Francesca Marongiu

Diclofenac nanosuspensions: influence of preparation procedure and crystal form on drug dissolution behaviour.

The aim of this paper was to ascertain the role of drug crystalline form and preparation procedure in nanosuspension formulations in order to optimise dissolution properties of lipophilic, poorly soluble drugs, thus improving their oral bioavailability. The non-steroidal anti-inflammatory drug diclofenac acid (DCF), which is known to exist in different crystal forms, was chosen as a model drug. To this purpose, the influence of homogenization technique was studied by preparing several nanosuspensions with two different crystalline forms of the drug (DCF1 and DCF2). Particle size and size distribution, morphology, microstructure, and thermal behaviour of the different formulations were studied by photon correlation spectroscopy (PCS), scanning electron microscopy (SEM), X-ray powder diffraction (XRD) and differential scanning calorimetry (DSC). Solubility studies of the bulk drug crystalline forms and dissolution experiments of nanosuspensions in comparison with different controls (bulk drug, physical mixtures, coarse suspensions) were carried out in different media: distilled water, simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Besides well known factors capable of affecting drug nanoparticle dissolution, results showed that drug dissolution rate in nanosuspensions is strongly affected by the drug solubility, which depends on the crystal form, and preparation procedure (high pressure homogenization process). Results demonstrated that this process partially transformed DCF2 in DCF1 while it did not have any effect on the DCF1 crystals.

Novel nanosized formulations of two diclofenac acid polymorphs to improve topical bioavailability

In this work, nanocrystal formulations, containing two different diclofenac acid crystal forms, were developed with the aim to improve dermal drug bioavailability. Nanosuspensions were obtaining using wet media milling technique and were characterized in terms of size distribution, morphology, zeta potential, differential scanning calorimetry and X-ray powder diffractometry. The ability of the nanocrystals to improve dermal drug bioavailability was investigated in vitro using Franz diffusion vertical cells and newborn pig skin, in comparison with diclofenac acid coarse suspensions and a commercial topical formulation containing diclofenac sodium. Nanocrystals exhibited a mean diameter ranging between 279 and 315 nm and a PI lower than 0.25, as shown by PCS measurements. The XRDP and DSC analysis clearly indicated that the preparation process did not modify the diclofenac polymorphic forms. In vitro transdermal delivery experiments showed an improved skin deposition and permeation of the nanocrystals compared to coarse suspensions and diclofenac sodium commercial topical formulation. These results highlight the fundamental role of the crystal size on drug solubility and, thus, on the ability of a poorly soluble drug to cross the skin and accumulate in the deeper skin layers.

Cavitation effect on chitosan nanoparticle size: a possible approach to protect drugs from ultrasonic stress.

The objective of this study was to verify the influence of different modes of ultrasonic radiation on both the mean diameter and the polydispersity index (PI) of chitosan (CH) nanoparticles, which were prepared by means of the ionotropic gelation method. The variations in duration, intensity and mode of cycle of ultrasonic radiation allowed us to highlight several optimal treatments in order to obtain a potential carrier for site-specific drug delivery. Despite the high utility, ultrasound may be a risk factor for sensitive drug-loaded nanoparticles; in order to protect the drug from thermal or mechanical stress, the effects of ultrasonic radiation only on the CH dispersion (instead of the chitosan/tripolyphosphate (TPP) mixture) were studied, without damaging the drug added to the TPP solution. The increase of the wave amplitude, mode of cycle and time of sonication decreased the particle mean diameter; moreover, the mode of cycle showed a greater effect than the other parameters on the PI of the nanoparticle system. Both the mean diameter and the PI of CH nanoparticles increased with increasing CH concentration. The application of ultrasound only on the CH dispersion showed interesting results, particularly in regard to formulations prepared from low and medium molecular weight chitosan.

Liposomes for (trans)dermal delivery of tretinoin: influence of drug concentration and vesicle composition

The influence of drug concentration and vesicle composition on the (trans)dermal delivery of tretinoin (TRA) was studied. To this purpose tretinoin was incorporated at different concentrations in unilamellar liposomes (UVs) prepared by sonication using phospholipids with different transition temperature (Tc): hydrogenated soy phosphatidylcholine (Tc = 51°C) or dipalmitoylphosphatidylcholine (Tc = 41°C). Vesicle dispersions were characterized in terms of morphology, size distribution and incorporation efficiency by using respectively optical and polarized light microscopy, transmission electron microscopy, dynamic laser light scattering, and HPLC. The effect of the vesicular incorporation of tretinoin at different molar ratios was investigated by zeta potential measurements and differential scanning calorimetry analysis. These analyses indicated that tretinoin principally interacts with the lipid groups until bilayer saturation. At higher concentration the ionized drug interacts with the polar head. Interactions between new-born pig skin and vesicle containing different amount of tretinoin were evaluated in vitro using Franz cells. The results obtained confirmed that liposomes saturated with TRA (molar fraction = 0.3) are capable of significantly promoting drug accumulation in the pig skin and that (trans)cutaneous delivery is strongly dependent on vesicular stability on the skin.

Diclofenac acid nanocrystals as an effective strategy to reduce in vivo skin inflammation by improving dermal drug bioavailability

In this work a diclofenac acid nanosuspension formulation was produced as a novel approach for the treatment of skin inflammation. Drug nanocrystals, prepared by the wet media milling technique and stabilized using Poloxamer 188, were characterized by different techniques: scanning electron microscopy, differential scanning calorimetry, X-ray powder diffractometry, Fourier transform infrared spectroscopy and photon correlation spectroscopy. The ability of nanocrystals to improve dermal drug bioavailability was investigated ex vivo by using Franz diffusion vertical cells and mouse skin, in comparison with both diclofenac acid coarse suspensions and a commercial formulation. The topical anti-inflammatory activity of the drug nanosuspension was assessed in vivo by testing its effect compared to common inflammatory endpoints: i.e. the inhibition of chemically induced oedema and leucocyte infiltration (reflected in myeloperoxidase activity). Following the milling procedure, diclofenac nanocrystals exhibited a mean diameter of approximately 279nm, a low polydispersity index (∼0.17) and maintained the same polymorphic form of the starting bulk powder. When the drug nanosuspension was applied on the mouse skin it produced a higher accumulation of diclofenac in the skin compared to both the coarse suspensions and the commercial formulation, as demonstrated by ex vivo transdermal delivery experiments. Moreover, the nanosuspension provided an in vivo oedema inhibition of 50%, which was not statistically different from the commercial formulation. On the contrary, the nanosuspension showed a higher inhibition of myeloperoxidase activity in the damaged tissue (86%) than the commercial formulation (16%).

Characterization and cytotoxicity studies on liposome–hydrophobic magnetite hybrid colloids

The aim of this study was to highlight the main features of magnetoliposomes prepared by TLE, using hydrophobic magnetite, and stabilized with oleic acid, instead of using the usual hydrophilic magnetite surrounded by sodium citrate. These biocompatible magnetoliposomes (MLs) were prepared with the purpose of producing a magnetic carrier capable of loading either hydrophilic or lipophilic drugs. The effect of different liposome/magnetite weight ratios on the stability of magnetoliposomes was evaluated by monitoring the mean diameter of the particles, their polydispersity index, and zeta potential over time. The prepared magnetoliposomes showed a high liposome-magnetite association, with magnetoliposomes containing PEG (polyethylene glycol) showing the best magnetite loading values. To verify the position of magnetite nanoparticles in the vesicular structures, the morphological characteristics of the structures were studied using transmission electron microscopy (TEM). TEM studies showed a strong affinity between hydrophobic magnetite nanoparticles, the surrounding oleic acid molecules, and phospholipids. Furthermore, the concentration above which one would expect to find a cytotoxic effect on cells as well as morphological cell-nanoparticle interactions was studied in situ by using the trypan blue dye exclusion assay, and the Prussian Blue modified staining method.

Drug silica nanocomposite: preparation, characterization and skin permeation studies

The aim of this work was to evaluate silica nanocomposites as topical drug delivery systems for the model drug, caffeine. Preparation, characterization, and skin permeation properties of caffeine-silica nanocomposites are described. Caffeine was loaded into the nanocomposites by grinding the drug with mesoporous silica in a ball mill up to 10 h and the efficiency of the process was studied by XRPD. Formulations were characterized by several methods that include FTIR, XRPD, SEM and TEM. The successful loading of caffeine was demonstrated by XRPD and FTIR. Morphology was studied by SEM that showed particle size reduction while TEM demonstrated formation of both core-shell and multilayered caffeine-silica structures. Solid-state NMR spectra excluded chemical interactions between caffeine and silica matrix, thus confirming that no solid state reactions occurred during the grinding process. Influence of drug inclusion in silica nanocomposite on the in vitro caffeine diffusion into and through the skin was investigated in comparison with a caffeine gel formulation (reference), using newborn pig skin and vertical Franz diffusion cells. Results from the in vitro skin permeation experiments showed that inclusion into the nanocomposite reduced and delayed caffeine permeation from the silica nanocomposite in comparison with the reference, independently from the amount of the tested formulation.

Freeze-dried eudragit-hyaluronan multicompartment liposomes to improve the intestinal bioavailability of curcumin.

This work aimed at finding an innovative vesicle-type formulation able to improve the bioavailability of curcumin upon oral administration. To this purpose, phospholipid, Eudragit® S100 and hyaluronan sodium salt were combined to obtain eudragit-hyaluronan immobilized vesicles using an easy and environmentally-friendly method. For the first time, the two polymers were combined in a system intended for oral delivery, to enhance curcumin stability when facing the harsh environment of the gastrointestinal tract. Four different formulations were prepared, keeping constant the amount of the phospholipid and varying the eudragit-hyaluronan ratio. The freeze-drying of the samples, performed to increase their stability, led to a reduction of vesicle size and a good homogeneity of the systems, after simple rehydration with water. X-ray diffraction study demonstrated that after the freeze-drying process, curcumin remained successfully incorporated within the vesicles. All the vesicles displayed similar features: size ranging from 220 to 287nm, spherical or oval shape, multilamellar or large unilamellar morphology with a peculiar multicompartment organization involving 1-4 smaller vesicles inside. In vitro studies demonstrated the ability of the combined polymers to protect the vesicles from the harsh conditions of the gastro-intestinal tract (i.e., ionic strength and pH variation), which was confirmed in vivo by the greater deposition of curcumin in the intestinal region, as compared to the free drug in dispersion. This enhanced accumulation of curcumin provided by the eudragit-hyaluronan immobilized vesicles, together with an increase in Caco-2 cell viability exposed to hydrogen peroxide, indicated that vesicles can ensure a local protection against oxidative stress and an increase in its intestinal absorption.

Nanoincorporation of bioactive compounds from red grape pomaces: In vitro and ex vivo evaluation of antioxidant activity

In this study, the active components of grape pomaces were first extracted by maceration in ethanol and propylene glycol, then in extra virgin olive oil. The main components of the hydrophilic extractive solutions were flavonoids, while monounsaturated fatty acids were the most abundant constituents of the extractive oil, with high levels of oleic acid, which were identified by HPLC/DAD and GC/MS, respectively. The hydrophilic extractive solutions and the lipophilic extractive oil were used to prepare phospholipid vesicles, avoiding the energetically and economically expensive steps required to obtain solid matrixes or pure compounds. The obtained grape bioactive enriched penetration enhancer containing vesicles (PEVs) were multilamellar, around 200nm in size, and more viscous than the corresponding solutions. The antioxidant activity, evaluated by the Folin-Ciocalteu and DPPH assays, was potentiated when the extractive solutions were loaded in PEVs. Further, the grape enriched PEVs were able to ensure an optimal protection against oxidative stress in an ex vivo human erythrocytes-based model.

Combination of grape extract-silver nanoparticles and liposomes: A totally green approach

&NA; In the present work, silver nanoparticles were prepared using a totally green procedure combining silver nitrate and an extract of grape pomace as a green source. Additionally, nanoparticles were stabilized using phospholipid and water and/or a mixture of water and propylene glycol (PG). To the best of our knowledge, grape‐silver nanoparticle stabilized liposomes or PG‐liposomes were formulated, for the first time, combining the residual products of wine‐made industry, silver nitrate and phospholipids, avoiding the addition of hazardous substances to human health and the environment, in an easy, scalable and reproducible method. The structure and morphology of grape‐silver nanoparticle stabilized vesicles were evaluated by transmission electron microscopy (TEM), UV‐vis spectroscopy and photon correlation spectroscopy. Samples were designed as possible carrier for skin protection because of their double function: the grape extract acts as antioxidant and the colloidal silver as antimicrobial agent, which might be helpful in eliminating dangerous free radicals and many pathogenic microorganisms. Obtained nanoparticles were small in size and their combination with phospholipids did not hamper the vesicle formation, which were multilamellar and sized ˜ 100 nm. TEM images shows a heterogeneous distribution of nanoparticles, which were located both in the intervesicular medium and in the vesicular structure. Further, grape‐silver nanoparticles, when stabilized by liposomes, were able to inhibit the proliferation of both Staphylococcus aureus and Pseudomonas aeruginosa and provided a great protection of keratinocytes and fibroblasts against oxidative stress avoiding their damage and death. Graphical abstract Figure. No caption available.