3D DNA origami pincers that multitask on giant unilamellar vesicles.

Proteins self-assemble to function in living cells. They may execute essential tasks in the form of monomers, complexes, or supramolecular cages via oligomerization, achieving a sophisticated balance between structural topology and functional dynamics. The modularity and programmability make DNA origami unique in mimicking these key features. Here, we demonstrate three-dimensional reconfigurable DNA origami pincers (DOPs) that multitask on giant unilamellar vesicles (GUVs). By programmably adjusting their pinching angle, the DOPs can dynamically control the degree of GUV remodeling. When oligomerized on the GUV to form origami cages, the DOP units interact with one another and undergo reorganization, resulting in the capture, compartmentalization, and detachment of lipid fragments. This oligomerization process is accompanied with membrane disruptions, enabling the passage of cargo across the membrane. We envisage that interfacing synthetic cells with engineered, multifunctional DNA nanostructures may help to confer customized cellular properties, unleashing the potential of both fields.

Welded Gold Nanoparticle Assemblies Defined Plasmonic Coupling.

Nanoparticle assemblies with interparticle ohmic contacts are crucial for nanodevice fabrication. Despite tremendous progress in DNA-programmable nanoparticle assemblies, seamlessly welding discrete components into welded continuous three-dimensional (3D) configurations remains challenging. Here, we introduce a single-stranded DNA-encoded strategy to customize welded metal nanostructures with tunable morphologies and plasmonic properties. We demonstrate the precise welding of gold nanoparticle assemblies into continuous metal nanostructures with interparticle ohmic contacts through chemical welding in solution. We find that the welded gold nanoparticle assemblies show a consistent morphology with welded efficiency over 90%, such as the rod-like, triangular, and tetrahedral metal nanostructures. Next, we show the versatility of this strategy by welding gold nanoparticle assemblies of varied sizes and shapes. Furthermore, the experiment and simulation show that the welded gold nanoparticle assemblies exhibit defined plasmonic coupling. This single-stranded DNA encoded welding system may provide a new route for accurately building functional plasmonic nanomaterials and devices.

Amelogenin-Inspired Autoadaptive Peptide in a Caries Microenvironment Facilitates Long-Term Protection of the Dentin-Pulp Complex.

Dental caries, one of the most prevalent infectious diseases, is the primary contributor to the early loss of natural teeth and is a significant public health issue. Known as the tooth's bioactive core, the dentin-pulp complex (DPCX) comprises tightly connected hard and soft tissues that not only serve as a biological barrier for the inner tooth tissue but also produce reparative dentin following mild disruptions. While efforts to preserve DPCX are numerous, most strategies focus on temporary antibacterial measures, inflammation reduction, or tissue regeneration, lacking a comprehensive, long-lasting solution. In this study, TVH-19, an autoadaptive peptide mimicking the pH- and ion-responsive capacity of amelogenin, was designed to exert multifaceted preservation of DPCX, providing a comprehensive strategy for preserving vital pulp. Leveraging its unique amphiphilicity-related cell penetration and ion/pH-responsive self-assembly properties, TVH-19 outperforms conventional pulp preservation materials by being capable of rapid cell penetration, minimizing diffused side effects, providing environment-responsive self-assembly/disassembly for balanced long-term antibacterial and cell protection, and facilitating the formation of lysosomal-escaping intracellular aggregates for the continuous activation of PDGFRα+ dental pulp stem cells.

Ultraphotostable Phosphorescent Nanosensors for Sensing the Lysosomal pH at the Single-Cell Level over Long Durations.

Ultraphotostable phosphorescent nanosensors have been designed for continuously sensing the lysosome pH over a long duration. The nanosensors exhibited excellent photostability, high accuracy, and capability to measure pH values during cell proliferation for up to 7 days. By arranging a metal-ligand complex of long phosphorescence lifetime and pH indicator in silica nanoparticles, we discover efficient Förster resonance energy transfer, which converts the pH-responsive UV-vis absorption signal of the pH indicator into a phosphorescent signal. Both the phosphorescent intensity and lifetime change at different pH values, and intracellular pH values can be accurately measured by our custom-built rapid phosphorescent lifetime imaging microscopy. The excellent photostability, high accuracy, and good biocompatibility prove that these nanosensors are a useful tool for tracing the fluctuation of pH values during endocytosis. The methodology can be easily adapted to design new nanosensors with different pKa or for different kinds of intracellular ions, as there are hundreds of pH and ion indicators readily available.

Unveiling the mechanism of an amelogenin-derived peptide in promoting enamel biomimetic remineralization.

Amelogenin and its derived peptides have exhibited excellent efficacy in promoting enamel biomimetic remineralization. However, little is known about their specific action mechanisms. Herein, by combining experiments and computer simulation, the mechanism of an amelogenin-derived peptide QP5 in regulating enamel biomimetic remineralization is unveiled for the first time. In experiments, peptide QP5 was separated into (QPX)5 and C-tail domains, the interactions of peptide-minerals in nucleation solution and the regulation of peptide on enamel biomimetic remineralization were explored. QP5 exhibited an unordered conformation when mineral ions existed, and it could adsorb on minerals through its two domains, thereby inhibiting spontaneous nucleation. The remineralized enamel regulated by C-tail showed better mechanical properties and formed more biomimetic crystals than that of (QPX)5, indicating the C-tail domain of QP5 played an important role in forming enamel-like crystals. The simulation results showed that the conformation of QP5 changed greatly, mainly exhibiting ß-bend, ß-turn, and coil structures, and it eventually adsorbed on enamel through negatively charged residues of the C-tail domain, then captured Ca2+ from solution to promote enamel remineralization. This study improved the evaluation methods of the mechanism of biomimetic peptides, and laid a theoretical basis for the amelioration and clinical transformation of peptide QP5.

DNA-mediated regioselective encoding of colloids for programmable self-assembly.

How far we can push chemical self-assembly is one of the most important scientific questions of the century. Colloidal self-assembly is a bottom-up technique for the rational design of functional materials with desirable collective properties. Due to the programmability of DNA base pairing, surface modification of colloidal particles with DNA has become fundamental for programmable material self-assembly. However, there remains an ever-lasting demand for surface regioselective encoding to realize assemblies that require specific, directional, and orthogonal interactions. Recent advances in surface chemistry have enabled regioselective control over the formation of DNA bonds on the particle surface. In particular, the structural DNA nanotechnology provides a simple yet powerful design strategy with unique regioselective addressability, bringing the complexity of colloidal self-assembly to an unprecedented level. In this review, we summarize the state-of-art advances in DNA-mediated regioselective surface encoding of colloids, with a focus on how the regioselective encoding is introduced and how the regioselective DNA recognition plays a crucial role in the self-assembly of colloidal structures. This review highlights the advantages of DNA-based regioselective modification in improving the complexity of colloidal assembly, and outlines the challenges and opportunities for the construction of more complex architectures with tailored functionalities.

Mineralization promotion and protection effect of carboxymethyl chitosan biomodification in biomimetic mineralization.

Biomimetic mineralization emphasizes reversing the process of dental caries through bio-inspired strategies, in which mineralization promotion and collagen protection are equally important. In this study, carboxymethyl chitosan (CMC) was deemed as an analog of glycosaminoglycan for biomimetic modification of collagen, both of the mineralization facilitation and collagen protection effect were evaluated. Experiments were carried out simultaneously on two-dimensional monolayer reconstituted collagen model, three-dimensional reconstituted collagen model and demineralized dentin model. In three models, CMC was successfully cross-linked onto collagen utilizing biocompatible 1-Ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxy sulfosuccinimide sodium salt to achieve biomodification. Results showed that CMC biomodification increased collagen's hydrophilicity, calcium absorption capacity and thermal degradation resistance. In demineralized dentin model, the activity of endogenous matrix metalloproteinases was significantly inhibited by CMC biomodification. Furthermore, CMC biomodification significantly improved cross-linking and intrafibrillar mineralization of collagen, especially in the two-dimensional monolayer reconstituted collagen model. This study provided a biomimetic mineralization strategy with comprehensive consideration of collagen protection, and enriched the application of chitosan-based materials in dentistry.

Promoting effect of a calcium-responsive self-assembly ß-sheet peptide on collagen intrafibrillar mineralization.

Recently, a de novo synthetic calcium-responsive self-assembly ß-sheet peptide ID8 (Ile-Asp-Ile-Asp-Ile-Asp-Ile-Asp) has been developed to serve as the template inducing hydroxyapatite nucleation. The aim of this study was to evaluate the effect of ID8 on intrafibrillar mineralization of collagen making full use of its self-assembly ability. The mineralization experiments were carried out in vitro on both bare Type I collagen and fully demineralized dentin samples. The calcium-responsive self-assembly of ID8 was revealed by circular dichroism spectrum, 8-anilino-1-naphthalenesulfonic acid ammonium salt hydrate assay, attenuated total reflection Fourier transform infrared spectrum (ATR-FTIR) and transmission electron microscope (TEM). Polyacrylic acid (450 kDa) with a concentration of 100 µg ml-1 was selected as the nucleation inhibitor based on the determination of turbidimetry and TEM with selected area electron diffraction (TEM-SAED). The results showed that collagen intrafibrillar mineralization was significantly promoted with the pretreatment of self-assembly ID8 detected by TEM-SAED, SEM, X-ray diffraction and ATR-FTIR. The pretreatment of collagen utilizing self-assembly ID8 not only enhanced intermolecular hydrogen bonding but also contributed to calcium retention inside collagen and significantly increased the hydrophilicity of collagen. These results indicated that peptides with self-assembly properties like ID8 are expected to be potential tools for biomimetic mineralization of collagen.

Single-Stranded DNA-Encoded Gold Nanoparticle Clusters as Programmable Enzyme Equivalents.

Nanozymes have emerged as a class of novel catalytic nanomaterials that show great potential to substitute natural enzymes in various applications. Nevertheless, spatial organization of multiple subunits in a nanozyme to rationally engineer its catalytic properties remains to be a grand challenge. Here, we report a DNA-based approach to encode the organization of gold nanoparticle clusters (GNCs) for the construction of programmable enzyme equivalents (PEEs). We find that single-stranded (ss-) DNA scaffolds can self-fold into nanostructures with prescribed poly-adenine (polyA) loops and double-stranded stems and that the polyA loops serve as specific sites for seed-free nucleation and growth of GNCs with well-defined particle numbers and interparticle spaces. A spectrum of GNCs, ranging from oligomers with discrete particle numbers (2-4) to polymer-like chains, are in situ synthesized in this manner. The polymeric GNCs with multiple spatially organized nanoparticles as subunits show programmable peroxidase-like catalytic activity that can be tuned by the scaffold size and the inter-polyA spacer length. This study thus opens new routes to the rational design of nanozymes for various biological and biomedical applications.

The Amelogenin-Derived Peptide TVH-19 Promotes Dentinal Tubule Occlusion and Mineralization.

In this study, the amelogenin-derived peptide, TVH-19, which has been confirmed to promote mineralization, was evaluated to derive its potential to induce dentinal tubule occlusion. The binding capability of fluorescein isothiocyanate (FITC)-labeled TVH-19 to the demineralized dentin surface was analyzed by confocal laser scanning microscopy (CLSM). Additionally, the sealing function of the peptide was studied through the remineralization of demineralized dentin in vitro. The adsorption results showed that TVH-19 could bind to the hydroxyapatite and demineralized dentin surfaces, especially to periodontal dentin. Scanning electron microscopy analysis further revealed that TVH-19 created mineral precipitates. The plugging rate in the TVH-19 group was higher than that in the PBS group. Moreover, energy-dispersive X-ray spectroscopy (EDX) results indicated that the calcium/phosphorus (Ca/P) ratio of the new minerals induced by TVH-19 was close to that of the hydroxyapatite. Attenuated total internal reflection-Fourier transform infrared (ATR-FTIR) spectrometry and X-ray diffraction (XRD) results indicated that the hydroxyapatite crystals formed via remineralization elongated the axial growth and closely resembled the natural dentin components. These findings indicate that TVH-19 can effectively promote dentin sealing by binding to the periodontal dentin, promoting mineral deposition, and reducing the space between the dentin tubules.

Evaluating the potential of an amelogenin-derived peptide in tertiary dentin formation.

Several novel biomaterials have been developed for dental pulp capping by inducing tertiary dentin formation. The aim of this study was to evaluate the effect of QP5, an amelogenin-based peptide, on the mineralization of dental pulp cells (DPCs) in vitro and in vivo. The cell viability of human DPCs (hDPCs) after treatment with QP5 was determined using the Cell Counting Kit-8 (CCK-8). Migration of hDPCs was assessed using scratch assays, and the pro-mineralization effect was determined using alkaline phosphatase (ALP) staining, alizarin red staining and the expression of mineralization-related genes and proteins. The results showed that QP5 had little effect on the cell viability, and significantly enhanced the migration capability of hDPCs. QP5 promoted the formation of mineralized nodules, and upregulated the activity of ALP, the expression of mRNA and proteins of mineralization-related genes. A pulp capping model in rats was generated to investigate the biological effect of QP5. The results of micro-computed tomography and haematoxylin and eosin staining indicated that the formation of tertiary dentin in QP5-capping groups was more prominent than that in the negative control group. These results indicated the potential of QP5 as a pulp therapy agent.

TVH-19, a synthetic peptide, induces mineralization of dental pulp cells in vitro and formation of tertiary dentin in vivo.

Functional peptides derived from the active domains of odontogenesis-related proteins have been reported to promote dental hard tissue regeneration. The purpose of this study was to evaluate the effects of an artificially synthesized peptide, TVH-19, on odontoblast differentiation and tertiary dentin formation in indirect pulp capping (IPC) using in vitro and in vivo experiments. TVH-19 did not exhibit any effect on the proliferation of human dental pulp cells (hDPCs) but significantly promoted cell migration, compared with the control (p < 0.05). TVH-19-treated hDPCs showed significantly higher alkaline phosphatase (ALP) activity and stronger alizarin red staining (ARS) reactivity than the control group (p < 0.05). TVH-19 also upregulated the mRNA and protein expression levels of odontogenic genes. After generating IPC in rats, the samples of teeth were studied using micro-computed tomography (Micro-CT), hematoxylin & eosin (HE) staining, and immunohistochemical staining to investigate the functions of TVH-19. The in vivo results showed that TVH-19 induced the formation of tertiary dentin, and reduced inflammation and apoptosis, as evident from the downregulated expression of interleukin 6 (IL-6) and cleaved-Caspase-3 (CL-CASP3). Overall, the results of our study suggest that TVH-19 induces differentiation of hDPCs, promotes tertiary dentin formation, relieves inflammation, and reduces apoptosis, indicating the potential applications of TVH-19 in IPC.

Long-Term Quantitatively Imaging Intracellular Chloride Concentration Using a Core-/Shell-Structured Nanosensor and Time-Domain Dual-Lifetime Referencing Method.

Luminescence lifetime-based nanosensors for chloride ions were designed by incorporating a luminescent ruthenium dye [Ru(1,10-phenanthroline)3] inside silica nanoparticles and chemically labelling their outer surface with chloride ion-sensitive fluorescent dyes (N,N'-bis(carboxypropyl)-9,9'-biacridine). The nanosensor surface was further functionalized with positively charged amino groups to facilitate intracellular uptake via endocytosis and target lysosomes. The nanosensors have an average diameter of 52 nm and are monodispersed in aqueous solutions. Because of the long lifetime of the reference ruthenium dye, the sensor response can be analyzed using the time-domain dual-lifetime referencing (td-DLR) approach. The use of pulsed excitation in td-DLR rather than intense continuous illumination in ratiometric measurements greatly prevents the dye from photobleaching which significantly improves its measurement stability and reproducibility for long-term monitoring. At optimum conditions, the sensor can measure chloride concentration in the range of 0-200 mM with a large ratiometric signal change from 140.9 to 40.2. Combined with our custom-built microscopic td-DLR system, variations of intracellular chloride concentration in lysosomes were imaged quantitatively with a high spatial resolution and accuracy.

Luminescent Oxygen-Sensitive Ink to Produce Highly Secured Anticounterfeiting Labels by Inkjet Printing.

A new covert luminescent anticounterfeiting (AC) technology was developed by employing combinatorial chemistry and concentration-dependent stimulus-responsive luminescent patterns. Oxygen-sensitive materials are carefully tailed to be inkjet printable and to form luminescent color inks. The inks are placed in the tanks of a jet printer. The printed luminescent patterns exhibited multilevel and highly secured AC features. Unlike conventional luminescent AC technology that solely relies on luminescent molecules/nanoparticles, the new technique utilizes the following features to fight counterfeiting: (1) the combination of luminescent oxygen-sensitive probes (OSPs) and the oxygen-permeable matrix (OPM), (2) the unique nonlinear oxygen-responsive behavior, (3) the local oxygen concentration, and (4) a luminescence lifetime reading device. The virtually unlimited number of codes is mainly due to the following features: (a) an almost endless number of combinations of OSPs and OPMs and (b) the nonlinearity of the Stern-Volmer plots that describe quenching of luminescence by oxygen. This combinatorial chemistry strategy makes it very difficult for counterfeiters to find the right composition even when the chemical composition of the luminescent molecules/nanoparticles was known. Information encrypted via this new methodology exhibits extremely high security, as counterfeiters need to identify all (not part of them) the following security measures: (1) the right combination of OSPs and OPMs, (2) the right chemical stimulus (here oxygen), (3) the proper oxygen concentration, and (4) the correct luminescence lifetime values.

Integrating Time-Resolved Imaging Information by Single-Luminophore Dual Thermally Activated Delayed Fluorescence.

The fact that the lifetime of photoluminescence is often difficult to access because of the weakness of the emission signals, seriously limits the possibility to gain local bioimaging information in time-resolved luminescence probing. We aim to provide a solution to this problem by creating a general photophysical strategy based on the use of molecular probes designed for single-luminophore dual thermally activated delayed fluorescence (TADF). The structural and conformational design makes the dual TADF strong in both diluted solution and in an aggregated state, thereby reducing sensitivity to oxygen quenching and enabling a unique dual-channel time-resolved imaging capability. As the two TADF signals show mutual complementarity during probing, a dual-channel means that lifetime mapping is established to reduce the time-resolved imaging distortion by 30-40 %. Consequently, the leading intracellular local imaging information is serialized and integrated, which allows comparison to any single time-resolved signal, and leads to a significant improvement of the probing capacity.

Remineralization of enamel caries by an amelogenin-derived peptide and fluoride in vitro.

Dental caries is one of the most common oral diseases in the world. This study was tantamount to investigate the combinatory effects of an amelogenin-derived peptide (called QP5) and fluoride on the remineralization of artificial enamel caries. The peptide QP5 was synthesized and characterized, and the binding capability of the peptide on hydroxyapatite (HA) and demineralized tooth enamel surface was analysed. Then, the mineralization function of the peptide and fluoride was studied through the spontaneous mineralization testing and remineralization on enamel caries in vitro. First, the novel peptide QP5 could bind on the hydroxyapatite and demineralized tooth enamel surfaces. Second, QP5 can transitorily stabilize the formation of amorphous calcium phosphate and direct the transformation into hydroxyapatite crystals alone and in combination with fluoride. In addition, compared to blocks treated by peptide QP5 alone or fluoride, the sample blocks showed significantly higher surface microhardness, lower mineral loss and shallower lesion depth after treatment with a combination of QP5 and fluoride at high or low concentrations. The peptide QP5 could control the crystallization of hydroxyapatite, and combinatory application of peptide QP5 and fluoride had a potential synergistic effect on the remineralization of enamel caries.

Tuftelin-derived peptide facilitates remineralization of initial enamel caries in vitro.

With the gradual discovery of functional domains in natural proteins, several biologically inspired peptides have been designed for use as biomaterials for hard tissue regeneration and repair. In this study, we designed a tuftelin-derived peptide (TDP) and tested its effects on hydroxyapatite crystallization and remineralization of initial enamel carious lesions in vitro. Using circular dichroism spectroscopy, we found that TDP contained 36.1% ß-sheets and ß-turns, which could be influenced by calcium ions. We verified the ability of TDP to crystallize hydroxyapatite using transmission electron microscopy and its ability to bind to the enamel surface and hydroxyapatite using confocal laser scanning microscopy and Langmuir adsorption isotherms (K = 881.56, N = 1.41 × 10-5 ). Artificial enamel lesions were generated on human enamel blocks and subjected to a 12-day pH cycling model and were treated with 25 µM TDP, 1 g/L sodium fluoride (NaF), or deionized water. We analyzed the results of remineralization by surface microhardness testing, polarized light microscopy, and transverse microradiography. The TDP group showed significantly higher surface microhardness recovery (49.21 ± 1.66%), shallower lesions (34.89 ± 4.05 µm), and less mineral loss (871.33 ± 81.49 vol%·µm) after pH cycling than the deionized water group (p < .05). There were no significant differences between the TDP and NaF groups. Our experiment indicated that TDP could regulate hydroxyapatite crystallization and promote remineralization of enamel caries in vitro.

Antibacterial Effect of Caffeic Acid Phenethyl Ester on Cariogenic Bacteria and Streptococcus mutans Biofilms.

Dental caries is the most common disease in the human mouth. Streptococcus mutans is the primary cariogenic bacterium. Propolis is a nontoxic natural product with a strong inhibitory effect on oral cariogenic bacteria. The polyphenol-rich extract from propolis inhibits S. mutans growth and biofilm formation, as well as the genes involved in virulence and adherence, through the inhibition of glucosyltransferases (GTF). However, because the chemical composition of propolis is highly variable and complex, the mechanism of its antimicrobial action and the active compound are controversial and not completely understood. Caffeic acid phenethyl ester (CAPE) is abundant in the polyphenolic compounds from propolis, and it has many pharmacological effects. In this study, we investigated the antibacterial effects of CAPE on common oral cariogenic bacteria (Streptococcus mutans, Streptococcus sobrinus, Actinomyces viscosus, and Lactobacillus acidophilus) and its effects on the biofilm-forming and cariogenic abilities of S. mutans CAPE shows remarkable antimicrobial activity against cariogenic bacteria. Moreover, CAPE also inhibits the formation of S. mutans biofilms and their metabolic activity in mature biofilms. Furthermore, CAPE can inhibit the key virulence factors of S. mutans associated with cariogenicity, including acid production, acid tolerance, and the bacterium's ability to produce extracellular polysaccharides (EPS), without affecting bacterial viability at subinhibitory levels. In conclusion, CAPE appears to be a new agent with anticariogenic potential, not only via inhibition of the growth of cariogenic bacteria.

Dual-Phase Thermally Activated Delayed Fluorescence Luminogens: A Material for Time-Resolved Imaging Independent of Probe Pretreatment and Probe Concentration.

Developing luminescent probes with long lifetime and high emission efficiency is essential for time-resolved imaging. However, the practical applications usually suffer from emission quenching of traditional luminogens in aggregated states, or from weak emission of aggregation-induced emission type luminogens in monomeric states. Herein, we overcome this dilemma by a rigid-and-flexible alternation design in donor-acceptor-donor skeletons, to achieve a thermally activated delayed fluorescence luminogen with high emission efficiency both in the monomeric state (quantum yield up to 35.3 %) and in the aggregated state (quantum yield up to 30.8 %). Such a dual-phase strong and long-lived emission allows a time-resolved luminescence imaging, with an efficiency independent of probe pretreatment and probe concentration. The findings open opportunities for developing luminescent probes with a usage in larger temporal and spatial scales.