Mitoxantrone-engineered multifunctional nanoplatforms for precision imaging-guided synergistic cancer therapy: recent advances and future perspectives

Abstract

Mitoxantrone (MTO)-based nanoplatforms represent a significant advancement in oncological therapeutics by synergistically combining precision drug delivery with multimodal treatment strategies. Recent progress in precision pharmacokinetics, modular multifunctionality, and intelligent stimuli-responsive systems has established MTO as a cornerstone of next-generation combinatorial cancer therapy. The implementation of sophisticated nanocarrier designs has enabled high-efficiency drug encapsulation, spatiotemporally controlled release, and integration with complementary treatment modalities. These developments collectively address three major challenges in cancer therapy, including systemic toxicity, tumor microenvironment adaptation, and multidrug resistance mechanisms. This comprehensive review systematically explores the molecular pharmacodynamics underpinning MTO's multifaceted antitumor activity, the structural classification and functional engineering of advanced nanocarriers for MTO delivery, and the emergent therapeutic synergies and translational potential of MTO within nano-enabled combination therapy frameworks. Furthermore, the current technological limitations and clinical translation barriers are critically evaluated, proposing a roadmap of innovative solutions to inform future research endeavors. By converging multivalent nanocarrier systems with precision oncology principles, this work establishes a transformative framework that transcends conventional chemotherapy modalities and catalyzes the development of patient-specific cancer theranostics.

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Juan Yu

Juan Yu is a graduate student at Shanxi Medical University under the supervision of Professor Yanan Li. She received her Bachelor's degree from Henan Medical University in 2023. As part of her undergraduate training, she completed a one-year clinical practicum at Fuwai Huazhong Cardiovascular Hospital. Her current research centers on mitochondria-targeting nanodrugs for synergistic antitumor photochemotherapy.

Zijun Qiu

Zijun Qiu is a graduate student at Shanxi Medical University under the supervision of Professor Yanan Li. She holds dual bachelor's degrees awarded jointly by Tianjin University of Traditional Chinese Medicine and the University of Nottingham (UK). Her research primarily focuses on tumor pharmacology and near-infrared fluorescence imaging-guided multimodal therapy for cancer.

Susu Yan

Susu Yan is a graduate student at Shanxi Medical University under the supervision of Professor Yanan Li. She earned her Bachelor's degree from Henan Medical University in 2022 and completed a one-year clinical internship at Qinggang County People's Hospital in Heilongjiang Province during her undergraduate studies. Her research interests center on the construction of nanodrugs and their application in tumor-targeted therapy.

Aishan Lin

Aishan Lin is a Master of Pharmacy candidate at Shanxi Medical University under the supervision of Professor Yanan Li. She obtained her Bachelor's degree from Zhaoqing University in 2017. Her research centers on the fabrication of nanomedicine and its application in targeted cancer therapy.

Yanan Li

Yanan Li is an Associate Professor at Shanxi Medical University, specializing in molecular imaging and targeted therapy for cancer. She obtained her PhD in 2013 from the Technical Institute of Physics and Chemistry, Chinese Academy of Sciences. Following her doctoral studies, she completed postdoctoral training successively at Shantou University and Tsinghua University. Since joining Shanxi Medical University in 2018, she has been actively engaged in research focused on the development of novel strategies for cancer diagnosis and treatment.

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1. Introduction

Cancer persists as a formidable global health challenge, maintaining persistently high morbidity and mortality rates despite decades of therapeutic innovation.¹ Within the pharmacological armamentarium against malignancies, anthraquinone-based chemotherapeutics have emerged as cornerstone agents, among which mitoxantrone (MTO) distinguishes itself as a uniquely versatile candidate. As a synthetic amino dihydroxy-anthraquinone derivative, MTO has demonstrated robust clinical efficacy across a spectrum of hematological and solid tumors, including acute myeloid leukemia, aggressive lymphomas, and advanced-stage hormone-refractory prostate and breast carcinomas.² Its therapeutic superiority stems from a multifaceted pharmacodynamic profile: cytotoxicity mediated by topoisomerase II inhibition and DNA intercalation,³ dose-dependent immunomodulation through macrophage polarization and T cell regulation,⁴ long-term suppression of metastatic recurrence via microenvironmental remodeling,⁵ a comparatively mitigated cardiotoxicity burden relative to classical anthracyclines,⁶ and recently discovered near-infrared (NIR) photoresponsive properties enabling theranostic applications.⁷ These attributes collectively underscore MTO's enduring significance in translational oncology.

However, the clinical implementation of MTO as a monotherapeutic regimen faces substantial pharmacological hurdles, including intrinsic or acquired resistance mechanisms in refractory malignancies,⁸ non-negligible cumulative cardiotoxicity despite improved safety profiles,⁹ and suboptimal pharmacokinetic characterized by rapid plasma clearance and uneven biodistribution.¹⁰ The evolution of nanomedicine has ushered in breakthrough solutions to these limitations through rationally engineered platforms that enhance drug solubility,¹¹ enable active tumor targeting beyond passive enhanced permeability and retention (EPR)-dependent accumulation,¹² and permit spatiotemporal release control via endogenous (pH, redox, enzymes) or exogenous (light, magnetic fields) triggers.^13,14 Such nano-enabled strategies have dramatically expanded the therapeutic window of MTO while minimizing off-target effects.

A particularly groundbreaking dimension of MTO's pharmacology lies in its intrinsic photophysicochemical properties, which transcend conventional cytotoxic mechanisms.¹⁵ The molecule demonstrates exceptional photothermal conversion efficiency (η > 40%) and robust reactive oxygen species (ROS) generation capacity, enabling concurrent photothermal therapy (PTT) and photodynamic therapy (PDT) within a single molecular framework.¹⁶ This dual phototherapeutic capacity has inspired a new generation of combinatorial nanoformulations that integrate MTO's chemotherapeutic, photothermal, and photodynamic actions into unified treatment modalities, achieving synergistic antineoplastic effects through precisely orchestrated multimodal synergy.

Given MTO's expanding role in next-generation cancer therapeutics, this review systematically examine the structure–activity relationships governing MTO's antitumor mechanisms, the classification and functional engineering of contemporary MTO nanocarriers, and recent breakthroughs in MTO-based combination therapies, including their integration with radiotherapy, PTT/PDT, molecular targeted therapy, immunotherapy, and gene therapy, with particular emphasis on mechanistic underpinnings and translational potential. Furthermore, the prevailing challenges and translational barriers facing MTO-based nanotherapeutics in both preclinical optimization and clinical implementation were critically analyzed. By synthesizing these multidisciplinary insights, this work aims to catalyze the development of precision oncology platforms that transcend traditional therapeutic boundaries (Fig. 1).

Fig. 1 Schematic illustration of multifunctional MTO-based nanosystems engineered for combinatorial cancer therapy.

2. Pharmacological mechanism of MTO

2.1. Molecular structure and structure–activity relationship

MTO represents a second-generation synthetic anthracenedione derivative with the systematic IUPAC nomenclature of 1,4-dihydroxy-5,8-bis[2-(2-hydroxyethylamino)ethylamino]anthracene-9,10-dione (molecular formula: C₂₂H₂₈N₄O₆, molecular weight: 444.49 g mol⁻¹). Developed through rational drug design in the 1980s as a structural analog of doxorubicin, MTO exhibits significant molecular modifications from classical anthracyclines (Fig. 2). The compound features a planar anthraquinone core that facilitates DNA intercalation, with two strategically positioned aminoalkyl side chains at the 5- and 8-positions replacing the natural glycoside moieties characteristic of first-generation anthracyclines.

Fig. 2 Molecular structure and key functional moieties of MTO.

These deliberate structural modifications confer three distinct pharmacological advantages: enhanced DNA binding affinity through optimized π–π stacking interactions between the planar chromophore and DNA base pairs, significantly reduced cardiotoxicity due to the elimination of quinone-mediated free radical generation, and improved cellular uptake through the protonation of terminal nitrogen atoms under physiological conditions. X-ray crystallographic studies have demonstrated that the dihydroxyanthraquinone system maintains an optimal intercalation distance of 6.5–7.0 Å between adjacent DNA base pairs, while the basic side chains facilitate electrostatic interactions with the phosphate backbone.^2,17 The presence of hydroxyl groups at positions 1 and 4 further contributes to hydrogen bonding with nucleobases, collectively resulting in a DNA-binding constant (K_b) of approximately 10⁶ M⁻¹, comparable to doxorubicin but with superior selectivity for CG (cytosine-guanine)-rich sequences.

2.2. Pharmacological mechanism and molecular targets

MTO exerted its potent antitumor activity through a multimodal mechanism of action, targeting critical cellular processes essential for cancer cell survival and proliferation. The drug's pharmacological effects can be categorized into five principal mechanisms, each contributing to its cytotoxic and antiproliferative efficacy:

(1) Topoisomerase II inhibition. MTO functioned as a potent topoisomerase II poison, stabilizing the topoisomerase II-DNA cleavage complex and preventing religation of double-stranded DNA breaks. This inhibition disrupted DNA replication and transcription, leading to replication fork collapse and lethal DNA damage. Unlike catalytic topoisomerase II inhibitors, MTO trapped the enzyme in a covalent intermediate state, resulting in persistent DNA strand breaks and genomic instability.^2,18

(2) DNA intercalation and structural disruption. The planar anthracenedione core of MTO intercalated preferentially at CG-rich regions of the DNA minor groove, inducing significant helical distortion. This intercalation not only impeded DNA polymerase and RNA polymerase processivity but also promoted DNA cross-linking and double-strand breaks. Spectroscopic and biochemical studies confirmed that MTO-DNA binding altered chromatin topology, suppressing transcriptional activity and inducing apoptosis through p53-dependent and -independent pathways.^18,19

(3) ROS generation. MTO underwent redox cycling in the presence of cellular oxidoreductases, catalyzing single-electron transfer to molecular oxygen and generating cytotoxic ROS. These oxidative radicals, including superoxide and hydroxyl radicals (˙OH), inflicted extensive damage to nucleic acids, lipids, and proteins. The resultant oxidative stress overwhelmed cellular antioxidant defenses, triggering ferroptosis and necroptosis in addition to classical apoptosis.²⁰

(4) Immunogenic cell death (ICD) induction. MTO elicited ICD through the coordinated emission of damage-associated molecular patterns (DAMPs). It induced the surface translocation of calreticulin (CRT) which served as an “eat-me” signal to facilitate the phagocytosis of tumor antigens by dendritic cells (DCs). This was accompanied by the early release of extracellular adenosine triphosphate (ATP), which recruited additional DCs to the tumor microenvironment (TME). Subsequently, MTO promoted the release of high-mobility group box 1 (HMGB1) which delivered a critical activation signal to DCs. These three DAMPs acted synergistically to drive DC maturation and robustly activate tumor-specific cytotoxic T lymphocytes, resulting in targeted tumor elimination and the establishment of long-term antitumor immunity.^21,22

(5) Mitochondrial dysfunction and metabolic disruption. MTO accumulated in mitochondria due to its cationic properties, where it disrupted electron transport chain complexes, depleted ATP, and collapsed mitochondrial membrane potential. Additionally, MTO chelated iron, exacerbating oxidative stress and impairing iron–sulfur cluster biogenesis, a critical process for DNA repair and metabolic homeostasis. These effects synergized to induce intrinsic apoptosis via cytochrome c release and caspase-9 activation.^23,24

The convergence of these mechanisms resulted in a highly effective antitumor response. The multimodal action of MTO not only circumvented conventional drug resistance but also provided a compelling rationale for its inclusion in combination therapy regimens.

3. Design of multifunctional nanocarriers for optimized MTO delivery

Recent advancements in nanomedicine have fostered the development of sophisticated delivery systems for MTO, leveraging diverse nanocarrier architectures to enhance therapeutic precision and overcome biological barriers. Compared to free MTO, nanocarriers can reverse multidrug resistance (MDR) through multiple mechanisms. These include bypassing P-glycoprotein (P-gp)-mediated drug efflux by utilizing endocytic pathways for cellular uptake; inducing direct disruption of the mitochondrial membrane potential via functionalized modifications on the carrier surface; and co-delivering P-gp inhibitors such as tamoxifen, curcumin, or small interfering RNA (siRNA).²⁵ These carrier materials included but were not limited to lipid-based systems, polymeric nanoparticles (NPs), and inorganic nanostructures.²⁶ Each platform exhibited unique physicochemical characteristics that critically influence biodistribution, tumor targeting, and chemotherapeutic outcomes.²⁷ Furthermore, state-of-the-art MTO-based nanocomposites have been extensively investigated for their potential in combinatorial oncotherapy and theranostic applications.²⁸ The recent progress will be critically discussed in this section, bridging fundamental research with translational challenges in clinical cancer management.

3.1. Liposomes

Liposomes represent a class of biomimetic nanovesicles spontaneously self-assembled from cholesterol and phospholipids in aqueous media. This distinctive architecture features a hydrophilic exterior shell, a hydrophobic bilayer membrane, and an aqueous inner core. The structural duality enables efficient encapsulation of hydrophobic chemotherapeutic agents like MTO within the lipid bilayer, while hydrophilic compounds can be loaded in the aqueous interior.²⁹ The liposomal platform offers multiple therapeutic advantages for MTO delivery, including enhanced pharmacokinetics, prolonged systemic circulation,³⁰ precision targeting, reduced renal clearance and reticuloendothelial system uptake,^31,32 improved safety,^33,34 and combinatorial potential.³⁵ A novel MTO-loaded liposomal NPs (LbL-LNPs) system was developed by Ramasamy et al.³⁶ using a layer-by-layer (LbL) self-assembly technique. The nanoparticle core was functionalized with a polyelectrolyte shell, which mitigated rapid clearance and thereby promoted tumor-specific accumulation through the EPR effect. These advanced systems showed particular promise for treating malignancies and have positioned liposomal MTO as a cornerstone of modern nanochemotherapy.

3.2. Polymeric nanoparticles

Biocompatible amphiphilic polymers represented a versatile platform for drug delivery, capable of self-assembling into diverse nanostructures in physiological environments. Polymeric NPs were typically engineered through well-established techniques including solvent evaporation, nanoprecipitation, and salting-out methods.³⁷ These methods enabled precise control over particle characteristics, with systems broadly categorized by material origin (natural polymers, synthetic polymers) and morphological diversity (lamellar structures, rod-shaped and worm-like micelles, core–shell nanocapsules).³⁸ The drug loading mechanisms in polymeric matrices primarily involved hydrophobic core encapsulation, surface adsorption, and covalent conjugation.³⁹ And Compared to liposomal systems, polymeric NPs demonstrated enhanced stability,⁴⁰ precision drug release, surface engineering capability,^41,42 and MDR overcoming.⁴³ Typically, a hybrid network nanogel was designed to achieve controlled drug release characterized by precise and sustained release at the tumor site with minimal leakage in normal tissues. This targeted profile resulted from the combined effects of slow digestion by hyaluronidase, moderate gel shrinkage at 37 °C, and the diffusional resistance imposed by the gel network pores.⁴⁴ In addition, Liu et al.⁴⁵ developed vitamin E succinate-grafted F68 polymeric micelles that achieved 22.4% higher tumor accumulation and 60.8% inhibition of MDA-MB-231 breast cancer growth. Wang et al.⁴⁶ engineered pH-responsive polymer vesicles co-encapsulating MTO hydrochloride and chloroquine, which effectively inhibited P-pg-mediated drug efflux and successfully reversed MDR in K562/ADR cells.

3.3. Inorganic nanomaterials

Inorganic nanomaterials have garnered significant attention as advanced drug delivery platforms due to their unique physicochemical properties and multifunctional capabilities. These materials were broadly categorized into two categories: metallic NPs (Au, Ag, Fe, Ti, Zn) and non-metallic nanomaterials (carbon-based nanostructures, silicon dioxide).⁴⁷ The exceptional attributes, such as high drug loading capacity, remarkable stability against enzymatic degradation, and versatile surface chemistry for precise functionalization, rendered them particularly advantageous for therapeutic applications.^48–50 A representative study by Heleg-Shabtai et al.⁵¹ demonstrated the therapeutic potential of these systems, where boric acid-functionalized mesoporous silica NPs were co-loaded with MTO and gossypol. This platform exhibited pH-responsive drug release and selective cytotoxicity against triple-negative breast cancer cells, underscoring the therapeutic promise of inorganic nanocarriers. Moreover, metallic NPs can be meticulously engineered with a variety of targeting ligands, such as growth factors, antibodies, bioactive peptides, and folic acid (FA) derivatives. These modifications not only enhanced tumor-specific delivery but also facilitated synergistic theranostic applications by integrating diagnostic imaging with targeted therapy.⁵²

3.4. Intelligent responsive MTO nanoplatforms

Stimuli-responsive nanoplatforms are a class of “smart” nanomaterials or nanocarrier systems. These platforms maintain stability under physiological conditions, enabling the safe transport of therapeutic agents to tumor tissues, while releasing their payload in a controlled manner upon exposure to specific internal or external stimuli.⁵³ Commonly exploited endogenous stimuli in the TME include weakly acidic pH, elevated ROS levels, and overexpression of specific enzymes.^54,55

In a recent study, leveraging the overproduction of ROS in the TME and the self-immolative behavior of prodrugs, Zhang et al.⁵⁶ co-loaded a novel ROS-activated self-immolative prodrug (CASDB) and MTO into poly(lactic-co-glycolic acid) (PLGA) NPs, designated as CMPs. Upon entering the TME, ROS triggered the self-immolation of CASDB, resulting in the simultaneous release of cinnamaldehyde (CA) and quinone methide (QM). CA localized to mitochondria, inducing mitochondrial dysfunction and generating substantial amounts of ROS, thereby amplifying intracellular oxidative stress. Concurrently, QM depleted glutathione (GSH) in tumor cells and weakened their antioxidant defense capacity. The synergistic action of CA and QM significantly sensitized tumor cells to the co-delivered chemotherapeutic agent MTO. As a result, this strategy markedly enhanced the chemotherapeutic efficacy of MTO in a 4T1 tumor-bearing mouse model.

In addition, pH-sensitive hydrazone-bonded prodrug NPs and matrix metalloproteinase (MMP)-triggered delivery systems, although less explored in the context of MTO nanoplatforms, hold considerable potential. The hydrazone bond undergoes specific cleavage in the acidic TME, enabling precise MTO release and minimizing off-target toxicity. MMP-activated systems utilize tumor-overexpressed MMPs to trigger drug release, thereby effectively addressing tumor heterogeneity. Both strategies overcome the limitations of conventional MTO therapy such as low bioavailability and high systemic toxicity, and can be readily integrated with targeting ligands or combination therapies. Although most of these systems remain at the preclinical stage, they represent promising avenues for precision oncology as technology continues to advance.^53,57

3.5. Emerging engineered MTO nanoplatforms

Metal–organic frameworks (MOFs), a class of porous materials formed through coordination-driven self-assembly of metal clusters and organic linkers, have attracted considerable attention. This structure represents a biomimetic advancement beyond conventional metal oxides.⁵⁸ Capitalizing on the high drug loading capacity and inherent bone-targeting potential of MOFs, Huang et al.⁵⁹ developed a nano-regulator designated MTO@PA/Fe³⁺ MOF. This system was constructed by encapsulating MTO within a nanoscale MOF assembled from phytic acid (PA) and Fe³⁺ ions, with the aim of improving immunotherapy for prostate cancer bone metastases. Upon intravenous injection, PA-mediated targeting promoted nanoparticle accumulation at bone metastatic sites. The released MTO exerted a dual antitumor effect. First, it directly induced ICD in prostate cancer cells, triggering the exposure and release of DAMPs such as CRT, HMGB1, and ATP, thereby activating antitumor immunity. Second, it directly bound to transforming growth factor-β (TGF-β) receptors on immune cells, inhibiting the TGF-β signaling pathway and restoring immune cell sensitivity against tumors. When combined with an anti-Cytotoxic T-Lymphocyte-Associated protein 4 (αCTLA-4), this nanoparticle formulation significantly suppressed tumor volume over a 12-day period compared with αCTLA-4 monotherapy, while also mitigating bone-related adverse events. Thus, the emergence and successful application of MOFs have facilitated a transition in MTO carrier design from conventional porous materials toward more sophisticated functional platforms.

In addition, leveraging the innate advantages of extracellular vesicles (EVs), such as high biocompatibility, low immunogenicity, and inherent tissue/cell targeting capabilities, several studies have developed EV-based nanoplatforms for MTO delivery.⁶⁰ In a recent investigation, Brancolini et al.⁶¹ constructed EV-MTO composite NPs by loading MTO into EVs derived from human embryonic kidney cells using a passive co-incubation method. Although the final results indicated that the cytotoxicity of the EV-MTO formulation was not significantly superior to that of free MTO, the system successfully maintained drug activity throughout the delivery process. This finding critically validated the feasibility of using EVs as effective carriers for MTO and other chemotherapeutic agents at the cellular level, thereby laying a crucial foundation for subsequent strategies aimed at enhancing therapeutic efficacy in vivo.

Overall, the evolution of MTO-based NPs has progressed through four distinct stages (Fig. 3). Initially constrained by dose-dependent toxicity as a free cytotoxic agent, MTO was subsequently integrated into conventional nanocarriers. This approach mitigated cardiotoxicity but suffered from low drug loading capacity. A major advance occurred with the introduction of self-assembling MTO prodrug NPs, which achieved high drug loading and stimuli-responsive release. The most recent advancements, involving functionalization and combination strategies, have further enhanced targeting precision and therapeutic efficacy. For the multifunctional optimization of MTO nanoplatforms, tumor-specific receptors can be engaged using targeted ligands such as FA for folate receptor (FR)-positive cells, peptides containing the Arg-Gly-Asp (RGD)-containing peptides for integrin αvβ3 overexpression, transferrin (Tf) for receptors on proliferating cells, and hyaluronic acid (HA) for CD44-mediated targeting and matrix degradation.⁶²

Fig. 3 Development stages of multifunctional MTO-based nanocarriers.

Accordingly, the delivery efficiency of MTO-based nanocarriers has been progressively enhanced throughout their development. Given that nanodrug delivery constitutes a multistep and cascade-like process influenced by nanoparticle physicochemical properties, biological barriers, targeting mechanisms, and drug release kinetics, an efficient nanodelivery platform should integrate multiple advantageous features to maximize overall delivery efficiency. Therefore, this evolution not only reflects a shift in MTO-based drug delivery from passive loading to active targeting, but also underscores the considerable promise and future potential of MTO nanoplatforms in cancer therapy.²⁵

4. MTO-based multifunctional nanoplatforms for combinatorial cancer therapy

4.1. Chemotherapy combined with radiotherapy

Radiotherapy stands as a cornerstone in clinical oncology, frequently employed in combination with chemotherapy to achieve enhanced therapeutic outcomes in tumor management. This modality facilitates localized tumor eradication through precisely targeted high-energy radiation, which selectively eradicates malignant cells while minimizing damage to adjacent healthy tissues.^63,64 However, the hypoxic conditions prevalent within the TME significantly attenuate the radiosensitivity of cancer cells, thereby diminishing the therapeutic efficacy of radiotherapy.^65,66 Nanotechnology-augmented chemoradiotherapy has emerged as a transformative strategy to circumvent this limitation, capitalizing on the unique advantages of nanocarriers, including superior targeting precision, EPR effect, prolonged systemic circulation, and accurate lesion localization. These attributes synergistically potentiate therapeutic efficacy, culminating in markedly improved antitumor outcomes. Among the diverse nanomaterials investigated for radiotherapy, iron-, titanium-, and gold-based NPs have garnered particular attention due to their favorable physicochemical and radiosensitizing properties. A foundational study by Bakhshizadeh et al.⁶⁷ exemplified this approach, wherein polymer-modified TiO₂ nanocores were engineered for the co-delivery of MTO. Under 6 MV X-ray irradiation, the TiO₂ NPs facilitated the generation of a substantial burst of ROS via MTO activation. This dual mechanism, combining chemotherapy-induced cytotoxicity with radiation-triggered ROS production, resulted in a pronounced synergistic enhancement of tumor cell death, underscoring the potential of nanomaterial-mediated chemoradiotherapy to overcome the limitations of conventional treatment modalities.

4.2. Chemotherapy combined with PTT

The integration of chemotherapy with PTT has emerged as a transformative strategy in oncological treatment, offering synergistic therapeutic advantages through enhanced tumor targeting, improved drug accumulation, and reduced systemic toxicity.^68,69 This combinatorial approach capitalized on the photothermal-EPR effect, wherein localized hyperthermia promoted vascular permeability and subsequent drug extravasation into tumor tissues. PTT induced selective tumor cell destruction through photothermal conversion, where photothermal agents absorbed NIR light and converted photon energy into heat via radiative and non-radiative decay processes (e.g., vibrational relaxation).⁷⁰ The resultant hyperthermia exerted dose-dependent cytotoxic effects. Temperatures exceeding 42 °C induced irreversible cellular necrosis, while higher temperatures (>50 °C) triggered catastrophic biomolecular damage, including chromosomal fragmentation, protein denaturation, and enzymatic inactivation.⁷¹ This thermal ablation synergized with chemotherapeutic agents to potentiate tumor cell death while sparing healthy tissues. Commonly employed photothermal agents included carbon-based NPs,⁷² noble metal nanostructures (e.g., Au, Ag) leveraging localized surface plasmon resonance,⁷³ magnetic NPs,⁷⁴ organic small molecules (e.g., cyanine dyes, porphyrins),⁷⁵ and semiconducting polymer NPs.⁷⁶ A representative study demonstrated the efficacy of a light-responsive nanoplatform co-encapsulating MTO and the NIR dye IR775 within diphenylporphyrin liposomes.⁷⁵ Upon 750 nm laser irradiation, this system achieved rapid intratumoral temperature elevation (>50 °C), resulting in synergistic tumor eradication through combined photothermal and chemotherapeutic actions.

Recent innovations have focused on exploiting TME characteristics to spatiotemporally control drug release.^56,77–79 For instance, Zhang et al.⁸⁰ engineered ROS-responsive NPs that disassemble in the presence of endogenous ROS, enabling the triggered release of MTO and CA. Under 660 nm laser irradiation, this system achieved potent suppression of 4T1 tumor growth through dual photothermal effects and mitochondria-targeted oxidative stress. A groundbreaking approach introduced a tumor-targeting nanolock system (MTO-Cu(II)-cRGD), wherein GSH-mediated reduction triggered Cu(II)-to-Cu(I) conversion.⁸¹ This process initiated a Fenton-like reaction, generating cytotoxic ˙OH, while the photothermal effect of MTO accelerated GSH depletion. The resultant ferroptosis sensitized tumor cells to chemotherapy, effectively overcoming drug resistance through a triple-combination mechanism (chemotherapy/PTT/ferroptosis).

In short, the combination of MTO-based chemotherapy and PTT establishes a novel therapeutic modality which exhibits consistently superior efficacy to conventional monotherapies. Despite compelling advantages evidenced by numerous preclinical studies (Table 1),^82–92 comprehensive clinical translation remains underway. Future research should focus on optimizing light dosimetry, improving tumor penetration, and elucidating heat shock protein-mediated resistance.

Table 1 Design parameters and performance metrics of multifunctional nanocarriers enabling MTO-based chemo-PTT

Photothermal agents	Nanoparticles	Response methods	Excitation wavelength (nm)	Surface modifications	Payloads	Tumor models	Ref.
Abbreviations: Ho-MPDA, Ho (III)-doped mesoporous polydopamine; rGO, reduced graphene oxide; 2D BP, two-dimensional black phosphorus; W, tungsten; Mo, molybdenum; Se, selenium; HMM@T, MTO@Ho-MPDA@4T1; MCMA NPs, the nanoplatform encapsulating MTO and MnCO; GOx, glucose oxidase; Lipo, liposome; IMQ, TLR7 agonist-imiquimod; MTX, MTO; SB, a TGF-β inhibitor; mHFn, murine heavy chain ferritin; AuNV, virus-like spikes gold nanoparticle; SLN, solid lipid nanoparticle; PEG, polyethylene glycol; Tmab, trastuzumab; CHI, chitosan; DSPE-PEG2000, N-(carbonyl-methoxy polyethylene glycol 2000)-1,2-distearoyl-sn-glycerol-3-phosphoethanolamine; MnCO, manganese carbonyl; AuNR, gold nanorod; 4T1, mouse breast cancer cells; MCF-7, human breast cancer cells; CT26 and MC38, murine colon adenocarcinoma cells.
Ho-MPDA	HMM@T	pH/GSH/NIR	808	4T1 cell membranes	MTO	4T1	82
MTO	MCMA NPs	H2O2	660	PLGA–PEG	MTO, MnCO	MCF-7	83
MTO	MTO-micelles	NIR	665	DSPE-PEG2000	MTO	4T1	84
MTO	MTO-GOx@rFe2O3	pH	660	DSPE-mPEG2000	MTO, GOx	4T1	85
MTO	HA/Lipo @MTO@IMQ	pH	650	HA	MTO, IMQ	4T1	86
rGO	rGO/MTX/SB	NIR/Lysosome	805	—	MTO, SB	4T1	87
MTO	mHFn@MTO	pH/thermal	660	—	MTO	CT26	88
						MC38
Au	AuNV–MTO	GSH	1064	MTO	—	MC38	89
Au	SLN	NIR	808	FA	MTO, AuNR	MCF-7	90
2D BP	BP–PEG–MTO–Tmab	pH	808	Tmab	MTO	4T1	91
W–Mo–Se	CHI–Mo0.8–W0.2–Se2–MTO	pH/NIR	808	CHI	MTO	4T1	92

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4.3. Chemotherapy combined with PDT

PDT has emerged as a groundbreaking non-invasive therapeutic approach in oncology, distinguished by its unique mechanism of action and exceptional tumor selectivity. This modality leverages photosensitizers that exhibit preferential accumulation in neoplastic tissues, which upon precise light activation catalyze the ROS generation.⁹³ The resultant oxidative stress cascade induces comprehensive cellular damage, including mitochondrial dysfunction, genomic instability, and activation of programmed cell death pathways.^94–96 The integration of nanotechnology has revolutionized PDT by enabling the engineering of photosensitizer-encapsulated NPs with optimized physicochemical characteristics, enhanced tumor-specific targeting, and expanded therapeutic applicability across diverse malignancies.^97–99 A notable advancement in this field was demonstrated through the development of an innovative nanotheranostic platform. This platform conjugated anti-epithelial cell adhesion molecule antibodies to PEGylated NaYF4:Yb, Er@NaGdF4 (UCNPs) co-encapsulating MTO.¹⁰⁰ Under 980 nm NIR irradiation, these UCNPs exhibited efficient photon upconversion, emitting at 650 nm to activate MTO-mediated ROS generation for precision PDT. This multifunctional system, incorporating dual-modality magnetic resonance and upconversion luminescence imaging, achieved remarkable tumor-targeting accuracy and demonstrated synergistic therapeutic outcomes in hepatocellular carcinoma.

While conventional nanocarriers are often hampered by low drug loading capacity and carrier-induced toxicity, carrier-free nanodrugs emerge as a distinct class of therapeutics that effectively circumvent these limitations. These supramolecular nanostructures, formed by the self-assembly of active pharmaceutical agents, offer unique advantages such as ultra-high drug loading, precisely tunable release kinetics, enhanced biocompatibility, and simplified fabrication processes.¹⁰¹ For instance, a “drug-delivering-drug” strategy was used to construct self-assembled NPs by leveraging π–π stacking interactions between MTO and pyropheophorbide a.¹⁰² The resulting monodisperse, spherical NPs exhibited exceptional photodynamic activity and chemotherapeutic performance. In a complementary approach, Li et al.¹⁰³ employed an amidation reaction to synthesize an amphiphilic polysaccharide (phA-PEG₅₀₀₀), which functioned dually as both nanocarrier and therapeutic agent when complexed with MTO. The resultant MTO@phA-PEG₅₀₀₀ NPs exhibited potent antitumor efficacy against colorectal cancer through combined chemo-photodynamic action upon 670 nm laser irradiation.

Recent scientific breakthroughs have further enhanced PDT efficacy through strategic induction of mitochondrial dysfunction, a vulnerable node in cancer cell biology.¹⁰⁴ For instance, rhodamine B-decorated polyprodrug NPs were developed by co-encapsulating amphiphilic cisplatin and self-assembled MTO.¹⁰⁵ These mitochondria-targeting nanoconstructs achieved unprecedented tumor growth suppression in ovarian cancer models by simultaneously exploiting chemotherapy and PDT mechanisms (Fig. 4).

Fig. 4 Mitochondria-targeting composite polyprodrug NPs (Mito-CMPNs) for synergistic chemo-PDT. (A) Schematic illustration of Mito-CMPN self-assembly from molecular precursors. (B) Proposed therapeutic mechanism: mitochondrial targeting, laser-triggered (660 nm) ROS generation, stimuli-responsive drug release, and mitochondrial dysfunction induction. (C) In vitro validation of H₂O₂-responsive MTO release profile and quantitative ROS generation capacity. (D) Subcellular localization studies demonstrate mitochondrial-specific accumulation. (E) Mitochondrial stress evaluation (ATP depletion and ultrastructural cristae cavitation). Reproduced from ref. 105 with permission from Elsevier B.V. © 2023.

The convergence of PDT with cutting-edge nanotechnology is redefining the landscape of precision oncology. By systematically addressing the limitations of conventional therapies through therapeutic synergy with attenuated systemic toxicity, innovative carrier-free nanodrug design, and precise TME modulation, this integrated approach holds transformative potential for cancer treatment. While numerous investigations have elucidated the synergistic benefits of MTO-based chemo-PTT-PDT combinations,^106–108 their comprehensive analysis extends beyond the purview of this discussion.

4.4. Chemotherapy combined with molecular targeted therapy

Molecular targeted therapy has revolutionized contemporary oncology by introducing unprecedented precision in tumor eradication while preserving normal tissue integrity. This therapeutic breakthrough was achieved through highly specific interactions with molecular targets governing tumor progression, metastatic dissemination, and microenvironmental reprogramming, or by precise modulation of oncogenic signaling networks.^109,110 The advent of nanotechnology has further propelled this field by enabling spatiotemporal control over drug delivery, multi-target engagement of critical signaling cascades,¹¹¹ enhanced recognition of tumor-associated antigens,¹¹² and precision targeting of overexpressed cell surface receptors.¹¹³ These advancements have successfully reconciled the traditional dichotomy between broad-spectrum cytotoxicity and molecular specificity in cancer treatment.^114,115

Among potential therapeutic targets, the Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway has garnered particular attention due to its central role in malignant transformation, where it coordinately regulates tumor invasiveness, metastatic potential, and immunosuppressive TME evolution. A breakthrough nanoparticle system functionalized with aminoethyl anisamide and co-encapsulating MTO with napabucasin exemplified this approach.¹¹¹ This sophisticated platform mediated a combined therapeutic mechanism including conventional cytotoxic chemotherapy via MTO, Cyclic GMP-AMP Synthase-Stimulator of Interferon Genes (cGAS-STING) pathway activation, and STAT3 pathway blockade through napabucasin. The resultant synergy between TME reprogramming and enhanced tumor cell chemosensitivity yielded exceptional anti-hepatocellular carcinoma efficacy.

The indoleamine 2,3-dioxygenase (IDO) pathway represented another critical therapeutic node, whose overactivation facilitated tumor immune evasion through tryptophan depletion and cell cycle checkpoint dysregulation.¹¹⁶ To counter this mechanism, researchers developed folate receptor-targeted, dual pH/GSH-responsive nanomicelles co-delivering MTO with the IDO inhibitor 1-methyl tryptophan.¹¹⁷ This intelligent system operated through tumor-selective accumulation via receptor-mediated endocytosis, triggered drug release in response to TME stimuli, direct tumoricidal effects from MTO, and IDO-mediated immunosuppression reversal. The concomitant suppression of tryptophan catabolism and kynurenine production effectively reverted the immunosuppressive TME phenotype and overcame MDR in breast cancer models. In a parallel innovation, Mei et al.¹¹⁸ engineered cholesterol-conjugated prodrug NPs incorporating both indoximod (an IDO-1 inhibitor) and MTO. This liposomal platform achieved comprehensive metabolic interference by simultaneously disrupting the tryptophan-kynurenine axis via IDO-1 inhibition and the tumor-associated aryl hydrocarbon receptor pathway, culminating in potent suppression of colorectal cancer progression.

While MTO-based nanotherapeutic platforms integrated with molecular targeting represented a promising frontier in oncology, their clinical translation remains in its infancy. Future investigations should prioritize the rational design of intelligent and multi-targeted nanosystems with dynamic TME responsiveness, systems-level modulation of interconnected oncogenic pathways, and development of tumor-agnostic therapeutic strategies with pan-cancer applicability. As nanotechnological innovations converge with expanding knowledge of cancer biology, these multifunctional platforms are anticipated to redefine precision oncology, offering enhanced therapeutic indices, reduced off-target effects, and personalized treatment regimens tailored to individual tumor molecular profiles. The coming decade will likely witness these approaches transitioning from experimental models to clinical reality, potentially establishing a new gold standard in cancer therapeutics.

4.5. Chemotherapy combined with immunotherapy

Immunotherapy represents a transformative approach in oncology by harnessing the host's immune system to selectively target and eradicate malignant cells.^119,120 Despite its promise, clinical efficacy is frequently constrained by tumor immune evasion mechanisms, suboptimal immune activation, and immunosuppressive TME.¹²¹ Nanoparticle-mediated drug delivery systems have emerged as a groundbreaking strategy to augment immunotherapy by remodeling the TME, potentiating immune effector cells, depleting immunosuppressive populations, and enhancing antigen presentation.^122–124 Among the most compelling approaches is the combinatorial regimen of immune checkpoint inhibitors with chemotherapeutic agents such as MTO, which demonstrates profound synergistic antitumor activity.^125,126 A foundational study by Hu et al.¹²⁷ illustrated this concept through the development of poly(L-glutamic acid)-g-methoxy poly(ethylene glycol) (PLG-g-mPEG) NPs co-loaded with MTO and the programmed death-ligand 1 (PD-L1) inhibitor JQ1 via electrostatic and hydrophobic interactions. Upon irradiation with a 671 nm laser, MTO-mediated mild photothermal heating (43 °C) not only potentiated intratumoral T cell activation but also augmented the production of antitumor cytokines, effectively overcoming PD-1/PD-L1 resistance in breast cancer models. Furthermore, localized hyperthermia enhanced tumor vascular perfusion, ameliorated hypoxia, and suppressed metastatic dissemination.

While blockade has revolutionized cancer treatment, therapeutic resistance often emerges due to compensatory upregulation of alternative checkpoints (e.g., CTLA-4, TIM-3, LAG-3), which attenuate T cell function through secondary immune adaptation. MTO circumvents this limitation by inducing ICD, thereby eliciting robust adaptive immunity and establishing long-term immune memory.¹²⁸ Capitalizing on this mechanism, an acid-responsive polymeric nanoparticle system incorporating MTO, proanthocyanidin, and optimized peptide checkpoint inhibitors was shown to promote ICD, evidenced by upregulated CRT exposure and HMGB1 release, which enhanced DC-mediated antigen presentation and alleviated CD8⁺ T cell suppression.¹²⁹ This strategy achieved potent chemo-immunotherapeutic synergy with minimal systemic toxicity. Notably, a growing body of evidence underscores the enhanced antitumor efficacy of MTO when combined with immunotherapy via ICD induction (Table 2).^{21,105,117,118,130–134}

Table 2 MTO-based nanoplatforms enabling synergistic chemo-immunotherapy

Nanocarriers	Particle size (nm)	Payloads	Tumor models	Therapeutic effects	Ref.
Abbreviations: MT-CNPs, MTO and αTIM-3-loaded coronated NPs; Mito-CMPNs, mitochondria-targeted polyprodrug nanoinducers; MX, MTO; 1-MT, 1-methyl tryptophan; IND(Indoximod), a non-competitive inhibitor of the IDO-1 pathway; MMH, MIL-100/MTO/HA NPs; APS, aminoethylanisamide-polymer-disulfide bond; SPIONs, superparamagnetic iron oxide NPs; SAPC NPs, the polysialylated nanocarriers; αTIM-3, anti-TIM-3 antibody; Pt, cisplatin; CEL, celastrol; Vad, the STING agonist vadimezan; ID8, mouse ovarian cancer cells; MCF-7/ADR, human breast cancer cells/adriamycin-resistant; CT26, murine colon carcinoma cells; EMT6, mouse mammary carcinoma cells; RENCA, mouse renal carcinoma cells; BPD 6, murine melanoma cells; HT-29, colon carcinoma cells; Raji, Raji burkitt lymphoma cells; Trp, tryptophan; Kyn, kynurenine; NK cells, natural killer cells.
MT-CNPs	171.3 ± 14.0	MTO, αTIM-3	4T1	ICD induction and ICD perception	21
				Induce innate and adaptive immune responses
Mito-CMPNs	129.5	MTO, Pt	ID8	Induce mitochondrial stress	105
				M1 macrophage polarization
				Down-regulate immunosuppressive Treg cells
PEG-SS-MX-1-MT	169.0 ± 1.6	MTO, 1-MT	MCF-7/ADR	Inhibition of IDO activity	117
				Inhibit Trp degradation and Kyn production
L-MTO/IND	98.0 ± 1.0	MTO, IND	CT 26	Recruite NK cells, increase perforin release	118
			EMT6, 4T1	Inhibit the IDO-1 pathway
			RENCA	Enhance immune response
MMH NPs	173.2 ± 3.9	MTO	CT 26	Remodel the tumor immune microenvironment	130
APS NPs	∼100–200	MTO, CEL	BPD 6	Reduce collagen deposition and fibrosis	131
				Long-term immune memory
SPION^MTO	55.7 ± 1.0	MTO	HT-29	Magnetically accumulation	132
				Promote DAMPS release
				Chemotactic migration of monocytes
MTO@SAPC NPs	300.0 ± 31.0	MTO	Raji	Splenic effect T cell proliferation and activation	133
				Induced CD22^+ effective cell apoptosis
MV@Lip	111.0	MTO, Vad	4T1	Activate STING pathway, amplify innate immunity	134
				Recruit NK cells and produce ROS

---

Recent advances highlighted the therapeutic potential of concurrently activating multiple immune pathways, particularly the STING pathway, to counteract chronic inflammation and tumor immune evasion through amplified downstream immune cascades.^135,136 In one innovative approach, pH-responsive zinc-phenolate nanocapsules were engineered for MTO delivery, inducing mitochondrial DNA release under cellular stress.¹³⁷ The liberated DNA was subsequently detected by the cGAS sensor, triggering STING pathway activation via TANK-binding kinase 1 and interferon regulatory factor 3 phosphorylation. This cascade elicited robust production of proinflammatory cytokines and type I interferons, culminating in DC activation, CD8⁺ T cell priming, and potent adaptive immunity that significantly suppressed triple-negative breast cancer progression. In a complementary strategy, a one-pot synthesized MOF co-delivered MTO and immunomodulatory thymus pentapeptide.¹³⁸ MTO-induced DNA damage activated the cGAS-STING axis, promoting DC maturation, T cell activation, and inflammatory cytokine secretion while reprogramming the TME. Intriguingly, the concomitant release of Zn²⁺ ions exerted multimodal antitumor effects by inhibiting cancer cell glycolysis, activating the adenylate-activated protein kinase pathway, and downregulating PD-L1 expression, thereby obstructing immune escape. This multifaceted intervention achieved significant suppression of colon cancer proliferation.

In summary, immunotherapy has redefined cancer treatment by reversing immunosuppressive TME dynamics, enhancing immune cell activation and infiltration, and fostering durable immunological memory. Concurrently, chemotherapy not only delivered direct cytotoxic effects but also potentiated antitumor immunity through ICD, further amplifying the overall antitumor immune response. The strategic convergence of chemotherapy and immunotherapy epitomizes an innovative therapeutic modality, meriting extensive preclinical and clinical exploration to fully realize its translational potential.

4.6. Chemotherapy combined with gene therapy

Gene therapy has emerged as a powerful oncotherapeutic strategy by enabling the precise delivery of genetic materials to target cells, including apoptosis-regulating genes,¹³⁹ antisense oligonucleotides (ASOs),¹⁴⁰ anti-angiogenic factors,¹⁴¹ immunomodulatory genes, and cytokines. These agents functioned to correct genetic aberrations, modulate dysregulated signaling pathways, and amplify antitumor immune responses.^142,143 The introduction of CRISPR-Cas9-based gene editing has transformed the field by enabling high-precision genome interventions. This technology offers the potential to eradicate malignancies with minimal off-target effects, representing a fundamental advance toward curative therapies for cancers that are refractory to conventional treatments.

The combinatorial application of gene therapy and chemotherapy has demonstrated superior efficacy compared to monotherapeutic regimens. A compelling example involved biomimetic NPs which were engineered to co-encapsulate MTO and B-cell lymphoma 2 (Bcl-2)-targeting ASO within homologous tumor cell membranes.¹⁴⁰ In this system, MTO not only exerted direct cytotoxic effects but also facilitated ASO delivery via acidic sphingomyelinase-mediated lysosomal escape, while the ASOs silenced Bcl-2 family mRNA, effectively reversing MDR and restoring chemosensitivity in breast cancer models. Similarly, Chen et al.¹⁴⁴ developed hybrid membrane-coated NPs by fusing ovarian cancer and erythrocyte membranes to co-deliver MTO and human epidermal growth factor receptor 2 (Her-2)-targeting ASOs. These pH-responsive Mito-Her-2 NPs exhibited TME-triggered ASO release, synergistically suppressing Her-2 overexpression while augmenting MTO-induced cytotoxicity.

siRNA-mediated gene silencing has also shown remarkable potential in targeted oncogene suppression.^145,146 An innovative pH-responsive polymeric nanoparticle system was designed for the co-delivery of an MTO prodrug and polo-like kinase 1 (PLK1)-specific siRNA (siPLK1).¹⁴⁷ These NPs released siPLK1 in the acidic TME, followed by esterase-mediated activation, inducing MTO-mediated nuclear DNA damage and PLK1 silencing, which collectively promoted apoptosis in breast cancer models. Extending this strategy, an advanced approach employed PLGA NPs to co-encapsulate MTO·2HCl with CD47-targeting siRNA (siCD47), effectively disrupting immune evasion signals and improving tumor cell phagocytosis.¹⁴⁸ By blocking the “don’t eat me” signal on malignant cells, this strategy enhanced phagocytic clearance in colorectal cancer and melanoma. In another breakthrough, multi-layered cationic NPs incorporating MTO-palmitoyl conjugates and myeloid cell leukemia sequence 1 (Mcl-1)-targeting siRNA (siMcl-1) achieved sustained siRNA release and profound Mcl-1 suppression, significantly impeding oral epidermoid carcinoma progression (Fig. 5).¹⁴⁹

Fig. 5 Engineered nanoplatforms for MTO-siRNA combination therapy. (A) Biomimetic erythrocyte membrane-coated NPs co-encapsulating MTO and Her-2 ASO for targeted ovarian cancer therapy. Reproduced from ref. 144 with permission from American Chemical Society © 2023. (B) pH/esterase dual-responsive NPs (SA-MTO) undergoing TME-triggered dissociation into siRNA-MTO conjugates, followed by enzymatic hydrolysis-mediated simultaneous release of MTO and siPLK1. Reproduced from ref. 147 with permission from American Chemical Society © 2019. (C) Vesicular nanoparticle system (VNPsiCD47) delivering siCD47 to potentiate MTO-induced ICD. Reproduced from ref. 148 with permission from American Chemical Society © 2021. (D) LbL assembled polyelectrolyte NPs for charge-mediated co-delivery of MTO and siMcl-1. Reproduced from ref. 149 with permission from Elsevier B.V. © 2011.

By integrating programmable genome-editing tools with optimized MTO delivery systems, such as biomimetic nanovectors, researchers can achieve spatiotemporal gene regulation, enhance tumor-selective cytotoxicity, and minimize systemic toxicity. Especially, the integration of CRISPR-Cas9 gene editing technology with advanced nanotherapeutic platforms represents a pivotal strategy for advancing personalized precision medicine. Leveraging patient-specific genomic data, this modular framework allows for the discerning selection of targeted ligands (e.g., anti-HER2, epidermal growth factor receptor (EGFR)-specific, and glypican-3 (GPC3)-specific antibodies), the rational design of TME-responsive mechanisms, and the development of customized nucleic acid therapies. These developments are anticipated to establish a new approach in targeted oncology, addressing the intractable challenges of therapeutic resistance and metastatic disease.

4.7. Chemotherapy combined with other therapies

Chemodynamic therapy (CDT) exploits transition metal-based NPs (e.g., Fe, Cu, Mn) to catalyze Fenton or Fenton-like reactions within the TME.¹⁵⁰ This mechanism capitalizes on the aberrantly elevated H₂O₂ concentrations in malignant tissues, facilitating redox cycling for the generation of highly cytotoxic ˙OH. The resultant oxidative stress induces non-selective biomolecular damage, culminating in irreversible tumor cell dysfunction.^151,152 Recent advancements have demonstrated remarkable synergism between CDT and chemotherapeutics, such as MTO. A typical strategy involved the engineering of γ-Fe₂O₃ NPs co-encapsulating MTO and glucose oxidase (GOx), with DSPE-mPEG₂₀₀₀ surface functionalization to optimize biocompatibility and pharmacokinetics.⁷⁰ Following cellular internalization, lysosomal degradation liberated Fe³⁺, which underwent GSH-mediated reduction to Fe²⁺, thereby amplifying Fenton reaction kinetics and ˙OH yield. Concurrently, GOx depleted endogenous glucose reserves while generating supplemental H₂O₂, establishing a self-sustaining cycle for enhanced CDT efficacy. This combinatorial system leveraged MTO's DNA intercalation capability to induce double-strand breaks, operating in concert with oxidative damage to elicit dual apoptotic pathways, achieving superior tumor growth inhibition in breast cancer models.

Sonodynamic therapy represents a non-invasive modality wherein ultrasound activates sonosensitizers (e.g., hematoporphyrin derivatives) to generate spatiotemporally controlled cytotoxic ROS with deep tissue penetration.^153,154 To transcend the constraints of monotherapy, an advanced nanoplatform was devised to co-load MTO and the sonosensitizer hematoporphyrin monomethyl ether (HMME) within a porous organic polymer framework.¹⁵⁵ Further biomimetic modification with 4T1 breast cancer cell membranes conferred active tumor-targeting specificity. The nanoconstruct exhibited GSH-responsive payload release within the TME. Under ultrasound irradiation, HMME-mediated ROS production synergized with MTO's dual mechanisms, direct genomic disruption and ICD induction. This orchestrated action stimulated DC maturation and cytotoxic T lymphocyte recruitment, establishing an integrated chemo-sonodynamic-immunotherapeutic strategy. The multimodal regimen achieved comprehensive tumor suppression through three-pronged mechanisms, localized ROS ablation, chemotherapy-driven apoptosis, and systemic antitumor immune priming.

MTO's intrinsic photothermal properties have been harnessed to develop theranostic platforms integrating photoacoustic imaging-guided PTT.^81,85 A groundbreaking innovation involved the molecular co-assembly of MTO, mPEG, and 3,3′-dithiodipropionic acid to fabricate redox-responsive prodrug NPs.¹⁵⁶ These NPs demonstrated TME-selective activation, where aberrant GSH levels triggered disulfide cleavage, enabling spatiotemporally controlled MTO release and in situ formation of therapeutic aggregates. This system achieved precision drug delivery through GSH-mediated drug liberation, yielding a 1.3-fold enhancement in tumoral MTO accumulation versus free drug administration. Furthermore, the NPs exhibited strong NIR absorption at 680 nm, facilitating real-time photoacoustic imaging for quantitative pharmacokinetic monitoring. This closed-loop theranostic platform permitted dynamic irradiation parameter optimization based on imaging feedback, thereby maximizing therapeutic index via spatiotemporally controlled drug activation while mitigating off-target effects.

While these multimodal strategies remain in nascent developmental stages compared to conventional therapies, they represent a transformative shift in oncological intervention. Current research is focused on optimizing combinatorial ratios, refining stimuli-responsive activation thresholds, and elucidating synergistic mechanisms at the molecular level. Such efforts aim to overcome the limitations of traditional chemotherapy while establishing new standards for therapeutic efficacy and precision in cancer treatment.

4.8. Clinical translation: status and challenges

The compelling efficacy and considerable developmental potential of the aforementioned combination therapies have catalyzed the emergence of numerous MTO-based multifunctional NPs. A notable milestone in clinical translation was recently achieved in China, with the market approval of MTO hydrochloride liposome injection for the treatment of head and neck squamous cell carcinoma.¹⁵⁷ Despite such progress, the inherent complexity of innovative drug loading systems and multifunctional modifications presents substantial challenges to clinical translation and regulatory approval.

To overcome these barriers, several strategic approaches are being actively pursued. These include the development of patient-specific nanoparticle complexes tailored to individual genetic and physiological profiles, advancements in light source technologies to enhance the in vivo localization and activation of nanomedicines, and the integration of machine learning to build predictive databases for nanomaterial performance and biosafety.^158–160 Furthermore, given the unique physicochemical and biological behaviors of nanomedicines, there is a pressing need to establish specific testing priorities for different nanomaterial categories, refine unified regulatory standards, and proactively evaluate the potential ethical implications of novel nanotherapeutic interventions. Concerted efforts in these directions will establish a robust foundation for the standardized oversight and successful clinical integration of nanomedicines.¹⁶¹

5. Concluding remarks and future perspectives

Despite its foundational role in clinical oncology, conventional chemotherapy remains constrained by intrinsic challenges including notably tumor heterogeneity, the evolution of drug resistance, and dose-limiting systemic toxicities. While empirical dose escalation may yield transient therapeutic gains, this approach invariably exacerbates adverse effects, undermining treatment adherence and long-term patient outcomes. The advent of biomedical innovation has catalyzed the development of next-generation anticancer modalities, including molecularly targeted agents, immune checkpoint inhibitors, gene therapies, and CRISPR-based genome editing systems. Contemporary research has underscored the transformative potential of combinatorial regimens that synergize traditional chemotherapeutics, such as MTO, with these cutting-edge interventions. An important advancement involved engineered MTO-loaded nanomedicines, which potentiated therapeutic efficacy through three synergistic axes: enhanced tumor targeting via EPR effect coupled with active targeting ligands, spatiotemporal therapeutic control via integration of NIR-responsive phototherapeutic agents enabling on-demand treatment activation, and substantially attenuated cardiotoxicity profiles compared to conventional MTO formulations. Furthermore, nano-engineering refinements have markedly improved pharmacokinetic stability and prolonged systemic circulation while minimizing nonspecific biodistribution.

Despite demonstrated preclinical advantages, four major challenges hinder the clinical translation of MTO-based nanomedicines:

(1) Suboptimal drug payload. While nanoencapsulation enhances MTO's aqueous solubility and circulation half-life, conventional polymeric matrices face inherent limitations such as low drug loading efficiency (<30% w/w) due to carrier inertness and potential immunogenicity risks. Emerging solutions include carrier-free nanoassemblies driven by π–π stacking and hydrophobic interactions to achieve >90% drug loading via molecular self-organization, hybrid inorganic–organic systems (e.g., mesoporous silica and MOFs) with tunable pore architectures for high-capacity MTO encapsulation, and stimuli-responsive nanocarriers incorporating pH-sensitive Schiff base linkages or redox-cleavable disulfide bonds for tumor-selective payload release.¹⁶²

(2) Thermodynamic instability. MTO-based chemo-PTT platforms, while synergistic, faced critical thermal stability challenges including structural denaturation of nanocarriers at therapeutic hyperthermia (42–45 °C), premature drug leakage, and photothermal agent degradation. To address these limitations, advanced thermal management strategies are being developed, including the design of plasmonic nanostructures with high photothermal efficiency for rapid and confined heating, the use of phase-change materials that buffer thermal stress via solid–liquid transitions to maintain payload integrity within a 40–42 °C therapeutic window, and the engineering of thermal homeostasis to stabilize drug release profiles and establish self-reinforcing therapeutic loops.^163,164

(3) Mechanistic ambiguity in polytherapy synergy. Whereas monotherapeutic agents typically exhibit well-characterized mechanisms of action, combination therapies introduce emergent pharmacodynamic complexities arising from multifaceted drug–drug and drug-carrier interfacial dynamics, spatiotemporal heterogeneity within the TME, and nonlinear crosstalk between distinct therapeutic modalities. To deconvolute these higher-order interactions, an integrative systems pharmacology framework is imperative, one that synergizes multi-omics data integration, computational modeling of drug-TME phase diagrams, and high-throughput phenotypic screening platforms. Crucially, multiscale validation framework that bridge molecular-resolution mechanistic insights with macroscopic pharmacodynamic readouts are needed to decode the polytherapy's currently opaque mechanistic “black box”.^89,165

(4) Concerns regarding long-term biosafety. Conventional techniques such as cytotoxicity tests, in vivo acute toxicity studies, and in vitro cell viability assays are useful for rapidly identifying acute safety risks of nanomaterials. However, growing evidence indicates that NPs toxicity can arise from multiple underlying mechanisms. Typically, lipid-based NPs may induce complement activation-related pseudoallergies, protein-based NPs can elicit immune responses upon denaturation, and polymeric NPs may provoke inflammatory reactions modulated by their degradation kinetics and byproducts.¹⁶⁶ To address these concerns, it is imperative to enhance long-term biosafety studies, toxicity screening, and immunogenicity assessments for NPs. Key measures include implementing long-term immunological profiling, repeated blood biochemistry monitoring, and extended animal dosing studies (e.g., over 6–12 months). Such comprehensive evaluations are essential for detecting delayed physiological responses resulting from cumulative biological interactions and for enabling a systematic assessment of chronic systemic toxicity.^161,167

In conclusion, MTO-based multifunctional nanocomposites have demonstrated superior antitumor efficacy through four pivotal mechanisms: biocompatibility-by-design through rational material selection, adaptive therapeutic selectivity via stimuli-responsive activation, precision targeting leveraging both passive and active mechanisms, and synergistic polytherapy integrating chemo-, photo-, and immuno-modalities. To fully realize this potential, future efforts must prioritize translational optimization, mechanistic elucidation, and regulatory innovation. These advancements will not only revitalize MTO's clinical utility but also establish a versatile framework for advancing precision oncology where diagnostic precision and therapeutic efficacy converge.

Author contributions

Juan Yu: writing – original draft, visualization, formal analysis; Zijun Qiu: writing – review & editing, validation; Susu Yan: data curation, resources; Aishan Lin: formal analysis; Yanan Li: conceptualization, writing – review & editing, investigation, supervision, project administration.

Conflicts of interest

There are no conflicts to declare.

Data availability

No primary research results, software or code have been included, and no new data were generated or analysed as part of this review.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81903662), the Basic Research Program of Shanxi Province (202303021211116), the Startup Foundation for Doctors of Shanxi Medical University (XD1824), and the Educational Innovation Program of Shanxi Medical University (XJ2024065).

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