Journal of Drug Targeting

ISSN: 1061-186X (Print) 1029-2330 (Online) Journal homepage: http://www.tandfonline.com/loi/idrt20

Mitochondria-targeted drug delivery system for cancer treatment Zhi-Peng Chen, Man Li, Liu-Jie Zhang, Jia-Yu He, Li Wu, Yan-Yu Xiao, Jin-Ao Duan, Ting Cai & Wei-Dong Li To cite this article: Zhi-Peng Chen, Man Li, Liu-Jie Zhang, Jia-Yu He, Li Wu, Yan-Yu Xiao, Jin-Ao Duan, Ting Cai & Wei-Dong Li (2015): Mitochondria-targeted drug delivery system for cancer treatment, Journal of Drug Targeting, DOI: 10.3109/1061186X.2015.1108325 To link to this article: http://dx.doi.org/10.3109/1061186X.2015.1108325

Published online: 07 Nov 2015.

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Date: 07 December 2015, At: 09:08

http://informahealthcare.com/drt ISSN: 1061-186X (print), 1029-2330 (electronic) J Drug Target, Early Online: 1–11 ! 2015 Taylor & Francis. DOI: 10.3109/1061186X.2015.1108325

REVIEW ARTICLE

Mitochondria-targeted drug delivery system for cancer treatment Zhi-Peng Chen1*, Man Li1*, Liu-Jie Zhang1, Jia-Yu He1, Li Wu1, Yan-Yu Xiao2, Jin-Ao Duan1, Ting Cai2, and Wei-Dong Li1 Department of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, P.R. China and 2Department of Pharmacy, China Pharmaceutical University, Nanjing, P.R. China

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Abstract

Keywords

Mitochondria are one type of the major organelles in the cell, participating in a variety of important physiological and biochemical processes, such as tricarboxylic acid cycle, fatty acid metabolism and oxidative phosphorylation. Meanwhile, it also happens to be the key regulator of apoptosis by triggering the complex cell-death processes through a variety of mechanisms. Since it plays a pivotal role in cell-death, a mitochondria-targeted treatment strategy could be promising for cancer therapy. In this comprehensive review, we focused on the mechanisms of mitochondrial targeting and a variety of strategies to realize the purpose of mitochondrial targeting, including that based on the use of lipophilic cations, and mitochondrial targeting signal peptides (MTS) as well as cell-penetrating peptides (CPPs). Then on this basis we present some several developed strategies for multifunctional mitochondria-targeted agents so as to achieve the good anti-cancer therapeutic effects.

Mitochondria-targeted, multifunctional, multilevel targeting, nanomedicine, peptides

Introduction As is well known, we can improve the efficacy of a drug by targeting the drug to the diseased tissues or cells [1]. Additionally, with the development of medical science, the demands on the drug delivery system have become much higher recently. They require not only the good curative effects, fewer side effects, but also preferably a smaller dose for therapeutic purpose. Thus, organelle-specific delivery of bioactive molecules becomes more important to achieve maximum therapeutic effects and minimum side effects [2]. Some pharmaceutical agents, if able to be directed specifically towards the organelle of interest, such as nucleic acid materials to the nuclei [3], pro-apoptotic compounds to the mitochondria [4] and lysosomal drugs to the lysosomes [5,6], their therapeutic effects can be enhanced manifolds compared to the random interactions with the desired sites of action. Therefore, development of organelle-targeted drug delivery system has become one of the new research fields in modern drug delivery systems [7,8]. Mitochondria are one type of the major organelles in the cell, and the main organelles that mediate cell apoptosis [9]. It can trigger the complex cell-death processes by a variety of mechanisms including translocation of the pro-apoptotic *These authors contributed equally to this work. Address for correspondence: Wei-Dong Li, Department of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210046, P.R. China. E-mail: [email protected] Ting Cai, Department of Pharmacy, China Pharmaceutical University, Nanjing, P.R. China. E-mail: [email protected]

History Received 13 August 2015 Revised 3 October 2015 Accepted 12 October 2015 Published online 5 November 2015

proteins, such as cytochrome-c, apoptosis-inducing factor, from the mitochondrial inter-membrane space to the cytosol [10]. These pro-apoptotic proteins can activate the deathsignal proteins such as caspases [11]. In recent years, mitochondrial targeting of small molecules/nanocarriers has gained much attention [12]. The progressive evidence shows that mitochondrial dysfunction is responsible for a variety of human disorders including neurodegenerative, neuromuscular diseases, diabetes, obesity and cancer [13–15]. ‘‘Mitochondrial dysfunction is responsible for cancer’’ – this insight was first reported over 75 years ago by Otto Warburg [16]. Changes of mitochondrial structure and function not only will interfere with growth, metabolism and proliferation of tumor, cells; but also will eventually trigger the apoptosis of cancer cells [17]. Along with the association between mitochondria and the apoptosis of tumor cells was found [18], mitochondria-targeted drugs have become one of active fields in organelle-targeted drug delivery system development. Mitochondria-targeted drugs can directly take effects on the mitochondrial membrane or matrix, they can also act on the mitochondrial apoptosis pathway or regulation pathway of signal molecules. Many mitochondria-targeted drugs have been involved in clinical studies, or already applied clinically in the fields of cancer and other diseases, such as cardiovascular and cerebrovascular diseases [19–21]. Researchers have devised or found a series of mitochondria-targeted drugs which can be used in tumor therapy, including Lonidamine (LND) [22], CD437 [23,24], Betulinic acid [25,26], and so on. In this review, several mechanisms that have been

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proposed for mitochondrial targeted as well as some strategies which are known to have the ability to target mitochondria are introduced. In addition, the challenges in the field together with the future directions would be discussed.

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The mechanism of mitochondrial targeting The major function of mitochondria in human cells is to provide adenosine triphosphate (ATP) by oxidative phosphorylation (OXPHOS). However, mitochondria have many other roles including the modulation of intracellular calcium concentration and the regulation of apoptotic cell death. In addition, the mitochondrial respiratory chain is the major source of highly-reactive oxygen species (ROS) [27]. The ROS-induced damage, including mitochondrial DNA (mtDNA) mutations, lipid peroxidation, protein oxidation, alters the function of many metabolic enzymes in the mitochondrial matrix, as well as those comprising the electron transport chain, damaging the cell and whole organism [15]. Therefore, searching for effective and convenient measures that could transport medicine to and into mitochondria is a critical approach to prevent or repair mitochondrial injury, artificially adjust of mitochondrial function. Most of the current mitochondria-targeted drug delivery system (MTDDS) for transporting chemotherapeutics to and into mitochondria within living mammalian cancer cells are based on two distinct mitochondrial features: the high membrane potential across the inner mitochondrial membrane (4 m) and the organelle’s protein import machinery [28,29]. Contemporary drug delivery strategies based on both features, either independently or in combination, will be discussed in detail below. Mitochondrial transmembrane potential (4wm) Each mitochondrion is composed of two membranes, which together create four compartments each with quite different compositions, activities and functions: a porous outer mitochondrial membrane (OMM), permeable to molecules smaller than 5 kDa (large molecules, such as proteins, cross the outer membrane via a unique protein importing apparatus, which will be discussed below) [30]; an narrow inter mitochondrial membrane space (IMS) containing a number of specialized proteins, which is chemically equivalent to the cytosol with respect to small molecules [31]; a convoluted and invaginated inner mitochondrial membrane (IMM), which is highly impermeable and characterized by an unusually high content of membrane proteins as well as a unique lipid composition [30]; the internal mitochondrial matrix, containing enzymes and coenzymes for many different metabolic pathways, including the citric acid cycle, fatty acid metabolism and the oxidative phosphorylation [32]. In most of mammalian tissues, 80–90% of ATP is derived from mitochondrial oxidative phosphorylation. The electron transport chain as a whole serves to oxidize NADH, with oxygen serving as the final electron acceptor (respiration). The complexity (complexes I, III, IV, and complex II, if one includes the oxidation of succinate) is designed to make use of the considerable free energy released from the oxidation of NADH (Figure 1) [33]. The mitochondrial inner membrane proteins are components of the respiratory chain, including the ATP synthase and a variety of transport proteins.

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The impermeability of the inner membrane is a prerequisite for the establishment of an imbalance in the distribution of protons between the mitochondrial matrix and the cytosol. Mitochondria make ATP by passing electrons derived from the oxidation of food down the respiratory chain to react with oxygen, using the redox energy to translocate protons across the mitochondrial inner membrane [34]. This establishes a proton electrochemical potential gradient across the inner membrane comprising a transmembrane electric potential of up to 200 mV (negative inside) and an internal pH of approximately eight (basic inside) [35]. Given that the mitochondrial membrane potential is much higher than that of other cellular organelles, lipophilic cations can be attracted by mitochondria through electrostatic interactions and accumulates in the mitochondrial matrix in response to the negative membrane potential. Mitochondrial protein import machinery Another mechanism used to selectively target mitochondria involves harnessing the mitochondrial protein import machinery, which is naturally utilized by cells for the delivery of nuclear-encoded mitochondrial proteins [36]. There are about 1000 different types of proteins in the mitochondria. Only a very small set, 13 polypeptides that are part of the protein complexes involved in the oxidative phosphorylation, is being synthesized inside mitochondria. Greater than 99% of all known mitochondrial proteins are synthesized in cytosolic ribosomes as precursor proteins that must be imported. Import of these proteins into mitochondria is performed by protein translocator complexes in the outer and inner membranes, which are sorted to be one of four intra-mitochondria locations: the outer membrane, the inner membrane, the intermembrane space, and the matrix [37–39]. Typically, proteins are synthesized in the cytosol and transported into mitochondria through a cleavable N-terminal targeting sequence (mitochondrial targeting signal peptide, N-MTS). Generally, an N-MTS consists of approximately 2 0–40 amino acids in length with structural motifs recognized by the mitochondrial import machinery [40]. Although no amino acid sequence homology has been observed among N-MTS peptides of different mitochondrial preproteins, two common attributes have been noted. MTS peptides usually display a net positive charge, and they have the ability to form amphiphilic a-helices [41]. Proteins bearing an MTS peptide are escorted through the cytosol by chaperones to the TOM complex, which is a translocase localized in the OMM [42]. After crossing the OMM, so-called TIM complexes, which are translocases of the IMM, mediate the further transport into the mitochondrial matrix. Once inside the matrix, the MTS is cleaved in one or two proteolytic steps by mitochondrial processing peptidases that with the help of matrixlocalized chaperones such as hsp70, the protein refolds into its mature form [43]. In the mitochondria, many proteins are involved in playing some vital functions. Dysfunction of these proteins may induce cell death and cause diseases. Therefore, delivering proteins or drugs to mitochondria by the mitochondrial protein import mechanism could be an effective approach for some mitochondrial diseases. As for tumor, it will be a key

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Figure 1. Mitochondrial electron transfer system.

means to kill cancer cells by transporting some therapeutic macromolecules to and into mitochondria. Thus, we can take advantage of MTS or combine with cell penetrating peptides (CPPs), which has protein transduction domain (PTD) to transport some macromolecular proteins or small molecule drugs into the mitochondria [44].

The strategy of mitochondrial targeting The mitochondria targeting strategy based on the lipophilic cations Delocalized lipophilic cations (DLCs), due to large hydrophobic surface areas and a delocalized positive charge, are readily absorbed through mitochondrial membranes. The lipophilic nature of DLCs allows for rapid passage through membrane bilayers, while the permanent cationic charge, eventually may lead to their accumulation inside the mitochondrial matrix driven by the negative membrance potential [28,45]. The mitochondrial membrane potential of tumor cells is much higher than that of normal epithelial cells. Based on the analysis of more than 200 cell lines/types of adenocarcinoma, melanoma, transitional cell carcinoma, squamous cell carcinoma, and normal epithelial cells, it was demonstrated in 1988 that cancer cells possess an elevated mitochondrial membrane potential in comparison to mitochondria in non-malignant cells [46]. Only about 2% of all cells tested so far disobey this apparently dominant precept. DLCs can accumulate in cancerous cells to a greater degree than in normal cells due to the more negative mitochondrial membrane potentials typically observed in tumor cells. This offers a unique means by which drugs can be delivered preferentially to tumors and normal healthy cells can be spared in hopes of reducing drug related toxicities. According to the Nernst equation as follows:   ½cationin D m½mv ¼ 61:5log10 ½cationout

Every 61.5 mV increase in membrane potential causes a ten-fold increase in accumulation of the membrane-permeant cation. This means that the matrix concentration of this cation will be 100–1000 times higher than the concentration in the cytoplasm. It is evident that these mitochondriotropic molecules have two structural features in common. First, they are all amphiphilic, that they combine a hydrophilic charged center with a hydrophobic core. Second, in all structures the -electron charge density extends over at least three atoms instead of being limited to the internuclear region between the heteroatom and the adjacent carbon atom. This causes a distribution of the positive-charge density between two or more atoms, which means that, the positive charge is delocalized. Both structural features have been recognized to be crucial for the accumulation of these organic cations inside the matrix of mitochondria. Sufficient lipophilicity, combined with delocalization of their positive charges, which can reduce the free energy change when moving from an aqueous to a hydrophobic environment, are prerequisites for their mitochondrial accumulation in response to the mitochondrial membrane potential. The most common DLCs are methyltriphenylphosphonium (TPP), dequaliniumchloride (DQA), Rhodamin123 (Rh123) and so on [47], as shown in Figure 2. Methyltriphenylphosphonium The most notable example of a delocalized lipophilic cation that has been used for mitochondrial targeting is the methyltriphenylphosphonium (TPP) cation [45]. This small targeting vector contains a single positive charge that is resonance-stabilized over three phenyl groups [48]. To further increase the therapeutic activity of drugs known to act on intracellular target sites, Sarathi et al. conjugated TPP to a stearyl residue to get stearyltriphenyl phosphonium (STPP), an amphiphilic molecule that can be incorporated into lipid bilayers, called STPP-modified nanocarrier [49]. Thus the

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Figure 2. Structure of several Delocalized lipophilic cations (DLCs).

mitochondria-targeted STPP formulation efficiently delivered Rh-PE (a fluorescently-labeled phospholipid that remains associated with the lipid bilayer in much the same way as a drug molecule would do) to the mitochondria, significantly inhibited tumor growth rate and improved animal survival time [7]. Based on this, Biswas et al. synthesized a novel polyethylene glycol-phosphatidylethanolamine (PEG-PE) conjugate with the TPP group attached to the distal end of the PEG block (TPP-PEG-PE) [50]. This conjugate was incorporated into the liposomal lipid bilayer to prepare, TPPPEG-PE-modified liposomes (TPP-PEG-L), which were less cytotoxic compared to STPP-L or PEGylated STPP-L. These TPP-PEG-L have been, demonstrated to be efficient for mitochondrial targeting in cancer cells. They enhanced PTXinduced cytotoxicity and anti-tumor efficacy in cell culture and mouse experiments compared to PTX-loaded unmodified plain liposomes (PL). In Biswas’ later studies, TPP was conjugated to the surface of the dendrimer and developed a novel mitochondria-targeted generation 5 poly(amidoamine) (PAMAM) dendrimer (G(5)-D) [51]. The newly developed TPP-anchored dendrimer (G(5)-D-Ac-TPP) was efficiently taken up by the cells and demonstrated good mitochondrial targeting, thus could be used for the treatment of diseases associated with mitochondrial dysfunction. Marrachea et al. reported a rationally designed mitochondria-targeted polymeric nanoparticle (NP) system and its optimization for efficient delivery of various mitochondria-acting therapeutics by blending a targeted poly (D, L-lactic-co-glycolic acid)block (PLGA-b)-poly (ethylene glycol) (PEG)-triphenylphosphonium (TPP) polymer (PLGA-b-PEG-TPP) with non-targeted either PLGA-b-PEG-OH or PLGA-COOH [52]. They first synthesized a PLGA-b-PEG copolymer with a single terminal lipophilic triphenylphosphonium (TPP) cation, which is known to be able to cross the mitochondrial matrix space, to determine how the attached cation and variations in NP size and charge affect intracellular trafficking of blended NPs formed by mixing PLGA-b-PEG-TPP

with either PLGA-b-PEG-OH or PLGA-COOH. A robust fluorescent reporter quantum dot (QD) has been performed to investigate the distribution of the targeted and non-targeted NPs in human cervical cancer HeLa cells. Confocal microscopy analysis of the treated cells indicated significantly greater uptake of targeted NPs than of non-targeted NPs in the mitochondria of cells. Mitochondria-acting chemotherapeutics lonidamine (LND), which inhibits mitochondrial glycolysis, and a-tocopheryl succinate (a-TOS), a tumor-selective drug, were selected to demonstrate the applicability of this system in cancer. The selectivity and efficiency of LND and a-TOS against cancer cells depend on their ability to target the mitochondria of cells. To evaluate the efficacy of the targeted NPs in delivering LND and a-TOS, we performed MTT assays in HeLa cells. Preferential localization in the target organelle accounts for the enhanced cytotoxicity of both LND and a-TOS encapsulated in the targeted NPs. Lung cancer is the leading cause of cancer-related death in humans. Meanwhile, the multidrug resistance (MDR) is the major obstacle to successful chemotherapy of lung cancer. Zhou et al. synthesized a D-a-tocopheryl polyethylene glycol 1000 succinate-triphenylphosphine conjugate (TPGS1000TPP) as a mitochondria-targeted molecule, and was incorporated onto the surface of paclitaxel liposomes to treat the drug-resistant lung cancer [53]. The targeting paclitaxel liposomes could significantly enhance the cellular uptake, be selectively accumulated into the mitochondria, and cause the release of cytochrome C, thereby enhance the apoptosis by acting on the mitochondrial signaling pathways. The liposomes, exhibited the strongest anticancer efficacy in vitro and in the drug-resistant A549/cDDP xenografted tumor model. A wide variety of small molecules have been conjugated to TPP to achieve mitochondrial targeting. The conjugation of these molecules to TPP typically occurs through a simple SN2 reaction. TPP can be designed to include a reversible tag that is only cleaved by a mitochondria-specific enzyme in order to release the chemically unaltered cargo. Millard et al. designed

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and evaluated a triphenylphosphonium derivative of the nitrogen mustard chlorambucil (Mito-Chlor) in vitro and in vivo [54]. The results showed that Mito-Chlor localized to cancer cell mitochondria where it acts on mtDNA to arrest cell cycle and induce cell death. Significantly, Mito-Chlor delayed tumor progression in a mouse xenograft model of human pancreatic cancer. Pathak et al. described a targeted molecular scaffold for construction of a multiple dichloroacetate (DCA)-loaded compound, Mito-DCA, incorporation of a lipophilic triphenylphosphonium cation through a biodegradable linker in Mito-DCA allowing for mitochondrial targeting, with three orders of magnitude enhanced potency and cancer cell specificity compared to DCA [55]. Effective delivery of DCA to the mitochondria could lead to induction of an efficient antitumor immune response, thus introducing the concept of combining glycolysis inhibition with immune system to destroy tumor.

anticancer drug DOX to tumor cells, using folate-terminated polyrotaxanes along with DQA, which have been proven to be able to overcome the cancer resistance [62]. Compared with free DOX, DOX hydrochloride, DOX nanoparticles, and targeted DOX nanoparticles, the functional DOX nanoparticles significantly increased the intracellular uptake of DOX, selectively accumulated in mitochondria exhibited the strongest anticancer efficacy in vitro and in the drug-resistant MCF-7/Adr xenograft tumor model. More importantly, the functional nanoparticles exhibited the most significant antitumor activity among all formulations at comparable doses of DOX. The tumor growth was markedly inhibited in the group of functional DOX nanoparticles. The addition of DQA and folate to the functional DOX nanoparticles enhanced the cellular permeability and uptake by the resistant tumors, which in turn enhanced cytotoxicity to the drug resistant cancer cells.

Dequalinium chloride

The mitochondrial targeting strategy based on the mitochondrial targeting signal peptides

Dequalinium chloride (DQA) is a dicationic mitochondriotropic compound resembling bolaform electrolytes, which means that, it is a symmetrical molecule with two charge centers separated at a relatively large distance [56]. It has confirmed that dequalinium could self-assemble into stable vesicles. Electron microscope images showed that dequalinium, upon sonication in aqueous medium, formed vesiclelike aggregates (named DQAsomes) with diameters between about 70 and 700 nm. Due to its high stability, DQAsomes do not seem to precipitate, to fuse with each other, or to aggregate in solution over a period of several days. More importantly, they still have the specialty of penetrating the lipid bilayer and then gather into the mitochondria [57–59]. Wang et al. synthesized a dequalinium polyethylene glycol-distearoylphosphati-dylethanolamine (DQA-PEG2000DSPE) conjugate as a mitochondriotropic molecule. Then mitochondrial targeting resveratrol liposomes were developed by modifying DQA-PEG2000-DSPE on the surface of liposomes for overcoming the cancer resistance [60]. The mitochondrial-targeting liposomes significantly enhanced the cellular uptake, and selectively accumulated into mitochondria, induced apoptosis of both non-resistant and resistant cancer cells, thus eventually significantly enhanced the anticancer efficacy. Based on this, Zhang et al. constructed a kind of mitochondrial targeting daunorubicin plus quinacrine liposomes by physically mixing DQA with lipid components for treating and preventing the recurrence of breast cancer arising from the cancer stem cells [61]. The results showed that this mitochondrial targeting liposomes evidently increased the mitochondrial uptake of drugs, selectively accumulated into mitochondria, activated the pro-apoptotic Bax protein, dissipated the mitochondrial membrane potential, opened the mitochondrial permeability transition pores, released cytochrome C by translocation, and initiated a cascade of caspase 9 and 3 reactions, thereby induced apoptosis of MCF-7 cancer stem cells. The mitochondrial targeting liposomes showed the strongest efficacy in treating MCF-7 cancer cells in vitro, and the relapsed tumor in mice. Wang et al. developed functional doxorubicin (DOX) nanoparticles for targeted delivery of the classical cytotoxic

According to the unique transporting process as described above, the mitochondrial targeting signal peptides (MTS) has the potential to become a useful device for selective and effective delivery of therapeutic proteins to mitochondria in patients suffering from mitochondrial diseases. The use of MTS for mitochondrial delivery has been successful with a variety of cargo molecules, including proteins, nucleic acids, and endonucleases [63]. Targeting of these candidate proteins to the mitochondria would protect mtDNA and nuclear DNA from reactive oxygen species (ROS) and from excessive apoptosis [64,65]. This approach is therefore a powerful means to transport biomolecular species. Flierl et al. reported that oligonucleotides could be introduced into the mitochondria of living mammalian cells by annealing them to peptide nucleic acids (PNA) coupled to mitochondrial targeting peptides [66]. In brief, an N-terminal mitochondrial targeting peptide is covalently coupled to a PNA encoding a portion of the sequence to be introduced into the mitochondrion. This targeting peptide-PNA conjugate is then annealed to an oligonucleotide with the desired sequence and the complex transferred into the cytosol using either polycations or transient membrane channels. This procedure efficiently delivers labeled oligonucleotides into the mitochondrial matrix, both in vitro and in vivo. Cha et al. used Amyloid b1–42 (Ab1–42) with a mitochondria-targeting sequence induced the identical morphological alteration of mitochondria as that was observed when Ab1–42 was mitochondria-targeted accumulated in HT22 cells in the APP/PS Alzheimer’s disease (AD) mouse model and exogenous Ab1–42-treated HT22 cells [67]. To introduce DNA into mitochondria efficiently, Yu et al. fused adenoassociated virus (AAV) capsid proteins VP2 with a mitochondrial targeting sequence to carry the mitochondrial gene encoding the human NADH ubiquinone oxidoreductase subunit 4 (ND4) [68]. Their studies showed that this MTS-modified AAV could effectively import DNA into Mitochondria. Shan et al. designed a new delivery technology based on functionalized vertical silicon nanoneedle (SiNNs) arrays to transfer intact 3D DNA nanocages (DNA-NCs) directly to the cytoplasm

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without endocytosis [69]. They demonstrated that this delivery approach offers high-uptake efficiency together with enhanced stability and low cytotoxicity of intact DNA-NCs in cells, as well as little damage of the cellular membrane. Remarkably, to investigate the subcellular targeting of DNANCs after entering cell cytoplasm, the mitochondrial targeting sequence (MTS) peptide N-(bA) LLYRSSCLTRTAPKFF RISQRLSLM, was conjugated to DNA-NCs. It showed that the site-selective peptide-functionalized DNA-NCs were localized in the specific mitochondria compartment. Although the MTS strategy appears promising, it may not be applicable in certain cases. Mitochondrial delivery using MTS is severely limited by the size of the agents, thus macromolecules cannot be transported by this pathway. Secondly, as the most effective MTS molecules are quite long, the synthesis of fusion constructs can be challenging. Furthermore, the aqueous solubility and cellular permeability of these constructs are limiting factors for exogenous delivery, as many of these MTS display hydrophobic stretches of amino acids. In addition, the current types of MTS that can be used for mitochondrial transporting are relatively limited, and the price of MTS is quite expensive. All these restrict the application of MTS [70]. The mitochondrial-targeting strategy based on the cell-penetrating peptides Although a variety of vectors have been chosen as candidates for cargo translocation, cell-penetrating peptides (CPPs) are one of the most popular and efficient vectors for achieving high cellular uptake, even more deeply organelle targeting. CPPs are a class of diverse peptides, typically with 5–30 amino acids, and unlike most peptides, they can directly cross the cellular membrane, may not rely on the classic endocytosis [71,72]. It has been 20 years since the discovery of the first CPP and the concept of protein transduction into cell presented in 1988 by Frankel and Green in parallel [73]. They discovered that the Trans-Activator of Transcription (TAT) protein of HIV can cross cell membranes and be efficiently internalized by cells in vitro, which results in transactivation of the viral promoter. From then on, the list of available CPPs has grown rapidly and CPPs have been employed for a variety of applications [74]. CPPs serving as vectors can successfully transport cargoes intracellularly such as siRNA [75], nucleic acids [76], small molecule therapeutic agents [77], proteins [78], quantum dots [79], and MRI contrast agents[80], both in vitro and in vivo. In addition, this efficient transport system has lower cytotoxicity in a variety of cell lines compared with other delivery methods. Although highly efficient in mediating the in vitro cell uptake of different molecules into most cell lines, the in vivo use of CPPs appears much more complicated mainly because of a complete lack of cell specificity [81]. Indeed, CPPs, and the therapeutic molecules attached to them, are dispersed almost all over the body independent of the way of administration. For cancer treatment, such spreading will bring a great deal of side effects, thus reduce the potential of CPPs as a therapeutic approach. To date, many efforts have been made to improve the cell uptake for tumor cells. CPPs have received attractive attention for medical applications,

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not only because of their high internalization ability but also due to their potential for variable modification design [82,83]. One widely used strategy to achieve effective intracellular access is to modify the CPPs according to the tumor microenvironment. Chen et al. provided a novel strategy for design of cancer-targeted drug delivery system, this carrier system consists of three components, PEG-DOPE (polyethylene glycol-2000-1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine), TAT-PEG-DOPE, and pH-sensitive compound stearyl sulfadiazine (SA-SD) [84]. At normal blood pH (pH 7.4), the sulfadiazine is negatively charged, and when mixed with the TAT-micelles, shields the TAT by electrostatic interaction. Only PEG is exposed to the outside which can make the carrier circulating longer. When the system experiences a decrease in pH (near tumor pH 6.8) sulfonamide will lose charge and detach, thus exposing TAT for interaction with tumor cells. By this way, TAT peptides lead the drug-loaded micelles into the tumor cells and killed them selectively. Based on this and in order to combine the advantages of pH-responsive CPPs for efficient intracellular delivery, hyaluronic acid (HA) for improved blood persistence and both for tumor targeting, Jiang et al. developed dual-decorated liposomes for tumor-targeted drug delivery [85]. In blood, HA endues HA-coated pH-responsive CPP-modified liposomes (HA-CPP-L) with a strongly negative charge and a protection by HA hydrophilic shell away from the attack of plasma protein for enhanced stability and duration. HA-CPP-L exhibits a high accumulation at the tumor site by means of the enhanced permeability and retention (EPR) effect and the affinity of HA with its binding receptors. At the tumor milieu, HA-CPP-L disassemble to be CPP-L as a result of the HA degradation by hyaluronidase (HAase) for exposing CPP on the liposomal surface. The exposed CPP has pH-response to the mildly acidic tumor microenvironment to increase the uptake of CPP-L into the cells. When CPP-L are internalized into endosomes and lysosomes, the imidazole group of histidine in CPP produces the proton sponge effect accompanied by membrane penetrating ability of CPP, leading to endosomal/lysosomal escape and cytoplasmic release for efficient intracellular trafficking. Newly developed strategies for multifunctional mitochondria-targeted agents Failure in the treatment of cancer frequently arises from resistance to cell death inducted by conventional radio- or chemotherapeutic protocols. As such, anti-apoptotic proteins from the Bcl-2 family, contribute to aberrant cell survival and drug resistance by counteracting physiological, inducible, programmed cell death signals, which should have resulted in the loss of mitochondrial integrity, activation of caspases and ultimate cell elimination [64]. Hence, the 4H-chromene derivative, ethyl [2-amino-6-bromo-4-(1-cyano-2-ethoxy-2oxoethyl)]-4H-chromene-3-carboxylate (HA14-1) was identified by using structure-based computer screening for molecular interactions with the hydrophobic binding pocket of Bcl-2. Based on this, Weyland et al. developed a safe nanocarrier (SV30-LNCs) which formulated from lipid nanocapsules loaded with SV30, a new analog of the proapoptotic molecule HA14-1 [86]. They found that SV30 itself

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Figure 3. Different fates and effects of AuNRs in cancer cell, normal cell, and stem cell due to distinct pathways for cellular trafficking.

conferred lipid nanocapsules (LNCs) on improved mitochondrial targeting skills significantly. Their results showed that SV30 alone or in combination with paclitaxel, etoposide or beam radiation could trigger cell death in a similar fashion to HA14-1.The effect was coincident to caspase-3 activation, thus indicated that it should be able to target the mitochondria. Confocal laser scanning microscopy revealed that NR (nile red) -SV30-LNCs could dramatically target the mitochondria compared with NR-LNCs, together with reducing the mitochondrial membrane potential, and eventually accelerating the apoptosis of F98 cells. The inorganic nanocarrier also has the property of mitochondrial targeting, such as TiO2 nanoparticles and gold nanorods (Au NRs). In a study by Wang et al., they investigated the effects of serum protein-coated Au NRs on carcinoma cells (A549 cells), normal bronchial epithelial cells (16HBE cells) and primary adult stem cells (MSC cells) [87]. Their study revealed that at concentrations between 25 and 100 mM, Au NRs could kill A549 cells but caused little damage to 16HBE and MSC cells. After which they went on investigating why the same nanorods behaved so differently in these three cells, by examining their uptake mechanism and intracellular localization. They found that most Au NRs in stem cells were resident in lysosomes and could be finally cleared from MSC cells after a time lag, resulting in the maintenance of normal metabolism. In contrast, large numbers of Au NRs coated by cetyltrimethylammonium bromide (CTAB) in A549 cells were difficult to exclude due to their translocation from endosomes/lysosomes to mitochondria, which caused cell death. Their study demonstrated that intracellular localization, not uptake pathway, determined the final fate of both Au NRs and cells. Due to the enhanced permeation of the lysosomal membrane after Au NRs uptake,

Au NRs are released into the cytoplasm of A549 cells and translocated from endosomes/lysosomes to mitochondria, inducing decreased mitochondrial membrane potentials, increased oxidation stress and finally reduced cell viability. However, Au NRs showed almost no toxicity in 16HBE and MSC cells since their lysosomal membranes remained more intact and Au NRs were localized in lysosomes. These differences in intracellular trafficking for three kinds of cells are shown schematically in Figure 3. In recent years, a myriad of intravenously administrated nanoparticle (NP)-based drug delivery systems such as liposomes [88], micelles [89], and albumin nanoparticles [90] have been used for preclinical and clinical cancer therapy. The anticancer efficacy of NPs depends on their ability to reach the target sites of action. Delivery of NPs from the injection site to the final antitumor targets consists of various transport steps with multiple physiological and biological barriers, including transport via blood to the tumor extracellular matrix (organ level), binding to the cell membrane (tissue level), internalization (cellular level), and intracellular delivery (cellular level and subcellular level) in turn [91]. Mo et al. designed, synthesized, and evaluated a novel mitochondria-targeted nanocarrier system based on zwitter ionic oligopeptide liposomes (HHG2C18 -L) with multistage pH responses to break down the series of barriers mentioned above in the whole-process delivery [92]. Charge conversion is the first-stage pH response to the mildly acidic tumor extracellular matrix. The proton sponge effect and hydrolysis of the pH-cleavable linker to increase the positive surface charge are the second-and third-stage pH responses, respectively, based on the intracellular compartmental acidity. The strength of the smart liposomes includes enhanced tumor

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Figure 4. (A) Schematic fabrication of smart MNPs with pH-response staged dissociation to the tumor intracellular compartments for mitochondriatargeted anticancer drug delivery. (B) Co-localization of C6/CTS-NPs into the mitochondria of HepG2 cells at 2 h, as observed by CLSM. The mitochondria were stained by MitoTracker Red. White arrows indicate C6 accumulated on the mitochondrial surface or into the mitochondria. (C) In vitro cytotoxicity assay profiles of various brucine/CTS-NPs and brucine solution at different concentrations against HepG2 cells after a 24 h incubation. Data are presented as the mean ± standard deviation (n ¼ 6). *p50.05, **p50.01 and ***p50.001. (D) Antitumor efficacy against Heps xenograft tumors after intravenous administration of different formulations of brucine (5 mg/kg). The arrows indicate the time of intravenous administration. Saline treatment was used as a control.

cellular uptake, improved cytoplasmic distribution, and good mitochondrial targeting, thus the goal of overcoming sequential physiological and biological barriers and efficient delivery of anticancer drugs is achieved. According to the physiological environment of the tumor and the requirement of mitochondrial targeting drug delivery, our group recently reported two multifunctional, structurally simple, chitosan derivatives N-glycyrrhetinic acid-polyethylene glycol (PEG)-chitosan (NGPC) and N-quaternary ammonium-chitosan (NQC) as basic construction unit of the mitochondrial-targeted nanocarrier system [93]. In this system, NQC carries quaternary amine groups and NGPC contains PEG (pH-cleavable Schiff’s base) as hydrophilic blocks, both carrying two chitosan chains as hydrophobic blocks. Both NQC and NGPC assemble into the multifunctional nanocomposite particles (MNPs) by crosslinking of tripolyphosphate (Figure 4A). The MNPs possess various functions such as stealth, hepatocyte targeting, multistage pHresponse, lysosomal escape and mitochondrial targeting (Figure 4B), which lead to targeted drug release after the

progressively shedding of functional groups. Thus the efficient intracellular delivery and mitochondrial localization can be realized. Compared with free brucine solution, the MNPs carried brucine, remarkably inhibited HepG2 cell growth (Figure 4C), inhibited the growth of tumor, elevated the antitumor efficacy (Figure 4D), and reduced the toxicity of anticancer drugs.

Conclusions and expert opinion As important cellular organelles, mitochondria exert both vital and lethal functions in diverse physiological and pathological conditions associated with cell survival and death. According to mitochondrial dysfunctions that have been linked to multiple aspects of tumorigenesis and tumor progression, mitochondrialy-targeted compounds represent a promising approach to target tumors selectively. This review summarized the mechanisms of mitochondrial targeting and several strategies to realize the purpose of mitochondrial targeting. Then we introduced a variety of newly developed

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mitochondria-targeted therapeutic strategies and discuss the perspectives and future directions of developing multifunctional nanostructures as below that can target and kill tumors to advance current mitochondria-targeted cancer therapy. It is our hope that we can identify and develop more potent mitochondrial-targeting preparations that can be used in the delivery of anti-cancer drugs, thus achieving the objective of better cancer treatment. Among the tactics mentioned above, the coupling methods of DQAsomes, MTS, and PTD are only applicable to delivery of biomacromolecules like genes or proteins, but not small molecular weight chemicals. Moreover, not only is MTS hard to find, it has also other constraints like complexity in technology and difficulty in implementation. The coupling method of lipophilic cationic compound TPP needs appropriate active groups from drugs to be covalently binding with TPP. In addition, drugs and TPP do not dissociate in mitochondria, but exist and take effects in the forms of conjugates, which could have impacts on the curative effects of drugs. Carriers based on target ligands, such as cationic arginine polypeptide CPPs, TPP, Rh123, and DQA, will achieve the aim of mitochondrial targeting after entering the cells. It does not solve the key issues in the application of cationic carrier. Cationic carrier does not have specificity on tumor cells. It has strong interaction with normal cells, affects the integrity of cell membranes, causes the rupture and death of normal cells, and leads to higher toxicity. In addition, under physiological conditions, the surface of cationic carrier bears a large amount of positive charges. It will readily have electrostatic attraction with proteins bearing negative charges (including enzyme, albumin and immune globulin) in the process of blood circulation, which will affect the function of drug transfer, e.g., carrier being damaged, drug leaking, and long circulation no longer maintained. Therefore, cationic carriers like cationic liposome, polyethyleneimine, and dendrimer have been applied only in cell or animal experiments due to toxicity and other issues even though many studies have been done. Nowadays, some nano-particles for intravenous injection have been applied clinically or in the preclinical antitumor therapy. The drugs need to be localized to their target sites to take effects, and these target effect sites are mostly located inside the cells. In general, nano-carriers needs to overcome physiological and biological barriers as below before they reach the ultimate antitumor target sites from the sites of intravenous injection: (1) get to tumor location through blood circulation (tissue level), (2) combine with the surface of tumor cells in tumor microenvironment and enter tumor cells by endocytosis (cell level), and (3) transfer inside tumor cells and finally reach the target effect sites (organelle level). Even the EPR effect of nano-carriers plays an important role in the course of reaching tumor location via blood circulation, the safety and stability of nano-carriers produced by surface charges in blood circulation is the indispensable requirement of its practical application considering its surface chemical property. The entry of nano-carriers into cells is mainly by clathrin-mediated endocytosis, getting into the endosome of the cells firstly and then the lysosome. Endosomes/lysosomes have been regarded as the major barriers of nano-carrier transfer inside cells because the acids and enzymes in the

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endosomes/lysosomes will lead to the degradation of the carriers and the drugs. As a result, in order to help the drugs reach the ultimate target effect sites, appropriate strategies must be designed to enable the escape of the carriers from lysosomes and finally realize the effective cytoplasmic transport. Therefore, it is important to render the carriers some specific related functions effects when being designed. It’s more important to make the safety evaluation based on the biocompatibilities of drug carriers, hematotoxicities triggered by physicochemical property, accumulation, and metabolism of the carriers, and consequent safety issues, which is the prerequisite of nano-carriers being clinically applied. It can be seen from the design of various multifunctional nano-drugs that in order to overcome all kinds of physiological barriers to finally reach the target sites and release the drugs, the multiple functions need to be assembled in an orderly way and realized through new multifunctional materials. However, as mentioned above, most of these synthesized multifunctional materials have certain acute or chronic toxicities. Poor drug loading (510%) is another issue generally existing in multifunctional nano-drugs. Meanwhile, big differences among various batches and uncontrollable carrier forms and drug loadings will be found since the polydispersities of nano-drugs are influenced by the lengths, drugloading rates, and the binding rates of functional polymer materials. Moreover, the effects of multiple functions cannot be well realized if the groups that have completed their functions cannot break away from the transfer system effectively. What happened there will affect other functional groups to take effects and in turn will affect the releases of drugs at the target effect sites. In conclusion, the disadvantages in multifunctional materials themselves such as the toxicities, poor-drug loadings, variation among batches, and interplays among the multiple functions, have severely restricted the further investigation on multifunctional nanodrugs. Therefore as pharmaceutical researchers, we should focus on the study of aspects mentioned above in a quite long term while exploring new materials. Only in this way, we can develop novel nano-drugs in the real sense, which can be practically applied in clinic.

Declaration of interest This work was supported by National Natural Science Foundation of China (No: 81473147, 81274100), 2011 Collaborative Innovation Center of Chinese Medicinal Resources Industrialization (No: ZDXM-2–3), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), sponsored by Qing Lan Project, Six talent peaks project in Jiangsu Province, Outstanding talent training program of Nanjing University of Chinese Medicine of Pharmacy. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

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