Recent Advances in Polymeric Nanoparticles for Enhanced Fluorescence and Photoacoustic Imaging.

In recent years, polymeric nanoparticles (NPs) have become increasingly popular for in vitro and in vivo bioimaging because of their excellent versatility and exceptional optical properties. Following a brief introduction on fluorescence and photoacoustic imaging, as well as the merits of using polymeric NPs over other types of fluorescent probes, this Minireview gives a concise summary of key advances made in recent years (2017-2020) in the use of polymeric NPs for fluorescence and photoacoustic imaging. A particular focus is placed on various strategies used for enhanced bioimaging applications, including polymeric NPs that encapsulate near-infrared dyes, aggregation-induced-emission fluorogens, cationic dyes doped with bulky hydrophobic counterions, and semiconducting polymers. Next, the current limitations in some of these polymeric NP systems are summarized and potential solutions offered to overcome them. Finally, some critical considerations in regard to the design of polymeric NPs are given for future bioimaging and sensing applications.

Competition-Based Universal Photonic Crystal Biosensors by Using Antibody-Antigen Interaction.

Sensors capable of detecting different types of biomolecules have widespread applications in the field of biomedical research, but despite many years of research, the development of biosensors suitable for point-of-care (POC) applications in resource-limited areas is still extremely challenging. Sensors based on photonic crystal hydrogels (PCHs) hold much promise in this regard because of their numerous advantages over other existing bioanalytical methods. All current PCH biosensors are however restricted in the types of analytes they can detect sensitively with good selectivity. By taking advantage of the powerful and ubiquitous antibody-antigen interaction, we report herein the first-ever competition-based PCH biosensors capable of naked-eye detection of various biomolecules (e.g., proteins, peptides, and small molecules) with high sensitivity and selectivity and minimal background and excellent reversibility. We showed such PCH designs could be extended to the fabrication of different enzyme-detecting biosensors. The universal feature of these novel biosensors thus enables future development of POC biosensors in disease diagnostics for other bioanalytes.

Light-Triggered PEGylation/dePEGylation of the Nanocarriers for Enhanced Tumor Penetration.

Nanocarriers-derived anticancer therapeutics typically suffers from poor tumor penetration and suboptimal antitumor efficacy. Although PEGylation improves the stability of nanoparticles and prolongs drug circulation, it further increases the size of nanoparticles and adversely affects the tumor penetration. Here, we developed a light-triggered PEGylation/dePEGylation strategy, whereby near-infrared (NIR)-/pH- dual responsive dePEGylation activates iRGD for tumor targeting. The embedded up-conversion nanoparticles (UCNPs) could efficiently convert NIR to UV-vis which cleaved the linker to remove PEG. NIR-induced dePEGylation remarkably improved vascular extravasation of drugs and deep tumor penetration. Therefore, the stimuli-responsive nanocarriers facilitated the tumor-targeted delivery of drugs through blood circulation and enhanced the antitumor effects.

Mitochondria-Targeting, Intracellular Delivery of Native Proteins Using Biodegradable Silica Nanoparticles.

Mitochondria are key organelles in mammalian cells whose dysfunction is linked to various diseases. Drugs targeting mitochondrial proteins provide a highly promising strategy for potential therapeutics. Methods for the delivery of small-molecule drugs to the mitochondria are available, but these are not suitable for macromolecules, such as proteins. Herein, we report the delivery of native proteins and antibodies to the mitochondria using biodegradable silica nanoparticles (BS-NPs). The modification of the nanoparticle surface with triphenylphosphonium (TPP) and cell-penetrating poly(disulfide)s (CPD) facilitated their rapid intracellular uptake with minimal endolysosomal trapping, providing sufficient time for effective mitochondrial localization followed by glutathione-triggered biodegradation and of native, functional proteins into the mitochondria.

Highly efficient, conjugated-polymer-based nano-photosensitizers for selectively targeted two-photon photodynamic therapy and imaging of cancer cells.

Two-photon photodynamic therapy (2P-PDT) is a promising noninvasive treatment of cancers and other diseases with three-dimensional selectivity and deep penetration. However, clinical applications of 2P-PDT are limited by small two-photon absorption (TPA) cross sections of traditional photosensitizers. The development of folate receptor targeted nano-photosensitizers based on conjugated polymers is described. In these nano-photosensitizers, poly{9,9-bis[6''-(bromohexyl)fluorene-2,7-ylenevinylene]-co-alt-1,4-(2,5-dicyanophenylene)}, which is a conjugated polymer with a large TPA cross section, acts as a two-photon light-harvesting material to significantly enhance the two-photon properties of the doped photosensitizer tetraphenylporphyrin (TPP) through energy transfer. These nanoparticles displayed up to 1020-fold enhancement in two-photon excitation emission and about 870-fold enhancement in the two-photon-induced singlet oxygen generation capability of TPP. Surface-functionalized folic acid groups make these nanoparticles highly selective in targeting and killing KB cancer cells over NIH/3T3 normal cells. The 2P-PDT activity of these nanoparticles was significantly improved, potentially up to about 1000 times, as implied by the enhancement factors of two-photon excitation emission and singlet oxygen generation. These nanoparticles could act as novel two-photon nano-photosensitizers with combined advantages of low dark cytotoxicity, targeted 2P-PDT with high selectivity, and simultaneous two-photon fluorescence imaging capability; these are all required for ideal two-photon photosensitizers.

"Minimalist" cyclopropene-containing photo-cross-linkers suitable for live-cell imaging and affinity-based protein labeling.

Target identification of bioactive compounds within the native cellular environment is important in biomedical research and drug discovery, but it has traditionally been carried out in vitro. Information about how such molecules interact with their endogenous targets (on and off) is currently highly limited. An ideal strategy would be one that recapitulates protein-small molecule interactions in situ (e.g., in living cells) and at the same time enables enrichment of these complexes for subsequent proteome-wide target identification. Similarly, small molecule-based imaging approaches are becoming increasingly available for in situ monitoring of a variety of proteins including enzymes. Chemical proteomic strategies for simultaneous bioimaging and target identification of noncovalent bioactive compounds in live mammalian cells, however, are currently not available. This is due to a lack of photoaffinity labels that are minimally modified from their parental compounds, yet chemically tractable using copper-free bioorthogonal chemistry. We have herein developed novel minimalist linkers containing both an alkyl diazirine and a cyclopropene. We have shown chemical probes (e.g., BD-2) made from such linkers could be used for simultaneous in situ imaging and covalent labeling of endogenous BRD-4 (an important epigenetic protein) via a rapid, copper-free, tetrazine-cyclopropene ligation reaction (k2 > 5 M(-1) s(-1)). The key features of our cyclopropenes, with their unique C-1 linkage to BRD-4-targeting moiety, are their tunable reactivity and solubility, relative stability, and synthetic accessibility. BD-2, which is a linker-modified analogue of (+)-JQ1 (a recently discovered nanomolar protein-protein-interaction inhibitor of BRD-4), was subsequently used in a cell-based proteome profiling experiment for large-scale identification of potential off-targets of (+)-JQ1. Several newly identified targets were subsequently confirmed by preliminary validation experiments.

Conjugated-polymer-based red-emitting nanoparticles for two-photon excitation cell imaging with high contrast.

Two-photon fluorescence microscopy is a widely used noninvasive bioimaging technique because of unique advantages such as a large penetration depth and 3D mapping capability. Ideal two-photon fluorophores require large two-photon absorption cross sections and red emission with high quantum yields. Here we report red-emitting-dye-doped conjugated polymer nanoparticles that display high two-photon excitation brightness. In these nanoparticles, conjugated polymer (PFV) was chosen as a two-photon light-harvesting material, and red-emitting dyes (MgPc and Nile red) were chosen as the energy acceptors and red-emitting materials. Two-photon excitation fluorescence of MgPc and Nile red was enhanced by up to ∼53 and ∼240 times, respectively. We have successfully demonstrated the application of these conjugated polymer-based nanoparticles in two-photon excitation cancer cell imaging with an excellent contrast ratio. This concept could become a general approach to the preparation of two-photon excitation red-emitting materials for deep-tissue live-cell imaging with high contrast.

In vivo targeted delivery of antibodies into cancer cells with pH-responsive cell-penetrating poly(disulfide)s.

Cell-penetrating poly(disulfide)s (CPDs) are promising vehicles for cytosolic delivery of proteins. However, currently available arginine-rich CPD has rarely been reported for systemic delivery due to its "always" positive charge. Herein, we developed pH-responsive CPDIMD that executes tumor targeting delivery via protonation of imidazole groups within the acidic tumor microenvironment.

Enhanced two-photon singlet oxygen generation by photosensitizer-doped conjugated polymer nanoparticles.

We have prepared photosensitizer-doped conjugated polymer nanoparticles by using a reprecipitation method. The conjugated polymer, poly[9,9-dibromohexylfluorene-2,7-ylenethylene-alt-1,4-(2,5-dimethoxy)phenylene] (PFEMO), was used as the host matrix to disperse tetraphenylporphyrin (TPP). These TPP-doped PFEMO nanoparticles are stable and have a uniform size of ∼50 nm. Efficient intraparticle energy transfer from PFEMO to TPP has been observed. The TPP emission of the nanoparticles was found to be enhanced by 21-fold by PFEMO under two-photon excitation. Enhanced two-photon excitation singlet oxygen generation efficiency in the TPP-doped PFEMO nanoparticles has been demonstrated. Our results suggest that these photosensitizer-doped conjugated polymer nanoparticles can act as novel photosensitizing agents for two-photon photodynamic therapy and related applications.

Broad-Spectrum Polymeric Nanoquencher as an Efficient Fluorescence Sensing Platform for Biomolecular Detection.

Colloidal inorganic nanostructures (metal, carbon, and silica) have been widely used as "nanoquenchers" for construction of nanosensors; however, inherent drawbacks such as insufficient fluorescence quenching efficiency, false positive signals, and uncertain long-term cytotoxicity have limited their further utility. Herein, by taking advantages of polymeric nanoparticles (PNPs) in terms of high loading capacity, facile surface modification chemistry, and good biocompatibility, we report a broad-spectrum (400-750 nm) polymeric fluorescence-quenching platform for sensor fabrication. Our newly developed polymeric nanoquenchers (qPNPs) were constructed by concurrently encapsulating various alkylated black-hole quenchers into nanoparticles made of poly(methyl methacrylate-co-methacrylic acid) and were found to have an excellent fluorescence quenching effect (>400-fold) on common fluorophores (FAM, TMR, and Cy5) together with high stability under physiological conditions. As a proof of concept, the feasibility of these qPNPs for fluorescence sensing was validated by successful construction of two nanosensors (FAMDEVD@qPNP and Cy5SurC@qPNP), which could be used as promising nanosensors for live-cell imaging of the apoptosis-related protease caspase-3 and cancer-related survivin mRNA, respectively. As expected, in the FAM channel, the FAMDEVD@qPNP showed fast and selective fluorescence responses toward caspase-3 in buffers and could be used to image the activation of drug-induced endogenous caspase-3. In the Cy5 channel, the Cy5SurC@qPNP could be used to distinguish normal cells (MCF10A) from cancer cells (HeLa) by quantitatively detecting the endogenous survivin mRNA level. It could be further used to monitor changes in the endogenous survivin mRNA expression levels in drug-treated HeLa cells. Altogether, by virtue of their high quencher loading and broad-spectrum quenching efficiency and good signal-to-background ratio, these qPNPs might be particularly attractive alternatives to other conventional nanoquenchers for the construction of more complex biosensors in the future.

Screening Mammalian Cells on a Hydrogel: Functionalized Small Molecule Microarray.

Mammalian cell-based microarray technology has gained wide attention, for its plethora of promising applications. The platform is able to provide simultaneous information on multiple parameters for a given target, or even multiple target proteins, in a complex biological system. Here we describe the preparation of mammalian cell-based microarrays using selectively captured of human prostate cancer cells (PC-3). This platform was then used in controlled drug release and measuring the associated drug effects on these cancer cells.

Controlled proliferation and screening of mammalian cells on a hydrogel-functionalized small molecule microarray.

A hydrogel-functionalized small molecule microarray has been developed, on which PC-3 cancer cells were selectively grown. Subsequent controlled release of immobilized bioactive compounds enabled cell-based screening to be directly carried out on this platform.

Red-emitting DPSB-based conjugated polymer nanoparticles with high two-photon brightness for cell membrane imaging.

New red-emitting conjugated polymers have been successfully synthesized by incorporating classical two-photon absorption (TPA) units, electron-rich units, and a small amount of electron-deficient units along the polymer backbones. Water-dispersible nanoparticles (NPs) based on these polymers were also fabricated for applications in two-photon excitation fluorescence imaging of cell membrane. Through optimization of the polymer/matrix mass ratio and the initial feed concentration of the polymer solution, a high quantum yield (QY) of 24% was achieved for the red-emitting NPs in water. TPA cross section and two-photon action cross section values of these polymers at 750 nm reached up to 1000 GM and 190 GM per repeat unit in aqueous media, 2.5 × 10(5) GM and 4.7 × 10(4) GM per NP, respectively. Furthermore, these NPs displayed excellent photostability and biocompatibility. Their applications as two-photon excitation fluorescence probes for cell membrane imaging have been demonstrated in three different cell lines with excellent imaging contrast. These results demonstrated that these polymer NPs hold great potentials as excellent two-photon excitation fluorescence probes in various biological applications.

Site-specific peptide immobilization strategies for the rapid detection of kinase activity on microarrays.

The massive throughput offered by array-based technologies can only be realized with the development of equally powerful strategies that offer reproducible consistency. The competence of arrays and efficacy of screening come under scrutiny, with most existing immobilization schemes that do not site-specifically ligate peptides on the arrays. Thus, it is crucial in array-based experiments to orientate peptides in an ordered and uniform fashion. Two new approaches were developed for the directed immobilization of peptides on a microarray, by exploiting measures involving native chemical ligation reactions as well as biotin-streptavidin interactions. This makes it possible to stably immobilize peptides in a consistent manner and in a predetermined orientation on the microarray. The first scheme employs glass slides that are functionalized with avidin for attachment of terminally biotinylated peptides. The second uses slides containing thioester moieties to ligate N-terminal cysteine containing peptides. The authors successfully immobilized peptides on chip using these strategies, and, in extending their method to the study of kinase activity on microarrays, they also developed a novel detection scheme that abrogates the dependence on traditional radioactivity-based kinase screening assays. This method employs fluorescently labeled antiphosphoserine and antiphosphotyrosine antibodies in assessing and monitoring kinase activity on arrays. The above methodologies provide for a fast and sensitive approach with which to conveniently assess kinase activity using peptide microarrays.

Gold nanorods as dual photo-sensitizing and imaging agents for two-photon photodynamic therapy.

Gold nanorods with three different aspect ratios were prepared and their dual capabilities for two-photon imaging and two-photon photodynamic therapy have been demonstrated. These gold nanorods exhibit large two-photon absorption action cross-sections, about two orders of magnitude larger than small organic molecules, which makes them suitable for two-photon imaging. They can also effectively generate singlet oxygen under two-photon excitation, significantly higher than traditional photosensitizers such as Rose Bengal and Indocyanine Green. Such high singlet oxygen generation capability under two-photon excitation was ascribed to their large two-photon absorption cross-sections. Polyvinylpyrrolidone (PVP) coated gold nanorods displayed excellent biocompatibility and high cellular uptake efficiency. The two-photon photodynamic therapy effect and two-photon fluorescence imaging properties of PVP coated gold nanorods have been successfully demonstrated on HeLa cells in vitro using fluorescence microscopy and indirect XTT assay method. These gold nanorods thus hold great promise for imaging guided two-photon photodynamic therapy for the treatment of various malignant tumors.

Photosensitizer-doped conjugated polymer nanoparticles for simultaneous two-photon imaging and two-photon photodynamic therapy in living cells.

Photosensitizer doped conjugated polymer nanoparticles have been prepared by incorporating polyoxyethylene nonylphenylether (CO-520) into the nanoparticles using a re-precipitation method. The conjugated polymer, poly[9,9-dibromohexylfluorene-2,7-ylenethylene-alt-1,4-(2,5-dimethoxy)phenylene] (PFEMO), was used as the host matrix to disperse tetraphenylporphyrin (TPP) and an energy donor to enhance the two-photon excitation properties of TPP. These CO-520 incorporated, TPP-doped PFEMO nanoparticles are stable and have low cytotoxicity in the dark. The TPP emission of the nanoparticles was found to be enhanced by about 20 times by PFEMO under two-photon excitation. The nanoparticles showed significantly enhanced two-photon excitation singlet oxygen generation efficiency and two-photon photodynamic therapy activity in cancer cells. These composite nanoparticles display features required for ideal photosensitizers, such as low cytotoxicity in the dark and efficient two-photon photodynamic activity under laser radiation. In addition, these novel nano-photosensitizers allow simultaneous in vivo monitoring by two-photon fluorescence imaging during two-photon photodynamic treatment. These photosensitizer-doped conjugated polymer nanoparticles can act as novel photosensitizing agents for two-photon photodynamic therapy and related applications.