Prevention and treatment of HPV-related cancer through a mRNA vaccine expressing APC-targeting antigen.

Persistent human papillomavirus (HPV) infection is associated with multiple malignancies. Developing therapeutic vaccines to eliminate HPV-infected and malignant cells holds significant value. In this study, we introduced a lipid nanoparticle encapsulated mRNA vaccine expressing tHA-mE7-mE6. Mutations were introduced into E6 and E7 of HPV to eliminate their tumourigenicity. A truncated influenza haemagglutinin protein (tHA), which binds to the CD209 receptor on the surface of dendritic cells (DCs), was fused with mE7-mE6 in order to allow efficient uptake of antigen by antigen presenting cells. The tHA-mE7-mE6 (mRNA) showed higher therapeutic efficacy than mE7-mE6 (mRNA) in an E6 and E7+ tumour model. The treatment resulted in complete tumour regression and prevented tumour formation. Strong CD8+ T-cell immune response was induced, contributing to preventing and curing of E6 and E7+ tumour. Antigen-specific CD8+ T were found in spleens, peripheral blood and in tumours. In addition, the tumour infiltration of DC and NK cells were increased post therapy. In conclusion, this study described a therapeutic mRNA vaccine inducing strong anti-tumour immunity in peripheral and in tumour microenvironment, holding promising potential to treat HPV-induced cancer and to prevent cancer recurrence.

Multistage Systemic and Cytosolic Protein Delivery for Effective Cancer Treatment.

Current clinical applications of protein therapy are largely limited to systemically accessible targets in vascular or extracellular areas. Major obstacles to the widespread application of protein therapeutics in cancer treatment include low membrane permeability and endosomal entrapment. Herein, we report a multistage nanoparticle (NP) strategy for systemic and cytosolic protein delivery to tumor cells, by encapsulating a protein conjugate, tetra-guanidinium (TG)-modified saporin, into tumor microenvironment (TME) pH-responsive polymeric NPs. Upon reaching the tumor site after systemic circulation, the polymeric NPs respond rapidly to the acidic tumor microenvironment and release the TG-saporin conjugates, which penetrate the tumor tissue and enter into tumor cells via TG-mediated cytosolic transportation. The TG-saproin NPs showed potent inhibition of lung cancer cell growth in vitro and in vivo. We expect that this multistage NP delivery strategy with long blood circulation, deep tumor penetration, and efficient cytosolic transport may be applicable to various therapeutic proteins for effective cancer treatment.

Intercalation-Driven Formation of siRNA Nanogels for Cancer Therapy.

RNA interference (RNAi) is a powerful approach in the treatment of various diseases including cancers. The clinical translation of small interfering RNA (siRNA)-based therapy requires safe and efficient delivery vehicles. Here, we report a siRNA nanogels (NG)-based delivery vehicle, which is driven directly by the intercalation between nucleic acid bis-intercalator and siRNA molecules. The intercalation-based siRNA NG exhibits good physiological stability and can enter cells efficiently via different endocytosis pathways. Furthermore, the siRNA NG can not only silence the target genes in vitro but also significantly inhibit the tumor growth in vivo. Therefore, this study provides an intercalation-based strategy for the development of a siRNA delivery platform for cancer therapy. To the best of our knowledge, this is the first report of the intercalation-driven siRNA NG.

mRNA vaccines encoding fusion proteins of monkeypox virus antigens protect mice from vaccinia virus challenge.

The recent outbreaks of mpox have raised concerns over the need for effective vaccines. However, the current approved vaccines have either been associated with safety concerns or are in limited supply. mRNA vaccines, which have shown high efficacy and safety against SARS-CoV-2 infection, are a promising alternative. In this study, three mRNA vaccines are developed that encode monkeypox virus (MPXV) proteins A35R and M1R, including A35R extracellular domain -M1R fusions (VGPox 1 and VGPox 2) and a mixture of encapsulated full-length mRNAs for A35R and M1R (VGPox 3). All three vaccines induce early anti-A35R antibodies in female Balb/c mice, but only VGPox 1 and 2 generate detectable levels of anti-M1R antibodies at day 7 after vaccination. However, all three mRNA vaccine groups completely protect mice from a lethal dose of vaccinia virus (VACV) challenge. A single dose of VGPox 1, 2, and 3 provide protection against the lethal viral challenge within 7 days post-vaccination. Long-term immunity and protection were also observed in all three candidates. Additionally, VGPox 2 provided better passive protection. These results suggest that the VGPox series vaccines enhance immunogenicity and can be a viable alternative to current whole-virus vaccines to defend against mpox.

Heterologous boost with mRNA vaccines against SARS-CoV-2 Delta/Omicron variants following an inactivated whole-virus vaccine.

The coronavirus SARS-CoV-2 has mutated quickly and caused significant global damage. This study characterizes two mRNA vaccines ZSVG-02 (Delta) and ZSVG-02-O (Omicron BA.1), and associating heterologous prime-boost strategy following the prime of a most widely administrated inactivated whole-virus vaccine (BBIBP-CorV). The ZSVG-02-O induces neutralizing antibodies that effectively cross-react with Omicron subvariants. In naïve animals, ZSVG-02 or ZSVG-02-O induce humoral responses skewed to the vaccine's targeting strains, but cellular immune responses cross-react to all variants of concern (VOCs) tested. Following heterologous prime-boost regimes, animals present comparable neutralizing antibody levels and superior protection against Delta and Omicron BA.1variants. Single-boost only generated ancestral and omicron dual-responsive antibodies, probably by "recall" and "reshape" the prime immunity. New Omicron-specific antibody populations, however, appeared only following the second boost with ZSVG-02-O. Overall, our results support a heterologous boost with ZSVG-02-O, providing the best protection against current VOCs in inactivated virus vaccine-primed populations.

Mitochondrion, lysosome, and endoplasmic reticulum: Which is the best target for phototherapy?

Photodynamic therapy (PDT) is a robust cancer treatment modality, and the precise spatiotemporal control of its subcellular action site is crucial for its effectiveness. However, accurate comparison of the efficacy of different organelle-targeted PDT approaches is challenging since it is difficult to find a single system that can achieve separate targeting of different organelles with separable time windows and similar binding amounts. Herein, we conjugated chlorin e6 (Ce6) with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000] (ammonium salt) (DSPE-PEG5000-NH2) to afford DSPE-PEG-Ce6, which could migrate from mitochondrion to lysosome and ultimately to endoplasmic reticulum (ER) after cellular internalization. Benefiting from the dynamic subcellular distribution of DSPE-PEG-Ce6 with tunable organelle-binding amounts, we accurately determined the PDT efficacy order of the molecule, i.e., mitochondrion > ER > lysosome. This work proposes an ideal model system for accurately evaluating the specific organelle-targeted PDT efficacy and may promote the future development of effective PDT strategies.

Bacterial Template Synthesis of Multifunctional Nanospindles for Glutathione Detection and Enhanced Cancer-Specific Chemo-Chemodynamic Therapy.

Biological synthetic methods of nanoparticles have shown great advantages, such as environmental friendliness, low cost, mild reaction conditions, and enhanced biocompatibility and stability of products. Bacteria, as one of the most important living organisms, have been utilized as bioreducing nanofactories to biosynthesize many metal nanoparticles or compounds. Here, inspired by the disinfection process of KMnO4, we for the first time introduce bacteria as both the template and the reducing agent to construct a novel tumor microenvironment-responsive MnO x -based nanoplatform for biomedical applications in various aspects. It is found that the bacterium/MnO x -based nanospindles (EM NSs) can efficiently encapsulate the chemotherapeutic agent doxorubicin (DOX), leading to the fluorescence quenching of the drug. The as-formed DOX-loaded EM NSs (EMD NSs) are proven to be decomposed by glutathione (GSH) and can simultaneously release DOX and Mn2+ ions. The former can be utilized for sensitive fluorescence-based GSH sensing with a limit of detection as low as 0.28 µM and selective cancer therapy, while the latter plays important roles in GSH-activated magnetic resonance imaging and chemodynamic therapy. We also demonstrate that these nanospindles can generate oxygen in the presence of endogenous hydrogen peroxide to inhibit P-glycoprotein expression under hypoxia and can achieve excellent tumor eradication and tumor metastasis inhibition performance. Taken together, this work designs a multifunctional bacterially synthesized nanomissile for imaging-guided tumor-specific chemo-chemodynamic combination therapy and will have implications for the design of microorganism-derived smart nanomedicines.

Nucleolus-Targeted Red Emissive Carbon Dots with Polarity-Sensitive and Excitation-Independent Fluorescence Emission: High-Resolution Cell Imaging and in Vivo Tracking.

Red-emitting carbon dots (CDs) have attracted tremendous attention due to their wide applications in areas including imaging, sensing, drug delivery, and cancer therapy. However, it is still highly challenging for red-emitting CDs to simultaneously achieve high quantum yields (QYs), nucleus targeting, and super-resolution fluorescence imaging (especially the stimulated emission depletion (STED) imaging). Here, it is found that the addition of varied metal ions during the hydrothermal treatment of p-phenylenediamine (pPDA) leads to the formation of fluorescent CDs with emission wavelengths up to 700 nm. Strikingly, although metal ions play a crucial role in the synthesis of CDs with varied QYs, they are absent in the formed CDs, that is, the obtained CDs are metal-free, and the metal ions play a role similar to a "catalyst" during the CD formation. Besides, using pPDA and nickel ions (Ni2+) as raw materials, we prepare Ni-pPCDs which have the highest QY and exhibit various excellent fluorescence properties including excitation-independent emission (at ∼605 nm), good photostability, polarity sensitivity, and ribonucleic acid responsiveness. In vitro and in vivo experiments demonstrate that Ni-pPCDs are highly biocompatible and can realize real-time, wash-free, and high-resolution imaging of cell nuclei and high-contrast imaging of tumor-bearing mice and zebrafish. In summary, the present work may hold great promise in the synthesis and applications of red emissive CDs.

Metal-doped carbon nanoparticles with intrinsic peroxidase-like activity for colorimetric detection of H2O2 and glucose.

Nanomaterial-based enzyme mimics (nanozymes) are attracting increasing attention because of their low production cost, high stability against denaturation, and resistance to high concentrations of substrates. Here, carbon nanoparticles doped with a small amount (<5 mol%) of Pt (denoted as PtCNPs) are synthesized via a facile, cost-effective hydrothermal treatment of p-phenylenediamine (PPD) and K2PtCl4. The obtained PtCNPs possess high aqueous stability, excellent water-dispersibility, and suitable size (∼15 nm). More interestingly, the PtCNPs exhibit an intrinsic peroxidase-like activity that can quickly catalyze 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2) and produce a blue color. Importantly, since satisfactory catalytic properties were also observed when K2PtCl4 was replaced with CuCl2, NiCl2, or Na2PdCl4 during the synthesis, the PPD- and inorganic metal salt-involved hydrothermal synthetic approach may be developed as a general and simple way to fabricate new nanozymes. Besides, the steady-state kinetics reveals that the PtCNPs have a stronger affinity for TMB and a weaker affinity for H2O2 compared with horseradish peroxidase. On the basis of the color reaction, a colorimetric detection method for H2O2 and glucose has been successfully established with a detection limit of 0.15 and 0.30 µM, respectively. Further, the method has also been successfully applied for glucose detection in human serum samples. To sum up, this work develops a new synthetic method of metal-doped carbon nanomaterials and demonstrates their capability for the sensitive and selective detection of H2O2 and glucose, which may foster the development of new nanozymes for biosensing applications.

Endoplasmic reticulum-targeted phototherapy using one-step synthesized trace metal-doped carbon-dominated nanoparticles: Laser-triggered nucleolar delivery and increased tumor accumulation.

Lysosomal entrapment and liver accumulation are the two main obstacles faced by many anticancer drugs for achieving satisfactory therapeutic outcomes. Here, we develop a facile one-step hydrothermal synthetic route to prepare trace metal (M)-, N-, and O-doped carbon-dominated nanoparticles (termed as MNOCNPs, M = Ni, Pd, or Cu, metal content: <0.1 mol%) with exceptional photothermal properties (e.g., the ultrahigh extinction coefficient of 32.7 L g-1 cm-1), which can simultaneously realize preferable endoplasmic reticulum (ER) targeting and specific tumor enrichment without noticeable liver accumulation after poly(ethylene glycol) (PEG) conjugation. More interestingly, the PEG-modified MNOCNPs with nanoscale lengths exhibit considerable nucleolar delivery and increased tumor accumulation upon laser irradiation. After fluorescence labeling, these PEG-modified MNOCNPs are suitable for fluorescence/photoacoustic/thermal triple-modal imaging-guided photothermal cancer treatment. Additionally, the ultralow metal content ensures the exceptional biosafety of the nanoagents. The present work provides a novel, facile, and general synthetic method of carbon-dominated nanoparticles with superior photothermal properties for highly efficient tumor ablation, and the large-organelle (ER and nucleus)-targeted cancer therapeutic strategy may represent an alternative solution for optimizing the anticancer efficacy of nanomaterials. STATEMENT OF SIGNIFICANCE: Limited wire-like nanomaterials have been used for biomedical applications due to their lack of intrinsic photothermal properties, poor cellular uptake and tumor accumulation, and potential biotoxicity arising from their micrometer lengths and/or massive heavy metal doping. Besides, the clinical applications of many nanoagents are hindered by their tendency to accumulate in liver, which may cause severe liver toxicity. Herein, we develop for the first time a one-step hydrothermal method to prepare wire-like trace metal-, N-, and O-doped carbon-dominated nanoparticles with excellent photothermal properties, massive cellular uptake, preferable ER localization, selective tumor targeting with negligible liver deposition, laser irradiation-enhanced nucleolar delivery and tumor accumulation, and multimodal imaging-guided cancer therapy. This work opens a new window for simultaneously overcoming lysosomal entrapment and liver accumulation in cancer therapy.

Multifunctional quaternized carbon dots with enhanced biofilm penetration and eradication efficiencies.

Biofilm formation can lead to the treatment failure of persistent bacterial infections. Although a variety of antibacterial agents have been developed, the restricted drug penetration and the embedded bacteria's potentiated recalcitrance to these agents synergistically lead to the unsatisfactory anti-biofilm effect. Herein, we report the applications of metal-free quaternized carbon dots (CDs) in imaging and eliminating bacterial biofilms. The CDs prepared by the solvothermal treatment of dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (abbreviated as Si-QAC) and glycerol possess ultrasmall size (ca. 3.3 ± 0.4 nm) and strong positively charged (zeta potential: ca. +33.1 ± 2.5 mV) surfaces with long alkyl chain-linked quaternary ammonium groups. The small size of the CDs endows them with the penetration ability into the interior of Gram-negative and Gram-positive bacterial biofilms, which enables excellent fluorescence imaging of the biofilms. Due to the different surfaces of the two types of bacteria, the positively charged CDs selectively interact with the more negatively charged Gram-positive bacteria via electrostatic and hydrophobic interactions, which inactivates the Gram-positive bacteria and ultimately eradicates the Gram-positive bacterial biofilms. In addition, we synthesize a new type of quaternized CDs without long alkyl chains (termed TTPAC CDs), and validate that the long alkyl chains potentiate the hydrophobic adhesion between CDs and Gram-positive bacteria. Meanwhile, the crystal violet staining results reveal that the cationic CDs inhibit the formation of Gram-positive bacterial biofilms. Collectively, our work highlights the feasibility of using cationic and ultrasmall metal-free CDs to eliminate and inhibit Gram-positive bacterial biofilms, which represents a highly effective strategy to cope with refractory biofilm-associated infections.

Fluorescent Carbon Quantum Dots with Intrinsic Nucleolus-Targeting Capability for Nucleolus Imaging and Enhanced Cytosolic and Nuclear Drug Delivery.

Nucleolus tracking and nucleus-targeted photodynamic therapy are attracting increasing attention due to the importance of nucleolus and the sensitivity of nucleus to various therapeutic stimuli. Herein, a new class of multifunctional fluorescent carbon quantum dots (or carbon dots, CDs) synthesized via the one-pot hydrothermal reaction of m-phenylenediamine and l-cysteine was reported to effectively target nucleolus. The as-prepared CDs possess superior properties, such as low-cost and facile synthesis, good water dispersibility, various surface groups for further modifications, prominent photostability, excellent compatibility, and rapid/convenient/wash-free staining procedures. Besides, as compared with SYTO RNASelect (a commonly used commercial dye for nucleolus imaging) that can only image nucleolus in fixed cells, the CDs can realize high-quality nucleolus imaging in not only fixed cells but also living cells, allowing the real-time tracking of nucleolus-related biological behaviors. Furthermore, after conjugating with protoporphyrin IX (PpIX), a commonly used photosensitizer, the resultant CD-PpIX nanomissiles showed remarkably increased cellular uptake and nucleus-targeting properties and achieved greatly enhanced phototherapeutic efficiency because the nuclei show poor tolerance to reactive oxygen species produced during the photodynamic therapy. The in vivo experiments revealed that the negatively charged CD-PpIX nanomissiles could rapidly and specifically target a tumor site after intravenous injection and cause efficient tumor ablation with no toxic side effects after laser irradiation. It is believed that the present CD-based nanosystem will hold great potential in nucleolus imaging and nucleus-targeted drug delivery and cancer therapy.

Ultrasmall All-In-One Nanodots Formed via Carbon Dot-Mediated and Albumin-Based Synthesis: Multimodal Imaging-Guided and Mild Laser-Enhanced Cancer Therapy.

Integration of multiple diagnostic/therapeutic modalities into a single system with ultrasmall size, excellent photothermal/photodynamic properties, high cellular uptake efficiency, nuclear delivery capacity, rapid renal clearance, and good biosafety is highly desirable for cancer theranostics, but still remains challenging. Here, a novel type of multifunctional nanodots (denoted as BCCGH) was synthesized by mixing bovine serum albumin, carbon dots, and metal ions (Cu2+ and Gd3+), followed by the conjugation with a photosensitizer (HPPH). The nanodots hold great promise for fluorescence/photoacoustic/magnetic resonance/photothermal imaging-guided synergistic photothermal/photodynamic therapy (PDT) because of their appealing properties such as high photothermal conversion efficiency (68.4%), high longitudinal relaxivity (11.84 mM-1 s-1, 7 T), and superior colloidal stability with negligible Gd3+ release. Benefiting from the massive cellular uptake, endoplasmic reticulum/mitochondrion-targeting ability, and mild near-infrared laser irradiation-promoted nuclear delivery of BCCGH, a high anticancer therapeutic efficiency is achieved in the subsequent in vitro PDT. Besides, as revealed by the in vivo/ex vivo results, the nanodots also exhibit excellent tumor accumulation, efficient renal clearance, complete tumor ablation, and exceptional biosafety. To summarize, this work develops a carbon dot-mediated and albumin-based synthetic approach for constructing ultrasmall and multifunctional nanodots, which may hold great potential for cancer theranostics and beyond.

Platinum-doped carbon nanoparticles inhibit cancer cell migration under mild laser irradiation: Multi-organelle-targeted photothermal therapy.

Tumor growth and metastasis are two main causes of cancer-related deaths. Here, we simultaneously investigated the effects of nanoparticles on cancer cell viability and migration using polyethylene glycol (PEG)-modified, platinum-doped (<4 mol %) carbon nanoparticles (denoted as PEG-PtCNPs). The bare PtCNPs were prepared by the facile one-step hydrothermal treatment of p-phenylenediamine and K2PtCl4 in aqueous solution. After PEGylation, the obtained PEG-PtCNPs can serve as an excellent photothermal nanoagent for cell migration inhibition, laser-triggered nuclear delivery, effective tumor accumulation, and imaging-guided tumor ablation with improved therapeutic efficacy and reduced side effects. In the absence of laser exposure, the positively charged PEG-PtCNPs with a hydrodynamic diameter of ∼19 nm easily entered the cells by endocytosis and were located in multiple organelles (including mitochondrion, endoplasmic reticulum, lysosome, and Golgi apparatus), causing a slight increase in the expression level of nuclear protein lamin A/C. Upon mild laser irradiation (0.3 W cm-2), the fragmented cytoskeletal structures and overexpression of lamin A/C were observed, thus inhibiting cancer cell migration. Furthermore, hyperthermia induced by PEG-PtCNPs plus laser irradiation at a higher power density (1.0 W cm-2) could cause irreversible damage to the nuclear membranes and then facilitate the nuclear delivery of the nanoagents without the introduction of nuclear targeting ligands. Taken together, this work develops a facile synthetic approach of platinum-based carbon nanoparticles with excellent photothermal properties, and demonstrates their potential applications for modulating tumor metastasis and realizing multi-organelle-targeted tumor ablation.

Hyperthemia-Promoted Cytosolic and Nuclear Delivery of Copper/Carbon Quantum Dot-Crosslinked Nanosheets: Multimodal Imaging-Guided Photothermal Cancer Therapy.

Copper-containing nanomaterials have been applied in various fields because of their appealing physical, chemical, and biomedical properties/functions. Herein, for the first time, a facile, room-temperature, and one-pot method of simply mixing copper ions and sulfur-doped carbon dots (CDs) is developed for the synthesis of copper/carbon quantum dot (or CD)-crosslinked nanosheets (CuCD NSs). The thus-obtained CuCD NSs with the size of 20-30 nm had a high photothermal conversion efficiency of 41.3% and good photothermal stability. Especially, after coating with thiol-polyethylene glycol and fluorescent molecules, the resultant CuCD NSs could selectively target tumor tissues and realize multimodal (photoacoustic, photothermal, and fluorescence) imaging-guided cancer therapy. More importantly, our CuCD NSs exhibited laser-triggered cytosolic delivery, lysosomal escape, and nuclear-targeting properties, which greatly enhanced their therapeutic efficacy. The significantly enhanced tumor accumulation of CuCD NSs after in situ tumor-site laser irradiation was also observed in in vivo experiments. These in vitro and in vivo events occurring during the continuous laser irradiation have not been observed. Overall, this work develops a CD-assisted synthetic method of photothermal nanoagents for triple-modal imaging-guided phototherapy and deepens our understanding of the action mechanism of photothermal therapy, which will promote the development of nanomedicine and beyond.

Carbon quantum dots with intrinsic mitochondrial targeting ability for mitochondria-based theranostics.

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.

Bacteria-derived fluorescent carbon dots for microbial live/dead differentiation.

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.

Enhanced cell membrane enrichment and subsequent cellular internalization of quantum dots via cell surface engineering: illuminating plasma membranes with quantum dots.

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.

Universal Cell Surface Imaging for Mammalian, Fungal, and Bacterial Cells.

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.

Synthesis of ultrastable copper sulfide nanoclusters via trapping the reaction intermediate: potential anticancer and antibacterial applications.

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.