Smart Biointerfaces via Click Chemistry-Enabled Nanopatterning of Multiple Bioligands and DNA Force Sensors.

Nanoscale biomolecular placement is crucial for advancing cellular signaling, sensor technology, and molecular interaction studies. Despite this, current methods fall short in enabling large-area nanopatterning of multiple biomolecules while minimizing nonspecific interactions. Using bioorthogonal tags at a submicron scale, we introduce a novel hole-mask colloidal lithography method for arranging up to three distinct proteins, DNA, or peptides on large, fully passivated surfaces. The surfaces are compatible with single-molecule fluorescence microscopy and microplate formats, facilitating versatile applications in cellular and single-molecule assays. We utilize fully passivated and transparent substrates devoid of metals and nanotopographical features to ensure accurate patterning and minimize nonspecific interactions. Surface patterning is achieved using bioorthogonal TCO-tetrazine (inverse electron-demand Diels-Alder, IEDDA) ligation, DBCO-azide (strain-promoted azide-alkyne cycloaddition, SPAAC) click chemistry, and biotin-avidin interactions. These are arranged on surfaces passivated with dense poly(ethylene glycol) PEG brushes crafted through the selective and stepwise removal of sacrificial metallic and polymeric layers, enabling the directed attachment of biospecific tags with nanometric precision. In a proof-of-concept experiment, DNA tension gauge tether (TGT) force sensors, conjugated to cRGD (arginylglycylaspartic acid) in nanoclusters, measured fibroblast integrin tension. This novel application enables the quantification of forces in the piconewton range, which is restricted within the nanopatterned clusters. A second demonstration of the platform to study integrin and epidermal growth factor (EGF) proximal signaling reveals clear mechanotransduction and changes in the cellular morphology. The findings illustrate the platform's potential as a powerful tool for probing complex biochemical pathways involving several molecules arranged with nanometer precision and cellular interactions at the nanoscale.

Amyloid-ß aggregates activate peripheral monocytes in mild cognitive impairment.

The peripheral immune system is important in neurodegenerative diseases, both in protecting and inflaming the brain, but the underlying mechanisms remain elusive. Alzheimer's Disease is commonly preceded by a prodromal period. Here, we report the presence of large Aß aggregates in plasma from patients with mild cognitive impairment (n = 38). The aggregates are associated with low level Alzheimer's Disease-like brain pathology as observed by 11C-PiB PET and 18F-FTP PET and lowered CD18-rich monocytes. We characterize complement receptor 4 as a strong binder of amyloids and show Aß aggregates are preferentially phagocytosed and stimulate lysosomal activity through this receptor in stem cell-derived microglia. KIM127 integrin activation in monocytes promotes size selective phagocytosis of Aß. Hydrodynamic calculations suggest Aß aggregates associate with vessel walls of the cortical capillaries. In turn, we hypothesize aggregates may provide an adhesion substrate for recruiting CD18-rich monocytes into the cortex. Our results support a role for complement receptor 4 in regulating amyloid homeostasis.

Protein ligand and nanotopography separately drive the phenotype of mouse embryonic stem cells.

Biochemical and biomechanical signals regulate stem cell function in the niche environments in vivo. Current in vitro culture of mouse embryonic stem cells (mESC) uses laminin (LN-511) to provide mimetic biochemical signaling (LN-521 for human systems) to maintain stemness. Alternative approaches propose topographical cues to provide biomechanical cues, however combined biochemical and topographic cues may better mimic the in vivo environment, but are largely unexplored for in vitro stem cell expansion. In this study, we directly compare in vitro signals from LN-511 and/or topographic cues to maintain stemness, using systematically-varied submicron pillar patterns or flat surfaces with or without preadsorbed LN-511. The adhesion of cells, colony formation, expression of the pluripotency marker,octamer-binding transcription factor 4 (Oct4), and transcriptome profiling were characterized. We observed that either biochemical or topographic signals could maintain stemness of mESCs in feeder-free conditions, indicated by high-level Oct4 and gene profiling by RNAseq. The combination of LN-511 with nanotopography reduced colony growth, while maintaining stemness markers, shifted the cellular phenotype indicating that the integration of biochemical and topographic signals is antagonistic. Overall, significantly faster (up to 2.5 times) colony growth was observed at nanotopographies without LN-511, suggesting for improved ESC expansion.

Serial intravital 2-photon microscopy and analysis of the kidney using upright microscopes.

Serial intravital 2-photon microscopy of the kidney and other abdominal organs is a powerful technique to assess tissue function and structure simultaneously and over time. Thus, serial intravital microscopy can capture dynamic tissue changes during health and disease and holds great potential to characterize (patho-) physiological processes with subcellular resolution. However, successful image acquisition and analysis require significant expertise and impose multiple potential challenges. Abdominal organs are rhythmically displaced by breathing movements which hamper high-resolution imaging. Traditionally, kidney intravital imaging is performed on inverted microscopes where breathing movements are partly compensated by the weight of the animal pressing down. Here, we present a custom and easy-to-implement setup for intravital imaging of the kidney and other abdominal organs on upright microscopes. Furthermore, we provide image processing protocols and a new plugin for the free image analysis software FIJI to process multichannel fluorescence microscopy data. The proposed image processing pipelines cover multiple image denoising algorithms, sample drift correction using 2D registration, and alignment of serial imaging data collected over several weeks using landmark-based 3D registration. The provided tools aim to lower the barrier of entry to intravital microscopy of the kidney and are readily applicable by biomedical practitioners.

Advanced bioengineering of female germ cells to preserve fertility.

Oogenesis and folliculogenesis are considered as complex and species-specific cellular differentiation processes, which depend on the in vivo ovarian follicular environment and endocrine cues. Considerable efforts have been devoted to driving the differentiation of female primordial germ cells toward mature oocytes outside of the body. The recent experimental attempts have laid stress on offering a suitable microenvironment to assist the in vitro folliculogenesis and oogenesis. Despite developing a variety of bioengineering techniques and generating functional mature gametes through in vitro oogenesis in earlier studies, we still lack knowledge of appropriate microenvironment conditions for building biomimetic culture systems for female fertility preservation. Therefore, this review paper can provide a source for a large body of scientists developing cutting-edge in vitro culture systems for female germ cells or setting up the next generation of reproductive medicine as feasible options for female infertility treatment. The focal point of this review outlines advanced bioengineering technologies such as 3D biofabricated hydrogels/scaffolds and microfluidic systems utilized with female germlines for fertility preservation through in vitro folliculogenesis and oogenesis.

The Role of Nanoscale Distribution of Fibronectin in the Adhesion of Staphylococcus aureus Studied by Protein Patterning and DNA-PAINT.

Staphylococcus aureus is a widespread and highly virulent pathogen that can cause superficial and invasive infections. Interactions between S. aureus surface receptors and the extracellular matrix protein fibronectin mediate the bacterial invasion of host cells and is implicated in the colonization of medical implant surfaces. In this study, we investigate the role of distribution of both fibronectin and cellular receptors on the adhesion of S. aureus to interfaces as a model for primary adhesion at tissue interfaces or biomaterials. We present fibronectin in patches of systematically varied size (100-1000 nm) in a background of protein and bacteria rejecting chemistry based on PLL-g-PEG and studied S. aureus adhesion under flow. We developed a single molecule imaging assay for localizing fibronectin binding receptors on the surface of S. aureus via the super-resolution DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique. Our results indicate that S. aureus adhesion to fibronectin biointerfaces is regulated by the size of available ligand patterns, with an adhesion threshold of 300 nm and larger. DNA-PAINT was used to visualize fibronectin binding receptor organization in situ at ∼7 nm localization precision and with a surface density of 38-46 µm-2, revealing that the engagement of two or more receptors is required for strong S. aureus adhesion to fibronectin biointerfaces.

Protein Ligand Nanopattern Size Selects for Cellular Adhesion via Hemidesmosomes over Focal Adhesions.

Hemidesmosomes (HDs) are multiprotein complexes that firmly anchor epidermal cells to the basement membrane of skin through the interconnection of the cytoplasmic intermediate filaments with extracellular laminin 332 (Ln332). Considerably less attention has been paid to HDs compared to focal complexes/focal adhesions (FC/FAs) in mechanistic single-cell structures due to the lack of suitable in vitro model systems. Here nanopatterns of Ln332 (100-1000 nm) are created to direct and study the formation of HD in adherent HaCaT cells. It is observed that HaCaT cells at Ln 332 nanopatterns adhere via hemidesmosomes, in stark contrast to cells at homogeneous Ln332 surfaces that adhere via FC/FAs. Clustering of α6 integrin is observed at nanopatterned Ln332 of 300 nm patches and larger. Cells at 500 nm diameter patterns show strong colocalization of α6 integrin with ColXVII or pan-cytokeratin compared to 300 nm/1000 nm indicating a threshold for HD initiation >100 nm but a pattern size selection for maturation of HDs. It is demonstrated that the pattern of Ln332 can determine the cellular selection of adhesion types with a size-dependent initiation and maturation of HDs. The protein nanopatterning approach that is presented provides a new in vitro route to study the role of HDs in cell signaling and function.

Co-delivery of STAT3 siRNA and methotrexate in breast cancer cells.

Co-delivery of anticancer drugs and biologics can provide synergetic effects and outperform single delivery therapies. Here, a nanoparticle (NP) system for co-delivery of methotrexate (MTX) and STAT3 siRNA has been developed and tested in vitro. Mesoporous silica nanoparticles (MSNs) were functionalized with chitosan (ch) by covalent grafting mediated by aminopropyl triethoxysilane (APTES) via glutaraldehyde as the linker. Co-delivery of MTX and STAT3 siRNA to MCF7 cells was demonstrated in cells by flow cytometric analysis and confocal laser scanning fluorescence microscopy for use in breast cancer treatment. MTX either competitively inhibits the dihydrofolate reductase (DHFR) receptor or suppresses the STAT3 metabolic pathway. STAT3 protein plays an essential role in cell division, proliferation and survival. Reduction of the protein by both MTX and STAT3 siRNA, achieved by chMSNs, significantly decreased the viability of breast cancer cells compared to single treatments alone. Cellular uptake of modified NPs was increased over time when additional free MTX was added implicating the DHFR receptor in uptake. In addition, protein corona compositions coated the NPs outer surface, were different between the NPs with and without drug potentially modulating cellular uptake. This study is the first report on co-delivery of MTX and STAT3 siRNA by chitosan modified MSNs.

A Protein Corona Modulates Interactions of α-Synuclein with Nanoparticles and Alters the Rates of the Microscopic Steps of Amyloid Formation.

Nanoparticles (NPs) can modulate protein aggregation and fibril formation in the context of amyloid diseases. Understanding the mechanism of this action remains a critical next step in developing nanomedicines for the treatment or prevention of Parkinson's disease. α-Synuclein (α-Syn) can undergo interactions of different strength with nanoparticles, and these interactions can be prevented by the presence of a protein corona (PC) acquired during the exposure of NPs to serum proteins. Here, we develop a method to attach the PC irreversibly to the NPs, which enables us to study in detail the interaction of α-Syn and polyethylenimine-coated carboxyl-modified polystyrene NPs (PsNPs-PEI) and the role of the dynamics of the interactions. Analysis of the kinetics of fibril formation reveals that the NPs surface promotes the primary nucleation step of amyloid fibril formation without significantly affecting the elongation and fragmentation steps or the final equilibrium. Furthermore, the results show that even though α-Syn can access the surface of NPs that are precoated with a PC, due to the dynamic nature of the PC proteins, the PC nevertheless reduces the acceleratoring effect of the NPs. This effect is likely to be caused by reducing the overall amount of weakly interacting α-Syn molecules on the NP surface and the access of further α-Syn required for fibril elongation. Our experimental approach provides microscopic insight into how serum proteins can modulate the complex interplay between NPs and amyloid proteins.

Advanced bioengineering of male germ stem cells to preserve fertility.

In modern life, several factors such as genetics, exposure to toxins, and aging have resulted in significant levels of male infertility, estimated to be approximately 18% worldwide. In response, substantial progress has been made to improve in vitro fertilization treatments (e.g. microsurgical testicular sperm extraction (m-TESE), intra-cytoplasmic sperm injection (ICSI), and round spermatid injection (ROSI)). Mimicking the structure of testicular natural extracellular matrices (ECM) outside of the body is one clear route toward complete in vitro spermatogenesis and male fertility preservation. Here, a new wave of technological innovations is underway applying regenerative medicine strategies to cell-tissue culture on natural or synthetic scaffolds supplemented with bioactive factors. The emergence of advanced bioengineered systems suggests new hope for male fertility preservation through development of functional male germ cells. To date, few studies aimed at in vitro spermatogenesis have resulted in relevant numbers of mature gametes. However, a substantial body of knowledge on conditions that are required to maintain and mature male germ cells in vitro is now in place. This review focuses on advanced bioengineering methods such as microfluidic systems, bio-fabricated scaffolds, and 3D organ culture applied to the germline for fertility preservation through in vitro spermatogenesis.

Characterization of DNA-protein complexes by nanoparticle tracking analysis and their association with systemic lupus erythematosus.

Nanotechnology enables investigations of single biomacromolecules, but technical challenges have limited the application in liquid biopsies, for example, blood plasma. Nonetheless, tools to characterize single molecular species in such samples represent a significant unmet need with the increasing appreciation of the physiological importance of protein structural changes at nanometer scale. Mannose-binding lectin (MBL) is an oligomeric plasma protein and part of the innate immune system through its ability to activate complement. MBL also serves a role as a scavenger for cellular debris, especially DNA. This may link functions of MBL with several inflammatory diseases in which cell-free DNA now appears to play a role, but mechanistic insight has been lacking. By making nanoparticle tracking analysis possible in human plasma, we now show that superoligomeric structures of MBL form nanoparticles with DNA. These oligomers correlate with disease activity in systemic lupus erythematosus patients. With the direct quantification of the hydrodynamic radius, calculations following the principles of Taylor dispersion in the blood stream connect the size of these complexes to endothelial inflammation, which is among the most important morbidities in lupus. Mechanistic insight from an animal model of lupus supported that DNA-stabilized superoligomers stimulate the formation of germinal center B cells and drive loss of immunological tolerance. The formation involves an inverse relationship between the concentration of MBL superoligomers and antibodies to double-stranded DNA. Our approach implicates the structure of DNA-protein nanoparticulates in the pathobiology of autoimmune diseases.

Biodegradable nanocarriers based on chitosan-modified mesoporous silica nanoparticles for delivery of methotrexate for application in breast cancer treatment.

Nanocarriers have demonstrated great promise in the delivery of hydrophobic drugs particularly to tumor spaces by enhanced permeability and retention (EPR) effects. Mesoporous silica nanoparticles (MSNs) are the attractive nanocarrier system to reduce the drug's toxic side effects, enable controlled drug release, prevent drug degradation and provide a biocompatible and biodegradable high surface area carrier. Surface-modified MSNs have been applied to increase drug loading and efficiency. In this study, functionalized MSNs loaded with methotrexate (MTX) were designed for use as a cytotoxic agent. The MSNs were first modified with 3-triethoxysilylpropylamine (APTES) and then with chitosan through covalent coupling mediated by glutaraldehyde. The physicochemical properties of the nanoparticles were optimized for each step. The loading percentage (12.2%) and release profile of MTX as an anti-breast cancer drug, loaded at amine-modified MSNs, were measured via high performance liquid chromatography (HPLC). Moreover, the uptake profiles of fluorescein isothiocyanate (FITC)-labeled MSN-APTES-chitosan with or without MTX were monitored on MCF7 cancer cells via confocal microscopy. Following exposure of nanoparticles to body fluids, they were surrounded by specific proteins that may affect their cellular uptake. Hence, the adsorption profiles of protein corona on the surface of MSN, amine-modified MSN and MTX-loaded MSN-APTES-chitosan were analyzed. The cytotoxic potential for killing breast cancer cells was also studied. The MTX loaded MSN-APTES-chitosan showed a positive effect at a low dose (0.5 µM MTX). In this study, we introduce a new method to synthesize biodegradable MSNs with small and uniform particle size, achieve high MTX loading via covalent amine and chitosan-functionalization, monitor the cellular uptake and demonstrate the potential to decrease the viability of breast cancer cells at low dose.

In vivo percutaneous permeation of gold nanomaterials in consumer cosmetics: implication in dermal safety assessment of consumer nanoproducts.

The increasing emergence of nano-cosmetics in the marketplace provokes safety concerns with respect to percutaneous permeation and toxicity of nanomaterials inside the human body. In this study, in vivo percutaneous permeation and dermal safety of cosmetic cream containing Au nanosheets and extracted Au nanosheets from cosmetic creams are investigated with guinea pigs. Quantitative percutaneous permeation data suggests that Au nanosheets in cosmetic creams permeate into the skin epidermis, dermis, and subcutaneous layer after 10 d cutaneous exposure, but cannot enter the systemic circulation. However, more Au nanosheets are accumulated in the skin and the permeation of Au nanosheets increased after embedded into the cream matrix. Synchrotron radiation X-ray fluorescence (SRXRF) imaging reveals that Au nanosheets in cosmetics penetrate mainly through hair follicles in a time-dependent manner. Cosmetic creams rather than extracted Au nanosheets decrease the cell viability of keratinocytes and slightly induce apoptosis/necrosis of keratinocytes and skin dermal fibroblasts. Intriguingly, the growth of hair is inhibited by the cosmetic cream and the extracted Au nanosheets revealed by HE staining and immunohistochemistry (IHC) assay. Altogether this study provides insights into the comprehensive understanding of percutaneous permeation and dermal safety of cosmetic creams containing Au nanosheets. This work provides reliable methods to study the skin permeation, biodistribution, and dermal safety of nano-cosmetics and reminds the community of the crucial need to combine the assays at molecular, cellular, and organ levels in nanotoxicology research.

Mapping and identification of soft corona proteins at nanoparticles and their impact on cellular association.

The current understanding of the biological identity that nanoparticles may acquire in a given biological milieu is mostly inferred from the hard component of the protein corona (HC). The composition of soft corona (SC) proteins and their biological relevance have remained elusive due to the lack of analytical separation methods. Here, we identify a set of specific corona proteins with weak interactions at silica and polystyrene nanoparticles by using an in situ click-chemistry reaction. We show that these SC proteins are present also in the HC, but are specifically enriched after the capture, suggesting that the main distinction between HC and SC is the differential binding strength of the same proteins. Interestingly, the weakly interacting proteins are revealed as modulators of nanoparticle-cell association mainly through their dynamic nature. We therefore highlight that weak interactions of proteins at nanoparticles should be considered when evaluating nano-bio interfaces.

Inhibitors of α-Synuclein Fibrillation and Oligomer Toxicity in Rosa damascena: The All-Pervading Powers of Flavonoids and Phenolic Glycosides.

There is an intense search for natural compounds that can inhibit the oligomerization and fibrillation of α-synuclein (α-Syn), whose aggregation is key to the development of Parkinson's disease (PD). Rosa damascena is a medicinal herb widely used in Middle Eastern food, ceremonies, and perfumes. The herb is known to contain many different polyphenols. Here we investigated the existence of α-Syn fibrillation inhibitors in R. damascena extract. Different HPLC fractions of the extract were assessed in α-Syn fibrillation and toxicity assays. The most active fractions led to the formation of more α-Syn oligomers but with less toxicity to SH-SY5Y cells, according to MTT and LDH assays. LC-MS analysis identified gallic acid, kaempferol 3-glucoside, kaempferol-3-O-ß-rutinoside, and quercetin which were subsequently shown to be strong α-Syn fibrillation inhibitors. Our results highlight the benefits of R. damascena extract to combat PD at the population level.

Tracing the In Vivo Fate of Nanoparticles with a "Non-Self" Biological Identity.

Nanoparticles can acquire a biomolecular corona with a species-specific biological identity. However, "non-self" incompatibility of recipient biological systems is often not considered, for example, when rodents are used as a model organism for preclinical studies of biomolecule-inspired nanomedicines. Using zebrafish embryos as an emerging model for nanobioimaging, here we unravel the in vivo fate of intravenously injected 70 nm SiO2 nanoparticles with a protein corona preformed from fetal bovine serum (FBS), representing a non-self biological identity. Strikingly rapid sequestration and endolysosomal acidification of nanoparticles with the preformed FBS corona were observed in scavenger endothelial cells within minutes after injection. This led to loss of blood vessel integrity and to inflammatory activation of macrophages over the course of several hours. As unmodified nanoparticles or the equivalent dose of FBS proteins alone failed to induce the observed pathophysiology, this signifies how the corona enriched with a differential repertoire of proteins can determine the fate of the nanoparticles in vivo. Our findings thus reveal the adverse outcome triggered by incompatible protein coronas and indicate a potential pitfall in the use of mismatched species combinations during nanomedicine development.

Development of a Label-Free LSPR-Apta Sensor for Staphylococcus aureus Detection.

The risk of foodborne diseases has increased over the last years. We have developed a simple, portable, and label-free optical sensor via aptamer recognition of Staphylococcus aureus at nanostructured plasmonic elements. The developed aptamers conjugated to a localized surface plasmon resonance (LSPR) sensing device were applied in both pure culture and artificially contaminated milk samples enabling a limit of detection of 103 CFU/mL for S. aureus in milk. There was no need for a pre-enrichment step, and the total analysis time decreased from 30 min to 120 s. Finite-difference time-domain was used to simulate the experimentally measured optical responses for a range of different sensor designs (100 and 200 nm disks), addressing the role of the near field and intrinsic refractive index sensitivity. A comparison of the aptamer to antibody-based recognition approaches showed that the thickness of the sensing layer was critical with a significantly larger response for the thinner aptamer layer. Comparison of differently sized metal nanostructures showed a significantly higher sensitivity for 200 nm diameter compared to 100 nm diameter disk structures resulting from both increases in bulk refractive index sensitivity and the extent to which the local field extends out from the metal surface. These findings confirmed that the developed gold nanodisk-based LSPR sensing chips could facilitate sensitive detection of S. aureus in food samples.

Nanoparticle Adsorption on Antifouling Polymer Brushes.

Polymer brushes have been widely used to functionalize surfaces and provide antifouling capabilities against proteins and cells. Many efforts have focused on methods for functionalization of antifouling polymer brush surfaces for interactions with specific cells, proteins, and bacteria, but none have focused on immobilizing nanoparticles (NPs) on these surfaces. This article demonstrates that both pristine NPs and protein-coated NPs can adsorb onto well-functioning antifouling polymer brush coatings formed from poly-l-lysine-graft-poly(ethylene glycol) (PLL-g-PEG) and methoxy PEG-thiol. The role of ionic strength in solution, substrate surface material, and NP surface charge in the interaction was investigated to explore the forces behind the interaction. The adsorption of different types of NPs onto the surface was studied, determining that polystyrene, gold, carbon black, and silica particles can adsorb onto PLL-g-PEG. We show that the approach can be applied in, and studied by, both surface plasmon resonance and fluorescence imaging and suggest its application as a means to study NP-protein interactions, such as the protein corona. NPs self-assembled at antifouling polymer brush surfaces provide a novel platform for both scientific studies and applications in biotechnology.

Chiral plasmonic nanocrescents: large-area fabrication and optical properties.

Large-area arrays of substrate-supported plasmonic gold crescents are fabricated by using the new colloidal lithography technique, which is based on an in situ-deposited silica resistance layer. The method provides the means to control the particles' asymmetry just by changing the mutual deposition angle of gold and silica. Asymmetric crescent structures exhibit a pronounced circular dichroism in near-infrared region, with the chiral asymmetry factor reaching 0.2. According to the simulation, the optical chirality enhancement reaches between one and two orders of magnitude and is localized near the crescents' tips.

A saliva molecular imprinted localized surface plasmon resonance biosensor for wine astringency estimation.

Wine astringency was evaluated based on the interaction of two complex matrices (red wine and saliva) by combining localized surface plasmon resonance (LSPR) and molecular imprinted polymers (MIP) at gold nanodisks as an alternative to sensorial analysis. The main objective of the work was to simulate wine astringency inside the mouth by mimicking this biological system. The LSPR/MIP sensor provided a linear response for astringency expressed in pentagalloyl glucose (PGG) units in concentrations ranging from 1 to 140µmol/L. The sensor was also applied to wine samples correlating well with sensorial analysis obtained by a trained panel. The correlation of astringency and wine composition was also evaluated showing that anthocyanins may have an important role, not only for pigmentation but also in astringency.