A major challenge to achieve effective X-ray radiation therapy is to use a relatively low and safe radiation dose. Various radiosensitizers, which can significantly enhance the radiotherapeutic performance, have been developed. Gold-based nanomaterials, as a new type of nanoparticle-based radiosensitizers, have been extensively used in researches involving cancer radiotherapy. However, the cancer therapeutic effect using the gold nanoparticle-based radiotherapy is usually not significant because of the low cellular uptake efficiency and the autophagy-inducing ability of these gold nanomaterials. Herein, using gold nanospikes (GNSs) as an example, we prepared a series of thiol-poly(ethylene glycol)-modified GNSs terminated with methoxyl (GNSs), amine (NH2-GNSs), folic acid (FA) (FA-GNSs), and the cell-penetrating peptide TAT (TAT-GNSs), and evaluated their effects on X-ray radiotherapy. For the in vitro study, it was found that the ionizing radiation effects of these GNSs were well correlated with their cellular uptake amounts, with the same order of GNSs < NH2-GNSs < FA-GNSs < TAT-GNSs. The sensitization enhancement ratio (SER), which is commonly used to evaluate how effectively radiosensitizers decrease cell proliferation, reaches 2.30 for TAT-GNSs. The extremely high SER value for TAT-GNSs indicates the superior radiosensitization effect of this nanomaterial. The radiation enhancement mechanisms of these GNSs involved the increased reactive oxygen species (ROS), mitochondrial depolarization, and cell cycle redistribution. Western blotting assays confirmed that the surface-modified GNSs could induce the up-regulation of autophagy-related protein (LC3-II) and apoptosis-related protein (active caspase-3) in cancer cells. By monitoring the degradation of the autophagy substrate p62 protein, GNSs caused impairment of autolysosome degradation capacity and autophagosome accumulation. Our data demonstrated that autophagy played a protective role against caner radiotherapy, and the inhibition of protective autophagy with inhibitors would result in the increase of cell apoptosis. Besides the above in vitro experiments, the in vivo tumor growth study also indicated that X-ray + TAT-GNSs treatment had the best tumor growth inhibitory effect, which confirmed the highest radiation sensitizing effect of TAT-GNSs. This work furthered our understanding on the interaction mechanism between gold nanomaterials and cancer cells and should be able to promote the development of nanoradiosensitizers for clinical applications.Keywords: autophagy inhibitor; cell penetrating peptide; gold nanostructures; ionizing radiation; sensitization enhancement ratio (SER);

Immunofluorescence staining is a crucial tool for studying the structure and behavior of intracellular proteins and organelles. During the staining process, the permeabilization treatment is usually required to enhance the penetration of a fluorescent antibody into the cells. However, since most of the membrane imaging dyes as well as the membrane lipids will detach from the cell surface after permeabilization, membrane labeling using these dyes is not compatible with immunofluorescence staining. Herein, by linking cholesterol-polyethylene glycol (PEG-Chol) and fluorescein isothiocyanate (FITC) with the amine-rich glycol chitosan (GC), we prepared a multifunctional polymeric construct, GC-PEG Chol-FITC, and realized permeabilization-tolerant plasma membrane imaging. Owing to the presence of abundant amine groups in the labeling reagent and the membrane proteins/lipids, the addition of paraformaldehyde in the fixation step induces the amine-cross-linking between the labeling reagents and the membrane proteins/lipids, thus preventing the detachment of fluorophores from the cell surface after permeabilization. Besides, the large molecular weight effect of the imaging reagent may also account for its antipermeabilization property. Furthermore, by combining immunofluorescence staining with the plasma membrane labeling by GC-PEG Chol-FITC, we simultaneously imaged the plasma membrane and cytoskeletons, and clearly observed metaphase cells and binucleated cells. The concept of using amine-rich polymeric dyes for plasma membrane imaging will inspire the development of more permeabilization-resistant membrane labeling dyes with better performance, which can realize simultaneous membrane and intracellular protein imaging and facilitate the future studies of membrane–intracellular protein interactions.Keywords: cell surface engineering; cellular imaging; glycol chitosan; immunofluorescence staining; permeabilization;

Benefiting from their inherent localized and controlled release properties, hydrogels are ideal delivery systems for therapeutic drugs or nanoparticles. In particular, applications of hydrogels for the delivery and release of photoresponsive drugs or nanoparticles are receiving increasing attention. However, the effect of the hydrogel matrix on the fluorescence emission and singlet oxygen generation efficiency of the embedded photosensitizers (PSs) has not been clarified. Herein, meso-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP) as a water-soluble PS was encapsulated into an injectable hydrogel formed by glycol chitosan and dibenzaldehyde-terminated telechelic poly(ethylene glycol). Compared to free TMPyP solution, the TMPyP encapsulated in the hydrogel exhibits three distinct advantages: (1) more singlet oxygen was generated under the same laser irradiation condition; (2) much longer tumor retention was observed due to the low fluidity of the hydrogel; and (3) the fluorescence intensity of TMPyP was significantly enhanced in the hydrogel due to its decreased self-quenching effect. These excellent characteristics lead to remarkable anticancer efficacy and superior fluorescence emission property of the TMPyP–hydrogel system, promoting the development of imaging-guided photodynamic therapy.

Because mitochondria are the key regulators for many cellular behaviors and are susceptible to hyperthermia and reactive oxygen species, mitochondria-specific reagents for simultaneous targeting, imaging, and treatment are highly desirable in cancer theranostics. Herein, we developed a mitochondria-targeted cyanine dye IR825-Cl, which possesses two separated excitation wavelength channels for both red fluorescence imaging and near-infrared (NIR) photothermal therapy (PTT). For imaging, IR825-Cl rapidly entered cells and selectively targeted mitochondria. Although IR825-Cl was completely quenched in water, interestingly, this dye had a turn-on response of red fluorescence (610 nm) in mitochondria under 552 nm excitation due to its polarity-responsive fluorescence emission. More interestingly, IR825-Cl realized the selective mitochondrial staining of cancer cells over normal cells and thus served as an ideal fluorescent probe for identifying cancer cells in normal tissues, which is extremely beneficial for cancer theranostics. For PTT, we demonstrated that under 808 nm NIR laser irradiation, this dye efficiently converted optical energy into heat, realizing mitochondria-targeted photothermal cancer therapy. Collectively, this molecule realized both high fluorescence emission (quantum yield > 43%) and effective light-to-heat conversion (17.4%), enabling its applications for wash-free fluorescence imaging for mitochondria and highly efficient fluorescence imaging-guided PTT.Keywords: cell differentiation; mitochondrial imaging/tracking; near-infrared heptamethine cyanine dye; photothermal therapy; turn-on response of visible light fluorescence;

The shape effect of gold (Au) nanomaterials on the efficiency of cancer radiotherapy has not been fully elucidated. To address this issue, Au nanomaterials with different shapes but similar average size (∼50 nm) including spherical gold nanoparticles (GNPs), gold nanospikes (GNSs), and gold nanorods (GNRs) were synthesized and functionalized with poly(ethylene glycol) (PEG) molecules. Although all of these Au nanostructures were coated with the same PEG molecules, their cellular uptake behavior differed significantly. The GNPs showed the highest cellular responses as compared to the GNSs and the GNRs (based on the same gold mass) after incubation with KB cancer cells for 24 h. The cellular uptake in cells increased in the order of GNPs > GNSs > GNRs. Our comparative studies indicated that all of these PEGylated Au nanostructures could induce enhanced cancer cell-killing rates more or less upon X-ray irradiation. The sensitization enhancement ratios (SERs) calculated by a multitarget single-hit model were 1.62, 1.37, and 1.21 corresponding to the treatments of GNPs, GNSs, and GNRs, respectively, demonstrating that the GNPs showed a higher anticancer efficiency than both GNSs and GNRs upon X-ray irradiation. Almost the same values were obtained by dividing the SERs of the three types of Au nanomaterials by their corresponding cellular uptake amounts, indicating that the higher SER of GNPs was due to their much higher cellular uptake efficiency. The above results indicated that the radiation enhancement effects were determined by the amount of the internalized gold atoms. Therefore, to achieve a strong radiosensitization effect in cancer radiotherapy, it is necessary to use Au-based nanomaterials with a high cellular internalization. Further studies on the radiosensitization mechanisms demonstrated that ROS generation and cell cycle redistribution induced by Au nanostructures played essential roles in enhancing radiosensitization. Taken together, our results indicated that the shape of Au-based nanomaterials had a significant influence on cancer radiotherapy. The present work may provide important guidance for the design and use of Au nanostructures in cancer radiotherapy.Keywords: anticancer; gold nanostructures; radiosensitizing effect; shape-dependent; X-ray radiotherapy;

A novel method to produce controllable asymmetric lipid vesicles using Ca2+ is reported. The enrichment of negatively charged phosphatidylserine (PS) molecules in the inner leaflet is found not due to charge–charge attraction, but rather a modulation effect on the occupying size of the headgroups of PS molecules.

In this work, we demonstrate that ultrasmall, photostable and multifunctional carbon quantum dots (or carbon dots, CDs) passivated with polyamine-containing organosilane molecules can realize simultaneous cell imaging and anticancer drug delivery. The presence of abundant surface amine groups makes these CDs be able to covalently link with the anticancer drug, doxorubicin (DOX), with an extremely high drug loading capacity (62.8%), while the surface hydroxyl groups ensure the good water-dispersibility of the CDs–DOX. Besides the use as a drug carrier, the fluorescent CDs also enable the dynamic tracing of the drug release process. When the CDs–DOX complexes were internalized by the human breast cancer cells (MCF-7), DOX could gradually detach from the surface of CDs and enter into the cell nucleus, while the CDs themselves still resided in the cytoplasm. In addition, the in vivo experiments showed that the CDs–DOX complexes exhibited a better tumor inhibition performance than free DOX molecules, which may be ascribed to the prolonged drug accumulation in tumor tissues. Furthermore, the as-synthesized CDs also exhibited negligible cytotoxicity/systemic side effects, and could successfully illuminate mammalian, bacterial and fungal cells, making them good candidates as not only drug delivery vehicles but also universal cell imaging reagents. The present work may have implications for the fabrication of functional carbon-based nanomaterials and foster the development of carbon dots as novel nanotheranostics for various biomedical applications.

Hydrogels constitute a group of polymeric materials which can hold a large amount of water in their three-dimensional networks due to their hydrophilic structures. In the past few years, they have been researched for various biomedical applications, such as drug/cell carriers, tissue engineering, and biosensors. Particularly, the hydrogels used as drug delivery systems have shown distinct advantages in phototherapy. This review presents recent advancements of hydrogel’s use in phototherapeutic applications by focusing on three kinds of phototherapeutic methods including photodynamic therapy (PDT), photothermal therapy (PTT), and phototherapy-containing combination therapy (PCCT). The applications of these therapies in anticancer and antibacterial fields have also been summarized. We hope that this review will inspire researchers to further develop promising materials for phototherapy applications.水凝胶构成了一类高分子材料, 它们的亲水结构使得其能够在三维网络中保有大量的水. 在过去几年中, 水凝胶在生物医学领域的 应用获得了很大的关注, 如作为药物或细胞的载体、组织工程和生物传感器等. 特别地, 水凝胶作为药物输运系统在光疗中拥有显著优点. 本综述总结了水凝胶在光疗应用中的最新进展, 尤其重点讨论了三种光疗方法(包括光动力治疗、光热治疗和组合治疗)及其在抗癌和抗 菌领域的应用. 我们希望本综述将有助于启发未来的相关研究以进一步拓展这种材料在光疗领域的新应用.

We prepare for the first time a novel type of fluorescent carbon quantum dot (or carbon dot, CD) with intrinsic mitochondrial targeting ability by a one-step hydrothermal treatment of chitosan, ethylenediamine and mercaptosuccinic acid. The as-prepared CDs can realize mitochondrial imaging and mitochondria-targeted photodynamic cancer therapy without further modifications of other mitochondriotropic ligands (such as triphenylphosphine, TPP). Currently, many commercial mitochondrial probes suffer from the lack of modifiable groups, poor photostability, short tracking time, high cost and/or complicated staining procedures, which severely limit their applications in live-cell mitochondrial imaging. Compared to commercial mitochondrial probes such as MitoTrackers, our CDs exhibit remarkable features including ultra-simple and cost-effective synthesis, excellent photostability, facile storage, easy surface modification, wash-free and long-term imaging capability and negligible cytotoxicity. Besides, since mitochondria are susceptible to the reactive oxygen species generated during chemo-, photo- or radiotherapy, mitochondria-targeted cancer therapy has attracted much attention due to its satisfying anticancer efficiency. To test if the CDs can be used for mitochondria-targeted drug delivery, they were conjugated with a photosensitizer rose bengal (RB) and the resultant CDs–RB nanomissiles achieved efficient cellular uptake and mitochondrial targeting/accumulation, realizing mitochondria-targeted photodynamic therapy. We believe that the CD-based nanotheranostics holds great promise in various biomedical applications.

Microbial viability assessment plays a key role in many areas such as pathogen detection, infectious disease treatment and antimicrobial drug development. Many conventional viability dyes (such as propidium iodide, PI) used for differentiating live/dead microbes suffer from notable cytotoxicity, poor photostability and are of high cost. Thus their applications for accurate microbial viability determination are limited. Herein, for the first time we report the successful synthesis of fluorescent carbon dots (CDs) from bacteria via one-step hydrothermal carbonization. Benefiting from their highly negative surface charge (the zeta potential is as high as around −42 mV) and suitable size, the CDs can selectively stain dead microbial cells (bacteria and fungi) but not live ones. Importantly, compared to the widely used commercial dye PI, the developed CDs possess many great advantages including low cytotoxicity, multicolor imaging ability, excellent photostability and high selectivity. Moreover, because the synthetic method is simple, inexpensive and eco-friendly, this type of CD is suitable for large-scale production, making it an excellent candidate for microbial live/dead differentiation and viability assessment. The present work explores the feasibility of using bacteria to fabricate novel CDs and broadens the applications of CDs for biomedical applications.

Photodynamic therapy (PDT) has drawn extensive attention as a promising cancer treatment modality. However, most PDT nanoagents suffer from insufficient drug loading capacity, a severe self-quenching effect, premature release of drugs and/or potential toxicity. Herein, we rationally designed an inorganic–organic nanohybrid with high drug loading capacity and superior chemical stability for enhanced PDT. Polyhedral oligomeric silsesquioxane (POSS), an amine-containing cage-shaped building block, was crosslinked with chlorin e6 (Ce6), a carboxyl-containing photosensitizer, via the amine–carboxyl reaction. Polyethylene glycol (PEG) polymers were further modified on the surface of the nanoparticle to improve the aqueous dispersibility and prolong the circulation time of the final nanoconstruct (POSS-Ce6-PEG). The as-prepared POSS-Ce6-PEG has a considerably high loading rate of Ce6 (19.8 wt%) with desirable fluorescence emission and singlet oxygen generation. Besides, in vitro experiments revealed that the nanoagent exhibited enhanced cellular uptake and a preferred intracellular accumulation within mitochondria and the endoplasmic reticulum, resulting in high anticancer efficiency under light irradiation. Furthermore, in vivo imaging-guided PDT was also successfully achieved, showing the effective tumor targeting and ablation ability of POSS-Ce6-PEG. More importantly, the nanoagent possesses negligible dark cytotoxicity and systemic side effects. Therefore, POSS-Ce6-PEG as an eligible PDT theranostic agent holds great potential in clinical applications.

In this study, a series of fluorescent carbon quantum dots (or carbon dots, CDs) with inherent mitochondrial targeting/imaging and cancerous/normal cell differentiation capabilities were prepared by a one-pot solvothermal treatment of glycerol and a silane molecule. Glycerol acted as a solvent and carbon source, and the silane molecule acted as a passivation agent. The as-prepared CDs could specifically and stably (for at least 24 h) visualize mitochondria of various types of cells without the introduction of mitochondria-targeting ligands (such as triphenylphosphonium). In addition, the CDs exhibited extraordinary features including facile synthesis, good water solubility, favorable biocompatibility, and excellent photostability as compared to commercial mitochondrial probes. Moreover, the CDs could efficiently distinguish cancerous cells from normal cells with high fluorescence contrast due to differences in their mitochondrial membrane potentials and substance uptake efficiencies. More importantly, to the best of our knowledge, the present study provides the first example of using CDs to distinguish cancerous cells from normal cells. The remarkable features of mitochondria-targeted imaging and cancerous cell recognition make the CDs an excellent fluorescent probe for various biomedical applications.

In this work, the interaction between a commonly used multifunctional fluorescent drug chlorpromazine (CPZ) and model cell membranes (liposomes, giant unilamellar vesicles and supported lipid membranes) was investigated using various fluorescence-based techniques (steady-state and time-resolved spectroscopy, confocal microscopy and flow cytometry). It was found that CPZ could substantially quench the fluorescence of lipid membrane fluorophores while the drug’s own fluorescence signal was significantly enhanced upon membrane interaction. The drug contact-induced fluorescence quenching response of the membrane fluorophores can be explained by the coexistence of both static and dynamic quenching processes, while the fluorescence enhancement of CPZ is attributed to the change in environmental polarity. Both fatty acid (tail)- and headgroup-labeled fluorophores in the lipid membranes were quenched immediately after the introduction of CPZ, indicating that the CPZ molecules reside at the lipid interfacial region and can interact with both the headgroups and tails of the lipids. In addition, fluorescence quenching was partly recoverable by water washing, indicating that the association of the CPZ molecules with the lipid membrane is not very strong. On the other hand, the significantly enhanced fluorescence intensity of CPZ after insertion into the less polar lipid bilayer enabled us to directly visualize the location of the drug molecules interacting with cell membranes. The current study provides important molecular-level knowledge about CPZ–cell membrane interactions using various model cell membranes, and represents a typical example of deciphering the drug and cell membrane interaction mechanisms using various fluorescence-based techniques.

Carbon nanomaterials have found numerous applications in various fields. However, their synthesis and functionalization usually require complicated procedures or tough experimental conditions. Herein, we report for the first time the synthesis of a new type of functional nanomaterial, quaternized carbon nanospheres (QCNSs), with superior antibacterial activity via a one-pot hydrothermal treatment of chitosan and hexadecylbetaine (abbreviated as BS-16). During the hydrothermal process, the direct reaction and carbonization between the amine-containing chitosan and the carboxyl-containing BS-16 were realized within only one step. The as-prepared QCNSs feature a well-defined spherical morphology and a homogeneous size distribution with an average diameter of ∼110 nm. In particular, the QCNSs could effectively kill Gram-positive bacteria with a minimum inhibitory concentration (MIC) of 2.0–5.0 μg mL−1. Meanwhile, the QCNSs showed excellent cytocompatibility towards normal human liver and lung cells and good hemocompatibility towards red blood cells. Moreover, in bacteria-infected macrophage cells, the QCNSs could selectively kill bacteria while the macrophage cells remained unaffected, which further confirmed their biocompatibility. Besides, we have also elucidated the antibacterial mechanism of the QCNSs by disrupting the bacterial cell walls/membranes via the bacterial adsorption and insertion of the long alkyl chain-containing quaternary ammonium groups on the particle surface. The present work provides a novel method for the preparation of functional carbon nanomaterials, which may promote the development of metal-free antibacterial agents.

To address the issue of low cellular uptake of photosensitizers by cancer cells in photodynamic therapy (PDT), we designed a smart plasma membrane-activatable polymeric nanodrug by conjugating the photosensitizer protoporphyrin IX (PpIX) and polyethylene glycol (PEG) with glycol chitosan (GC). The as-prepared GC-PEG-PpIX can self-assemble into core-shell nanoparticles (NPs) in aqueous solution and the fluorescence of PpIX moieties in the inner core is highly quenched due to strong π–π stacking. Interestingly, when encountering plasma membranes, the GC-PEG-PpIX NPs can disassemble and stably attach to plasma membranes due to the membrane affinity of PpIX moieties, which effectively suppresses the self-quenching of PpIX, leading to significantly enhanced fluorescence and singlet oxygen (1O2) production upon laser irradiation. The massively produced 1O2 can compromise the integrity of the plasma membrane, enabling the influx of extracellular nanoagents into cells to promote cell death upon further laser irradiation. Through local injection, the membrane anchored GC-PEG-PpIX enables strong physical association with tumor cells and exhibits highly enhanced in vivo fluorescence at the tumor site. Besides, excellent tumor accumulation and prolonged tumor retention of GC-PEG-PpIX were realized after intravenous injection, which ensured its effective imaging-guided PDT.Download high-res image (451KB)Download full-size image

Efficient cellular uptake of nanoparticles is crucial for modulating the cell behaviors as well as dictating the cell fate. In this work, by using two commercial reagents (the membrane modification reagent “cholesterol–PEG–biotin” and the avidin-modified quantum dots (QDs) “QD–avidin”), we achieved the enhanced plasma membrane enrichment and endocytosis of fluorescent QDs in cancer cells through cell surface engineering. The QD–cell interaction involved two stages: adsorption and internalization. After incubation with cholesterol–PEG2k–biotin, the cell membrane was engineered with biotin groups that would actively recruit QD–avidin to the cell surface within 1 min. This fast adsorption process could realize high quality and photostable plasma membrane imaging, which is simple, low-cost and generally applicable as compared with the previously reported membrane protein/receptor labeling-based QD imaging. After that, the QDs attached on the cell surface underwent the internalization process and 12 h later, almost all the QDs were internalized through endocytosis. Notably, we found that the internalization of QDs was not via common endocytosis pathways (such as clathrin- or caveolae-mediated endocytosis or macropinocytosis) but more likely via lipid raft-dependent endocytosis. In contrast, without cell surface engineering, the QD–avidin showed negligible cellular uptake. The results demonstrate that cell surface engineering is an efficient strategy to image the plasma membrane and increase cellular uptake of nanoparticles, and will be potentially applied to enhance the efficacy of nanomedicines when therapeutic nanoparticles are used.

Metallic nanostructures as excellent candidates for nanosensitizers have shown enormous potentials in cancer radiotherapy and photothermal therapy. Clinically, a relatively low and safe radiation dose is highly desired to avoid damage to normal tissues. Therefore, the synergistic effect of the low-dosed X-ray radiation and other therapeutic approaches (or so-called “combined therapeutic strategy”) is needed. Herein, we have synthesized hollow and spike-like gold nanostructures by a facile galvanic replacement reaction. Such gold nanospikes (GNSs) with low cytotoxicity exhibited high photothermal conversion efficiency (η = 50.3%) and had excellent photostability under cyclic near-infrared (NIR) laser irradiations. We have demonstrated that these GNSs can be successfully used for in vitro and in vivo X-ray radiation therapy and NIR photothermal therapy. For the in vitro study, colony formation assay clearly demonstrated that GNS-mediated photothermal therapy and X-ray radiotherapy reduced the cell survival fraction to 89% and 51%, respectively. In contrast, the cell survival fraction of the combined radio- and photothermal treatment decreased to 33%. The synergistic cancer treatment performance was attributable to the effect of hyperthermia, which efficiently enhanced the radiosensitizing effect of hypoxic cancer cells that were resistant to ionizing radiation. The sensitization enhancement ratio (SER) of GNSs alone was calculated to be about 1.38, which increased to 1.63 when the GNS treatment was combined with the NIR irradiation, confirming that GNSs are effective radiation sensitizers to enhance X-ray radiation effect through hyperpyrexia. In vivo tumor growth study indicated that the tumor growth inhibition (TGI) in the synergistically treated group reached 92.2%, which was much higher than that of the group treated with the GNS-enhanced X-ray radiation (TGI = 29.8%) or the group treated with the GNS-mediated photothermal therapy (TGI = 70.5%). This research provides a new method to employ GNSs as multifunctional nanosensitizers for synergistic NIR photothermal and X-ray radiation therapy in vitro and in vivo.Keywords: gold nanospikes; photothermal therapy; radiosensitizing; synergistic effect; X-ray radiation therapy

Long-time stable plasma membrane imaging is difficult due to the fast cellular internalization of fluorescent dyes and the quick detachment of the dyes from the membrane. In this study, we developed a two-step synergistic cell surface modification and labeling strategy to realize long-time plasma membrane imaging. Initially, a multisite plasma membrane anchoring reagent, glycol chitosan–10% PEG2000 cholesterol–10% biotin (abbreviated as “GC-Chol-Biotin”), was incubated with cells to modify the plasma membranes with biotin groups with the assistance of the membrane anchoring ability of cholesterol moieties. Fluorescein isothiocyanate (FITC)-conjugated avidin was then introduced to achieve the fluorescence-labeled plasma membranes based on the supramolecular recognition between biotin and avidin. This strategy achieved stable plasma membrane imaging for up to 8 h without substantial internalization of the dyes, and avoided the quick fluorescence loss caused by the detachment of dyes from plasma membranes. We have also demonstrated that the imaging performance of our staining strategy far surpassed that of current commercial plasma membrane imaging reagents such as DiD and CellMask. Furthermore, the photodynamic damage of plasma membranes caused by a photosensitizer, Chlorin e6 (Ce6), was tracked in real time for 5 h during continuous laser irradiation. Plasma membrane behaviors including cell shrinkage, membrane blebbing, and plasma membrane vesiculation could be dynamically recorded. Therefore, the imaging strategy developed in this work may provide a novel platform to investigate plasma membrane behaviors over a relatively long time period.

Because of the distinct surface structures of different cells (mammalian cells, fungi, and bacteria), surface labeling for these cells requires a variety of fluorescent dyes. Besides, fluorescent dyes (especially the commercial ones) for staining Gram-negative bacterial cell walls are still lacking. Herein, a conformation-adjustable glycol chitosan (GC) derivative (GC-PEG cholesterol-FITC) with “all-in-one” property was developed to realize universal imaging for plasma membranes of mammalian cells (via hydrophobic interaction) and cell walls of fungal and bacterial cells (via electrostatic interaction). By comparing the different staining behaviors of GC-PEG cholesterol-FITC and three other analogs (GC-PEG-FITC, GC-FITC, and cholesterol-PEG-FITC), we have elucidated the different roles the hydrophobic and electrostatic interactions play in the staining performance of these different cells. Such a simple, noncytotoxic, economic, and universal cell surface staining reagent will be very useful for investigating cell surface-related biological events and advancing cell surface engineering of various types of cells.Keywords: bioimaging; cell surface engineering; cell surface−biomaterial interaction; electrostatic interaction; glycol chitosan; hydrophobic interaction

The folding/unfolding behavior of proteins (enzymes) in confined space is important for their properties and functions, but such a behavior remains largely unexplored. In this article, we reported our finding that lysozyme and a double hydrophilic block copolymer, methoxypoly(ethylene glycol)5K-block-poly(l-aspartic acid sodium salt)10 (mPEG5K-b-PLD10), can form a polyelectrolyte complex micelle with a particle size of ∼30 nm, as verified by dynamic light scattering and transmission electron microscopy. The unfolding and refolding behaviors of lysozyme molecules in the presence of the copolymer were studied by microcalorimetry and circular dichroism spectroscopy. Upon complex formation with mPEG5K-b-PLD10, lysozyme changed from its initial native state to a new partially unfolded state. Compared with its native state, this copolymer-complexed new folding state of lysozyme has different secondary and tertiary structures, a decreased thermostability, and significantly altered unfolding/refolding behaviors. It was found that the native lysozyme exhibited reversible unfolding and refolding upon heating and subsequent cooling, while lysozyme in the new folding state (complexed with the oppositely charged PLD segments of the polymer) could unfold upon heating but could not refold upon subsequent cooling. By employing the heating–cooling–reheating procedure, the prevention of complex formation between lysozyme and polymer due to the salt screening effect was observed, and the resulting uncomplexed lysozyme regained its proper unfolding and refolding abilities upon heating and subsequent cooling. Besides, we also pointed out the important role the length of the PLD segment played during the formation of micelles and the monodispersity of the formed micelles. Furthermore, the lysozyme–mPEG5K-b-PLD10 mixtures prepared in this work were all transparent, without the formation of large aggregates or precipitates in solution as frequently observed in other protein–polyelectrolyte systems. Hence, the present protein–PEGylated poly(amino acid) mixture provides an ideal water-soluble model system to study the important role of electrostatic interaction in the complexation between proteins and polymers, leading to important new knowledge on the protein–polymer interactions. Moreover, the polyelectrolyte complex micelle formed between protein and PEGylated polymer may provide a good drug delivery vehicle for therapeutic proteins.

Lipid rafts are highly ordered small microdomains mainly composed of glycosphingolipids, cholesterol, and protein receptors. Optically distinguishing lipid raft domains in cell membranes would greatly facilitate the investigations on the structure and dynamics of raft-related cellular behaviors, such as signal transduction, membrane transport (endocytosis), adhesion, and motility. However, current strategies about the visualization of lipid raft domains usually suffer from the low biocompatibility of the probes, invasive detection, or ex situ observation. At the same time, naturally derived biomacromolecules have been extensively used in biomedical field and their interaction with cells remains a long-standing topic since it is closely related to various fundamental studies and potential applications. Herein, noninvasive visualization of lipid raft domains in model lipid bilayers (supported lipid bilayers and giant unilamellar vesicles) and live cells was successfully realized in situ using fluorescent biomacromolecules: the fluorescein isothiocyanate (FITC)-labeled glycol chitosan molecules. We found that the lipid raft domains in model or real membranes could be specifically stained by the FITC-labeled glycol chitosan molecules, which could be attributed to the electrostatic attractive interaction and/or hydrophobic interaction between the probes and the lipid raft domains. Since the FITC-labeled glycol chitosan molecules do not need to completely insert into the lipid bilayer and will not disturb the organization of lipids, they can more accurately visualize the raft domains as compared with other fluorescent dyes that need to be premixed with the various lipid molecules prior to the fabrication of model membranes. Furthermore, the FITC-labeled glycol chitosan molecules were found to be able to resist cellular internalization and could successfully visualize rafts in live cells. The present work provides a new way to achieve the imaging of lipid rafts and also sheds new light on the interaction between biomacromolecules and lipid membranes.

Cholesterol-containing molecules or nanoparticles play a significant role in achieving favorable plasma membrane imaging and efficient cellular uptake of drugs by the excellent membrane anchoring capability of the cholesterol moiety. By linking cholesterol to a water-soluble component (such as poly(ethylene glycol), PEG), the resulting cholesterol-PEG conjugate can form micelles in aqueous solution through self-assembly, and such a micellar structure represents an important drug delivery vehicle in which hydrophobic drugs can be encapsulated. However, the understanding of the subcellular fate and cytotoxicity of cholesterol-PEG conjugates themselves remains elusive. Herein, by using cholesterol-PEG2000-fluorescein isothiocyanate (Chol-PEG-FITC) as a model system, we found that the Chol-PEG-FITC molecules could attach to the plasma membranes of mammalian cells within 10 min and such a firm membrane attachment could last at least 1 h, displaying excellent plasma membrane staining performance that surpassed that of commonly used commercial membrane dyes such as DiD and CellMask. Besides, we systematically studied the endocytosis pathway and intracellular distribution of Chol-PEG-FITC and found that the cell surface adsorption and endocytosis processes of Chol-PEG-FITC molecules were lipid-raft-dependent. After internalization, the Chol-PEG-FITC molecules gradually reached many organelles with membrane structures. At 5 h, they were mainly distributed in lysosomes and the Golgi apparatus, with some in the endoplasmic reticulum (ER) and very few in the mitochondrion. At 12 h, the Chol-PEG-FITC molecules mostly aggregated in the Golgi apparatus and ER close to the nucleus. Finally, we demonstrated that Chol-PEG-FITC was toxic to mammalian cells only at concentrations above 50 μM. In summary, Chol-PEG-FITC can be a promising plasma membrane imaging reagent to avoid the fast cellular internalization and quick membrane detachment problems faced by commercial membrane dyes. We believe that the investigation of the dynamic subcellular fate of Chol-PEG-FITC can provide important knowledge to facilitate the use of cholesterol–PEG conjugates in fields such as cell surface engineering and drug delivery.

In this work, we prepared quaternized carbon dots (CDs) with simultaneous antibacterial and bacterial differentiation capabilities using a simple carboxyl–amine reaction between lauryl betaine and amine-functionalized CDs. The obtained quaternized CDs have several fascinating properties/abilities: (1) A long fluorescence emission wavelength ensures the exceptional bacterial imaging capability, including the super-resolution imaging ability; (2) the polarity-sensitive fluorescence emission property leads to significantly enhanced fluorescence when the quaternized CDs interact with bacteria; (3) the presence of both hydrophobic hydrocarbon chains and positively charged quaternary ammonium groups makes the CDs selectively attach to Gram-positive bacteria, realizing the bacterial differentiation; (4) excellent antimicrobial activity is seen against Gram-positive bacteria with a minimum inhibitory concentration of 8 μg/mL for Staphylococcus aureus. Besides, the quaternized CDs are highly stable in various aqueous solutions and exhibit negligible cytotoxicity, suggesting that they hold great promise for clinical applications. Compared to the traditional Gram staining method, the selective Gram-positive bacterial imaging achieved by the quaternized CDs provides a much simpler and faster method for bacterial differentiation. In summary, by combining selective Gram-positive bacterial recognition, super-resolution imaging, and exceptional antibacterial activity into a single system, the quaternized CDs represent a novel kind of metal-free nanoparticle-based antibiotics for antibacterial application and a new type of reagent for efficient bacterial differentiation.Keywords: bacterial differentiation; metal-free NP-based antibiotics; polarity-sensitive fluorescence; quaternized carbon dots; super-resolution imaging;

Plasma membrane imaging has received substantial attention due to its capability for dynamically tracing significant biological processes including cell trafficking, vesicle transportation, apoptosis, etc. However, cellular internalization of staining molecules poses challenges to the development of fluorescent dyes to specifically label plasma membranes rather than intracellular organelles. In this work, glycol chitosan, a multifunctional biomaterial derived from natural polymers, was used for the first time to image the plasma membranes based on a strategy of multisite membrane anchoring. A glycol chitosan derivative, glycol chitosan–cholesterol–FITC (Chito–Chol–FITC), was synthesized by using glycol chitosan as the backbone, and PEG–cholesterols and FITC molecules as side chains. The cholesterol groups and FITC molecules serve as hydrophobic anchoring units and fluorescent units, respectively. Benefitting from the strategy, this molecular probe could rapidly stain the cell membrane within 5 min as well as effectively restrain the cellular uptake process—it could tolerate an incubation time of 6 h without substantial cellular internalization. Its imaging performance far exceeds that of the current commercial plasma membrane imaging reagents based on small molecules (such as DiD and FM families), which will be easily internalized by the cells within 10–15 min. The present work shows the biomacromolecular assembly of the glycol chitosan derivative on the cell surface, which may shed new light on the interactions of biomaterials with biological systems. Besides, the multisite membrane anchoring strategy developed herein also provides a novel platform for future cell surface engineering studies.

Copper-based nanomaterials have broad applications in electronics, catalysts, solar energy conversion, antibiotics, tissue imaging, and photothermal cancer therapy. However, it is challenging to prepare ultrasmall and ultrastable CuS nanoclusters (NCs) at room temperature. In this article, a simple method to synthesize water-soluble, monodispersed CuS NCs is reported based on the strategy of trapping the reaction intermediate using thiol-terminated, alkyl-containing short-chain poly(ethylene glycol)s (HS-(CH2)11-(OCH2CH2)6-OH, abbreviated as MUH). The MUH-coated CuS NCs have superior stability in solutions with varied pH values and are stable in pure water for at least 10 months. The as-prepared CuS NCs were highly toxic to A549 cancer cells at a concentration of higher than 100 μM (9.6 μg/mL), making them be potentially applicable as anticancer drugs via intravenous administration by liposomal encapsulation or by direct intratumoral injection. Besides, for the first time, CuS NCs were used for antibacterial application, and 800 μM (76.8 μg/mL) CuS NCs could completely kill the E. coli cells through damaging the cell walls. Moreover, the NCs synthesized here have strong near-infrared (NIR) absorption and can be used as a candidate reagent for photothermal therapy and photoacoustic imaging. The method of trapping the reaction intermediate for simple and controlled synthesis of nanoclusters is generally applicable and can be widely used to synthesize many metal-based (such as Pt, Pd, Au, and Ag) nanoclusters and nanocrystals.Keywords: antibacterial; copper sulfide nanocluster; reaction intermediate; short-chain PEG; ultrastable

A simple and highly efficient method for dopamine (DA) detection using water-soluble silicon nanoparticles (SiNPs) was reported. The SiNPs with a high quantum yield of 23.6% were synthesized by using a one-pot microwave-assisted method. The fluorescence quenching capability of a variety of molecules on the synthesized SiNPs has been tested; only DA molecules were found to be able to quench the fluorescence of these SiNPs effectively. Therefore, such a quenching effect can be used to selectively detect DA. All other molecules tested have little interference with the dopamine detection, including ascorbic acid, which commonly exists in cells and can possibly affect the dopamine detection. The ratio of the fluorescence intensity difference between the quenched and unquenched cases versus the fluorescence intensity without quenching (ΔI/I) was observed to be linearly proportional to the DA analyte concentration in the range from 0.005 to 10.0 μM, with a detection limit of 0.3 nM (S/N = 3). To the best of our knowledge, this is the lowest limit for DA detection reported so far. The mechanism of fluorescence quenching is attributed to the energy transfer from the SiNPs to the oxidized dopamine molecules through Förster resonance energy transfer. The reported method of SiNP synthesis is very simple and cheap, making the above sensitive and selective DA detection approach using SiNPs practical for many applications.

The problem of stability hinders the practical applications of nanomaterials. In this research, an innovative and simple synthetic method was developed for preparing ultrastable and multifunctional gold nanoclusters (Au NCs). HS–C11–EG6–X is a class of molecules consisting of four components: a mercapto group (–SH), an alkyl chain (C11), a short chain of polyethylene glycols (EG6) and a functional group (X, X = OH, COOH, NH2, GRGD, etc). The present work demonstrated the importance of using HS–C11–EG6–X to prepare Au NCs with excellent properties and the role each component in this molecule played for synthesizing Au NCs. Au NCs with tunable surface functionalities were successfully synthesized and characterized. It was found that Au NC precursors had a fluorescent quantum yield of 0.4%; in contrast, after capping with HS–C11–EG6–X, the quantum yield significantly increased to 1.3–2.6%. The HS–C11–EG6–X capped Au NCs exhibited superior stability under various solution conditions (including extreme pH, high salt concentration, phosphate buffered saline and cell medium) for at least 6 months, even after conjugation with anticancer drug doxorubicin. Besides, we have also demonstrated that other commonly employed thiol-containing ligands failed to prepare stable fluorescent Au NCs. Moreover, the Au NCs showed negligible toxicity to A549 lung cancer cells up to 100 μM, and the application of the ultrastable Au NCs for anticancer drug delivery has also been demonstrated.

Sum frequency generation (SFG) vibrational spectroscopy was employed to study the interaction between memantine (a water-soluble drug for treating Alzheimer’s disease) and lipid bilayers (including zwitterionic PC and negatively charged PG lipid bilayers) at the molecular level in real time and in situ. SFG results revealed how the memantine affected these lipid bilayers in terms of the lipid dynamics, average tilt angle (θ), as well as angle distribution width (σ). It was found that memantine could adsorb onto the zwitterionic PC surface but did not affect the flip-flop rate of the PC bilayer even in the presence of 5.0 mM memantine, indicating the negligible interaction between memantine and the PC bilayer. However, for the negatively charged PG bilayer, it was found that the outer PG leaflet could be significantly destroyed by memantine at a relatively low memantine concentration (1.0 mM), while the inner PG leaflet remained intact. Besides, the θ and σ of CD3 groups in the outer PG lipid leaflet were calculated to be ∼82.0° and ∼19.5° after adding 5 mM memantine, respectively, indicating that these CD3 groups were prone to lie down at the membrane surface (versus the surface normal) with the addition of 5 mM memantine while nearly standing up without the addition of drug molecules. These monolayer- and molecular-level results could hardly be obtained by other techniques. To the best of our knowledge, this is the first experimental attempt to quantify the drug-induced orientational changes of lipid molecules within a lipid bilayer. The present work provided an in-depth understanding on the interaction between memantine and model cell membranes, which will potentially benefit the development of new drugs for neurodegenerative diseases involving drug–membrane interaction.

Sum frequency generation (SFG) vibrational spectroscopy was applied to study molecular interactions between amantadine and substrate supported lipid bilayers serving as model cell membranes. Both isotopically asymmetric and symmetric lipid bilayers were used in the research. SFG results elucidated how the water-soluble drug, amantadine, influenced the packing state of each leaflet of a lipid bilayer and how the drugs affected the lipid flip-flop process. It is difficult to achieve such detailed molecular-level information using other analytical techniques. Especially, from the flip-flop rate change of isotopically asymmetric lipid bilayer induced by amantadine, important information on the drug–membrane interaction mechanism can be derived. The results show that amantadine can be associated with zwitterionic PC bilayers but has a negligible influence on the flip-flop behavior of PC molecules unless at high concentrations. Different effects of amantadine on the lipid bilayer were observed for the negatively charged DPPG bilayer; low concentration amantadine (e.g., 0.20 mM) in the subphase could immediately disturb the outer lipid leaflet and then the lipid associated amantadine molecules gradually reorganize to cause the outer leaflet to return to the original orderly packed state. Higher concentration amantadine (e.g., 5.0 mM) immediately disordered the packing state of the outer lipid leaflet. For both the high and low concentration cases, amantadine molecules only bind to the outer PG leaflet and cannot translocate to the inner layer. The presence of amantadine within the negatively charged lipid layers has certain implications for using liposomes as drug delivery carriers for amantadine. Besides, by using PC or PG bilayers with both leaflets deuterated, we were able to examine how amantadine is distributed and/or oriented within the lipid bilayer. The present work demonstrates that SFG results can provide an in-depth understanding of the molecular mechanisms of interactions between water-soluble drugs and model cell membranes.

Sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy were applied to study interactions between an antipsychotic agent, chlorpromazine (CPZ), and model cell membranes consisting of either distearoylphosphatidylcholine (DSPC) or dipalmitoylphosphatidylglycerol (DPPG). The PC and PG lipids represent the zwitterionic and anionic components of the cell membranes, respectively. For an isotopically asymmetric bilayer composed of a deuterated lipid leaflet and a hydrogenated lipid leaflet, the time-dependent SFG signals from the lipids revealed that CPZ can significantly accelerate the flip-flop process of the neutral DSPC bilayer and such an acceleration effect is more pronounced at higher CPZ concentrations. While for the negatively charged DPPG bilayer, it was found that CPZ molecules can immediately bind to and disrupt the outer lipid leaflet and then gradually reduce the ordering of the inner lipid leaflet. A higher CPZ concentration in the subphase leads to a faster disordering effect on the inner leaflet. The association of CPZ to the lipid membranes can be verified by the change in the SFG spectra of the OH stretching vibration of the interfacial water molecules. ATR-FTIR results revealed that addition of CPZ to the subphase did not exert significant effect on the dDSPC/dDSPC bilayer, especially at low CPZ concentrations (<2 mM). It was found that CPZ can cause gel-to-fluid phase transition of the dDPPG/dDPPG bilayer at CPZ concentrations below 2 mM, and higher CPZ concentrations can lead to dissolution of the bilayer. This work demonstrated that SFG (along with ATR-FTIR) is a powerful in situ and label-free technique that can be used to study various aspects of the drug–membrane interactions at the molecular level.

Plasma membrane imaging has received substantial attention due to its capability for dynamically tracing significant biological processes including cell trafficking, vesicle transportation, apoptosis, etc. However, cellular internalization of staining molecules poses challenges to the development of fluorescent dyes to specifically label plasma membranes rather than intracellular organelles. In this work, glycol chitosan, a multifunctional biomaterial derived from natural polymers, was used for the first time to image the plasma membranes based on a strategy of multisite membrane anchoring. A glycol chitosan derivative, glycol chitosan–cholesterol–FITC (Chito–Chol–FITC), was synthesized by using glycol chitosan as the backbone, and PEG–cholesterols and FITC molecules as side chains. The cholesterol groups and FITC molecules serve as hydrophobic anchoring units and fluorescent units, respectively. Benefitting from the strategy, this molecular probe could rapidly stain the cell membrane within 5 min as well as effectively restrain the cellular uptake process—it could tolerate an incubation time of 6 h without substantial cellular internalization. Its imaging performance far exceeds that of the current commercial plasma membrane imaging reagents based on small molecules (such as DiD and FM families), which will be easily internalized by the cells within 10–15 min. The present work shows the biomacromolecular assembly of the glycol chitosan derivative on the cell surface, which may shed new light on the interactions of biomaterials with biological systems. Besides, the multisite membrane anchoring strategy developed herein also provides a novel platform for future cell surface engineering studies.

Efficient cellular uptake of nanoparticles is crucial for modulating the cell behaviors as well as dictating the cell fate. In this work, by using two commercial reagents (the membrane modification reagent “cholesterol–PEG–biotin” and the avidin-modified quantum dots (QDs) “QD–avidin”), we achieved the enhanced plasma membrane enrichment and endocytosis of fluorescent QDs in cancer cells through cell surface engineering. The QD–cell interaction involved two stages: adsorption and internalization. After incubation with cholesterol–PEG2k–biotin, the cell membrane was engineered with biotin groups that would actively recruit QD–avidin to the cell surface within 1 min. This fast adsorption process could realize high quality and photostable plasma membrane imaging, which is simple, low-cost and generally applicable as compared with the previously reported membrane protein/receptor labeling-based QD imaging. After that, the QDs attached on the cell surface underwent the internalization process and 12 h later, almost all the QDs were internalized through endocytosis. Notably, we found that the internalization of QDs was not via common endocytosis pathways (such as clathrin- or caveolae-mediated endocytosis or macropinocytosis) but more likely via lipid raft-dependent endocytosis. In contrast, without cell surface engineering, the QD–avidin showed negligible cellular uptake. The results demonstrate that cell surface engineering is an efficient strategy to image the plasma membrane and increase cellular uptake of nanoparticles, and will be potentially applied to enhance the efficacy of nanomedicines when therapeutic nanoparticles are used.