Denis Labarre

Relationship between complement activation, cellular uptake and surface physicochemical aspects of novel PEG-modified nanocapsules.

The aim of our work was to examine the relationship between modifications of the surface of nanocapsules (NC) by adsorption or covalent grafting of poly(ethylene oxide) (PEG), and changes in their phospholipid (PL) content on complement activation (C3 cleavage) and on uptake by macrophages. The physicochemical characterization of the NC included an investigation of their properties, such as surface charge, size, hydrophilicity, morphology and homogeneity. This is the first time that such properties have been correlated with biological interactions for NC, a novel carrier system with a structure more complex than nanospheres. C3 crossed immunoelectrophoresis revealed the reduced activation for NC with longer PEG chain and higher density, although all formulations induced C3 cleavage to a lesser or greater extent. NC bearing PEG covalently bound to the surface were weaker activators of complement than plain PLA [poly(D,L-lactide)] NC or nanospheres (NS). Furthermore, the fluorescent/confocal microscopy of J774A1 cells in contact with NC reveal a dramatically reduced interaction with PEG-bearing NC. However, the way in which PEG was attached (covalent or adsorbed) seemed to affect the mechanism of uptake. Taken together, these results suggest that the low level of protein binding to NC covered with a high density of 20kDa PEG chains is likely to be due to the steric barriers surrounding these particles, which prevents protein adsorption and reduces their interaction with macrophages.

Novel polysaccharide-decorated poly(isobutyl cyanoacrylate) nanoparticles.

AbstractPurpose. The aim of this work was to synthesize new surface-modified nanoparticles using a radical emulsion polymerization of an alkyl cyanoacrylate. Methods. Isobutyl cyanoacrylate was polymerized in nitric acid 0.2 M containing a polysaccharide (0.1375 g) and cerium (8 × 10∖-2 M). After 1 h, the pH was adjusted to 7.0, and the nanoparticles were purified by dialysis. Nanoparticle characterization included scanning electron microscopy, quasi-elastic light scattering, zeta potential determination, measurements of the complement activation induced by different polysaccharide-coated nanoparticles and of the antithrombic activity of heparin. Results. Dispersions of spherical particles were obtained using various polysaccharides. The particle diameter varied from 90 nm to several micrometers, and the zeta potential depended on the molecular weight and the nature and charge of the polysaccharide. Surface analysis performed by ESCA confirmed the presence of polysaccharides at the nanoparticle surface. The nanoparticles were very stable, and the biologic activity of the polysaccharide was preserved. Complement activation was influenced by the polysaccharide characteristics. Conclusions. A new method based on radical emulsion polymerization of isobutyl cyanoacrylate initiated by polysaccharides and cerium was developed to prepare nanoparticles. It leads, in a single step, to nanoparticles with surface properties defined by the polysaccharide. This method is a new concept for the development of biomimetic drug carriers with multiple functions.

Anticoagulant effects of sulphonated polyurethanes.

Sulphonated polyurethanes have been shown to have excellent blood contacting properties. In this paper, similar polyurethanes which are water soluble have been investigated to determine their influence on thrombus formation. These polymers were shown to delay clotting times in the following ways: by direct complex formation between the polymer and thrombin; by interference with fibrin polymerization; and by complex interactions between polymer, thrombin, plasma antiproteases and fibrinogen in plasma.

Heparin-like activity of insoluble sulphonated polystyrene resins. Part I: Influence of the surface density, nature and binding of substituted anionic groups.

It was previously demonstrated that copolystyrene (sulphonate-amino acid sulphamide) resins possessed an anticoagulant heparin-like activity in the presence of blood plasma. Taking into account the variable surfaces of swollen resins developed by these dry resins, it is now shown that the antithrombic activity of crosslinked sulphonated polystyrene is linearly dependent on the surface density of the sulphonate groups. This fact implies that the presence of such isolated groups is sufficient to obtain a catalytic site for increasing the rate of inactivation of thrombin by plasmatic proteins. It is also shown that replacing sulphonate groups either by directly backbone-bonded carboxylate groups or by methionine linked by amide bonds to polystyrene backbone is not sufficient to endow the resulting resins with a significant anticoagulant activity.

Heparin-like activity of insoluble sulphonated polystyrene resins Part III: Binding of dicarboxylic amino acids

It has been demonstrated previously that polystyrene sulphonate possesses anticoagulant properties and that the binding of some amino acids could enhance the heparin-like properties of such resins. These properties depend on the surface density of the active groups, the nature and binding of the group and on the net change borne by the polymer. In this paper, we describe the preparation of copolystyrene (sulphonate-dicarboxylic amino acid sulphamide) resins. By measuring their antithrombotic-surface-activity, we demonstrate that the activity developed by each carboxyl group is at least roughly the same as the activity of one sulphonate group, except in the case of aspartic acid sulphamide resin for which a cooperative effect is shown. The anticoagulant properties of resins bearing phosphonate or monocarboxylic amino acid sulphamides are also examined.

Artificial oxygen carrier based on polysaccharides-poly(alkylcyanoacrylates) nanoparticle templates.

Biomimetic nanoparticles based on polysaccharides-poly(alkylcyanoacrylates) copolymers were initially developed in view of drug delivery. Core-shell nanoparticles covered with a sufficiently long brush of polysaccharides were shown to be very low complement activators and have the potential for long circulation times in the bloodstream. Such nanoparticles bearing haemoglobin were envisaged as potential red cell substitutes. Different core-shell nanoparticles with a brush shell made of dextran, dextran-sulphate, or heparin were prepared and haemoglobin (Hb) could be adsorbed on their surface. Benzene tetracarboxylic acid (BTCA) was used as a coupling agent for Hb to dextran-coated nanoparticles; the Hb loading capacity of the dextran nanoparticles showed a 9.3 fold increased. The coupled Hb maintained the allosteric properties of free Hb. While modification of nanoparticles by BTCA slightly increased complement activation, the further addition of Hb totally reversed this effect providing Hb-loaded nanoparticles with a very low level of complement activation. Such nanoparticles could be a suitable alternative to haemoglobin solutions in the development of a blood substitute.

Complement activation and adsorption of protein fragments by functionalized polymer surfaces in human serum

The interactions between blood and polymer surfaces used in extracorporeal circulations result in variable activations of the immune system of complement. Measuring concentrations of C3a or C5a in supernatant blood or serum after contact with the surface has been the most usual way of assessing this activation. Most polymer surfaces bearing various chemical groups were found to adsorb C3a and sometimes C5a. After taking into account adsorption, a good correlation was found between total C3a generated and CH50 units consumed by most of the polymer samples tested. Measuring only C3a remaining in the fluid phase should not be considered sufficient to conclude that a material surface is not an activator of complement.

Plug-in Spectrometry with Optical Fibers as a Novel Analytical Tool for Nanoparticles Technology: Application to the Investigation of the Emulsion Polymerization of the Alkylcyanoacrylate

This paper advances a simple technique to follow, in situ, the emulsion polymerization of isobutylcyanoacrylate (IBCA) performed in different conditions. The method is based on the monitoring of the optical density of the polymerization medium using a plug-in spectrometer equipped with optical fibers since during polymerization the optical density of the medium increases. The technique is demonstrated using poly(alkylcyanoacrylate) nanoparticles developed as carrier for the delivery of drugs in vivo. These nanoparticles are produced by emulsion polymerization of alkylcyanoacrylates (ACA) which are among the highest reactive monomers. The results showed that the spontaneous anionic polymerization of IBCA induced by water could be controlled at pH 1. It was initiated after a delay whereas an immediate and extremely rapid polymerization could be monitored when it was initiated using a combination of cerium IV and dextran allowing the radical polymerization to occur. At higher pH, the spontaneous anionic polymerization could not be controlled. The method developed in this study appeared suitable to monitor emulsion polymerization of ACA and to highlight different mechanisms of polymerization.

Interactions of functionalized polystyrene derivatives with the complement system in human serum

The interactions between blood and insoluble polysaccharidic surfaces result in activation of the immune system of complement. When substituted with carboxymethyl groups, Sephadex loses its capacity to activate complement, whereas Sephadex sulphate has been described as an activator. In order to elucidate the molecular mechanisms of complement activation and inhibition, a simpler polymer model has been chosen: it consists of an insoluble polystyrene backbone on which either isolated hydroxymethyl or sulphonate groups or both are present. The surfaces bearing the isolated groups consume complement but the mechanisms involved are quite different. In contrast, a surface bearing equal proportions of both types of groups is a non-activator. Such model surfaces can be very useful for designing artificial surfaces able to control in situ complement activation.

Heparin-like activity of insoluble sulfonated polystyrene resins: II. Properties of polyampholytes

Abstract Insoluble sulfonated polystyrene resins are endowed with heparin-like activity toward coagulation of blood plasma induced by thrombin (catalysis of the neutralization of thrombin by antithrombin). By introducing variable amounts of amino groups and sulfonate groups on the same chains of polystyrene resins (Polyampholytes = PA), the effects of the net charge borne by the PA resins on the binding of hydrogen ions, heparin-like properties and the adsorption of thrombin were studied. It was demonstrated that the apparent acidity constant of the amino groups was strongly dependent upon the electrical environment of these groups: they behaved like strong bases when surrounded by negative charges and like weak bases when surrounded by positive charges. This behavior was in agreement with the theory of Katchalsky concerning PA. This model was used to explain why the affinity of thrombin for sulfonate groups strongly decreased when these groups were surrounded by positively charged amino groups. The adsorption of active thrombin was strongly involved in the heparin-like properties of the hydrophilic resins bearing a net negative charge. In the presence of the hydrophobic resins bearing a net positive charge, no adsorption of active thrombin was detected. The gross effect of one positively charged amino group was to neutralize the heparin-like activity of one sulfonate group.