Water‐Dispersible,pH‐Stable and Highly‐Luminescent Organic Dye Nanoparticles with Amplified Emissions for In Vitro and In Vivo Bioimaging

A new strategy is presented for using doped small‐molecule organic nanoparticles (NPs) to achieve high‐performance fluorescent probes with strong brightness, large Stokes shifts and tunable emissions for in vitro and in vivo imaging. The host organic NPs are used not only as carriers to encapsulate different doped dyes, but also as fluorescence resonance energy transfer donors to couple with the doped dyes (as acceptors) to achieve multicolor luminescence with amplified emissions (AE). The resulting optimum green emitting NPs show high brightness with quantum yield (QY) of up to 45% and AE of 12 times; and the red emitting NPs show QY of 14% and AE of 10 times. These highly‐luminescent doped NPs can be further surface modified with poly(maleic anhydride‐alt‐1‐octadecene)‐polyethylene glycol (C18PMH‐PEG), endowing them with excellent water dispersibility and robust stability in various bio‐environments covering wide pH values from 2 to 10. In this study, cytotoxicity studies and folic acid targeted cellular imaging of these multicolor probes are carried out to demonstrate their potential for in vitro imaging. On this basis, applications of the NP probes in in vivo and ex vivo imaging are also investigated. Intense fluorescent signals of the doped NPs are distinctly, selectively and spatially resolved in tumor sites with high sensitivity, due to the preferential accumulation of the NPs in tumor sites through the passive enhanced permeability and retention effect. The results clearly indicate that these doped NPs are promising fluorescent probes for biomedical applications.     

Eccentric Loading of Fluorogen with Aggregation‐Induced Emission in PLGA Matrix Increases Nanoparticle Fluorescence Quantum Yield for Targeted Cellular Imaging

A simple strategy is developed to prepare eccentrically or homogeneously loaded nanoparticles (NPs) using poly (DL‐lactide‐co‐glycolide) (PLGA) as the encapsulation matrix in the presence of different amounts of polyvinyl alcohol (PVA) as the emulsifier. Using 2,3‐bis(4‐(phenyl(4‐(1,2,2‐triphenylvinyl)‐phenyl)amino)‐phenyl)‐fumaronitrile (TPETPAFN), a fluorogen with aggregation‐induced emission (AIE) characteristics, as an example, the eccentrically loaded PLGA NPs show increased fluorescence quantum yields (QYs) as compared to the homogeneously loaded ones. Field emission transmission electron microscopy and fluorescence lifetime measurements reveal that the higher QY of the eccentrically loaded NPs is due to the more compact aggregation of AIE fluorogens that restricts intramolecular rotations of phenyl rings, which is able to more effectively block the non‐radiative decay pathways. The eccentrically loaded NPs show far red/near infrared emission with a high fluorescence QY of 34% in aqueous media. In addition, by using poly([lactide‐co‐glycolide]‐b‐folate [ethylene glycol]) (PLGA‐PEG‐folate) as the co‐encapsulation matrix, the obtained NPs are born with surface folic acid groups, which are successfully applied for targeted cellular imaging with good photostability and low cytotoxicity. Moreover, the developed strategy is also demonstrated for inorganic‐component eccentrically or homogeneously loaded PLGA NPs, which facilitates the synthesis of polymer NPs with controlled internal architectures.     

Fluorescent Polymer Nanoparticles for Cell Barcoding In Vitro and In Vivo

Fluorescent polymer nanoparticles for long‐term labeling and tracking of living cells with any desired color code are developed. They are built from biodegradable poly(lactic‐co‐glycolic acid) polymer loaded with cyanine dyes (DiO, DiI, and DiD) with the help of bulky fluorinated counterions, which minimize aggregation‐caused quenching. At the single particle level, these particles are ≈20‐fold brighter than quantum dots of similar color. Due to their identical 40 nm size and surface properties, these nanoparticles are endocytosed equally well by living cells. Mixing nanoparticles of three colors in different proportions generates a homogeneous RGB (red, green, and blue) barcode in cells, which is transmitted through many cell generations. Cell barcoding is validated on 7 cell lines (HeLa, KB, embryonic kidney (293T), Chinese hamster ovary, rat basophilic leucemia, U97, and D2A1), 13 color codes, and it enables simultaneous tracking of co‐cultured barcoded cell populations for >2 weeks. It is also applied to studying competition among drug‐treated cell populations. This technology enabled six‐color imaging in vivo for (1) tracking xenografted cancer cells and (2) monitoring morphogenesis after microinjection in zebrafish embryos. In addition to a robust method of multicolor cell labeling and tracking, this work suggests that multiple functions can be co‐localized inside cells by combining structurally close nanoparticles carrying different functions.     

Tuning Cellular Response to Nanoparticles via Surface Chemistry and Aggregation

The aggregation of gold nanoparticles (Au NPs) in cell media is a common phenomenon that can influence NP‐cell interactions. Here, we control Au NP aggregation in cell media and study the impact of Au NP aggregation on human dermal fibroblast (HDF) cells. By first adding Au NPs to fetal bovine serum (FBS) and then subsequently to a buffer, aggregation can be avoided. Aggregation of Au NPs also can be avoided by coating Au NPs with other biomolecules such as lipids. The aggregation state of the Au NPs influences cellular toxicity and Au NP uptake: non‐aggregated cationic Au NPs are four‐fold less toxic to HDF cells than aggregated cationic Au NPs, and the uptake of non‐aggregated anionic citrate Au NPs is three orders of magnitude less than that of aggregated citrate Au NPs. Upon uptake of Au NPs, cellular F‐actin fiber formation is disrupted and actin dots are predominant. When lipid‐coated Au NPs are doped with a fluorescent lipid (F‐lipid) and incubated with HDF cells, the fluorescence from the F‐lipid was found throughout the cell, showing that lipids can dissociate from the Au NP surface upon entering the cell.     

Intravital Multiphoton Imaging of the Selective Uptake of Water‐Dispersible Quantum Dots into Sinusoidal Liver Cells

Although many studies reporting the organ‐level biodistribution of nanoparticles (NPs) in animals, very few have addressed the fate of NPs in organs at the cellular level. The liver appears to be the main organ for accumulation of NPs after intravenous injection. In this study, for the first time, the in vivo spatiotemporal disposition of recently developed mercaptosuccinic acid (MSA)‐capped cadmium telluride/cadmium sulfide (CdTe/CdS) quantum dots (QDs) is explored in rat liver using multiphoton microscopy (MPM) coupled with fluorescence lifetime imaging (FLIM), with subcellular resolution (～1 μm). With high fluorescence efficiency and largely improved stability in the biological environment, these QDs show a distinct distribution pattern in the liver compared to organic dyes, rhodamine 123 and fluorescein. After intravenous injection, fluorescent molecules are taken up by hepatocytes and excreted into the bile, while negatively charged QDs are retained in the sinusoids and selectively taken up by sinusoidal cells (Kupffer cells and liver sinusoidal endothelial cells), but not by hepatocytes within 3 h. The results could help design NPs targeting the specific types of liver cells and choose the fluorescent markers for appropriate cellular imaging.     

Organic Dye Based Nanoparticles for Cancer Phototheranostics

Phototheranostics, which simultaneously combines photodynamic and/or photothermal therapy with deep‐tissue diagnostic imaging, is a promising strategy for the diagnosis and treatment of cancers. Organic dyes with the merits of strong near‐infrared absorbance, high photo‐to‐radical and/or photothermal conversion efficiency, great biocompatibility, ready chemical structure fine‐tuning capability, and easy metabolism, have been demonstrated as attractive candidates for clinical phototheranostics. These organic dyes can be further designed and fabricated into nanoparticles (NPs) using various strategies. Compared to free molecules, these NPs can be equipped with multiple synergistic functions and show longer lifetime in blood circulation and passive tumor‐targeting property via the enhanced permeability and retention effect. In this article, the recent progress of organic dye‐based NPs for cancer phototheranostic applications is summarized, which extends the anticancer arsenal and holds promise for clinical uses in the near future.     

Enriching PMMA nanospheres with adjustable charges as novel templates for multicolored dye@PMMA nanocomposites

Multicolored fluorescent dye loaded PMMA nanospheres were synthesized by the electrostatic adsorption of dye molecules on the charged PMMA nanospheres, whose charges were adjusted by choosing different initiators. The charged PMMA nanospheres have a wider capacity and advantage for combining the charged dyes. The fluorescent dye@PMMA composite nanospheres possess the advantages of higher brightness, longer lifetime and stronger resistance to photobleaching relative to dye molecules. Dye leakage remained lower than 5% over one week. These fluorescent nanospheres have been used in biological labels in cell imaging. They can easily stain blood cancer cells without further surface modification.     

Polyvalent Display and Packing of Peptides and Proteins on Semiconductor Quantum Dots: Predicted Versus Experimental Results

Quantum dots (QDs) are loaded with a series of peptides and proteins of increasing size, including a <20 residue peptide, myoglobin, mCherry, and maltose binding protein, which together cover a range of masses from <2.2 to ≈44 kDa. Conjugation to the surface of dihydrolipoic acid‐functionalized QDs is facilitated by polyhistidine metal affinity coordination. Increasing ratios of dye‐labeled peptides and proteins are self‐assembled to the QDs and then the bioconjugates are separated and analyzed using agarose gel electrophoresis. Fluorescent visualization of both conjugated and unbound species allows determination of an experimentally derived maximum loading number. Molecular modeling utilizing crystallographic coordinates or space‐filling structures of the peptides and proteins also allow the predicted maximum loadings to the QDs to be estimated. Comparison of the two sets of results provides insight into the nature of the QD surface and reflects the important role played by the nanoparticle's hydrophilic solubilizing surface ligands. It is found that for the larger protein molecules steric hindrance is the major packing constraint. In contrast, for the smaller peptides, the number of available QD binding sites is the principal determinant. These results can contribute towards an overall understanding of how to engineer designer bioconjugates for both QDs and other nanoparticle materials.     

Engineered Multifunctional Albumin‐Decorated Porous Silicon Nanoparticles for FcRn Translocation of Insulin

The last decade has seen remarkable advances in the development of drug delivery systems as alternative to parenteral injection‐based delivery of insulin. Neonatal Fc receptor (FcRn)‐mediated transcytosis has been recently proposed as a strategy to increase the transport of drugs across the intestinal epithelium. FcRn‐targeted nanoparticles (NPs) could hijack the FcRn transcytotic pathway and cross the epithelial cell layer. In this study, a novel nanoparticulate system for insulin delivery based on porous silicon NPs is proposed. After surface conjugation with albumin and loading with insulin, the NPs are encapsulated into a pH‐responsive polymeric particle by nanoprecipitation. The developed NP formulation shows controlled size and homogeneous size distribution. Transmission electron microscopy (TEM) images show successful encapsulation of the NPs into pH‐sensitive polymeric particles. No insulin release is detected at acidic conditions, but a controlled release profile is observed at intestinal pH. Toxicity studies show high compatibility of the NPs with intestinal cells. In vitro insulin permeation across the intestinal epithelium shows approximately fivefold increase when insulin is loaded into FcRn‐targeted NPs. Overall, these FcRn‐targeted NPs offer a toolbox in the development of targeted therapies for oral delivery of insulin.     

Bright Far‐Red/Near‐Infrared Conjugated Polymer Nanoparticles for In Vivo Bioimaging

A highly emissive far‐red/near‐infrared (FR/NIR) fluorescent conjugated polymer (CP), poly[(9,9‐dihexylfluorene)‐co‐2,1,3‐benzothiadiazole‐co‐4,7‐di(thiophen‐2‐yl)‐2,1,3‐benzothiadiazole] (PFBTDBT10) is designed and synthesized via Suzuki polymerization. Formulation of PFBTDBT10 using 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[methoxy(polyethylene glycol)‐2000] (DSPE‐PEG₂₀₀₀) and DSPE‐PEG₅₀₀₀‐folate as the encapsulation matrix yielded CP‐loaded DSPE‐PEG‐folic acid nanoparticles (CPDP‐FA NPs) with bright FR/NIR fluorescence (27% quantum yield) and a large Stoke's shift of 233 nm in aqueous solution. CPDP‐FA NPs show improved thermal/photostabilities and larger Stoke's shifts as compared to commercially available quantum dots (Qdot 655) and organic dyes such as Alexa Fluor 555 and Rhodamine 6G. In vivo studies of CPDP‐FA NPs on a hepatoma H22 tumor‐bearing mouse model reveal that they could serve as an efficient FR/NIR fluorescent probe for targeted in vivo fluorescence imaging and cancer detection in a high contrast and specific manner. Together with the negligible in vivo toxicity, CPDP‐FA NPs are promising FR/NIR fluorescent probes for future in vivo applications.     

Monolayer Silane‐Coated,Water‐Soluble Quantum Dots

A one‐step method to produce ≈12 nm hydrodynamic diameter water‐soluble CdSe/ZnS quantum dots (QDs), as well as CdS/ZnS, ZnSe/ZnMnS/ZnS, AgInS₂/ZnS, and CuInS₂/ZnS QDs, by ligand exchange with a near‐monolayer of organosilane caps is reported. The method cross‐links the surface‐bound silane ligands such that the samples are stable on the order of months under ambient conditions. Furthermore, the samples may retain a high quantum yield (60%) over this time. Several methods to functionalize aqueous QD dispersions with proteins and fluorescent dyes have been developed with reaction yields as high as 97%.     

Amorphous Silica Nanoparticles Promote Monocyte Adhesion to Human Endothelial Cells: Size‐Dependent Effect

There is evidence that nanoparticles can induce endothelial dysfunction. Here, the effect of monodisperse amorphous silica nanoparticles (SiO₂‐NPs) of different diameters on endothelial cells function is examined. Human endothelial cell line (EA.hy926) or primary human pulmonary artery endothelial cells (hPAEC) are seeded in inserts introduced or not above triple cell co‐cultures (pneumocytes, macrophages, and mast cells). Endothelial cells are incubated with SiO₂‐NPs at non‐cytotoxic concentrations for 12 h. A significant increase (up to 2‐fold) in human monocytes adhesion to endothelial cells is observed for 18 and 54 nm particles. Exposure to SiO₂‐NPs induces protein expression of adhesion molecules (ICAM‐1 and VCAM‐1) as well as significant up‐regulation in mRNA expression of ICAM‐1 in both endothelial cell types. Experiments performed with fluorescent‐labelled monodisperse amorphous SiO₂‐NPs of similar size evidence nanoparticle uptake into the cytoplasm of endothelial cells. It is concluded that exposure of human endothelial cells to amorphous silica nanoparticles enhances their adhesive properties. This process is modified by the size of the nanoparticle and the presence of other co‐cultured cells.     

Highly Active Metastable Ruthenium Nanoparticles for Hydrogen Production through the Catalytic Hydrolysis of Ammonia Borane

Late transition metal nanoparticles (NPs) with a favorably high surface area to volume ratio have garnered much interest for catalytic applications. Yet, these NPs are prone to aggregation in solution, which has been mitigated through attachment of surface ligands, additives or supports; unfortunately, protective ligands can severely reduce the effective surface area on the NPs available for catalyzing chemical transformations. The preparation of ‘metastable’ NPs can readily address these challenges. We report herein the first synthesis of monodisperse metastable ruthenium nanoparticles (RuNPs), having sub 5 nm size and an fcc structure, in aqueous media at room temperature, which can be stored for a period of at least 8 months. The RuNPs can subsequently be used for the catalytic, quantitative hydrolysis of ammonia‐borane (AB) yielding hydrogen gas with 21.8 turnovers per min at 25 °C. The high surface area available for hydrolysis of AB on the metastable RuNPs translated to an E_a of 27.5 kJ mol^‐1, which is notably lower than previously reported values for RuNP based catalysts.     

Ratiometric Nanoparticle Probe Based on FRET‐Amplified Phosphorescence for Oxygen Sensing with Minimal Phototoxicity

Luminescent oxygen probes enable direct imaging of hypoxic conditions in cells and tissues, which are associated with a variety of diseases, including cancer. Here, a nanoparticle probe that addresses key challenges in the field is developed, it: i) strongly amplifies room temperature phosphorescence of encapsulated oxygen‐sensitive dyes; ii) provides ratiometric response to oxygen; and iii) solves the fundamental problem of phototoxicity of phosphorescent sensors. The nanoprobe is based on 40 nm polymeric nanoparticles, encapsulating ≈2000 blue‐emitting cyanine dyes with fluorinated tetraphenylborate counterions, which are as bright as 70 quantum dots (QD525). It functions as a light‐harvesting nanoantenna that undergoes efficient Förster resonance energy transfer to ≈20 phosphorescent oxygen‐sensitive platinum octaethylporphyrin (PtOEP) acceptor dyes. The obtained nanoprobe emits stable blue fluorescence and oxygen‐sensitive red phosphorescence, providing ratiometric response to dissolved oxygen. The light harvesting leads to ≈60‐fold phosphorescence amplification and makes the single nanoprobe particle as bright as ≈1200 PtOEP dyes. This high brightness enables oxygen detection at a single‐particle level and in cells at ultra‐low nanoprobe concentration with no sign of phototoxicity, in contrast to PtOEP dye. The developed nanoprobe is successfully applied to the imaging of a microfluidics‐generated oxygen gradient in cancer cells. It constitutes a promising tool for bioimaging of hypoxia.     

Bright Single‐Chain Conjugated Polymer Dots Embedded Nanoparticles for Long‐Term Cell Tracing and Imaging

Single‐chain conjugated polymer (CP) dots embedded nanoparticles (NPs) bearing cell penetration peptide (TAT) as surface ligands are synthesized for long term cancer cell tracing applications. The CPNPs are fabricated by matrix‐encapsulation method and the embedded CPs can be modulated into spherical dots with different size upon alteration of feed concentrations. Single‐chain CP dots are formed upon decreasing feed concentration to 0.2 mg/mL, where CPNPs exhibit highest fluorescence quantum yield of 32%. Maleimide is introduced as the new NP surface functional group, which favors easy conjugation with cell penetration peptide via click chemistry to preserve its biofunctions. The obtained CPNPs show high brightness and good biocompatibility, which allow cell tracing for over 9 generations, superior to commercial cell tracker Qtracker 585.     

Preparation and in vitro evaluation of novel cross‐linked chondroitin sulphate nanoparticles by aluminium ions for encapsulation of green tea flavonoids

Chondroitin sulphate is a sulphated glycosaminoglycan biopolymer composed over 100 individual sugars. Chondroitin sulphate nanoparticles (NPs) loaded with catechin were prepared by an ionic gelation method using AlCl₃ and optimised for polymer and cross‐linking agent concentration, curing time and stirring speed. Zeta potential, particle size, loading efficiency, and release efficiency over 24 h (RE₂₄ %) were evaluated. The surface morphology of NPs was investigated by scanning electron microscopy and their thermal behaviour by differential scanning calorimetric. Antioxidant effect of NPs was determined by chelating activity of iron ions. The cell viability of mesenchymal stem cells was determined by 3‐[4, 5‐dimethylthiazol‐2‐yl]‐2, 5‐diphenyl tetrazolium bromide assay and the calcification of osteoblasts was studied by Alizarin red staining. The optimised NPs showed particle size of 176 nm, zeta potential of −20.8 mV, loading efficiency of 93.3% and RE₂₄ % of 80.6%. The chatechin loaded chondroitin sulphate NPs showed 70‐fold more antioxidant activity, 3‐fold proliferation effect and higher calcium precipitation in osteoblasts than free catechin.Inspec keywords: nanoparticles, encapsulation, biomedical materials, particle size, nanofabrication, nanomedicine, electrokinetic effects, cellular biophysics, polymer blends, molecular biophysics, molecular configurations, biochemistry, curing, surface morphology, scanning electron microscopy, differential scanning calorimetry, dyes, precipitationOther keywords: in vitro evaluation, cross‐linked chondroitin sulphate nanoparticles, aluminium ions, nanoparticles, green tea flavonoids, sulphated glycosaminoglycan biopolymer, sugars, catechin, ionic gelation method, cross‐linking agent concentration, curing time, size 176 nm, time 24 h, calcium precipitation, 3‐fold proliferation effect, antioxidant activity, chatechin loaded chondroitin sulphate NPs, Alizarin red staining, osteoblasts, calcification, 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide assay, mesenchymal stem cells, cell viability, chelating activity, differential scanning calorimetry, thermal behaviour, scanning electron microscopy, surface morphology, release efficiency, loading efficiency, particle size, zeta potential, stirring speed     

Fluorescence‐Encoded Gold Nanoparticles: Library Design and Modulation of Cellular Uptake into Dendritic Cells

In order to harness the unique properties of nanoparticles for novel clinical applications and to modulate their uptake into specific immune cells we designed a new library of homo‐ and hetero‐functional fluorescence‐encoded gold nanoparticles (Au‐NPs) using different poly(vinyl alcohol) and poly(ethylene glycol)‐based polymers for particle coating and stabilization. The encoded particles were fully characterized by UV‐Vis and fluorescence spectroscopy, zeta potential and dynamic light scattering. The uptake by human monocyte derived dendritic cells in vitro was studied by confocal laser scanning microscopy and quantified by fluorescence‐activated cell sorting and inductively coupled plasma atomic emission spectroscopy. We show how the chemical modification of particle surfaces, for instance by attaching fluorescent dyes, can conceal fundamental particle properties and modulate cellular uptake. In order to mask the influence of fluorescent dyes on cellular uptake while still exploiting its fluorescence for detection, we have created hetero‐functionalized Au‐NPs, which again show typical particle dependent cellular interactions. Our study clearly prove that the thorough characterization of nanoparticles at each modification step in the engineering process is absolutely essential and that it can be necessary to make substantial adjustments of the particles in order to obtain reliable cellular uptake data, which truly reflects particle properties.     

Three‐Dimensional Gold Nanoparticle Clusters with Tunable Cores Templated by a Viral Protein Scaffold

Assembling nanoparticles (NPs) into ordered architectures remains a challenge in the field of nanotechnology. Templated strategies have been widely utilized for NP assembly. As typical biological nanostructures, virus‐based NPs (VNPs) have shown great promise in templating NP assembly. Here it is illustrated that the VNP of simian virus 40 (SV40) is a powerful scaffold in directing the assembly of 3D hybrid nanoarchitectures with one NP encapsulated inside as a core and a cluster of gold NPs (AuNPs) on the outer surface of the SV40 VNP as a shell, in which the core NPs can be CdSe/ZnS quantum dots (QDs), Ag₂S QDs, or AuNPs. The assembling of AuNPs onto the SV40 VNP surface is determined by the interactions between the AuNPs and the amine groups on the outer surface of SV40 VNPs. It is expected that the VNP guided 3D hybrid nanoarchitectures provide ideal models for NP interaction studies and open new opportunities for integrating various functionalities in NP assemblies.     

Surface Engineering of Spherical Metal Nanoparticles with Polymers toward Selective Asymmetric Synthesis of Nanobowls and Janus‐Type Dimers

New synthetic methods capable of controlling structural and compositional complexities of asymmetric nanoparticles (NPs) are very challenging but highly desired. A simple and general synthetic approach to designing sophisticated asymmetric NPs by anisotropically patterning the surface of isotropic metallic NPs with amphiphilic block copolymers (BCPs) is reported. The selective galvanic replacement and seed‐mediated growth of a second metal can be achieved on the exposed surface of metal NPs, resulting in the formation of nanobowls and Janus‐type metal–metal dimers, respectively. Using Ag and Au NPs tethered with amphiphilic block copolymers of poly(ethylene oxide)‐block‐polystyrene (PEO‐b‐PS), anisotropic surface patterning of metallic NPs (e.g., Ag and Au) is shown to be driven by thermodynamical phase segregation of BCP ligands on isotropic metal NPs. Two proof‐of‐concept experiments are given on, i) synthesis of Au nanobowls by a selective galvanic replacement reaction on Janus‐type patched Ag/polymer NPs; and ii) preparation of Au–Pd heterodimers and Au–Au homodimers by a seed‐mediated growth on Janus‐type patched Au/polymer NPs. The method shows remarkable versatility; and it can be easily handled in aqueous solution. This synthetic strategy stands out as the new methodology to design and synthesis asymmetric metal NPs with sophisticated topologies.