Lipid based liquid-crystalline stabilized formulations for the sustained release of bioactive hydrophilic molecules.

Lipid based formulations, endowed of long term stability as a result of the formation of lamellar liquid crystals, were prepared using the natural lipids lecithin and glycerol trioleate in water, and characterized using optical microscopy, SAXRD and NMR. The formulations, designed as possible carriers for lysozyme and caffeine, were evaluated for structural features and stability after the loading of the guest molecules. Release experiments were performed at 37 °C using the PBS medium. No burst release was observed either for lysozyme or caffeine. Although lysozyme released from the lipid formulations does not fully retain its biological activity, the investigated liquid crystal stabilized formulations display a promising potential as drug and cosmetic carriers for topical applications, due to their high biocompatibility.

Purification, Conformational Analysis, and Properties of a Family of Tigerinin Peptides from Skin Secretions of the Crowned Bullfrog Hoplobatrachus occipitalis.

Four host-defense peptides belonging to the tigerinin family (tigerinin-1O: RICTPIPFPMCY; tigerinin-2O: RTCIPIPLVMC; tigerinin-3O: RICTAIPLPMCL; and tigerinin-4O: RTCIPIPPVCF) were isolated from skin secretions of the African crowned bullfrog Hoplobatrachus occipitalis. In aqueous solution at pH 4.8, the cyclic domain of tigerinin-2O adopts a rigid amphipathic conformation that incorporates a flexible N-terminal tail. The tigerinins lacked antimicrobial (MIC > 100 µM) and hemolytic (LC50 > 500 µM) activities but, at a concentration of 20 µg/mL, significantly (P < 0.05) inhibited production of interferon-γ (IFN-γ) by peritoneal cells from C57BL/6 mice without affecting production of IL-10 and IL-17. Tigerinin-2O and -4O inhibited IFN-γ production at concentrations as low as 1 µg/mL. The tigerinins significantly (P ≤ 0.05) stimulated the rate of insulin release from BRIN-BD11 clonal ß-cells without compromising the integrity of the plasma membrane. Tigerinin-1O was the most potent (threshold concentration 1 nM) and the most effective (395% increase over basal rate at a concentration of 1 µM). Tigerinin-4O was the most potent and effective peptide in stimulating the rate of glucagon-like peptide-1 release from GLUTag enteroendocrine cells (threshold concentration 10 nM; 289% increase over basal rate at 1 µM). Tigerinin peptides have potential for development into agents for the treatment of patients with type 2 diabetes.

Improvement of quercetin protective effect against oxidative stress skin damages by incorporation in nanovesicles.

Quercetin was incorporated in glycerosomes, new phospholipid-glycerol vesicles, and their protective effect against oxidative stress skin damages was extensively evaluated. In particular, the concentration-dependent effect of glycerol (from 10 to 50%) on vesicle suitability as cutaneous carriers of quercetin was carefully assessed. All vesicles were unilamellar and small in size (∼80-110 nm), as confirmed by cryo-TEM observation, with a drug incorporation efficiency ranging between 81 and 91%. SAXS studies, performed to investigate the bilayer arrangement, indicated a strong, dose-dependent interaction of glycerol with the polar portions of the phospholipid molecules, while quercetin did not significantly change the bilayer packing. In vitro studies on newborn pig skin underlined the concentration-dependent ability of glycerosomes to promote quercetin accumulation in the different layers, also confirmed by confocal microscopic observation of skin treated with fluorescent vesicles. Quercetin incorporated into liposomal and glycerosomal nanoformulations showed a strong ability to scavenge free radicals (DPPH test) and protect human keratinocytes in vitro against hydrogen peroxide damage. Moreover, quercetin-loaded vesicles were avidly taken up by keratinocytes in vitro. Overall, results indicate 40 and 50% glycerosomes as promising nanosystems for the improvement of cutaneous quercetin delivery and keratinocyte protection against oxidative stress damage.

Cancer-cell-targeted theranostic cubosomes.

This work was devoted to the development of a new type of lipid-based (cubosome) theranostic nanoparticle able to simultaneously host camptothecin, a potent anticancer drug, and a squarain-based NIR-emitting fluorescent probe. Furthermore, to confer targeting abilities on these nanoparticles, they were dispersed using mixtures of Pluronic F108 and folate-conjugated Pluronic F108 in appropriate ratios. The physicochemical characterization, performed via SAXS, DLS, and cryo-TEM techniques, proved that aqueous dispersions of such cubosomes can be effectively prepared, while the photophysical characterization demonstrated that these nanoparticles may be used for in vivo imaging purposes. The superior ability of these innovative nanoparticles in targeting cancer cells was emphasized by investigating the lipid droplet alterations induced in HeLa cells upon exposure to targeted and nontargeted cubosomes.

Molecular arrangements and interconnected bilayer formation induced by alcohol or polyalcohol in phospholipid vesicles.

A self-assembled hybrid phospholipid vesicular system containing various penetration enhancers - ethanol, Transcutol and propylenglycol - was prepared and characterized. The effects of the different alcohol or polyalcohols structure and their concentration on the features of the assembled vesicles were evaluated using a combination of different techniques, including cryo-transmission electron microscopy, laser light scattering, differential scanning calorimetry, small- and wide-angle X-ray scattering and rheological analysis. These techniques allow explaining the structural rearrangements of the bilayer assembly due to the alcohol or polyalcohol addition. X-ray scattering studies showed that such addition at the highest concentration (20%) allowed structure modification to oligolamellar vesicles and a bilayer transition to interdigitated phase. Rheological studies confirmed the importance of alcohol or polyalcohol in the structuring dispersions probably due to a partial tilting of phosphatidylcholine acyl chains forming interdigitated and interconnected bilayer vesicles.

From self-assembly fundamental knowledge to nanomedicine developments.

This review highlights the key role of NMR techniques in demonstrating the molecular aspects of the self-assembly of surfactant molecules that nowadays constitute the basic knowledge which modern nanoscience relies on. The aim is to provide a tutorial overview. The story of a rigorous scientific approach to understand self-assembly in surfactant systems and biological membranes starts in the early seventies when the progresses of SAXRD and NMR technological facilities allowed to demonstrate the existence of ordered soft matter, and the validity of Tanford approach concerning self-assembly at a molecular level. Particularly, NMR quadrupolar splittings, NMR chemical shift anisotropy, and NMR relaxation of dipolar and quadrupolar nuclei in micellar solutions, microemulsions, and liquid crystals proved the existence of an ordered polar-apolar interface, on the NMR time scale. NMR data, rationalized in terms of the two-step model of relaxation, allowed to quantify the dynamic aspects of the supramolecular aggregates in different soft matter systems. In addition, NMR techniques allowed to obtain important information on counterion binding as well as on size of the aggregate through molecular self-diffusion. Indeed NMR self-diffusion proved without any doubt the existence of bicontinuous microemulsions and bicontinuous cubic liquid crystals, suggested by pioneering and brilliant interpretation of SAXRD investigations. Moreover, NMR self-diffusion played a fundamental role in the understanding of microemulsion and emulsion nanostructures, phase transitions in phase diagrams, and particularly percolation phenomena in microemulsions. Since the nineties, globalization of the knowledge along with many other technical facilities such as electron microscopy, particularly cryo-EM, produced huge progresses in surfactant and colloid science. Actually we refer to nanoscience: bottom up/top down strategies allow to build nanodevices with applications spanning from ICT to food technology. Developments in the applied fields have also been addressed by important progresses in theoretical skills aimed to understand intermolecular forces, and specific ion interactions. Nevertheless, this is still an open question. Our predictive ability has however increased, hence more ambitious targets can be planned. Nanomedicine represents a major challenging field with its main aims: targeted drug delivery, diagnostic, theranostics, tissue engineering, and personalized medicine. Few recent examples will be mentioned. Although the real applications of these systems still need major work, nevertheless new challenges are open, and perspectives based on integrated multidisciplinary approaches would enable both a deeper basic knowledge and the expected advances in biomedical field.

An OFF-ON chemosensor for biological and environmental applications: sensing Cd2+ in water using catanionic vesicles and in living cells.

A new OFF-ON fluorescent chemosensor (L(1)) for Cd(2+) recognition based on a 5-chloro-8-hydroxyquinoline pendant arm derivative of 1,4,7-triazacyclononane ([9]aneN3) will be presented and its photochemical features in an MeCN-H2O 1 : 1 (v/v) mixture, in pure water, after inclusion within catanionic vesicles, and in living cells will be discussed. The coordination properties of L(1) both in solution and in the solid state were preliminarily studied and its selectivity towards Cd(2+)versus a set of different metal ions (Cu(2+), Zn(2+), Cd(2+), Pb(2+), Al(3+), Hg(2+), Co(2+), Ni(2+), Mn(2+), Mg(2+), K(+), Ca(2+), Ag(+), and Na(+)) was verified in MeCN-H2O 1 : 1 (v/v). In water, upon addition of increasing amounts of Cd(2+) to L(1) an enhancement of the fluorescence emission was detected. To overcome this serious drawback, L(1) was dissolved in an innovative catanionic vesicular solution based on sodium bis(2-ethylhexyl) sulfosuccinate, a traditional surfactant, and 1-dodecyl-3-methylimidazolium bromide, an ionic liquid. When enclosed within the vesicle bilayers in water, L(1) restored its fluorescence emission property upon addition of Cd(2+). Remarkably, L(1) enters the cellular membrane of living cells thus allowing the detection of intracellular Cd(2+). These findings encourage the application of this new fluorescent chemosensor in real samples for histological and environmental analyses.

Effect of diclofenac and glycol intercalation on structural assembly of phospholipid lamellar vesicles.

The aim of the current study was to improve the knowledge of drug-glycol-phospholipid-interactions and their effects in lamellar vesicle suitability as drug delivery systems. Liposomes were prepared using hydrogenated soy phosphatidylcholine (P90H, 60 mg/ml) and diclofenac sodium salt at two concentrations (5-10 mg/ml). To obtain innovative vesicles two permeation enhancers with glycol group, diethyleneglycol monoethyl ether and propylene glycol, were added to the water phase at different ratios (5%, 10%, and 20%). Vesicle organization was deeply investigated by physico-chemical characterization, including differential scanning calorimetry and small-angle diffraction signal analysis while macroscopic structure behavior was evaluated by rheological studies. Results evidenced that the presence of the penetration enhancer and diclofenac sodium salt led to structural rearrangements within and among vesicles forming a tridimensional and complex architecture in which vesicles were closely packed and interconnected. This new design allowed a change in the physical state of dispersions that became highly viscous liquid or soft-solid-like, thus forming an ideal system for topical application able of both adhering to the skin and delivering the drug.

Close-packed vesicles for diclofenac skin delivery and fibroblast targeting.

Concentrated and interconnected penetration enhancer containing vesicles (PEVs) are proposed as carriers for dermal delivery of diclofenac. PEVs were prepared by using a commercial phosphatidylcholine mixture (180 mg/m) and transcutol in different amounts. Conventional liposomes were also prepared and tested as control. All vesicles showed a mean size ranging from 75 to 253 nm with fairly narrow size distribution, negative zeta potential value, and drug loading capacity between 48 and 70%. SWAXS studies showed that composition affected vesicle structure and morphology: 10 and 30% transcutol PEVs were unilamellar while liposomes and 20% transcutol PEVs were multilamellar. Rheological studies demonstrated that control liposomes and 10 and 30% transcutol containing PEVs behaved as Newtonian fluids while 20% transcutol containing PEVs showed a plastic behavior. Ex vivo (trans)dermal delivery experiments showed an improved skin deposition of diclofenac when PEVs were used. Vesicle toxicity and uptake of fibroblasts, target of inflammation treatment, were evaluated by MTT test and fluorescence microscopy. Control liposomes and PEVs were both able to interact and being internalized by the 3T3 fibroblasts at all time exposure tested. Furthermore, PEVs showed to be able to reduce the in vitro drug toxicity.

Nanocarriers for antioxidant resveratrol: formulation approach, vesicle self-assembly and stability evaluation.

In this work we studied various nanoformulations of resveratrol in phospholipid vesicles. Conventional phophatidylcholine liposomes were prepared and characterized in parallel with PEVs (Penetration Enhancer-containing Vesicles) obtained by adding one of eight selected amphiphilic penetration enhancers (PEs; 0.2% w/v; HLB range 1-16) to the composition. All vesicles were around 100 nm, negatively charged (∼-30 mV) and able to incorporate resveratrol in good yields (>74%). The structure and the lamellar self-organization of the vesicles were investigated by Transmission Electron Microscopy (TEM) and Small and Wide Angle X-ray Scattering (SWAXS). These analyses showed that the lamellarity of the vesicles depended on the formulation composition. This work also addressed the stability of our colloidal dispersions, which was measured by means of the analytical centrifuge LUMiSizer(®): this procedure disclosed the absence of any demixing phenomena and estimated a 3- to 6-month shelf-life. Moreover, the antioxidant activity of resveratrol was determined by assessing its ability to scavenge free radicals (DPPH assay), and showed that it was not affected by the vesicular formulation.

Drug-loaded fluorescent cubosomes: versatile nanoparticles for potential theranostic applications.

In this work, monoolein-based cubosomes were doped with two fluorescent probes, namely, fluorescein and dansyl, properly modified with a hydrocarbon chain to increase their encapsulation efficiency within the monoolein palisade. The same nanocarriers were also loaded with quercetin, a hydrophobic molecule with potential anticancer activity. Particularly, the cubosomes doped with the modified fluorescein probe were successfully exploited for single living cell imaging. The physicochemical and photophysical characterizations reported here, along with the well-known ability of cubosomes in hosting molecules with pharmaceutical interest, strongly encourage the use of these innovative fluorescent nanocarriers for theranostic purposes.

Physicochemical, cytotoxic, and dermal release features of a novel cationic liposome nanocarrier.

A novel cationic liposome nanocarrier, having interesting performance in topical drug delivery, is here presented and evaluated for its features. Two penetration enhancers, namely monoolein and lauroylcholine chloride, are combined to rapidly formulate (15 min) a cationic liposome nanostructure endowed of excellent stability (>6 months) and skin penetration ability, along with low short-term cytotoxicity, as evaluated via the MTT test. Cytotoxicity tests and lipid droplet analysis give a strong indication that monoolein and lauroylcholine synergistically endanger long-term cells viability. The physicochemical features, investigated through SAXS, DLS, and cryo-TEM techniques, reveal that the nanostructure is retained after loading with diclofenac in its acid (hydrophobic) form. The drug release performances are studied using intact newborn pig skin. Analysis of the different skin strata proves that the drug mainly accumulates into the viable epidermis with almost no deposition into the derma. Indeed, the flux of the drug across the skin is exceptionally low, with only 1% release after 24 h. These results validate the use of this novel formulation for topical drug release when the delivery to the systemic circulation should be avoided.

In vitro release of lysozyme from gelatin microspheres: effect of cross-linking agents and thermoreversible gel as suspending medium.

This study was aimed to characterize the microstructure and the performance of gelatin microspheres (GMs) cross-linked by two different cross-linkers viz. d-glucose and glutaraldehyde. New formulations were obtained, suspending the GMs in a thermoreversible Pluronic F127 (PF127) liquid-crystalline gel. Lysozyme was used as a model biomacromolecular drug to evaluate release features. Both types of cross-linked GMs were prepared by thermal gelation method. The lysozyme-loaded microspheres were characterized by scanning electron microscopy (SEM) for size distribution, shape, and surface texture. SEM revealed that both types of lysozyme-loaded GMs were spherical in shape and that the surface of glutaraldehyde cross-linked GMs was smoother than that of the glucose cross-linked GMs. The degree of cross-linking of microspheres was investigated using ATR-FTIR technique. The prepared GMs were suspended in 20% w/v aqueous PF127 gel for which the usual sol-gel transition temperature of 22 °C did not change in the presence of GMs, as indicated by rheological measurements. SAXS study of the PF127 gel confirmed the occurrence of a discrete cubic liquid-crystalline phase of the Fm3m type whose lattice parameter slightly decreased as a result of GMs addition. The in vitro release of lysozyme from both types of cross-linked GMs was successfully controlled when they were suspended in PF127 gel, thus suggesting the potential use of this new combined formulation as a drug-depot system.

Aerosol-OT in water forms fully-branched cylindrical direct micelles in the presence of the ionic liquid 1-butyl-3-methylimidazolium bromide.

A recent investigation on the sodium bis(2-ethylhexyl)sulfosuccinate/water/1-butyl-3-methylimidazolium tetrafluoborate (NaAOT/W/bmimBF(4)) system showed that the anionic two-tailed surfactant NaAOT, that is known to form reverse micelles or planar interfaces (typically lamellar liquid crystals), can originate discrete spherical micelles of normal curvature because of strong interactions with the ionic liquid. The goal of the present paper was to detect macro- and microscopic modifications within such a system upon substitution of the ionic liquid's counter-ion tetrafluoroborate with bromide. Firstly, the phase diagram of the NaAOT/water/bmimBr system was determined. Then, the monophasic regions were investigated by means of NMR self-diffusion and SAXRD experiments. The results obtained proved this system to be surprisingly different from that containing bmimBF(4). This study focused mainly on the characterization of the micellar region, which turned out to be constituted of a bicontinuous nanostructure. This finding can be accounted for suggesting a decreasing of the NaAOT effective surfactant packing parameter, as in the case of NaAOT/water/bmimBF(4) system, although the effect in the presence of Br(-) is less pronounced. Data modeling showed the same degree of interfacial adsorption for the bmim(+) cation in both systems, regardless of the particular counterion used-either BF(4)(-) or Br(-). Thus, the remarkable differences between the two systems appear to be mainly due to a specific counterion effect. This result highlights once again the ions specificity, which is found ubiquitously in chemistry and biology.

Nucleotide recognition and phosphate linkage hydrolysis at a lipid cubic interface.

Mononucleotides, when entrapped within a mono-olein-based cubic Ia3d liquid crystalline phase, have been found to undergo hydrolysis at the sugar-phosphate ester bond in spite of their natural inertness toward hydrolysis. Here, kinetics of the hydrolysis reaction and interactions between the lipid matrix and the mononucleotide adenosine 5'-monophosphate disodium salt (AMP) and its 2'-deoxy derivative (dAMP) are thoroughly investigated in order to shed some light on the mechanism of the nucleotide recognition and phosphate ester hydrolysis. Experiments evidenced that molecular recognition occurs essentially through the sn-2 and the sn-3 alcoholic OH groups of mono-olein. As deduced from the apparent activation energies, the mechanism underlying the hydrolysis reaction is the same for AMP and dAMP. Nevertheless, the reaction proceeds slower for the latter, highlighting a substantial difference in the chemical behavior of the two nucleotides. A model that explains the hydrolysis reaction is presented. Remarkably, the hydrolysis mechanism appears to be highly specific for the Ia3d phase.

Nanoparticles from lipid-based liquid crystals: emulsifier influence on morphology and cytotoxicity.

Here, monoolein-based nanoparticles (NPs), obtained through fragmentation of bulk liquid crystalline phases, and stabilized by two different emulsifiers, namely, Pluronic F127 (PF127) and lauroylcholine chloride (LCh), are investigated for structural features and for short-term in vitro cytotoxicity. Depending on the emulsifiers, different morphologies of the lipid NPs (cubosomes and liposomes) are obtained, as demonstrated by cryo-TEM images. Although NPs offer many advantages in medical applications and various chemicals used for their preparation are under investigation, so far there are no standardized procedures to evaluate cell biocompatibility. Two different protocols to evaluate the impact of these lipid NPs on biological systems are presented. Results show that nanoparticles stabilized by PF127 (cubosomes) display a relevant toxicity toward different cell lines, whereas those stabilized by LCh (liposomes) affect cell viability at a much lesser extent.

Orientation and specific interactions of nucleotides and nucleolipids inside monoolein-based liquid crystals.

The entrapment of AMP, GMP, CMP, and UMP nucleotides along with two different AMP-based nucleolipids (hydrophobically functionalized nucleotides) inside the liquid crystalline phases of the monoolein/water system is investigated through optical microscopy, small-angle X-ray diffraction (SAXRD), and nuclear magnetic resonance (NMR) techniques. As ascertained mainly through (31)P NMR experiments, when included within the cubic phase, the various nucleotides undergo a slow hydrolysis of the sugar-phosphate ester bond, induced by specific interactions at the monoolein-water interface. Upon aging, the degradation of the nucleotides causes a cubic-to-hexagonal phase transition. Differently, neither hydrolysis nor alterations of the monoolein self-assembly are observed when the nucleotides are included as lipid derivatives within the cubic liquid crystalline phase. A model that explains both the hydrolysis and the consequent phase transition is presented.

Aerosol-OT forms oil-in-water spherical micelles in the presence of the ionic liquid bmimBF4.

The phase diagram sodium bis(2-ethylhexyl) sulfosuccinate (NaAOT)/water/1-butyl-3-methylimidazolium tetrafluoborate (bmimBF(4)), a polar room temperature ionic liquid, is explored through optical microscopy in polarized light, SAXRD and NMR PGSTE techniques. The analysis of SAXRD and self-diffusion data reveals that the bmim(+) cation is strongly adsorbed at the interface. Data are accounted for by Hill's model for cooperative binding. The overall process is described as a comicellization of AOT(-) and bmim(+) involving roughly two cations for AOT(-) ion. The bmim(+) is severely involved in the structural arrangement of the interface. Indeed, a huge modification of the interfacial geometry resulting in the occurrence of micelles having positive curvature is inferred from the analysis of the self-diffusion coefficients. The analysis of the water diffusion data in the L(1) phase (according to the effective cell model) allows one to exclude the presence of oblate and/or discoid micelles. Finally, the study of the oil diffusion in samples doped with p-xylene permits one to assess furthermore not only the formation of AOT aggregates of the oil-in-water type but also the occurrence of dynamic percolation phenomena.