Self-assembly of perovskite nanoplates in colloidal suspensions.

In recent years, perovskite nanocrystal superlattices have been reported with collective optical phenomena, offering a promising platform for both fundamental science studies and device engineering. In this same avenue, superlattices of perovskite nanoplates can be easily prepared on different substrates, and they too present an ensemble optical response. However, the self-assembly and optical properties of these aggregates in solvents have not been reported to date. Here, we report on the conditions for this self-assembly to occur and show a simple strategy to induce the formation of these nanoplate stacks in suspension in different organic solvents. We combined wide- and small-angle X-ray scattering and scanning transmission electron microscopy to evaluate CsPbBr3 and CsPbI3 perovskite nanoplates with different thickness distributions. We observed the formation of these stacks by changing the concentration of nanoplates and the viscosity of the colloidal suspensions, without the need for antisolvent addition. We found that, in hexane, the concentration for the formation of the stacks is rather high and approximately 80 mg mL-1. In contrast, in decane, dodecane, and hexadecane, we observe a much easier self-assembly of the nanoplates, presenting a clear correlation between the degree of aggregation and viscosity. We, then, discuss the impact of the self-assembly of perovskite nanoplates on Förster resonant energy transfer. Our predictions suggest an energy transfer efficiency higher than 50% for all the donor-acceptor systems evaluated. In particular, we demonstrate how the aggregation of these particles in hexadecane induces FRET for CsPbBr3 nanowires. For the n = 2 nanowires (donor) to the n = 3 nanowires (acceptor), the FRET rate was found to be 4.1 ns-1, with an efficiency of 56%, in agreement with our own predictions.

The diffusion-driven microfluidic process to manufacture lipid-based nanotherapeutics with stealth properties for siRNA delivery.

Our study investigated the manufacturing of lipid-based nanotherapeutics with stealth properties for siRNA delivery by employing a diffusion-driven microfluidic process in one or two-steps strategies to produce siRNA-loaded lipid nanocarriers and lipoplexes, respectively. In the one-step synthesis, siRNA in the aqueous phase is introduced from one inlet, while phospholipids dispersed in anhydrous ethanol are introduced from other inlets, generating the lipid nanocarriers. In the two-steps strategies, the pre-formed liposomes are complexed with siRNA. The process configuration with an aqueous diffusion barrier exerts a significant effect on the nanoaggregates synthesis. Dynamic light scattering data showed that lipid nanocarriers had a bigger particle diameter (298 ± 24 nm) and surface charge (43 ± 6 mV) compared to lipoplexes (194 ± 7 nm and 37.0 ± 0.4 mV). Moreover, DSPE-PEG(2000) was included in the formulation to synthesize lipid-based nanotherapeutics containing siRNA with stealth characteristics. The inclusion of PEG-lipid resulted in an increase in the surface charge of lipoplexes (from 33.7 ± 4.4-54.3 ± 1.6 mV), while a significant decrease was observed in the surface charge of lipid nanocarriers (30.3 ± 8.7 mV). The different structural assemblies were identified for lipoplex and lipid nanocarriers using Synchrotron SAXS. Lipid nanocarriers present a lower amount of multilayers than lipoplexes. Lipid-PEG insertion significantly influenced lipid nanocarriers' characteristics, drastically decreasing the number of multilayers. This effect was not observed in lipoplexes. The association between process configuration, lipid composition, and its effect on the characteristics of lipid-based vector systems can generate fundamental insights, contributing to gene-based nanotherapeutics development.

One-step Production of Sterically Stabilized Anionic Nanoliposome Using Microfluidic Device.

Anionic liposomes (AL) are very attractive for nanomedicine and some formulations have already been launched for clinical development. Despite the excellent potential, their application presents two major challenges: laborious production methods and rapid degradation and elimination from blood by the immune system. In this work, we optimized the production of AL and its stealth form (SAL) using a onestep microfluidic process. We obtained unilamellar and near-monodisperse (< 10%) AL composed by the commercial composition (DMPC:DMPG) with mean size small as 53.7 nm, which is optimized for application in drug delivery. We also obtained SAL with similar characteristics using the microfluidic technique, overcoming the limitation of conventional methods where SAL presents high polydispersity (> 30%). This study demonstrates the great potential of the microfluidic technique for one-step production of stealth anionic nanoliposomes with controlled sizes and reproducible characteristics.

Hybrid polymer/lipid vesicle synthesis: Association between cationic liposomes and lipoplexes with chondroitin sulfate.

The association of cationic carriers with different anionic mucoadhesive biopolymers has been widely explored as an alternative to improve their delivery routes and specific targeting. This work presents a complete analysis of the association between chondroitin sulfate (CS) and cationic liposomes (CLs)/lipoplex (CL-pDNA). In this study, plasmid DNA (pDNA) was used as a genetic cargo for association with carriers. Firstly, we measured the stoichiometry of pseudo complexes and evaluated their colloidal properties, structural and morphological characteristics. Optimized CL-pDNA lipoplexes (positive z-potential) and CL-CS / CL-pDNA-CS (negative z-potential with CS mass ratio of 9% (w/w)) were further studied in detail. Small-angle X-ray scattering analysis and cryo-transmission electron microscopy micrographs revealed that the electrostatic interaction between CS and CL / CL-pDNA easily reorganized the lipid bilayers resulting in nanoscale uni/multilamellar vesicles. A high CS mass ratio (9% (w/w)) led to the reassembly of liposomal structure, wherein the pDNA was easily exchanged for CS chains, forming more than 50% of dense multilamellar vesicles. This data evidenced that the association between CS and CLs is not a conventional coating process since it generates complex and hybrid structures. We believe that these obtained colloidal data may be used in the future to investigate polymer-tailored nanocarriers and their production process. In brief, the colloidal study of hybrid structures may open interesting perspectives for developing novel carriers for drug and gene delivery applications.

Periodic bilayer organization in the complexes of Beta-2 Glycoprotein I with anionic lipid membranes.

ß2 glycoprotein I (ß2GPI) is a soluble protein that participates in blood coagulation, clearance of apoptotic bodies and generation of antigens in antiphospholipid syndrome among many other functions. We studied the aggregates formed by ß2GPI with the anionic phospholipids palmitoyloleoylphosphatidyl glycerol, dimyristoylphosphatidyl glycerol, dipalmitoylphosphatidyl glycerol and cardiolipin using small angle X-ray scattering. The complexes obtained in a medium containing 0.01 M NaCl showed Bragg peaks up to the sixth order in a well-defined integer sequence indicating the presence of a lamellar stacking with a periodicity of 17.8 nm and with largely reduced membrane fluctuations. Modeling the complex signal allowed us to conclude that the coherence length was only two bilayers and that about 15% of the total surface was actually stacked. The space between bilayers allows accommodating an extended ß2GPI molecule making a bridge between the interacting bilayers. The interactions between membranes mediated by ß2GPI was favored when the membranes were in the liquid crystalline state.

3D micromixer for nanoliposome synthesis: a promising advance in high mass productivity.

This paper addresses an important breakthrough in the high mass production of liposomes by microfluidics technology. We investigated the synthesis of liposomes using a high flow rate microfluidic device (HFR-MD) with a 3D-twisted cross-sectional microchannel to favor chaotic advection. A simple construction scaffold technique was used to manufacture the HFR-MD. The synthesis of liposomes combined the effects of high flow and high concentration of lipids, resulting in high mass productivity (2.27 g of lipid per h) which, to our knowledge, has never been registered by only one microdevice. We assessed the effects of the flow rate ratio (FRR), total flow rate (TFR), and lipid concentration on the liposome physicochemical properties. HFR-MD liposomes were monodisperse (0.074) with a size around 100 nm under the condition of an FRR of 1 (50% v/v ethanol) and TFR of 5 ml min-1 (expandable to 10 ml min-1). We demonstrated that the mixing conditions are not the only parameter controlling liposome synthesis using experimental and computational fluid dynamics analysis. A vacuum concentrator was used for ethanol removal, and there is no further modification after processing in accordance with the structural (SAXS) and morphological (cryo-TEM) analysis. Hence, the HFR-MD can be used to prepare nanoliposomes. It emerges as an innovative tool with high mass production.

Unmodified Clay Nanosheets at the Air-Water Interface.

Quasi-two-dimensional (2D) nanolayers, such as graphene oxide or clay layers, adhere to gas-liquid or liquid-liquid interfaces. Particularly, clays are of wide general interest in this context because of their extensive and crucial use as Pickering emulsion stabilizers, as well as for their ability to provide colloidosome capsules. So far, clays could only be localized at oil-water or air-saline-water interfaces in aggregated states, while our results now show that clay nanosheets without any modification can be located at air-deionized-water interfaces. The clay mineral used in the present work is synthetic fluorohectorite with a very high aspect ratio and superior quality in homogeneity and charge distribution compared to other clay minerals. This clay mineral is more suitable for achieving unmodified clay anchoring to fluid interfaces compared to other clay minerals used in previous works. In this context, we studied clay nanosheet organization at the air-water interface by combining different experimental methods: Langmuir-Blodgett trough studies, scanning electron microscopy (SEM) studies of film deposits, grazing-incidence X-ray off-specular scattering (GIXOS), and Brewster angle microscopy (BAM). Clay films formed at the air-water interface could be transferred to solid substrates by the Langmuir-Schaefer method. The BAM results indicate a dynamic equilibrium between clay sheets on the interface and in the subphase. Because of this dynamic equilibrium, the Langmuir monolayer surface pressure does not change significantly when pure clay sheets are spread on the liquid surface. However, also, GIXOS results confirm that there are clay nanosheets at the air-water interface. In addition, we find that clay sheets modified by a branched polymer are much more likely to be confined to the interface.

Physical and biological effects of paclitaxel encapsulation on disteraroylphosphatidylethanolamine-polyethyleneglycol polymeric micelles.

Simple size observations of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-mPEG2000) polymeric micelles (PM) with different compositions including or not paclitaxel (PTX) are unable to evidence changes on the nanocarrier structure. In such system a detailed characterization using highly sensitive techniques such as X-ray scattering and asymmetric flow field flow fractionation coupled to multi-angle laser light scattering and dynamic light scattering (AF4-MALS-DLS) is mandatory to observe effects that take place by the addition of PTX and/or more lipid-polymer at PM, leading to complex changes on the structure of micelles, as well as in their supramolecular organization. SAXS and AF4-MALS-DLS suggested that PM can be found in the medium separately and highly organized, forming clusters of PM in the latter case. SAXS fitted parameters showed that adding the drug does not change the average PM size since the increase in core radius is compensated by the decrease in shell radius. SAXS observations indicate that PEG conformation takes place, changing from brush to mushroom depending on the PM composition. These findings directly reflect in in vivo studies of blood clearance that showed a longer circulation time of blank PM when compared to PM containing PTX.

Evidence of photoinduced lipid hydroperoxidation in Langmuir monolayers containing Eosin Y.

Photodynamic therapy (PDT) efficiency depends on many factors including the incorporation of the photosensitizer (PS) in cell membranes and possible lipid hydroperoxidation. In this study, we show that hydroperoxidation may be photoinduced when eosin Y is incorporated into Langmuir monolayers that serve as cell membrane models. This occurs for Langmuir monolayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which have unsaturation in their hydrophobic chains. In contrast, light irradiation had no effect on monolayers of saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Evidence of hydroperoxidation was obtained from the area increase in eosin-containing DOPC and POPC monolayers upon irradiation, which was accompanied by a decrease in monolayer thickness according to grazing incidence X-ray off-specular scattering (GIXOS) data. Furthermore, the changes in polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) induced by irradiation were consistent with hydroperoxide migration toward the lipid hydrophilic heads.. In summary, this combination of experimental methods allowed us to determine the effects of eosin Y interaction with cell membrane models under irradiation, which may be associated with the underlying mechanisms of eosin Y as photosensitizer in PDT.

Lecithin and phytosterols-based mixtures as hybrid structuring agents in different organic phases.

In this study the effect of lecithin (L) addition and solvent quality in a well-established oleogel system formed by ß-sitosterol and γ-oryzanol (BG) was investigated. Medium chain triglycerides (MCT) and sunflower oil (SFO) were used as triglycerides and hexadecane (HEX) as a model of linear hydrocarbon. Lecithin was proposed due to its natural and versatile properties, showing different functionalities such as emulsifier and co-oleogelator. A study based on hierarchical organization of structured oil was performed applying techniques for bulk, meso and nanoscale. Self-sustained structures could no longer be observed after 40 wt% of BG replacement by lecithin. Small-angle X-ray scattering showed that the formed nanostructures (building blocks) were dependent on type of solvent and BG:L ratio in the mixture of oleogelators. Differential scanning calorimetry showed that stability against temperature was improved decreasing the polarity of the oil, and a time-dependent self-assembly of hybrid systems was observed from thermal and rheological measurements. Microscopy images exhibited changes on typical fibril aggregation of BG as lecithin was added, which promoted to a certain extent the suppression of ribbons. Oscillatory shear and uniaxial compression measurements were influenced by BG:L ratio and solvent mainly at higher lecithin amount. The combination of BG and MCT appeared to be the most affected by lecithin incorporation whereas SFO rendered harder oleogels. These results could contribute to understand the role of both lecithin and solvent type influencing the host oleogelator structure. It was hypothesized that intermolecular BG complex formation is hindered by lecithin, besides this phospholipid also might coexist as a different phase, causing structural changes in the gel network. Addressing the role of co-oleogelator it can provide the opportunity to tune soft materials with adjusted properties.

Refractive index and thickness determination in Langmuir monolayers of myelin lipids.

Langmuir monolayers at the air/water interface are widely used as biomembrane models and for amphiphilic molecules studies in general. Under controlled intermolecular organization (lateral molecular area), surface pressure, surface potential, reflectivity (R) and other magnitudes can be precisely determined on these planar monomolecular films. However, some physical parameters such as the refractive index of the monolayer (n) still remain elusive. The refractive index is very relevant because (in combination with R) it allows for the determination of the thickness of the film. The uncertainties of n determine important errors that propagate non-linearly into the calculation of monolayers thickness. Here we present an analytical method for the determination of n in monolayers based on refractive index matching. By using a Brewster angle microscopy (BAM) setup and monolayers spread over subphases with variable refractive index (n2), a minimum in R is search as a function of n2. In these conditions, n equals n2. The results shown correspond to monolayers of myelin lipids. The n values remain constant at 1.46 upon compression and equals the obtained value for myelin lipid bilayers in suspension. The values for n and R allow for the determination of thickness. We establish comparisons between these thicknesses for the monolayer and those obtained from two X-ray scattering techniques: 1) GIXOS for monolayers at the air/water interface and 2) SAXS for bilayers in bulk suspension. This allows us to conclude that the thickness that we measure by BAM includes the apolar and polar headgroup regions of the monolayer.

Poloxamer 407/188 binary thermosensitive hydrogels as delivery systems for infiltrative local anesthesia: Physico-chemical characterization and pharmacological evaluation.

In this study, we reported the development and the physico-chemical characterization of poloxamer 407 (PL407) and poloxamer 188 (PL188) binary systems as hydrogels for delivering ropivacaine (RVC), as drug model, and investigate their use in infiltrative local anesthesia for applications on the treatment of post-operative pain. We studied drug-micelle interaction and micellization process by light scattering and differential scanning calorimetry (DSC), the sol-gel transition and hydrogel supramolecular structure by small-angle-X-ray scattering (SAXS) and morphological evaluation by Scanning Electron Microscopy (SEM). In addition, we have presented the investigation of drug release mechanisms, in vitro/in vivo toxic and analgesic effects. Micellar dimensions evaluation showed the formation of PL407-PL188 mixed micelles and the drug incorporation, as well as the DSC studies showed increased enthalpy values for micelles formation after addition of PL 188 and RVC, indicating changes on self-assembly and the mixed micelles formation evoked by drug incorporation. SAXS studies revealed that the phase organization in hexagonal structure was not affected by RVC insertion into the hydrogels, maintaining their supramolecular structure. SEM analysis showed similar patterns after RVC addition. The RVC release followed the Higuchi model, modulated by the PL final concentration and the insertion of PL 188 into the system. Furthermore, the association PL407-PL188 induced lower in vitro cytotoxic effects, increased the duration of analgesia, in a single-dose model study, without evoking in vivo inflammation signs after local injection.

Microfluidic Assembly of pDNA/Cationic Liposome Lipoplexes with High pDNA Loading for Gene Delivery.

Microfluidics offers unique characteristics to control the mixing of liquids under laminar flow. Its use for the assembly of lipoplexes represents an attractive alternative for the translation of gene delivery studies into clinical trials on a sufficient throughput scale. Here, it was shown that the microfluidic assembly of pDNA/cationic liposome (CL) lipoplexes allows the formation of nanocarriers with enhanced transfection efficiencies compared with the conventional bulk-mixing (BM) process under high pDNA loading conditions. Lipoplexes generated by microfluidic devices exhibit smaller and more homogeneous structures at a molar charge ratio (R±) of 1.5, representing the ratio of lipid to pDNA content. Using an optimized model to fit small-angle X-ray scattering (SAXS) curves, it was observed that large amounts of pDNA induces the formation of aggregates with a higher number of stacked bilayers (N ∼ 5) when the BM process was used, whereas microfluidic lipoplexes presented smaller structures with a lower number of stacked bilayers (N ∼ 2.5). In vitro studies further confirmed that microfluidic lipoplexes achieved higher in vitro transfection efficiencies in prostate cancer cells at R ± 1.5, employing a reduced amount of cationic lipid. The correlation of mesoscopic characteristics with in vitro performance provides insights for the elucidation of the colloidal arrangement and biological behavior of pDNA/CL lipoplexes obtained by different processes, highlighting the feasibility of applying microfluidics to gene delivery.

The Link Between Self-Assembly and Molecular Conformation of Amphiphilic Block Copolymers Monolayers at the Air/Water Interface: The Spreading Parameter.

Two amphiphilic block copolymers, namely polystyrene-b-poly(methyl-methacrylate) (PS-b-PMMA), at the air/water interface were studied combining the in situ structural characterization, by means of X-ray reflectivity (XRR) and grazing incidence small-angle X-ray scattering (GISAXS), and the ex situ morphological one, by atomic force microscopy (AFM). We observed that the conformation of the hydrophilic block spread on the water surface is dramatically influenced by the morphology of the monolayer. Our results showed that the hydrophilic PMMA block adopts an expanded conformation when thermodynamically stable nanodots are formed, whereas a coiled conformation is observed when the block copolymer formed kinetically frozen nanostrands. This effect is interpreted in terms of the spreading parameter, which changes with the relative length ratio between the two blocks. Finally, we proved that, even for block copolymer monolayer with fixed block length ratio, it is possible to change the morphology, and in turn the conformation of the hydrophilic block, by changing the spreading parameter value.

Characterization of a Pt mirror to be used to deflect synchrotron radiation beam onto Langmuir monolayers.

A homemade mirror for X-rays has been built to prepare a diffraction beamline for liquid surface diffraction and scattering measurements. This simple approach is in operation at the XRD2 bending-magnet beamline at the Brazilian Synchrotron Light Laboratory.

Association between cationic liposomes and low molecular weight hyaluronic acid.

This work presents a study of the association between low molecular weight hyaluronic acid (16 kDa HA) and cationic liposomes composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). The cationic liposome/HA complexes were evaluated to determine their mesoscopic structure, average size, zeta potential, and morphology as a function of the amount of HA in the system. Small angle X-ray scattering results revealed that neighboring cationic liposomes either stick together after a partial coating of low concentration HA or disperse completely in excess of HA, but they never assemble as multilamellar vesicles. Cryo-transmission electron microscopy images confirm the existence of unilamellar vesicles and large aggregates of unilamellar vesicles for HA fractions up to 80% (w/w). High concentrations of HA (> 20% w/w) proved to be efficient for coating extruded liposomes, leading to particle complexes with sizes in the nanoscale range and a negative zeta potential.

Correlation of the physicochemical and structural properties of pDNA/cationic liposome complexes with their in vitro transfection.

In this study, we characterized the conventional physicochemical properties of the complexes formed by plasmid DNA (pDNA) and cationic liposomes (CL) composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (50/25/25% molar ratio). We found that these properties are nearly unaffected at the studied ranges when the molar charge ratio (R(±)) between the positive charge from the CL and negative charge from pDNA is not close to the isoneutrality region (R(±) = 1). However, the results from in vitro transfection of HeLa cells showed important differences when R(±) is varied, indicating that the relationships between the physicochemical and biological characteristics were not completely elucidated. To obtain information regarding possible liposome structural modifications, small-angle X-ray scattering (SAXS) experiments were performed as a function of R(±) to obtain correlations between structural, physicochemical, and transfection properties. The SAXS results revealed that pDNA/CL complexes can be described as being composed of single bilayers, double bilayers, and multiple bilayers, depending on the R(±) value. Interestingly, for R(±) = 9, 6, and 3, the system is composed of single and double bilayers, and the fraction of the latter increases with the amount of DNA (or a decreasing R(±)) in the system. This information is used to explain the transfection differences observed at an R(±) = 9 as compared to R(±) = 3 and 6. Close to the isoneutrality region (R(±) = 1.8), there was an excess of pDNA, which induced the formation of a fraction of aggregates with multiple bilayers. These aggregates likely provide additional resistance against the release of pDNA during the transfection phenomenon, reflected as a decrease in the transfection level. The obtained results permitted proper correlation of the physicochemical and structural properties of pDNA/CL complexes with the in vitro transfection of HeLa cells by these complexes, contributing to a better understanding of the gene delivery process.