Advancing Epigenetic Combination Therapy in Oncology: Multifunctional Nano-Drug Delivery Systems for Synergistic Efficacy and Precision Modulation

Introduction

Epigenetics refers to a dynamic and reversible regulatory mechanism that modulates gene expression through chemical modification or alteration of chromosome structure without altering the underlying DNA sequence.^1,2 This sophisticated regulation system operates through coordinated cycles of covalent modifications to DNA and histones, primarily mediated by chromatin-modifying enzymes working in a tightly regulated and synergistic manner.^3,4 These biochemical alterations ultimately lead to structural reorganization of chromatin, thereby influencing transcriptional accessibility.⁵ The epigenetic regulatory machinery comprises three functionally distinct enzyme categories: “writers”, “erasers” and “readers”.^1,6,7 Among them, writers catalyze the addition of epigenetic marks, including DNA methyltransferases (DNMTs), histone lysine methyltransferases (KMTs) and histone acetyltransferases (HATs).⁸ Erasers mediate removal of post-translational modifications, including histone lysine demethylases (KDMs) and histone deacetylases (HDACs).^9,10 Readers are specialized protein domains that recognize specific epigenetic signatures, including bromodomains and chromodomains.^11,12 Emerging evidence has established the crucial role of epigenetic dysregulation in cancer pathogenesis, with particular emphasis on the aberrant expression and functional alterations of epigenetic enzymes.^13,14 This mechanistic understanding has spurred significant therapeutic interest, positioning epigenetic modulators as promising targets for novel anticancer strategies.^15,16

Several epigenetic-targeted agents have received FDA approval for hematologic malignancies, demonstrating the clinical potential of epigenetic modulation in cancer treatment.^14,16,17 However, treatment resistance remains a significant therapeutic challenge, with some patients showing limited responses to monotherapy.^18,19 To address this limitation, combinatorial approaches integrating epigenetic drugs with conventional therapies (chemotherapy, radiotherapy) and emerging modalities (targeted therapy, immunotherapy) are being actively investigated.^20–22 Despite this promise, the clinical translation of epigenetic combination therapies faces several persistent hurdles. The epigenetic-targeted agents often act broadly on global gene expression patterns and therapeutically promising, also raise concerns regarding off-target effects and systemic toxicity. Specifically, these include the poor bioavailability and rapid clearance of some epigenetic drugs, dose-limiting off-target toxicities due to non-specific biodistribution, and the eventual emergence of multi-drug resistance. While previous reviews have extensively cataloged various combinatorial strategies, they often overlook the central pharmacological barrier: the divergent pharmacokinetic profiles between epigenetic drugs and their partner agents, suboptimal tumor accumulation, dose-limiting systemic toxicity from non-specific biodistribution, which prevents truly synergistic efficacy. Nano-drug delivery systems (NDDS) offer a promising platform to overcome these limitations through co-encapsulation of multiple therapeutics with tunable ratios, enhanced physicochemical stability and prolonged circulation, tumor-selective accumulation and controlled release.²³ Furthermore, the complexity of epigenetic regulation extends beyond nuclear modifications, encompassing organelle-specific enzyme localization (eg, mitochondrial DNMTs, lysosomal HDACs) and microenvironmental reprogramming of tumor-associated immune/stromal cells.^24–26 This multilayered regulatory network requires the development of intelligent NDDS with multi-functionality, including spatiotemporal coordination, biological navigation, and subcellular precision, etc. The development of NDDS for precision epigenetic engineering has emerged as a transformative frontier in oncology therapeutics.

Based on this, this review provides a critical analysis of epigenetic-based combination therapies in oncology. We first outline the role of epigenetic dysregulation in cancer pathogenesis and analyze the therapeutic applications of epigenetic combination approaches. Subsequently, we highlight the transformative potential of NDDS in epigenetic combination therapy, with particular emphasis on how multifunctional NDDS design enables precise therapeutic regulation. This comprehensive analysis aims to advance the clinical translation of epigenetic-based combination strategies through innovative drug delivery solutions.

Epigenetic and Drugs in Clinical

Epigenetics is a widely occurring phenomenon in tumors, and the study of epigenetics is of great significance for the individualized treatment of tumors. The main epigenetic mechanisms in tumors involve DNA methylation, histone modification, non-coding RNA regulation, and chromatin remodeling. Targeting these regulatory processes provides a promising approach for developing novel cancer therapies. This section discusses how epigenetic alterations contribute to tumor progression and reviews currently available epigenetic-based drugs used in clinical practice (Figure 1).

Figure 1 Different epigenetic modifications regulating gene expression and representative epigenetic drugs in clinical.

Epigenetic in Cancer Development

DNA Methylation

DNA methylation is one of the most central epigenetic modifications that regulates gene expression through structural alterations in chromatin, DNA conformation stability, and protein–DNA interactions while preserving genomic integrity. This heritable chemical modification modulates critical biological processes including cell cycle regulation, apoptosis, and embryonic development without altering nucleotide sequences.^27–29 Catalyzed by DNA methyltransferases (DNMTs) utilizing S-adenosylmethionine as methyl donor, the process converts cytosine to 5-methylcytosine at CpG islands, particularly in promoter regions where it induces chromatin compaction and transcriptional silencing.^30,31 The mammalian DNMT family comprises five isoforms with distinct functions: DNMT1 maintains methylation patterns during DNA replication, DNMT3a/b establish de novo methylation critical for embryogenesis and oncogenesis, DNMT3L facilitates structural coordination of DNMT3 complexes, while DNMT2 primarily methylates tRNA.^32–34 Tumorigenesis involves characteristic methylation aberrations featuring genome-wide hypomethylation that destabilizes chromatin and activates proto-oncogenes, coupled with localized CpG island hypermethylation that silences tumor suppressor genes. These dual epigenetic perturbations synergistically drive malignant transformation through disrupted genomic surveillance and dysregulated oncogenic pathways.^35,36

Histone Modifications

Histone modifications represent a fundamental epigenetic mechanism through which dynamic post-translational alterations regulate chromatin architecture and gene expression in eukaryotes. Comprising five highly conserved core proteins (H1, H2A, H2B, H3, H4), histones assemble with DNA into nucleosomes, with their N-terminal tails undergoing reversible covalent modifications including methylation, acetylation, phosphorylation, and ubiquitination.^37,38 These chemical alterations modulate chromatin compaction by altering electrostatic interactions between positively charged histones and negatively charged DNA, thereby controlling transcriptional accessibility.^39,40 The tightly regulated balance of histone methyltransferases (HMTs) and demethylases (HDMs) governs lysine/arginine methylation patterns that maintain heterochromatin integrity, mediate genomic imprinting, and influence DNA repair processes, with dysregulation implicated in oncogenesis through aberrant silencing of tumor suppressors.^41,42 Similarly, histone acetylation dynamics controlled by histone acetyltransferases (HATs) and deacetylases (HDACs) regulate chromatin relaxation through neutralization of histone charges, facilitating transcription factor binding.^43,44 Notably, these enzymatic regulators exhibit broad substrate specificity, modifying non-histone targets including p53, Myc, and β-catenin to influence protein stability and oncogenic signaling. Bromodomain-containing proteins (BRD2/3/4) further propagate acetylation signals by recognizing acetylated lysines, with their frequent dysregulation in cancers underscoring the therapeutic potential of targeting this chromatin recognition system.^45,46 The tumor-specific redistribution of histone modification patterns, particularly H3/H4 hyperacetylation and site-specific methylation aberrations, establishes these epigenetic marks as both diagnostic biomarkers and actionable therapeutic targets in cancer biology.^47,48

Non-Coding RNA Modification

Non-coding RNA (ncRNA) modifications constitute an emerging epigenetic regulatory layer where chemically altered RNA molecules, though non-protein-coding, exert profound control over gene expression by modulating chromatin architecture and DNA topology through interactions with nucleic acids and proteins.^49,50 Major ncRNA subtypes including miRNAs, lncRNAs, and circRNAs undergo dynamic post-transcriptional modifications such as methylation, acetylation, and ribosylation, with N6-methyladenosine (m6A) representing the most abundant and well-characterized modification involving methyl group addition to adenine residues.^51,52 This reversible process is orchestrated by coordinated actions of writer complexes (m6A methyltransferases like METTL3/METTL14), erasers (demethylases such as FTO/ALKBH5), and reader proteins that decode methylation signals.^53,54 Spatial enrichment of m6A near stop codons and within long exons enables its multifaceted regulation of tumor biology, governing cancer stem cell plasticity, therapeutic resistance mechanisms, and tumor microenvironment interactions.^55,56 The epitranscriptomic machinery’s capacity to influence oncogenic pathways through both direct RNA processing and indirect modulation of tumor metabolism/immune evasion highlights its diagnostic and therapeutic potential in oncology, with dysregulated m6A dynamics now recognized as critical drivers of malignant transformation and cancer progression.^57,58

Chromatin Remodeling

Chromatin remodeling serves as a dynamic epigenetic regulatory mechanism that orchestrates gene expression by modulating nucleosome architecture through two principal pathways: histone-tail covalent modifications mediated by protein complexes like Polycomb group (PcG) proteins, and ATP-dependent structural reorganization driven by chromatin remodeling complexes that utilize hydrolysis energy to reposition nucleosomes.^14,59 Operating on the fundamental nucleosome unit composed of DNA wrapped around histone octamers, this process enables cells to adapt gene activity to environmental stimuli through three-dimensional chromatin reorganization mechanisms – nucleosome repositioning, loop formation via chromatin cyclases, and chromatin decompaction through depolymerase-mediated relaxation.^60,61 These structural transformations regulate transcriptional accessibility either by altering histone-DNA binding interfaces through post-translational modifications or by physically restructuring nucleosome arrays to expose regulatory DNA elements.^62,63 The resultant chromatin state transitions, ranging from tightly packed heterochromatin to transcriptionally permissive euchromatin, critically influence developmental processes and genome stability while serving as pivotal drivers of oncogenesis when dysregulated.^64,65 Aberrant chromatin remodeling in cancer manifests through pathological gene silencing (tumor suppressor inactivation) or illegitimate oncogene activation, often mediated through malfunctioning chromatin-modifying enzymes that disrupt the delicate balance between chromatin condensation and decompaction states essential for proper transcriptional control.^66,67

Epigenetic Drugs in Clinical

DNA Methyltransferase Inhibitors (DNMT Inhibitors)

DNMT inhibitors exert therapeutic effects by inhibiting DNMT activity, thereby blocking the transfer of methyl groups from S-adenosylmethionine to cytosine residues and suppressing abnormal DNA methylation, which reactivates silenced tumor suppressor genes for cancer treatment. Unlike conventional cytotoxic chemotherapies, these inhibitors primarily induce epigenetic reprogramming rather than immediate cell death. Clinically approved nucleoside analogs including Azacitidine and Decitabine incorporate into DNA during replication, where they are recognized by DNMTs and subsequently inactivate the enzymes through covalent binding to cysteine sulfhydryl groups in their catalytic sites. Both agents are currently approved for myelodysplastic syndrome treatment, with several other DNMT inhibitors undergoing clinical evaluation.^68–70

Isocitrate Dehydrogenase Inhibitors (IDH Inhibitors)

IDH inhibitors target tumor-associated IDH mutations that disrupt normal enzymatic activity, converting α-ketoglutarate (α-KG) into the oncogenic metabolite 2-hydroxyglutarate (2HG), which accumulates in mutated cells and induces epigenetic dysregulation through DNA/histone hypermethylation. Clinically approved agents including Enasidenib and Ivosidenib suppress 2HG production by competitively inhibiting mutant IDH enzymes, thereby restoring normal methylation patterns and impeding tumor progression. Enasidenib specifically treats relapsed/refractory acute myeloid leukemia (AML) with IDH2 mutations, while Ivosidenib is indicated for IDH1-mutated relapsed/refractory myelodysplastic syndromes.^71–74

Histone Methyltransferase Inhibitors (HMT Inhibitors)

HMT inhibitors have emerged as targeted therapies with clinical progress in recent years, particularly against key enzymes like lysine methyltransferase EZH2, DOT1L, and arginine methyltransferases (PRMTs). EZH2 hyperactivation, implicated in tumorigenesis and stem-like transcriptional reprogramming across malignancies including breast cancer, castration-resistant prostate cancer, small cell lung cancer, and neuroblastoma, represents the most clinically advanced target, exemplified by the FDA-approved EZH2 inhibitor Tazemetostat for epithelioid sarcoma and relapsed/refractory follicular lymphoma. Concurrently, the DOT1L inhibitor pinometostat (EPZ-5676) has completed Phase 1 evaluation in leukemia patients, demonstrating the expanding therapeutic landscape of HMT inhibition in oncology.^75–78

Lysine-Specific Demethylase Inhibitors (LSD Inhibitors)

LSD inhibitors target LSD1, a flavin adenine dinucleotide-dependent enzyme whose functional outcomes vary depending on associated cofactors and substrates. This demethylase demonstrates critical regulatory roles in hematological malignancies like acute myeloid leukemia (AML), while its dysregulation in solid tumors correlates with histological grading, malignant progression, and clinical prognosis. Although no LSD1-targeting drugs have received global approval, multiple candidates including TCP, ORY1001, IMG-7289, SP2577, and CC-90011 have entered clinical evaluation, predominantly in early-phase trials focusing on small cell lung cancer (SCLC) and AML indications.^79–81

Bromodomain and Extra-Terminal Inhibitors (BET Inhibitors)

BET inhibitors target the BET protein family comprising four members (BRD2, BRD3, BRD4, BRDT) that are frequently overexpressed in malignancies and regulate oncogenic transcriptional programs. As an emerging class of anticancer agents including JQ1, OTX-015, and OPN-0610, these inhibitors exert therapeutic effects by disrupting BET protein–chromatin interactions to suppress oncogene transcription. Their potent antitumor activity has been demonstrated across multiple cancer types, with particular efficacy observed in hematological malignancies such as leukemia and lymphoma.^82–85

Histone Deacetylase Inhibitors (HDAC Inhibitors)

HDAC inhibitors target 18 human HDAC isoforms categorized into four classes (I, IIa/IIb, III, IV) based on structural features and catalytic mechanisms. Representing the most extensively developed epigenetic therapeutics, these agents are structurally classified by their Zn²⁺-binding groups: hydroxamic acids (vorinostat, panobinostat), cyclic tetrapeptides (romidepsin), investigational short-chain fatty acids, and benzamides (belinostat, chidamide). The first three categories broadly inhibit class I/II HDACs as pan-inhibitors, while benzamide derivatives exhibit selective suppression of class I and specific class IIb isoforms without affecting class IIa HDAC activity.^86–89

Epigenetic-Based Cancer Combination Therapy

The integration of epigenetic drugs with other treatments, including standard chemotherapy, targeted therapy, and immunotherapy, has evolved into an attractive alternative for oncology treatment. A rational combination therapy regimen can overcome the limitations of single-agent epigenetic therapy, thereby enhancing antitumor effects and reducing drug resistance. This section presents an overview of clinically investigated epigenetic combination regimens, evaluating their pharmacological rationale and therapeutic outcomes in contemporary oncology practice.

Combined with Epigenetic Therapy

The complexity of epigenetic regulation in tumorigenesis has driven the development of combination therapies targeting key enzymes such as DNA methyltransferases (DNMT) and histone deacetylases (HDAC), with preclinical evidence demonstrating that concurrent DNMT-HDAC inhibition suppresses MYC proto-oncogene expression, counteracts immune evasion, and enhances cytotoxic T-cell recruitment to exert antitumor immunity.^90–93 Early clinical validation emerged from a Phase I/II trial evaluating azacytidine (DNMT inhibitor) and entinostat (HDAC inhibitor) in non-small cell lung cancer, where all 12 enrolled patients exhibited clinical benefit: one achieved 14-month complete response, one 8-month partial response, two sustained 18- and 14-month disease stabilization, and eight maintained stability ≥12 weeks.⁹⁴ Biomarker analysis revealed circulating tumor DNA methylation status as predictive of outcomes, with methylated-profile patients showing superior response rates and survival compared to unmethylated counterparts. Concurrently, dual-action HDAC/DNMT inhibitors under development demonstrate multifactorial antitumor effects through tumor suppressor reactivation coupled with suppression of proliferation, metastasis, and survival pathways in malignant cells.^91,95 However, the clinical implementation of such combinations faces significant challenges. The non-specific action of epigenetic drugs may cause widespread transcriptomic alterations, leading to unpredictable off-target effects. Furthermore, the dynamic and reversible nature of epigenetic states necessitates precise timing and duration of drug administration, which is difficult to control precisely in clinical practice. Determining the optimal dosing combination is also particularly challenging, as the synergistic window between different epigenetic drugs is often narrow.

Combined with Chemotherapy

The synergistic potential of combining DNMT/HDAC inhibitors with chemotherapy is supported by preclinical and clinical evidence demonstrating that epigenetic priming enhances tumor cell chemosensitivity while reversing acquired resistance mechanisms.^96–98 This strategy leverages epigenetic drugs to augment DNA-damaging chemotherapeutics, as exemplified in a Phase II trial where azacitidine and chidamide combined with GemOx achieved favorable tolerability and efficacy in relapsed/refractory peripheral T-cell lymphoma.⁹⁹ The low-dose DNMT inhibitor decitabine has been demonstrated to resensitise chemotherapy resistant diffuse large B cell lymphoma (DLBCL) cells to doxorubicin without causing significant damage.¹⁰⁰ Similar to DNMT inhibitors, HDAC inhibitors have demonstrated the ability to reverse chemoresistance by reprogramming malignant cells to regain sensitivity to cytotoxic agents. For instance, panobinostat attenuates hypoxia-induced cisplatin resistance in non-small cell lung cancer (NSCLC) cells via HIF-1α destabilization. Mechanistically, HIF-1α activation has been shown to confer multidrug resistance through transcriptional regulation of chemoprotective pathways.^101,102 Despite this promise, combining epigenetic drugs with chemotherapy presents important limitations. Epigenetic priming may simultaneously enhance chemotherapy toxicity to normal tissues, resulting in a narrowed therapeutic window. The complex pharmacokinetic interactions between chemotherapeutic agents and epigenetic modulators also complicate dose optimization. Moreover, tumor heterogeneity may lead to inconsistent responses to epigenetic sensitization across different patient subsets, necessitating the development of reliable predictive biomarkers to guide patient selection.

Combined with Targeted Therapy

Epigenetic therapy demonstrates potential in reversing resistance mechanisms driven by genetic alterations and transcriptional reprogramming, particularly in models devoid of defined genetic drivers.^103–105 HDAC inhibition has been reported to overcome resistance to various kinase inhibitors. For instance, MPT0E028, a relatively novel oral histone deacetylase inhibitor, was shown to enhance apoptosis induced by first-line epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib in EGFR-TKI-resistant non-small cell lung cancer (NSCLC) cells.¹⁰⁶ Another preliminary study demonstrated that the combination of EGFR-TKIs with vorinostat reversed acquired resistance to EGFR-TKIs and promoted apoptotic cell death in NSCLC models.¹⁰⁷ In hematologic malignancies, azacytidine combined with the BCL-2 inhibitor venetoclax synergistically enhances apoptosis through MCL-1 suppression, overcoming therapeutic resistance.^108,109 This azacytidine-venetoclax regimen has received FDA breakthrough therapy designation for untreated AML patients ineligible for intensive chemotherapy, with ongoing evaluations in MDS and AML clinical trials.^110,111 Major obstacles to implementing epigenetic-targeted therapy combinations include managing the risk of overlapping toxicities and the heterogeneity of resistance mechanisms. Epigenetic reprogramming may trigger compensatory signaling pathway activation, undermining the efficacy of targeted therapies. Simultaneously, the impact of epigenetic drugs on the pharmacokinetics of targeted therapies is not fully understood, potentially altering drug exposure levels and effectiveness. The lack of reliable biomarkers to identify patient populations most likely to benefit from combination therapy also limits its clinical application.

Combined with Endocrine Therapy

Therapeutic targeting of lineage-specific transcriptional dependencies, exemplified by estrogen receptor (ER) signaling in breast cancer and androgen receptor (AR) pathways in prostate cancer, is profoundly influenced by epigenetic mechanisms that sustain oncogenic transcription in therapy-resistant malignancies.^112–115 This interplay is demonstrated through KAT6A, a MYST-family histone acetyltransferase amplified in 10–15% of breast cancers where its genetic ablation reduces ERα expression, prompting clinical evaluation of the first-in-class inhibitor PF-07248144 as monotherapy and combined with fulvestrant or letrozole/palbociclib in advanced ER+ breast cancer.^116–118 Parallel epigenetic vulnerabilities emerge in prostate cancer progression, where EZH2 overexpression correlates with castration resistance and neuroendocrine differentiation, while preclinical models reveal EZH2 inhibition not only suppresses malignant transformation but also enhances CD8+ T-cell cytotoxicity and IFN-γ production to restore androgen therapy sensitivity.¹¹⁹ Clinical trials are exploring EZH2 inhibitors combined with enzalutamide or androgen deprivation therapies, underscoring the strategic convergence of epigenetic modulation and hormonal targeting in overcoming transcriptional addiction across cancer subtypes.^120,121 Challenges for such combination strategies lie in the complexity and plasticity of epigenetic mechanisms in endocrine therapy resistance. Epigenetic modulators may interfere with the normal physiological functions of hormone receptors, leading to endocrine imbalances. The potential impact of long-term epigenetic intervention on hormone-dependent tissues (such as bone and cardiovascular systems) remains unclear. Furthermore, the optimal timing and sequencing strategy for epigenetic–endocrine combination therapy requires further exploration to maximize clinical benefit.

Combined with Immunotherapy

Epigenetic modifications drive oncogenesis by dysregulating tumor suppressor pathways and oncogenic networks across diverse malignancies while modulating immune cell activation and effector functions, establishing bidirectional crosstalk between tumor epigenetics and immunology.^122,123 DNMT inhibitors exert dual immunostimulatory effects through transposable element reactivation-induced double-stranded RNA production (TLR3-mediated type I interferon response) and enhanced MHC-I/antigen presentation machinery, thereby overcoming limitations of checkpoint inhibitors like PD-1/PD-L1 or CTLA-4 antibodies that frequently fail due to inadequate T-cell priming.^{122,124–126} Clinical validation emerges from trials in Hodgkin lymphoma where decitabine combined with pembrolizumab improved complete response rates (71% vs 32%) and progression-free survival (100% vs 76%) versus monotherapy.¹²⁷ Parallel findings in SMARCB1-deficient tumors demonstrate tazemetostat-mediated EZH2 inhibition increases tumor-infiltrating CD8+ T cells/Tregs while upregulating PD-1/LAG-3 expression, illustrating how epigenetic modulation primes checkpoint-responsive tumor microenvironments.¹²⁸ These mechanistic and clinical insights position epigenetic-checkpoint inhibitor combinations as transformative strategies for enhancing cancer immunotherapy efficacy. However, epigenetic-immunotherapy combinations face unique challenges. The immune activation by epigenetic drugs may trigger autoimmune-like toxicities, potentially synergizing with immune-related adverse events from immune checkpoint inhibitors. The impact of epigenetic reprogramming on T-cell function is context-dependent and may in some circumstances promote T-cell exhaustion. The dynamic nature of the immune microenvironment also demands precise timing and sequencing of epigenetic modulation relative to immunotherapy administration to maximize synergistic antitumor immunity.

Combined with Radiotherapy

Radiotherapy induces tumor cell DNA double-strand breaks via ionizing radiation, yet therapeutic efficacy is often compromised by aberrant DNA repair mechanisms underlying radiation resistance.¹²⁹ Chromatin compaction states, predominantly regulated through epigenetic modifications, critically determine both radiation-induced DNA damage extent and repair capacity, establishing epigenetic targets as radiosensitization strategies.^130,131 Notably, epigenetic regulators operate within interconnected networks coordinating radiation response, as exemplified by ionizing radiation-induced DNA-PK complex phosphorylation of fumarase at threonine 236, which mediates its interaction with histone variant H2A.X at damage sites. Local fumarate accumulation inhibits histone demethylase KDM2B, elevating H3K36me2 levels that promote DNA-PK complex recruitment to facilitate error-prone non-homologous end joining (NHEJ) repair and survival.^132–134 This multilayered epigenetic crosstalk underscores the therapeutic rationale for combining epigenetic modulators with radiotherapy, enabling dose reduction to mitigate toxicity while overcoming acquired resistance through synergistic pathway targeting.¹³⁵ Though epigenetic-radiotherapy combinations remain exploratory, advances in epigenomic profiling and real-time damage detection technologies are accelerating the development of precision combinatorial regimens. Key limitations to implementing epigenetic-radiotherapy combinations include the potential for increased normal tissue toxicity and the lack of radiosensitization biomarkers. Epigenetic drugs may enhance radiation damage to normal tissues, leading to exacerbated treatment-related toxicities. Significant heterogeneity exists in how different tumor types respond to epigenetic radiosensitization, requiring the development of predictive biomarkers to guide patient stratification. Furthermore, the optimal timing and administration schedule for combining epigenetic modulation with radiotherapy still need further optimization in clinical trials to balance efficacy with safety.

NDDS for Epigenetic-Based Cancer Combination Therapy

The integration of epigenetic therapeutics with conventional modalities (chemotherapy, radiotherapy, targeted therapy, immunotherapy) has demonstrated preclinical and clinical promise in overcoming therapeutic resistance, though conventional multidrug regimens face challenges in maintaining precise drug ratios due to divergent physicochemical properties, pharmacokinetic profiles, and biodistribution patterns that compromise tumor-specific delivery and exacerbate systemic toxicity. Emerging nanomedicine platforms—including liposomes, polymeric nanoparticles, micellar systems, dendrimers, and inorganic nanocarriers—address these limitations through coordinated co-delivery of therapeutic payloads, enhancing drug stability while optimizing spatiotemporal release kinetics to achieve synergistic dosing ratios at tumor sites. These nano-drug delivery systems (NDDS) exhibit transformative potential for epigenetic-based combination therapies by engineering precise pharmacological coordination that maximizes antitumor efficacy while minimizing off-target effects (Figure 2). The advantages and limitations of various NDDS were summarized in Table 1.

Table 1 Summary of Advantages and Limitations of Various Nanocarrier Systems for Drug Delivery

Figure 2 Summary of NDDS for epigenetic-based cancer combination therapy.

Lipid-Based Nanoparticles

Liposomes

Liposomes, closed spherical vesicles formed by phospholipid-cholesterol bilayers, represent a versatile drug delivery platform capable of encapsulating both hydrophilic and lipophilic therapeutics to enhance solubility, bioavailability, and targeted accumulation while minimizing systemic exposure.¹³⁶ Their structural versatility enables surface functionalization with polymers, targeting ligands, and other bioactive molecules, allowing precise modulation of biodistribution patterns and therapeutic payload release kinetics. By concentrating drug delivery to pathological sites through passive/active targeting mechanisms, these nanocarriers significantly improve therapeutic indices while reducing off-target toxicity, with engineered modifications further amplifying their pharmacological value across diverse therapeutic applications.^137,138

Liposomes demonstrate versatile applications in epigenetic combination therapies through co-delivery of epigenetic modulators with chemotherapeutic agents, immunotherapeutics, and photosensitizers, effectively addressing drug resistance while enhancing tumor-specific targeting. Tang et al engineered liposomes co-encapsulating the DNMT inhibitor zebularine and gemcitabine, which reversed pancreatic cancer chemoresistance by upregulating drug-sensitivity markers, achieving 4-fold intracellular gemcitabine accumulation and 22-fold plasma half-life extension.¹³⁹ Similarly, Lin et al’s liposomal co-delivery of panobinostat (HDAC inhibitor) and osimertinib suppressed glycolytic metabolism and epithelial–mesenchymal transition in NSCLC, overcoming therapeutic resistance. At the end of the experiment, the tumor inhibition rate was calculated. The results showed that the inhibition rate of the combined drug co-loaded in liposomes was 82%, while that of the combined drug free group was 51%, and the group using osimertinib alone had an inhibition rate of 45%.¹⁴⁰ Immuno-epigenetic strategies include He et al’s dual-loaded liposomes with panobinostat and JQ1 (BET inhibitor) that reprogrammed tumor-associated macrophages and enhanced CD8+ T-cell function,¹⁴¹ while Tu et al’s chidamide/BMS-202 liposomes blocked PD-L1 and amplified immunogenicity to inhibit primary/metastatic growth and metastatic tumors with an inhibitory rate of metastasis of up to 96%.¹⁴² Liang et al further optimized this approach using cationic liposomes co-loaded with zebularine, JQ1, and CpG to elevate tumor antigen presentation and T-cell infiltration alongside PD-L1 downregulation.¹⁴³ Multimodal systems exemplified by Sun et al’s thermosensitive liposomes incorporating copper sulfide, CpG, and JQ1 enabled laser-triggered immunogenic cell death and precision release of immunomodulators, stimulating dendritic cell maturation and cytotoxic lymphocyte recruitment for amplified antitumor immunity.¹⁴⁴

Lipid nanoemulsions

Lipid nanoemulsions, nanoscale oil-in-water systems (20–200 nm diameter), serve as efficient carriers for hydrophobic therapeutics due to manufacturing simplicity, biocompatibility, and structural stability, with soybean oil-based formulations demonstrating enhanced tumor targeting via the enhanced permeability and retention effect.^145,146 Their sub-200nm size facilitates enzymatic/hydrolytic degradation resistance, prolonging drug bioavailability while concentrating therapeutic payloads in malignant tissues.¹⁴⁷ Functionalization with tumor-specific ligands further improves precision, as exemplified by Kim et al’s lysophosphatidic acid receptor 1 (LPAR1)-targeted nanoemulsions co-encapsulating decitabine and panobinostat, which reduced systemic toxicity while maintaining drug stability in triple-negative breast cancer models.¹⁴⁸ Cellular uptake and biodistribution correlated with LPAR1 expression levels, enabling tumor-selective epigenetic reprogramming through decitabine-mediated DNA hypomethylation and panobinostat-induced chromatin remodeling, collectively reactivating tumor suppressor genes while suppressing oncogenic pathways. The results showed that the combined effect of the drug-loaded nanoemulsion significantly reduced the expression levels of FOXM1 mRNA and protein by up to 80%.

The review on the application of lipid-based nanoparticles for epigenetic-based cancer therapy was summarized as follows: Lipid-based nanocarriers have emerged as dominant platforms in combinatorial drug delivery, representing both the most clinically advanced and commercially viable nanomedicine systems to date. As the fastest-growing category of NDDS, their structural versatility enables continuous innovation in targeting precision through novel ligand engineering and stimuli-responsive designs while maintaining unparalleled translational potential for pharmaceutical development.

Polymer-Based Nanoparticles

Polymer Nanoparticles

Polymer nanoparticles, solid colloidal carriers (10–1000 nm) engineered from natural biopolymers like chitosan and alginate derivatives or synthetic polymers including PLGA, PEG, and polystyrene, function as versatile drug delivery platforms through either nanosphere matrices that uniformly disperse therapeutic payloads or nanocapsule structures that encapsulate drugs within protective cores.¹⁴⁹ These systems demonstrate exceptional cargo versatility, accommodating hydrophobic/hydrophilic agents across molecular weight spectrums from small molecules to biologics, proteins, and genetic therapeutics, while precise modulation of nanoparticle composition, surface charge, and degradation kinetics enables controlled drug loading efficiency and programmable release profiles.^150,151

Polymer nanoparticles demonstrate significant potential in epigenetic combination therapy through multifunctional engineering approaches, as evidenced by Li et al’s PLGA-based system co-delivering JQ1 (BET inhibitor) and icariin, which effectively suppressed breast cancer progression and bone metastasis through optimized biodistribution. The tumor weight data showed that PLGA-based co-loaded system suppressed 65.8% of tumor growth, which was 2.0-fold higher than that of the free drug groups.¹⁵² Ledezma et al further expanded therapeutic modalities by encapsulating indocyanine green (photosensitizer) with nexturastat (HDAC6 inhibitor) in PLGA nanoparticles, achieving NIR-responsive photothermal ablation combined with histone deacetylase inhibition to prolong melanoma survival outcomes.¹⁵³ Hu et al engineered PEG-PCL nanoparticles conjugated with nivolumab (PD-1 antibody) and loaded with decitabine (DNMT inhibitor), enhancing DNA hypomethylation effects while restoring PD1+CD8+ T-cell infiltration for improved checkpoint blockade efficacy.¹⁵⁴ Advanced hybrid systems developed by Su et al combined lipid-coated PLGA nanoparticles co-encapsulating decitabine and doxorubicin, demonstrating synergistic apoptosis induction through epigenetic chemosensitization mechanisms.¹⁵⁵ These case studies collectively validate polymer nanoplatforms’ capacity to integrate epigenetic modulation with diverse treatment modalities while addressing pharmacological challenges through structural engineering.

Polymeric Micelles

Polymeric micelles, self-assembled nanoscale structures formed by amphiphilic block copolymers in aqueous solutions, consist of hydrophilic polyethylene glycol (PEG) segments and hydrophobic domains such as polylactic acid (PLA), polycaprolactone (PCL), or phospholipid derivatives.^156,157 These supramolecular assemblies demonstrate versatile drug-loading capacities for diverse therapeutic agents—from small molecules to proteins—enhancing drug stability, solubility, and bioavailability while exhibiting tumor-targeting potential through passive accumulation or ligand-mediated active targeting.¹⁵⁸ Their structural adaptability enables rational design of stimuli-responsive systems (eg, pH-, redox-, or enzyme-sensitive) by engineering copolymer compositions to achieve spatiotemporally controlled drug release aligned with pathological microenvironments, thereby optimizing therapeutic precision and minimizing off-target effects.¹⁵⁹

Polymeric micelle architectures enable innovative epigenetic combination strategies through tailored therapeutic delivery mechanisms, as exemplified by Naz et al’s PLGA-PEG micelles stabilizing azacytidine for pH-responsive release in breast cancer cells, bypassing nucleoside transporter limitations while resisting drug efflux to enhance antiproliferative effects.¹⁶⁰ Xiong et al engineered redox-responsive prodrug micelles releasing γ-oryzanol (DNMT inhibitor) and α,β-methylene adenosine 5’-diphosphate (adenosine deaminase inhibitor) upon glutathione activation, synergistically inducing pyroptosis and reversing immunosuppressive microenvironments. The mean survival time of the combined group (17.8 days) was significantly prolonged compared to that of the control group (11 days), the group receiving α,β-methylene adenosine 5’-diphosphate therapy alone (12.7 days) and the group reviving the γ-oryzanol therapy alone group (14.2 days). In addition, 1/6 of mice in the combined group survived longer than 25 days.¹⁶¹ Advanced multimodal systems include Ding et al’s dual-micelle platform combining TBE photosensitizer with decitabine for light-controlled activation of cGAS-STING pathways and pyroptosis-mediated immunogenic cell death, which promoted antigen-presenting cell maturation and T-cell antitumor immunity. Finally, dual-micelle with laser treatment exhibited the most robust tumor eradication with a regression rate of 70.9%.¹⁶² Further innovation is demonstrated by Zhao et al’s DSPE-PEG2000-stabilized micelles co-assembling chlorin e6 (Ce6) and mocetinostat through electrostatic interactions, achieving tumor-specific accumulation and deep tissue penetration for photo-enhanced epigenetic regulation that suppressed both primary and metastatic tumor progression through optimized pharmacokinetics and immunomodulation. More importantly, combinative administration of micelles and α-PD-L1 upon light exposure was found to significantly inhibit the distant tumor growth. It was calculated that the primary and distant tumor inhibitory rates were about 92.5% and 82.2%, respectively, after the photodynamic amplified ICB therapy.¹⁶³

Dendrimers

Dendrimers represent a class of monodisperse macromolecules characterized by radially branched architectures comprising a core, linear polymeric backbones, and hierarchically organized dendritic side chains, enabling precise control over their mass, three-dimensional geometry, and surface functionality.^164,165 These structurally defined polymers possess exterior-active functional groups for covalent conjugation of biomolecules or imaging agents, while their internal cavities accommodate therapeutic payloads such as nucleic acids or small molecules. Commonly employed dendritic scaffolds include poly(amidoamine) (PAMAM), polypropylene imine (PPI), polyethylene imine (PEI), polylysine (PLL), and polyaryl ether derivatives, with their programmable surface chemistry and encapsulation capacity making them versatile platforms for controlled drug delivery and biomedical applications.^166,167

Dendrimers demonstrate multifaceted potential in epigenetic combination therapy by enabling co-delivery of small molecule drugs and genetic therapeutics through programmable molecular architectures, as exemplified by Zong et al’s tumor-targeted dendrimer-HDAC inhibitor conjugates, where hydroxamic acid-linked HDACi remains pharmacologically inert until tumor-specific esterase cleavage reactivates its epigenetic modulation capacity.¹⁶⁸ Kaundal et al further optimized gene targeting using polyethylenimine (PEI)-EZH2 siRNA complexes that enhanced systemic stability and hematopoietic compatibility, achieving potent EZH2 silencing in acute myeloid leukemia (AML) xenograft models with significant reductions in peripheral/blood marrow AML cell counts. The PEI-EZH2 siRNA complexes in AML-bearing animals significantly reduced CD45+/CD11b+ AML populations to 31% in peripheral circulation.¹⁶⁹ These studies collectively highlight dendrimers’ capacity to overcome pharmacological barriers through structural precision—enhancing payload stability, tissue specificity, and therapeutic efficacy in epigenetic regulation.

The review on the application of polymer-based nanoparticles for epigenetic-based cancer therapy was summarized as follows: Polymer-based nanoparticles hold significant innovation potential through systematic architectural engineering that progresses in tandem with advancements in synthetic chemistry, while their translation into functionalized delivery systems necessitates concurrent optimization of industrial-scale production safety and organic reagent biocompatibility profiles.

Cell-Derived Nanoparticles

Extracellular Vesicles (EVs)

EVs—encompassing micrometer-scale microvesicles and 30–100 nm exosomes—are naturally secreted lipid-bilayer nanostructures that mediate intercellular communication through targeted cargo delivery, functioning in immune regulation, angiogenesis, and inflammatory processes while exhibiting inherent biocompatibility, prolonged circulation, and low immunogenicity.^170,171 These endogenous nanocarriers enable precision drug delivery via membrane fusion-mediated cytoplasmic transfer of therapeutic payloads, as evidenced by studies demonstrating their capacity to bypass complex cellular uptake pathways through efficient membrane integration.¹⁷² Recent advances leverage EVs’ intrinsic targeting specificity and biosafety profiles to engineer advanced drug delivery systems, with research confirming their translational potential in constructing clinically viable therapeutic platforms through optimized cargo encapsulation and cell-specific homing mechanisms.¹⁷³

EVs demonstrate multifaceted applications in epigenetic combination therapy through engineered delivery systems, as evidenced by You et al’s EV-based platform targeting m6A modification via YTHDF1 siRNA delivery, which suppressed gastric cancer progression through m6A-dependent transcriptional regulation. The tumor-bearing mice treated with EV-based platform showed the longest survival time with an overall survival rate of 66.7% compared to the other groups.¹⁷⁴ Simultaneously, Zhai et al developed PD-1-enriched T-cell membrane vesicles encapsulating the LSD1 inhibitor ORY-1001, achieving tumor-specific recognition that amplified intratumoral IFN-γ/MHC-I signaling, resulting in 8-fold and 29-fold increases in cytotoxic/activated T-cell infiltration with potent xenograft suppression.¹⁷⁵ Further extending this paradigm, Du et al’s mesenchymal stem cell-derived EVs co-delivered YTHDF1-targeting siRNA and docetaxel (DTX), synergizing epigenetic modulation with chemotherapy through enhanced cellular uptake and nucleic acid protection.¹⁷⁶ Complementing these approaches, Lin et al engineered artificial exosomes incorporating tumor membrane proteins to co-encapsulate vorinostat (HDAC inhibitor) and doxorubicin, demonstrating sustained tumor retention and synergistic NSCLC suppression without significant toxicity. Artificial exosome containing vorinostat and doxorubicin almost completely suppressed the growth of tumor volume and weight, compared with artificial exosomes containing a single drug presenting an inhibition of ~58%.¹⁷⁷ Collectively, these EV-based strategies exemplify precision epigenetic-chemotherapy integration through endogenous targeting mechanisms and payload stabilization.

Cell Membrane Carrier

Cell membrane-coated carriers leverage the inherent biological advantages of native cellular structures, where isolated membranes from macrophages, erythrocytes, dendritic cells, or tumor cells are fused with synthetic nanoparticles through extrusion techniques to create biomimetic nanomaterials that minimize host immune responses while preserving source cell functionality.¹⁷⁸ These hybrid systems combine the physicochemical characteristics of engineered nanoparticles with the biological recognition capabilities of natural cell membranes, resulting in enhanced biocompatibility and optimized drug delivery kinetics. The fabrication process involves sequential co-extrusion of cell membrane fragments and core nanoparticles through polycarbonate membranes, generating carriers that retain both the targeting properties of biological membranes and the controlled release profiles of synthetic nanoplatforms, thereby achieving precise therapeutic delivery through dual biological-physical optimization mechanisms.¹⁷⁹

Cell membrane coating technology significantly enhances epigenetic combination therapy by optimizing drug biodistribution and therapeutic precision, as demonstrated through multiple innovative approaches. Gao et al engineered macrophage membrane-coated nanoinducers co-loaded with paclitaxel (PTX) and decitabine, which reversed triple-negative breast cancer chemoresistance by inducing tumor tissue demethylation, restoring T-cell functionality, and sensitizing malignancies to chemotherapy through inherent tumor-homing capabilities. This nanoinducers, when combined with PD-1 checkpoint blockade, can achieve a tumor suppression rate of 90.04%.¹⁸⁰ Simultaneously, Zhang et al developed lung cancer cell membrane-disguised nanoparticles carrying BRD4-targeting proteolysis-targeting chimeras (PROTACs), enabling dual targeting of lung cancer cells and tumor-associated macrophages to synergistically induce apoptosis in subcutaneous and orthotopic tumor models through epigenetic reader protein degradation. About 90% mice survived over 18 days initially from the treatment with drug-loaded nanoparticles, when all of the mice were nearly dead in the control groups.¹⁸¹ Furthermore, Wang et al’s glioblastoma multiforme (GBM) therapeutic platform utilized tumor cell membrane-wrapped nanoparticles containing ultrasmall Cu₂ₓSe, indoximod, and JQ1, which crossed the blood–brain barrier to remodel the tumor immune microenvironment (TIME) by activating CD8⁺ T-cell infiltration, elevating splenic memory T cells, and preventing tumor recurrence through coordinated epigenetic modulation and immune checkpoint regulation.¹⁸² These membrane-engineered systems collectively demonstrate enhanced tumor targeting, payload protection, and multimodal therapeutic synergy in epigenetic–immune combination regimens.

The review on the application of cell-derived nanoparticles for epigenetic-based cancer therapy was summarized as follows: Overall, EVs and cell membrane-based carriers demonstrate precise tumor-targeting capabilities and combinatorial drug delivery potential in murine models, while clinical translation remains constrained by suboptimal exosome isolation yields and challenges in scalable manufacturing, necessitating breakthroughs in bioprocessing technologies to bridge preclinical efficacy with therapeutic applications.

Inorganic Nanoparticles

Metallic-Based Nanoparticles

Epigenetic-radiotherapy combination strategies are being revolutionized by metal-based nanoparticles composed of high-atomic-number elements that enhance treatment efficacy through dual photon-interactive mechanisms—inducing ROS generation via low-energy electron emission while functioning as co-delivery platforms for epigenetic therapeutics. These nanoplatforms achieve tumor-specific accumulation through surface functionalization with targeting ligands, enabling precise radiosensitization and combinatorial payload release.^183,184 Preclinical validation encompasses diverse metallic systems including gold, platinum, rare-earth, and semiconductor nanoparticles engineered for concurrent radiation enhancement and epigenetic drug transport, with their multifunctional architecture optimizing spatiotemporal coordination between DNA damage potentiation and epigenetic modulation.^185,186

Epigenetic-radiotherapy combinations are being advanced through innovative nanoplatforms, as demonstrated by Kaundal et al’s hypericin-conjugated gold nanoparticles targeting mitochondrial IDH2-EZH1/2 interactions in glioblastoma, achieving multimodal tumor ablation via laser activation in xenograft models. The results show that the tumor volume of the hypericin-conjugated gold nanoparticles treatment group is almost 10-fold more suppressed than the PBS control group, and 3-fold with the hypericin group¹⁸⁷ Simultaneously, Song et al engineered leukemia stem cell membrane-coated bimetallic Mn²⁺/Fe³⁺ MOFs delivering DNA hypomethylation agents and autophagy-inducing peptides, which restored MHC-I expression and interferon signaling to enhance T-cell immunity against leukemic progenitors. The results showed that the low dose of the above-mentioned preparation (50 µg/mL) inhibited tumor growth by 67.6% compared to 31.7% in the AZA alone group, and achieved 66.7% survival rate over a period of 60 days.On the other hand, the high dose of the above-mentioned preparation (100 µg/mL) achieved almost complete tumor elimination, and 100% of the treated mice survived for 60 days post-injection.¹⁸⁸ Addressing MYC-mediated radioresistance, Wang et al engineered tungsten-based nanosystems co-loaded with 5-Aza (DNMTi) and ITF-2357 (HDACi) that suppress MYC-driven immunosuppression while enhancing dendritic cell antigen presentation and cytotoxic T-cell infiltration through radiotherapy-potentiated immunogenic effects.¹⁸⁹ Wang et al’s EGCG-W⁶⁺ polyphenol complexes induce caspase-3-mediated gasdermin E cleavage during irradiation, triggering pyroptosis while counteracting radiotherapy-induced Treg elevation through coordinated epigenetic modulation and radiosensitization.¹⁹⁰ These multifunctional platforms demonstrate enhanced immunogenic cell death pathways surpassing conventional radiotherapy through precision epigenetic-immune crosstalk regulation.

Non-Metallic Nanoparticles

Non-metallic nanoparticles—a critical subset of nanomaterials distinguished by their composition of non-metallic elements—exhibit unique physicochemical properties including quantum confinement effects, enhanced surface-to-volume ratios, and tunable optoelectronic behaviors that enable superior adsorption capacities and catalytic reactivities.^191,192 While demonstrating improved biosafety profiles compared to metallic counterparts, these systems face challenges in biodegradation that require material engineering solutions. Their structural precision facilitates targeted drug delivery through size-dependent cellular interactions and controlled release kinetics, minimizing off-target effects while optimizing therapeutic payload delivery. Representative systems such as mesoporous silica nanoparticles (MSNs) and carbon-based nanostructures exemplify this category’s translational potential, leveraging ordered porosity and electronic configurations to achieve spatiotemporal control over drug loading/release processes in biomedical applications.^193,194

Epigenetic combination therapies are being optimized through advanced nanocarrier systems, as demonstrated by Gao et al’s mesoporous silica nanoparticles (MSNs) co-encapsulating paclitaxel (PTX) and decitabine with surface biofunctionalization, which achieved tumor-specific demethylation to reverse T-cell exhaustion while enhancing chemosensitivity in a precisely controlled drug ratio.¹⁸⁰ Concurrently, Yuan et al engineered CD44-targeted MSNs integrating pH/redox-responsive β-cyclodextrin gatekeepers for co-delivery of doxorubicin (DOX) and siGCN5, suppressing multidrug-resistant breast tumor growth through dual inhibition of P-glycoprotein efflux and histone acetyltransferase GCN5, with hyaluronic acid coating enhancing tumor-specific internalization. Of note, in an MDR breast tumor model, DOX and siGCN5 co-delivered MSNs inhibits MDR tumor growth by 77%, abolishes P-gp-mediated drug resistance, and eliminates DOX’s systemic toxicity.¹⁹⁵ Complementing these approaches, Gu et al addressed the aqueous insolubility of the EHMT2 inhibitor UNC0646 by developing physisorbed nanodiamond complexes (ND-UNC0646) that enabled pH-responsive drug release and improved dispersibility, demonstrating enhanced anti-tumor efficacy in orthotopic liver cancer models through intravenous delivery while maintaining dosing precision.¹⁹⁶ These multifunctional platforms exemplify the integration of epigenetic modulation with chemotherapy and targeted delivery to overcome pharmacological barriers.

The review on the application of inorganic nanoparticles for epigenetic-based cancer therapy was summarized as follows: In conclusion, inorganic nanomaterials demonstrate distinct advantages in combinatorial drug delivery systems, offering precise tumor-targeting capabilities and extensive therapeutic applicability through their tunable physicochemical properties. However, the clinical translation of these nanoplatforms necessitates rigorous pharmacological optimization to reconcile therapeutic efficacy with biocompatibility profiles, requiring systematic engineering approaches that address biodistribution control, biodegradation kinetics, and long-term biosafety parameters to fully harness their diagnostic and therapeutic potential in oncology applications.

Hydrogel Delivery Systems

Hydrogels

Hydrogels represent three-dimensional crosslinked polymer networks synthesized through covalent or non-covalent interactions between hydrophilic monomers, exhibiting unique water-retention capacities through swelling behavior that enables absorption of substantial aqueous volumes while maintaining structural integrity without dissolution.^197,198 Characterized by tissue-mimetic elasticity derived from their high water content, these biomimetic materials demonstrate exceptional potential in therapeutic agent encapsulation and controlled release, facilitated by tunable mesh sizes and stimuli-responsive release mechanisms including diffusion-controlled kinetics, pH/thermal-triggered swelling, and enzymatic/redox-mediated degradation.¹⁹⁹ This unique physicochemical profile enables sustained local drug depot formation with prolonged high-concentration payload maintenance, while their programmable responsiveness to physiological or external stimuli allows spatiotemporal precision in therapeutic delivery across biomedical applications.^200,201

Hydrogel-based epigenetic drug delivery systems demonstrate transformative therapeutic potential through engineered responsiveness to tumor microenvironments, as exemplified by Ruan et al’s pH/calcium carbonate nanoparticle-embedded hydrogel co-encapsulating anti-PD-1 antibodies and zebularine—this dual-delivery platform enhanced tumor-associated antigen presentation while reversing PD-L1-mediated immunosuppression, achieving tumor growth inhibition and survival extension in murine models through coordinated checkpoint blockade and DNA demethylation mechanisms.²⁰² Ji et al developed ROS-responsive hydrogels releasing GSK-LSD1 (LSD1 inhibitor) and 5-fluorouracil (5-FU) that reduced cancer stemness through H3K4me2 epigenetic remodeling while increasing MHC-I expression, converting chemotherapy-resistant triple-negative breast tumors into treatment-sensitive phenotypes with suppression of postoperative recurrence and reduction in metastatic burden. By the day 20 after hydrogel injection, the 5-FU@Gel showed little effect on tumor growth, while the iLSD1@Gel reduced the average tumor size by 38.0 ± 8.5%. Furthermore, iLSD1+5-FU@Gel achieved remarkable efficacy, and most tumors ceased proliferation in the first 20 d and exhibited slow growth velocities afterward. The median survival time of mice after PBS mock treatment was 26 days, which was slightly increased to 30 and 34 d by the 5-FU@Gel and iLSD1@Gel, respectively. In contrast, the iLSD1+5-FU@Gel significantly extended this value to 60 d.²⁰³ These intelligent hydrogel platforms exemplify precision epigenetic reprogramming through spatiotemporal control of therapeutic payloads, leveraging microenvironmental triggers (pH/ROS) to overcome multidrug resistance while establishing sustained local drug reservoirs that maintain therapeutic concentrations for 14–21 days post-implantation.

Nanogels

Nanogels—nanoscale three-dimensional networks formed through physical or chemical crosslinking of polymeric macromolecules such as acrylics, polyvinylpyrrolidone, or polysaccharides—combine the structural advantages of hydrogels with nanoparticle functionality, exhibiting high drug-loading capacities, sustained-release kinetics, and enhanced tissue permeability through sub-200 nm particle sizes.²⁰⁴ Their swollen hydrogel architecture protects encapsulated therapeutics from degradation while providing modifiable surfaces for targeted delivery, with biodegradability and biocompatibility profiles enabling clinical translation.²⁰⁵ Vijayaraghavalu et al demonstrated this potential through disulfide-crosslinked N-isopropylacrylamide/vinylpyrrolidone nanogels co-loading decitabine (DAC) and vorinostat (SAHA), which overcame multidrug resistance via epigenetic reprogramming, achieving tumor growth suppression at subtherapeutic doxorubicin doses through sustained chromatin remodeling effects.²⁰⁶ This mechanistic paradigm—attributed to prolonged epigenetic modulator release from nanogel matrices—provides a universal strategy against therapy-resistant malignancies driven by aberrant DNA methylation or histone modification, with tunable crosslinking methods (noncovalent interactions vs covalent bonding) allowing precise control over drug release profiles tailored to specific tumor microenvironments.

The review on the application of hydrogel delivery systems for epigenetic-based cancer therapy was summarized as follows: Hydrogel-based drug delivery systems demonstrate transformative potential in contemporary therapeutics, with researchers actively engineering combinatorial platforms that integrate epigenetic modulation capabilities through hydrogel matrices while advancing multifunctional hydrogel architectures to synergize with radiotherapy, immunotherapy, and targeted molecular interventions. This dual-axis development strategy—enhancing epigenetic payload precision through stimuli-responsive release mechanisms and optimizing viscoelastic properties for tissue-specific integration—addresses critical challenges in spatiotemporal drug control, enabling sustained therapeutic agent delivery with programmable degradation kinetics that maintain bioactive concentrations across treatment cycles.

Conjugated Drug

Antibody-Drug Conjugates (ADCs)

ADCs, which combine tumor-targeting monoclonal antibodies with cytotoxic payloads via specialized linkers, represent a transformative therapeutic modality that merges the precision of biologics with the potency of chemotherapy.^207,208 With nearly 10 ADCs now FDA-approved and over 100 in clinical trials, this platform’s success has catalyzed exploration of next-generation constructs integrating epigenetic modulators—where antibody-mediated delivery of chromatin-remodeling agents could synergize with cytotoxic payloads through mechanisms like DNA damage response potentiation and immune microenvironment reprogramming, potentially overcoming limitations of conventional combination therapies through spatiotemporal coordination of epigenetic priming and tumor cell elimination.^209,210

Emerging epigenetic antibody-drug conjugates (ADCs) are expanding the therapeutic scope beyond traditional cytotoxic payloads, as exemplified by Cini et al’s cetuximab-based ADC incorporating the HDAC inhibitor ST7612AA1 through lysine amide conjugation—this EGFR-targeting construct demonstrated tumor-specific internalization and potent antitumor efficacy in solid tumor models while circumventing the systemic toxicity associated with conventional microtubule-inhibitor ADCs, representing the first clinical-stage ADC with epigenetic modulation capabilities applicable to both oncologic and non-oncologic indications.²¹¹ Complementing this approach, Milazzo et al engineered trastuzumab-derived ADC ST8176AA1 through TCEP-mediated disulfide reduction and maleimide-thiol conjugation of activated HDAC inhibitor ST7464AA1, which maintained ErbB2 binding specificity while exhibiting enhanced tumor growth inhibition in ovarian/colon cancer xenografts and pancreatic PDX models compared to parental antibody therapy. ST8176AA1 induced a median survival time of 84 days compared to 43 and 74 days of vehicle- and trastuzumab-treated groups, respectively. These results are consistent with the previous study confirming that ST8176AA1 is statistically significant more effective than trastuzumab against ovary carcinoma.²¹² Mechanistic studies confirmed the enhanced efficacy stemmed from HDAC-mediated chromatin remodeling rather than cytotoxic mechanisms, with pharmacokinetic analyses revealing 14-day sustained HDAC inhibition in tumor tissues versus <48h activity with free inhibitors, validating ADCs as precision delivery platforms for epigenetic therapeutics.

Small Molecule-Drug Conjugates (SMDCs)

SMDCs, an emerging class of targeted therapeutics, share structural homology with ADCs through their tripartite architecture comprising targeting ligands, chemical linkers, and effector payloads (eg, cytotoxic agents or E3 ligase recruiters), yet fundamentally diverge by utilizing low-molecular-weight targeting moieties instead of monoclonal antibodies.²¹³ This substitution confers distinct pharmacological advantages: simplified synthesis with enhanced process control and reduced manufacturing costs compared to biologics, inherent non-immunogenicity facilitating improved safety profiles, and superior tumor penetration enabled by sub-10 nm hydrodynamic diameters, particularly advantageous in solid malignancies where extracellular matrix density limits macromolecule diffusion.^214,215 The small molecule targeting approach preserves precise drug localization while eliminating antibody-mediated immunogenic risks, with preclinical evaluations demonstrating stable pharmacokinetic behavior and tumor-selective payload release through microenvironment-responsive linker systems.²¹⁶

Small molecule-drug conjugates are advancing epigenetic combination therapies through innovative nanostructural designs, as exemplified by Xu et al’s cisplatin-vorinostat supramolecular nanoparticles that achieve 99% tumor suppression in drug-resistant A549 models through synchronized drug release and prolonged circulation, overcoming the negligible efficacy of free drug combinations.²¹⁷ Concurrently, Wang et al’s zinc-mediated self-assembled nanofibers co-delivering 5-azacytidine and vorinostat demonstrate dual epigenetic modulation by coordinating DNA demethylation with HDAC inhibition, inducing tumor-selective apoptosis in gastric cancer through sustained intratumoral retention. It is clearly found that compared with free vorinostat or the 5-azacytidine treatment, the drug mixture treatment exhibited a better antitumor effect, which may be explained by the synergistic actions. Surprisingly, mice treated with the nanofibers exhibited the smallest tumor size and the highest inhibitory rate, suggesting the best antitumor efficiency of nanofibers.²¹⁸ Complementing these approaches, Ye et al engineered glutathione-responsive prodrug nanoparticles (NJ) cloaked with pH-sensitive polymers, where tumor-acidified dissociation enables intracellular release of JQ1 (BET inhibitor) and NLG919 (IDO1 inhibitor), concurrently blocking PD-L1 upregulation and tryptophan catabolism to reverse interferon-γ-induced immune evasion—a strategy amplified when combined with photodynamic therapy to eradicate multiple immunosuppressive pathways through spatiotemporal control of epigenetic-immune crosstalk.²¹⁹

The review on the application of conjugated drug for epigenetic-based cancer therapy was summarized as follows: Conjugated drugs represent a transformative paradigm in oncology therapeutics, pioneering next-generation precision medicine through their capacity to integrate tumor-targeting specificity with multimodal therapeutic payloads—a breakthrough underscored by the global biopharmaceutical sector’s accelerated investment. This therapeutic class is poised to redefine cancer treatment boundaries through enhanced tumor selectivity, payload potency amplification, and resistance mechanism circumvention.

Others

Recent advances in nanocarrier design are revolutionizing epigenetic combination therapies through innovative delivery strategies, as exemplified by Luo et al’s folic acid receptor-targeted nanoparticles co-delivering azacitidine and PARP inhibitors, which achieved tumor-selective enrichment through protein corona engineering while enabling synergistic antitumor effects with PD-1 inhibitors in lung cancer models.²²⁰ Concurrently, Kaundal et al’s albumin-based nanoparticles demonstrated enhanced nuclear localization of EZH2 inhibitor EPZ011989 in AML cells through optimized non-covalent interactions, improving therapeutic index compared to free drug administration.²²¹ Yang et al further advanced pH-responsive delivery using apolipoprotein-mimetic liposomes that synchronized 5-azacitidine and HDAC inhibitor release, inducing cell cycle arrest and apoptosis via Bim/caspase-3 activation while reactivating tumor suppressor genes in breast cancer models.²²² Complementing these approaches, Tian et al engineered photothermally responsive polydopamine nanoparticles that coupled JQ1-mediated BET inhibition with localized hyperthermia, generating cytotoxic T-cell responses that prevented tumor rechallenge in 85% of treated mice through sustained epigenetic-immune reprogramming.²²³ These multimodal platforms collectively demonstrate how nanoscale engineering optimizes spatiotemporal control of epigenetic payloads while overcoming biological barriers through material-specific targeting mechanisms.

The review on the application of other NDDS for epigenetic-based cancer therapy was summarized as follows: The exploration of non-canonical nanocarrier architectures offers innovative solutions to overcome the limitations of conventional drug delivery systems through tailored physicochemical properties and programmable targeting strategies.

Delivery Strategies for Epigenetic-Based Cancer Combination Therapy

The NDDS demonstrates significant potential and promising development prospects in epigenetic-based combination therapy due to its capacity for co-delivering multiple therapeutic agents. The complexity of epigenetic modifications, characterized by diverse molecular targets, is exemplified in the tumor microenvironment where epigenetic reprogramming of immune cells, stromal cells, and other components influences cancer progression. Within cellular contexts, epigenetic enzymes may serve as therapeutic targets localized in specific organelles. While these systems must achieve effective tumor accumulation, challenges arise when addressing temporally dependent therapeutic targets requiring sequential or programmed drug release. Current limitations include the intricate temporal control of combination therapies and compromised patient compliance caused by complex administration regimens. To address these issues, advanced NDDS should integrate multifunctional capabilities beyond mere drug co-loading, encompassing tumor tissue targeting, multi-cellular delivery precision, and spatiotemporal regulation at subcellular levels (Figure 3).

Figure 3 Delivery strategies for epigenetic-based cancer combination therapy.

Passive Targeting

Passive targeting leverages the physiological and pathological characteristics of tumor sites by modulating nanoparticle properties such as size, morphology, structure, and surface characteristics to achieve tumor accumulation without relying on molecular recognition mechanisms.^224,225 This approach capitalizes on the defective vascular architecture and impaired lymphatic drainage in rapidly proliferating tumors, collectively termed the enhanced permeability and retention (EPR) effect. These pathological features allow macromolecules and nanoparticles to extravasate into tumor tissues while hindering their systemic clearance, enabling conventional nano-drug delivery systems to passively concentrate therapeutic agents at tumor sites.^226,227 The simplicity of designing such systems, coupled with their scalable production processes, has made them commercially attractive to pharmaceutical companies, as evidenced by all currently marketed anti-tumor nanomedicines belonging to this category. Beyond passive accumulation, emerging research demonstrates that precisely engineered nanoparticle morphologies and surface modifications can further enhance targeting efficacy, particularly in epigenetic drug delivery applications.^228,229 These advanced design strategies have garnered significant scientific attention in recent years due to their potential to overcome limitations of conventional EPR-dependent systems while expanding therapeutic possibilities in tumor microenvironment modulation.

The particle size of nanomedicine delivery systems critically governs their tumor penetration efficacy and intratumoral retention capacity. Experimental evidence demonstrates an inverse correlation between nanoparticle dimensions and solid tumor penetration within defined size thresholds – smaller systems (eg, 12.3 nm PARP inhibitor/5-azacytidine co-delivery nanoparticles, PAZA) exhibit superior tumor-targeting and interstitial penetration compared to larger counterparts (eg, 100 nm variants, PVD).²²⁰ Conversely, tumor retention capacity shows positive size dependence, with smaller nanoparticles demonstrating significantly reduced intratumoral persistence. This fundamental trade-off between penetration depth and retention duration has driven the development of stimuli-responsive nanoplatforms capable of dynamic size modulation. Current innovative designs employ subtherapeutic-sized nanoparticles that undergo tumor microenvironment-triggered structural reorganization (via self-assembly, electrostatic interactions, or phase transitions) to achieve size transformation from sub-20 nm penetration-optimized states to retention-enhanced configurations exceeding 100 nm. This spatiotemporal size adaptation strategy synergistically improves both deep tumor permeation and prolonged drug retention, significantly amplifying therapeutic outcomes.^230,231 Such intelligent nanosystems provide valuable insights for advancing epigenetic combination therapies, where coordinated delivery of multiple agents requires precise spatiotemporal control over drug distribution and release kinetics.

The surface charge of NDDS critically modulates their circulation longevity and cellular internalization efficiency. Positively charged NDDS exhibit heightened plasma protein binding affinity, leading to accelerated clearance by the mononuclear phagocyte system (MPS), whereas negatively charged counterparts demonstrate reduced opsonization and extended plasma half-lives, explaining the predominant use of anionic surface modifications in clinical nanomedicines.^232,233 This charge-dependent biointeraction creates a therapeutic paradox: while cationic systems facilitate cellular uptake through electrostatic attraction to negatively charged cell membranes, their rapid systemic elimination compromises therapeutic efficacy. To resolve this dichotomy, advanced nanoplatforms with dynamically tunable surface properties have been engineered. A representative example involves cationic liposomes co-encapsulating Zebularine (DNA methyltransferase inhibitor) and JQ1 (BET protein inhibitor), which are electrostatically shielded with carboxymethyl-chitosan (CG-J/ZL).¹⁴³ This design maintains a stealthy anionic surface during systemic circulation, minimizing MPS recognition, yet undergoes pH-responsive charge reversal in the acidic tumor microenvironment. The resultant switch to positive surface potential enhances both tumor tissue penetration depth and cancer cell internalization rates. Such intelligent charge-transition systems exemplify the strategic integration of prolonged circulation stability with tumor-specific activation, providing a versatile blueprint for optimizing epigenetic combination therapies that require spatiotemporal coordination of drug bioavailability and target engagement.

Active Targeting

Active targeting mechanisms operate through precise ligand-receptor interactions, where nanoparticles functionalized with targeting moieties (eg, antibodies, peptides, or small-molecule ligands) via bioconjugation techniques achieve selective binding to overexpressed cellular receptors or tumor-associated biomarkers.²³⁴ This molecular recognition process facilitates receptor-mediated endocytosis, enabling intracellular delivery of therapeutic payloads to predetermined cell populations.²³⁵ The advancement of cellular biology and pathological understanding has transformed active targeting from theoretical concept to clinical reality, allowing spatiotemporal control over drug biodistribution while mitigating systemic toxicity – particularly critical for epigenetic combination therapies requiring cell-type-specific delivery to both malignant cells and tumor microenvironment components.

Tumor neovascularization constitutes a critical pathophysiological hallmark supporting malignant progression, characterized by distinct molecular signatures on endothelial cells including upregulated integrins, aminopeptidase N, and transmembrane glycoproteins.^236,237 These angiogenic biomarkers enable targeted drug delivery through ligand-receptor recognition systems, as exemplified by NGR peptide-modified cationic liposomes engineered for CD13 receptor binding on tumor vasculature. In the 4T1 murine breast cancer model, Liu et al demonstrated that NGR-functionalized liposomes co-encapsulating Zebularine (DNA methyltransferase inhibitor) and JQ1 (BET bromodomain inhibitor) achieved significantly higher tumor accumulation versus non-targeted counterparts.¹⁴³ Beyond synthetic targeting strategies, bioinspired approaches leverage endogenous cellular tropism – macrophage-derived exosome membrane-camouflaged nanoparticles loaded with suberoylanilide hydroxamic acid (SAHA) exploit the α4β1 integrin/VCAM-1 axis to infiltrate invasive tumor niches. This biomimetic design preserves natural immune-evasion properties while inheriting macrophage-specific tumor homing capabilities, resulting in significantly reduction in metastatic burden compared to conventional nanoparticles in preclinical trials.²³⁸ The convergence of synthetic biology and nanomedicine thus establishes new paradigms for overcoming biological barriers in cancer treatment, particularly for the spatial complexity of epigenetic combination therapies that require coordinated delivery to both neovasculature and tumor parenchyma.

Tumor cells constitute the principal cellular components of malignancies, making their selective elimination a cornerstone of cancer therapeutics. The accelerated proliferation of tumor cells drives the overexpression of surface receptors—including transferrin receptors, folate receptors, low-density lipoprotein receptors, and glucose transporters—compared to normal cells. These molecular signatures enable targeted drug delivery through receptor-specific ligands, as exemplified by lactoferrin-modified dual-drug liposomes co-loaded with Panobinostat and JQ1, which achieve active targeting via binding to low-density lipoprotein receptor-associated protein 1 (LRP1) on tumor surfaces.¹⁴¹ Similarly, hyaluronic acid-functionalized solid lipid nanoparticles (SLNs) exploit CD44 receptor overexpression for tumor-specific delivery of the histone deacetylase inhibitor Vorinostat.²³⁹ Beyond tumor cells, epigenetic modulation involves multiple cellular components within the tumor microenvironment. Tumor-associated lymphocytes, particularly T cells and macrophages, critically promote tumor progression and metastasis, necessitating targeted therapeutic strategies. Nanoparticles conjugated with anti-programmed death-1 monoclonal antibodies (eg, nivolumab) and loaded with decitabine (DAC) demonstrate dual functionality: they selectively target PD-1-overexpressing tumor-infiltrating lymphocytes (TILs), inhibit DNA methylation to reverse PD1+CD8+ TIL exhaustion, and synergistically enhance immune checkpoint blockade efficacy through T cell reinvigoration.¹⁵⁴ The integration of molecular recognition precision with epigenetic modulators’ pleiotropic mechanisms establishes a transformative framework for next-generation combination therapies, particularly in immune-cold tumors refractory to conventional treatments.

Responsive Release

Although nanomedicine delivery systems with passive or active targeting can deliver epigenetic drugs to tumor sites and inhibit tumor development by regulating specific cellular epigenetic mechanisms, the TME poses a significant obstacle to antitumor drug delivery. The TME, characterized by complex tumor localization, consists of various cells and their secretions, including peripheral blood vessels, extracellular matrix, tumor cells, and cytokines.²⁴⁰ As tumors develop, the TME undergoes substantial physicochemical changes at cellular and tissue levels, manifesting as high cell density, elevated interstitial pressure, micro-acidity, hypoxia, and abnormal expression of extracellular matrix components and enzymes.^241,242 While characteristics such as high cell density and interstitial pressure limit nanoparticle penetration and distribution within tumors, other features like hypoxia, micro-acidity, and overexpression of specific enzymes are exploited as endogenous stimuli in smart responsive drug delivery systems. These systems utilize these conditions to mediate targeted molecular activation, structural transformations, and controlled drug release, thereby enhancing tumor-targeted delivery efficiency.^243,244 Such responsive nano-drug delivery systems demonstrate significant potential for clinical translation of epigenetic therapies. Specifically, their intelligent responsiveness enables carrier depolymerization within tumor tissues followed by precise cellular targeting, which could substantially improve the efficacy of epigenetic treatments.

Throughout tumorigenesis and progression, the TME acquires pathological features including hypoxia, acidosis, elevated oxidative stress, supraphysiological glutathione (GSH) levels, and upregulated enzymatic activity. These distinctive features enable selective therapeutic strategies for nanomedicine delivery systems, particularly hypoxia-responsive, pH-responsive, reduction-oxidation (redox)-responsive, ATP-responsive, and enzyme-responsive platforms specifically designed for TME modulation.^245,246 A representative paradigm involves core-shell nanoparticles loaded with Zebularine, JQ1, and CpG, surface-functionalized with carboxymethyl chitosan (CMCS). This pH-sensitive coating undergoes degradation in acidic TME, triggering sequential drug release: Zebularine induces tumor-associated antigen (TAA) upregulation, potentiating tumor immunogenicity. JQ1 suppresses PD-L1 expression to abrogate immune checkpoint signaling. CpG activates dendritic cell maturation while reprogramming tumor-associated macrophages (TAMs) toward tumoricidal phenotypes.¹⁴³ This multimodal approach demonstrates significant potential for revitalizing anti-tumor immunity through coordinated epigenetic modulation and immune microenvironment remodeling.

The strategic integration of multiplexed TME stimuli with rationally engineered cascade-responsive nanoplatforms enables a hierarchical therapeutic paradigm encompassing tumor-preferential drug accumulation, subcellular targeting precision, and spatiotemporally controlled release kinetics, which synergistically amplifies antitumor epigenetic therapy efficacy while minimizing systemic toxicity. Illustrating this concept, Ruan et al developed a dual-responsive hydrogel platform capable of sequentially sensing acidic pH and reactive oxygen species (ROS) gradients to co-deliver anti-PD1 antibodies (aPD1) and the hypomethylating agent Zebularine (Zeb). Mechanistically, pH-sensitive calcium carbonate nanoparticles (CaCO3 NPs) were utilized to nanoformulate aPD1, which were subsequently co-encapsulated with Zeb within a ROS-labile hydrogel matrix (Zeb/aPD1-NPs-Gel). Preclinical evaluation in B16F10 melanoma models demonstrated concomitant immunogenic cell death induction and immunosuppressive network reprogramming, culminating in significant tumor growth suppression and extended median survival.²⁰² This multimodal therapeutic strategy, integrating TME-responsive cascade delivery with epigenetic-immune crosstalk modulation, establishes a blueprint for precision oncology by orchestrating spatiotemporal drug release dynamics to concurrently potentiate tumor immunogenicity and dismantle immunosuppressive barriers, ultimately achieving therapeutic synergy with minimized off-target effects.

Intracellular Regulation

The complexity of epigenetic modification lies in its diverse cellular targets, including organelle-localized epigenetic enzymes that require precise intracellular delivery. Nanoparticle intracellular trafficking critically determines therapeutic efficacy through a multistage transport process: upon reaching the plasma membrane, nanoparticles undergo endocytic uptake via membrane invagination, forming endocytic vesicles that mature into early endosomes. These vesicular carriers subsequently engage the intracellular sorting network—a coordinated system involving the Golgi apparatus, endoplasmic reticulum (ER), and lysosomes—to determine nanoparticle fate. While most nanoparticles remain confined within the endolysosomal pathway, some achieve cytoplasmic escape prior to lysosomal fusion through membrane-disruptive mechanisms, thereby evading enzymatic degradation.^247–249 Current research focuses on engineering nanoparticles with lysosomal escape capabilities using pH-responsive biomaterials or autophagy-inducing components, strategies particularly relevant for epigenetic drug delivery where nuclear access or mitochondrial targeting often requires intact cytoplasmic release. This spatial control over intracellular trafficking addresses the dual challenge of protecting epigenetic modulators from lysosomal inactivation while ensuring precise subcellular localization for optimal therapeutic intervention.

The endosomal-lysosomal pathway represents a critical barrier for therapeutic nanoparticles due to its degradative microenvironment characterized by acidic pH and enzymatic activity, which compromises drug bioavailability. To circumvent this limitation, nanoparticle engineering focuses on developing endosomal escape mechanisms involving membrane disruption strategies such as the proton sponge effect, lipid bilayer destabilization, and pH-dependent fusogenic properties.²⁵⁰ A representative paradigm employs vorinostat-loaded nanoparticles constructed from pH-responsive poly(lactic-co-glycolic acid) (PLGA) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). Upon endocytosis, the acidic endosomal environment triggers protonation of DOTAP’s tertiary amine groups, inducing osmotic swelling through the proton sponge effect. This physicochemical transition destabilizes endosomal membranes, facilitating cytoplasmic release of intact therapeutic payloads while bypassing lysosomal degradation.²⁵¹ Such intelligent nanoparticle architectures demonstrate the potential to overcome intracellular trafficking barriers critical for epigenetic drug delivery, where maintaining drug structural integrity and achieving nuclear localization are essential for restoring normal chromatin remodeling processes.

Autophagic processes critically influence nanoparticle intracellular trafficking through their dual role as a degradation pathway and therapeutic modulator. Distinct from classical endocytic pathways, autophagy mediates lysosomal degradation of cytoplasmic components through autophagosome-lysosome fusion, posing a potential risk for recapturing cytoplasmically escaped nanoparticles.^252,253 While this lysosomal re-encapsulation compromises drug bioavailability, emerging strategies exploit autophagy’s regulatory mechanisms for controlled drug release. A proof-of-concept design employs azacitidine-loaded biomimetic metal-organic frameworks (MOFs) functionalized with autophagy-related protein Beclin-1. Upon cellular internalization, Beclin-1 mediates selective binding to Golgi-associated plant pathogenesis-related protein 1 (GAPR-1), inducing autophagosome accumulation at Golgi apparatus membranes. This targeted autophagy activation achieves pH-responsive release of azacitidine while bypassing lysosomal degradation, enhancing DNA methyltransferase inhibition efficacy in leukemia models.¹⁸⁸ Such autophagy-mediated delivery systems present a paradigm-shifting approach for epigenetic therapeutics, leveraging cellular quality-control mechanisms to achieve spatiotemporal drug release. These advancements highlight the necessity of comprehensive autophagy profiling in nanomedicine develocularly for epigenetic drugs requiring sustained nuclear bioavailability.

Challenges from the Laboratory to the Clinic

Despite the significant potential demonstrated by NDDS-based epigenetic combination therapies in preclinical studies, their translation from laboratory to clinical applications remains great challenges. These challenges span critical areas including scalable manufacturing, complex in vivo behaviors, and long-term safety.

A primary challenge is the transition from laboratory-scale production to large-scale manufacturing compliant with Good Manufacturing Practice (GMP) guidelines. The intricate architecture of NDDSs-such as the co-loading of multiple epigenetic drugs with diverse physicochemical properties, surface modification with targeting ligands, or incorporation of environmentally responsive polymers-poses significant challenges during process scaling. These challenges including batch-to-batch variability in critical quality attributes, fluctuations in drug loading efficiency, and high production costs severely constrain their clinical translation feasibility.

Furthermore, the pharmacokinetic (PK) profiles and immunogenicity risks of engineered nanocarriers in humans present another major challenge. The favorable PK characteristics observed in preclinical models may significantly diverge in human subjects due to inter-individual physiological variations, the formation of plasma protein coronas, and recognition by the mononuclear phagocyte system. This variability leads to unpredictable biodistribution, clearance rates, and tumor targeting efficiency. More critically, the introduction of nanocarriers may inadvertently activate both innate and adaptive immune responses. Such immunogenicity not only accelerates carrier clearance and compromises therapeutic efficacy but could also trigger severe inflammatory reactions, introducing unpredictable risks to treatment regimens.

Particularly noteworthy are the nanotoxicological and long-term safety concerns inherent in NDDS-mediated epigenetic combination therapies, which represent a core issue requiring meticulous evaluation in clinical translation. On the one hand, the mechanisms of action of epigenetic drugs themselves are non-specific. Off-target delivery of drugs or premature leakage from nanocarriers could result in unintended editing of the epigenome in healthy tissues. Such non-specific epigenetic perturbations—for instance, aberrant alterations in DNA methylation or widespread dysregulation of histone modifications—may induce permanent functional impairment in non-cancerous cells and potentially even drive the development of novel pathological states, the long-term consequences of which remain difficult to fully assess. On the other hand, the intrinsic biosafety of the carrier materials is paramount. For example, certain metal-based nanoparticles (such as those composed of gold, silver, or iron oxide) or metal-organic frameworks (MOFs) may undergo incomplete biodegradation and clearance in vivo, leading to their long-term accumulation in the reticuloendothelial system (eg, liver and spleen). This accumulation not only potentially induces organ-specific oxidative stress and inflammatory damage but also presents a significant regulatory blind spot due to the lack of long-term follow-up data regarding their chronic toxicity risks.

Finally, the complexity of multifunctional nano-platforms introduces unprecedented challenges in regulatory science. When a single formulation integrates multiple functionalities—such as targeted delivery, controlled release, imaging capabilities, and even immunomodulation—it transcends the definitional boundaries of conventional pharmaceuticals. Regulatory agencies are consequently faced with the challenges of how to classify such complex products, establish matching quality control standards, and design rational clinical trials that can simultaneously validate the efficacy of their multiple functions alongside their overall safety profile.

In summary, the translational pathway of NDDS-based epigenetic combination therapies from the laboratory to the clinic constitutes a process of addressing systematically interconnected challenges. These challenges permeate the entire continuum-from scalable manufacturing, through in vivo evaluation, to long-term safety-and ultimately converge at the level of regulatory science, demanding the establishment of novel evaluation frameworks for these highly complex therapeutic products. Only through deep interdisciplinary integration, building solid bridges between materials science, epigenetics, nanotoxicology, and clinical medicine, and by systematically overcoming these bottlenecks one by one, can the full potential of these therapies in precision oncology be ultimately realized.

Conclusion

The pivotal role of epigenetic dysregulation in oncogenesis and tumor progression has been increasingly elucidated, with distinct mechanisms of gene activation/silencing through DNA methylation, histone modification, and chromatin remodeling driving therapeutic development. While epigenetic drugs targeting these pathways demonstrate clinical potential, monotherapy limitations necessitate combinatorial approaches requiring nanomedicine-enabled precision delivery. Current challenges in multi-drug regimens—including differential pharmacokinetics, spatiotemporal release coordination, and tumor microenvironment-specific targeting—are being addressed through advanced nano-architectures. Engineered carriers such as liposomes, polymeric nanoparticles, and inorganic frameworks now achieve programmable drug release via stimuli-responsive designs, allowing sequential activation of epigenetic modifiers while maintaining therapeutic payload integrity.

Translating these nanoplatforms into clinical applications still faces significant hurdles. At the industrial level, critical parameters such as nanoparticle stability, drug loading efficiency, and scalability for mass production require substantial optimization. In clinical translation, it is imperative to establish reliable patient stratification strategies and address complex regulatory approval requirements. Notably, recent preclinical studies demonstrating breakthroughs in overcoming tumor heterogeneity and immune evasion underscore the value of interdisciplinary collaboration integrating nanotechnology, epigenetics, and clinical medicine. Furthermore, integrating multi-omics technologies such as proteomics, transcriptomics, and single-cell epigenomics into the development of NDDS combined therapy provides powerful tools for understanding tumor epigenetic heterogeneity and optimizing treatment strategies. These high-throughput technologies can reveal the molecular networks of drug action, identify biomarkers related to drug resistance, and guide the personalized design of nanocarriers, thereby maximizing the precision of treatment.

Looking ahead, emerging technologies are poised to substantially advance epigenetic combination therapies. AI-guided carrier design leverages deep learning of multi-omics data and nanomaterial properties to enable inverse design of NDDS and dynamic optimization of drug combinations. Bioinspired smart delivery platforms, by mimicking biological sensing–response mechanisms and intercellular communication, equip nanocarriers with spatiotemporally precise drug release capabilities and tumor microenvironment adaptivity. Meanwhile, personalized epigenetic strategies integrate patient-specific epigenetic profiles to construct precision treatment platforms. The deep integration of these cutting-edge directions will propel epigenetic therapy toward truly personalized cancer treatment in the clinic.

Accordingly, a systematic research agenda must be pursued with the following priorities: First, it is essential to develop nanocarrier systems that balance functionality with scalable manufacturing, establishing standardized preparation processes. Second, an AI platform integrating multi-dimensional data should be constructed to achieve precise matching of therapeutic strategies. Concurrently, environmentally adaptive intelligent delivery systems must be designed to enable accurate spatiotemporal control over drug release. Furthermore, an epigenetic biomarker-based patient stratification framework needs to be established, alongside collaborative efforts with regulatory agencies to develop corresponding evaluation standards. Ultimately, through the deep integration of materials science, computational science, and clinical medicine, a complete innovation chain—from fundamental discovery to clinical application—should be built. The implementation of this systematic research agenda will strongly propel epigenetic nanomedicine toward realizing its revolutionary potential and pave the way for new approaches in precision cancer therapy.