Rutin/Sulfobutylether-ß-Cyclodextrin as a Promising Therapeutic Formulation for Ocular Infection.

Ocular pathologies present significant challenges to achieving effective therapeutic results due to various anatomical and physiological barriers. Natural products such as flavonoids, alone or in association with allopathic drugs, present many therapeutic actions including anticancer, anti-inflammatory, and antibacterial action. However, their clinical employment is challenging for scientists due to their low water solubility. In this study, we designed a liquid formulation based on rutin/sulfobutylether-ß-cyclodextrin (RTN/SBE-ß-CD) inclusion complex for treating ocular infections. The correct stoichiometry and the accurate binding constant were determined by employing SupraFit software (2.5.120) in the UV-vis titration experiment. A deep physical-chemical characterization of the RTN/SBE-ß-CD inclusion complex was also performed; it confirmed the predominant formation of a stable complex (Kc, 9660 M-1) in a 1:1 molar ratio, with high water solubility that was 20 times (2.5 mg/mL) higher than the free molecule (0.125 mg/mL), permitting the dissolution of the solid complex within 30 min. NMR studies revealed the involvement of the bicyclic flavonoid moiety in the complexation, which was also confirmed by molecular modeling studies. In vitro, the antibacterial and antibiofilm activity of the formulation was assayed against Staphylococcus aureus and Pseudomonas aeruginosa strains. The results demonstrated a significant activity of the formulation than that of the free molecules.

Characterization and In Vivo Antiangiogenic Activity Evaluation of Morin-Based Cyclodextrin Inclusion Complexes.

Morin (MRN) is a natural compound with antiangiogenic, antioxidant, anti-inflammatory, and anticancer activity. However, it shows a very low water solubility (28 µg/mL) that reduces its oral absorption, making bioavailability low and unpredictable. To improve MRN solubility and positively affect its biological activity, particularly its antiangiogenic activity, in this work, we prepared the inclusion complexes of MNR with sulfobutylether-ß-cyclodextrin (SBE-ß-CD) and hydroxypropyl-ß-cyclodextrin (HP-ß-CD). The inclusion complexes obtained by the freeze-drying method were extensively characterized in solution (phase-solubility studies, UV-Vis titration, and NMR spectroscopy) and in the solid state (TGA, DSC, and WAXD analysis). The complexation significantly increased the water solubility by about 100 times for MRN/HP-ß-CD and 115 times for MRN/SBE-ß-CD. Furthermore, quantitative dissolution of the complexes was observed within 60 min, whilst 1% of the free drug dissolved in the same experimental time. 1H NMR and UV-Vis titration studies demonstrated both CDs well include the benzoyl moiety of the drug. Additionally, SBE-ß-CD could interact with the cinnamoyl moiety of MRN too. The complexes are stable in solution, showing a high value of association constant, that is, 3380 M-1 for MRN/HP-ß-CD and 2870 M-1 for MRN/SBE-ß-CD. In vivo biological studies on chick embryo chorioallantoic membrane (CAM) and zebrafish embryo models demonstrated the high biocompatibility of the inclusion complexes and the effective increase in antiangiogenic activity of complexed MRN with respect to the free drug.

Amphiphilic Cyclodextrin Nanoparticles as Delivery System for Idebenone: A Preformulation Study.

Idebenone (IDE), a synthetic short-chain analogue of coenzyme Q10, is a potent antioxidant able to prevent lipid peroxidation and stimulate nerve growth factor. Due to these properties, IDE could potentially be active towards cerebral disorders, but its poor water solubility limits its clinical application. Octanoyl-ß-cyclodextrin is an amphiphilic cyclodextrin (ACyD8) bearing, on average, ten octanoyl substituents able to self-assemble in aqueous solutions, forming various typologies of supramolecular nanoassemblies. Here, we developed nanoparticles based on ACyD8 (ACyD8-NPs) for the potential intranasal administration of IDE to treat neurological disorders, such as Alzheimer's Disease. Nanoparticles were prepared using the nanoprecipitation method and were characterized for their size, zeta potential and morphology. STEM images showed spherical particles, with smooth surfaces and sizes of about 100 nm, suitable for the proposed therapeutical aim. The ACyD8-NPs effectively loaded IDE, showing a high encapsulation efficiency and drug loading percentage. To evaluate the host/guest interaction, UV-vis titration, mono- and two-dimensional NMR analyses, and molecular modeling studies were performed. IDE showed a high affinity for the ACyD8 cavity, forming a 1:1 inclusion complex with a high association constant. A biphasic and sustained release of IDE was observed from the ACyD8-NPs, and, after a burst effect of about 40%, the release was prolonged over 10 days. In vitro studies confirmed the lack of toxicity of the IDE/ACyD8-NPs on neuronal SH-SY5Y cells, and they demonstrated their antioxidant effect upon H2O2 exposure, as a general source of ROS.

Bicalutamide Anticancer Activity Enhancement by Formulation of Soluble Inclusion Complexes with Cyclodextrins.

Bicalutamide (BCL) is a nonsteroidal antiandrogen drug that represents an alternative to castration in the treatment of prostate cancer, due to its relatively long half-life and tolerable side effects. However, it possesses a very low water solubility that can affect its oral bioavailability. In this work, we developed inclusion complexes of BCL with the highly soluble hydroxypropyl-ß-cyclodextrin (HP-ß-CyD) and sulfobutylether-ß-cyclodextrin (SBE-ß-CyD) to increase the water solubility and anticancer activity of BCL. The inclusion complexes were prepared using the freeze-drying method and were then characterized in a solid state via differential scanning calorimetry and X-ray analysis and in solution via phase-solubility studies and UV-vis and NMR spectroscopy. The BCL/HP-ß-CyD and BCL/SBE-ß-CyD inclusion complexes were amorphous and rapidly dissolved in water. Both the 1H-NMR spectra and molecular modeling studies confirmed the penetration of the 2-(trifluoromethyl)benzonitrile ring of BCL within the cavity of both cyclodextrins (CyDs). Due to the consistent improvement of the water solubility of BCL, the inclusion complexes showed higher antiproliferative activity toward the human prostate androgen-independent cell lines, DU-145 and PC-3, with respect to free BCL. These results demonstrate the ability of HP-ß-CyD and SBE-ß-CyD to complex BCL, permitting the realization of liquid formulations with potentially high oral bioavailability and/or possible parenteral administration.

Chitosan-Hyaluronan Nanoparticles for Vinblastine Sulfate Delivery: Characterization and Internalization Studies on K-562 Cells.

In the present study, we developed chitosan/hyaluronan nanoparticles (CS/HY NPs) for tumor targeting with vinblastine sulfate (VBL), that can be directed to the CD44 transmembrane receptor, over-expressed in cancer cells. NPs were prepared by coating with HY-preformed chitosan/tripolyphosphate (CS/TPP) NPs, or by polyelectrolyte complexation of CS with HY. NPs with a mean hydrodynamic radius (RH) of 110 nm, 12% polydispersity index and negative zeta potential values were obtained by a direct complexation process. Transmission Electron Microscopy (TEM) images showed spherical NPs with a non-homogeneous matrix, probably due to a random localization of CS and HY interacting chains. The intermolecular interactions occurring between CS and HY upon NPs formation were experimentally evidenced by micro-Raman (µ-Raman) spectroscopy, through the analysis of the spectral changes of characteristic vibrational bands of HY during NP formation, in order to reveal the involvement of specific chemical groups in the process. Optimized NP formulation efficiently encapsulated VBL, producing a drug sustained release for 20 h. In vitro studies demonstrated a fast internalization of labeled CS/HY NPs (within 6 h) on K-562 human myeloid leukemia cells. Pre-saturation of CD44 by free HY produced a slowing-down of NP uptake over 24 h, demonstrating the need of CD44 for the internalization of HY-based NPs.

Rutin-Loaded Solid Lipid Nanoparticles: Characterization and In Vitro Evaluation.

This study was aimed at preparing and characterizing solid lipid nanoparticles loading rutin (RT-SLNs) for the treatment of oxidative stress-induced diseases. Phospholipon 80H® as a solid lipid and Polysorbate 80 as surfactant were used for the SLNs preparation, using the solvent emulsification/diffusion method. We obtained spherical RT-SLNs with low sizes, ranging from 40 to 60 nm (hydrodynamic radius) for the SLNs prepared starting from 2% and 5% (w/w) theoretical amount. All prepared formulations showed negative zeta-potential values. RT was efficiently encapsulated within SLNs, obtaining high encapsulation efficiency and drug content percentages, particularly for SLNs prepared with a 5% theoretical amount of RT. In vitro release profiles and analysis of the obtained data applying different kinetic models revealed Fickian diffusion as the main mechanism of RT release from the SLNs. The morphology of RT-SLNs was characterized by scanning electron microscopy (SEM), whereas the interactions between RT and the lipid matrix were investigated by Raman spectroscopy, evidencing spectral modifications of characteristic bands of RT due to the establishment of new interactions. Finally, antioxidant activity assay on human glioblastoma astrocytoma (U373) culture cells showed a dose-dependent activity for RT-SLNs, particularly at the highest assayed dose (50 µM), whereas the free drug showed the lesser activity.

Gemcitabine anticancer activity enhancement by water soluble celecoxib/sulfobutyl ether-ß-cyclodextrin inclusion complex.

We investigated the complexation of celecoxib (CCB) into sulfobuthyl-ether-ß-cyclodextrin (SBE-ß-CD) for the realization of an inhalable dry-powder formulation containing gemcitabine (GEM) for lung anticancer therapy. Complexation increased the water solubility of CCB (0.003 mg/mL and 0.834 mg/mL for CCB free and complexed, respectively) and produced a quantitative dissolution of the drug within 15 min. The CCB/SBE-ß-CD inclusion complex showed a high stability constant (8131 M-1) not influenced by the presence of GEM in solution. Two-dimensional NMR experiments and computational studies demonstrated that the pyrazole ring of CCB penetrates deeper into SBE-ß-CD from the secondary rim. The aromatic rings are positioned at the edge of the cavity, establishing hydrogen bonds with the SBE-ß-CD that stabilized the complex. CCB showed limited cytotoxic activity on A549 cell lines. Complexation significantly increased activity passing from 30% to 45% cell mortality. Moreover, CCB/SBE-ß-CD strongly improved the cytotoxicity of GEM, observing about 60% of cell mortality for the combined formulation.

Physicochemical characterization of pH-responsive and fusogenic self-assembled non-phospholipid vesicles for a potential multiple targeting therapy.

In order to obtain nanocarriers suitable for the delivery of drugs in the treatment of cancer, pH-responsive nanovesicles capable of facilitating fusion (fusogenic nanovesicles) were synthesized and then their physicochemical characteristics were modified. These nanovesicles were made by combining polysorbates having different physicochemical features with the aim of realizing multiple-targeting nanoformulations suitable for in vitro treatment of cancer cells. Tween21 and Tween80 were self-assembled at different molar concentrations resulting in pH-responsive fusogenic nanovesicles with an average size of less than 150nm, and a narrow size distribution (polydispersity index) value of less than 0.2. Hydrophobic and hydrophilic fluorescent probes were loaded inside the nanovesicles in order to study their pH-responsiveness and fusogenic properties and it was noted that this process did not modify their physicochemical features. The pH-responsiveness and fusogenic assay demonstrated that the nanovesicles containing Tween21 at different molar ratios were pH-responsive and interacted with a synthetic model of a biological membrane supplemented with Ca2+ in the incubation medium. Fifty percent (molar ratio) of Tween21 was replaced with Tween80, since Tween80 can promote the adsorption of apolipoproteins (A-E) onto the surfaces of nanovesicles without altering their pH-responsiveness or fusogenic properties. In fact this equivalent molar concentration of Tween21 and Tween80 also maintained their degree of interaction with the apolipoproteins (A-E). Doxorubicin hydrochloride-loaded nanovesicles were synthesized and physicochemically characterized in order to obtain nanoformulations suitable for anticancer treatment. The therapeutic nanovesicles showed physicochemical properties similar to those of empty nanoformulations, and maintained pH-responsiveness, fusogenic properties and targeting versus the apolipoproteins (A-E). The doxorubicin hydrochloride was loaded into the nanovesicles using both passive and pH gradient remote loading procedures. The latter provided the nanovesicles with an entrapment efficiency percentage of over 30%, which was much higher than the 10% that was obtained using the passive loading procedure. The entrapment efficiency improved up to 60% for the nanovesicles made from the same molar concentration of Tween21 and Tween80. The anticancer activity of doxorubicin hydrochloride-loaded nanovesicles was further tested in vitro using human neuroblastoma (SH-SY5Y) cells which respond to treatment with this chemotherapeutic drug, but the nanovesicles carrying it must cross the BBB by means of specific receptors before the drug can provide a therapeutic effect in vivo. The anticancer activity of these doxorubicin hydrochloride-loaded nanovesicles was time- and dosage- dependent, and the surfactant components making up the nanoformulations was also a determining factor in the efficiency of their activity. These nanovesicles could provide innovative nanotherapeutics for potential in vitro multidrug targeting therapy.

Solute-Solvent Interactions in Aqueous Solutions of Sulfobutyl Ether-ß-cyclodextrin As Probed by UV-Raman and FTIR-ATR Analysis.

A vibrational study by means of UV-Raman and FTIR-ATR measurements has been performed on sulfobutyl ether ß-cyclodextrin (SBE-ß-CD)-water solutions, as a function of concentration and temperature, with the aim to provide a molecular-scale explanation of the enhanced performances as carrier agent exhibited by this modified macrocycle with respect to natural cyclodextrin. The attention has been mainly paid to the modifications induced on the vibrational band assigned to the O-H stretching intramolecular mode, in turn related to the dynamical rearrangement occurring in the hydrogen bonding (HB) network of water molecules. The results of our measurements clearly showed a characteristic "structure-breaker" effect on the tetrahedral HB arrangements induced on water molecules by increasing of both temperature and solute concentration, allowing us to also extract thermodynamic parameters. These results could be a key step for a clearer understanding of the connection between the dynamical properties of hydration water and the complexing ability of this cyclodextrin derivative.

A characterization study of resveratrol/sulfobutyl ether-ß-cyclodextrin inclusion complex and in vitro anticancer activity.

A resveratrol/sulfobutylether-ß-cyclodextrin inclusion complex was prepared using the freeze-drying method and characterized in solution through UV-vis spectroscopy, solubility phase studies and Job's plot methods. At the solid state it was characterized using the FTIR-ATR technique. Sulfobutylether-ß-cyclodextrin has a high affinity for the drug, and forms an inclusion complex with a 1:1 molar ratio both in solution and as a solid sample. It also has a high stability constant (Kc, 10,114 M(-1)). Complexation strongly increases the water solubility of resveratrol (from 0.03 mg/ml to 1.1 mg/ml, at 25 °C) and positively influences its in vitro anticancer activity which was observed on a human breast cancer cell line (MCF-7). In solid phase, FTIR-ATR revealed itself as being a useful technique in elucidating the complexation mechanism, which it did by emphasizing the functional groups involved in the activation of non-covalent "host-guest" interactions.

Liposomal chemotherapeutics.

Currently, six liposomal chemotherapeutics have received clinical approval and many more are in clinical trials or undergoing preclinical evaluation. Liposomes exhibit low toxicity and improve the biopharmaceutical features and therapeutic index of drugs, thereby increasing efficacy and reducing side effects. In this review we discuss the advantages of using liposomes for the delivery of chemotherapeutics. Gemcitabine and paclitaxel have been chosen as examples to illustrate how the performance of a metabolically unstable or poorly water-soluble drug can be greatly improved by liposomal incorporation. We look at the beneficial effects of liposomes in a variety of solid and blood-borne tumors, including thyroid cancer, pancreatic cancer, breast cancer and multiple myeloma.

Anticancer activity of liposomal bergamot essential oil (BEO) on human neuroblastoma cells.

Citrus extracts, particularly bergamot essential oil (BEO) and its fractions, have been found to exhibit anticancer efficacy. However, the poor water solubility, low stability and limited bioavailability have prevented the use of BEO in cancer therapy. To overcome such drawbacks, we formulated BEO liposomes that improved the water solubility of the phytocomponents and increased their anticancer activity in vitro against human SH-SY5Y neuroblastoma cells. The results warrant further investigation of BEO liposomes for in vivo applications.

Gemcitabine-loaded chitosan microspheres. Characterization and biological in vitro evaluation.

This study investigates the potential of chitosan microspheres as delivery systems for the anticancer drug gemcitabine. The microspheres were obtained by spray-drying chitosan-gemcitabine solutions containing different amounts of the polyanion dextran sulphate. Morphological characterization by SEM and FIB analyses showed the presence of porous spherical particles having sizes ranging from about 1 to 5 µm. Dextran sulphate improves the technological parameters of the systems by producing very high encapsulation efficiency (about 96%, w/w) and by influencing the in vitro release of gemcitabine. The immediate drug release observed in the system prepared without dextrane sulphate (complete drug release within 30 min) was somewhat reduced by the polyanion (immediate release of 70% (w/w) of the encapsulated drug within 30 min, and subsequent continued release of the remaining 30% of the drug for over 4 days). The anti-tumoral efficacy of the various formulations was tested in vitro on human lung cancer cells (A549) comparing the effects with those of the free drug or drug/dextran sulfate complex. The carriers improved the cytotoxic activity of the drug, particularly the formulation containing the lowest amount of dextran sulphate after 72 h incubation.

Improved in vitro anti-tumoral activity, intracellular uptake and apoptotic induction of gemcitabine-loaded pegylated unilamellar liposomes.

Anaplastic thyroid carcinoma is one of the most aggressive and lethal solid carcinomas affecting humans. A major limit of the chemotherapeutic agents is represented by their low therapeutic index. In this work, we investigated the possibility of improving the anti-tumoral activity of gemcitabine by using pegylated unilamellar liposomes. Liposomes were made up of 1,2-dipalmitoyl-sn-glycero-3-phospocholine monohydrate/cholesterol/N-(carbonyl-methoxypolyethylene glycol-2000)-1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (6:3:1 molar ratio) and they were prepared with a pH gradient to improve the gemcitabine loading capacity. The anti-tumoral efficacy of the liposomal formulation was tested in vitro on human anaplastic thyroid carcinoma cells (ARO) in culture, comparing the effects with those of the free drug. Gemcitabine-loaded unilamellar liposomes had a mean size approximately 200 nm with a zeta potential approximately -2 mV. The liposomal carrier noticeably improved the anti-tumoral activity of gemcitabine against ARO cells in terms of both dose-dependent cytotoxic effect and of drug exposition effect. Namely, gemcitabine-loaded liposomes showed a cytotoxic effect (58.2% increase of cell mortality at 1 microM with respect to free drug) after 12 h incubation, while the free drug showed a significant activity only after 72 h incubation. Moreover, a significant effect on the cell mortality appeared at 0.1 microM and 100% mortality was detected at a concentration of 1 microM of gemcitabine-loaded liposomes, while the free drug elicited the same effect at a concentration of 100 microM. The improved anti-tumoral activity of gemcitabine determined by the liposomal carrier was due to a greater intracellular uptake. The intracellular gemcitabine levels as a function of time showed a sinusoidal profile with peaks after 2 h, 6 h and 11 h, related to the cellular cycle of ARO. PARP cleavage and DNA fragmentation analysis provided clear evidence of the apoptosis induction in ARO cells by treatment with liposomally entrapped gemcitabine after 72 h incubation. Thus, gemcitabine-loaded liposomes may have a potential therapeutic relevance for the treatment of anaplastic thyroid carcinoma.

Lecithin microemulsions for the topical administration of ketoprofen: percutaneous adsorption through human skin and in vivo human skin tolerability.

The potential application of highly biocompatible o/w microemulsions as topical drug carrier systems for the percutaneous delivery of anti-inflammatory drugs, i.e. ketoprofen, was investigated. Microemulsions were made up of triglycerides as oil phase, a mixture of lecithin and n-butanol as a surfactant/co-surfactant system and an aqueous solution as the external phase. To evaluate the percutaneous enhancing effect of oleic acid, this compound was used as a component of some o/w microemulsions. The topical carrier potentialities of lecithin-based o/w microemulsions were compared with respect to conventional formulations, i.e. a w/o emulsion, a o/w emulsion and a gel. Physicochemical characterisation of microemulsions was carried out by light scattering and zeta potential analyses. Microemulsions showed mean droplet size < 35 nm and a negative zeta potential, that is -39.5 mV for the oleic acid-lecithin microemulsion and -19.7 mV for the lecithin-based microemulsion. The percutaneous adsorption of the various topical formulations was evaluated through healthy adult human skin, which was obtained from abdominal reduction surgery. Ketoprofen-loaded microemulsions showed an enhanced permeation through human skin with respect to conventional formulations. No significant percutaneous enhancer effect was observed for ketoprofen-loaded oleic acid-lecithin microemulsions. The human skin tolerability of various microemulsion formulations was evaluated on human volunteers. Microemulsions showed a good human skin tolerability.

Preparation, characterization, molecular modeling and in vitro activity of paclitaxel-cyclodextrin complexes.

Paclitaxel (PTX) was complexed with beta-cyclodextrin (1), 2,6-dimethyl-beta-cyclodextrin (2) and 2,3,6-trimethyl-beta-cyclodextrin (3). PTX-CYD complexes were characterized both at the solid and liquid states. Experimental findings are in agreement with molecular modeling analysis, which showed different PTX-CYD interaction as a function of macrocyle methylation. The complexation of PTX within the CYD cavity preserved its antitumoral activity.