Annalisa Cutrignelli

The use of Eudragit RS 100/cyclodextrin nanoparticles for the transmucosal administration of glutathione

The aim of this work was to develop and characterize new nanoparticle systems based on Eudragit RS 100 and cyclodextrins (CDs) for the transmucosal administration of glutathione (GSH). For this purpose, nanoparticles (NPs) with the mucoadhesive properties of Eudragit RS 100 and the penetration enhancing and peptide protective properties of CDs were prepared and evaluated. The quasi-emulsion solvent diffusion technique was used to prepare the NPs with natural and chemically modified (HP-beta-CD and Me-beta-CD) CDs. The NPs prepared showed homogeneous size distribution, mean diameters between 99 and 156nm, a positive net charge and spherical morphology. Solid state FT-IR, thermal analysis (DSC), and X-ray diffraction studies suggest that the nanoencapsulation process produces a marked decrease in crystallinity of GSH. The encapsulation efficiency of the peptide was found to be between 14.8% and 24%. The results indicate that mean diameters, surface charges and drug-loaded NPs were not markedly affected by the CD, whereas the presence of the latter influences drug release and to some extent peptide stability and absorption. Finally, it has been shown that CD/Eudragit RS 100 NPs may be used for transmucosal absorption of GSH without any cytotoxicity using the epithelial human HaCaT and murine monocyte macrophage RAW264.7 cell lines.

Translocator protein ligand-PLGA conjugated nanoparticles for 5-fluorouracil delivery to glioma cancer cells.

Translocator protein 18 kDa (TSPO) is a promising target for molecular imaging and for targeted drug delivery to tumors overexpressing TSPO. In our previous work, new macromolecular conjugates with a high affinity and selectivity for TSPO were prepared by conjugating the biodegradable poly(d,l-lactic-co-glycolic acid) (PLGA) polymer with two potent and selective TSPO ligands, namely, compounds 1 and 2. Based on this, nanoparticle delivery systems (NPs), employing TSPO ligand-PLGA conjugated (PLGA-TSPO) polymers, were prepared. Furthermore, to evaluate the ability of the new NPs to be used as a drug delivery systems for anticancer therapy, PLGA-TSPO NPs were loaded with 5-fluorouracil (5-FU), chosen as a model hydrophilic anticancer drug. The main goal of this work was to investigate the synergistic potential of using NP conjugates PLGA-TSPO, TSPO ligands being pro-apoptotic agents, to simultaneously deliver a cytotoxic anticancer drug. To better highlight the occurrence of synergistic effects, dual drug loaded PLGA NPs (PLGA NPs/5-FU/1) and dual drug loaded PLGA-TSPO NPs (PLGA-TSPO NPs/5-FU/1), with 5-FU and TSPO ligand 1 physically incorporated together, were also prepared and characterized. The particle size and size distribution, surface morphology, and drug encapsulation efficiency, as well as the drug release kinetics, were investigated. In vitro cytotoxicity studies were carried out on C6 glioma cells overexpressing TSPO, and to evaluate the potential uptake of these nanoparticulate systems, the internalization of fluorescent labeled PLGA-TSPO NPs (FITC-PLGA-TSPO NPs) was also investigated by fluorescence microscopy. Results demonstrated that PLGA-TSPO NPs/5-FU and dual drug loaded PLGA NPs/5-FU/1 and PLGA-TSPO NPs/5-FU/1 could significantly enhance toxicity against human cancer cells due to the synergistic effect of the TSPO ligand 1 with the anticancer drug 5-FU.

In vitro targeting and imaging the translocator protein TSPO 18-kDa through G(4)-PAMAM-FITC labeled dendrimer

Mitochondria represent an attractive subcellular target due to its function particularly important for oxidative damage, calcium metabolism and apoptosis. However, the concept of mitochondrial targeting has been a neglected area so far. The translocator protein (TSPO) represents an interesting subcellular target not only to image disease states overexpressing this protein, but also for a selective mitochondrial drug targeting. Recently, we have delivered in vitro and in vivo small molecule imaging agents into cells overexpressing TSPO by using a family of high-affinity conjugable ligands characterized by 2-phenyl-imidazo[1,2-a]pyridine acetamide structure. As an extension, in the present work we studied the possibility to target and image TSPO with dendrimers. These nano-platforms have unique features, in fact, are prepared with a level of control not reachable with most linear polymers, leading to nearly monodisperse, globular macromolecules with a large number of peripheral groups. As a consequence, they are an ideal delivery vehicle candidate for explicit study of the effects of polymer size, charge, composition, and architecture on biologically relevant properties such as lipid bilayer interactions, cytotoxicity, cellular internalization, and subcellular compartments and organelles interactions. Here, we present the synthesis, characterization, cellular internalization, and mitochondria labeling of a TSPO targeted fourth generation [G(4)-PAMAM] dendrimer nanoparticle labeled with the organic fluorescent dye fluorescein. We comprehensively studied the cellular uptake behavior of these dendrimers, into glioma C6 cell line, under the influence of various endocytosis inhibitors. We found that TSPO targeted-G(4)-PAMAM-FITC dendrimer is quickly taken up by these cells by endocytosis pathways, and moreover specifically targets the mitochondria as evidenced from subcellular fractionation experiments and co-localization studies performed with CAT (Confocal-AFM-TIRF) microscopy.

Complexation of phenytoin with some hydrophilic cyclodextrins: effect on aqueous solubility, dissolution rate, and anticonvulsant activity in mice

The main objective of this study was to evaluate the influence of hydroxypropylated beta- and gamma-cyclodextrins and Me-beta-cyclodextrin (HP-beta-CD, HP-gamma-CD, and Me-beta-CD, respectively) on the dissolution rate and bioavailability of the antiepileptic agent, phenytoin (DPH). The corresponding solid complexes were prepared by a freeze-drying method and characterized by infrared spectroscopy, X-ray powder diffraction, and differential scanning calorimetry studies. Evidence of inclusion complex formation in the case of HP-beta-CD was obtained by (1)H- and (13)C-nuclear magnetic resonance spectroscopy. Drug solubility and dissolution rate in 0.05 M potassium phosphate buffer (pH 6) were notably improved by employing the beta-CDs. Thus a 45% w/v HP-beta-CD or Me-beta-CD solution gave rise to an increase of dissolved drug of 420- and 578-fold, respectively. The Q(10) (i.e. percentage of dissolved DPH at 10 min) was 5.2% for the pure drug and 93, 98, and 96% for DPH/HP-beta-CD, DPH/HP-gamma-CD, and DPH/Me-beta-CD complexes, respectively. Moreover, it was found that in the maximal electroshock seizure test in mice the DPH/Me-beta-CD complex exhibited anticonvulsant activity similar to DPH sodium salt (NaDPH).

A New Complex of Curcumin with Sulfobutylether‐β‐Cyclodextrin: Characterization Studies and In Vitro Evaluation of Cytotoxic and Antioxidant Activity on HepG‐2 Cells

Curcumin (CR) is a natural polyphenol with antioxidative, anti-inflammatory, and anticancer properties but its therapeutic potential is substantially hindered by the rather low-water solubility and bioavailability. Thus, in this work, a new soluble inclusion complex of CR with sulfobutylether-β-cyclodextrin (SBE-β-CD) was prepared in solution and at the solid state using different preparation techniques and characterized by Fourier transform infrared, nuclear magnetic resonance, differential scanning calorimetry, scanning electron microscopy, phase solubility studies, and Jobs plot method. Results clearly indicate that CR reacts with SBE-β-CD to form a host-guest complex with an apparent formation constant of 1455 M(-1) . Moreover, SBE-β-CD strongly increases water solubility of CR (from 0.56 to 102.78 μg/mL, at 25°C), and lyophilization method seems to be the best preparation technique to obtain the complex at the solid state. Finally, an in vitro test on a human hepatic cancer cell line (HepG-2) shows that complexation positively influences CR anticancer and antioxidant activity.

Spray-dried mucoadhesives for intravesical drug delivery using N-acetylcysteine- and glutathione-glycol chitosan conjugates.

UNLABELLED This work describes N-acetylcysteine (NAC)- and glutathione (GSH)-glycol chitosan (GC) polymer conjugates engineered as potential platform useful to formulate micro-(MP) and nano-(NP) particles via spray-drying techniques. These conjugates are mucoadhesive over the range of urine pH, 5.0-7.0, which makes them advantageous for intravesical drug delivery and treatment of local bladder diseases. NAC- and GSH-GC conjugates were generated with a synthetic approach optimizing reaction times and purification in order to minimize the oxidation of thiol groups. In this way, the resulting amount of free thiol groups immobilized per gram of NAC- and GSH-GC conjugates was 6.3 and 3.6mmol, respectively. These polymers were completely characterized by molecular weight, surface sulfur content, solubility at different pH values, substitution and swelling degree. Mucoadhesion properties were evaluated in artificial urine by turbidimetric and zeta (ζ)-potential measurements demonstrating good mucoadhesion properties, in particular for NAC-GC at pH 5.0. Starting from the thiolated polymers, MP and NP were prepared using both the Büchi B-191 and Nano Büchi B-90 spray dryers, respectively. The resulting two formulations were evaluated for yield, size, oxidation of thiol groups and ex-vivo mucoadhesion. The new spray drying technique provided NP of suitable size (<1μm) for catheter administration, low degree of oxidation, and sufficient mucoadhesion property with 9% and 18% of GSH- and NAC-GC based NP retained on pig mucosa bladder after 3h of exposure, respectively. STATEMENT OF SIGNIFICANCE The aim of the present study was first to optimize the synthesis of NAC-GC and GSH-GC, and preserve the oxidation state of the thiol moieties by introducing several optimizations of the already reported synthetic procedures that increase the mucoadhesive properties and avoid pH-dependent aggregation. Second, starting from these optimized thiomers, we studied the feasibility of manufacturing MP and NP by spray-drying techniques. The aim of this second step was to produce mucoadhesive drug delivery systems of adequate size for vesical administration by catheter, and comparable mucoadhesive properties with respect to the processed polymers, avoiding thiolic oxidation during the formulation. MP with acceptable size produced by spray-dryer Büchi B-191 were compared with NP made with the apparatus Nano Büchi B-90.

A Novel PET Imaging Probe for the Detection and Monitoring of Translocator Protein 18 kDa Expression in Pathological Disorders

A new fluorine-substituted ligand, compound 1 (CB251), with a very high affinity (Ki = 0.27 ± 0.09 nM) and selectivity for the 18-kDa translocator protein (TSPO), is presented as an attractive biomarker for the diagnosis of neuroinflammation, neurodegeneration and tumour progression. To test compound 1 as a TSPO PET imaging agent in vivo, 2-(2-(4-(2-[18F]fluoroethoxy)phenyl)-6,8-dichloroimidazo[1,2-a]pyridin-3-yl)-N,N-dipropylacetamide ([18F]1; [18F]CB251) was synthesized by nucleophilic aliphatic substitution in a single-step radiolabelling procedure with a 11.1 ± 3.5% (n = 14, decay corrected) radiochemical yield and over 99% radiochemical purity. In animal PET imaging studies, [18F]CB251 provided a clearly visible image of the inflammatory lesion with the binding potential of the specifically bound radioligand relative to the non-displaceable radioligand in tissue (BPND 1.83 ± 0.18), in a neuroinflammation rat model based on the unilateral stereotaxic injection of lipopolysaccharide (LPS), comparable to that of [11C]PBR28 (BPND 1.55 ± 0.41). [18F]CB251 showed moderate tumour uptake (1.96 ± 0.11%ID/g at 1 h post injection) in human glioblastoma U87-MG xenografts. These results suggest that [18F]CB251 is a promising TSPO PET imaging agent for neuroinflammation and TSPO-rich cancers.

Relationship between dissolution efficiency of Oxazepam/carrier blends and drug and carrier molecular descriptors using multivariate regression analysis

Quantitative structure-property relationships were developed for predicting the enhancement of dissolution rate of the model lipophilic drug Oxazepam (Oxa) from blends (BLs) with 12 structurally different carriers at three different drug/carrier weight ratios (1/5, 1/10, and 1/20). To this end, 36 BLs were prepared by the solvent-evaporation method and characterized by spectroscopic (FT-IR), thermoanalytical (DSC) and X-ray diffraction studies. The dissolution rate of the examined systems was quantified by logDE/DE(Oxa), where DE and DE(Oxa) are the dissolution efficiencies of the BL and pure drug, respectively. Twenty molecular descriptors, including parameters for size, lipophilicity, cohesive energy density (CED), and hydrogen bonding capacity were calculated and together with the experimental melting point (MP), were used in multivariate analysis. Twelve pertinent variables were detected after looking at the results of principal component analysis (PCA) and cluster analysis, and reliable six-descriptor models generated by Partial Least Squares-Projection to Latent Structures (PLS) method. Satisfactory coefficient of determination values were obtained (i.e., R(2) equal to 0.794 and Q(2) equal to 0.705). The equations generated can predict with reasonable accuracy the dissolution rate increase of the model lipophilic drug/carrier BLs.

2-Phenylimidazo[1,2-a]pyridine-containing ligands of the 18-kDa translocator protein (TSPO) behave as agonists and antagonists of steroidogenesis in a mouse leydig tumor cell line.

Ligands of 18-kDa translocator protein (TSPO) are known for their ability to potently and dose-dependently stimulate steroid biosynthesis in steroidogenic cells. In this study, we investigated a number of 2-phenyl-imidazo[1,2-a]pyridine acetamide derivatives, analogs of alpidem, for their ability to bind TSPO and to affect steroidogenesis in a mouse Leydig tumor cell line. We observed that not only some compounds behaved as agonists, stimulating steroidogenesis (e.g., 3 and 4) with EC50 values (15.9 and 6.99μM) close to that determined for FGIN-1-27 used as positive control (7.24μM), but two compounds, namely 5 and 6, which on the other hand are the most lipophilic ones in the investigated series, behaved as antagonists, by significantly inhibiting steroid production at concentrations at least twenty times lower than the cytotoxic ones. To our surprise, the newly synthesized compound 3, which is a strict analog of alpidem bearing at the para position of the 2-phenyl group a methoxy group instead of chlorine, achieved a ten-fold stimulation of the steroid production (for comparison FGIN-1-27 achieved 1.6-fold stimulation). Within the limits of the examined property space, some unprecedented SARs were unveiled, which can help in understanding the key molecular factors underlying the transition from agonism to antagonism in the steroidogenesis process. Besides the substitution pattern and the physicochemical features (mainly hydrogen bonding potential) of the substituents at the positions C(6) and C(8) of the imidazo[1,2-a]pyridine nucleus, and at the para position of the 2-phenyl group, the structure-activity relationship analysis suggested lipophilicity, whose increase seems to be generally related to steroidogenesis inhibition, and steric hindrance, which appeared as a stimulation-limiting factor, as two main properties to control in the design or optimization of novel imidazo[1,2-a]pyridine-based TSPO ligands endowed with potential in modulating the steroidogenesis process.

Synthesis, Characterization, and in Vitro Evaluation of a New TSPO-Selective Bifunctional Chelate Ligand.

The 18-kDa translocator protein (TSPO) is overexpressed in many types of cancers and is also abundant in activated microglial cells occurring in inflammatory neurodegenerative diseases. Thus, TSPO has become an extremely attractive subcellular target not only for imaging disease states overexpressing this protein, but also for a selective mitochondrial drug delivery. In this work we report the synthesis, the characterization, and the in vitro evaluation of a new TSPO-selective ligand, 2-(8-(2-(bis(pyridin-2-yl)methyl)amino)acetamido)-2-(4-chlorophenyl)H-imidazo[1,2-a]pyridin-3-yl)-N,N-dipropylacetamide (CB256), which fulfils the requirements for a bifunctional chelate approach. The goal was to provide a new TSPO ligand that could be used further to prepare coordination complexes of a metallo drug to be used in diagnosis and therapy. However, the ligand itself proved to be a potent tumor cell growth inhibitor and DNA double-strand breaker.