Layer‐by‐Layer Assembly of Functional Nanoparticles for Hepatocellular Carcinoma Therapy

Cancer treatments with conventional approaches often result in limited clinical outcomes due to inefficient therapeutic efficacy and cumulative toxicity against normal tissue. Recently, most research has focused on combined therapeutic studies by functional carriers. In this study, functional nanoparticles (FNPs) are assembled in a layer‐by‐layer fashion. FNPs are loaded with two drugs (10‐hydroxycamptothecin and apoptin plasmid) with dual hepatocellular carcinoma‐targeting ligands (lactobionic acid and biotin) on the surface. Cytotoxicity studies and acute toxicity experiments in BAL b/c mice show that blank FNPs demonstrate good biocompatibility. Flow cytometry analysis and cytotoxicity studies demonstrate that the dual‐targeting FNPs allow for better specificity and selectivity of the tumor mass. FNPs can escape from endosomal/lysosomal compartments effectively, as is demonstrated using the Cell Navigator lysosome staining kit. When the drugs are released into the cytosol, the nuclear localization signal can enhance the nuclear delivery of 10‐hydroxycamptothecin loaded carriers and apoptin plasmids, as is demonstrated by confocal laser scanning microscopy. In vivo experiments show the circulation time and tissue distribution of FNPs, which greatly improve the therapeutic efficacy of BAL b/c nude mice with subcutaneous tumors. Taken together, the results suggest that FNPs are a promising candidate for hepatocellular carcinoma therapy.     

Fluorescent Conjugated Polyelectrolytes for Bioimaging

This Feature Article summarizes the recent advances of water‐soluble fluorescent conjugated polyelectrolytes (CPEs) in bioimaging. Apart from a brief overview of traditional linear CPEs, a special emphasis is placed on CPEs that can self‐assemble into or are born with three‐dimensional nano‐architectures, including grafted CPEs, hyperbranched CPEs, and polyhedral oligomeric silsesquioxanes(POSS)‐based CPE derivatives. These CPEs naturally form nanoparticles with sizes ranging from 3 to 100 nm in aqueous media, and possess reactive functional groups for bioconjugation or complexation with desired biorecognition elements. The tunable size, low cytotoxicity, good photostability, and ease of surface modification ultimately enable these CPEs with wide applications in in vitro intracellular protein sensing, cell detection, in vivo cell imaging and drug tracking. Moreover, traditional linear CPEs can be transformed into uniform nanoparticles by complexation with oppositely charged biomolecules to allow for cell detection and in situ drug release monitoring. The work featured herein not only reveals the important molecular design principles of CPEs for different imaging tasks, but also highlights the promising directions for the further development of CPE‐based imaging materials.     

Tetrahydro[5]helicene‐Based Nanoparticles for Structure‐Dependent Cell Fluorescent Imaging

Four new fluorescent dyes containing tetrahydro[5]helicene moiety characterized by three‐primary emission colors (blue‐green‐red) are designed and synthesized, and their structures are characterized by NMR, MS, and single crystal X‐ray crystallography. Organic nanoparticles based on the fluorescent dyes are then prepared by re‐precipitation method, and their photophysical properties are investigated. These nanoparticles retain the strong emissions of the organic dyes, and multicolor nanoparticles were also prepared by simply tuning the ratios of the three‐primary colors dyes. These organic nanoparticles exhibit low cytotoxicity, good photostability, and high quantum yields. Moreover, the nanoparticles can also be applied in the cell fluorescence imaging. Especially, it is interestingly found that the stained regions of these nanoparticles from membrane to cytoplasm for HeLa cells show obvious structure‐dependent properties. This strategy provides a new perspective to fluorescence probe by molecular design for specific location imaging of living cells.     

Biodegradable Fluorescent Nanoparticles for Endoscopic Detection of Colorectal Carcinogenesis

Early and comprehensive endoscopic detection of colonic dysplasia—the most clinically significant precursor lesion to colorectal adenocarcinoma—provides an opportunity for timely, minimally invasive intervention to prevent malignant transformation. Here, the development and evaluation of biodegradable near‐infrared fluorescent silica nanoparticles (FSN) that have the potential to improve adenoma detection during fluorescence‐assisted white‐light colonoscopic surveillance in rodent and human‐scale models of colorectal carcinogenesis is described. FSNs are biodegradable (t_1/2 of 2.7 weeks), well‐tolerated, and enable detection and delineation of adenomas as small as 0.5 mm² with high tumor‐to‐background ratios. Furthermore, in the human scale, APC^1311/+ porcine model, the clinical feasibility and benefit of using FSN‐guided detection of colorectal adenomas using video‐rate fluorescence‐assisted white‐light endoscopy is demonstrated. Since nanoparticles of similar size (e.g., 100–150 nm) or composition (i.e., silica and silica/gold hybrid) have already been successfully translated to the clinic, and clinical fluorescent/white‐light endoscopy systems are becoming more readily available, there is a viable path towards clinical translation of the proposed strategy for early colorectal cancer detection and prevention in high‐risk patients.     

Ultrabright Fluorescent and Lasing Microspheres from a Conjugated Polymer

Fluorescent microspheres are used for biomarkers, assay substrates, chemical diagnostics, flow cytometry, and biological imaging. These applications demand the highest fluorescence intensity achievable; however, concentration quenching limits the amount of dye that can be practically incorporated in conventional fluorescent microspheres. Conjugated polymers (CPs) can be less susceptible to concentration quenching, suggesting that they can be excellent candidates for a new class of light‐emitting microspheres. Due to their long‐chain‐conjugated backbone, however, CPs can be resistant to forming smoothly curved or spherical structures. Here, strongly fluorescent CP microspheres as large as 100 µm in diameter are synthesized. Whispering gallery modes (WGMs) appear in the fluorescence spectra, and the microspheres show clear evidence of lasing above a threshold pump intensity. These conjugated polymer beads are up to 50 times larger than CP microspheres obtained by other methods, and they exceed the emission intensity of conventional fluorescent microspheres by more than an order of magnitude.     

Fluorescent Protein Nanovessels: A New Platform to Generate Bio–Abiotic Hybrid Materials for Bioimaging

In this report, a new platform to generate fluorescent protein nanovessels is described. Based on systemic analyses and reconstitution experiments, a combination of protein scaffold and organic dye is identified. Briefly, certain proteins such as bovine serum albumin (BSA) can rapidly form cube‐like scaffold upon heating. This protein scaffolds intrinsically interact with nonfluorescent dyes such as bromophenol blue (BPB), forming BSA‐BPB nanocubes (BBNCs). Moreover, it turns out that the commercially available dye BPB contains aggregation‐induced emission (AIE) properties, allowing the BBNCs emissive upon irradiation. The fluorescent protein nanovessels are highly biocompatible and can be readily internalized by different type of cells. The fluorescent signal of the materials is well‐penetrable from mouse tissues and can be detected at near‐infrared region, making it a useful tool for various biological imaging studies. This platform for making fluorescent protein nanovessels is green, rapid, and cost‐effective and can be extended to other protein scaffolds and possibly other dye/AIE molecules.     

Oligotriphenylene Nanofiber Sensors for Detection of Nitro‐Based Explosives

Blue light‐emitting oligotriphenylene nanofibers are synthesized by oxidizing triphenylene using ferric chloride. By adjusting the monomer concentration, the acid used, and the temperature employed, the average diameter and length of the nanofibers can be readily tuned from 50 to 200 nm and 0.5 to 5 μm, respectively. Structural characterization, electrical conductivity, thermal stability, and fluorescence of oligotriphenylene, along with a proposed nanofiber formation mechanism, are presented. Both oligotriphenylene nanofiber dispersions and oligotriphenylene/polysulfone composite films are developed as fluorescent sensors for detecting traces of nitro‐based explosives including nitromethane, nitrobenzene, and 2,4,6‐trinitrophenol, as well as an electron‐deficient metal ion, Fe(III). The sensors exhibit much better selectivity and sensitivity compared to conventional sensors, with detection limits down to 1.0 nm with a detection range covering ～4 orders of magnitude. The detection mechanism of the fluorescent sensors is also disscussed.     

InGaP@ZnS‐Enriched Chitosan Nanoparticles: A Versatile Fluorescent Probe for Deep‐Tissue Imaging

InGaP QDs overcoated with several monolayers of ZnS are covalently bound to chitosan to address the challenges of developing highly biologically stable and fluorescent nanoparticle probes for deep‐tissue imaging. Transmission electron microscopy images reveal that the average diameter of these luminescent nanoparticles is approximately 29 nm, and they contain multiple InGaP@ZnS QDs that have an average diameter between 4 and 5 nm. These new InGaP@ZnS–chitosan nanoparticles emit near the near IR region at 670 nm and are able to penetrate three times deeper into tissue (e.g., even through a mouse skull) while revealing a higher uptake efficiency into PC12 cells with a robust signal. Additionally, a cell viability assay demonstrates that these new fluorescent nanoparticles have good biocompatibility and stability with PC12 cells and neural cells. As a result, these near‐IR‐emitting nanoparticles can be used for real‐time and deep‐tissue examination of diverse specimens, such as lymphatic organs, kidneys, hearts, and brains, while leaving the tissue intact.     

Polydopamine Encapsulation of Fluorescent Nanodiamonds for Biomedical Applications

Fluorescent nanodiamonds (FNDs) are promising bioimaging probes compared with other fluorescent nanomaterials such as quantum dots, dye‐doped nanoparticles, and metallic nanoclusters, due to their remarkable optical properties and excellent biocompatibility. Nevertheless, they are prone to aggregation in physiological salt solutions, and modifying their surface to conjugate biologically active agents remains challenging. Here, inspired by the adhesive protein of marine mussels, encapsulation of FNDs within a polydopamine (PDA) shell is demonstrated. These PDA surfaces are readily modified via Michael addition or Schiff base reactions with molecules presenting thiol or nitrogen derivatives. Modification of PDA shells by thiol terminated poly(ethylene glycol) (PEG‐SH) molecules to enhance colloidal stability and biocompatibility of FNDs is described. Their use as fluorescent probes for cell imaging is demonstrated; it is found that PEGylated FNDs are taken up by HeLa cells and mouse bone marrow‐derived dendritic cells and exhibit reduced nonspecific membrane adhesion. Furthermore, functionalization with biotin‐PEG‐SH is demonstrated and long‐term high‐resolution single‐molecule fluorescence based tracking measurements of FNDs tethered via streptavidin to individual biotinylated DNA molecules are performed. This robust polydopamine encapsulation and functionalization strategy presents a facile route to develop FNDs as multifunctional labels, drug delivery vehicles, and targeting agents for biomedical applications.     

A Simple and Versatile Pathway for the Synthesis of Visible Light Photoreactive Nanoparticles

This work pioneers the design of visible (415 nm) and UV‐B light (300 nm) reactive nanoparticles via radical polymerization in aqueous heterogeneous media based on methyl methacrylate (MMA) and unique acrylates bearing tetrazole functionalities in a simple and straightforward two step reaction. Stable colloidal nanoparticles with an average diameter of 150 nm and inherent tetrazole functionality (varying from 2.5 to 10 wt% relative to MMA) are prepared via one‐pot miniemulsion polymerization. In a subsequent step, fluorescent pyrazoline moieties serving as linkage points are generated on the nanoparticles by either photoinduced nitrile imine‐mediated tetrazole‐ene cycloaddition (NITEC) or nitrile imine carboxylic acid ligation (NICAL) in water, thus enabling the particles as fluorescent tracers. Through in‐depth molecular surface analysis, it is demonstrated that the photoreactive nanoparticles undergo ligation to a variety of substrates bearing functionalities including maleimides, acrylates, or carboxylic acids, illustrating the versatility of the particle modification process. Critically, the unique ability of the photoreactive nanoparticles to be activated with visible light allows for their decoration with UV light–sensitive molecules. Herein, the ligation of folic acid—a vitamin prone to degradation under UV light—to the photoreactive nanoparticles using visible light is exemplified, demonstrating the synthetic power of our photoreactive fluorescent nanoparticle platform technology.     

Visible-Light-Driven Photoswitchable Fluorescent Polymers for Photorewritable Pattern,Anti-Counterfeiting,and Information Encryption

Photoswitchable fluorescent polymers (PFPs) are emerging as a class of promising systems for photowritable pattern, optical anti-counterfeiting, and information encryption. They typically use harmful UV light as the stimulus, which often leads to inevitable photobleaching and poor reversibility. To address this issue, visible-light-driven PFPs by copolymerization of novel negative photochromic spiropyran monomer and methyl acrylate are developed. The obtained copolymers display bright red fluorescence, which can be reversibly turned off by visible light irradiation. Many appealing features are observed in this system, including fast photo-responsibility, prominent photo-reversibility and photostability, simple preparation process, and processability. Their applications in photorewritable patterns, optical anti-counterfeiting, and information encryption are also demonstrated.     

A Dendrimer‐Based Electropolymerized Microporous Film: Multifunctional,Reversible, and Highly Sensitive Fluorescent Probe

A dendrimer PYTPAG2 composed of a central pyrene “core” and four exterior “arms” capped with electroactive triphenylamine is developed as an electroactive precursor to prepare fluorescent films through electropolymerization (EP). The fluorescence emission comes from the central pyrene “core” and the steric hindrance of the exterior “arms” is beneficial for the formation of microporous morphology. The stable and highly cross‐linked fluorescent EP films can be obtained even as free‐standing films. Further, these dendrimer EP films are first studied as the multifunctional fluorescent probe: the emission of EP films exposed to trinitrotoluene vapor is quenched by 82% in 120 s; while the fluorescence is increased to nearly 400% in 120 s upon exposure to benzene vapor, EP films also act as the fluorescent sensor to Fe³⁺ in solution and the limit of detection is obtained to be 8.5 × 10^?8 m . All the above detection processes exhibit remarkable reversibility. These excellent performances are attributed to both the specific molecular features of PYTPAG2 and the intrinsic properties of EP films.     

PbS/CdS/ZnS Quantum Dots: A Multifunctional Platform for In Vivo Near‐Infrared Low‐Dose Fluorescence Imaging

Over the past decade, near‐infrared (NIR)‐emitting nanoparticles have increasingly been investigated in biomedical research for use as fluorescent imaging probes. Here, high‐quality water‐dispersible core/shell/shell PbS/CdS/ZnS quantum dots (hereafter QDs) as NIR imaging probes fabricated through a rapid, cost‐effective microwave‐assisted cation exchange procedure are reported. These QDs have proven to be water dispersible, stable, and are expected to be nontoxic, resulting from the growth of an outer ZnS shell and the simultaneous surface functionalization with mercaptopropionic acid ligands. Care is taken to design the emission wavelength of the QDs probe lying within the second biological window (1000–1350 nm), which leads to higher penetration depths because of the low extinction coefficient of biological tissues in this spectral range. Furthermore, their intense fluorescence emission enables to follow the real‐time evolution of QD biodistribution among different organs of living mice, after low‐dose intravenous administration. In this paper, QD platform has proven to be capable (ex vivo and in vitro) of high‐resolution thermal sensing in the physiological temperature range. The investigation, together with the lack of noticeable toxicity from these PbS/CdS/ZnS QDs after preliminary studies, paves the way for their use as outstanding multifunctional probes both for in vitro and in vivo applications in biomedicine.     

Magnetic/Fluorescent Barcodes Based on Cadmium‐Free Near‐Infrared‐Emitting Quantum Dots for Multiplexed Detection

Magnetic/fluorescent barcodes, which combine quantum dots (QDs) and superparamagnetic nanoparticles in micrometer‐sized host microspheres, are promising for automatic high‐throughput multiplexed biodetection applications and “point of care” biodetection. However, the fluorescence intensity of QDs sharply decreases after addition of magnetic nanoparticles (MNPs) due to absorption by MNPs, and thus, the encoding capacity of QDs becomes more limited. Furthermore, the intrinsic toxicity of cadmium‐based QDs, the most commonly used QD in barcodes, has significant risks to human health and the environment. In this work, to alleviate fluorescence quenching and intrinsic toxicity, cadmium‐free NIR‐emitting CuInS₂/ZnS QDs and Fe₃O₄ MNPs are successfully incorporated into poly(styrene‐co‐maleic anhydride) microspheres by using the Shirasu porous glass membrane emulsification technique. A “single‐wavelength” encoding model is successfully constructed to guide the encoding of NIR QDs with wide emission spectra. Then, a “single‐wavelength” encoding combined with size encoding is used to produce different optical codes by simply changing the wavelength and the intensity of the QDs as well as the size of the barcode microspheres. 48 barcodes are easily created due to the greatly reduced energy transfer between the NIR‐emitting QDs and MNPs. The resulting bifunctional barcodes are also combined with a flow cytometer using one laser for multiplexed detection of five tumor markers in one test. Assays based on these barcodes are significantly more sensitive than non‐magnetic and traditional ELISA assays. Moreover, validating experiments also show good performance of the bifunctional barcodes‐based suspension array when dealing with patient serum samples. Thus, magnetic/fluorescent barcodes based on NIR‐emitting CuInS₂/ZnS QDs are promising for multiplexed bioassay applications.     

Generation of Monodisperse,Shape‐Controlled Single and Hybrid Core–Shell Nanoparticles via a Simple One‐Step Process

In nano‐biotechnology, optoelectronics, and energy research areas, various fabrication methods have been developed for hybrid nanoparticles. A method is developed here for fabricating highly monodisperse three‐dimensional hybrid nanoparticles using a unique top‐down method based on secondary sputtering lithography. Nanostructures that have been formed on a PEDOT sacrificial layer are transferred from the substrate to an aqueous solution in a process that could be used to successfully disperse a variety of nanoparticle shapes and hybrid nanoparticles. By this method, a fluorescent dye could be encapsulated within the fabricated hybrid nanoparticles for use in bio‐sensing and drug‐delivery applications     

Synthesis,Characterization, and Application of Antibody Functionalized Fluorescent Silica Nanoparticles

Fluorescent silica nanoparticles (FSNs) are prepared by incorporating dye into a mesoporous silica nanoparticle (MSN) synthesis procedure. FSNs containing sulforhodamine B, hydrophobically modified sulforhodamine B, and Cascade Blue hydrazide are made. The MSN‐based FSNs do not leach dye under simulated physiological conditions and have strong, stable fluorescence. FSNs prepared with sulforhodamine B are compared to FSNs prepared with hydrophobically modified sulforhodamine B. The data indicate that FSNs prepared with sulforhodamine B are equally as stable but twice as fluorescent as particles made with hydrophobically modified sulforhodamine B. The fluorescence of a FSN prepared with sulforhodamine B is 10 times more intense than the fluorescence of a 4.5 nm core–shell CdSe/ZnS quantum dot. For diagnostic applications, a method to selectively and covalently bind antibodies to the surface of the FSNs is devised. FSNs that are functionalized with antibodies specific for Neisseria gonorrhoeae specifically bind to Neisseria gonorrhoeae in flow cytometry experiments, thus demonstrating the functionality of the attached antibodies and the potential of MSN‐based FSNs to be used in diagnostic applications.     

Generic Method of Preparing Multifunctional Fluorescent Nanoparticles Using Flash NanoPrecipitation

There is increased demand for nanoparticles with a high fluorescence yield that have the desired excitation wavelength, surface functionalization, and particle size to act as biological probes. Here, a simple, rapid, and robust method, Flash NanoPrecipitation (FNP), to produce such fluorescent nanoparticles is described. This process involves encapsulation of a hydrophobic fluorophore with an amphiphilic biocompatible diblock copolymer in a kinetically frozen state. FNP is used to produce nanoparticles ranging from 30 to 800 nm with fluorescence emission peaks ranging from, but not limited to, 370 nm to 720 nm. Such fluorescent nanoparticles remain stable in aqueous solutions, and, in contrast to soluble dyes, show no photobleaching. Fluorophores and drugs are incorporated into a single nanoparticle, allowing for simultaneous drug delivery and biological imaging. In addition, functionalization of nanoparticle surfaces with disease‐specific ligands permits precise cell targeting. These features make FNP‐produced fluorescent nanoparticles highly desirable for various biological applications.     

Nanoparticle‐Based Fluoroionophore for Analysis of Potassium Ion Dynamics in 3D Tissue Models and In Vivo

The imaging of real‐time fluxes of K⁺ ions in live cell with high dynamic range (5–150 × 10^?3m ) is of paramount importance for neuroscience and physiology of the gastrointestinal tract, kidney, and other tissues. In particular, the research on high‐performance deep‐red fluorescent nanoparticle‐based biosensors is highly anticipated. It is found that boron‐dipyrromethene (BODIPY)‐based K⁺‐sensitive fluoroionophore FI3 encapsulated in cationic polymer RL100 nanoparticles displays unusually strong efficiency in staining of broad spectrum of cell models, such as primary neurons and intestinal organoids. Using comparison of brightness, photostability, and fluorescence lifetime imaging microscopy, it is confirmed that FI3 nanoparticles display distinctively superior intracellular staining compared to the free dye. FI3 nanoparticles in real‐time live cell imaging are evaluated and it is found highly useful for monitoring intra‐ and extracellular K⁺ dynamics in cultured neurons. Proof‐of‐concept in vivo brain imaging confirms applicability of the biosensor for visualization of epileptic seizures. Collectively, these data make fluoroionophore FI3 a versatile cross‐platform fluorescent biosensor, broadly compatible with diverse experimental models, and crown‐ether‐based polymer nanoparticles can provide a new venue for the design of efficient fluorescent probes.     

Preparation of Uniform,Water‐Soluble,and Multifunctional Nanocomposites with Tunable Sizes

Novel, thiol‐functionalized, and superparamagnetic, silica composite nanospheres (SH‐SSCNs) with diameters smaller than 100 nm are successfully fabricated through the self‐assembly of Fe₃O₄ nanoparticles and polystyrene₁₀₀‐block‐poly(acrylic acid)₁₆ and a subsequent sol‐gel process. The size and magnetic properties of the SH‐SSCNs can be easily tuned by simply varying the initial concentrations of the magnetite nanoparticles in the oil phase. By incorporating fluorescent dye molecules into the silica network, the composite nanospheres can be further fluorescent‐functionalized. The toxicity of the SH‐SSCNs is evaluated by choosing three typical cell lines (HUVEC, RAW264.7, and A549) as model cells, and no toxic effects are observed. It is also demonstrated that SH‐SSCNs can be used as a new class of magnetic resonance imaging (MRI) probes, having a remarkably high spin–spin (T₂) relaxivity (r₂* = 176.1 mM ^?1 S^?1). The combination of the sub‐100‐nm particle size, monodispersity in aqueous solution, superparamagnetism, and fluorescent properties of the SH‐SSCNs, as well as the non‐cytotoxicity in vitro, provides a novel and potential candidate for an earlier MRI diagnostic method of cancer.     

Multimodal Magnetic Nanoclusters for Gene Delivery,Directed Migration,and Tracking of Stem Cells

This study develops multimodal magnetic nanoclusters (M‐MNCs) for gene transfer, directed migration, and tracking of human mesenchymal stem cells (hMSCs). The M‐MNCs are designed with 5 nm iron oxide nanoparticles and a fluorescent dye (i.e., Rhodamine B) in the matrix of the Food and Drug Administration approved polymer poly(lactide‐co‐glycolide) using a nanoemulsion method. The synthesized M‐MNCs have a hydrodynamic diameter of ≈150 nm, are internalized by stem cells via endocytosis, and deliver genes with high efficiency. The cellular internalization and gene expression efficiency of the clustered nanoparticles are significantly higher than that of single nanoparticles. The M‐MNC‐labeled hMSCs migrate upon application of a magnetic force and can be visualized by both optical and magnetic resonance (MR) imaging. In animal models, the M‐MNC‐labeled hMSCs are also successfully tracked using optical and MR imaging. Thus, the M‐MNCs not only allow the efficient delivery of genes to stem cells but also the tracking of cells in animal models. Taken together, the results show that this new type of nanocomposite can be of great help in future stem cell research and in the development of cell‐based therapeutic agents.