Direct Functionalization of Polysaccharide-Based Xylan Phenyl Carbonate Nanoparticles with Tumor Cell Specific Antibodies.

An efficient and easy-to-use approach is presented for obtaining biocompatible polysaccharide-based nanoparticles (NP) that can act as tumor-specific drug delivery agents. Two antibodies are directly immobilized onto reactive xylan phenyl carbonate (XPC) NP; namely Cetuximab (CTX) that binds to human epidermal growth factor receptor (EGFR) and Atezolizumab (ATZ) that binds to programmed death-ligand 1 (PD-L1). High coupling efficiency (up to 100 %) are achieved without any pre-activation and no aggregation occurs during antibody immobilization. By quartz crystal microbalance experiments with dissipation monitoring (QCM-D), flow cytometry assays, and confocal laser scanning microscopy imaging it is demonstrated that the functionalized XPC-NP specifically bind to cells carrying the corresponding antigens. Moreover, the NP retain the antibody specific bioactivities (growth inhibition for CTX and induction of T-cell cytotoxicity for ATZ).

Ultrathin Ambipolar Polyelectrolyte Capacitors Prepared via Layer-by-Layer Assembling.

Miniaturized solid state capacitors leveraging migration of unipolar ions in a single polyelectrolyte layer sandwiched between metal electrodes, namely, polyelectrolyte capacitors (PECs), have been recently reported with areal capacitance up to 100-200 nF mm-2 . Nonetheless, application of PECs in consumer and industrial electronics has been hindered so far by their small operational frequency range, up to a few kHz, due to the resistive behavior (phase angle >-45°) of PECs in the range kHz-to-MHz. Here, it is reported on multilayer polyelectrolyte capacitors (mPECs) that leverage as dielectric an ambipolar nanometer-thick (down to 10 nm) stack of anionic and cationic polyelectrolytes assembled layer-by-layer between metal electrodes to eliminate the resistive behavior at frequencies from kHz to MHz. This significantly extends the operational range of mPECs over PECs. mPECs with areal capacitance as high as 25 nF mm-2 at 20 Hz and full capacitive behavior from 100 mHz to 10 MHz are demonstrated using different assembling conditions and anionic/cationic polyelectrolyte pairs. The mPECs reliably operate over time for >300 million cycles, at different biasing voltages up to 3 V, and temperatures up to 80 °C, showing a reversible capacitive behavior without significant hysteresis. Application of mPECs in flexible electronics, also operating at high frequency, is envisaged.

A multi-pulse ultrasound technique for imaging of thick-shelled microbubbles demonstrated in vitro and in vivo.

Contrast enhanced ultrasound is a powerful diagnostic tool and ultrasound contrast media are based on microbubbles (MBs). The use of MBs in drug delivery applications and molecular imaging is a relatively new field of research which has gained significant interest during the last decade. MBs available for clinical use are fragile with short circulation half-lives due to the use of a thin encapsulating shell for stabilization of the gas core. Thick-shelled MBs can have improved circulation half-lives, incorporate larger amounts of drugs for enhanced drug delivery or facilitate targeting for use in molecular ultrasound imaging. However, methods for robust imaging of thick-shelled MBs are currently not available. We propose a simple multi-pulse imaging technique which is able to visualize thick-shelled polymeric MBs with a superior contrast-to-tissue ratio (CTR) compared to commercially available harmonic techniques. The method is implemented on a high-end ultrasound scanner and in-vitro imaging in a tissue mimicking flow phantom results in a CTR of up to 23 dB. A proof-of-concept study of molecular ultrasound imaging in a soft tissue inflammation model in rabbit is then presented where the new imaging technique showed an enhanced accumulation of targeted MBs in the inflamed tissue region compared to non-targeted MBs and a mean CTR of 13.3 dB for stationary MBs. The presence of fluorescently labelled MBs was verified by confocal microscopy imaging of tissue sections post-mortem.

Bioresorbable Nanostructured Chemical Sensor for Monitoring of pH Level In Vivo.

Here, the authors report on the manufacturing and in vivo assessment of a bioresorbable nanostructured pH sensor. The sensor consists of a micrometer-thick porous silica membrane conformably coated layer-by-layer with a nanometer-thick multilayer stack of two polyelectrolytes labeled with a pH-insensitive fluorophore. The sensor fluorescence changes linearly with the pH value in the range 4 to 7.5 upon swelling/shrinking of the polymer multilayer and enables performing real-time measurements of the pH level with high stability, reproducibility, and accuracy, over 100 h of continuous operation. In vivo studies carried out implanting the sensor in the subcutis on the back of mice confirm real-time monitoring of the local pH level through skin. Full degradation of the pH sensor occurs in one week from implant in the animal model, and its biocompatibility after 2 months is confirmed by histological and fluorescence analyses. The proposed approach can be extended to the detection of other (bio)markers in vivo by engineering the functionality of one (at least) of the polyelectrolytes with suitable receptors, thus paving the way to implantable bioresorbable chemical sensors.

Operation of Hybrid Membranes for the Removal of Pharmaceuticals and Pollutants from Water and Wastewater.

Hybrid ceramic membranes (i.e., membranes with a layer-by-layer (LbL) coating) are an emerging technology to remove diverse kinds of micropollutants from water. Hybrid ceramic membranes were tested under laboratory conditions as single-channel (filter area = 0.00754 m2) and multi-channel (0.35 m2) variants for the removal of pharmaceuticals (sulfamethoxazole, diclofenac, clofibric acid, and ibuprofen) and typical wastewater pollutants (i.e., COD, TOC, PO4-P, and TN) from drinking water and treated wastewater. The tests were conducted with two low transmembrane pressures (TMP) of 2 and 4 bar and constant temperatures and flow velocities, which showed rejections above 80% for all the tested pharmaceuticals as well for organic pollutants and phosphorous in the treated wastewater. Tests regarding sufficient cleaning regimes also showed that the LbL coating is stable and resistant to pHs between 2 and 10 with the use of typical cleaning agents (citric acid and NaOH) but not to higher pHs, a commercially available enzymatic solution, or backwashing. The hybrid membranes can contribute to the advanced treatment of water and wastewater with low operational costs, and their application at a larger scale is viable. However, the cleaning of the membranes must be further investigated to assure the stability and durability of the LbL coating.

Electrospun PCL Scaffolds as Drug Carrier for Corneal Wound Dressing Using Layer-by-Layer Coating of Hyaluronic Acid and Heparin.

Due to its ability to reduce scarring and inflammation, human amniotic membrane is a widely used graft for wound dressings after corneal surgery. To overcome donor dependency and biological variances in the donor tissue, artificial nanofibrous grafts acting as drug carrier systems are promising substitutes. Electrospun nanofibrous scaffolds seem to be an appropriate approach as they offer the properties of permeable scaffolds with a high specific surface, the latter one depending on the fiber diameter. Electrospun scaffolds with fiber diameter of 35 nm, 113 nm, 167 nm and 549 nm were manufactured and coated by the layer-by-layer (LbL) technology with either hyaluronic acid or heparin for enhanced regeneration of corneal tissue after surgery. Studies on drug loading capacity and release kinetics defined a lower limit for nanofibrous scaffolds for effective drug loading. Additionally, scaffold characteristics and resulting mechanical properties from the application-oriented characterization of suture pullout from suture retention tests were examined. Finally, scaffolds consisting of nanofibers with a mean fiber diameter of 113 nm were identified as the best-performing scaffolds, concerning drug loading efficiency and resistance against suture pullout.

Glycopolymer Based LbL Multilayer Thin Films with Embedded Liposomes.

Layer-by-layer (LbL) self-assembly emerged as an efficient technique for fabricating coating systems for, e.g., drug delivery systems with great versatility and control. In this work, protecting group free and aqueous-based syntheses of bioinspired glycopolymer electrolytes aredescribed. Thin films of the glycopolymers are fabricated by LbL self-assembly and function as scaffolds for liposomes, which potentially can encapsulate active substances. The adsorbed mass, pH stability, and integrity of glycopolymer coatings as well as the embedded liposomes are investigated via whispering gallery mode (WGM) technology and quartz crystal microbalance with dissipation (QCM-D) monitoring , which enable label-free characterization. Glycopolymer thin films, with and without liposomes, are stable in the physiological pH range. QCM-D measurements verify the integrity of lipid vesicles. Thus, the fabrication of glycopolymer-based surface coatings with embedded and intact liposomes is presented.

Hybrid Ceramic Membranes for the Removal of Pharmaceuticals from Aqueous Solutions.

Layer-by-Layer (LbL) technology was used to coat alumina ceramic membranes with nanosized polyelectrolyte films. The polyelectrolyte chains form a network with nanopores on the ceramic surface and promote the rejection of small molecules such as pharmaceuticals, salts and industrial contaminants, which can otherwise not be eliminated using standard ultrafiltration methods. The properties and performance of newly developed hybrid membranes are in the focus of this investigation. The homogeneity of the applied coating layer was investigated by confocal fluorescence microscopy and scanning transmission electron microscopy (STEM). Properties such as permeability, bubble point, pore size distribution and Zeta potential were determined for both pristine and LbL coated membranes using various laboratory tests. Subsequently, a thorough comparison was drawn. The charging behavior at solid-liquid interface was characterized using streaming potential techniques. The retention potential was monitored by subjecting widely used pharmaceuticals such as diclofenac, ibuprofen and sulfamethoxazol. The results prove a successful elimination of pharmaceutical contaminants, up to 84% from drinking water, by applying a combination of polyelectrolyte multilayers and ceramic membranes.

The impact of skin massage frequency on the intrafollicular transport of silica nanoparticles: Validation of the ratchet effect on an ex vivo porcine skin model.

The human hair follicle (HF) represents a promising drug delivery target as an anatomical entity by itself, but also as a gateway enabling dermal or systemic bioavailability of active cosmetic and pharmaceutical ingredients. Due to its morphological characteristics, the HF provides a mechanically driven transport process of nanoparticles (NPs) when external forces are applied. This mechanism was presented as the so-called ratchet effect within the framework of an in silico study published recently. To investigate the influence of massage frequency on the penetration depth of NPs, and, by this, to validate the results obtained in silico, we implemented a corresponding application protocol on an ex vivo porcine skin model. In this connection, we compared three different skin massage frequencies (4.2 Hz, 50 Hz, 100 Hz) for the topical application of cyanine 5-labeled silica NPs (Cy5-SNPs). To elucidate the interplay of frequency and particle size, we furthermore applied Cy5-SNPs of three different diameters (300 nm, 676 nm, 1000 nm). Confocal laser scanning microscopy was utilized to investigate the follicular penetration depth of Cy5-SNPs on cryohistological slices. By this, we could demonstrate that the massage frequency and the follicular penetration depth exhibit an inverse relation pattern. Thus, the highest follicular penetration depth was observed within the 4.2 Hz group, while the lowest follicular penetration depth was found within the 100 Hz group for each Cy5-SNP size category. Additionally, we found that 676 nm Cy5-SNPs penetrated significantly deeper into HFs than 300 nm Cy5-SNPs and 1000 nm Cy5-SNPs, respectively. Summarizing, our results show that a low massage frequency including a dominant radial direction component leads to deeper follicular penetration depths of NPs than automated 3D-oscillation massage at 50 Hz or 100 Hz. Thus, our findings are in line with recent in silico results. Regarding translational purposes, our results are of high interest, since a massage executed at 250BPM (4.2 Hz) is within a realizable range for manual application, e.g. for the implementation into clinical routines or the domestic use of drugs or cosmetics. Furthermore, the application of different massage frequencies offers the opportunity of patho-specific targeting as different anatomical parts of the HF can be reached.

Performance of Layer-by-Layer-Modified Multibore® Ultrafiltration Capillary Membranes for Salt Retention and Removal of Antibiotic Resistance Genes.

Polyether sulfone Multibore® ultrafiltration membranes were modified using polyelectrolyte multilayers via the layer-by-layer (LbL) technique in order to increase their rejection capabilities towards salts and antibiotic resistance genes. The modified capillary membranes were characterized to exhibit a molecular weight cut-off (at 90% rejection) of 384 Da. The zeta-potential at pH 7 was -40 mV. Laboratory tests using single-fiber modified membrane modules were performed to evaluate the removal of antibiotic resistance genes; the LbL-coated membranes were able to completely retain DNA fragments from 90 to 1500 nt in length. Furthermore, the pure water permeability and the retention of single inorganic salts, MgSO4, CaCl2 and NaCl, were measured using a mini-plant testing unit. The modified membranes had a retention of 80% toward MgSO4 and CaCl2 salts, and 23% in case of NaCl. The modified membranes were also found to be stable against mechanical backwashing (up to 80 LMH) and chemical regeneration (in acidic conditions and basic/oxidizing conditions).

Determination of the pH Gradient in Hair Follicles of Human Volunteers Using pH-Sensitive Melamine Formaldehyde-Pyranine Nile Blue Microparticles.

Nanoparticles can be applied to the hair follicles, which can serve as reservoirs for triggered drug release. A valid measurement method for the determination of the pH within the hair follicle in vivo has not been shown yet. Here, melamine formaldehyde particles up to 9 µm in size were applied on 40 freshly plucked scalp hairs of eight individuals to determine the pH along the hair shaft down to the root area of the hair. For fluorescent pH indicators, pyranine and Nile blue were incorporated into the particles. Measurements were conducted using confocal laser scanning microscopy. A pH decay gradient could be found from the hair sheath towards the external hair shaft (p = 0.012) with pH values at the hair sheath of 6.63 ± 0.09, at the hair sheath end at 6.33 ± 0.11, and at the external hair shaft at 6.17 ± 0.09 (mean ± SE). The pH difference between the hair sheath end and the external hair shaft was found to be significant (p = 0.036). The results might be comparable with the pH within the hair follicle in vivo indicating a pH increase towards the hair root.

Molecular Imaging of a New Multimodal Microbubble for Adhesion Molecule Targeting.

INTRODUCTION: Inflammation is an important risk-associated component of many diseases and can be diagnosed by molecular imaging of specific molecules. The aim of this study was to evaluate the possibility of targeting adhesion molecules on inflammation-activated endothelial cells and macrophages using an innovative multimodal polyvinyl alcohol-based microbubble (MB) contrast agent developed for diagnostic use in ultrasound, magnetic resonance, and nuclear imaging. METHODS: We assessed the binding efficiency of antibody-conjugated multimodal contrast to inflamed murine or human endothelial cells (ECs), and to peritoneal macrophages isolated from rats with peritonitis, utilizing the fluorescence characteristics of the MBs. Single-photon emission tomography (SPECT) was used to illustrate 99mTc-labeled MB targeting and distribution in an experimental in vivo model of inflammation. RESULTS: Flow cytometry and confocal microscopy showed that binding of antibody-targeted MBs to the adhesion molecules ICAM-1, VCAM-1, or E-selectin, expressed on cytokine-stimulated ECs, was up to sixfold higher for human and 12-fold higher for mouse ECs, compared with that of non-targeted MBs. Under flow conditions, both VCAM-1- and E-selectin-targeted MBs adhered more firmly to stimulated human ECs than to untreated cells, while VCAM-1-targeted MBs adhered best to stimulated murine ECs. SPECT imaging showed an approximate doubling of signal intensity from the abdomen of rats with peritonitis, compared with healthy controls, after injection of anti-ICAM-1-MBs. CONCLUSIONS: This novel multilayer contrast agent can specifically target adhesion molecules expressed as a result of inflammatory stimuli in vitro, and has potential for use in disease-specific multimodal diagnostics in vivo using antibodies against targets of interest.

Decoration of Porous Silicon with Gold Nanoparticles via Layer-by-Layer Nanoassembly for Interferometric and Hybrid Photonic/Plasmonic (Bio)sensing.

Gold nanoparticle layers (AuNPLs) enable the coupling of morphological, optical, and electrical properties of gold nanoparticles (AuNPs) with tailored and specific surface topography, making them exploitable in many bioapplications (e.g., biosensing, drug delivery, and photothermal therapy). Herein, we report the formation of AuNPLs on porous silicon (PSi) interferometers and distributed Bragg reflectors (DBRs) for (bio)sensing applications via layer-by-layer (LbL) nanoassembling of a positively charged polyelectrolyte, namely, poly(allylamine hydrochloride) (PAH), and negatively charged citrate-capped AuNPs. Decoration of PSi interferometers with AuNPLs enhances the Fabry-Pérot fringe contrast due to increased surface reflectivity, resulting in an augmented sensitivity for both bulk and surface refractive index sensing, namely, about 4.5-fold using NaCl aqueous solutions to infiltrate the pores and 2.6-fold for unspecific bovine serum albumin (BSA) adsorption on the pore surface, respectively. Sensitivity enhancing, about 2.5-fold, is also confirmed for affinity and selective biosensing of streptavidin using a biotinylated polymer, namely, negatively charged poly(methacrylic acid) (b-PMAA). Further, decoration of PSi DBR with AuNPLs envisages building up a hybrid photonic/plasmonic optical sensing platform. Both photonic (DBR stop-band) and plasmonic (localized surface plasmon resonance, LSPR) peaks of the hybrid structure are sensitive to changes of bulk (using glucose aqueous solutions) and surface (due to BSA unspecific adsorption) refractive index. To the best of our knowledge, this is the first report about the formation of AuNPLs via LbL nanoassembly on PSi for (i) the enhancing of the interferometric performance in (bio)sensing applications and (ii) the building up of hybrid photonic/plasmonic platforms for sensing and perspective biosensing applications.

Polymer Fibers Covered by Soft Multilayered Films for Sensing Applications in Composite Materials.

This paper presents the possibility of applying a soft polymer coating by means of a layer-by-layer (LbL) technique to highly birefringent polymer optical fibers designed for laminating in composite materials. In contrast to optical fibers made of pure silica glass, polymer optical fibers are manufactured without a soft polymer coating. In typical sensor applications, the absence of a buffer coating is an advantage. However, highly birefringent polymer optical fibers laminated in a composite material are much more sensitive to temperature changes than polymer optical fibers in a free space as a result of the thermal expansion of the composite material. To prevent this, we have covered highly birefringent polymer optical fibers with a soft polymer coating of different thickness and measured the temperature sensitivity of each solution. The results obtained show that the undesired temperature sensitivity of the laminated optical fiber decreases as the thickness of the coating layer increases.

The Future of Layer-by-Layer Assembly: A Tribute to ACS Nano Associate Editor Helmuth Möhwald.

Layer-by-layer (LbL) assembly is a widely used tool for engineering materials and coatings. In this Perspective, dedicated to the memory of ACS Nano associate editor Prof. Dr. Helmuth Möhwald, we discuss the developments and applications that are to come in LbL assembly, focusing on coatings, bulk materials, membranes, nanocomposites, and delivery vehicles.

Controlled release of nisin from Neusilin particles to enhance food safety of sour curd cheese.

Nisin is frequently added as food additive to soft cheese to increase food safety against foodborne pathogens like Listeria monocytogenes. The goal of this study was the extension of the antimicrobial activity of nisin in sour curd cheese (SCC) by self-releasing adsorbed nisin from Neusilin UFL2 over production-based pH shift. First, the antimicrobial activity of nisin adsorbed to Neusilin UFL2 (UFL2-N) and free nisin was investigated in BHI broth at a pH range from 7.5 to 4.5 for each of six L. monocytogenes field isolates. UFL2-N showed similar minimal inhibition concentration to L. monocytogenes over time as free nisin. Distribution of nebulized, fluorescence-labelled UFL2 was homogenous on SCC surface. Thereafter, SCC surface was inoculated with L. monocytogenes and 0.004, 0.013, 0.026, and 0.132 mg mL-1 UFL2-N or free nisin. In SCC, L. monocytogenes was below quantification limit at 0.132 mg mL-1 UFL2-N or free nisin after 2 days of ripening. Collectively, UFL2-N enabled a slow release and antilisterial activity in vitro as well as in cheese manufacturing.

Layer-by-layer biofunctionalization of nanostructured porous silicon for high-sensitivity and high-selectivity label-free affinity biosensing.

Nanostructured materials premise to revolutionize the label-free biosensing of analytes for clinical applications, leveraging the deeper interaction between materials and analytes with comparable size. However, when the characteristic dimension of the materials reduces to the nanoscale, the surface functionalization for the binding of bioreceptors becomes a complex issue that can affect the performance of label-free biosensors. Here we report on an effective and robust route for surface biofunctionalization of nanostructured materials based on the layer-by-layer (LbL) electrostatic nano-assembly of oppositely-charged polyelectrolytes, which are engineered with bioreceptors to enable label-free detection of target analytes. LbL biofunctionalization is demonstrated using nanostructured porous silicon (PSi) interferometers for affinity detection of streptavidin in saliva, through LbL nano-assembly of a bi-layer of positively-charged poly(allylamine hydrochloride) (PAH) and negatively-charged biotinylated poly(methacrylic acid) (b-PMAA). High sensitivity in streptavidin detection is achieved, with high selectivity and stability, down to a detection limit of 600 fM.

New application of depth filters for the immobilization of Candida antarctica lipase B.

The objective of this study was to use for the first time depth filters, which are usually intended for clarification of cell culture broth, as a direct immobilization support/matrix for industrially relevant enzymes. With this method, it is not only possible to immobilize pure enzymes; it can be also used for capturing recombinant enzymes directly out of culture supernatant. Therefore, the depth filters were coated with different anionic and cationic polymer layers by Layer-by-Layer (LbL) technology. The immobilization behavior of the model enzyme Candida antarctica lipase B (CalB) was examined. Optimal conditions for lipase immobilization were found for anionic surfaces with Poly (allylamin hydrochlorid) (PAH)/Poly (sodium-4-styrene sulfonate) (PSS) coating in 20 mM acetate buffer pH 4. Stability studies showed that immobilized CalB is 1.7-fold more stable when storage is carried out in buffer at 4 °C, compared to storage in buffer at room temperature or storage after drying at 30 °C for 24 h. The calculated half-life period is 108 days until half of the activity was lost. Furthermore, the possibility of direct capture of the CalB either from sonicated culture broth (Escherichia coli) or from cell-free supernatant was tested. Filter blocking prevented the immobilization of lipase from sonicated culture broth, but immobilization from cell-free supernatant could be performed successfully at moderate biomass content (OD600 = 7.0).

Biological in situ characterization of polymeric microbubble contrast agents.

Polymeric microbubbles (MBs) are gas filled particles composed of a thin stabilized polymer shell that have been recently developed as valid contrast agents for the combined use of ultrasonography (US), magnetic resonance imaging (MRI) and single photon emission computer tomography (SPECT) imaging. Due to their buoyancy, the commonly available approaches to study their behaviour in complex media are not easily applicable and their use in modern medicine requires such behaviour to be fully elucidated. Here we have used for the first time flow cytometry as a new high throughput approach that allows characterisation of the MB dispersion, prior to and after exposure in different biological media and we have additionally developed a method that allows characterisation of the strongly bound proteins adsorbed on the MBs, to fully predict their biological behaviour in biological milieu.

A medical-toxicological view of tattooing.

Long perceived as a form of exotic self-expression in some social fringe groups, tattoos have left their maverick image behind and become mainstream, particularly for young people. Historically, tattoo-related health and safety regulations have focused on rules of hygiene and prevention of infections. Meanwhile, the increasing popularity of tattooing has led to the development of many new colours, allowing tattoos to be more spectacular than ever before. However, little is known about the toxicological risks of the ingredients used. For risk assessment, safe intradermal application of these pigments needs data for toxicity and biokinetics and increased knowledge about the removal of tattoos. Other concerns are the potential for phototoxicity, substance migration, and the possible metabolic conversion of tattoo ink ingredients into toxic substances. Similar considerations apply to cleavage products that are formed during laser-assisted tattoo removal. In this Review, we summarise the issues of concern, putting them into context, and provide perspectives for the assessment of the acute and chronic health effects associated with tattooing.