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.     

Cell-Penetrating Mitochondrion-Targeting Ligands for the Universal Delivery of Small Molecules,Proteins and Nanomaterials

Mitochondria are key organelles that perform vital cellular functions such as those related to cell survival and death. The targeted delivery of different types of cargos to mitochondria is a well-established strategy to study mitochondrial biology and diseases. Of the various existing mitochondrion-transporting vehicles, most suffer from poor cytosolic entry, low delivery efficiency, limited cargo types, and cumbersome preparation protocols, and none was known to be universally applicable for mitochondrial delivery of different types of cargos (small molecules, proteins, and nanomaterials). Herein, two new cell-penetrating, mitochondrion-targeting ligands (named Mito^Ligand) that are capable of effectively “tagging” small-molecule drugs, native proteins and nanomaterials are disclosed, as well as their corresponding chemoselective conjugation chemistry. Upon successful cellular delivery and rapid endosome escape, the released native cargos were found to be predominantly localized inside mitochondria. Finally, by successfully delivering doxorubicin, a well-known anticancer drug, to the mitochondria of HeLa cells, we showed that the released drug possessed potent cell cytotoxicity, disrupted the mitochondrial membrane potential and finally led to apoptosis. Our strategy thus paves the way for future mitochondrion-targeted therapy with a variety of biologically active agents.     

Intracellular Delivery of Native Proteins Facilitated by Cell‐Penetrating Poly(disulfide)s

Intracellular delivery of therapeutic proteins is highly challenging and in most cases requires chemical or genetic modifications. Herein, two complementary approaches for endocytosis‐independent delivery of proteins to live mammalian cells are reported. By using either a “glycan” tag naturally derived from glycosylated proteins or a “traceless” tag that could reversibly label native lysines on non‐glycosylated proteins, followed by bioorthogonal conjugation with cell‐penetrating poly(disulfide)s (CPDs), we achieved intracellular delivery of proteins (including antibodies and enzymes) which, upon spontaneous degradation of CPDs, led to successful release of their “native” functional forms with immediate bioavailability.     

Fluorescent Coumarin–Artemisinin Conjugates as Mitochondria‐Targeting Theranostic Probes for Enhanced Anticancer Activities

Mitochondria‐targeting theranostic probes that enable the simultaneously reporting of and triggering of mitochondrial dysfunctions in cancer cells are highly attractive for cancer diagnosis and therapy. Three fluorescent mitochondria‐targeting theranostic probes have been developed by linking a mitochondrial dye, coumarin‐3‐carboximide, with a widely used traditional Chinese medicine, artemisinin, to kill cancer cells. Fluorescence images showed that the designed coumarin–artemisinin conjugates localized mainly in mitochondria, leading to enhanced anticancer activities over artemisinin. High cytotoxicity against cancer cells correlated with the strong ability to accumulate in mitochondria, which could efficiently increase the intracellular reactive oxygen species level and induce cell apoptosis. This study highlights the potential of using mitochondria‐targeting fluorophores to selectively trigger and directly visualize subcellular drug delivery in living cells.     

Sonodynamic Therapeutic Effects of Sonosensitizers with Different Intracellular Distribution Delivered by Hollow Nanocapsules Exhibiting Cytosol Specific Release

Sonodynamic therapy (SDT) is a novel promising noninvasive therapy involving utilization of low‐intensity ultrasound and sonosensitizer, which can generate reactive oxygen species (ROS) by sonication. In SDT, a high therapeutic effect is achieved by intracellular delivery and accumulation at the target sites of sonosensitizer followed by oxidative damage of produced ROS by sonication. Here, pH‐ and redox‐responsive hollow nanocapsules are prepared through the introduction of disulfide cross‐linkages to self‐assembled polymer vesicles formed from polyamidoamine dendron‐poly(l‐ lysine) for the efficient delivery of sonosensitizer. As sonosensitizer, doxorubicin (DOX), an anticancer drug accumulating into cell nucleus, is selected. Also, the conjugate of DOX and triphenylphosphonium (TPP‐DOX) is synthesized as sonosensitizer with mitochondrial targeting ability. DOX and TPP‐DOX are delivered to nucleus and mitochondria by nanocapsules. Furthermore, DOX‐ or TPP‐DOX‐loaded nanocapsules exhibit in vitro sonodynamic therapeutic effect to HeLa cells with sonication, which might be through oxidative damage to nucleus and mitochondria.     

Protein phosphorylation in mitochondria – A study on fermentative and respiratory growth of Saccharomyces cerevisiae

Phosphorylation as a posttranslational protein modification is a common subject of proteomic studies, but phosphorylation in mitochondria is still poorly investigated. The study presented here applied 2‐DE to characterize phosphorylation in the yeast mitochondrial proteome and identified 59 spots corresponding to 34 phosphorylated mitochondrial or mitochondria‐associated proteins. Most of these proteins presented putative substrates of mitogen‐activated protein and target of rapamycin kinases, cAMP‐dependent protein kinase, cyclin‐dependent kinases and Snf1p suggesting them as key players in the phosphorylation of mitochondrial or mitochondria‐associated proteins. The dynamic behaviour of the phosphoproteome under a major metabolic change, the shift from fermentation to respiration (diauxic shift), was further studied. Eight proteins (Ald4p, Eft1p/2p, Eno1p, Eno2p, Om14p, Pda1p, Qcr2p, Sdh1p) had growth dependent changes in their phosphorylation, indicating a role of phosphorylation‐dependent regulation of translation, metabolic pathways (e.g. glucose fermentation, tricarboxylic acid cycle, pyruvate dehydrogenase and its bypass) and respiratory chain.     

Mitochondria‐Targeted Cancer Therapy Using a Light‐Up Probe with Aggregation‐Induced‐Emission Characteristics

Subcellular organelle‐specific reagents for simultaneous tumor targeting, imaging, and treatment are of enormous interest in cancer therapy. Herein, we present a mitochondria‐targeting probe (AIE‐mito‐TPP) by conjugating a triphenylphosphine (TPP) with a fluorogen which can undergo aggregation‐induced emission (AIE). Owing to the more negative mitochondrial membrane potential of cancer cells than normal cells, the AIE‐mito‐TPP probe can selectively accumulate in cancer‐cell mitochondria and light up its fluorescence. More importantly, the probe exhibits selective cytotoxicity for studied cancer cells over normal cells. The high potency of AIE‐mito‐TPP correlates with its strong ability to aggregate in mitochondria, which can efficiently decrease the mitochondria membrane potential and increase the level of intracellular reactive oxygen species (ROS) in cancer cells. The mitochondrial light‐up probe provides a unique strategy for potential image‐guided therapy of cancer cells.     

A Synthetic Fluorescent Mitochondria‐Targeted Sensor for Ratiometric Imaging of Calcium in Live Cells

Ca²⁺ handling by mitochondria is crucial for cell life and the direct measure of mitochondrial Ca²⁺ concentration in living cells is of pivotal interest. Genetically‐encoded indicators greatly facilitated this task, however they require demanding delivery procedures. On the other hand, existing mitochondria‐targeted synthetic Ca²⁺ indicators are plagued by several drawbacks, for example, non‐specific localization, leakage, toxicity. Here we report the synthesis and characterization of a new fluorescent Ca²⁺ sensor, named mt‐fura‐2, obtained by coupling two triphenylphosphonium cations to the molecular backbone of the ratiometric Ca²⁺ indicator fura‐2. Mt‐fura‐2 binds Ca²⁺ with a dissociation constant of ≈1.5 μm in vitro. When loaded in different cell types as acetoxymethyl ester, the probe shows proper mitochondrial localization and accurately measures matrix [Ca²⁺] variations, proving its superiority over available dyes. We describe the synthesis, characterization and application of mt‐fura‐2 to cell types where the delivery of genetically‐encoded indicators is troublesome.     

Die zitterfreie Thermogenese: Wärmeproduktion bei Kältestress

Non‐shivering thermogenesis is based on the increase of the permeability of the inner membrane of mitochondria to H⁺. An increase in the H⁺‐flux from the cytosolic to the matrix compartiment dissipates the electrochemical proton gradient and, therefore, activates the mitochondrial respiratory chain. Permeability of H⁺ is stimulated by artificial protonophores such as 2,4‐dinitrophenol or the interaction of non‐esterified fatty acids with certain proteins of the inner membrane of mitochondria, called uncoupling proteins (UCPs). UCPs are expressed in various tissues of mammalians, but also in riping plants. Despite of their role in thermogenesis, activated UCPs suppress the formation of the superoxid anion radical. Therefore, UCPs are also physiological tools to diminish oxidative stress within the cell.     

Engineered Cell-Penetrating Peptides for Mitochondrion-Targeted Drug Delivery in Cancer Therapy

Mitochondrion is a promising target in cancer therapy. However, gaining access to this organelle is difficult due to the obstacles to cross the complicated mitochondrial membrane. Cell-penetrating peptides (CPPs) with mitochondrion-targeting ability, named mitochondrion-targeting peptides (MTPs), are efficient tools to deliver exogenous therapeutics into mitochondria. Herein, we report several new MTPs, which can be readily synthesized via resin-based solid-phase peptide synthesis. In particular, MTP3 (compound 5 ), consisting of three positively charged arginines and two D- and L- alternating naphthylalanines, demonstrated excellent mitochondrion-targeting ability with high Pearson's correlation coefficient, suggesting that MTP3 has good potential for mitochondrion-targeted drug delivery. As proof-of-concept, the feasibility of MTP3 was validated by the preparation of a mitochondrion-targeting prodrug (compound 17 , doxorubicin-based prodrug). This prodrug was subsequently confirmed to be specifically transported to the mitochondria of tumor cells, where it was able to release the native doxorubicin upon intracellular GSH activation, leading to mitochondrial depolarization and eventually cell death. Importantly, compound 17 showed good cytotoxicity against human tumor cells while negligible toxicity towards normal cells, indicating its potential as a potent mitochondrial medicine for targeted cancer therapy. Our study thus opens a way for engineered CPPs to be used to deliver bioactive cargos in mitochondrion-targeted cancer therapy.     

Functional assays in high-resolution clear native gels to quantify mitochondrial complexes in human biopsies and cell lines

We introduce high resolution clear native electrophoresis (CNE) as a powerful technique to resolve enzymatically active mitochondrial complexes from cultured human cell lines and skeletal muscle biopsy samples. Quantitative enzymatic assays can be performed using small amounts of cultured cells with low mitochondria content, for example, around 10 mg of sedimented osteosarcoma cells (wet weight) which is equivalent to around 10 million cells. High resolution CNE offers general advantages for in-gel catalytic activity assays compared to blue native electrophoresis. It seems especially suited for assaying mitochondrial ATP synthase and respiratory chain complexes I and II in cell models of human mitochondrial disorders and for detailed analyses of patient cells and tissues with defects in oxidative phosphorylation.     

Intracellular Delivery of Functional Native Antibodies under Hypoxic Conditions by Using a Biodegradable Silica Nanoquencher

Antibodies are important biopharmaceuticals, but almost all existing antibody‐based drugs are limited to targeting antigens located at the cell exterior because of the inability of antibodies to enter the cell interior. Available methods for intracellular delivery of antibodies have major shortcomings. Herein, we report an approach to encapsulate native antibodies in a biodegradable silica nanoquencher (BS‐qNP), which could undergo efficient cellular uptake and intracellular degradation to release antibodies only under hypoxic conditions. By coating the surface of BS‐qNP with cell‐penetrating poly(disulfide)s (CPD), the delivered antibodies (or other proteins) avoided endolysosomal trapping. Doping of the silica coating with a fluorescent dye and a dark hole quencher further endowed BS‐qNP with hypoxia‐responsive fluorescence turn‐on property. Our antibody delivery system thus provides the first platform capable of stable encapsulation, efficient uptake, on‐demand antibody release, and imaging of release/cell state.     

A Multi‐responsive Fluorescent Probe Reveals Mitochondrial Nucleoprotein Dynamics with Reactive Oxygen Species Regulation through Super‐resolution Imaging

Understanding the biomolecular interactions in a specific organelle has been a long‐standing challenge because it requires super‐resolution imaging to resolve the spatial locations and dynamic interactions of multiple biomacromolecules. Two key difficulties are the scarcity of suitable probes for super‐resolution nanoscopy and the complications that arise from the use of multiple probes. Herein, we report a quinolinium derivative probe that is selectively enriched in mitochondria and switches on in three different fluorescence modes in response to hydrogen peroxide (H₂O₂), proteins, and nucleic acids, enabling the visualization of mitochondrial nucleoprotein dynamics. STED nanoscopy reveals that the proteins localize at mitochondrial cristae and largely fuse with nucleic acids to form nucleoproteins, whereas increasing H₂O₂ level leads to disassociation of nucleic acid–protein complexes.     

A Multisite‐Binding Switchable Fluorescent Probe for Monitoring Mitochondrial ATP Level Fluctuation in Live Cells

Adenosine triphosphate (ATP), commonly produced in mitochondria, is required by almost all the living organisms; thus fluorescent probes for monitoring mitochondrial ATP levels fluctuation are essential and highly desired. Herein, we report a multisite‐binding switchable fluorescent probe, ATP‐Red 1 , which selectively and rapidly responds to intracellular concentrations of ATP. Live‐cell imaging indicated that ATP‐Red 1 mainly localized to mitochondria with good biocompatibility and membrane penetration. In particular, with the help of ATP‐Red 1 , we successfully observed not only the decreased mitochondrial ATP levels in the presence of KCN and starvation state, but also the increased mitochondrial ATP levels in the early stage of cell apoptosis. These results indicate that ATP‐Red 1 is a useful tool for investigating ATP‐relevant biological processes.     

Alterations of the mitochondrial proteome caused by the absence of mitochondrial DNA: A proteomic view

The proper functioning of mitochondria requires that both the mitochondrial and the nuclear genome are functional. To investigate the importance of the mitochondrial genome, which encodes only 13 subunits of the respiratory complexes, the mitochondrial rRNAs and a few tRNAs, we performed a comparative study on the 143B cell line and on its Rho-0 counterpart, i.e., devoid of mitochondrial DNA. Quantitative differences were found, of course in the respiratory complexes subunits, but also in the mitochondrial translation apparatus, mainly mitochondrial ribosomal proteins, and in the ion and protein import system, i.e., including membrane proteins. Various mitochondrial metabolic processes were also altered, especially electron transfer proteins and some dehydrogenases, but quite often on a few proteins for each pathway. This study also showed variations in some hypothetical or poorly characterized proteins, suggesting a mitochondrial localization for these proteins. Examples include a stomatin-like protein and a protein sharing homologies with bacterial proteins implicated in tyrosine catabolism. Proteins involved in apoptosis control are also found modulated in Rho-0 mitochondria.     

Light Might Directly Affect Retinal Ganglion Cell Mitochondria to Potentially Influence Function†

Visible light (360–760 nm) entering the eye impinges on the many ganglion cell mitochondria in the non‐myelinated part of their axons. The same light also disrupts isolated mitochondrial function in vitro and kills cells in culture with the blue light component being particularly lethal whereas red light has little effect. Significantly, a defined light insult only affects the survival of fibroblasts in vitro that contain functional mitochondria supporting the view that mitochondrial photosensitizers are influenced by light. Moreover, a blue light insult to cells in culture causes a change in mitochondrial structure and membrane potential and results in a release of cytochrome c. Blue light also causes an alteration in mitochondria located components of the OXPHOS (oxidative phosphorylation system). Complexes III and IV as well as complex V are significantly upregulated whereas complexes I and II are slightly but significantly up‐ and downregulated, respectively. Also, blue light causes Dexras1 and reactive oxygen species to be upregulated and for mitochondrial located apoptosis‐inducing factor to be activated. A blue light detrimental insult to cells in culture does not involve the activation of caspases but is known to be attenuated by necrostatin‐1, typical of a death mechanism named necroptosis.     

Promotion of Proapoptotic Signals by Lysosomal Photodamage

We previously reported that low‐level lysosomal photodamage enhanced the efficacy of subsequent mitochondrial photodamage, resulting in a substantial promotion of apoptotic cell death. We now extend our analysis of the sequential PDT protocol to include two additional lysosomal‐targeting photosensitizers. These agents, because of enhanced permeability, are more potent than the agent (N‐aspartyl chlorin E6, NPe6) used in the initial study. Addition of the cell‐permeable cysteine protease inhibitor E‐64d and calcium chelator BAPTA‐AM almost completely suppressed sequential PDT‐induced loss of mitochondrial membrane potential and activation of procaspases‐3 and ‐7. These inhibitors did not, however, suppress the proapoptotic effect of a BH3 mimetic or mitochondrial photodamage. Knockdowns of ATG7 or ATG5, proteins normally associated with autophagy, suppressed photodamage induced by the sequential PDT protocol. These effects appear to be independent of the autophagic process as pharmacological inhibition of autophagy offered no such protection. Effects of ATG7 and ATG5 knockdowns may reflect the role that ATG7 plays in regulating lysosome permeability, and the likelihood that a proteolytic fragment of ATG5 amplifies mitochondrial proapoptotic processes. Our results suggest that low‐dose photodamage that sequentially targets lysosomes and mitochondria may offer significant advantages over the use of single photosensitizers.     

Efficient Uptake of 177Lu‐Porphyrin‐PEG Nanocomplexes by Tumor Mitochondria for Multimodal‐Imaging‐Guided Combination Therapy

The benefits to intracellular drug delivery from nanomedicine have been limited by biological barriers and to some extent by targeting capability. We investigated a size‐controlled, dual tumor‐mitochondria‐targeted theranostic nanoplatform (Porphyrin‐PEG Nanocomplexes, PPNs). The maximum tumor accumulation (15.6 %ID g^?1, 72 h p.i.) and ideal tumor‐to‐muscle ratio (16.6, 72 h p.i.) was achieved using an optimized PPN particle size of approximately 10 nm, as measured by using PET imaging tracing. The stable coordination of PPNs with ¹⁷⁷Lu enables the integration of fluorescence imaging (FL) and photodynamic therapy (PDT) with positron emission tomography (PET) imaging and internal radiotherapy (RT). Furthermore, the efficient tumor and mitochondrial uptake of ¹⁷⁷Lu‐PPNs greatly enhanced the efficacies of RT and/or PDT. This work developed a facile approach for the fabrication of tumor‐targeted multi‐modal nanotheranostic agents, which enables precision and radionuclide‐based combination tumor therapy.     

Oligonucleotide loaded polypeptide‐peptide nanoparticles towards mitochondrial‐targeted delivery

Nanoparticles (NPs) consisting of biodegradable and biocompatible polymers may have the ability to deliver a cargo to specific tissue, cell type, and organelle. Various diseases, which are linked to mitochondrial genome (mtDNA) mutations and have no effective treatments, may be approached by gene therapy strategies. In this study, we adapted the recently developed mitochondria delivering polypeptide‐peptide nanoparticles (PoP‐NPs) system to carry an oligonucleotide cargo to the proximity of the mitochondria. PoP‐NPs are formulated by self‐assembly of the negatively charged polypeptide, poly gamma glutamic acid (γ‐PGA), with an amphiphilic and cationic β‐sheet peptide (PFK). Here, we show that PFK interacts favorably with oligonucleotides and thereby enables the formation of DNA‐loaded PoP‐NPs (DNA‐PoP‐NPs). DNA‐PoP‐NPs could be assembled with different peptide to oligonucleotide (N/P) ratios, and their targeting to the proximity of mitochondria in cell culture could be facilitated through NPs coating with PFK peptide.