Gabriela Calderó

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.

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.

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.

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.

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.

Studies on the formation of polymeric nano-emulsions obtained via low-energy emulsification and their use as templates for drug delivery nanoparticle dispersions.

Ethylcellulose nanoparticles have been obtained from O/W nano-emulsions of the water/polyoxyethylene 10 oleyl ether/[ethyl acetate+4wt% ethylcellulose] system by low energy-energy emulsification at 25°C. Nano-emulsions with droplet sizes below 200nm and high kinetic stability were chosen for solubilising dexamethasone (DXM). Phase behaviour, conductivity and optical analysis studies of the system have evidenced for the first time that both, the polymer and the drug play a role on the structure of the aggregates formed along the emulsification path. Nano-emulsion formation may take place by both, phase inversion and self-emulsification. Spherical polymeric nanoparticles containing surfactant, showing sizes below 160nm have been obtained from the nano-emulsions by organic solvent evaporation. DXM loading in the nanoparticles was high (>90%). The release kinetics of nanoparticle dispersions with similar particle size and encapsulated DXM but different polymer to surfactant ratio were studied and compared to an aqueous DXM solution. Drug release from the nanoparticle dispersions was slower than from the aqueous solution. While the DXM solution showed a Fickian release pattern, the release behaviour from the nanoparticle dispersions was faster than that expected from a pure Fickian release. A coupled diffusion/relaxation model fitted the results very well, suggesting that polymer chains undergo conformational changes enhancing drug release. The contribution of diffusion and relaxation to drug transport in the nanoparticle dispersions depended on their composition and release time. Surfactant micelles present in the nanoparticle dispersion may exert a mild reservoir effect. The small particle size and the prolonged DXM release provided by the ethylcellulose nanoparticle dispersions make them suitable vehicles for controlled drug delivery applications.

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<pKa diffusion is determined by the interfacial film. This demonstrates that solubility of the diffusing molecule can have a higher effect on release from HIPREs than the interfacial film properties.

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.

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.

Electrolytes as a tuning parameter to control nano-emulsion and nanoparticle size

Currently, polymeric nanoparticles are widely used in a wide variety of biomedical applications, which requires different nanoparticle sizes. Their preparation by solvent evaporation from polymeric nano-emulsions has been described as an appropriate methodology. Nano-emulsions can be prepared by the phase inversion composition method, a low-energy emulsification method, apt for biomedical applications since it can be performed at low conditions. Using this methodology, nano-emulsions droplets and resulting nanoparticle properties depend on the selected components. Many studies have been performed varying some of them. In this work, a specific study has been performed for the first time to find out the effect that the presence of electrolytes in the aqueous phase of the template nano-emulsion has on nano-emulsion and nanoparticle physicochemical properties. PLGA nano-emulsions were prepared using the system: aqueous solution (W)/polysorbate 80 (S)/[4 wt% PLGA in ethyl acetate] (O). The addition of increasing concentrations of electrolytes (PBS) in the aqueous phase produced a decrease on droplet hydrodynamic radii from around 200 nm to around 35 nm. Nanoparticle sizes were determined by the droplet sizes of their template nano-emulsions. Nanoparticles dialysis to an electrolyte solution of 0.16 M, required for many biomedical applications to accomplish the physiological pH and osmolality, did not produce changes in nanoparticle sizes. Therefore, it has been demonstrated for the first time that nano-emulsion and nanoparticle size can be tuned by only varying the electrolyte concentration of the template nano-emulsions. Nano-emulsion templating is a versatile technology that enables the obtaining of tunable nanoparticle sizes; appropriate for a wide range of biomedical applications.