Ethylcellulose nanoparticles prepared from nano-emulsion templates as new folate binding haemocompatible platforms.

Ethylcellulose is a biocompatible polymer attracting increasing interest for biomedical applications. In the present work, the formation of folate-ethylcellulose nanoparticle complexes from nano-emulsion templates prepared by a low-energy approach, using aqueous components suitable for biomedical applications has been investigated. The composition of the aqueous component is shown to be crucial for the formation of stable nano-emulsions and influences the zeta potential values. The ethylcellulose nanoparticles with mean sizes around 100 nm were obtained from the nano-emulsions by solvent evaporation and showed positive zeta potential values above +20 mV due to the presence of the cationic surfactant. The nanoparticles were successfully complexed with folate, as evidenced by both particle size and zeta potential measurements. The complexes prepared with HEPES buffered glucose solution showed excellent haemocompatibility, which make them promising for parenteral therapeutic applications and also for those in which easy access to systemic circulation may occur, like in lungs.

Functionalized PLGA nanoparticles prepared by nano-emulsion templating interact selectively with proteins involved in the transport through the blood-brain barrier.

During the last few decades, extensive efforts has been made to design nanocarriers to transport drugs into the central nervous system (CNS). However, its efficacy is limited due to the presence of the Blood-Brain Barrier (BBB) which greatly reduces drug penetration making Drug Delivery Systems (DDS) necessary. Polymeric nanoparticles (NPs) have been reported to be appropriate for this purpose and in particular, poly(lactic-co-glycolic acid) (PLGA) has been used for its ability to entrap small molecule drugs with great efficiency and the ease with which it functionalizes NPs. Despite the fact that their synthetic identity has been studied in depth, the biological identity of such manufactured polymers still remains unknown as does their biodistribution and in vivo fate. This biological identity is a result of their interaction with blood proteins, the so-called "protein corona" which tends to alter the behavior of polymeric nanoparticles in the body. The aim of the present research is to identify the proteins bounded to polymeric nanoparticles designed to selectively interact with the BBB. For this purpose, four different PLGA NPs were prepared and analyzed: (i) "PLGA@Drug," in which a model drug was encapsulated in its core; (ii) "8D3-PLGA" NPs where the PLGA surface was functionalized with a monoclonal anti-transferrin receptor antibody (8D3 mAb) in order to specifically target the BBB; (iii) "8D3-PLGA@Drug" in which the PLGA@Drug surface was functionalized using the same antibody described above and (iv) bare PLGA NPs which were used as a control. Once the anticipated protein corona NPs were obtained, proteins decorating both bare and functionalized PLGA NPs were isolated and analyzed. Apart from the indistinct interaction with PLGA NPs with the most abundant serum proteins, specific proteins could also be identified in the case of functionalized PLGA NPs. These findings may provide valuable insight into designing novel vehicles based on PLGA NPs for crossing the BBB.

Biomedical perfluorohexane-loaded nanocapsules prepared by low-energy emulsification and selective solvent diffusion.

Perfluorohexane-loaded nanocapsules are interesting materials for many biomedical applications such as oxygen delivery systems or contrast agents. However, their formulation into stable colloidal systems is challenging because of their hydro- and lipophobicity, high density and high vapour pressure. In this study, perfluorohexane-loaded polymeric nanocapsules are prepared for the first time by low-energy emulsification and selective solvent diffusion. The colloidal stability of the perfluorohexane nano-emulsion templates has been improved by the incorporation of an apolar low-density oil (isopropyl myristate) in the dispersed phase, thus addressing droplet coarsening and migration phenomena. The perfluorohexane-loaded nanocapsules prepared from the nano-emulsions show sizes smaller than the corresponding emulsion templates (below 150 nm by dynamic light scattering) and exhibit good stability under storage conditions. Hyperspectral enhanced dark field microscopy revealed a layered core/shell structure and allowed also to confirm the encapsulation of perfluorohexane which was quantified by elemental microanalysis. Although isopropyl myristate has an unfavourable biocompatibility profile, cell viability is enhanced when perfluorohexane is present in the nanocapsules, which is attributed to its high oxygen transport capacity.

Protein-nanoparticle interactions evaluation by immunomethods: Surfactants can disturb quantitative determinations.

The adsorption of proteins on nanoparticle surface is one of the first events that occur when nanoparticles enter in the blood stream, which influences nanoparticles lifetime and further biodistribution. Albumin, which is the most abundant protein in serum and which has been deeply characterized, is an interesting model protein to investigate nanoparticle-protein interactions. Therefore, the interaction of nanoparticles with serum albumin has been widely studied. Immunomethods were suggested for the investigation of adsorption isotherms because of their ease to quantify the non-adsorbed bovine serum albumin without the need of applying separation methods that could modify the balance between the adsorbed and non-adsorbed proteins. The present work revealed that this method should be applied with caution. Artifacts in the determination of free protein can be generated by the presence of surfactants such as polysorbate 80, widely used in the pharmaceutical and biomedical field, that are needed to preserve the stability of nanoparticle dispersions. It was shown that the presence of traces of polysorbate 80 in the dispersion leads to an overestimation of the amount of bovine serum albumin remaining free in the dispersion medium when determined by both radial immunodiffusion and rocket immunoelectrophoresis. However, traces of poloxamer 188 did not result in clear perturbed migrations. These methods are not appropriate to perform adsorption isotherms of proteins on nanoparticle dispersions containing traces of remaining free surfactant. They should only be applied on dispersions that are free of surfactant that is not associated with nanoparticles.

Interactions of PLGA nanoparticles with blood components: protein adsorption, coagulation, activation of the complement system and hemolysis studies.

The intravenous administration of poly(lactic-co-glycolic) acid (PLGA) nanoparticles has been widely reported as a promising alternative for delivery of drugs to specific cells. However, studies on their interaction with diverse blood components using different techniques are still lacking. Therefore, in the present work, the interaction of PLGA nanoparticles with blood components was described using different complementary techniques. The influence of different encapsulated compounds/functionalizing agents on these interactions was also reported. It is worth noting that all these techniques can be simply performed, without the need for highly sophisticated apparatus or skills. Moreover, their transference to industries and application of quality control could be easily performed. Serum albumin was adsorbed onto all types of tested nanoparticles. The saturation concentration was dependent on the nanoparticle size. In contrast, fibrinogen aggregation was dependent on nanoparticle surface charge. The complement activation was also influenced by the nanoparticle functionalization; the presence of a functionalizing agent increased complement activation, while the addition of an encapsulated compound only caused a slight increase. None of the nanoparticles influenced the coagulation cascade at low concentrations. However, at high concentrations, cationized nanoparticles did activate the coagulation cascade. Interactions of nanoparticles with erythrocytes did not reveal any hemolysis. Interactions of PLGA nanoparticles with blood proteins depended both on the nanoparticle properties and the protein studied. Independent of their loading/surface functionalization, PLGA nanoparticles did not influence the coagulation cascade and did not induce hemolysis of erythrocytes; they could be defined as safe concerning induction of embolization and cell lysis.

Design and in vitro evaluation of biocompatible dexamethasone-loaded nanoparticle dispersions, obtained from nano-emulsions, for inhalatory therapy.

Polymeric nanoparticle dispersions containing dexamethasone (DXM) have been prepared from O/W nano-emulsions of the water/polysorbate 80/[4 wt% poly(lactide-co-glycolide) acid+0.18 wt% DXM in ethyl acetate] system by a low-energy method at 25°C. Nano-emulsions were formed at O/S ratios between 45/55 and 72/25 and water contents above 70 wt% by the phase inversion composition (PIC) method. The mean hydrodynamic diameter of nano-emulsions with a constant water content of 90 wt% and O/S ratios from 50/50 to 70/30 was below 350 nm as assessed by dynamic light scattering. The nanoparticles obtained from these nano-emulsions (by solvent evaporation) showed mean diameters of around 130 nm, as determined by transmission electron microscopy image analysis. Therapeutic concentrations of DXM were encapsulated in the nano-emulsions prior to nanoparticle preparation. DXM entrapment efficiency of the nanoparticle dispersion (above 74 wt%) decreased at increasing O/S ratios of the precursor nano-emulsion while DXM loading, which was around 10 mg/100 mL, showed the reverse tendency. DXM release from nanoparticle dispersions was about an order of magnitude slower than from an aqueous solution. In vitro studies performed in a lung carcinoma cell line and in vitro haemolysis studies performed in red blood cells revealed a dose-dependent toxicity and haemolytic response, respectively. The as-prepared nanoparticle dispersions were non-toxic up to a concentration of 40 µg/mL and non-haemolytic up to a concentration of 1 mg/mL. After purification, nanoparticle dispersions were non-toxic up to a concentration of 90 µg/mL. These results allow concluding that these polymeric nanoparticle dispersions are good candidates for inhalatory therapy.

Novel non-viral gene delivery systems composed of carbosilane dendron functionalized nanoparticles prepared from nano-emulsions as non-viral carriers for antisense oligonucleotides.

The development of novel and efficient delivery systems is often the limiting step in fields such as antisense therapies. In this context, poly(d,l-lactide-co-glycolide) acid (PLGA) nanoparticles have been obtained by a versatile and simple technology based on nano-emulsion templating and low-energy emulsification methods, performed in mild conditions, providing good size control. O/W polymeric nano-emulsions were prepared by the phase inversion composition method at 25°C using the aqueous solution/polysorbate80/[4 wt% PLGA in ethyl acetate] system. Nano-emulsions formed at oil-to-surfactant (O/S) ratios between 10/90-90/10 and aqueous contents above 70 wt%. Nano-emulsion with 90 wt% of aqueous solution and O/S ratio of 70/30 was chosen for further studies, since they showed the appropriate characteristics to be used as nanoparticle template: hydrodynamic radii lower than 50 nm and enough kinetic stability. Nanoparticles, prepared from nano-emulsions by solvent evaporation, showed spherical shape, sizes about 40 nm, negative surface charges and high stability. The as-prepared nanoparticles were functionalized with carbosilane cationic dendrons through a carbodiimide-mediated reaction achieving positively charged surfaces. Antisense oligonucleotides were electrostatically attached to nanoparticles surface to perform gene-silencing studies. These complexes were non-haemolytic and non-cytotoxic at the concentrations required. The ability of the complexes to impart cellular uptake was also promising. Therefore, these novel nanoparticulate complexes might be considered as potential non-viral carriers in antisense therapy.

Phototherapeutic functionality of biocompatible graphene oxide/dendrimer hybrids.

Hydroxyl-terminated fourth generation poly(amido amine) dendrimer and folic acid were chemically bound on graphene oxide. The resultant hybrids exhibited one-photon and two-photon fluorescence emission, since the excitation irradiation at 390 and 780nm on the hybrids brought a fluorescence emission in the visible region around 450nm. In addition, the photocytotoxicity study revealed that under the two-photon excitation at 780nm, the hybrids can absorb near-infrared light and generate reactive oxygen species which can oxidize the HeLa cells and cause their death, suggesting the phototherapeutic behavior. Cytotoxicity measurement revealed the high biocompatibility of the hybrids toward HeLa cells. Thus, the present biocompatible hybrids consisting of only dendrimer, folic acid and graphene oxide have potentials as photodynamic therapeutic agents for medical treatment.

Conductive microemulsions for template CoNi electrodeposition.

Microemulsions have been revealed as feasible templates to grow magnetic nanostructures using an electrodeposition method. Reducing agents are not required and the applied potential has been used as driving force of the nanostructure growth. A systematic study of conductive microemulsion systems to allow the CoNi electrodeposition process has been performed. Different surfactants and organic components have been tested to form microemulsions with a CoNi electrolytic bath as an aqueous component in order to define the microemulsions showing enough conductivity to perform an electrodeposition process from the aqueous component. By using microemulsions of the aqueous electrolyte solution-Triton X-100-diisopropyl adipate system, CoNi electrodeposition has been achieved, the structure of the deposits being dependent on the composition and structure of the microemulsion, which can act as a soft-template to obtain different discontinuous deposits. The magnetic properties of the CoNi deposits vary with their structure.

New insights on the mechanisms of drug release from highly concentrated emulsions.

High kinetic stability water-in-oil high internal phase ratio emulsions (W/O-HIPREs) have been obtained in a 0.5% Theophylline (TP) aqueous solution/Cremophor WO7/liquid paraffin system at 25 °C. The release of TP has been studied from HIPREs with pH values of the dispersed phase ranging between 2 and 12. Although the release from aqueous solutions was not influenced by pH, the release from HIPREs depended strongly on the pH of the dispersed phase. Increasing the solubility of TP in the dispersed phase, its apparent diffusion coefficient decreased over two orders of magnitude. Two different physico-chemical models have been applied to describe the diffusion of TP, showing an excellent agreement with experiments and confirming the role of the structure of the emulsions and the solubility of the drug. It has been shown that only non-ionized species are able to cross the interfacial film. Therefore, at pH>pKa diffusion is limited by the concentration of non-ionized species inside the emulsion droplets, while at pH

Two-photon confocal imaging study: cell uptake of two photon dyes-labeled PAMAM dendrons in HeLa cells.

A two-photon excitation difluoroboron dye activated in the near infrared region for biological image analysis was synthesized in this study. Cell affinity, membrane interaction, and the endocytosis pathway of PAMAM dendrons were investigated using only covalent two-photon dyes (TPD) at the periphery of the PAMAM dendrons. Generation 3 TPD-labeled PAMAM dendrons (BG3) exhibited multivalency binding on the HeLa cell membranes from the cell affinity study in the fixation of HeLa cells. Photo-stimulation on the membrane of the living HeLa cell was observed by confocal optical imaging in situ, using the two-photon model, when incubated with BG3. Analyses of cell membrane integrity via lactate dehydrogenase (LDH) assay confirmed membrane damage at two photon excitation model. However, no variation in the cell was observed using the one-photon excitation model. These results indicated a high degree of dendrons uptake by cells through binding to the cell membrane following the endocytotic pathway. Furthermore, the wide excitation fluorescence spectrum of difluoroboron dye provides dual imaging with which to study the endocytosis of TPD-labeled PAMAM dendrons using a single near infrared laser.

Formation of polymeric nano-emulsions by a low-energy method and their use for nanoparticle preparation.

Formation of polymeric O/W nano-emulsions has been studied in the water/polyoxyethylene 4 sorbitan monolaurate/ethylcellulose solution system by the phase inversion composition (PIC) method. These nano-emulsions were used for the preparation of nanoparticles by solvent evaporation. Composition variables such as O/S ratio or final water content as well as emulsification path have been found to play a key role in the formation of stable, nanometer sized emulsions. Nano-emulsions with a constant water content of 90 wt.% and O/S ratios from 50/50 to 70/30 showed an average droplet size of about 200 nm as assessed by dynamic light scattering. Mean nanoparticle diameters, as determined by transmission electron microscopy image analysis, were of the order of 50 nm and showed a slight increase as well as a broader size distribution at increasing O/S ratios. The findings verify that the low-energy emulsification methods are not only valid for aliphatic and semipolar oils, but also for a highly polar solvent such as ethylacetate containing a preformed polymer.

Studies on controlled release of hydrophilic drugs from W/O high internal phase ratio emulsions.

Formation of high internal phase ratio emulsions (HIPREs) has been studied in water/Cremophor WO7/soybean oil and water/Cremophor WO7/liquid paraffin systems. Two hydrophilic model drugs, clindamycin hydrochloride (CH) and theophylline (TP), were incorporated in HIPREs with a water concentration of 90% and an oil/surfactant (O/S) weight ratio of 60:40 and their release was determined in vitro at 25 degrees C. The release of both model drugs from HIPREs was much slower than from aqueous solutions. In aqueous solution the release pattern of both actives was identical. In contrast, a clearly distinct release pattern from HIPREs was observed: The release of CH, which is freely soluble in water, was very slow, regardless of the emulsion system, while the release of TP, which is slightly soluble in water, was faster. By changing the pH of the dispersed phase of HIPREs, which in turn affects solubility, drug release was modulated. An increase in the solubility of TP in the dispersed phase by a factor of roughly 4.5 produced a decrease in the diffusion coefficient of two orders of magnitude. These results show for the first time the key role of drug solubility in the release from W/O-HIPREs.