Data supporting the physico-chemical characterization, cellular uptake and cytotoxicity of lipid nanocapsules.

The aim of this data article is to provide data for a basic knowledge of the properties of lipid nanocapsules, a new colloidal system with very promising applications in drug delivery. Firstly, we pay attention on how it is possible to determine their surface composition by means of electrokinetics measurements. On the other hand, we provide experimental evidences for a better understanding of the factors that determine the interactions of these nanoparticles with cells as a necessary step to guide the design of the most effective formulations. Additionally, we supply information about encapsulation efficiency of docetaxel, a potent chemotherapy drug, inside nanocapsules supporting the experimental cytotoxicity results of these nanosystems.

Balancing the effect of corona on therapeutic efficacy and macrophage uptake of lipid nanocapsules.

Several studies have shown the potential of biocompatible lipid nanocapsules as hydrophobic drug delivery systems. Understanding the factors that determine the interactions of these oil-in-water nanoemulsions with cells is a necessary step to guide the design of the most effective formulations. The aim of this study was to probe the ability of two surfactants with a markedly different nature, a non-ionic poloxamer, and a charged phospholipid, to prepare formulations with shells of different composition and different surface properties. Thus we determined their effects on the interaction with biological environments. In particular, we investigated how the shell formulation affected the adsorption of biomolecules from the surrounding biological fluids on the nanocapsule surface (corona formation). A complete physicochemical characterization including an isothermal titration calorimetry (ITC) study revealed that the use of poloxamer led to nanocapsules with a marked reduction in the number of protein-binding sites. Surface hydrophilicity and changes in corona formation strongly correlated to changes in uptake by cancer cells and by macrophages. Our results indicate that the nature and concentration of surfactants in the nanocapsules can be easily manipulated to effectively modulate their surface architecture with the aim of controlling the environmental interactions, thus optimizing functionality for in vivo applications. In particular, addition of surfactants that reduce protein binding can modulate nanoparticle clearance by the immune system, but also screens the desired interactions with cells, leading to lower uptake, thus lower therapeutic efficacy. The two effects need to be balanced in order to obtain successful formulations.

Chitosan nanocapsules: Effect of chitosan molecular weight and acetylation degree on electrokinetic behaviour and colloidal stability.

In recent years, chitosan nanocapsules have shown promising results as carriers for oral drug or peptide delivery. The success in their applicability strongly depends on the stability of these colloidal systems passing through the digestive tract. In gastric fluids, clear stability comes from the high surface charge density of the chitosan shell, which is completely charged at acidic pH values. However, in the intestinal fluid (where the pH is almost neutral) the effective charge of these nanocapsules approaches zero, and the electrostatic forces cannot provide any stabilization. Despite the lack of surface charge, chitosan nanocapsules remain stable in simulated intestinal fluids. Recently, we have demonstrated that this anomalous stability (at zero charge) is owed to short-range repulsive forces that appear between hydrophilic particles when immersed in saline media. The present work examines the influence of the chitosan hydrophobicity, as well as molecular weight, in the stability of different chitosan nanocapsules. A study has been made of the size, polydispersity, electrophoretic mobility, and colloidal stability of eight core-shell nanocapsule systems, in which the chitosan-shell properties have been modified using low-molecular-weight (LMW) and high-molecular-weight (HMW) chitosan chains having different degrees of acetylation (DA). With regard to the stability mediated by repulsive hydration forces, the LMW chitosan provided the best results. In addition, contrary to initial expectations, greater stability (also mediated by hydration forces) was found in the samples formed with chitosan chains of high DA values (i.e. with less hydrophilic chitosan). Finally, a theoretical treatment was also tested to quantify the hydrophilicity of the chitosan shells.

Nanoparticles made from novel starch derivatives for transdermal drug delivery.

The goal of this paper was aimed to the formulation of nanoparticles by using two different propyl-starch derivatives - referred to as PS-1 and PS-1.45 - with high degrees of substitution: 1.05 and 1.45 respectively. A simple o/w emulsion diffusion technique, avoiding the use of hazardous solvents such as dichloromethane or dimethyl sulfoxide, was chosen to formulate nanoparticles with both polymers, producing the PS-1 and PS-1.45 nanoparticles. Once the nanoparticles were prepared, a deep physicochemical characterization was carried out, including the evaluation of nanoparticles stability and applicability for lyophilization. Depending on this information, rules on the formation of PS-1 and PS-1.45 nanoparticles could be developed. Encapsulation and release properties of these nanoparticles were studied, showing high encapsulation efficiency for three tested drugs (flufenamic acid, testosterone and caffeine); in addition a close to linear release profile was observed for hydrophobic drugs with a null initial burst effect. Finally, the potential use of these nanoparticles as transdermal drug delivery systems was also tested, displaying a clear enhancer effect for flufenamic acid.

Insulin-loaded PLGA nanoparticles for oral administration: an in vitro physico-chemical characterization.

The aim of this work was to study poly(d,l-lactic-co-glycolic) acid (PLGA) nanoparticles--formulated by a modified solvent diffusion technique--applied as model nanocarriers for insulin in potential oral administrations. These nanostructures consisted of a blend matrix formed by PLGA copolymer and polyoxyethylene derivatives. Two types of blend formulations, PLGA:poloxamer (Pluronic F68) and PLGA:poloxamine (Tetronic T904), were analyzed, and the results compared to those obtained with pure PLGA nanoparticles. The work has been divided into two parts. (a) Firstly, the stability of the unloaded nanoparticles in simulated gastric and intestinal fluids was studied. Degradation studies reflected a strong interaction between the pure PLGA nanoparticles and the digestive enzymes. However, this interaction was considerably reduced in the blend formulations, although the PLGA:poloxamine system became colloidally unstable in the simulated gastric fluid. (b) Secondly, the effect of the net charge of the encapsulated macromolecule in the final properties of the blend formulations was studied by encapsulating insulin below and above its corresponding isoelectric point. The net charge of the encapsulated protein showed a clear effect in the final size of the nanoparticles, while the encapsulation efficiency was controlled by the polyoxyethylene derivative presents in the blend formulation. The obtained results show that those carriers formed with encapsulated insulin in PLGA-Pluronic F68 particles are capable, at least in vitro, to overcome the gastrointestinal barrier. Therefore, these nanocarriers seem to be appropriate for oral administration of insulin, although performing in vivo studies becomes necessary to corroborate such statement.

Electrophoretic mobility and colloidal stability of PLGA particles coated with IgG.

Drug delivery systems based on polymeric nanocarriers have been widely exploited during the last years. However, one of the basic problems that is still not totally solved in this kind of systems is the ability of delivering drugs to specific target cells. Coating the nanocarrier with reactive antibodies against specific molecules presented in the external membrane of the target cells is a usual recommendation. In this paper, an ideal delivery system has been studied. Nanoparticles made of poly(d,l-lactic acid/glycolic acid) 50/50 (PLGA) polymers have been coated with polyclonal IgG. In the first part of the paper, some basic characteristics of these IgG-PLGA complexes have been analysed (i.e. size, electrophoretic mobility and colloidal stability). Then, the immunoreactivity of the immobilized IgG molecules was tested by using an optical device, monitoring the binding of a standard molecule (C-reactive protein, CRP) to the antibody (antiCRP-IgG) adsorbed on the PLGA particles. This allowed us to estimate the percentage of active IgG molecules on the PLGA particles by applying a simple kinetic model to the immunoreactivity results. According to this model, the PLGA-IgG particles supply a good immunoresponse even if only less than 5% of the total IgG molecules on the surface were active. Despite the simplicity of the system, the results may be of potential interest for developing more realistic nanocarriers with targeting ability. That is, it can be inferred that it is possible to obtain a high targeting specificity in IgG-sensitized nanocarriers even working with a low coverage of active antibody molecules. The results have been compared with those similarly obtained with polystyrene (PS) particles used as a reference system.

Colloidal stability of pluronic F68-coated PLGA nanoparticles: a variety of stabilisation mechanisms.

Poloxamers are a family of polypropylene oxide (PPO) and polyethylene oxide (PEO) tri-block copolymers that are usually employed in the micro- and nanoparticulate engineering for drug delivery systems. The aim of this work is to study the electrophoretic mobility (mu(e)) and colloidal stability of complexes formed by adsorbing a poloxamer (Pluronic F68) onto poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles. A variety of stabilisation mechanisms have been observed for the Pluronic-coated PLGA nanoparticles, where DLVO interactions, solvent-polymer segment interactions and hydration forces play different roles as a function of the adsorbed amount of Pluronic. In addition, the mu(e) and stability data of these complexes have been compared to those obtained previously using a PLGA-Pluronic F68 blend formulation. As both the mu(e) and the stability data are identical between the two systems, a phase separation of both components in the PLGA-Pluronic blend formulation is suggested, being the PLGA located in the core of the particles and the Pluronic in an adsorbed shell.

Electrokinetic behavior and colloidal stability of polystyrene latex coated with ionic surfactants.

This work is focused on analyzing the electrokinetic behavior and colloidal stability of latex dispersions having different amounts of adsorbed ionic surfactants. The effects of the surface charge sign and value, and the type of ionic surfactant were examined. The analysis of the electrophoretic mobility (mu(e)) versus the electrolyte concentration up to really high amounts of salt, much higher than in usual studies, supports the colloidal stability results. In addition, useful information to understand the adsorption isotherms was obtained by studying mu(e) versus the amount of the adsorbed surfactant. Aggregation studies were carried out using a low-angle light scattering technique. The critical coagulation concentrations (ccc) of the particles were obtained for different surfactant coverage. For latex particles covered by ionic surfactants, the electrostatic repulsion was, in general, the main contribution to the colloidal stability of the system; however, steric effects played an important role in some cases. For latices with not very high colloidal stability, the adsorption of ionic surfactants always improved the colloidal stability of the dispersion above certain coverage, independently of the sign of both, latex and surfactant charge. This was in agreement with higher mobility values. Several theoretical models have been applied to the electrophoretic mobility data in order to obtain different interfacial properties of the complexes (i.e., zeta potential and density charge of the surface charged layer).

Hofmeister effects on the colloidal stability of an IgG-coated polystyrene latex.

The effect of various ions related to the Hofmeister series (HS) on different properties of a cationic latex covered with a protein (IgG) is analyzed in this study. NaNO3, NH4NO3, and Ca(NO3)2 were used to compare the specificity of the cations, and NaCl, NaSCN, NaNO3, and Na2SO4, to compare the specificity of the anions. Two pH values, 4 and 10, were chosen to analyze the behavior of these ions acting as counter- and co-ions. At pH 4, the total surface charge is positive, whereas at pH 10 it is negative. Three different phenomena have been studied in the presence of these Hofmeister ions: (1) colloidal aggregation, (2) electrophoretic mobility, and (3) colloidal restabilization. The specific effect of the ions was clearly observed in all experiments, obtaining ion sequences ordered according to their specificity. The most important parameter for ion ordering was the sign of the charge of the colloidal particle. Positively charged particles displayed an ion order opposite that observed for negatively charged surfaces. Another influential factor was the hydrophobic/hydrophilic character of the particle surface. IgG-latex particle surfaces at pH 10 were more hydrophilic than those at pH 4. The SCN- ion had a peculiar specific effect on the phenomena studied (1)-(3) at pH 10. With respect to the restabilization studies at high ionic strengths, new interesting results were obtained. Whereas it is commonly known that cations may provoke colloidal restabilization in negative particles when they act as counterions, our experiments demonstrated that such restabilization is also possible with positively charged particles. Likewise, restabilization of negative surfaces induced by the specific effect of chaotropic anions (acting as co-ions) was also observed.

Physicochemical characterization of chitosan nanoparticles: electrokinetic and stability behavior.

Some physical properties of nanogel particles formed by chitosan ionically cross-linked by tripolyphosphate (TPP) have been studied. Electrokinetic properties and colloidal stability were analyzed as a function of pH and ionic strength of the medium. Chitosan particles showed volume phase transitions (swelling/shrinking processes) when the physicochemical conditions of the medium were changed. Experimental data were mainly obtained by electrophoretic mobility measurements and by photon correlation spectroscopy and static light scattering techniques. Chitosan chains possess glucosamine groups that can be deprotonated if the pH increases. Therefore, modification of pH from acid to basic values caused a deswelling process based on a reduction of the intramolecular electric repulsions inside the particle mesh. Electrophoretic mobility data helped to corroborate the above electrical mechanism as responsible for the size changes. Additionally, at those pH values around the isoelectric point of the chitosan-TPP particles, the system became colloidally unstable. Ionic strength variations also induced important structural changes. In this case, the presence of KCl at low and moderate concentrations provoked swelling, which rapidly turned on particle disintegration due to the weakness of chitosan-TPP ionic interactions. These last results were in good agreement with the predictions of gel swelling theory by salt in partially ionized networks.

Hofmeister effects in the restabilization of IgG--latex particles: testing Ruckenstein's theory.

The hydration interaction is responsible for the colloidal stability observed in protein-coated particles at high ionic strengths. The origin of this non-DLVO interaction is related not only to the local structure of the water molecules located at the surface but also to the structure of those molecules involved in the hydration of the ions that surround the colloidal particles. Ruckenstein and co-workers have recently developed a new theory based on the coupling of double-layer and hydration interactions. Its validity was contrasted by their fitting of experimental data obtained with IgG-latex particles restabilized at high salt concentration. The theory details the important role played by the counterions in the stability at high salt concentrations by proposing an ion pair reaction forming surface dipoles. These surface dipoles are responsible of repulsive interactions between two approaching surfaces. This paper checks the theory with recent data where some ions associated with the Hofmeister series (NO(3)(-), SCN(-) and Ca(2+)) restabilize the same kind of IgG-latex systems by means of hydration forces. Surprisingly, these ions induce stability acting even as co-ions, likely by modifying the water structure at the surface, but not forming surface ion pairs. Therefore, this experimental evidence would question Ruckenstein's theory based on the surface dipole formation for explaining the observed restabilization phenomena.

Adsorption of different amphiphilic molecules onto polystyrene latices.

In order to know the influence of the surface characteristics and the chain properties on the adsorption of amphiphilic molecules onto polystyrene latex, a set of experiments to study the adsorption of ionic surfactants, nonionic surfactants and an amphiphilic synthetic peptide on different latex dispersions was performed. The adsorbed amount versus the equilibrium surfactant concentration was determined. The main adsorption mechanism was the hydrophobic attraction between the nonpolar tail of the molecule and the hydrophobic regions of the latex surface. This attraction overcame the electrostatic repulsion between chains and latex surface with identical charge sign. However, the electrostatic interactions chain-surface and chain-chain also played a role. General patterns for the adsorption of ionic chains on charged latex surfaces could be established. Regarding the shape, the isotherms presented different plateaus corresponding to electrostatic effects and conformational changes. The surfactant size also affects the adsorption results: the higher the hydrophilic moiety in the surfactant molecule the lower the adsorbed amount.

A review of factors affecting the performances of latex agglutination tests.

The present review describes the different strategies followed to improve the performance of latex agglutination tests. The analysis is mainly focused on the diverse parameters that affect the final colloidal stability of the immunoprotein-latex system. These parameters include: the surface properties of polymer carriers; the different kind of antibodies usually employed; the use of BSA as stabilizer; the co-adsorption of various macromolecules (BSA, surfactants and lipids) and antibodies; recent approaches to colloidal stability at high ionic strengths due to hydration forces; and the covalent coupling of antibodies on functionalized latex particles. Special emphasis is given to the relation between electrophoretic mobility and the colloidal stability of the sensitized particles and how this knowledge can be utilized for a better understanding of the immunoagglutination kinetic.

An Enthalpic Analysis on the Aggregation of Colloidal Particles Studied by Microcalorimetry.

There are different theories concerning the stability of colloidal suspensions. Most of them arise from the well-known DLVO theory which relates colloidal stability to intermolecular forces between particles. Experimental corroboration of these theories has been obtained mainly by using different optical techniques that analyze changes in the optical properties of the solution while particles aggregate. However, no attention has been paid to studying the aggregation process thermodynamically. This is why we have focussed on studying the heat released during the agglutination of polystyrene particles. The enthalpy change in this aggregation process was detected by using a highly sensitive and modern technique called isothermal titration calorimetry. In addition, some results about repeptization, that is, reversibility in the aggregation process, are also shown. Copyright 2001 Academic Press.

A comparative study between the adsorption of IgY and IgG on latex particles.

The use of egg yolk antibodies (IgY) instead of IgG from mammalian species may present several advantages in the development of routine diagnostic immunoassays. On the one hand, the animal suffering is reduced, as antibodies are obtained directly from the egg. On the other hand, the use of IgY avoids the rheumatoid factor interference. The rheumatoid factor interacts with IgG molecules in many immunoassays causing false positive results. Despite these advantages, IgY antibodies are scarcely used. As part of an aim to develop a diagnostic test based on IgY-latex agglutination, a preliminary study on some characteristics of the IgY-latex complexes is carried out. In this work, protein adsorption and desorption, isoelectric point, electrokinetic mobility, and colloidal stability are analysed. Results are compared to those obtained by IgG. Interesting differences are observed (which mainly arise from the difference in molecular structure between IgY and IgG), suggesting that IgY is a more hydrophobic molecule than IgG. In addition, colloidal dispersions of IgY-covered latex particles are more stable (at pH 8) than those sensitized by IgG.

Forces acting on particle-enhanced immunoassays.

The aim of the present work is to study the role of the different forces involved in the agglutination of immuno gamma-globulin (IgG) covered latex particles due to antigen-antibody reaction. An experimental investigation on the adsorption of IgG molecules on three latexes with different surface charge densities is described. Photon correlation spectroscopy was used to determine the hydrodynamic layer thickness of the IgG molecules adsorbed on the latexes. In order to get an insight into the forces acting between two antibody-covered particles approaching each other, the colloidal stability and immunoreactivity of these biocomplexes were studied. They can be stabilized by electrostatic or hydration forces. The immunological agglutination of IgG-immobilized latex particles due to the addition of the antigen was quantified through scattered light intensity measurements. The immunoresponse increases with ionic strength of the medium until a maximum value is achieved. Above this maximum, the immunoreactivity decreases.

Stagnant versus dynamic conditions: a comparative adsorption study of blood proteins.

Haemodynamic parameters of flowing blood, such as diffusion, convection, flow and shear rates, are important as they determine the interaction of cells with vessel walls and prosthetic implants in the cardiovascular system. Most of the studies under flow conditions have been performed with platelets or other cells, and less attention has been paid to the effects that these parameters may cause on the adsorption of proteins. For this reason we studied how different shear rates affect the adsorption of human albumin, fibrinogen, total serum proteins, and complement factors 1q and 3c from human serum to silicon surfaces. The most relevant results indicate that during non-flow conditions the amount of adsorbed proteins is always lower than under flow. The different shear rates (225, 915, 1800 and 2700 s(-1)) all gave similar results, indicating that such a parameter is not very critical for single protein deposition. The differences in kinetics of complex protein solutions are conveniently highlighted by use of specific polyclonal antibodies. The difference between non-flow or low shear rate conditions and physiological flow conditions was enhanced for the complement cascade system.

A comparative study of optical techniques applied to particle-enhanced assays of C-reactive protein.

This work is based on the well-established immunoassay principle of agglutination of latex particles covered by immunoproteins. In our experiments, positively charged particles act as carriers for the F(ab')2 fragment, obtained from rabbit polyclonal IgG, active against C-reactive protein (CRP). The presence of the antigen CRP in the immunolatex system causes agglutination and the aim of the present study was to compare different optical techniques (turbidimetry, nephelometry, angular anisotropy and photon correlation spectroscopy) capable of detecting the agglutination. The sensitivity and detection limit largely depend on the optical method. We have analyzed for each optical technique the following aspects: sensitivity, reproducibility, detection limit, reaction time, amount of sample wasted and availability of the required detection device. The results presented in this paper show that both angular anisotropy and photon correlation spectroscopy offer lower detection limits, and use little reagent, but have longer assay times than the classical optical techniques of turbidimetry and nephelometry.

Particle enhanced immunoaggregation of F(ab')2 molecules.

The immunological agglutination reactions of physically absorbed F(ab')2 molecules onto anionic and cationic latex particles have been investigated by means of optical absorbance measurements. These measurements have been conducted under different conditions to determine the most influential factors. Surface F(ab')2 and BSA densities, particle concentration in the reaction medium and polyethylene glycol concentration are some of these factors. Sensitized cationic and anionic latexes differ considerably with respect to their colloidal stability and reactivity. As a general rule, the sensitized cationic latex has a relatively higher colloidal stability and hence, it provides reagents with a better optical response. Less than 0.025 microgram/ml of C-reactive protein has been detected using this particle enhanced optical immunoassay.

Sequential adsorption of F(ab')(2) and BSA on negatively and positively charged polystyrene latexes.

The aim of the present work is to study the sequential adsorption of F(ab')(2) and bovine serum albumin (BSA) molecules adsorbed onto positively and negatively charged polystyrene latexes. Cationic and anionic latexes were prepared by emulsifier-free emulsion polymerization. Adsorptions of F(ab')(2) on both latexes at a low ionic strength and different pHs were performed. The cationic latex showed a higher adsorption of F (ab')(2) molecules over a range of pH, which could be due to the formation of multilayers. Sequential adsorption of anti-CRP F(ab')(2) and monomeric BSA were performed at two different pre-adsorbed F(ab')(2) amounts on both types of latex. Displacement of F(ab')(2) occurred only when the preadsorbed amounts were larger than a certain critical value, which depends on the adsorption pH. A greater displacement of larger preadsorbed amounts might be the result of a weaker contact between the protein molecules and the polystyrene surface. The displacement of F(ab')(2) previously adsorbed onto both latexes occurred due to pH changes, an increase of ionic strength and the presence of BSA molecules. The effect caused by these three factors was studied independently. The main factors in the desorption of F(ab')(2) on the anionic latex are the changes in pH and ionic strength, whereas on the cationic latex the desorption is mainly caused by the increase of the ionic strength and the presence of BSA. The colloidal stability of the immunotatex was improved by BSA adsorption, especially on cationic latex. (c) 1995 John Wiley & Sons, Inc.