Polystyrene Brush Evolution by Grafting to Reaction on Deglazed and Not-Deglazed Silicon Substrates.

Two model substrates for the grafting to reaction are considered: not-deglazed silicon, whose surface is coated by a thin oxide layer with reactive silanol groups on its surface; and deglazed silicon, where the oxide layer is removed by treatment with hydrofluoric acid. The reactive polymers are hydroxy-terminated polystyrenes with molecular weights ranging from 3.9 to 13.9 kg mol⁻1. The grafting to reaction is carried out at different temperatures and for different periods of time on the two different substrates. The thickness and the thermal stability of the resulting brushes are evaluated. Furthermore, the grafting of a highly dispersed system is simulated by blending two polymers with different molecular weights. Although the brush thickness growth is found to be faster on deglazed silicon, the preferential grafting of short chains occurs with equal chain selection propensity on both substrates.

Development of an enrichment method for endogenous phosphopeptide characterization in human serum.

The work describes the development of an enrichment method for the analysis of endogenous phosphopeptides in serum. Endogenous peptides can play significant biological roles, and some of them could be exploited as future biomarkers. In this context, blood is one of the most useful biofluids for screening, but a systematic investigation of the endogenous peptides, especially phosphorylated ones, is still lacking, mainly due to the lack of suitable analytical methods. Thus, in this paper, different phosphopeptide enrichment strategies were pursued, based either on metal oxide affinity chromatography (MOAC, in the form of commercial TiO2 spin columns or magnetic graphitized carbon black-TiO2 composite), or on immobilized metal ion affinity chromatography (IMAC, in the form of Ti4+-IMAC magnetic material or commercial Fe3+-IMAC spin columns). While MOAC strategies proved completely unsuccessful, probably due to interfering phospholipids displacing phosphopeptides, the IMAC materials performed very well. Different sample preparation strategies were tested, comprising direct dilution with the loading buffer, organic solvent precipitation, and lipid removal from the matrix, as well as the addition of phosphatase inhibitors during sample handling for maximized endogenous phosphopeptide enrichment. All data were acquired by a shotgun peptidomics approach, in which peptide samples were separated by reversed-phase nanoHPLC hyphenated with high-resolution tandem mass spectrometry. The devised method allowed the identification of 176 endogenous phosphopeptides in fresh serum added with inhibitors by the direct dilution protocol and the Ti4+-IMAC magnetic material enrichment, but good results could also be obtained from the commercial Fe3+-IMAC spin column adapted to the batch enrichment protocol.

Composition of ultrathin binary polymer brushes by thermogravimetry-gas chromatography-mass spectrometry.

In the present paper, a reliable and rugged thermogravimetry-gas chromatography-mass spectrometry (TGA-GC-MS) method was developed to determine the composition of ultrathin films consisting of binary blends of functional polystyrene (PS) and polymethylmethacrylate (PMMA) grafted to a silicon wafer. A general methodology will be given to address the composition determination problem for binary or even multicomponent polymer brush systems using the PS/PMMA-based samples as a paradigmatic example. In this respect, several distinct tailor-made materials were developed to ensure reliable calibration and validation stages. The analytical method was tested on unknown samples to follow the composition evolution in PS/PMMA brushes during the grafting reaction. A preferential grafting of the PMMA was revealed in full agreement with its preferential interaction with the SiO2 polar surface.

Self-cleaning properties in engineered sensors for dopamine electroanalytical detection.

Fouling and passivation are the major drawbacks for a wide applicability of electroanalytical sensors based on nanomaterials, especially in biomedical and environmental fields. The production of highly engineered devices, designed ad hoc for specific applications, is the key factor in the direction of overcoming the problem and accessing effective sensors. Here, the fine-tuning of the system, composed of a highly ordered distribution of silver nanoparticles between a bottom silica and a top titania layer, confers multifunctional properties to the device for a biomedical complex challenge: dopamine detection. The crucial importance of each component towards a robust and efficient electroanalytical system is studied. The total recovery of the electrode performance after a simple UV-A cleaning step (self-cleaning), due to the photoactive interface and the aging resistance, is deeply investigated.

Bessel-like photonic nanojets from core-shell sub-wavelength spheres.

It is accepted so far that the formation of photonic nanojets requires the use of large dielectric spheres (several wavelengths in diameter). Here we show both numerically and experimentally that similar effects can be obtained with properly engineered sub-wavelength core-shell colloids. The design of the spheres is strongly inspired by a far-field approach for the generation of Bessel beams.

Evolution of lateral ordering in symmetric block copolymer thin films upon rapid thermal processing.

This work reports experimental findings about the evolution of lateral ordering of lamellar microdomains in symmetric PS-b-PMMA thin films on featureless substrates. Phase separation and microdomain evolution are explored in a rather wide range of temperatures (190-340 °C) using a rapid thermal processing (RTP) system. The maximum processing temperature that enables the ordering of block copolymers without introducing any significant degradation of macromolecules is identified. The reported results clearly indicate that the range of accessible temperatures in the processing of these self-assembling materials is mainly limited by the thermal instability of the grafted random copolymer layer, which starts to degrade at T > 300 °C, inducing detachment of the block copolymer thin film. For T ⩽ 290 °C, clear dependence of correlation length (ξ) values on temperature is observed. The highest level of lateral order achievable in the current system in a quasi-equilibrium condition was obtained at the upper processing temperature limit after an annealing time as short as 60 s.

Thermally induced self-assembly of cylindrical nanodomains in low molecular weight PS-b-PMMA thin films.

The phase behaviour in thin films of an asymmetric polystyrene-b-polymethylmethacrylate (PS-b-PMMA) block copolymer with a molecular weight of 39 kg mol(-1) was assessed at a wide range of temperatures and times. Cylindrical PMMA structures featuring a diameter close to 10 nm and perpendicularly oriented with respect to the substrate were obtained at 180 °C in relatively short annealing times (t ≤ 30 min) by means of a simple thermal treatment performed in a standard rapid thermal processing machine.

Persistent dystrophin protein restoration 90 days after a course of intraperitoneally administered naked 2'OMePS AON and ZM2 NP-AON complexes in mdx mice.

In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of 2'-O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules.

Sulfonates-PMMA nanoparticles conjugates: a versatile system for multimodal application.

We report herein the viability of a novel nanoparticles (NPs) conjugated system, namely the attachment, based on ionic and hydrophobic interactions, of different sulfonated organic salts to positively charged poly(methylmethacrylate) (PMMA)-based core-shell nanoparticles (EA0) having an high density of ammonium groups on their shells. In this context three different applications of the sulfonates@EA0 systems have been described. In detail, their ability as cytotoxic drugs and pro-drugs carriers was evaluated in vitro on NCI-H460 cell line and in vivo against human ovarian carcinoma IGROV-1 cells. Besides, 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS) was chosen for NPs loading, and its internalization as bioimaging probe was evaluated on Hep G2 cells. Overall, the available data support the interest for these PMMA NPs@sulfonates systems as a promising formulation for theranostic applications. In vivo biological data strongly support the potential value of these core-shell NPs as delivery system for negatively charged drugs or biologically active molecules. Additionally, we have demonstrated the ability of these PMMA core-shell nanoparticles to act as efficient carriers of fluorophores. In principle, thanks to the high PMMA NPs external charge density, sequential and very easy post-loading of different sulfonates is achievable, thus allowing the preparation of nanocarriers either with bi-modal drug delivery behaviour or as theranostic systems.

Cationic PMMA nanoparticles bind and deliver antisense oligoribonucleotides allowing restoration of dystrophin expression in the mdx mouse.

For subsets of Duchenne muscular dystrophy (DMD) mutations, antisense oligoribonucleotide (AON)-mediated exon skipping has proven to be efficacious in restoring the expression of dystrophin protein. In the mdx murine model systemic delivery of AON, recognizing the splice donor of dystrophin exon 23, has shown proof of concept. Here, we show that using cationic polymethylmethacrylate (PMMA) (marked as T1) nanoparticles loaded with a low dose of 2'-O-methyl-phosphorothioate (2'OMePS) AON delivered by weekly intraperitoneal (IP) injection (0.9 mg/kg/week), could restore dystrophin expression in body-wide striated muscles. Delivery of an identical dose of naked AON did not result in detectable dystrophin expression. Transcription, western, and immunohistochemical analysis showed increased levels of dystrophin transcript and protein, and correct localization at the sarcolemma. This study shows that T1 nanoparticles have the capacity to bind and convoy AONs in body-wide muscle tissues and to reduce the dose required for dystrophin rescue. By immunofluorescence and electron microscopy studies, we highlighted the diffusion pathways of this compound. This nonviral approach may valuably improve the therapeutic usage of AONs in DMD as well as the delivery of RNA molecules with many implications in both basic research and medicine.

Core-shell silica-rhodamine B nanosphere for synthetic opals: from fluorescence spectral redistribution to sensing.

Photonic crystals are a unique tool to modify the photoluminescence of light-emitting materials. A variety of optical effects have been demonstrated by infiltrating opaline structures with photoactive media. On the other hand, the fabrication of such structures includes complex infiltration steps, that often affect the opal lattice and decrease the efficiency of light emission control. In this work, silica nanospheres were directly functionalized with rhodamine B to create an emitting shell around the dielectric core. Simple tuning of the microsphere preparation conditions allows selecting the appropriate sphere diameter and polydispersity index approaching 5%. These characteristics allow facile self-assembling of the nanospheres into three-dimensional photonic crystals whose peculiar density of photonic states at the band-gap edges induces spectral redistribution of the rhodamine B photoluminescence. The possibility to employ the new stable structure as sensor is also investigated. As a proof of principle, we report the variation of light emission obtained by exposure of the opal to vapor of chlorobenzene.

Directed Self-Assembly of Polystyrene Nanospheres by Direct Laser-Writing Lithography.

In this work, we performed a systematic study on the effect of the geometry of pre-patterned templates and spin-coating conditions on the self-assembling process of colloidal nanospheres. To achieve this goal, large-scale templates, with different size and shape, were generated by direct laser-writer lithography over square millimetre areas. When deposited over patterned templates, the ordering dynamics of the self-assembled nanospheres exhibits an inverse trend with respect to that observed for the maximisation of the correlation length ξ on a flat surface. Furthermore, the self-assembly process was found to be strongly dependent on the height (H) of the template sidewalls. In particular, we observed that, when H is 0.6 times the nanospheres diameter and spinning speed 2500 rpm, the formation of a confined and well ordered monolayer is promoted. To unveil the defects generation inside the templates, a systematic assessment of the directed self-assembly quality was performed by a novel method based on Delaunay triangulation. As a result of this study, we found that, in the best deposition conditions, the self-assembly process leads to well-ordered monolayer that extended for tens of micrometres within the linear templates, where 96.2% of them is aligned with the template sidewalls.

Effect of Trapped Solvent on the Interface between PS-b-PMMA Thin Films and P(S-r-MMA) Brush Layers.

The orientation of block copolymer (BCP) features in thin films can be obtained by spin-coating a BCP solution on a substrate surface functionalized by a polymer brush layer of the appropriate random copolymer (RCP). Although this approach is well established, little work reporting the amount and distribution of residual solvent in the polymer film after the spin-coating process is available. Moreover, no information can be found on the effect of trapped solvent on the interface between the BCP film and RCP brush. In this work, systems consisting of poly(styrene)-b-poly(methyl methacrylate) thin films deposited on poly(styrene-r-methyl methacrylate) brush layers are investigated by combining neutron reflectivity (NR) experiments with simulation techniques. An increase in the amount of trapped solvent is observed by NR as the BCP film thickness increases accompanied by a significant decrease of the interpenetration length between the BCP and RCP, thus suggesting that the interpenetration between grafted chains and block copolymer chains is hampered by the solvent. Hybrid particle-field molecular dynamics simulations of the analyzed system confirm the experimental observations and demonstrate a clear correlation between the interpenetration length and the amount of trapped solvent.

Effect of shell structure of Ti-immobilized metal ion affinity chromatography core-shell magnetic particles for phosphopeptide enrichment.

Magnetic materials in sample preparation for shotgun phosphoproteomics offer several advantages over conventional systems, as the enrichment can be achieved directly in solution, but they still suffer from some drawbacks, due to limited stability and selectivity, which is supposed to be affected by the hydrophilicity of the polymeric supports used for cation immobilization. The paper describes the development of an improved magnetic material with increased stability, thanks to a two-step covering of the magnetic core, for the enrichment of phosphopeptides in biological samples. Four materials were prepared featuring a polymeric shell with tunable hydrophilicity, obtained by "grafting from" polymerization of glycidyl methacrylate with 0-8.3% of polyethylene glycol methacrylate (PEGMA), the latter used to modulate the hydrophilicity of the material surface. Finally, the materials were functionalized with iminodiacetic acid for Ti4+ ion immobilization. The materials were analyzed for their composition by a combination of CHN elemental analysis and thermogravimetric analysis, also hyphenated to gas chromatography and mass spectrometric detection. Surface characteristics were evaluated by water contact angle measurements, scanning electron microscopy and energy dispersive X-ray spectrometry. These materials were applied to the enrichment of phosphopeptides from yeast protein digests. Peptides were identified by proteomics techniques using nano-high performance liquid chromatography coupled to mass spectrometry and bioinformatics. Qualitatively the peptides identified by the four systems were comparable, with 1606-1693 phosphopeptide identifications and a selectivity of 47-54% for all materials. The physico-chemical features of the identified peptides were also the same for the four materials. In particular, the grand average of hydropathy index values indicated that the enriched phosphopeptides were hydrophilic (ca. 90%), and only some co-enriched non-phosphorylated peptides were hydrophobic (21-28%), regardless of the material used for enrichment. Peptides had a pI ≤ 7, which indicated a well-known bias for acidic peptides binding, attributed to the interaction with the metal center itself. The results indicated that the enrichment of phosphopeptides and the co-enrichment of non-phosphorylated peptides is mainly driven by interactions with Ti4+ and does not depend on the amount of PEGMA chains in the polymer shell.

Functional polymeric nano/microparticles for surface adsorption and delivery of protein and DNA vaccines.

The use of particulate polymeric carriers holds great promise for the development of effective and affordable DNA and protein subunit vaccines. Rational development of such vaccine formulations requires a detailed understanding of their physico-chemical properties, cell-free and in vitro behaviour, in addition to particle uptake and processing mechanisms to antigen presenting cells capable of stimulating safe and effective immune responses. We here provide an overview on functional polymeric nano- and micro-particles designed for surface adsorption of proteins and DNA antigens currently under investigation for the formulation of new vaccines, including comments on their preparation method, antigen delivery strategy, cell-free and in vitro behaviour. In addition, we focus on their influence in activating antigen-specific humoral and/or cellular immune responses and on their potential for the development of new vaccines.

Control of Doping Level in Semiconductors via Self-Limited Grafting of Phosphorus End-Terminated Polymers.

An effective bottom-up technology for precisely controlling the amount of dopant atoms tethered on silicon substrates is presented. Polystyrene and poly(methyl methacrylate) polymers with narrow molecular weight distribution and end-terminated with a P-containing moiety were synthesized with different molar mass. The polymers were spin coated and subsequently end-grafted onto nondeglazed silicon substrates. P atoms were bonded to the surface during the grafting reaction, and their surface density was set by the polymer molar mass, according to the self-limiting nature of the "grafting to" reaction. Polymeric material was removed by O2 plasma hashing without affecting the tethered P-containing moieties on the surface. Repeated cycles of polymer grafting followed by plasma hashing led to a cumulative increase, at constant steps, in the dose of P atoms grafted to the silicon surface. P injection in the silicon substrate was promoted and precisely controlled by high-temperature thermal treatments. Sheet resistance measurements demonstrated effective doping of silicon substrate.

Hierarchical Order in Dewetted Block Copolymer Thin Films on Chemically Patterned Surfaces.

We investigated the dewetting process on flat and chemically patterned surfaces of ultrathin films (thickness between 2 and 15 nm) of a cylinder forming polystyrene- block-poly(methyl methacrylate) (PS- b-PMMA) spin coated on poly(styrene- r-methyl methacrylate) random copolymers (RCPs). When the PS- b-PMMA film dewets on a 2 nm-thick RCP layer, the ordering of the hexagonally packed PMMA cylinders in the dewetted structures extends over distances far exceeding the correlation length obtained in continuous block copolymer (BCP) films. As a result, micrometer-sized circular droplets featuring defectless single grains of self-assembled PS- b-PMMA with PMMA cylinders perpendicularly oriented with respect to the substrate are generated and randomly distributed on the substrate. Additionally, alignment of the droplets along micrometric lines was achieved by performing the dewetting process on large-scale chemically patterned stripes of 2 nm thick RCP films by laser lithography. By properly adjusting the periodicity of the chemical pattern, it was possible to tune and select the geometrical characteristics of the dewetted droplets in terms of maximum thickness, contact angle and diameter while maintaining the defectless single grain perpendicular cylinder morphology of the circular droplets.

Influence of the long-range ordering of gold-coated Si nanowires on SERS.

Controlling the location and the distribution of hot spots is a crucial aspect in the fabrication of surface-enhanced Raman spectroscopy (SERS) substrates for bio-analytical applications. The choice of a suitable method to tailor the dimensions and the position of plasmonic nanostructures becomes fundamental to provide SERS substrates with significant signal enhancement, homogeneity and reproducibility. In the present work, we studied the influence of the long-range ordering of different flexible gold-coated Si nanowires arrays on the SERS activity. The substrates are made by nanosphere lithography and metal-assisted chemical etching. The degree of order is quantitatively evaluated through the correlation length (ξ) as a function of the nanosphere spin-coating speed. Our findings showed a linear increase of the SERS signal for increasing values of ξ, coherently with a more ordered and dense distribution of hot spots on the surface. The substrate with the largest ξ of 1100 nm showed an enhancement factor of 2.6 · 103 and remarkable homogeneity over square-millimetres area. The variability of the signal across the substrate was also investigated by means of a 2D chemical imaging approach and a standard methodology for its practical calculation is proposed for a coherent comparison among the data reported in literature.

New Ti-IMAC magnetic polymeric nanoparticles for phosphopeptide enrichment from complex real samples.

The work describes the preparation of a new magnetic phase for batch enrichment of phosphopeptides. The material exploits the advantages of magnetic solid phase extraction and couples them with the most employed approach for phosphopeptide enrichment, i.e. Ti4+-IMAC. In order to immobilize Ti4+ ions on the surface of the magnetite nanoparticles, they were first covered by a silica shell and then modified to expose at the surface bromine containing groups. Glycidyl methacrylate was subsequently polymerized from these groups using the "grafting from" approach by the activator regenerated by electron transfer-atom transfer radical polymerization (ARGET-ATRP) technique. Finally, the glycidyl groups were reacted with iminodiacetic acid to functionalize the material with moieties suitable for coordination. The prepared material was extensively characterized and subsequently tested for enrichment of a bovine serum albumin mixture with casein to ascertain its potential. With positive results, the new magnetic polymeric material was further employed to set up an enrichment method on yeast protein digest based on shotgun proteomics. The sample to phase ratio was optimized and the best condition compared to a commercial TiO2 spin column. At the end of the comparison, the new material proved better and could enrich a larger total number of phosphopeptides with increased selectivity. All these conclusions and the test performed on a real complex sample within the final shotgun application further support the applicability of the new material in phosphopeptide analysis of real matrices.

Effect of Entrapped Solvent on the Evolution of Lateral Order in Self-Assembled P(S-r-MMA)/PS-b-PMMA Systems with Different Thicknesses.

Block copolymers (BCPs) are emerging as a cost-effective nanofabrication tool to complement conventional optical lithography because they self-assemble in highly ordered polymeric templates with well-defined sub-20-nm periodic features. In this context, cylinder-forming polystyrene-block-poly(methyl methacrylate) BCPs are revealed as an interesting material of choice because the orientation of the nanostructures with respect to the underlying substrate can be effectively controlled by a poly(styrene-random-methyl methacrylate) random copolymer (RCP) brush layer grafted to the substrate prior to BCP deposition. In this work, we investigate the self-assembly process and lateral order evolution in RCP + BCP systems consisting of cylinder-forming PS-b-PMMA (67 kg mol-1, PS fraction of ∼70%) films with thicknesses of 30, 70, 100, and 130 nm deposited on RCP brush layers having thicknesses ranging from 2 to 20 nm. The self-assembly process is promoted by a rapid thermal processing machine operating at 250 °C for 300 s. The level of lateral order is determined by measuring the correlation length (ξ) in the self-assembled BCP films. Moreover, the amount of solvent (Φ) retained in the RCP + BCP systems is measured as a function of the thicknesses of the RCP and BCP layers, respectively. In the 30-nm-thick BCP films, an increase in Φ as a function of the thickness of the RCP brush layer significantly affects the self-assembly kinetics and the final extent of the lateral order in the BCP films. Conversely, no significant variations of ξ are observed in the 70-, 100-, and 130-nm-thick BCP films with increasing Φ.