Immunostimulatory DNA Hydrogel Enhances Protective Efficacy of Nanotoxoids against Bacterial Infection.

While vaccines have been highly successful in protecting against various infections, there are still many high-priority pathogens for which there are no clinically approved formulations. To overcome this challenge, researchers have explored the use of nanoparticulate strategies for more effective antigen delivery to the immune system. Along these lines, nanotoxoids are a promising biomimetic platform that leverages cell membrane coating technology to safely deliver otherwise toxic bacterial antigens in their native form for antivirulence vaccination. Here, in order to further boost their immunogenicity, nanotoxoids formulated against staphylococcal α-hemolysin are embedded into a DNA-based hydrogel with immunostimulatory CpG motifs. The resulting nanoparticle-hydrogel composite is injectable and improves the in vivo delivery of vaccine antigens while simultaneously stimulating nearby immune cells. This leads to elevated antibody production and stronger antigen-specific cellular immune responses. In murine models of pneumonia and skin infection caused by methicillin-resistant Staphylococcus aureus, mice vaccinated with the hybrid vaccine formulation are well-protected. This work highlights the benefits of combining nanoparticulate antigen delivery systems with immunostimulatory hydrogels into a single platform, and the approach can be readily generalized to a wide range of infectious diseases.

Protease-Responsive Potential-Tunable AIEgens for Cell Selective Imaging of TMPRSS2 and Accurate Inhibitor Screening.

Transmembrane protease serine 2 (TMPRSS2) is a plasma membrane protease that activates both spike protein of coronaviruses for cell entry and oncogenic signaling pathways for tumor progression. TMPRSS2 inhibition can reduce cancer invasion and metastasis and partially prevent the entry of SARS-CoV-2 into host cells. Thus, there is an urgent need for both TMPRSS2-selective imaging and precise screening of TMPRSS2 inhibitors. Here, we report a TMPRSS2-responsive surface-potential-tunable peptide-conjugated probe (EGTP) with aggregation-induced emission (AIE) features for TMPRSS2 selective imaging and accurate inhibitor screening. The amphiphilic EGTP was constructed with tunable surface potential and responsive efficiency with TMPRSS2 and its inhibitor. The rational construction of AIE luminogens (AIEgens) with modular peptides indicated that the cleavage of EGTP led to a gradual aggregation with bright fluorescence in high TMPRSS2-expressing cells. This strategy may have value for selective detection of cancer cells, SARS-CoV-2-target cells, and screening of protease inhibitors.

Supramolecular Loading of DNA Hydrogels with Dye-Drug Conjugates for Real-Time Photoacoustic Monitoring of Chemotherapy.

A longstanding problem with conventional cancer therapy is the nonspecific distribution of chemotherapeutics. Monitoring drug release in vivo via noninvasive bioimaging can thus have value, but it is difficult to distinguish loaded from released drug in live tissue. Here, this work describes an injectable supramolecular hydrogel that allows slow and trackable release of doxorubicin (Dox) via photoacoustic (PA) tomography. Dox is covalently linked with photoacoustic methylene blue (MB) to monitor Dox before, during, and after release from the hydrogel carrier. The conjugate (MB-Dox) possesses an IC50 of 161.4 × 10-9  m against human ovarian carcinoma (SKOV3) cells and loads into a DNA-clad hydrogel with 91.3% loading efficiency due to MB-Dox's inherent intramolecular affinity to DNA. The hydrogel is biodegradable by nuclease digestion, which causes gradual release of MB-Dox. This release rate is tunable based on the wt% of the hydrogel. This hydrogel maintains distinct PA contrast on the order of days when injected in vivo and demonstrates activatable PA spectral shifts   during hydrogel degradation. The released and loaded payload can be imaged relative to live tissue via PA and ultrasound signal being overlaid in real-time. The hydrogel slowed the rate of the murine intraperitoneal tumor growth 72.2% more than free Dox.

DNA Origami Disguises Herpes Simplex Virus 1 Particles and Controls Their Virulence.

DNA nanostructures are well-established vectors for packaging diversified payloads for targeted cellular delivery. Here, DNA origami rectangular sheets were combined with Herpes Simplex Virus 1 (HSV1) capsids to demonstrate surface coverage of the particle via electrostatic interactions. The optimized origami:HSV1 molar ratios led to characteristic packaging geometries ranging from dispersed "HSV1 pockets" to agglomerated "HSV1 sleeves". "Pockets" were disguised from cells in HeLa and B16F10 cells and were 44.2% less infective than naked HSV1 particles. However, the pockets were 117% more infective than naked HSV1 particles when the origami sheets were coated with folic acid. We observed infectivity from naked origami, but they are 99.1% less infective with respect to HSV1 and 99.6% less infective with respect to the pocket complexes. This work suggests that DNA origami can selectively modulate virus infectivity.

Protease-Responsive Peptide-Conjugated Mitochondrial-Targeting AIEgens for Selective Imaging and Inhibition of SARS-CoV-2-Infected Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a serious threat to human health and lacks an effective treatment. There is an urgent need for both real-time tracking and precise treatment of the SARS-CoV-2-infected cells to mitigate and ultimately prevent viral transmission. However, selective triggering and tracking of the therapeutic process in the infected cells remains challenging. Here, we report a main protease (Mpro)-responsive, mitochondrial-targeting, and modular-peptide-conjugated probe (PSGMR) for selective imaging and inhibition of SARS-CoV-2-infected cells via enzyme-instructed self-assembly and aggregation-induced emission (AIE) effect. The amphiphilic PSGMR was constructed with tunable structure and responsive efficiency and validated with recombinant proteins, cells transfected with Mpro plasmid or infected by SARS-CoV-2, and a Mpro inhibitor. By rational construction of AIE luminogen (AIEgen) with modular peptides and Mpro, we verified that the cleavage of PSGMR yielded gradual aggregation with bright fluorescence and enhanced cytotoxicity to induce mitochondrial interference of the infected cells. This strategy may have value for selective detection and treatment of SARS-CoV-2-infected cells.

One-Step Supramolecular Multifunctional Coating on Plant Virus Nanoparticles for Bioimaging and Therapeutic Applications.

Plant viral nanoparticles (plant VNPs) are promising biogenetic nanosystems for the delivery of therapeutic, immunotherapeutic, and diagnostic agents. The production of plant VNPs is simple and highly scalable through molecular farming in plants. Some of the important advances in VNP nanotechnology include genetic modification, disassembly/reassembly, and bioconjugation. Although effective, these methods often involve complex and time-consuming multi-step protocols. Here, we report a simple and versatile supramolecular coating strategy for designing functional plant VNPs via metal-phenolic networks (MPNs). Specifically, this method gives plant viruses [e.g., tobacco mosaic virus (TMV), cowpea mosaic virus, and potato virus X] additional functionalities including photothermal transduction, photoacoustic imaging, and fluorescent labeling via different components in MPN coating [i.e., complexes of tannic acid (TA), metal ions (e.g., Fe3+, Zr4+, or Gd3+), or fluorescent dyes (e.g., rhodamine 6G and thiazole orange)]. For example, using TMV as a viral substrate by choosing Zr4+-TA and rhodamine 6G, fluorescence is observed peaking at 555 nm; by choosing Fe3+-TA coating, the photothermal conversion efficiency was increased from 0.8 to 33.2%, and the photoacoustic performance was significantly improved with a limit of detection of 17.7 µg mL-1. We further confirmed that TMV@Fe3+-TA nanohybrids show good cytocompatibility and excellent cell-killing performance in photothermal therapy with 808 nm irradiation. These findings not only prove the practical benefits of this supramolecular coating for designing multifunctional and biocompatible plant VNPs but also bode well for using such materials in a variety of plant virus-based theranostic applications.

Ultrasmall gold nanorod-polydopamine hybrids for enhanced photoacoustic imaging and photothermal therapy in second near-infrared window.

Gold nanorods (GNRs) have attracted great interest for photo-mediated biomedicines due to their tunable and high optical absorption, high photothermal conversion efficiency and facile surface modifiability. GNRs that have efficient absorption in second near-infrared (NIR-II) window hold further promise in bio-applications due to low background signal from tissue and deep tissue penetration. However, bare GNRs readily undergo shape deformation (termed as 'melting effect') during the laser illumination losing their unique localized surface plasmon resonance (LSPR) properties, which subsequently leads to PA signal attenuation and decreased photothermal efficiency. Polydopamine (PDA) is a robust synthetic melanin that has broad absorption and high photothermal conversion. Herein, we coated GNRs with PDA to prepare photothermally robust GNR@PDA hybrids for enhanced photo-mediated theranostic agents. Ultrasmall GNRs (SGNRs) and conventional large GNRs (LGNRs) that possess similar LSPR characteristics as well as GNR@PDA hybrids were compared side-by-side in terms of the size-dependent photoacoustic (PA) imaging, photothermal therapy (PTT), and structural stability. In vitro experiments further demonstrated that SGNR@PDA showed 95% ablation of SKOV3 ovarian cancer cells, which is significantly higher than that of LGNRs (66%) and SGNRs (74%). Collectively, our PDA coating strategy represents a rational design for enhanced PA imaging and efficient PTT via a nanoparticle, i.e., nanotheranostics.

Enhanced Photoacoustic Detection of Heparin in Whole Blood via Melanin Nanocapsules Carrying Molecular Agents.

Photoacoustic (PA) imaging has proved versatile for many biomedical applications from drug delivery tracking to disease diagnostics and postoperative surveillance. It recently emerged as a tool for accurate and real-time heparin monitoring to avoid bleeding complications associated with anticoagulant therapy. However, molecular-dye-based application is limited by high concentration requirements, photostability, and a strong background hemoglobin signal. We developed polydopamine nanocapsules (PNCs) via supramolecular templates and loaded them with molecular dyes for enhanced PA-mediated heparin detection. Depending on surface charge, the dye-loaded PNCs undergo disassembly or aggregation upon heparin recognition: both experiments and simulation have revealed that the increased PA signal mainly results from dye-loaded PNC-heparin aggregation. Importantly, Nile blue (NB)-loaded PNCs generated a 10-fold higher PA signal than free NB dye, and such PNC enabled the direct detection of heparin in a clinically relevant therapeutic window (0-4 U/mL) in whole human blood (R2 = 0.91). Furthermore, the PA signal of PNC@NB obtained from 17 patients linearly correlated with ACT values (R2 = 0.73) and cumulative heparin (R2 = 0.83). This PNC-based strategy for functional nanocapsules offers a versatile engineering platform for robust biomedical contrast agents and nanocarriers.

A Photoacoustic Contrast Agent for miR-21 via NIR Fluorescent Hybridization Chain Reaction.

Nucleic acids are well-established biomarkers of cancer with immense value in diagnostics and basic research. However, strategies to monitor these species in tissue can be challenging due to the need for amplification of imaging signal from low analyte concentrations with high specificity. Photoacoustic (PA) imaging is gaining traction for molecular imaging of proteins, small biomolecules, and nucleic acids by coupling pulsed near-infrared (NIR) excitation with broadband acoustic detection. This work introduces a PA nucleic acid contrast agent that harnesses NIR fluorophore and quencher-tagged hybridization chain reaction (HCR) for signal amplification. This HCR probe was designed to enable contact quenching between NIR dye-quencher pairs by coercing their direct alignment when miR-21, a microRNA cancer biomarker, is detected. The probe demonstrated a ratiometric PA limit of detection of 148 pM miR-21, sequence specificity against one- and two-base mutations, and selectivity over other microRNAs. It was further tested in live human ovarian cancer (SKOV3) and noncancerous (HEK 293T) cells to exemplify in situ PA activation based on differences in endogenous miR-21 regulation (p = 0.0002). The probe was lastly tested in tissue mimicking phantoms to exemplify sustained contrast in centimeter-range depths and 85.3% photostability after 15 min of laser irradiation. The probe's miR-21-specific activation and its ability to maintain contrast in biologically relevant absorbing and scattering media support its consideration for live-cell PA microscopy and potential cancer diagnostics. Results from this probe also underscore the combined detection power between ratiometric PA signaling and strand amplification for more sensitive DNA-based PA sensors.

A Dual-Color Fluorescent Probe Allows Simultaneous Imaging of Main and Papain-like Proteases of SARS-CoV-2-Infected Cells for Accurate Detection and Rapid Inhibitor Screening.

The main protease (Mpro ) and papain-like protease (PLpro ) play critical roles in SARS-CoV-2 replication and are promising targets for antiviral inhibitors. The simultaneous visualization of Mpro and PLpro is extremely valuable for SARS-CoV-2 detection and rapid inhibitor screening. However, such a crucial investigation has remained challenging because of the lack of suitable probes. We have now developed a dual-color probe (3MBP5) for the simultaneous detection of Mpro and PLpro by fluorescence (or Förster) resonance energy transfer (FRET). This probe produces fluorescence from both the Cy3 and Cy5 fluorophores that are cleaved by Mpro and PLpro . 3MBP5-activatable specificity was demonstrated with recombinant proteins, inhibitors, plasmid-transfected HEK 293T cells, and SARS-CoV-2-infected TMPRSS2-Vero cells. Results from the dual-color probe first verified the simultaneous detection and intracellular distribution of SARS-CoV-2 Mpro and PLpro . This is a powerful tool for the simultaneous detection of different proteases with value for the rapid screening of inhibitors.

A Charge-Switchable Zwitterionic Peptide for Rapid Detection of SARS-CoV-2 Main Protease.

The transmission of SARS-CoV-2 coronavirus has led to the COVID-19 pandemic. Nucleic acid testing while specific has limitations for mass surveillance. One alternative is the main protease (Mpro ) due to its functional importance in mediating the viral life cycle. Here, we describe a combination of modular substrate and gold colloids to detect Mpro via visual readout. The strategy involves zwitterionic peptide that carries opposite charges at the C-/N-terminus to exploit the specific recognition by Mpro . Autolytic cleavage releases a positively charged moiety that assembles the nanoparticles with rapid color changes (t<10 min). We determine a limit of detection for Mpro in breath condensate matrices <10 nM. We further assayed ten COVID-negative subjects and found no false-positive result. In the light of simplicity, our test for viral protease is not limited to an equipped laboratory, but also is amenable to integrating as portable point-of-care devices including those on face-coverings.

Versatile Polymer Nanocapsules via Redox Competition.

Polymer nanocapsules have demonstrated significant value in materials science and biomedical technology, but require complicated and time-consuming synthetic steps. We report here the facile synthesis of monodisperse polymer nanocapsules via a redox-mediated kinetic strategy from two simple molecules: dopamine and benzene-1,4-dithiol (BDT). Specifically, BDT forms core templates and modulates the oxidation kinetics of dopamine into polydopamine (PDA) shells. These uniform nanoparticles can be tuned between ≈70 and 200 nm because the core diameter directly depends on BDT while the shell thickness depends on dopamine. The supramolecular core can then rapidly disassemble in organic solvents to produce PDA nanocapsules. Such nanocapsules exhibit enhanced physicochemical performance (e.g., loading capacity, photothermal transduction, and anti-oxidation) versus their solid counterparts. Particularly, this method enables a straightforward encapsulation of functional nanoparticles providing opportunities for designing complex nanostructures such as yolk-shell nanoparticles.

Activatable Carbocyanine Dimers for Photoacoustic and Fluorescent Detection of Protease Activity.

Activatable contrast agents are of ongoing research interest because they offer low background and high specificity to the imaging target. Engineered sensitivity to protease activity is particularly desirable because proteases are critical biomarkers in cancer, infectious disease, inflammatory disorders, and so forth. Herein, we developed and characterized a set of peptide-linked cyanine conjugates for dual-modal detection of protease activity via photoacoustic (PA) and fluorescence imaging. The peptide-dye conjugates were designed to undergo contact quenching via intramolecular dimerization and contained n dyes (n = 2, 3, or 4) with n - 1 cleavable peptide substrates. The absorption peaks of the conjugates were blue-shifted 50 nm relative to the free dye and had quenched fluorescence. This effect was sensitive to solvent polarity and could be reversed by solvent switching from water to dimethyl sulfoxide. Employing trypsin as a model protease, we observed a 2.5-fold recovery of the peak absorbance, 330-4600-fold fluorescent enhancement, and picomolar detection limits following proteolysis. The dimer probe was further characterized for PA activation. Proteolysis released single dye-peptide fragments that produced a 5-fold PA enhancement through the increased absorption at 680 nm with nanomolar sensitivity to trypsin. The peptide substrate could also be tuned for protease selectivity; as a proof-of-concept, we detected the main protease (Mpro) associated with the viral replication in SARS-CoV-2 infection. Last, the activated probe was imaged subcutaneously in mice and signal was linearly correlated to the cleaved probe. Overall, these results demonstrate a tunable scaffold for the PA molecular imaging of protease activity with potential value in areas such as disease monitoring, tumor imaging, intraoperative imaging, in vitro diagnostics, and point-of-care sensing.

Hybridizing clinical translatability with enzyme-free DNA signal amplifiers: recent advances in nucleic acid detection and imaging.

Nucleic acids have become viable prognostic and diagnostic biomarkers for a diverse class of diseases, particularly cancer. However, the low femtomolar to attomolar concentration of nucleic acids in human samples require sensors with excellent detection capabilities; many past and current platforms fall short or are economically difficult. Strand-mediated signal amplifiers such as hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) are superior methods for detecting trace amounts of biomolecules because one target molecule triggers the continuous production of synthetic double-helical DNA. This cascade event is highly discriminatory to the target via sequence specificity, and it can be coupled with fluorescence, electrochemistry, magnetic moment, and electrochemiluminescence for signal reporting. Here, we review recent advances in enhancing the sensing abilities in HCR and CHA for improved live-cell imaging efficiency, lowered limit of detection, and optimized multiplexity. We further outline the potential for clinical translatability of HCR and CHA by summarizing progress in employing these two tools for in vivo imaging, human sample testing, and sensing-treating dualities. We finally discuss their future prospects and suggest clinically-relevant experiments to supplement further related research.