Ultrastable High Internal Phase Pickering Emulsions: Forming Mechanism, Processability, and Application in 3D Printing.

High internal phase Pickering emulsions (HIPPEs) are versatile platforms for various applications owing to their low-density, solid-like structure, and large specific surface area. Here, naturally occurring polysaccharide-protein hybrid nanoparticles (PPH NPs) were used to stabilize HIPPEs with an internal phase fraction of 80% at a PPH NP concentration of 1.5%. The obtained HIPPEs displayed a gel-like behavior with excellent stability against centrifugation (10000g, 10 min), temperature (4-121 °C), pH (1.0-11.0), and ionic strength (0-500 mM). Confocal laser scanning microscope and cryo-scanning electron microscopy results showed that PPH NPs contributed to the stability of HIPPEs by effectively adsorbing and anchoring on the surface of the emulsion droplets layer by layer to form a dense 3D network barrier to inhibit droplet coalescence. The rheological analysis showed that the HIPPEs possessed a higher viscosity and lower frequency dependence with increasing PPH NP concentration, suggesting the potential application of such HIPPEs in three-dimensional (3D) printing, which was subsequently confirmed by a 3D printing experiment. This work provides highly stable and processable HIPPEs, which can be developed as facile and reusable materials for numerous applications. They can also be directly used for future food manufacturing, drug and nutrient delivery, and tissue reconstruction.

See the Unseen: Red-Emissive Carbon Dots for Visualizing the Nucleolar Structures in Two Model Animals and In Vivo Drug Toxicity.

Nucleolus, which participates in many crucial cellular activities, is an ideal target for evaluating the state of a cell or an organism. Here, bright red-emissive carbon dots (termed CPCDs) with excitation-independent/polarity-dependent fluorescence emission are synthesized by a one-step hydrothermal reaction between congo red and p-phenylenediamine. The CPCDs can achieve wash-free, real-time, long-term, and high-quality nucleolus imaging in live cells, as well as in vivo imaging of two common model animals-zebrafish and Caenorhabditis elegans (C. elegans). Strikingly, CPCDs realize the nucleolus imaging of organs/flowing blood cells in zebrafish at a cellular level for the first time, and the superb nucleolus imaging of C. elegans suggests that the germ cells in the spermatheca probably have no intact nuclei. These previously unachieved imaging results of the cells/tissues/organs may guide the zebrafish-related studies and benefit the research of C. elegans development. More importantly, a novel strategy based on CPCDs for in vivo toxicity evaluation of materials/drugs (e.g., Ag+ ), which can visualize the otherwise unseen injuries in zebrafish, is developed. In conclusion, the CPCDs represent a robust tool for visualizing the structures and dynamic behaviors of live zebrafish and C. elegans, and may find important applications in cell biology and toxicology.

Cationic Liposomes with Different Lipid Ratios: Antibacterial Activity, Antibacterial Mechanism, and Cytotoxicity Evaluations.

Due to their strong bacterial binding and bacterial toxicity, cationic liposomes have been utilized as effective antibacterial materials in many studies. However, few researchers have systematically compared their antibacterial activity with their mammalian cell cytotoxicity or have deeply explored their antibacterial and cytotoxicity mechanisms. Here, we prepared a series of cationic liposomes (termed CLs) using dimethyldioctadecylammonium chloride (DODAC) and lecithin at different molar ratios. CLs have the ability to effectively bind with Gram-positive and Gram-negative bacteria through electrostatic and hydrophobic interactions. Further, the CLs with high molar ratios of DODAC (30 and 40 mol%) can disrupt the bacterial wall/membrane, efficiently inducing the production of reactive oxygen species (ROS). More importantly, we carefully compared the antibacterial activity and the mammalian cell cytotoxicity of various CLs differing in DODAC contents and liposomal concentrations and revealed that, whether they are bacterial or mammalian cells, an increasing DODAC content in CLs can lead to an elevated cytotoxicity level. Further, there exists a critical DODAC contents (>20 mol%) in CLs to endow them with effective antibacterial ability. However, the variation in the DODAC content and liposomal concentration of CLs has different degrees of influence on the antibacterial activity or cytotoxicity. For example, CLs at high DODAC content (i.e., CL0.3 and CL0.4) could effectively kill both types of bacterial cells but only cause negligible toxicity to mammalian cells. We believe that a systematic comparison between the antibacterial activity and the cytotoxicity of CLs with different DODAC contents will provide an important reference for the potential clinical applications of cationic liposomes.

Fluorescent dendrimer-based probes for cell membrane imaging: Zebrafish epidermal labeling-based toxicity evaluation.

Visualizing the plasma membrane of living mammalian cells both in vitro and in vivo is crucial for tracking their cellular activities. However, due to the complex and dynamic nature of the plasma membrane, most commercial dyes for membrane staining can only realize very limited imaging performance. Thus, precise and stable plasma membrane imaging remains technically challenging. Here, by taking advantage of the small, well-defined, and amine-rich dendrimers, we prepared poly(ethylene glycol)-cholesterol (PEG-Chol)-conjugated and cyanine dye (e.g., cyanine2, cyanine3, and cyanine5)-labeled dendrimer nanoprobes (termed DPC-Cy2, DPC-Cy3, and DPC-Cy5 NPs). It was revealed that these probes enabled universal, wash-free, long-term (at least 8 h), and multicolor (green, yellow, and red) plasma membrane labeling of a variety of live mammalian cells. Further, we confirmed that the nanoprobes (using DPC-Cy5 as a representative) could achieve high-quality, wash-free, and stable cell surface labeling of live zebrafish embryos. More importantly, we demonstrated that our probes could act as biosensors to visualize the toxicity of metal-organic frameworks (MOFs) toward the epidermal cells of zebrafish embryos, and thus they hold great potential for identifying the toxic effect of drugs/materials at the single-cell scale or in live animals. The present work highlights the advantages of utilizing dendrimers for constructing functional imaging materials, and it is also believed that the fluorescent dendrimer nanoprobes developed in this work may find wide applications like cell imaging, drug toxicity evaluation, and cellular state monitoring.

Antibacterial and Fluorescence Staining Properties of an Innovative GTR Membrane Containing 45S5BGs and AIE Molecules In Vitro.

This study aimed to add two functional components-antibacterial 45S5BGs particles and AIE nanoparticles (TPE-NIM+) with bioprobe characteristics-to the guided tissue regeneration (GTR) membrane, to optimize the performance. The PLGA/BG/TPE-NIM+ membrane was synthesized. The static water contact angle, morphologies, and surface element analysis of the membrane were then characterized. In vitro biocompatibility was tested with MC3T3-E1 cells using CCK-8 assay, and antibacterial property was evaluated with Streptococcus mutans and Porphyromonas gingivalis by the LIVE/DEAD bacterial staining and dilution plating procedure. The fluorescence staining of bacteria was observed by Laser Scanning Confocal Microscope. The results showed that the average water contact angle was 46°. In the cytotoxicity test, except for the positive control group, there was no significant difference among the groups (p > 0.05). The antibacterial effect in the PLGA/BG/TPE-NIM+ group was significantly (p < 0.01), while the sterilization rate was 99.99%, better than that in the PLGA/BG group (98.62%) (p < 0.01). Confocal images showed that the membrane efficiently distinguished G+ bacteria from G- bacteria. This study demonstrated that the PLGA/BG/TPE-NIM+ membrane showed good biocompatibility, efficient sterilization performance, and surface mineralization ability and could be used to detect pathogens in a simple, fast, and wash-free protocol.

Naphthalimide-based multifunctional AIEgens: Selective, fast, and wash-free fluorescence tracking and identification of Gram-positive bacteria.

Pathogenic infections, particularly caused by Gram-positive bacteria (G+), pose a serious threat to human health, and therefore the fast and accurate discrimination of G+ bacteria from Gram-negative bacteria (G-) and fungi is highly desirable. Organic molecules with facile synthesis, robust photostability, good biocompatibility, and high selectivity toward pathogens are urgently needed in the clinical diagnosis and therapy. To this end, herein we report the synthesis of two naphthalimide-based bioprobes named tetraphenylethylene-naphthalimide (TPE-NIM) and triphenylamine-naphthalimide (TPA-NIM) with aggregation-induced emission (AIE) characteristic. First, the staining capacity of the designed AIEgens toward six kinds of bacteria and two kinds of fungi was evaluated. Both TPE-NIM and TPA-NIM showed a high degree of binding/imaging selectivity for G+ bacteria over G- bacteria and fungi via a wash-free protocol. Second, the two AIEgens had the ability to visualize the biofilms formed by G+ bacteria (Staphylococcus aureus) and can quickly track the G+ bacteria (Staphylococcus aureus) in red blood cell suspensions. Third, we have revealed that electrostatic attraction and hydrophobic interaction both contribute to the selective binding of the AIEgens toward G+ bacteria. In view of the high binding/imaging specificity toward G+ bacteria, low hemolysis rates, and low toxicity toward the bacterial cells, these AIEgens can be applied for the clinical detection of pathogenic infections caused by G+ bacteria and broaden the theranostic applications of AIE materials.

Rational Design of Self-Assembled Cationic Porphyrin-Based Nanoparticles for Efficient Photodynamic Inactivation of Bacteria.

Bacterial infection has become an urgent health problem in the world. Especially, the evolving resistance of bacteria to antibiotics makes the issue more challenging, and thus new treatments to fight these infections are needed. Antibacterial photodynamic therapy (aPDT) is recognized as a novel and promising method to inactivate a wide range of bacteria with few possibilities to develop drug resistance. However, the photosensitizers (PSs) are not effective against Gram-negative bacteria in many cases. Herein, we use conjugated meso-tetra(4-carboxyphenyl)porphine (TCPP) and triaminoguanidinium chloride (TG) to construct self-assembled cationic TCPP-TG nanoparticles (NPs) for efficient bacterial inactivation under visible light illumination. The TCPP-TG NPs can rapidly adhere to both Gram-negative and Gram-positive bacteria and display promoted singlet oxygen (1O2) generation compared with TCPP under light irradiation. The high local positive charge density of TCPP-TG NPs facilitates the interaction between the NPs and bacteria. Consequently, the TCPP-TG NPs produce an elevated concentration of local 1O2 under light irradiation, resulting in an extraordinarily high antibacterial efficiency (99.9999% inactivation of the representative bacteria within 4 min). Furthermore, the TCPP-TG NPs show excellent water dispersity and stability during 4 months of storage. Therefore, the rationally designed TCPP-TG NPs are a promising antibacterial agent for effective aPDT.

Efficient cell surface labelling of live zebrafish embryos: wash-free fluorescence imaging for cellular dynamics tracking and nanotoxicity evaluation.

Imaging the dynamics and behaviors of plasma membranes is at the leading edge of life science research. We report here the development of a universal red-fluorescent probe Chol-PEG-Cy5 for wash-free plasma membrane labelling both in vitro and in vivo. In aqueous solutions, the fluorescence of Chol-PEG-Cy5 is significantly quenched due to the intermolecular resonance energy transfer (RET) between neighbouring Cy5 moieties; however, upon membrane anchoring, the probes undergo lateral diffusion in lipid bilayers, resulting in weakened RET and turn-on fluorescence emission. We demonstrate that Chol-PEG-Cy5 enables rapid, stable and high-quality in vitro cell surface imaging in a variety of mammalian cells. Additionally, with the assistance of three-dimensional (3D) image reconstruction, we achieve for the first time the whole-mount in situ fluorescence imaging of the epidermal cell surfaces of live zebrafish embryos, which cannot be realized by conventional plasma membrane probes due to the presence of the surface-covering mucus barrier. This novel technique encourages us to track the cellular dynamics of the epidermis during embryonic development with 3D visualization. Moreover, we also develop a new method to evaluate the epidermal toxicity of nanomaterials (e.g., gold nanoparticles and graphene oxide nanosheets) toward zebrafish embryos using this fluorescent probe.

Cholesterol-Modified Dendrimers for Constructing a Tumor Microenvironment-Responsive Drug Delivery System.

Poly(amidoamine) (PAMAM) dendrimers are widely used as templates for synthesizing small nanoparticles. Herein, the surfaces of amine-terminated generation 4 (G4-NH2) poly(amidoamine) (PAMAM) dendrimers are modified with poly(ethylene glycol)-cholesterol to form self-assembled nanoparticles (termed DPC NPs). It is found that the cellular uptake of DPC NPs increases in a cholesterol-content-dependent manner. Benefiting from the hydrophobic cores of dendrimers, the DPC NPs are developed as drug carriers to encapsulate the photosensitizer chlorin e6 (Ce6) to obtain DPCC NPs for photodynamic therapy (PDT). To improve the PDT efficacy of the DPCC NPs in hypoxic tumor tissues, MnO2 was synthesized in DPCC NPs in situ to obtain Ce6/MnO2@DPC NPs (termed DPCCM NPs) based on the strong coordination between dendrimer and Mn2+. The as-designed DPCCM NPs behave like nanozymes, which can catalyze H2O2 to produce O2 and achieve enhanced PDT effect upon 670 nm laser irradiation. The in vivo imaging experiments for cyanine7 (a near-infrared fluorescent dye)-conjugated DPCCM NPs reveal the excellent tumor accumulation performance of the nanozyme in tumor-bearing mice after intravenous administration. Finally, we have demonstrated the satisfactory in vivo antitumor therapeutic outcome and the good biosafety of the nanozyme. This work provides a new strategy for increasing the cellular uptake of drugs and proposes a method of synthesizing smart nanozymes based on dendrimers.

Plasma membrane-anchorable photosensitizing nanomicelles for lipid raft-responsive and light-controllable intracellular drug delivery.

The past decade has witnessed a growing number of nanoparticulate drug delivery systems for cancer treatment. However, insufficient cellular uptake by cancer cells and the undesirable endo/lysosomal entrapment of internalized therapeutic drugs remain the "Achilles heel" of many developed nanoagents. Here, we develop a novel lipid raft-responsive and light-controllable polymeric drug for efficient cytosolic delivery of photosensitizers. Conjugating a photosensitizer protoporphyrin IX (PpIX) to a polyethylene glycol-cholesterol polymer affords the amphiphilic drug (denoted as Chol-PEG-PpIX) that forms micelles in aqueous solutions. The Chol-PEG-PpIX with two hydrophobic units (cholesterol and PpIX) showed robust binding to plasma membranes and enabled significant cellular uptake via two pathways: (1) cholesterol moiety triggered the lipid raft-mediated endocytosis of Chol-PEG-PpIX with minimized endo/lysosomal trafficking after internalization; (2) the membrane-bound PpIX acted as a light-controlled trigger and can augment the permeability of plasma membranes upon laser irradiation, allowing the rapid influx of extracellular Chol-PEG-PpIX within 5 min. For systemic drug delivery, Chol-PEG-PpIX was anchored on the surface of liposomes via in situ membrane modification, which substantially avoided nonspecific binding of Chol-PEG-PpIX to red blood cells during circulation. Besides, the Chol-PEG-PpIX-anchored liposomes exhibited enhanced in vivo fluorescence, reduced liver uptake, prolonged tumor retention, and effective tumor ablation by photodynamic therapy. This work illustrates a new strategy for direct and efficient cytosolic delivery of photosensitizers, which may hold great promise in cancer therapy.

Turning Toxicants into Safe Therapeutic Drugs: Cytolytic Peptide-Photosensitizer Assemblies for Optimized In Vivo Delivery of Melittin.

Melittin (MEL) is recognized as a highly potent therapeutic peptide for treating various human diseases including cancer. However, the clinical applications of MEL are largely restricted by its severe hemolytic activity and nonspecific cytotoxicity. Here, it is found that MEL can form a stable supramolecular nanocomplex of ≈60 nm with the photosensitizer chlorin e6 (Ce6), which after hyaluronic acid (HA) coating can achieve robust, safe, and imaging-guided tumor ablation. The as-designed nanocomplex (denoted as MEL/Ce6@HA) shows largely reduced hemolysis and selective cytolytic activity toward cancer cells. Upon laser irradiation, the loaded photosensitive Ce6 can synergistically facilitate the membrane-lytic efficiency of melittin and greatly increase the tumor penetration depth of the complexes in multicellular tumor spheroids. In vivo experiments reveal that MEL/Ce6@HA can realize enhanced tumor accumulation, reduced liver deposition, and rapid body clearance, which are beneficial for highly efficient and safe chemo-photodynamic dual therapy. This work develops a unique supramolecular strategy for optimized in vivo delivery of melittin and may have implications for the development of peptide-based theranostics.