Complex Biological Milieu Research Articles

Ultrasmall Gold Nanoparticles Coated with Zwitterionic Glutathione Monoethyl Ester: A Model Platform for the Incorporation of Functional Peptides.

Ultrasmall gold nanoparticles (AuNPs) are an emerging class of nanomaterials exhibiting distinctive physicochemical, molecular, and in vivo properties. Recently, we showed that ultrasmall AuNPs encompassing a zwitterionic glutathione monoethyl ester surface coating (AuGSHzwt) were highly resistant to aggregation and serum protein interactions. Herein, we performed a new set of biointeraction studies to gain a more fundamental understanding into the behavior of both pristine and peptide-functionalized AuGSHzwt in complex media. Using the model Strep-tag peptide (WSHPQFEK) as an integrated functional group, we established that AuGSHzwt could be conjugated with increasing numbers of Strep-tags by simple ligand exchange, which provides a generic approach for AuGSHzwt functionalization. It was found that the strep-tagged AuGSHzwt particles were highly resistant to nonspecific protein interactions and retained their targeting capability in biological fluid, displaying efficient binding to Streptactin receptors in nearly undiluted serum. However, AuGSHzwt functionalized with multiple Strep-tags displayed somewhat lower resistance to protein interactions and lower levels of binding to Streptactin than monofunctionalized AuGSHzwt under given conditions. These results underscore the need for optimizing ligand density onto the surface of ultrasmall AuNPs for improved performance. Collectively, our findings support ultrasmall AuGSHzwt as an attractive platform for engineering functional, protein-mimetic nanostructures capable of specific protein recognition within the complex biological milieu.

Amyloid-Like Protein Aggregates: A New Class of Bioinspired Materials Merging an Interfacial Anchor with Antifouling.

Surfaces that resist nonspecific protein adsorption in a complex biological milieu are required for a variety of applications. However, few strategies can achieve a robust antifouling coating on a surface in an easy and reliable way, regardless of material type, morphology, and shape. Herein, the preparation of an antifouling coating by one-step aqueous supramolecular assembly of bovine serum albumin (BSA) is reported. Based on fast amyloid-like protein aggregation through the rapid reduction of the intramolecular disulfide bonds of BSA by tris(2-carboxyethyl)phosphine, a dense proteinaceous nanofilm with controllable thickness (≈130 nm) can be covered on virtually arbitrary material surfaces in tens of minutes by a simple dipping or spraying. The nanofilm shows strong stability and adhesion with the underlying substrate, exhibiting excellent resistance to the nonspecific adsorption of a broad-spectrum of contaminants including proteins, serum, cell lysate, cells, and microbes, etc. In vitro and in vivo experiments show that the nanofilm can prevent the adhesion of microorganisms and the formation of biofilm. Compared with native BSA, the proteinaceous nanofilm coating exposes a variety of functional groups on the surface, which have more-stable adhesion with the surface and can maintain the antifouling in harsh conditions including under ultrasound, surfactants, organic solvents, and enzymatic digestion.

Precision-Guided Missile-Like DNA Nanostructure Containing Warhead and Guidance Control for Aptamer-Based Targeted Drug Delivery into Cancer Cells in Vitro and in Vivo.

It is crucial to deliver anticancer drugs to target cells with high precision and efficiency. While nanomaterials have been shown to enhance the delivery efficiency once they reach the target, it remains challenging for precise drug delivery to overcome the nonspecific adsorption and off-target effect. To meet this challenge, we report herein the design of a novel DNA nanostructure to act as a DNA nanoscale precision-guided missile (D-PGM) for highly efficient loading and precise delivery of chemotherapeutic agents to specific target cells. The D-PGM consists of two parts: a warhead (WH) and a guidance/control (GC). The WH is a rod-like DNA nanostructure as a drug carrier, whose trunk is a three-dimensionally self-assembled DNA nanoscale architecture from the programmed hybridization among two palindromic DNA sequences in the x-y dimension and two common DNA oligonucleotides in the z direction, making the WH possess a high payload capacity of drugs. The GC is an aptamer-based logic gate assembled in a highly organized fashion capable of performing cell-subtype-specific recognition via the sequential disassembly, mediated by cell-anchored aptamers. Because of the cooperative effects between the WH and the GC, the GC logic gates operate like the guidance and control system in a precision-guided missile to steer the doxorubicin (DOX)-loaded DNA WH toward target cancer cells, leading to selective and enhanced therapeutic efficacy. Moreover, fluorophores attached to different locations of D-PGM and DOX fluorescence dequenching upon release enable intracellular tracing of the DNA nanostructures and drugs. The results demonstrate that by mimicking the functionalities of a military precision-guided missile to design the sequential disassembly of the GC system in multistimuli-responsive fashion, our intrinsically biocompatible and degradable D-PGM can accurately identify target cancer cells in complex biological milieu and achieve active targeted drug delivery. The success of this strategy paves the way for specific cell identity and targeted cancer therapy.

Rapid detection of urokinase plasminogen activator using flexible paper-based graphene-gold platform.

Many studies have shown that urokinase plasminogen activator (uPA) is causally involved in promoting cancer invasion and metastasis. Thus, monitoring uPA levels could be very useful in cancer diagnosis, identification of initial metastasis, and guiding cancer treatment. Here, the authors developed a novel and scalable uPA sensor based on a graphene-gold nanoparticle platform that uses fluorescence of quantum dots to rapidly (<1 h) detect uPA up to 100 pM. Indeed, the authors' sensor is highly selective and showed an ability to sense up to 100 pM uPA even in the presence of complex biological milieu such as the fetal bovine serum.

Small-Molecule-Based Fluorescent Sensors for Selective Detection of Reactive Oxygen Species in Biological Systems.

Reactive oxygen species (ROS) encompass a collection of intricately linked chemical entities characterized by individually distinct physicochemical properties and biological reactivities. Although excessive ROS generation is well known to underpin disease development, it has become increasingly evident that ROS also play central roles in redox regulation and normal physiology. A major challenge in uncovering the relevant biological mechanisms and deconvoluting the apparently paradoxical roles of distinct ROS in human health and disease lies in the selective and sensitive detection of these transient species in the complex biological milieu. Small-molecule-based fluorescent sensors enable molecular imaging of ROS with great spatial and temporal resolution and have thus been appreciated as excellent tools for aiding discoveries in modern redox biology. We review a selection of state-of-the-art sensors with demonstrated utility in biological systems. By providing a systematic overview based on underlying chemical sensing mechanisms, we wish to highlight the strengths and weaknesses in prior sensor works and propose some guiding principles for the development of future probes.

Gold Nanoparticle Couples with Entropy-Driven Toehold-Mediated DNA Strand Displacement Reaction on Magnetic Beads: Toward Ultrasensitive Energy-Transfer-Based Photoelectrochemical Detection of miRNA-141 in Real Blood Sample.

Highly stable circulating microRNAs (miRNAs) are currently recognized as a novel potential biomarker for clinical cancer diagnosis in the early stage. However, limited by its low concentration, high sequence similarity, as well as the numerous interferences in body fluids, detection of miRNA in whole blood with sufficient selectivity and sensitivity is still challenging. Herein, we reported the integration of entropy-driven toehold-mediated DNA strand displacement (ETSD) reaction with magnetic beads (MB) toward the energy-transfer-based photoelectrochemical (PEC) detection of the prostate carcinoma (PCa) biomarker miRNA-141 in a real blood sample. In this protocol, the ETSD reaction was divided into two steps, and cooperated with magnetic separation, target extraction and amplification could be realized in a single test and ultrasensitive detection of miRNA-141 could be achieved in undiluted whole blood sample. This work proposed a new solution for sensitive biomolecular detection in a complex biological milieu and exhibited great promise for future clinical cancer diagnosis.

New Potentiometric Sensor Based on Molecularly Imprinted Polymer for Dipicolinic Acid Detection in Aqueous Media

A new molecularly imprinted polymer (MIP) was introduced herein for specific detection of dipicolinic acid (DPA) in the complex biological milieu. The MIP was synthesized by precipitation polymerization after a systematic investigation of influential parameters on its adsorption performance. The appropriate functional monomer was selected to be acrylic acid (AA) based on the results of molecular dynamics simulation. The ratio of DPA to AA and to cross-linker was experimentally examined using the full factorial method. The optimal molar ratio of 1:8:24 resulted in the highest adsorption capacity of 47.69 mg g−1 and imprinting factor of 4.43. The synthesized polymers were characterized by a scanning electron microscopy, a Fourier transform infrared spectroscopy, and an atomic force microscopy. The spherical MIPs possessing a diameter of 483 nm and an imprinting factor of 4.43 was found to play the major role in the development of an efficient MIP-modified carbon paste electrode. This electrode can be cheaply constructed in bulk and be applied for potentiometric DPA sensing in aqueous solutions with a response time and detection limit of 5 min and $0.1~\mu \text{M}$ , respectively.

Bacteria-Based Materials for Stem Cell Engineering.

Materials can be engineered to deliver specific biological cues that control stem cell growth and differentiation. However, current materials are still limited for stem cell engineering as stem cells are regulated by a complex biological milieu that requires spatiotemporal control. Here a new approach of using materials that incorporate designed bacteria as units that can be engineered to control human mesenchymal stem cells (hMSCs), in a highly dynamic-temporal manner, is presented. Engineered Lactococcus lactis spontaneously colonizes a variety of material surfaces (e.g., polymers, metals, and ceramics) and is able to maintain growth and induce differentiation of hMSCs in 2D/3D surfaces and hydrogels. Controlled, dynamic, expression of fibronectin fragments supports stem cell growth, whereas inducible-temporal regulation of secreted bone morphogenetic protein-2 drives osteogenesis in an on-demand manner. This approach enables stem cell technologies using material systems that host symbiotic interactions between eukaryotic and prokaryotic cells.

Nitric Oxide Detection Using Electrochemical Third‐generation Biosensors – Based on Heme Proteins and Porphyrins

AbstractNitric oxide radical (NO) is a signalling molecule involved in virtually all forms of life. Its relevance has been leading to the development of different analytical methodologies to assess the temporal and spatial fluxes of NO under the complex biological milieu. Third‐generation electrochemical biosensors are promising tools for in loco and in vivo NO quantification and, over the past years, heme proteins and porphyrins have been used in their design. Since there are some limitations with the biorecognition element directly adsorbed onto the electrode surface, nanomaterials (carbon nanotubes, gold nanoparticles, etc.) and polymers (cellulose, chitosan, nafion®, polyacrylamide, among others) have been explored to achieve high kinetics and better biosensor performance. In this review, a broad overview of the field of electrochemical third‐generation biosensors for NO electroanalysis is presented, discussing their main characteristics and aiming new outlooks and advances in this field.

Kinetic analyses and structure-activity relationship studies of synthetic lysine acetylation catalysts

Lysine acylation of proteins is a crucial chemical reaction, both as a post-translational modification and as a method for bioconjugation. We previously developed a chemical catalyst, DSH, which activates a chemically stable thioester including acyl-CoA, allowing the site-selective lysine acylation of histones under physiological conditions. However, a more active catalyst is required for efficient lysine acylation in more complex biological milieu, such as in living cells, but there are no rational guidelines for developing efficient lysine acylation catalysts for use under physiological conditions as opposed to in organic solvents. We, herein, conducted a kinetic analysis of the ability of DSH and several derivatives to mediate lysine acetylation to better understand the structural elements essential for high acetylation activity under physiological conditions. Interestingly, the obtained trend in reactivity was different from that observed in organic solvents, suggesting that a different principle is necessary for designing chemical catalysts specifically for use under physiological conditions compared to catalysts for use in organic solvents. Based on the obtained information, we identified a new catalyst scaffold with high activity and structural flexibility for further modification to improve this catalyst system.

Differential Recognition of Nanoparticle Protein Corona and Modified Low-Density Lipoprotein by Macrophage Receptor with Collagenous Structure.

Key practical challenges such as understanding the immunological processes at the nanoscale and controlling the targeting and accumulation of nano-objects in vivo now further stimulate efforts to underpin phenomenological knowledge of the nanoscale with more mechanistic and molecular insight. Thus, the question as to what constitutes nanoscale biological identity continues to evolve. Certainly nanoparticles in contact with a complex biological milieu develop a biological identity, differing from the original nanomaterial, now referred to as the "biomolecular corona". However, this surface-adsorbed layer of biomolecules may in some circumstance lead to different forms of receptor-particle interactions not evident only from the identity of the surface-adsorbed biomolecules and hard to predict or detect by current physicochemical methods. Here we show that scavenger receptors may recognize complex as yet unidentified biomolecular surface layer motifs, even when no current physicochemical analysis is capable of doing so. For instance, fluorescently labeled SiO2 nanoparticles in a biological milieu are strongly recognized by the macrophage receptor with collagenous structure (MARCO) in even dense biological media (human serum) apparently using a form of binding with which most of the MARCO's known ligands ( e. g., LPS, modified LDL) fail to compete. Such observations may suggest the need for a much stronger emphasis on nanoscale receptor-corona and other biomolecular interaction studies if one wishes to unravel how biomolecular recognition drives outcomes in the nanoscale biological domain.

Modular Protein Cages for Size-Selective RNA Packaging in Vivo.

Protein cages have recently emerged as an important platform for nanotechnology development. Of the naturally existing protein cages, viruses are among the most efficient nanomachines, overcoming various barriers to achieve component replication and efficient self-assembly in complex biological milieu. We have designed an artificial system that can carry out the most basic steps of viral particle assembly in vivo. Our strategy is based on patchwork capsids formed from Aquifex aeolicus lumazine synthase and a circularly permuted variant with appended cationic peptides. These two-component protein containers self-assemble in vivo, capturing endogenous RNA molecules in a size-selective manner. By varying the number and design of the RNA-binding peptides displayed on the lumenal surface, the length of guest RNA can be further controlled. Using a fluorescent aptamer, we also show that short-lived RNA species are captured by the protein cage. This platform has potential as a model system for investigating virus assembly, as well as developing RNA regulation or sampling tools to augment biotechnology.

Dual-Mode Optical Sensing of Histamine at Nanomolar Concentrations in Complex Biological Fluids and Living Cells.

An easily synthesized fluorescein-based luminescent dye has been utilized for the dual-mode detection of histamine at nanomolar concentrations at pH 7.0 in water. The specific response to histamine was achieved by imidazole-catalyzed 'imine formation' reaction. The protocol was subsequently applied for the estimation of histamine in complex biological milieu such as human blood serum and urine samples. Furthermore, the dose-dependent cellular uptake of histamine and de novo synthesis (by thapsigargin treatment) was visualized in RAW 264.7, a mouse macrophage cell line. We have also developed portable paper strips for rapid, on-site detection of histamine without involving costly instruments.

In situ characterization of nanoparticle biomolecular interactions in complex biological media by flow cytometry.

Nanoparticles interacting with, or derived from, living organisms are almost invariably coated in a variety of biomolecules presented in complex biological milieu, which produce a bio-interface or ‘biomolecular corona' conferring a biological identity to the particle. Biomolecules at the surface of the nanoparticle–biomolecule complex present molecular fragments that may be recognized by receptors of cells or biological barriers, potentially engaging with different biological pathways. Here we demonstrate that using intense fluorescent reporter binders, in this case antibodies bound to quantum dots, we can map out the availability of such recognition fragments, allowing for a rapid and meaningful biological characterization. The application in microfluidic flow, in small detection volumes, with appropriate thresholding of the detection allows the study of even complex nanoparticles in realistic biological milieu, with the emerging prospect of making direct connection to conditions of cell level and in vivo experiments.

Development and Investigation of Ultrastable PbS/CdS/ZnS Quantum Dots for Near‐Infrared Tumor Imaging

Achieving bright, reliable, robust, and stable probes for in vivo imaging is becoming extremely urgent for the cancer imaging research community. To date very few works have reported on elucidating in the varied and chemically complex biological milieu. The authors report detailed investigations of the synthesis of near‐infrared, water dispersive, strongly luminescent, and highly stable PbS/CdS/ZnS core/shell/shell quantum dots (QDs). These QDs are extremely stable, they could keep their initial morphology, dispersion status, and photoluminescence (PL) in phosphate buffered saline buffer for as long as 14 months. The QDs also show excellent photostability and could keep ≈80% of their initial PL intensity after 1 h continuous, strong UV illumination. More interestingly, they show negligible toxicity to cultured cells even at high QDs concentration. Given these outstanding properties, the QDs are explored for in vivo, tumor imaging in mice. With one order of magnitude lower QD concentration (0.04 mg mL–1), significantly weaker laser intensity (0.04 W cm–2 vs ≈1 W cm–2), and considerably shorter signal integration time (≤1 ms vs hundreds of ms) as compared to the best reported rare earth doped nanoparticles, the QDs show high emission intensity even at injection depth of ≈2.5 mm.

88 - HKPerox-1: A Highly Selective and Sensitive Fluorescent Probe for Hydrogen Peroxide Detection in Live Cells

Hydrogen peroxide (H2O2) plays important roles in mediating diverse physiological and pathological responses. Recent research clearly demonstrates that H2O2 acts as a key regulator in tumor cell survival, metastasis and proliferation, and increasing evidence shows that high concentration of H2O2 are present in a wide range of pathological samples, such as human urine, exhaled air, and saliva. However, it is always challenging to achieve high selectivity and sensitivity in H2O2 detection in complex biological milieu by using small-molecule fluorescent probes. Here we report a novel fluorescent probe HKPerox-1 for highly selective and sensitive detection and imaging of H2O2 in biological systems. In confocal imaging, HKPerox-1 has been robustly applied to detect H2O2 in multiple cell types. It will not only provide valuable insights into the physiological and pathological roles of H2O2 in human diseases but also offer new approaches for drug discovery and therapeutic intervention.

A benzoboroxole-based affinity ligand for glycoprotein purification at physiological pH.

Developing ligands capable of carbohydrate recognition has become increasingly important as the essential roles of glycoproteins and glycolipids in a diverse array of cellular signaling, pathophysiology, and immune response mechanisms are elucidated. Effective ligands for the glycan portion of glycoproteins and glycolipids are needed for pre-enrichment proteomics strategies, as well as for the purification of individual glycoproteins from complex biological milieu encountered both in biochemistry research and bio-pharmaceutical development. In this work, we developed a carbohydrate specific affinity ligand for glycoprotein purification using a one-pot, multi-component synthesis reaction (Ugi synthesis) and an amine-functionalized benzoboroxole moiety immobilized on agarose beads. Benzoboroxoles are unique boronic acid derivatives that have recently been found to bind specifically to the cis-diol groups of carbohydrates at physiological pH, with superior affinity to any other Wulff-type boronic acid. The solid-phase affinity ligand developed herein specifically binds the carbohydrate moiety of the glycoprotein glucose oxidase, as well as a fluorescein isothiocyanate-dextran, as shown through deglycosylation binding studies. Additionally, the ligand is able to purify glucose oxidase from crude Escherichia coli lysate, at physiological pH, equitably to commercially available boronic acid-functionalized agarose beads that required alkaline pH conditions. Thus, this affinity ligand is a marked improvement on current, commercially available boronic acid-based glycoprotein enrichment matrices and has the potential to exhibit high individual glycoprotein specificity because of the additional functional groups available for variation on the Ugi scaffold.

Efficient one-pot synthesis of hydrophilic and fluorescent molecularly imprinted polymer nanoparticles for direct drug quantification in real biological samples.

Efficient one-pot synthesis of hydrophilic and fluorescent molecularly imprinted polymer (MIP) nanoparticles and their application as optical chemosensor for direct drug quantification in real, undiluted biological samples are described. The general principle was demonstrated by preparing tetracycline (Tc, a broad-spectrum antibiotic)-imprinted fluorescent polymer nanoparticles bearing hydrophilic polymer brushes via poly(2-hydroxyethyl methacrylate) (PHEMA) macromolecular chain transfer agent-mediated reversible addition-fragmentation chain transfer (RAFT) precipitation polymerization in the presence of a fluorescent monomer. The introduction of hydrophilic PHEMA brushes and fluorescence labeling onto/into the MIP nanoparticles proved to not only significantly improve their surface hydrophilicity and lead to their obvious specific binding and high selectivity toward Tc in the undiluted bovine serum, but also impart them with strong fluorescent properties. In particular, significant fluorescence quenching was observed upon their binding with Tc in such complex biological milieu, which makes these Tc-MIP nanoparticles useful optical chemosensor with a detection limit of 0.26 μM. Furthermore, such advanced functional MIP nanoparticles-based chemosensor was also successfully utilized for the direct, sensitive, and accurate determination of Tc in another biological medium (i.e., the undiluted pig serum) with average recoveries ranging from 98% to 102%, even in the presence of several interfering drugs.

Spherical nucleic acid nanoparticle conjugates enhance G-quadruplex formation and increase serum protein interactions.

To understand the effect of three-dimensional oligonucleotide structure on protein corona formation, we studied the identity and quantity of human serum proteins that bind to spherical nucleic acid (SNA) nanoparticle conjugates. SNAs exhibit cellular uptake properties that are remarkably different from those of linear nucleic acids, which have been related to their interaction with certain classes of proteins. Through a proteomic analysis, this work shows that the protein binding properties of SNAs are sequence-specific and supports the conclusion that the oligonucleotide tertiary structure can significantly alter the chemical composition of the SNA protein corona. This knowledge will impact our understanding of how nucleic acid-based nanostructures, and SNAs in particular, function in complex biological milieu.

Spherical Nucleic Acid Nanoparticle Conjugates Enhance G‐Quadruplex Formation and Increase Serum Protein Interactions

AbstractTo understand the effect of three‐dimensional oligonucleotide structure on protein corona formation, we studied the identity and quantity of human serum proteins that bind to spherical nucleic acid (SNA) nanoparticle conjugates. SNAs exhibit cellular uptake properties that are remarkably different from those of linear nucleic acids, which have been related to their interaction with certain classes of proteins. Through a proteomic analysis, this work shows that the protein binding properties of SNAs are sequence‐specific and supports the conclusion that the oligonucleotide tertiary structure can significantly alter the chemical composition of the SNA protein corona. This knowledge will impact our understanding of how nucleic acid‐based nanostructures, and SNAs in particular, function in complex biological milieu.