Engineered Neutrophils in Translational Medicine: Gene Editing, Nanotechnology, and AI‐Driven Clinical Breakthroughs

ABSTRACT

Engineered neutrophils, modified via advanced biotechnological tools, are emerging as pivotal agents in translational medicine. By integrating gene editing (e.g., CRISPR‐Cas9), nanotechnology, and artificial intelligence (AI), these cells are redefining precision diagnostics and therapeutics. Gene editing enables precise reprogramming to enhance tumor‐targeting, antimicrobial activity, and immune modulation. Nanotechnology facilitates neutrophil‐bound drug delivery, improving targeting to inflamed or tumor sites while reducing off‐target effects. Simultaneously, AI‐driven platforms analyze multi‐omics data to optimize engineering strategies and personalize treatments. These innovations demonstrate significant promise across clinical domains: in oncology, they deliver cytotoxic payloads and remodel the tumor microenvironment; in infectious diseases, they enhance pathogen clearance; and in autoimmune disorders, they dampen aberrant inflammatory responses. Despite this progress, challenges including off‐target edits, short in vivo persistence, and immune rejection persist. The convergence of microfluidics and high‐throughput screening, coupled with AI‐driven monitoring, is essential to accelerate clinical translation. As interdisciplinary collaboration deepens, engineered neutrophils stand at the forefront of next‐generation medicine, offering versatile, patient‐tailored strategies to bridge the gap between bioengineering breakthroughs and clinical reality.

A Unified Paradigm for Engineered Neutrophil Therapeutics. This framework integrates gene editing, nanotechnology, and AI‐driven modeling to reprogram neutrophil precursors (HSPCs, iPSCs) for precision medicine. By leveraging microfluidics and multimodal bioimaging, cell manufacturing and in vivo tracking can be optimized. These engineered neutrophils exhibit versatile efficacy in oncology, infectious diseases, and autoimmunity, bridging the gap between innovative bioengineering and clinical implementation.

1. Introduction

Neutrophils, as pivotal components of the innate immune system, are recognized as “immune sentinels” due to their employment of multifaceted antimicrobial mechanisms, including phagocytosis, intracellular degradation, granule secretion, and neutrophil extracellular trap (NET) formation [1]. Nevertheless, their short lifespan (typically <24 h in circulation), nonspecific activation patterns, and rapid responsiveness pose substantial challenges for in‐depth mechanistic investigations [2]. In recent years, advancements in biomedical engineering have led to engineered neutrophils being recognized as a promising therapeutic tool with broad clinical potential (Figure 1).

FIGURE 1.

Timeline of major breakthroughs in engineered neutrophil research and translational development. The timeline summarizes the major scientific and technological milestones that have driven the evolution of engineered neutrophils from basic immunology research to emerging clinical translation. Key advances include the discovery of neutrophil heterogeneity and NETosis, the emergence of nanotechnology‐based drug delivery systems, the introduction of CRISPR‐mediated engineering, the integration of microfluidics and artificial intelligence, and the initiation of early clinical trials. The timeline highlights the interdisciplinary convergence of immunology, nanotechnology, bioengineering, and computational medicine that is shaping the future of engineered neutrophil‐based precision therapeutics. Note: Figures 1 and 2 were originally generated using Python codes I wrote (utilizing the Matplotlib library, https://github.com/xzhu2692/EngineeredNeutrophils_py) and subsequently enhanced with scientific icons from BioRender's built‐in library (https://www.biorender.com/). This usage fully complies with the respective licensing terms and presents no intellectual property issues.

Engineered neutrophils are derived from native neutrophils and designed to possess enhanced or novel functions for clinical diagnostic and therapeutic purposes [3, 4, 5] through gene editing [6, 7], nanotechnology [8, 9, 10], and other biotechnological means (Figure 2). Research in this field encompasses the source, function, engineering methods, and applications of neutrophils in various diseases. Through neutrophil modification, better management of infection, tumors, immune disorders, and other diseases can be achieved; clinical diagnostic and therapeutic outcomes can be enhanced; and the clinical value of these approaches can be increased via innovative technical means [4, 11, 12].

FIGURE 2.

Conceptual Framework of Engineered Neutrophils in Translational Medicine. A comprehensive paradigm integrating multi‐disciplinary technologies to advance engineered neutrophils for next‐generation precision diagnostics and live‐cell therapeutics. Core Engineering Strategies: Neutrophils are structurally or functionally reconfigured via three primary pillars: Gene Editing (e.g., CRISPR‐Cas9): Applied to mature neutrophils or upstream expandable precursor cells (such as hematopoietic stem and progenitor cells [HSPCs] and induced pluripotent stem cells [iPSCs]) to achieve precise genetic reprogramming for enhanced tumor‐targeting, potentiated antimicrobial activity, and tailored immune modulation. Nanotechnology: Utilizing nanoparticle‐bound cellular carriers, intracellular loading techniques, and biomimetic membrane‐camouflaged systems to establish active site‐specific drug delivery (e.g., to tumors or inflamed tissues) while dramatically mitigating off‐target toxicities. Artificial Intelligence (AI): Integrating multi‐omics analytics (e.g., transcriptomics) and computational modeling to predict disease progression, optimize nanoparticle formulations, and significantly accelerate the iterative “Design‐Build‐Test‐Learn” engineering cycle. Technological Convergences: Multi‐pronged bioengineering frontiers converge to enhance cell processing and evaluation: Microfluidics enables high‐purity cell sorting and provides automated biomimetic platforms for extended culture to mitigate the short ex vivo lifespan of neutrophils; Multimodal Bioimaging (including photoacoustic, and magnetic resonance tracking) facilitates real‐time monitoring of in vivo homing and trafficking dynamics; High‐Throughput Screening accelerates functional assessments and clinical translation. Downstream Clinical Applications: Fully reconfigured neutrophils demonstrate profound therapeutic and diagnostic potential across major disease categories: Oncology: Targeted delivery of cytotoxic payloads, remodeling of the immunosuppressive tumor microenvironment (TME), and synergistic integration with traditional immunotherapies. Infectious Diseases: Acceleration of pathogen clearance via enhanced phagocytosis and smart, localized antimicrobial drug delivery. Autoimmune Disorders: Attenuation of hyper‐inflammatory cascades through cytokine/chemokine modulation and targeted inhibition of aberrant NETosis. Tissue Repair & Regeneration: Promotion of structural healing and active restoration of tissue homeostasis. Translational Bottlenecks & Future Outlook: Current hurdles in engineering “immune sentinels” involve off‐target edits, brief in vivo persistence, and host immune rejection. Future clinical implementation relies on scaling up automated Good Manufacturing Practice (GMP) manufacturing workflows, refining cellular biocompatibility, and fostering deep interdisciplinary collaboration to deliver versatile, patient‐tailored strategies bridging bioengineering breakthroughs and clinical reality.

This review introduces the basic preparation methods of engineered neutrophils, their innovative applications in the frontier fields of microfluidic technology, artificial intelligence, and nanomaterials, and explores their clinical application prospects in cancer, autoimmune diseases, and tissue repair (Figure 2). In addition, the technical and clinical challenges of engineered neutrophils are discussed, and future development is considered, including strategies to overcome immune rejection and to improve cell survival time and homing efficiency, thereby promoting their wide clinical application.

2. Biological Characteristics and Functions of Neutrophils

2.1. Origin and Development of Neutrophils

Neutrophils are primarily derived from multipotent hematopoietic stem cells in the bone marrow. In the bone marrow, these stem cells proliferate and differentiate into mature neutrophils, which are a type of polymorphonuclear granulocyte (PMN) [13]. Multipotent hematopoietic stem cells first differentiate into common myeloid progenitors, and these subsequently differentiate into myeloblasts [14]. Subsequently, via the promyelocyte, myelocyte, and metamyelocyte stages, they become band granulocytes and, ultimately, segmented granulocytes, the latter representing mature neutrophils [15, 16]. Upon reaching full maturity, these neutrophils are then released from the bone marrow into the peripheral bloodstream, where they circulate actively and perform essential immune surveillance.

The generation and development of neutrophils are primarily regulated by transcription factors, cytokines, and signaling pathways (Figure 1). Transcription factors such as PU.1, C/EBPα, and C/EBPε drive myeloid differentiation from hematopoietic stem cells and subsequently contribute primarily to the specialization of cells into neutrophils [17, 18]. Cytokines such as granulocyte colony‐stimulating factor (G‐CSF) influence the proliferation, maturation, and release of neutrophils. Signaling pathways such as the Notch signaling pathway help maintain the balance between neutrophil proliferation and differentiation [19].

2.2. Distribution of Neutrophils

Neutrophils are primarily distributed in the bone marrow, blood, and tissues. They are produced and mature in the bone marrow, where approximately 2.5 × 10¹² mature neutrophils are stored [20]. Mature neutrophils may enter the blood, where some circulate freely, while others adhere to vascular endothelial cells and migrate across the endothelium into tissues [21]. Once neutrophils migrate into tissues, they cannot re‐enter the bloodstream. Normally, a dynamic equilibrium exists between circulating and marginated neutrophil pools within the vasculature, and this equilibrium is maintained within a defined physiological range. Significant deviations above or below this range serve as clinical indicators of underlying infectious processes [13, 22].

The traditional view held that neutrophils are terminally differentiated homogeneous cells; however, recent studies have confirmed that neutrophils exhibit significant heterogeneity both during their development in the bone marrow and after their release into the bloodstream. This heterogeneity largely accounts for their functional diversity.

2.3. Functional Heterogeneity of Neutrophils and Precision Engineering Strategies

With the rapid advancement of single‐cell RNA sequencing (scRNA‐seq) technology, the heterogeneity of neutrophils has been further resolved into multiple functionally distinct subsets. Based on maturation status, neutrophils can be categorized into three stages: immature, mature, and senescent. Among these, the immature subset is significantly enriched under pathological conditions such as cancer and exerts immunosuppressive functions [23]; its abundance correlates with poor prognosis and resistance to immunotherapy, making it a potential therapeutic target. Based on functional characteristics, neutrophils can be further classified into interferon‐responsive, immunosuppressive, and functionally silent subsets [24, 25] (Figure 1). The interferon‐responsive subset is associated with antiviral responses and may contribute to autoimmune disease activity; the immunosuppressive subset promotes tumor immune evasion; and the functionally silent subset represents a resting state that could be primed for activation [25]. These distinctions provide a molecular basis for developing subset‐specific biomarkers and targeted interventions.

Single‐cell sequencing studies have identified subsets with well‐defined molecular signatures in various disease states (Figure 1). Marteau et al. [26] employed scRNA‐seq to identify blood neutrophil (BN1‐BN3) and tumor‐associated neutrophil (TAN1‐TAN4) subtypes in colorectal cancer, among which the LOX‐1+ TAN subset serves as a pro‐tumor phenotypic marker, whereas the HLA‐DRA+ TAN subset exhibits antigen‐presenting capacity and is associated with favorable patient prognosis. Through single‐cell transcriptomic and surface proteomic analyses, Nishide et al. [27] found that the immature neutrophil subset and the type II interferon signature neutrophil subset (Neu_T2ISG) were significantly increased in the peripheral blood of patients with microscopic polyangiitis (MPA). This subset differentiates upon co‐stimulation with IFN‐γ and TNF, promotes ANCA‐induced NET formation, and serum IFN‐γ levels correlate with disease relapse, thereby serving as a potential biomarker for predicting relapse.

The marked heterogeneity of neutrophils provides a theoretical basis for precision engineering strategies. Based on the molecular characteristics of distinct subsets, strategies for selective targeting or functional reprogramming are being explored. Examples include reprogramming pro‐tumor N2‐like neutrophils into an anti‐tumor N1‐like phenotype via TGF‐β inhibitors, and blocking tumor infiltration of pro‐tumor subsets with CXCR2 antagonists. These findings collectively highlight the critical role of neutrophil heterogeneity in tumor immunity and autoimmune diseases and open new avenues for the development of precision immunotherapy strategies targeting specific subsets [28] (Figure 1).

2.4. Function of Neutrophils

2.4.1. Phagocytosis and Bactericidal Action

As professional phagocytes, neutrophils constitute a key line of defense against infection. Their core functions include chemotaxis, adhesion, phagocytosis, and bactericidal activity (Figure 3B). Mediated by opsonins, neutrophils recognize and phagocytose pathogens [29]. This efficient phagocytic machinery enables neutrophils to rapidly internalize and eliminate invading microbes, thereby containing infection at an early stage [30]. Degranulation initiates non‐oxidative microbial killing and provides the basis for oxidative killing. This function serves as an important foundation for engineering neutrophils for anti‐infective applications [31].

FIGURE 3.

Application of engineered neutrophils in clinical diagnosis and therapy. (A) Engineered neutrophil‐mediated bioimaging. (B) Schematic illustration of engineered neutrophils homing to murine tumor sites and executing tumor tissue destruction. Utilizing engineered techniques, drug‐loaded nanocarrier particles are encapsulated within neutrophils and subsequently administered to mice. These engineered neutrophils actively migrate to tumor sites, releasing antitumor payloads to induce targeted tumor cell death.

Loading nanoparticles with antimicrobial agents or using gene editing techniques to enhance the expression of phagocytosis‐related receptors (e.g., FcγR, CR3), are expected to further improve the targeted recognition and bactericidal efficiency of engineered neutrophils. These strategies offer potential molecular targets for developing anti‐infective live cell therapies [32].

2.4.2. Oxygen Burst

A sharp increase in intracellular oxygen consumption, accompanied by activation of the membrane‐bound NADPH oxidase, is termed the oxidative burst. In the resting state, components of the NADPH oxidase complex are distributed in the cytoplasm and cell membrane. Upon stimulation by pathogens, the NADPH oxidase complex on the neutrophil membrane is activated, catalyzing the generation of superoxide anion and subsequent reactive oxygen species (ROS) such as hydrogen peroxide and hydroxyl radicals [33]. These ROS exert antimicrobial effects by damaging pathogen membranes, proteins, and nucleic acids. The regulatory mechanisms governing ROS production provide potential targets for the functional engineering of neutrophils [34] (Figure 1).

Engineering strategies, including modulating NADPH oxidase activity by introducing conditionally activatable switches triggered by specific inflammatory signals and co‑delivering ROS‑scavenging systems, hold promise for enhancing the bactericidal function of neutrophils while curbing excessive inflammatory damage, thereby enabling precise on‑demand ROS release from engineered neutrophils.

2.4.3. Formation and Regulation of Neutrophil Extracellular Traps (NETs)

Activated neutrophils can release NETs through inflammatory cell death (NETosis) [35], which is a specific, programmed death of neutrophils distinct from cell necrosis and apoptosis [36] (Figure 1). NETs are web‐like structures composed of DNA fibers, histones, and antimicrobial proteins that can capture bacteria, fungi, protozoa, and viruses [37, 38], and employ their cytotoxic components to directly kill pathogens (Figure 3B). However, their abnormal accumulation or incomplete clearance can lead to chronic inflammation [28], autoimmune diseases [39], thrombosis [40, 41], and can promote tumor recurrence and metastasis [42]. This represents a key functional regulatory target to be addressed in neutrophil engineering [33, 34]. Thus, better control of NET formation and clearance could be beneficial for the application of engineered neutrophils.

Various engineering strategies have been explored to address this target [43]. For example, designing NET clearance systems may help preserve anti‑infective capacity while simultaneously mitigating NET‑related pathological damage. Maiocchi et al. [44] encapsulated DNase I into polymeric nanoparticles and utilized the intrinsic migratory capacity of neutrophils to deliver these nanoparticles to thrombus sites. In vitro experiments confirmed that nanoparticle‑loaded neutrophils maintained key biological functions, including viability and oxidative burst capacity, and were capable of delivering DNase I to thrombus sites. Such strategies represent potential research directions for engineered therapies in autoimmune and thrombotic diseases.

2.4.4. Secretion of Cytokines and Chemokines

Neutrophils can also play an important role in the immune response by secreting cytokines and chemokines. Upon pathogen infection or inflammatory stimulation, neutrophils secrete pro‐inflammatory cytokines (IL‐1, IL‐6, IL‐12, TNF‐α, IFN‐γ), anti‐inflammatory cytokines (e.g., IL‐4 and IL‐10), and chemokines such as CXCL8 (IL‐8) [45]. By regulating immune cell recruitment and modulating local inflammatory responses [31], neutrophils participate in immune responses [46]. Precise regulation of their secretory functions represents a core direction for engineering neutrophils for immunotherapy [47] (Figures 1 and 3B).

Studies have shown that modifying the cytokine expression profile via genetic engineering (e.g., overexpressing IL‑12 or knocking out TGF‑β) can reprogram neutrophils toward an anti‑tumor N1 phenotype or an anti‑inflammatory phenotype, thereby adapting them to different scenarios such as cancer immunotherapy or autoimmune diseases [48]. Furthermore, engineered neutrophils can serve as cell‑based delivery vehicles for the local, targeted release of immunomodulators at lesion sites, thereby reducing systemic toxicity [49].

2.4.5. Immunoregulatory Function: Interaction Network With Other Immune Cells

Neutrophils can positively or negatively regulate dendritic cell (DC) functions, promoting DC recruitment, antigen presentation, and T cell activation [50].

They also directly modulate T cell activation and differentiation by releasing NETs, presenting antigens, and secreting cytokines, while suppressing overactivation via TGF‑β or PD‑L1 expression [51, 52, 53]. Based on these interactive mechanisms, one direct neutrophil engineering strategy involves polarizing neutrophils via attachment of discoidal polymer micropatches to their surface. When administered intravenously, these micropatch‑loaded neutrophils accumulate in the spleen and tumor‑draining lymph nodes, activate natural killer cells and T cells, enhance the infiltration of dendritic cells and natural killer cells, and thereby generate potent systemic anti‑tumor immune responses that reduce tumor burden and improve survival rates [54].

Furthermore, neutrophils and macrophages exert synergistic bactericidal effects [55], and neutrophil‑derived granule proteins and cytokines influence macrophage polarization toward M1 or M2 phenotypes [19]. These interactions provide a theoretical basis for the coordinated engineering of neutrophils and macrophages.

2.5. Short Life Cycle, High Mobility, and High Reactivity of Neutrophils

Neutrophils are characterized by a short lifespan (circulating half‐life of 6–8 h) [56], high migratory capacity, and high reactivity. Their rapid apoptosis helps maintain immune homeostasis but poses challenges for ex vivo gene editing and cell manufacturing. Their high migratory capacity enables them to rapidly reach sites of inflammation or tumors, which makes them ideal “living cell drug” carriers. Their high reactivity necessitates fine regulation of their activation threshold during engineering design to avoid excessive immune responses and tissue damage [30]. These characteristics collectively define the core challenges in neutrophil engineering: how to preserve their natural functional advantages while overcoming inherent limitations such as terminal differentiation, poor transfectability, and short ex vivo survival, so as to construct safe and effective engineered neutrophil therapeutic strategies [2].

To address the short lifespan bottleneck, current engineering strategies often employ precursor cells (e.g., hematopoietic stem and progenitor cells, HSPCs) for gene editing followed by directed differentiation, or utilize microfluidic biomimetic microenvironments to prolong the ex vivo survival of mature neutrophils [57, 58]. To leverage their high migratory capacity, neutrophils can serve as “living cell missiles” for active targeted drug delivery. To manage their high reactivity, safety switches (e.g., inducible suicide genes) or regulatory nanoscale switches can be introduced to enable timely elimination after treatment, thereby avoiding excessive inflammatory responses [59].

Building on these strategies, emerging technologies, including nanotechnology, microfluidics, artificial intelligence, and advanced bioimaging, are now being leveraged to further enhance the functionality, controllability, and therapeutic precision of engineered neutrophils, as elaborated in the following sections.

3. Basic Research on Engineered Neutrophils

3.1. Concept of Engineered Neutrophils

Engineered neutrophils are cells obtained through the modification of natural neutrophils using gene editing technology [60] (such as CRISPR‐Cas9), cell surface modification, and other biological engineering methods. These modifications are designed to confer or enhance specific functions of neutrophils, such as enabling them to more precisely target diseased cells (e.g., tumor cells) [61], more effectively identify and eliminate pathogens [62], and enhance immune regulation [63]. Consequently, they can be utilized in various medical applications, including disease diagnosis, treatment, and immune regulation.

3.2. Preparation Basis of Engineered Neutrophils

3.2.1. Basic Preparation Conditions

First, with respect to the sample source, mature neutrophils are primarily isolated from peripheral blood. Highly purified neutrophils are obtained following centrifugation, washing, and red blood cell lysis using Ficoll‐Hypaque density gradient centrifugation and Histopaque density gradient centrifugation [64].

To maintain the survival and function of neutrophils in vitro, conditions must include a temperature close to human physiology (typically 37°C) and a 5% CO₂ atmosphere to preserve the pH stability of the culture medium. In addition, specific media and additives are typically used. Commonly used media include RPMI‐1640, Dulbecco's Modified Eagle Medium (DMEM), and others; such media are routinely supplemented with defined concentrations of serum, antibiotics, and additional cytokines to promote cell growth and expansion. GM‐CSF (granulocyte‐macrophage colony‐stimulating factor) and G‐CSF (granulocyte colony‐stimulating factor) are two key factors that can stimulate neutrophil production; consequently, they are frequently used as culture additives [65, 66, 67].

3.2.2. Stem Cells and Differentiation Culture Technology

3.2.2.1. Stem Cells are Induced to Differentiate into Neutrophils

First, an appropriate source of stem cells should be selected, including bone marrow, peripheral blood, or umbilical cord blood. Hematopoietic stem cells (HSCs) have the potential to differentiate into various blood cell lineages, including neutrophils. CD34⁺ cells from different sources exhibit differences in ex vivo expansion and differentiation efficiency [68]; among these, umbilical cord blood‐derived cells possess the highest proliferative capacity and are therefore more suitable as starting cells for engineering modifications. Supplementing the in vitro culture system with specific cytokine combinations (e.g., GM‐CSF, IL‐3) mimics the in vivo hematopoietic microenvironment, initiates the directed differentiation of stem cells toward the granulocytic lineage, and progressively generates morphologically and functionally mature terminal neutrophils [55, 56]. This in vitro induced differentiation technology provides a sufficient and controllable source of cellular starting material for subsequent engineering modifications, including genetic engineering, nanomaterial modification, and microfluidic manipulation.

3.2.2.2. Engineering Transformation: Endowing Neutrophils With New Functions

During or after the differentiation of stem cells into neutrophils, these cells can be engineered through gene transduction and cellular reprogramming. For example, tools such as CRISPR‐Cas9 are used to knock out or repair relevant genes in neutrophils or their precursors, thereby exploring potential strategies to enhance their anti‐tumor capacity or restore normal function [69]. Furthermore, nanotechnology enables the loading of functional nanoparticles (e.g., drug carriers, immunomodulators) onto the surface or into the interior of neutrophils [70], thereby achieving targeted delivery [71].

On this basis, combining technologies such as microfluidics, artificial intelligence, and bioimaging allows for efficient single‐cell sorting, drug delivery, and functional analysis of neutrophils, which optimizes engineering designs, predicts cellular behavior, and accelerates screening workflows, thus further advancing the engineering modification of neutrophils [72, 73, 74]. These engineering strategies complement each other and collectively expand the therapeutic applications of neutrophils, providing a feasible path for their clinical translation in cancer, immune‐mediated diseases, infectious diseases, and related fields.

3.2.2.3. Quality Testing and Screening

After completing the previous steps, the prepared engineered neutrophils need to be tested for purity and viability. Purity testing determines the proportion of engineered neutrophils in the whole cell population, while viability assessment ensures proper in vivo functionality. In addition, the effect of genetic modification should be verified, including whether the transferred genes are correctly expressed and whether the cells function as expected. To further enhance the screening process, advanced technologies such as microfluidics, artificial intelligence (AI), and bioimaging can be integrated. Microfluidics enables high‐throughput single‐cell sorting and functional analysis [75, 76]; AI accelerates phenotypic screening and predicts cellular behavior patterns [77]; and bioimaging allows real‐time monitoring of cell viability, migration, and functional status. These technologies collectively improve screening efficiency, accuracy, and throughput. Finally, based on the test results, cells that meet the requirements are selected, unqualified cells are discarded, and high‐performing engineered neutrophils are retained for subsequent research or treatment.

3.2.3. Technical Difficulties and Breakthroughs in Neutrophil Culture In Vitro

Neutrophils are terminally differentiated cells, which makes their survival in vitro face many challenges [78]. In vitro, neutrophils exhibit a short survival time and are highly susceptible to apoptosis [56]. They have stringent requirements regarding the culture environment (including pH, nutrients, and other factors), and the purification process is laborious, involving complex, time‑consuming procedures. Consequently, traditional culture conditions often fail to fully meet these requirements [79]. As a result, the function and activity of neutrophils rapidly decline over time during in vitro culture, which limits further in‑depth investigation and clinical application of these cells.

Furthermore, microfluidic chip technology and 3D cell culture systems based on microfluidic chips offer new possibilities for optimizing neutrophil culture conditions [80]. These technologies enable precise regulation of nutrient concentrations and fluid flow rates within the culture microenvironment, potentially achieving more stable cell culture conditions that more closely mimic the in vivo state on a smaller scale [81, 82]. In theory, such a microenvironment that more closely resembles physiological conditions including more biomimetic cell‑cell and cell‑matrix interactions may help maintain neutrophil viability and function [81, 83, 84]. However, the actual efficacy of these approaches still requires validation in neutrophil‐specific experiments.

3.3. Neutrophils and Genetic Engineering

3.3.1. Gene Editing Technology

Gene engineering technologies [85] such as gene editing (e.g., CRISPR‐Cas9 [58], TALEN), gene transduction, and cellular reprogramming have provided powerful tools for studying neutrophil functions and developing novel immunotherapies [86, 87] (Figures 1 and 2). Theoretically, gene engineering techniques can be used to isolate and collect mature neutrophils from peripheral blood by density gradient centrifugation [64]. This is followed by ex vivo incubation of the collected mature neutrophils with editing tools that function like “molecular scissors” to precisely cut and modify specific genes. Finally, successfully edited cells are selected and cultured (Figure 4A). However, due to the inherent limitations of mature peripheral blood neutrophils as terminally differentiated cells, namely a short ex vivo half‑life, lack of proliferative capacity, and poor transfection efficiency, the current mainstream strategy in this field is not to directly edit primary mature neutrophils but rather to select cell models with proliferative capacity for gene editing, followed by directed differentiation into functional neutrophils [88].

FIGURE 4.

Basic research of engineered neutrophils. (A) Genetic engineering. (B) The nanoparticle is enveloped by a neutral cell membrane. (C) Neutrophils modified by nanoparticles. (D) Neutrophil‐conjugated nanoparticle‐based targeted drug delivery system.

Two types of editing targets are commonly used. The first is immortalized neutrophil cell lines (e.g., HL‐60, NB4) (Figures 1 and 2), which are derived from leukemia patients, can proliferate indefinitely in vitro, are easy to culture and genetically manipulate, and are suitable for model establishment and mechanistic studies (Table 1). For example, Jennings et al. used CRISPR‐Cas9 to generate a ΔF508 CFTR neutrophil model in HL‐60 cells [60]; the neutrophils differentiated from these cells recapitulated the bactericidal dysfunction phenotype observed in neutrophils from patients with cystic fibrosis. The second type is CD34⁺ hematopoietic stem or progenitor cells (HSPCs) (Figure 2), which possess multilineage differentiation potential and self‐renewal capacity (Table 1). Their ability to achieve long‐term myeloid reconstitution in vivo after gene editing makes them ideal target cells for gene‐based therapies for hereditary and immune disorders. To address the limited availability of HSPCs, Sakurai et al. found that a combination of a PI3K activator, a TPO receptor agonist, and UM171 can stimulate ex vivo expansion of umbilical cord blood‐derived HSCs, providing a source of precursor cells for subsequent gene engineering and directed neutrophil differentiation [89].

TABLE 1.

Comparative summary of major engineering strategies for engineered neutrophils. The table summarizes the core principles, representative technologies, advantages, limitations, translational applications, and clinical potential of current engineering approaches used in neutrophil engineering. These strategies collectively integrate gene editing, nanotechnology, biomaterials, artificial intelligence, and precision manufacturing technologies to enhance the therapeutic and diagnostic capabilities of neutrophils.

Engineering Strategy	Core Principle	Representative technologies	Advantages	Limitations	Representative applications	Translational potential	Ref.
Gene Editing	Direct genomic modification of neutrophils or progenitors	CRISPR‐Cas9, TALEN, Prime Editing, CAR engineering	Precise functional reprogramming; long‐term effects; customizable phenotypes	Off‐target effects; low transfection efficiency; regulatory concerns	CAR‐neutrophils, CGD therapy, anti‐tumor reprogramming	Moderate–High	[58, 60, 64, 85, 86, 87, 88, 89, 90]
Cell Reprogramming	Phenotypic conversion of neutrophils into functional states	N1 polarization, cytokine engineering, transcriptional modulation	Dynamic immune modulation; adaptable therapeutic responses	Functional instability; phenotype reversion	Tumor immunotherapy, inflammation control	Moderate	[91]
Gene Transduction	Delivery of exogenous genes into neutrophils or progenitors	Viral vectors, lentiviral systems, electroporation	Stable expression of therapeutic genes	Low integration efficiency; safety concerns	CAR‐neutrophils, cytokine delivery	Moderate	[69]
Ex Vivo Hitchhiking	Drug/nanoparticle loading into isolated neutrophils before reinfusion	Surface conjugation, intracellular loading	High targeting efficiency; personalized therapy	Complex preparation; short cell lifespan; batch variability	Anti‐tumor delivery, thrombus targeting	Moderate	[5, 44, 61, 108, 119]
In Situ Hitchhiking	Nanoparticles bind endogenous circulating neutrophils in vivo	Anti‐Ly6G antibodies, anti‐CD11b ligands	Simplified workflow; scalable manufacturing	Off‐target binding; dependence on neutrophil count	Acute inflammation, infection treatment	High	[54, 109, 120, 121, 122]
Neutrophil Membrane Camouflage	Coating nanoparticles with neutrophil membranes	Membrane extrusion, sonication, biomimetic nanocarriers	Immune evasion; prolonged circulation; biomimetic targeting	Membrane standardization challenges	Cancer therapy, inflammatory diseases	High	[5, 10, 112, 113, 114, 115, 116, 117, 118, 123]
Smart Nanomaterials	Stimulus‐responsive therapeutic activation	pH‐responsive systems, photothermal agents, ROS‐responsive materials	Controlled release; reduced toxicity; precision therapy	Material toxicity concerns; manufacturing complexity	Intracerebral hemorrhage, phototherapy	High	[125, 136, 137]
Polymeric Biomaterials	Biomaterial‐mediated functional support and delivery	Hydrogels, electrospun fibers, GelMA scaffolds	Enhanced survival; improved tissue repair	Limited long‐term validation	Regenerative medicine, wound healing	Moderate–High	[138, 139]
Microfluidic Engineering	Precision cell sorting and nanoformulation manufacturing	Microfluidic chips, single‐cell sorting, deformability cytometry	High‐throughput processing; standardized manufacturing	Limited industrial‐scale throughput	Precision manufacturing platforms	High	[57, 72, 96, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151]
AI‐Driven Engineering	Computational optimization of engineering parameters	Machine learning, deep learning, Bayesian optimization	Accelerated design; personalized prediction; automation	Limited neutrophil‐specific datasets	Multi‐omics analysis, nanoformulation optimization	Very High	[29, 73, 152, 153, 154, 155, 156, 157, 158, 159, 160]
Bioimaging Engineering	Imaging‐guided monitoring and tracking	MRI, 19F‐MRI, fluorescence imaging, multimodal imaging	Real‐time monitoring; therapeutic evaluation	Signal decay; labeling‐associated toxicity	Tumor tracking, inflammatory imaging	High	[11, 161, 162, 163, 164, 165, 166, 167, 168, 169]

In the field of gene editing‐based therapy, preliminary clinical studies have been reported. Gori et al. [88] used a high‐precision gene editing strategy (Prime Editing) to repair, ex vivo, autologous hematopoietic stem cells derived from two patients with p47phox‐deficient chronic granulomatous disease (CGD) (Table 1). After treatment, neutrophil oxidase activity was restored in both patients, and no new infections or CGD‐related complications occurred during follow‐up periods of 4 and 6 months, respectively (Table 2). This study provides preliminary human evidence for gene editing‐based treatment of primary immunodeficiencies (Table 3). Furthermore, for severe congenital neutropenia caused by ELANE gene mutations, Nasri et al. used CRISPR‐Cas9D10A nickase to edit the ELANE promoter region. In patient‐derived CD34⁺ hematopoietic stem and progenitor cells (Table 2), they achieved an editing efficiency exceeding 90% and successfully restored neutrophil differentiation function without detectable off‐target effects (Table 3). These results remain at the preclinical stage [90].

TABLE 2.

The engineered neutrophils in disease diagnosis and therapeutic applications.

Study	Research achievement	Function	Ref.
Gori et al.	PM359: an autologous CD34‐positive hematopoietic stem cell gene therapy product based on prime editing, designed to correct the pathogenic mutation in p47phox‐deficient chronic granulomatous disease.	Restores NADPH oxidase activity in neutrophils after ex vivo repair of patient autologous hematopoietic stem cells, providing a clinical‐grade solution for genetic correction of neutrophil dysfunction.	[88]
Nasri et al.	MILESTONE technology: precise editing in the ELANE gene promoter region using the CRISPR‑Cas9D10A nickase.	Achieves >90% editing efficiency in CD34‑positive hematopoietic stem and progenitor cells from patients with severe congenital neutropenia, successfully restoring neutrophil differentiation without detectable off‑target effects, offering a highly efficient and safe new strategy for gene therapy of neutrophil development disorders.	[90]
Yang et al.	L/G@HMPs: injectable dextran‑based porous hydrogel microspheres co‑loaded with the TLR4 agonist lipopolysaccharide and the PAD4 inhibitor GSK484.	Through sustained local release, reprograms radiotherapy‑recruited tumor‑associated neutrophils into an anti‑tumor N1 phenotype, while inhibiting NETosis and extending neutrophil lifespan to >72 hours, providing a new strategy for modulating neutrophil fate in radioimmunotherapy of solid tumors.	[91]
Chang et al.	CAR‐neutrophils derived from human pluripotent stem cells (chimeric antigen receptor neutrophils).	Large‐scale differentiation of functional CAR‐neutrophils from human pluripotent stem cells; exhibits superior specific cytotoxicity against glioblastoma and prostate cancer cells in vitro and in vivo, opening a new avenue for cancer immunotherapy based on engineered neutrophils.	[69]
Fan et al.	NPEOz: a pH‐responsive neutrophil membrane‐mimetic nanoplatform co‐loading the ADAM17 inhibitor GW280264X and the LXR agonist desmosterol.	Releases therapeutic agents precisely in the acidic microenvironment of intracerebral hemorrhage; promotes erythrophagocytosis by inhibiting ADAM17‐mediated MERTK/AXL shedding, facilitating hematoma clearance and improving neurological recovery, providing a new strategy for neutrophil membrane‐mimetic therapy in central nervous system diseases.	[137]
Maiocchi et al.	DNase‐1‐loaded polymeric nanoparticles prepared by inverse flash nanoprecipitation and loaded into neutrophils via co‐incubation.	Loaded neutrophils retain viability and oxidative burst; they associate with ex vivo thrombi and deliver DNase‐1 protein to degrade neutrophil extracellular traps, offering an innovative platform for neutrophil‐based thrombus‐targeted drug delivery.	[44]
Zhang et al.	Anti‐Ly‐6G antibody‐modified hollow MnO2 nanoreactor anchored to circulating neutrophils via an in situ hitchhiking strategy.	Responsively scavenges reactive oxygen species within neutrophils in the acidic microenvironment of acute pancreatitis, inhibits N1/N2 polarization and generates O2, thereby alleviating inflammation, providing a new approach for targeted regulation of neutrophils in inflammatory diseases.	[110]
Kumbhojkar et al.	Disc‐shaped polymer micropatches modified with anti‐CD11b antibody fragments that anchor onto the neutrophil surface without internalization.	Polarizes neutrophils to an anti‐tumor N1 phenotype, activates splenic natural killer cells and T cells, and reduces tumor burden; combination with CTLA‐4 inhibitors achieves complete tumor regression in a subset of mice, advancing a new strategy for neutrophil phenotype modulation in cancer immunotherapy.	[54]
Shen et al.	Neutrophil membrane‐coated Prussian blue nanozymes, constructing a biomimetic targeting platform.	Actively targets inflamed liver, exerts synergistic antioxidant, anti‐inflammatory, metabolic regulatory and immunomodulatory effects, and promotes liver repair after hepatic ischemia reperfusion injury, providing an example for the application of neutrophil biomimetic nanozymes in organ injury repair.	[116]
Cao et al.	NM@Ce6/QZn: neutrophil‐membrane‐coated quercetin‐zinc coordination nanocomplex integrating photothermal, photodynamic and metabolic interference functions.	Achieves triple synergistic therapy for colorectal cancer through direct tumor killing, elimination of cancer stem cells and activation of anti‐tumor immunity, demonstrating a multipronged comprehensive therapeutic approach based on neutrophil membrane biomimetic technology.	[115]
Li et al.	Neutrophil membrane‐wrapped sponge‐like biodegradable polymer nanosystem.	Inhibits expansion and function of myeloid‐derived suppressor cells, alleviates the tumor immunosuppressive microenvironment, and suppresses pre‐metastatic niche formation to reduce lung metastasis, providing a new strategy for neutrophil membrane biomimetic systems in tumor metastasis prevention.	[138]
Zhuang et al.	Cell membrane camouflaged metal–organic framework system with enhanced biocompatibility and functionality.	Efficiently encapsulates therapeutic enzymes while maintaining their biological activity; the surface receptor membrane and enzymes act synergistically to achieve safe in vivo drug delivery, offering a technical foundation for the construction of neutrophil membrane‐camouflaged metal–organic framework platforms.	[117]
Bakhtiari et al.	3D extracellular matrix‐inflammation microfluidic chip that isolates high‐purity, high‐viability neutrophils from small volumes of unprocessed whole blood.	Provides high‐quality seed cells for engineering and reveals the nonlinear relationship between platelet count and neutrophil transendothelial migration, delivering an advanced in vitro analytical tool for basic neutrophil research and engineering applications.	[140]
Yang et al.	CSFD platform: a fully integrated cell sorting and functional detection microfluidic platform that assesses neutrophil motility in sepsis patients within 30 min.	Constructs a neutrophil motility function index for early sepsis diagnosis and real‐time immune monitoring, providing a rapid evaluation technique for functional quality control of engineered neutrophil therapies.	[72]
Wang et al.	Helical focusing flow microreactor: produces homogeneous lipid nanoparticles with PDI < 0.1 and achieves complete membrane coating via microfluidic electroporation.	Enables continuous homogeneous production and surface functionalization of nanoparticles; technology can be extended to the scalable preparation of neutrophil membrane‐coated biomimetic nanoparticles, supporting GMP‐level manufacturing of engineered neutrophil biomimetic platforms.	[144]
Ashley et al.	Microfluidic chip‐based multi‐frequency impedance detection system coupled with antibody‐conjugated microspheres for highly sensitive neutrophil recognition.	Integrates nanoparticle functionalization with rapid evaluation and screening of targeting efficiency, providing a high‐throughput detection technology for performance characterization of neutrophil‐targeted nanoplatforms.	[145]
Migone et al.	Microfluidic electroporation technology for preparing biomimetic nanoparticles coated with red blood cell and macrophage membranes.	Validates the applicability of microfluidic technology for the preparation of cell membrane‐coated nanoparticles, offering a general platform technology that can be adapted for constructing neutrophil membrane biomimetic systems.	[146]
Hasenberg et al.	Catchup mouse model: a neutrophil‑specific fluorescent reporter mouse generated by knocking tdTomato into the Ly6G locus via CRISPR/Cas9.	Enables real‑time two‑photon microscopy tracking of endogenous neutrophil homing dynamics and therapeutic responses in vivo, providing an ideal animal model platform for monitoring the fate of engineered neutrophils.	[163]
Wu et al.	Doxorubicin‐loaded magnetic mesoporous silica nanoparticle‐engineered neutrophils prepared by co‐incubating drug‐loaded magnetic nanoparticles with neutrophils.	Enables real‐time magnetic resonance imaging tracking of engineered neutrophils crossing the blood‐brain barrier to home to brain tumors; significantly increases intratumoral drug concentration and delays glioma recurrence, providing proof‐of‐concept for engineered neutrophil‐targeted drug delivery in brain tumors.	[167]
Li et al.	Neu@PFC: perfluorocarbon nanoemulsion‐loaded engineered neutrophils.	Achieves quantitative imaging without fluorine background, precisely defines pulmonary inflammatory areas and assesses acute lung injury severity, offering a novel tool for neutrophil‐mediated diagnostic imaging of inflammatory diseases.	[11]
Yu et al.	Neutrophils loaded with biomineralization‐inspired multimodal nanoformulations for MRI/PAI/NIRF imaging.	Multimodal complementarity provides complete spatiotemporal information on engineered cell homing in a rheumatoid arthritis model, delivering an advanced imaging strategy for in vivo tracking of neutrophil‐based therapies in autoimmune diseases.	[168]
Wang et al.	ICG and magnetic nanoparticle‑engineered neutrophils: magnetic silica near‑infrared sensitive nanoparticles bound to neutrophils.	Demonstrates triple functionality of engineered neutrophils‐magnetic targeting, real‑time MRI tracking, and photothermal therapy; all materials are clinically approved, facilitating translational application.	[4]
Sun et al.	Acouscyte/O2: oxygen‑carrying perfluorocarbons and Temoporfin loaded into cRGD‑modified multilayered liposomes, carried by neutrophils.	Constructs an engineered cell with both acoustic function and oxygen‑carrying capacity, enabling real‑time dual‑modality imaging (fluorescence + ultrasound) and oxygen‑enhanced sonodynamic therapy of tumors.	[175]
Wang et al.	CM@AIE NPs: aggregation‑induced emission nanorobots coated with neutrophil membrane.	Biomimetic neutrophil membrane coating grants precise targeting of inflamed sites, achieving photothermal bacterial clearance and accelerated wound healing.	[70]
Yu et al.	Neutrophil‑camouflaged stealth nanocarrier: anti‑CD11b and IR820‑conjugated nanoparticles disguised by neutrophils.	Exploits neutrophil biotropism for tumors to achieve “hitchhiking” delivery of nanocarriers with real‑time tracking of distribution and migration.	[190]
Zhang et al.	SPIONs‑modified neutrophil exosome‑like nanovesicles (loaded with doxorubicin).	Confirms that engineered neutrophil exosomes can serve as targeted drug delivery vehicles; magnetic guidance enables tumor‑selective accumulation, growth inhibition, and prolonged survival after a single treatment.	[186]
Zhang et al.	uPB‑Exo: ultrasmall Prussian blue nanozyme‑functionalized neutrophil exosomes.	Constructs a theranostic exosome targeting activated fibroblast‑like synoviocytes, combining anti‑inflammatory effects (ROS scavenging, Th17/Treg modulation) with MRI imaging capability.	[193]
Cruz et al.	α1‑antitrypsin‑derived peptide‑modified hydroxychloroquine nanoparticles.	Achieves selective binding to activated neutrophil elastase, delivering drug to inhibit neutrophil activation and venous thrombosis in mice.	[12]
Luo et al.	Neutrophil “hitchhiking” delivery platform: drug‑loaded PLGA nanoparticles combined with neutrophils.	Utilizes natural bone‑marrow homing of neutrophils to cross the bone‑marrow‑blood barrier, delivering chemotherapy for bone metastases or osteogenic peptide for osteoporosis.	[108]
Gao et al.	GelMA 3D hydrogel scaffold integrated with growth factors from N2 neutrophils.	Uses pro‑angiogenic factors secreted by anti‑inflammatory (N2‑polarized) neutrophils to construct an immunomodulatory scaffold promoting rapid vascularization of ischemic tissue.	[195]
Meng et al.	iSEND: cholesterol‑engineered neutrophil nanovesicles loaded with dexamethasone – an inhalable delivery system.	Enables inhaled self‑immunoregulatory delivery of dexamethasone via engineered neutrophil nanovesicles, effectively alleviating SARS‑CoV‑2‑induced pneumonia at one‑tenth of the conventional dose.	[179]
Guo et al.	nMOF‑activated radiotherapy‑radiodynamic therapy (RT‑RDT).	Reprograms tumor‑infiltrating neutrophils into atypical antigen‑presenting cells during radiotherapy, enabling cross‑presentation of tumor antigens and remodeling of the tumor microenvironment.	[188]
Tamassia et al.	Electroporation‑mediated transfection of poly(I:C) into human neutrophils.	Reveals that neutrophils recognize viral RNA via their helicase system and upregulate type I interferons and antiviral genes, providing a theoretical basis for using engineered neutrophils in antiviral therapy.	[180]
Eruslanov et al.	Identification of an APC‑like tumor‑associated neutrophil subset in early‑stage lung cancer patients (phenotype: CD11b+CD66b+CD15+HLA‑DR+CD14+).	Proves that engineered neutrophils can be reprogrammed into functional antigen‑presenting cells that directly induce anti‑tumor T‑cell responses, opening new avenues for cancer immunotherapy.	[185]

TABLE 3.

Engineered neutrophil strategies by disease area and engineering technology.

Disease area	Engineering technology strategy	Specific platform/method	Authors and Key effect summary	Ref.
Hematologic & Immunologic disorders	Gene Editing (Prime Editing)	PM359: prime editing of autologous HSPCs to restore neutrophil NADPH oxidase activity	Gori et al. demonstrated that treated CGD patients showed restored neutrophil function with no new infections or complications.	[88]
	Gene Editing (CRISPR‐Cas9D10A)	MILESTONE: editing the ELANE gene promoter region	Nasri et al. achieved >90% editing efficiency in SCN patient CD34+ cells and successfully restored neutrophil differentiation.	[90]
Cancer	Gene Transduction (CAR)	Human pluripotent stem cell‐derived CAR‐neutrophils	Chang et al. proved specific anti‐tumor cytotoxicity in glioma and prostate cancer mouse models.	[69]
	In Situ Hitchhiking (micropatch)	Discoidal polymer micropatches (CAMP) anchored to neutrophil surface via anti‐CD11b antibody fragments	Kumbhojkar et al. discovered that neutrophils were polarized to an anti‐tumor N1 phenotype, activating splenic NK and T cells; combination with CTLA‐4 inhibitor achieved complete tumor regression in a subset of mice.	[54]
	Ex Vivo Hitchhiking	Doxorubicin‐loaded magnetic mesoporous silica nanoparticles co‐incubated with neutrophils	Wu et al. constructed engineered neutrophils that crossed the BBB to target glioma; MRI tracking revealed increased intratumoral drug concentration and delayed recurrence.	[167]
	Ex Vivo Hitchhiking	PLGA nanoparticles loaded into neutrophils (exploiting bone‐marrow homing)	Luo et al. developed a platform that delivered cabazitaxel to suppress bone metastasis or teriparatide to increase bone density in an osteoporosis model.	[108]
	Ex Vivo Drug Loading	Autologous drug‐loaded neutrophil therapy (NCT07198659) for advanced pancreatic cancer, loading MMAE ex vivo followed by reinfusion combined with radiotherapy	Preclinical studies validated targeted efficacy: MMAE‐loaded neutrophils precisely home to tumor sites, release drug to inhibit tumor growth; the therapy has been approved for clinical trial, offering a new strategy for advanced pancreatic cancer.	NCT07198659
	Neutrophil Membrane Coating	NM@Ce6/QZn: neutrophil‐membrane‐veiled quercetin‐zinc coordination nanocomplex	Cao et al. realized triple synergistic therapy (photothermal, photodynamic, metabolic interference) for colorectal cancer and cancer stem cells, activating anti‐tumor immunity.	[115]
	Neutrophil Membrane Coating	Neutrophil membrane‐wrapped biodegradable polymer nanosystem	Li et al. showed inhibition of MDSC expansion and function, alleviation of immunosuppressive TME, and reduction of lung metastasis.	[138]
	Biomimetic Nanovesicles (exosome‐like)	SPIONs‐modified neutrophil exosome‐like nanovesicles (DOX‐loaded, DOX@NNVs)	Zhang et al. demonstrated magnetic guidance enabled tumor‐selective accumulation, effective tumor growth inhibition, and prolonged mouse survival.	[186]
	In Situ Phenotype Reprogramming (non‐genetic)	nMOF‐activated radiotherapy‐radiodynamic therapy (RT‐RDT)	Guo et al. reprogrammed tumor‐infiltrating neutrophils into atypical antigen‐presenting cells, enabling cross‐presentation of tumor antigens and remodeling of the TME.	[188]
	Artificial Intelligence (analysis)	Machine learning integrating single‐cell transcriptomics to identify engineering targets	Wu et al. identified an HLA‐DR+CD74+ neutrophil subset whose antigen‐presenting capacity is enhanced by leucine metabolic reprogramming, providing a candidate for engineering.	[29]
	Artificial Intelligence (analysis)	NeuRGI (random forest‐based neutrophil regulating gene identifier)	Wang et al. discovered that MAP4K4 knockout leads to neutropenia, offering a new engineering target for neutrophil functional regulation.	[155]
Infectious diseases	Ex Vivo Hitchhiking	Neutrophil‐membrane‐coated AIE nanorobots (CM@AIE NPs)	Wang et al. achieved targeted bacterial clearance under 980 nm laser, promoted angiogenesis, and wound healing.	[70]
	Ex Vivo Hitchhiking (inhalation)	Cholesterol‐engineered neutrophil nanovesicles loaded with dexamethasone (iSEND) for inhalation	Meng et al. found that inhalation at one‐tenth of the intravenous dose effectively alleviated SARS‐CoV‐2‐induced acute pneumonia and lung injury.	[179]
	Functional Gene Discovery	Electroporation‐mediated transfection of poly(I:C) into human neutrophils	Tamassia et al. revealed that neutrophils recognize viral RNA via their helicase system and upregulate type I interferons, providing a theoretical basis for antiviral therapy.	[180]
Autoimmune/Inflammation	Neutrophil Exosome Engineering	Ultrasmall Prussian blue nanozyme‐conjugated neutrophil exosomes (uPB‐Exo)	Zhang et al. proved targeted rheumatoid arthritis synovium, scavenged ROS, regulated Th17/Treg balance, and alleviated inflammatory stress.	[193]
	In Situ Hitchhiking	Anti‐Ly‐6G antibody‐modified hollow MnO2 nanoreactor (in situ anchoring to circulating neutrophils)	Zhang et al. developed a system that responsively scavenges intracellular ROS in acute pancreatitis, inhibits N1/N2 polarization, generates O2, and alleviates inflammation.	[110]
	Ex Vivo Hitchhiking	DNase‐1‐loaded polymeric nanoparticles (loaded into neutrophils via co‐incubation)	Maiocchi et al. showed that loaded neutrophils retained viability and oxidative burst, associated with ex vivo thrombi, and delivered DNase‐1 to degrade NETs for thrombus targeting.	[44]
	Peptide‐Targeted Nanoparticles	α1‐antitrypsin‐derived peptide‐modified hydroxychloroquine nanoparticles	Cruz et al. achieved specific binding to activated neutrophil elastase, delivering hydroxychloroquine to inhibit neutrophil activation and venous thrombosis in mice.	[12]
Tissue repair /Regeneration	Hydrogel Scaffold with Neutrophil Factors	GelMA hydrogel integrated with growth factors from N2‐polarized anti‐inflammatory neutrophils	Gao et al. promoted HUVEC migration and capillary‐like structure formation; achieved rapid vascularization in ischemic tissue by modulating inflammation and promoting vascular anastomosis.	[195]
	Neutrophil Membrane‐Coated Nanozymes	Neutrophil membrane‐coated Prussian blue nanozymes (Met@PBN@Neu‐CVs)	Shen et al. demonstrated active targeting of inflamed liver, exerting synergistic antioxidant, anti‐inflammatory, metabolic regulatory and immunomodulatory effects, promoting liver repair after ischemia‐reperfusion injury.	[116]
	pH‐Responsive Nanoplatform	pH‐responsive neutrophil membrane‐mimetic nanoplatform (NPEOz) co‐loading ADAM17 inhibitor GW280264X and LXR agonist desmosterol	Fan et al. realized precise drug release in the acidic microenvironment of intracerebral hemorrhage, inhibiting ADAM17‐mediated MERTK/AXL shedding, promoting erythrophagocytosis, and improving neurological recovery.	[137]

3.3.2. Cell Reprogramming and Gene Transduction

The combined application of cellular reprogramming, gene transduction, and gene editing technologies offers potential research strategies for engineering neutrophils. Neutrophils exhibit remarkable phenotypic plasticity within the tumor microenvironment, enabling a functional switch between the anti‑tumor N1 phenotype and the pro‑tumor N2 phenotype through artificial reprogramming techniques (Table 1). Yang et al. [91] designed an injectable dextran‑based porous hydrogel microsphere (L/G@HMPs) co‑loaded with the TLR4 agonist LPS and the PAD4 inhibitor GSK484 (Table 2). In a 4T1 breast cancer mouse model, these microspheres combined with fractionated radiotherapy reprogrammed radiotherapy‑recruited tumor‑associated neutrophils toward an anti‑tumor N1 phenotype and cleared NETs (Table 3). The reprogrammed neutrophils formed immune synapses with CD8⁺ T cells via CD54, promoting T cell activation and infiltration, thereby bridging innate and adaptive immunity to achieve long‑term anti‑tumor effects (Figure 2).

Gene transduction technology has further expanded the approaches for constructing engineered neutrophils. Chang et al. performed CAR gene modification via gene transduction at the human pluripotent stem cell (hPSC) stage (Table 3), followed by a chemically defined differentiation protocol to convert these cells into functional CAR‑neutrophils (Figure 1, Table 2). The study demonstrated that these hPSC‑derived CAR‑neutrophils exhibited superior and specific anti‑tumor activity against glioblastoma [69] (Table 1).

3.3.3. The Challenges of Genetic Engineering

3.3.3.1. Off‐Target Effects of Gene Editing

Although gene editing technologies such as CRISPR/Cas9 have shown great potential in research, off‑target effects remain one of the key issues limiting their safe application [54, 92]. In neutrophils, off‑target gene editing may lead to unintended cleavage and modification of genomic loci, thereby causing a series of adverse consequences (Table 1). From a safety perspective, off‑target effects may activate oncogenes or suppress tumor suppressor genes, increasing the risk of cellular malignant transformation. From an efficacy perspective, off‑target effects may interfere with the expression of genes related to normal physiological functions, affecting neutrophil chemotaxis, phagocytosis, and bactericidal capacity, thereby ultimately weakening therapeutic outcomes [93]. Of note, current strategies more frequently employ editing of precursor cells, such as hematopoietic progenitor cells or induced pluripotent stem cells (iPSCs), followed by induced differentiation into neutrophils [94]. Under this strategy, gene editing occurs at the stage of proliferative precursor cells; therefore, the risk of malignant transformation cannot be considered negligible simply because terminally differentiated neutrophils do not proliferate in vivo. During the expansion and differentiation of edited precursor cells, off‑target mutations may be amplified [95]. Nevertheless, because the final differentiated neutrophils have a short lifespan and do not continuously proliferate in vivo, their risk level may still be lower than that of long‑lived cells such as T cells; however, this risk still requires careful evaluation [69]. In the context of therapeutic applications, off‑target editing not only poses a potential risk of oncogenesis but may also compromise the intended anti‑tumor or immunomodulatory functions of engineered neutrophils, thereby undermining clinical efficacy.

3.3.3.2. Low Efficiency of Gene Transduction and Unstable Cellular Reprogramming

Current viral and non‑viral vectors struggle to efficiently integrate target genes into the neutrophil genome during transduction. This means that only a minority of cells acquire the desired genetic modification, a yield that cannot meet the demand for the large numbers of functional cells required for clinical therapy [96, 97] (Table 1). This not only increases production costs and time but also limits the large‑scale application of engineered neutrophils. To address this challenge, novel vector systems are being continuously explored and transduction methods are being improved, for example by optimizing electroporation parameters, developing expression vectors driven by neutrophil‑specific promoters, and exploring nanomaterial‑assisted gene delivery strategies [98, 99].

During the reprogramming process, cellular states are prone to fluctuation, and stable acquisition of novel functional phenotypes is difficult to maintain. When inducing neutrophils to convert into cell types with enhanced immunomodulatory functions, the resulting cells may revert to their original state or exhibit functional decline. Furthermore, insufficient or aberrant expression of homing‑related receptors on the cell surface (e.g., chemokine receptors such as CXCR2 and CXCR4, and integrin family molecules) [100], combined with physical barriers and competing chemotactic signals present in the in vivo microenvironment [101], results in poor precision targeting of engineered neutrophils to lesions, leading to waste of therapeutic resources and diminished efficacy [101] (Table 1). Current research focuses on identifying more stable combinations of reprogramming factors and exploring microenvironmental factors that maintain the reprogrammed state, such as specific extracellular matrix components and cytokine cocktails [73, 89].

3.4. Binding of Neutrophils to Biomaterials

3.4.1. Nanoparticle Modification of Neutrophils

3.4.1.1. Surface Modification and Intracellular Loading Techniques

Nanoparticle modification techniques can be divided into two categories: surface modification of cells and intracellular loading (Figure 1). These techniques exploit the natural chemotactic ability of neutrophils to precisely deliver anticancer drugs to the tumor microenvironment, thereby reducing toxicity and enhancing efficacy, or to enhance pathogen recognition and killing capacity to accelerate infection clearance (Figure 4C) [102]. Additionally, nanoparticles can be loaded with immunostimulatory agents such as bacterial DNA or specific antigens to further potentiate immune responses [103, 104]. Based on these advantages, this technology shows significant application potential in anti‑infective, anti‑tumor, and immunomodulatory fields [105, 106] (Figure 2).

Surface modification of neutrophils with nanoparticles involves immobilizing drug‑loaded or ligand‑functionalized nanoparticles onto the neutrophil surface via electrostatic adsorption, chemical conjugation, or other methods to enhance targeted recognition, immune response, and drug delivery efficiency [107] (Figure 2).

Intracellular loading techniques deliver functional nanoparticles into the interior of neutrophils (cytoplasm or endocytic vesicles) through co‑incubation, electroporation, or phagocytosis induction, using neutrophils as “living cell carriers” for protective transport and targeted delivery of drugs [44] (Figure 2). Unlike surface modification strategies, intracellular loading hides the nanoparticles inside the cells, effectively preventing premature release and degradation of nanoparticles in the bloodstream while leveraging the natural chemotaxis of neutrophils for precise drug delivery to sites of inflammation or tumors.

3.4.1.2. “Ex Vivo Hitchhiking Strategy” and “In Situ Hitchhiking Strategy”

The ex vivo hitchhiking strategy typically involves: first, isolating autologous neutrophils from the patient's peripheral blood or obtaining neutrophils through in vitro induced differentiation; second, ex vivo, combining the neutrophils with drug molecules or nanoparticles via surface modification or intracellular loading (Figure 2); and third, reinfusing the nanoparticle‑modified neutrophils into the patient [5, 108] (Table 1).

In preclinical animal studies, Maiocchi et al. demonstrated that neutrophils loaded ex vivo with DNase‑1 nanoparticles maintained cell viability and biological functions, including oxidative burst, and were capable of associating with ex vivo‑formed blood clots to deliver nanoparticles and DNase‑1 protein [44]. Notably, a novel autologous drug‑loaded neutrophil therapy for advanced pancreatic cancer (Clinical Trial NCT07198659) is about to enter clinical trials. In prior laboratory studies, this therapy validated its targeted therapeutic efficacy by loading monomethyl auristatin E (MMAE) into neutrophils and reinfusing them into subjects (Table 3).

The “in situ hitchhiking strategy” involves functionalizing the surface of nanoparticles so that they can actively anchor to circulating neutrophils in vivo (Figure 2). This strategy eliminates the need for ex vivo cell isolation and manipulation. Instead, specific ligands on the nanoparticle surface (e.g., anti‑Ly6G antibodies and anti‑CD11b antibodies) recognize and bind to antigens on the neutrophil surface, allowing the nanoparticles to “hitch a ride” on the natural homing ability of neutrophils as they actively chemotax to sites of inflammation or tumors, achieving precise drug delivery [54, 109] (Table 1). Zhang et al. [110] constructed a hollow manganese dioxide nanoreactor modified on its surface with Ly‑6G antibodies (Table 2). This nanoreactor can “hitchhike” onto neutrophils in vivo by recognizing surface antigens on neutrophils, actively target pancreatic inflammatory lesions, degrade responsively in the local weakly acidic microenvironment, scavenge reactive oxygen species within neutrophils, inhibit their polarization, and thereby alleviate inflammatory responses (Table 3). Kumbhojkar et al. [54] developed discoidal polymer micropatches (CAMP) that evade phagocytosis due to their high aspect ratio and specifically anchor to neutrophils using surface‑modified anti‑CD11b antibody fragments (Table 2). These patches continuously activate neutrophils and promote their polarization toward an anti‑tumor N1 phenotype, achieving effective anti‑tumor therapy in animal models and synergizing with immune checkpoint inhibitors to enhance efficacy. This strategy transforms neutrophils from “therapeutic carriers” into “active in vivo transport vehicles,” offering a new direction for neutrophil‑based targeted delivery with greater potential for clinical translation (Table 3).

Compared with direct ex vivo modification of neutrophils, this strategy avoids the loss of activity and immunogenicity risks associated with ex vivo cell manipulation, simplifies the preparation process, and is more amenable to large‑scale production and clinical application.

3.4.2. Neutrophil Membrane Encapsulation Technology

Neutrophil membrane‑coated nanocarrier technology involves isolating and purifying neutrophils, extracting their cell membranes, and then coating the neutrophil membranes (which contain functional membrane proteins) onto nanomaterial surfaces using methods such as sonication or co‑extrusion [111] (Figure 2), thereby constructing biomimetic delivery systems with both biocompatibility and targeting capability [112] (Figures 1 and 4B). This strategy leverages the biocompatibility of the neutrophil membrane and its natural characteristics, such as adhesion molecules and chemokine receptors, to endow the carriers with immune evasion capacity, prolong circulation half‑life, and inherit the inflammatory and tumor‑homing properties of neutrophils [112], demonstrating broad application prospects in anti‑tumor, anti‑infective, and immunomodulatory fields [10, 113, 114] (Table 1).

Cao et al. [115] developed a neutrophil membrane‑camouflaged nanoplatform named NM@Ce6/QZn (Table 2). By integrating the photothermal effect and metabolic interference capability of a quercetin‑zinc coordination complex with the photodynamic effect of a photosensitizer, this platform achieved triple synergistic therapy for colorectal cancer: direct tumor cell killing, elimination of cancer stem cells, and activation of anti‑tumor immunity (Table 3). Shen et al. [116] developed neutrophil membrane‑coated Prussian blue nanozymes (Met@PBN@Neu‑CVs). The coating of neutrophil membranes enabled the nanoparticles to actively target the inflamed liver (Table 2). This study combined antioxidant defense, anti‑inflammatory effects, metabolic regulation, and immunomodulation for the treatment of hepatic ischemia‑reperfusion injury, promoting liver repair (Table 3).

Currently, neutrophil membrane encapsulation technology has been widely used to construct biomimetic nanocarriers loaded with chemotherapeutic drugs, immunomodulators, and nucleic acids, and its efficacy has been validated in various tumor and inflammatory disease models. This approach reduces the complexity of live cell manipulation and offers advantages in terms of stability, reproducibility, and large‑scale production [5, 117]. With the standardization of membrane sources, optimization of preparation processes, and improvement of safety evaluation systems, biomimetic nanomedicines are expected to substantially advance toward clinical application, becoming the next generation of precision targeted delivery platforms [118] (Table 1).

3.4.3. Potential for Clinical Translation Among Different Strategies

The above‑mentioned strategies each have distinct characteristics in terms of mechanism and operation, but they share common as well as unique challenges in clinical translation.

The “ex vivo hitchhiking strategy” requires isolation of autologous or donor neutrophils, ex vivo nanoparticle modification (surface or intracellular loading), and reinfusion. It faces bottlenecks such as limited cell sources, short ex vivo half‑life (approximately 7 h), complex and time‑consuming manipulation, large batch‑to‑batch variability, and the risk of immune rejection after reinfusion of intact cells, as natural surface antigens can be recognized by the host. These factors make standardized, large‑scale production difficult [61, 119].

The “in situ hitchhiking strategy” eliminates ex vivo manipulation of neutrophils, simplifies the preparation process, and reduces immunogenicity risks. However, its targeting efficiency is significantly affected by blood flow shear stress, protein corona formation, and cell status. There are potential risks of off‑target cross‑binding (e.g., with monocytes) and unintended neutrophil activation (e.g., triggering excessive inflammation) [120, 121]. Moreover, this strategy is highly dependent on neutrophil counts and thus has limited efficacy in patients with severe neutropenia. Although preclinical evidence demonstrates the effectiveness of cell hitchhiking strategies in cancer, immune‑mediated, and inflammatory diseases, whether they can translate into higher remission rates or lower relapse rates in patients remains to be validated by prospective clinical studies [122].

The neutrophil membrane encapsulation strategy extracts natural membranes to coat nanoparticles, preserving functions such as inflammatory homing and immune evasion. However, the membrane extraction process is relatively complex, and large‑scale production remains a key challenge for the clinical translation of membrane‑coated nanoparticles. Critical bottlenecks still need to be overcome, including standardization of membrane sources, batch‑to‑batch consistency, long‑term immunogenicity, and good manufacturing practice (GMP) preparation processes. Furthermore, the fusion efficiency and stability between the cell membrane and the nanoparticle core still require further improvement [123].

The three strategies described above are applicable to different clinical scenarios. The “ex vivo hitchhiking strategy” is suitable for personalized autologous therapy scenarios, such as rare diseases or cases requiring complex genetic modifications, offering tailored treatment options for patients. The “in situ hitchhiking strategy” requires no ex vivo manipulation and enables “off‑the‑shelf” targeted therapy, making it suitable for acute inflammation, infections, or rapid postoperative intervention [124]. The “neutrophil membrane encapsulation technology”, combining biomimetic targeting with the stability of synthetic nanomaterials, has the greatest potential to achieve standardized product development first, with significant advantages in clinical translation potential for diseases such as cancer, inflammatory disorders, and autoimmune disorders [105, 118, 125, 126, 127, 128].

It is worth noting that although the results of animal studies involving the above strategies are generally positive, these preclinical models have limitations in translating to human clinical applications. In the early stages of development, animal models often fail to replicate the heterogeneity and complexity of the human body. In the future, with the maturation of iPSC‑derived neutrophils, automated cell processing equipment, and reproducible membrane preparation processes, neutrophil engineering technologies are expected to jointly advance clinical implementation in the three major fields of inflammation, cancer, and immunity [94, 129] (Figure 1).

3.4.4. Targeted Drug Delivery System With Neutrophil Binding to Nanoparticles

Organic nanomaterials have attracted much attention in the field of biomedicine due to their versatility, while inorganic nanomaterials have also shown broad research prospects due to their unique physicochemical properties. At the same time, owing to their excellent tumor and inflammation‑homing ability, neutrophils can be used as drug delivery carriers to transport therapeutic agents, thereby achieving targeted cancer and inflammation treatment and significantly improving the targeting of traditional drug delivery systems [125, 126] (Figures 2 and 4D).

The combination of various functional nanomaterials and neutrophil‐derived drug carriers not only expands the therapeutic field of nano‐drug delivery systems (NDDS) but also overcomes the respective limitations of each. This combination significantly improves the biocompatibility, targeting, stability, circulation time, and versatility of drug delivery systems (DDS), enabling their combination with various treatment modalities such as chemotherapy, radiotherapy, immunotherapy, and phototherapy [70, 130], thereby providing new ideas for the development of innovative drug delivery platforms to combat cancer and inflammatory diseases [131, 132, 133] (Figure 2). In addition, the concealing nature of neutrophil membrane endowed the constructed nanoplatforms with many unique properties resembling living cells, which endows the nanoplatforms with great research and clinical application potential in the development of novel nanocarriers [134]. These neutrophil‑inspired platforms may offer a viable strategy toward the clinical translation of next‑generation targeted therapies. Further optimization of their in vivo behavior and safety profiles will be critical for eventual clinical adoption.

3.4.5. Binding of Neutrophils to Smart Nanomaterials

Smart nanomaterials are responsive to environmental changes such as temperature, pH, light, and magnetic fields and can be activated under specific conditions to modulate therapeutic effects [135]. They can respond to different environmental conditions, such as hypoxia in the tumor microenvironment or low pH, to activate drug release or neutrophil function (Figure 1). Through the responsive design of nanomaterials, the activity, migration ability, and targeting of neutrophils can be altered to optimize the therapeutic effects [125, 136] (Table 1). For example, in the treatment of intracerebral hemorrhage, Fan et al. [137] developed a pH‐responsive neutrophil membrane nanoplatform (NPEOz) (Table 2), which can release therapeutic agents in an acidic environment and enhance erythrocyte phagocytosis, thereby improving neurological recovery (Table 3). These examples show that the combination of neutrophils and smart nanomaterials can not only adjust neutrophil functions according to the external environment but also improve treatment precision and reduce side effects, providing new strategies and methods for precisely targeted therapy, anti‑tumor immunotherapy, and dynamic drug delivery systems (Table 1).

3.4.6. Use of Polymeric Materials to Enhance Neutrophil Survival and Activity

Certain polymeric materials can incorporate functional substances through specific technical means to interact with neutrophils, thereby significantly enhancing neutrophil viability and reducing their damage and death in vivo. With regard to strategies for enhancing neutrophil function, Li et al. [138] used neutrophil membranes to wrap biodegradable polymer nanoparticles, constructing a biomimetic nano‑delivery system that inhibits the expansion, recruitment, and activation of myeloid‑derived suppressor cells (MDSCs), thereby alleviating the tumor immunosuppressive microenvironment and enhancing the tumor‑killing effect of T lymphocytes (Table 2). Additionally, polymeric materials can enhance neutrophil activity from multiple dimensions by altering neutrophil surface characteristics and promoting adhesion, migration, and activation (Table 1). Kumbhojkar et al. [54] found that discoid polymer micropatches attached to the neutrophil surface can polarize neutrophils toward an anti‑tumor phenotype, activate splenic natural killer (NK) cells and T cells (Table 3), promote the accumulation of dendritic cells and NK cells, significantly reduce tumor burden, and improve survival rates (Table 2). These studies demonstrate the diverse strategies and application prospects of polymeric materials in regulating neutrophil function.

In the broader field of immune engineering, polymer‑based biomaterials, particularly electrospun fibers, indirectly provide favorable conditions for the maintenance of neutrophil function by mimicking the extracellular matrix microenvironment and modulating local immune responses. Electrospun fibers, which possess an extracellular matrix‑like structure, a high surface‑to‑volume ratio, and reliable drug‑loading capacity, can regulate immune cell behavior through surface modification, drug loading, physicochemical parameter modulation, and bioconjugation strategies, thereby ameliorating the inflammatory microenvironment at the injury site and creating a suitable local niche for neutrophil survival and functional maintenance [139] (Table 1).

4. The Combination of Neutrophils and Biomedical Engineering Technology

4.1. Precision Manufacturing Platforms for Engineered Neutrophils

4.1.1. Microfluidic Sorting of Neutrophils

In the context of engineered neutrophil research, microfluidics can be regarded as a precision manufacturing platform (Figure 1). It primarily contributes to two aspects: the acquisition of high‑quality neutrophils and the controlled synthesis of functional nanocarriers (Table 1). Integrated microfluidic technologies enable label‑free, rapid, and high‑viability sorting of neutrophils while simultaneously analyzing their biophysical functions, including migration, phagocytosis, adhesion, and deformability [72] (Figure 2). For cell sorting, Bakhtiari et al. developed a 3D ECM‑inflammation microfluidic chip that can directly isolate high‑purity and high‑viability neutrophils from small volumes of unprocessed whole blood (Table 2). This chip provides high‑quality seed cells for subsequent engineering modification and anti‑inflammatory drug development [140]. For functional assessment, microfluidic approaches based on cell deformability cytometry are particularly noteworthy. By measuring cell deformation through narrow channels, this method effectively reflects the physiological state and pathological alterations of cells. It offers advantages such as operational simplicity and high throughput and provides important biophysical parameters for neutrophil functional assessment [141] (Table 1). Yang et al. [72] established a fully integrated cell separation and functional detection platform using a microfluidic cell sorting chip (Table 2). This platform enables comprehensive assessment of neutrophil motility function in sepsis patients within 30 min, highlighting the potential of microfluidic technology for early diagnosis of sepsis and real‑time monitoring of immune dysfunction (Table 1).

However, even when high‑viability mature neutrophils are obtained, their inherent limitations, such as a short half‑life (circulating half‑life of only 6–8 h) and poor tolerance to gene editing manipulations, continue to constrain their direct use in engineering modification [96]. Notably, Gerhardson et al. [57] utilized the high‑throughput microphysiological system PREDICT96 based on microfluidic technology (Table 2). By optimizing pump control strategies to reduce shear‑induced cell damage and adjusting culture medium density to maintain cell suspension, they successfully achieved continuous 24‑h recirculation culture of neutrophils within a vascular tissue model, with cell viability exceeding 90% and low activation levels. This achievement provides a valuable strategy for mitigating the extremely short ex vivo survival time of neutrophils, specifically by extending functional maintenance through a microfluidic biomimetic microenvironment and thereby gaining a larger time window for subsequent engineering manipulations. Nevertheless, this strategy has not yet been applied to specific engineering modifications of neutrophils such as gene editing or drug loading. Therefore, effectively overcoming the inherent limitations of mature neutrophils remains an urgent challenge to be addressed.

4.1.2. Microfluidic Regulation of Nanoparticles

In response to the above challenges, microfluidic technology can play a key role at another level beyond directly prolonging the ex vivo survival time of neutrophils (Figure 1). By precisely controlling the synthesis and functionalization of nanoparticles, microfluidics enables intravenous injection of surface‑functionalized nanoparticles that specifically bind to neutrophils in the bloodstream (Figure 2). This provides technical support for the in situ hitchhiking strategy [122] (Table 1). However, conventional methods for nanoparticle preparation suffer from inhomogeneous size distribution and poor batch‑to‑batch reproducibility, which compromise targeting efficiency [142]. Research has demonstrated that microfluidic technology can achieve continuous and homogeneous production of nanoparticles through precise control of fluid mixing and shear forces. It can also enable surface functionalization within an integrated workflow to achieve active targeting of neutrophils [143] (Table 1). For example, Wang et al. [144] showed that lipid nanoparticles prepared using a helical focusing flow microreactor exhibited a polydispersity index below 0.1, which is far superior to conventional preparation methods (Table 2). Ashley et al. [145] utilized a multi‑frequency impedance detection system within a microfluidic chip to couple antibodies against neutrophil surface markers CD11b and CD66b to polystyrene microspheres coated with metal oxides of varying thicknesses, achieving highly sensitive recognition of neutrophils (Table 2). In this study, microfluidic technology was used not only for precise control of the antibody‑microsphere conjugation process but also for the rapid evaluation and screening of targeting efficacy via impedance cytometry.

Notably, the above microfluidic surface functionalization strategies can also be extended to the preparation of biomimetic nanoparticles. Migone et al. [146] validated the feasibility of microfluidic technology in preparing biomimetic nanoparticles using other cell membrane types such as red blood cell membranes and macrophage membranes (Table 2). Through microfluidic electroporation, they precisely controlled fluid mixing and shear forces to completely coat extracted cell membranes onto nanoparticle cores, achieving uniformity of membrane coating and high‑throughput preparation (Table 1). These findings provide a technological platform for further extending microfluidic technology to neutrophil membrane‑coated nanoparticles (Figure 2).

4.1.3. Challenges and Perspectives for Clinical Translation

Although microfluidic technology shows great potential in the field of neutrophil engineering, its clinical translation still faces multiple challenges. For example, at the level of large‑scale production, existing microfluidic platforms have limited throughput, and chip materials (e.g., PDMS) tend to adsorb small molecules and exhibit poor tolerance, making them inadequate for clinical‑grade production requirements [147]. At the level of functional regulation, subtle variations in synthesis conditions may affect the integrity and activity of surface ligands [148]. For cell membrane biomimetic systems, the native orientation of membrane proteins is difficult to precisely control during the coating process, which compromises targeting function [149]. For quality control, issues remain such as insufficient biosafety evaluation and the lack of comprehensive quality control standards throughout the entire process [150]. Moreover, the large volume of imaging data generated by these microfluidic technologies requires advanced computational methods. In this context, the introduction of artificial intelligence brings new opportunities for microfluidics. Machine learning methods can be applied for automated analysis of microfluidic images, optimization of preparation parameters, and rapid assessment of cellular functions, thereby providing critical information for clinical decision‑making [151].

In summary, microfluidic technologies address two fundamental challenges in neutrophil engineering: the procurement of high‑quality viable neutrophils and the precise fabrication of functionalized nanocarriers. By providing a biomimetic microenvironment that extends the short ex vivo lifespan of neutrophils, microfluidics creates a critical time window for subsequent engineering steps such as gene editing and drug loading. These capabilities position microfluidics as an indispensable manufacturing platform that directly enables the transition from bench‑scale experiments to clinically relevant production of engineered neutrophils. Notably, the integration of microfluidics with artificial intelligence holds promise for further automating and optimizing these manufacturing processes, as discussed in the following section (Figure 1). While microfluidics provides the manufacturing tools for neutrophil engineering, artificial intelligence serves as the intelligent design engine that integrates, analyzes, and leverages complex biological data to guide the entire engineering process.

4.2. Artificial Intelligence‑Driven Design of Engineering Strategies

4.2.1. Application of Artificial Intelligence in Multi‑Omics Data Analysis for Neutrophil Engineering

Artificial intelligence may, to some extent, serve as an intelligent design engine, offering supportive roles in target discovery, delivery system optimization, and process control for engineered neutrophils (Figure 2). Artificial intelligence technologies such as deep learning and deep convolutional neural networks can be applied to the integrated analysis and deep mining of multi‑omics data including genomics, transcriptomics, proteomics, and metabolomics (Table 1). Through preprocessing and noise reduction of massive biological information, AI can extract key features closely related to neutrophil functions such as chemotaxis, phagocytosis, and antigen presentation. These features include biomarker discovery and cell phenotype prediction [73, 152, 153, 154] (Figure 1, Table 1).

Wu et al. [29] employed AI to integrate single‑cell neutrophil transcriptomic data from 17 cancer types comprising 225 samples from 143 patients. Their work not only revealed the antigen‑presenting function of neutrophils in the tumor microenvironment but also precisely identified a subset with engineering application potential, namely the HLA‑DR⁺CD74⁺ neutrophil subset (Table 1). This subset relies on leucine metabolic reprogramming to enhance antigen‑presenting capacity. Wang et al. [155] developed a machine learning workflow based on random forests and named it NeuRGI, which stands for Neutrophil Regulating Gene Identifier. They found that knockout of MAP4K4 in mouse hematopoietic stem and progenitor cells leads to neutropenia and impairs neutrophil differentiation in the bone marrow. This study provides a reliable predictive model and establishes a paradigm for identifying engineering targets in neutrophils from a computational biology perspective (Table 1). Pang et al. [156] constructed a neutrophil prognostic model by integrating single‑cell RNA‑sequencing data to predict the response to immunotherapy in patients with non‑small cell lung cancer. They revealed the central role of neutrophil differentiation‑related genes in patient prognosis and immunotherapy response, suggesting that these genes may serve as candidate targets for engineering modification.

4.2.2. AI‑Enabled Optimization of Neutrophil Engineering Strategies

4.2.2.1. AI‑Driven Design and Optimization of Nano‑Delivery Systems

In nanocarrier‑mediated neutrophil engineering systems, AI provides an efficient solution for the rational design of delivery systems. The composition, ratio, morphology, and surface properties of nanocarriers directly influence their loading efficiency into neutrophils, intracellular release behavior, and biosafety. Conventional optimization approaches often consume substantial experimental resources (Figure 2).

In recent years, intelligent optimization strategies combining automated experimental platforms and machine learning algorithms have emerged (Figure 1). These strategies enable rapid convergence to optimal combinations within vast formulation spaces. For example, Zhang et al. [157] combined an automated liquid handling platform with machine learning to construct a dataset containing 1275 formulations, increasing the success rate of nanoparticle preparation by 42.9%. This technology provides direction for optimizing neutrophil nanocarrier delivery (Table 1). Chan et al. [158] constructed LANCE, the largest dataset of lipid nanoparticles, and developed COMET, a Transformer‑architecture‑based model capable of end‑to‑end prediction of lipid nanoparticle formulation delivery efficiency. This model not only accurately predicts the efficacy of conventional lipid nanoparticles but also adapts to non‑standard formulations containing two ionizable lipids or polymeric materials. It can perform cross‑cell‑line predictions and evaluate lyophilization stability. Experimental validation demonstrated that COMET can identify lipid nanoparticles with excellent performance both in vitro and in vivo, providing a powerful tool for accelerating the development of nucleic acid therapeutics. Its methodology can be applied to nano‑delivery systems for engineered neutrophils, facilitating the optimization of engineering strategies (Table 1).

4.2.2.2. AI‑Integrated Microfluidic Technology

Recent breakthroughs have been achieved in the integration of artificial intelligence with microfluidic technology (Figure 1). Microfluidics provides a miniature stage for particle manipulation, with microchannel networks that precisely constrain fluid flow and leverage unique flow characteristics at the microscale, such as laminar flow effects, to create a controllable environment for particle motion (Figure 1). Deep learning addresses the challenges of complex microfluidic chip design and inefficient prediction. The synergy between the two offers new possibilities for hardware platform and intelligent design co‑development, enabling precise navigation of particles within microchannels [159] (Figure 2). For example, Cho et al. [160] developed an autonomous Bayesian optimization‑based control system called ABCD combined with computer vision and convolutional neural networks. This system can recognize phenotypes such as droplet generation and fluid state in real time and obtain optimal process parameters including flow rates and ratios within an average of 15 iterations, while exhibiting strong anti‑interference and generalization capabilities (Table 2). For precision manipulation, reinforcement learning‑based control strategies can integrate multiple factors including channel geometry, fluid viscosity, and flow rate to achieve dynamic prediction and closed‑loop control of droplet size and cell encapsulation efficiency. Such intelligent optimization strategies can be applied to neutrophil microfluidic sorting, single‑cell culture, and in vitro simulation systems to reduce experimental variable fluctuations and improve the consistency and scalability of engineered neutrophil preparation.

4.2.3. Challenges and Perspectives of AI‑Enabled Clinical Decision‑Making for Engineered Neutrophils

The above sections have demonstrated existing applications of AI in the biomedical field across three levels: screening of cellular engineering targets, design of nano‑delivery carriers, and optimization of microfluidic processes. However, it must be noted that none of the above studies directly targeted engineered neutrophils, nor did they involve clinical decision support for such therapies. For example, the TuNa‑AI and COMET models optimized conventional nanomedicines or LNP delivery systems without modeling key engineering parameters specific to neutrophils, such as selective uptake, intracellular release, or preservation of cell viability [158]. Although the ABCD and reinforcement learning systems can optimize microfluidic droplet generation, they have not yet been applied to real‑world scenarios of neutrophil sorting, single‑cell encapsulation, or functional screening, nor have they established associations with clinical outcomes. Nevertheless, these methods are, in principle, transferable to the field of engineered neutrophils [160]. Therefore, further research is needed to optimize AI‑guided engineering strategies for neutrophils.

In summary, AI integrates multi‑omics data and high‑dimensional experimental parameters to predict optimal engineering targets, nanocarrier formulations, and microfluidic operating conditions. This capability not only accelerates the design‑build‑test cycle but also lays the foundation for personalized engineered neutrophil therapies through patient‑specific predictive modeling (Figure 1). However, direct applications of AI to engineered neutrophil systems remain limited. Prospective collection of patient‑derived neutrophil response profiles, in vivo behavior parameters, and biomarker dynamics will be essential to train clinically relevant AI models in the future.

4.3. Neutrophil‑Based Bioimaging Technologies as Auxiliary Diagnostic Tools

4.3.1. Genetic Engineering Labeling

Neutrophil‑based bioimaging technologies can function as auxiliary diagnostic tools, providing support for real‑time monitoring of in vivo distribution and therapeutic response during the clinical translation of engineered neutrophils (Figure 2). To track the homing dynamics, targeting efficiency, and therapeutic response of engineered neutrophils in vivo, molecular labeling strategies such as genetically encoded fluorescent proteins and antibody labeling are required. These strategies are combined with imaging modalities including fluorescence imaging and MRI [161, 162]. Neutrophil labeling strategies are mainly divided into two categories: genetic engineering labeling and antibody labeling (Figure 3A).

For genetic engineering labeling, reporter genes such as green fluorescent protein or red fluorescent protein can be precisely inserted into specific locations in the neutrophil genome using gene editing tools like CRISPR/Cas9 (Table 1). For example, Hasenberg et al. generated the Catchup mouse model by knocking tdTomato into the neutrophil‑specific Ly6G locus, which allows in vivo two‑photon microscopy imaging [163] (Table 2).

4.3.2. Antibody Labeling

Compared with genetic engineering labeling, antibody labeling is simpler to perform and does not require transgenic animals, making it suitable for clinically derived human neutrophils (Figure 2). Researchers can use fluorophore‑conjugated antibodies that bind to neutrophil‑specific surface molecules such as human CD66b or mouse Ly6G to achieve in vitro labeling and tracking of cells [164] (Table 1). However, the signal persistence of antibody labeling is limited because antibodies may dissociate from the cell surface or be internalized and degraded, which leads to signal decay over time [165]. Therefore, this approach is more suitable for short‑term tracking experiments lasting hours to days.

4.3.3. Nanomaterial Loading and Multimodal Imaging

To minimize the potential impact of the labeling process on neutrophil function, fluorescent or magnetic nanomaterials can be loaded onto the neutrophil surface. Imaging techniques are then used to assess the biodistribution and targeted accumulation of engineered cells [166, 167] (Figures 2 and 3A).

4.3.3.1. MRI Imaging

Wu et al. [167] incubated doxorubicin‑loaded magnetic mesoporous silica nanoparticles with neutrophils, allowing the cells to actively phagocytose these nanoparticles (Table 2). The resulting drug‑loaded engineered neutrophils were then injected into a mouse model of surgically treated glioma. Using MRI, they successfully tracked the migration paths of the cells in the brain and their accumulation at the tumor site. The results showed that the drug‑loaded neutrophils were capable of crossing the blood‑brain barrier and releasing drugs in the residual tumor area, significantly increasing intratumoral drug concentration and delaying recurrence (Table 1).

4.3.3.2. 2 ¹⁹F‑MRI Imaging

Because there is virtually no fluorine background signal in vivo, ^{1 9}F‑MRI enables interference‑free quantitative imaging. Li et al. [11] loaded perfluorocarbon nanoemulsions into neutrophils to obtain Neu@PFC engineered cells, which were then tracked by ¹⁹F‑MRI (Table 2). In a mouse model of acute lung injury, after intratracheal delivery of Neu@PFC, the researchers were able to precisely define the inflammatory area and assess the severity of injury based on the intensity and extent of the fluorine signal in the lungs. These studies suggest that imaging techniques can not only reveal the location of engineered cells but also quantify to some extent the number of cells at the lesion site, thereby inferring their therapeutic potential (Table 1).

4.3.3.3. Multimodal Imaging

Yu et al. [168] developed biomineralization‑inspired multimodal nanoformulations and loaded them into neutrophils (Table 2). They simultaneously achieved MRI, photoacoustic imaging, and near‑infrared fluorescence imaging for dynamic monitoring of engineered cell homing in a rheumatoid arthritis model. Different imaging modalities complement each other. Fluorescence has high sensitivity but limited penetration depth. MRI has no depth limitation but relatively low resolution. Photoacoustic imaging provides high resolution in superficial tissues (Table 1). Multimodal integration can provide more complete spatiotemporal information for a comprehensive assessment of engineered cell behavior in vivo (Figure 1).

4.3.4. Challenges and Perspectives for Clinical Translation

The clinical translation of engineered neutrophils requires not only effective therapeutic action but also the ability to non‑invasively monitor their fate in patients. Therefore, imaging technologies are indispensable for visualizing the biodistribution and persistence of engineered neutrophils, thereby bridging the gap between preclinical proof‑of‑concept and clinical therapy. With this perspective, we discuss below the key challenges that must be addressed to realize this translational potential.

Impact of labeling on cell function: Whether loading nanomaterials or performing gene editing, these procedures may alter neutrophil viability, chemotactic capacity, or bactericidal function. For example, high concentrations of magnetic nanoparticles or prolonged incubation with fluorescent dyes may induce cytotoxicity or functional impairment. Therefore, control experiments are required in each study to confirm that key functional parameters of labeled engineered cells are not inferior to those of unlabeled cells [169].

Signal specificity and persistence: Although antibody labeling is simple and convenient, antibodies may dissociate from the cell surface or be internalized and degraded, leading to signal decay over time. Moreover, if engineered cells die in vivo and are cleared by phagocytes, the released nanomaterials or fluorophores may be taken up by other cells. This can cause false‑positive signals, meaning the observed signal may not represent viable engineered cells. This issue is particularly concerning for imaging modalities such as MRI, which cannot distinguish between live cells and dead cell remnants [170]. In contrast, genetically encoded fluorescent proteins and stably integrated nanomaterials generally offer better persistence [165].

Feasibility of clinical translation: For therapeutic engineered neutrophils intended for human use, most nanomaterials and gene editing strategies are still at the preclinical stage. Regulatory agencies such as the FDA have strict safety requirements regarding residual nanomaterials or exogenous genes in cells injected into humans [171, 172]. Therefore, current imaging methods mainly serve proof‑of‑concept and mechanistic studies; there remains a gap before clinical application. However, with the future development of safer labeling probes and more sensitive imaging devices, imaging technologies are expected to become standard quality control tools in clinical trials of engineered neutrophils.

In summary, imaging technologies serve not merely as tracking tools but as essential companion diagnostics, providing real‑time feedback on biodistribution, homing efficiency, and therapeutic response of engineered neutrophils (Figure 1). Overcoming challenges in signal specificity, including distinguishing live cells from dead cell remnants, minimizing the functional impact of labeling probes, and establishing regulatory standards for nanoformulations will facilitate the integration of imaging into clinical trials. Multimodal imaging and AI‑based image analysis represent promising future directions to maximize information gain while reducing the burden on engineered cell products.

5. Engineered Neutrophils and Clinical Diagnosis

With the continuous development of biomedical engineering technologies, new diagnostic tools are continuously being explored. Genetic engineering can enhance the specific recognition ability of neutrophils and their capacity to respond to pathogens, thereby improving the efficiency and accuracy of diagnosing infectious diseases. Among these tools, nanoengineered neutrophils have emerged as a novel diagnostic platform. For example, Li et al. [11] designed a nanoengineered neutrophil (Neu@PFC) based on the internalization of perfluorocarbon nanoemulsions, which can serve as a ¹⁹F‐MRI tracer for early warning diagnosis and severity assessment of acute lung injury (ALI) (Table 2). Neu@PFC has been shown to target inflammatory sites and enable real‑time imaging and quantitative assessment of inflamed areas by ¹⁹F‐MRI (Table 3).

In recent years, engineered neutrophils have been applied to cancer diagnosis, particularly in the combination of tumor‑targeted imaging and cancer immunotherapy [166, 173]. As one of the main immune cells in the body, neutrophils can migrate to tumor tissues through chemotaxis and exert anti‑tumor effects [174]. Through engineering approaches, their ability to recognize the tumor microenvironment (TME) can be enhanced, thereby improving targeted localization and tumor‑killing efficiency. Wang et al. [4] developed an engineered composite system based on indocyanine green (ICG)‐loaded magnetic silica near‑infrared‑sensitive nanoparticles (NSNPs) bound to neutrophils (Table 2). This system achieves dual precision delivery via neutrophil targeting and magnetic targeting, allowing real‑time tracking of neutrophil distribution and migration by magnetic resonance imaging (MRI) while significantly enhancing tumor cell killing (Table 3). In a mouse model of pancreatic cancer, this therapy led to complete tumor regression with no evidence of recurrence. Notably, all materials used in this system have been licensed for clinical use, laying a foundation for further translational application. Sun et al. [175] employed nanoengineering techniques to combine customized liposomes with neutrophils, constructing Acouscyte/O₂ — a nanosensitive cell with both acoustic function and oxygen‑carrying capacity (Table 2). This system was built by encapsulating oxygen‑carrying perfluorocarbons (PFC) and temoporfin in cRGD peptide‑modified multilayered liposomes (C‑ML/HPT/O₂), which were then loaded into neutrophils in vivo (Table 3). Real‑time dynamic monitoring and precise tumor localization were achieved through temoporfin‑mediated fluorescence imaging and perfluorocarbon microbubble‑enhanced ultrasound imaging.

In addition, liquid biopsy focuses on the analysis of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) in the blood of cancer patients. Because it is non‑invasive and can be repeated at different time points, liquid biopsy has attracted considerable attention for its clinical implications, such as monitoring disease progression [176]. The analysis of CTCs and ctDNA provides a new avenue for diagnosis and serves as the cornerstone of liquid biopsy‑based diagnostics.

6. Application of Engineered Neutrophils in Clinical Therapy

6.1. Application of Engineered Neutrophils in Antibacterial and Antiviral Therapy

Neutrophils are the most abundant white blood cells in circulation and constitute the first line of defense against infection. In preclinical studies of antibacterial and antiviral therapies, engineered neutrophils have demonstrated considerable application potential [124, 177, 178] (Figure 3B). Wang et al. [70] developed a biomimetic neutrophil AIE nanorobot (CM@AIE NPs) capable of precisely localizing to inflamed wound sites in vivo and generating a photothermal effect under 980 nm laser irradiation to achieve effective bacterial clearance (Table 2). Experiments showed that CM@AIE NPs promoted angiogenesis and accelerated wound healing by upregulating VEGF and CD31 expression (Table 3). Meng et al. [179] constructed the iSEND delivery system based on cholesterol‑engineered neutrophil nanovesicles for inhaled administration of dexamethasone (DEX) (Table 2). This system significantly improved therapeutic efficacy in severe COVID‑19 and reduced the toxic side effects of DEX (Table 3). In a mouse model of acute pneumonia, iSEND‑encapsulated nano‑DEX enhanced anti‑inflammatory effects, and in non‑human primates, inhaled low‑dose (1/10 of the intravenous dose) nano‑DEX was more effective in ameliorating pulmonary inflammation and injury caused by SARS‑CoV‑2. This study provides a novel strategy for safe and efficient aerosol delivery of drugs for COVID‑19 and other respiratory diseases. Tamassia et al. [180] employed transfection to introduce poly(I:C) into neutrophils and seeded the cells via electroporation at a density of 5 × 10⁶ cells/mL into 6‑well or 24‑well tissue culture plates (Table 2). One hour after transfection, morphological changes in neutrophils were observed under microscopy. The results indicated that neutrophils can recognize and activate antiviral immune responses through their helicase system, playing a critical role in antiviral immune defense (Table 3). This finding reveals a previously unrecognized function of neutrophils in antiviral immunity and provides a theoretical basis for their application in antiviral therapy (Figure 2).

6.2. Precision Tumor Immunotherapy

6.2.1. Enhance the Antitumor Function of Neutrophils in the Tumor Microenvironment

Neutrophils are an important component of the TME and key regulators of cancer. The TME has become a prominent topic in cancer research, as it not only participates in tumor initiation, progression, and metastasis but also profoundly influences the sensitivity and efficacy of cancer therapy [181, 182, 183]. In this context, engineered neutrophils are gaining widespread attention as an emerging therapeutic strategy [184, 185, 186]. Engineered neutrophils can exert anti‑tumor functions in the TME by enhancing antigen‑presenting capacity, remodeling the TME, exerting cytotoxic effects, and synergizing with other immunotherapies (Figures 2 and 3B).

In an analysis of samples from patients with early‑stage lung cancer, Eruslanov et al. [185] found that tumor‑associated neutrophils (TANs) in early‑stage lung cancer are not immunosuppressive but instead stimulate T‑cell responses. They identified a subset of TANs (CD11b⁺CD66b⁺CD15⁺HLA‑DR⁺CD14⁺) exhibiting a hybrid phenotype of granulocytes and antigen‑presenting cells (APCs). In patients with stage I/II lung cancer, these APC‑like TANs were superior to classical TANs in inducing anti‑tumor T‑cell responses. This finding suggests that reprogramming neutrophils through nanomaterials or gene editing technologies may represent a new direction for anti‑tumor therapy [187]. Guo et al. [188] demonstrated that nanoscale metal‑organic framework (nMOF)‐activated radiotherapy‑radiodynamic therapy (RT‑RDT) reprograms tumor‑infiltrating neutrophils into atypical antigen‑presenting cells in animal models (Table 3). This reprogramming enables cross‑presentation of tumor antigens and remodels the tumor microenvironment to generate anti‑tumor immune responses (Table 2). Importantly, this strategy does not involve any genetic modification of neutrophils; instead, it leverages physical cues (low‑dose X‑ray) to re‑educate endogenous neutrophils for antigen cross‑presentation, offering a potentially safer translational path.

6.2.2. Precise Tumor Targeting Using Neutrophil Nano‐Delivery Systems

Neutrophils can be used as carriers for targeted drug delivery (Figure 2). Luo et al. [108] found that neutrophils efficiently take up drug‑loaded poly (lactic‑co‑glycolic acid) (PLGA) nanoparticles (Table 2). After ex vivo loading and reinfusion into animals, these particles crossed the bone marrow‑blood barrier and increased drug concentrations in the bone marrow (Table 3). In a mouse model of bone metastatic cancer, bone marrow neutrophils efficiently delivered cabazitaxel nanoformulations to suppress tumor growth; in an osteoporosis mouse model, they effectively delivered teriparatide nanoparticles and increased bone mineral density. This study exploited the natural homing property of neutrophils to the bone marrow to develop a novel bone marrow‑targeted drug delivery platform, offering new approaches for the treatment of orthopedic diseases.

Leveraging the natural affinity of neutrophils for tumors, nanocarriers can be camouflaged by neutrophils and can utilize a “hitchhiking” effect for tumor‑targeted delivery [104, 189] (Figure 2). Yu et al. [190] developed a stealth nano‑delivery system based on neutrophil camouflage technology (Table 2), enabling real‑time tracking of nanocarriers using the fluorescent signal molecule IR820. Animal experiments showed that under laser irradiation, this system upregulated Gasdermin E expression and activated the caspase‑3 pathway, effectively inhibiting lung metastasis and inducing anti‑tumor immune memory in a mouse model of triple‑negative breast cancer (Table 3).

Neutrophil‑derived exosomes can also serve as targeted delivery vehicles (Figures 1 and 2). Zhang et al. [186] confirmed that neutrophil exosomes (N‑Ex) exert anti‑tumor effects in animal models by delivering cytotoxic proteins and activating apoptotic signaling pathways (Table 2). Modification of N‑Ex with superparamagnetic iron oxide nanoparticles (SPIONs) further enhanced their tumor‑targeting efficacy. Moreover, doxorubicin (DOX)‐loaded neutrophils inhibited tumor cell proliferation more effectively in animal models than did liposomal DOX or naïve neutrophils. Under an external magnetic field, DOX‑loaded, SPION‑modified neutrophils selectively accumulated at the tumor site and prolonged mouse survival (Table 3).

6.3. Treatment of Autoimmune Diseases

Preclinical studies have shown that neutrophils can modulate the release of neutrophil extracellular traps (NETs), secrete extracellular vesicles or specific metabolites, and regulate the activity of other immune cells such as macrophages and T cells, thereby reducing the intensity of inflammatory responses in animal models [34, 38, 191]. Additionally, neutrophils can target inflammatory sites and promote tissue repair and restoration of immune homeostasis (Figure 2). These mechanisms provide new strategies for treating autoimmune diseases with engineered neutrophils [192].

Leveraging the high tropism of neutrophil exosomes for the inflammatory microenvironment, Zhang et al. [193] conjugated ultra‑small Prussian blue nanozymes with anti‑inflammatory activity to neutrophil exosomes (Table 2). In a mouse model of rheumatoid arthritis, this system selectively accumulated in activated fibroblast‑like synoviocytes, neutralized pro‑inflammatory cytokines, scavenged reactive oxygen species, and alleviated inflammatory stress responses, demonstrating therapeutic potential (Table 3).

In autoimmune diseases, aberrant activation of the immune system promotes the formation of complexes between platelets and immune cells such as neutrophils, exacerbating inflammatory responses and thrombosis [194]. Cruz et al. [12] constructed a nanoplatform based on neutrophil‑platelet complexes, utilizing an α‑1 antitrypsin‑derived peptide to achieve specific binding to activated neutrophil elastase (Table 2). In vitro experiments showed that hydroxychloroquine delivered by this nanoparticle inhibited neutrophil activation, and in vivo experiments demonstrated therapeutic efficacy against venous thrombosis in mice. This targeting strategy offers potential treatment options for various neutrophil‑driven pathological processes (Table 3).

6.4. Tissue Repair and Regenerative Medicine

Neutrophils play an important role in the initiation, regulation, and resolution of inflammation, participate in wound healing, and are essential for maintaining tissue homeostasis [177] (Figure 2). Gao et al. [195] employed porous gelatin methacryloyl (GelMA) hydrogels as scaffolds to integrate neutrophil‑derived growth factors, constructing 3D scaffolds for ischemic tissue regeneration (Table 2). Experimental results showed that anti‑inflammatory phenotype neutrophils obtained by N2 polarization promoted the migration of human umbilical vein endothelial cells and induced the formation of capillary‑like structures in vitro. Further animal experiments demonstrated that this composite hydrogel system achieved rapid vascularization by modulating inflammatory responses and promoting vascular anastomosis (Table 3). These findings are currently at the preclinical validation stage.

6.5. Clinical Translation of Engineered Neutrophils: Current Status

Considerable progress has been made in preclinical studies of engineered neutrophil platforms, yet clinical trials are limited. As of April 2026, only two interventional trials are actively recruiting patients, and two additional products are expected to enter Phase I studies in the near future.

One ongoing trial is PM359 (NCT06559176), an autologous hematopoietic stem/progenitor cell (HSPC) product edited with prime editing to correct the NCF1 mutation in patients with chronic granulomatous disease (CGD) [88] (Figure 1). After engraftment, the gene‑corrected HSPCs differentiate in vivo to generate functional neutrophils. Preliminary clinical reports from a limited number of patients suggest acceptable safety and initial signs of efficacy (Table 3). The other active trial is EDIT‑301 (NCT04853576), which uses gene editing to modify HSPCs for the treatment of severe hemoglobinopathies such as sickle cell disease and β‑thalassemia. Although the primary indication of this trial is not neutrophil‑related disorders, it provides supportive evidence from the perspective of hematopoietic differentiation that gene‑edited HSPCs can give rise to fully functional neutrophils, offering a valuable reference for the clinical translation of engineered neutrophils.

Two additional neutrophil‑related products are approaching clinical testing. LIfT BioSciences’ IMAN is an allogeneic, off‑the‑shelf neutrophil‑like cell product derived from healthy donor stem cells, intended for solid tumors; its Phase I trial is expected to start in early 2027 (Figure 1). NeuMed's autologous drug‑loaded neutrophils (NCT07198659) have been registered for a Phase I study that plans to recruit three patients with advanced pancreatic cancer, using neutrophils loaded ex vivo with the anti‑tumor peptide monomethyl auristatin E (MMAE) for targeted therapy (Figure 1).

The scarcity of clinical cases of engineered neutrophils can be attributed to several translational barriers. First, the physiological half‑life of circulating neutrophils is only 6–24 h [56], which severely limits the therapeutic window when relying solely on adoptive transfer. In vivo continuous generation through HSPC gene editing represents a key strategy to overcome this limitation. Second, manufacturing under GMP conditions faces significant technical hurdles: primary neutrophils are fragile, prone to spontaneous activation during ex vivo handling, and difficult to cryopreserve, resulting in low yield and high cost under current compliant production systems [196, 197]. Third, there is a regulatory gap, as no dedicated guidance for engineered neutrophil‑based therapies has been issued by any regulatory agency worldwide, leaving clinical development and filing without clear standardized references.

Despite these challenges, PM359 and EDIT‑301 have advanced to human trials and have generated critical safety and feasibility data. These early‑phase studies, together with the forthcoming IMAN and NeuMed trials, provide an important foundation for further mechanistic optimization, product formulation, and clinical application of engineered neutrophil‑based therapies (Figure 1).

7. Challenges and Prospects of Engineered Neutrophils in Clinical Application

7.1. Scalability and Manufacturing Issues

From a technical perspective, the direct harvesting of mature neutrophils is constrained by their short half‐life and inability to expand in vitro. Current engineering strategies primarily rely on immortalized neutrophil cell lines (e.g., HL‐60, NB4) and CD34⁺ hematopoietic stem and progenitor cells (HSPCs) [94]. However, the differentiation cycle of these cells is long, and the stability and reproducibility of engineering modifications (e.g., gene editing, nanomaterial loading) still require rigorous validation [60] (Figure 2).

At the manufacturing process level, current methods are largely dependent on manual operations or small‐scale laboratory equipment, which are insufficient to meet large‐scale clinical demands. The adoption of automated, standardized manufacturing equipment (e.g., high‐throughput cell sorters, microfluidic chips) is crucial for improving scalability, as it can significantly enhance production efficiency and quality control [198]. Furthermore, the high costs of nanomaterials, reagents, and culture media pose additional barriers to large‐scale manufacturing. To address this, the optimization of material selection, the reduction of reagent consumption, and the development of low‑cost alternatives are imperative (Figure 2).

The manufacturing of advanced therapy medicinal products (ATMPs) must strictly comply with good manufacturing practice (GMP) requirements. For engineered neutrophils, which exhibit extremely short ex vivo survival times, traditional release testing (e.g., sterility, viability, potency) must be completed within a few hours. This imposes extremely high demands on the proximal layout of production facilities and clinical centers [197]. The International Society for Cell & Gene Therapy (ISCT) has emphasized that GMP should be integrated from the early stages of engineered neutrophil development to ensure product scalability, reliability, and long‐term success [199].

7.2. Immunogenicity of Engineered Neutrophils

When engineered neutrophils are infused into the human body, two major types of immunogenicity issues may arise. On one hand, exogenous modifications on the cell surface (e.g., nanomaterial coatings) or neoantigens introduced by gene editing may be recognized as foreign substances by the immune system, triggering immune rejection [200, 201] (Figure 2). On the other hand, engineered neutrophils may aberrantly activate the host immune system. For instance, nanomaterial‐modified neutrophils may induce excessive inflammatory responses or even cytokine storms through the release of damage‐associated molecular patterns (DAMPs) or neutrophil extracellular traps (NETs) [202]. Moreover, allogeneic neutrophils carry HLA molecules that can be recognized by recipient T cells and NK cells, leading to the rapid clearance of engineered neutrophils. In contrast, autologous neutrophils face challenges of long preparation times and high costs [96]. Biomimetic strategies, such as neutrophil membrane coating, thus represent an important research direction [123]. However, whether these strategies compromise the inherent chemotactic and phagocytic functions of neutrophils while reducing immune recognition requires further evaluation.

7.3. Biodistribution and Toxicity of Neutrophil‑Based Nanocarriers

Engineering neutrophils with nanomaterials (e.g., nanoparticles, liposomes, or polymers) is a common strategy for targeted drug delivery and imaging tracing. The natural chemotactic properties of neutrophils enable them to accumulate at lesion sites in response to chemokines secreted by tumor tissues or inflammatory signals released at inflammatory foci [101] (Figure 2).

However, this targeting capability is not absolute. On one hand, the in vivo distribution of nanocarriers is also influenced by the reticuloendothelial system (RES); organs rich in macrophages, such as the liver and spleen, nonspecifically take up nanoparticles, resulting in a certain degree of off‐target accumulation [203]. On the other hand, after delivering nanomedicines to lesion sites, neutrophils themselves may become activated and release reactive oxygen species (ROS), proteases, and NETs [204, 205]. If not properly regulated, this may instead cause local tissue damage or systemic inflammatory responses.

Furthermore, some nanomaterials (e.g., cationic liposomes, certain metal nanoparticles) possess intrinsic cytotoxicity [206], which may accelerate neutrophil apoptosis or induce excessive activation. In addition, engineering manipulations themselves may alter the biological behavior of neutrophils [207]. Therefore, when developing novel neutrophil‐based nanocarriers, it is essential to systematically evaluate their pharmacokinetics, tissue distribution, elimination pathways, and acute/chronic toxicity, as well as to establish targeted in vitro and in vivo biosafety evaluation standards [123].

7.4. Long‑Term Safety of Engineered Neutrophils

Gene editing of neutrophil precursors carries the risk of off‐target mutations, which may pose a carcinogenic risk [95] (Figure 2). Additionally, the persistent presence of engineered neutrophils in vivo may induce long‐term alterations in immune memory or the production of autoantibodies, leading to delayed‐type hypersensitivity or autoimmune diseases (Table 1). Therefore, for engineered neutrophil products that have entered or are about to enter clinical trials, follow‐up protocols longer than those for conventional drugs must be designed [208].

During the observation period, patient safety should be closely monitored to promptly detect potential risks, such as the development of hematologic malignancies, autoimmune diseases, or chronic inflammation. Meanwhile, a traceable patient registry system should be established to facilitate timely causal analysis in the event of adverse events [209].

7.5. Multidisciplinary Collaboration and Precision Medicine

The development of engineered neutrophils relies heavily on multidisciplinary collaboration, which not only broadens their application prospects in medicine, biology, and materials science but also promotes their integration into precision medicine (Table 1). In tumor therapy, Mi et al. [210] developed a “Trojan horse” strategy by integrating multidisciplinary technologies, including neutrophils, nanotechnology, and immunology. This strategy uses Salmonella to target tumor regions and enhances anti‐tumor efficacy through silver nanoparticles, providing a novel approach for tumor therapy and new insights for other combination therapies.

In the treatment of inflammation and cardiovascular diseases, Liu et al. [211] constructed a Neu‐balloon that combines ultrasound imaging with targeted drug delivery technology to achieve precise treatment of vulnerable atherosclerotic plaques (Table 2). Furthermore, the Neu‐balloon was found to have a high and broad drug‐loading capacity, enabling the in vivo delivery of indocyanine green and miR‐126a‐5p to vulnerable plaques for precise therapy.

In the field of nervous system diseases, engineered neutrophils and their exosomes can be used to regulate the immune microenvironment and alleviate neuroinflammation. Qiu et al. [212] utilized emerging nanomaterial technology to develop neutrophil‐derived nanovesicles (NNVs) and found that NNVs can regulate neuroinflammation by clearing myelin debris, representing a promising therapeutic strategy for multiple sclerosis (Table 2). This work exemplifies the cross‐integration of brain science and materials science in the research of brain diseases.

These studies not only demonstrate the potential of engineered neutrophils in the treatment of various diseases but also highlight the importance of multidisciplinary collaboration. By integrating the advantages of materials science, nanotechnology, immunology, medicine, and biology, engineered neutrophils are expected to provide more effective solutions for precision medicine and personalized treatment in the future (Figures 1 and 2).

7.6. Regulatory and Ethical Oversight of Cell‐Based Therapies

7.6.1. Evolution of the Global Regulatory Framework

Global regulatory frameworks for cell and gene therapies have undergone significant advancements, establishing tailored approval pathways that provide a clearer basis for the clinical translation of engineered neutrophils (Figure 1). The FDA's Center for Biologics Evaluation and Research designated cell and gene therapy as a priority in its 2026 Guidance Agenda, planning to release product‐specific guidance documents and introducing a “plausible mechanism” review pathway to accelerate the approval of ultra‐rare disease therapies. In April 2026, the FDA released a draft guidance on the safety assessment of genome‐edited gene therapy products using next‐generation sequencing, establishing standards for off‐target editing detection directly relevant to engineered neutrophil products such as PM359 [88]. In the European Union, advanced therapy medicinal products continue to be governed by Regulation (EC) No 1394/2007. While ongoing legislative revisions and the PRIME initiative seek to expedite reviews, the number of approved products remains limited. At the international level, the ICH S12 guideline on the biodistribution of gene therapy products is actively being discussed, and the WHO's guidance on cell and gene therapies is promoting global standards harmonization. As important participants in this global landscape, China has established a comprehensive translational governance system: the Regulations on the Administration of Clinical Research and Clinical Translational Application of Emerging Biomedical Technologies (State Council Order No. 818, effective May 1, 2026) introduced the legal definition of “advanced therapeutic products” and mandates dual academic and ethical review.

7.6.2. Classification Challenges for Engineered Neutrophils

Despite ongoing global regulatory improvements, engineered neutrophils occupy an ambiguous classification space. Depending on the modification strategy, a single product may fall under multiple categories such as somatic cell therapy, gene therapy, or cell‐drug combination products; under the EU ATMP Regulation, it would be classified as a combined ATMP, while in the U.S., categories such as HCT/P and regenerative medicine therapy also exhibit definitional overlap. To date, no jurisdiction worldwide has issued dedicated guidance for neutrophil‐based therapies. Developers are thus forced to extrapolate from CAR‐T or myeloid cell therapy guidelines. However, the unique biological properties of neutrophils—short half‐life (6–24 h), susceptibility to ex vivo activation, and distinct inflammatory potential—introduce substantial uncertainty when applying existing frameworks to this cell type [213].

7.6.3. Ethical Oversight Requirements

Engineered neutrophils involve gene editing of human cells; therefore, prior to clinical translation, they must undergo review and continuous oversight by an independent ethics committee. Ethical review should focus on the following aspects. First, fully assess potential risks—including toxicity, off‑target effects, tumorigenicity, and immunogenicity—based on animal studies, reasonably exclude high‑risk populations, and ensure risk minimization. Second, safeguard patients' rights to informed consent and autonomy, fully disclose foreseeable and unforeseeable risks (e.g., the health impacts of off‑target effects), and obtain re‑consent if new risk information emerges during the trial. Ensure that subjects receive timely treatment and legal compensation for research‑related injuries, with compensation not contingent upon liability attribution [214]. Third, strictly protect participant privacy and genetic data security, clearly defining the scope of data storage, use, and sharing [215, 216]. Fourth, establish an adverse event reporting system; serious adverse events must be reported to the ethics committee within the prescribed timeframe. Moreover, research should comply with the Declaration of Helsinki, CIOMS ethical guidelines, and relevant international guidelines.

7.6.4. Future Directions: The Regulatory Path Forward for Engineered Neutrophils

Recent global regulatory updates have moved the field for engineered neutrophils from a state of “regulatory vacuum” toward the construction of tailored frameworks. To accelerate clinical translation, four priority tasks lie ahead: developing neutrophil‐specific guidance documents that establish appropriate quality control and evaluation standards; clarifying classification and review pathways for combination products and establishing multidisciplinary review teams; advancing global standards harmonization through ICH and WHO frameworks; and integrating advanced analytical technologies, such as next‐generation sequencing, into regulatory science to build high‐precision risk assessment systems. With continued refinement of these frameworks, engineered neutrophil therapies are poised to overcome translational hurdles and achieve clinical application (Figure 1).

8. Conclusion

Engineered neutrophils represent a transformative frontier in biomedical research, merging immunology, nanotechnology, and artificial intelligence to redefine diagnostics and therapeutics. By harnessing gene editing tools like CRISPR‐Cas9, biomimetic nanomaterials, and AI‐driven multi‐omics analytics, researchers have unlocked unprecedented precision in enhancing neutrophil functions—from targeted tumor eradication and pathogen neutralization to dynamic modulation of immune responses. These advancements highlight the versatility of neutrophils as both therapeutic agents and diagnostic tools, particularly in oncology, infectious diseases, and autoimmune disorders. However, challenges such as off‐target genetic modifications, short in vivo survival, and immune rejection underscore the need for further innovation in cell engineering and delivery systems. Future efforts must prioritize scalable production, clinical validation, and ethical frameworks to ensure safe translation. As interdisciplinary collaboration accelerates, engineered neutrophils stand poised to revolutionize precision medicine, offering tailored solutions that bridge the gap between laboratory breakthroughs and patient‐centered care.

Author Contributions

Jingru Chen, Jiaqi Xu, and Subinuer Aikebaier wrote the first draft of the manuscript. Youcai Liang, Xiaorong Zhou, Xiao Zhu, and Xiaoling Ding edited the manuscript. Xiaoling Ding and Xiao Zhu supervised the manuscript. All authors read and approved the final manuscript.

AI Disclosure

The authors did not use generative AI or AI‐assisted technologies in the preparation of this work.

Ethics Statement

The author has nothing to report.

Consent

The author has nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.