Laurent Leclercq

Multilayered textile coating based on a β-cyclodextrin polyelectrolyte for the controlled release of drugs

The aim of this work was to develop the formation of multilayered coating incorporating a cyclodextrin polyelectrolyte onto a non-woven polyethylene terephthalate (PET) textile support in order to obtain reservoir and sustained release properties towards bioactive molecules. We optimized the multilayer assembly immobilization onto the PET surface according to the layer-by-layer (LbL) deposition process. After a pre-treatment of the textile support aiming to offer a sufficient ionic character to the surface, it was alternatively immersed into two polyelectrolytes aqueous solutions consisting of chitosan (CHT) as polycation on the one hand, and a β-cyclodextrin polymer (polyCTR-βCD) as polyanion on the other hand. In a second approach, a TBBA/polyCTR-βCD complex (4-tert-butylbenzoic acid, TBBA) was used in order to load the system with a drug model whose kinetics of release was assessed. Gravimetry, microscopy, OWLS, colorimetric titration, infrared and zetametry were used as characterization techniques. An effective deposition on the textile surface due to ionic interactions with alternation of up to 10 layers of each of both polyelectrolytes was clearly evidenced. However, we observed that layer formation occurred to a lesser extent when TBBA/polyCTR-βCD complex was applied instead of polyCTR-βCD alone. The release study showed that drug reservoir properties and release kinetics could be controlled by the number of layers in the system and that TBBA release was faster than the multilayered coating degradation.

A Physico-chemical Approach of Polyanion-Polycation Interactions Aimed at Better Understanding the In Vivo Behaviour of Polyelectrolyte-based Drug Delivery and Gene Transfection

Polyanions and polycations are known to interact electrostatically and form soluble or insoluble polyelectrolyte complexes. Body fluids, blood and cells are composed of many polyelectrolytic systems such as proteins, glycoproteins, poly(glycosamino glycane)s, polynucleotides, etc. under physiological conditions. Nowadays synthetic polyelectrolytes are proposed as carriers of bioactive compounds, such as drugs and genes, and are thus to be injected into body fluids. For the sake of better understanding the complex behaviour of such artificial polyelectrolytic systems in the pool of natural polyelectrolytes forming living systems, interactions of bi- and multi-components mixtures of synthetic polyanions with the same synthetic polycation, namely poly[(dimethylaminoethyl) methacrylate], HCl, were investigated under the conditions imposed by physiological media, namely pH=7.4, ionic strength μ =0.15 and T =37°C. The selected artificial polyanions were the sodium salts of poly(acrylic acid), poly(methacrylic acid), poly (L -lysine citramide) and poly(styrene sulfonic acid) which have different acid strength, charge density and ionogenic group. The influence of ionic strength and pH on complex formation and stability was investigated by turbidimetry at λ =520 nm. Phase separation occurred regardless of ionic strength in the case of sodium polystyrene sulfonate. For the other polyanions, redissolution was observed at critical NaCl concentrations much higher than the physiological ionic strength. In the case of mixtures of two or three polyanions with the polycation, the complex formation appeared polyanion-selective at physiological ionic strength. Data are discussed with regard to phenomena that can occur in vivo.

Study of interactions between oppositely charged dendrigraft poly-l-lysine and human serum albumin by continuous frontal analysis capillary electrophoresis and fluorescence spectroscopy

Dendrigraft poly-L-lysine (DGL) are biomacromolecules of great interest for many applications including antibacterial activity, drug delivery systems, gene therapy and production of antibodies. As human serum albumin (HSA) is the most abundant serum protein, the study of interactions between these two compounds is crucial for the use of DGL in drug or gene delivery systems. The present work aims at determining the number of binding sites and the corresponding successive equilibrium constants between DGL of generation 3 (G3) and HSA in physiological conditions. To meet this end, continuous frontal analysis capillary electrophoresis (FACCE) and fluorescence spectroscopic methods were implemented and compared. FACCE was performed on a polycationic modified capillary in combination with a co-pressure that allowed for selectively introducing the free G3 from the G3/HSA mixtures. FACCE studies demonstrated that HSA has 2 binding sites with DGL G3 with the following successive constants K1=31.2×10(3) M(-1) and K2=30.6×10(3) M(-1). For a 1 g/L concentration in G3 and assuming a plasmatic HSA concentration of 40g/L, these binding constants lead to only 5% free DGL in the medium. It was also shown that the interactions between G3 and HSA corresponded to a model of cooperative sites. These results are in good agreement with the presence of two negatively charged domains in the HSA. Good fitting of the fluorescence spectroscopy data was obtained using the equilibrium constants derived from FACCE. Nevertheless, due to the high number of fitting parameters, it was difficult to fit the fluorescence spectroscopic data independently of the results obtained by FACCE.

Correlation of Length of Linear Oligo(ethanamino) Amides with Gene Transfer and Cytotoxicity

The optimization of synthetic carriers for gene transfer remains a major challenge. Cationic polymers such as polyethylenimine (PEI) often show increasing gene transfer activity with increasing molecular weight, but this favorable effect is accompanied by an undesired increase in cytotoxicity. Moreover, the polydispersity of polymers prevents accurate determination of optimum size. Herein we describe the step‐by‐step elongation of precise linear oligo(ethanamino) amides by making use of the artificial amino acid succinoyl‐tetraethylene pentamine (Stp) for solid‐phase‐assisted synthesis. This procedure enabled us to identify the optimal oligomer Stp30‐W (8.4 kDa) with a length of 30 Stp units, with which effective gene transfer occurs in the absence of cytotoxicity. The transfection efficiency of Stp30‐W exceeded that of standard linear PEI (22 kDa) by sixfold; nevertheless, Stp30‐W exhibited tenfold lower cytotoxicity. In addition to the lower molecular weight, the succinate spacer between the oligoamine units may also contribute to the favorable biocompatibility. The cytotoxicity of the cationic polymer PEI is a major concern for use as a carrier for gene delivery, so this comparison between linear PEI and the new Stp oligomers is particularly relevant.

Polyelectrolyte multilayer coatings for the separation of proteins by capillary electrophoresis: Influence of polyelectrolyte nature and multilayer crosslinking

The present work aims at studying the influence of the nature of the polyelectrolytes used in successive multiple ionic polymers on the performances of protein separation in acetic acid volatile background electrolyte. A broad library of polyelectrolyte multilayers was compared on the basis of 9 different weak/strong polyanions and 8 different weak/strong polycations. More than 20 couples of different polyelectrolytes were investigated. The separation efficiencies (expressed as the N/l ratio, where N is the plate number and l is the capillary effective length) were systematically compared for the separation of a protein test mixture. The coating stability was evaluated by the relative standard deviation of the migration times. For weak polyelectrolyte multilayers, the influence of the polymer crosslinking on the coating stability and separation efficiency has been studied. Intra-day repeatability of 100 successive runs, and capillary-to-capillary reproducibility were tested on coatings of each category (crosslinked and non crosslinked). The main (not obvious) result rising from this study is that the nature of the polyanion constituting the multilayers is of primary importance for the performance in terms of separation efficiency and stability, even when the mulilayers finish with a polycation.

Release of Polyanions from Polyelectrolyte Complexes by Selective Degradation of the Polycation

One of the major problems associated with analyzing polyelectrolyte complexes is the separation of strongly bound oppositely charged polymeric components. As part of a work aimed at better understanding the factors that affect polyelectrolyte complex formation and stability, an investigation of the possibility to release and analyze the polyanion, after hydrolytic or enzymatic degradation of the partner polycation, was made. Mixtures of poly(acrylic acid) or poly(L-lysine citramide) polyanions with poly(L-lysine) or poly(amino serinate) polycations were investigated. For each polycation-polyanion couple, four complex fractions were obtained by adding the polycation to the polyanion according to a titration protocol. The selective degradation of the polycation within the different complex fractions was investigated after the complex was disrupted with a NaCl solution. The molecular weights of the recovered polyanionic macromolecules were assessed by both static light scattering and size exclusion chromatography. The data supported previous findings that complexation was selective according to the molecular weight of the polyanion for a given polycation. The lower the degree of neutralization of the polyanion negative charges by the polycation positive charges, the greater the molecular weight of the complexed polyanionic macromolecules.

Effect of Dendrimer Generation on the Interactions between Human Serum Albumin and Dendrigraft Polylysines

This work aims at studying the interaction between human serum albumin and different generations of dendrigraft poly-L-lysine (DGL) in physiological conditions. The binding constants and stoichiometry of the interaction were successfully determined using frontal analysis continuous capillary electrophoresis. The effect of generation on the interaction was evaluated for the five first generations of DGL. An increase of the binding constant accompanied with a decrease of the HSA:DGL (1:n) stoichiometry and a decrease of the cooperativity with dendrimer generation was observed. These findings were in good agreement with the increase of ligand (DGL) size, the increase of electrostatic ligand-ligand repulsion, and the localization of two negatively charged interaction sites on the HSA. The effect of the ligand topology (linear vs dendrigraft) on the HSA interaction revealed that linear poly(L-lysine) leads to much lower stoichiometry compared to DGL of similar molar mass due to much higher flexibility and contour length.

Affinity Chromatography as a Tool to Analyze Polyanion–Polycation Complexes: the Case of Poly(Llysine Citramide)–Poly(L-lysine) Systems

Artificial polymeric carriers for bioactive compounds, such as drugs and genes, are increasingly being reported in the literature. Most are polyelectrolytes aimed at being injected into body fluids that are composed of charged macromolecules (proteins, glycoproteins, poly(glycosamino glycane)s, polynucleotides, etc.). Many interactions can occur and lead to dramatic phenomena, such as polyelectrolyte complexation or substitution, cell aggregation and hemolysis. For the purpose of modeling and better understanding these interactions and their effects, a library of polyanions of the poly(L-lysine citramide)-type was interacted with the polycation poly(L-lysine) in aqueous media. Different fractions of complexes were generated by successive additions of aliquots of the polycation solution to the polyanion library solution by a titrating process. The two components, the polycation and the complex population of polyanions, were separated from the collected complex fractions by affinity chromatography and then analyzed by aqueous SEC. Molecular weight selectivity was shown, the high molecular weight polyanion macromolecules precipitated first, while complexes involving smaller molecules were found to remain in solution. The data show the potential of affinity chromatography for studying interpolyelectrolyte complexes and emphasizes the critical role that polydispersity can play when therapeutic charged macromolecules are injected into blood.

Build-up of an antimicrobial multilayer coating on a textile support based on a methylene blue-poly(cyclodextrin) complex.

The aim of this work was to develop an antibacterial multilayer coating activated with methylene blue (MB) and based on chitosan (CHT) and cyclodextrin polyelectrolyte (polyCD) onto a non-woven polyethylene terephthalate (PET) textile support. The MB-free and MB-loaded systems were built-up by applying the dip-coating technique, alternating soak cycles of the PET textile preliminarily modified with carboxylate groups in CHT and in polyCD or polyCD/MB complex solutions. The layer-by-layer assembly build-up was followed by optical waveguide lightmode spectroscopy on the one hand and by gravimetry once it was applied on the textile substrate on the other hand. Two chitosan grades were used, low molecular weight (CHT-L) and medium molecular weight (CHT-M). The influence of the molar ratio CD/MB in the polyCD solutions was varied and finally the system underwent a post reticulation with genipin. Such parameters influences were investigated with regard to the loading capacity in MB of the systems, the release kinetics profiles of MB in pure water, phosphate buffer and MEM media, and the degradation of the self-assembled coating in the same media. Finally, biological and microbiological tests were performed to demonstrate the cytocompatibility of the systems and their ability to display a sustained antibacterial effect of the device through the MB prolonged release.

Dynamics of polyelectrolyte complex formation and stability when a polycation is progressively added to a polyanion under physico-chemical conditions modeling blood

The formation of polyelectrolyte complexes is known to depend on many factors, especially pH, temperature, and ionic strength, as well as acid—base properties and mixing conditions. In an approach aimed at by-passing the complexity of blood, the formation and the stability of complexes between oppositely charged polymers were studied in salted media (0.15N NaCl and 0.13 M, pH 7.4 PBS) at room temperature. Different molar masses of poly(L-lysine) were reacted with polyanions with different chemical structures and charge densities, namely: poly(acrylic acid), poly(L-lysine citramide), poly(L-lysine citramide imide), and poly(malic acid). A stepwise protocol was used to investigate the fractionation phenomena reported previously. After each addition, the precipitate was separated and analyzed. The polyanion macromolecules were fractionated according to their structure; no significant fractionation was observed for the polycation. The NaCl concentration, required to destabilize the complexes in the isolated fractions, was found to depend on the polycation molar mass and to vary linearly with log(polyanion Mw). Based on these data, the possible fate of polycationic species, and of polycation-based polyelectrolytic complex, when injected into blood, are addressed.