Harnessing macrophages for precision drug delivery and cancer therapy: Strategies, advances and challenges

Abstract

Macrophages possess inherent tumor tropism, the capacity to traverse biological barriers, and potent immunomodulatory activity, positioning them as living carriers for precision drug delivery in cancer therapy. By leveraging these properties, macrophage-based drug delivery systems mitigate persistent limitations of conventional modalities, including poor intratumoral penetration, off-target toxicity, and rapid systemic clearance. This review highlights the unique capabilities of macrophages in drug delivery, discussing their sources, preparation, and engineering strategies for enhanced therapeutic efficacy. Key advancements in drug loading methods and applications in cancer treatment are also discussed. Despite these advancements, macrophage-based systems face challenges such as immune compatibility, large-scale production, and precise control of drug release. We conclude by outlining future opportunities to harness macrophages for precision medicine, emphasizing their transformative potential in improving cancer therapy outcomes.

Graphical abstract

Abbreviations

AMs	Alveolar macrophages
BMDMs	Bone marrow-derived macrophages
BBB	Blood-brain barrier
CCL-2	C-C motif ligand 2
CSF-1	Colony-stimulating factor 1
CNS	Central nervous system
CAR-Ms	Chimeric antigen receptor macrophages
CAR-iMACs	Induced pluripotent stem cell-derived CAR-macrophages
DOX	Doxorubicin
EPR	Enhanced permeability and retention effect
ECM	Extracellular matrix
GM-CSF	Granulocyte-macrophage colony-stimulating factor
iPSCs	Induced pluripotent stem cell-derived
CAR-iMACs	IPSCs CAR-macrophages
IL-12	Interleukin-12
IL-10	Interleukin-10
LPS	Lipopolysaccharide
MPS	Mononuclear phagocyte system
MDSCs	Myeloid-derived suppressor cells
MDMs	Monocyte-derived macrophages
M-CSF	Macrophage colony-stimulating factor
NIR	Near-infrared
NPs	Nanoparticles
NO	Nitric oxide
PGE2	Prostaglandin E2
PRRs	Pattern recognition receptors
PAMs	Porcine alveolar macrophages
ROS	Reactive oxygen species
TME	Tumor microenvironments
TAMs	Tumor-associated macrophages
TCR	T-cell receptor
TNF-α	Tumor necrosis factor-alpha
TLRs	Toll-like receptors
TLR4	Toll-like receptor 4
TIR	Toll/IL-1R

1. Introduction

Nanotechnology has revolutionized drug delivery by providing transformative solutions to long-standing challenges in medicine. Nanoparticles (NPs), with their tunable size, modifiable surface properties, and functional versatility, offer considerable advantages over conventional small-molecule drugs [[1], [2], [3], [4]]. These systems enhance drug solubility, protect therapeutic agents from premature degradation, prolong systemic circulation time, and enable controlled drug release [[5], [6], [7], [8]]. A key mechanism underlying their efficacy is the enhanced permeability and retention (EPR) effect, whereby the leaky vasculature and impaired lymphatic drainage of tumors facilitate the preferential accumulation of NPs in the tumor microenvironment (TME) [[9], [10], [11]]. These advances have led to the development of clinically successful nanomedicines, including Doxil® (liposomal doxorubicin) and Abraxane® (albumin-bound paclitaxel), which exhibit improved efficacy and reduced systemic toxicity in cancer treatment [12,13]. Despite this potential, conventional nanoparticle-based delivery systems face several critical limitations that hinder their widespread application and therapeutic impact. Although the EPR effect provides a basis for tumor targeting, its heterogeneity significantly constrains clinical outcomes. Quantitative analyses indicate that typically less than 1 % of the injected nanoparticle dose accumulates in solid tumors, with considerable variation across tumor types [14]. Clinical and preclinical studies report that EPR-mediated accumulation ranges from as low as 0.7 % to over 50 % of the injected dose per gram of tissue, depending on the tumor model and vascular properties [15]. Advanced imaging and machine learning approaches have further elucidated this heterogeneity, revealing more than 100-fold variations in vascular permeability among different tumor types and even within the same tumor [16]. A major challenge lies in the limited penetration of NPs into tumor tissues [17]. Although the EPR effect offers a targeting advantage, its efficiency is highly variable among patients [18], resulting in spatially uneven drug distribution. This leaves certain tumor regions untreated and can promote therapeutic resistance. The clinical significance of this variability is underscored by the fact that only about 14 % of nanomedicines entering clinical trials demonstrate clear therapeutic benefits, largely attributable to inconsistent EPR effects across patient populations [19]. Furthermore, nanoparticles are frequently recognized and rapidly cleared by the mononuclear phagocyte system (MPS), particularly by resident macrophages in the liver and spleen, which significantly reduces their circulation time and diminishes therapeutic efficacy [20]. While surface modifications such as polyethylene glycol (PEG) conjugation have been employed to enhance "stealth" properties, immune recognition and opsonization remain persistent challenges [21,22]. Another critical limitation is the lack of targeting specificity. Passive targeting via the EPR effect provides some tumor selectivity but is insufficient for precise drug delivery. Consequently, off-target accumulation in healthy tissues can cause adverse effects. The complex and heterogeneous nature of the TME further complicates delivery; tumors are often characterized by abnormal vasculature, high interstitial fluid pressure, and dense extracellular matrices, all of which pose significant physical and biological barriers to effective nanoparticle penetration. Beyond the TME, systemic biological barriers such as the blood-brain barrier (BBB) restrict nanoparticle access to critical regions like the central nervous system (CNS), limiting their utility in treating brain tumors and other CNS-related diseases [23].

Given these challenges, researchers have turned to alternative strategies aimed at enhancing the efficacy of drug delivery, with particular focus on leveraging the intrinsic biological properties of living cells. Cell-based drug delivery systems have thus emerged as an innovative solution, offering distinct advantages over synthetic nanoparticles [[24], [25], [26]]. Living cells possess inherent biological functionalities, such as active tumor-homing, immune modulation, and the ability to navigate complex biological barriers, which make them ideal candidates for advanced therapeutic applications [[27], [28], [29]]. Although various immune cells, including neutrophils and T cells, have been explored for drug delivery, macrophages exhibit a unique combination of attributes that make them especially versatile carriers. Neutrophils, the most abundant white blood cells, exhibit rapid chemotaxis toward acute inflammatory sites. However, their extremely short circulatory half-life (typically <24 h) and predisposition to undergo rapid, programmed cell death following isolation severely limit their utility as sustained drug delivery vehicles [30]. In contrast, macrophages exhibit a longer persistence in circulation and within the TME, allowing for sustained drug release. Moreover, while neutrophils are excellent for targeting acute inflammation, macrophages are key players in chronic inflammation, which is a hallmark of established solid tumors, making them more suitable for long-term therapy [31]. T cells, especially with the advent of CAR-T therapy, have demonstrated unparalleled efficacy in targeting hematological malignancies. Their exquisite specificity for tumor antigens is a major advantage. However, this strength is also a limitation for broad-spectrum drug delivery. T cell infiltration into solid tumors is highly dependent on the presence of specific antigens and a permissive, non-immunosuppressive TME, which is often not the case [32]. Furthermore, efficiently loading T cells with therapeutic payloads without impairing their critical T-cell receptor (TCR) signaling and cytotoxic functions is a significant technical challenge. Macrophages, on the other hand, employ a more general "sense-and-move" homing mechanism, responding to a wide array of tumor-derived chemokine, which enables them to target a broader range of solid tumors, including those that are poorly immunogenic or immunosuppressive. In addition, as professional phagocytes, macrophages can naturally engulf and carry large payloads without compromising their core function, making them inherently suitable as drug carriers. These combined advantages—broad tumor-homing capability and high payload capacity—are precisely why macrophages have garnered particular attention among the various cell types explored for drug delivery [33].

Macrophages are highly dynamic immune cells that can migrate to and accumulate in tumors, driven by chemotactic signals such as C-C motif ligand 2 (CCL-2), vascular endothelial growth factor (VEGF), and colony-stimulating factor 1 (CSF-1) secreted by tumor cells and the surrounding microenvironment [34,35]. These cells originate from multiple sources, including yolk-sac progenitors during embryogenesis (tissue-resident macrophages) and circulating monocytes derived from bone marrow hematopoietic stem cells, which differentiate upon entering tissues. Key surface markers for identifying macrophage lineage include CD14 (human monocytes/macrophages), CD11b (integrin alpha M), and F4/80 (mouse macrophages), which are commonly used to isolate and characterize these cells [36]. The tumor-homing ability allows macrophages to access regions of the tumor that are typically inaccessible to synthetic nanoparticles, including hypoxic and necrotic cores. Furthermore, macrophages can penetrate biological barriers such as the BBB, offering a unique advantage for delivering therapeutic agents to hard-to-reach sites like brain tumors. Beyond their innate tumor-targeting capabilities, macrophages also play a central role in modulating the immune system [37,38]. A critical functional attribute is their plasticity, which enables polarization into distinct functional phenotypes: classically activated (M1-type) or alternatively activated (M2-type) macrophages. M1 macrophages, induced by IFN-γ and LPS, exhibit pro-inflammatory, anti-tumor properties and are characterized by high surface expression of markers such as CCR7 and CD86, along with secretion of cytokines like tumor necrosis factor-alpha (TNF-α) and interleukin-12 (IL-12). In contrast, M2-type macrophages, induced by IL-4 or IL-13, display anti-inflammatory, pro-tumorigenic functions and are identified by high expression of CD206 (mannose receptor) and CD163, as well as secretion of IL-10 and TGF-β. This polarization is not static; M1 and M2 phenotypes can switch themselves, and this plasticity can be controlled using drugs/chemicals or engineering strategies [39,40]. They can be engineered to either deliver cytotoxic agents directly to tumor cells or reprogram the TME to enhance anti-tumor immunity, thus serving as both delivery vehicles and active participants in cancer therapy.

Herein, this review provides a comprehensive overview of macrophage-based drug delivery systems, emphasizing their unique capabilities and applications in cancer therapy. While several excellent reviews have surveyed the broad challenges and general principles of macrophage-based drug delivery [41], this article is positioned to provide a focused and forward-looking analysis of the engineering strategies driving the field toward precision medicine, particularly in oncology. We discuss the sources and preparation of macrophages, including immortalized cell lines and primary macrophages, as well as strategies for selecting specific macrophage types targeted to therapeutic needs. Recent advancements in drug loading techniques, such as physical encapsulation, surface binding, and functional engineering through biochemical and genetic modifications, are also discussed in detail. Additionally, we explore the advantages and applications of macrophage-based delivery systems in cancer therapy, and conclude with an in-depth discussion of the future challenges and opportunities in this promising field (see Scheme 1).

Scheme 1.

Schematic illustration of harnessing macrophages for precision drug delivery and cancer therapy, including the properties (phagocytosis, tumor-homing abilities, modulate the immune response) and derivation of macrophages (immortalized cell lines &primary macrophages), drug loading technologies (physical encapsulation or phagocytosis, surface covalent binding or non-covalent binding) and functional engineering (biochemical modifications, genetic modification).

2. Design and engineering of macrophage-based delivery systems

2.1. Sources and preparation of macrophages

Macrophages, which play essential roles in immune regulation and tissue homeostasis, have gained increasing attention as versatile platforms for drug delivery [42]. The development of macrophage-based delivery systems generally involves three critical steps: selecting a suitable cell source, optimizing drug loading strategies, and proper functional engineering to enhance their functionality and therapeutic potential. The choice of macrophage source significantly influences their biological properties, scalability, and translational potential. Macrophages are commonly can be classified into immortalized macrophage cell lines and primary macrophages, including bone marrow-derived, tissue-resident and autologous monocyte-derived macrophages [41,43,44]. Each type possesses distinct characteristics that render it suitable for specific experimental or therapeutic contexts (Table .1).

Table 1.

Comparison of macrophage sources for drug delivery.

Macrophage Source	Key Advantages	Major Limitations	Suitable Applications	Primary Challenges
Immortalized Cell Lines (e.g., Raw 264.7)	•Scalability & Reproducibility: Easy to culture and expand indefinitely, suitable for high-throughput screening. •Genetic Manipulation: Amenable to efficient transfection and genetic engineering. •Cost-Effective: Lower cost and effort compared to primary cell isolation.	•Altered Phenotype: May not fully replicate primary macrophage physiology and in vivo behavior. •Immunogenicity & Tumor Risk: Potential to induce immune responses or form tumors in vivo, limiting clinical translation. •Limited Donor Diversity: Lack of genetic and phenotypic diversity.	•Early-stage proof-of-concept studies and mechanistic research. •High-throughput screening of drug carriers and nanoparticles. •Investigating basic cell-nanoparticle interactions.	•Overcoming immunogenicity for safe in vivo application. •Ensuring phenotypic stability and functional relevance.
Monocyte-Derived Macrophages (MDMs)	•Autologous Potential: Can be derived from patient's own blood, minimizing immune rejection. •Human-Relevant Model: Directly relevant for human disease modeling and therapy. •Clinical Feasibility: Leukapheresis allows for obtaining substantial cell numbers.	•Donor Variability: Function and drug loading efficiency can vary between donors. •Limited Proliferation: Differentiated macrophages are post-mitotic, restricting expansion. •Complex Isolation: Requires access to human blood and specific isolation procedures.	•Personalized cancer immunotherapy and drug delivery. •Autologous cell therapy applications. •Studying human-specific pathogen-macrophage interactions.	•Managing donor-to-donor variability. •Developing cost-effective and scalable Good Manufacturing Practice (GMP) processes for autologous therapies.
Bone Marrow-Derived Macrophages (BMDMs)	•High Yield & Homogeneity: Can generate a large number of relatively homogeneous cells from one donor. •Physiological Relevance: More representative of native macrophage biology than cell lines. •Flexible Polarization: Can be polarized to various phenotypes (M1/M2) in vitro.	•Species-Specific: Typically isolated from mice, raising questions about human translatability. •Phenotypic Instability: May lose polarized phenotype upon infusion in vivo. •Time-Consuming Process: Isolation and differentiation require 7–10 days.	•Preclinical studies in mouse models. •Investigating polarization-dependent drug delivery and therapy. •Exploring macrophage interactions with the tumor microenvironment (TME).	•Improving phenotypic persistence in the complex in vivo environment. •Scaling up and standardizing differentiation protocols for clinical use.
Tissue-resident macrophages (Kupffer, alveolar, microglia, splenic/peritoneal)	•Native Tissue Homing & Retention: Inherently reside in and are adapted to specific organ. •Specialized Functions: Perform tissue-specific functions (e.g., surfactant clearance by alveolar macrophages). •Optimal for Organ-Specific Targeting: Ideal for treating diseases of their native organ.	•Extremely Low Yield: Difficult to isolate in large quantities. •Phenotype Maintenance: Challenging to maintain specialized phenotype ex vivo. •Complex Isolation: Require sophisticated tissue processing techniques.	•Organ-specific drug delivery (e.g., lungs, liver, brain). •Understanding the role of specific macrophage populations in disease. •Ex vivo engineering for reinfusion into the same tissue.	•Developing methods for efficient isolation and expansion. •Preserving unique tissue-specific markers and functions during culture and engineering.

2.1.1. Immortalized cell lines

Immortalized macrophage cell lines, such as the widely used Raw 264.7 line, play a pivotal role in preclinical studies. Originating from a murine leukemia model induced by the Abelson murine leukemia virus in the 1970s, these cells offer significant advantages in scalability, ease of culture, and experimental reproducibility [45]. Such properties make them an ideal platform for exploring macrophage-based drug delivery systems. For example, Xiao and co-authors reported an updated hybrid nanogel (NGs) system designed for macrophage (Raw 264.7 cells)-mediated tumor delivery and enhanced tumor therapy [46]. In their study, cystamine dihydrochloride-crosslinked hyaluronic acid (HA) NGs were prepared using a double-emulsion method, loaded with polypyrrole nanoparticles (PPy NPs) through in-situ oxidative polymerization of pyrrole monomers, and encapsulated with doxorubicin (DOX) via π-π stacking with the hydrophobic PPy NPs. Once endocytosed by macrophages, the HA NGs formed macrophage-nanogel complexes, maintaining the macrophages' normal biofunctional properties (Fig. 1A). The hybrid macrophage-nanogel system leverages the innate tumor-homing ability of macrophages to deliver the therapeutic payload precisely to the tumor site. In vitro studies demonstrated that these complexes could target cancer cells effectively while preserving macrophage functionality. In vivo, the macrophage-nanogel system exhibited enhanced tumor accumulation, taking advantage of macrophages' intrinsic migratory and targeting properties. Furthermore, the system enabled combination photothermal-chemotherapy when exposed to laser irradiation, offering a promising platform for advanced cancer therapy. Another study by Hu et al. developed a biomimetic delivery system using Raw264.7 macrophage-like cells loaded with DOX to improve cancer therapy [47]. The system was constructed through simple incubation, achieving significant drug loading without affecting cell viability. The DOX-loaded macrophages maintained their tumor-targeting ability toward 4T1 mouse breast cancer cells in vitro and in vivo (Fig. 1B). In addition, in an innovative departure from conventional phagocytosis-dependent strategies, Hu et al. demonstrated that macrophages can be engineered to function as "backpacks" for nanoparticle (NP) delivery by anchoring drug-loaded NPs to their surface [48]. The authors designed a pH-responsive system wherein DOX-loaded nanomicelles (DMPM) were conjugated to Raw264.7 macrophages via CCR2/MCP-1 axis interactions, creating a macrophage-DMPM (MA-DMPM) complex (Fig. 1C). In vitro, MA-DMPM exhibited superior tumor-homing efficiency (220 % vs. control), while in vivo experiments using NCI-H1299 tumor-bearing mice demonstrated significant tumor accumulation via fluorescence imaging. Notably, pH-responsive DOX release in the acidic tumor microenvironment (TME) enhanced drug delivery, resulting in a 63.33 % tumor cell apoptosis rate and potent antitumor efficacy. Similarly, Chen and colleagues engineered Raw264.7 macrophages as living carriers for tumor-targeted combination therapy [49]. By covalently conjugating β-elemene-loaded germanium-sulfide nanosonosensitizers (GeSNSs@ELE) to macrophage surfaces via maleimide-thiol chemistry, the team created a dual-functional platform that leverages macrophages' intrinsic tumor-homing ability while avoiding phagocytic clearance (Fig. 1D). This innovative "hitchhiking" strategy enabled efficient delivery of both sonodynamic therapy (GeSNSs) and chemotherapy (β-elemene) to tumors, capitalizing on macrophages' natural capacity to navigate biological barriers and modulate the tumor microenvironment.

Fig. 1.

Immortalized Cell Lines (Raw 264.7 cells)-based for drug delivery. (A) Schematic design of MAs internalized with HA/DOX@PPy NGs (MAs-NGs) for MA-mediated tumor delivery and combination PTT/chemotherapy of tumors. Reprinted with permission from ref.46, copyright©2021 Ivyspring International Publisher. (B) Transwell test to demonstrate the migration ability of Raw264.7 cells towards 4T1 tumor cells in vitro and bioluminescence test to track Raw264.7 cells with or without DOX loading in lung metastasis of breast cancer bearing mouse model. Reprinted with permission from ref.47, copyright©2015 Elsevier Publisher. (C) Schematic of macrophage-mediated targeted DOX delivery via surface-bound functionalized Pluronic nanoparticles. Reprinted with permission from ref.48, copyright©2024 American Chemical Society Publisher. (D) Schematic representation of macrophage-facilitated GeSNSs@ELE delivery, combining β-elemene's chemotherapeutic effects with GeS-mediated sonodynamic therapy to simultaneously eradicate tumor cells and remodel the immunosuppressive tumor microenvironment. Reprinted with permission from ref.49, copyright©2025 American Association for the Advancement of Science Publisher.

The above studies highlight the significant potential of immortalized macrophage cell lines as versatile and scalable platforms for developing innovative macrophage-based drug delivery systems, offering enhanced tumor targeting and therapeutic efficacy in cancer treatment. Despite the potential of using Raw264.7 cells as drug delivery carriers, several limitations exist, primarily due to their immunogenicity [50]. Raw 264.7 cells are inherently immune-active, possessing numerous pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), that make them highly responsive to stimuli. The robust immunogenicity of Raw 264.7 cells primarily stems from their origin—they were derived from a tumor induced by the Abelson murine leukemia virus in a male BALB/c mouse. This background equips them with a complete and sensitive innate immune recognition system. For instance, transcriptome analyses have revealed that live Edwardsiella tarda infection in Raw 264.7 cells trigger significant differential expression of hundreds of immune-related genes, including those encoding cytokines and interferon-related factors, compared to stimulation with dead bacteria. This highlights the cells' capacity for potent, pathogen-specific immune responses. While this characteristic is advantageous in their natural role as immune modulators, it becomes a challenge in drug delivery systems. Activation of these receptors can lead to the secretion of pro-inflammatory cytokines and chemokines. For example, studies have shown that lipopolysaccharide (LPS)-activated Raw 264.7 macrophages significantly upregulate the production of nitric oxide (NO), prostaglandin E2 (PGE2), and pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 [51]. This activation is often mediated through key signaling pathways like NF-κB and MAPK. Conversely, certain pathogens, like Mycobacterium bovis BCG, can exploit bacterial kinases such as PknG to manipulate the host macrophage's phosphoproteome, thereby reprogramming normal macrophage functions including cytoskeletal organization and phagosome maturation to evade immune clearance [52]. This demonstrates that the very immune pathways that make Raw 264.7 cells responsive can also be hijacked, leading to unpredictable behavior. Therefore, while Raw 264.7 cells have undoubtedly been invaluable in advancing macrophage-based drug delivery research, their immunogenicity represents a significant barrier that necessitates thoughtful mitigation strategies for broader application. Future directions may involve genetic engineering to knockout key PRRs or cytokines, pre-conditioning the cells to induce a more tolerant state, or using targeted pharmacological inhibitors to suppress undesirable activation pathways upon administration, thereby improving their safety and efficacy profile as drug carriers in vivo.

2.1.2. Primary macrophages

Primary macrophages, isolated directly from natural sources, have emerged as a cornerstone in the field of macrophage-based drug delivery systems [53]. Their use leverages their physiological relevance, immune functionality, and innate adaptability, distinguishing them from immortalized cell lines, which often deviate from native phenotypes due to genetic modifications and prolonged culture. Based on origin, primary macrophages are commonly classified into monocyte-derived macrophages (MDMs), bone marrow-derived macrophages (BMDMs), and tissue-resident macrophages. Each type presents distinct advantages, limitations, and research applications, all of which impact their suitability for preclinical research and therapeutic development.

Monocyte-derived macrophages (MDMs) represent a highly versatile and accessible platform for cell-based drug delivery. Isolated from peripheral blood monocytes via density gradient centrifugation or immunomagnetic separation, these cells can be differentiated into functional macrophages using cytokines such as macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) [54,55]. Their compatibility with autologous use offers significant advantages, minimizing the risks of immune rejection and systemic side effects. Recent studies have further demonstrated the potential of MDMs in enhancing the efficacy of drug delivery systems. Lang et al. developed Ly6Chi monocytes loaded with pH-sensitive paclitaxel micelles (PM@MC), which homed to both primary tumors and metastatic sites in a breast tumor model (Fig. 2A) [56]. The study reported a 15-fold increase in intratumoral drug accumulation compared to commercial paclitaxel formulations, achieving nearly complete suppression of lung metastasis and primary tumor growth with minimal systemic toxicity. The results highlighted the potential of MDMs to efficiently deliver chemotherapeutics to tumors while maintaining biocompatibility. Furthermore, Bryan Ronain et al. explored the tumor-targeting mechanisms of Ly6Chi monocytes using single-walled carbon nanotubes [57]. Their findings showed that these nanotubes were selectively taken up by Ly6Chi monocytes and delivered to tumor sites with enhanced precision. Conjugating targeting ligands, such as RGD peptides, significantly increased monocyte-mediated delivery, demonstrating an immune-cell-based strategy for improving nanoparticle penetration into tumors (Fig. 2B). Additionally, Wang et al. investigated nano-doxorubicin-loaded monocytes (Nano-DOX-MC) for glioblastoma treatment [58]. Their work revealed that Nano-DOX-MC crossed endothelial barriers, infiltrated glioblastoma spheroids, and released therapeutic agents within the tumor. Furthermore, the study uncovered lysosomal sequestration and exocytosis as key pathways enabling monocytes to tolerate and deliver Nano-DOX effectively. The treatment induced autophagy and damage-associated molecular patterns in glioblastoma cells, enhancing drug efficacy and tumor-specific responses.

Fig. 2.

Primary macrophages-based for drug delivery. (A) The tumor-targeting drug delivery system based on the Ly6Chi monocytes. The pH-sensitive amphiphilic block copolymer PDB was synthesized to prepare paclitaxel-loaded micelles (PM), which were then internalized by mouse Ly6Cᵒʰᵢ monocytes. These PM-loaded monocytes (PM@MC) retained the innate tropism for tumor tissues and lung metastases in vivo, entering tumor cells via endocytosis. Following lysosomal fusion and acidification, the protonation of PDB's hydrophobic block triggered micelle dissociation, resulting in cytoplasmic drug release. Reprinted with permission from ref.56, copyright©2017 Wiley-VCH Publisher. (B) Schematic of SWNTs non-covalently coated with a block amphiphile phospholipid PEG on the surface. Reprinted with permission from ref.57, copyright©2014 Springer Nature Publisher. (C) The microrobot composed of splenocyte-derived MΦs loaded with CA-MNPs and DOX-TSLPs, are guided to the tumor site by both an electromagnetic field and tumor cell-secreted chemical gradients, thus enabling targeted tumor cell destruction through NIR-triggered drug release. Reprinted with permission from ref.64, copyright©2021 American Chemical Society Publisher. (D) Scheme of the AMs-mediated 19F-MR imaging strategy. Reprinted with permission from ref.65, copyright©2023 American Chemical Society Publisher.

Bone marrow-derived macrophages (BMDMs) are another widely utilized source in macrophage-based therapeutic research. Typically obtained from animal models, BMDMs are generated by isolating bone marrow hematopoietic progenitor cells and inducing their differentiation into mature macrophages through cytokine-driven culture in vitro [59,60]. These cells are instrumental in elucidating macrophage polarization mechanisms and drug delivery interactions. BMDMs are often employed in preclinical models for their ability to replicate physiological macrophage behavior. Recent research underscores the value of BMDMs in overcoming critical barriers in drug delivery, particularly in challenging tumor microenvironments. For instance, Michael et al. proposed a strategy leveraging the chemotactic and phagocytic abilities of bone marrow-derived macrophages to deliver hypoxia-activated prodrugs (HAPs) to the hypoxic cores of solid tumors [61]. By encapsulating tirapazamine (TPZ) within nanoparticles carried by macrophages, the system demonstrated enhanced penetration and accumulation in hypoxic regions of 4T1 breast tumors. In vivo studies showed that this approach improved drug concentration in poorly oxygenated tumor regions and achieved significant tumor growth suppression, especially when combined with irinotecan. These findings highlight the potential of BMDMs to address the clinical limitations of HAPs by enhancing their intratumoral delivery and therapeutic efficacy.

Tissue-resident macrophages, such as Kupffer cells in the liver, alveolar macrophages (AMs) in the lungs, histiocytes in the spleen, microglia in the brain and peritoneal macrophages in the abdominal cavity, represent a specialized subset of primary macrophages [62]. These cells originate from embryonic progenitors during development and exhibit unique phenotypic and functional properties tailored to their tissue-specific environment [63]. The specialization makes them highly attractive candidates for organ-specific drug delivery applications, where they can leverage their innate tissue-homing abilities and unique roles within the immune microenvironment. For instance, Van Du et al. demonstrated the potential of spleen-derived macrophages engineered into microrobots for targeted cancer therapy [64]. By incorporating magnetic nanoparticles (MNPs) and DOX-loaded thermosensitive liposomes, these macrophage-based microrobots achieved precise spatiotemporal drug release at tumor sites (Fig. 2C). In vitro studies confirmed their tumor infiltration abilities, particularly under the influence of a magnetic field, while in vivo experiments showed significant tumor growth inhibition with a subtherapeutic dose of DOX. This approach highlights the utility of tissue-resident macrophages, such as spleen-derived macrophages, in integrating multiple targeting and drug release mechanisms to enhance therapeutic outcomes. In a different context, Wang and colleagues explored the role of alveolar macrophages in mediating intratracheally delivered nanoparticles for lung tumor imaging (Fig. 2D) [65]. Alveolar macrophages effectively engulfed perfluoro-15-crown-5-ether (PFCE) nanoparticles and migrated to tumor margins, increasing local PFCE concentrations and enhancing 19F-MRI signals. The macrophage-mediated translocation was pivotal in improving tumor visualization and reducing background signals in lung-draining lymph nodes, thus offering a new pathway for lung cancer diagnostics via imaging nanoparticles. Together, these studies demonstrate the versatility of tissue-resident macrophages as drug carriers, with applications ranging from organ-specific targeting to multifunctional therapeutic delivery systems. By leveraging their tissue-specific characteristics and interactions with the local microenvironment, tissue-resident macrophages present a promising platform for both therapeutic and diagnostic innovations.

Despite their considerable advantages, the broader application of primary macrophages in drug delivery is constrained by several practical challenges. The isolation processes, whether from blood, bone marrow, or tissues, are time-consuming, yield-limited, and resource-intensive [66]. For instance, a comprehensive protocol for murine BMDMs highlights that while reproducible, the process requires specific differentiation conditions and careful handling to maintain cell integrity [67]. Furthermore, maintaining the viability and native functionality of primary macrophages during culture, cryopreservation and after drug-loading remains a critical hurdle. Studies on porcine alveolar macrophages (PAMs) have shown that optimized cryopreservation media (e.g., 70 % RPMI-1640, 20 % newborn calf serum, and 10 % DMSO) can achieve high post-thaw viability rates up to 98.09 %, with preserved phagocytic function demonstrated by ink phagocytosis assays. Similarly, cryopreserved and resuscitated murine BMDMs maintained high viability and retained their ability to clear Staphylococcus aureus infections, indicating functional preservation [67]. For tissue-resident macrophages, preserving their specialized phenotypes and ensuring reproducibility across different batches remain significant obstacles. Another critical challenge is the scalability of primary macrophages for clinical use, especially in autologous therapies where donor-derived cells are required. The expansion cycle for autologous macrophages typically takes 2–3 weeks, which poses a significant barrier for immediate clinical application. The inherent heterogeneity of primary macrophage populations further complicates their application, as variations in donor age, health, and genetic background can influence macrophage function and therapeutic efficacy. Moreover, primary macrophages are inherently short-lived in vitro, which limits their use in extended research studies or large-scale drug delivery platforms. To address these scalability and standardization challenges, researchers are exploring alternative sources for generating large numbers of functional macrophages. One promising approach involves the large-scale generation of macrophages from human pluripotent stem cells. For instance, one study demonstrated that starting with a single T150 dish of 10 human induced pluripotent stem cells (iPSCs), over 10 billion mature macrophages (iMacs) could be generated within one month, providing a potential off-the-shelf strategy for cancer therapy [68]. This pluripotent stem cell-based approach can potentially provide a virtually unlimited supply of standardized macrophages, overcoming the key limitations of donor variability and low yield associated with primary isolation. In conclusion, while primary macrophages provide a more physiologically relevant and versatile platform compared to immortalized cell lines, their use in drug delivery systems is not without limitations. Continued advancements in isolation techniques, culture methods, and cryopreservation protocols are essential to overcome these barriers. Efforts to optimize the large-scale production of functional macrophages, coupled with innovations in engineering macrophages to enhance their therapeutic potential, will be critical in unlocking the full potential of primary macrophages as next-generation drug delivery carriers.

2.1.3. Strategic selection of macrophage types for cancer therapy

The selection of the appropriate macrophage type for drug delivery largely depends on the specific characteristics of the tumor and its microenvironment. For solid tumors with a complex immune landscape, such as breast cancer or melanoma, MDMs or BMDMs are particularly effective due to their ability to migrate toward tumors and modulate the tumor microenvironment. These macrophages can be engineered to express anti-tumor agents or reprogrammed to enhance their cytotoxic activities. In contrast, for tumors within specific organs, leveraging tissue-resident macrophages offers a more targeted approach. For instance, in liver cancer, utilizing Kupffer cells for drug delivery can take advantage of their natural residency in the liver and their capacity to interact with hepatic tumor cells. Similarly, in brain tumors such as glioblastoma, microglia-derived macrophages are ideal due to their ability to infiltrate the brain parenchyma. Immortalized macrophage cell lines, such as Raw264.7 cells are frequently utilized in early-stage preclinical studies to screen nanoparticle formulations due to their ease of manipulation and scalability, though their clinical relevance is limited by genetic instability. Additionally, the polarization status of macrophages plays an essential role in selecting the appropriate macrophage type. M1-type macrophages are effective in eliciting anti-tumor immunity and promoting tumor cell death, whereas M2-type macrophages, which are typically involved in tumor progression and immune suppression, may need to be reprogrammed to M1-type for effective treatment [69,70].

Overall, the selection between immortalized cell lines and primary macrophages hinges on the stage of research and therapeutic application. Immortalized cell lines are cost-effective and reproducible for mechanistic studies and high-throughput screening but lack clinical applicability. Conversely, primary macrophages, including BMDMs, MDMs and tissue-resident macrophages, provide more physiologically relevant results for translational research and personalized therapies. Advances in stem cell technologies, such as induced pluripotent stem cell-derived macrophages, also offer promising avenues for generating disease-specific macrophage populations tailored for particular therapeutic needs [71,72]. The decision matrix for macrophage selection thus requires a comprehensive understanding of the disease pathophysiology, delivery site, and the macrophage's biological properties to optimize therapeutic outcomes.

2.2. Drug loading techniques

The effectiveness of macrophage-based drug delivery systems heavily depends on the strategies employed to load therapeutic agents. The chosen loading method directly influences drug stability, release kinetics, and targeting efficiency, making it a fundamental aspect of system design. This section summarizes the primary approaches for drug loading, including physical encapsulation or phagocytosis and surface binding (covalent and non-covalent), detailing their principles, applications, and challenges. For a comparative overview, key characteristics of each method are summarized in Table 2, Table 3.

Table 2.

Quantitative comparison of primary drug loading methods for macrophage-based delivery systems.

Feature	Physical Encapsulation/Phagocytosis	Covalent Surface Binding	Non-Covalent Surface Binding
Typical Loading Efficiency	Highly variable (20–80 %), depends on NP properties & cell type [73,74]	Typically high (>80–90 %)	Variable, often moderate to high (60–90 %)
Release Kinetics	Sustained release over days; can be slow and incomplete due to lysosomal trapping	Controlled and sustained release over 48–72 h; release triggered by enzymatic cleavage or TME cues	Often rapid, stimulus-responsive (e.g., pH, enzyme); release within hours at target site
Key Advantages	•High Loading Capacity: Utilizes large internal cell volume. •Protection of Payload: Shields drugs from premature degradation in circulation. •Leverages Native Function: Uses the macrophage's natural phagocytic ability.	•High Stability: Prevents premature drug detachment during circulation. •Controlled Release: Enables spatiotemporally controlled drug release via designed linkers. •Avoids Lysosomal Pathway: Bypasses lysosomal degradation of the payload.	•Cell-Friendly: Minimally disruptive to cell membrane integrity and function. •Simplicity and Speed: Often involves simple incubation steps. •Reversibility: Allows for dynamic and context-dependent release.

Table 3.

Comparison of drug loading techniques for macrophage-based delivery systems.

Drug Loading Technique	Key Parameters	Impact on Cell Function	Triggered-Release Mechanisms	Major Limitations
Physical Encapsulation/Phagocytosis	•Particle Size: Uptake is most efficient for particles between 100 and 500 nm. Smaller particles may be exocytosed. •Surface Charge: Positively charged particles often show higher uptake due to electrostatic interaction with the negatively charged cell membrane. •Shape: Elongated or rod-shaped particles may be internalized more efficiently than spherical ones.	•High drug payloads can induce cytotoxicity and apoptosis. •Can impair migratory capacity and other cellular functions due to metabolic burden or specific drug effects (e.g., cytoskeleton disruption). •Particles are trafficked to lysosomes, risking drug degradation.	•Passive Diffusion: Slow, uncontrolled release. •Cell Death: Drug release upon carrier macrophage death in the TME. •Lysosomal Degradation: Drug release upon degradation of the carrier particle.	•Premature Release: Drug leakage during circulation can cause systemic toxicity. •Lysosomal Degradation: The harsh lysosomal environment can degrade bioactive drugs. •Potential Functional Impairment of the carrier cell.
Covalent Surface Binding	•Binding Stability: Forms strong, stable bonds (e.g., amide, thioether) using chemistry like maleimide-thiol. •Conjugation Efficiency: Depends on the density of reactive groups (e.g., amines, thiols) on the cell surface.	•Can potentially alter membrane protein function if conjugation sites are critical. •Generally, does not impede phagocytosis or migration if optimize. •The conjugation process itself must be optimized to maintain cell viability.	•Enzymatic Cleavage: Use of enzyme-cleavable linkers (e.g., matrix metalloproteinase-sensitive peptides) in the TME. •Acid-Labile Linkers: Cleavage in the acidic TME. •Redox-Sensitive Linkers: Cleavage in the high redox potential environment of the TME.	•Complex Synthesis: Requires chemical modification of both the drug/carrier and the cell surface. •Potential Immunogenicity: Introduced chemical moieties might provoke immune responses. •Limited Drug Loading Capacity: Only a small fraction of the membrane surface can be modified without affecting function.
Non-Covalent Surface Binding	•Interaction Strength: Relies on weaker forces like electrostatic interactions, host-guest chemistry (e.g., cucurbit[7]uril-adamantane), or biotin-avidin cross-linking. •Binding Affinity: Must be strong enough to withstand shear stress in circulation but allow release at the target site.	•Minimal Impact: Generally preserves cell viability, proliferation, and native functions like migration and chemotaxis. •Rapid and Simple: Attachment processes are often faster and less invasive than covalent methods.	•Environmental Triggers: Detachment in response to TME cues like low pH or specific enzymes. •External Triggers: Release upon application of light (e.g., NIR) or other external energy. •Competitive Displacement: Use of competitive agents to trigger release.	•Lower Stability: Risk of premature detachment during systemic circulation compared to covalent methods. •Batch-to-Batch Variability: Binding efficiency can be inconsistent. •Potential Use of Immunogenic Molecules: Avidin, for example, can be immunogenic.

2.2.1. Physical encapsulation or phagocytosis

Physical encapsulation or phagocytosis capitalizes on the innate endocytic and phagocytic capacity of macrophages, which are naturally adept at engulfing and processing foreign particles such as pathogens, apoptotic cells, and other debris [75]. The natural mechanism has been successfully adapted for drug delivery systems, allowing macrophages to internalize therapeutic agents, often in the form of nanoparticles or free drugs. Loading free drugs or particles into the cell cytoplasm offers several advantages, including providing a protected intracellular environment that shields the drug from premature degradation in the bloodstream, slowing the release of drugs and facilitating targeted delivery to diseased tissues. However, the efficiency of macrophage-mediated drug loading depends on several factors, including particle size, shape, surface charge, and functionalization, which can influence the internalization process and the subsequent therapeutic outcome [76]. Firstly, the size of nanoparticles plays a significant role in the internalization efficiency of macrophages [77]. Particles in the range of 100–500 nm are optimal for efficient phagocytic uptake and particles with smaller size may exhibit higher levels of exocytosis after being engulfed by macrophages [78]. Quantitative studies indicate that particles within the range of 100–500 nm are preferentially endocytosed by macrophages, with optimal phagocytic uptake observed around 100–200 nm. Precise data from carboxylated polystyrene nanoparticles (COOH-PS) of varying sizes (30, 50, 100, and 500 nm) revealed distinct uptake mechanisms and cellular responses. For instance, 30 nm COOH-PS rapidly entered Raw264.7 cells via direct membrane penetration, distributing uniformly around the nucleus, while larger particles (50, 100, and 500 nm) primarily utilized actin-mediated endocytosis and co-localized with acidic vesicles like lysosomes. Furthermore, the size significantly influences cellular toxicity. Smaller glucan nanoparticles (130 nm) demonstrated a greater ability to reduce human peripheral blood mononuclear cell and macrophage viability and to induce reactive oxygen species (ROS) production compared to their 355 nm counterparts [79]. The shape of the drug-loaded particles can also influence the internalization process by macrophages. It has been demonstrated that macrophages are more likely to internalize elongated or rod-shaped particles compared to spherical ones [80]. The extended shape of these particles may facilitate better interactions with macrophage membrane receptors, increasing the efficiency of particle engulfment. Elongated particles provide a larger surface area for receptor binding and may improve the fit with the macrophage's membrane during phagocytosis [41,81]. Notably, non-spherical shapes can also hinder uptake; for instance, worm-like polymeric particles with high aspect ratios have been shown to reduce phagocytosis by up to 4-fold compared to spherical particles of the same volume, offering a strategy to minimize carrier cell clearance by resident macrophages [82]. In addition, surface charge plays a pivotal role in determining how nanoparticles interact with the macrophage membrane. In general, particles with a charge tend to be taken up by macrophages more quickly than neutral particles, likely because of electrostatic interactions between the particles and the phagocytic cells [83,84]. Then, the negative charge on the cell membrane of macrophages allows for an electrostatic interaction with positively charged nanoparticles, which can enhance their internalization. For example, a study quantifying the uptake of gold nanoparticles found that positively charged particles were internalized ∼2–3 times more efficiently than their neutral or negatively charged counterparts [85]. Positively charged particles often experience greater uptake due to their affinity for the negatively charged components of the macrophage membrane, such as phospholipids. However, while a positive charge can improve internalization efficiency, excessively high surface charge densities may lead to cytotoxicity or unwanted immune responses [86]. Finally, surface modifications with ligands such as antibodies, peptides, or other targeting moieties can further enhance the specificity and efficiency of this process. For instance, particles functionalized with mannose or scavenger receptor ligands can target macrophages expressing these receptors, enabling selective uptake.

2.2.2. Surface covalent binding or non-covalent binding

Surface modification of macrophages through covalent or non-covalent binding of therapeutic agents has emerged as a versatile strategy for drug delivery. Covalent binding involves the direct attachment of therapeutic agents to reactive groups on the macrophage membrane, ensuring high stability of the conjugate during systemic circulation [87]. Reactive chemical groups such as amines, thiols, or carboxyls present on the macrophage surface serve as anchoring points for covalent conjugation [88]. Widely used chemical reactions include thiol-maleimide coupling, amide bond formation, and bioorthogonal click chemistry. Zhou et al. demonstrated the utility of covalent binding by constructing a biomimetic system (MPLP) in which Poly I:C-encapsulated poly(lactic-co-glycolic acid) nanoparticles (PLP NPs) were loaded onto the surface of BMDMs using maleimide-thiol conjugation (Fig. 3A) [89]. The system allowed for a controlled drug release and localized activation of immune responses in tumors. Importantly, the conjugation process did not impair the activity or functionality of the macrophages. Once delivered to the tumor microenvironment, the MPLP system reprogrammed macrophages into tumoricidal M1 phenotypes and triggered potent antitumor immune responses, effectively inhibiting tumor growth and metastasis without adverse immune reactions. Similarly, Gao et al. highlighted a complementary approach that leveraged supramolecular host-guest chemistry using cucurbit [7]uril (CB [7]) and adamantane as conjugation agents (Fig. 3B) [90]. Although less invasive than traditional covalent conjugation, this method ensured stable linkage between macrophages and therapeutic liposomes while preserving the physiological function of the macrophages. The resulting macrophage-liposome conjugate (M-L) demonstrated targeted accumulation in inflamed tissues and effective therapeutic delivery in various inflammatory and cancer models. When loaded with chemotherapeutic agents such as doxorubicin, the system significantly enhanced the efficacy of chemoimmunotherapy while maintaining the migratory and invasive capabilities of the macrophages. Covalent conjugation strategies provide highly stable drug-macrophage systems with precise control over drug release and biodistribution. However, potential limitations include the complexity of synthesis and the possibility of altering cell function depending on the binding method.

Fig. 3.

Drug loading techniques on macrophages in representative literature. (A) Schematic depiction of PLP NPs loaded on BMDM surface (MPLP), which could be activated in situ, as a tumor-targeting living cell delivery system for efficient tumor immunotherapy. Reprinted with permission from ref.89, copyright©2021 Elsevier Publisher. (B) M-L is'married’ via artificial host-guest interactions between CB [7] modified peritoneal macrophage and ADA modified liposome. Reprinted with permission from ref.90, copyright©2023 Wiley-VCH Publisher. (C) Schematic illustration of the construction of macrophages with surface-attached DOX-NP via MPN. High bioactivities are maintained compared to the conventional intracellular loading via endocytosis. Reprinted with permission from ref.92, copyright©2021 Wiley-VCH Publisher. (D) Schematic illustration of the macrophage–liposome (MA-DOX-Lip) system for tumor therapy. Biotin-modified, DOX-loaded liposomes are anchored onto streptavidin-engineered macrophages via avidin–biotin linkage. This system facilitates deep tumor penetration and subsequent DOX release, which induces immunogenic cell death and, in synergy with the macrophages, elicits potent anti-tumor immunity, leading to effective suppression of 4T1 triple-negative breast tumor growth in mice. Reprinted with permission from ref.93, copyright©2022 American Chemical Society Publisher.

Non-covalent binding, in contrast, relies on weaker interactions such as electrostatic forces, hydrophobic effects, or hydrogen bonding to attach drugs or nanoparticles to the macrophage surface. This approach preserves the structural and functional integrity of macrophages while simplifying the modification process [91]. Non-covalent methods are also advantageous for dynamic and reversible modifications, allowing drugs to detach at the target site under specific environmental triggers, such as changes in pH or ionic strength. For example, Zhu et al. demonstrated this approach by employing a divalent metal ion-phenolic network (MPN) to adhere poly(lactic-co-glycolic acid) nanoparticles (NPs) onto the surface of macrophages without inducing cytotoxicity or affecting cell proliferation [92]. This method significantly reduced intracellular uptake, which is often associated with drug-induced toxicity, and preserved the bioactivity of the macrophage carrier, including its viability and chemotactic migration (Fig. 3C). The resulting system, DOX-NP@Mφ, utilized doxorubicin-loaded nanoparticles attached to the macrophage surface and exhibited enhanced drug release at the tumor site. The photothermal properties of the MPN facilitated the release of drug-associated vesicles upon reaching the tumor, thereby improving chemotherapeutic efficacy. The simplicity and efficiency of the extracellular nanoparticle attachment process, which was achieved in an ice bath within 2 min, highlight the feasibility of this method for large-scale applications. The strategy exemplifies how non-covalent binding can minimize adverse interactions between drugs and carrier cells, maintaining cell functionality while optimizing drug release at the target site.

Non-covalent binding strategies also include ligand-receptor interactions and biotin-avidin (or biotin-streptavidin) crosslinking, both of which provide versatile and biologically relevant methods for attaching therapeutic agents or nanoparticles to the macrophage surface. These interactions exploit naturally occurring or engineered molecular recognition systems to achieve selective and reversible drug attachment, enhancing the specificity and functionality of macrophage-based delivery systems. For example, Yang et al. developed a macrophage–liposome (MA-Lip) system by loading doxorubicin-carrying liposomes onto the macrophage surface using biotin–avidin interactions [93]. The system demonstrated superior drug accumulation in tumor sites compared to conventional liposomes and exhibited deeper tumor tissue penetration, thereby significantly enhancing the antitumor immune response (Fig. 3D). In a 4T1 tumor-bearing mouse model, the MA-Lip system markedly improved survival rates with minimal systemic toxicity, underscoring its therapeutic potential. The biocompatibility and clinical approval of the materials used in this system further highlight its safety and feasibility for translational applications. Despite these advantages, the stability of non-covalent interactions under physiological conditions can pose challenges, as premature drug release may occur. Additionally, variations in binding efficiency, due to differences in interaction strength and receptor density on the macrophage surface, may impact therapeutic outcomes. Nonetheless, with appropriate optimization, non-covalent strategies provide a promising avenue for enhancing the precision and efficacy of macrophage-based drug delivery systems.

2.3. Functional engineering

Functional engineering of macrophages enhances homing to cancer sites, increases drug payload, or improves tumor microenvironment. This approach involves biochemical modifications and genetic modifications and these strategies enable the development of “designer macrophages” with enhanced targeting capabilities, improved drug retention, and the ability to overcome physiological barriers.

2.3.1. Biochemical modifications

Biochemical modifications represent a powerful strategy to enhance the functionality of macrophages as drug delivery vehicles by tailoring their surface properties or embedding active biomolecules. These modifications enable macrophages to interact more effectively with specific tissues, improve their stability, and expand their therapeutic applications while preserving their intrinsic biological functions. A notable approach of biochemical modification involves the surface decoration of macrophages with targeting ligands, such as antibodies, peptides, or aptamers, which confer specificity to pathological sites. Ning et al. demonstrated this approach by engineering anti-HER2 affibodies onto the extracellular membrane of macrophages [94]. These engineered macrophages, termed AE-Mφ, were further loaded with DOX-encapsulated poly (lactic-co-glycolic acid) nanoparticles (NPs). The resulting system, NPs(DOX)@AE-Mφ, exhibited a high degree of HER2⁺ cancer cell specificity, leveraging the affibody-mediated targeting mechanism (Fig. 4A). Crucially, this enhanced targeting was quantitatively validated in vivo. In HER2⁺ 4T1 tumor-bearing mice, AE-Mφ loaded with near-infrared dye (IR780)-encapsulated NPs (NPs(IR780)@AE-Mφ) showed progressively increasing fluorescence at the tumor site over 48 h, achieving approximately a 4-fold higher accumulation compared to non-targeted macrophages (NPs(IR780)@Mφ) or free nanoparticles, as quantified by ex vivo imaging of tumors and major organs. Mechanistically, this "affibody-guided" targeting operates in concert with the macrophage's innate tumor-homing ability. While unmodified macrophages rely on general chemokine gradients, the affibody provides a precise molecular anchor, increasing the avidity and retention of the carrier within the specific tumor sub-regions that express HER2. The targeted macrophage system achieved superior accumulation at HER2⁺ tumor sites, ensuring enhanced delivery of chemotherapeutic agents. This specificity not only amplified the antitumor efficacy of the treatment but also minimized systemic toxicity. Furthermore, the combination of affibody-mediated targeting and macrophage-mediated cell therapy enabled a synergistic effect, significantly improving therapeutic outcomes against solid tumors. The work highlights the potential of integrating targeting ligands into macrophages to create innovative, living drug delivery platforms capable of combining targeted chemotherapy with cell-mediated immunotherapy. In addition, a recent study introduced a bacteria-based backpack strategy, providing an innovative approach for the modification of macrophages. An and co-authors developed a bacteria-based backpack (Mø@bac) that adheres to macrophages to enhance their functionality by leveraging the innate immunogenicity of bacteria [95]. Using a layer-by-layer assembly method, bacteria were coated with polysaccharides to form an adhesive nanocoating, facilitating their integration with macrophages (Fig. 4B). These bacterial backpacks were enriched with pro-inflammatory factors and exhibited the ability to proliferate within tumor tissues, enabling sustained activation of macrophages toward the M1 phenotype. Moreover, Mø@bac demonstrated the ability to repolarize endogenous tumor-associated macrophages (TAMs), remodeling the immunosuppressive tumor microenvironment (TME). The sustained and localized release of pro-inflammatory factors from the backpacks creates a chemotactic gradient and a supportive niche that may enhance the recruitment and retention of both the engineered and endogenous macrophages within the tumor, amplifying the overall anti-tumor immune response. In subcutaneous and orthotopic 4T1 murine models, Mø@bac exhibited significantly enhanced anti-tumor responses compared to macrophages alone or combined with free bacteria. Importantly, Mø@bac showed improved biocompatibility with a reduced systemic inflammatory response. This innovative bacteria-based backpack strategy provides a novel pathway to modulate cellular phenotypes, offering a powerful tool for adoptive macrophage therapy against solid tumors.

Fig. 4.

Functional Engineering on macrophages in representative literature. (A) Schematic of engineered macrophages as a "living targeted drug" platform. A gene sequence encoding an anti-HER2⁺ antibody was genomically integrated, leading to surface expression on macrophages, which were subsequently loaded with DOX-loaded nanoparticles (NPs). The resulting macrophages specifically bind HER2 on tumor cells, enabling targeted drug delivery and exerting combined inhibitory effects through both antibody-mediated action and enhanced chemotherapy. Reprinted with permission from ref.94, copyright©2024 Elsevier Publisher. (B) Preparation of Mø@bac and regulation of tumor immunosuppressive microenvironment mediated by Mø@bac. Reprinted with permission from ref.95, copyright©2024 Wiley-VCH Publisher. (C) Targeted phagocytosis of cancer cells by CAR-macrophages and activation of T cells -mediated adaptive immune response.

2.3.2. Genetic modification

Genetic modification of macrophages has paved the way for transformative advancements in immunotherapy, particularly through the development of chimeric antigen receptor (CAR)- macrophages (CAR-Ms). CAR-Ms, inspired by the success of CAR-T cell therapy, are engineered to recognize tumor-specific antigens, enabling targeted immune responses against malignant cells. This approach has shown promise in addressing the challenges posed by the immunosuppressive tumor microenvironment (TME), particularly in solid tumors, where conventional CAR-T therapies often fall short (Fig. 4C). A critical determinant of CAR-Ms efficacy lies in the design of its intracellular signaling domains, which directly dictates the magnitude and quality of macrophage activation upon antigen engagement. Early CAR-Ms designs primarily borrowed components from CAR-T technology. In 2020, Jin Zhang and colleagues reported the first generation of CAR-macrophages, using CD3ζ as the intracellular signaling domain of the CAR construct [96]. While this modification successfully enabled antigen-dependent phagocytosis of tumor cells, the use of CD3ζ—a T-cell-specific signaling component with limited native expression in macrophages—may lead to suboptimal and non-physiological activation, potentially limiting its therapeutic potential. This study nonetheless marked a significant milestone, highlighting the potential of iPSCs CAR-macrophages (CAR-iMACs), which demonstrated antigen-specific tumor phagocytosis, cytokine secretion, pro-inflammatory polarization, and in vivo anticancer activity. Notably, the use of iPSCs provided an unlimited source of engineered macrophages, addressing scalability challenges associated with personalized cell therapies. Building on this breakthrough, second-generation CAR-Ms emerged, incorporating signaling domains more native to myeloid cell biology to enhance potency. Zhang and colleagues developed second-generation CAR-macrophages with enhanced functionality [97]. This iteration incorporated a tandem CD3ζ and Toll-like receptor 4 (TLR4) intracellular Toll/IL-1R (TIR) signaling domain into the CAR construct, significantly amplifying the antitumor effects. These second-generation CAR-iMACs exhibited not only improved tumor phagocytosis but also antigen-dependent M1 polarization, resistance to M2 phenotypic conversion, and modulation of the TME via NF-κB signaling pathways. Additionally, this design enabled CAR-induced efferocytosis, effectively eliminating apoptotic tumor cells and their associated immunosuppressive effects. The integration of the TLR4/TIR domain represented a pivotal shift towards macrophage-centric engineering, leveraging innate immune signaling pathways to drive a more robust and sustainable anti-tumor response.

Most recently, the field has witnessed a sophisticated evolution in activation domain engineering aimed at achieving superior control over CAR-Ms function. A groundbreaking study introduced a fully macrophage-optimized signaling component, fusing the transmembrane domain of α1β1 integrin with the intracellular signaling domain of Fc-gamma receptor I (FcγRI) [98]. This design is conceptually superior for several reasons: it replaces the non-native CD3ζ with α1β1 integrin, which natively mediates macrophage adhesion and inflammation, and couples it with FcγRI, a master regulator of phagocytosis. This engineered α1β1-FcγRI component (termed ACT CAR-Ms) was shown to induce enhanced macrophage activation, specific phagocytosis, and potent pro-inflammatory (M1-type) polarization in vitro. In vivo, it demonstrated significantly improved tumor control and survival in multiple cancer models compared to conventional CD3ζ-based CAR-Ms. Transcriptomic analysis revealed that this novel signaling axis enhanced critical antitumor pathways, including Toll-like receptor, TNF, and IL-17 signaling. The approach exemplifies the next frontier in CAR-Ms design: creating synthetic signaling pathways that are meticulously tailored to the host cell's biology to maximize efficacy while potentially improving safety through more controlled activation. In summary, the trajectory of CAR-Ms development, from adopting T-cell domains to engineering macrophage-specific signaling components, underscores a maturation in the field towards tailored design. This preclinical progress is now substantiated by the first clinical evidence from a phase 1 trial of CT-0508, an anti-HER2 CAR-Ms, in patients with advanced HER2-overexpressing tumors (NCT04660929) [99]. The interim analysis of this pioneering study, which enrolled patients without the use of lymphodepletion chemotherapy, confirmed the safety and manufacturability of the approach. Critically, no dose-limiting toxicities, severe (≥grade 3) cytokine release syndrome, or immune effector cell-associated neurotoxicity syndrome were observed. Regarding antitumor activity, 44 % (4/9) of patients with HER2 3⁺ tumors achieved stable disease as their best overall response at 8 weeks post-treatment, while no meaningful activity was seen in HER2 2⁺ patients, highlighting the importance of target antigen density. Most significantly, correlative analyses of serial biopsies provided direct human validation of the core mechanism: CT-0508 successfully trafficked to and remodeled the tumor microenvironment, leading to an expansion of CD8⁺ T cells. These findings represent a landmark demonstration that CAR-Ms can be safely administered, infiltrate human tumors, and instigate local immune activation as designed, thereby de-risking the platform and paving the way for its next-phase clinical development. A critical analysis of their potential, must focus on three pivotal aspects: in vivo persistence, tumor penetration, and immune microenvironment reconstruction. As terminally differentiated cells, macrophages have a finite lifespan. Myeloid-optimized signaling domains (e.g., TLR4/TIR, FcγRI) are therefore engineered not only to activate but also to enhance the survival and resilience of CAR-Ms within the hostile TME, countering exhaustion and repolarization. Tumor penetration is a postulated major advantage. Macrophages inherently possess the ability to infiltrate stromal-rich solid tumors, a site where T cells often fail. It is hypothesized that antigen recognition via the CAR could further guide and enhance this intrinsic tropism, though direct imaging evidence quantifying this process is still emerging. Most critically, CAR-Ms function as microenvironmental engineers. Their efficacy extends beyond direct phagocytosis. By secreting pro-inflammatory cytokines (e.g., IFN-γ, IL-12), they recruit T cells and NK cells, effectively turning "cold" tumors "hot." Simultaneously, they can repolarize endogenous TAMs towards an antitumor M1-type and promote antigen presentation, thereby initiating a broad, in situ antitumor immune cascade. This multifaceted capacity to persist, penetrate, and reprogram the TME positions CAR-Ms therapy as a compelling strategy to overcome the core limitations of cell therapies in solid tumors.

In summary, despite the promising advancements, genetic modification of macrophages faces several challenges that limit its clinical application. A primary challenge lies in achieving high-efficiency gene editing in primary macrophages. These cells are notoriously difficult to transfect and transduce due to their innate immune surveillance mechanisms, which readily recognize and degrade foreign nucleic acids. While viral vectors, particularly lentiviruses, offer relatively high efficiency, they raise safety concerns regarding insertional mutagenesis and immunogenicity. Beyond delivery, the inherent risk of off-target effects poses a substantial safety concern. The problem is compounded in macrophages, where the transcriptome and epigenetic landscape can vary significantly between activation states, potentially influencing guide RNA specificity. Finally, ensuring stable and durable transgene expression in vivo is exceptionally difficult. Macrophages engineered ex vivo and adoptively transferred face a hostile physiological environment. The transient nature of expression is a key issue, especially when using non-integrating vectors. Furthermore, the genetically modified cells are subject to immune-mediated clearance by the host's immune system, which may recognize the newly expressed protein (e.g., a CAR) or the viral vector components as foreign antigens. Additionally, epigenetic silencing of the transgenic promoter can lead to the gradual loss of expression over time, undermining long-term therapeutic efficacy. Achieving persistent function requires innovative approaches to shield the cells from immune rejection and to maintain open chromatin states at the transgene integration site. However, as illustrated by the latest advances in activation domain engineering (e.g., α1β1-FcγRI), many limitations of earlier constructs, such as suboptimal activation and lack of controlled phagocytic response, are being actively addressed through innovative molecular design. Addressing the fundamental challenges of editing efficiency, off-target effects, and stable in vivo expression is now the central focus of ongoing research, and success in these areas is essential for translating genetically engineered macrophages into safe and viable clinical therapies.

3. Advantages and application of macrophage-based drug delivery systems

Macrophage based drug delivery systems represent a novel and promising platform for cancer therapies. Unlike traditional nanoparticles, macrophages leverage their biological properties and engineered versatility to overcome key barriers in drug delivery. Below, we provide a detailed exploration of the unique advantages and application of macrophages, supported by recent advancements and literature.

3.1. Navigating multi-level biological barriers for targeted delivery

Macrophages excel at traversing a spectrum of biological barriers, from the internal microstructure of solid tumors to systemic anatomical boundaries, enabling drug delivery to sites that are typically inaccessible. Their capacity to penetrate deeply into tumors allows them to reach poorly vascularized, hypoxic, and necrotic regions, which are often resistant to conventional therapies [100]. For instance, in a study by Choi et al., they highlighted the potential of exploiting monocytes, the precursors of macrophages, for targeted delivery into hypoxic tumor zones [101]. These monocytes, recruited by tumor-derived signals, can differentiate into macrophages upon tumor infiltration and retain nanoparticle-based therapeutic payloads. Once in the hypoxic regions, these "Trojan Horse" macrophages can release their cargo in response to environmental triggers, such as near-infrared (NIR) illumination (Fig. 5A). This targeted approach not only eliminates TAMs, which are critical to tumor survival, but also disrupts adjacent malignant cells, significantly reducing tumor regrowth and metastatic potential. Similarly, Muthana et al. demonstrated the application of macrophages engineered to carry hypoxia-regulated oncolytic viruses [102]. Administered post-chemotherapy or radiation therapy, these macrophages selectively delivered the virus to hypoxic tumor regions, effectively halting tumor regrowth and preventing metastatic dissemination. This strategy capitalized on natural homing properties of macrophages to hypoxic environments, offering enhanced therapeutic efficacy in otherwise treatment-resistant tumor areas. Additionally, traditional nanoparticle-based therapies primarily accumulate in well-vascularized tumor areas due to the EPR effect, leaving substantial portions of the tumor untreated. Macrophages, however, actively infiltrate these inaccessible zones, ensuring uniform drug distribution throughout the tumor. For example, Zhang and colleagues investigated the tumor-targeting potential of macrophages loaded with DOX nanoparticles (DOX NPs) in a mouse model of breast cancer (Fig. 5B) [103]. Their findings demonstrated that intravenously administered DOX NP-loaded macrophages exhibited a stronger fluorescent signal in the tumor region and a weaker signal in the liver compared to the group receiving DOX NPs alone. This highlights the enhanced ability of macrophages to deliver therapeutic agents to challenging tumor regions with poor vascularization, offering a promising strategy for overcoming drug delivery barriers in such environments.

Fig. 5.

Representative studies based on macrophage delivery systems for targeting tumors. (A) Schematic of Trojan Horse therapeutic nanoparticle delivery into the hypoxic region of tumor. Reprinted with permission from ref.101, copyright©2007 American Chemical Society Publisher. (B) Schematic illustration of the functional macrophages-based delivery system for chemo-photothermal combination therapy. Reprinted with permission from ref.103, copyright©2020 Elsevier Publisher. (C) Macrophages loaded with Fe₃O₄-Cy5.5 was used for multimodal imaging to help in the accurate diagnosis of gliomas, guide surgical resection, and perform postoperative photothermal therapy. Reprinted with permission from ref.105, copyright©2021 American Chemical Society Publisher. (D) Schematic illustration of RILO@MG for solid tumor therapy via specific phagocytosis and immune activation. Preparation of RILO@MG: RILO-loaded M1-type macrophages (RILO@M) were first prepared, followed by surface anchoring of DSPE-PEG5k-GTP to form RILO@MG. Upon intravenous administration, RILO@MG accumulates in tumors through chemotaxis and GPC3 targeting, directly killing tumor cells via phagocytosis. The system also generates RI-exosomes (containing R848 and INCB) that reprogram TAMs and enhance cytotoxic T-cell (CTL) activity, collectively reversing the immunosuppressive TME and exerting potent antitumor efficacy. Reprinted with permission from ref.107, copyright©2024 Springer Nature Publisher.

Beyond the tumor mass, macrophages uniquely cross major systemic barriers. These include the BBB, peritoneal barriers, and dense stromal matrices in solid tumors. The BBB is a highly selective barrier that restricts the entry of most drugs and nanoparticles into the CNS. However, macrophages can cross this barrier via transcytosis or by exploiting inflammatory signals that temporarily disrupt the BBB's integrity. Recent studies have highlighted the potential of macrophages as drug carriers for effective BBB penetration in glioma treatment. For instance, Pang et al. demonstrated that M1-type macrophages, known for their enhanced phagocytic capacity and resistance to the immunosuppressive tumor microenvironment, could serve as efficient vehicles for nanoparticle delivery [104]. In their study, M1-type macrophages loaded with poly (lactide-co-glycolide) (PLGA) nanoparticles (M1-NPs) exhibited robust tumor tropism and successfully traversed the endothelial barrier to reach glioma tissues. In vivo imaging further confirmed that M1-NPs achieved superior brain tumor accumulation compared to free nanoparticles, resulting in significantly enhanced therapeutic efficacy and prolonged survival in glioma models. Similarly, Wang et al. explored a macrophage-mediated nanoplatform for glioma diagnosis and treatment [105]. By loading macrophages with a photothermal nanoprobe (MFe₃O₄-Cy5.5), they demonstrated the system's ability to cross the BBB and accumulate in deep gliomas (Fig. 5C). This platform enabled multimodal imaging, combining fluorescence, photoacoustic, and magnetic resonance imaging to guide precise glioma resection. Additionally, the photothermal properties of the nanoprobe provided effective postoperative tumor suppression. The integration of diagnostic and therapeutic capabilities in macrophage-mediated delivery systems emphasizes their transformative potential in overcoming the limitations posed by the BBB in glioma management. Macrophages also excel in traversing the peritoneal barrier, which is particularly relevant for cancers with peritoneal metastases, such as ovarian or gastric cancers. They can efficiently deliver drugs to micrometastases that conventional therapies often fail to reach due to poor vascularization. Choi et al. showcased the potential of peritoneal macrophages loaded with liposomal doxorubicin (LP-DOX) as active drug carriers [106]. These macrophages effectively traversed the peritoneal barrier and targeted tumor sites. The study emphasized the macrophages' ability to infiltrate tumors while preserving their biological function and the cytotoxic activity of the encapsulated doxorubicin. Magnetic resonance imaging revealed successful tumor migration of macrophages containing iron oxide nanoparticles. Systemic administration of macrophage-LP-DOX significantly enhanced drug delivery in both subcutaneous and metastatic tumor models, leading to notable tumor growth inhibition. These results emphasize the utility of peritoneal macrophages in overcoming the peritoneal barrier to access challenging tumor microenvironments. In addition, solid tumors are characterized by dense ECM and poor vascularization, which significantly impede drug penetration and distribution. Macrophages, with their inherent migratory capabilities, can effectively navigate through these physical barriers, offering a promising solution for drug delivery to solid tumors. Recent work by Liu et al. highlights a novel approach leveraging macrophages to target solid tumors [107]. Their study engineered macrophages by incorporating a surface glypican-3-targeting peptide, enhancing tumor cell recognition and selective targeting (Fig. 5D). These macrophages were also loaded with a dual cargo comprising the TLR7/TLR8 agonist R848 and the IDO1 inhibitor INCB024360, encapsulated within C16-ceramide-fused outer membrane vesicles (OMVs) derived from Escherichia coli, termed as RILO. The OMVs facilitated cargo internalization via caveolin-mediated endocytosis, while C16-ceramide promoted membrane invagination and exosome formation, ensuring efficient release of therapeutic payloads. In a mouse model of glypican-3-expressing H22 hepatocellular carcinoma, these RILO-loaded macrophages demonstrated significant therapeutic efficacy, underscoring the potential of this cytotherapeutic strategy to overcome the physical barriers posed by ECM and poor vascularization. This approach complements conventional treatments, offering a targeted and efficient delivery system for combating solid tumors.

3.2. Immune evasion and prolonged circulation time

Macrophage-based drug delivery systems offer distinct advantages in immune evasion and prolonged circulation time, which significantly enhance their therapeutic efficacy [81]. These systems exploit the natural properties of macrophages, which are inherently biocompatible and capable of mimicking endogenous immune cells, enabling them to evade the body's immune surveillance and achieve extended circulation within the bloodstream. One of the main challenges for traditional drug delivery systems, particularly synthetic nanoparticles, is their rapid recognition and clearance by the mononuclear phagocyte system, which includes macrophages, dendritic cells, and other immune cells. This clearance can result in a shortened circulation time and diminished therapeutic efficacy. In contrast, macrophages, as part of the body's immune system, are equipped with various "self-markers" such as CD47, which serve as a mechanism to avoid phagocytosis by other immune cells. CD47 interacts with inhibitory receptors on phagocytes, thereby preventing the premature clearance of macrophages from circulation. This feature allows macrophage-based systems to persist longer in the bloodstream, giving them more time to reach and accumulate at target sites, particularly tumors, where they can exert their therapeutic effects. Li et al. further elucidate these advantages by developing a macrophage-mediated drug delivery system for the targeted delivery of Bi₂Se₃ nanosheets, which possess excellent NIR photothermal properties [108]. In their study, macrophages were used to encapsulate Bi₂Se₃ nanosheets, leading to the formation of macrophage-laden nanoparticles that exhibited high cellular uptake and negligible cytotoxicity (Fig. 6A). Importantly, the macrophage-based delivery system demonstrated prolonged blood circulation compared to free Bi₂Se₃ nanosheets. This prolonged circulation time is especially beneficial in overcoming challenges associated with the tumor microenvironment, such as hypoxia and limited blood supply, which often hinder the efficient delivery of therapeutic agents. Furthermore, the macrophage-laden Bi₂Se₃ nanosheets exhibited excellent biocompatibility, with most of the material being excreted from the body within 25 days, indicating the system's safety and feasibility for clinical application. In summary, macrophage-based drug delivery systems provide significant advantages in immune evasion and prolonged circulation, making them ideal candidates for overcoming some of the limitations faced by conventional drug delivery methods. By exploiting the natural immune properties of macrophages, these systems can enhance drug delivery efficiency, improve therapeutic outcomes, and reduce systemic toxicity, paving the way for more effective and safer cancer treatments.

Fig. 6.

Representative studies based on macrophage delivery systems. (A) Schematic illustration of the macrophages loaded Bi₂Se₃ nanosheets delivery system expected to overcome the hypoxia-associated drug delivery barrier to enhance tumor coverage and PTT efficiency. Reprinted with permission from ref.108, copyright©2017 Elsevier Publisher. (B) Schematic of Met@Man-MPs as a combinational agent to enhance anti-PD-1 therapy. Met@Man-MPs target M2-type macrophage via MMP activity, repolarizing them to an M1-typ e phenotype to recruit CD8⁺ T cells and alleviate immunosuppression. Concurrently, their collagen degradation promotes deeper T-cell infiltration and improves anti-PD-1 antibody penetration. Together, this synergy potently inhibits tumor growth and induces long-term immune memory. Reprinted with permission from ref.110, copyright©2021 Springer Nature Publisher. (C) Preparation of M1/SLNP and schematic of M1/SLNP for targeted HCC therapy, in which M1-type macrophages serve dual roles as both a targeted delivery vehicle and an active therapeutic agent, enhancing treatment efficacy. Reprinted with permission from ref.111, copyright©2020 American Chemical Society Publisher. (D) Schematic illustration of the preparation of Oxa(IV)@ZnPc@M and the mechanism of Oxa(IV)@ZnPc@M mediated chemo-photodynamic therapy to trigger robust antitumor immune responses and potentiate PD-L1 blockade immunotherapies for anti-primary and bone metastatic tumors. Reprinted with permission from ref.110, copyright©2021 Springer Nature Publisher. (E) Schematic illustration of inflammatory monocytes loading legumain-sensitive nanoparticles (M-SMNs) to target lung metastasis of breast cancer and initiate metastatic-specific drug release to achieve the anti-metastasis efficacy. Reprinted with permission from ref.113, copyright©2021 American Chemical Society Publisher.

3.3. Immune modulation within the tumor microenvironment

Macrophages are key players in the tumor microenvironment, capable of influencing tumor progression and the host immune response. While TAMs often exhibit a pro-tumorigenic M2-type, promoting growth, angiogenesis, and immune suppression, they can also be reprogrammed into an anti-tumorigenic M1 phenotype [109]. Macrophage-based drug delivery systems (M-DDS) leverage this duality to remodel the TME, either by delivering therapeutic agents to target cancer cells directly or by inducing immune responses that inhibit tumor progression. The polarization of macrophages into M1-type or M2-type is a critical determinant of their functional role in the TME. M1-type macrophages, characterized by the secretion of pro-inflammatory cytokines such as IL-12 and TNF-α, enhance anti-tumor immunity and T-cell activation. In contrast, M2-type macrophages produce anti-inflammatory cytokines such as IL-10 and VEGF, which support tumor growth, immune evasion, and angiogenesis. Strategies that promote M1 polarization within the TME have the potential to restore immune surveillance and inhibit tumor progression. For example, Wei et al. developed an innovative approach using mannose-modified macrophage-derived microparticles (Man-MPs) loaded with metformin (Met@Man-MPs) to target and repolarize M2-type macrophage into the M1-type [110]. This macrophage-mediated immunomodulation reshaped the TME by increasing infiltration of CD8⁺ T cells while reducing the presence of immunosuppressive components, including myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs, Fig. 6B). The unique collagen-degrading properties of Man-MPs further enhanced the penetration and accumulation of immune checkpoint inhibitors, such as anti-PD-1 antibodies, in tumor tissues. This combination therapy not only improved checkpoint blockade efficacy but also induced long-term immune memory, providing durable protection against tumor recurrence. Similarly, Hou et al. exploited M1-type macrophages as both immune effectors and drug carriers in a "cell-chemotherapy" strategy [111]. Sorafenib-loaded lipid nanoparticles (M1/SLNPs) were delivered using M1-type macrophages, which enhanced tumor specificity and facilitated localized drug release within the TME (Fig. 6C). The treatment with M1/SLNPs significantly increased the M1-type to M2-type macrophage ratio and boosted CD4⁺ and CD8⁺ T-cell infiltration, effectively alleviating the immunosuppressive characteristics of the TME. This dual therapeutic approach resulted in robust inhibition of tumor growth, demonstrating the synergistic potential of combining chemotherapy and immunotherapy within a single delivery platform.

The ability of macrophage-based systems to modulate the immune landscape within the TME offers a powerful tool for cancer therapy. By shifting the balance toward an immunostimulatory state, these systems not only inhibit tumor progression but also enhance the efficacy of concurrent therapeutic modalities such as checkpoint blockade and chemotherapy. The integration of immune modulation with drug delivery highlights the versatility of macrophage-based systems in addressing the multifaceted challenges of tumor therapy, paving the way for more effective and comprehensive treatment strategies.

3.4. Applications in metastatic and residual cancer

Metastatic and residual cancer remain major therapeutic challenges, contributing significantly to cancer-associated mortality. Traditional treatment approaches often fail to effectively target and treat metastatic lesions due to poor drug delivery to distant sites and the heterogeneity of metastatic tumors. Macrophage-based drug delivery systems have emerged as promising candidates for improving the delivery and efficacy of cancer therapies, particularly in the context of metastatic and residual cancers.

A particularly innovative strategy involves engineering macrophages to not only deliver drugs but also actively remodel the metastatic microenvironment itself. A landmark study developed a "uPA-scavenger" macrophage (uPAR-MΦ) platform to confront cancer metastasis through a dual-mechanism approach [112]. Recognizing that the urokinase-type plasminogen activator (uPA) system is a key driver of metastasis, researchers genetically engineered macrophages to stably express high levels of uPAR on their surface. These engineered cells act as living scavengers, dynamically capturing and clearing uPA from the peripheral blood and the tumor colonization microenvironment. This process effectively disrupts the "soil" required for metastatic seeding and outgrowth by inhibiting extracellular matrix degradation and cancer cell migration. To simultaneously attack the metastatic "seeds," these uPAR-MΦs were loaded with gemcitabine-encapsulated nanoparticles (GEM@PLGA). This combined design resulted in a robust antimetastatic effect, significantly reducing metastatic lesions and prolonging survival in murine models of breast cancer. This platform represents a paradigm shift, demonstrating how macrophages can be engineered as sophisticated living drugs to simultaneously neutralize a critical metastatic pathway and deliver cytotoxic agents, offering a potent strategy against cryptic metastasis that is often intractable at the time of diagnosis.

Similarly, as demonstrated by Huang et al., engineered macrophages (Oxa(IV)@ZnPc@M), which are designed to deliver a combination of oxaliplatin prodrug and photosensitizer, show efficient targeting of primary and bone metastatic tumors (Fig. 6D) [113]. The macrophage-based delivery system exploits the inherent ability of macrophages to home to tumors, even in metastatic sites, overcoming the limitations of conventional drug delivery systems. The system not only delivers the therapeutic agents to the tumor site but also triggers a synergistic effect through NIR-activated chemo-photodynamic therapy, which enhances the anticancer efficacy while inducing immunogenic cell death. Moreover, combining this system with immune checkpoint inhibitors like anti-PD-L1 results in a potent antitumor immune response, leading to the effective elimination of both primary and metastatic tumors with reduced systemic toxicity. The integrated approach represents a promising strategy for treating metastatic cancers, especially in patients with limited response to traditional therapies. Similarly, He et al. highlight the potential of macrophage-based systems in targeting lung metastases [114]. In their study, inflammatory monocytes loaded with legumain-activated nanoparticles (SMNs) were utilized for anti-metastasis therapy (Fig. 6E). SMNs are designed to respond to the microenvironment at metastatic sites, where legumain, a protease overexpressed in tumors, facilitates the controlled release of the cytotoxic agent, mertansine. The inflammatory monocytes, upon differentiation into macrophages, transport these nanoparticles directly to the lung metastases, where they are activated to release the anticancer drugs. This targeted drug release leads to a significant inhibition of the proliferation, migration, and invasion of metastatic cancer cells. Moreover, the system improves the penetration of drugs into the tumor microenvironment, resulting in a notable reduction in lung metastases and enhanced therapeutic outcomes.

The above findings emphasize the advantage of macrophage-based drug delivery systems in actively targeting metastatic sites and facilitating intelligent drug release for effective treatment. The ability of macrophage-based delivery systems to target both primary and metastatic lesions is critical in addressing the challenge of residual cancer, where small, dispersed tumor cells evade detection and treatment. By leveraging the natural migration patterns of macrophages, macrophage-based drug delivery systems can reach and treat residual cancer cells that might otherwise be missed by conventional therapies. The macrophage's innate ability to adapt to and interact with the tumor microenvironment further enhances the efficacy of such systems, especially in the context of complex and heterogenous metastatic lesions. In conclusion, the application of macrophage-based drug delivery systems in metastatic and residual cancer therapies holds significant promise. Their ability to target and treat distant tumor sites, deliver drugs intelligently, reprogram the metastatic niche and activate immune responses makes them an attractive approach for improving therapeutic outcomes in patients with metastatic cancer. These systems offer a novel strategy for overcoming the challenges posed by metastatic and residual tumors, potentially leading to more effective and less toxic treatment options for cancer patients.

4. Challenges and future perspectives

Macrophage-based drug delivery systems represent a transformative platform in cancer therapy; however, their clinical translation is impeded by multifaceted challenges across biological, technical, and regulatory domains. These challenges highlight the complexity of harnessing macrophages as therapeutic carriers and necessitate innovative solutions to overcome their limitations while leveraging their unique capabilities.

One of the primary obstacles lies in the biological characteristics of macrophages within the tumor microenvironment. Tumors exhibit remarkable heterogeneity, with differences in immune cell infiltration, ECM composition, and vascularization that can hinder the uniform distribution of therapeutic agents. While macrophages naturally home to tumors via chemotactic signals such as CCL-2 and CSF-1, their ability to penetrate deep into the tumor core remains variable, particularly in dense ECM regions. Collagen crosslink–driven tumor stiffening physically limits immune-cell migration and favors peritumoral “stacking,” creating a distribution gap even when recruitment signals are intact. Reversing collagen crosslinking reduces stiffness, improves lymphocyte migration, and enhances anti-PD-1 efficacy in vivo—an ECM-aware principle that is directly applicable to macrophage carriers [115]. Therefore, addressing this requires strategies that modulate the TME, such as co-delivery of agents like TGF-β inhibitors or ECM-targeting enzymes, to enhance macrophage penetration and sustain their anti-tumoral activity.

Efficient drug loading and retention within macrophages pose significant technical challenges that directly impact therapeutic efficacy and safety. A critical, and often underappreciated, challenge is achieving high drug loading without compromising macrophage viability, native homing function, and safety profile—a prerequisite for clinical translation. Many chemotherapeutic drugs (e.g., doxorubicin, paclitaxel) are inherently cytotoxic and can induce significant stress, apoptosis, or functional impairment in the carrier macrophages during the loading process and subsequent carriage. For example, free doxorubicin is directly toxic to THP-1/macrophage-like cells, lowering survival and impairing function [116]. This can deplete the therapeutic cell population before they even reach the tumor. Conventional methods like passive incubation often result in suboptimal payloads. Advanced physical methods like electroporation and sonoporation can improve loading efficiency by creating transient pores in the cell membrane. For instance, one study demonstrated enhanced doxorubicin loading via electroporation [117]. However, these techniques must be meticulously optimized, as excessive energy can cause irreversible membrane damage and high rates of cell death, undermining the therapy's foundation. The field is exploring several strategies to mitigate these issues: 1)Nano-encapsulation: pre-encapsulating drugs within nanoparticles provides a protective barrier, shielding the macrophage from direct drug toxicity and enabling higher, more tolerable payloads; 2) Using Pro-drugs: loading inactive, less toxic pro-drugs that are selectively activated within the TME minimizes damage to the carrier cell; 3) Exploiting natural transporters: utilizing specific nutrient or ion transporters for active drug uptake presents a more gentle and physiologically compatible loading alternative. Therefore, optimizing drug loading is not solely about maximizing payload, but about defining the "therapeutic window" for the carrier cell itself.

Furthermore, perhaps the significant pharmacological hurdle is achieving precise control over drug release. The "off-target" release of drugs during systemic circulation is a major safety concern that can lead to dose-limiting systemic toxicities, negating the targeting advantage of cell-based delivery. Conversely, failure to efficiently release the payload within the tumor results in sub-therapeutic drug concentrations. Drugs can leak from macrophages during circulation due to spontaneous diffusion, extracellular matrix interactions, or shear stress, particularly for small molecules not stably encapsulated. After intravenous dosing, macrophage carriers can undergo first-pass/RES sequestration in lung, liver, or spleen, diluting exposure at extra-pulmonary lesions. For example, a macrophage “cellular vector” carrying doxorubicin provided continued release in the lung—desirable for pulmonary targets but mis-targeted for others. Likewise, cell-membrane–coated nanoparticles often show enhanced 24 h spleen accumulation: circulation is prolonged, yet tumor-site exposure can fall unless additional homing/trigger constraints are engineered. Mitigations include route retargeting (e.g., regional/intra-arterial for liver lesions), tight control of size/ligand/opsonin (∼150–250 nm), and dual-lock designs that require both a navigation cue and a TME trigger to activate release [118]. Therefore, recently significant research is focused on engineering "smart" release mechanisms triggered by the TME: 1) Endogenous triggers: leveraging the TME's unique properties, such as mild acidity, overexpressed enzymes (e.g., MMPs, cathepsins), or elevated ROS. Drug carriers can be designed with acid-labile linkers or enzyme-cleavable peptides that degrade specifically in the TME; 2) Exogenous triggers: applying external stimuli like NIR light or focused ultrasound offers unparalleled spatiotemporal control to induce drug release on demand at the tumor site, though this requires specialized equipment. The ultimate goal is to design macrophage systems that are "leak-proof" in circulation but transform into efficient "drug pumps" upon receiving a tumor-specific signal.

While addressing the technical challenges of drug loading and controlled release, it is crucial to critically evaluate the unique profile of macrophage-based delivery against conventional nanoparticle systems (Table .4). The principal advantage of macrophages lies in their superior biodistribution and tumor retention, achieved through active chemotaxis that enables homing to and deep penetration into tumor regions inaccessible to most NPs relying on passive EPR effects. Their biological nature further confers a more favorable pharmacokinetic profile with prolonged circulation. In contrast, conventional NPs maintain distinct advantages in precise drug loading and controllable release, offering higher, more defined payloads and engineerable release kinetics without the complexities of live-cell carriers. This fundamental dichotomy positions macrophage-based systems as particularly promising for treating invasive, stromal-rich malignancies where active targeting and penetration are paramount, while NPs remain advantageous for well-accessible targets requiring precise pharmaceutical control.

Table 4.

Comparison between macrophage-based and conventional nanoparticle drug delivery systems.

Feature	Macrophage-based Drug Delivery	Conventional Nanoparticles
Pharmacokinetics	Long circulation; evades rapid clearance.	Short-moderate half-life; prone to MPS clearance.
Biodistribution & Targeting	Active tropism via innate chemotaxis.	Passive accumulation via EPR effect.
Tumor Retention & Penetration	High retention & deep penetration via active motility.	Heterogeneous retention; limited penetration.
Drug Payload & Control	Limited by cell viability; complex loading.	High, tunable payload; precise release kinetics.
Manufacturing	Complex, costly live-cell processes.	Scalable, reproducible synthesis.

The clinical safety of macrophage-based delivery systems also hinges on mitigating adverse immune responses and systemic toxicities. A key concern is uncontrolled pro-inflammatory activation, which could trigger a systemic cytokine release syndrome. This risk is being addressed by engineering safety switches, such as inducible suicide genes, to eliminate overactive cells. Additionally, "on-target, off-tumor" toxicity remains a challenge, driving the development of more specific targeting strategies like logic-gated CARs that require multiple tumor antigens for activation. For allogeneic approaches, host immune rejection of the transferred macrophages must be managed, potentially through the creation of hypoimmunogenic cells. Finally, as noted previously, premature drug leakage during circulation must be minimized through robust, stimulus-responsive release systems to avoid off-target damage.

In addition, the scalability and standardization of macrophage-based therapies remain critical hurdles for clinical translation. Autologous macrophage therapies require patient-specific isolation, differentiation, and engineering, which are labor-intensive, costly, and prone to variability. Immortalized macrophage lines and stem-cell-derived macrophages offer scalable alternatives but raise concerns about immunogenicity and genetic instability. Advances in 3D bioreactor technology have facilitated the large-scale production of macrophages while preserving their phenotype and functionality, but further refinement is needed to achieve Good Manufacturing Practice (GMP) compliance. Establishing standardized protocols for macrophage isolation, culture, and engineering is essential to ensure consistency and reproducibility in clinical applications. Meanwhile, the regulatory landscape for macrophage-based systems adds another complexity. Unlike conventional small molecules or biologics, macrophage therapies involve living cells that interact dynamically with the immune system and tumor environment. This complexity complicates preclinical safety and efficacy evaluations, as traditional toxicology models may not adequately predict clinical outcomes. Regulatory agencies require robust evidence of long-term safety, stability, and scalability, which often necessitates extensive preclinical studies. International collaboration among researchers, industry stakeholders, and regulatory bodies could help establish standardized evaluation frameworks and accelerate the clinical translation of macrophage therapies.

Looking ahead, the field is poised for transformative advances through the integration of cutting-edge biotechnologies that address current limitations and unlock new functionalities. Beyond optimizing existing parameters, several disruptive approaches are emerging on the horizon. First, the derivation of macrophages from iPSCs presents a paradigm shift for manufacturing. iPSC-derived macrophages offer a genetically uniform, inexhaustible, and clinically scalable source of cells, effectively overcoming the donor variability and expansion limitations of primary cells. This platform is exceptionally powerful for CRISPR-based genetic engineering, as edits can be introduced at the stem cell level, clonally selected, and then differentiated into massive quantities of engineered macrophages (e.g., CAR-Ms) with perfect consistency. This not only streamlines production but also enables the creation of complex, multi-gene edited constructs that would be exceedingly difficult to implement in primary cells. Second, the concept of hybrid "cell-bot" or "cyborg" systems is gaining traction, merging the biological intelligence of cells with the synthetic versatility of nanomaterials. Future systems may involve macrophages that are not merely loaded with nanoparticles but are intricately fused with them. This could include embedding inorganic nanosensors within the cell to report on its physiological status (e.g., pH, reactive oxygen species) in vivo, or attaching nanoscale actuators that can be remotely triggered by magnetic fields or ultrasound to precisely control cell behavior, such as inducing drug release or directional migration. These bio-digital hybrids aim to create macrophages whose innate abilities are augmented by engineered components, offering a new level of control over these living therapeutics. The next chapter of macrophage-based delivery will likely be written at the intersection of cell biology, materials science, and synthetic biology.

5. Conclusion

In conclusion, macrophage-based drug delivery systems represent a pivotal advancement in oncology therapeutics, embodying a transformative yet complex paradigm. Their considerable potential stems from a unique biological profile unattainable by conventional synthetic carriers: an innate capacity to actively home to and penetrate diseased tissues, dynamically respond to environmental cues, and function not merely as passive vectors but as active therapeutic agents. Through phagocytosis, immune modulation, and microenvironment remodeling, macrophages contribute directly to treatment efficacy. Recent advances in engineering strategies—ranging from precision surface modification with targeting ligands to the development of genetically engineered CAR-Ms—have significantly augmented these intrinsic capabilities, paving the way for “living drug” platforms capable of targeted chemotherapy, immunotherapy, and sophisticated combination regimens.

However, the path to routine clinical application is paved with significant and interconnected challenges. The very biological complexity that defines their advantage also underpins the major hurdles. Technically, achieving high, non-toxic drug loading and spatiotemporally precise release remains a formidable pharmacological barrier to ensuring both safety and efficacy. Biologically, the heterogeneity of both tumors and macrophage populations demands strategies to ensure consistent and robust performance across diverse patient populations. From a translational standpoint, the scalable, GMP-compliant manufacturing and standardized quality control of these living therapeutics present a substantial leap from traditional drug development, necessitating close collaboration with regulatory bodies to establish new evaluation frameworks. The preliminary but promising clinical data for CAR-Ms, demonstrating safety and proof-of-mechanism, is a crucial first step that simultaneously validates the platform and underscores the need to overcome these challenges to achieve meaningful efficacy.

Looking forward, the successful clinical translation of macrophage-based delivery will hinge on interdisciplinary convergence. The integration of stem cell biology for creating off-the-shelf, consistently engineered products, the development of bio-hybrid systems that merge cellular intelligence with nanoscale engineering, and the implementation of smart control and monitoring technologies represent the next frontier. By systematically addressing the current limitations through such innovative approaches, macrophage-based systems are poised to evolve from a compelling therapeutic concept into a cornerstone of precision medicine, ultimately offering a powerful new arsenal against cancers that have thus far remained intractable.

CRediT authorship contribution statement

Muse Ji: Writing – original draft. Hongbing Liu: Writing – original draft. Xinxin Liang: Resources. Mingli Wei: Resources. Dongmei Shi: Resources. Jingxin Gou: Supervision. Tian Yin: Supervision. Haibing He: Supervision. Xing Tang: Conceptualization. Yu Zhang: Conceptualization.

Declaration of competing interest

The authors have declared no conflict of interest.

Data availability

Data will be made available on request.