Enzymatic Delivery of Magnetic Nanoparticles into Mitochondria of Live Cells

Abstract

Delivering magnetic nanoparticles (MNPs) into mitochondria provide a facile approach to manipulate cell life because mitochondria play essential roles in cell survival and death. Here we report the use of enzyme-responsive peptide assemblies to deliver MNPs into mitochondria of live cells. The mitochondria-targeting peptide (Mito-Flag), as the substrate of enterokinase (ENTK), assembles with MNPs in solution. The MNPs that are encapsulated by Mito-Flag peptides selectively accumulate to the mitochondria of cancer cells, rather than normal cells. The mitochondrial localization of MNPs reduces the viability of the cancer cells, but hardly affects the survival of the normal cell. This work demonstrates a new and facile strategy to specifically transport MNPs to the mitochondria in cancer cells for exploring the applications of MNPs as the targeted drug for biomedicine and cancer therapy.

Graphical Abstract

Self-assembling with magnetic nanoparticles (MNPs) in solution, Mito-Flag peptides deliver MNPs to mitochondria for mitochondrial dysfunction in a cancer-specific manner.

Mitochondria, as a complex organelle, play a central role in many cellular processes,^[1] including metabolism, stress responses, and cell death.^{[1f, 2]} Mitochondria are becoming one of the most important drug targets for treating a wide range of diseases, such as cancer,^[3] cardiovascular,^[4] inflammation,^[5] and neurological disorders.^[6] Therefore, considerable efforts have focused on delivering varies cargos into mitochondria. For example, Murphy et al. pioneered the use of triphenyl phosphonium (TPP) as a facile molecular motif for targeting the mitochondrial matrix,^[7] Kelly et al. demonstrated a unique class of mitochondria penetrating peptides that are cationic and hydrophobic,^[8] Wipf et. al. reported gramicidin S derivatives for targeting mitochondria,^[9] and Kim et al. utilized ethidium for targeting mitochondria.^[10] Recently, Ryu et. al. reported that pyrene-conjugated peptides also target mitochondria.^[11] While these works have illustrated useful approaches to deliver molecules into mitochondria, there are few reports on the delivery of magnetic nanoparticles (MNPs) into mitochondria,^[12] and the dosages required remain high. Thus, there is a need to explore new approaches for delivering magnetic nanoparticles^[13] into mitochondria of live cells.

The mitochondria targeting approaches mentioned above largely rely on cationic, hydrophobic moieties binding to mitochondria matrices for targeting. Recently, we and other have shown that it is feasible to use enzyme-instructed self-assembly (EISA) for mitochondria targeting.^[14] For example, we demonstrated that EISA of peptide assemblies is able to deliver molecules into mitochondria of live cells.^[15] The key feature is that an enzyme (i.e., enterokinase, ENTK^[16]) on the surface of the mitochondria of certain cells (e.g., HeLa) enables morphological transition of negatively charged peptide assemblies, from micelles to nanofibers.^{[14a, 15b]} This subcellular (mitochondrial) EISA process delivers drugs and proteins to mitochondria efficiently in live cells.^{[15a, 17]} Sun et al. also reported the use of SIRT5, a mitochondria-localized enzyme to desuccinylate a peptide precursor to form supramolecular nanofibers for targeting mitochondria. Moreover, our recent study also demonstrated the use of mitochondrial EISA to deliver DNA plasmids (e.g., pGLO^[18] or CRISPR/Cas9^[19]) or viral gene vectors for protein expression or gene knockout in the mitochondria.^[15b] These successes encourage us to use enzymatic reaction for delivering magnetic nanoparticles into mitochondria of live cells.

MNPs have been widely reported and used for biomedical applications.^{[13e, 20]} So far, many studies have utilized MNPs as the carrier for delivering drugs, proteins, and DNA into cells. The MNPs-based drug delivery systems have advanced considerably in the application of anticancer therapy.^[21] However, few studies take MNPs as the cargo to be distributed into cells, let alone the development of delivery agents for the intracellular delivery of MNPs. It is reported that the intracellular MNPs enhance the production of cytotoxic reactive oxygen species (ROS) for cancer inhibition.^[22] Since mitochondria are the center of hydrogen peroxide production, ^[23] the delivery of MNPs to the mitochondria of cancer cells can in situ activate the generation of ROS, which causes mitochondrial dysfunction and cancer cell death. These advances also stimulate us to develop a facile method to deliver MNPs to mitochondria of live cells. As shown in Scheme 1, the mitochondria-targeting peptides, denoted as Mito-Flag, form assembly with MNPs in solution. After cell entry, the MNPs enveloped by Mito-Flag selectively adhere to the mitochondria in cancer cell for localization. Upon the ENTK-catalyzed hydrolysis, the amorphous assembly of Mito-Flag and MNPs transforms into a peptidic network containing MNPs, which stabilizes the mitochondrial retention of MNPs in cancer cell. The cancer-specific delivery of the extraneous MNPs to mitochondria reduces the viability of cancer cell likely though causing mitochondrial disfunction. However, the survival of HS-5, a normal cell line, is hardly changed in the presence of Mito-Flag and MNPs, because few MNPs get delivered to the mitochondria in HS-5 cell by Mito-Flag. This work illustrates a new type of molecular motif and process for targeting mitochondria and delivering nanoparticles to mitochondria, which is significant for exploring the applications of nanoscale material and protease-instructed assembly for biomedicine.

Scheme 1.

Mito-Flag delivers MNPs to mitochondria.

Unless specially mentioned, all the MNPs in this work are composed of Fe₃O₄. To validate that Mito-Flag forms assembly with MNPs, we used transmission electron microscope (TEM) to characterize the mixture of Mito-Flag and MNPs. As shown in Figure 1A, MNPs alone, coated by carboxylic groups, are monodispersed in the solution. The mixture of MNPs and Mito-Flag exhibits amorphous nanoaggregates containing MNPs that are engulfed by peptide assembles (Figure 1B and S1), confirming that Mito-Flag peptides associate with the MNPs in solution and self-assemble into nanoclusters which incorporate MNPs through noncovalent interaction. Since Mito-Flag is carboxylic group rich, the interaction between MNPs and Mito-Flag likely originates from the carboxylic groups interacting with the surface of MNPs.^[24] Upon the addition of enterokinase (ENTK) that catalyzes the cleavage of FLAG tag into the mixture of Mito-Flag and MNPs, the enzymatic product of Mito-Flag peptides (Scheme S1) self-assembles into nanofilaments, generating a peptidic network that retains MNPs (Figure 1C).

Figure 1.

TEM images of (A) free MNPs (10 μg/mL), (B) the mixture of Mito-Flag (200 μM) and MNPs, and (C) the mixture of Mito-Flag and MNPs in the presence of ENTK (10 U/mL, 24 h). Scale bar = 100 nm.

To ensure the ability of intracellular delivery of MNPs via Mito-Flag, we mixed Mito-Flag with the MNPs that are tagged by the Cy5-capped polyethylene glycol (Cy5-PEG), followed by the incubation (4 h) with live HeLa cells. As shown by Figure 2A, the HeLa cells incubated with the combination of Mito-Flag peptides and the Cy5-PEG labeled MNPs exhibit bright red fluorescence within the cells. The coassembly of Mito-Flag and MNPs can enter the cells through clathrin-dependent endocytosis, and pH sponge effect likely facilitates the endosomal escape.^{[14a, 15, 17]} Additional staining using mitochondria tracker (MitoTracker) confirms that the fluorescence of Cy5 largely originates from the mitochondria in HeLa cells. However, the HeLa cells that are incubated with the MNPs alone display very little intracellular fluorescence (Figure 2B). TEM images of the mitochondria isolated from HeLa cells incubated with the mixture of Mito-Flag and MNPs (24 h) also reveal the presence of MNPs on mitochondria that are surrounded by nanofibers (Figure 2C). The sparse MNPs on the isolated mitochondria likely results from that MNPs shed in the process of mitochondria isolation. These results suggest that (i) free Cy5-PEG labeled MNPs rarely enter the cells, (ii) Mito-Flag peptides, when being assembled with MNPs, not only efficiently enter the cells, but also can deliver MNPs to the mitochondria of HeLa cells, and (iii) the proteolysis of Mito-Flag at mitochondria produce peptidic nanofibers which facilitate the retention of MNPs at mitochondria.

Figure 2.

Mito-Flag delivers MNPs to the mitochondria of HeLa cells, a cancer cell line. (A) Fluorescence images of HeLa cells incubated with MNPs (PEG-Cy5 modified, 10 μg/mL, 4 h) in the presence of Mito-Flag (200 μM, 4 h). (B) Fluorescence images of HeLa cells incubated with MNPs (PEG-Cy5 modified, 10 μg/mL, 4 h) alone. Scale bar = 20 μm. (C) TEM images of the isolated mitochondria from the HeLa cells incubated with the mixture of Mito-Flag (200 μM) and MNPs (10 μg/mL, 24 h). The boundary of mitochondria is depicted by dash line.

We used the same mixture of Cy5-PEG MNPs and Mito-Flag to incubate with HS-5 cell, a normal cell line. The subcellular location of the Mito-Flag encapsulated MNPs in the HS-5 cell differ significantly different from that of HeLa cells. Although Mito-Flag can deliver the Cy5-PEG-coated MNPs to the HS-5 cells, the red fluorescence of Cy5 rarely coincides with the green fluorescence of MitoTracker (Figure 3). This result indicates that after the intracellular delivery, Mito-Flag peptides hardly transfer MNPs to the mitochondria of the HS-5 cells. In addition, the punctate fluorescence of MNPs in the HS-5 cell indicates a retention in endosomes or lysosomes. The results above accord with the cancer-selective delivery of drugs, DNA, and proteins using Mito-Flag and its derivatives in our previous reports.^{[14a, 15, 17]} The selectivity likely originates from the higher ENTK level (around 2.5 times) in HeLa than in HS-5 cells and the more polarized mitochondria in cancer cells.^{[14a, 15, 17]}

Figure 3.

Mito-Flag hardly delivers MNPs to the mitochondria of HS-5 cells, a normal cell line. (A) Fluorescence images of HS-5 cells incubated with MNPs (PEG-Cy5 modified, 10 μg/mL, 4 h) in the presence of Mito-Flag (200 μM, 4 h). (B) High magnification images of (A). Scale bar = 20 μm.

The difference in the subcellular distribution between cancerous (HeLa) and normal (HS-5) cells of MNPs after the intracellular delivery by Mito-Flag results in a cancer cell-selective inhibition. While the cells incubated with either Mito-Flag or MNPs alone show very little cell death (Figure 4A), the combination of Mito-Flag and MNPs exhibits significant suppression on the viability of HeLa cells in 48 h (Figure 4A). Conversely, the addition of MNPs in Mito-Flag hardly increases the cytotoxicity of Mito-Flag against HS-5 cells (Figure 4A). To investigate the mechanism of cancer cell inhibition, we stain the mitochondria of the HeLa cells using MitoTracker. As shown in Figure 4B, although the HeLa cells incubated with Mito-Flag or MNPs (48 h), respectively, display tubular mitochondria, globular mitochondria appear in the HeLa cells treated by the mixture of Mito-Flag and MNPs (48 h). The morphological changes in mitochondria from a tubular to globular pattern refers to mitochondria fragmentation which is the consequence of mitochondria dysfunction.^[25] Since mitochondria are the center of generating hydrogen peroxide,^[23] the delivery of Fe₃O₄ MNPs to mitochondria can activate the production of mitochondria-damaging ROS through fenton-like reaction.^[26] Thus, the results above suggest that Mito-Flag peptides deliver MNPs to the mitochondria of the HeLa cells, which harms mitochondria likely through the promoted production of ROS, therefore, induces cancer inhibition. In contrast, Mito-Flag and MNPs likely remain to be sequestered in the endosome/lysosome of HS-5 cells (Figure 3), hence, hardly leads to the mitochondria dysfunction in HS-5 cells.

Figure 4.

Mito-Flag delivers MNPs to mitochondria causes cancer cell death through mitochondrial disfunction. (A) Cell viability assay of HeLa and HS-5 cells incubated with Mito-Flag (200 μM), MNPs (50 μg/mL, -COOH modified), and the mixture of Mito-Flag and MNPs for 48 h. (B) MNPs (50 μg/ml) delivery to mitochondria via Mito-Flag (200 μM, 48 h) cause mitochondrial fragmentation. Mitochondria are labelled by MitoTracker (red). Scale bar = 20 μm.

In conclusion, Mito-Flag peptides, after assembling with MNPs, selectively deliver MNPs to the mitochondria of cancer cells for inducing mitochondrial dysfunction and the suppression of the growth of cancer cells. The cancer selectivity likely originates from the overexpression of ENTK at the mitochondria of cancer cells.^{[14a, 15, 17]} The combination of the EISA of peptide with anticancer agents has become a powerful approach for increasing selectivity in cancer therapy.^[27] Among all the therapeutic methods, mitochondria-targeting strategy is especially unique and efficient due to the critical roles played by mitochondria in cell metabolism. Inducing mitochondria fragmentation by selective transport of MNPs to mitochondria expands the feasibility of MNPs in cancer treatment and demonstrates a fundamentally new approach to deliver nanoparticles to the mitochondria of cancer cells for damaging mitochondria and cancer inhibition. Hence, this work ultimately may lead to the discovery of novel nanomaterials and protease-drive molecular assembly for the applications in biomedicine.