In Vitro Evaluation of Nanoerythrosome Cytotoxicity and Uptake in Pancreatic Endothelial Cells: Implications for ß-Cell Imaging Applications.

Biomolecule-targeted imaging represents one of the most difficult challenges in medicine. Nanoerythrosomes (NERs) are nanovesicles obtained after lysis of red blood cells, and they are promising tools for drug delivery and imaging. In this work, a formulation based on NERs functionalized with 7-amino-3-methylcoumarin via cross-linking was tested on rat INS-1E and mouse MIN6 ß-cells and endothelial MSI cell lines. First, the morphology, size, ζ-potentials, and spectroscopic properties of the aggregates were investigated, highlighting that the functionalization did not significantly affect the nanoparticles' physicochemical features. In vitro, the nanoparticles did not significantly affect the proliferation and function of INS-1E and MIN6 ß-cells at different concentrations. Only at the highest concentration tested on the MSI cell line, the formulation inhibited proliferation. Furthermore, NER aggregates were not internalized in both INS-1E and MIN6 cell lines, while a diffuse fluorescence was noticed in the cytosol of the MSI cell line at the highest concentrations. These findings proved that NER formulations might represent a new nanotool for ß-cell imaging as a part of a strategy aimed to prevent any intracellular accumulation, thus reducing/avoiding side effects.

Role of the Solvent in the Reactivity of Bis-4-imidazoline-2-selone Derivatives toward I2: An Experimental and Theoretical Approach.

The reactivity of 1,1'-bis(3-methyl-4-imidazolin-2-selone)methane (L1) and 1,2-bis(3-methyl-4-imidazolin-2-selone)ethane (L2) toward I2 has been explored in MeCN under different experimental conditions and compared with that in CH2Cl2. The compounds [L1'](I)2 (I), [L1I]n(I)n (II), [L1(µ-Se)](I)2·1/2H2O (III), [L1I](I3)·2I2 (IV), and [L2](I)2·MeCN (V) were obtained and characterized. X-ray diffraction analyses point out an ionic nature for these compounds, which is presumably favored by the polarity of the solvent used. In particular, [L1I]n(I)n (II) represents the first example of an iodonium complex of imidazoline-2-selone derivatives, while [L1(µ-Se)](I)2·1/2H2O (III) represents a unique example of a dicationic [RSeSeSeR] triselane. Density functional theory calculations have allowed us to better understand the nature of the obtained compounds and to justify their formations in polarizing reaction conditions rather than in low polar solvents.

Improving Dermal Delivery of Rose Bengal by Deformable Lipid Nanovesicles for Topical Treatment of Melanoma.

Cutaneous melanoma is one of the most aggressive and metastatic forms of skin cancer. However, current therapeutic options present several limitations, and the annual death rate due to melanoma increases every year. Dermal delivery of nanomedicines can effectively eradicate primary melanoma lesions, avoid the metastatic process, and improve survival. Rose Bengal (RB) is a sono-photosensitizer drug with intrinsic cytotoxicity toward melanoma without external stimuli but the biopharmaceutical profile limits its clinical use. Here, we propose deformable lipid nanovesicles, also known as transfersomes (TF), for the targeted dermal delivery of RB to melanoma lesions to eradicate them in the absence of external stimuli. Considering RB's poor ability to cross the stratum corneum and its photosensitizer nature, transfersomal carriers were selected simultaneously to enhance RB penetration to the deepest skin layers and protect RB from undesired photodegradation. RB-loaded TF dispersion (RB-TF), prepared by a modified reverse-phase evaporation method, were nanosized with a ζ-potential value below -30 mV. The spectrophotometric and fluorimetric analysis revealed that RB efficiently interacted with the lipid phase. The morphological investigations (transmission electron microscopy and small-angle X-ray scattering) proved that RB intercalated within the phospholipid bilayer of TF originating unilamellar and deformable vesicles, in contrast to the rigid multilamellar unloaded ones. Such outcomes agree with the results of the in vitro permeation study, where the lack of a burst RB permeation peak for RB-TF, observed instead for the free drug, suggests that a significant amount of RB interacted with lipid nanovesicles. Also, RB-TF proved to protect RB from undesired photodegradation over 24 h of direct light exposure. The ex vivo epidermis permeation study proved that RB-TF significantly increased RB's amount permeating the epidermis compared to the free drug (78.31 vs 38.31%). Finally, the antiproliferative assays on melanoma cells suggested that RB-TF effectively reduced cell growth compared to free RB at the concentrations tested (25 and 50 µM). RB-TF could potentially increase selectivity toward cancer cells. Considering the outcomes of the characterization and cytotoxicity studies performed on RB-TF, we conclude that RB-TF represents a valid potential alternative tool to fight against primary melanoma lesions via dermal delivery in the absence of light.

Rational Design of Sustainable Liquid Microcapsules for Spontaneous Fragrance Encapsulation.

The high volatility, water-immiscibility, and light/oxygen-sensitivity of most aroma compounds represent a challenge to their incorporation in liquid consumer products. Current encapsulation methods entail the use of petroleum-based materials, initiators, and crosslinkers as well as mixing, heating, and purification steps. Hence, more efficient and eco-friendly approaches to encapsulation must be sought. Herein, we propose a simple method by making use of a pre-formed amphiphilic polymer and employing the Hansen Solubility Parameters approach to determine which fragrances could be encapsulated by spontaneous coacervation in water. The coacervates do not precipitate as solids but they remain suspended as colloidally stable liquid microcapsules, as demonstrated by fluorescence correlation spectroscopy. The effective encapsulation of fragrance is proven through confocal Raman spectroscopy, while the structure of the capsules is investigated by means of cryo FIB/SEM, confocal laser scanning microscopy, and small-angle X-ray scattering.

Multifunctional cubic liquid crystalline nanoparticles for chemo- and photodynamic synergistic cancer therapy.

With the aim of engineering multifunctional nanoparticles useful for cancer therapy, a diketopyrrolopyrrole-porphyrin based photosensitizer was here conjugated to a block copolymer (Pluronic F108), and used to stabilize in water lipidic cubic liquid crystalline nanoparticles (cubosomes), also loaded with the antineoplastic agent docetaxel. The physicochemical characterization by SAXS, DLS, and cryo-TEM demonstrated that the formulation consisted of cubosomes, about 150 nm in size, possessing a bicontinuous cubic structure (space group Pn3m). The cellular imaging experiments proved that these nanoparticles localized in lysosomes and mitochondria, while cytotoxicity tests evidenced a slight but significant synergistic effect which, after irradiation, increased the toxicity induced by docetaxel alone, allowing further reduction of cell viability.

Cubosomes for in vivo fluorescence lifetime imaging.

Herein we provided the first proof of principle for in vivo fluorescence optical imaging application using monoolein-based cubosomes in a healthy mouse animal model. This formulation, administered at a non-cytotoxic concentration, was capable of providing both exogenous contrast for NIR fluorescence imaging with very high efficiency and chemospecific information upon lifetime analysis. Time-resolved measurements of fluorescence after the intravenous injection of cubosomes revealed that the dye rapidly accumulated mainly in the liver, while lifetimes profiles obtained in vivo allowed for discriminating between free dye or dye embedded within the cubosome nanostructure after injection.

Needle-free jet injection of intact phospholipid vesicles across the skin: a feasibility study.

Needle-free liquid jet injectors are devices developed for the delivery of pharmaceutical solutions through the skin. In this paper, we investigated for the first time the ability of these devices to deliver intact lipid vesicles. Diclofenac sodium loaded phospholipid vesicles of two types, namely liposomes and transfersomes, were prepared and fully characterized. The lipid vesicles were delivered through a skin specimen using a jet injector and the collected samples were analyzed to assess vesicle structural integrity, drug retention and release kinetics after the injection. In this regard, data concerning size, size distribution, surface charge of vesicles and bilayer integrity and thickness, before and after the injections, were measured by dynamic light scattering experiments, cryo-electron microscopy, and X-ray scattering techniques. Finally, the effect of vesicle fast jet injection through the skin on drug release kinetics was checked by in vitro experiments. The retention of the morphological, physico-chemical, and technological features after injection, proved the integrity of vesicles after skin crossing as a high-speed liquid jet. The delivery of undamaged vesicular carriers beneath the skin is of utmost importance to create a controlled release drug depot in the hypoderm, which may be beneficial for several localized therapies. Overall results reported in this paper may broaden the range of application of liquid jet injectors to lipid vesicle based formulations thus combining beneficial performance of painless devices with those of liposomal drug delivery systems.

Docetaxel-Loaded Fluorescent Liquid-Crystalline Nanoparticles for Cancer Theranostics.

Here, we describe a novel monoolein-based cubosome formulation engineered for possible theranostic applications in oncology. The Docetaxel-loaded nanoparticles were stabilized in water by a mixture of commercial Pluronic (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer) F108 (PF108) and rhodamine- and folate-conjugated PF108 so that the nanoparticles possess targeting, therapeutic, and imaging properties. Nanoparticles were investigated by DLS, cryo-TEM, and SAXS to confirm their structural features. The fluorescent emission characterization of the proposed formulation indicated that the rhodamine conjugated to the PF108 experiences an environment less polar than water (similar to chloroform), suggesting that the fluorescent fragment is buried within the poly(ethylene oxide) corona surrounding the nanoparticle. Furthermore, these nanoparticles were successfully used to image living HeLa cells and demonstrated a significant short-term (4 h incubation) cytotoxicity effect against these cancer cells. Furthermore, given their analogy as nanocarriers for molecules of pharmaceutical interest and to better stress the singularities of these bicontinuous cubic nanoparticles, we also quantitatively evaluated the differences between cubosomes and multilamellar liposomes in terms of surface area and hydrophobic volume.

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.

Polyphosphoester-stabilized cubosomes encapsulating a Ru(II) complex for the photodynamic treatment of lung adenocarcinoma.

The clinical translation of photosensitizers based on ruthenium(II) polypyridyl complexes (RPCs) in photodynamic therapy of cancer faces several challenges. To address these limitations, we conducted an investigation to assess the potential of a cubosome formulation stabilized in water against coalescence utilizing a polyphosphoester analog of Pluronic F127 as a stabilizer and loaded with newly synthesized RPC-based photosensitizer [Ru(dppn)2(bpy-morph)](PF6)2 (bpy-morph = 2,2'-bipyridine-4,4'-diylbis(morpholinomethanone)), PS-Ru. The photophysical characterization of PS-Ru revealed its robust capacity to induce the formation of singlet oxygen (1O2). Furthermore, the physicochemical analysis of the PS-Ru-loaded cubosomes dispersion demonstrated that the encapsulation of the photosensitizer within the nanoparticles did not disrupt the three-dimensional arrangement of the lipid bilayer. The biological tests showed that PS-Ru-loaded cubosomes exhibited significant phototoxic activity when exposed to the light source, in stark contrast to empty cubosomes and to the same formulation without irradiation. This promising outcome suggests the potential of the formulation in overcoming the drawbacks associated with the clinical use of RPCs in photodynamic therapy for anticancer treatments.

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.

A new family of bis-ureidic receptors for pyrophosphate optical sensing.

A new family of bis-ureidic receptors (L(1)-L(6)) has been synthesised. The binding properties of L(1)-L(6) towards different anions (acetate, benzoate, glutarate, malonate, dihydrogen phosphate, hydrogen pyrophosphate, triphosphate, AMP and ADP) have been studied by means of (1)H-NMR, UV-Vis and fluorescence spectroscopies and a remarkable affinity for HPpi(3-) has been observed in the case L(3) (in DMSO-d6 and DMSO-d6-5% H2O) which also acts as a fluorimetric chemosensor, even to the naked eye, for this anion. Theoretical calculations helped us explain the binding properties observed.

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.

Quantification of specific anion binding to non-ionic Triton X-100 micelles.

Anion binding to nonionic micelles was quantified by self-diffusion. Four anions were probed by multinuclear PGSTE NMR measurements in a Triton X-100 micellar aqueous solution. The salt concentration used was sufficiently low to avoid any micellar growth affecting surface curvature. The micellar aggregates that provide a model surface are uncharged with hydrophilic headgroups so that electrostatic ion surface interactions play little or no role in prescribing specific anion binding. Anionic affinity to the micellar surface followed a Hofmeister series, (CH(3))(2)AsO(2)(-) ≫ CH(3)COO(-) > H(2)PO(4)(-) > F(-). The observed ion specificity is rationalized by calling into play the nonelectrostatic interactions occurring between the anions and the micellar surface.

Lipid vesicular gels for topical administration of antioxidants.

The application of a formulation on the skin represents an effective way to deliver bio-active molecules for therapeutical purposes. Moreover, the outermost skin layer, the stratum corneum, can be overcome by employing chemical permeation enhancers and edge activators as components. Several lipids can be considered as permeation enhancers, such as the ubiquitous monoolein, one of the most used building blocks for the preparation of lipid liquid crystalline nanoparticles which are applied as drug carriers for nanomedicine applications. Recent papers highlighted how bile salts can affect the phase behavior of monoolein to obtain drug carriers suitable for topical administration, given their role as edge activators into the formulation. Herein, the encapsulation of natural antioxidants (caffeic acid and ferulic acid) into lipid vesicular gels (LVGs) made by monoolein and sodium taurocholate (TC) in water was studied to produce formulations suitable for topical application. TC induces a bicontinuous cubic to multilamellar phase transition for monoolein in water at the given concentrations, and by increasing its content into the formulations, unilamellar LVGs are formed. The encapsulation of the two antioxidants did not affect significantly the structure of the gels. The oscillating rheological studies showed that ferulic acid has a structuring effect on the lipid matrix, in comparison with the empty dispersion and the one containing caffeic acid. These gels were then tested in vitro on new-born pig skin to evaluate their efficacy as drug carriers for topical administration, showing that caffeic acid is mostly retained in the gel whereas ferulic acid is released at a higher degree. The data herein reported provide some further information on the effect of bile salts on the lipid self-assembly to evaluate useful compositions for topical administration of natural antioxidants.

Understanding the self-assembly of the polymeric drug solubilizer Soluplus®.

HYPOTHESIS: Soluplus® is one of the most widely used amphiphilic copolymers in drug delivery and has been reported to strongly enhance the adsorption of model drugs. However, there is still a limited understanding of its micellar behavior as it responds to the different routes of administration, which involve important changes in concentration. EXPERIMENTS: The microstructure of Soluplus aqueous solutions has been investigated at a wide range of polymer concentrations (2 × 10-6 - 0.2 g/mL) by a combination of diffusion NMR (dNMR), small angle X-ray scattering (SAXS), static (SLS) dynamic (DLS) light scattering and viscosity measurements. These techniques have been coupled with surface tension measurements to frame the polymer's critical micellar concentration (cmc). FINDINGS: We demonstrate the presence at all tested concentrations of two forms of Soluplus, with hydrodynamic radii of 3 and 26 nm, where the fraction of smaller objects accounts for as much as 60-70%. dNMR, SAXS, DLS and SLS indicate that Soluplus spontaneously self-assembles into large spherical particles with a core-shell structure. However, self-assembly takes place three orders of magnitude above the cmc evaluated via surface tension measurements. Instead of the traditional cooperative micellization process, we propose a thermal-activated isodesmic self-assembly of the small aggregates into core-shell micelles.

Study of the DNA binding mechanism and in vitro activity against cancer cells of iron(III) and aluminium(III) kojic acid derivative complexes.

Metal ions have unique electrochemical and spectroscopical properties that cannot be attained by purely organic compounds. Most of the metal ions are toxic to humans, but paradoxically, metallodrugs are used in medicine as therapeutics and theranostics. Metallodrugs are eliminated in urine and faeces, and therefore release toxic metals and ligands into aquatic ecosystems, thereby raising concerns regarding environmental risks. The use of metallodrugs based on essential metal ions (i.e., iron, copper and zinc), instead of toxic ions, is a new alternative with minor hazards. Kojic acid is an Asperigillus oryzae metabolite of low toxicity used in the food and cosmetics industries. Its derivatives form stable complexes with iron(III) ions, which bind effectively to DNA and inhibit DNA polymerization. The iron(III)/S2 ligand complexes reduce in vitro colon carcinoma (Caco2) cell viability and significantly decrease the cell number. The kojic acid derivative complexes with iron(III) presented here are an alternative to the currently used platinum complexes in cancer therapy.

Hybrid Theranostic Cubosomes for Efficient NIR-Induced Photodynamic Therapy.

In recent years, lipid bicontinuous cubic liquid-crystalline nanoparticles known as cubosomes have been under investigation because of their favorable properties as drug nanocarriers useful for anticancer treatments. Herein, we present organic/inorganic hybrid, theranostic cubosomes stabilized in water with a shell of alternate layers of chitosan, single strand DNA (model genetic material for potential gene therapy), and folic acid-chitosan conjugate (the outmost layer), coencapsulating up-converting Er3+ and Yb3+ codoped NaYF4 nanoparticles and daunorubicin. The latter acts as a chemotherapeutic drug of photosensitizing activity, while up-converting nanoparticles serve as energy harvester and diagnostic agent. Cellular uptake and NIR-induced photodynamic therapy were evaluated in vitro against human skin melanoma (MeWo) and ovarian (SKOV-3) cancer cells. Results evidenced the preferential uptake of the theranostic cubosomes in SKOV-3 cells in comparison to uptake in MeWo cells, and this effect was enhanced by the folic acid functionalization of the cubosomes surface. Nanocarriers coloaded with the hybrid fluorophores exhibited a superior NIR-induced photodynamic activity, also confirmed by the improved mitochondrial activity and the most affecting f-actin fibers of cytoskeleton. Similar results, but with higher photocytotoxicity, were detected when folic acid-functionalized cubosomes were incubated with SKOV-3 cells. Taken on the whole, these results prove these hybrid cubosomes are good candidates for the photodynamic treatment of tumor lesions.

Potential of curcumin-loaded cubosomes for topical treatment of cervical cancer.

Cervical cancer is one of the most common cancers affecting women worldwide. There are an estimated 570.000 new cases of cervical cancer each year and conventional treatments can cause severe side effects. In this work, we developed a platform for vaginal administration of lipophilic drugs for cervical cancer treatment. We formulated mucoadhesive cubosomes for the delivery of curcumin, a lipophilic drug for cervical cancer treatment, to increase its bioavailability and local absorption. This study tests the use of cubosomes for vaginal drug administration and assesses their potential efficiency using the CAM (chick embryo chorioallantoic membrane) model. SAXS (small-angle X-ray scattering), cryo-TEM (cryo-transmission electron microscopy), and dynamic light scattering (DLS) were employed to characterise the system. With ex vivo permeation and retention studies, we find that the curcumin released from our system is retained in the vaginal mucosa. In vitro cytotoxicity assay and cellular uptake showed an increased cytotoxic effect of curcumin against HeLa cell line when incorporated into the cubosomes. The curcumin-loaded cubosomes also demonstrated an antiangiogenic effect evaluated in vivo by the CAM model.

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.