Szczepan Zapotoczny

Hypochlorous Acid: A Natural Adjuvant That Facilitates Antigen Processing, Cross-Priming, and the Induction of Adaptive Immunity

The production of hypochlorous acid (HOCl) is a characteristic of granulocyte activation, a hallmark of the early phase of innate immune responses. In this study, we show that, in addition to its well-established role as a microbicide, HOCl can act as a natural adjuvant of adaptive immunity. HOCl enhances the T cell responses to the model Ag OVA, facilitating the processing and presentation of this protein via the class II MHC pathway. HOCl modification also enhances cross-presentation of the tumor Ag tyrosinase-related protein 2 via class I MHC. The adjuvant effects of HOCl are independent of TLR signaling. The enhanced presentation of HOCl-modified OVA is mediated via modification of the N-linked carbohydrate side chain rather than formation of protein aldehydes or chloramines. HOCl-modified OVA is taken up more efficiently by APCs and is degraded more efficiently by proteinases. Atomic force microscopy demonstrated that enhanced uptake is mediated via specific receptor binding, one candidate for which is the scavenger receptor lectin-like oxidized low-density lipoprotein receptor, which shows enhanced binding to chlorinated OVA. A function of HOCl is therefore to target glycoprotein Ags to scavenger receptors on the APC surface. This additional mechanism linking innate and adaptive immunity suggests novel strategies to enhance immunity to vaccines.

Buried, Covalently Attached RGD Peptide Motifs in Poly(methacrylic acid) Brush Layers : The Effect of Brush Structure on Cell Adhesion

Iniferter-mediated surface-initiated photopolymerization was used to graft poly(methacrylic acid) (PMAA) brush layers obtained from surface-attached iniferters in self-assembled monolayers to a gold surface. The tethered chains were subsequently functionalized with the cell-adhesive arginine-glycine-aspartic acid (RGD) motif. The modified brushes were extended by reinitiating the polymerization to obtain an additional layer of PMAA, thereby burying the peptide-functionalized segments inside the brush structure. Contact angle measurements and Fourier transform infrared (FTIR) spectroscopy were employed to characterize the wettability and the chemical properties of these platforms. Time of flight secondary ion mass spectroscopy (TOF-SIMS) measurements were performed to monitor the chemical composition of the polymer layer as a function of the distance to the gold surface and obtain information concerning the depth of the RGD motifs inside the brush structure. The brush thickness was evaluated as a function of the polymerization (i.e., UV-irradiation) time with atomic force microscopy (AFM) and ellipsometry. Cell adhesion tests employing human osteoblasts were performed on substrates with the RGD peptides exposed at the surface as well as covered by a PMAA top brush layer. Immunofluorescence studies demonstrated a variation of the cell morphology as a function of the position of the peptide units along the grafted chains.

Characterization and molecular engineering of surface-grafted polymer brushes across the length scales by atomic force microscopy

With the advent of regulated, surface initiated polymerizations, specifically using controlled radical approaches, the choice of polymerizable compounds and the control over grafting chemistry have seen tremendous advancement. New analysis techniques and approaches are now needed to characterize these brushes with molecular precision. In addition, spatial structure control at the nanoscale, and tuning of thickness as well as composition of the brushes, have become feasible by utilizing recently developed enabling molecular nanofabrication approaches. Atomic force microscopy (AFM) is a powerful analytical tool for the characterization of polymer brushes, as well as for the fabrication of brush structures across the length scales. AFM has been used to investigate polymer brushes in a number of ways including imaging surface morphologies, measuring brush thickness, estimating the value of number average molar mass, observing stimulus responsive behavior and probing surface mechanical properties. In addition, AFM based methods such as nanoscratching, dip-pen nanolithography (DPN) and scanning probe oxidation (SPO) have been also employed for the nanofabrication of patterned polymer brushes. This feature article gives a short account of this field and highlights recent advances.

Polymeric photosensitizers. Part 4. Synthesis of poly(sodium styrenesulfonate-block-N-vinylcarbazole) by nitroxide-mediated free radical polymerization

Abstract Block amphiphilic copolymer, poly(sodium styrenesulfonate- block - N -vinylcarbazole) (PSSS- block -VCz), was synthesized by nitroxide-mediated “living” free radical polymerization in homogenous solution. The procedure of the synthesis of poly(sodium styrenesulfonate) (PSSS) via the same technique was modified to reduce the amount of mediator and initiator needed. Camphorsulphonic acid (CSA) and acetic anhydride (AA) were used as rate-accelerating additives in polymerization of PSSS and PSSS- block -VCz, respectively. The physicochemical and photophysical properties of PSSS-block-VCz copolymer were determined and discussed.

Robust “one-component” chitosan-based ultrathin films fabricated using layer-by-layer technique

“One-component” chitosan-based multilayer films have been formed and characterized for the first time. Two derivatives of chitosan containing oppositely charged strong ionic groups were synthesized for that purpose. Low molecular weight chitosan was modified by glycidyltrimethylammonium chloride to obtain the cationic form (CCh) of the polymer. The anionic form of chitosan (ACh) was obtained by sulfonation of carboxymethylchitosan with trimethylamine-sulfur trioxide. The two derivatives of chitosan were used to prepare stable multilayer polyelectrolyte films through a layer-by-layer (LbL) deposition technique. The films revealed smooth surfaces and linear growth of the thickness during LbL adsorption as measured by atomic force microscope (AFM). Contact angle measurements revealed a very hydrophilic nature of the formed films. In contrast, the hybrid films, made of one of the chitosan derivatives and the synthetic oppositely charged polyelectrolyte, demonstrated non-linear growths and much higher surface roughnesses. So far, the multilayer films built of natural polymers like chitosan and alginate as well as the mentioned hybrid films have shown difficulties in regular formation related to the differences in polymer backbones and/or the charge distribution along them. The approach proposed here omits the inherent entropic barrier and leads to the controlled formation of robust “one-component” multilayer films. The entropy-driven formation of such films was also supported by the calorimetric studies on the model polyelectrolyte complexes in solution. Thanks to biocompatibility and bacteriostatic properties of chitosan and its derivatives, CCh/ACh multilayer films may be potentially useful for many biomedical and environmental protection applications.

Preparation and characterization of macromolecular “hedge” brushes grafted from Au nanowires

Linear polymer brush structures of poly(methacrylic acid) chains, exhibiting a width from several hundred to 20–30 nm and a controllable height (nano-“hedge”), were grafted from designer substrates by photopolymerization using thiol-based iniferters assembled on Au nano-lines. The Au nano-lines were obtained using H-terminated silicon substrates by AFM tip assisted (“dip-pen”) nanolithography of HAuCl4 which yielded Au nano-wires upon reduction in contact with the substrate. The polymeric nano-“hedge” structures were characterized by AFM.

Properties of polyethylene glycol supported tetraarylporphyrin in aqueous solution and its interaction with liposomal membranes.

5,10,15,20-Tetrakis(4-hydroxyphenyl)porphyrin was functionalized by covalent attachment of poly(ethylene glycol) (PEG) chains of various molecular weights, 350, 2000, and 5000 Da. The properties of PEG-functionalized tetraarylporphyrins in aqueous solution and their interactions with liposomes have been studied. Electronic absorption spectroscopy, dynamic light scattering, atomic force microscopy, and fluorescence quenching were used to monitor aggregation of porphyrin chromophores and behavior of the attached PEG chains in the aqueous solution. The tendency for aggregation of porphyrin chromophores in aqueous solution and the efficiency of fluorescence quenching by KI decrease with increasing length of PEG chain linked to the porphyrin ring. The experimental results indicate that polymer clusters are present in aqueous solution of all pegylated porphyrins. The interactions between the pegylated porphyrins and phosphatidylcholine liposomes in the aqueous solution were studied using the fluorescence methods. The apparent binding constants of porphyrin chromophores to liposomes were determined. The degree of binding was found to be dependent on the molecular weight of the attached polymer.

Nanostructured micellar films formed via layer-by-layer deposition of photoactive polymer.

Photoactive nanostructured micellar films were prepared from the amphiphilic copolymer poly(sodium styrenesulfonate- stat-2-vinylnaphthalene) (PSSS- stat-VN) and cationic polyelectrolyte poly(diallyldimethylammonium chloride) (PDADMAC) or poly(allylamine hydrochloride) (PAH) on quartz and silicon substrates via layer-by-layer (LbL) electrostatic self-assembly. The macromolecules of this amphiphilic copolymer adopt a coiled micellar conformation in aqueous solution that is preserved in the films as evidenced by atomic force microscopy (AFM) and spectroscopic studies. The hydrophobic domains present in the film can serve as host sites for various organic molecules. The probe molecules reside in those isolated nanosize domains. Their aggregation and quenching of their emission is eliminated. The experiments showed a regular growth of multilayer thickness and the content of solubilized compounds in the films. Thus, a defined amount of the hydrophobic compounds of interest may be introduced into these water-processable polymeric films. Some stratification of the films was induced by the presence of stiff nanoparticle-like micelles. That makes these films an important new material for studies of photoinduced energy and electron transfer.

Chitosan-Based Ultrathin Films as Antifouling, Anticoagulant and Antibacterial Protective Coatings

Abstract Ultrathin antifouling and antibacterial protective nanocoatings were prepared from ionic derivatives of chitosan using layer-by-layer deposition methodology. The surfaces of silicon, and glass protected by these nanocoatings were resistant to non-specific adsorption of proteins disregarding their net charges at physiological conditions (positively charged TGF-β1 growth factor and negatively charged bovine serum albumin) as well as human plasma components. The coatings also preserved surfaces from the formation of bacterial (Staphylococcus aureus) biofilm as shown using microscopic studies (SEM, AFM) and the MTT viability test. Moreover, the chitosan-based films adsorbed onto glass surface demonstrated the anticoagulant activity towards the human blood. The antifouling and antibacterial actions of the coatings were correlated with their physicochemical properties. The studied biologically relevant properties were also found to be dependent on the thickness of those nanocoatings. These materials are promising for biomedical applications, e.g., as protective coatings for medical devices, anticoagulant coatings and protective layers in membranes.

Nanoheterogeneous multilayer films with perfluorinated domains fabricated using the layer-by-layer method.

Nanoheterogenous ultrathin films containing perfluorinated domains were prepared via the layer-by-layer (LbL) electrostatic self-assembly method. The films are constructed from the amphiphilic cationic copolymer with perfluorinated side chains and poly(sodium styrenesulfonate) (PSS). The LbL process was optimized by the application of sonication which allowed linear growth of the film. The resulting film exhibited micellar structure with isolated fluorocarbon hydrophobic domains. The remarkable features of the films were their switchable wettability and friction properties. The obtained water-processable films can find a number of potential applications, e.g., as smart and low friction coatings.