Size and phase preservation of amorphous calcium carbonate nanoparticles in aqueous media using different types of lignin for contrast-enhanced ultrasound imaging.

HYPOTHESIS: Calcium carbonate (CaCO3) nanoparticles could have great potential for contrast-enhanced ultrasound imaging (CEUS) due to their gas-generating properties and sensitivity to physiological conditions. However, the use of nano CaCO3 for biomedical applications requires the assistance of stabilizers to control the size and avoid the fast dissolution/recrystallization of the particles when exposed to aqueous conditions. EXPERIMENTS: Herein, we report the stabilization of nano CaCO3 using lignin, and synthesized core-shell amorphous CaCO3-lignin nanoparticles (LigCC NPs) with a diameter below 100 nm. We have then investigated the echogenicity of the LigCC NPs by monitoring the consequent generation of contrast in vitro for 90 min in linear and non-linear B-mode imaging. FINDINGS: This research explores how lignin type and structure affect stabilization efficiency, lignin structuration around CaCO3 cores, and particle echogenicity. Interestingly, by employing lignin as the stabilizer, it becomes possible to maintain the echogenic properties of CaCO3, whereas the use of lipid coatings prevents the production of signal generation in ultrasound imaging. This work opens new avenue for CEUS imaging of the vascular and extravascular space using CaCO3, as it highlights the potential to generate contrast for extended durations at physiological pH by utilizing the amorphous phase of CaCO3.

Hydrophobized MFC as Reinforcing Additive in Industrial Silica/SBR Tire Tread Compound.

Silica is used as reinforcing filler in the tire industry. Owing to the intensive process of silica production and its high density, substitution with lightweight bio-based micro fibrillated cellulose (MFC) is expected to provide lightweight, sustainable, and highly reinforced tire composite. MFC was modified with oleoyl chloride, and the degree of substitution (DS) was maintained between 0.2 and 0.9. Subsequently, the morphology and crystallinity of the modified MFC were studied and found to be significantly dependent on the DS. The advantages associated with the use of the modified MFC in synergy with silica for the reinforcement of styrene butadiene rubber (SBR) nanocomposite was investigated in comparison with silica/SBR compound. The structural changes occasioned by the DS values influenced the processability, curing kinetics, modulus-rolling resistance tradeoff, and tensile properties of the resultant rubber compounds. We found that the compound made with modified MFC at a DS of 0.67 (MFC16) resulted to the highest reinforcement, with a 350% increase in storage modulus, 180% increase in Young`s modulus, and 15% increase in tensile strength compared to the referenced silica-filled compounds. Our studies show that MFC in combination with silica can be used to reinforce SBR compound for tire tread applications.

Solvents drive self-assembly mechanisms and inherent properties of Kraft lignin nanoparticles (<50 nm).

HYPOTHESIS: Strikingly, Kraft lignin nanoparticles (KLNPs) can substitute polluting nanoparticles in diverse applications. An attractive method for synthesizing KLNPs is Solvent shifting. We hypothesized that by a detailed understanding of the solvent properties and influence of the process parameters, one could derive new fundamental and technical information about the lignin nanoparticle formation process. EXPERIMENTS: DMSO and THF were chosen best solvents based on the Hansen solubility parameter of lignin. The four synthesis parameters such as lignin concentration, (anti-solvent) water volume, temperature, and stirring speed were used to investigate the size, polydispersity index (PDI), morphology as well as the thermal, mechanical and optical properties of KLNPsDMSO & KLNPsTHF. FINDINGS: KLNPsTHF follows the well-known nucleation and growth (NG) mechanism, resulting in spherical KLNPs (43 ± 12 nm: 0.20 PDI). Surprisingly, KLNPsDMSO follows a unique mechanism resembling spinodal decomposition (SD), which generates rare bicontinuous-to-spherical KLNPs (17 ± 8 nm: 0.20 PDI). Remarkably, we show that the difference in the KLNPs mechanism modulates their intrinsic properties, such as glass transition temperature (Tg), specific surface area (SSA), elastic modulus (EM) and optical properties. Beyond the new mechanism, our synthesis resulted in reproducible ultra-small KLNPs with an excellent % yield. Such findings have vast implications in high-performance nanocomposites.

Dual-Silane Premodified Silica Nanoparticles-Synthesis and Interplay between Chemical, Mechanical, and Curing Properties of Silica-Rubber Nanocomposites: Application to Tire Tread Compounds.

In silica-rubber based nanocomposites, a single organo-silicon is often used to compatibilize and covalently link silica to rubber. In this work, we have investigated the impact, at micro- and macroscales, of the decoupling of the hydrophobization and the coupling activity of silane by pretreating silica with two different silane chemistries. The first one, a mercaptosilane, is the coupling agent that promotes a covalent link between silica and rubber during the sulfur-mediated vulcanization reaction. The second one, an alkylsilane, aims to improve the silica dispersion. For both kind of silanes, we have varied the chain length and studied at macroscale the dynamic mechanical properties through the key indicators that are E'' as loss modulus, E' as storage modulus, and their respective ratio tan δ. The shorter silanes combination yielded an improvement in terms of wet grip indicators with tan δ at 0 °C increasing from 0.205 to 0.237 while maintaining rolling resistance indicators at the same level. We have evaluated the impact of the silane chemistry onto the cross-linking reactivity within the fabricated rubber-based nanocomposites by using moving-dye rheometer measurements (MDR). By purposely using atomic force microscopy (AFM), we have studied the silica dispersion in the matrix and the rubber/silica interface and provided the rationale explanation of the mechanical properties observed at the macroscale. AFM observation pointed out the existence of a soft interface around silica fillers when long alkylsilanes were used. We infer that this interface impacts the polymer-filler dynamic and subsequently affects the mechanical properties of the composite material.

Original Basic Activation for Enhancing Silica Particle Reactivity: Characterization by Liquid Phase Silanization and Silica-Rubber Nanocomposite Properties.

Silica fillers are used in various nanocomposites in combination with silanes as a reinforcing filler. In tire technology, silica is generally functionalized before (pre-treated) or during mixing (in-situ silanization or post-treated). In both cases, a soft base catalyst (e.g., triethylamine or diphenyl guanidine, DPG) is typically used to accelerate and increase the yield of the silane/silica coupling reaction. In this study, we investigated how pre-treatments of silica particles with either strong amine or hydride bases impact the silanization of silica prior to or during SBR mixing for silica-rubber nanocomposite fabrication. Our findings are supported by molecular characterization (solid state 29Si NMR, 1H NMR and TGA), and scanning electron microscopy. In addition, the impact of these silica pre-treatments on a nanocomposite's mechanical properties was evaluated using dynamic mechanical analysis (DMA).

Semiquantitative Solid-State NMR Study of the Adsorption of Soybean Oils on Silica and Its Significance for Rubber Processing.

Soybean oil (SBO) is a renewable material used as an alternative to conventional petroleum-derived oils in the processing of rubber composites. Upon chemical modifications, such as epoxidation, its performance in the processing of rubber can be significantly improved, as indicated by a considerable reduction of the mixing energy. Although it has been hypothesized that hydrogen bonding between functional groups (e.g., epoxy) of SBOs and silanols present on the silica surface plays a key role, there is still a lack of direct evidence supporting this hypothesis. In this work, it is demonstrated that there is an overall correlation between the epoxy concentration of SBOs and the mixing energy, consistent with the long-held hypothesis. In particular, a correlation between the SBO-silica adsorption affinity and the degree of epoxidation is revealed by a set of surface-selective solid-state nuclear magnetic resonance (ssNMR) experiments. In addition, the surface-selective ssNMR technique demonstrated in this work could also be used to evaluate the adsorption affinity of other oils and/or additives more broadly.

Hybrid Silica-Based Fillers in Nanocomposites: Influence of Isotropic/Isotropic and Isotropic/Anisotropic Fillers on Mechanical Properties of Styrene-Butadiene (SBR)-Based Rubber.

The improvement of mechanical properties of polymer-based nanocomposites is usually obtained through a strong polymer-silica interaction. Most often, precipitated silica nanoparticles are used as filler. In this work, we study the synergetic effect occurring between dual silica-based fillers in a styrene-butadiene rubber (SBR)/polybutadiene (PBD) rubber matrix. Precipitated Highly Dispersed Silica (HDS) nanoparticles (10 nm) have been associated with spherical Stöber silica nanoparticles (250 nm) and anisotropic nano-Sepiolite. By imaging filler at nano scale through Scanning Transmission Electron Microscopy, we have shown that anisotropic fillers align only in presence of a critical amount of HDS. The dynamic mechanical analysis of rubber compounds confirms that this alignment leads to a stiffer nanocomposite when compared to Sepiolite alone. On the contrary, spherical 250 nm nanoparticles inhibit percolation network and reduce the nanocomposite stiffness.

Positive Effect of Cold Atmospheric Nitrogen Plasma on the Behavior of Mesenchymal Stem Cells Cultured on a Bone Scaffold Containing Iron Oxide-Loaded Silica Nanoparticles Catalyst.

Low-temperature atmospheric pressure plasma was demonstrated to have an ability to generate different reactive oxygen and nitrogen species (RONS), showing wide biological actions. Within this study, mesoporous silica nanoparticles (NPs) and FexOy/NPs catalysts were produced and embedded in the polysaccharide matrix of chitosan/curdlan/hydroxyapatite biomaterial. Then, basic physicochemical and structural characterization of the NPs and biomaterials was performed. The primary aim of this work was to evaluate the impact of the combined action of cold nitrogen plasma and the materials produced on proliferation and osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (ADSCs), which were seeded onto the bone scaffolds containing NPs or FexOy/NPs catalysts. Incorporation of catalysts into the structure of the biomaterial was expected to enhance the formation of plasma-induced RONS, thereby improving stem cell behavior. The results obtained clearly demonstrated that short-time (16s) exposure of ADSCs to nitrogen plasma accelerated proliferation of cells grown on the biomaterial containing FexOy/NPs catalysts and increased osteocalcin production by the cells cultured on the scaffold containing pure NPs. Plasma activation of FexOy/NPs-loaded biomaterial resulted in the formation of appropriate amounts of oxygen-based reactive species that had positive impact on stem cell proliferation and at the same time did not negatively affect their osteogenic differentiation. Therefore, plasma-activated FexOy/NPs-loaded biomaterial is characterized by improved biocompatibility and has great clinical potential to be used in regenerative medicine applications to improve bone healing process.

Do the pristine physico-chemical properties of silver and gold nanoparticles influence uptake and molecular effects on Gammarus fossarum (Crustacea Amphipoda)?

The specific and unique properties of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs), make them of high interest for different scientific and industrial applications. Their increasing use will inevitably lead to their release in the environment and aquatic ecosystems where they may represent a threat to aquatic organisms. Being a widespread and important component of the aquatic macroinvertebrate assemblage, amphipods and more specifically Gammarus fossarum will certainly be exposed to AgNPs and AuNPs. For these reasons, G. fossarum was selected as model organism for this study. The aim of the present work was the evaluation of the influence of both size (20, 40 and 80 nm) and surface coating (citrate CIT, polyethylene glycol PEG) on the acute toxicity of AgNPs and AuNPs on G. fossarum. We investigated the effects of AgNPs and AuNPs on the uptake by G. fossarum, NP tissue distribution and the expression of stress related genes by the use of ICP-MS, NanoSIMS50, Cytoviva®, and Rt-qPCR, respectively. Ag and Au bioaccumulation revealed a significant surface-coating dependence, with CIT-AgNPs and CIT-AuNPs showing the higher bio-accumulation potential in G. fossarum as compared to PEG-NPs. Opposite to that, no size-dependent effects on the bioaccumulation potential was observed. SIMS imaging and CytoViva® revealed an influence of the type of metal on the tissue distribution after uptake, with AgNPs detected in the cuticle and the gills of G. fossarum, while AuNPs were detected in the gut area. Furthermore, AgNPs were found to up-regulate CuZnSOD gene expression while AuNPs led to its down-regulation. Modulation of SOD may indicate generation of reactive species of oxygen and a possible activation of antioxidant defence in order to prevent and defend the organism from oxidative stress. However, further investigations are still needed to better define the mechanisms underlying the observed AgNPs and AuNPs effects.

Electrochemical Control of pH in Nanoliter Volumes.

The electrochemical management of the proton concentration in miniaturized dimensions opens the way to control and parallelize multistep chemical reactions, but still it faces many challenges linked to the efficient proton generation and control of their diffusion. Here we present a device operated electrochemically that demonstrates the control of the pH in a cell of ∼140 nL. The device comprises a microfluidic reactor integrated with a pneumatic mechanism that allows the exchange of reagents and the isolation of protons to decrease the effect of their diffusion. We monitored the pH with a fluorescence marker and calculated the final value from the redox currents. We demonstrate a large pH amplitude control from neutral pH values beyond the fluorescence marker range at pH 5. On the basis of the calculations from the Faradaic currents, the minimum pH reached should undergo pH ∼ 0.9. The pH contrast between neutral and acid pH cells can be maintained during periods longer than 15 min with an appropriate design of a diffusion barrier.

Influence of polymerisation on the reversibility of low-energy proton exchange reactions by Para-Aminothiolphenol.

The reversibility of redox processes is an important function for sensing and molecular electronic devices such as pH reporters or molecular switches. Here we report the electrochemical behaviour and redox reversibility of para-aminothiolphenol (PATP) after different polymerisation methods. We used electrochemical and photo-polymerisation in neutral buffers and plasma polymerisation in air to induce reversible redox states. The chemical stoichiometry and surface coverage of PATP in the polymerized layers were characterized by X-ray photoelectron spectroscopy (XPS), while cyclic voltammetry (CV) was used to measure the charge transfer, double layer capacitance and electrochemical rate of the layers during successive potential cycles. Our results show that the surface coverage of the redox active species is higher on electro-polymerised samples, however, after consecutive cycles all the methods converge to the same charge transfer, while the plasma polymerised samples achieve higher efficiency per molecule and UV polymerised samples have a higher electron transfer rate.

Solubilization of α-galactosylceramide in aqueous medium: Impact on Natural Killer T cell activation and antitumor responses.

The potent antitumor effect of α-galactosylceramide (α-GalCer) is based on its recognition by invariant Natural Killer T cells (iNKT) after its capture and presentation by antigen presenting cells including dendritic cells (DCs). Synthetic α-GalCer has already been tested in advanced cancer patients but no or only moderate clinical responses were obtained. To optimize α-GalCer efficacy, we have postulated that alternative formulations impacting its molecular organization in aqueous medium could modify DC uptake and iNKT-based immune responses. To this end, we have developed two strategies: (1) the formulation of α-GalCer in non-cationic liposomes and (2) the synthesis of a water-soluble α-GalCer analogue by anchoring a polyethyleneglycol moiety on its sugar head. The biological activities of these new preparations were compared to that induced by the classically used Polysorbate 20 α-GalCer micelles. Both formulations retained their uptake by DCs and activated iNKT cells both in vitro and in vivo. Despite a lower cytokine production, the formulations induced a potent immune response able to control lung murine carcinoma. In conclusion, it is possible to increase α-GalCer solubility in aqueous solution without limiting its antitumor properties.

Photoinduced effects of m-tetrahydroxyphenylchlorin loaded lipid nanoemulsions on multicellular tumor spheroids.

BACKGROUND: Photosensitizers are used in photodynamic therapy (PDT) to destruct tumor cells, however, their limited solubility and specificity hampers routine use, which may be overcome by encapsulation. Several promising novel nanoparticulate drug carriers including liposomes, polymeric nanoparticles, metallic nanoparticles and lipid nanocomposites have been developed. However, many of them contain components that would not meet safety standards of regulatory bodies and due to difficulties of the manufacturing processes, reproducibility and scale up procedures these drugs may eventually not reach the clinics. Recently, we have designed a novel lipid nanostructured carrier, namely Lipidots, consisting of nontoxic and FDA approved ingredients as promising vehicle for the approved photosensitizer m-tetrahydroxyphenylchlorin (mTHPC). RESULTS: In this study we tested Lipidots of two different sizes (50 and 120 nm) and assessed their photodynamic potential in 3-dimensional multicellular cancer spheroids. Microscopically, the intracellular accumulation kinetics of mTHPC were retarded after encapsulation. However, after activation mTHPC entrapped into 50 nm particles destroyed cancer spheroids as efficiently as the free drug. Cell death and gene expression studies provide evidence that encapsulation may lead to different cell killing modes in PDT. CONCLUSIONS: Since ATP viability assays showed that the carriers were nontoxic and that encapsulation reduced dark toxicity of mTHPC we conclude that our 50 nm photosensitizer carriers may be beneficial for clinical PDT applications.

Preparation and characterization of mTHPC-loaded solid lipid nanoparticles for photodynamic therapy.

Among various attempts to enhance the therapeutic efficacy of photodynamic therapy (PDT), the specific delivery of photosensitizer (PS) in the tumor tissue is expected to improve its clinical applications. The aim of this study was to engineer lipid nanoparticles (LNP) with different sizes and various PS contents, using simple solvent-free and easily scale up manufacturing processes. Meso-(tetrahydroxyphenyl) chlorin (mTHPC) is one of the most potent photoactive compounds for clinical use. We demonstrated that mTHPC was efficiently incorporated into the lipid core of LNP, leading to a large range of stable and reproducible mTHPC-loaded LNP with narrow size distribution. Photophysical and physico-chemical properties of mTHPC-loaded LNP were assessed as well as absorption spectra and singlet oxygen emission, colloidal stability, particle size and zeta potential. The photocytotoxicity of selected mTHPC-loaded solid LNP was demonstrated on MCF-7 cells under irradiation at 652nm with a range of light fluence from 1.0 to 10J/cm(2). All physico-chemical, photophysical and biological results allow us to conclude that solid LNP appear as a very promising nano-mTHPC delivery system for PDT.

Novel glycolipid TLR2 ligands of the type Pam2Cys-α-Gal: synthesis and biological properties.

A more complete understanding of the mechanism of action of TLR agonists has fueled the investigation of new synthetic immunoadjuvants. In this context, we designed and synthesized glycolipids of the type Pam(2)Cys-α-Galactose as novel immunoadjuvants. Their synthesis required modifying a hydrophobic tBoc-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteinyl moiety, i.e. the minimal structure required for TLR2 agonist activity, by addition of a hydrophilic head, either an α-Galactosylpyranose or an α-Galactosylfuranose to gain respectively Pam(2)CGalp and Pam(2)CGalf. While preparing a carbohydrate building block, an unexpected stereoselectivity was observed during a halide ion-catalytic process on a protected galactofuranose: the alpha anomer was obtained with surprisingly high selectivity (α/ß ratio>9) and with good isolated yield (51%). The TLR2 binding properties of Pam(2)CGalp and Pam(2)CGalf were then fully evaluated. Their efficiency in triggering the proliferation of BALB/c mouse splenocytes was also compared to that of Pam(2)CAG and Pam(3)CAG, two well-established ligands of TLRs. Moreover, the maturation state of murine dendritic cells previously incubated with either Pam(2)CGalp or Pam(2)CGalf was monitored by flow cytometry and compared to that induced by lipopolysaccharide. Pam(2)CGalp and Pam(2)CGalf were found to be equivalent TLR2 agonists, and induced splenocyte proliferation and DC maturation. With very similar activity, Pam(2)CGalp and Pam(2)CGalf were also 10-fold to 100-fold better than Pam(2)CAG and Pam(3)CAG at inducing B cell proliferation. This represents the first time a glucidic head has been added to the tBoc-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteinyl moiety whilst maintaining the immunomodulating activity. This should greatly enrich the data available on Pam(2)C structure/activity relationships.

Pyridylthiourea-grafted polyethylenimine offers an effective assistance to siRNA-mediated gene silencing in vitro and in vivo.

Success of synthetic interfering nucleic acids (siRNAs)-based therapy relies almost exclusively on effective, safe and preferably nanometric delivery systems which can be easily prepared, even at high concentrations. We prepared by chemical synthesis various self-assembling polymers to entrap siRNAs into stable polyplexes outside cells but with a disassembly potential upon sensing endosomal acidity. Our results revealed that pyridylthiourea-grafted polyethylenimine (πPΕΙ) followed the above-mentioned principles. It led to above 90% siRNA-mediated gene silencing in vitro on U87 cells at 10 nM siRNA concentration and did not have a hemolytic activity. Assembly of siRNA/πPΕΙ at high concentration was then studied and 4.5% glucose solution, pH 6.0, yielded stable colloidal solutions with sizes slightly below 100 nm for several hours. A single injection of these concentrated siRNA polyplexes into luciferase-expressing human glioblastoma tumors, which were subcutaneously xenografted into nude mice, led to a significant 30% siRNA-mediated luciferase gene silencing 4 days post-injection. Our results altogether substantiate the potential of self-assembling cationic polymers with a pH-sensitive disassembly switch for siRNA delivery in vitro and also in vivo experiments.

Antitumor activity of liposomal ErbB2/HER2 epitope peptide-based vaccine constructs incorporating TLR agonists and mannose receptor targeting.

Synthetic and molecularly defined constructs containing the minimal components to mimic and amplify the physiological immune response are able to induce an efficient cytotoxic response. In the current study this approach was applied to the development of highly versatile liposomal constructs to co-deliver peptide epitopes in combination with TLR agonists in order to induce a specific anti-tumor cellular immune response against ErbB2 protein-expressing tumor cells. Liposomes containing ErbB2 p63-71 cytotoxic T lymphocyte (CTL) and HA307-319 T- helper (Th) peptide epitopes associated to innovative synthetic TLR2/1 (Pam(3)CAG) or TLR2/6 agonists (Pam(2)CAG and Pam(2)CGD), were injected in mice bearing ErbB2 protein-expressing tumor cells. Mannosylated ligands were also incorporated into the constructs to target antigen-presenting cells. We showed that the TLR2/6 agonists were more efficient than the TLR2/1 agonists for the eradication of tumors expressing ErbB2 protein. Furthermore, mannose-targeted liposomes displayed higher therapeutic efficiency against tumor allowing treatment with decreased quantities of both TLR ligands and peptide epitopes. Our results validated that antigen-associated mannosylated liposomes combined with efficient TLR ligands are effective vectors for vaccination against tumor. In this study we developed useful tools to evaluate the vaccination efficiency of various adjuvants and/or targeting molecules and their potential synergy.

Covalent layer-by-layer assemblies of polyelectrolytes and homobifunctional spacers.

The step-by-step buildup of organic films through physical or covalent bonds is usually performed by the alternating adsorption of two types of polymeric chains. Overcompensation of the interacting groups after each deposition step (e.g., charge overcompensation in the case of polyelectrolyte multilayers) allows the buildup process to proceed. This overcompensation is intimately linked to the polymeric nature of the interacting species. We report here another type of film architecture also based on step-by-step construction but involving the covalent bonding, through the Sharpless click reaction, between polyelectrolytes (i.e., polyanions) and neutral bifunctional molecules. The films are built by the Cu(I)-catalyzed click reaction of poly(acrylic acid) (PAA) functionalized with ethylene glycol (EG) arms, each ending with either an alkyne or an azide group, and bifunctionalized EG spacers ended with either alkyne or azide functions. We prove that these systems lead to the regular buildup of films that cover the whole substrate surface and whose roughness varies as the thickness of the film core. The effects of various parameters on film buildup are investigated. The grafting density of reactive moieties along the PAA chains has no influence on the thickness increment per bilayer. EG spacers bifunctionalized with alkyne groups reacting with PAA chains functionalized with azide arms give films that grow more rapidly than those obtained with azide-functionalized EG spacers and alkyne-functionalized PAA chains. The influence of the length of the EG arm (grafted on PAA) and of the EG spacer on the film buildup is also investigated: longer arms or longer spacers lead to larger thickness increments per bilayer, except for very large spacers of 50 EG units for which the thickness is the smallest probably because of size exclusion effects during the deposition.

Polymer multilayer films obtained by electrochemically catalyzed click chemistry.

We report the covalent layer-by-layer construction of polyelectrolyte multilayer (PEM) films by using an efficient electrochemically triggered Sharpless click reaction. The click reaction is catalyzed by Cu(I) which is generated in situ from Cu(II) (originating from the dissolution of CuSO(4)) at the electrode constituting the substrate of the film. The film buildup can be controlled by the application of a mild potential inducing the reduction of Cu(II) to Cu(I) in the absence of any reducing agent or any ligand. The experiments were carried out in an electrochemical quartz crystal microbalance cell which allows both to apply a controlled potential on a gold electrode and to follow the mass deposited on the electrode through the quartz crystal microbalance. Poly(acrylic acid) (PAA) modified with either alkyne (PAA(Alk)) or azide (PAA(Az)) functions grafted onto the PAA backbone through ethylene glycol arms were used to build the PEM films. Construction takes place on gold electrodes whose potentials are more negative than a critical value, which lies between -70 and -150 mV vs Ag/AgCl (KCl sat.) reference electrode. The film thickness increment per bilayer appears independent of the applied voltage as long as it is more negative than the critical potential, but it depends upon Cu(II) and polyelectrolyte concentrations in solution and upon the reduction time of Cu(II) during each deposition step. An increase of any of these latter parameters leads to an increase of the mass deposited per layer. For given buildup conditions, the construction levels off after a given number of deposition steps which increases with the Cu(II) concentration and/or the Cu(II) reduction time. A model based on the diffusion of Cu(II) and Cu(I) ions through the film and the dynamics of the polyelectrolyte anchoring on the film, during the reduction period of Cu(II), is proposed to explain the major buildup features.