreonam (AZT) against P. aeruginosa was elucidated. Meanwhile, cytotoxicity studies on A549 cells were carried out. Results clearly demonstrate that the dry powders have promising aerosolization properties and afford a prolonged in vitro release of both dec-ODN and PEI. The encapsulation of PEI into LPP results in a 2-fold reduction of the minimum inhibitory concentration of AZT, while reducing the cytotoxic effect of PEI. Of note, the developed ODN/PLGA/PEI LPP persisted at lung at least for 14 days after intratracheal administration in rats where they can provide sustained and combined release of dec-ODN and PEI. dec-ODN will likely act as an anti-inflammatory drug, while PEI may enhance the therapeutic activity of inhaled antibiotics, which are commonly employed for the treatment of concomitant lung infections. We have recently demonstrated that the specific inhibition of nuclear factor-kappa B by a decoy oligonucleotide (dec-ODN) delivered through inhalable large porous particles (LPP) made of poly(lactic-co-glycolic acid) (PLGA) may be highly beneficial for long-term treatment of lung inflammation. Nevertheless, besides chronic inflammation, multifunctional systems aimed to control also infection are required in chronic lung diseases, such as cystic fibrosis (CF). In this work, we tested the hypothesis that engineering PLGA-based LPP with branched poly(ethylenimine) (PEI) may improve LPP properties for pulmonary delivery of dec-ODN, with particular regard to the treatment of Pseudomonas aeruginosa lung infections. After getting insight into the role of PEI on the technological properties of PLGA-based LPP for delivery of dec-ODN, the putative synergistic effect of PEI free or PEI released from LPP on in vitro antimicrobial activity of tobramycin (Tb) and aztreonam (AZT) against P. aeruginosa was elucidated. Meanwhile, cytotoxicity studies on A549 cells were carried out. Results clearly demonstrate that the dry powders have promising aerosolization properties and afford a prolonged in vitro release of both dec-ODN and PEI. The encapsulation of PEI into LPP results in a 2-fold reduction of the minimum inhibitory concentration of AZT, while reducing the cytotoxic effect of PEI. Of note, the developed ODN/PLGA/PEI LPP persisted at lung at least for 14 days after intratracheal administration in rats where they can provide sustained and combined release of dec-ODN and PEI. dec-ODN will likely act as an anti-inflammatory drug, while PEI may enhance the therapeutic activity of inhaled antibiotics, which are commonly employed for the treatment of concomitant lung infections.

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.

// Annapina Russo 1 , Sara Maiolino 2 , Valentina Pagliara 1 , Francesca Ungaro 2 , Fabiana Tatangelo 3 , Alessandra Leone 3 , Giulia Scalia 4 , Alfredo Budillon 3 , Fabiana Quaglia 2, * , Giulia Russo 1, * 1 Laboratory of Biochemistry, Department of Pharmacy, University of Napoli Federico II, 80131 Napoli, Italy 2 Laboratory of Drug Delivery, Department of Pharmacy, University of Napoli Federico II, 80131 Napoli, Italy 3 Istituto Nazionale Tumori “Fondazione Pascale”-IRCCS, 80131 Napoli, Italy 4 CEINGE-Biotecnologie Avanzate, 80145 Napoli, Italy * These authors contributed equally to this work Correspondence to: Annapina Russo, email: annapina.russo@unina.it Keywords: 5-FU, p53, ribosomal protein rpL3, colon cancer, apoptosis Received: July 29, 2016 Accepted: October 14, 2016 Published: November 08, 2016 ABSTRACT Colon cancer is one of the leading causes of cancer-related death worldwide and the therapy with 5-fluorouracil (5-FU) is mainly limited due to resistance. Recently, we have demonstrated that nucleolar stress upon 5-FU treatment leads to the activation of ribosome-free rpL3 (L3) as proapoptotic factor. In this study, we analyzed L3 expression profile in colon cancer tissues and demonstrated that L3 mRNA amount decreased with malignant progression and the intensity of its expression was inversely related to tumor grade and Bcl-2/Bax ratio. With the aim to develop a combined therapy of 5-FU plus plasmid encoding L3 (pL3), we firstly assessed the potentiation of the cytotoxic effect of 5-FU on colon cancer cells by L3. Next, 10 μM 5-FU and 2 μg of pL3 were encapsulated in biocompatible nanoparticles (NPs) chemically conjugated with HA to achieve active tumor-targeting ability in CD44 overexpressing cancer cells. We showed the specific intracellular accumulation of NPs in cells and a sustained release for 5-FU and L3. Analysis of cytotoxicity and apoptotic induction potential of combined NPs clearly showed that the 5-FU plus L3 were more effective in inducing apoptosis than 5-FU or L3 alone. Furthermore, we show that the cancer-specific chemosensitizer effect of combined NPs may be dependent on L3 ability to affect 5-FU efflux by controlling P-gp (P-glycoprotein) expression. These results led us to propose a novel combined therapy with the use of 5-FU plus L3 in order to establish individualized therapy by examining L3 profiles in tumors to yield a better clinical outcomes.

Background Peptide based vaccines may suffer from limited stability and inefficient delivery to professional antigen-presenting cells (APCs), such as dendritic cells (DCs). In order to overcome such limitations, several types of biodegradable nanoparticles (NPs) have been developed as carrier system for antigens. The present study describes for the first time the extensive biological characterization of cationic NPs made of poly (D,L-lactide-co-glycolide) (PLGA) and polyethylenimine (PLGA/PEI) as delivery system for protein/peptide antigens, with potential in therapeutic cancer vaccine development. Results Flow cytometry as well as confocal laser scanning microscopy (CLSM) showed that PLGA/PEI NPs are more readily taken up than PLGA NPs by both human CD14+ monocytes and mouse Hepa 1–6 hepatoma cell line. No signs of toxicity were observed in either cellular setting. Sequential image acquisition by TEM showed an intracellular apical localization for PLGA NPs and a perinuclear localization for PLGA/PEI NPs. Both NPs showed a clathrin-dependent as well as a caveolin-dependent internalization pathway and, once in the cells, they formed multivesicular endosomes (MVE). Finally, an ex vivo priming experiment showed that PLGA/PEI NPs are comparable to PLGA NPs in delivering a non-self antigen (i.e., ovalbumin - OVA) to immature dendritic cells (imDCs), which matured and induced autologous naïve CD4+ T cells to differentiate to memory (i.e., central memory and effector memory) cells. Such a differentiation was associated with a Th1 phenotype suggesting a downstream activation and amplification of a CD8+ T cell cytotoxic response. The same OVA antigen in a soluble form was unable to induce maturation of DCs, indicating that both NP formulations provided an intrinsic adjuvanting effect combined to efficient antigen delivery. Conclusions Our study represents the first report on side-by-side comparison of PLGA and PLGA/PEI NPs as strategy for protein antigen delivery. PLGA/PEI NPs are superior for cellular uptake and antigen delivery as compared to PLGA NPs. Such an evidence suggests their great potential value for vaccine development, including therapeutic cancer vaccines. Electronic supplementary material The online version of this article (doi:10.1186/s13046-015-0231-9) contains supplementary material, which is available to authorized users.

The aim of this work was to produce lycopene-containing powders from tomato products by a solvent-free method making use of β-cyclodextrin (βCD). Powders were prepared by spray-drying a tomato concentrate (TC), one of the most bioavailable form of lycopene, after mechanical treatment with βCD in different weight ratios. The obtained product was centrifuged to eliminate partly food matrix and characterized for the amount of lycopene hydrodispersed/hydrosolubilized in the aqueous fraction. The chemical antioxidant activity of sera was evaluated too. Powders obtained by spray-drying sera exhibited good flow properties, a lycopene content between 0.4 and 1.09 mg/g and excellent water dispersability. The process developed, which makes use of βCD for the treatment of tomato products, turns to be of great interest to obtain a bulk material for nutraceuticals displaying superior biovailability of lycopene.

The aim of this work was to investigate the potential of small nanoparticles (NPs) made of a poly(ethylene glycol)-poly(ε-caprolactone)-amphiphilic diblock copolymer (PEG-. b-PCL, PEG. =. 2. kDa and PCL. =. 4.2. kDa) as drug carrier system through the skin. Zinc(II) phthalocyanine (ZnPc), selected as lipophilic and fluorescent model molecule, was loaded inside NPs by a melting/sonication procedure. Loaded NPs with a hydrodynamic diameter around 60. nm, a slightly negative zeta potential and a ZnPc entrapment dependent on polymer/ZnPc ratio were obtained. Spectroscopic investigations evidenced that ZnPc was entrapped in monomeric form maintaining its emission properties. The transport of ZnPc through porcine ear skin was evaluated on Franz-type diffusion cells after treatment with different vehicles (water or PEG 0.4. kDa) containing free ZnPc or ZnPc-loaded NPs without and with (2-hydroxypropyl)-β-cyclodextrin (HPβCD) as permeation enhancer. Independently of the sample tested, ZnPc was transported in the skin without reaching receptor compartment. On the other hand, ZnPc was found in the skin in large amount and also in the viable epidermis when delivered through NPs associated with HPβCD, especially in conditions limiting water evaporation. Fluorescence images of skin samples after 24. h of permeation were in line with ZnPc dosage in the skin and demonstrated the ability of NPs covalently tagged with rhodamine to penetrate the skin and to locate in the intercellular spaces. Insight into skin chemical properties upon application of NPs by confocal Raman spectroscopy demonstrated that HPβCD caused an alteration of water profile in the skin, highly reducing the degree of hydration at stratum corneum/viable epidermis interface which can promote NP transport. Taken together, these results highlight PEG-. b-PCL NPs coupled with HPβCD as a novel vehicle for the skin delivery of highly lipophilic compounds paving the way to several applications.

The objective of this study was to design and in vitro–in vivo evaluate oral nanocapsules prepared from amphiphilic cyclodextrins (CDs) or poly-ε-caprolactone (PCL) for the effective oral delivery of an anticancer agent, camptothecin (CPT). CPT-loaded anionic and Chitosan (CS)-coated cationic nanocapsules were prepared and characterized in vitro. Morphological analysis was performed by scanning electron microscope (SEM). CPT release profile was evaluated using dialysis method under sink conditions. To determine the protective effect and drug stability provided by nanocapsules, all the formulations were incubated in simulated gastrointestinal media. Measurement of mucoadhesive tendency of CPT-loaded nanocapsules was realized by turbidimetric method. Penetration of nanocapsules was performed through an artificial mucus model. The permeability of CPT in solution form and bound to nanocapsule formulations were demonstrated across Caco-2 cell line. Finally, the intestinal uptake of nanocapsules was evaluated in vivo, in a mouse model. Both anionic and cationic formulations were in the range of 180–220 nm with a narrow size distribution and desired zeta potential values. CPT-loaded nanocapsules were found to be stable in simulated gastrointestinal media. Turbidimetric measurements confirmed the interaction between nanoparticles and mucin. Penetration of CPT through an artificial mucus gel layer was higher with CS-coated nanocapsules in accordance with the results obtained from permeability studies across Caco-2 cell line. In vivo animal studies confirmed that the intestinal uptake of nanocapsules was significantly higher with cationic nanocapsules. CPT-loaded positively charged CD nanocapsules might be an attractive and promising treatment for oral chemotherapy.

Inhaled antivirulence drugs are currently considered a promising therapeutic option to treat Pseudomonas aeruginosa lung infections in cystic fibrosis (CF). We have recently shown that the anthelmintic drug niclosamide (NCL) has strong quorum sensing (QS) inhibiting activity against P. aeruginosa and could be repurposed as an antivirulence drug. In this work, we developed dry powders containing NCL nanoparticles that can be reconstituted in saline solution to produce inhalable nanosuspensions. NCL nanoparticles were produced by high-pressure homogenization (HPH) using polysorbate 20 or polysorbate 80 as stabilizers. After 20 cycles of HPH, all formulations showed similar properties in the form of needle-shape nanocrystals with a hydrodynamic diameter of approximately 450 nm and a zeta potential of -20 mV. Nanosuspensions stabilized with polysorbate 80 at 10% w/w to NCL (T80_10) showed an optimal solubility profile in simulated interstitial lung fluid. T80_10 was successfully dried into mannitol-based dry powder by spray drying. Dry powder (T80_10 DP) was reconstituted in saline solution and showed optimal in vitro aerosol performance. Both T80_10 and T80_10 DP were able to inhibit P. aeruginosa QS at NCL concentrations of 2.5-10 mu M. NCL, and these formulations did not significantly affect the viability of CF bronchial epithelial cells in vitro at microbiologically active concentrations (i.e.

In this paper we propose an in situ forming ionically cross-linked alginate (Alg) hydrogel delivering a Tea Tree Oil microemulsion (MeTTO) and potentially useful as an advanced dressing for infected wounds. Alg hydrogels were prepared by a spray-by-spray deposition method with the aim to minimize the discomforts during application. From pseudoternary phase diagrams, it was found that proper combination of TTO, water, polysorbate 80 and ethanol gave stable spherical MeTTO with good antimicrobial activity. On this basis, MeTTO at 20% TTO was selected for further inclusion in an Alg hydrogel prepared by alternating sprays of Alg/MeTTO and calcium chloride solutions. Homogeneous dispersion of MeTTO inside cross-linked Alg was assessed by different macroscopic and microscopic methods demonstrating the superior propensity of MeTTO to be integrated in the water-based hydrogel as compared to TTO. Antimicrobial effect of Alg/MeTTO hydrogels on Escherichia Coli strains was remarkable, highlighting the potential of the system as bioactive wound dressing.

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.

Pulmonary drug delivery represents the best way of treating lung diseases, since it allows direct delivery of the drug to the site of action, with few systemic effects. Meanwhile, the lungs may be used as a portal of entry to the body, allowing systemic delivery of drugs via the airway surfaces into the bloodstream. In both cases, the therapeutic effect of the inhaled drug can be optimized by embedding it in appropriately engineered inhalable carriers, which can protect the drug against lung defense mechanisms and promote drug transport across the extracellular and cellular barriers. To this purpose, the attention has been very recently focused on polymeric nanoparticles (NPs). The aim of this review is to offer an overview on the recent advances in NPs for pulmonary drug delivery. After a description of the main challenges encountered in developing novel inhaled products, the design rules to engineer polymeric NPs for inhalation, and in so doing to overcome barriers imposed by the lungs anatomy and physiology, are described. Then, the state-of-art on inhalable biocompatible polymeric NPs based on enzymatically-degradable natural polymers and biodegradable poly(ester)s is presented, with a special focus on NP-based dry powders for inhalation. Finally, the in vitro/in vivo models useful to address the never-ending toxicological debate related to the use of NPs for inhalation are described.

In the attempt to develop novel concepts in designing targeted nanoparticles for combination therapy of cancer, we propose here CD44-targeted hyaluronan-decorated double-coated nanoparticles (dcNPs) delivering the lipophilic chemotherapeutic docetaxel (DTX) and an anionic porphyrin (TPPS4). dcNPs are based on electrostatic interactions between a negative DTX-loaded nanoscaffold of poly(lactide-co-glycolide), a polycationic shell of polyethyleneimine entangling negatively-charged TPPS4 and finally decorated with hyaluronan (HA) to promote internalization through CD44 receptor-mediated endocytosis. DTX/TPPS4-dcNPs, prepared through layer-by-layer deposition, showed a hydrodynamic diameter of around 180 nm, negative zeta potential and efficient loading of both DTX and TPPS4. DTX/TPPS4-dcNPs were freeze-dried with trehalose giving a powder that could be easily dispersed in different media. Excellent stability of dcNPs in specific salt- and protein-containing media was found. Spectroscopic behavior of DTX/TPPS4-dcNPs demonstrated a face-to-face arrangement of the TPPS4 units in non-photoresponsive H-type aggregates accounting for an extensive aggregation of the porphyrin embedded in the shell. Experiments in MDA-MB-231 cells overexpressing the CD44 receptor demonstrated a 9.4-fold increase in the intracellular level of TPPS4 delivered from dcNPs as compared to free TPPS4. Light-induced death increased tremendously in cells that had been treated with a combination of TPPS4 and DTX delivered through dcNPs as compared with free drugs, presumably due to efficient uptake and co-localization inside the cells. In perspective, the strategy proposed here to target synergistic drug combinations through HA-decorated nanoparticles seems very attractive to improve the specificity and efficacy of cancer treatment.

Background Nuclear factor-κB (NF-κB) transcription factor regulates the expression of genes involved in immune response and inflammation. NF-κB activity can be efficiently inhibited by double-stranded oligodeoxynucleotides (ODNs). In the present study, we investigated the potential of poly(DL-lactic-co-glycolic acid) (PLGA) microspheres as delivery system for an ODN against NF-κB in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). Methods Microspheres encapsulating ODN were prepared by the multiple emulsion/solvent evaporation technique and characterised in terms of size, morphology, encapsulation efficiency and in vitro release profile. In vitro uptake after 4 h and activity of ODN released from microspheres were evaluated in RAW 264.7 macrophages stimulated with LPS for 24, 48 and 72 h. Results We prepared microspheres with a high encapsulation efficiency showing a very slow and almost constant in vitro release of ODN for up to 1 month. ODN slowly released from microspheres translocated better into LPS-stimulated cells as compared with naked ODN. Incubation of cells with ODN-encapsulating microspheres resulted in a decrease of tumor necrosis factor-alpha (TNF-α) and nitrite production, inducible nitric oxide synthase (iNOS) protein expression, as well as NF-κB/DNA-binding activity. Similar results were obtained with naked ODN only at about 80 times higher concentrations. Conclusions Our results suggest that PLGA microspheres could be a useful tool to improve pharmacokinetics of a ODN decoy to NF-κB and may represent a promising strategy to effectively inhibit the transcriptional activity of NF-κB in inflammatory process. Copyright © 2005 John Wiley & Sons, Ltd.

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.

With the aim to find novel therapeutical approaches for triple-negative breast cancer (TNBC) treatment, we have developed a powder for i.v. injection based on cyclodextrins and docetaxel (DTX)-loaded polyethyleneglycol-poly(epsilon-caprolactone) nanoparticles (DTX-NPs). Nanoparticles are designed to concentrate at tumor level by enhanced permeability and retention effect and release drug cargo at a sustained rate in the blood and in tumor interstitium. DTX-NPs of around 70 nm, shielding proteins and allowing a sustained DTX release for about 30 days, were produced by melting sonication technique. DTX-NPs were associated to hydroxypropyl-β-cyclodextrin to give a powder for injection with excellent dispersibility and suitable for i.v. administration. DTX-NPs were as efficient as free DTX in inhibiting cell growth of MDA-MB231 cells, even at low concentrations, and displayed a comparable in vivo antitumor efficacy and better survival in a TNBC animal model as compared with DTX commercial formulation (Taxotere®). In conclusion, PEGylated biodegradable DTX-NPs highlighted their potential in the treatment of aggressive TNBC providing a foundation for future clinical studies.

Photodynamic therapy (PDT) is a clinically approved, minimally invasive procedure that can exert a cytotoxic activity toward malignant cells. The procedure involves administration of a photosensitizer (PS) followed by irradiation with light at wavelengths within of the PS absorption band. In the presence of oxygen, a series of events lead to direct tumor cell death, damage to the microvasculature, and induction of a local inflammatory reaction. Clinical studies reveal that PDT can be curative, particularly in early stage tumors, can prolong survival in patients with inoperable cancers, and can significantly improve quality of life. Unfortunately, most PS lack specificity for tumor cells and this can result in undesirable side effects in healthy tissues. Furthermore, due to their mostly planar structure, PS form aggregates with low photoactivity in an aqueous environment. Nanotechnology offers a great opportunity in PDT based on the concept that a nanocarrier can drive therapeutic concentrations of PS to the tumor cells without generating any harmful effect in vivo. Currently, several nanoscale carriers made of different materials such as lipids, polymers, metals, and inorganic materials have been proposed in nano-PDT. Each type of system highlights pros and cons and should be selected on the basis of delivery requirements. In the following, we describe the principle of PDT and its application in the treatment of cancer. Then, we illustrate the main systems proposed for nano-PDT that demonstrated potential in preclinical models together with emerging concepts for their advanced design.

A β-cyclodextrin nanosponge cross-linked with pyromellitic dianhydride (βNS-PYRO) is reported for the first time as multifunctional ingredient in semisolid formulations for drug delivery to the skin. The role of βNS-PYRO on solubilization and stabilization of the photosensitizer benzoporphyrin-derivative monoacid ring A (BPDMA) and all-trans retinoic acid (atRA) as well as its effect on skin permeation of diclofenac (DIC) was investigated. Aqueous solutions, gels, and cream-gels were prepared from mixtures of βNS-PYRO with a conventional gelling agent at specific ratios. The incorporation of BPDMA in βNS-PYRO water solutions prevented its aggregation and gave kinetically stable complexes with high photostability and singlet oxygen generation upon irradiation. atRA incorporated in the βNS-PYRO-containing gel demonstrated a remarkable stability as compared with the formulation without βNS-PYRO, resulting in an eightfold increase of its lifetime. Skin permeation studies highlighted that βNS-PYRO in gels and cream-gels containing DIC significantly decreased the amount of drug permeated through the skin while increasing its amount in stratum corneum and viable epidermis. Overall, swellable βNS-PYRO turns to be a multifunctional coingredient with potential in topical monophasic and biphasic formulations to stabilize light-sensitive drugs and to localize the action of highly penetrating drugs in the external layers of skin.

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.

Due to their poor solubility and propensity to aggregate in aqueous media, therapeutic application of several photosensiting agents, such as phthalocyanines, in photodynamic therapy (PDT) of solid tumors is severely hampered. With the aim to propose a novel nanotechnological approach, in this paper biodegradable nanoassemblies based on heptakis (2-oligo(ethyleneoxide)-6-hexadecylthio-)-??-CD (SC16OH) and zinc-phthalocyanine (ZnPc) were developed and tested. Nanoassemblies, prepared by the emulsion???solvent evaporation technique, displayed a hydrodynamic diameter around 200 nm, a negative zeta potential and a satisfactory entrapment efficiency of ZnPc. Steady-state and time resolved fluorescence emission spectroscopy studies showed the entrapment of ZnPc as a monomer in the carrier, with a low tendency to self-aggregate and consequently a fairly good propensity to generate singlet oxygen after photoactivation. The interaction of ZnPc with SC16OH was elucidated by 1H-NMR, which suggested the formation of complexes between drug and both hydrophobic and hydrophilic moieties of the amphiphile. Finally, in vitro potential of the nanoassembly was evaluated in HeLa cells by following cellular uptake and photobiological activity. Overall, results suggest the suitability of the nanoassembly based on SC16OH for delivering ZnPc to cancer cells, thus inducing photodynamic anticancer effects.

Several PEGylated polyester-based nanoncologicals have been proposed in the literature, some of them nowadays being under preclinical/clinical trials or marketed. In this review, we describe the main features of PEGylated polyesters and their correspondent nanocarriers. A first part is devoted to intravenously injectable PEGylated nanocarriers, which represent the systems most investigated so far. After describing fundamental design rules dictated by the administration route, PEGylated nanocarriers currently under preclinical/clinical investigation or in the market will be described from a technological point of view and related therapeutic implications discussed. Finally, new perspective of use of PE-Gylated nanocarriers for oral and pulmonary delivery of anticancer drugs will be considered. © 2014 Bentham Science Publishers.

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.

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(epsilon-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 60-100 nm 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 610 nm within a 45 min 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 30 min with a halogen lamp. After 72 h, 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. (C) 2013 Elsevier B.V. All rights reserved.

Inspired by the multiple roles cyclodextrins can play in polymeric systems, here we engineered poly(ethylene oxide) (PEO) films with (2-hydroxypropyl)- β-cyclodextrin (CD) as multipurpose ingredient. To shed light on the potential of CD in formulating PEO buccal films for the delivery of poorly water-soluble drugs, we preliminarily assessed thermal and mechanical properties as well as wettability of films prepared at different PEO/CD ratios. PEO/CD platform containing 54% by weight of CD was chosen as the optimized composition since it matched acceptable mechanical properties, in terms of tensile strength and elasticity, with a good wettability. The platform was tested as buccal delivery system for triamcinolone acetonide (TrA), a lipophilic synthetic corticosteroid sparely water soluble. Confocal Raman imaging clearly showed that CD was homogeneously (i.e. molecularly) dispersed in PEO. Nevertheless, homogenous drug distribution in the film without TrA crystallization occurred only in the presence of CD. Finally, CD-containing PEO film placed in simulated buccal fluids provided a useful speed-up of TrA release rate while showing slower dissolution as compared to PEO film. These results, as well as compliance with quality specifications of pharmaceutical manufacturing products, strongly support the soundness of the strategy and prompt toward further applications of PEO/CD films in buccal drug delivery. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.

New vessel formation is the result of the complex orchestration of various elements, such as cells, signalling molecules and extracellular matrix (ECM). In order to establish the suitable conditions for an effective cell response, the influence of vascular endothelial growth factor (VEGF) complexation with heparin sodium salt (Hp) on its pro-angiogenic activity has been evaluated by an in vitro capillary-like tube formation assay. VEGF with or without Hp was embedded into collagen gels, and the activated matrices were characterized in terms of VEGF activity and release kinetics. Taking into account the crucial role of Hp in VEGF stability and activity, VEGF/Hp complex was then encapsulated into microspheres based on poly(lactide-co-glycolide) (PLGA), and microsphere properties, VEGF/Hp release kinetics and VEGF in vitro activity over time were evaluated. Integrated microsphere/collagen matrices were developed in order to provide a continuous release of active VEGF/Hp inside the matrix but also a VEGF gradient at the boundary, which is an essential condition for endothelial cell attraction and scaffold invasion. The results confirmed a strong influence of Hp on VEGF configuration and, consequently, on its activity, while the encapsulation of VEGF/Hp complex in PLGA-microspheres guaranteed a sustained release of active VEGF for more than 30 days. This paper confirms the importance of VEGF stability and signal presentation to cells for an effective proangiogenic activity and highlights how the combination of two stabilizing approaches, namely VEGF/Hp complexation and entrapment within PLGA-based microspheres, may be a very effective strategy to achieve this goal. © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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. Key findings Biodegradable polymer carriers, such as PLGA particles, may permit effective protection and long-term delivery of the inhaled drug and, when adequately engineered, its efficient transport to the target. The carrier can be designed for inhalation on the basis of several strategies through the adequate combination of available particle technologies and excipients. In so doing, the properties of PLGA particles can be finely tuned at micro-size and nano-size level to fulfill specific therapeutic needs. This means not only to realize optimal in vitro/in vivo lung deposition of the formulation, which is still crucial, but also to control the fate of the drug in the lung after particle landing. Summary Although many challenges still exist, PLGA carriers may be highly beneficial and present a new scenario for patients suffering from chronic lung diseases and for pharmaceutical companies working to develop novel inhaled products.

Amphiphilic block copolymers of poly(ɛ-caprolactone) and poly(ethylene oxide) were assembled in core-shell nanoparticles (NPs) by a melting-sonication technique (MeSo). The entrapment of the poorly water-soluble anticancer drug docetaxel (DTX), nanocarrier cytotoxicity toward different cells and toxicity in mice were investigated. The encapsulation mechanism was rationalized and related to copolymer properties such as crystallinity and drug solubility in the copolymer phase. DTX release from NPs occurred in 2 drug pulses over 30 days. DTX entrapment in NPs strongly decreased haemolysis of erythrocytes in comparison with a commercial DTX formulation. In comparison with free DTX, NPs were both more efficient in inhibiting cell growth of breast and prostate cancer cells and less toxic in experimental animal models. The results of this study indicate that MeSo is an interesting technique for the achievement of peculiar core-shell nanocarriers for the passive targeting and sustained release of poorly water-soluble anticancer drugs. From the Clinical Editor In this study, stealth nanoparticles of PEO/PCL block copolymers for passive targeting of docetaxel to solid tumors were developed using a novel technique. The studied properties of NPs suggest strong potential as anticancer drug-delivery system.

A specific and promising approach to limit inflammation and mucin iperproduction in chronic lung diseases relies on specific inhibition of nuclear Factor-κB (NF-κB) by a decoy oligonucleotide (dec-ODN). To fulfill the requirements dictated by translation of dec-ODN therapy in humans, inhalable dry powders were designed on a rational basis to provide drug protection, sustained release and to optimize pharmacological response. To this end, large porous particles (LPP) for dec-ODN delivery made of a sustained release biomaterial (poly(lactic-co-glycolic) acid, PLGA) and an "adjuvant" hydrophilic polymer (polyethylenimine, PEI) were developed and their effects on LPS-stimulated human airway epithelial cells evaluated. The composite PLGA/PEI particles containing dec-ODN (i.e., LPP(PEI)) were successfully engineered for widespread deposition in the lung and prolonged release of intact dec-ODN in vitro. LPP(PEI) caused a prolonged inhibition of IL-8 and MUC2 expression in CF human bronchial epithelial cells and human epithelial pulmonary NCI-H292 cells, respectively, as compared to naked dec-ODN. Nonetheless, as compared to previously developed LPP, the presence of PEI was essential to construct a dec-ODN delivery system able to act in mucoepidermoid lung epithelial cells. In perspective, engineering LPP with PEI may become a key factor for tuning carrier properties, controlling lung inflammation and mucin production which, in turn, can foster in vivo translation of dec-ODN therapy.

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 success of any tissue engineering implant relies upon prompt vascularization of the cellular construct and, hence, on the ability of the scaffold to broadcast specific activation of host endothelium and guide vessel ingrowth. Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulator, and if released in a controlled manner it may enhance and guide scaffold vascularization. Therefore, the aim of this work was to realize a scaffold with integrated depots able to release VEGF in a controlled rate and assess the ability of this scaffold to promote angiogenesis. VEGF-loaded poly(lactide-co-glycolide) (PLGA) microspheres were produced and included in a collagen scaffold. The release of VEGF from microspheres was tailored to be sustained over several weeks and occurred at a rate of ∼0.6 ng/day per mg of microspheres. It was found that collagen scaffolds bioactivated with VEGF-loaded microspheres strongly enhanced endothelial cell activation and vascular sprouting both in vitro and in vivo as compared with a collagen scaffold bioactivated with free VEGF. This report demonstrates that by finely tuning VEGF release rate within a polymeric scaffold, sprouting of angiogenic vessels can be guided within the scaffolds interstices as well as broadcasted from the host tissues. © 2009 Wiley Periodicals, Inc.

The aim of this study was to develop nanoparticles made of the amphiphilic cyclodextrin heptakis (2- O -oligo(ethyleneoxide)-6-hexadecylthio-)-β-CD (SC16OH) entrapping docetaxel (Doc) and establish their in vivo potential. Doc-loaded SC16OH nanoparticles were prepared by the emulsion–solvent evaporation technique and fully characterized for size, zeta potential, amount of entrapped drug, release rate and degradation rate. Spherical vesicular nanoparticles displaying a hydrodynamic radius of about 95 nm which did not change upon storage as an aqueous dispersion, a negative zeta potential and entrapment efficiency of Doc very close to 100% were produced. DSC study highlighted the crystalline nature of SC16OH, unloaded and Doc-loaded SC16OH nanoparticles which resulted in their very slow dissolution during release stage and well-modulated release of entrapped Doc for about 8 weeks. Doc-loaded SC16OH nanoparticles were not hemolytic toward red blood cells as compared to a commercial Doc formulation (Taxotere ® ) which shows a dose-dependent toxicity. After exposure of HEp-2 cells to equivalent doses of free Doc and Doc-loaded SC16OH nanoparticles, superior cell killing and cell damage were observed for nanoparticles. Finally, cell damage was attributed to aberrant mitosis which was found to be significantly higher for HEp-2 cells treated with Doc-loaded SC16OH nanoparticles as compared to free Doc likely due to the ability of nanoparticles to slowly release the drug allowing prolonged cell arrest in mitosis. Taken together, these results highlights a great potential of nanoparticles based on SC16OH in solid tumors therapy.

Nuclear factor-kappaB (NF-kappaB) plays a key role in the expression of several genes involved in the immune and inflammatory process. Previously, we demonstrated that NF-kappaB activation can be significantly inhibited by a double stranded oligodeoxynucleotide (ODN). Nevertheless, the therapeutic use of ODN requires a delivery system able to improve poor crossing of cell membranes and rapid in vivo enzymatic degradation. Poly(D,L-lactide-co-glycolide) (PLGA) microspheres can increase ODN stability in biological environment and release the encapsulated drug in long time frames. Here, we used a decoy ODN against NF-kappaB and we investigated its effect, when administered in naked form or when delivered by PLGA microspheres, in a rat model of chronic inflammation. The subcutaneous implant of lambda-carrageenin-soaked sponges caused leukocyte infiltration and formation of granulation tissue which were inhibited up to 15 days by co-injection of microspheres releasing decoy ODN whereas naked decoy ODN showed this effect only up to 5 days. Molecular analysis performed on granulation tissue demonstrated an inhibition of NF-kappaB activation correlated to a decrease of tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS) expression. Our results suggest that microspheres could be an useful tool to improve pharmacokinetics of decoy ODN and may represent a strategy to inhibit NF-kappaB activation in chronic inflammation.

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.

The influence of Hyaluronic acid (HA) on the gelation properties of Poloxamers blends has been studied with the aim of engineering thermosensitive and mucoadhesive polymeric platforms for drug delivery. The gelation temperature (Tgel), viscoelastic properties and mucoadhesive force of the systems were investigated and optimised by means of rheological analyses. Poloxamers micellar radius was evaluated by Photon Correlation Spectroscopy (PCS). Moreover in order to explore the feasibility of these platforms for drug delivery, the optimised systems were loaded with acyclovir and its release properties studied in vitro. By formulating Poloxamers/HA platforms, at specific concentrations, it was possible to obtain a thermoreversible gel with a Tgel close to body temperature. The addition of Low Molecular Weight HA did not hamper the self assembling process of Poloxamers just delaying the gelation temperature of few Celsius degrees. Furthermore, HA presence led to a strong increase of the Poloxamer rheological properties thus indicating possible HA interactions with micelles through secondary bonds, such as hydrogen ones, which reinforce the gel structure. These interactions could also explain PCS results which show, in systems containing HA, aggregates with hydrodynamic diameters much higher than those of Poloxamer micelles. Mucoadhesion experiments showed a rheological synergism between Poloxamers/HA gels and mucin dispersion which led to a change of the flow behaviour from a quite Newtonian one of the separate solutions to a pseudoplastic one of their mixture. In vitro release experiments indicated that the optimised platform was able to prolong and control acyclovir release for more than 6 hours

The aim of this work was to develop dry powders intended for insulin pulmonary delivery. To this purpose, large porous particles (LPP) made of poly(lactide-co-glycolide) (PLGA) were produced by the double emulsion-solvent evaporation technique. Hydroxypropyl-beta-cyclodextrin (HPbetaCD), also known as absorption enhancer for pulmonary protein delivery, was tested as aid excipient to optimize the aerodynamic behaviour of the microparticles. Several microsphere formulations, differing in HPbetaCD and insulin loadings, were produced and their properties compared. A contemporary release of insulin and HPbetaCD from the system can be achieved by selecting appropriate formulation conditions. HPbetaCD-containing LPP with flow properties and dimensions suitable for aerosolization and deposition in deep regions of the lung following inhalation were produced. In conclusion, the developed system turns to be of great potential for the combined delivery of the protein and the adsorption promoter in the respiratory tract.

Anthracyclines are among the most active antineoplastic drugs developed to date, used both in the treatment of solid cancers, such as breast and ovarian cancer and sarcomas, and of hematologic cancers. However, their clinical use is limited by cardiotoxicity, which is observed at a range of 0.4-41\%. The risk of this side effect can be minimized by using cardioprotective agents, planning dosing schedules to lower the anthracycline peak plasma concentration, identifying and monitoring high-risk patients, keeping in mind that early anthracycline-induced histologic changes may be identified successfully by cardiac biopsy. Nonetheless, the challenge to increase the tumor response to chemotherapy while keeping low the cardiac risk may be now met by the use of a recently introduced polyethylene glycol-coated (pegylated) liposomal doxorubicin (PLD). Here, we review the pharmacologic properties of PLD as well as the results of phases I, II and III trials demonstrating activity and low cardiac toxicity associated with the use of this novel drug.

This study was carried out with the aim to optimize the pharmacological profile of gliquidone (GLI)--a poorly bioavailable hypoglycaemic agent sparingly soluble in water--through complexation with cyclodextrins. In order to increase the apparent solubility of GLI, two cyclodextrins, namely beta-cyclodextrin (betaCD) and hydroxypropyl-beta-cyclodextrin (HPbetaCD), were tested. The effect of cyclodextrin addition on the aqueous solubility of GLI was evaluated by the phase solubility method at different pH values. The amount of GLI in solution increased upon CD addition according to A type plots. The aqueous solubility of GLI was enhanced more at higher pH values and using HPbetaCD. On the basis of its performance, HPbetaCD was selected as host to prepare GLI oral formulations. GLI/HPbetaCD solid systems were prepared at 1:2 molar ratio by co-grinding, spray-drying and freeze-drying and characterized by DSC, FTIR and X-ray powder diffractometry. Powders were amorphous and showed an improved dissolution rate in comparison with GLI. GLI/HPbetaCD co-ground and freeze-dried products were the most interesting systems, since they dissolved 62 and 94% of total drug after 15 min, respectively. The hypoglycaemic effect of the most rapidly dissolving binary systems was evaluated after oral administration in fasted rats by measuring plasma glucose level in the time interval 0.5-36 h and compared to free GLI. Our findings indicate that cyclodextrin-containing formulations not only provide an onset of hypoglycaemic effect faster than GLI, but also enhance significantly the pharmacological effect due to improved biopharmaceutics. The association GLI/HPbetaCD allows a reduction of the oral dose and is expected to provide a better control over drug side effects, contributing to improve safety and efficacy of GLI.

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.