Circulating Tumor Cells Adhesion: Application in Biosensors.

The early and non-invasive diagnosis of tumor diseases has been widely investigated by the scientific community focusing on the development of sensors/biomarkers that act as a way of recognizing the adhesion of circulating tumor cells (CTCs). As a challenge in this area, strategies for CTCs capture and enrichment currently require improvements in the sensors/biomarker's selectivity. This can be achieved by understanding the biological recognition factors for different cancer cell lines and also by understanding the interaction between surface parameters and the affinity between macromolecules and the cell surface. To overcome some of these concerns, electrochemical sensors have been used as precise, fast-response, and low-cost transduction platforms for application in cytosensors. Additionally, distinct materials, geometries, and technologies have been investigated to improve the sensitivity and specificity properties of the support electrode that will transform biochemical events into electrical signals. This review identifies novel approaches regarding the application of different specific biomarkers (CD44, Integrins, and EpCAm) for capturing CTCs. These biomarkers can be applied in electrochemical biosensors as a cytodetection strategy for diagnosis of cancerous diseases.

Automated, High-Throughput Screening of Hybrid Elastin-like Polypeptide/Polysaccharide Multilayer Film Deposition.

The self-assembled layer-by-layer technique has attracted a great deal of attention as a method for engineering bio-functional surfaces under mild chemical conditions. The production of multilayer films, starting from newly designed building blocks, may be laborious, considering the inherent limitations for anticipating how minimal changes in the macromolecular composition may impact both film deposition and performance. This paper presents an automated, high-throughput approach to depositing polyelectrolyte multilayers (PEMs) in multiwell plates, enabling the screening of nearly 100 film formulations in the same process. This high-throughput layer-by-layer (HT-LbL) method runs in an affordable, fully commercial platform using Python-coded routines that can be easily adapted for the materials science lab settings. The HT-LbL system was validated by investigating the deposition of polysaccharide-based films in multiwell plates, probing the absorbance signal of ionically stained polyelectrolyte multilayers (PEMs) prepared in one single batch. The HT-LbL method was also used to investigate the deposition of PEMs with a small library of genetically engineered elastin-like polypeptides (ELPs) with different levels of ionizable and hydrophobic amino acid residues. The deposition of ELP/chitosan films was assessed based on the signal of fluorescently labeled species (chitosan or ELP-mCherry), demonstrating that both electrostatic and hydrophobic residues are essential for film buildup. The growth and surface properties of ELP-mCherry/chitosan films also seemed susceptible to the assembly pH, forming a higher film growth and a rougher and more hydrophobic surface for both polyelectrolytes deposited under a low ionization degree. Overall, this study illustrates the challenge of predicting the growth and properties of multilayer films and how the HT-LbL can accelerate the development of multilayer films that demand high levels of testing and optimization.

Three-Dimensional Printing and Its Potential to Develop Sensors for Cancer with Improved Performance.

Cancer is the second leading cause of death globally and early diagnosis is the best strategy to reduce mortality risk. Biosensors to detect cancer biomarkers are based on various principles of detection, including electrochemical, optical, electrical, and mechanical measurements. Despite the advances in the identification of biomarkers and the conventional 2D manufacturing processes, detection methods for cancers still require improvements in terms of selectivity and sensitivity, especially for point-of-care diagnosis. Three-dimensional printing may offer the features to produce complex geometries in the design of high-precision, low-cost sensors. Three-dimensional printing, also known as additive manufacturing, allows for the production of sensitive, user-friendly, and semi-automated sensors, whose composition, geometry, and functionality can be controlled. This paper reviews the recent use of 3D printing in biosensors for cancer diagnosis, highlighting the main advantages and advances achieved with this technology. Additionally, the challenges in 3D printing technology for the mass production of high-performance biosensors for cancer diagnosis are addressed.

Antimicrobial peptides and their potential application in antiviral coating agents.

Coronavirus pandemic has evidenced the importance of creating bioactive materials to mitigate viral infections, especially in healthcare settings and public places. Advances in antiviral coatings have led to materials with impressive antiviral performance; however, their application may face health and environmental challenges. Bio-inspired antimicrobial peptides (AMPs) are suitable building blocks for antimicrobial coatings due to their versatile design, scalability, and environmentally friendly features. This review presents the advances and opportunities on the AMPs to create virucidal coatings. The review first describes the fundamental characteristics of peptide structure and synthesis, highlighting the recent findings on AMPs and the role of peptide structure (α-helix, ß-sheet, random, and cyclic peptides) on the virucidal mechanism. The following section presents the advances in AMPs coating on medical devices with a detailed description of the materials coated and the targeted pathogens. The use of peptides in vaccine formulations is also reported, emphasizing the molecular interaction of peptides with different viruses and the current clinical stage of each formulation. The role of several materials (metallic particles, inorganic materials, and synthetic polymers) in the design of antiviral coatings is also presented, discussing the advantages and the drawbacks of each material. The final section offers future directions and opportunities for using AMPs on antiviral coatings to prevent viral outbreaks.

Amino acid-functionalized chitosan beads for in vitro copper ions uptake in the presence of histidine.

One of the hallmarks of Alzheimer's Disease (AD) is the anomalous binding involving amyloid-ß (Aß) peptide and metal ions, such as copper, formed through histidine (His) residues. Herein, adsorption experiments were performed to test the in vitro ability of chitosan to uptake copper ions in the presence of histidine. The characterization of the beads was assessed before and after the adsorption process by scanning electron microscope, X-ray diffraction and Fourier-transform infrared spectroscopy. Amino acid functionalization of chitosan-based beads promoted an increase in the copper ions adsorption capacity (2.47 mmol of Cu(II)/gram of adsorbent). Nevertheless, depending on the order of addition of histidine to the system, different adsorption behaviors were observed. The kinetics showed that, once the Cu(II)-His bond was established, functionalized beads were less efficient to capture Cu(II), which promoted a decrease in the overall adsorption capacity. However, when chitosan and histidine were simultaneously added to the Cu(II) solution, there was no decrease in adsorption capacity. To sum up, chitosan-based materials are an interesting model to provide a better understanding on the biomolecules­copper interactions that occur in AD, as well as a possible chelating agent that can interfere in the bonds between Aß residues and copper ions.

Probing axial metal distribution on biopolymer-based layer-by-layer films for antimicrobial use.

This study presents the axial molar composition of polysaccharide-based polyelectrolyte multilayer (PEM) films loaded with silver ions for antimicrobial applications. Individual polymers (chitosan, hyaluronan or alginate) and silver composition were determined using X-Ray Photoelectron Spectroscopy coupled with C60+ cluster ion sputtering technique, while the influence of silver loading on film topography was assessed using Atomic Force Microscopy. Despite the use of the layer-by-layer approach for film assembly, these PEM films present a non-stratified, nanoblend-like, polymer composition, with a nearly uniform metal distribution over the axial direction. Results also show surface antimicrobial activity towards Staphylococcus aureus bacteria and Candida albicans fungi over 20 h for hyaluronan/chitosan PEM, which is associated with its higher silver loading capacity. The interplay of bulk film composition and surface properties may provide valuable insights for engineering advanced materials with controlled spatio-temporal behavior.

Interplay of the Assembly Conditions on Drug Transport Mechanisms in Polyelectrolyte Multilayer Films.

The layer-by-layer film deposition is a suitable strategy for the design and functionalization of drug carriers with superior performance, which still lacks information describing the influence of assembly conditions on the mechanisms governing the drug release process. Herein, traditional poly(acrylic acid)/poly(allylamine) polyelectrolyte multilayers (PEM) were explored as a platform to study the influence of the assembly conditions such as pH, drug loading method, and capping layer deposition on the mechanisms that control the release of calcein, the chosen model drug, from PEM. Films with 20-40 bilayers were assembled at pH 4.5 or 8.8, and the drug loading process was carried out during- or post-film assembly. Release data were fitted to three release models, namely, Higuchi, Ritger-Peppas, and Berens-Hopfenberg, to investigate the mechanism governing the drug transport, such as the apparent diffusion and the relaxation time. The postassembly drug loading method leads to a higher drug loading capacity than the during-assembly method, attributed to the washing out of calcein during film assembly steps in the latter method. Higuchi's and Ritger-Peppas' model analyses indicate that the release kinetic constant increased with the number of bilayers for the postassembly method. The opposite trend is observed for the during-assembly method. The Berens-Hopfenberg release model enabled the decoupling of each drug transport mechanism's contribution, indicating the increase of the diffusion contribution with the number of bilayers for the postassembly method at pH 4.5 and the increase of the polymer relaxation contribution for the during-assembly method at pH 8.8. Deborah's number, which represents the ratio of the polymer relaxation time to the diffusion time, follows the trends observed for the relaxation contribution for the conditions investigated. The deposition of the capping phospholipid layer over the payload also favored the polymer relaxation contribution in the drug release, featuring new strategies to investigate the drug release in PEM.

Tracking Sulfonated Polystyrene Diffusion in a Chitosan/Carboxymethyl Cellulose Layer-by-Layer Film: Exploring the Internal Architecture of Nanocoatings.

Since chitosan presents the ability to interact with a wide range of molecules, it has been one of the most popular natural polymers for the construction of layer-by-layer thin films. In this study, depth-profiling X-ray photoelectron spectroscopy (XPS) was employed to track the diffusion of sulfonated polystyrene (SPS) in carboxymethyl cellulose/chitosan (CMC/Chi) multilayers. Our findings suggest that the CMC/Chi film does not constitute an electrostatic barrier sufficient to block diffusion of SPS, and that diffusion can be controlled by adjusting the diffusion time and the molecular weight of the polymers that compose the CMC/Chi system. In addition to monitoring the diffusion, it was also possible to observe a process of preferential interaction between Chi and SPS. Thus, the nitrogen N 1s peak, due to functional groups found exclusively in chitosan chains, was the key factor to identifying the molecular interactions involving chitosan and the different polyanions. Accordingly, the presence of a strong polyanion such as SPS shifts the N 1s peak to a higher level of binding energy. Such results highlight that understanding the fundamentals of polymer interactions is a major step to fine-tuning the internal architecture of LbL structures for specific applications (e.g., drug release).

Copper Ion Uptake by Chitosan in the Presence of Amyloid-ß and Histidine.

Alzheimer's disease (AD) is related to the anomalous binding that occurs between amyloid-ß peptide (Aß) and copper ion, through imidazole ring of histidine (His), as stated in the literature. It is also known that high-affinity metal ion chelators can be pharmacologically used as a possible therapeutic approach. In this work, we tested the ability "in vitro" of chitosan (Chi) to reduce Aß aggregation and Thioflavin T binding assay indicated that chitosan has affinity for Aß and interferes in its aggregation. We also tested the ability of Chi to uptake copper ions in the presence of Aß or His. Equilibrium data reveals that chitosan acted as an effective chelating agent competing with Aß and histidine for copper binding. The addition of histidine or Aß in the system promotes an unfolding of chitosan chains, as verified by small-angle X-ray scattering. Extended X-ray absorption fine structure and XPS spectra show that new copper interactions with groups containing nitrogen in the presence of histidine may occur. These results can help understanding fundamental chemical interactions among species detected in AD and biopolymers, opening up possibilities for new treatment approaches for this disease.

Silk fibroin/nanohydroxyapatite hydrogels for promoted bioactivity and osteoblastic proliferation and differentiation of human bone marrow stromal cells.

Silk fibroin (SF) is a natural, biocompatible, and biodegradable polymer having a great potential for the successful regeneration of damaged bone tissue. In the present work, nanohydroxyapatite (nanoHA) was incorporated into SF polymer to form a bioactive composite hydrogel for applications as bone implants. The degradation and bioactive properties of SF/nanoHA composite hydrogels were evaluated. Additionally, biological investigations of human bone marrow stromal cells (hBMSCs) viability, proliferation and differentiation to the osteoblastic phenotype were conducted. The incorporation of nanoHA in SF polymer matrices improved the bioactivity of the hydrogels. The biological results highlighted that the SF/nanoHA composite hydrogels are suitable for hBMSCs attachment and proliferation, while a test for alkaline phosphatase (ALP) and bone morphogenetic protein 2 (BMP-2) expression suggested osteoblast differentiation. Additionally, a cell staining method for ALP allowed to observe cell infiltration with active production of ALP by the infiltrated cells, paving the way to use the proposed composite hydrogel for bone tissue regeneration.

Investigation of the Internal Chemical Composition of Chitosan-Based LbL Films by Depth-Profiling X-ray Photoelectron Spectroscopy (XPS) Analysis.

Chitosan-based thin films were assembled using the layer-by-layer technique, and the axial composition was accessed using X-ray photoelectron spectroscopy with depth profiling. Chitosan (CHI) samples possessing different degrees of acetylation ([Formula: see text]) and molecular weight ([Formula: see text]) produced via the ultrasound-assisted deacetylation reaction were used in this study along with two different polyanions, namely, sodium polystyrenesulfonate (PSS) and carboxymethylcellulose (CMC). When chitosan, a positively charged polymer in aqueous acid medium, was combined with a strong polyanion (PSS), the total positive charge of chitosan, directly related to its [Formula: see text], was the key factor affecting the film formation. However, for CMC/CHI films, the pH of the medium and [Formula: see text] of chitosan strongly affected the film structure and composition. Consequently, the structure and the axial composition of chitosan-based films can be finely adjusted by choosing the polyanion and defining the chitosan to be used according to its DA and [Formula: see text] for the desired application, as demonstrated by the antibacterial tests.

Roughness dynamic in surface growth: Layer-by-layer thin films of carboxymethyl cellulose/chitosan for biomedical applications.

Surfaces are responsible for important interactions of biomaterials since they create the interface with the biological environment and affect the response that the body will have to the material. Surface roughness and morphology have great impact on the material performance, affecting cell, bacterial, and biomolecular adhesion. Thin films of chitosan and carboxymethyl cellulose were produced by layer-by-layer deposition at different pH values and had their surface growth process studied throughout roughness measurements. Both polymers are nontoxic and biocompatible to the human biological system, with biomedical applications from tissue engineering to drug delivery. Growth exponents are presented, and it is suggested that fractal-based growth models are suitable for describing surface evolution and morphology of carboxymethyl cellulose/chitosan layer-by-layer thin film growth during deposition, primarily nonlinear models.

Phase Behaviour and Miscibility Studies of Collagen/Silk Fibroin Macromolecular System in Dilute Solutions and Solid State.

Miscibility is an important issue in biopolymer blends for analysis of the behavior of polymer pairs through the detection of phase separation and improvement of the mechanical and physical properties of the blend. This study presents the formulation of a stable and one-phase mixture of collagen and regenerated silk fibroin (RSF), with the highest miscibility ratio between these two macromolecules, through inducing electrostatic interactions, using salt ions. For this aim, a ternary phase diagram was experimentally built for the mixtures, based on observations of phase behavior of blend solutions with various ratios. The miscibility behavior of the blend solutions in the miscible zones of the phase diagram was confirmed quantitatively by viscosimetric measurements. Assessing the effects of biopolymer mixing ratio and salt ions, before and after dialysis of blend solutions, revealed the importance of ion-specific interactions in the formation of coacervate-based materials containing collagen and RSF blends that can be used in pharmaceutical, drug delivery, and biomedical applications. Moreover, the conformational change of silk fibroin from random coil to beta sheet, in solution and in the final solid films, was detected by circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR), respectively. Scanning electron microscopy (SEM) exhibited alterations of surface morphology for the biocomposite films with different ratios. Surface contact angle measurement illustrated different hydrophobic properties for the blended film surfaces. Differential scanning calorimetry (DSC) showed that the formation of the beta sheet structure of silk fibroin enhances the thermal stability of the final blend films. Therefore, the novel method presented in this study resulted in the formation of biocomposite films whose physico-chemical properties can be tuned by silk fibroin conformational changes by applying different component mixing ratios.

Antibacterial silk fibroin/nanohydroxyapatite hydrogels with silver and gold nanoparticles for bone regeneration.

The rapid emergence of antibiotic resistance is becoming an imminent problem in bone tissue engineering, and therefore biomaterials must be modified to promote the tissue integration before bacterial adhesion. In this work, silk fibroin/nanohydroxyapatite hydrogel was modified with in situ synthesized silver and gold nanoparticles (AgNPs and AuNPs), taking advantage of the tyrosine amino acid. The presence of AgNPs and AuNPs in the hydrogels was characterized by UV spectrophotometer, transmission electron microscopy and thermogravimetric analysis. In vitro antimicrobial studies revealed that hydrogels with AgNPs and AuNPs exhibited significant inhibition ability against both gram-positive and gram-negative bacteria. Cytocompatibility studies carried out using osteoblastic cells revealed that up to 0.5 wt% of AgNPs, and for all concentrations of AuNPs, the hydrogels can be effectively used as antimicrobial materials, without compromising cell behavior. On the basis of the aforementioned observations, these hydrogels are very attractive for bone tissue engineering.

Nanofilms of hyaluronan/chitosan assembled layer-by-layer: An antibacterial surface for Xylella fastidiosa.

In this work, nanofilms of hyaluronan/chitosan (HA/CHI) assembled layer by layer were synthesized; their application as a potential antimicrobial material was demonstrated for the phytopathogen Xylella fastidiosa, a gram-negative bacterium, here used as a model. For the synthesis, the influence of pH and ionic strength of these natural polymer stem-solutions on final characteristics of the HA/CHI nanofilms was studied in detail. The antibacterial effect was evaluated using widefield fluorescence microscopy. These results were correlated with the chemical properties of the nanofilms, studied by FTIR and Raman spectroscopy, as well as with their morphology and surface properties characterized using SEM and AFM. The present findings can be extended to design and optimize HA/CHI nanofilms with enhanced antimicrobial behavior for other type of phytopathogenic gram-negative bacteria species, such as Xanthomonas citri, Xanthomas campestri and Ralstonia solanacearum.

Liposome-Loaded Cell Backpacks.

Cell backpacks, or micron-scale patches of a few hundred nanometers in thickness fabricated by layer-by-layer (LbL) assembly, are potentially useful vehicles for targeted drug delivery on the cellular level. In this work, echogenic liposomes (ELIPs) containing the anticancer drug doxorubicin (DOX) are embedded into backpacks through electrostatic interactions and LbL assembly. Poly(allylamine hydrochloride)/poly(acrylic acid) (PAH/PAA)n , and poly(diallyldimethylammonium chloride)/poly(styrene sulfonate) (PDAC/SPS)n film systems show the greatest ELIP incorporation of the films studied while maintaining the structural integrity of the vesicles. The use of ELIPs for drug encapsulation into backpacks facilitates up to three times greater DOX loading compared to backpacks without ELIPs. Cytotoxicity studies reveal that monocyte backpack conjugates remain viable even after 72 h, demonstrating promise as drug delivery vehicles. Because artificial vesicles can load many different types of drugs, ELIP containing backpacks offer a unique versatility for broadening the range of possible applications for cell backpacks.

Optimization of amine-rich multilayer thin films for the capture and quantification of prostate-specific antigen.

It is demonstrated that poly(allylamine hydrochloride)/poly(styrenesulfonate) (PAH/SPS) multilayer films can be successfully tailored for the capture and detection of small biomolecules in dilute concentrations. Based on in vitro results, these films could be potentially applied for rapid and high-throughput diagnosis of dilute biomarkers in serum or tissue. PAH presents functional amino groups that can be further reacted with desired chemistries in order to create customizable and specific surfaces for biomolecule capture. A variety of film assembly characteristics were tested (pH, molecular weight of PAH, and ionic strength) to tune the biotinylation and swelling behavior of these films to maximize detection capabilities. The resultant optimized biotinylated PAH/SPS 9.3/9.3 system was utilized in conjunction with quantum dots (Qdots) to capture and detect a dilute biomarker for prostate cancer, prostate-specific antigen (PSA). Compared to previous work, our system presents a good sensitivity for PSA detection within the clinically relevant range of 0.4-100 ng/mL.

Removal of glyphosate herbicide from water using biopolymer membranes.

Enormous amounts of pesticides are manufactured and used worldwide, some of which reach soils and aquatic systems. Glyphosate is a non-selective herbicide that is effective against all types of weeds and has been used for many years. It can therefore be found as a contaminant in water, and procedures are required for its removal. This work investigates the use of biopolymeric membranes prepared with chitosan (CS), alginate (AG), and a chitosan/alginate combination (CS/AG) for the adsorption of glyphosate present in water samples. The adsorption of glyphosate by the different membranes was investigated using the pseudo-first order and pseudo-second order kinetic models, as well as the Langmuir and Freundlich isotherm models. The membranes were characterized regarding membrane solubility, swelling, mechanical, chemical and morphological properties. The results of kinetics experiments showed that adsorption equilibrium was reached within 4 h and that the CS membrane presented the best adsorption (10.88 mg of glyphosate/g of membrane), followed by the CS/AG bilayer (8.70 mg of glyphosate/g of membrane). The AG membrane did not show any adsorption capacity for this herbicide. The pseudo-second order model provided good fits to the glyphosate adsorption data on CS and CS/AG membranes, with high correlation coefficient values. Glyphosate adsorption by the membranes could be fitted by the Freundlich isotherm model. There was a high affinity between glyphosate and the CS membrane and moderate affinity in the case of the CS/AG membrane. Physico-chemical characterization of the membranes showed low values of solubility in water, indicating that the membranes are stable and not soluble in water. The SEM and AFM analysis showed evidence of the presence of glyphosate on CS membranes and on chitosan face on CS/AG membranes. The results showed that the glyphosate herbicide can be adsorbed by chitosan membranes and the proposed membrane-based methodology was successfully used to treat a water sample contaminated with glyphosate. Biopolymer membranes therefore potentially offer a versatile method to eliminate agricultural chemicals from water supplies.

Sugar-mediated disassembly of mucin/lectin multilayers and their use as pH-Tolerant, on-demand sacrificial layers.

The layer-by-layer (LbL) assembly of thin films on surfaces has proven to be an extremely useful technology for uses ranging from optics to biomedical applications. Releasing these films from the substrate to generate so-called free-standing multilayer films opens a new set of applications. Current approaches to generating such materials are limited because they can be cytotoxic, difficult to scale up, or have undesirable side reactions on the material. In this work, a new sacrificial thin film system capable of chemically triggered dissolution at physiological pH of 7.4 is described. The film was created through LbL assembly of bovine submaxillary mucin (BSM) and the lectin jacalin (JAC) for a (BSM/JAC) multilayer system, which remains stable over a wide pH range (pH 3-9) and at high ionic strength (up to 5 M NaCl). This stability allows for subsequent LbL assembly of additional films in a variety of conditions, which could be released from the substrate by incubation in the presence of a competitive inhibitor sugar, melibiose, which selectively disassembles the (BSM/JAC) section of the film. This novel multilayer system was then applied to generate free-standing, 7 µm diameter, circular ultrathin films, which can be attached to a cell surface as a "backpack". A critical thickness of about 100 nm for the (BSM/JAC) film was required to release the backpacks from the glass substrate, after incubation in melibiose solution at 37 °C for 1 h. Upon their release, backpacks were subsequently attached to murine monocytes without cytotoxicity, thereby demonstrating the compatibility of this mucin-based release system with living cells.

The role of dialysis and freezing on structural conformation, thermal properties and morphology of silk fibroin hydrogels.

Silk fibroin has been widely explored for many biomedical applications, due to its biocompatibility and biodegradability. The aim of this work was to study the role of dialysis and freezing on structural conformation, thermal properties and morphology of silk fibroin hydrogels. Hydrogels were prepared after 3 and 7 days of dialysis and the effect of freezing was analyzed. For that purpose, a part of the fibroin hydrogels underwent freezing at -20 °C for 24 h, followed by lyophilization and the rest of the hydrogels were kept at 8 °C for 24 h, with further lyophilization. The fibroin hydrogels were characterized by X-ray diffraction (XRD), Fourier transformed infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). Measurements by XRD and FTIR indicated that silk I and silk II structures were present in the fibroin hydrogels and that the secondary structure of fibroin is transformed mostly to ß-sheet during the gelation process. Thermal analysis indicated that fibroin hydrogels are thermally stable with the degradation peak at around 330-340 °C. SEM micrographs showed porous structures and the fibroin hydrogels subjected to freezing presented a much larger pore size. Results indicate that the dialysis time and freezing did not alter the material crystallinity, conformation or thermal behavior; however, hydrogel microstructure was strongly affected by dialysis time and freezing, showing controlled pores size. This study provides fundamental knowledge on silk fibroin hydrogels preparation and properties and the studied hydrogels are promising to be used in the biomaterial field.