Targeted mitochondrial therapy for pancreatic cancer

Highlights

• •The article elucidates the importance of mitochondria in PC therapy.
• •We have summarized drug carriers targeting mitochondrial delivery.
• •Mitochondrial targeted therapy can improve PC treatment.

Abstract

Pancreatic cancer (PC) is a highly invasive tumor characterized by delayed diagnosis, rapid progress, and resistance to chemotherapy. Mitochondria, as the "power chamber" of cells, not only play a central role in energy metabolism but also participate in the production of reactive oxygen species (ROS), calcium signaling, regulation, and differentiation of the cell cycle. The abnormal activity of mitochondria is closely related to the development of PC. In this paper, we discussed the key role of mitochondria in PC, including mitochondrial DNA, mitochondrial biogenesis, mitochondrial dynamics, metabolic regulation, ROS generation, and mitochondrial-dependent apoptosis. We elaborated on the importance of these mitochondrial mechanisms in the development of PC and emphasized the potential of targeted mitochondrial therapy strategies for these mechanisms in the treatment of PC. In addition, this article also reviews the latest developments in innovative drug carriers such as cell-penetrating peptides, nucleic acid aptamers, and nanomaterials, which can achieve precise localization of mitochondria and drug delivery. Therefore, this article comprehensively analyzed the important role of mitochondria in the treatment of PC and clarified the effectiveness and necessity of targeting mitochondria in the treatment of PC.

Graphical abstract

Introduction

The pancreas is an approximately 15-cm-long, tube-shaped, spongy organ located in the upper abdomen between the stomach and spine. Pancreatic cancer (PC) arises from the proliferation of pancreatic cells due to abnormal DNA mutations, leading to the formation of tumors [1]. Currently, there is only one curative measure available for patients with PC, surgical resection. However, approximately 80 % of PC cases are unresectable at the point of diagnosis due to their aggressive nature and rapid growth, which leads to metastases and local-regional advances [2,3]. Therefore, in order to effectively prevent and treat PC, there is an urgent need to develop more effective drugs and explore new treatment methods.

In 1857, Rudolf Albert von Kölliker discovered a granular structure in muscle cells, and in 1898, Carle Benda named this granular structure "mitochondria". Mitochondria are important organelles within cells, with complex structures and functions. Structurally, mitochondria comprise two separate and functionally distinct membranes, the outer membrane (OM) and inner membrane (IM), that encapsulate the intermembrane space (IMS) and matrix compartments [4]. Mitochondria are the energy factories of cells, primarily responsible for energy metabolism and cellular respiration. Unlike normal cells, tumor cells tend to convert glucose to lactate through glycolysis even under aerobic conditions [5]. Although this metabolic pathway is less efficient, it can quickly generate energy to support the rapid proliferation of tumor cells. Due to the metabolic differences between cancer cells and normal cells, cancer cells can resist apoptosis signals by maintaining a high proliferation rate [6]. The primary molecular defense mechanism against the establishment and progression of cancer is apoptosis, and resistance to apoptosis is essential for the onset, growth, and metastasis of cancer [7]. Mitochondria play a crucial role in this process, influencing tumor development by regulating the apoptotic signaling pathway. The functional status of mitochondria directly affects the metabolic reprogramming of tumor cells, which involves energy production, supply of biosynthetic precursors, and cellular respiration [8]. In addition, mitochondria play a balancing role in the production of reactive oxygen species (ROS) and antioxidant defense, which is crucial for maintaining the redox homeostasis of tumor cells [9]. The multifaceted functions of mitochondria make them a promising target for cancer treatment, as they can influence tumor progression and response to treatment. Therefore, a deeper understanding of the role of mitochondria in cancer is of great significance for developing new therapeutic strategies.

In recent years, mitochondrial research has become a hot topic in treating PC. However, existing research has mostly focused on a single level of mitochondrial function, lacking comprehensiveness. For this reason, this article reviews the latest literature in the PubMed database, systematically summarizes the function and mechanism of mitochondria in PC, and deeply discusses the treatment strategy of a new vector targeting mitochondria to clarify the importance of mitochondria in the treatment of PC and to provide new ideas for drug development and targeted therapy of PC in the future.

Important functions of mitochondria in pancreatic cancer

Pancreatic mitochondrial dysfunction

The structural feature of mitochondria is a bilayer membrane system composed of an outer membrane, an intermembrane space, and an inner membrane. Mitochondria are the core of cellular metabolism, regulating glucose, glutamine, and lipid metabolism; maintaining the tricarboxylic acid cycle (TCA) and aerobic respiration through OXPHOS; and meeting cellular energy requirements [10]. However, in PC, mitochondrial dysfunction such as metabolic enzyme changes, mitochondrial autophagy abnormalities, excessive production of ROS, apoptosis resistance, and mtDNA mutations will promote tumor progression [11]. Mild dysfunction may increase ROS production and stimulate the growth and invasion of pancreatic cancer cells; serious dysfunction may lead to cell death in pancreatic cancer and inhibit tumor formation [10]. Kras mutations are extremely common in PC and are one of the main driving mutations [12]. It enhances cell proliferation, migration, and invasion by activating signaling pathways such as NF-κB and MAPK while affecting mitochondrial metabolism and ROS production, leading to mitochondrial dysfunction [13] [14]. Mitochondrial dysfunction can make PC cells show anti-apoptosis, chemotherapy resistance, and invasive phenotype. Therefore, further studying the role of mitochondrial dysfunction in the occurrence, development, and treatment of PC is of great significance for the development of new treatment strategies (Fig. 1).

Fig. 1.

The operational mechanism of normal mitochondria in cells (left), as well as a series of changes that occur after mitochondrial dysfunction in cancer cells (right), provide some targets for cancer treatment (By Figdraw).

Mitochondrial DNA

Mitochondria are organelles that exist in eukaryotes and contain genetic material, namely mtDNA [15]. Since the discovery of mtDNA in 1963, humans have conducted in-depth research on it [16]. MtDNA exists in the mitochondrial matrix, which is related to the mitochondrial inner membrane and distributed throughout the entire mitochondrial network [17]. Human mtDNA is a double-stranded circular molecule containing 16,569 base pairs, divided into the light chain (L) and heavy chain (H), encoding 13 respiratory chain complex subunits, 2 rRNA, and 22 tRNAs [18,19]. The process of oxidative phosphorylation (OXPHOS) involves 13 proteins encoded by mtDNA and over 50 proteins encoded by nuclear DNA [11]. MtDNA also includes a noncoding region, the D-loop, which contains key regulatory elements for transcription and replication. Mutations in the D-loop may severely affect mitochondrial function and interfere with mtDNA transcription [19,20].Due to the importance of mtDNA in maintaining the normal function of organelles, mitochondrial dysfunction caused by mtDNA mutation may induce cell aging and cell apoptosis [21].

Human cancer has been found to have a wide range of mtDNA alterations, with copy number variation and point mutations being the two most prevalent modifications [20]. Because mtDNA is only inherited by mothers, most mtDNA copies are identical from birth, a condition called homoplasmy. Heteroplasmy is the state in which mutant and normal mtDNA coexist as a result of new mtDNA mutations [22]. In heterogeneous cells, the ratio of mutant and wild-type genomes determines the phenotype of pathogenic mtDNA mutations. Before tissue dysfunction and clinical symptoms manifest, there must be a significant accumulation of mutant mitochondrial DNA, known as the threshold effect [23]. At low heterogeneity frequencies, the harmful effects of mutant mtDNA are mostly masked by coexisting wild-type copies, but once the threshold is exceeded, mutant mtDNA will lead to phenotypic changes [[24], [25], [26]]. The frequency threshold varies depending on mutations and tissues [27]. There are thousands of copies of mammalian mitochondrial DNA in every cell, and mutations usually affect only a fraction of them [28]. As mtDNA codes only for oxidative system proteins, mutations that affect its phenotypic expression typically modify the activity of the OXPHOS system, leading to altered calcium homeostasis, reduced ATP production, increased ROS generation, and reorganization of the mitochondrial network [29].

Virtually all human malignancies, including PC, have somatic mtDNA mutations in them. There is an association between increased mitochondrial DNA copy number and decreased risk of PC [30]. In addition to mtDNA copy number variation, PC is strongly associated with mtDNA copy number variation. Tuchalska-Czuro W et al. reported that the mitochondrial DNA content in PC cells is reduced in comparison with neighboring normal pancreatic tissues, which can impair mitochondrial activity [31].

Mitochondrial biogenesis

Mitochondrial biogenesis is the process of producing new mitochondria from existing mitochondria [32]. Mitochondrial biogenesis relies on timely and coordinated transcriptional control of mitochondrial and nuclear coding genes [33]. Regulatory factors can be divided into two categories. One type includes some DNA-binding factors, such as peroxisome proliferator-activated receptor (PPAR), estrogen-related receptor (ERR), cAMP response element-binding protein (CREB), c-Myc, nuclear respiratory factor 1 (NRF-1), and GABP [34]. These factors target different but overlapping sets of mitochondrial genes, and when they are upregulated, mitochondrial biosynthesis is activated. Another important category includes some co-activators. Represented by members of the PPAR coactivator-1 family (PGC-1α, PGC-1β, and PRC), they enhance the transcriptional activity of DNA-binding factors and induce mitochondrial biogenesis. These co-activating factors are overexpressed in multiple cell types and can compensate for functional loss with each other [34]. These regulatory factors form several feedforward and feedback loops, which keep mitochondrial organisms in a state of equilibrium.

The regulatory factors of mitochondrial biogenesis are crucial to maintaining cell energy balance and may play a role in the treatment of PC. By adjusting these factors, new therapies can be developed to enhance mitochondrial function or inhibit energy metabolism in cancer cells. LINC00842 is a long intergenic noncoding RNA that promotes the development and invasion of pancreatic ductal adenocarcinoma by targeting PGC-1α [35]. The mechanism of action of LINC00842 includes inhibiting the acetylation of PGC-1α and preventing SIRT1 from deacetylating it, leading to metabolic remodeling in pancreatic ductal adenocarcinoma cells [35]. C-Myc oncoprotein is related to the pathogenesis of PC [36]. It was found that the activation of c-Myc is sufficient to transform inert pancreatic intraepithelial tumors into mouse PC [37]. PC has a poor response to chemotherapy, and chemotherapy resistance is the main obstacle to chemotherapy for PC. Overexpression of C-Myc will enhance the resistance of PC cells to chemotherapy drugs, while down-regulation or inhibition of C-Myc can improve the effect of chemotherapy drugs (such as nab-paclitaxel and gemcitabine) and help overcome the chemotherapy resistance of PC. Therefore, the inhibition of C-Myc is of great significance for improving the chemosensitivity of PC [[36], [37], [38]].

Mitochondrial dynamics

In living cells, mitochondria are extremely dynamic organelles that can change in shape to form long or short fragments. Mitochondrial dynamics is the term used to describe this highly regulated process [15]. Under physiological and pathological conditions, mitochondria are more likely than other organelles to change shape [39]. Fusion, fission, selective degradation, and transport are all included in the definition of mitochondrial dynamics [40]. Mitochondrial fission 1 (FIS1), mitochondrial fission factor (MFF), dynein-related/like protein 1 (DRP1), mitochondrial fusion 1 and 2 (MFN1 and MFN2), and optic atrophy protein family (OPA) GTPases play key roles in mitochondrial dynamics [41]. The proper balance between fission and fusion events determines the size, number, and shape of organelles to suit the requirements of cell metabolism. Additionally, mitophagy eliminates damaged organelles as a mechanism for mitochondrial quality control. All of these processes are governed by mitochondrial dynamics [15].

The balance between fusion and fission mediated by the large GTPase of the motor protein family determines mitochondrial morphology [42]. The study by Kashatus JA et al. showed that activation of the MAPK pathway or expression of oncogenic RAS leads to an increase in mitochondrial debris. Knocking out DRP1 can block this phenomenon and inhibit tumor growth. Mitochondrial fragments and ERK2-mediated DRP1 phosphorylation of serine 616 are key factors in this effect, and PC amplifies these two processes, indicating that oncogenic KRAS regulates mitochondrial fission/fusion periods [42]. Cdk5 and CAMKII promote division by phosphorylating DRP1-S616, while PKA phosphorylates DRP1-S637 to inhibit its activity, and calcineurin dephosphorylation of S637 promotes DRP1 recruitment to mitochondria and division [43]. PKA phosphorylates DRP1 by enhancing complex I activity, improving mitochondrial glutamine utilization, reducing intracellular ROS generation, and stimulating autophagy, highlighting the close relationship between mitochondrial dynamics and PC metabolism [44].

Numerous cellular functions rely on the equilibrium between fission and fusion, which is essential for maintaining mitochondrial metabolic activity [43]. In the initial step of fission, DRP1 is recruited to the mitochondrial surface, regulated by the ratio of serine 616 to serine 637 phosphorylation [45]. S616 phosphorylation is an activator of fission, while S637 phosphorylation is an inhibitor of fission [45]. Mitigation of DRP1 by genetics or pharmacology, or overexpression of MFN2, can induce mitochondrial fusion [46]. Yu M et al. found that oral flutamide promoted the expression of MFN2 in tumors by 2-fold and can be used as a chemotherapeutic agent. Moreover, it was found that enhancing mitochondrial autophagy is the main mechanism by which mitochondrial fusion inhibits tumours, which can proportionally reduce mitochondrial mass and ATP production. In this study, mitochondrial fusion was demonstrated to be a specific and druggable inhibitor of PC growth [46]. Many studies have shown that the mitochondrial fission protein Drp1 promotes tumor migration and pathogenesis [47]. Murata D and other researchers found that targeting OPA1 can effectively inhibit the activity of PC [48]. Of particular note is that when used in combination with mutation-specific KRAS inhibitors, OPA1 inhibition exhibits a synergistic effect [48]. These results suggest that targeting both mitochondrial dynamics and KRAS signal transduction can provide a therapeutic strategy for PC. At the same time, OPA1 was also found to overexpress PC stem cells and regulate the formation of tumor globules [49]. Further research shows that PC stem cells undergo mitochondrial remodeling in the process of stem acquisition [49]. A growing body of evidence indicates that mitochondrial dynamics can play a crucial role in the treatment of PC.

Mitochondrial regulation of metabolism

In normally differentiated cells, mitochondrial OXPHOS is the main pathway for energy production. Under physiological oxygen concentration and functional mitochondria, tumor cells transition from oxidative phosphorylation to aerobic glycolysis, characterized by high glucose uptake and lactate production, known as the Warburg effect. Even with sufficient oxygen supply, cancer cells can increase energy supply through aerobic glycolysis due to improper proliferation and the influence of oncogenes [50]. According to current theories, the hypoxic tumor microenvironment might promote the glycolytic transition by HIF-1α activation, p53 inactivation, aberrant signaling from oncogene activity (Ras, PI3K/mTOR, c-Myc), or alterations in the OXPHOS pathway [51]. Glycolysis has been shown to be promoted by the Ras oncogene in particular [52]. Carcinogenic Kras mutations are involved in regulating the metabolic pathways of PC cells [53], including increasing glucose absorption and glycolysis content, expanding the use of glutamine to support cell growth and proliferation, and increasing the NADPH/NADP+ ratio to maintain cellular redox status [53]. In PC cells, activated Kras activates two key downstream signal transduction pathways, MAPK/ERK, and PI3K/Akt/mTOR, by combining with GTP to regulate the development, cell growth, epigenetic disorders, differentiation, and survival of PC in a KRAS-dependent manner [54,55].

Desmoplasia in pancreatic tumours creates a hypoxic microenvironment [56]. Compared to the surrounding stroma, pancreatic tumour cells express higher levels of HIF-1a and its downstream glucose metabolism genes [57]. Shukla SK et al. showed that the expression of HIF-1a in gemcitabine-resistant PC cells increased, while the glycolytic phenotype and glucose dependence increased [58]. By suppressing HIF-1α and Stat3 signaling, LB-1, a new triptolide (LA) derivative, exhibits antitumor action in PC, as demonstrated by Niu F et al. [59]. MUC1 (Mucin 1), A large type I transmembrane protein overexpressed in PC can change the response of PC cells to hypoxia by regulating the expression, stability, and activity of HIF-1α [57]. Under the condition of hyperglycemia, the accumulation of HIF-1α is promoted and lactate dehydrogenase A (LDHA), hexokinase 2 (HK2), and platelet phosphofructose kinase (PFKP) are activated, thus promoting the glycolysis and disease progression of PC [60].

The initial stage of glycolysis, glucose transfer across the plasma membrane, is mediated by glucose transporters. The tumor suppressor p53 inhibits GLUT1 (glucose transporter 1) while the oncogenes cMyc, HIF-1α, and Kras transactivate it [61]. By inhibiting glucose transporters such as GLUT1, glucose entry into tumor cells can be reduced, thereby inhibiting glycolysis [61] [62]. In addition, targeting key enzymes in the glycolysis process, such as HK2, PFK1, and LDHA, can reduce the energy supply to tumor cells and the production of biosynthetic precursors[52].According to Yang Y et al., miR-135 inhibits glycolysis and promotes the adaptation of PC cells to metabolic stress by targeting PFK1 [63]. In contrast to glycolysis, phosphorylated pyruvate dehydrogenase kinase 1 (PDHK1) suppresses mitochondrial OXPHOS in PC cells by inhibiting the pyruvate dehydrogenase (PDH) complex [64]. According to Anderson M et al., HK2 regulates lactate production in PC to promote tumour growth and metastasis [65].

More and more studies have shown that cancer cells not only generate energy through aerobic glycolysis, but certain subtypes strongly rely on OXPHOS to maintain their energy needs [66]. For example, in PC, pancreatic tumor cells with SETD2 mutation or low expression show activated mitochondrial OXPHOS, which is also reflected in other tumor types [67]. This finding reveals the dependence of specific PC subtypes on OXPHOS and emphasizes the potential importance of treatment strategies for OXPHOS in these subtypes. During the OXPHOS process, mitochondria generate a high proton gradient across the membrane, driving ATP synthase to synthesize ATP [68]. A higher OXPHOS level can enhance PC cell growth [69]. The TCA cycle is a key pathway for OXPHOS in cells and a key node for redox homeostasis, energy metabolism, and macromolecule synthesis [70]. TCA cycle intermediates are able to influence cancer development and progression by modulating cellular activities such as metabolism and signalling [71].

It is a new strategy to develop corresponding inhibitors for the key enzymes of the OXPHOS metabolic pathway, such as isocitrate dehydrogenase (IDH), succinate dehydrogenase (SDH), and ATP synthase, to treat PC. Many new IDH inhibitors are currently under research. For example, Enasidenib targeting IDH2 mutations has been approved for the treatment of relapsed or refractory acute myeloid leukemia (AML) patients carrying IDH2 mutations; Vorasidenib, an inhibitor targeting IDH1 mutations, is used to treat low-grade gliomas and secondary glioblastomas carrying IDH1 mutations [72]. These IDH inhibitors mainly inhibit the activity of mutant IDH enzymes, reducing the accumulation of 2-hydroxyglutarate (2-HG) in cells. This metabolite accumulates in IDH mutant cancer cells and is associated with tumor development [72]. IDH inhibitors can induce tumor cell differentiation, reduce tumor growth, and to some extent reverse high methylation of DNA and histones [72]. SDH, as an important enzyme complex that participates in both the TCA and electron transport chain, its functional loss leads to the accumulation of succinic acid in cells, triggering a series of epigenetic and metabolic changes, promoting tumor occurrence and development [73]. DT-010, As a conjugate of danishes and tetramethylpyrazine, has an anti-tumor effect in breast cancer cells in vivo and in vitro by inhibiting ROS production and mitochondrial dysfunction mediated by mitochondrial complex II [74]. Temozolomide may be effective in treating SDH - deficient gastrointestinal stromal tumors (GISTs) [75]. ATP synthase inhibitors inhibit the growth and survival of cancer cells by blocking ATP synthesis. Gboxin is an OXPHOS inhibitor that can specifically inhibit the growth of glioblastoma cells [76]. Due to the irreversible toxic effects of Gboxin, improving delivery or coupling with other anti-tumor drugs may help completely eradicate tumor growth [76].

Another pathway by which cancer cells derive energy is glutamine catabolism. During rapid cell growth, Gln can be converted to lactic acid, which provides cancer cells with anabolic support [77]. Transporters for glutamine, like SLC1A5 or ASCT2, facilitate the entry of glutamine into the cytoplasm, initiating its catabolism by converting it to glutamate [61]. NEDD4L (an E3 ubiquitin ligase) suppresses mitochondrial metabolism by reducing cellular ASCT2 levels, thereby suppressing PC growth and survival [78]. An alternative strategy to disrupt glutamine metabolism is to use glutamine analogues [79]. The disruption of glutamine pathways by glutamine analogues was shown to significantly enhance the chemosensitivity of drug-resistant PC cells to gemcitabine therapy [79]. Hamada S et al. observed increased sensitivity of PC cells containing Kras and Nrf2 active forms to glutaminase inhibition [80]. This suggests that glutamine protease inhibition has potential as a new therapeutic intervention for PC.

Mitochondrial regulation of ROS

ROS is a substance with significant oxidative activity. ROS inside cells oxidizes lipids, proteins, and DNA, leading to damage to various organelles. It contains higher levels of ROS in cancer cells in nutrient-restricted environments than in normal cells [81,82]. In the treatment of PC, the most promising strategy is to increase the level of intracellular ROS, making PC cells more vulnerable to oxidative stress-induced cell death [83]. The NADPH inhibitor diphenylene iodonium (DPI) inhibits ROS formation in PC cells and induces apoptosis [84]. Among the quinazolidinedione (QD) family of compounds, QD325 is considered the most effective candidate for redox regulators in the treatment of pancreatic ductal adenocarcinoma due to its activation of Nrf2 mediated oxidative stress response and inhibition of mitochondrial activity (by inhibiting mitochondrial DNA transcription and downregulating mitochondrial DNA encoded OXPHOS enzymes [85].

Uncoupling proteins (UCP) are members of the mitochondrial inner membrane anion transport carrier superfamily, which control the production of ROS and heat generation [86]. According to some studies, UCP2 is overexpressed in PC cells compared with adjacent normal tissues [86]. UCP2 inhibits oxidative stress by increasing the entry of protons into the mitochondrial matrix and decreasing electron leakage and mitochondrial superoxide production [87]. The inhibition of UCP2 by genipin or the mRNA silencing of UCP2 enhances GEM's ability to induce mitochondrial superoxide accumulation and apoptosis, reducing cancer cell proliferation synergistically [87]. In addition, the use of arsenic trioxide combined with small beta lactide has been proven to induce ROS production and apoptosis through the mitochondrial pathway in human PC cells [88]. In addition, in the treatment of PC, an appropriate amount of ROS helps to activate the immune response and promote apoptosis of cancer cells, but an excessive amount may damage normal cells [83]. Therefore, the rational use of antioxidants to balance ROS levels and protect normal cells is a key step in treatment.

Mitochondrial apoptosis

Apoptosis is the most important molecular defense mechanism against cancer occurrence and development, playing a crucial role in the occurrence, progression, and metastasis of cancer. The regulation of apoptosis involves three main pathways: endoplasmic reticulum stress-induced apoptosis pathway, death receptor-induced exogenous pathway, and mitochondrial-mediated endogenous pathway [89]. Although apoptosis is triggered by different pathways, the mitochondrial pathway is the key signaling pathway for inducing apoptosis [90].

Caspase and Bcl-2 families play a central role in the induction of mitochondrial-dependent cell apoptosis [89]. The Bcl-2 family consists of anti-apoptotic proteins (such as Bcl-2, Bcl xL, and Mcl-1) and pro-apoptotic proteins (such as Bak, Bax, and proteins containing only the BH3 domain such as Bad, Bid, Bim, Puma, and Noxa) [91]. These proteins not only maintain the stability of mitochondrial membranes but also regulate the intrinsic apoptotic pathway mediated by mitochondria. The apoptosis of mitochondrial cells is related to many important factors, such as mitochondrial outer membrane permeability (MOMP), mitochondrial pyruvate hydrolase (MPTP), and mitochondrial transcription [92]. Under stress conditions, pro-apoptotic proteins Bax and Bak oligomerize on the outer mitochondrial membrane, promoting the release of cytochrome C and activating the caspase-9/caspase-3 cascade reaction, leading to cell apoptosis [93].

In PC, the strong resistance of cells to apoptosis is an important factor leading to poor prognosis of patients and low efficiency of chemotherapy and radiotherapy programs [94]. This resistance is usually associated with the upregulation of anti-apoptotic molecules in the Bcl-2 family [94]. The release of cytochrome C, as a key signaling event in the apoptotic pathway, is mainly regulated by Bcl-2 family proteins [95]. For example, rottlerin shows pro-apoptosis and anti-tumor activity by destroying the interaction between Bcl-2 and BH3 proteins in PC [95]. Mcl-1 is crucial for the survival of PaCa cells, and its knockout can significantly induce cell apoptosis [94]. In addition, RAB14, as a protein regulating Bcl-2 family proteins, promotes the proliferation of PC cells and inhibits gemcitabine-induced apoptosis by up-regulating Bcl-2 and maintaining mitochondrial membrane potential [96]. As a small molecule BH3 analog, ABT-737 can significantly enhance the mitochondrial apoptosis signal induced by TRAIL. Its mechanism includes releasing Bim from Bcl-2 or Bcl xL, and relieving the inhibition of Bcl xL on Bak, thus promoting the apoptosis process of PC cells [97]. The discovery of Bcl-2 family inhibitors and BH3 mimics provide new potential targets for the development of therapeutic strategies for PC.

Delivery to mitochondria: a narrower approach for broader therapeutics

Chemotherapeutics need to specifically target a tumor site and then enter cells at a concentration high enough to kill cancer cells without endangering nearby healthy cells in order to be effective [98]. The chemotherapeutic efficiency of a drug delivery system is largely dependent on its capacity to target and penetrate tumor masses [99]. Since healthy mitochondria are necessary for the survival and effective operation of cells, mitochondrial malfunction can result in a variety of illnesses [100]. To get around various barriers to targeting mitochondria, researchers have developed a variety of pharmaceutical preparations, such as liposomes, polymeric nanoparticles, and inorganic nanoparticles modified by mitochondriotropic moieties, such as dequalinium (DQA), triphenylphosphonium (TPP), mitochondrial penetrating peptides (MPPs), and mitochondrial protein import machinery [101].In vitro and in vivo, the targeted formulations outperformed their untargeted counterparts in terms of pharmacological effects and therapeutic outcomes [101]. To get around the difficulties of targeting mitochondria, a variety of nanoformulations have been developed recently [100]. These include liposomes, polymeric nanoparticles, and inorganic nanoparticles coupled to DQA, MPPs, TPP, and other mitochondriotropic moieties as well as mitochondrial protein import machinery [100].

The structure of the mitochondria is unusual compared to that of other subcellular organelles and comprises four important parts: the OMM, IMM, intermembrane space (IMS) and matrix [102]. Because of the comparatively wide transition pore in the membrane where the medications would be driven by passive diffusion, therapeutic molecules can pass through the OMM with ease [101]. The shorter transition slits between the intermembrane space and the mitochondrial matrix of the highly folded, hard IMM, however, make it more difficult for many therapeutic compounds to penetrate the mitochondrial matrix [100]. The primary challenge in both fundamental research investigations and therapeutic development is to the challenging task of permeating the mitochondrial membranes [103]. Translational research and the development of mitochondrial therapy are hampered by this highly impermeable barrier, which stands in the way of medication delivery [103]. There are two different approaches to mitochondrial targeting: attaching a mitochondrial targeting moiety to a nanocarrier or directly coupling a targeting moiety to a therapeutic drug [102].

Cell-permeable peptide-based drug delivery to mitochondria

While little is known about creating molecules that can pass through the mitochondrial membrane, the discovery of a number of short peptide sequences with effective cellular absorption represented a significant advancement in the search for substances that can pass through the plasma membrane [104]. The Tat peptide, which is generated from the HIV transactivator protein, is one that has drawn a lot of interest [104]. This peptide's discovery, along with others showing membrane-crossing properties, formed the basis of a recently established sector devoted to the application of cell-penetrating peptides (CPPs) as molecular transporters [104]. The transactivator of transcription (TAT) protein of HIV-1 was shown to be effectively internalized by cells in vitro in 1988, according to two separate investigations [105]. In 1991, after a few more years, it was also demonstrated that the Drosophila melanogaster homeoprotein Antennapedia's homeodomain could penetrate cells [105]. Many of the peptides that are popularly referred to as CPPs have translocation capacities. This was found a few years after the internalization features of the Antennapedia homeodomain and TAT were found [105].

CPPs are a family of diverse peptides that may cross tissue and cell membranes by energy-dependent or energy-independent methods without interacting with particular receptors [105]. Typically, CPPs consist of five to thirty amino acids [105]. CPPs fall into three basic types based on their properties: hydrophobic, amphipathic, and cationic [106]. The low toxicity of peptides in comparison to other drug carriers is a critical benefit of CPP-based therapies, in addition to their superior efficacy resulting from quick and powerful delivery [107]. However, they lack specificity and are highly unstable [107]. Using a nonspecific CPP to transport a cargo with focused activity or a cell-specific CPP to carry the therapeutic substance can result in higher specificity [107]. These peptides are referred to as peptidic delivery factors because of the substantial evidence demonstrating their ability to transfer a wide range of physiologically active conjugates (cargoes) into cells, such as proteins, peptides, DNAs, siRNAs, and tiny medicines [105]. Either covalent bonding or noncovalent complex formation can be used to conjugate cargo to CPPs [105]. Furthermore, mitochondrial penetrating peptides (MPPs) have been developed by the Kelley laboratory [108]. It has been demonstrated that MPPs can be utilized to carry several medications, including doxorubicin, desferrioxamine, platinum-based anticancer medicines, and chlorambucil, to the mitochondria [108].

MPPs are made up of four to eight hydrophobically modified, positively charged amino acids that alternate [109]. By damaging mitochondria, Li Q et al. created a mitochondrial targeted drug delivery system (P-D-R8MTS) based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers. In vitro, P-D-R8MTS repressed the migration and invasion of MDA-MB-231 and 4T1 breast cancer cells in addition to inhibiting their proliferation [110]. Their research offered a viable approach to the delivery of drugs targeted to the mitochondria in metastatic cancer [110].

Delocalized lipophilic cation-based drug delivery to mitochondria

Cancer cells have a higher intrinsic mitochondrial membrane potential than normal cells [111]. This property makes directing bioactive agents through conjugation with lipophilic cations a feasible strategy for targeting cancer cells [111]. Positively charged molecules can be transported into mitochondria using the substantial transmembrane potential of 140–180 mV (negative inside) that exists in mitochondria [100]. Lipophilic cations, also referred to as mitochondriotropic ligands, are mitochondrial carriers that were first used by Murphy and associates [100]. The first compounds that targeted mitochondria were called delocalized lipophilic cations (DLCs), and examples of these include rhodamine 123, DQA, and TPP [112]. Positively charged substances build up in the mitochondrial matrix against their concentration gradient due to the negative membrane potential of the inner membrane of the mitochondria [113].

To enhance the bioactive chemical of interest's mitochondrial uptake, a variety of lipophilic cations can be linked to it, such as cationic peptides, cyanine cations, rhodamine, and alkyltriphenylphosphonium cations [113].Chen W et al. showed that mitochondrion-targeting chlorambucil, Mito-Chlor, efficiently inhibits the transcription of mitochondrial DNA [111]. Despite a lengthy history of safety and effectiveness in chemotherapy regimens, nitrogen mustards like chlorambucil still carry a significant risk of drug resistance and subsequent neoplasms [114]. Based on the changed mitochondrial membrane potential, Δψmt, Millard M. et al. created Mito-Chlor, a triphenylphosphonium derivative of chlorambucil, to target the mitochondria of cancer cells [114]. In panels of PC and breast cell lines, this kind of targeting has erased drug resistance [114].

Nanoparticle-based drug delivery to mitochondria

The unique bilayer structure of mitochondria and their extremely negative potential make it difficult for medicinal compounds to enter mitochondria [101]. Many pharmacological preparations, including liposomes, polymeric nanoparticles, and inorganic nanoparticles modified by mitochondriotropic moieties like DQA, TPP, and MPPs, have been produced by researchers to target mitochondria despite a number of obstacles [101]. In vitro and in vivo, the targeted formulations outperformed their untargeted counterparts in terms of pharmacological effects and therapeutic outcomes [101]. Nanocarriers have a lot of promise for treating illnesses linked to the mitochondria and can be used to deliver biotherapeutics to particular mitochondria [101]. Over the past thirty years, nanotechnology has garnered significant interest in the field of medicine, with certain nanoparticles being studied for their potential to target mitochondria [112].

Dequalinium-based drug delivery to mitochondria

The divalent cation DQA is a potent and selective blocker of Ca²⁺-activated, small conductance K⁺-channels, acts as an antimicrobial agent, and has remarkable cancerostatic activities [102]. The first documented mitochondrial targeting vectors for use as a gene delivery mechanism are DQAsomes (1998) [101]. Weissig V et al. recently succeeded in preparing cationic vesicles made of DQA, termed DQAsomes [115]. DNA is bound by DQAsomes, which shield it from DNase action [115]. DQAsomes have proven to be efficient gene carriers and can also be utilized to encapsulate anticancer medications, such as curcumin and paclitaxel [101]. These investigations found that, both in vitro and in vivo, encapsulated paclitaxel or curcumin in DQAsomes exhibited more tumoricidal or antioxidant activity than free paclitaxel or curcumin [101]. In the present study, an amphiphilic polymer composed of glycol chitosan (GC) and DQA was synthesized via a Michael addition reaction using a methyl acrylate linker and used to target mitochondria [116]. DQA was selected as the mitochondrion-targeting moiety as well as the lipophilic component of the polymer, which self-assembled into nanoparticles in aqueous solvent [116]. The experimental results indicated that the GC-DQA polymer is suitable for a wide range of hydrophobic drugs and has potential applications in mitochondrial medicine [116].

DQAsomes are well-known conventional transfection agents that form DQAsomes/DNA complexes (DQAplexes) that have both nucleotropic and mitochondriotropic properties [117]. However, it was discovered that this form of carrier was not appropriate for the successful transfection or mitochondrial delivery of medicines because of their issues stemming from physical instability, low effectiveness of transfection, and intramitochondrial delivery [117]. A novel nanosome made of DQA-DOTAP-DOPE (1,2-dioleoyl-3-trimethylammonium-propane-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) (DQA80s) was described by Bae Y et al. [117] as a possible delivery vector that targets mitochondria. The recently developed nanosomes, known as DQA80s, were shown by Bae Y et al. to have much improved stability as well as good potential for successful mitochondrial targeting and intracellular uptake in HeLa cells [117]. The DQA/DOTAP/DOPE nanosomes, also known as DQA80s, are a liposome-based carrier system that has been described by Bae Y et al. [118] and may be used as an anticancer reagent in addition to a carrier for mitochondria-specific targeting. According to the current study, treating cancer cells with DQA80 causes cellular uptake, mitochondrial targeting, lysosomal escape, and metabolic activity [118]. These findings clearly imply that DQA80s' dual function as anticancer agents and drug delivery vehicles shows sufficient promise to further in vivo research toward the ultimate objective of targeted mitochondrial disease therapy [118].

The chemotherapeutic efficiency of a drug delivery system is largely dependent on its capacity to target and penetrate tumor masses [99]. Two of these objectives have been met by conventional liposomes: improved tumor delivery and fewer side effects. Nevertheless, instead of actively penetrating cancer cells, conventional liposomes aggregate passively in the tumor region [99]. Numerous liposome methods have been developed for delivery of mitochondrion-targeting compounds since the encouraging success of DQAsomes. To obtain a predefined drug delivery outcome, liposome surfaces can be easily changed with ligands and other membrane-fusing agents [119].

Liposome-based drug delivery to mitochondria

Since their discovery in 1964, liposomes—lipid bilayer membrane vesicles—have been widely employed as nanocarriers for drugs and other bioactive materials [120]. The ability to encapsulate and transport bioactive compounds without changing their chemical structure or bioactivity is one benefit of the liposomal encapsulation technique over lipophilic cations [109]. In order to effectively deliver a nanocarrier to mitochondria, Yamada Y et al. reported the development of a dual-ligand liposomal system consisting of octaarginine (R8), a moiety that improves cellular absorption, and an RP aptamer for mitochondrial targeting [121]. A diterpene of the labdane class called sclareol was used in another effort at mitochondrial delivery [119]. In addition to inducing cell cycle arrest and apoptosis and downregulating the expression of the proto-oncogene cmyc, it has been shown to exhibit growth-inhibiting and cytotoxic effects against a range of human cancer cell lines [119]. The intracellular transport of sclareol via mitochondriotropic liposomes resulted in a notable enhancement of its apoptotic and cytotoxic effect [119]. Scleneol is a poorly soluble anticancer medication [119].

The primary drawback of the liposome system for the delivery of mitochondrion-targeted antioxidants (MTAs) is its ability to evade endosome degradation, which restricts the endosomes' ability to spontaneously degrade in the mitochondria and cytoplasm [109]. In order to get over this restriction, the MITO-Porter was created to transport bioactive materials to mitochondria using a lipid envelope and condensed plasmid DNA [109]. Abe J et al. activated cardiac progenitor cells (CPCs) by delivering resveratrol to mitochondria using a mitochondrial drug delivery system (MITO-Porter system) [122]. In doxorubicin-induced cardiomyopathy, the transplantation of MITO cells, which possess activated mitochondria, is more efficient than conventional CPC transplantation [122].

pH-responsive nanoparticle-based drug delivery to mitochondria

Tumour mitochondria exhibit some specific features that differ from those of other cellular compartments and normal mitochondria [123]. TPP, a lipophilic cation, has been used in mitochondrion-targeted drug delivery systems. In this study, CTPP-CSOSA/Cela (4-carboxybutyl) (triphenylphosphonium bromide-glucolipid-like conjugates/Celastrol) micelles were developed for mitochondrial targeting and alkaline pH-responsive drug release to treat cancer [123]. By minimizing drug leakage in the cytoplasmic neutral pH and lysosomal acidic pH prior to transport to mitochondrial target sites, a mitochondrial alkaline pH-responsive drug delivery system can, in comparison to the traditional mitochondrial targeted navigation of therapeutic drugs, realize fast drug release at mitochondrial target sites to improve the tumour inhibition efficiency and significantly reduce system toxicity [123].

Mitochondrion-targeted photodynamic therapy (PDT) has emerged as one of the most efficient antitumour strategies [124]. Qi T et al. successfully developed a pH-activatable nanoparticle with precise dual-targeting of early endosomes and mitochondria, as well as theranostic signal amplification for efficient cancer treatment [124]. Yang G et al. constructed a nanoplatform with a glutathione (GSH)-activatable and mitochondrion-targeted pro-photosensitizer encapsulated by an ultrasensitive pH-responsive polymer to achieve imaging-guided tumour-specific PDT [125]. These well-designed GSH-activatable and mitochondrion-targeted P@DCy7 nanoparticles achieved precise tumour theranostics with reduced nonspecific side effects, and this design could provide a new paradigm for organelle-targeted cancer-specific treatments [125].

Aptamer-based drug delivery to mitochondria

The majority of antitumor drugs are not able to specifically recognize or target tumor tissues, tumor cells, or even subcellular organelles [126]. The therapeutic impact is far from satisfactory as a result of their incapacity to differentiate between healthy and tumor cells [126]. Aptamers are single-stranded DNA or RNA oligonucleotides that bind with high affinity to specific non-nucleic acid target molecules (e.g., peptides, proteins, drugs, organic and inorganic molecules, or even whole cells) through folding into unique three-dimensional (3D) structures [127]. hree often utilized aptamers include AS1411, MUC1 aptamer, and A10 RNA aptamer [126]. A variety of cancer cells have highly expressed MUC1 protein and nucleolin on their membranes, which can be recognized and bound to by MUC1 aptamer or AS1411, respectively [126]. Aptamers have gained rapid traction as intracellular targeting agents and intracellular imaging tools due to their exceptional structural recognition of intracellular epitopes [128]. Since ATP is the main energy unit in living things and is therefore crucial to a wide range of biological activities, including energy conversion and the synthesis of several bioproducts, it is one of the most significant targets of aptamer sensors in cells [129].

Due to their sensitivity to ROS and significant involvement in supporting living activities, mitochondria have emerged as a prospective pharmacological target for photodynamic therapy (PDT) [130]. For targeted tumor imaging and photodynamic therapy (PDT), Xiong H et al. developed a novel approach in which mitochondrion-targeted aptamer-pyro conjugates (ApPCs) combine with in situ biosynthesized Au NCs [130]. An in vivo investigation revealed that fluorescent Au NC-ApPC assemblies were only produced in tumors and were retained for an extended period of time; following 15 days of PDT treatment, tumor development was markedly suppressed [130]. All of this data points to the Au NC-ApPC assembly that is biosynthesized in situ as a powerful mitochondrion-targeted nanoprobe that can increase the PDT efficacy of malignancies [130].

The potential of fluorescent aptamer sensors for intracellular imaging of small-molecule metabolites has been demonstrated [129]. Hong S et al. described an aptamer sensor with a photocleavable linker that targets mitochondria for spatiotemporally regulated monitoring of ATP in the mitochondria of living cells [129], building on earlier success in the temporal control of aptamer-based sensors. This work is the first instance of a DNA aptamer sensor being successfully delivered to mitochondria, opening up a new avenue for targeted delivery to subcellular organelles for the purpose of monitoring energy-producing activities and disorders associated with mitochondrial malfunction in different cell types [129]. The ability to identify cancers with DNA aptamer-targeted ICG-loaded NJs was evaluated against that of untargeted NJs using in vivo near-infrared optical imaging and ex vivo fluorescence analysis [131].Tumor-targeted nanoparticles carrying either ICG or rhodamine WT were evenly disseminated throughout the matrix of both pancreatic and prostate tumors via particular interactions with CCK-B receptors. Enhancing tumor identification is possible with tumor-targeted NJs that contain different imaging agents [131].

In order to overcome various obstacles in targeting mitochondria, researchers have developed various drug formulations, such as liposomes, polymer nanoparticles, and inorganic nanoparticles modified by mitochondrial promoter moieties, such as Cell qualified peptides, Delocalized lipophilic cations, nanoparticles, and Aptamers. This article reviews the latest developments in these innovative drug carriers, which can achieve precise localization of mitochondria and drug delivery (Table 1).

Table 1.

Summary of new carriers.

New delivery carriers		Targeted mitochondrial drugs	Targeted delivery mechanism
Cell-permeable peptides	——	P-D-R8MTS	penetrate the mitochondrial membrane	[110]
Delocalized lipophilic cations	——	Mito-Chlor	inhibit the transcription of mitochondrial DNA	[111]
Nanoparticle	Dequalinium	DQAsomes	mitochondrial targeting vectors as gene delivery system; encapsulate drugs to enhance their efficacy	[110,116]
		DQA80s	mitochondrial targeting	[117,118]
	Liposomes	MITO-Porter	deliver bioactive components to mitochondria	[109,122]
	pH-responsive nanoparticles	CTPP-CSOSA/Cela micelles	improve tumour inhibition efficiency	[123]
Aptamer	——	Au NCs-ApPCs	achieve long-term retention	[130]
	——	DNA aptamer-targeted ICG-loaded NJs	enhance tumour detection	[131]

Analysis and challenges

One of the main challenges facing mitochondrial targeted therapy is drug resistance in cancer cells, which may be achieved by adjusting mitochondrial function and metabolic pathways to resist drugs. Cancer resistance is a complex phenomenon influenced by multiple mechanisms, including drug inactivation, changes in drug targets, drug efflux, DNA damage repair, cell death inhibition, epithelial-mesenchymal transition, intrinsic cellular heterogeneity, epigenetic effects, and any combination of these mechanisms. To overcome this problem, researchers need to delve into the role of mitochondria in tumors and seek combination therapies or new drugs to inhibit resistance pathways. Drug resistance is a vast and complex topic that has not been thoroughly explored in this article. We plan to further explore this issue in future research.

Secondly, the heterogeneity and metabolic plasticity within tumors, as well as the diversity of cellular bioenergy spectra within the same tumor mass, also depend on the microenvironment/nutritional niche in which cancer cells are located. The metabolic reprogramming of tumor cells is guided by a complex microenvironment, while cancer cell metabolism dynamically changes the tumor microenvironment in turn. When striving to eradicate as many cancer cells as possible and prevent chemotherapy-resistant cells from surviving, this should be taken into consideration. In conclusion, further research is urgently needed to expand our understanding of the metabolic characteristics of PC cells compared with normal cells and other tissue types, the metabolic adaptability during treatment, and the degree of metabolic heterogeneity and plasticity within the tumor and between different patients, which will help to more effective precision medicine.

Off-target effects and toxicity to normal tissues are another important issue in mitochondrial-targeted therapy. In order to reduce these risks, it is necessary to conduct in-depth research on the pharmacokinetics and toxicity characteristics of drugs and develop more accurate delivery systems. For example, the use of nanotechnology can improve the targeting of drugs and reduce their impact on normal tissues. Meanwhile, by optimizing the dosage and treatment duration, side effects can be minimized to the greatest extent possible, thereby improving the safety of treatment. Reasonable drug development and accidental discovery of drug molecules enable clinicians to choose targeted drugs to treat specific clinical diagnoses and/or avoid worsening of accompanying disease states due to their impact on signaling pathways.

Traditional drug delivery methods may not be effective in directly delivering drugs to mitochondria, resulting in poor therapeutic efficacy. Chemotherapy requires specific targeting of the tumor site and then entering the cells at a sufficiently high concentration to kill cancer cells without endangering nearby healthy cells, in order to be effective. The chemotherapy efficiency of drug delivery systems largely depends on their ability to target and penetrate tumor masses. This article reviews some novel delivery vehicles targeting mitochondria that can improve the intracellular delivery efficiency of drugs, thereby enhancing the effectiveness of mitochondrial-targeted therapy.

Funding Information

This work was financially supported by grants from Liaoning Provincial Department of Science and Technology Applied Basic Research Program Project (No. 2023JH2/101300101).

Ethics statement

- Approval of the research protocol by an Institutional Reviewer Board. N/A.

- Informed Consent. N/A.

- Registry and the Registration No. of the study/trial. N/A.

- Animal Studies. N/A.

CRediT authorship contribution statement

Xinya Zhao: Writing – review & editing, Writing – original draft. Guoyu Wu: Writing – original draft, Conceptualization. Xufeng Tao: Visualization, Supervision, Conceptualization. Deshi Dong: Supervision, Funding acquisition. Jing Liu: Visualization, Supervision.

Declaration of competing interest

The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.