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.

Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants continue to spread worldwide, causing millions of cumulative cases and deaths.[1,2] Despite the availability of accurate screening and reliable vaccines, there is still a lack of effective therapies for this disease.[3−6] Several antiviral therapies, targeting the transmembrane protease serine 2 (TMPRSS2) and/or cell surface angiotensin-converting enzyme 2 (ACE2), are under study. Some of them have emergency use authorization or are in clinical trials to prevent infection of host cells via protease inhibitors, therapeutic antibodies, engineered aptamers, and cross-linking peptides.[7−12] Upon infection with SARS-CoV-2, cells produce several key proteases through the coronavirus RNA genome for viral replication.[13] Among them, main protease (Mpro, also known as 3CLpro) is an essential nonstructural protein that can effectively cleave the viral precursor polyprotein at specific sites to form functional proteins. Mpro is an attractive biomarker and a promising target for virus detection and inhibition.[14−17] Monitoring Mpro activity has attracted broad interest due to its potential value in identifying infected cells and monitoring inhibitors, which led to the development of green fluorescent protein (GFP)-derived FlipGFP reporters, fluorescence (or Förster) resonance energy transfer-based probes, and colorimetric sensing platforms.[18−20] Beyond detection, systems that offer both selective imaging and inhibition of SARS-CoV-2-infected cells could improve the management of the current epidemic situation and understanding of viral progression.

Enzyme-instructed self-assembly (EISA) has been a paradigm shift in cancer therapy and can be leveraged separately and in combination with existing therapies.[21−25] Indeed, the controlled formation and accumulation of supramolecular nanostructures can affect the behavior and fate of targeted cells. By jointly utilizing overexpressed proteases, responsive peptides, and self-assembling precursors, the tunable aggregates can be precisely constructed and selectively inhibit target cancer cells or even specific organelles without damaging normal cells.[26−28] This similar strategy has been applied to combat inflammatory cells and kill bacteria.[29,30] On the other hand, in order to accurately monitor treatment by EISA, various contrast agents have been developed for real-time and long-term tracking of delivery processes.[31−36] In particular, fluorescent probes offer a rapid signal, high sensitivity, and easy labeling to monitor the treatment process and enhance aggregate formation via hydrophobic aromatic cyclic hydrocarbon dyes.[37−41] However, the fluorescence intensity of conventional fluorophores is often quenched at high concentrations because of the notorious aggregation-caused quenching (ACQ) effect.[42] As an alternative, aggregation-induced emission (AIE) luminogens (AIEgens) with bright fluorescence, a large Stokes shift, and superior photostability have been widely used for optical devices, luminescent sensors, and imaging systems.[43] Notably, the fluorescent intensity of AIEgens can be regulated by the customized sequences of peptides, nucleic acids, and glycans via changes in the molecular aggregation state without self-quenching.[44] Specifically, the modular-peptide-conjugated AIEgens have many advantages for cell-selective imaging, targeted gene delivery, and synergistic therapy.[45−48] At least four typical principles can be used to activate AIEgen fluorescence via functional peptides—these all restrict intramolecular motion: (1) trapping AIEgens into a confinement groove of specific proteins with ligands,[45] (2) delivering numerous AIEgens into a narrow space of organelles with targeted peptides,[46] (3) cleaving the hydrophilic peptides from AIEgens with proteases to enhance hydrophobic aggregation,[47] and (4) creating AIEgen assemblies with self-assembling peptides.[37] Therefore, combining EISA and the AIE effect can be a feasible method to selectively image and kill cells of interest. To the best of our knowledge, no study has yet reported this approach for effective treatment of SARS-CoV-2-infected cells through rational construction of peptide-conjugated AIEgens.

Herein, we report a Mpro-responsive, modular-peptide-conjugated, and mitochondrial-targeting AIEgen (PSGMR) for selective imaging and inhibition of SARS-CoV-2-infected cells (Scheme ). PSGMR consists of five segments: The first is an AIEgen (PyTPE, P for short in PSGMR). PyTPE as a typical AIEgen with a pyridinium moiety has bright fluorescence, excellent biocompatibility, and good photostability for biomarker detection and mitochondrial-targeting imaging.[49,50] Second, the self-assembling peptide (KLVFF, S) is a β-sheet-forming peptide derived from a β-amyloid (Aβ) protein.[51,52] KLVFF can spontaneously self-assemble into amyloid fibrils and kill cells when aggregated into insoluble fibrils through the intermolecular interactions in the absence of additional hydrophilic residues.[53−55] Third, the spacer trimylglycine (GGG, G) is designed to enhance probe flexibility and reduce steric hindrance for Mpro–substrate interactions.[56,57] The fourth component is the Mpro-responsive peptide (SAVLQ/SGFRKMA, M), which can be cleaved by Mpro after the LQ sequence.[58,59] The fifth is a positive charged hexamolyarginine (RRRRRR, R) that increases both the solubility and cell-penetrating ability of PSGMR and shields the self-assembly capability of PyTPE and KLVFF.[60−63] These five components were covalently coupled through a Fmoc-based solid-phase peptide synthesis and a copper-catalyzed azide–alkyne click reaction. In the absence of Mpro, PSGMR as an amphiphilic molecule with highly water-soluble and electrostatic repulsion can form loose nanoparticles with limited fluorescence in an aqueous solution (Scheme a). It shows the positive charged hexamolyarginine residues on the surface and the hydrophobic core of PyTPE. After being cleaved by Mpro, the hydrophilic hexamolyarginine is separated from PSG, and the self-assembling peptides with C-terminal carboxyl are exposed to the surface of smaller nanoparticles. Finally, the decreasing hydrophilicity and increasing self-assembly of KLVFF as well as electrostatic attraction led to PSG gradual aggregation with strong fluorescence (Scheme S1). Thus, after incubation with PSGMR, the cells transfected with a Mpro plasmid or infected by SARS-CoV-2 to produce Mpro can induce PSG aggregation inside the mitochondria and selectively inhibit the growth of the cells to prevent virus replication (Scheme b). This theranostic probe will provide a controllable avenue for selective imaging and inhibition of the SARS-CoV-2-infected cells.

Scheme 1

Structure and function of PSGMR

(a) Molecular structure and schematic illustration of PSGMR with main protease (Mpro). (b) PSGMR is used for Mpro-responsive mitochondrial imaging and selective inhibition of Mpro plasmid-transfected or SARS-CoV-2-infected cells. (i) PSGMR effectively crosses the cell membrane; (ii) Mpro plasmid-transfected or SARS-CoV-2-infected cells can produce Mpro; (iii) Mpro binds and cleaves the substrate of PSGMR; (iv) after being cleaved by Mpro, PSG fragments decrease in size and are targeted for delivery to the mitochondria; (v) PSG aggregation induces mitochondrial interference and selectively inhibits the growth of SARS-CoV-2-infected cells with strong fluorescence. The potentially charged amino acids are labeled with the charge symbol after Mpro cleavage.

Results and Discussion

Design, Synthesis, and Characterization of PSGMR and Its Derivatives

PSGMR and its derivatives were designed based on the requirements of EISA and the AIE effect via a Mpro trigger. Importantly, SAVLQ/SGFRKMA as a substrate for Mpro cleavage can regulate the ratio of hydrophobicity and hydrophilicity and surface potential. This sequence has an area of high enzyme digestion efficiency located in the center. After the Mpro cleavage, it was divided into the hydrophobic SAVLQ and hydrophilic SGFRKMA. For the proper arrangement of these segments, the hydrophobic PyTPE and KLVFF were located at the N-terminal, whereas the hydrophilic hexamolyarginine part was placed at the C-terminal. Trimylglycine was added at both ends of the Mpro substrate to leave enough space for enzyme and substrate binding. All peptide domains were covalently linked via Fmoc-based solid-phase peptide synthesis. Propargylglycine (Pra) was used as a linker to couple with azide-functionalized PyTPE under mild conditions via a copper-catalyzed click reaction. Two control probes without a spacer (PSMR) and self-assembling peptide (PMR) were synthesized to verify Mpro accessibility and self-assembly of the probes.

All of the probes were synthesized according to previous reports with minor improvement (Schemes S2–S5 and Table S1) and characterized by high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) to confirm their purity (at least 95%) and chemical structures (Figures S1–S9). We also tested the high-resolution mass spectra (HRMS) to verify the accuracy of multiple charge peaks of PSMR in the ESI-MS (Figures S3 and S5). Taking PSGMR as an example, Figure S7 shows a strong peak at 768.2742 that is attributed to the [M + 5H]5+ ion of PSGMR (calcd 767.8326), a strong peak at 640.3345 attributed to the [M + 6H]6+ ion of PSGMR (calcd 640.0285), a strong peak at 549.0886 attributed to the [M + 7H]7+ ion of PSGMR (calcd 548.7398), and a strong peak at 480.9327 attributed to the [M + 8H]8+ ion of PSGMR (calcd 480.2733). The mass data of PSMR and PMR also matched well with the calculated data. These data indicated that PSGMR, PSMR, and PMR were synthesized successfully.

Responsiveness to Enzyme in Solutions

We first evaluated whether the peptides and probes can be specifically cleaved by recombinant Mpro as predicted. We found that the specially customized peptides CGAVLQDDD and AVLQFFVLKC were not cleaved by Mpro, but RVRRSAVLQSGFRKMAC and CGKLVFFGTSAVLQSGFRGDDD were cleaved by Mpro between Q and S (Figures a–d and S10–S12). The docking scores of Mpro with different peptides also showed the enhanced binding abilities among them (Figure S13). HPLC and ESI-MS analysis also confirmed that PSGMR and PMR but not PSMR can be cleaved by Mpro between Q and S after incubation for 1 h at 37 °C in 20 mM Tris-HCl buffer (pH 8.0) (Figures e,f and S14–S17). After 24 h of incubation, PSGMR was fully cleaved by Mpro, whereas PSMR remained intact. These data suggest that the hydrophobic peptide or PyTPE prevent Mpro from binding to the substrate. The trimylglycine spacer can improve the digestion efficiency of Mpro for this substrate (SAVLQ/SGFRKMA).

Figure 1

Characteristics of PSGMR and its derivatives. (a) HPLC and (b–d) ESI-MS results of the designed peptide incubation with Mpro. (e) Molecular composition, (f) HPLC, (g) absorption, and (h) fluorescence spectra of PSGMR, PSMR, and PMR showed the good purity and solubility enhancement with the decreased fluorescence intensity. (i) Fluorescence spectra and (j) kinetics of PSGMR with different concentration of Mpro and 10 μM GC376 at 590 nm showed the fluorescence increase because of Mpro. (k) Probe specificity of PSGMR with 200 nM different proteins including Mpro, papain-like protease, thrombin, bovine serum albumin, and hemoglobin. (l) Hydrodynamic sizes, (m) zeta-potential values, (n) photographs and transmission electron microscope images of PSGMR with Mpro. In panel j, vials contain 10, 50, 100, 200, and 400 μM PSGMR solutions under UV light (365 nm, 16 W), and 10 μM of PSGMR, PSMR, PMR, and PyTPE was dissolved in Tris-HCl buffer with 1% DMSO at λex = 405 nm.

We then explored the spectral properties of PyTPE, PSGMR, PSMR, and PMR. They showed absorption spectral profiles at 350–450 nm in Tris-HCl buffer with 1% DMSO at room temperature (Figure g); 405 nm was chosen as the optimal excitation wavelength. The fluorescence spectral profiles of PyTPE, PSGMR, PSMR, and PMR were 500–750 nm (Figure h). The fluorescence intensity of PSGMR, PSMR, and PMR decreased after being modified with hydrophilic peptides. The critical micelle concentration values of PSGMR and PSGMR after incubation with 200 nM Mpro decreased from 8.95 to 2.06 μM (Figure S18). The fluorescence changes of PSGMR and PMR incubation were monitored upon incubation with Mpro in different media: 10 μM offered a significant fluorescence enhancement and was used for subsequent experiments (Figures S19–S21). Particularly, the fluorescence intensity of PMR and PSMR after incubation with Mpro was much weaker than that of PSGMR because of the lack of a self-assembling peptide and spacer.

To validate the enzyme digestion efficiency, PSGMR was incubated with different concentrations of Mpro. The fluorescence intensity of PSGMR gradually increased with increasing Mpro concentration (Figure i). When PSGMR was incubated with more Mpro (from 100 to 400 nM), the fluorescence intensity of PSGMR increased but not as much as the PyTPE at the same concentration (Figure S22). This is because of some hydrophilic residues (K, S, and Q) that are still linked with PyTPE. Subsequent kinetic studies were performed by incubating PSGMR with 200 nM Mpro and 10 μM Mpro inhibitor GC376 over time.[18,19] In the absence of GC376, the fluorescence intensity of PSGMR at 590 nm obviously increased with time and plateaued within 40 min with Mpro incubation (Figure j). No increase in the fluorescence was detected in the presence of the GC376, thus showing that the fluorescence increase was due to Mpro-mediated peptide cleavage. PSGMR was also treated under identical conditions with several commercial proteins to investigate probe specificity: papain-like protease, thrombin, bovine serum albumin (BSA), and hemoglobin. The fluorescence intensity of PSGMR was clearly enhanced selectively with Mpro (Figure k). The fluorescence intensity of PyTPE was only slightly elevated when BSA was added (Figure S23). BSA has a low isoelectric point, and thus it or other negatively charged proteins may cause PyTPE and PSGMR to aggregate due to the positively charged pyridinium and hexamolyarginine.

Dynamic light scattering tests, transmission electron microscopy (TEM), circular dichroism spectroscopy of PSGMR were performed to determine the change of particle size, surface potential distribution, and secondary structure after incubation with Mpro. The average hydrodynamic size of PSGMR increased from 142 to 396 nm (Figure l), and the mean zeta-potential value decreased from 23.97 to 12.46 mV (Figure m), suggesting nanoparticle aggregation and a reduction of arginine on the nanoparticle surface. The morphology change of PSGMR before and after incubation with Mpro was confirmed by TEM (Figure n). The β-sheet structure of PSGMR, but not in Tris-HCl buffer and PMR, was observed after incubation with Mpro or at high concentrations (Figures S24–S27). These data proved that PSGMR is responsive to Mpro, leading to aggregation state changes and fluorescence enhancement.

Mitochondrial Imaging and Imaging Comparison of HeLa Cells with PyTPE, PSGMR, and PMR

The mitochondrial-targeting capability of PyTPE was tested via HeLa cell colocalization imaging. HeLa cells were cocultured with commercial dyes Mitotracker green (MTG), Hoechst 33258 (nucleus staining of all cells), and propidium iodide (PI, nucleus staining of dead cells) for confocal laser scanning microscopy (CLSM) observation. First, the photobleaching results showed that PyTPE, PSGMR, and PMR had photostability better than that of MTG (Figure S28). HeLa cells were incubated with different concentrations of PyTPE for 3 h and 1 μM MTG and 5 μM Hoechst 33258 for 1 h (Figure S29). The yellow fluorescence of PyTPE overlapped well with the green fluorescence of MTG with a Pearson correlation coefficient of 81.71% (5 μM) to 85.09% (10 μM). High-resolution cell imaging and fluorescence intensities of PyTPE and MTG showed exact overlap on each other along the red line, thus confirming that PyTPE can target mitochondria with high specificity (Figure a). Previous work showed that positively charged pyridinium units and hydrophobic alkyl chain of PyTPE can target mitochondria based on the hydrophobic effect and electrostatic interactions.[49,50,64,65] Compared to the fluorescence of PyTPE with PSGMR and PMR at a 5 μM concentration, PSGMR and PMR displayed weak fluorescence and poor overlap with MTG because of the hydrophilic polypeptides (Figure S30). The strong yellow fluorescence of 10 μM PSGMR was seen inside the HeLa cells due to nonspecific aggregation. This phenomenon is more clear in the Z-stack imaging for PyTPE and PSGMR (Figure S31).

Figure 2

Mitochondrial imaging of HeLa cells with PyTPE and PSGMR. (a) CLSM images and the normalized fluorescence intensities based on the red dotted lines of HeLa cells with Hoechst 33258, PyTPE and a commercial dye MTG for colocalization imaging. (b) Experimental scheme of HeLa cells after incubation with lipopolysaccharide (LPS) for 3 h and PyTPE, PSGMR, Hoechst 33258, and PI. The enlarged portion shows the chemical construction of LPS. CLSM images and average fluorescence intensities of HeLa cells with (c) PyTPE, Hoechst 33258, and PI, (d) LPS, PyTPE, Hoechst 33258, and PI, (e) LPS, PSGMR, Hoechst 33258, and PI. The MTG is activated for mitochondria in the green channel. The PI is activated in the red channel when cells are dead.

To study the crowding effects that might happen during apoptosis and induce the fluorescence, HeLa cells were incubated with 30 μg/mL lipopolysaccharide (LPS, known as endotoxin) for 3 h. We then added PyTPE (or PSGMR), Hoechst 33258, and PI for 1 h (Figure b). The yellow and green fluorescence had good overlap, but no red fluorescence was seen in the HeLa cells without LPS incubation, suggesting the cells were alive (Figure c). After pretreatment with LPS, some of HeLa cells were dead and showed red fluorescence for both PyTPE and PSGMR incubation (Figure d,e). The yellow and green fluorescence in dead cells was weaker than that in living cells, especially for the PSGMR, which had obvious extracellular yellow fluorescence. We found that a stronger red fluorescence of dead cells implied a lower yellow and green fluorescence. The membrane potential decreased after cell death and prevented the macromolecule PSGMR from interacting with the cells. Similarly, small molecule dyes PyTPE and MTG could not enter the cells because of the negatively charged LPS and its toxicity.

Imaging and Inhibition of Mpro Plasmid-Transfected HEK 293T Cells with PSGMR and Mpro Reporter

To image Mpro in living cells, HEK 293T cells were transfected with a Mpro plasmid, influenza virus protein (PR8) plasmid, and a Mpro-related FlipGFP reporter plasmid to produce proteins of interest (Figure a).[18,19] The Mpro-related FlipGFP reporter plasmid was cotransfected into the Mpro plasmid or PR8 plasmid-transfected cells to assess Mpro expression in plasmid-transfected HEK 293T cells. The green fluorescence of the FlipGFP reporter only activates after cleavage by Mpro. The transfected cells were further incubated with PSGMR and PI for CLSM cell imaging. First, to examine the cytotoxicity and optimize the concentration of probes for cell imaging, different concentrations between 1 and 40 μM of PyTPE, SGMR, PSGMR, and PMR were incubated with HEK 293T cells for 48 h under standard cell culture conditions (Figure S32). PSGMR showed negligible toxicity to HEK 293T cells with almost 100% cell viability at low concentrations (1–10 μM). A high concentration of PyTPE (>5 μM) and PSGMR (>40 μM) can cause significant cytotoxicity. Compared with the cell viability of Mpro plasmid-transfected HEK 293T cells, over 50% of the cells were dead with 10 mM PSGMR incubation, unlike that with 5 mM PSGMR. According to the CLSM images of cells for 3 h incubation, blue fluorescence of Hoechst 33258 was observed in the nucleus, and yellow fluorescence of PSGMR appeared at higher concentrations (≥5 μM), leading to strong background signals (Figure S33). Most of HEK 293T cells with a high concentration (20 μM) of PSGMR had red fluorescence from PI, indicating that high concentrations of PSGMR could cause significant cytotoxicity due to the mitochondrial-targeting damage by PyTPE. Therefore, to avoid nonspecific aggregation and nonspecific cleavage by proteolytic enzymes in the complex cellular microenvironment, 5 μM of probe was used for Mpro imaging for 30 min incubation and 10 μM probes for cell inhibition experiments.

Figure 3

Validation of PSGMR via plasmids and reporter. (a) Experimental scheme of different plasmid-transfected HEK 293T cells after incubation with PI and PSGMR. The enlarged portion shows the imaging mechanism of FlipGFP. CLSM images of the HEK 293T cells with (b) PSGMR, (c) Mpro-related FlipGFP reporter plasmid and PSGMR, (d) Mpro-related FlipGFP reporter plasmid, PR8 plasmid, and PSGMR, (e) Mpro-related FlipGFP reporter plasmid, Mpro plasmid and PSGMR, and Mpro plasmid and PSGMR for (f) 1 h, (g) 4 h, and (h) 8 h. Average fluorescence intensities of (i) GFP, (j) PI, and (k) PSGMR in each panel. The FlipGFP and PSGMR are activated in the green and yellow channels when cells are transfected with the Mpro plasmid. The PI is activated in the red channel when cells are dead.

The untransfected HEK 293T cells showed almost no yellow fluorescence just for 1 h incubation with 5 μM PSGMR (Figure b). Only weak green and yellow fluorescence was observed in the FlipGFP reporter plasmid-transfected and PR8 plasmid and FlipGFP reporter plasmid cotransfected HEK 293T cells (Figure c,d). Notably, the transfection agent was toxic to cells and led to the red fluorescence.[66,67] Strong green, yellow, and red fluorescence was observed in the cells that were cotransfected with Mpro plasmid and FlipGFP reporter plasmid (Figure e,j). Only cotransfection of Mpro and FlipGFP plasmids produced a strong fluorescence signal of PSGMR, FlipGFP, and PI, thus indicating that Mpro and the FlipGFP reporter were functional in these cells, and more cells were dead because of PSGMR. The flow cytometric results showed fluorescence intensities of FlipGFP and PI in the Mpro and FlipGFP plasmid-cotransfected HEK 293T cells higher than those in the control experiments (Figure S34). We further investigated the FlipGFP reporter and PSGMR independent for Mpro sensing. Both approaches could be used for intracellular Mpro imaging (Figure S35). While the corresponding plasmid needed to be transfected into cells and expressed to FlipGFP reporter, which would take a long-time incubation (24–48 h) and complicated operation with limited yield.[18] PSGMR not only can easily enter the cells for Mpro imaging with good photostability but also kill the targeted cells (Figure S36). We clearly observe the aggregation of PSGMR with increasing fluorescence inside the cells. The yellow fluorescence was first displayed from the cytoplasm near the nucleus and then near the cell membrane (Figure S37).

To validate the cell inhibitory effect of probes, 10 μM PSGMR was incubated with Mpro plasmid-transfected cells for 1, 4, and 8 h (Figure f–h). In particular, compared with the fluorescence of HeLa cells incubated with LPS and PSGMR, there was strong yellow fluorescence inside the Mpro plasmid-transfected HEK 293T cells but not in the culture medium. The red and yellow fluorescence intensities were markedly enhanced with the prolonged incubation time (Figure j,k). We further confirmed that PSGMR overlapped well with MTG with a Pearson correlation coefficient of 85.26% in the Mpro plasmid-transfected HEK 293T cell (Figure S38). All of these data proved that PSGMR can be used for selective mitochondrial imaging and inhibition of Mpro plasmid-transfected cells.

Imaging and Inhibition of SARS-CoV-2-Infected TMPRSS2-Vero Cells with PSGMR and GC376

After validating that PSGMR could selectively image and induce cytotoxicity in Mpro plasmid-transfected HEK 293T cells, we then examined whether PSGMR could similarly image and kill SARS-CoV-2-infected cells. Thus, TMPRSS2-Vero cells were infected with SARS-CoV-2 (USA-WA1/2020) at a multiplicity of infection of 0.02 for 24 h before PSGMR, Hoechst 33258, and PI were added. These cells were further labeled by staining viral proteins using fluorescent antibodies after cells were fixed, including anti-SARS-CoV-2 nucleocapsid (Capsid) primary antibody and anti-SARS-CoV-2 Mpro primary antibody; AlexaFluor488-labeled secondary antibody (Alexa488) was used for both (Figure a). At 24 h postinfection, these probes were separately incubated with the noninfected and infected TMPRSS2-Vero cells for cell imaging. Cyan fluorescence of Alexa488 (either Capsid or Mpro) was observed in infected cells but not in uninfected cells, thus confirming that these cells were infected by SARS-CoV-2 and produced Mpro (Figure b,c). This result has also been confirmed with Western blot analysis in our previous work.[19] In addition, strong yellow and red fluorescence was displayed only in infected cells treated with PSGMR rather than the uninfected cells with PSGMR or infected cells without PSGMR, thus showing that PSGMR can selectively kill SARS-CoV-2-infected cells (Figure d,e). The Pearson correlation coefficient of colocalization between PSGMR and Alexa488 for Capsid or Mpro increased from 33.56 to 61.57% (Figure S39). Alexa488-labeled secondary antibody for Mpro detects primary antibody bound to Mpro, which is mainly in the cytoplasm. PSGMR can target mitochondria and be cleaved by Mpro but not bind to Mpro. This leads to a low Pearson correlation coefficient of colocalization between PSGMR and Alexa488. Upon increasing PSGMR concentration to 10 μM, the yellow and red fluorescence of the infected cells was significantly enhanced (Figure S40). When 10 μM PMR was added to infected cells, the yellow and red fluorescence intensities were weaker than that of PSGMR because of the absence of a self-assembling peptide (Figure S41).

Figure 4

Selective imaging and inhibition of SARS-CoV-2-infected TMPRSS2-Vero cells with PSGMR. (a) Experimental scheme of SARS-CoV-2-infected TMPRSS2-Vero cell incubation with PSGMR, anti-SARS-CoV-2 nucleocapsid (Capsid) primary antibody, anti-SARS-CoV-2 Mpro primary antibody, and AlexaFluor488-labeled secondary antibody (Alexa488). The enlarged portion shows the imaging mechanism of Alexa488. CLSM images and average fluorescence intensities of (b) noninfected cells with PSGMR, (c) infected cells with capsid primary antibody/Alexa488, (d) infected cells with capsid primary antibody/Alexa488 and PSGMR, and (e) infected cells with Mpro primary antibody/Alexa488 and PSGMR. The PI is activated in the red channel when cells are dead. The PSGMR and Alexa488 are, respectively, activated in the yellow and cyan channels when cells are infected by SARS-CoV-2. The red and pink arrows show clear fluorescence only from Alexa488, which represents PSGMR protease activity, and the Alexa488-labeled Capsid or Mpro antibody showed the protein location.

Further exploration of the intracellular distribution of probes in infected cells can help the study of the mechanism of action of these probes in inducing cell death (Figure a). As can be seen in Figure S42, the blue and red fluorescence overlapped well in the nucleus, and the yellow fluorescence of PSGMR was close to the cell membrane. During cell apoptosis, the decreased mitochondrial membrane potential could result in the diffusion of AIEgens from intracellular mitochondria to cytosol and extracellular medium due to the concentration gradient.[49,68] Importantly, the cyan fluorescence of Alexa488 for Capsid was mainly located in the cell membrane and nucleus (Movies S1 and S2). The cyan fluorescence of Alexa488 for Mpro was displayed in the cell membrane and cytoplasm (Movies S3 and S4). This result clearly revealed the different locations of each protein in the infected cells. Moreover, compared with the differences between PSGMR and PMR in the noninfected and infected cells, the noninfected cells with PSGMR had weak red and yellow fluorescence with normal cell morphology (Figure S43). The infected cells with PSGMR showed bright red and yellow fluorescence with obvious cell morphologic dilatation. The red and yellow fluorescence as well as cell morphology change of infected cells with PMR was less than that of the infected cells with PSGMR (Figure b,c). The average red and yellow fluorescence intensities of infected cells with PSGMR were 600 and 569% higher than that of the infected cells with PMR (Figures S43 and S44). These results confirmed that PSGMR was superior to PMR at selectively imaging and killing the SARS-CoV-2-infected TMPRSS2-Vero cells.

Figure 5

Imaging and inhibition of SARS-CoV-2-infected TMPRSS2-Vero cells with PSGMR and Mpro inhibitor. (a) Experimental scheme of SARS-CoV-2-infected TMPRSS2-Vero cells with PSGMR and Mpro inhibitor. CLSM images of SARS-CoV-2-infected cells with (b) 10 μM PSGMR, (c) 10 μM PMR, and 10 μM PSGMR with (d) 2 μM GC376, (e) 6 μM GC376, and (f) 12 μM GC376. Mean fluorescence intensity of (g) PI and (h) PSGMR in infected cells incubated with 10 μM PSGMR and different concentrations of GC376. The number on the dotted line represents the fold-enhanced fluorescence intensity versus the 12 μM Mpro inhibitor. In (b,c), the transverse and vertical dotted lines denote the XZ and YZ plane cutting line of Z-stack images. The white boxes in the 2.5D images are magnified. Red scale bar = 20 μm.

We then evaluated the inhibitory effect of infected cells with PSGMR and GC376. After 24 h postinfection, 10 μM PSGMR and different concentrations (2, 6, and 12 μM) of GC376 were incubated with the infected cells. Both red and yellow fluorescence markedly decreased with increasing concentrations of GC376 (Figure d–f). Their average red and yellow fluorescence intensities decreased from 32.9 to 11.7 times and 6.2 to 3.6 times, respectively (Figure g,h). These data suggested that PSGMR can measure Mpro inhibition, and the cytotoxicity of PSGMR can be regulated by GC376. PSGMR and GC376 could be used as a combination therapy to prevent virus replication and track the treatment process.

Conclusions

Mpro is a vital protease only expressed in the infected cells for coronavirus replication. The Mpro-related substrate is applied to accurately detect SARS-CoV-2 and quickly screen protease inhibitors to stop the epidemic. To take advantage of this particular property of Mpro and its substrate, we anticipate designing a series of Mpro-responsive probes to selectively image and even kill SARS-CoV-2-infected cells as well as achieve precision treatment through the long-term fluorescence change. The key issue is to construct an efficient and controllable integrated probe for diagnosis and treatment of the SARS-CoV-2-infected cells rather than normal cells.

In summary, this work exploited the potential advantages of the EISA and AIE effect for selective detection and treatment of SARS-CoV-2-infected cells. When combined with SARS-CoV-2 replication characteristics, a Mpro-responsive and modular-peptide-conjugated mitochondrial-targeting AIEgen PSGMR offered selective imaging and inhibition of the Mpro plasmid-transfected HEK 293T cells and SARS-CoV-2-infected TMPRSS2-Vero cells. We utilized the mitochondrial-targeting function of the PyTPE to realize both mitochondrial selective delivery and long-term tracking. Compared to control probes without spacers (PSMR) and self-assembling peptides (PMR), PSGMR can be more effectively cleaved by Mpro and form aggregates in the mitochondria of targeted cells with bright yellow fluorescence. Importantly, the formation process and therapeutic effect of aggregation were controlled by the rational composition of the modular peptides and visualized by the fluorescence change. We verified the theranostic property of PSGMR and PMR with Mpro-related FlipGFP reporter, PI, Alexa488 staining of SARS-CoV-2 Capsid and Mpro, and Mpro inhibitor GC376. Under the combined influence of AIEgens and self-assembling peptides, 10 μM PSGMR fragments had significant toxicity on cells but only in the presence of Mpro. This strategy for protease-responsive organelle-targeting, modular-peptide-conjugated probes could lead to effective theranostic agents against SARS-CoV-2 and other emerging diseases. In addition, using AIEgens that absorb or emit light in the NIR window with multiple-responsive peptides can achieve deeper tissue optical imaging with higher signal-to-background ratios and better spatial resolution as well as accurate targeted delivery for precise diagnosis and treatment.

Experimental Section

Chemistry Methods and Characterization

The synthesis protocols and details are provided in the Supporting Information.

Enzymatic Assay with PSGMR, PSMR, and PMR

The stock solution of PyTPE, PSGMR, PSMR, and PMR in 20 mM Tris-HCl buffer (pH 8.0) was diluted with Mpro assay buffer (20 mM Tris-HCl buffer (pH 8.0) with 150 mM NaCl, 1 mM DTT, and 5% glycerol) to make 5 and 10 μM working solutions. Recombinant Mpro was added into the working solution and then diluted to a total of 100 or 200 μL with deionized water. The reaction mixture was incubated at 37 °C for 1 h for UV–vis absorption and photoluminescence measurement. The solution was excited at 405 nm, and the emission was collected from 430 to 800 nm.

Determination of Critical Micelle Concentration (CMC) of PSGMR and PSGMR after Incubation with Mpro

PSGMR solutions with or without pretreatment with 200 nM Mpro for 1 h were tested. The fluorescence intensity of PyTPE was analyzed as a function of the PSGMR concentration (ex = 405 nm, em = 590 nm). When extrapolating the intensity of the PSGMR concentration region, the CMC values were determined as crossing points.

Cell Culture and Plasmid Transfection

HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (FBS) and 1% penicillin streptomycin (PS, 10000 IU penicillin and 1000 μg/mL streptomycin, multicell) in cell culture plates at 37 °C in a humidified atmosphere containing 5% CO2. For plasmid transfection, cells were treated with 100–1000 μL of poly-l-lysine for 20 min before being seeded with HEK 293T cells. After 24 h incubation, Opti-MEM, plasmids (1–3 μg/μL), and TransIT-LT (Mirus) were successively mixed and incubated at room temperature for 15 min before being added to cells dropwise according to the manufacturer’s instructions.

Incubation of Living Cells with Probes

For confocal laser scanning microscopy imaging, HEK 293T cells or the plasmid-transfected HEK 293T cells were seeded into cell culture dishes at a density of 2.0 × 105 in growth medium (DMEM supplemented with 10% FBS, 200 mL). After overnight incubation, the cells were washed with phosphate-buffered saline (PBS, pH 7.4) three times. A solution of the indicated probe in medium or PBS was then added, and the cells were incubated in a 5% CO2 atmosphere at 37 °C for further use. Hoechst 33258, PI, and AlexaFluor488 were subsequently added for probe incubation. The supernatant was then discarded, and the cells were washed gently twice with PBS and fixed with 2% paraformaldehyde at room temperature for 20 min prior to optical imaging.

Viral Infection

SARS-CoV-2 isolate WA1 (USA-WA1/2020, BEI NR-52281) was passaged once through primary human bronchial epithelial cells differentiated at the air–liquid interface to select against Furin site mutations. Virus was then expanded by one passage through TMPRSS2-Vero cells. Supernatants were clarified and stored at −80 °C, and titers were determined by fluorescent assay on TMPRSS2-Vero cells. TMPRSS2-Vero cells were infected with a multiplicity of infection of 0.02 FFU per cell 24 h before incubation with probes. The SARS-CoV-2-noninfected and -infected TMPRSS2-Vero cells were washed with Dulbecco’s phosphate-buffered saline to remove FBS-containing media and then incubated with 5 or 10 μM probes for 30 min and fixed with 4% formaldehyde for 30 min. Cells were then stained using the nucleocapsid antibody or SARS-CoV-2 Mpro antibody and 5 μM Hoechst 33258. All work with SARS-CoV-2 was conducted in Biosafety Level 3 conditions at the University of California San Diego.

Immunofluorescence

Fixed cells were washed with PBS and then with PBS including 1% BSA and 0.1% TritonX-100. Cells were incubated with primary antibody against SARS-CoV-2 nucleocapsid protein (1:2000, GeneTex, GTX135357) or Mpro (1:100, Cell Signaling Technology #51661) in PBS including 1% BSA and 0.1% TritonX-100 overnight at 4 °C. Cells were washed and incubated with Alexa488 goat anti-rabbit secondary antibody (Thermo Fisher Scientific) in PBS including 1% BSA for 1 h at room temperature followed by three PBS washes.

Confocal Laser Scanning Microscopy

The fluorescence signals of cells were detected using a Zeiss LSM880 confocal laser scanning microscope (Zeiss), equipped with a 63/1.42 numerical aperture oil-immersion objective lens. A 405 nm laser was chosen for the excitation of Hoechst 33258, and the emission was collected at 420–460 nm. A 405 nm laser was chosen for the excitation of AIEgens, and the emission was collected at 550–620 nm. A 488 nm laser was chosen for the excitation of GFP, and the emission was collected at 500–530 nm. A 488 nm laser was chosen for the excitation of PI, and the emission was collected at 595–650 nm. A 488 nm laser was chosen for the excitation of AlexaFluor488, and the emission was collected at 500–550 nm. All fluorescence images were analyzed with Zeiss Image software (Zeiss).

Cytotoxicity Assay

The cytotoxic potential of PyTPE, SGMR, PSGMR, and PMR were assessed using the HEK 293T cells for 48 h incubation in quadruplicate in a 96-well plate. The fluorescence of Resazurin solution at 690 nm using an excitation wavelength of 560 nm was recorded by a Synergy H1 microplate reader (BioTek) with standard operation procedures.