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.     

Ultralong Phosphorescence of Water‐Soluble Organic Nanoparticles for In Vivo Afterglow Imaging

Afterglow or persistent luminescence eliminates the need for light excitation and thus circumvents the issue of autofluorescence, holding promise for molecular imaging. However, current persistent luminescence agents are rare and limited to inorganic nanoparticles. This study reports the design principle, synthesis, and proof‐of‐concept application of organic semiconducting nanoparticles (OSNs) with ultralong phosphorescence for in vivo afterglow imaging. The design principle leverages the formation of aggregates through a top‐down nanoparticle formulation to greatly stabilize the triplet excited states of a phosphorescent molecule. This prolongs the particle luminesce to the timescale that can be detected by the commercial whole‐animal imaging system after removal of external light source. Such ultralong phosphorescent of OSNs is inert to oxygen and can be repeatedly activated, permitting imaging of lymph nodes in living mice with a high signal‐to‐noise ratio. This study not only introduces the first category of water‐soluble ultralong phosphorescence organic nanoparticles but also reveals a universal design principle to prolong the lifetime of phosphorescent molecules to the level that can be effective for molecular imaging.     

Continuous Coaxial Electrohydrodynamic Atomization System for Water‐Stable Wrapping of Magnetic Nanoparticles

An electrohydrodynamic atomization (EHDA) system that generates an electrospray can achieve particle formation and encapsulation by accumulating an electric charge on liquid flowing out from the nozzle. A novel coaxial EHDA system for continuous fabrication of water‐stable magnetic nanoparticles (MNPs) is established, based on a cone‐jet mode of electrospraying. Systemic variables, such as flow rates from dual nozzles and inducing voltages, are controlled to enable the preparation of water‐soluble MNPs coated by polysorbate 80. The PEGylated MNPs exhibit water stability. The magnetic resonance imaging potential of these MNPs is confirmed by in vivo imaging using a gastric cancer xenograft mouse model. Thus, this advanced coaxial EHDA system demonstrates remarkable capabilities for the continuous encapsulation of MNPs to render them water‐stable while preserving their properties as imaging agents.     

A Porphyrin‐Based Conjugated Polymer for Highly Efficient In Vitro and In Vivo Photothermal Therapy

Conjugated polymers have been increasingly studied for photothermal therapy (PTT) because of their merits including large absorption coefficient, facile tuning of exciton energy dissipation through nonradiative decay, and good therapeutic efficacy. The high photothermal conversion efficiency (PCE) is the key to realize efficient PTT. Herein, a donor–acceptor (D–A) structured porphyrin‐containing conjugated polymer (PorCP) is reported for efficient PTT in vitro and in vivo. The D–A structure introduces intramolecular charge transfer along the backbone, resulting in redshifted Q band, broadened absorption, and increased extinction coefficient as compared to the state‐of‐art porphyrin‐based photothermal reagent. Through nanoencapsulation, the dense packing of a large number of PorCP molecules in a single nanoparticle (NP) leads to favorable nonradiative decay, good photostability, and high extinction coefficient of 4.23 × 10⁴m ^?1 cm^?1 at 800 nm based on porphyrin molar concentration and the highest PCE of 63.8% among conjugated polymer NPs. With the aid of coloaded fluorescent conjugated polymer, the cellular uptake and distribution of the PorCP in vitro can be clearly visualized, which also shows effective photothermal tumor ablation in vitro and in vivo. This research indicates a new design route of conjugated polymer‐based photothermal therapeutic materials for potential personalized theranostic nanomedicine.     

Cytotoxicity of BSA‐Stabilized Gold Nanoclusters: In Vitro and In Vivo Study

Gold nanoclusters (Au NCs) are one of the most promising fluorescent nanomaterials for bioimaging, targeting, and cancer therapy due to their tunable optical properties, yet their biocompatibility still remains unclear. Herein, the cytotoxicity of bovine serum albumin (BSA)‐stabilized Au NCs is studied by using three tumor cell lines and two normal cell lines. The results indicate that Au NCs induce the decline of cell viabilities of different cell lines to varying degrees in a dose‐ and time‐dependent manner, and umbilical vein endothelial cells which had a higher intake of Au NCs than melanoma cells show more toxicity. Addition of free BSA to BSA‐Au NCs solutions can relieve the cytotoxicity, implying that BSA can prevent cell damage. Moreover, Au NCs increase intracellular reactive oxygen species (ROS) production, further causing cell apoptosis. Furthermore, N‐acetylcysteine, a ROS scavenger, partially reverses Au NCs‐induced cell apoptosis and cytotoxicity, indicating that ROS might be one of the primary reasons for the toxicity of BSA‐Au NCs. Surprisingly, Au NCs with concentrations of 5 and 20 nM significantly inhibit tumor growth in the xenograft mice model of human liver cancer, which might provide a new avenue for the design of anti‐cancer drug delivery vehicles.     

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.     

In Vitro and In Vivo Interrogation of Bio-sprayed Cells

Bio-sprays can directly form pre-organized cell-bearing structures for applications ranging from engineering functional tissues to the forming of cultures, most useful for modeling disease, to the discovery and development of drugs. Bio-electrosprays and aerodynamically assisted bio-jets, are leading approaches that have been demonstrated as having far-reaching ramifications for regenerative biology and medicine.     

Development of Monolithic Osmotic Pump Tablet System for Isosorbide-5-Mononitrate Delivery and Evaluation of it In Vitro and In Vivo

The objective of this study is to develop the monolithic osmotic pump tablet system (MOTS) containing isosorbide-5-mononitrate (5-ISMN), and to evaluate its in vitro and in vivo properties. The influences of tablet formulation variables, size and location of the delivery orifice, membrane variables, and pH value of the dissolution medium on 5-ISMN release from MOTS have been investigated. These results demonstrated that the tablet core played an important role in MOTS, and membrane variables could affect the 5-ISMN release rate. The optimal formulation of 5-ISMN MOTS was determined by uniform design. Furthermore, the dog pharmacokinetics and relative bioavailability of the test formulation (5-ISMN MOTS) have been compared with the reference formulation (Imdur^®: 60 mg/tablet, a sustained release, SR, tablet system) following an oral single dose of 60 mg given to each of six Beagle dogs. The mean drug fraction absorbed by the dog was calculated by the Wagner–Nelson technique. The results showed that drug concentration in plasma could be maintained more stable and longer after the administration of 5-ISMN MOTS compared with the matrix tablets of Imdur^®, and a level A “in vitro–in vivo correlation” was observed between the percentage released in vitro and percentage absorbed in vivo. It is concluded that 5-ISMN MOTS is more feasible for a long-acting preparation than 5-ISMN SR tablet system as once-a-day treatment, and it is very simple in preparation, and can release 5-ISMN at the rate of approximately zero order for the combination of hydroxypropylmethyl cellulose as retarder and NaCl as osmogent.     

In Vivo Repeatedly Activated Persistent Luminescence Nanoparticles by Radiopharmaceuticals for Long‐Lasting Tumor Optical Imaging

Persistent luminescence nanoparticles (PLNPs) with rechargeable near‐infrared afterglow properties attract much attention for tumor diagnosis in living animals since they can avoid tissue autofluorescence and greatly improve the signal‐to‐background ratio. Using UV, visible light, or X‐ray as excitation sources to power up persistent luminescence (PL) faces the challenges such as limited tissue penetration, inefficient charging capability, or tissue damage caused by irradiation. Here, it is proved that radiopharmaceuticals can efficiently excite ZnGa₂O₄:Cr³⁺ nanoparticles (ZGCs) for both fluorescence and afterglow luminescence via Cerenkov resonance energy transfer as well as ionizing radiation. ¹⁸F‐FDG, a clinically approved tumor‐imaging radiopharmaceutical with a short decay half‐life around 110 min, is successfully used as the internal light source to in vivo excite intravenously injected ZGCs for tumor luminescence imaging over 3 h. The luminescence with similar decay time can be re‐obtained for multiple times upon injection of ¹⁸F‐FDG at any time needed with no health concern. It is believed this strategy can not only provide tumor luminescence imaging with high sensitivity, high contrast, and long decay time at desired time, but also guarantee the patients much less radiation exposure, greatly benefiting image‐guided surgery in the future.     

Expanding the Toolbox of Upconversion Nanoparticles for In Vivo Optogenetics and Neuromodulation

Optogenetics is an optical technique that exploits visible light for selective neuromodulation with spatio‐temporal precision. Despite enormous effort, the effective stimulation of targeted neurons, which are located in deeper structures of the nervous system, by visible light, remains a technical challenge. Compared to visible light, near‐infrared illumination offers a higher depth of tissue penetration owing to a lower degree of light attenuation. Herein, an overview of advances in developing new modalities for neural circuitry modulation utilizing upconversion‐nanoparticle‐mediated optogenetics is presented. These developments have led to minimally invasive optical stimulation and inhibition of neurons with substantially improved selectivity, sensitivity, and spatial resolution. The focus is to provide a comprehensive review of the mechanistic basis for evaluating upconversion parameters, which will be useful in designing, executing, and reporting optogenetic experiments.     

Branched‐Tail Lipid Nanoparticles Potently Deliver mRNA In Vivo due to Enhanced Ionization at Endosomal pH

The potential of mRNA therapeutics will be realized only once safe and effective delivery systems are established. Unfortunately, delivery vehicle development is stymied by an inadequate understanding of how the molecular properties of a vehicle confer efficacy. Here, a small library of lipidoid materials is used to elucidate structure–function relationships and identify a previously unappreciated parameter—lipid nanoparticle surface ionization—that correlates with mRNA delivery efficacy. The two most potent materials of the library, 306O₁₀ and 306O_i10, induce substantial luciferase expression in mice following a single 0.75 mg kg^?1 mRNA dose. These lipidoids, which have ten‐carbon tails and identical molecular weights, vary only in that the 306O₁₀ tail is straight and the 306O_i10 tail has a one‐carbon branch. Remarkably, this small difference in structure conferred a tenfold improvement in 306O_i10 efficacy. The enhanced potency of this branched‐tail lipidoid is attributed to its strong surface ionization at the late endosomal pH of 5.0. A secondary lipidoid library confirms that O_i10 materials ionize more strongly and deliver mRNA more potently than lipidoids containing linear tails. Together, these data highlight the exquisite control that lipid chemistry exerts on the mRNA delivery process and show that branched‐tail lipids facilitate protein expression in animals.     

Red‐Emitting Upconverting Nanoparticles for Photodynamic Therapy in Cancer Cells Under Near‐Infrared Excitation

Upconverting nanoparticles (UCNPs) have attracted considerable attention as potential photosensitizer carriers for photodynamic therapy (PDT) in deep tissues. In this work, a new and efficient NIR photosensitizing nanoplatform for PDT based on red‐emitting UCNPs is designed. The red emission band matches well with the efficient absorption bands of the widely used commercially available photosensitizers (Ps), benefiting the fluorescence resonance energy transfer (FRET) from UCNPs to the attached photosensitizers and thus efficiently activating them to generate cytotoxic singlet oxygen. Three commonly used photosensitizers, including chlorine e6 (Ce6), zinc phthalocyanine (ZnPc) and methylene blue (MB), are loaded onto the alpha‐cyclodextrin‐modified UCNPs to form Ps@UCNPs complexes that efficiently produce singlet oxygen to kill cancer cells under 980 nm near‐infrared excitation. Moreover, two different kinds of drugs are co‐loaded onto these nanoparticles: chemotherapy drug doxorubicin and PDT agent Ce6. The combinational therapy based on doxorubicin (DOX)‐induced chemotherapy and Ce6‐triggered PDT exhibits higher therapeutic efficacy relative to the individual means for cancer therapy in vitro.