Optimizing the synthesis of dimeric peptides: influence of the reaction medium and effects that modulate kinetics and reaction yield.

Peptides are remarkably interesting alternatives to several applications. In particular, antimicrobial sequences have raised major interest of the scientific community due to the resistance acquired by commonly used antibiotics. Amongst these, some dimeric peptides have shown very promising characteristics as strong biological activities and resistance against degradation by peptidases. However, despite such promising characteristics, a relatively small number of studies address dimeric peptides, mainly due to the synthesis-related obstacles in their production, whereas the well-implemented routines of solid phase peptide synthesis-which includes the possibility of automation-makes life significantly easier. Here, we present kinetic investigations of the dimerization of a cysteine-containing sequence to obtain the homodimeric antimicrobial peptide homotarsinin. Based on the structural and membrane interaction data already available for the dimer and its monomeric chain, we have proposed distinct dimerization protocols in selected environments, namely, aqueous buffer, TFE:H2O and micellar solutions. The experimental results were adjusted by a theoretical model. Both the kinetic profiles and the reaction yields are dependent on the reaction medium, clearly indicating that aggregation, peptide structure, and peptide-membrane interactions play major roles in the formation of the disulfide bond. Finally, the rationalization of the different aspects addressed here is expected to contribute to research and applications that demand the obtainment of dimeric peptides.

Microfluidic mixing system for precise PLGA-PEG nanoparticles size control.

In this study, a microfluidic device was employed to produce polymeric nanoparticles (NPs) with well-controlled sizes. The influence of several parameters in the synthesis process, namely, polymer concentration, flow rate and flow rate ratio between the aqueous and organic solutions was investigated. To evaluate the NPs size effect, three diameters were selected (30, 50 and 70 nm). Their cytocompatibility was demonstrated on endothelial cells and macrophages. Additionally, their efficacy to act as drug carriers was assessed in an in vitro inflammatory scenario. NPs loaded and released diclofenac (DCF) in a size-dependent profile (smaller sizes presented lower DCF content and higher release rate). Moreover, 30 nm NPs were the most effective in reducing prostaglandin E2 concentration. Therefore, this study demonstrates that microfluidics can generate stable NPs with controlled sizes, high monodispersity and enhanced batch-to-batch reproducibility. Indeed, NPs size is a crucial parameter for drug encapsulation, release and overall biological efficacy.

Microfluidic-Driven Biofabrication and the Engineering of Cancer-Like Microenvironments.

Despite considerable advances in cancer research and oncological treatments, the burden of the disease is still extremely high. While past research has been cancer cell centered, it is now clear that to understand tumors, the models that serve as a framework for research and therapeutic testing need to improve and integrate cancer microenvironment characteristics such as mechanics, architecture, and cell heterogeneity. Microfluidics is a powerful tool for biofabrication of cancer-relevant architectures given its capacity to manipulate cells and materials at very small dimensions and integrate varied living tissue characteristics. This chapter outlines the current microfluidic toolbox for fabricating living constructs, starting by explaining the varied configurations of 3D soft constructs microfluidics enables when used to process hydrogels. Then, we analyze the possibilities to control material flows and create space varying characteristics such as gradients or advanced 3D micro-architectures. Envisioning the trend to approach the complexity of tumor microenvironments also at higher dimensions, we discuss microfluidic-enabled 3D bioprinting and recent advances in that arena. Finally, we summarize the future possibilities for microfluidic biofabrication to tackle important challenges in cancer 3D modelling, including tools for the fast quantification of biological events toward data-driven and precision medicine approaches.

Glycotriazole-peptides derived from the peptide HSP1: synergistic effect of triazole and saccharide rings on the antifungal activity.

This work proposes a strategy that uses solid-phase peptide synthesis associated with copper(I)-catalyzed azide alkyne cycloaddition reaction to promote the glycosylation of an antimicrobial peptide (HSP1) containing a carboxyamidated C-terminus (HSP1-NH2). Two glycotriazole-peptides, namely [p-Glc-trz-G1]HSP1-NH2 and [p-GlcNAc-trz-G1]HSP1-NH2, were prepared using per-O-acetylated azide derivatives of glucose and N-acetylglucosamine in the presence of copper(II) sulfate pentahydrate (CuSO4·5H2O) and sodium ascorbate as a reducing agent. In order to investigate the synergistic action of the carbohydrate motif linked to the triazole-peptide structure, a triazole derivative [trz-G1]HSP1-NH2 was also prepared. A set of biophysical approaches such as DLS, Zeta Potential, SPR and carboxyfluorescein leakage from phospholipid vesicles confirmed higher membrane disruption and lytic activities as well as stronger peptide-LUVs interactions for the glycotriazole-peptides when compared to HSP1-NH2 and to its triazole derivative, which is in accordance with the performed biological assays: whereas HSP1-NH2 presents relatively low and [trz-G1]HSP1-NH2 just moderate fungicidal activity, the glycotriazole-peptides are significantly more effective antifungal agents. In addition, the glycotriazole-peptides and the triazole derivative present strong inhibition effects on ergosterol biosynthesis in Candida albicans, when compared to HSP1-NH2 alone. In conclusion, the increased fungicidal activity of the glycotriazole-peptides seems to be the result of (A) more pronounced membrane-disruptive properties, which is related to the presence of a saccharide ring, together with (B) the inhibition of ergosterol biosynthesis, which seems to be related to the presence of both the monosaccharide and the triazole rings.

Co-axial hydrogel spinning for facile biofabrication of prostate cancer-like 3D models.

Glandular cancers are amongst the most prevalent types of cancer, which can develop in many different organs, presenting challenges in their detection as well as high treatment variability and failure rates. For that purpose, anticancer drugs are commonly tested in cancer cell lines grown in 2D tissue culture on plastic dishesin vitro, or in animal modelsin vivo. However, 2D culture models diverge significantly from the 3D characteristics of living tissues and animal models require extensive animal use and time. Glandular cancers, such as prostate cancer-the second leading cause of male cancer death-typically exist in co-centrical architectures where a cell layer surrounds an acellular lumen. Herein, this spatial cellular position and 3D architecture, containing dual compartments with different hydrogel materials, is engineered using a simple co-axial nozzle setup, in a single step utilizing prostate as a model of glandular cancer. The resulting hydrogel soft structures support viable prostate cancer cells of different cell lines and enable over-time maturation into cancer-mimicking aggregates surrounding the acellular core. The biofabricated cancer mimicking structures are then used as a model to predict the inhibitory efficacy of the poly ADP ribose polymerase inhibitor, Talazoparib, and the antiandrogen drug, Enzalutamide, in the growth of the cancer cell layer. Our results show that the obtained hydrogel constructs can be adapted to quickly obtain 3D cancer models which combine 3D physiological architectures with high-throughput screening to detect and optimize anti-cancer drugs in prostate and potentially other glandular cancer types.

Shining a Light on Cancer-Photonics in Microfluidic Tumor Modeling and Biosensing.

Microfluidic platforms represent a powerful approach to miniaturizing important characteristics of cancers, improving in vitro testing by increasing physiological relevance. Different tools can manipulate cells and materials at the microscale, but few offer the efficiency and versatility of light and optical technologies. Moreover, light-driven technologies englobe a broad toolbox for quantifying critical biological phenomena. Herein, the role of photonics in microfluidic 3D cancer modeling and biosensing from three major perspectives is reviewed. First, optical-driven technologies are looked upon, as these allow biomaterials and living cells to be manipulated with microsized precision and present opportunities to advance 3D microfluidic models by engineering cancer microenvironments' hallmarks, such as their architecture, cellular complexity, and vascularization. Second, the growing field of optofluidics is discussed, exploring how optical tools can directly interface microfluidic chips, enabling the extraction of relevant biological data, from single fluorescent signals to the complete 3D imaging of diseased cells within microchannels. Third, advances in optical cancer biosensing are reviewed, focusing on how light-matter interactions can detect biomarkers, rare circulating tumor cells, and cell-derived structures such as exosomes. Photonic technologies' current challenges and caveats in microfluidic 3D cancer models are overviewed, outlining future research avenues that may catapult the field.

Pushing the Natural Frontier: Progress on the Integration of Biomaterial Cues toward Combinatorial Biofabrication and Tissue Engineering.

The engineering of fully functional, biological-like tissues requires biomaterials to direct cellular events to a near-native, 3D niche extent. Natural biomaterials are generally seen as a safe option for cell support, but their biocompatibility and biodegradability can be just as limited as their bioactive/biomimetic performance. Furthermore, integrating different biomaterial cues and their final impact on cellular behavior is a complex equation where the outcome might be very different from the sum of individual parts. This review critically analyses recent progress on biomaterial-induced cellular responses, from simple adhesion to more complex stem cell differentiation, looking at the ever-growing possibilities of natural materials modification. Starting with a discussion on native material formulation and the inclusion of cell-instructive cues, the roles of shape and mechanical stimuli, the susceptibility to cellular remodeling, and the often-overlooked impact of cellular density and cell-cell interactions within constructs, are delved into. Along the way, synergistic and antagonistic combinations reported in vitro and in vivo are singled out, identifying needs and current lessons on the development of natural biomaterial libraries to solve the cell-material puzzle efficiently. This review brings together knowledge from different fields envisioning next-generation, combinatorial biomaterial development toward complex tissue engineering.

Engineered living bioassemblies for biomedical and functional material applications.

Recent breakthroughs in biofabrication of bioasemblies, consisting of the engineered structures composed of biological or biosynthetic components into a single construct, have found a wide range of practical applications in medicine and engineering. This review presents an overview of how the bottom-up assembly of living entities could drive advances in medicine, by developing tunable biological models and more precise methods for quantifying biological events. Moreover, we delve into advances beyond biomedical applications, where bioassemblies can be manipulated as functional robots and construction materials. Finally, we address the potential challenges and opportunities in the field of engineering living bioassemblies, toward building new design principles for the next generation of bioengineering applications.

Large-Scale Functionalized Metasurface-Based SARS-CoV-2 Detection and Quantification.

Plasmonic metasurfaces consist of metal-dielectric interfaces that are excitable at background and leakage resonant modes. The sharp and plasmonic excitation profile of metal-free electrons on metasurfaces at the nanoscale can be used for practical applications in diverse fields, including optoelectronics, energy harvesting, and biosensing. Currently, Fano resonant metasurface fabrication processes for biosensor applications are costly, need clean room access, and involve limited small-scale surface areas that are not easy for accurate sample placement. Here, we leverage the large-scale active area with uniform surface patterns present on optical disc-based metasurfaces as a cost-effective method to excite asymmetric plasmonic modes, enabling tunable optical Fano resonance interfacing with a microfluidic channel for multiple target detection in the visible wavelength range. We engineered plasmonic metasurfaces for biosensing through efficient layer-by-layer surface functionalization toward real-time measurement of target binding at the molecular scale. Further, we demonstrated the quantitative detection of antibodies, proteins, and the whole viral particles of SARS-CoV-2 with a high sensitivity and specificity, even distinguishing it from similar RNA viruses such as influenza and MERS. This cost-effective plasmonic metasurface platform offers a small-scale light-manipulation system, presenting considerable potential for fast, real-time detection of SARS-CoV-2 and pathogens in resource-limited settings.

Engineering Hydrogel-Based Biomedical Photonics: Design, Fabrication, and Applications.

Light guiding and manipulation in photonics have become ubiquitous in events ranging from everyday communications to complex robotics and nanomedicine. The speed and sensitivity of light-matter interactions offer unprecedented advantages in biomedical optics, data transmission, photomedicine, and detection of multi-scale phenomena. Recently, hydrogels have emerged as a promising candidate for interfacing photonics and bioengineering by combining their light-guiding properties with live tissue compatibility in optical, chemical, physiological, and mechanical dimensions. Herein, the latest progress over hydrogel photonics and its applications in guidance and manipulation of light is reviewed. Physics of guiding light through hydrogels and living tissues, and existing technical challenges in translating these tools into biomedical settings are discussed. A comprehensive and thorough overview of materials, fabrication protocols, and design architectures used in hydrogel photonics is provided. Finally, recent examples of applying structures such as hydrogel optical fibers, living photonic constructs, and their use as light-driven hydrogel robots, photomedicine tools, and organ-on-a-chip models are described. By providing a critical and selective evaluation of the field's status, this work sets a foundation for the next generation of hydrogel photonic research.

Emerging biofabrication approaches for gastrointestinal organoids towards patient specific cancer models.

Tissue engineered organoids are simple biomodels that can emulate the structural and functional complexity of specific organs. Here, we review developments in three-dimensional (3D) artificial cell constructs to model gastrointestinal dynamics towards cancer diagnosis. We describe bottom-up approaches to fabricate close-packed cell aggregates, from the use of biochemical and physical cues to guide the self-assembly of organoids, to the use of engineering approaches, including 3D printing/additive manufacturing and external field-driven protocols. Finally, we outline the main challenges and possible risks regarding the potential translation of gastrointestinal organoids from laboratory settings to patient-specific models in clinical applications.

Wearable Collector for Noninvasive Sampling of SARS-CoV-2 from Exhaled Breath for Rapid Detection.

Airborne transmission of exhaled virus can rapidly spread, thereby increasing disease progression from local incidents to pandemics. Due to the COVID-19 pandemic, states and local governments have enforced the use of protective masks in public and work areas to minimize the disease spread. Here, we have leveraged the function of protective face coverings toward COVID-19 diagnosis. We developed a user-friendly, affordable, and wearable collector. This noninvasive platform is integrated into protective masks toward collecting airborne virus in the exhaled breath over the wearing period. A viral sample was sprayed into the collector to model airborne dispersion, and then the enriched pathogen was extracted from the collector for further analytical evaluation. To validate this design, qualitative colorimetric loop-mediated isothermal amplification, quantitative reverse transcription polymerase chain reaction, and antibody-based dot blot assays were performed to detect the presence of SARS-CoV-2. We envision that this platform will facilitate sampling of current SARS-CoV-2 and is potentially broadly applicable to other airborne diseases for future emerging pandemics.

Engineering Polysaccharide-Based Hydrogel Photonic Constructs: From Multiscale Detection to the Biofabrication of Living Optical Fibers.

Solid-state optics has been the pillar of modern digital age. Integrating soft hydrogel materials with micro/nanooptics could expand the horizons of photonics for bioengineering. Here, wet-spun multilayer hydrogel fibers are engineered through ionic-crosslinked natural polysaccharides that serve as multifunctional platforms. The resulting flexible hydrogel structure and reversible crosslinking provide tunable design properties such as adjustable refractive index and fusion splicing. Modulation of the optical readout via physical stimuli, including shape, compression, and multiple optical inputs/outputs is demonstrated. The unique permeability of the hydrogels is also combined with plasmonic nanoparticles for molecular detection of SARS-CoV-2 in fiber-coupled biomedical swabs. A tricoaxial 3D printing nozzle is then employed for the continuous fabrication of living optical fibers. Light interaction with living cells enables the quantification and digitalization of complex biological phenomena such as 3D cancer progression and drug susceptibility. These fibers pave the way for advances in biomaterial-based photonics and biosensing platforms.

KRAS Oncogenic Signaling Extends beyond Cancer Cells to Orchestrate the Microenvironment.

KRAS is one of the most frequently mutated oncogenes in cancer, being a potent initiator of tumorigenesis, a strong inductor of malignancy, and a predictive biomarker of response to therapy. Despite the large investment to understand the effects of KRAS activation in cancer cells, pharmacologic targeting of KRAS or its downstream effectors has not yet been successful at the clinical level. Recent studies are now describing new mechanisms of KRAS-induced tumorigenesis by analyzing its effects on the components of the tumor microenvironment. These studies revealed that the activation of KRAS on cancer cells extends to the surrounding microenvironment, affecting the properties and functions of its constituents. Herein, we discuss the most emergent perspectives on the relationship between KRAS-mutant cancer cells and their microenvironment components. Cancer Res; 78(1); 7-14. ©2017 AACR.

Screening Platform for Cell Contact Guidance Based on Inorganic Biomaterial Micro/nanotopographical Gradients.

High-throughput screening (HTS) methods based on topography gradients or arrays have been extensively used to investigate cell-material interactions. However, it is a huge technological challenge to cost efficiently prepare topographical gradients of inorganic biomaterials due to their inherent material properties. Here, we developed a novel strategy translating PDMS-based wrinkled topography gradients with amplitudes from 49 to 2561 nm and wavelengths between 464 and 7121 nm to inorganic biomaterials (SiO2, Ti/TiO2, Cr/CrO3, and Al2O3) which are frequently used clinical materials. Optimal substratum conditions promoted human bone-marrow derived mesenchymal stem cell alignment, elongation, cytoskeleton arrangement, filopodia development as well as cell adhesion in vitro, which depended both on topography and interface material. This study displays a positive correlation between cell alignment and the orientation of cytoskeleton, filopodia, and focal adhesions. This platform vastly minimizes the experimental efforts both for inorganic material interface engineering and cell biological assessments in a facile and effective approach. The practical application of the HTS technology is expected to aid in the acceleration of developments of inorganic clinical biomaterials.

Structure and membrane interactions of the homodimeric antibiotic peptide homotarsinin.

Antimicrobial peptides (AMPs) from amphibian skin are valuable template structures to find new treatments against bacterial infections. This work describes for the first time the structure and membrane interactions of a homodimeric AMP. Homotarsinin, which was found in Phyllomedusa tarsius anurans, consists of two identical cystine-linked polypeptide chains each of 24 amino acid residues. The high-resolution structures of the monomeric and dimeric peptides were determined in aqueous buffers. The dimer exhibits a tightly packed coiled coil three-dimensional structure, keeping the hydrophobic residues screened from the aqueous environment. An overall cationic surface of the dimer assures enhanced interactions with negatively charged membranes. An extensive set of biophysical data allowed us to establish structure-function correlations with antimicrobial assays against Gram-positive and Gram-negative bacteria. Although both peptides present considerable antimicrobial activity, the dimer is significantly more effective in both antibacterial and membrane biophysical assays.