Correction to “Fluorescent Carbon Quantum Dots with Intrinsic Nucleolus-Targeting Capability for Nucleolus Imaging and Enhanced Cytosolic and Nuclear Drug Delivery”

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

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 <i>m</i>-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

Interaction of Polyethylenimine with Model Cell Membranes Studied by Linear and Nonlinear Spectroscopic Techniques

Polyethylenimine (PEI) has been widely used as a transfection agent for gene delivery, but it is cytotoxic and can lead to cell apoptosis. Although several apoptotic mechanisms have been proposed, a molecular level understanding of PEI/cell membrane interaction can help develop further insight into such cytotoxicity. We combined sum frequency generation (SFG) vibrational spectroscopy and attenuated total-internal reflection Fourier transform infrared (ATR-FTIR) spectroscopy to study the effect of PEI on lipid transbilayer movement in supported bilayers (serving as model cell membranes) as a function of lipid composition, PEI concentration, and temperature. For both dipalmitoylphosphatidylglycerol (DPPG) and distearoylphosphatidylcholine (DSPC) bilayers, PEI molecules showed no significant effect on lipid translocation at room temperature (21 °C). In contrast, significant lipid translocation was observed near the physiological temperature (39 °C), indicating the ability of PEI to induce lipid translocation in both negatively charged and zwitterionic lipid bilayers, without the assistance of membrane proteins. Furthermore, results showed that PEI had strong interactions with negatively charged DPPG and weak interactions with zwitterionic DSPC. Concentration-dependent studies indicated that the lipid translocation rate had a linear dependence on the PEI concentration in the subphase. The effects of branched and linear PEIs were compared in the study, showing that branched PEI had a greater effect on the lipid translocation rate due to the higher charge density, which might be a possible indication of higher toxicity. ATR-FTIR spectroscopy verified that the results observed in SFG were mainly caused by lipid translocation, not bilayer damage or removal from the substrate. The combined SFG and ATR-FTIR study provides a powerful method to examine molecular interactions between lipid bilayers and polyelectrolytes at a molecular level. The results can help to develop further understanding on PEI’s cytotoxicity in biological systems

Folding Behaviors of Protein (Lysozyme) Confined in Polyelectrolyte Complex Micelle

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-<i>block</i>-poly­(l-aspartic acid sodium salt)₁₀ (mPEG_5K-<i>b</i>-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 mPEG_5K-<i>b</i>-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–mPEG_5K-<i>b</i>-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

Cholesterol-Assisted Bacterial Cell Surface Engineering for Photodynamic Inactivation of Gram-Positive and Gram-Negative Bacteria

Antibacterial photodynamic therapy (PDT), which enables effective killing of regular and multidrug-resistant (MDR) bacteria, is a promising treatment modality for bacterial infection. However, because most photosensitizer (PS) molecules fail to strongly interact with the surface of Gram-negative bacteria, this technique is suitable for treating only Gram-positive bacterial infection, which largely hampers its practical applications. Herein, we reveal for the first time that cholesterol could significantly facilitate the hydrophobic binding of PSs to the bacterial surface, achieving the hydrophobic interaction-based bacterial cell surface engineering that could effectively photoinactivate both Gram-negative and Gram-positive bacteria. An amphiphilic polymer composed of a polyethylene glycol (PEG) segment terminated with protoporphyrin IX (PpIX, an anionic PS) and cholesterol was constructed (abbreviated Chol-PEG-PpIX), which could self-assemble into micelle-like nanoparticles (NPs) in aqueous solution. When encountering the Gram-negative Escherichia coli cells, the Chol-PEG-PpIX NPs would disassemble and the PpIX moieties could effectively bind to the bacterial surface with the help of the cholesterol moieties, resulting in the significantly enhanced fluorescence emission of the bacterial surface. Under white light irradiation, the light-triggered singlet oxygen (¹O₂) generation of the membrane-bound PpIX could not only severely damage the outer membrane but also facilitate the entry of external Chol-PEG-PpIX into the bacteria, achieving >99.99% bactericidal efficiency. Besides, as expected, the Chol-PEG-PpIX NPs also exhibited excellent antibacterial performance against the Gram-positive Staphylococcus aureus. We also verified that this nanoagent possesses negligible dark cytotoxicity toward mammalian cells and good hemocompatibility. To the best of our knowledge, this study demonstrates for the first time the feasibility of constructing a fully hydrophobic interaction-based and outer membrane-anchored antibacterial PDT nanoagent

Enhanced Fluorescence Emission and Singlet Oxygen Generation of Photosensitizers Embedded in Injectable Hydrogels for Imaging-Guided Photodynamic Cancer Therapy

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

Subcellular Fate of a Fluorescent Cholesterol-Poly(ethylene glycol) Conjugate: An Excellent Plasma Membrane Imaging Reagent

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

Complexation of Lysozyme with Sodium Poly(styrenesulfonate) via the Two-State and Non-Two-State Unfoldings of Lysozyme

To provide an in-depth understanding of the complexation mechanism of protein and polyelectrolyte, a heating–cooling–reheating protocol was employed to study the unfolding and refolding behaviors of a model protein, lysozyme, in the presence of a negatively charged polyelectrolyte, sodium poly­(styrenesulfonate) (PSS). It was found that, with elevated PSS concentration, a new state (state I) was first formed via a “two-state” conversion process and this state could further convert to a completely unfolded state (state II) via a “non-two-state” conversion. This non-two-state conversion process occurs without the coexistence of states I and II but involves the formation of various intermediate unfolded protein structures. Different from the pure lysozyme that exhibited refolding upon cooling from its heat-denatured state, lysozyme in state I could undergo unfolding upon heating but no refolding upon cooling, while lysozyme in state II did not undergo unfolding or refolding upon thermal treatments. In addition, the effects of ionic strength and molecular weight of polyelectrolyte on the unfolding and refolding behaviors of lysozyme were also investigated. The present work provides a better understanding of the principles governing protein–polyelectrolyte interactions and may have implications for the fabrication of biocolloids and biofilms

Long-Time Plasma Membrane Imaging Based on a Two-Step Synergistic Cell Surface Modification Strategy

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

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