Lipid nanocapsules maintain full integrity after crossing a human intestinal epithelium model

Lipid nanocapsules (LNCs) have demonstrated great potential for the oral delivery of drugs having very limited oral bioavailability (BCS class II, III and IV molecules). It has been shown previously that orally-administered LNCs can permeate through mucus, increase drug absorption by the epithelial tissue, and finally, increase drug bioavailability. However, even if transport mechanisms through mucus and the intestinal barrier have already been clarified, the preservation of particle integrity is still not known. The aim of the present work is to study in vitro the fate of LNCs after their transportation across an intestinal epithelium model (Caco-2 cell model). For this, two complementary techniques were employed: Förster Resonance Energy Transfer (FRET) and Nanoparticle Tracking Analysis (NTA). Results showed, after 2 h, the presence of nanoparticles in the basolateral side of the cell layer and a measurable FRET signal. This provides very good evidence for the transcellular intact crossing of the nanocarriers

Two-Dimensional Molecular Patterning by Surface-Enhanced Zn-Porphyrin Coordination

In this contribution, we show how zinc-5,10,15,20-meso-tetradodecylporphyrins (Zn-TDPs) self-assemble into stable organized arrays on the surface of graphite, thus positioning their metal center at regular distances from each other, creating a molecular pattern, while retaining the possibility to coordinate additional ligands. We also demonstrate that Zn-TDPs coordinated to 3-nitropyridine display a higher tendency to be adsorbed at the surface of highly oriented pyrolytic graphite (HOPG) than noncoordinated ones. In order to investigate the two-dimensional (2D) self-assembly of coordinated Zn-TDPs, solutions with different relative concentrations of 3-nitropyridine and Zn-TDP were prepared and deposited on the surface of HOPG. STM measurements at the liquid-solid interface reveal that the ratio of coordinated Zn-TDPs over noncoordinated Zn-TDPs is higher at the n-tetradecane/HOPG interface than in n-tetradecane solution. This enhanced binding of the axial ligand at the liquid/solid interface is likely related to the fact that physisorbed Zn-TDPs are better binding sites for nitropyridines.

Inter-nanocarrier and nanocarrier-to-cell transfer assays demonstrate the risk of an immediate unloading of dye from labeled lipid nanocapsules

Release studies constitute a fundamental part of the nanovector characterization. However, it can be difficult to correctly assess the release of lipophilic compounds from lipid nanocarriers using conventional assays. Previously, we proposed a method including an extraction with oil to measure the loading stability of lipophilic dyes in lipid nanocapsules (LNCs). The method indicated a rapid release of Nile Red from LNCs, while the loading of lipophilic carbocyanine dyes remained stable. This method, although interesting for a rapid screening of the fluorescence labeling stability of nanocarriers, is far from what happens in vivo, where lipid acceptor phases are nanostructured. Here, lipophilic dye loading stability has been assessed, by monitoring dye transfer from LNCs toward stable colloidal lipid nanocompartments, i.e. non-loaded LNCs, using new methodology based on size exclusion chromatography (SEC) and Förster Resonance Energy Transfer (FRET). Dye transfer between LNCs and THP-1 cells (as model for circulating cells) has also been studied by FACS. The assays reveal an almost instantaneous transfer of Nile Red between LNCs, from LNCs to THP-1 cells, between THP-1 cells, and a reversal transfer from THP-1 cells to LNCs. On the contrary, there was no detectable transfer of the lipophilic carbocyanine dyes. Dye release was also analyzed using dialyses, which only revealed a very slow release of Nile Red from LNCs, demonstrating the weakness of membrane based assays for investigations of the lipophilic compound loading stability in lipid nanocarriers. These results highlight the importance of using relevant release assays, and the potential risk of an immediate unloading of lipophilic fluorescent dyes from lipid nanocarriers, in the presence of a lipid acceptor nanocompartment. Some misinterpretations of cellular trafficking and in vivo biodistribution of fluorescent nanoparticles should be avoided

Neutral fluorescence probe with strong ratiometric response to surface charge of phospholipid membranes

AbstractWe report on dramatic differences in fluorescence spectra of 4′-dimethylamino-3-hydroxyflavone (probe F) studied in phospholipid membranes of different charge (phosphatidyl glycerol, phosphatidylcholine (PC), their mixture and the mixture of PC with a cationic lipid). The effect consists in variations of relative intensities at two well-separated band maxima at 520 and 570 nm belonging to normal (N*) and tautomer (T*) excited states of flavone chromophore. Based on these studies we propose a new approach to measure electrostatic potential at the surface layer of phospholipid membranes, which is based on potential-dependent changes of bilayer hydration and involves very sensitive and convenient ratiometric measurements in fluorescence emission

N-(Di)icosyl-substituted benzo[a]phenoxazinium chlorides : synthesis and evaluation as near-infrared membrane probes

Five benzo[a]phenoxazinium chlorides containing alkyl chains with twenty carbon atoms on 5- or 9-positions of the tetracyclic ring were efficiently synthesised and characterised by UV/Vis/NIR spectroscopy. The absorption and emission maxima in ethanol lie in the range 627-641 nm and 645-676 nm, respectively, with quantum yields varying from 0.14 to 0.38. Preliminary photophysical studies with these fluorochromophores in zwitterionic (2,3- bis(palmitoyl-oxy)propyl-2-(trimethylammonio)ethyl phosphate, DPPC) and cationic (N,N-dimethyl-N-octadecyloctadecan-1-aminium bromide, DODAB) vesicles were carried out. The results showed that the new benzo[a]phenoxazinium derivatives are able to detect the gel to liquid-crystalline lipid phase transition through variations, either in the H-aggregation extent or in an acid-base equilibrium.Fundação para a Ciência e Tecnologia (FCT) - REDE/1517/RMN/2005Fundo Europeu de Desenvolvimento Regional (FEDER) - POCI 201

Fighting Aggregation‐Caused Quenching and Leakage of Dyes in Fluorescent Polymer Nanoparticles: Universal Role of Counterion

Dye‐loaded polymer nanoparticles (NPs) emerge as a powerful tool for bioimaging applications, owing to their exceptional brightness and controlled small size. However, aggregation‐caused quenching (ACQ) and leakage of dyes at high loading remain important challenges of these nanomaterials. The use of bulky hydrophobic counterions has been recently proposed as an effective approach to minimize ACQ and dye leakage, but the role of counterion structure is still poorly understood. Here, a systematic study based on ten counterions, ranging from small hydrophilic perchlorate up to large hydrophobic tetraphenylborate derivatives, reveals how counterion nature can control encapsulation and emission of a cationic dye (rhodamine B octadecyl ester) in NPs prepared by nanoprecipitation of a biodegradable polymer, poly‐lactide‐co‐glycolide (PLGA). We found that increase in counterion hydrophobicity enhances dye encapsulation efficiency and prevents dye adsorption at the particle surface. Cellular imaging studies revealed that ≥95 % encapsulation efficiency, achieved with most hydrophobic counterions (fluorinated tetraphenylborates), is absolutely required because non‐encapsulated dye species at the surface of NPs are the origin of dye leakage and strong fluorescence background in cells. The size of counterions is found to be essential to prevent ACQ, where the largest species, serving as effective spacer between dyes, provide the highest fluorescence quantum yield. Moreover, we found that the most hydrophobic counterions favor dye–dye coupling inside NPs, leading to ON/OFF fluorescence switching of single particles. By contrast, less hydrophobic counterions tend to disperse dyes in the polymer matrix favoring stable emission of NPs. The obtained structure‐property relationships validate the counterion‐based approach as a mature concept to fight ACQ and dye leakage in the development of advanced polymeric nanomaterials with controlled optical properties

Solvatochromic and Fluorogenic Dyes as Environment-Sensitive Probes: Design and Biological Applications.

Fluorescent environment-sensitive probes are specially designed dyes that change their fluorescence intensity (fluorogenic dyes) or color (e.g., solvatochromic dyes) in response to change in their microenvironment polarity, viscosity, and molecular order. The studies of the past decade, including those of our group, have shown that these molecules become universal tools in fluorescence sensing and imaging. In fact, any biomolecular interaction or change in biomolecular organization results in modification of the local microenvironment, which can be directly monitored by these types of probes. In this Account, the main examples of environment-sensitive probes are summarized according to their design concepts. Solvatochromic dyes constitute a large class of environment-sensitive probes which change their color in response to polarity. Generally, they are push-pull dyes undergoing intramolecular charge transfer. Emission of their highly polarized excited state shifts to the red in more polar solvents. Excited-state intramolecular proton transfer is the second key concept to design efficient solvatochromic dyes, which respond to the microenvironment by changing relative intensity of the two emissive tautomeric forms. Due to their sensitivity to polarity and hydration, solvatochromic dyes have been successfully applied to biological membranes for studying lipid domains (rafts), apoptosis and endocytosis. As fluorescent labels, solvatochromic dyes can detect practically any type of biomolecular interactions, involving proteins, nucleic acids and biomembranes, because the binding event excludes local water molecules from the interaction site. On the other hand, fluorogenic probes usually exploit intramolecular rotation (conformation change) as a design concept, with molecular rotors being main representatives. These probes were particularly efficient for imaging viscosity and lipid order in biomembranes as well as to light up biomolecular targets, such as antibodies, aptamers and receptors. The emerging concepts to achieve fluorogenic response to the microenvironment include ground-state isomerization, aggregation-caused quenching, and aggregation-induced emission. The ground-state isomerization exploits, for instance, polarity-dependent spiro-lactone formation in silica-rhodamines. The aggregation-caused quenching uses disruption of the self-quenched dimers and nanoassemblies of dyes in less polar environments of lipid membranes and biomolecules. The aggregation-induced emission couples target recognition with formation of highly fluorescent dye aggregates. Overall, solvatochromic and fluorogenic probes enable background-free bioimaging in wash-free conditions as well as quantitative analysis when combined with advanced microscopy, such as fluorescence lifetime (FLIM) and ratiometric imaging. Further development of fluorescent environment-sensitive probes should address some remaining problems: (i) improving their optical properties, especially brightness, photostability, and far-red to near-infrared operating range; (ii) minimizing nonspecific interactions of the probes in biological systems; (iii) their adaptation for advanced microscopies, notably for superresolution and in vivo imaging.journal article2017 Feb 212017 01 09importe

Characterizing Counterion-Dependent Aggregation of Rhodamine B by Classical Molecular Dynamics Simulations

The aggregation in a solution of charged dyes such as Rhodamine B (RB) is significantly affected by the type of counterion, which can determine the self-assembled structure that in turn modulates the optical properties. RB aggregation can be boosted by hydrophobic and bulky fluorinated tetraphenylborate counterions, such as F5TPB, with the formation of nanoparticles whose fluorescence quantum yield (FQY) is affected by the degree of fluorination. Here, we developed a classical force field (FF) based on the standard generalized Amber parameters that allows modeling the self-assembling process of RB/F5TPB systems in water, consistent with experimental evidence. Namely, the classical MD simulations employing the re-parametrized FF reproduce the formation of nanoparticles in the RB/F5TPB system, while in the presence of iodide counterions, only RB dimeric species can be formed. Within the large, self-assembled RB/F5TPB aggregates, the occurrence of an H-type RB-RB dimer can be observed, a species that is expected to quench RB fluorescence, in agreement with the experimental data of FQY. The outcome provides atomistic details on the role of the bulky F5TPB counterion as a spacer, with the developed classical FF representing a step towards reliable modeling of dye aggregation in RB-based materials