Nano into Micro Formulations of Tobramycin for the Treatment of Pseudomonas aeruginosa Infections in Cystic Fibrosis.

Here, nano into micro formulations (NiMs) of tobramycin for the treatment of Pseudomonas aeruginosa airway infections in cystic fibrosis (CF) are described. NiMs were produced by spray drying a solution containing polymers or sugars and a nanometric polyanion-tobramcyin complex (PTC), able to achieve a prolonged antibiotic release. NiMs properties were compared to TOBIPodhaler(Novartis), the only one commercially available dry powder inhalatory formulation based on porous microparticles. Produced NiMs showed adequate characteristics for pulmonary administration, as spherical shape, micrometric size, and high cytocompatibility toward human bronchial epithelial cells. Contrarily to TOBIPodhaler, some of produced NiMs, thanks to their specific chemical composition, are able to facilitate the drug diffusion through the mucus secretion, achieving, at the same time, a sustained tobramycin delivery. Moreover, NiMs showed pronounced antimicrobial activity against P. aeruginosa pathogens and their biofilm, if compared to free tobramycin and TOBIPodhaler, demonstrating the potential of obtained formulations as drug delivery systems for the treatment of pulmonary infections in CF patients.

Pegylated Polyaspartamide-Polylactide-Based Nanoparticles Penetrating Cystic Fibrosis Artificial Mucus.

Here, the preparation of mucus-penetrating nanoparticles for pulmonary administration of ibuprofen in patients with cystic fibrosis is described. A fluorescent derivative of α,ß-poly(N-2-hydroxyethyl)-D,L-aspartamide is synthesized by derivatization with rhodamine, polylactide, and poly(ethylene glycol), to obtain polyaspartamide-polylactide derivatives with different degrees of pegylation. Starting from these copolymers, fluorescent nanoparticles with different poly(ethylene glycol) content, empty and loaded with ibuprofen, showed spherical shape, colloidal size, slightly negative ζ potential, and biocompatibility toward human bronchial epithelial cells. The high surface poly(ethylene glycol) density of fluorescent nanoparticles and poly(ethylene glycol) brush-like conformation assumed on their surface, conferred to pegylated nanoparticles the mucus-penetrating properties, properly demonstrated by assessing their ability to avoid interactions with mucus components and to penetrate cystic fibrosis artificial mucus. Finally, ibuprofen release profile and uptake capacity within human bronchial epithelial cells in the presence of cystic fibrosis artificial mucus showed how these mucus-penetrating nanoparticles could rapidly diffuse through the mucus barrier reaching the mucosal surface, where they could offer a sustained delivery of ibuprofen at the site of disease.

Improvements in Rational Design Strategies of Inulin Derivative Polycation for siRNA Delivery.

The advances of short interfering RNA (siRNA)-mediated therapy provide a powerful option for the treatment of many diseases, including cancer, by silencing the expression of targeted genes involved in the progression of the pathology. On this regard, a new pH-responsive polycation derived from inulin, Inulin-g-imidazole-g-diethylenetriamine (INU-IMI-DETA), was designed and employed to produce INU-IMI-DETA/siRNA "Inulin COmplex Nanoaggregates" (ICONs). The experimental results showed that INU-IMI-DETA exhibited strong cationic characteristics and high solubility in the pH range 3-5 and self-aggregation triggered by pH increase and physiological salt concentration. INU-IMI-DETA showed as well a high buffering capacity in the endosomal pH range of 7.4-5.1. In the concentration range between 25 and 1000 µg/mL INU-IMI-DETA had no cytotoxic effect on breast cancer cells (MCF-7) and no lytic effect on human red blood cells. ICONs were prepared by two-step procedure involving complexation and precipitation into DPBS buffer (pH 7.4) to produce siRNA-loaded nanoaggregates with minimized surface charge and suitable size for parenteral administration. Bafilomycin A1 inhibited transfection on MCF-7 cells, indicating that the protonation of the imidazole groups in the endolysosome pathway favors the escape of the system from endolysosomal compartment, increasing the amount of siRNA that can reach the cytoplasm.

Polyaspartamide-Polylactide Graft Copolymers with Tunable Properties for the Realization of Fluorescent Nanoparticles for Imaging.

Here, the synthesis and the characterization of novel amphiphilic graft copolymers with tunable properties, useful in obtaining polymeric fluorescent nanoparticles for application in imaging, are described. These copolymers are obtained by chemical conjugation of rhodamine B (RhB) moieties, polylactic acid (PLA), and O-(2-aminoethyl)-O'-methyl poly(ethylene glycol) (PEG) on α,ß-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA). In particular, PHEA is first functionalized with RhB to obtain PHEA-RhB with a derivatization degree in RhB (DDRhB ) equal to 0.55 mol%. By varying the reaction conditions, different amounts of PLA are grafted on PHEA-RhB to obtain PHEA-RhB-PLA with DDPLA equal to 1.9, 4.0, and 6.2 mol%. Then, PEG chains are grafted on PHEA-RhB-PLA derivatives to obtain PHEA-RhB-PLA-PEG graft copolymers. The preparation of polymeric fluorescent nanoparticles with tunable properties and spherical shape is described by using PHEA-RhB-PLA-PEG with DD in PLA and PEG equal to 4.0 and 4.9 mol%, by following easily scaling up processes, such as emulsion-solvent evaporation and high pressure homogenization (HPH)-solvent evaporation techniques.

MRI-visible poly(ε-caprolactone) with controlled contrast agent ratios for enhanced visualization in temporary imaging applications.

Hydrophobic macromolecular contrast agents (MMCAs) are highly desirable to provide safe and efficient magnetic resonance (MR) visibility to implantable medical devices. In this study, we report on the synthesis and evaluation of novel biodegradable poly(ε-caprolactone)-based MMCAs. Poly(α-propargyl-ε-caprolactone-co-ε-caprolactone)s containing 2, 5, and 10 mol % of propargyl groups have been prepared by ring-opening copolymerization of ε-caprolactone and the corresponding propargylated lactone. In parallel, a diazido derivative of the clinically used diethylenetriaminepentaacetic acid (DTPA)/Gd(3+) complex has been synthesized. Finally, MRI-visible poly(ε-caprolactone)s (PCLs) were obtained by the efficient click ligation of these compounds via a Cu(I)-catalyzed [3 + 2] cycloaddition. ICP-MS analyses confirmed the efficient coupling of the complex on the PCL backbone with the MRI-visible PCLs containing 1.0, 2.6, and 3.6 wt % of Gd(3+). The influence of the Gd(3+) grafting density on the T1 relaxation times and on the MRI visibility of the novel biodegradable MMCAs was evaluated. Finally, their stability and cytocompatibility were assessed with regard to their potential as innovative MRI-visible biomaterials for biomedical applications.

Inhalable nano into micro dry powders for ivacaftor delivery: The role of mannitol and cysteamine as mucus-active agents.

In this paper the innovative approach of nano into micro dry powders (NiM) was applied to incorporate into mannitol or mannitol/cysteamine micromatrices ivacaftor-loaded nanoparticles for pulmonary delivery in CF. Nanoparticles composed by a mixture of two polyhydrohydroxyethtylaspartamide copolymers containing loaded with ivacaftor at 15.5% w/w were produced. The nanoparticles were incorporated into microparticles to obtain NiM that were fully characterized in terms of size, morphology, interactions with artificial Cf mucus (CF-AM) as well as for aerodynamic behaviour. Finally the activity of ivacaftor-containing NiM was evaluated by in vitro preliminary experiments. NiM at matrix composed by a mixture of mannitol:cysteamine showed greater ability to reduce CF-AM viscosity whereas that based on just mannitol showed better aerodynamic properties with a FPF of about 25%. All produced NiM showed very good cytocompatibility and the released ivacaftor was able to restore the chroride transport in vitro.

Combining Inulin Multifunctional Polycation and Magnetic Nanoparticles: Redox-Responsive siRNA-Loaded Systems for Magnetofection.

Superparamagnetic Iron Oxide Nanoparticles (SPIONs) are recognized as one of the most promising agents for theranostic applications. Among methods designed for siRNA delivery, magnetofection, that is, nucleic acid cell uptake under the influence of a magnetic field acting on magnetic nucleic acid vectors, is emerging as a unique approach to combining advantages such as strong improvement of the kinetics of the delivery process and the possibility of localizing nucleic acid delivery to an area where the magnetic field is applied. This paper reports on the preparation of siRNA loaded magnetoplexes-named ICD@SS@SPIONs/siRNA-by controlled crosslinking, in the presence of SPIONs, of the polycation INU-C-DETA, synthesized starting from the polysaccharide inulin by grafting diethylenetriamine and cystamine molecules. The obtained ICD@SS@SPIONs/siRNA have suitable chemical-physical characteristics to be employed for iv administration and are also able to release siRNA in a redox-triggered manner thanks to intracellular glutathione (GSH) mediated reduction of disulphide bridges formed during the crosslinking process. Moreover, ICD@SS@SPIONs/siRNA are able to produce magnetic targeting in vitro on breast cancer cells, without appreciable cyto- and hemo-toxic effects, in a wide range of concentrations. Finally, protein binding to nanoparticles revealed that obtained systems are potentially longer circulating and applicable as a smart multifunctional agents for cancer therapy.

Nanometric ion pair complexes of tobramycin forming microparticles for the treatment of Pseudomonas aeruginosa infections in cystic fibrosis.

Sustained pulmonary delivery of tobramycin from microparticles composed of drug/polymer nanocomplexes offers several advantages against traditional delivery methods. Namely, in patients with cystic fibrosis, microparticle delivery can protect the tobramycin being delivered from strong mucoadhesive interactions, thus avoiding effects on its diffusion toward the infection site. Polymeric ion-pair complexes were obtained starting from two synthetic polyanions, through impregnation of their solid dissociated forms with tobramycin in aqueous solution. The structure of these polymeric systems was characterized, and their activities were examined against various biofilm-forming Pseudomonas aeruginosa. Once dried, the nanocomplexes can change their aggregation state, to form microparticle-based aggregates with a spherical shape and a micrometer size. In aqueous dispersions, the ion-pair complexes produced had nanometric size, negative ζ potential, and high biocompatibility toward human bronchial epithelium cells. The antibiofilm activity of these formulations was more efficient than for free tobramycin, with the antibiofilm activity against P. aeruginosa mucoid and nonmucoid end-stage strains isolated from cystic fibrosis lungs being of particular relevance.

Mucus and Cell-Penetrating Nanoparticles Embedded in Nano-into-Micro Formulations for Pulmonary Delivery of Ivacaftor in Patients with Cystic Fibrosis.

Here, mucus-penetrating nanoparticles (NPs) for pulmonary administration of ivacaftor in patients with cystic fibrosis (CF) were produced with the dual aim of enhancing ivacaftor delivery to the airway epithelial cells, by rapid diffusion through the mucus barrier, and at the same time, promoting ivacaftor lung cellular uptake. Pegylated and Tat-decorated fluorescent nanoparticles (FNPs) were produced by nanoprecipitation, starting from two synthetic copolymers, and showed nanometric sizes (∼70 nm), a slightly negative ζ potential, and high cytocompatibility toward human bronchial epithelium cells. After having showed the significant presence of poly(ethylene glycol) chains and Tat protein onto the FNP surface, the FNP mucus-penetrating ability, ivacaftor release profile, and lung cellular uptake were studied in the presence of CF-artificial mucus as a function of the FNP surface chemical composition. Moreover, microparticle-based pulmonary drug-delivery systems composed of mucus-penetrating FNPs loaded with ivacaftor and mannitol were prepared by using the nano-into-micro strategy and realized by spray-drying, thereby providing optimal preservation and stabilization of FNP technological and fluorescence properties.

Polymeric nanoparticles for siRNA delivery: Production and applications.

Gene therapy through the use of siRNA and a polymeric carrier are becoming an efficient therapeutic option to conventional pharmaceutical formulations for the treatment of deadly diseases, such as cancer, pulmonary, ocular and neurodegenerative diseases. However, several considerations regarding the stability, formulation, and efficacy have to be faced up until these systems could be considered to be a marketable pharmaceutical products for to extend siRNA application to clinical practice. This review is focused on the key challenges of siRNA therapeutics, with special attention on the faced obstacles and on the formulation-related difficulties, providing a list of requirements needed for obtaining an ideal carrier for siRNA. Moreover, the current non-viral polymers investigated for the realization of efficient carriers for siRNA are described, with a special attention on synthetic polyamines such as polyethylenimine (PEI), polysaccharides such as chitosan and inulin (INU), and polyaminoacids such as α,ß-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) and poly-l-lysine (PLL).

Polyanion-tobramycin nanocomplexes into functional microparticles for the treatment of Pseudomonas aeruginosa infections in cystic fibrosis.

AIM: Efficacy of antibiotics in cystic fibrosis (CF) is compromised by the poor penetration through mucus barrier. This work proposes a new 'nano-into-micro' approach, used to obtain a combinatorial effect: achieve a sustained delivery of tobramycin and overcome mucus barrier. METHODS: Mannitol microparticles (MPs) were loaded with a tobramycin polymeric nanocomplex and characterized in presence of CF artificial mucus. RESULTS & DISCUSSION: MPs are able to alter the rheological properties of CF artificial mucus, enhancing drug penetration into it and allowing a prolonged drug release. MPs resulted to be effective in Pseudomonas aeruginosa infections if compared with free tobramycin. CONCLUSION: MPs resulted to be a formulation of higher efficacy, with potential positive implications, as lower required dose, administration frequency, side effects and antibiotic resistance problems.

Nanoparticles of a polyaspartamide-based brush copolymer for modified release of sorafenib: In vitro and in vivo evaluation.

In this paper, we describe the preparation of polymeric nanoparticles (NPs) loaded with sorafenib for the treatment of hepatocellular carcinoma (HCC). A synthetic brush copolymer, named PHEA-BIB-ButMA (PBB), was synthesized by Atom Trasnfer Radical Polymerization (ATRP) starting from the α-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) and poly butyl methacrylate (ButMA). Empty and sorafenib loaded PBB NPs were, then, produced by using a dialysis method and showed spherical morphology, colloidal size, negative ζ potential and the ability to allow a sustained sorafenib release in physiological environment. Sorafenib loaded PBB NPs were tested in vitro on HCC cells in order to evaluate their cytocompatibility and anticancer efficacy if compared to free drug. Furthermore, the enhanced anticancer effect of sorafenib loaded PBB NPs was demonstrated in vivo by using a xenograft model, by first allowing Hep3B cells to grow subcutaneously into nude mice and then administering sorafenib as free drug or incorporated into NPs via intraperitoneal injection. Finally, in vivo biodistribution studies were performed, showing the ability of the produced drug delivery system to accumulate in a significant manner in the solid tumor by passive targeting, thanks to the enhanced permeability and retention effect.

Galactosylated polyaspartamide copolymers for siRNA targeted delivery to hepatocellular carcinoma cells.

The limited efficacy of available treatments for hepatocellular carcinoma (HCC) requires the development of novel therapeutic approaches. We synthesized a novel cationic polymer based on α,ß-poly-(N-2-hydroxyethyl)-d,L-aspartamide (PHEA) for drug delivery to HCC cells. The copolymer was synthesized by subsequent derivatization of PHEA with diethylene triamine (DETA) and with a polyethylene glycol (PEG) derivative bearing galactose (GAL) molecules, obtaining the cationic derivative PHEA-DETA-PEG-GAL. PHEA-DETA-PEG-GAL has suitable chemical-physical characteristics for a potential systemic use and can effectively deliver a siRNA (siE2F1) targeted against the transcription factor E2F1, a gene product involved in HCC. The presence of GAL residues in the polyplexes allows the targeting of HCC cells that express the asialo-glycoprotein receptor (ASGP-R). In these cells, but not in ASGP-R non-expressing cells, PHEA-DETA-PEG-GAL/siE2F1 polyplexes induce the reduction of the mRNA and protein levels of E2F1 and of E2F1-regulated genes, all involved in the promotion of the G1/S phase transition. This results in a decrease of cell proliferation with a G1/G0 phase cells accumulation. Notably, removal of GAL residue almost completely abrogates the targeting capacity of the developed polyplexes. In conclusion, the generated polyplexes demonstrate the potential to effectively contributing to the development of novel anti-HCC therapeutic approaches via a siRNA-targeted delivery.

From Genesis to Revelation: The Role of Inflammatory Mediators in Chronic Respiratory Diseases and their Control by Nucleic Acid-based Drugs.

Asthma, chronic obstructive pulmonary disease, cystic fibrosis, and idiopathic pulmonary fibrosis, are among the most common chronic diseases and their prevalence is increasing. Each of these diseases is characterized by the secretion of cytokines and pro-inflammatory molecules which are thought to play a critical role in their pathogenesis. Moreover, immune cells, particularly neutrophils, macrophages and dendritic cells as well structural cells such as epithelial and airway smooth muscle cells are also involved in the pathogenic cycle of these diseases. There is a pressing need for the development of new therapies for these pulmonary diseases, particularly as no existing treatment has been shown to reduce disease progression. HMGB1 (high-mobility group box 1), originally identified as a nuclear non histone protein with DNA-binding domains can be secreted by living and dying cells and it is now regarded as an important endogenous danger signaling molecule. Besides as a signal of tissue injury, HMGB1 is considered a powerful mediator of inflammation and high levels of HMGB1 are found in chronic lung diseases. The role of HMGB1 in respiratory diseases is still elusive nevertheless these studies suggest an involvement of this cytokine in their pathogenesis. Nucleic acid-based drugs (NABDs) are a novel class of pharmaceuticals including antisense oligonucleotides, DNA-zymes, and RNA interference as mediated by small interfering RNA (siRNA), which are used to dampen the expression of disease-causing genes having therapeutic potential for controlling chronic airway diseases. Due to their inherent difficulties, such as for example sensitivity to endonucleases, their delivery in vivo should be assured by vectors. Non-viral lipid- and polymer-based nanosystems have acquired much importance in this context. In this review, we will discuss these emerging tools in gene therapy of chronic lung diseases, particularly the use of siRNA in the down-regulation of critical molecules in the pathogenesis of chronic lung diseases, with particular emphasis on HMGB1 as therapeutic target.

Toward potent antibiofilm degradable medical devices: a generic method for the antibacterial surface modification of polylactide.

The effects of biomaterials on their environment must be carefully modulated in most biomedical applications. Among other approaches, this modulation can be obtained through the modification of the biomaterial surface. This paper proposes a simple and versatile strategy to produce non-leaching antibacterial polylactide (PLA) surfaces without any degradation of the polyester chains. The method is based on a one-pot procedure that provides a "clickable" PLA surface via anionic activation which is then functionalized with an antibacterial quaternized poly(2-(dimethylamino)ethyl methacrylate) (QPDMAEMA) by covalent immobilization on the surface. The anti-adherence and antibiofilm activities of modified PLA surfaces are assessed for different QPDMAEMA molecular weights and different quaternization agents. Antibacterial PLA surfaces are shown to be very active against Gram-negative and Gram-positive strains, with adherence reduction factors superior to 99.999% and a marked reduction in biofilm on the most potent surfaces. In addition to this substantial antibacterial activity, the proposed PLA surfaces are also cytocompatible, as demonstrated through the proliferation of L929 fibroblasts.