As highlighted in the previous section, ROS plays a central regulatory role in the TME and drives cancer development and progression (39). Tumor cells adapt to the high reactive oxygen environment and avoid cell death by inducing the secretion of inflammatory cytokines, stabilizing hypoxia-inducible factor-1α (HIF-1α), activating AMP-activated protein kinase (AMPK) signaling, and promoting the production of nicotinamide adenine dinucleotide phosphate (NADPH), which in turn promotes tumor metastasis and angiogenesis (40). In addition, ROS regulates the activation status of immune cells in the TME that affect cancer progression. High ROS levels oxidize major histocompatibility complex (MHC) class I molecules, which impairs antigen peptide loading and T-cell receptor(TCR)-MHC/peptide complex stability (41). Tumor cells and immunosuppressive cells in the microenvironment act synergistically to induce mitochondrial ROS (mtROS) generation, aiding in the establishment of immune tolerance (42). Lon protease in the mitochondrial quality control system induces ROS generation by interacting with multiple proteins, mediating activation of the NF-κB signaling axis and enhancing downstream signaling activity to promote tumorigenesis (43). Highly expressed HIF-1α promotes mtROS production by inducing Lon protease expression (44). Lon protease binds PYCR1, a key enzyme in proline metabolism, enhancing NADPH consumption and promoting electron leakage in the ETC, thereby elevating mtROS (45).

To avoid the deleterious effects of high ROS levels on immune cells, there exists a set of strict regulatory mechanisms in the organism to maintain a delicate balance between immune cell activity and ROS levels (46). Precise control of ROS levels in NK cells and T lymphocytes prevents their damage to other lymphocytes ( Figure 3 ). In the tumor microenvironment, IL-15 has been shown to induce NK cells to enhance their resistance to oxidative stress and protect against ROS via the thioredoxin system (47). During anti-tumor immunity, activated T lymphocytes and NK cells recruit neutrophils and macrophages by increasing ROS production, ultimately killing tumor cells (48). On the other hand, elevated ROS inhibits prolyl hydroxylases (PHDs), stabilizing HIF-1α to drive myeloid-derived suppressor cell (MDSC) differentiation (49). For example, tumor-associated fibroblasts promote the transformation of peripheral monocytes into MDSCs by increasing their oxidative stress, thereby inhibiting the proliferation of CD8⁺ T cells and promoting tumor progression (50).

ROS plays an important regulatory role in T cell activation, promotion of T cell antigen-specific proliferation and apoptosis (51). Moderate levels of ROS are essential for the normal activation and differentiation of T lymphocytes, whereas high levels of ROS promote T cell apoptosis by up-regulating the apoptosis-related factor Fas and down-regulating expression of the anti-apoptotic protein Bcl-2 (52). In addition, extracellular ROS affects T cell activation by altering the immunogenicity of antigenic peptides in APCs (53). During immunogenic cell death (ICD), intracellular damage-associated molecular patterns (DAMPs) such as ATP, endoplasmic reticulum calmodulin, and high mobility group protein B1 leaks to the extracellular space, which in turn activates DCs by interacting with its receptors and triggers anti-tumor immune responses in T lymphocytes (54). Nanoparticle-delivered catalase scavenges extracellular H₂O₂, enhancing T cell infiltration and reversing immunosuppression (55). In addition, reduced glutathione deficiency in regulatory T cells (Treg) leads to abnormal serine metabolism and down-regulates transcription factor forkhead box P3 (Foxp3), which ultimately attenuates the immunosuppressive function of Treg (56). These studies suggest that ROS levels and sustained generation capacity have a key role in ICD.

ROS have long been considered to be harmful metabolites of mitochondria, but recent studies have shown that mtROS have a necessary signaling role in preventing excessive immune responses, and in particular ROS play a key role in regulating macrophage immune responses (57). Under normal conditions, ROS affects macrophage polarization by modulating relevant signaling pathways (58). ROS also have important regulatory roles in macrophages subsets. For example, M1-type macrophages generate ROS through NADPH-oxidase (NOX) 2 signaling, which activates NF-κB signaling and enhances cellular phagocytosis (59). In contrast, high levels of ROS have harmful effects on macrophages (60). During tumorigenesis, macrophages become an important immune cell population for maintaining immune homeostasis in the TME. Tumor cells remodel the peripheral and distal TME by secreting tumor-derived factors, which stimulate the activation of both monocytes and macrophages in the microenvironment and accelerate tumor progression (61). Although TAM can exhibit both pro-inflammatory M1-type and anti-inflammatory M2-type polarized forms, it is generally accepted that TAM exhibit similar functions to M2-type macrophages, promoting tumor growth, metastasis, angiogenesis, and immunosuppression by secreting cytokines, chemokines, and proteases (62). Mitochondrial Lon protease is upregulated in M2-type macrophages, suggesting that during tumorigenesis macrophages may regulate Lon expression through multiple signals, inducing mtROS generation and participating in the TAM differentiation process (63).

DCs differentiated from monocytes have potent antigen presentation properties, promote T-cell activation, and play an important role in initiating and regulating immune responses (64). DC maturation is regulated by different types of stimuli. When immature DCs are stimulated by the pro-inflammatory cytokine IL-6 or the TLR ligand lipopolysaccharide, they are transformed into mature DCs that express CD80, CD86 and IL-6, and initiate effector T cell responses (65). In contrast, when DCs are stimulated by the regulatory factors IL-10, transforming growth factor beta (TGF-β), vitamin D3 and corticosteroids, they are transformed into tolerogenic DCs, which ultimately contribute to impaired differentiation of effector T cells and activation of Treg (66). The TME establishes an immunosuppressive state by inducing the differentiation of regulatory DCs and MDSCs, thus helping the tumor to escape immune surveillance (67). In addition, TGF-β and IL-10 secreted by tumor cells and TAM inhibit DC-mediated antigen presentation and adaptive immune responses (68). Ultimately, the concentration of ROS in the TME has an important role in regulating the cytotoxic or immunosuppressive effects of immune cells.

Mitochondria are the main ROS-producing organelles in cells, producing ROS and OXPHOS through ETC, which have a dual role in tumors: low levels are important cell signaling molecules, high levels cause oxidative damage and promote tumor development, and ROS balance is key to cell homeostasis.

In tumors, ROS drives cancer progression, and tumor cells can adapt to a high ROS environment, and also regulate the activation state of TME immune cells, such as damaging the stability of T cell-related complexes, synergizing with immunosuppressive cells to help build immune tolerance, and Lon protease is also involved in promoting mtROS production. At the same time, ROS affects a variety of immune cells: regulates the activity of NK cells and T lymphocytes, promotes MDSC differentiation, affects macrophage polarization and function, regulates DC maturation and antigen presentation, and ROS concentration in the TME plays an important role in regulating immune cell function.

The chemical structure of TPP contains three phenyl groups, which makes it highly lipid-soluble (115). At the same time, the positively charged phosphorus ions can be delocalized to the three benzene rings, allowing them to pass smoothly through the lipid bilayer (116). TPP drives drug accumulation within mitochondria due to the the negative mitochondrial membrane potential (ΔΨm) (117) ( Figure 6 ).

Chemotherapy drugs have long been the mainstay of therapy for many cancer types. However, severe side effects, low bioavailability, poor stability, and acquired drug resistance limit their clinical application. Mitochondria-targeted monofunctional platinum complexes can accumulate in the mitochondria, induce significant changes in mitochondrial ultrastructure and membrane, release cytochrome c(Cytc) from mitochondria, and disrupt mitochondrial OXPHOS and glycolysis (118). Paclitaxel (PTX) modified with TPP cations reduced the decrease in ΔΨm and significantly inhibited the growth of MCF-7 cells. Doxorubicin (DOX) resistance is a common problem in cancer treatment (119). Addition of TPP to DOX-PLGA/CPT nanoparticles leads to effective mitochondrial localization of DOX-PLGA/CPT, releases DOX to target mtDNA, induces tumor cell apoptosis and overcomes DOX resistance in MCF-7/ADR breast cancer cells (120) ( Figure 7 ).

Photodynamic therapy (PDT) is being used to treat some cancers, and it has become a promising approach for the treatment of malignant brain tumors. Mitochondria-targeted triphenylphosphine can enhance PDT efficacy in brain cancer. The TPP-conjugated photosensitizer chloramphenicol e6 (Ce6) selectively accumulates in mitochondria, colocalizes with 88% of mitochondria, and has potent cytotoxic activity, thereby significantly enhancing PDT efficacy (121, 122). The PDT effect of the mitochondria-targeting photodynamic therapeutic (MitDt) agent is amplified after laser irradiation because mitochondria are susceptible to ROS, triggering apoptotic anticancer effects (123). TPP-modified photosensitizer zinc phthalocyanine (ZnPc) selectively accumulates in mitochondria, showing excellent mitochondrial targeting for ROS-activated chemotherapy and PDT (124). TPP-modified liposomes encapsulating black phosphorus (BP) and calcium peroxide (CaO₂) accumulate in tumor mitochondria and are activated by near-infrared (NIR) laser irradiation to generate abundant PO4^3- and Ca²⁺ to accelerate in situ mitochondrial mineralization, leading to mitochondrial dysfunction and cancer cell death (125).

Radiotherapy has been an important form of cancer treatment for many years. TPP can significantly increase the efficacy of radiation enhancers and improve the effect of radiotherapy. Smaller doses of radiation (4Gy vs standard 12Gy) can be given in combination with TPP-based PDT to control tumor growth, reducing radiation side effects (126). 4-hydroxy-2,2,6,6-tetramethyl-1-oxy-piperidin(Tempol) coupled with TTP enhances X-irradiation-induced germ cell death, reduces basal ΔΨm and inhibits X-ray-induced increase in ATP production (127).

Photothermal therapy (PTT) is a new non-invasive tumor treatment that uses photothermal agents (PTA) to convert light energy into heat energy to kill tumor cells under irradiation with external light sources such as NIR light. Micro-nanoparticles loaded with TPP and S-nitrosothiol can release NO generated by surface overheating and elicit PTT upon NIR laser irradiation. The released NO can also destroy collagen fibers by activating matrix metalloproteinases (MMPs), thereby loosening the dense extracellular matrix (ECM) to enhance immune cell infiltration. The highly toxic reactive nitrogen species (RNS) peroxynitrite (ONOO^-) is produced, resulting in mitochondrial damage and induction of cell apoptosis (128). Nanoparticles with core-shell-disulfide-shell nanoparticles burst in the high GSH environment of tumors to achieve targeted drug release. The loaded DOX can quickly enter mitochondria, subsequently destroying mitochondrial DNA and leading to cell apoptosis. The synergistic effect of PTT and chemotherapy targeting mitochondria significantly enhances cancer treatment (129). The heat stress-damaged mitochondria produced can cause ICD in tumor cells, release damage-related factors, reactivate the immune response of macrophages against tumor cells, and effectively activate tumor-associated macrophages to fight against tumor cells (130).

TPP conjugates enhance the accumulation of chemopreventive agents in tumor cell mitochondria, enhancing efficacy and reducing toxicity to normal tissues (131). Metformin (Met), a commonly used hypoglycemic drug, has certain mitochondria-targeting effects and anti-tumor ability, but its clinical performance is not ideal. In pancreatic ductal adenocarcinoma (PDAC) cell lines, the IC₅₀ concentration required to inhibit proliferation by Mito-Met is nearly 1,000 times lower than that of Met (132). Mito-Met induces superoxide (O2•^-) production through complex I, inducing ROS-disrupting membrane potential, and activating calcineurin- and Cn-dependent retrograde signaling pathways in multiple cells (133). Atovaquone(ATO), an antimalarial drug, was discovered to have anti-tumor potential in the form of TPP-modified and PEGylated mitochondrial-targeted ATO (Mito-(PEG)n-ATO). Mito-ATO analogs inhibit mitochondrial complex I and complex III-induced OXPHOS in human pancreatic and brain cancer cells. Combined use with inhibitors of monocarboxylate transporters (MCT), Krebs cycle redox metabolism, or glutaminolysis, the Mito-ATO analogs can synergistically eliminate tumor cell proliferation (134). TPP mitochondrial targeting increases the drug's involvement in metabolic processes within mitochondria, inhibits tumor cell development, and promotes tumor cell death (135).

TPP has a high fat-soluble transportable positively charged phosphorus ion, which can drive the accumulation of drugs in mitochondria with the help of mitochondrial negative membrane potential. Drugs such as metformin and atropine can be used in chemotherapy to increase efficacy and overcome resistance, in radiotherapy to increase efficacy and reduce side effects, and in PTT to aid the initiation of treatment and destruction of ectoplasm, induction of apoptosis. TPP-derived compounds exhibit good antitumor activity, but there are few clinical studies to verify their anticancer efficacy, and further clinical studies are needed in the future (117). Although the TPP cation itself has low toxicity, some of the TPP-derived compounds administered systemically have non-specific toxicity, and the current strategy is mainly to modify the structure of TPP cations, encapsulate the modified compounds in liposomes, and reduce the toxicity of TPP cations, enhance its targeting selectivity to tumor cells and mitochondria to reduce toxicity (131).

F16 is a delocalized DLC that can target and aggregate in the mitochondrial matrix of tumor cells. F16 induces mPTP opening by inhibiting the interaction between mitochondrial inner membrane adenine nucleotide translocase (ANT) and cyclophilin D. F16 can form conjugates with other substances to enhance anti-tumor effects. At the same time, the reduced availability of intracellular adenosine 5'-triphosphate induced by the uncoupling effect of F16 is the main factor in the enhanced cytotoxicity mediated by F16 (136). F16 conjugates show higher cytotoxicity at low doses, and F16 conjugates initiate cell cycle arrest at the G0/G1 phase leading to mitochondrial dysfunction and excessive production of ROS, thereby inducing apoptosis (137–139). The F16-modified compounds accumulate in cancer cell mitochondria to depolarize ΔΨm, increase ROS and attack mtDNA, effectively killing cancer cells and overcoming multi-drug resistance (140, 141). Fluorescent mitochondria-targeted organic arsenic accumulates in mitochondria and inhibits the activity of pyruvate dehydrogenase complex (PDHC), leading to ATP synthesis inhibition and heat production disorders. The inhibition of respiratory chain complexes accelerates mitochondrial dysfunction and causes cell apoptosis (142).

F16 can also be used for fluorescence imaging of mitochondria. CyM is a multifunctional organic biological probe that can facilitate NIR imaging and PDT in vivo and in vitro ( 143). There are two F16 isomers that can specifically display mitochondria in the green and red channels, respectively, due to their unique fluorescence properties, providing new ways of studying mitochondrial targeting by F16. The above studies suggest that F16 and its derivatives can be of great value in cancer treatment and tumor imaging. However, the clinical application of F16 is limited by its toxicity to normal cells (144). Therefore, scientists have focused on enhancing the selectivity of F16 and its derivatives for tumor cells, and future research will likely uncover new drugs that can specifically target tumor cell mitochondria and reduce toxic effects on normal cells.

In summary, F16 acts as a DLC that can be targeted to cluster in the mitochondrial matrix of tumor cells, and its conjugate can depolarize δψm, increase Ros to attack mtDNA to kill cancer cells, and overcome multiple drug resistance. F16 can also be used for mitochondrial fluorescence imaging.

Rhodamine is an organic fluorescent dye based on xanthene that can be substituted with different 3- and 6-amino groups. It has a darker color and stronger fluorescence signal. Rhodamine can penetrate the cell membrane and selectively stain the mitochondria of living cells. Rhodamine dyes have photophysical properties such as high fluorescence quantum yield, high molar extinction coefficient and good water solubility, and low biological toxicity, making them attractive for wide use as biomarkers and fluorescent probes.

Rhodamine conjugates can be delivered to tumor mitochondria and functional proteins through organic cation transporters, improving their tumor inhibitory effects. Rhodamine B mitochondria-targeted multi-drug nanoparticles focus on mitochondrial stress-induced ICD to improve their therapeutic effect on treatment of ovarian cancer (145). Centella asiatica-rhodamine B conjugates are highly cytotoxic to human tumor cell lines, affect cell apoptosis, and can overcome resistance to chemotherapeutic drugs (146). Rhodamine B-conjugated oleanolic acid derivatives (RhodOA) reduce tumor cell viability, reduce cell migration and disrupt mitochondrial function (147). Hybrid peptide-fused rhodamine B increases anticancer activity by up to 37.5 fold, targeting the nucleus and triggering apoptosis to enhance anticancer cell activity (148). Enrichment of rhodamine B-modified catalase in cancer tissues can effectively inhibit mouse xenograft human lung tumors (149). Differences in ΔΨm and ATPase sensitivity in tumor cells contribute to the selective cytotoxicity of rhodamine123 against certain cell types in vitro ( 150). Mitochondrial targeting by rhodamine enhances the preferential cellular uptake of paclitaxel and SN-38 in cancer cells by 2–3 fold (151). Multi-walled carbon nanotubes (MWCNTs) with mitochondria-targeted fluorescent rhodamine-110 colocalize 80% with mitochondria and exhibit superior efficacy to drugs without PtBz (152). Ciacic acid-rhodamine 101 conjugates induce proliferation or growth arrest of MDA-MB-231 breast cancer cells at low doses and induce apoptosis at higher doses (153).

Rhodamine can significantly improve the effect of PDT on tumors and is by itself a potential PDT agent. The combination of rhodamine organic dyes and luminescent transition metal centers exhibits low cytotoxicity, increases tumor cell uptake, and enhances antitumor efficacy (154). Rhodamine 6G-based organic salts are stable under physiological conditions and show excellent fluorescence photostability. More importantly, they have tunable chemotherapeutic properties. Rhodamine fluorescent groups synthesized from Rh-6G and amines show pH-dependent anticancer bioactivity and trigger cell apoptosis through mitochondrial pathways, showing anticancer bioactivity in bladder cancer (155). Rhodamine 6G-based organic salts can produce nanoparticles that are toxic to cancer cells but not normal cells (156). The apoptotic index of Dasatinib (DST) contained nanoparticles is 7.5 times higher than that of free DST and is non-toxic to normal cells (157). Rhodamine-mediated novel supramolecular assemblies can efficiently capture phosphorescence energy transfer (PET) processes and have potential applications in delayed fluorescence cell imaging (158). Mitochondria-targeted silicon rhodamine-based photosensitizer (SiR-PXZ) can be rapidly taken up by mitochondria and effectively induce cancer cell apoptosis, showing excellent anti-tumor effects and potential value in photodynamic cancer therapy (159). Burst-specific PDT in mitochondria by the rhodamine derivative UCNP-GQD/TRITC induces a sharp drop in ΔΨm, thereby irreversibly initiating tumor cell apoptosis (160).

Rhodamine, with its ability to penetrate cell membranes and selectively stain mitochondria in living cells, is often used as a biomarker and fluorescent probe, its conjugates can target tumor mitochondria to inhibit tumor cells, overcome drug resistance and enhance anticancer drug uptake through a variety of mechanisms. Rhodamine can improve the effect of tumor PDT and is a potential PDT agent, showing potential value in fields such as photodynamic cancer therapy and delayed fluorescence cell imaging.

DQA is a DLC with two positive charge centers. It can selectively accumulate in mitochondria driven by transmembrane potential, allowing anti-tumor drugs to target mitochondria in tumor cells. DQA-coupled FMPSi-Cis@GO targets mitochondria in cancer cells and destroys their function (161). DQA-containing micelles deliver DOX to the mitochondria and nucleus of tumor cells, significantly inhibiting the growth of DOX-resistant tumors without obvious systemic toxicity (162). Amphiphilic polymer GC-DQA nanoparticles were synthesized as carriers to efficiently deliver curcumin to mitochondria (163). The emulsion of DQA and α-tocopheryl succinate (α-TOS) targeting mitochondria has good stability and can effectively target mitochondria and inhibit the growth of HeLa cells (164).

DQA modification destroys mitochondrial structure and induces cell death by generating ROS and dissipating ΔΨm. DQA causes loss of mitochondrial transmembrane potential, O2*^- accumulation and ATP depletion in this tumor cell line, alters mitochondrial function and induces cell death (165). Hinokiflavone (HF) hybrid micelles increase ROS levels, reduce ΔΨm, and induce mitochondria-mediated apoptosis (166). DQA chloride vesicles (HPS-DQAsomes) of DOX increase cytotoxicity to MCF-7/ADR cell lines, can target the delivery of therapeutic agents to mitochondria and induce mitochondria-driven apoptosis (167). DQA- polyethylene glycol (PEG)-modified resveratrol liposomes DLS (Res) selectively accumulate in mitochondria, inducing cytotoxicity of cancer cells by generating ROS and dissipating ΔΨm (168).

DQA is an inhibitor of apoptotic proteins that can directly inhibit the activity of caspases, regulate apoptosis through multiple pathways, and promote the degradation of Bcl-2 as an E3 ligase, thereby exerting anti-tumor effects. DQA hybrid micelles enhance the uptake of paclitaxel by drug-resistant breast cancer cells. Induction of tumor cell apoptosis is related to the activation of pro-apoptotic proteins Bax, Cytc, caspases-3, 9 and the inhibition of Bcl-2 and Mcl-1 (169). Targeted lonidamine liposomes selectively accumulate in mitochondria of drug-resistant A549 lung cancer cells, dissipate ΔΨm, open mitochondrial permeability transition pores, and release Cytc through translocation. A cascade of caspases 9 and 3 reactivity is initiated, which activates the pro-apoptotic Bax protein and inhibits the anti-apoptotic Mcl-1 protein, thereby enhancing cytotoxicity by acting on mitochondrial signaling pathways (170). The development of targeted resveratrol liposomes modified with DQA-PEG (2000)-DSPE on the liposome surface significantly enhance cellular uptake and selectively accumulate in mitochondria. They induce apoptosis in non-resistant and resistant cancer cells by dissipating ΔΨm, releasing Cytc, and increasing the activity of caspases 9 and 3 (171).

DQA can be combined with other drugs to exert a wide range of anti-tumor activities through targeting of mitochondria and is a safe and economical cancer treatment. However, the existing combination of nanomaterials and drugs has not yet achieved breakthrough results, and further research is needed on the role of DQA in targeting mitochondria and its synergistic effect with other drugs.

Due to the high mitochondrial membrane potential of tumor cells, DLC can penetrate the lipid bilayer to accumulate mitochondria, and is often covalently linked to drugs for targeted delivery, mainly including TPP and its derivatives, F16, rhodamine, and DQA. TPP and its derivatives are highly lipid-soluble and positively charged, which can help the accumulation of mitochondrial cells of drugs, enhance the efficacy of chemotherapy, PDT, radiotherapy, PTT, etc., and can also improve the effect of chemopreventive agents. F16 targets the mitochondrial stroma and induces mPTP opening, and its conjugates enhance cytotoxicity and aid tumor imaging, but are toxic to normal cells. Rhodamine can stain mitochondria, and the conjugate can enhance tumor suppression and improve PDT effect. DQA has two positive charge centers that help drugs target mitochondria, disrupt mitochondrial function, induce cell death, and can be combined with other drugs to fight tumors.

MPPs are synthetic mitochondrial localization peptides composed of 4 to 16 amino acids, containing cationic and hydrophobic residues. Similar to DLC, MPPs can finely regulate the localization of mitochondria by changing lipophilicity and charge, and have a significant inhibitory effect on growth of tumor cells in vivo and in vitro ( 172).

MPP-modified DOX copolymers can promote cell apoptosis and inhibit tumor metastasis by destroying mitochondria, inhibit the growth of breast cancer 4T1 cells in vivo, and overcome tumor resistance (173). MPP-modified DOX significantly enhances drug accumulation in mitochondria by 11.6 fold, resulting in a significant increase in ROS generation and a decrease in the production of ATP that can inhibit drug efflux and the growth of drug-resistant cancer cells (174). Nanoparticles (NPs) consisting of membrane-permeable peptide amphiphiles (MMPA) and small interfering RNA (siRNA) can specifically accumulate in mitochondria and inhibit tumor growth by inhibiting ATP production and repolarizing TAMs (175). Nanocomplexes of mitochondrial-penetrating peptides mtCPP1 and PepFect14 affect biological functions in cytoplasm and mitochondria and can effectively target mitochondrial genes (176). Poly(lactide-co-glycolide) (PLGA) conjugates with 6-mer mitochondrial penetrating peptides (MPP) can be used for mitochondrial targets without cytotoxicity. DOX modified with mitochondrial penetrating peptides (MPP) delivers the drug to cancer cell mitochondria, mediating apoptosis and enhancing therapeutic outcomes for multidrug resistant tumors (177). DOX modified with MPP promotes apoptosis and inhibits tumor metastasis by disrupting mitochondria (178).

Compared with DLC, MPP may have more potential as a ligand targeting mitochondria due to its advantages including good biocompatibility and low toxicity. Molecules modified by MPP include RNA, DNA and proteins which can be exploited for human cancer treatment (179).

MPP can regulate lipophilicity and charge to achieve mitochondrial localization, promote apoptosis, inhibit tumor growth and metastasis, and overcome drug resistance. Compared with DLC, MPP has the advantages of good Biocompatibility and low toxicity, and has the potential to be a mitochondrial targeting ligand. Its modified RNA, DNA and protein molecules can be used for human cancer treatment.

Szeto-Schiller (SS) peptides are usually composed of four positively charged amino acids and are a new small peptide targeting strategy. They can specifically target mitochondrial cardiolipin, enhance mitochondrial plasticity and re-establish optimal bioenergetics.

SS peptides can reduce mtROS production, inhibit the opening of mitochondrial permeability transition pores, and have significant effects in preventing oxidative stress or inhibiting mitochondrial ETC-induced cell apoptosis and necrosis (180). The peptide SS-31 specifically localizes in the mitochondrial inner membrane by interacting with cardiolipin and can be used in the treatment of patients with abnormal ΔΨm (181). The SS-31-modified DOX-loaded liposome delivery system LS-DOX can effectively cross the blood brain barrier (BBB) to target gliomas, and mitochondrial targeting of SS-31 can enhance cellular uptake (182). SS-31 selectively binds to cardiolipin through electrostatic and hydrophobic interactions. By interacting with cardiolipin, SS-31 prevents cardiolipin from converting Cytc to peroxidase while protecting its electron-carrying function. Therefore, SS-31 protects the structure of mitochondrial cristae and promotes OXPHOS (183). Similarly, with excellent mitochondrial targeting ability, SS-20 peptide modification is a promising strategy for mitochondria-targeted drug delivery systems (184). Conjugation of α-TOS with SS-20 achieves delivery to mitochondria, increases ROS generation, opens the mitochondrial permeability transition pore, reduces ΔΨm, and promotes cell apoptosis (185). SS peptides are therefore expected to be studied more extensively in the future as strategic molecules for targeting cancer.

Peptide targeting sequences mainly include mitochondrial penetrating peptides (MPPs) and Stuart Schiller (SS) peptides. MPP is composed of 4–16 amino acids, contains cations and hydrophobic residues, can regulate lipophilicity and charge to locate mitochondria, can enhance the accumulation of mitochondria, promote apoptosis of tumor cells, inhibit metastasis and drug resistance, and has good biocompatibility and low toxicity, and the modified molecule can be used in cancer treatment. SS peptides often contain four positively charged amino acids, which can target mitochondrial cardiolipids, reduce mtROS production, inhibit the opening of mitochondrial permeability transition pores, and protect mitochondrial structure and function.

Liposomes are artificial membranes with bilayers, which are generally prepared by high-pressure homogenization, ethanol injection, rotary evaporation and ultrasound, and microfluidics. They can carry hydrophilic and lipophilic drugs, with the former distributed in the core compartment and the latter distributed in the bilayer membrane (187). Liposomes have attracted extensive attention due to their excellent drug delivery capabilities, biocompatibility, biodegradability, and ease of manufacture. Mitochondria-targeted liposomes have advantages in tumor-targeted therapies (188).

Liposomes enhance mitochondrial uptake of DOX and the chemosensitizer lonidamine (LND) by cancer cells, inhibiting tumor cell proliferation and inducing cell apoptosis. Lip-SPG significantly alters mitochondrial functions including reduced production of intracellular ATP, induction of ROS production, and enhancing ΔΨm depolarization (189). Milpoxetine (MPt)-loaded liposomes target mitochondria and trigger mtDNA replication blockage to induce mitophagy (190). DOX-loaded liposomes localize to mitochondria, and generate higher ROS levels (191).

Mitochondria-targeted photosensitizer liposomes exhibit high photodynamic therapy efficiency. Nanophotosensitizers can monitor abnormal mitochondrial morphology during photodynamic therapy under the guidance of fluorescence imaging. Liposome-encapsulated photosensitizers enhance cellular uptake, localize in mitochondria, and enhance anti-angiogenesis in PDT treatment (192). After TPP-modified liposomes are internalized by cells, a large amount of ROS can be generated upon laser irradiation, and a stimulatory effect on STING activation and enhanced infiltration of anti-tumor immune cells is observed, which can be used for PDT treatment (193).

MSNs are an ordered mesoporous material prepared by sol-gel methodologies including microwave-assisted technology, chemical etching technology, and template methods. The material has characteristics of good biocompatibility, high specific surface area, controllable size, and degradability. MSN can improve the targeting of drugs in tumor mitochondria through direct coupling with drugs and enhance the killing effect of tumors. MSNs can efficiently deliver DOX and α-TOS to tumor cell mitochondria, enhancing cancer cell killing effects (194, 195). MSN modified Bcl-2 conversion peptides enter mitochondria and bind to Bcl-2, exposing the BH3 domain and inducing apoptosis of DOX-resistant cells (196). MSN increases the accumulation of folate membrane cell receptors (folate) in tumor cells and targets mitochondria (197). MicroRNA-31 coupled to MSNs loaded with DOX increases active transport and promotes intracellular accumulation of drugs. MicroRNA-31 not only directs targeted mtEF4 to promote cell death, but also has a synergistic effect when used in combination with DOX (198). Phenylboronic acid (PBA)-labeled MSN carriers induce mitochondria-dependent apoptosis in MCF-7 cells through oxidative stress (199).

MSNs can also be combined with mitochondria targeting strategies such as DLC or MPP to further improve the targeting effect of drugs in tumor mitochondria. Pt-loaded MSNs achieve ROS burst in mitochondria, leading to cell apoptosis (200). Mesoporous connections of MSNs can deliver DOX to mitochondria and enhance copper consumption by producing H₂O₂ (201). After blocking surface pores through disulfide bonds, MSNs can target cancer cells with DOX, penetrate the cell membrane and quickly release anticancer drugs and mitochondria-targeted peptides, and induce significant synergistic anticancer effects (202).

MSNs can target the delivery of photosensitive and thermosensitive drugs to mitochondria, increase ROS, and enhance the efficacy and tumor imaging of new treatments such as tumor PDT. α-Tocopherol succinate and indocyanine green (IDG) MSNs reduce innate oxygen consumption by blocking the mitochondrial respiratory chain, leading to endogenous mitochondrial ROS burst and imaging-guided PDT (203). IR780-loaded MSNs nanoparticles can accumulate in tumors, destroy mitochondria and inhibit cellular respiration by decomposing H₂O₂, resulting in sustained reduction of hypoxia in tumor tissues, thereby enhancing the therapeutic effect of PDT (204). Redox-responsive drugs delivered by MSNs target mitochondria in living cells and induce apoptosis derived from mitochondrial membrane depolarization (205).

Dendrimers have a hyperbranched structure that can fill hydrophobic drug small molecules into polymer gaps and graft drugs onto polymer chains. Targeted dendrimer curcumin (TDC) colocalizes with mitochondria of cancer cells, inducing potent apoptosis and cell cycle arrest. It reduces ATP and glutathione and increases ROS levels in isolated mitochondria of rat hepatocytes (206). Poly(amidoamine) (PAMAM) is a common dendrimer targeting strategy with the ability to effectively regulate dendrimer targeting mitochondria. Active targeting of dendrimers induces P-glycoprotein (P-gp) overexpression and apoptosis in multidrug-resistant cells (207). TPP conjugated to PAMAM dendrimers, optimizes the density of surface TPP by adjusting the length of TPP-PEG linker, enhancing mitochondrial targeting ability and antitumor bioactivity (208).

Metal-organic frameworks (MOFs) are a class of porous materials formed by the coordination of inorganic metal ions and organic ligands. Compared with other nano-drug carriers, MOFs have the advantages of high porosity, adjustable structure, controllable size, and easy modification. In addition, MOFs exhibit unique advantages: (1) easy preparation and good stability, assembled from non-toxic metals (Fe, Zn, Ca, Mg, etc.) and low-toxic carboxylic acids or phosphonic acids; (2) biodegradable, especially when exposed to water; (3) an internal microenvironment suitable for the delivery of drug molecules with different activities (209). These properties make MOFs ideal materials for biomedical applications, such as the delivery of drugs or imaging agents. Surface modification of materials further enriches the approach of using MOF as a drug delivery platform to treat diseases, such as PTT combined with chemotherapy, ultrasound therapy combined with chemotherapy, and other combination treatment strategies (210).

MOFs encapsulated in macrophage-cancer hybrid membranes (MCHMs) enhance the cancer homing targeting ability of nanoparticles (NPs), damage ΔΨm, and lead to cancer cell apoptosis (211). The ZCProP nanoplatform triggers cell ferroptosis through cuproposis and inhibits the anti-ferroptosis protein glutathione peroxidase 4 (212). MOFs can deliver oxymatrine (Om) and astragaloside IV (As) into the HCC microenvironment, and increase the oxygen consumption rate and proton efflux rate of tumor-infiltrating lymphocytes (TILs) by regulating the mitochondrial function of CAFs and TILs (213). The mitochondrial targeting drug of gallium-based organic frameworks produces ROS and releases L-Arg, which reacts with ROS to generate NO, downregulates HIF-1α expression to improve tumor hypoxia, and enhances immune responses by increasing calreticulin (CRT), high-mobility group box 1 (HMGB1), and T cell proliferation (214). The metal-organic framework GCZMT targets mitochondria and releases NO under MW irradiation, interfering with the cell's energy supply and inhibiting tumor cell growth. Upregulation of heat shock protein (HSP)70 expression can facilitate CD4⁺ and CD8⁺ T cell activation to promote anti-tumor immunity (215).

Lipid-polymer nanoparticles (LPNPs) are composed of a polymer core and a biocompatible lipid shell. LPNPs have a longer half-life compared with conventional liposomes. After lipid-polymer hybrid nanoparticles are taken up by cancer cells, the surface charge of LPNPs is restored due to the separation of PEG under intracellular reducing conditions, resulting in rapid and precise targeting of mitochondria (216). Nanomicelles are nanocarriers with a core-shell structure formed by self-assembly of amphiphilic copolymers in aqueous media, which have the advantages of simple preparation and small particle size. Micelles not only significantly improve drug solubility, but also increase drug accumulation in tumor sites through enhanced permeability and retention (EPR), and they improve the effect of chemotherapy and can partially reverse tumor drug resistance (217). Mitochondria-targeted polymeric micelles (OPDEA-PDCA) target mitochondria and induce mitochondrial oxidative stress via inhibition of pyruvate dehydrogenase kinase 1(PDHK1), leading to immunogenic pyroptosis in osteosarcoma cell lines (218). Charge-reversible nanocopolymers are copolymers that are modified with anions to shield the positive charge of the nanosystem, avoiding nonspecific binding with other proteins and subsequent elimination (219). In normal physiological environments (pH 7.4), the copolymers are neutral or negatively charged, which can reduce the uptake of nanomedicines by macrophages in the reticuloendothelial system while ensuring their stability in the blood circulation. However, when they reach the tumor site, their potential undergoes a charge reversal, and their affinity with the tumor cell surface is significantly enhanced, leading to the accumulation of nanoparticles in tumor cells (220). Tumor acidity triggers charge reversal and mitochondrial targeting activation of TPP-containing nanomedicines, which is a simple and effective strategy for delivering DOX to cancer cell mitochondria and overcoming DOX resistance in MCF-7/ADR breast tumor cells in vitro and in vivo ( 120).

In summary, mitochondrial-targeted drug delivery systems can deliver drugs to tumor sites and enhance the effectiveness of cancer treatment through various mechanisms, including disrupting mitochondrial energy metabolism, regulating ROS levels, modulating cell death-related proteins, damaging mitochondrial DNA, and regulating mitophagy. Liposomes can carry both hydrophilic and lipophilic drugs, enhancing drug uptake and inducing mitophagy, while their photosensitizer liposomes can also assist in photodynamic therapy (PDT). MSN boasts good biocompatibility and high surface area, allowing for direct drug conjugation or the integration of other targeted strategies, thus enhancing drug targeting and cytotoxic effects, and can also deliver photothermal sensitizers. Dendritic polymers are suitable for carrying hydrophobic drugs. MOFs are porous, structurally tunable, and biodegradable, making them apt for delivering drugs or imaging agents, and they can also be employed in combination therapies. Additionally, LPNP has a long half-life, nanomicelles can reverse drug resistance, and charge-reversible nanocopolymers offer precise targeting. These systems can improve cancer treatment outcomes through various approaches, making them a promising therapeutic strategy, reducing the high risks associated with traditional treatments such as surgery.

Glucose is one of the main players in tumor progression and a promoter of tumor invasion and metastasis. Glycolysis rapidly produces ATP, which provides sufficient energy for tumor cell proliferation. Under aerobic or hypoxic conditions, tumor cells have enhanced glycolytic activity and reduced mitochondrial respiration. Therefore, reversing the high glycolytic state of tumor cells to induce cell death is a possible approach to cancer treatment. Key glycolytic genes include glucose transporter 1 (GLUT1), hexokinase 2 (HK2), pyruvate kinase-M2 splicing isoform (PKM2) and lactate dehydrogenase (LDH-A) ( Figure 9 ).

Glucose uptake provides a key metabolic control point by targeting the Glut family of glucose transporters (120). Interference with HK1/2 and GLUT1 function in hematopoietic cells inhibits glycolysis, reduces ATP production, enhances the apoptotic effect, and preserves normal CD34⁺ bone marrow progenitors (222). Glycosylated poly(amidoamine)/celastrol (PAMAM/Cel) complexes are characterized by high photothermal conversion efficiency, hypoxia-sensitive PEG outer layer detachment, and alkali-sensitive drug release. The complexes show specific cellular uptake and accumulation in tumor cell mitochondria in hypoxic environments that overexpress GLUT1 (223). Met administration elevates mtROS and cell surface Glut-1, leading to IFN-γ production in CD8⁺ TILs in tumor cells (224).

Hexokinase-2 (HK2) is located at mitochondrial-endoplasmic reticulum (ER) contact sites called mitochondrial associated membranes (MAMs). HK2 expression is significantly elevated in most HCC cell lines and tumor tissues compared to normal cell lines and tissues (225). Since HK2 is the major rate-limiting enzyme in the aerobic glycolysis pathway, inhibiting HK2 is an effective strategy to block glycolysis. HK2 degraders cause mitochondrial damage and then induce GSDME-dependent pyroptosis and ICD, resulting in increased anti-tumor immunity (226). HK2-targeting peptides trigger mitochondrial Ca2⁺ overload, leading to Ca2⁺-dependent calpain activation, mitochondrial depolarization and cell death, and can also cause massive death of chronic lymphocytic leukemia B cells (227). Dihydrotanshinone I (DHTS) reverses metabolic reprogramming in colon cancer by inhibiting hexokinase activity and free fatty acids (FFA) via the PTEN/AKT/HIF1α-mediated signaling pathway (228).

Pyruvate kinase (PK) is a key enzyme that regulates the last step of glycolysis, catalyzing phosphoenolpyruvate (PEP) and ADP to generate pyruvate and ATP. Pyruvate kinase includes erythrocyte/liver pyruvate kinase (PKLR) and muscle pyruvate kinase (PKM), and PKM has two isoforms, PKM1 and PKM2. Mitochondra-targeted dichloroacetate (Mito-DCA) enhances delivery of DCA to mitochondria, leading to a shift from glycolysis to glucose oxidation, cell death through apoptosis, and increses DCs secretion of IL-12 (229). ATO treatment inhibits oxygen consumption and metabolically induces aerobic glycolysis and oxidative stress, and also induces apoptosis in CD44⁺/CD24^low/- and ALDH⁺ cancer stem cells (230).

Another way to target glycolysis is to reduce levels of ATP. The mechanism of Met's anti-tumor effect may be that it directly reduces the production of ATP due to its function as an inhibitor of mitochondrial electron transfer chain complex I and an activator of AMPK (132). Metformin (Met) reduces tumor-infiltrating Treg (Ti-Treg), especially in the terminally differentiated CD103⁺KLRG1⁺ population, and also reduces expression of immune suppressive effector molecules such as CTLA4 and IL-10. Met inhibits the differentiation of naive CD4⁺ T cells into induced Treg (iTreg) by reducing forkhead box P3 (Foxp3) protein expression, which is determined by the increase of phosphorylated S6 (pS6), a downstream molecule of mTORC1. Rapamycin and compound C (AMPK inhibitor) restores the generation of iTreg, further indicating that mTORC1 and AMPK are involved (231).

A variety of strategies targeting key glycolytic genes (such as GLUT1, HK2, PK, etc.) can induce tumor cell death or enhance anti-tumor immunity by reducing ATP production and triggering mitochondrial damage. By inhibiting the mitochondrial electron transfer chain complex and activating AMPK, ATP production is directly reduced, which affects tumor immune cells and plays an anti-tumor role.

The "Warburg effect" is necessary for tumor cells to undergo malignant transformation and escape immune attack. Reversing the Warburg effect by using drugs to intervene in the metabolic behavior of tumor cells so that their main energy production pathway changes from glycolysis to OXPHOS, may be a promising therapeutic strategy for treating such tumors. Upregulating the expression of genes related to mitochondrial biogenesis, thereby increasing mitochondrial activity and reprogramming cellular energy metabolism, produces an "anti-Warburg effect", and is known to regulate the differentiation of glioblastoma cells towards normal cell phenotype (118). Mitochondria-targeted OXPHOS inhibitors reduce hypoxia in tumor cells in a dose-dependent manner, potentially sensitizing hypoxic tumor cells to radiotherapy (232). Mitoquinone (Mito-Q) adsorbed to the inner mitochondrial membrane blocks ATP synthase, dissipates ΔΨm in HepG2 cells, and induces uncoupling of autophagy from OXPHOS in cancer cells (233). Functionalized Ir(III) complexes selectively localize in mitochondria and generate singlet oxygen and superoxide anion radicals upon two-photon irradiation, leading to the disruption of the mitochondrial respiratory chain, thereby interfering with mitochondrial OXPHOS and glycolytic metabolism, triggering cell death by combining ICD and ferritin autophagy (234).

Metabolic reprogramming of T cells in the TME impairs effector T cell responses against tumor cells. Mitochondrial-targeted drugs modulate the immune microenvironment via OXPHOS. Mitochondria-targeted hydroxyurea (Mito-HU) reduces mitochondrial complex I and complex III-induced oxygen consumption, effectively inhibits monocytic MDSCs and suppressive neutrophils, and stimulates T cell responses (235). Mito-CI reduces mitochondrial complex I oxygen consumption and Akt-FOXO signaling, blocks cell cycle progression, melanoma cell proliferation, and inhibits tumor progression. Anti-proliferative properties of mitochondria-targeted complex I inhibitors (Mito-CI) inhibit differentiation, viability, and suppressive function of bone marrow-derived MDSCs and increase activation of T cells (236). Mitochondria-targeted ATO inhibits the expression of mitochondrial complex components, OXPHOS, and glycolysis genes in granulocytic-MDSCs and Treg (237). The resulting reduction in intra-tumoral granulocytic-MDSCs (G-MDSCs) and Treg could contribute to the observed increase in tumor-infiltrating CD4⁺ T cells. In contrast, Mito-ATO significantly inhibits OXPHOS and glycolysis in G-MDSCs. These observations support the predicted higher OXPHOS and glycolysis in effector memory CD8⁺ T cells and lower OXPHOS and glycolysis in G-MDSCs after Mito-ATO treatment (238). AMPK is considered a major intracellular energy sensor and key regulator of mitochondrial biogenesis that can control metabolic reprogramming in immune cells and enhance anti-tumor immunity. The AMPK activator Met is considered a candidate drug to improve cancer treatment efficacy by interfering with tumor metabolic reprogramming (239). Met increases the numbers of CD8⁺ T cells and protects them from apoptosis and exhaustion (240).

Mitochondria-targeted OXPHOS inhibitors can induce tumor cell death or enhance their sensitivity to radiotherapy by affecting mitochondrial function and interfering with energy metabolism. Mitochondria-targeted drugs can also improve TME, stimulate T cell responses, protect CD8⁺ T cells and enhance anti-tumor immunity by regulating OXPHOS.