Complex Biological Environments Research Articles

DNAzyme-driven tripedal DNA walker triggered hybridization chain reaction for label-free electrochemical detection of Alzheimer’s tau protein

The tau protein is considered to be a reliable molecular biomarker of Alzheimer's disease (AD). Developing sensitive and reliable techniques for tau protein detection is a vital task. In this work, we designed a label-free electrochemical aptasensor for tau protein quantification based on Mg2+-DNAzyme (MNAzyme)-driven tripedal DNA walker. With the assistance of MNAzyme, free-running tripedal DNA walkers could rapidly and continually scissor DNA molecular beacon (MBs), and the fragment as a primer initiated the hybridization chain reaction (HCR) process generating a huge amount of long DNA strand containing many adenines. Following that, the strand with long-repeated adenines (HCR product) may absorb numerous AgNPs on the surface of electrode for electrochemical stripping analysis. Benefiting from MNAzyme-tripedal DNA walker and HCR triple signal amplification, we achieved a superior detection limit of 0.43 fM as well as a broad linear range of 1 fM to 0.1 nM. After being applied to tau protein detection in human serum samples, this novel electrochemical aptasensor demonstrated a high degree of anti-interference, and reproducibility, indicating its great potential for bioanalysis in complex biological environments.

Rapid and ultrasensitive detection of acute kidney injury biomarkers CH3L1 and L-FABP using surface-enhanced Raman spectroscopy

Chitinase 3-like 1 (CH3L1) and liver fatty acid binding protein (L-FABP) are promising biomarkers for the early diagnosis of acute kidney injury (AKI). Here, a highly sensitive method for the quantitative detection of CH3L1 and L-FABP by surface-enhanced Raman spectroscopy (SERS) based on graphene oxide/gold and silver core–shell nanoparticles (GO/Au@Ag NPs) was proposed. The results showed that such GO/Au@Ag substrate can achieve rapid sensing of CH3L1 and L-FABP with a wide response range (2 × 10-1 to 2 × 10-8 mg/mL and 1.2 × 10-1 to 1.2 × 10-8 mg/mL, respectively) and high sensitivity. The detection limits of CH3L1 and L-FABP were 1.21 × 10-8 mg/mL and 0.62 × 10-8 mg/mL, respectively. In addition, the simultaneous detection of the two biomarkers in serum was demonstrated, showing the feasibility of this method in the complex biological environment. The detection of CH3L1 and L-FABP will greatly improve the early diagnosis and intervention of AKI.

Biomimetic Bilayered Scaffolds for Tissue Engineering: From Current Design Strategies to MedicalApplications.

Tissue damage due to cancer, congenital anomalies, and injuries needs new efficient treatments that allow tissue regeneration. In this context, tissue engineering shows a great potential to restore the native architecture and function of damaged tissues, by combining cells with specific scaffolds. Scaffolds made of natural and/or synthetic polymers and sometimes ceramics play a key role in guiding cell growth and formation of the new tissues. Monolayered scaffolds, which consist of uniform material structure, are reported as not being sufficient to mimic complex biological environment of the tissues. Osteochondral, cutaneous, vascular, and many other tissues all have multilayered structures, therefore multilayered scaffolds seem more advantageous to regenerate these tissues. In this review, recent advances in bilayered scaffolds design applied to regeneration of vascular, bone, cartilage, skin, periodontal, urinary bladder, and tracheal tissues are focused on. After a short introduction on tissue anatomy, composition and fabrication techniques of bilayered scaffolds are explained. Then, experimental results obtained in vitro and in vivo are described, and their limitations are given. Finally, difficulties in scaling up production of bilayer scaffolds and reaching the stage of clinical studies are discussed when multiple scaffold components are used.

Intelligent micro/nanorobots for improved tumor therapy

AbstractA major bottleneck underlying nanomaterial‐based tumor therapy lies in complex biological environment and physiological barriers. Micro/nanorobots with the features of self‐propulsion and controllable navigation have gradually become a research hotspot in the tumor therapeutic community, exhibiting their advantages in efficient cargo loading, controllable cargo delivery, stimuli‐triggered cargo release, deeper tumor tissue penetration, and enhanced cargo accumulation in tumor tissue. In this review, the self‐propulsion and controllable navigation are introduced as two major properties of micro/nanorobots, in which micro/nanorobots are propelled by chemical reactions, physical fields, and biological systems and could be navigated by chemotaxis, remote magnetic guidance, and light. Then, the recent advances of micro/nanorobots for chemotherapy, immunotherapy, photothermal therapy, photodynamic therapy, chemodynamic therapy, and multimodal tumor therapy would be discussed. Finally, the perspective and challenges are also mentioned. It is expected that this review gives an insight into intelligent micro/nanorobots for improved tumor therapy, aiming for more extensive and in‐depth investigations, and final applications in the clinic.

Excited-State Dynamics in Borylated Arylisoquinoline Complexes in Solution and in cellulo.

Two four-coordinate organoboron N,C-chelate complexes with different functional terminals on the PEG chains are studied with respect to their photophysical properties within human MCF-7 cells. Their excited-state properties are characterized by time-resolved pump-probe spectroscopy and fluorescence lifetime microscopy. The excited-state relaxation dynamics of the two complexes are similar when studied in DMSO. Aggregation of the complexes with the carboxylate terminal group is observed in water. When studying the light-driven excited-state dynamics of both complexes in cellulo, i. e., after being taken up into human MCF-7 cells, both complexes show different features depending on the nature of the anchoring PEG chains. The lifetime of a characteristic intramolecular charge-transfer state is significantly shorter when studied in cellulo (360±170 ps) as compared to in DMSO (∼960 ps) at 600 nm for the complexes with an amino group. However, the kinetics of the complexes with the carboxylate group are in line with those recorded in DMSO. On the other hand, the lifetimes of the fluorescent state are almost identical for both complexes in cellulo. These findings underline the importance to evaluate the excited-state properties of fluorophores in a complex biological environment in order to fully account for intra- and intermolecular effects governing the light-induced processes in functional dyes.

An ultra-sensitive dopamine measurement platform based on molecularly imprinted polymer-carbon hybrid nanomaterials for in vitro use

In the present study, we designed an ultrasensitive sensing platform for the evaluation of the physiologically relevant values of basal dopamine (DA) in a culture medium as a complex biological environment. The proposed sensing platform was fabricated via the integration of molecular imprinting technology with carbon hybrid nanomaterials. Carbon nanofibers (CNFs) were grown by using plasma-enhanced chemical vapor deposition (PECVD) on tetrahedral amorphous carbon (ta-C) thin films on silicon wafers. The prepared ta-C/CNFs sensing platforms were electrochemically coated with DA-imprinted polypyrrole as the molecularly imprinted polymer (MIP) or "artificial receptors". The three-dimensional MIP receptors were able to determine trace values of DA in phosphate-buffered saline solution (PBS) pH 7.4 (LOD = 5.43 nM) as well as in the absolute culture media such as DMEM/F-12 medium (LOD = 39 nM), DMEM/F-12 medium supplemented with 15% horse serum and 2.5% fetal bovine serum (LOD = 53.26 nM), and F-12 K cell culture medium (LOD = 62.57 nM), with highly physiologically relevant sensitivity and free of interference by other coexisting biomolecules and biological compounds. As all the fabrication steps of the composite electrode are compatible with common microsystem technology processes, the present results pave the way for integrating these ultra-sensitive electrodes to microelectrode arrays (MEA) platforms used for human dopaminergic neurons studies in vitro and enable continuous measurement of the basal DA concentration in real-time for instance in organoid studies.

Hydrogel-Coated Microelectrode Resists Protein Passivation of In Vivo Amperometric Sensors.

Passivation of electrodes caused by nonspecific adsorption of protein can dramatically reduce sensing sensitivity and accuracy, which is a great challenge for in vivo neurochemical monitoring. However, most antipassivation strategies are not suitable to carbon fiber microelectrodes (CFMEs) for in vivo measurement, and these methods also do not work on electrochemical biosensors that fix biometric elements. In this study, we demonstrate that chitosan hydrogel-coated microelectrodes can avoid the current passivation caused by protein adsorption on the surface of carbon fiber because the chitosan hydrogel prepared by local pH gradient caused by hydrogen evolution reaction has three-dimensional networks containing large amounts of water. The highly hydrophilic three-dimensional structure of hydrogel not only forms a biocompatible interface to confine enzymes but also keeps the fast mass transfer of analytes, such as dopamine, ascorbic acid, and glucose. The consistency of the precalibration and postcalibration of the prepared sensor enables in vivo amperometric detection of both electroactive species based on their redox property and electroinactive species based on the enzyme. This study provides a simple and versatile strategy to constitute an amperometric sensor interface to resist passivation of protein adsorption in a complex biological environment such as the brain.

Plant-Derived Polyphenol and LL-37 Peptide-Modified Nanofibrous Scaffolds for Promotion of Antibacterial Activity, Anti-Inflammation, and Type-H Vascularized Bone Regeneration

The regeneration of oral tissues is a challenging clinical problem because of the complex microbial and biological stress environments. Electrospun fibrous scaffolds have attracted significant interest as effective barrier membranes for guided bone regeneration (GBR); however, no mature strategy yet exists for the surface modification of fibers to provide versatility to satisfy clinical requirements. This study demonstrated a practical biosafety strategy: the combined use of plant polyphenols and LL-37 peptides to modify the fiber surface to endow the fibrous scaffold with antimicrobial activity, immunoregulation, and vascularized bone regeneration. We confirmed that the LL-37 peptides interacted with tannic acid (TA) through noncovalent bonds through experiments and molecular docking simulation analysis. In vitro experiments showed that the TA coating imparted strong antibacterial properties to the fibrous scaffold, but it also caused cytotoxicity. The grafting of LL-37 peptide promoted the spreading, migration, and osteogenic differentiation of mesenchymal stem cells and was also conducive to the M2 polarization of RAW264.7 cells. In vivo experiments further verified that the LL-37 peptide-grafted fibrous scaffold significantly enhanced angiogenesis, anti-inflammatory effects, and type-H vascularized bone regeneration. Overall, the fibrous scaffold modified by the LL-37 peptide through TA grafting has significant potential for GBR applications.

Tumor-derived extracellular vesicles hybridized liposomes in cancer therapy.

Extracellular vesicles (EVs) have gained widespread interest due to their potential in the treatment of inflammation, autoimmune diseases, and immuno-cancer therapy. Among various classes of EVs such as microvesicles (MVs), exosomes, and apoptotic bodies, exosomes are of endosomal origin that are released into the extracellular environment. Exosomes carry complex sets of cargo from their cells of origin, including proteins, lipids, mRNA, and DNA, hence exhibit “Mother Nature”. These are typically 30-150 nm lipidic vesicles rich in integrin proteins that facilitate intrinsic cellular targeting. Within the background, we hypothesized that when these exosomes are hybridized with synthetic liposomes, it would help navigate the hybrid construct in the complex biological environment to find its target. Toward this endeavor, we have re-engineered a synthetic liposome with the exosomes (herein called LEVs) derived from mouse breast cancer and incorporated a fluorescent dye to investigate its potential for cellular targeting. Using the membrane extrusion, we have successfully hybridized both entities resulting in the formation of LEVs and characterized for their colloidal properties and stability over a period. LEVs were monodispersed with an average hydrodynamic size of 120±6. Using Elisa and western-blot, we monitored and quantified exosome-specific protein CD63, which was taken as a handle to ensure the reproducibility of exosome and LEVs. These LEVs were rapidly taken up by immune cells and their parent cells thereby confirming their targeting properties, which can be exploited in drug delivery applications. We will discuss biomedical applications such as its distribution in an animal model, biocompatibility, and imaging in this presentation. We anticipate that this study can picture future opportunities for drug delivery solutions that are superior in target recognition.

Nanoscopy on drug-encapsulating nanosystems by phasor-based fluorescence lifetime analysis.

Understanding the supramolecular organization of active compounds (drugs) encapsulated within standard formulations is becoming increasingly important in nanomedicine. In fact, the synthetic identity of a drug-encapsulating nanosystem is supposed to change throughout its journey, from administration to final fate within the target tissue/cell. Here we addressed this issue by exploiting phasor-based fluorescence lifetime imaging (FLIM) of a drug intrinsic fluorescence signal. As case-study, we selected Irinotecan (CPT-11), a chemotherapy medication used to treat colon cancer, small lung cancer and pancreatic cancer, and focused on its liposomal formulation Onivyde®, launched in 2015 as an orphan drug for the treatment of metastatic ductal adenocarcinoma (mPDAC). After measuring the FLIM signature of the drug's individual physical states, we extracted the fractional intensities of each coexisting physical state of the active principle within the nanoparticle. Our analysis was boosted by means of dedicated custom algorithms based on preliminary data processing and multi-dimensional histogram inference to identify regions of interest in the phasor plot. Whereas, in the case of biological samples, we achieved autonomous cell segmentation by means of computer vision algorithms. Onivyde® in commercial vials was found to coexist in multiple physical states (i.e. gelated/precipitated, free and membrane-associated) and undergo extensive leakage in media mimicking complex biological environments. Moreover, we identified multiple mechanism of cellular uptake and were able to investigate subcellular localization and supramolecular organization of irinotecan in a model of pancreatic cells. In conclusion, we believe that Phasor-FLIM represents a useful platform to quantitatively address the synthetic identity of encapsulated fluorescent drugs, both in cuvette and cellular environments. In this context, we envision many potential applications ranging from batch-to-batch analysis, at the production level, to drug development optimization.

Chiral MoSe2 Nanoparticles for Ultrasensitive Monitoring of Reactive Oxygen Species In Vivo.

Reactive oxygen species (ROS) are involved in neurodegenerative diseases, cancer, and acute hepatitis, and quantification of ROS is critical for the early diagnosis of these diseases. In this work, a novel probe is developed,based on chiral molybdenum diselenide (MoSe2 ) nanoparticles (NPs) modified by the fluorescent molecule, cyanine 3 (Cy3). Chiral MoSe2 NPs show intensive circular dichroism (CD) signals at 390 and 550nm, whereas the fluorescence of Cy3 at 560nm is quenched by MoSe2 NPs. In the presence of ROS, the probe reacts with the ROS and then oxidates rapidly, resulting in decreased CD signals and the recovery of the fluorescence. Using this strategy, the limit of detection values of CD and fluorescent signals in living cells are 0.0093nmol/106 cells and 0.024nmol/106 cells, respectively. The high selectivity and sensitivity to ROS in complex biological environments is attributed to the Mo4+ and Se2- oxidation reactions on the surface of the NPs. Furthermore, chiral MoSe2 NPs are able to monitor the levels of ROS in vivo by the fluorescence. Collectively, this strategy offers a new approach for ROS detection and has the potential to inspire others to explore chiral nanomaterials as biosensors to investigate biological events.

Real-Time Ratiometric Optical Nanoscale Thermometry

All-optical nanothermometry has become a powerful, remote tool for measuring nanoscale temperatures in applications ranging from medicine to nano-optics and solid-state nanodevices. The key features of any candidate nanothermometer are brightness, sensitivity, and (signal, spatial, and temporal) resolution. Here, we demonstrate a real-time, diamond-based nanothermometry technique with excellent sensitivity (1.8% K-1) and record-high resolution (5.8 × 104 K Hz-1/2 W cm-2) based on codoped nanodiamonds. The distinct performance of our approach stems from two factors: (i) temperature sensors─nanodiamonds cohosting two group IV color centers─engineered to emit spectrally separated Stokes and anti-Stokes fluorescence signals under excitation by a single laser source and (ii) a parallel detection scheme based on filtering optics and high-sensitivity photon counters for fast readout. We demonstrate the performance of our method by monitoring temporal changes in the local temperature of a microcircuit and a MoTe2 field-effect transistor. Our work advances a powerful, alternative strategy for time-resolved temperature monitoring and mapping of micro-/nanoscale devices such as microfluidic channels, nanophotonic circuits, and nanoelectronic devices, as well as complex biological environments such as tissues and cells.

Well-dispersed strawberry-like PtCo nanocrystals/porous N-doped carbon nanospheres for multiplexed assays

Small biomolecules such as ascorbic acid (AA), dopamine (DA) and uric acid (UA) coexist in human body, and play decisive roles for evaluating human health. Given that, it is particularly important for rapid and effective detection of their levels in real samples. Enzyme-free electrochemical sensors are widely applied for direct, sensitive and selective monitoring of biomolecules in complex biological environment. Herein, well-dispersed strawberry-like PtCo nanocrystals/porous N-doped carbon nanospheres (PtCo/N-CNSs) were prepared by a one-step pyrolysis. The results showed that the electronic structures and incorporated compositions of the PtCo/N-CNSs tightly correlated to the enhanced electrocatalytic mechanism. Under the optimal conditions, this material based sensor achieved selective detection of AA, DA and UA with broad detection ranges of 30–1900 μM, 0.5–80 μM and 1–145 μM, along with low detection limits (S/N = 3) of 1.02 μM, 0.12 μM and 0.11 μM, respectively. It shows promising applications for actual disease prevention and early diagnosis in clinic.

Locomotion of bovine spermatozoa during the transition from individual cells to bundles

Various locomotion strategies employed by microorganisms are observed in complex biological environments. Spermatozoa assemble into bundles to improve their swimming efficiency compared to individual cells. However, the dynamic mechanisms for the formation of sperm bundles have not been fully characterized. In this study, we numerically and experimentally investigate the locomotion of spermatozoa during the transition from individual cells to bundles of two cells. Three consecutive dynamic behaviors are found across the course of the transition: hydrodynamic attraction/repulsion, alignment, and synchronization. The hydrodynamic attraction/repulsion depends on the relative orientation and distance between spermatozoa as well as their flagellar wave patterns and phase shift. Once the heads are attached, we find a stable equilibrium of the rotational hydrodynamics resulting in the alignment of the heads. The synchronization results from the combined influence of hydrodynamic and mechanical cell-to-cell interactions. Additionally, we find that the flagellar beat is regulated by the interactions during the bundle formation, whereby spermatozoa can synchronize their beats to enhance their swimming velocity.

Catalyst-Accelerated Circular Cascaded DNA Circuits: Simpler Design, Faster Speed, Higher Gain.

DNA cascaded circuits have great potential in detecting low abundance molecules in complex biological environment due to their powerful signal amplification capability and nonenzymatic feature. However, the problem of the cascaded circuits is that the design is relatively complex and the kinetics is slow. Herein, a new design paradigm called catalyst-accelerated circular cascaded circuits is proposed, where the catalyst inlet is implanted and the reaction speed can be adjusted by the catalyst concentration. This new design is very simple and only requires three hairpin probes. Meanwhile, the results of a series of studies demonstrate that the reaction speed can be accelerated and the sensitivity can be also improved. Moreover, endogenous mRNA can also be used as a catalyst to drive the circuits to amplify the detection of target miRNA in live cells and in mice. These catalyst-accelerated circular cascaded circuits can substantially expand the toolbox for intracellular low abundance molecular detection.

Peptides as Baits for the Coprecipitation of SH2 Domain-Containing Proteins.

Phosphotyrosine (pTyr)-containing amino acid sequences have regulatory effects on proteins that contain pTyr recognition motifs, such as Src Homology 2 (SH2) domains. Using pTyr-containing peptides as a bait for coprecipitation, by immobilization of the synthesized phosphopeptides to beads and incubation with cell lysates, enables to study the binding preference of the SH2 domain for the specific pTyr-sequence obtained from a pTyr-containing protein in a complex biological environment. Using phosphopeptides allows to not only assess the wild-type sequence, but also peptides that can contain modified sequences which carry a nonhydrolyzable pTyr or other modifications varying the binding strength and selectivity, for example, to create strong SH2 domain binders to inhibit their interaction with pTyr-containing proteins. This pulldown experiment can be used as an assay to evaluate the ability of a peptide to bind to the protein of interest in the cell lysate or investigate the selectivity of the peptide. Therefore, immobilizing phosphopeptides and using them as a pulldown tool has a wide range of applications.

Focused Engineering of Pyrrolysyl-tRNA Synthetase-Based Orthogonal Translation Systems for the Incorporation of Various Noncanonical Amino Acids.

The expansion of the genetic code has become a valuable tool for molecular biology, biochemistry, and biotechnology. The pyrrolysyl-tRNA synthetase (PylRS) variants with their cognate tRNAPyl derived from methanogenic archaea of the genus Methanosarcina are the most popular tools for ribosomally mediated site-specific and proteome-wide statistical incorporation of noncanonical amino acids (ncAAs) into proteins. The incorporation of ncAAs can be used for numerous biotechnological and even therapeutically relevant applications. Here we present a protocol of engineering PylRS for novel substrates with unique chemical functionalities. These functional groups can act as intrinsic probes, especially in complex biological environments such as mammalian cells, tissues, and even whole animals.

Fluorine-thiol displacement probes for acetaminophen's hepatotoxicity.

Chemicals possessing reactive electrophiles can denature innate proteins leading to undesired toxicity, and the overdose-induced liver injury by drugs containing electrophiles has been one of the major causes of non-approval and withdraw by the US Food and Drug Administration (FDA). Elucidating the associated proteins could guide the future development of therapeutics to circumvent these drugs' toxicities, but was largely limited by the current probing tools due to the steric hindrance of chemical tags including the common "click chemistry" labels. Taking the widely used non-steroidal anti-inflammatory drug acetaminophen (APAP) as an example, we hereby designed and synthesized an APAP analogue using fluorine as a steric-free label. Cell toxicity studies indicated our analogue has similar activity to the parent drug. This analogue was applied to the mouse hepatocellular proteome together with the corresponding desthiobiotin-SH probe for subsequent fluorine-thiol displacement reactions (FTDRs). This set of probes has enabled the labeling and pull-down of hepatocellular target proteins of the APAP metabolite as validated by Western blotting. Our preliminary validation results supported the interaction of APAP with the thioredoxin protein, which is an important redox protein for normal liver function. These results demonstrated that our probes confer minimal steric perturbation and mimic the compounds of interest, allowing for global profiling of interacting proteins. The fluorine-thiol displacement probing system could emerge as a powerful tool to enable the investigation of drug-protein interactions in complex biological environments.

Self-assembly of Au nanocubes for ultrasensitive detection of Alzheimer's disease biomarkers by SERS.

Since presently Alzheimer's disease (AD) is incurable, early diagnosis of AD is crucial. Aβ 1-42 and tau-441 proteins are promising core biomarkers for early diagnosis and early therapeutic intervention in AD. Here we constructed a surface-enhanced Raman spectroscopy (SERS) biosensor for highly sensitive quantitative detection of Aβ 1-42 and tau proteins by preparing gold nanocube (AuNC) superlattices through evaporation self-assembly. The results showed that the method has a wide response range (0.1-10 000 ng mL-1 and 0.01-1000 ng mL-1, respectively) and high sensitivity. The detection limits of Aβ1-42 and tau protein were 0.0416 ng mL-1 and 0.0087 ng mL-1, respectively. In addition, the method was able to rapidly and simultaneously detect the two biomarkers in serum, which showed the feasibility of the method in complex biological environments. The detection of Aβ 1-42 and tau protein has great potential for the accurate prediction and early diagnosis of Alzheimer's disease.

NIR light, pH, and redox-triple responsive nanogels for controlled release.

Herein we report a novel spiropyran (SP)-based organic-inorganic composite nanogel (NG), which was prepared using upconverting nanoparticles, spiropyran, acrylic acid and N,N'-bis(acryloyl)cystamine (BAC) compounds under emulsion polymerisation. Compared with other polymer nanoparticles, the crosslinked multi-stimulus responsive nanogels can adjust the release rate by altering more of the parameters and this can meet the needs of a complex biological environment to control the release of drugs. Doxorubicin hydrochlorides were used as a simulated drug to test the drug loading performance and controllable drug release performance of the composite NGs. Under near-infrared light (NIR) irradiation, an acidic environment or a reducing agent, the delivery of the loaded drugs was by controlled release over 24 hours. Under mild triple stimulation (NIR light, pH 6, and 4 mM reducing agent), the loaded drug could be released more efficiently. The organic-inorganic composite NGs with highly-efficient and controllable release performance for loaded drugs provide many choices for novel stimulus responsive nanocarriers.