Design and characterization of the Cy3.5@Ag NPs
As silver nanoparticles possess the merits of being benign and biocompatible, easily accessible and facilely decorated and previous literature reports have shown that silver nanoparticle treatment may cause lysosome injury[21], we designed Ag-based fluorescent nanoparticles to promote the efficient release of antitumor drugs from the lysosomes. Moreover, the common fluorescent dye Cy3.5 was also incorporated to modify the Ag nanoparticles (generating Cy3.5@Ag NPs) for imaging inside the cells. These nanoparticles were synthesized by forming a covalent bond between Cy3.5 and the polyethylene glycol (PEG)-encased silver nanocore (Ag NP) via a universally employed amide linkage that connects the amino group of Cy3.5 with the carboxyl group of the PEG chain (Figure 1A). These nanoparticles preferentially accumulate in lysosomes, instigating lysosomal passivation and lysosomal membrane permeabilization (LMP), which catalyzes the escape of these particles from the lysosomes (Figure 1B).
The cytotoxicity of both the Cy3.5 compound and the Cy3.5@Ag NPs was examined by treating HeLa cells with varying concentrations of these samples for a duration of 24 hours. Cytotoxicity was subsequently assessed using CCK-8 kits by measuring the samples' respective optical densities (ODs). The findings revealed that none of the samples inhibited the proliferation of HeLa cells within a specified concentration range (Figures S1 and S2). Consequently, this suggests that both the Cy3.5 compound and the Cy3.5@Ag NPs do not demonstrate any discernible inhibitory effects on cell proliferation, thereby ensuring their safety, and Cy3.5 could serve solely as a fluorescence marker.
Comprising three components—a 15 nm silver core, a biocompatible PEG spacer (5 kD) and the fluorescent dye Cy3.5 (Figure 1A)—the Cy3.5@Ag NPs exhibited homogeneous spherical structures. Their size predominantly ranged from 25-45 nm, as observed in the transmission electron microscopy (TEM) images (Figure S3A), while their hydrodynamic radii were approximately near 80-90 nm, as indicated by dynamic light scattering (DLS) (Figure S3B). The photophysical properties of these nanoparticles were scrutinized via UV‒vis absorbance spectroscopy and fluorescence spectroscopy, with the Cy3.5@Ag NPs showing a single peak at 406 nm in the absorbance spectrum (Figure S4A) and emitting intense red fluorescence at approximately 610 nm (Figure S4B).
Localization of the Cy3.5@Ag NPs in the lysosomes
To determine the ability of the nanoparticles to be used for live cell imaging, HeLa cells were incubated with Cy3.5@Ag NPs (0.5 μg/ml) for 1 h. We performed SIM imaging in single-channel mode with excitation at 488 nm. After 1 hour of incubation with Cy3.5@Ag NPs, we observed conspicuous red fluorescence signals, which presented as evenly dispersed globular particles within the cytoplasm (Figure 2A and 2B). The magnified images further showed distinctive red fluorescence appearing as individually scattered circular spots, consistent with previous literature [22]. Notably, when compared to the nanoparticles, HeLa cells treated with Cy3.5 (0.1 μM) for 1 h demonstrated consistent localization of Cy3.5 in mitochondria (Figure S5). Our results thus confirmed time-dependent variations in the cellular distribution of the Cy3.5@Ag NPs.
Given the observed fluorescence distribution of the Cy3.5@Ag NPs, we deduced that these NPs likely localized within lysosomes. To substantiate our hypothesis, we administered Cy3.5@Ag NPs (0.5 μg/mL) to HeLa cells, and after 1 hour, the cells were treated with the commercially available probe Lyso-Tracker Deep Red (LTDR, 100 nM). As anticipated, the red fluorescence of the Cy3.5@Ag NPs showed considerable overlap with the magenta fluorescence of LTDR after 1 hour of incubation, resulting in a Pearson's colocalization coefficient (PCC) of 0.68 (Figure 2C, 2D and 2E).
The confocal microscopy data lent further support to these findings (Figure S6). We also validated the subcellular distribution of the Cy3.5@Ag NPs within HT-1080 cells (Figure S7), which demonstrated pronounced colocalization with LTDR. These findings strongly support the hypothesis that Cy3.5@Ag NPs localize to lysosomes. Therefore, our study concludes that Cy3.5@Ag NPs penetrated living cells and were assimilated by lysosomes.
Cy3.5@Ag NPs induced lysosomal membrane permeabilization and lysosomal passivation
We also assessed how the Cy3.5@Ag NPs affect the behavior of lysosomes in live cells, focusing on their selective uptake and subsequent effects on lysosomal membrane integrity. Following treatment with these nanoparticles (0.5 μg/mL, 6 h), we observed sensitization of the HeLa cell lysosomal membranes to damage by photooxidation[23]. This was indicated by an increase in green fluorescence and a decrease in red fluorescence. Time-lapse confocal images of both untreated and treated cells revealed substantial changes in the fluorescence of the lysosomes (red signal) and cytoplasm (green signal) following exposure to the NPs (Figure 3A, 3B and 3C), suggesting that the stability of the lysosomal membrane was impacted. Our findings suggest that Cy3.5@Ag NPs induce lysosomal membrane instability and instigate lysosomal evasion (Figure 3D).
When lysosomes absorb nanoparticles, it can lead to a condition known as lysosomal stress, potentially causing the lysosomes to swell[24, 25]. Our analysis of the effect of Cy3.5@Ag NPs on lysosomal morphology[26] involved treating HeLa cells with 0.5 μg/mL NPs for 1 hour and LTDR for 30 minutes. Upon examination, we found no signs of lysosomal swelling caused by the NPs. The size distribution of the particles was consistent with standard lysosomal characteristics[22], supporting this conclusion (Figure S8). The morphology was further confirmed by TEM analysis (Figure S9).
To assess the impact of the Cy3.5@Ag NPs on lysosomal movement[1] within the cytoplasm, we tracked lysosomal motion using SIM time-lapse photography. Untreated HeLa cells exhibited random movements near the nucleus and more directed movements around the cell, consistent with previous findings[24]. In contrast, the presence of Cy3.5@Ag NPs caused significant changes in the movement patterns of the lysosomes, resulting in stable in situ trajectory structures (Figure 4A, 4B and 4C). There was also a noticeable reduction in both the track length and displacement of the lysosomal movements in the cells treated with the NPs (Figure 4D), pointing to a significant decrease in lysosomal motility due to the presence of Cy3.5@Ag NPs (Figure 4E).
Investigation of the specificity and selectivity of Cy3.5@Ag NPs for the lysosomes in HeLa cells was conducted after demonstrating their lysosomal escape behavior and their effect on inhibiting lysosome mobility. To evaluate the nanoparticles’ lysosomal uptake specificity postinternalization, HeLa cells were treated with bafilomycin A1 (BafA1, 100 nM) and chloroquine (CQ, 50.0 μM) for 3 hours to minimize lysosomal uptake[27-29]. Next, the cells were treated with Cy3.5@Ag NPs for an hour, after which observations were made via confocal laser scanning microscopy (CLSM). The CLSM images revealed that BafA1 or CQ treatment led to Cy3.5@Ag NPs displaying diffuse fluorescence signals in the cytoplasm, replacing the speckled patterns seen in nontreated cells (Figure S10), proving that these nanoparticles are specifically absorbed by lysosomes.
Moreover, the selectivity of these nanoparticles for lysosomes and their safety concerning other organelles were examined by costaining HeLa cells with Cy3.5@Ag NPs (λex= 488 nm) and other commercially available probes, such as a nucleus probe (Hoechst, λex=405 nm), a lipid droplet probe (Lipi-Blue, λex=405 nm), and an autophagolysosome probe (DALG, λex= 488 nm). The findings confirmed that the Cy3.5@Ag NPs did not invade other organelles (Figures S11-S13). Therefore, collectively, our findings affirm that these Cy3.5@Ag NPs selectively target lysosomes without impacting other organelles.
Enhancement of subcellular-targeted drug efficacy facilitated by Cy3.5@Ag NPs
Many existing antitumor drugs are unable to escape from lysosomes after being swallowed[6, 7], resulting in a decrease in efficacy and the development of drug resistance. To assess whether Cy3.5@Ag NPs can trigger lysosomal escape, thereby boosting drug release from the lysosomes and amplifying drug effectiveness, we paired them with various traditional antitumor drugs. We then used SIM imaging and cellular toxicity tests to confirm the potential of the Cy3.5@Ag NPs to enhance the potency of these antitumor drugs.
Previous research has shown that carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a mitochondria-targeted inhibitor, can inhibit STING-mediated IFN-β production by disrupting the mitochondrial membrane potential[30]. As illustrated in Figure 5A, HeLa cells were treated with CCCP (5.0 μM), and the mitochondria were labeled with MTG. The results showed that cellular mitochondria were swollen and even fragmented, and more mitochondrial swelling as well as more severe mitochondrial fragmentation was observed after CCCP coincubation with the Cy3.5@Ag NPs (0.5 μg/mL). This was evident in MTG-labeled mitochondria determined through aspect ratio analysis plots of the mitochondria in each group (Figure 5C and Figure S14). We speculate that this phenomenon was due to the increased permeability of the lysosomal membrane induced by Cy3.5@Ag NPs that facilitated the effective binding of CCCP to the mitochondria, resulting in an enhanced ability to induce mitochondrial autophagy. Moreover, the cellular value-added toxicity assay results also demonstrated that coincubation of Cy3.5@Ag NPs with CCCP resulted in lower cell survival than CCCP treatment alone (Figure 5E). Our findings provide compelling evidence that the presence of Cy3.5@Ag NPs amplifies the capacity of CCCP to induce mitochondrial autophagy.
Brequinar (BQR), a typical inhibitor of mitochondrial dihydroorotic acid dehydrogenase, can trigger lipid peroxidation-linked iron death by obstructing the fourth step in the pyrimidine de novo synthesis pathway[31, 32]. As illustrated in Figure 5B and 5D, HeLa cells were treated with BQR (40 μM), and mitochondria were labeled with MTG. The results showed that the cellular mitochondria were fibrillated and fragmented, and BQR coincubation with Cy3.5@Ag NPs (0.5 μg/mL) resulted in more mitochondrial fibrillation as well as more severe mitochondrial fragmentation. Moreover, the cellular value-added toxicity assay data also demonstrated that coincubation of Cy3.5@Ag NPs with BQR resulted in lower cell survival than BQR treatment alone (Figure 5E). All these results suggest that Cy3.5@Ag NPs can effectively promote the efficacy of mitochondria-targeted antitumor drugs (Figure 5F and 5G).
Furthermore, in addition to mitochondria-targeted drugs, Cy3.5@Ag NPs also have the ability to facilitate the release of nucleus-targeted drugs from the lysosomes. Herein, two nucleus-targeted drugs, adriamycin (ADR) and gemcitabine (GEM), which target DNA and RNA, respectively, were chosen to validate this hypothesis[33, 34]. HeLa cells were treated with ADR (1.0 μM) and Cy3.5@Ag NPs (0.5 μg/mL) for 24 h, and cytotoxicity was examined with CCK-8 kits. GEM (10 μM) was analyzed in the same way. The results demonstrated that the utilization of Cy3.5@Ag NPs can strengthen the cytotoxicity of ADR and GEM in comparison to ADR and GEM alone (Figure S15), suggesting that the Cy3.5@Ag NPs promote drug escape from the lysosome by altering the permeability of the lysosomal membrane. In conclusion, Cy3.5@Ag NPs can improve the efficacy of subcellular-targeted drugs while retaining biosafety.