Advances in the applications of stimulus-responsive nanomedicines for anti-cancer therapy.

1.
Introduction
Cancer, a prevalent and leading cause of global death, poses immense challenges due to its complex nature, diverse metabolic pathways, and multidrug resistance (MDR) [1–3]. Current therapies like surgery struggles with advanced, metastasized tumors, while radiotherapy, chemotherapy, and immunotherapy suffer from nonspecific drug distribution, leading to damage to healthy tissues and severe side effects, even molecularly targeted drugs exhibit suboptimal biodistribution and off-target toxicity [4,5].
Nanomedicine, defined as the use of nanoscale (several to several hundred nanometers) materials in medicine, offers a new direction for achieving specific drug accumulation in tumor sites. Utilization of different nanoparticles (NPs) such as liposomes, albumin NPs and polymeric micelles as carriers for payload drug in nanomedicine not only make use of the unique function and property of NPs (high surface-to-volume ratios, tunable size and shape, etc.) but also can enhance solubility and stability of hydrophobic drugs, extend the drug blood circulation time, facilitate drug release in a targeted site, and offer in vivo imaging to monitor drug distribution and/or tumor progression during treatment [6–8]. Accumulation of conventional nanomedicine after its administration and blood circulation is thought to be realized through passive targeting of the enhanced permeability and retention (EPR) effect, as a result of leaky tumor vasculature and poor lymphatic drainage (Figure 1). Another active targeting strategy employs modification of nanomedicine carriers with specific antibodies and ligands to ensure a specific recognition and binding of nanomedicine with pathological cells [5,9,10]. After reaching the tumor site, further tumor tissue penetration, tumor cell internalization, endosomal escape, and release of drug payload into the cytoplasm are also critical processes for nanomedicine to reduce the side effects of antitumor drugs and improve their therapeutic efficacy [6,9].

The stimulus-responsive nanomedicines, which release drug payload in response to distinct physical, chemical, or biological triggers, have gained prominence for their ability to overcome the spatiotemporal barriers of conventional drug delivery. This responsive strategy comes partly from the idea of prodrug, which remains a non-bioactive precursor until it is activated by certain enzymes or chemical reactions and is converted to biologically active drugs (since prodrug also suffer from rapid degradation and premature activation, prodrug-encapsulated nanomedicine will be viewed same as its counterpart containing small molecule chemical drug and be discussed in a stimulus-responsive manner here) [11]. The tumor microenvironment (TME) provides a unique biological backdrop for such systems, characterized by distinct features that differentiate it from normal tissues: acidic pH (6.5–7.2 versus 7.4 in healthy tissues), elevated levels of glutathione (GSH), overexpression of matrix metalloproteinase 2 (MMP-2), reactive oxygen species (ROS) accumulation, and aberrant vasculature. These internal (endogenous) cues, alongside external (exogenous) stimuli (light, heat, magnetic fields, ultrasound, etc.), enable stimulus-responsive nanomedicines to achieve site-specific drug release, minimizing off-target effects [9,12–19]. Internal stimulus-responsive systems depend on internal information like intracellular ionic strength, pH, and enzyme concentrations. They can work on different tumors in superficial or deeper parts of the body, but they are not reversible. The external stimulus-responsive system is based on external stimulus and needs an outer machine supply to generate responsive sources such as hyperthermia, light, magnetic field, etc. It usually has no relationship with ionic concentration in the TME. The limitation of external-responsive nanomedicine is that it is mainly applied in superficial tumors, because the outer stimulus are not able to reach deep tissues [13].
In this review, we will talk about the recent advances in how these internal stimulus and external stimulus are exploited for anti-cancer therapy in recent 5 years (searching literatures by combining each stimuli and keyword “cancer” on PubMed), focusing on how these versatile nanomedicines enable targeted drug release in tumors, improve tumor penetration, improve efficacy and reduce toxicity, overcome multidrug resistance, modulate the TME and exert synergistic anti-tumor effects with combinational therapies (chemo-immunotherapy, chemo-photodynamic therapy, etc.). Current barriers and challenges of stimulus-responsive nanomedicines will also be discussed, together with future directions and prospects to address these challenges.

2.
Internal stimulus-responsive nanomedicine
2.1.
pH-responsive nanomedicine
Normal tissues sustain a pH of around 7.4 to maintain cellular and tissue homeostasis; in contrast, the extracellular pH of cancerous tissue typically ranges from 6.5 to 7.2. This acidic TME arises from cancer cells’ so-called Warburg effect that they predominantly utilize glycolysis for energy production even in the presence of oxygen, leading to the overproduction of lactic acid that accumulates as a result of impaired clearance mechanisms [20–22]. Intracellularly, endosomes have a pH of 5.06.5, while lysosomes are even more acidic, with a pH of 4.5–5.0 [23,24]. pH-responsive nanomedicine has emerged as a promising approach in cancer therapy, they are engineered with pH-sensitive chemical bonds (ester bonds, amide bonds, hydrazone bond, imine bond, etc.) and protonable/deprotonable chemical groups (carboxyl, amino groups, etc.) [25] to form nanomaterials such as liposomes, micelles, hydrogels, polymers, inorganic NPs and so on, the hydrolytic cleavage of these chemical bonds and protonation/deprotonation of these chemical groups on these nanomaterials and the consequent dissociation, disintegration, detachment and surface charge change of the formed NPs (Figure S1) are events that are responsive to the pH trigger and then enable the release of encapsulated drug.
To exploit the pH gradients between the TME and normal tissues, as well as within different intracellular compartments, pH-responsive nanomedicine has been massively studied in recent years to enable targeted drug release in tumors, improved efficacy, and reduced toxicity [26–29]. One of these studies employed a HER2-targeted DNA-aptamer-modified tetrahedral framework nucleic acid (HApt-tFNA) as a drug delivery system and conjugated it to maytansine (DM1) to form HApt-tFNA@DM1 (HTD, drug/carrier of 3:1). HTD was wrapped in a hybrid membrane composed of a biomimetic red blood cell membrane and pH-sensitive Poly(2-ethyl-oxazoline)-liposome (PEOz-liposome). The byrid PEOz-erythrosome disassembled with oxazoline protonation after the EPR effect-mediated passive accumulation in acidic TME and HER2-mediated endocytosis into endosomes to release HTD and showed better inhibition of HER2-positive cancer over other drug formulations and superior biosafety [26]. In another study focusing on nanomedicine delivery across blood-brain barrier (BBB) for glioblastoma treatment, a pH-sensitive anti-polyethylene glycol (PEG) Fab was generated by substituting ionizable amino acid (histidine or glutamic acid) into the humanized anti-PEG 6.3 Fab and fused it with anti-transferrin receptor (TfR) domain to enable active targeting, the resulted pH-responsive bispecific antibody (pH-PEG engagerTfR) can bind PEGylated nanomedicine at physiological pH to facilitate TfR-mediated transcytosis across BBB and rapidly dissociate and release drugs in acidic endosomes to improve brain accumulation and antitumor effect [29].
With the size change of pH-sensitive modality after pH stimulation, improving tumor penetration with pH-responsive nanomedicine was also extensively investigated [28,30–36]. For example, Lin et al. developed a pH-responsive nanoplatform via co-assembly of ursolic acid and bioactive polypeptoid polyelectrolytes (as assembly cofactors), with lactobionic acid for targeting. At neutral pH, it formed helical fibers, but in an acidic TME it transformed into virus-like clusters to enhance penetration [36]. Another study used trivalent Copper Sulfide (CuS)-DNA nanodots (NDs) assembled by applying pH-sensitive i-motif linkers at physiological pH (7.4) into ~60 nm aggregates, ensuring prolonged blood circulation and enhanced tumor accumulation via EPR effect. The i-motif linkers folded in an acidic tumor microenvironment (pH 6.0–6.5) and triggered CuS-i disassembly into 4.35 nm NDs. These ultrasmall NDs penetrated deep into the tumor parenchyma and enabled in vivo near-infrared (NIR)-II fluorescence imaging and deep tumor photothermal therapy (PTT) [34]. Zhou et al. further exploited epigallocatechin3-gallate (EGCG) to disrupt the dense collagenous stroma and alleviate fibrosis (Figure 2) together with a methotrexate (MTX)-loaded dual phosphate and pH-responsive nanodrug to realize tumor deep penetration and validated this in 3D spheroids and in vivo experiments [33].

Except of direct tumor cell killing by chemotherapy, recent studies have also shown that modulation of the tumor microenvironment by pH-responsive systems can enhance other therapeutics [37–40]. One of these studies developed a pH-responsive, guanidinium-rich nanoagonist that can self-assemble to encapsulate the hydrophilic and negatively charged 2’,3’cyclic-GMP-AMP (cGAMP) at pH 7.4 and disassemble in acidic TME and endosomes. Along with sustained tumor regression, this nanomedicine also enabled precise endoplasmic reticulum targeting of cGAMP by guanidinium groups and elicited immunotherapy function of tumor-associated macrophage (TAM) polarization modulation and potent antigen-specific cellular immune response [38]. Thanapongpibul et al. also reported a versatile, ultralow-volume (10 μL) oxygen-tolerant photoinitiated polymerization-induced self-assembly synthetic platform for producing pH-responsive protein-loaded polymeromes and successfully cross-presented ovalbumin bone marrow-derived dendritic cells (BMDCs) with them and elicited the immune activation of BMDCs well [39]. Radiotherapy enhancement by immunosuppressive metabolic microenvironment reprogramming by pH-responsive nanomedicine was also illustrated in another study, with coated calcium carbonate (CaCO3) NPs to neutralize protons and reverse the radioresistance [37].
Combination therapies using pH-responsive carriers and other internal/external-stimulus responsive nanoplatforms (e.g., chemo-photodynamic) to achieve synergistic anti-tumor effects were also elucidated in recent studies [38,41–43]. Via seed-mediated growth of gold nanorods (AuNRs) and cuprous oxide (Cu2O) shell coating, pH-responsive hybrid core-shell nanorods (AuNRs@Cu2O NRs, ACNRs) were constructed. In an acidic tumor microenvironment, ACNRs release Cu+ to induce cuproptosis, while the remaining core exhibited NIR-II photothermal property, generating ROS to enhance apoptosis. It reprogrammed metabolism by inhibiting glycolysis and the pentose phosphate pathway, reducing lactate/adenosine triphosphate (ATP) and boosting immune response via dendritic cell (DC) maturation and CD8+ T cell infiltration. These PTT, cuproptosis and durable immune response therapies suppressed tumor growth, reduced lung metastasis, and prolonged survival in 4T1/B16F10 tumor-bearing mice, together with elevated cytokines [42].

2.2.
Redox-responsive nanomedicine
ROS are oxygen-containing oxidants endogenously produced in distinct biochemical, metabolic, and protein folding activities that happened in mitochondria, peroxisome, and endoplasmic reticulums, and they encompass radicals like superoxide anions (O2•−), hydroxyl radicals (HO•), as well as non-radicals such as hydrogen peroxide (H2O2). Redox (short for reduction-oxidation) denotes a set of biochemical reactions centered on electron transfer. Key mediators of redox processes in biological systems include small-molecule redox pairs (e.g., reduced GSH and oxidized glutathione(GSSG)), redox-active bonds (disulfide, diselenide bonds), and enzymatic systems (superoxide dismutase/SOD, glutathione peroxidase/GPX, catalase/CAT). Tumor cells generate abundant ROS due to high metabolic demand, as antioxidant defense mechanisms to scavenge ROS, elevated redox antioxidants like GSH (2–10 mM intracellularly) are also common in tumor tissue to counteract ROS toxicity [44–46]. Redox-responsive nanomedicine leverages the distinctive redox gradients between tumor and normal tissues, with increased levels of GSH in tumors serving as key triggers [47]. They are typically engineered with redox-sensitive moieties, such as disulfide bonds, selenide linkages, or metal-based redox couples, which undergo cleavage or structural rearrangement upon exposure to TME-specific redox signals.
As a general strategy, a massive recent study on redox-responsive nanomedicine has been used to enable targeted, controlled drug release in tumors. For example, one study coated cancer cell membranes (high CD44 expression) on poly(ε-caprolactone)-SS-methoxy poly(ethylene glycol) (PCL-SS-PEG) micelles loaded with phosphorus dendrimer–copper(II) complexes (1G3-Cu) and toyocamycin (Toy) and developed redox-responsive NPs. Their redox responsiveness lies in GSH-triggered disassembly of the disulfide bond-containing PCL-SS-PEG polymer in the TME, with released 1 G3-Cu inducing mitochondrial dysfunction and Toy amplifying endoplasmic reticulum stress [48]. Another redox-responsive nanomedicine DAS@CDC was also synthesized by conjugating cabazitaxel (CTX) to chondroitin sulfate (CS) via GSH-sensitive dithio¬maleimide (DTM) (forming CS-DTM-CTX, CDC) and co-assembling with dasatinib (DAS). After being delivered to tumor cells and cancer-associated fibroblasts (CAFs) via CD44 receptor-mediated targeting, DAS@CDC released DAS and CTX upon DTM cleavage in high-GSH TME. The CAFs reprogrammed to reduce the extracellular matrix (ECM) by DAS jointly blocked tumor-stromal crosstalk with CTX and effectively inhibited tumor proliferation and metastasis [49]. Other than concurrent passive, active, and redox-initiated targeting, both of these two studies also used amphiphilic polymers to load hydrophobic chemotherapeutics to enhance drug solubility and extend blood circulation as well as bioavailability.
Excessive intracellular ROS induces cytotoxicity via damage to proteins, lipid bilayers, and chromosomal DNA and poses a constraint on tumor survival and progression. Enhancing GSH biosynthesis establishes a strongly reductive intracellular environment not only to support tumor cell survival and metastasis but also to mediate chemotherapy resistance through scavenging of drug-induced ROS. Redox-responsive nanomedicine was also utilized to overcome drug resistance via GSH depletion and ROS amplification in recent research. For example, to reverse cisplatin (CisPt) resistance, Niu et al. loaded CisPt and GSH S-transferases (GST) inhibitor ethacrynic acid into disulfide-bridged degradable organosilica hybrid NPs and developed a redox-responsive nanomedicine, released drugs of which can deplete GSH and inhibit GST synergistically to prevent CisPt detoxification (called “pincer movement” strategy). It exhibited expected functions with lower IC50 than free CisPt in A375/DDP cells and superior tumor suppression and low toxicity in A375/DDP tumor-bearing BALB/c nude mice [50]. Yu et al. also developed a GSH-responsive cysteine polymer-based nanomedicine (GCFN) and paclitaxel-loaded GCFN (PTX@GCFN) via cysteine self-assembly and nanoprecipitation, respectively. GCFN depleted intracellular GSH through disulfide bonds and inhibited GSH Peroxidase 4 to induce lipid peroxidation and ferroptosis in AML cells. PTX@GCFN specifically targeted leukemic cells for controlled PTX release and spared normal hematopoietic cells. It showed higher cytotoxicity against AML cells than free PTX, with lower toxicity to normal cells and reduced leukemic burden, prolonged survival, and mitigated myeloablation in MLL-AF9 AML mice [51]. Another redox-responsive example called DMSN/Fe3O4-Mn@CB-839 (DFMC) integrated ultrasmall Fe3O4 NPs and glutaminase inhibitor CB-839 into dendritic mesoporous silica nanoparticles (DMSN) via Manganese (Mn) etching. DFMC exerted dual GSH depletion with Iron(III) Ion (Fe3+), Manganese(III) Ion (Mn3+), and Manganese(IV) Ion (Mn4+) oxidize existing GSH to GSSG, and CB-839 blocks endogenous GSH synthesis by inhibiting GHS metabolism. It amplified ROS via Fenton/Fenton-like reaction, induced apoptosis and ferroptosis, and restored oxaliplatin sensitivity [52].
Recent studies also showed that combining different cancer therapeutics such as chemotherapy, immunotherapy, and radiotherapy using redox-responsive platforms can exert synergistic and more efficacious anticancer effects. In a study using Chalcogens as sensitive redox-responsive reagents to bypass chemoresistance and global systemic toxicity, hybrid organosilica NPs with homonuclear chalcogen bonds (Tellurium-Tellurium Bond: Te-Te; Selenium–Selenium Bond: Se-Se; Sulfur–Sulfur Bond; S-S) were synthesized and loaded with Manganese(II) Ion (Mn2+) (Figure 3). Te-Te exhibited the most responsiveness to X-ray triggers and cleaved to generate toxic Tellurite Ion (TeO32−) for chemotherapy, while releasing Mn2+ to activate the Stimulator of Interferon Genes Pathway (STING) pathway, boosting antitumor immunity [53]. Zhang et al. also developed X-ray-triggered redox-responsive polymer micelles co-encapsulating anti-PD-L1 (aPD-L1) and PTX and modified them with angiopep2 for glioblastoma (GBM) therapy. This nanomedicine crossed the blood-tumor barrier (BTB) via angiopep2 targeting and released aPDL1 and PTX via disulfide bond cleavage. These induced immunogenic cell death (ICD) to amplify aPD-L1-mediated immune checkpoint blockade, synergistically activated cytotoxic T cells, and inhibited tumor growth, prolonging survival in GL261/G422 orthotopic GBM mice as expected [54].

2.3.
ROS-responsive nanomedicine
ROS elevation is classified as a hallmark of cancer progression, diverse immune cells, CAFs, endothelial cells, invasive tumor cells, and TME features such as angiogenesis and hypoxia, collectively contributing to aberrant ROS production [55–57]. Tumor cells exhibit 10-fold higher ROS levels compared to normal cells, and hydrogen peroxide, hydroxyl radicals, and superoxide are major components. ROS-responsive nanomedicine has emerged as a pivotal strategy in tumor therapy by leveraging the distinctive redox feature of tumor tissues [15,58]. ROS-responsive nanomedicine is engineered with ROS-sensitive moieties such as thioketal linkages, boronate esters, and diselenide bonds, which undergo cleavage under elevated ROS conditions, enabling precise cargo release within tumors [59–61]. For example, an amphiphilic biodegradable polymer containing ROS-sensitive thioketal was designed to encapsulate elesclomol (ES) and Cu, forming NP@ESCu to achieve the cuproptosis and immune checkpoint inhibitors combinational therapy [62]. Using ROS-labile charge-reversal polymer that contains phenylboronic ester bonds and Distearoylphosphatidylethanolamine (DSPE)-PEG-IRDye 700, Mao et al. also constructed NP700 and encapsulated ectonucleotidase inhibitor ARL67156 (ARL) with it, forming ROS-responsive NP700-ARL. Under NIR irradiation, photosensitizer IRDye 700 generated ROS, cleaving phenylborate esters to release ARL, which blocked ATP degradation into immunosuppressive adenosine and induced ICD [59]. Both these two studies integrated hydrophilic PEG segments into the nanocarrier structure to form a “stealth” corona to improve circulation stability and utilized both intracellular ROS as a predominant trigger and extracellular ROS as a secondary trigger for pre-activation weakening of the polymer structure, with strengthened cuproptosis and photodynamic function further amplified intracellular ROS, respectively, to enable targeted, controlled drug release and synergistic anticancer effects.
As aforementioned, elevated intracellular ROS possesses cellular cytotoxicity due to their potent biomolecule reactivity, thus ROS-replenishing and GSH-depleting are common strategies used in ROS-responsive nanomedicine to overcome TME barriers in recent studies. For example, Zhu et al. developed a novel MnOOH nanocatalyst with flake-like morphology (~80–100 nm), which can catalyze GSH autoxidation to deplete GSH and generate H2O2 concurrently, further H2O2 decomposition produced abundant ROS such as HO•, the released Mn2+ activated the cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, and promoted macrophage M1 polarization and innate immunity [63]. As an inhibitor of Nuclear Factor Erythroid 2-Related Factor 2 (NRF2), clobetasol propionate (CP) was co-loaded with ROS-cleavable docetaxel prodrug (DTX-L-DTX, L as thioketal linker) in a recent study, forming DTX-L-DTX/CP@PEGylated liposomal nanoparticle. NRF2 inhibition by CP reduced antioxidant gene expression and increased ROS, which triggered DTX release; further, radiotherapy can amplify ROS and result in a self-sustainable cycle. These synergistic targeted/chemo/radio therapy accomplished enhanced ROS, DTX release, and tumor inhibition performance both in vitro and in vivo, with alleviated systemic toxicity [64].
Immunomodulatory ROS-responsive nanomedicine was frequently seen in recent studies, primarily because ROS exhibit dual regulatory roles in the TME by regulating ICD and reprogramming immune cell functionality, and targeted release of different immunomodulatory components at the tumor region with already elevated or even amplified ROS can further activate anti-tumor immunity based on other chemotherapy/photo-dynamics (PDT)/sonodynamic therapeutics (SDT), thereby overcoming the immunosuppressive bottleneck of single therapy [65,66]. In following studies, nanocomplex of anti-mouse programmed death ligand 1 antibody (αPDL1)/glucose oxidase [67], carbon dots conjugated to immunoadjuvant-intensified Limosilactobacillus reuteri probiotic and cytidine phosphorylated guanine (CpG)-encapsulated mulberry leaf lipid nanoparticles [68] and copper ionophore stabilized by polydopamine and hydroxyethyl starch with folate targeting [69], were selectively delivered to cancer cells with several ROS-responsive nanoplatforms, along with Fenton reaction-mediated chemodynamic therapy (CDT) [67], NIR-mediated-photothermal/photodynamic therapy (PTT/PDT), promoting beneficial bacteria abundance [68] and enhanced cuproptosis [69], these nanomedicines also boosted TME modulation and anti-cancer immunotherapy function such as PDL1 blocking, DC maturation promoting and ICD.

2.4.
Enzyme-responsive nanomedicine
Enzyme-responsive nanomedicine relies heavily on specific enzymes that are overexpressed in the unique TME compared to normal tissues (e.g., cathepsin B, CTSB; MMP-2; γ-glutamyl transferase, GGT; adiponectin, APN). These enzymes catalyze chemical reactions for many crucial biological and metabolic processes, but their dysregulation and overexpression also play a pivotal role in tumor initiation, growth, angiogenesis, invasion, metastasis, as well as immune suppression [16]. This enzymatic heterogeneity enables precise activation of nanomedicine that is engineered with high selectivity enzyme-cleavable motifs, which undergo site-specific hydrolysis upon encountering target enzymes. Such cleavages trigger cascading effects from structural transformation (e.g., from nanoparticles to nanofibers) and charge reversal to achieve controlled drug release, thereby enhancing tumor accumulation, deep penetration, and intracellular delivery [70–73]. For example, fibroblast-activated protease (FAP) overexpressed on CAFs and GGT overexpressed on cancer cells were used to cleave a dual-enzyme-responsive polymer-conjugate containing SN38, the cleavage of responsive moieties in tumors generated cationic surfaces to induce mutual transcytosis between CAFs and cancer cells, facilitating deep penetration. It also achieved effective tumor growth suppression and metastasis inhibition with SN38 released by intracellular GSH triggers [70]. A CTSB-responsive branched glycopolymer nanoassembly for co-delivering PTX and AKT inhibitor capivasertib (CAP) was also synthesized in another study. Overexpressed CTSB cleaved Glycine-Phenylalanine-Leucine-Glycine linker in tumor lysosomes and released PTX and CAP; CAP inhibited PI3K/AKT pathway, enhanced cytotoxicity of PTX toward gastric cancer cells, and induced apoptosis synergistically, with reduction in toxicity of PTX and CAP in vitro and in vivo [71].
Enzyme-responsive nanomedicine was also used in synergistic therapies (chemo-PDT, immunotherapy, etc.) in recent studies to enhance anticancer efficacy. For example, a lymph node-targeting pH/enzyme dual-responsive nanomedicine for programmed antitumor immunity was established recently. It released dimeric amidobenzimidazole to activate DCs and T cells in acidic lymph nodes (pH 6.3–6.7), the remaining nanomedicines hitchhiked on PD-1+ T cells to tumors, where MMP-2 triggered aPD-1 release for checkpoint blockade, while GSK entered tumor cells to induce pyroptosis and ICD with a combination of granzyme B. This positive feedback loop of cancer-immunity cycles significantly inhibited tumor growth, prolonged survival, and suppressed recurrence/metastasis in 4T1-tumor BALB/c mice [74]. A self-assembling lonidamine (LND)-peptide conjugate compromising a mitochondria targeting peptide sequence, a CTSB-cleavable sequence and a hydrophilic PEG tail (LND-1-PEG) was also designed to enhance the bioavailability of a GSH-activatable photosensitizer PSD (protoporphyrin IX linked to disperse blue 3 via disulfide bond), the resulted dual responsive nanomedicine LND-1-PEG@PEG responded specifically to elevated CTSB levels within the cancer cells and release PSD and LND-1; PSD is activated by GSH for PDT, while LND-1 targeted mitochondria by forming nanofibers to enhance cytotoxicity via ROS, mitochondrial dysfunction and DNA damage [75].
Enzyme-responsive nanomedicine can also take advantage of the newly appeared nanozyme, which are specific nanomaterials endowed with intrinsic biocatalytic activities; They represent a relatively novel category of substances capable of mimicking the catalytic functions of natural enzymes [76]. Due to their inherent advantages of superior operational stability, cost-effectiveness, and tunable catalytic activity, a nanozyme-based system to amplify ROS and oxidative stress was also combined with enzyme-responsive as a strategy for tumor ablation. Cu-doped polypyrrole nanozymes (CuP) were synthesized using Copper(II) chloride (CuCl2) as oxidant and PEGylated to form CuPP (Figure S2). It exhibits trienzyme-like activities, including catalase, GSH peroxidase, and peroxidase via Cu+/Cu2 + redox cycles, with 1064 nm hyperthermia enhancing these activities, its immune response remodeling, and immune cells recruiting and activating function combined with αPD-L1 achieved nearly complete tumor inhibition in 4T1-tumor Balb/c mice [77].

2.5.
ATP-responsive nanomedicine
ATP is an indispensable energy-rich fuel and also a key signal molecule mediating inflammatory cascades, immune cell activation, and intercellular communication. Normal intracellular ATP concentration is maintained at approximately 5 mM, and extracellular ATP remains in the nanomolar range under non-stressed conditions, while the ATP levels in the tumor extracellular space are several orders of magnitude higher than in normal organs and reach millimolar concentrations. This disparity arises from necrotic tumor cells, controlled pannexin/connexin hemichannel secretion, ATP-containing vesicle exocytosis, and membrane transporter-mediated efflux, particularly under hypoxic, ischemic, inflammatory, as well as therapeutic drug tumor killing stress in tumors. Tumor extracellular ATP metabolism is tightly regulated by ectonucleotidases, mainly be hydrolyzed to adenosine 5’-monophosphate (5’-AMP) and adenosine (Ado) step by step by CD39 and CD73. This elevated ATP-Ado axis forms a TME immunomodulatory enzymatic cascade, with ATP boosting tumor cell growth/angiogenesis/metastasis via purinergic receptors and Ado impairing cytotoxic T cells and strengthening immunosuppressive cells [78]. ATP responsiveness of ATP-responsive nanomedicine is achieved through electrostatic interaction, host-guest recognition, and enzyme catalysis of structures such as ATP-binding aptamers, cationic polymers, and peptide/protein conjugates [79].
By manipulating ATP concentration heterogeneity, ATP-responsive nanomedicine can enable tumor-specific drug release, enhance the efficacy of tumor treatment, and reduce toxicity. For example, an ATP-sensitive celastrol (CEL)-Fe(III) chelate prodrug was synthesized and encapsulated in ROS-sensitive PEG-grafted polymer containing thioketal groups and polyethylenimine – modified F127, forming an ATP-ROS dual-responsive nanosystem (CEL-Fe NPs). CEL-Fe remained stable in normal tissues, reducing toxicity via weakened interaction with heat shock protein 90/cell division cycle. In tumors, ROS degraded the polymer shell and accelerated the release of CEL-Fe, with high ATP competitively bound Fe(III) to release active CEL and Fe(III) generated ·OH via Fenton reaction to enhance efficacy [80]. Tumor targeting of ATP-responsive systems was also integrated with imaging; their ability to serve diagnostically and therapeutically at the same time is called theranostics. Chen et al. developed an ATP-responsive zeolitic imidazolate framework (ZIF)-90-based fluorescence nanosystem by encapsulating doxorubicin (DOX) and the photosensitizer asymmetrical cyanine dye Cy via self-assembly. It remained stable in circulation and accumulated in tumors via the EPR effect. Intracellular high ATP disrupted ZIF-90 by competing with Zn2+ because of their high affinity for coordination, releasing DOX and Cy. Cy emitted NIR fluorescence for imaging and generated ROS under NIR irradiation for PDT. This synergistic therapy enhanced anti-tumor activity significantly in HeLa tumor-bearing nude mice with no major organ damage [81].
ATP-depleting nanomedicine was also used to reverse drug resistance and inhibit metastasis in recent studies. For example, Hou et al. developed an intracellular ATP sequestration system using the transformable nucleopeptide NLS-FF-T (Figure 4). It comprises coumarin, thymine (for ATP-sequestering), KLVFF (β-sheet-forming), PEG, and karyopherin subunit alpha-2 (KPNA2)-binding motifs. NLS-FF-T can self-assemble into nuclear-targeted NPs with encapsulated ATP-binding sites, transforming into nanofibers upon KPNA2 interaction to expose sites and sequester ATP, inducing mitochondriopathy-like damages to inhibit tumor [82]. Ding’s team also developed a pH/ROS dual-responsive self-adaptive nanocarrier encapsulating camptothecin. Exposing its acid-labile 2,3 2,3-dimethylmaleic anhydride-masked arginine residues to acidic TME can enhance cellular uptake, ROS can trigger cinnamaldehyde release and generate excess ROS that reacted with arginine to produce peroxynitrite (ONOO−) and activateed MMPs to degrade ECM for deep penetration. ONOO− also inhibited mitochondrial function, reducing ATP production and ATP-dependent tumor-derived microvesicles to suppress metastasis [83].

Modulation of the ATP-Ado axis via nanomedicine was another strategy to enhance antitumor immunity and overcome immunosuppression. For example, Wu et al. developed a lipid-coated micelle loaded with palmitoylated stapled oncolytic peptide (PalAno) and Ado 2A receptor (A2AR) inhibitor CPI-444. It targeted CD44-overexpressing tumors via A6 peptide, releasing cargo in acidic TME. PalAno can lyse tumor cells and release ATP and antigens to trigger ICD; CPI-444 blocks adenosine-A2AR signaling, reversing adenosine-mediated immunosuppression by modulating the ATP-adenosine axis [84]. Zhan’s team also developed a dual-cascade activatable nanopotentiator by crosslinking chlorin e6 (Ce6)-conjugated Manganese(IV) Oxide (MnO2) nanoparticles with Ado deaminase (ADA) via ROS-cleavable linkers. Under ultrasound in the tumor microenvironment, MnO2 reacted with GSH to generate Mn2+ that mediated CDT by producing ·OH, while sonosensitizer Ce6 enabled SDT to generate singlet oxygen (1O2). These ROS cleaved linkers and release ADA to degrade adenosine, modulating the ATP-adenosine axis and enhancing antitumor immunity, such as increasing CD4+/CD8+ T cells and cytokines [85]. Activation of the cGAS-STING pathway serves as a pivotal tactic for bacteria to elicit antitumor immunity. Yang et al. engineered an ATP-responsive manganese (Mn)-based bacterial hybrid material that degrades in the ATP-enriched TME, enabling the release of Mn2+ and the exposure of encapsulated bacteria. This dual-event cascade synergistically amplified cGAS-STING activation, as Mn2+ potentiated cGAS sensitivity toward bacterial-secreted extracellular DNA, effectively inhibited tumor growth in mice and rabbit [86].

3.
External stimulus-responsive nanomedicine
3.1.
Light-responsive nanomedicine
The field of externally responsive nanomedicines-particularly those activated by light stimuli-has witnessed remarkable advances in recent years, offering new avenues for precise, minimally invasive, and synergistic anticancer therapies. For the application of light-responsive nanomedicine in anticancer treatment, the three essential conditions are light, photosensitizers (PSs), and oxygen in the tissues. The two predominant light-responsive nanomedicine modalities in oncology are PDT and PTT [87]. Since thermal-responsive nanomedicine is usually combined with other stimulus-responsive nanomedicines in applications, it will be introduced alongside different types of externally responsive nanomedicines.
PDT relies on the activation of PSs that selectively accumulate in target tumor tissue. Upon irradiation with light of a specific wavelength on the location of the PSs, they are activated to react with oxygen, generating direct damage to the plasma membrane or causing cytotoxic ROS that induce tumor cell death [88,89]. PS has been successively developed and evaluated, evolving from the first generation represented by Hemaporphyrin derivative (HpD), which suffers from low light absorption rates and a long half-life, to the second generation. A typical example of the latter is 5-aminolevulinic acid (5-ALA), which addressed the limitations of first-generation PS but still exhibits drawbacks such as inherent toxicity and poor stability. The latest third-generation PS is primarily derived from modifications of the second generation: one approach involves incorporating functional components (e.g., peptides, antibodies, amino acids, and carbohydrates) on various easily degradable organic and inorganic materials such as liposomes [90], micelles, nanoemulsions, semiconducting polymers [91], graphene oxide, polydopamine, and exosomes [92] to enhance their stability and efficacy, offering excellent biocompatibility. While another type adopts encapsulation with nanoparticles to increase the stability and transport of hydrophobic PSs.
In contrast to PDT, PTT functions through light-mediated excitation of photothermal agents (PTA), converting absorbed light energy into localized heat and raising the temperature of the TME to a range of 41–48°C, which is sufficient to induce irreversible damage to cancer cells, including protein denaturation, membrane disruption and ultimately cell death and trigger the release of drugs or the ablation of tumors, while having minimal impact on normal cells [93]. PTA is capable of surpassing interference from biological chromophores during light absorption processes [82]. Presently, NIR radiation falling within the biological transparency window of 750–1,350 nm is the primary laser source employed in PTT, enabling enhanced penetration into deep-seated tissues. This NIR spectrum is further categorized into two subwindows: the NIR-I region (spanning 750–1,000 nm), which is restricted by shallow tissue penetration, and the NIR-II region (ranging from 1000–1,350 nm), which exhibits superior tissue penetration depth and reduced biological interference. Under such application scenarios, nanoscale PTAs demonstrate distinct advantages over small-molecule counterparts: they not only achieve higher accumulation within tumor tissues but also provide diverse imaging pathways and a range of favorable functional characteristics [94]. Materials including gold nanoparticles [95–97], metal oxide nanoparticles [98], silica, upconversion NPs, and quantum dots are characterized by high photothermal conversion coefficients and can be functionalized as PTAs to achieve targeted delivery and deep tumor penetration [91,95,96,99–103].
Light-responsive nanomaterials typically incorporate molecules or structures that react to light by changing their physical or chemical properties, offering precise control over drug delivery and activation using external light sources. They can also be loaded with a variety of PDT and/or PTT active molecules. Combining light-responsive nanomedicine with immunotherapy, like checkpoint blockade [104], modulation of immune cells or TME, and cancer vaccine, has emerged as a promising approach in cancer treatment, given its favorable synergistic effects in tackling both primary tumors and metastatic lesions [105]. For combination with checkpoint blockade, Fan’s team [106] developed a mild NIR laser-triggered photo-immunotherapy platform that consists of a PD-L1 conjugated with gadolinium-doped NIR-emitting carbon dot, which showed negligible cytotoxicity without light stimulation. Under NIR irradiation, it exerted photothermal/photodynamic effects to ablate tumors, induced ICD, and promoted CD8+ T cell infiltration. It outperformed single phototherapy or PD-L1 in enhancing T-cell infiltration, reducing tumor growth, and inhibiting metastasis.
For CAR-T cell therapies, particularly in solid tumors, light-responsive nanomedicine provides precise and controllable activation, as well as enhanced safety. Chen and coworkers [107] developed CAR-T biohybrids (CT-INPs) by conjugating CAR-T cells with indocyanine green NPs (INPs) to enhance solid tumor immunotherapy. These biohybrids retain CAR-T activity while gaining photothermal and tracing abilities. Under NIR laser irradiation, CT-INPs produced mild photothermal effects (<45°C) that disrupted the extracellular matrix, dilated blood vessels, loosened tumor tissue, and triggered chemokine release without harming CAR-T function, thereby remodeling the TME to break physical and immunological barriers and enhance CT-INPs’ infiltration. In Raji tumor models, CT-INPs outperformed CAR-T alone, INPs, or PBS in inhibiting tumors with good biocompatibility, offering a reliable strategy for solid tumor immunotherapy via TME reconstruction.
Light-responsive nanomedicine can also act as a nucleic acid cargo [108]. Jia et al. [109] addressed the shortcomings of low gene delivery/release efficiency and the inability to trigger release on demand by creating an intelligent light-responsive nanodelivery and release systems (NDRS). This system not only responded to NIR light, achieving selective siRNA delivery and controlled release, but also efficiently converted absorbed NIR light into heat, enabling gene-photothermal synergistic therapy in vitro and in vivo. Similarly, Chen et al. [110] developed a photosensitive spherical nucleic acid NDRS for the delivery and release of siRNA and antisense oligonucleotides, which can simultaneously facilitate the release of siRNA and antisense oligonucleotides to achieve cytoplasmic targeting via lysosomal escape. The system also inhibited the expression of hypoxia-inducible factor-1α and Bcl-2, thereby inhibiting tumor cell growth.
While clinical translation is advancing, challenges remain. The majority of current NPs used in PDT and PTT are nonbiodegradable and may gather in the body if they are not excreted, which confines their clinical application. The development of biodegradable nanostructures with high phototherapeutic properties should be the focus of future research.

3.2.
Magnetic field-responsive nanomedicine
Magnetic field-sensitive nanoscale therapeutic agents possess magnetic responsiveness with superparamagnetic behavior beyond distinct properties of very small particle size, and large surface area has emerged as a revolutionary approach in cancer treatment [111]. The most common components of magnetic nanoparticles (MNPs) are iron and iron oxides, which have low toxicity and a relatively higher abundance and low cost of the precursors. Depending on specific applications in the biomedical field, the composition of MNPs has evolved to include Zn, Ni, Co, and rare earth metals. MNPs are synthesized through chemical (coprecipitation method, thermal decomposition, microemulsion method), physical methods (mechanical milling, vapor deposition and lithography, electrical explosion of wires), and biological methods [112,113]. The biological method, also called the green synthesis method, is a bottom-up process that produces MNPs by oxidizing or reducing metallic ions with the help of secreted biomolecules such as proteins, enzymes, polysaccharides, and carbohydrates, which allows for the production of NPs economically, at milder temperatures, pressures, and pH levels [114,115].
Three primary areas of clinical research on MNPs are targeted drug delivery, imaging, and thermal therapy. Due to their ferro- and superparamagnetism, MNPs made from iron, nickel, cobalt, and other materials can be guided by a magnetic field [116]. Iron oxide-based MNPs, such as magnetite (Fe3O4) and maghemite (γ-Fe2O3), are commonly used in biomedical applications because of their low toxicity and high biocompatibility [117,118]. Additionally, superparamagnetic iron oxide nanoparticles (SPIONs) conjugated with therapeutic agents and manipulated by an external magnetic field have led to the development of another promising approach known as magnetic drug targeting (MDT) [119]. When combined with an MRI image, magnetic hyperthermia shows an advantage in that the distribution of MNPs in the body can be visualized, which enables precise thermal ablation of target tissue. Innovative methods also exploit magnetic field-induced ICD, ferroptosis, and chemodynamic therapy, expanding the therapeutic options for treating drug-resistant and metastatic cancers [120–122].
In recent years, there has been significant development in the creation of multifunctional magnetic nanoparticles for the detection and treatment of cancer. Magnetogenetics is an emerging field that enables the noninvasive, remote control of cellular functions by combining magnetic fields with genetically engineered MNPs. This technology offers deep tissue penetration and precise spatiotemporal control, making it a promising tool for neuroscience, immunotherapy, gene editing, and regenerative medicine. For instance, genetically engineered immune cells (e.g., T cells, NK cells) can be remotely activated or modulated using magnetic fields, enhancing their proliferation, activation, and tumor-targeting abilities. As Jiang’s team showed in their research, via penetration depth-unlimited mild magnetocaloric regulation (~40°C), their magnetogenetic nanoplatform, which was synthesized by loading a heat-inducible plasmid DNA (HSP70-IL-2-EGFP) on polyethyleneimine (PEI)- and folic acid (FA)-modified ZnCoFe2O4@ZnMnFe2O4 MNPs, induces IL-2 expression in orthotopic liver cancer, promotes proliferation and activation of tumor-infiltrating NK cells through the IL-2/IL-2 R pathway, and achieves significant tumor inhibition. Magnetogenetics also enables remote gene editing using CRISPR systems. Shim et al. used magnetomechanical force generated by the MPNs to activate pre-encoded Piezo1, the mechanosensitive ion channel, on the target cell. The activated Piezo1 further triggers the intracellular Ca2+ signaling pathway, inducing the pre-encoded genes to express genes of interest, which is the Cas9 protein for the CRISPR regulation of the target proteins. This breakthrough tool demonstrates remarkable potential in overcoming drug resistance, enhancing chemotherapy sensitivity, and improving immunotherapy strategies such as CRISPR-engineered CAR-T cells [123].
MNPs, especially iron oxide-based types, can also function as “nanozymes”-artificial enzymes that mimic the catalytic activity of natural enzymes. MNPs can catalyze the decomposition of hydrogen peroxide (H2O2) to hydroxyl-free radicals through a heterogeneous Fenton reaction, which occurs on iron oxide nanocrystal surfaces, with the reaction rate being affected by the interface between the nanocrystals and their surrounding medium. The pH-dependent and size-dependent catalytic activity of MNPs enables targeting of specific cell populations based on cellular uptake and iron metabolism. As Trujillo-Alonso’s team showed in their work, the FDA-approved ferumoxytol, an iron oxide MNP, displayed anti-leukemia efficacy against cells with low ferroportin levels, which show lower cellular uptake and iron metabolism [124].
In conclusion, magnetic field-responsive nanomedicine offers a powerful and versatile platform for cancer therapy, enabling targeted, multimodal, and image-guided treatments with promising preclinical efficacy. Despite extensive research, gaps remain in clinical translation [125]. MNPs have a complex internal structure owing to the complex preparation methods. One of the biggest problems in the field is the absence of standardized testing procedures for MNPs, which causes a lot of discrepancies in study results. The inconsistency limits the capacity to conduct significant cross-study comparisons, which are crucial for verifying results and constructing a cohesive repository of information.

3.3.
Ultrasound-responsive nanomedicine
Ultrasound-responsive nanomedicine utilizes various nanocarriers, including liposomes, polymersomes, micelles, nanobubbles, and biomimetic platforms [126–128]. These carriers are engineered to release drugs upon ultrasound stimulation via mechanisms including cavitation, sonoporation, and thermal effects, enabling spatiotemporal control over drug delivery [129–131]. The mechanical impacts of ultrasound are mainly derived from its inherent physical vibrational properties, exerting biological effects via cavitation phenomena and acoustic radiation force. Low-frequency ultrasound (below 1 MHz) combined with moderate intensity (ranging from 0.4 to 1.5 W/cm2) tends to induce pronounced mechanical effects, whereas short-pulse stimulation (less than 1 s) and low duty cycles (under 40%) can mitigate heat buildup and strengthen the predominance of mechanical actions. Some systems are dual- or multi-responsive, combining ultrasound with pH, redox, or enzyme sensitivity for enhanced selectivity [132–134]. Ultrasound-responsive nanomedicine has demonstrated significant efficacy in preclinical models of various cancers, including breast, pancreatic, glioblastoma, and lymphoma [135–138].
Numerous preclinical studies, along with a handful of clinical investigations, have demonstrated precise targeting capabilities and effective tumor ablation with minimal off-target impacts [139]. As ultrasound-responsive carriers, microbubbles and nanodroplets can encapsulate chemotherapeutic agents, and under ultrasound stimulation, they enhance drug delivery to tumor sites [140]. For instance, perfluorocarbon-based nanodroplets loaded with DOX undergo a liquid-to-gas phase transition when activated by ultrasound, which promotes drug release at the tumor site while reducing systemic toxicity [141]. When combined with high-intensity focused ultrasound (HIFU), local hyperthermia is induced, which increases the permeability of tumor vasculature and thereby facilitates the extravasation of nanodroplets [142]. Qin et al. [143] reported in their study that HIFU enhanced oncolytic virus therapy by enabling precise delivery and on-demand release, boosting oncolytic virus replication and tumor-killing efficiency, aiding intracellular survival, and augmenting anti-tumor immunity via synergistic effects.
Moreover, ultrasound-induced ICD can enhance tumor immunogenicity. For instance, combining HIFU with anti-PD-1 therapy has been shown to enhance T-cell infiltration and tumor suppression in murine models. Further research has confirmed this synergistic effect and highlighted its potential for clinical translation [144]. In contrast, low-intensity focused ultrasound (LIFU) operates at a much lower acoustic intensity (0.4–3.2 W/cm2 at the focal spot), minimizing damage to surrounding normal tissues while maintaining precise targeting of tumor tissue and reducing nonspecific-phase transformations that could harm healthy cells [145]. LIFU promotes dendritic cell recruitment by upregulating chemokines [146], and ultrasound-guided delivery of CpG-loaded nanoparticles can effectively activate DCs, enhancing their antigen-presenting function and strengthening immune responses, as validated in studies by Shih [147].
SDT uses ultrasound to activate sonosensitizers and generate ROS, offering precise tumor targeting with deeper tissue penetration and fewer side effects and making it a promising noninvasive alternative to existing cancer therapies with greater versatility and safety in clinical settings. By incorporating acoustically responsive nanomaterials, SDT enables controlled drug release and activation, further reducing collateral damage to healthy tissues; it can also be combined with chemotherapy, PTT, or immunotherapy to achieve synergistic effects [148]. SDT effectively induces and releases the tumor-associated antigens and damage-associated molecular patterns, thereby activating inflammatory responses in TME and draining lymph nodes, inducing systemic antitumor immunity and immune memory, and inhibiting tumor growth and recurrence [149], as explored in Xue and colleagues’ work [150], which underscores SDT’s potential to activate innate immunity (M2-to-M1 macrophage repolarization (Figure 5), NK cell response) and remodel the immunosuppressive TME to boost adaptive immunotherapy, achieving over 98% tumor inhibition with good biosafety and significant anti-metastatic effects.

Ultrasound imaging plays a key role in guiding nanoparticle delivery, with multimodal platforms integrating ultrasound-responsive nanomedicine with other imaging modalities. For example, iron oxide nanoparticles loaded with doxorubicin enable both BBB and multidrug-resistant glioma cells and provide site-specific magnetic targeting [151]. Scholars have also developed fluorescence-labeled nanoparticles to support real-time tracking. As shown in Chen’s study [152], they constructed indocyanine green (ICG)-doped microbubbles (MBs-ICG) for visualizing focused ultrasound-mediated BBB opening and enhancing PTT for glioblastoma. In Du’s work [153], they developed a real-time imaging-guided ultrasound-activatable tumor-targeted therapy, in which focused ultrasound was used to enable engineered bacteria (expressing Cytolysin A and miRFP720), optimizing therapy and PD-L1 efficacy. Such imaging data – including tumor size and nanoparticle localization – allows for adjustments to ultrasound parameters, and real-time monitoring facilitates timely modifications to treatment plans. As emphasized by Wang et al. [154], this personalized approach, driven by real-time imaging, is vital for tailoring therapies to individual patient needs.
In summary, ultrasound-responsive nanomedicine shows promise in targeted chemotherapy, SDT, immunotherapy enhancement, and theranostic platforms. Despite significant progress, gaps remain in clinical translation. There is a need for more studies on tumor heterogeneity, immune modulation, and standardized protocols for ultrasound parameters. A key bottleneck for ultrasound-mediated therapies across experimental settings is the lack of standardized protocols for critical parameters-including ultrasound intensity, pulse duration, tissue attenuation coefficient, frequency, single-treatment duration, treatment cycles, and thermal/cavitation effects. For example, in HIFU applications, ultrasound reflection by gas- or bone-containing tissues may cause unintended effects like skin burns. While ultrasonography shows promise in cancer treatment, its thermal and non-thermal effects can damage healthy tissues and trigger adverse reactions [155].

4.
Dual- and multiple-responsive nanomedicine
As mentioned above, dual- and multiple-stimulus-responsive nanomedicines are designed to integrate responsiveness to two or more triggers and have emerged as a prevailing trend to address the complexity and heterogeneity of the TME. They represent a significant leap forward in precision cancer therapy, offering enhanced selectivity, controlled drug release, and potential to overcome the limitations of traditional monotherapies. These smart nanocarriers are engineered to respond to two or more internal or external stimuli, and these responsive components can be constructed in forms of internal-internal, internal-external, and external-external, but most of the recent studies include at least one internal stimulus responsive element, and the last combination is relatively less seen.
Internal-internal dual-responsive nanomedicines achieve precise tumor targeting by integrating two complementary internal stimuli to ensure stable circulation in normal tissues while triggering rapid drug release at tumor sites. For example, the self-assembled GSH/ROS dual-responsive supramolecular nanomedicine developed by Hu et al. can co-deliver nitric oxide donor and IDO inhibitor NLG919. GSH triggered NO release and NLG919 liberation by cascade ROS activation, achieving remarkable tumor accumulation and bioavailability, with NO-induced tumor pyroptosis and NLG919 blocked IDO-mediated immunosuppression. This dual internal responsiveness synergistically enhances the dendritic cell maturation and CD8+ T cell infiltration, achieving robust antitumor immunity [156]. Tumor penetration is a critical factor for therapy success, especially in solid tumors with dense extracellular matrix (ECM). Dual-responsive systems can be engineered to improve penetration by responding to TME stimuli. Ding et al.’s pH/ROS dual-responsive nanocarrier exposes arginine residues in acidic TME to enhance cellular uptake, while ROS triggers cinnamaldehyde release to generate ONOO−, which degrades ECM via activation of matrix MMPs and promotes deep pancreatic cancer penetration, further maximizing intratumoral drug concentration, minimizing off-target toxicity specificity [83].
Internal-external dual-responsive systems combine TME-specific internal triggers with externally controllable stimuli, balancing specificity and flexibility. For example, Xing et al. reported a pH/light dual-activatable binary CRISPR nanomedicine consisting of a thioketal-cross-linked polyplex core and an acid-detachable polymer shell. In the acidic TME (pH ~ 6.5), the polymer shell disassociates to enhance cellular internalization, and 660 nm laser irradiation induces endosomal escape and payload release. This system co-delivers CRISPR-Cas9 (for PD-L1 disruption) and CRISPR-dCas9 (for MHC-1 upregulation), achieving precise gene editing in tumors while avoiding off-target effects [157]. For deep tumors, external stimuli like ultrasound (instead of light) can be paired with internal triggers to overcome penetration limitations, as demonstrated by Zhan et al.’s ultrasound/ROS dual-responsive nanopotentiator for chemodynamic/sonodynamic therapy [85]. A multiple responsive nanomedicine was also engineered by Shi et al. by a combination of pH/GSH dual-triggered systems with light-responsive modality, containing a core-shell nanostructure composed of hollow mesoporous silica loaded with DOX, coated with polydopamine and MnO2, and functionalized with hyaluronic acid. In the acidic and GSH-rich TME, MnO2 degrades to release Mn2+ for MRI imaging and depletes GSH, while polydopamine’s pH responsiveness and photothermal properties further regulate drug release. This multiple stimulus responsiveness ensured DOX is specifically released in tumors, with minimal leakage in normal tissues, enhancing therapeutic efficacy [158].
Hyperthermia induced by light/magnet/ultrasound is a kind of external-external dual responsiveness, as we discussed above [42,121,142]. These systems offer precise spatiotemporal control, but their application is limited to superficial tumors; thus, they are often combined with internal stimuli. Light suits superficial tumors, while ultrasound/magnetic fields enable deep-tumor targeting. Gold nanotetrapod-based nanoprobes respond to ultrasound (1–3 MHz) via cavitation-induced shell disruption and NIR light (808 nm) via photothermal-enhanced drug release, enabling deep-tumor therapy. This light-ultrasound-magnetic combinations leverage NIR light’s photothermal conversion and magnetic targeting/hyperthermia [159]. External stimuli also encompass challenges of limited penetration (e.g., NIR light attenuation in deep tissues, constrained magnetic/ultrasound reach), which can be mitigated by adopting long-wavelength NIR-II light or implantable micro-devices. Another problem is the complex synthesis and cumbersome scalable production of external-external stimulus-responsive multifunctional nanocarriers, which may be addressed by integrated copolymerization strategies or continuous microfluidic fabrication platforms
In summary, the choice of dual- and multiple stimulus combinations depends on tumor location and characteristics: light-based external stimuli are suitable for superficial tumors, while ultrasound or internal-internal combinations are preferred for deep tumors. Additionally, optimizing nanocarrier size and surface properties further enhances tumor penetration and targeting, ensuring overcoming multidrug resistance (MDR) in diverse tumor types.

5.
Conclusion
Overall, substantial advances in the application of stimulus-responsive nanomedicines for anti-cancer therapy have been observed, from single-stimulus systems to multi-responsive platforms that synergize with immunotherapy, radiotherapy, and other modalities (Figure S3). Stimulus-responsive nanomedicines represent a transformative advancement in precision cancer therapy, addressing the inherent limitations of conventional nanomedicines by introducing an additional layer of spatiotemporal targeting beyond passive (EPR effect) and active (antibody/ligand-mediated) strategies. Internal stimulus-responsive nanomedicines leverage TME-specific cues and offer inherent biocompatibility and deep-tissue applicability. Recent advances have illustrated their advantages of targeted drug release (via cleavage of pH-sensitive hydrazone bonds or GSH-triggered disulfide bond degradation), enhanced tumor penetration by size transformation and charge reversal for transcytosis, and reversal of MDR through GSH depletion, ROS amplification, and disrupting tumor-stromal crosstalk to improve drug access. Additionally, these systems modulate the TME by reprogramming immunosuppressive cells and inducing ICD, thereby activating adaptive immunity. For external stimulus-responsive nanomedicines, light-responsive systems (PDT/PTT) enable noninvasive ablation and synergistic chemo-immunotherapy via NIR-triggered ICD, while MNPs facilitate targeted delivery through magnetic guidance and multimodal imaging (MRI), enhancing penetration through magnetothermal-induced TME remodeling. Ultrasound-responsive systems achieve deep-tissue drug release and SDT-mediated ROS production, overcoming light penetration limitations. Dual/multi-stimulus-responsive platforms integrate internal and external triggers to address TME heterogeneity, further improving specificity and efficacy.
We also acknowledge additional stimuli-responsive nanomedicine modalities that were not elaborated on in detail herein, including lactate-responsive systems-primarily utilizing microorganism-derived lactate oxidase to confer responsiveness [160], nanoplatforms responsive to gastrointestinal microenvironmental characteristics (e.g., low pH, enzymes, and bile acids, et al.) that are associated with or produced by the microbiome (notably, the formal terminology “microbiome-responsive nanomedicines” has not yet been established) [161], and electric field-responsive nanomedicines engineered with functional materials such as conducting polymers and electrically responsive hydrogels, which are confronted with challenges including deep-tissue electric field attenuation, biocompatibility limitations, and safety considerations [162]. Notably, only a limited number of these stimuli-responsive nanomedicines have advanced to clinical trials in recent years, with merely three products successfully launched onto the market (summarized in Table S1). The first clinically approved thermosensitive ThermoDox® enhances tumor-site drug bioavailability via local hyperthermia but relies on specialized hyperthermia equipment and may cause risks of nonspecific healthy tissue damage, NanoTherm® combines magnetic targeting and thermotherapy, improves local drug concentration but harbors potential toxicity resulted from iron oxide nanoparticles and penetration limitation. Both the benefits and drawbacks of these two approved external stimulus-responsive nanomedicines are derived from the outer stimulus suppliers and physical materials used, while limitations of internal stimulus-responsive on-market Opaxio® and products in clinical trials are inherent to differential expression/concentration of triggers between on tumor surface and in its core.

6.
Future perspectives
To expedite their clinical translation and broaden their therapeutic impact, several critical challenges must be effectively addressed. Tumor heterogeneity and the dynamic complexity of the TME pose significant hurdles, as variability in stimulus levels (e.g., inconsistent EPR, acidity/ROS gradient) across patients and even within individual tumors can lead to off-target effects or insufficient drug release. To mitigate these issues, personalized nanomedicine design, guided by patient-specific TME profiling (e.g., via MRI or photoacoustic imaging), allows tailored responsiveness to individual tumor signatures. Multi-stimulus systems, such as dual-enzyme-responsive polymer-drug conjugates that exploit mutual transcytosis between cancer-associated fibroblasts and tumor cells, adapt to heterogeneous microenvironments for deeper penetration [70]. Additionally, combining nanomedicines with TME modulators (e.g., EGCG to disrupt collagen stroma [33]) stabilizes stimulus levels, enhancing activation consistency.
Long-term toxicity and immunogenicity of stimulus-responsive nanomaterials remain underexplored, with concerns over accumulation of non-degradable components in vital organs and immune reactions to synthetic surfaces (e.g., anti-PEG antibodies). Addressing these requires prioritizing biodegradable materials, such as redox-responsive cysteine polymers that degrade into amino acids [51], or biomimetic coatings (e.g., RBC membranes [26]) to evade immune clearance. Rigorous long-term toxicity screening, including multi-omics analyses of immune responses and organ function, complements acute toxicity assays to ensure safety.
Clinical translation is further limited by challenges in maintaining stability during storage and circulation, as well as scaling manufacturing to GMP standards – particularly for complex multi-responsive architectures prone to batch variability. Stabilization strategies, such as silica-coated metal-organic frameworks [33] or cross-linked matrices, prevent premature degradation, while continuous manufacturing technologies (e.g., microfluidics) enable precise control over size and responsiveness, reducing variability. Photo-initiated polymerization-induced self-assembly (Photo-PISA), for example, has enabled scalable synthesis of stable pH-responsive polymersomes [39].
Looking forward, stimulus-responsive nanomedicines hold immense potential to redefine precision cancer therapy. The integration of artificial intelligence for predictive TME modeling and nanocarrier design will accelerate the development of adaptive platforms. Combining multi-responsive systems with emerging modalities, such as CRISPR-based gene editing or microbiome modulation, could further synergize therapeutic efficacy. With advances in personalized design, biocompatible materials, and scalable manufacturing, these nanomedicines are poised to overcome current limitations, offering safer, more effective treatments for diverse cancer types.

Supplementary Material

Supplemental Material