Precisely Engineering Biomimetic Nanoparticles via Controlled Cell Membrane Coating

ABSTRACT

Cell membrane‐coated nanoparticles (CMNPs) represent an advanced class of biomimetic nanomaterials that integrate the functional complexity of natural cell membranes with the tunable properties of synthetic nanoparticle cores. By inheriting native membrane proteins, lipids, and glycans, CMNPs exhibit attractive properties such as enhanced immune evasion, prolonged circulation, and cell‐specific interactions, thereby supporting a wide range of therapeutic, diagnostic, and biomedical applications. However, precise control over CMNPs fabrication remains challenging, particularly with respect to particle size, membrane coating integrity, and membrane leaflet orientation, all of which critically influence biological performance. This review focuses on recent progress in engineering CMNPs with precise control over size, coating integrity, and membrane leaflet orientation. It first analyzes how synthetic parameters influence the particle size and size distribution of CMNPs, then outlines strategies to improve membrane coating integrity together with the corresponding evaluation methods and discusses approaches to regulate membrane leaflet orientation and the analytical techniques used for its assessment. Finally, this review highlights remaining challenges and future directions for the rational design of CMNPs, supporting their continued development toward translational nanomedicine applications.

Precise engineering of cell membrane‐coated nanoparticles (CMNPs) requires coordinated control over particle size, coating integrity, and membrane leaflet orientation. This review integrates key synthesis parameters, assembly strategies, and analytical methods that govern these features, revealing how structural control underpins biomimetic function and providing a practical framework for the rational design and translational development of CMNPs.

1. Introduction

Cell membrane‐coated nanoparticles (CMNPs), which are a group of advanced biomimetic materials with a nanoparticle core wrapped in natural cell membranes, enable superior immune evasion, targeted delivery, and detoxification, representing a major advance in biomimetic nanotechnology [1, 2, 3]. Over the past few decades, nanomedicine has sought to improve the efficiency of drugs by altering their pharmacokinetics and biodistribution [4, 5, 6]. Traditionally, stealth properties were achieved through surface modifications, most notably via surface functionalization with polyethylene glycol (PEG) [7, 8]. While PEGylation successfully reduces protein adsorption and extends circulation time, it does not provide specific biological recognition and has recently been associated with the production of anti‐PEG antibodies, which accelerate blood clearance upon repeated dosing [9, 10]. To overcome these synthetic limitations, researchers have turned to nature for inspiration. By cloaking synthetic nanoparticle cores with native plasma membranes, CMNPs inherit the compositional complexity of living cells, including membrane proteins, lipids, glycans, and associated microdomains [11]. Consequently, they present biologically relevant surface cues that mediate specific interactions with the extracellular environment [12, 13, 14]. Lacking specific targeting mechanisms, bare nanoparticles often face significant challenges, such as rapid clearance, increased macrophage uptake, and non‐specific accumulation, which can lead to off‐target toxicity [6]. Coating the nanoparticle core with cell membranes enables intercellular communication and facilitates homotypic and phenotypic recognition [15, 16]. This strategy not only enhances nanoparticle performance but also opens novel possibilities for their applications in various biomedical fields [17, 18].

The development of CMNPs can be traced to the seminal work of Zhang et al. in 2011, in which erythrocyte membrane‐coated PLGA nanoparticles were fabricated using sonication and extrusion to confer prolonged blood circulation [19]. This work laid the foundation for the CMNPs field by defining a biomimetic fabrication strategy in which natural cell membranes are transferred onto synthetic nanoparticle cores through sonication and extrusion, thereby endowing nanoparticles with prolonged systemic circulation. Initially conceived as a passive camouflage strategy, the platform rapidly expanded into a programmable nanotechnology. In 2013, lipid‐insertion functionalization enabled lipid–PEG–ligand conjugates to be incorporated into the membrane shell, allowing CMNPs to acquire receptor‐specific targeting capabilities [20]. This approach was later extended through streptavidin–biotin chemistry, in which lipid–PEG–streptavidin insertion provided a modular interface for conjugating biotinylated peptides or ligands [21]. Subsequent advances broadened the biological complexity of CMNPs. In 2017, hybrid membrane‐coated nanoparticles were introduced by combining membranes from distinct cell sources, such as erythrocytes and platelets, thereby enabling a single nanoplatform to integrate multiple membrane‐derived functions [22]. In parallel, genetic engineering emerged as a powerful route to customize membrane bioactivity. In 2018, Gu et al. demonstrated that source cells could be engineered to overexpress PD‐1 receptors before membrane extraction, producing CMNPs with tailored immune‐modulatory functions [23]. The emergence of CAR‐T cell membrane‐coated nanoparticles further highlighted the translational potential of patient‐derived and immune cell‐engineered CMNPs, particularly in the context of personalized cancer therapy [24]. More recently, the field has shifted from demonstrating biomimetic functionality toward understanding and controlling membrane structure, coating quality, and fabrication scalability. Alongside these functional engineering strategies, increasing attention has been directed toward controlling the structural quality of CMNPs, particularly membrane orientation and coating integrity. A molecular‐affinity strategy was recently reported to promote right‐side‐out membrane coating, demonstrating that membrane orientation could be actively engineered to enhance targeted drug delivery efficacy [25]. Since 2021, coating integrity has also been recognized as a critical determinant of CMNPs internalization and biological performance [26]. Strategies such as increasing membrane fluidity and depleting membrane cholesterol have been explored to improve coating completeness, membrane permeability, and functional activity [27]. In parallel, algae‐coated nanoparticle systems have illustrated the possibility of coupling biomimetic membrane coatings with motile biological components [28]. In 2023, studies on nanoparticle core geometry revealed that particle shape can regulate cell binding by altering contact area and the number of simultaneous ligand–receptor interactions [29]. Recent developments have increasingly emphasized modular engineering and translational manufacturability. In 2024, genetic engineering‐based universal ligand attachment systems, including SpyTag/SpyCatcher chemistry, enabled diverse functional ligands to be displayed on a common CMNPs platform [30]. In 2025, affinity chromatography‐based membrane isolation was reported as a scalable strategy for producing high‐purity cell membranes for CMNPs fabrication [31]. Collectively, these advances mark the evolution of CMNPs from passive immune‐evasive nanocarriers into structurally engineered, functionally programmable, and potentially scalable biomimetic nanomedicine platforms.

Despite the vast potential of CMNPs, their effective translation remains hindered by limited control over key physicochemical and interfacial properties, particularly particle size, coating integrity, and membrane orientation [26, 32]. The inherently stochastic nature of conventional top‐down fabrication methods can generate heterogeneity in these critical parameters, which in turn affects biological performance. For example, particle size governs biodistribution and tissue penetration, while also influencing receptor–ligand interactions through steric hindrance and surface curvature effects [33, 34]. Coating integrity is equally important, as incomplete membrane coverage can expose the synthetic core to the biological environment, affect cellular internalization, and compromise the intended biomimetic functions [26]. In addition, controlling membrane orientation is essential to ensure that functional membrane domains are correctly displayed on the nanoparticle surface; misoriented membranes may render key ligands inaccessible and impair specific cellular communication [35, 36].

Although several reviews have summarized the biological applications, cell membrane sources, and therapeutic potential of CMNPs, fewer have systematically examined CMNPs from an engineering perspective, particularly control over coating integrity and membrane orientation. This review, therefore, focuses specifically on the precise engineering of CMNPs, with emphasis on how synthetic methods and functionalization strategies regulate particle size, dispersity, coating integrity, and membrane orientation. We summarize emerging strategies to improve coating completeness and orientation control, together with analytical approaches used to evaluate these structural features (Figure 1). We first discuss the structural composition of cell membranes, membrane source selection, and major coating methods. We then examine how different fabrication strategies influence the key engineering parameters of CMNPs, highlighting current methods for improving and characterizing structural precision. Finally, we discuss the remaining challenges and future perspectives for the controlled engineering of CMNPs toward more reproducible and translatable biomimetic nanomedicines.

FIGURE 1.

Schematic illustration of the overview of precision engineering strategies for cell membrane‐coated nanoparticles (CMNPs). (a) Optimization of synthetic parameters governing CMNPs particle size and dispersity. The effects of fabrication methods, including sonication, membrane extrusion, and microfluidic processing, together with nanoparticle‐to‐membrane ratio, are considered to regulate hydrodynamic size, polydispersity, aggregation, and coating outcomes. (b) Engineered membrane assembly strategies for improving coating integrity and controlling membrane orientation. Approaches such as hybrid membrane assembly and electroporation facilitate the formation of fully coated CMNPs, whereas orientation control can be achieved through ligand‐ and lipid‐based interactions, including streptavidin–biotin binding, protein–ligand interactions, bioorthogonal reactions, and phosphatidylserine‐targeting peptides. (c) Verification of membrane assembly integrity and orientation. Coating integrity can be assessed using fluorescence quenching, nanoplasmonic methods, and streptavidin–biotin‐based assays, whereas membrane orientation can be evaluated through enzymatic assays, antibody binding, and fluorescence‐based techniques. Figure created in BioRender. Lim, JL. (https://BioRender.com/gvz403x) is licensed under CC BY 4.0.

2. Methodologies for Cell Membrane Coating and Their Functional Implications

2.1. Cell Membrane Coating Methods

The fabrication of CMNPs involves isolation of cell membranes and coating of cell membranes onto the nanoparticle core (Figure 2) [3, 37]. The initial step in creating cell membrane‐coated nanoparticles involves isolating intact plasma membranes from the donor cells. This process begins with harvesting the target cell population (cultured cell lines or primary cells obtained from blood or tissues), followed by controlled disruption and differential centrifugation to enrich the membrane fraction. Among the most common disruption methods is hypotonic lysis, where cells are suspended in a low ionic strength buffer (e.g., dilute Tris‐HCl or phosphate‐buffered saline) [38, 39, 40, 41, 42]. Osmotic swelling weakens the plasma membrane and causes cells to rupture under mild conditions, thereby preserving the native organization and functionality of membrane proteins. The downside of hypotonic lysis is inconsistent fragmentation of the cell membrane [43]. Mechanical homogenization, often performed with a Dounce or Potter‐Elvehjem homogenizer, applies calibrated shear forces to disaggregate cells and liberate membrane sheets [44, 45, 46, 47]. This approach complements hypotonic treatment by increasing yield without introducing excessive heat. In some protocols, ultrasonication or repeated freeze‐thaw cycles are incorporated to further improve membrane recovery [48]. Ultrasonic cavitation fragments cells and membrane vesicles rapidly but generates localized heating [49, 50, 51]. To mitigate thermal denaturation of critical proteins, sonication is conducted on ice in short pulses, and protease inhibitors are added to suppress proteolytic activity from released intracellular enzymes. Likewise, freeze‐thaw cycles, where cells are alternately frozen in liquid nitrogen and thawed at 37°C, can promote membrane rupture, especially when combined with hypotonic swelling, but they must be balanced to avoid extensive denaturation [52, 53, 54]. Following cell disruption, low‐speed centrifugation removes nuclei and large debris, and successive higher‐speed centrifugation pellets the plasma membrane fraction [20, 55, 56]. Throughout isolation, the entire procedure is conducted at 4°C, and broad‐spectrum protease inhibitors are included in all buffers to prevent degradation of membrane proteins by co‐released intracellular proteases. The resulting membrane pellet, enriched in lipid bilayers and associated proteins, provides the essential building blocks for subsequent vesicle formation and nanoparticle coating.

FIGURE 2.

Schematic illustration of methods of extracting cell membrane from various cell sources and formation of CMNPs. Figure created in BioRender. Lim, JL. (https://BioRender.com/jr9ss5z) is licensed under CC BY 4.0.

Once isolated, membrane fragments reassemble into vesicles wrapped on nanoparticle cores via various methods including extrusion, sonication, and electroporation. Extrusion and sonication remain the most commonly used methods for CMNPs synthesis [51, 57, 58, 59, 60]. In extrusion, cell membrane materials are first passed through polycarbonate filters to generate uniform vesicles, which are then coextruded with preformed nanoparticle cores through smaller pores, thereby driving the membranes to wrap around the particles. One study hypothesized that when membrane vesicles and nanoparticles are co‐extruded, membrane‐core fusion occurs within the extrusion membrane channels [61]. This method best preserves the native orientation and functionality of membrane proteins but can be time‐consuming and, if not carefully optimized, may induce particle aggregation. Ultrasonication offers a rapid, single‐step alternative in which acoustic energy promotes spontaneous membrane coating of nanoparticles [62, 63, 64]. However, the high mechanical stress and localized heating may widen nanoparticle size distributions and increase the risk of partial denaturation of sensitive membrane proteins. The formation of CMNPs requires extraction of the cell membrane which disrupts the original orientation of the cell membrane, causing the reassembled CMNPs' membrane structure to become randomly oriented during this process [32].

Microfluidic techniques have emerged as an alternative approach offering the advantages of batch‐to‐batch consistency and upscaling potential [65, 66, 67, 68, 69]. Microfluidic techniques can be categorized into active micromixers and passive micromixers. Active micromixers use external forces to facilitate mixing, such as electric fields, magnetic fields, and pressure fields, while passive micromixers use the architecture geometry of the microfluidic channel to achieve mixing [70]. A typical microfluidic electroporation chip for CMNPs synthesis contains dual inlets for membrane vesicles and nanoparticle cores, a Y‐shaped merging junction, and an S‐shaped or serpentine mixing region to ensure homogeneous reagent distribution. Some studies incorporate a confined electroporation chamber integrated with thin conductive electrodes (e.g., aluminum or gold) [66, 68]. Additional protocols may also introduce sonication or acoustic energy to assist membrane fusion [67]. The incorporation of external stimuli, including electroporation and sonication, has been shown to facilitate membrane fusion with nanoparticle cores and enhance overall coating efficiency.

2.2. Effect of Synthetic Parameters on Particle Size

The effect of synthetic factors such as extrusion, sonication, and cell membrane‐to‐nanoparticle ratio on particle size has been investigated. When comparing the particle size of CMNPs synthesized using either sonication or extrusion, the sonication method generally yielded particles with larger hydrodynamic diameters than those produced by extrusion [71]. This trend was also evident when directly comparing sonication‐only and extrusion‐only fabrication methods, where the increase in average particle size after membrane coating via sonication was greater than that via extrusion [19, 72, 73, 74, 75, 76, 77, 78]. Despite producing smaller particles, membrane extrusion presents limitations for large‐scale manufacturing, including pore clogging during processing of concentrated suspensions and restricted throughput due to the limited effective pore surface area [79]. In contrast, sonication offers greater scalability and processing flexibility, making it more amenable to upscaling despite its tendency to yield larger particle sizes.

To optimize CMNPs synthesis, a combined sonication and extrusion approach was reported to be effective [71]. In this hybrid process, sonication first generated smaller membrane vesicles, and subsequent extrusion then facilitated controlled vesicle‐core fusion while reducing large aggregates, ultimately producing particles with improved size uniformity [71, 80]. Moreover, when pre‐sonicated small vesicles passed through extrusion membranes with relatively larger membrane pore sizes, vesicle rupture was minimized, thereby enhancing coating integrity and promoting the formation of more uniform CMNPs [61, 80]. Although combining both sonication and extrusion could result in less aggregation and improved dispersity, introducing ultrasonic stress waves could affect the membrane proteins and glycoproteins on cell membrane vesicles [81, 82, 83]. Therefore, it is important to optimize the amplitude and duration of sonication to preserve the functionality of membrane proteins when sonication is introduced.

The polydispersity of CMNPs is affected by the membrane‐to‐nanoparticle ratio. A representative study reported that a 1:1 membrane‐to‐nanoparticle ratio yielded a low polydispersity index (PDI), producing monodispersed particles [71]. This behavior was attributed to improved membrane coating efficiency, as insufficient membrane coverage exposed the nanoparticle surface to ionic buffers, which promoted interparticle aggregation and led to elevated PDI [68, 84, 85]. Although higher membrane fractions could improve nanoparticle surface masking and reduce aggregation, excessive membrane fraction caused multilamellar membrane coating on the nanoparticles [36]. Current evidence indicates that a 1:1 membrane‐to‐nanoparticle ratio is a commonly adopted and suitable starting point for CMNPs fabrication, and prior discussion supports this ratio as an effective baseline [86, 87, 88, 89, 90, 91, 92]. Although this ratio is broadly validated and widely used, the optimal membrane proportion remains system‐dependent and must be optimized on a case‐by‐case basis, with 1:1 serving as a practical initial reference for method development.

When comparing microfluidic mixing platforms with and without sonication, the inclusion of sonication leads to higher membrane coating efficiency and CMNPs with smaller hydrodynamic size and lower PDI [67]. In microfluidic systems, the mechanism of sonication is attributed to the application of an acoustic pressure field that is sufficiently intense to transiently disrupt lipid membranes, enabling membrane rupture followed by spontaneous resealing around the nanoparticle core.

3. Cell Membrane Coating Integrity

The coating integrity of CMNPs remains an under‐examined yet critical parameter in the synthesis of biomimetic nanoparticles. In many conventional fabrication techniques, such as extrusion and sonication, not all nanoparticles achieve complete and uniform membrane coverage. Inadequate coating may compromise the intended biomimetic functions of CMNPs, especially concerning their interactions with target cells and the subsequent process of internalization [26]. The major strategies for improving coating integrity and the corresponding analytical methods used to evaluate membrane coverage are summarized in Figure 1b,c.

3.1. Strategies for Controlling Cell Membrane Coating Integrity

A recent comparative study investigated the integrity of membrane coatings on CMNPs that were synthesized using different methods, core materials, and membrane sources [26]. Notably, the study revealed that fully coated nanoparticles accounted for only a small fraction of the final product when conventional top‐down methods were used, with complete coating efficiencies of approximately 1.8% for sonication and 6.2% for extrusion. This finding challenges the common assumption that CMNPs obtained by these methods are uniformly encapsulated. Instead, conventional CMNP preparations may contain a heterogeneous mixture of fully coated, partially coated, and uncoated particles. Such heterogeneity is not merely a structural concern but has direct biological consequences, as particles with more complete membrane coverage showed markedly enhanced cellular internalization. In response to this challenge, Liu et al. put forward a novel approach that involves integrating synthetic lipids into the cell membrane fragment, thereby creating a hybrid membrane before applying the nanoparticle coating (Figure 3a1) [61]. This method is based on the premise that increasing membrane fluidity by incorporating external lipid components might facilitate a more thorough encapsulation of the nanoparticle core during the manufacturing process [95]. The synthesis of CMNPs utilizing this hybrid membrane and subsequent extrusion resulted in a significant increase in the percentage of fully coated particles; the percentage of hybrid membrane‐coated nanoparticles with a coating degree greater than 50% was 82.3%, 11‐fold higher than conventional CMNPs (Figure 3a2) [61]. The significant enhancement in coating integrity was associated with improved cellular uptake, which further supports the utility of lipid supplementation in the engineering of CMNPs. These results suggest that coating integrity is a critical, yet often under‐characterized, determinant of CMNPs' function. Many studies infer successful coating from ensemble‐level characterization methods, such as changes in hydrodynamic size, zeta potential, or protein retention, which may not accurately reflect the coating status of individual nanoparticles. Because only a limited proportion of systemically administered nanoparticles reach solid tumors [96, 97], the development of improved biomimetic nanoparticle designs may provide a strategy to enhance tumor targeting and intratumoral uptake. Beyond its role in improving coating integrity, membrane fluidity represents an underexplored design parameter in CMNPs engineering. Current studies suggest that lipid supplementation can enhance membrane coverage and cellular internalization, but the mechanistic contribution of fluidity remains insufficiently defined [61]. Future studies should clarify how membrane fluidity influences coating integrity, membrane orientation, receptor accessibility, and cellular uptake in CMNPs.

FIGURE 3.

Strategies to improve and analyze coating integrity of cell membrane. (a1) Schematic illustration of improving coating integrity using a hybrid membrane. (a2) Cell membrane coating degree distribution of hybrid membrane‐coated nanoparticles calculated from TEM images. Reproduced with permission [61]. Copyright 2022, Springer Nature. (b1) Schematic illustration of improving coating integrity using an electroporation strategy. (b2) Protein concentration of RAW 264.7 cells, conventional cell membrane coating nanoparticles (CMNP) and electroporation‐based membrane‐coating nanoparticles (EMNP). Reproduced with permission [93]. Copyright 2021, Ivyspring International Publisher. (c1) Schematic illustration of analyzing the coating integrity through streptavidin‐biotin aggregation assay. (c2) Size increase caused by streptavidin‐biotin aggregation due to different coating integrity. Reproduced with permission [85]. Copyright 2014, Royal Society of Chemistry. (d1) Red‐shifting in the plasmon resonance peak when gold nanoparticles interacted with membrane‐coated silica nanoparticles. (d2) Linear correlation of plasmonic spectral shift and coating integrity. Reproduced with permission [94]. Copyright 2023, Elsevier. (e1) Schematic illustration of analyzing the coating integrity through the fluorescence quenching method. (e2) Quenching of fluorescence intensity with different coating integrity of nanoparticles. Reproduced with permission [26]. Copyright 2021, Springer Nature.

Coating integrity is also affected by the membrane‐to‐nanoparticle ratio. Increasing the membrane fraction can improve masking of the nanoparticle core from the external environment, limiting direct contact between buffer and the core that would otherwise lead to aggregation. With sufficient coating integrity, the nanoparticle core is shielded from the surrounding ionic buffer, which supports colloidal stability in suspension. Yang et al. demonstrated that under constant agitation, uncoated nanoparticles exhibited significant increases in size variability and PDI, consistent with aggregation; by contrast, increasing the membrane‐to‐nanoparticle ratio suppressed these changes, indicating improved coating integrity and enhanced stability in solution [84, 98]. Luk et al. also demonstrated the relationship between membrane‐to‐nanoparticle ratio and membrane coating completeness using a biotin‐streptavidin aggregation assay [85]. In this method, biotinylated nanoparticles were incubated with streptavidin, and particles lacking full membrane coverage exposed biotin to the streptavidin‐containing environment, inducing aggregation. Increasing the membrane‐to‐nanoparticle ratio suppressed aggregation, indicating improved coating integrity. However, the membrane‐to‐nanoparticle ratio should not be interpreted simply as a higher is better parameter. Although increasing membrane availability can enhance surface coverage, excessive membrane input may also generate free membrane vesicles that are difficult to distinguish from genuinely complete coatings using ensemble techniques such as DLS or zeta potential [98].

Apart from conventional extrusion, a new electroporation strategy was developed to increase coating integrity (Figure 3b1) [93]. Unlike standard protocols that isolate membranes prior to coating, this approach retained membrane material directly from cell culture by first incubating nanoparticles with live cells, followed by magnetic‐field‐assisted extraction. Electroporation was then applied to complete membrane coating onto the nanoparticle core. When comparing both extrusion and novel electroporation methods, the novel methods showed more membrane protein coated onto the CMNPs, indicating increased coating integrity (Figure 3b2).

3.2. Analytical Methods of Coating Integrity

Several methods have been reported for measuring the coating integrity of CMNPs (Figure 1c and Table 1). One strategy utilizes streptavidin‐biotin interactions to examine coating integrity. When nanoparticles are coated with the cell membrane, the membrane shields the biotinylated nanoparticle core, preventing aggregation upon contact with streptavidin solution. Using this aggregation assay, coating integrity can be determined by measuring particle aggregate size, where a larger aggregate size correlates with lower coating integrity. For example, PLGA nanoparticle cores were functionalized with biotin before membrane extrusion (Figure 3c1) [85]. Following the coating process, fully coated PLGA cores masked their biotin tags, whereas partially coated PLGA cores exposed biotin to streptavidin due to incomplete membrane coating. Upon the addition of streptavidin, biotin‐functionalized PLGA cores with exposed surfaces crosslinked to form aggregates, and the measured aggregate size correlated with coating integrity (Figure 3c2).

TABLE 1.

Analytical methods for evaluating coating integrity of cell membrane‐coated nanoparticles (CMNPs).

Methods	Advantages	Limits	Applications	Refs.
Streptavidin‐biotin aggregation assay	Simple and robust; quantitative; sensitive to coating defects; does not require sophisticated instrumentation	Requires biotin functionalization of the core; indirect measurement; aggregation can be influenced by particle concentration and solution conditions	Quantitative assessment of coating completeness; comparison of coating efficiency under different synthesis conditions	[85]
Nanoplasmonic gold nanoparticle (AuNP) reporter assay	Label‐free; rapid; highly sensitive; allows quantitative estimation of coating degree	Requires careful control of AuNP size and surface chemistry; interpretation can be affected by nonspecific adsorption	High‐throughput and quantitative evaluation of membrane coverage; validation of coating uniformity	[94]
Fluorescence quenching	High sensitivity; distinguishes fully coated from partially coated CMNPs; simple fluorescence‐based readout	Requires dye labeling of the core; potential dye leakage; reducing agent compatibility must be validated	Quantification of fully coated CMNP fraction; optimization of coating protocols	[26, 61]

Analogous to the streptavidin‐biotin aggregation assay, a nanoplasmonic method employs gold nanoparticles as reporters. Nanoplasmonic methods rely on optical phenomena that occur in the electron cloud of nanoscale metal systems when they interact with their surrounding environment [99, 100]. When measuring cell membrane coating integrity using gold nanoparticles, plasmon coupling when gold nanoparticles interact with membrane‐coated surfaces, inducing a red‐shift in the plasmon resonance peak. For example, when citrate‐coated gold nanoparticles interacted with membrane‐coated silica nanoparticles, the clustering of gold nanoparticles onto the membrane caused a red‐shift in the plasmon resonance spectrum (Figure 3d1) [94]. A linear correlation was observed between the plasmonic spectral shift and the fraction of the nanoparticle surface covered by the membrane (Figure 3d2). Measuring the absorbance of gold nanoparticles provides a fast, label‐free, and non‐destructive readout of coating integrity. When combined with the analysis of Si and P content using inductively coupled plasma‐atomic emission spectrometry (ICP‐AES), the membrane coating degree of CMNPs can be estimated. Another method is the fluorescence quenching assay, which quantifies the fraction of fully coated CMNPs [26]. This employs a fluorescent dye conjugated to the nanoparticle core surface and a membrane‐impermeable reducing agent (Figure 3e1). Fluorescent dyes exposed on the outer surface are quenched by the reducing agent. Due to defects in the membrane, the reducing agent can penetrate partially coated membranes, leading to fluorescence quenching in partially coated or uncoated CMNPs. This assay was demonstrated using nitro‐2,1,3‐benzoxadiazol‐4‐yl (NBD) as the fluorescent dye and sodium dithionite (DT) as the reducing agent. In this method, membrane‐coated NBD nanoparticles were incubated with DT, and uncoated or partially coated particles lost most of their fluorescence, whereas fully coated particles retained theirs (Figure 3e2).

Collectively, these methods have substantially advanced the quantitative assessment of CMNPs coating integrity by providing sensitive and relatively high‐throughput measurements of membrane coverage. Notably, all three approaches rely on probing the accessibility of the nanoparticle surface through specific chemical or physical interactions, including streptavidin–biotin binding, gold nanoparticle adsorption, and fluorescence quenching. As a result, coating integrity is inferred from the extent to which the membrane coating blocks these interactions rather than being directly measured. This indirect measurement strategy is advantageous for rapid quantification and comparison across formulations, but it provides limited information regarding the spatial distribution, thickness, uniformity, and nanoscale architecture of the membrane shell. Future advances in coating integrity characterization may therefore benefit from integrating these interaction‐based assays with direct structural imaging techniques, such as cryogenic electron microscopy or high‐resolution atomic force microscopy. Such approaches could provide complementary physical evidence of membrane coverage and reveal structural heterogeneities that are difficult to resolve using ensemble biochemical assays alone. Combining functional interaction‐based measurements with direct nanoscale visualization may ultimately provide a more comprehensive understanding of CMNPs coating integrity.

4. Cell Membrane Coating Orientation

The plasma membrane consists of two leaflets: the outer leaflet and the inner leaflet. The outer leaflet contains lipids and proteins that are extracellular facing, while the inner leaflet faces the cytoplasm. Regarding the lipid component, the majority of negatively charged lipid species, including phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), and the phosphorylated derivatives of PI (mono‐, bis‐, and tris‐phosphate PIPs), are localized to the cytoplasmic leaflet [101]. These anionic lipids are known for their signaling functions and their strong preference for the intracellular face of the plasma membrane [102]. Conversely, glycolipid components are positioned within the outer leaflet, consistent with their extracellular orientation and their involvement in recognition and adhesion processes [103]. Zwitterionic phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM) are distributed across both leaflets but in a distinctly asymmetric manner. PC and SM predominantly localize to the outer leaflet, representing roughly 70% of their total abundance, which reflects their enrichment on the outer surface of mammalian membranes. In contrast, PE is concentrated mainly in the inner leaflet, comprising nearly 80% of its total content, consistent with its cytosolic orientation and its role in supporting membrane curvature and intracellular interactions [104, 105].

The orientation of the cell membrane leaflet is critical to CMNPs' functions such as homotypic targeting and immune evasion [106, 107, 108, 109, 110]. Proper orientation ensures that membrane‐bound moieties responsible for biological activity are presented in the desired configuration. A right‐side‐out orientation is essential for cell‐mimicking properties, whereas an inside‐out orientation can be engineered for applications such as ligand capture and drug sequestration [111, 112, 113, 114]. Current strategies for controlling membrane orientation primarily rely on ligand‐based anchoring, in which specific membrane proteins or lipids are selectively coupled to functionalized nanoparticle cores to promote a defined orientation during membrane coating. Representative approaches include streptavidin–biotin binding, protein–peptide interactions, and His‐tag/Ni–NTA coordination. Collectively, these strategies provide a means to regulate membrane leaflet orientation and thereby improve the functional performance of CMNPs (Table 2).

TABLE 2.

Summary of strategies for controlling the membrane orientation of cell membrane‐coated nanoparticles (CMNPs).

Strategy approach	Molecular basis	Representative applications/outcomes	Refs.
Streptavidin‐biotin interaction	Ultra‐high‐affinity noncovalent binding between biotinylated membrane components and streptavidin‐functionalized cores	Controlled inside‐out membrane orientation; manipulation of membrane proteins; correct orientation of tumor‐targeting ligands	[32, 112]
His6‐tag/Ni‐NTA coordination	Metal chelation between His‐tagged proteins and Ni2 +‐NTA‐modified surfaces	Precise protein immobilization; enhanced binding strength; improved surface coverage	[117]
Band 3‐protein 4.2 peptide interaction (RBC membrane)	Specific peptide–protein interaction exploiting native RBC cytoskeletal anchoring	Preservation of RBC membrane topology; enhanced immune evasion; improved cardiovascular targeting	[25, 115]
Azide‐alkyne click chemistry	Selective covalent coupling between bioorthogonal handles introduced into membrane biomolecules and functionalized cores	Site‐specific ligand anchoring; controlled inversion of membrane orientation; receptor‐targeted nanoplatforms	[113]
Phosphatidylserine (PS)‐targeted peptides	Recognition of lipid asymmetry (PS localized to cytoplasmic leaflet)	High‐fidelity orientation; preserved extracellular receptor presentation; improved targeting and therapeutic efficacy	[116, 118]

4.1. Strategies for Controlling Cell Membrane Coating Orientation

Ligand‐based anchoring methods have been developed to achieve oriented cell membrane coating by utilizing high‐affinity interactions between the nanoparticle core and the cell membrane leaflet via ligand anchoring. For example, streptavidin was conjugated to biotin through a well‐established, extremely strong noncovalent interaction, which was adopted to develop an inside‐out‐oriented cell membrane biomimetic magnetic nanoparticle (Figure 4a1) [112]. Bu et al. reported that cell membranes harvested from human lung adenocarcinoma cells were first biotinylated on their cell membrane using sulfosuccinimidobiotin and then incubated with streptavidin‐functionalized Fe₃O₄ magnetic cores [112]. By exploiting the ultra‐high affinity of biotin‐streptavidin, the system forced an “inside‐out” orientation where the cytoplasmic domains normally sequestered inside the cell were displayed externally on the nanoparticle surface (Figure 4a2). In addition to using sulfosuccinimidobiotin, another study employed biotin‐modified DNA probes to enrich right‐side‐out membrane vesicles (Figure 4b1) [32]. Cells were incubated with the DNA probe prior to membrane extraction, ensuring selective labeling of the extracellular leaflet. After membrane isolation, streptavidin‐coated magnetic beads were used to capture right‐side‐out vesicles via biotin‐streptavidin binding. The bound vesicles were subsequently released from the magnetic beads by disrupting the interaction between the DNA probe and streptavidin on the bead surface. Quantitative flow cytometry analysis showed successful capture and subsequent release of right‐side‐out vesicles from magnetic beads, with approximately 46% of labeled vesicles being recovered (Figure 4b2). Streptavidin‐biotin interaction was also demonstrated in the manipulation of membrane proteins [119]. Membrane proteins were manipulated by streptavidin‐coated fluorescent magnetic nanoparticles, which allowed control over the movement of membrane proteins conjugated with biotin‐PEG‐nanobodies. Tumor‐targeting ligands were also correctly orientated using streptavidin‐biotin interactions in red blood cell‐coated drug nanocrystals [120].

FIGURE 4.

Strategies to control orientation of cell membrane. (a1) Schematic illustration of controlling the orientation of biotinylated membrane using streptavidin‐functionalized Fe₃O₄ magnetic cores. (a2) TEM images of inside‐out coated CMNPs (IOCMMNPs), right‐side‐out coated (ROCMMNPs), and bare MNPs with extracellular or intracellular domains of CD47 indicated by immunogold labeling (scale bar = 50 nm). Reproduced with permission [112]. Copyright 2021, American Chemical Society. (b1) Schematic illustration of enrichment of right‐side‐out membrane vesicles using biotin‐modified DNA probes. (b2) Quantitative flow cytometry analysis to reveal the binding and release of right‐side‐out vesicles (RSVs) from magnetic beads. Reproduced with permission [32]. Copyright 2025, American Chemical Society. (c1) Schematic illustration of controlling the orientation of the cell membrane using Band 3 protein and Protein‐4.2‐derived peptide ligand. (c2) T1‐weighted magnetic resonance image of infected mouse model after different treatments. Reproduced with permission [25]. Copyright 2019, American Chemical Society. (d) Quantification of membrane orientation of probe‐RBC‐LVTNPs and probe‐RBC‐PLGA with Förster resonance energy transfer. Reproduced with permission [115]. Copyright 2023, Wiley. (e) Schematic illustration of controlling the orientation using a PS‐targeted peptide. Reproduced with permission [116]. Copyright 2024,. Royal Society of Chemistry.

Another example of the ligand­based approach employed His₆‐tag/Ni‐NTA interactions. The targeted protein was first histidine tagged, allowing the oligo‐histidine tags (His‐tags) to bind divalent metal ions anchored through chelators like nitrilotriacetic acid (NTA). This strategy approach utilizing metal ion chelation, offered a more targeted and less error‐prone method [121]. Ataka et al. attached cytochrome c oxidase to a chemically functionalized gold base layer [117]. The gold base layer allowed cytochrome c oxidase to be correctly attached to the surface via the interaction between the His₆‐tag on the subunit of cytochrome c oxidase and the Nickel NTA modified gold surface. The precise placement of His₆ tags profoundly influenced both binding strength and protein orientation on Ni^{2 +}–NTA surfaces [122]. TagRFP variants with multiple His₆ repeats displayed a tenfold increase in binding strength for each additional tag, and those carrying all His₆ repeats on a single “end” of the β‐barrel achieved 1.5‐fold higher surface coverage compared to constructs with tags on opposite sides.

Xie et al. proposed a “molecular affinity” strategy that uses intracellular domains of transmembrane receptors as anchoring “grippers” to direct membrane assembly (Figure 4c1) [25]. A cytoplasmic Protein‐4.2‐derived peptide ligand (LFVRRGQPFTIILYF), designed to selectively bind the cytoplasmic domain of Band 3, was immobilized on the liposome surface and drove right‐side‐out membrane orientation after membrane isolation and coating. These right‐side‐out‐orientated amphotericin B‐loaded RBC membrane‐coated liposomes (RBC‐LIP‐AmB) showed prolonged circulation, improved macrophage escape, enhanced targeting ability, and superior therapeutic effects compared with randomly oriented RBC‐LIP‐AmB (Figure 4c2). This interaction between Band 3 and Protein 4.2 was further applied to cardiovascular disease [115]. A ROS‐sensitive polymeric drug was synthesized by conjugating lovastatin with oxalyl chloride, which is ROS‐sensitive, and the end of the polymer is further conjugated with the Protein‐4.2 peptide sequence for right‐side‐out orientated coupling with the RBC inner membrane. By using this ligand‐based anchoring, the correct orientation increased from 51.3% to 92.8% (Figure 4d).

Bioorthogonal reactions had also been explored as a ligand‐based anchoring strategy to construct inside‐out cell membrane‐coated nanoparticles. Bioorthogonal chemistry involves reactions that function within biological settings but do not interact with or disrupt biomolecules or normal biochemical activity, typically following a two‐step strategy [123]: (1) a bioorthogonal handle such as an azide group is introduced into cell‐membrane biomolecules by incorporating modified metabolic precursors into native biosynthetic pathways such as glucose, lipid, or protein synthesis, and (2) the labelled biomolecule is selectively reacted with a ligand carrying a complementary functional group such as an alkyne that rapidly and specifically couples to the bioorthogonal handle. In the case of CMNPs, azide is functionalized into the cell membrane by incubating cells with azido sugar during cell culture [124]. The cell membranes are extruded with alkyne‐functionalized nanoparticle cores, where bioorthogonal reactions occur, resulting in CMNPs with inside‐out orientation. For example, HEK293 cells highly expressing VEGFR‐2 were pre‐treated with azido sugar to express azide groups on the cell membrane (N₃‐cell membrane), and the membranes were collected and dispersed with alkyne‐functionalized iron oxide nanoparticles (Fe₃O₄@SiO₂@NH₂@C≡CH), producing inside‐out CMNPs through bioorthogonal coupling [113].

Apart from protein‐ligand anchoring, recent work identified lipid‐ligand anchoring as a distinct strategy to enforce right‐side‐out membrane orientation in CMNPs [116, 118]. This approach leverages the intrinsic lipid asymmetry of biological membranes, in which specific phospholipids are confined predominantly to one leaflet of the bilayer [125]. Among these, phosphatidylserine (PS) is of particular relevance because it resides almost exclusively on the cytoplasmic inner leaflet under physiological conditions [126]. Exploiting this asymmetry, nanoparticles can be engineered to present PS‐targeted peptides to create a specific molecular recognition interface that binds selectively to the PS‐containing inner leaflet. This interaction acts as a molecular anchor that secures the inner leaflet to the nanoparticle surface, steering the membrane toward a configuration in which the extracellular leaflet faces outward. As a result, membrane inversion is disfavored, and the self‐assembly process yields a reproducible, right‐side‐out orientation. Evidence supporting this anchoring‐driven mechanism was demonstrated in recent studies involving ROS‐responsive membrane‐camouflaged nanotherapeutic and theranostic platforms for atherosclerosis [116]. ROS‐sensitive polymeric prodrugs functionalized with PS‐targeted peptides were coated with bioengineered cell membranes overexpressing integrin α9β1 (OEM‐ETBNPs), forming a right‐side‐out ROS‐responsive nanotherapeutic system (Figure 4e). The PS‐peptide interaction ensured that the membrane assembled with high orientation fidelity, preserving the native extracellular domain presentation necessary for targeting foam cells in atherosclerotic plaques. The controlled orientation also improved the therapeutic performance of the CMNPs, allowing the CMNPs to inhibit lipid uptake, promote lipid efflux, and support the phenotypic reprogramming of foam cells. In a direct comparison between PS‐guided right‐side‐out CMNPs (OEM‐ETBNPs) and randomly oriented CMNPs (OEM‐ETBPD), the OEM‐ETBNPs showed a 9.6‐fold reduction of plaque in atherosclerosis model mice, whereas the OEM‐ETBPD showed only a 2.6‐fold reduction. OEM‐ETBNPs also exhibited decreased lipid content within the plaques, resulting in lower inflammatory responses and smaller necrotic areas when compared with OEM‐ETBPD treatment. Liu et al. also employed a similar anchoring strategy to construct a right‐side‐out ROS‐responsive theranostic nanoplatform capable of lipid imaging and rapamycin delivery [118]. In this system, PS‐targeted peptides on the nanoparticle surface ensured selective coupling to the inner leaflet of macrophage membranes during the coating step, resulting in a uniformly right‐side‐out orientation. This precise membrane arrangement was essential for the nanoplatform's diagnostic reliability, as the correct topology preserved the surface markers required for plaque targeting and allowed a ROS‐responsive fluorescent probe to remain quenched in lipid‐poor environments. In lipid‐rich foam cells and atherosclerotic plaques, however, the probe generated a strong fluorescence signal that correlated directly with lipid burden, demonstrating that orientation control is critical not only for membrane functionality but also for accurate biological readouts. Furthermore, the maintained right‐side‐out orientation enhanced drug delivery efficiency, thereby supporting the therapeutic inhibition of plaque progression.

Collectively, ligand‐based anchoring strategies provide an important route to move CMNPs synthesis from stochastic membrane adsorption toward more programmable membrane orientation. These approaches exploit high‐affinity molecular recognition, including biotin–streptavidin binding, His₆/Ni‐NTA coordination, cytoplasmic protein–peptide interactions, bioorthogonal coupling, and lipid‐specific recognition of inner‐leaflet phosphatidylserine, to bias membrane assembly toward either inside‐out or right‐side‐out configurations. Importantly, these studies demonstrate that membrane orientation is not merely a passive consequence of extrusion or sonication, but can be actively engineered through defined molecular interfaces between the nanoparticle core and membrane leaflet.

However, several important questions remain for future investigation. First, most ligand‐based anchoring strategies are demonstrated in specific membrane–core systems, and it remains unclear whether the same anchoring logic can be generalized across different cell membrane sources, nanoparticle materials, sizes, curvatures, and surface chemistries. Second, the strength and density of the anchoring ligand may need careful optimization. Insufficient ligand density may fail to enforce orientation, whereas excessive anchoring could restrict membrane mobility, alter membrane spreading, or affect the lateral organization of membrane proteins. Third, some approaches require chemical modification of the membrane or nanoparticle surface, which may influence native membrane composition, protein conformation, or downstream biological function. Therefore, future studies should not only report improved orientation percentages, but also examine whether the engineered orientation preserves membrane bioactivity, receptor accessibility, and cellular interaction profiles.

A key future direction is to establish quantitative design framework linking ligand type, ligand density, binding affinity, nanoparticle curvature, and membrane source to orientation fidelity. This should be supported by orthogonal characterization methods, such as antibody‐based sidedness assays, fluorescence quenching, protease accessibility, cryo‐electron microscopy, or single‐particle imaging. Such studies would allow ligand‐based anchoring to develop from proof‐of‐concept orientation control into a more generalizable engineering framework for producing CMNPs with predictable topology and reproducible biological performance.

4.2. Analytical Methods of Cell Membrane Coating Orientation

Qualitative and quantitative analytical methods have been developed to evaluate cell membrane coating orientation, including enzymatic assays, antibody‐binding assays, and fluorescence‐based assays (Figure 1c). Each method interrogates a different molecular target such as proteins, glycans, or lipids and therefore provides partially orthogonal information. When used in combination, they allow for a robust evaluation of whether the coating is in the desired orientation.

Enzymatic assays provide a direct, activity‐independent measure of membrane sidedness because they rely on the physical principle that intact lipid bilayers are impermeable to enzymes; thus, only protein domains exposed on the external leaflet are accessible to cleavage. Typical enzymatic workflows involve incubating CMNPs with a membrane‐impermeable protease or glycosidase, pelleting the particles by ultracentrifugation, and assaying the supernatant for released fragments. Since intact RBC membranes are impermeable to trypsin, only proteins on the outer face of the cell membrane coating are released. For example, when RBC membrane‐coated nanoparticles were cleaved using trypsin followed by ultracentrifugation, glycoproteins were released into the supernatant and further analyzed by a glycoprotein quantification assay (Figure 5a1) [85]. The trypsinized RBC ghosts and RBC‐NPs yielded approximately 95% of the glycoprotein content observed from trypsinized RBC ghosts (Figure 5a2), and RBC ghosts and RBC‐NPs also showed closely matched sialic acid amounts after sialidase treatment (Figure 5a3), indicating that the vast majority of glycoproteins were presented on the particle exterior and supporting a right‐side‐out configuration. Similarly, surface glycans such as sialic acid were used as orientation reporters. A sialidase enzyme cleaved terminal sialic groups from exposed glycans without disrupting the lipid bilayer or the size of CMNPs. Bu et al. incubated the membrane‐coated magnetic nanoparticles with sialidase and then measured the released sialic acid in the supernatant [112]. The amount of sialic acid liberated from the particle surface was almost identical to that of native cell membranes, again confirming that the majority of the CMNPs were right‐side‐out oriented.

FIGURE 5.

Analytical methods to validate cell membrane coating orientation. (a1) Schematic illustration of validating cell membrane orientation using an enzymatic assay. (a2) Comparison of the relative glycoprotein content and (a3) sialic acid content recovered from equivalent amounts of RBC ghosts, RBC‐NPs, and bare NPs after trypsinization and sialidase treatment. Reproduced with permission [85]. Copyright 2014, Royal Society of Chemistry. (b) TEM images of RBC‐NP and bare‐NP under negative staining with uranyl acetate or under immunostaining with anti‐CD47 antibodies that target either the extracellular or the intracellular protein domains (scale bar: 30 nm). Reproduced with permission [36]. Copyright 2013, Royal Society of Chemistry. (c) Western blotting analysis of band 3 and CD47 in RBC ghost, treated RBCm, and RBC‐LIP. Reproduced with permission [25]. Copyright 2019, American Chemical Society. (d) Comparison of the fluorescence intensity measured from TNPs or T cells containing equal amounts of membrane content and stained with fluorescein‐isothiocyanate‐labeled CCR5 antibodies. Reproduced with permission [127]. Copyright 2018, Wiley. (e1) Flow cytometry of PS distribution in M‐TPCR6G using the probe Annexin V‐EGFP. (e2) Emission spectra of probe‐M‐TPCR and probe‐eM‐TPR in PBS before and after adding ssDNA quencher. Reproduced with permission [118] Copyright 2019, Elsevier.

Antibody‐binding assays offer direct visualization or quantitative detection of epitope accessibility and are therefore widely used to correlate with enzymatic findings. Two primary approaches of antibody‐binding assays include immunogold labeling combined with transmission electron microscopy (TEM) and antibody probing in solution with fluorescent or enzymatic reporters followed by flow cytometry or spectrophotometry. In the immunogold approach, CMNPs are adsorbed onto glow‐discharged carbon‐coated EM grids, blocked with bovine serum albumin, and incubated sequentially with antibodies that selectively recognize either extracellular or intracellular epitopes of a transmembrane marker. In most cases, CD47 was frequently probed because it contains well‐characterized extracellular and intracellular domains [25, 36, 112]. Following incubation with a gold‐conjugated secondary antibody and negative staining, TEM reveals the topological distribution of electron‐dense gold particles. The dense labeling with an extracellular‐domain antibody accompanied by minimal labeling with an intracellular‐domain antibody is interpreted as right‐side‐out coating, whereas the reverse pattern indicates inside‐out orientation. For example, when CMNPs derived from RBC were incubated with immunogold staining, abundant gold labeling was observed with anti‐CD47 extracellular antibodies on right‐side‐out CMNPs but not with anti‐CD47 intracellular antibodies, while deliberately inside‐out coated particles showed the opposite pattern (Figure 5b) [36, 112]. A complementary immunoblotting assay such as Western blotting or dot‐blot analysis of isolated CMNPs protein profiles can corroborate TEM findings. By comparing CMNPs and their source ghosts, this assay confirmed the presence of expected membrane proteins, indicating a comparable degree of right‐side‐out orientation (Figure 5c) [25, 127]. Wei et al. coated T‐cell membranes onto polymeric cores and subsequently probed intact particles with FITC‐labelled anti‐CCR5 antibodies (Figure 5d) [127]. The amount of antibody bound (or remaining unbound) can be quantified by flow cytometry to measure antigen exposure.

Fluorescence‐based assays encompass a broad range of methods that probe either lipid composition or functional presentation of surface molecules, and they are particularly useful for quantitative high‐throughput assessment. Annexin V‐PS binding was adopted to probe phosphatidylserine, which resides on the cytoplasmic leaflet. For instance, Annexin V‐Cy5 dye was used as a probe to validate the right‐side‐out orientation of a bioengineered cell membrane‐coated nanoparticle (OEM‐ETBNPs). The fluorescence signal was rarely observed under normal conditions, confirming the general distribution of PS on the inner leaflet of the cell membrane (which faces the core in right‐side‐out particles). However, under stress conditions, PS translocation was detected through the Annexin V‐Cy5 fluorescent probe [116]. Another fluorescent probe, Annexin V‐EGFP dye, was reported to validate right‐side‐out orientation by incubating with CMNPs and measuring via flow cytometry (Figure 5e1) [118].

Fan et al. developed a fluorescence‐based assay using an single‐stranded DNA (ssDNA) probe and ssDNA quencher [128]. In this approach, cell membranes are initially incubated with a fluorescently labeled ssDNA probe to label the outer leaflet. After membrane extrusion, a complementary ssDNA quencher is introduced. If the membrane is right‐side‐out, the probe is exposed on the surface and accessible to the quencher; if the membrane is inside‐out (or scrambled), the probe may be shielded inside the particle or inaccessible [115, 128]. In the representative study reported by Liu et al., intact cells were first incubated with a fluorescent ssDNA probe prior to membrane extraction, ensuring that only the extracellular leaflet became fluorescently labeled (Figure 5e1). After membrane collection, the vesicles were either fused with a PS‐targeted‐peptide‐functionalized core or assembled by standard extrusion. Comparison of fluorescence quenching demonstrated that the PS‐functionalized core promoted a higher proportion of outward‐facing membrane leaflets relative to extrusion alone (Figure 5e2) [118].

There are several key considerations when evaluating orientation. First, partial coating or incomplete coverage can yield mixed signals. Incomplete membrane coverage on the nanoparticle core may allow the fluorescent probe to penetrate exposed gaps at the membrane core interface and bind to membrane leaflets, which can distort the resulting fluorescence readout [26]. Second, enzymatic treatments must be carefully controlled to prevent membrane permeabilization or excessive proteolysis, as over‐digestion can induce membrane disruption and unintended release of membrane proteins [129, 130, 131]. Third, antibody accessibility can be affected by conformational masking or by tight packing of proteins and glycans [131, 132, 133]. Hence, a negative antibody binding result does not necessarily confirm inside‐out orientation, only antibody inaccessibility under the tested conditions. Finally, heterogeneous molecular compositions of cell membranes across different cell types, such as variable PS content and glycosylation patterns, indicate that a single assay may not be universally applicable [134, 135, 136].

Determining CMNPs orientation requires a multi‐approach strategy that combines enzymatic, immunological, and fluorescence‐based evidence, in which (1) enzymatic assays offer quantitative activity‐independent readouts of exposed glycans and proteins, (2) antibody‐binding provides high‐resolution topological mapping and epitope‐specific verification, and (3) fluorescence techniques deliver scalable, high‐throughput measurements and functional correlation.

5. Conclusion and Future Perspectives

This review outlines the current landscape of CMNPs, emphasizing how synthetic parameters, coating integrity, and membrane orientation collectively determine their physicochemical properties and biological performance. Major fabrication strategies are summarized alongside the influence of key parameters such as extrusion, sonication, microfluidic methods, and membrane‐to‐core ratios. Strategies for achieving controlled membrane orientation through ligand‐based anchoring or modulation of surface physicochemical environments are also discussed, together with methods aimed at improving coating integrity. In addition, analytical approaches for evaluating membrane orientation and coating completeness are reviewed.

Although a large body of application‐driven studies has demonstrated the therapeutic promise of CMNPs, several key questions remain insufficiently addressed. Future research should move beyond proof‐of‐concept efficacy studies and place greater emphasis on long‐term immunogenicity, fundamental mechanistic understanding, and the development of patient‐derived CMNP platforms.

Preclinical studies have generally reported favorable biocompatibility of CMNPs. For example, systematic in vivo toxicity assessments in mice, including studies in pregnant mice, fetuses, and juvenile offspring, have shown no obvious toxicity based on histological and hematological analyses after short‐term or repeated administration [84, 137, 138]. However, most existing studies remain limited to relatively short observation windows, such as 24 h or 21 days after injection. To support clinical translation, longer‐term safety studies are required, particularly repeated‐dose studies over several months to evaluate chronic toxicity, delayed immune responses, organ accumulation, and potential effects at the genetic or developmental level [139, 140]. In addition, safety evaluation in non‐rodent species would provide a more robust toxicological profile and better inform the translational potential of CMNPs‐based therapeutics.

A second important direction is the need for deeper mechanistic investigation. Despite rapid progress in CMNPs applications, the fundamental understanding of biological processes governing CMNPs behavior remain incomplete. For example, different studies have proposed distinct uptake mechanisms, including clathrin‐dependent endocytosis and membrane‐fusion‐mediated uptake [26, 141, 142]. These pathways have substantially different implications for intracellular trafficking, endosomal escape, core release and drug bioavailability. Therefore, systematic studies are needed to define how membrane source, coating integrity, membrane orientation, core physicochemical properties and surface functionalization collectively determine CMNPs uptake and intracellular fate. Establishing these structure–function relationships will be essential for the rational design of CMNPs with predictable therapeutic behavior. A further direction is to extend current design principles from solid‐core CMNPs to lipid‐based nanoparticle systems. Most mechanistic studies on coating integrity and membrane orientation have focused on solid‐core nanoparticles, which provide well‐defined substrates for evaluating membrane coverage, core shielding, and leaflet sidedness. However, lipid‐based nanoparticles present a more dynamic and compositionally similar interface to the cell membrane coating. Comparative studies between solid‐core and lipid‐based CMNPs could therefore provide valuable insight into how core rigidity, lipid miscibility, membrane fusion and interfacial fluidity influence coating completeness and membrane orientation. Such studies may clarify whether the design rules established using solid nanoparticles are directly transferable to lipid‐based systems, or whether distinct engineering principles are required for different core architectures. This would broaden the applicability of CMNPs engineering and provide a more comprehensive framework for designing biomimetic nanoplatforms across both solid and lipid‐based systems.

The evolution of CMNPs design reflects a broader transition from simple single‐membrane camouflage toward increasingly programmable biointerfaces. The field has expanded to include hybrid membrane coatings, lipid‐insertion strategies, and genetically engineered source cells. Lipid insertion enables the post‐assembly incorporation of targeting ligands, imaging probes or therapeutic motifs into the membrane shell, whereas genetic engineering allows source cells to express defined functional proteins before membrane extraction. In parallel, the rapid development of genetically engineered cellular nanovesicles has further demonstrated how membrane‐derived systems can be programmed to display immune checkpoints, cytokines, tumor antigens or receptor‐targeting proteins for cancer immunotherapy [143, 144]. These approaches move CMNPs beyond passive biomimicry, positioning the membrane coating as an engineerable biological interface capable of mediating active targeting, immune regulation and disease‐specific therapeutic responses.

Looking forward, patient‐derived and autologous membrane platforms may represent an important next stage in CMNPs development. Membranes derived from patient immune cells, engineered CAR‐T cells, tumor cells, or autologous blood cells could be used to generate personalized CMNPs with improved immune compatibility and patient‐specific biological functionality [24, 145, 146]. Recent perspectives on genetically engineered cellular nanovesicles further support this direction, highlighting how artificial cell membrane nanovesicles and extracellular vesicle‐inspired systems can be rationally designed through cell‐source selection, genetic programming, and membrane functionalization [144]. Such strategies may reduce immunogenicity while enabling individualized nanomedicine designs tailored to patient‐specific tumor antigens, immune status or disease microenvironments. Nevertheless, their translational value will depend on whether these personalized membrane platforms can overcome major challenges in source‐cell variability, manufacturing scalability, quality control and regulatory definition.

Notably, the clinical translation of CMNPs is beginning to progress. Some CMNP formulations have entered clinical trials or advanced stages of clinical development. For example, Cello Therapeutics has reported US FDA IND clearance for its platelet‐membrane‐coated CMNPs platform, CE120, for first‐in‐human immunotherapy studies [147]. These developments indicate that CMNPs are moving beyond experimental nanomedicine toward clinically relevant platforms. Nevertheless, successful translation will require rigorous long‐term safety validation, standardized characterization of coating integrity and membrane orientation, scalable manufacturing, and a clearer mechanistic understanding of how CMNPs' structure governs biological function.

In conclusion, CMNPs represent a highly adaptable and biologically inspired strategy for addressing persistent challenges in nanomedicine. Ongoing advances in controlled cell membrane coating technologies, coupled with emerging data‐driven approaches, are poised to unlock the full therapeutic and diagnostic potential of this next‐generation platform and to advance more precise, targeted, and biocompatible nanobiotechnologies.

Funding

This work was supported by the ARC Discovery Projects (DP240100770), NHMRC Ideas Grants (APP2038497), and Tour de Cure Pioneering Research Grant (RSP‐124‐2024)

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 analyzed during the current study