Recent Advances in Photosensitizer Materials for Light-Mediated Tumor Therapy.

Photodynamic therapy (PDT) as an emerging therapeutic method has drawn much attention in the treatment field for cancer. Photosensitizer, which can convert photon energy into cytotoxic species under light irradiation, is the core component in PDT. The design of photosensitizers still faces problems of light absorption, targeting, penetration and oxygen dependence. With the rapid progress of material science, various photosensitizers have been developed to produce cytotoxic species for treatment of tumor with high selectivity, safety, and noninvasiveness. Besides, the applications of photosensitizers have been expanded to diverse cancer treatments such as drug release, optogenetics and immune checkpoint blockade. In this review, we summarize the recent advances of photosensitizers in various therapeutic methods for cancer. Prevailing challenges and further prospects associated with photosensitizers are also discussed.

Advancing X-ray Luminescence for Imaging, Biosensing, and Theragnostics.

X-ray luminescence is an optical phenomenon in which chemical compounds known as scintillators can emit short-wavelength light upon the excitation of X-ray photons. Since X-rays exhibit well-recognized advantages of deep penetration toward tissues and a minimal autofluorescence background in biological samples, X-ray luminescence has been increasingly becoming a promising optical tool for tackling the challenges in the fields of imaging, biosensing, and theragnostics. In recent years, the emergence of nanocrystal scintillators have further expanded the application scenarios of X-ray luminescence, such as high-resolution X-ray imaging, autofluorescence-free detection of biomarkers, and noninvasive phototherapy in deep tissues. Meanwhile, X-ray luminescence holds great promise in breaking the depth dependency of deep-seated lesion treatment and achieving synergistic radiotherapy with phototherapy.In this Account, we provide an overview of recent advances in developing advanced X-ray luminescence for applications in imaging, biosensing, theragnostics, and optogenetics neuromodulation. We first introduce solution-processed lead halide all-inorganic perovskite nanocrystal scintillators that are able to convert X-ray photons to multicolor X-ray luminescence. We have developed a perovskite nanoscintillator-based X-ray detector for high-resolution X-ray imaging of the internal structure of electronic circuits and biological samples. We further advanced the development of flexible X-ray luminescence imaging using solution-processable lanthanide-doped nanoscintillators featuring long-lived X-ray luminescence to image three-dimensional irregularly shaped objects. We also outline the general principles of high-contrast in vivo X-ray luminescence imaging which combines nanoscintillators with functional biomolecules such as aptamers, peptides, and antibodies. High-quality X-ray luminescence nanoprobes were engineered to achieve the high-sensitivity detection of various biomarkers, which enabled the avoidance of interference from the biological matrix autofluorescence and photon scattering. By marrying X-ray luminescence probes with stimuli-responsive materials, multifunctional theragnostic nanosystems were constructed for on-demand synergistic gas radiotherapy with excellent therapeutic effects. By taking advantage of the capability of X-rays to penetrate the skull, we also demonstrated the development of controllable, wireless optogenetic neuromodulation using X-ray luminescence probes while obviating damage from traditional optical fibers. Furthermore, we discussed in detail some challenges and future development of X-ray luminescence in terms of scintillator synthesis and surface modification, mechanism studies, and their other potential applications to provide useful guidance for further advancing the development of X-ray luminescence.

Transcranial deep-tissue phototherapy for Alzheimer's disease using low-dose X-ray-activated long-afterglow scintillators.

Non-invasive phototherapy has been emerging as an ambitious tactic for suppression of amyloid-ß (Aß) self-assembly against Alzheimer's disease (AD). However, it remains a daunting challenge to develop efficient photosensitizers for Aß oxygenation that are activatable in a deep brain tissue through the scalp and skull, while reducing side effects on normal tissues. Here, we report an Aß targeted, low-dose X-ray-excitable long-afterglow scintillator (ScNPs@RB/Ab) for efficient deep-brain phototherapy. We demonstrate that the as-synthesized ScNPs@RB/Ab is capable of converting X-rays into visible light to activate the photosensitizers of rose bengal (RB) for Aß oxygenation through the scalp and skull. We show that the ScNPs@RB/Ab persistently emitting visible luminescence can substantially minimize the risk of excessive X-ray exposure dosage. Importantly, peptide KLVFFAED-functionalized ScNPs@RB/Ab shows a blood-brain barrier permeability. In vivo experimental results validated that ScNPs@RB/Ab alleviated Aß burden and slowed cognitive decline in triple-transgenic AD model mice at extremely low X-ray doses without side effects. Our study paves a new pathway to develop high-efficiency transcranial AD phototherapy. STATEMENT OF SIGNIFICANCE: Non-invasive phototherapy has been emerging as an ambitious tactic for suppression of amyloid-ß (Aß) self-assembly against Alzheimer's disease (AD). However, it remains a daunting challenge to develop efficient photosensitizers for Aß oxygenation that are activatable in a deep brain tissue through the scalp and skull, while reducing side effects on normal tissues. Herein, we report an Aß targeted, low-dose X-ray-excitable long-afterglow scintillators (ScNPs@RB/Ab) for efficient deep-brain phototherapy. In vivo experimental results validated that ScNPs@RB/Ab alleviated Aß burden and slowed cognitive decline in triple-transgenic AD model mice at extremely low X-ray doses without side effects.

Organic phosphorescent scintillation from copolymers by X-ray irradiation.

Scintillators that exhibit X-ray-excited luminescence have great potential in radiation detection, X-ray imaging, radiotherapy, and non-destructive testing. However, most reported scintillators are limited to inorganic or organic crystal materials, which have some obstacles in repeatability and processability. Here we present a facile strategy to achieve the X-ray-excited organic phosphorescent scintillation from amorphous copolymers through the copolymerization of the bromine-substituted chromophores and acrylic acid. These polymeric scintillators exhibit efficient X-ray responsibility and decent phosphorescent quantum yield up to 51.4% under ambient conditions. The universality of the design principle was further confirmed by a series of copolymers with multi-color radioluminescence ranging from green to orange-red. Moreover, we demonstrated their potential application in X-ray radiography. This finding not only outlines a feasible principle to develop X-ray responsive phosphorescent polymers, but also expands the potential applications of polymer materials with phosphorescence features.

Singlet Oxygen Generation in Dark-Hypoxia by Catalytic Microenvironment-Tailored Nanoreactors for NIR-II Fluorescence-Monitored Chemodynamic Therapy.

Singlet oxygen (1 O2 ) has a potent anticancer effect, but photosensitized generation of 1 O2 is inhibited by tumor hypoxia and limited light penetration depth. Despite the potential of chemodynamic therapy (CDT) to circumvent these issues by exploration of 1 O2 -producing catalysts, engineering efficient CDT agents is still a formidable challenge since most catalysts require specific pH to function and become inactivated upon chelation by glutathione (GSH). Herein, we present a catalytic microenvironment-tailored nanoreactor (CMTN), constructed by encapsulating MoO42- catalyst and alkaline sodium carbonate within liposomes, which offers a favorable pH condition for MoO42- -catalyzed generation of 1 O2 from H2 O2 and protects MoO42- from GSH chelation owing to the impermeability of liposomal lipid membrane to ions and GSH. H2 O2 and 1 O2 can freely cross the liposomal membrane, allowing CMTN with a built-in NIR-II ratiometric fluorescent 1 O2 sensor to achieve monitored tumor CDT.

High-resolution X-ray luminescence extension imaging.

Current X-ray imaging technologies involving flat-panel detectors have difficulty in imaging three-dimensional objects because fabrication of large-area, flexible, silicon-based photodetectors on highly curved surfaces remains a challenge1-3. Here we demonstrate ultralong-lived X-ray trapping for flat-panel-free, high-resolution, three-dimensional imaging using a series of solution-processable, lanthanide-doped nanoscintillators. Corroborated by quantum mechanical simulations of defect formation and electronic structures, our experimental characterizations reveal that slow hopping of trapped electrons due to radiation-triggered anionic migration in host lattices can induce more than 30 days of persistent radioluminescence. We further demonstrate X-ray luminescence extension imaging with resolution greater than 20 line pairs per millimetre and optical memory longer than 15 days. These findings provide insight into mechanisms underlying X-ray energy conversion through enduring electron trapping and offer a paradigm to motivate future research in wearable X-ray detectors for patient-centred radiography and mammography, imaging-guided therapeutics, high-energy physics and deep learning in radiology.

Lanthanide-Activated Nanoparticles: A Toolbox for Bioimaging, Therapeutics, and Neuromodulation.

Owing to their unique features, the past decade has witnessed rapid developments of lanthanide-activated nanoparticles for biological applications. These include highly tunable upconverting and downshifting photoluminescence when illuminated in deep tissue, excellent photostability against blinking and bleaching effects, biocompatibility through versatile surface modification, and ease of achieving multifunctionality, as well as satisfactory signal output. These attributes make lanthanide-doped nanoparticles an ideal toolbox for advanced bioimaging and next-generation therapeutics.The interest in lanthanide-doped nanoparticles for biomedical research arises from their unique optical properties in response to deep-tissue-penetrable light sources. Upon near-infrared irradiation, these nanoparticles with properly doped emitters display photon upconversion with large anti-Stokes shifts and broad-spectrum tunability from the ultraviolet to the visible. It is also possible to achieve orthogonal photoluminescence with variations in wavelength and lifetime. Coupled with surface ligands, dyes, biomolecules, or other types of functional nanomaterials, lanthanide-doped nanoparticles offer new opportunities for applications in bioimaging, advanced oncotherapy, and neuromodulation. Given the possibility of locating downshifting luminescence at "biological transmission windows", exquisite design of lanthanide-doped nanoparticles also enables deep-tissue imaging with high spatial resolution. In addition, these nanoparticles can respond to high-energy photons, such as X-rays, to trigger nonradioactive and radiative pathways, making it possible to develop high-sensitivity X-ray detectors. Precise control of paramagnetic lanthanide ions in nanocrystal lattices also provides advanced materials for high-performance magnetic resonance imaging in medical diagnostics and biomedical research. Full consideration of fundamental attributes of lanthanide-doped nanoparticles will facilitate the design of multifunctional and sensitive probes and improve diagnostic and therapeutic outcomes.In this Account, we categorize various lanthanide-activation strategies into three modes: near-infrared excitation, X-ray irradiation, and magnetic field stimulation. We introduce energy manipulations in upconverting, downshifting, and persistence luminescence in spectral and time domains and discuss how they can be applied in biological practices. We assess general design principles for lanthanide-activated nanosystems with multiple modalities of bioimaging, oncotherapy, and neuromodulation. We also review the current state-of-the-art in the field of lanthanide-based theranostic nanoplatforms, with particular emphasis on energy conversion and nano-/biointerfacing as well as emerging bioapplications. In this context, we also highlight recent advances in controlling optical properties of nanoplatforms for single- or multimodal bioimaging, stimulus-responsive phototherapy, and optogenetics. Finally, we discuss future opportunities and challenges of this exciting research field.

Microfluidic Formation of Coculture Tumor Spheroids with Stromal Cells As a Novel 3D Tumor Model for Drug Testing.

Three-dimensional (3D) tumor spheroids offer unprecedented capability for drug screening because of their unique features such as spatial 3D structure, relevant physiological responses, more complex intercellular network, and stroma-cancer cell interactions. Microfluidic technology provides a facile strategy to make uniform tumor spheroids with potential of high-throughput production. In this article, we develop a microfluidic approach to produce core-shell alginate particles, which allows the separate confinement of different cells in the core and shell structure. To reconstitute the complex tumor structure, we encapsulated tumor cells in the core and stromal fibroblast cells in the shell. These coculture tumor spheroids were applied for drug evaluation showing similar drug resistance as those prepared using conventional methods in well plates. These results demonstrated that our microfluidic approach are facile and versatile for making various tumor spheroids with uniform size but different components to better mimic tumor microenvironment. Moreover, the production rate of around 200 spheroids/min indicates the great potential of this approach for high-throughput drug screening.

Microfluidic technologies in cell isolation and analysis for biomedical applications.

Efficient platforms for cell isolation and analysis play an important role in applied and fundamental biomedical studies. As cells commonly have a size of around 10 microns, conventional handling approaches at a large scale are still challenged in precise control and efficient recognition of cells for further performance of isolation and analysis. Microfluidic technologies have become more prominent in highly efficient cell isolation for circulating tumor cells (CTCs) detection, single-cell analysis and stem cell separation, since microfabricated devices allow for the spatial and temporal control of complex biochemistries and geometries by matching cell morphology and hydrodynamic traps in a fluidic network, as well as enabling specific recognition with functional biomolecules in the microchannels. In addition, the fabrication of nano-interfaces in the microchannels has been increasingly emerging as a very powerful strategy for enhancing the capability of cell capture by improving cell-interface interactions. In this review, we focus on highlighting recent advances in microfluidic technologies for cell isolation and analysis. We also describe the general biomedical applications of microfluidic cell isolation and analysis, and finally make a prospective for future studies.

Controlled assembly of heterotypic cells in a core-shell scaffold: organ in a droplet.

This paper reports a droplet-based microfluidic approach to fabricate a large number of monodisperse, portable microtissues, each in an individual drop. We use water-water-oil double emulsions as templates and spatially assemble hepatocytes in the core and fibroblasts in the shell, forming a 3D liver model in a drop.

DNA-mediated cell surface engineering for multiplexed glycan profiling using MALDI-TOF mass spectrometry.

Glycans are crucial for many key biological processes and their alterations are often a hallmark of disease. Thus, multiplexed and sensitive analysis of glycans is of intense interest. Here we report a novel approach using DNA-mediated cell surface engineering for glycan profiling by MALDI-TOF mass spectrometry (MS). Following lectin binding, DNA amplification and hybridization, glycans on the cell surface are specifically labeled by short DNA probes, which can be facilely released, ionized and detected in MALDI-TOF MS. This strategy converts the analysis of glycans to the detection of DNA probes, overcoming the complicated composition and low ionization efficiency of glycans, enabling in situ detection and facilitating multiplex analysis. The amplification procedure also improves the sensitivity. This approach has been applied to evaluate glycomic alterations in cancer cells and provided the intrinsic distribution of glycans in tissues using MALDI imaging mass spectrometry.

Cell-patterned glass spray for direct drug assay using mass spectrometry.

In this work, the establishment of a glass spray mass spectrometry (GS-MS) platform for direct cell-based drug assay was described. Cell co-culture, drug-induced cell apoptosis, proliferation analysis and intracellular drug absorption measurement were performed simultaneously on this specifically designed platform. Two groups of co-cultured cells (NIH-3T3/HepG2 and HepG2/MCF-7) were cultivated and they showed high viability within 3 days. The biocompatibility of the platform facilitated the subsequent bioassays, in which, cyclophosphamide (CPA) and genistein were used as the model drugs. The distinctions of cell apoptosis and proliferation between the mono-cultured and co-cultured cells were clearly observed and well explained by in situ GS-MS measurements. A satisfactory linearity of the calibration curve between the relative MS intensity and CPA concentrations was obtained using stable isotope labeling method (y = 0.16545 + 0.0985x, R(2) = 0.9937). The variations in the quantity of absorbed drug were detected and the results were consistent with the concentration-dependence of cell apoptosis. All the results demonstrated that direct cell-based drug assay could be performed on the stable isotope labeling assisted GS-MS platform in a facile and quantitative manner.

Oxygen-induced cell migration and on-line monitoring biomarkers modulation of cervical cancers on a microfluidic system.

In this work, we report an integrated microfluidic device for cell co-culture under different concentrations of oxygen, in which the secreted protein VEGF165 was on-line qualitatively and semi-quantitatively analyzed by functional nucleic acid, hemin, ABTS and peroxide system. This microfluidic platform allowed investigation of various oxygen and distances effect on cell-to-cell communication. Besides, the microfluidic device was used for real-time analysis of VEGF165 protein by aptamer-functionalized microchannels. Under 5% O2 condition, we found that the migration of CaSki cells was faster than the migration of human umbilical vein endothelial cells. However, the migration of CaSki cells was slower than the migration of HUVECs under 15% O2 condition. Moreover, the shorter intercellular distances, the quicker cells migration. Furthermore, HIF-1α and VEGF165 genes, ROS were analyzed, and the results would provide new perspectives for the diagnosis and medical treatment of cervical cancer.

An in vitro liver model on microfluidic device for analysis of capecitabine metabolite using mass spectrometer as detector.

In this work, an in vitro liver model in a microfluidic device to imitate and detect prodrug metabolism was developed. A widely used prodrug capecitabine (CAP), which needs to be metabolized into active intermediate in the liver and then transformed into final effective drug in tumor cells, was selected as a model compound. The microfluidic device we exploited consists of a cell co-culture section, in which HepG2 cells were cultured to represent liver while MCF-7 cells were used to represent the tumor tissue, and an on-line solid phase extraction (SPE) section connecting to the ionization source of the ESI-Q-TOF mass spectrometer. The prodrug metabolism was realized and confirmed within this in vitro liver model as the intermediate product of the prodrug 5'-deoxy-5-fluorouridine (DFUR) was successfully detected with MS after the conditioning of HepG2 cells, and the anti-tumor effect of the active metabolite was observed through cell vitality assays of MCF-7 cells. The limit of detection (LOD) using on-chip SPE was at 10nM and semi-quantitative analysis could be realized. This system has been proved useful and practical, showing a potential to replace conventional drug screening methods.

Assay of multiplex proteins from cell metabolism based on tunable aptamer and microchip electrophoresis.

A simple and rapid method for multiplex protein assay based on tunable aptamer by microchip electrophoresis has been developed. Different lengths of aptamers can modulate the electrophoretic mobility of proteins, allowing the protein molecules to be effectively separated in hydroxyethyl cellulose buffer with 1.00 mM magnesium ion. A non-specific DNA was exploited as an internal standard to achieve the quantitative assay and to reduce the interference. A fluorescence dye SYBR gold was exploited to improve the sensitivity and to suppress the interference from sample matrix. Under optimum conditions, quantitative assay of PDGF-BB (R(2)=0.9986), VEGF165 (R(2)=0.9909), and thrombin (R(2)=0.9947) were achieved with a dynamic range in the 5.00-150.0 nM and RSDs in the 5.87-16.3% range. The recoveries were varied from 83.6% to 113.1%. Finally, the proposed method was successfully applied to analyze cell secretions, and then the concentration of PDGF-BB and VEGF165 were detected from 5.15 nM to 2.03 nM, and 3.14 to 2.53 nM, respectively, indicating the established method can be used to analyze cell secretions.

Microfluidic isolation of highly pure embryonic stem cells using feeder-separated co-culture system.

Engineered artificial tissues from stem cells show great potential in regenerative medicine, disease therapies and organ transplantation. To date, stem cells are typically co-cultured with inactivated feeder layers to maintain their undifferentiated state, and to ensure reliable cell purity. Herein, we propose a novel microfabricated approach for feeder-separated coculture of mouse embryonic stem (mES) cells on polydimethylsiloxane (PDMS) porous membrane-assembled 3D-microdevice. Normal mouse embryonic fibroblasts (mEFs) without inactivation were specifically co-cultured with mES cells, resulting in the formation of mES cell colonies on spatially controlled co-culture with feeder layers. An excellent undifferentiated state was confirmed by the expressions of Nanog, octamer binding protein 4 (Oct-4) and alkaline phosphatase (ALP) after 5 days culture. As a result, with the significant advantages of efficiency and simplicity, pure mES cell populations (a purity of 89.2%) from mEFs co-cultures were easily collected without any further purification or separation.

A simple and versatile microfluidic cell density gradient generator for quantum dot cytotoxicity assay.

In this work, a simple and versatile microfluidic cell density gradient generator was successfully developed for cytotoxicity of quantum dots (QDs) assay. The microfluidic cell density gradient generator is composed of eight parallel channels which are respectively surrounded by 1-8 microwells with optimized length and width. The cells fall into microwells by gravity and the cell densities are obviously dependent of microwell number. In a case study, HepG2 and MCF-7 cells were successfully utilized for generating cell density gradients on the microfluidic chip. The microfluidic cell density gradient generator was proved to be easily handled, cell-friendly and could be used to conduct the subsequent cell-based assay. As a proof-of-concept, QD cytotoxicity was evaluated and the results exhibited obvious cell density-dependence. For comparison, QD cytotoxicity was also investigated with a series of cell densities infused by pipette tips. Higher reproducibility was observed on the microfluidic cell density gradient generator and cell density was demonstrated to be a vital factor in cytotoxic study. With higher efficiency, controllability and reproducibility, the microfluidic cell density gradient generator could be integrated into microfluidic analysis systems to promote chip-based biological assay.

Targeted isolation and analysis of single tumor cells with aptamer-encoded microwell array on microfluidic device.

Microfluidic-based single cells analysis has been of great interest in recent years, promising disease diagnosis and personalized medicine. Current technologies are challenging in bioselectively isolating specific single cells from complex matrices. Herein, a novel microfluidic platform integrated with cell-recognizable aptamer-encoded microwells was specifically developed to isolate single tumor cells with satisfied single-cell occupancy and unique bioselectivity. In this work, the designed microwell-structures enable us to encourage strong 3D local topographic interactions of the target cell surface with biomolecules and regulate the single-cell resolution. Under the optimized size of microwells, the single-cell occupancy was significantly enhanced from 0.5% to 88.2% through the introduction of the aptamer. Analysis of the target cells was directly performed in short time periods (<5.0 min) with small volumes (4.5 µL). Importantly, such an aptamer-enabled microfluidic device shows an excellent selectivity for target single cells isolation compared with three control cells. Subsequently, targeted isolation and analysis of single tumor cells were demonstrated by using artificial complex cell samples at simulated conditions, and various cellular carboxylesterases were studied by time-course measurements of cellular fluorescence kinetics at individual-cell level. Thus, our technique will open up a new opportunity in single-cell level-based disease diagnosis and personalize medicine screening.

Cytotoxicity of quantum dots assay on a microfluidic 3D-culture device based on modeling diffusion process between blood vessels and tissues.

In this work, a novel quantum dot (QD) cytotoxicity assay platform on a microfluidic three-dimensional (3D) culture device via imitating the diffusion process between blood vessels and tissues was developed. The device is composed of a main channel and two sets of cell culture chambers. The cell culture chambers were located at different distances from the main channel and were divided into "close chambers" and "far chambers". HepG2 cells were cultured in an agarose matrix under 3D conditions and kept at high viability for at least three days. Fluorescein sodium and fluorescein isothiocyanate conjugated to bovine serum albumin (FITC-BSA) were used as models to demonstrate the diffusion process between main channel and cell culture chambers. QD cytotoxicity was evaluated by determining cell apoptosis, intracellular reactive oxygen species (ROS) and glutathione (GSH) with specific fluorescence probes. Cell autophagy inhibitor 3-methyladenine (3-MA) could reduce cell apoptosis at low concentrations of QDs, which proves that cell autophagy plays a key role in QD cytotoxicity. The effect of a series of 3-MA solutions on cell apoptosis at QD concentration of 40 µg mL(-1) was investigated, which showed that the percentage of cell apoptosis decreased ∼15% from 0 to 12 mM 3-MA. The device shows potential as a high-throughput, low-cost and time-saving platform and constructs a more vivid biomimetic microenvironment for the QD cytotoxicity study.