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.

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.

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.

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.

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.

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.

Improving the Phototherapeutic Efficiencies of Molecular and Nanoscale Materials by Targeting Mitochondria.

Mitochondria-targeted cancer phototherapy (PT), which works by delivering photoresponsive agents specifically to mitochondria, is a powerful strategy to improve the phototherapeutic efficiency of anticancer treatments. Mitochondria play an essential role in cellular apoptosis, and are relevant to the chemoresistance of cancer cells. Furthermore, mitochondria are a major player in many cellular processes and are highly sensitive to hyperthermia and reactive oxygen species. Therefore, mitochondria serve as excellent locations for organelle-targeted phototherapy. In this review, we focus on the recent advances of mitochondria-targeting materials for mitochondria-specific PT. The combination of mitochondria-targeted PT with other anticancer strategies is also summarized. In addition, we discuss both the challenges currently faced by mitochondria-based cancer PT and the promises it holds.

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.

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.

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.