Hybrid Cell Membrane‐Coated Nanoparticles for Biomedical Applications

There is growing interest in developing cell membrane‐coated nanoparticles (CNPs) for unique host cell mimicry and therapeutic applications. The continuous evolution of this technology has motivated the coating of nanoparticles with hybrid membranes originating from diverse cell types. The resulting hybrid cell membrane‐coated nanoparticles (hybrid CNPs) exhibit a higher level of synergy among multifunctionalities with better multitasking capabilities than their monotypic membrane‐coated counterparts. This advancement has catalyzed the initiation of numerous research opportunities, marking the advent of a promising frontier in therapeutic applications. This review outlines emerging biomedical applications of hybrid CNPs, focusing on drug targeting, immune modulation, biological neutralization, and disease diagnosis. Within each application, the review underscores how the strategic hybridization of distinct cell membranes augments the resulting nanoparticle therapeutic efficacy. Overall, the insights presented herein consolidate our understanding of current applications and may inspire novel designs with new biomedical applications.


Introduction
[3][4] Among the various NP platforms, cell membrane-coated NPs, fabricated by coating synthetic NP cores with natural cell membranes, have gained substantial attention. [5,6]] Compelled by their unique biomimicry, we have coined the term "cellular NPs" or "CNPs" to describe them.
For instance, some CNPs inherit "selfmarkers" from their source cell membrane, allowing them to evade immune clearance and circulate longer in the body. [10,11][14] Such binding capability also enables them to scavenge harmful molecules or pathogens to protect source cells without prior knowledge of the threat, offering a function-driven and broadspectrum biological neutralization strategy. [6,7,15]Some CNPs present whole-cell antigens mirroring parent cell membrane antigen profile but with transport and targeting advantages of sub-100 nm NPs, or they detain bacterial toxins, limiting their harm while preserving their structural integrity. [6,16,17]s a result, they become valuable tools for vaccine development, eliciting highly effective protective immunity.
While cell membrane coating effectively enhances NP functionality, additional features are often needed to enhance their efficacy in specific applications.For example, while cell membrane coating provides excellent immune evasion, incorporating targeting ligands can further improve precision in homing in on specific targets such as tumors. [18]Additionally, the homing ability acquired from different source cells may facilitate CNPs to overcome multiple barriers and reach the target site more efficiently.Furthermore, despite a faithful antigen presentation for immune processing, additional abilities to amplify the immune activation are desirable to modulate immunity for adequate protection.These scenarios suggest that the integration of functionalities beyond the innate characteristics of a cell membrane would significantly expand the range of applications for CNPs.
One strategy to enhance CNP functionality is to coat them with fused membranes from cells of different origins, thereby integrating distinct cellular functions into a single NP entity (Figure 1). [19]In this perspective, cell membrane fusion can be achieved by allowing living cells to fuse first, then collecting the membrane from the cell hybrids and coating it onto the NP cores.Fusogenic conditions such as polymer-induced cell agglutination or electrofusion have been extensively studied to create hybrid cells. [20,21]Alternatively, cell membranes from different cells can be collected and fused through sonication or extrusion, and then the hybrid membrane (HM) is coated onto the NP substrates. [19]Both methods have been shown to retain the functional characteristics of each cell type on the subsequent CNPs.Since its initial development, this unique type of CNP, known as "hybrid CNPs," has demonstrated compelling properties and therapeutic capabilities.Herein, this article provides a comprehensive review of the major applications of hybrid CNPs, including drug targeting, immune modulation, biological neutralization, and disease diagnosis.In each application, we highlight how functions of cell membranes from distinct cell types cooperate to enhance CNP biointerfacing capabilities and, therefore, accomplish unique biomedical tasks that are otherwise unattainable by CNPs with a single membrane type.

Hybrid CNPs for Targeted Drug Delivery
The RBC membrane harbors crucial immunomodulatory markers, including CD47, a potent "do not eat me" receptor that inhibits phagocytosis by ligation with CD172a on the macrophages, and various complement regulatory proteins, guarding against membrane damage for stealth and long circulation. [22][25][26] Meanwhile, some CNPs recognize and bind to specific tissues or cells through ligand-receptor interactions.Platelets (PLTs), in this regard, stand out as an ideal source of the membrane due to their unique presentation of membrane-binding antigens, including glycoprotein (GP) Ib, P-selectin, and GP IIb/IIIa. [14]PLT membrane-coated NPs (PNPs) inherit these ligands and have demonstrated efficient drug-targeting abilities to thrombosis, subendothelium sites, tumors, and bacteria. [13,27]he distinctive properties inherent in RBC and PLT membranes have inspired researchers to hybridize them for coating, effectively harnessing immune evasion and active targeting to improve therapeutic effectiveness.One area where the RBC-PLT hybrid CNPs have received much attention is in the treatment of cardiovascular diseases.In one study, RBC-PLT hybrid CNPs coated onto poly(lactic-co-glycolic acid) (PLGA) NP cores targeted atherosclerosis effectively due to combined immune evasion and active binding with P-selectin glycoprotein ligand-1 (PSGL-1). [19]In another study, similar RBC-PLT hybrid CNPs were applied to regenerating vasculature in ischemic tissues.The synergistic effect of the RBC membrane's stealth properties to evade the immune clearance and the PLT membrane's binding capabilities with the exposed subendothelial extracellular matrix (ECM) enabled efficient targeting of angiogenic drugs to ischemic injury sites. [26]BC-PLT hybrid CNPs have also been extensively studied for cancer treatment.In one study, the RBC-PLT HM was coated onto NP cores responsive to near-infrared irradiation (NIR) to target tumors via fibrin-GP receptor binding. [23]The immune stealth from the RBC membrane further amplified the targeting efficiency, effectively inhibiting tumors through heat-induced vessel injury and microthrombosis.In another study, chlorin e6 (Ce6), a sonosensitizer, and tirapazamine (TPZ), a hypoxiasensitive cytotoxic drug, were encapsulated into a pH-sensitive liposome.The liposome was coated with an RBC-PLT HM for targeted delivery. [28]In a mouse model of lung metastasis melanoma, these hybrid CNPs showed enhanced tumor accumulation due to the immune stealth and specific targeting by the HM coating.Upon exposure to the local ultrasound (US), Ce6 generated toxic reactive oxygen species (ROS).The resulting hypoxia microenvironment activated TPZ, creating a synergistic tumor inhibition.
The targeting ability of PLT membrane has also motivated its hybridization with membranes of other cell types for NP coating.For instance, to promote cardiomyocyte regeneration in myocardial ischemia-reperfusion (IR) injury, the PLT membrane was hybridized with the macrophage membrane and coated onto a pH-responsive NP core carrying Sav1 siRNA (Figure 2). [29]he hybrid design facilitated targeted delivery, with the macrophage membrane binding to chemokine receptor 2 (CCR2) to target inflammation and the PLT membrane binding to the ECM of the injured tissue to target microthrombi.Specifically, in IR-injured pigs, the collagen deposition (blue area) level was reduced to 30.3% after hybrid CNP treatment, significantly lower than that after phosphat-buffered saline (PBS) treatment (61.9%).The hybrid CNPs significantly decreased the infarct size from 20.9% to 12.0%.The reduced ejection fraction (EF) value after IR injury was largely recovered on day 7 after the hybrid CNP treatment.
In cancer treatment, the hybridization of the PLT membrane with the neutrophil membrane led to hybrid CNPs effective in inhibiting breast cancer metastasis.The PLT membrane's P-selectin binding with PSGL-1 or CD44 on primary tumor cells, in combination with the neutrophil membrane's β-integrin binding with adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) on circulating tumor cells (CTCs), contributed to the effective inhibition of breast cancer metastasis. [30,31]n another study, the hybridization of the PLT membrane with the cancer stem cell (CSC) membrane was utilized for enhanced photothermal therapy against head and neck squamous cell carcinoma (HNSCC).Here, the immune-evasive markers of the PLT membrane and the homotypic targeting ability from the CSC membrane mediated by cell surface cadherins combined to improve the efficacy of the photothermal therapy. [32]o address inflammatory disorders, researchers hybridized the membrane of PLT-derived extracellular vesicles (PEV) with the membrane of calreticulin (CRT)-expressing L929 cells and coated the HM onto PLGA cores.In this hybrid design, P-selectin on PEVs anchored the NPs onto activated neutrophils, while calreticulin mimics exogenous "aged" signal secreted by macrophages to trigger programmed neutrophil removal.PEVs target activated neutrophils through the interaction between PEV surface P-selectin with P-selectin glycol protein ligand-1(PSGL-1) on Figure 2. The development of PLT-macrophage hybrid CNPs for cardiomyocyte regeneration in myocardial IR injury.A) A schematic illustration of synthesizing hybrid CNPs coated with PLT-macrophage HM for delivering myocardial Sav1 siRNA (siSav1) and managing IR injury.B) Ex vivo imaging of rat hearts at 6 h after i.v.injection of Cy3-siSav1-containing hybrid CNPs.C) Relative mRNA levels of genes related to cell regeneration (Areg and Ctgf ) and apoptosis (Bim and Pten) in the ischemic myocardium in IR-injured rats.D) Calculated percentage of Ki67þ cardiomyocytes, apoptotic level, fibrotic area, infarct size, and EF of the left ventricle in IR-injured pigs.Reproduced with permission. [29]Copyright 2023, John Wiley and Sons. the activated neutrophils. [33]By targeting activated neutrophils and misled macrophages to recognize them as "aged" for removal, these NPs inhibited proinflammatory response and tissue damage in a mouse model of acute lung injury and severe acute pancreatitis. [34]mong various CNPs, cancer cell membrane-coated NPs (CCNPs) have been developed for cancer targeting due to homotypic recognition capabilities. [35]However, the nonhomologous nature of the cancer cell membrane due to its recognition by the body's own antitumor immunity results in a significant portion of CCNPs being recognized as "foreign" and subsequently cleared by the immune system, leading to inefficient tumor targeting. [36,37]To overcome this challenge, researchers have hybridized the cancer cell membrane with the RBC membrane to enhance the overall NP stealth abilities for better tumor targeting.For example, the membrane of B16-F10 cells, a melanoma cell line, was hybridized with RBC membrane and coated onto doxorubicin (DOX)-loaded hollow copper sulfide NPs against melanoma (Figure 3). [38]These NPs demonstrated about 7-9-fold higher mean fluorescence intensity (MFI) inside B16-F10 cells than that of other cells, indicating highly specific self-recognition to the source cell line in vitro.The hybrid CNP formulation exhibited a blood retention of 20.2% ID/g, comparable to that of RBC membrane-coated control CNPs but significantly longer than the cancer membrane-coated (14.5% ID/g) or uncoated controls (5.2%ID/g).For the DOX-loaded hybrid CNPs, the tumor growth inhibition (TGI) rate was around 14% without the NIR irradiation but reached nearly 100% with NIR irradiation.Control groups, including the DOX group (with a TGI rate of around 10%), NIR-irradiation-only group (with a TGI rate of around 3%), or the DOX-free hybrid CNPs under the NIR irradiation group (with a TGI rate of around 86%) were less effective.Following a similar approach, the membrane of MCF-7 cells, a breast cancer cell line, was hybridized with RBC membrane and ), 5: DCuS@[RBC-B16], 6: CuS@[RBC-B16] with NIR laser (1064 nm, 1 W cm À2 ), 7: DCuS@[RBC-B16] with NIR laser (1064 nm, 1 W cm 2 ).Reproduced with permission. [38]Copyright 2018, American Chemical Society.coated onto DOX-loaded gold nanocages. [36,39]The effective homotypic targeting facilitated by the cancer cell membrane and the immune evasion capability conferred by the RBC membrane led to highly efficient NP targeting and effective tumor inhibition.
The cancer cell membrane was also hybridized with the macrophage membrane for coating, where the macrophage membrane provides NPs with enhanced immune stealth due to less altered self-molecules. [28,40,41]Intriguingly, such hybrid CNPs allow for dual targeting, where the cancer cell membrane targets the primary tumor through homotypic recognition, and the macrophage membrane targets the metastatic cancer cells via interactions between the α4 integrins of the macrophage and the VCAM-1 of cancer cells.For example, breast cancer (4T1) cell membrane was hybridized with macrophage (RAW) membrane and coated onto liposomes loaded with emtansine, a cytotoxic anticancer drug.The 4T1 membrane targeted the primary breast cancer via homotypic recognition, while the RAW cell membrane adhered to the metastatic cancer cells through α4 integrin-VCAM-1 interaction.By targeting both primary and metastatic cells, the hybrid CNPs effectively inhibited lung metastasis of breast cancer. [42,43]esides the earlier examples, researchers also explored other cell membranes to make hybrid NPs for drug targeting.For example, the retinal endotheliocyte membrane was hybridized with the RBC membrane and coated onto PLGA cores. [44]The retinal endotheliocyte membrane allowed for homotypic targeting of the NPs to the retinal endotheliocytes due to homologous adhesion domains of membrane cadherins. [45]The retinal endotheliocyte membrane component also allowed NPs to serve as an antiangiogenic agent by neutralizing vascular endothelial growth factors.Meanwhile, the RBC membrane protected the hybrid NPs from phagocytosis by macrophages.With these mechanisms in place, the NPs targeted the choroidal neovascularization (CNV) region in a laser-induced wet age-related macular degeneration mouse model and significantly reduced the leakage and area of CNV.In another study, the glioblastoma cancer cell membrane was hybridized with the mitochondria membrane and coated onto NPs loaded with Gboxin, a glioblastoma inhibitor. [46]n this hybrid design, the glioblastoma membrane facilitated NP permeation across the blood-brain barrier (BBB) by binding with tight-junction proteins such as zonula occludens-1, claudin-5, and occludin.After crossing the BBB, the NPs were internalized by glioblastoma cells.Inside the cells, the mitochondria membrane allowed the NPs to target mitochondria through homotypic targeting.Such dual targeting to glioblastoma cells and to their mitochondria led to effective tumor inhibition in a glioblastoma mouse model.
So far, various hybrid CNPs have been developed for drugtargeting applications.In this context, hybrid CNPs enhance drug delivery efficacy by integrating immune stealth and active targeting, thereby increasing the likelihood of specific receptorligand interactions.Additionally, certain hybrid CNPs achieve this objective through dual targeting, either concurrently or sequentially, facilitated by membrane hybridization.These instances highlight the synergistic interplay between the two membrane components, potentially inspiring novel designs for more efficient drug-targeting approaches.

Hybrid CNPs for Immune Modulation
Researchers have explored CNPs as vaccines for immunotherapy.Among them, CCNPs have garnered significant attention due to several distinct advantages.For example, CCNPs mimic the host cancer cell membrane for immune presentation.This precise resemblance in the antigen profile likely promotes a more meaningful immune activation. [47]Additionally, through homotypic recognition, CCNPs targeted antigens to specific cancer cells, boosting the vaccine potency. [48]Furthermore, the encapsulation capacity of the inner cores allows for the simultaneous presentation of tumor antigens and adjuvants, potentially bolstering anticancer immunity.
While CCNPs have demonstrated significant potential in developing anticancer vaccines, researchers are advancing this platform by fusing cancer cell membranes with membranes from other cell types to incorporate additional functionalities.One such approach involves the hybridization of the cancer cell membrane with the dendritic cell (DC) membrane.This hybridization preserves the cancer cell membrane's ability for homotypic targeting, while facilitating the concurrent expression of tumor antigens and immunological costimulatory molecules, leading to DC-mediated simultaneous activation of polyclonal antigen-specific CD4 þ and CD8 þ T cells. [49,50]For instance, the fusion of murine mammary carcinoma (4T1) cells and bone marrow-derived DCs resulted in the formation of an HM, which was subsequently coated onto a PCN-224 metal-organic framework (MOF) core (Figure 4). [51]These hybrid CNPs are smaller than fusion cells, showing enhanced capacity to penetrate lymphoid organs.When incubated with T lymphocytes, the hybrid CNPs demonstrated superior T cell activation compared to NPs coated with either cancer cell membrane or DC membranes alone.In vivo, these CNPs were retained better than CNPs coated with a single membrane.The images of tumors indicate that the hybrid CNPs effectively prevented tumor occurrence.The levels of IFN-γ and IL-6 in mice treated with the hybrid CNPs increased by about 27 and 3 times, respectively, compared with the PBS group.Besides, the hybrid CNPs completely inhibited tumor growth, while groups treated with NPs coated with single-cell membranes developed tumors.The vaccination with the hybrid CNPs led to a significant increment of CD3 þ CD8 þ CTLs compared with PBS control.The corresponding percentage of CTLs in the hybrid CNP group reached 14.45%, which was higher than that of groups treated with PBS (8.47%), MOF NPs, (9.53%), MOF coated with cancer cell membrane (9.69%),and MOF coated with DC membrane (11.02%).
In a separate study, DCs were fused with mouse colon adenocarcinoma MC38 cells or mouse glioma GL261 cells, and the resulting HM was coated onto PLGA cores. [52]The MC38-DC hybrid CNPs efficiently penetrated lymphoid organs and activated T cells to eradicate tumors, providing protection benefits in an MC38 tumor engraftment model.Additionally, vaccination with GL261-DC hybrid CNPs delayed the engraftment of GL261 gliomas and significantly prolonged the survival of tumorbearing mice.Notably, recent research has incorporated TCPP porphyrin, a photosensitizing agent, into 4T1-DC hybrid CNPs, and the addition of photothermal therapy further enhanced the anticancer efficacy of these NPs. [51]esearchers have also explored the hybridization of the cancer cell membrane with bacterial outer membrane (OM) as a strategy to bolster antitumor efficacy (Figure 5). [53]This approach leverages the ability of the cancer cell membrane component to present tumor-specific antigens on the NP surface, directing the NP toward the tumor site.Simultaneously, the OMs exhibit bacterial antigens, serving as robust immune adjuvants to stimulate effective immune activation.For instance, the fusion of the membrane from B16-F10 melanoma cells with Salmonella bacterial OMs, achieved through extrusion techniques, led to the development of an HM.This HM was then coated onto PLGA cores containing indocyanine green, a photothermal agent.Notably, these NPs exhibited superior DC maturation compared to NPs coated with either B16-F10 cancer cell membrane or Salmonella bacterial OMs alone.Moreover, they induced more robust T cell activation than NPs solely coated with the cancer cell membrane, owing to the presence of OM components.When immunized mice were challenged with B16-F10 tumor, vaccination with monomembrane-coated CNPs showed negligible tumor inhibition.However, vaccination with hybrid CNPs suppressed 78.57% B16-F10 tumor growth and prolonged the tumor-free time (19 days after tumor challenge) compared with 15 days in the other groups.Furthermore, the hybrid CNPs prompted the generation of tumor-specific T lymphocytes, which OM-coated NPs failed to accomplish due to the absence of tumor-associated antigens.In a subcutaneous melanoma treatment model, combining these hybrid CNPs with photothermal tumor ablation led to enhanced tumor necrosis and improved mice survival compared to NPs coated with individual membranes, emphasizing the synergistic effects derived from the hybridized membrane coating.
The hybridization of cancer cell membrane with macrophage membrane has also garnered attention for its potential to activate antibacterial immunity.Through this hybridization, the macrophage membrane enables the NP to migrate to infection sites, facilitated by the recruitment of macrophages via damageassociated molecular patterns. [54]Simultaneously, the cancer cell membrane acts as a secondary adjuvant, enhancing the immunostimulatory response.In a particular study, the membrane from murine macrophage cell RAW264.7 was hybridized with the membrane of 4T1 carcinoma cells. [55]The resulting HM was coated onto a hollow manganese oxide (MnOx) NP that Reproduced with permission. [51]Copyright 2019, Springer Nature.encapsulated a sonosensitizer, protoporphyrin IX.Upon intravenous injection into injured mice, the hybrid CNPs exhibited strong fluorescence at the infection site, indicating effective targeting.In contrast, uncoated NPs were predominantly found in the kidneys and liver due to the lack of homing ability.The hybrid CNPs, combined with ROS generated by sonodynamic therapy, demonstrated more efficient bacterial eradication compared to uncoated MnOx particles.Such heightened bacterial killing led to an increased release of bacterial antigens, triggering a potent downstream antibacterial immune response.Overall, using hybrid CNPs proved more effective in protecting mice against osteomyelitis than uncoated MnOx particles.
Overall, investigation of the cancer immunotherapy potential of CNPs has led to an increased interest in utilizing hybrid CNPs to combine the antitumor benefits of membranes from different cells, including DCs and bacteria.These hybrid CNPs withhold the immune characteristics of the original cell membranes but outperform their counterparts coated with single-cell membrane under various experimental settings.We anticipate that their applications will broaden to contribute to the treatment of other diseases.

Hybrid CNPs for Biological Neutralization
Biological neutralization is a fundamental approach that employs therapeutic agents to bind antagonistically with harmful molecules or infectious pathogens, effectively impeding their bioactivity and preventing the onset of diseases. [7]In contrast to traditional neutralization methods, CNPs set themselves apart by replicating the functional characteristics of susceptible host cells rather than adapting to the structural attributes of the causative agents for binding and neutralization.This host function-driven approach enables CNPs to circumvent the structural diversity of the targeted agents, homing in on a select few host cell types.This unique approach significantly streamlines the development of neutralization platforms, resulting in CNPs offering versatile and function-driven solutions for achieving broad-spectrum neutralization.
For example, CNPs coated with RBC membrane effectively neutralize a wide spectrum of hemolytic toxins, demonstrating their efficacy regardless of the toxins' molecular structures. [56,57]urthermore, CNPs cloaked with white blood cell (WBC) membranes, including macrophages and neutrophils, simultaneously  [53] Copyright 2020, John Wiley and Sons.
neutralize inflammatory cytokines, exerting anti-inflammatory effects in animal models of diseases such as sepsis, rheumatoid arthritis, and gout. [15,58,59]64][65] As the single-membrane-coated CNPs gain attraction for bioneutralization, researchers have also made dual-membrane hybrid CNPs to harness synergistic bioneutralization capabilities.A notable example involves CNPs coated with RBC-PLT hybridized membrane (Figure 6). [66]In this hybridization, the PLT membrane targets and neutralizes bacteria via the binding interactions between PLT von Willebrand factor and fibrinogen with bacterial clumping factor A (clfA). [67]This targeting effect, in turn, positions the RBC membrane closer to the source of toxins, enhancing the efficiency of toxin neutralization.This hybridized membrane was then applied to fuel-free US-propelled gold nanorobots (RBC-PL robots).When subjected to US propulsion, these nanorobots effectively neutralized pore-forming toxins secreted by methicillin-resistant Staphylococcus aureus (MRSA), which typically targets RBCs.Furthermore, the US-propelled RBC-PL-robots exhibited enhanced performance in isolating MRSA USA300 strain model bacteria, known for their high adhesion to PLTs, achieving faster results compared to uncoated robots, RBC membrane-coated robots, or static RBC-PL-robots.The US-propelled RBC-PL robots produced lower hemolysis when compared with static RBC-PL robots (17% vs 40%).Additionally, the RBC membrane component conferred Figure 6.RBC-PLT HM-functionalized nanorobots (RBC-PL-robots) for concurrently neutralizing bacteria and bacterial toxins.A) A schematic of RBC-PLrobot preparation, including (i) gold nanowire modification, (ii) membrane fusion, and (iii) membrane coating.B) Comparison of the speed between bare robots and RBC-PL-robots in water and after 0 and 1 h incubation in whole blood.C) A representative scanning electron microscope (SEM) image of an RBC-PL-robot with a captured MRSA USA300 bacterium.D) Normalized fluorescence intensity of DAPI-stained MRSA USA300 bacteria attached on (i) PBS (no robots), (ii) bare robots, (iii) RBC-robots (without PL membrane), (iv) RBC-PL-robots under a static condition (without US), (v) RBC-PL-vesicles, (vi) US-propelled RBC-PL-robots, and (vii) PL-robots (without RBC membrane).E) MRSA toxin-induced RBC hemolysis after treating the RBCs with (i) PBS, (ii) static RBC-PL-robots, (iii) US-propelled RBC-PL-robots, and (iv) US-propelled RBC-robots.F) MRSA bacterial growth curves obtained by measuring OD600.G) Corresponding hemolysis profiles when RBCs were incubated with nontreated MRSA bacteria and MRSA bacteria treated with RBC-PLrobots.In (F, G), the arrows indicate the first measurement after the treatment.Reproduced with permission. [66]Copyright 2018, American Association for the Advancement of Science.
antibiofouling properties, enabling the nanorobots to navigate through whole blood without a decrease in speed.
The hallmark of CNPs is their remarkable ability to faithfully replicate the attributes of source cells, allowing them to neutralize a wide array of harmful molecules or infectious pathogens, while circumventing the substantial diversity and complexity of these agents.Compared to single-membrane CNPs, hybrid CNPs take one step forward by harnessing the collective biofunctions of natural protein receptors found on distinct cell membranes, leading to a level of synergy that would otherwise be unattainable.As the understanding of CNP-disease interactions deepens and we strategically select membranes for hybridization, hybrid CNPs are poised to unveil and showcase even more distinct strengths in biological neutralization applications.

Hybrid CNPs for Disease Diagnosis
The potential of hybrid CNPs has extended to disease diagnosis, mainly through the detection and isolation of rare cells from peripheral blood samples.One prominent application is the identification and isolation of CTCs.Various bioassay techniques, including NP platforms with antibody conjugation and immunomagnetic beads (IMBs), have been developed to precisely detect and selectively isolate CTCs from the peripheral blood of cancer patients. [68,69]For instance, the CellSearch system, approved by the U.S. Food and Drug Administration, employs IMBs for the collection and enrichment of CTCs. [70]However, the CTC recovery rate and the purity of these systems have been constrained by the nonspecific adsorption of WBCs onto the IMBs. [71,72]herefore, there is a growing need for novel approaches that ensure specific capture, reliable isolation, and accurate quantification of CTCs, particularly those with sparse abundance in peripheral blood.
To address these limitations, researchers recently fused the cancer cell membrane with the leukocyte membrane and coated the HM onto the magnetic beads (denoted "CM-LM-MBs") for the isolation of CTCs (Figure 7). [73]The HM was initially obtained by extruding membranes from MCF-7 cells, a human breast cancer cell line, and purified human leukocytes.Subsequently, the HM was coated onto magnetic beads, leading to the formation of CM-LM-MBs.These NPs relied on the homologous adhesion of the MCF-7 membrane to bind with MCF-7 cells in the blood.Simultaneously, the leukocyte membrane component in the coating reduced the nonspecific adsorption of background leukocytes due to the leukocyte homology.By designing NPs with increased affinity for CTCs and reduced background interference with leukocytes, the hybrid CNPs significantly enhanced CTC capture efficiency and specificity.Moreover, the magnetic core facilitated the rapid extraction of NP-bound CTCs from the blood using an external magnetic field.The study demonstrated higher separation purity and improved detection rates of CTCs in orthotopic 4T1 mouse metastatic mammary carcinoma models compared to conventional anti-EpCAM-based IMBs.Notably, the captured MCF-7 cells were completely released from the CM-LM-MBs within 2 min after trypsin-EDTA treatment, and the cell viability was preserved.EpCAMconjugated MBs (MBs-Ab), CM-coated MBs (CM-MBs), and CM-LM-MBs effectively captured the CTCs from the blood of the 4T1 tumor-bearing mice, while no CTCs were found in the healthy mice.The average number of the CTCs captured by the CM-MBs (5.25) and CM-LM-MBs (5.75) was greater than that of the CTCs captured by MBs-Ab (1.9) Hybrid membrane-coated magnetic NPs have also been integrated with other functionalities to enable ultrasensitive and in situ detection of CTCs. [74]In one study, the membranes of murine RAW264.87 macrophage cells and MCFf-7 cancer cells were fused.The resulting HM was conjugated with streptavidin and then coated onto magnetic NP cores.After the NP preparation, biotin-modified multivalent aptamer-Ag2S nanodots were grafted onto the hybrid cell membrane-magnetic NPs.This NP platform utilized membrane hybridization to facilitate cancer cell targeting while reducing interference from background WBCs.The aptamer provided an additional CTC targeting ability, thereby enhancing capture efficiency.Simultaneously, the nanodots offered in situ CTC detection through their strong NIR fluorescence.In a cell mixture, the NPs specifically recognized MCF-7 cells but not Hela or RAW264.7 cells.Their detection ability was further demonstrated by accurately detecting and capturing CTCs from blood samples of cancer patients while not showing such recognition in specimens from healthy subjects.
In a separate study, the cancer cell membrane was substituted with a PLT membrane, which was then hybridized with a leukocyte membrane for coating onto magnetic beads. [75]Similar to previous examples, the HM coating substantially enhanced the binding of NPs to CTCs, simultaneously preventing nonspecific binding with background WBCs.Notably, the NPs were additionally conjugated with anti-EpCAM antibodies, enhancing the selectivity for capturing EpCAM-positive cancer cells, such as MCF-7 or HCT116 cells.Upon testing with spiked blood samples, the hybrid CNPs exhibited significantly higher cell purity than commercially available IMBs.Furthermore, they accurately detected CTCs in clinical blood samples obtained from breast cancer patients.
Besides CTC detection and isolation, hybrid CNPs have recently been employed to isolate fetal nucleated RBCs (fNRBCs) in maternal peripheral blood (Figure 8). [76]If isolated with high efficiency and purity, these cells can provide comprehensive genetic information about the fetus, making them ideal for noninvasive prenatal diagnostics.To achieve this objective, researchers hybridized the RBC membrane with a leukocyte membrane and coated the HM onto magnetic cores.The NPs were then conjugated with anti-CD147, an antibody targeting fNRBCs for isolation.In this design, the RBC membrane component reduced nonspecific protein adsorption by the NPs, thus enhancing the potency of antibody binding with the fNRBCs.Simultaneously, the leukocyte membrane component minimized background leukocyte adsorption.In simulated blood samples, the NPs effectively isolated fNRBCs with a high purity of ≈90%.Furthermore, they were tested for the isolation of fNRBCs from a series of clinical maternal blood samples taken during the 11th-13th gestational weeks, isolating 11-24 fNRBCs per milliliter of blood, respectively.The high isolation efficiency and purity of the isolated cells facilitated the accurate diagnosis of various chromosomal aneuploidies, including Down syndrome, Trisomy 18 syndrome, Klinefelter's syndrome, XXX syndrome, and XYY syndrome.
Recent advancements in the use of hybrid CNPs for disease diagnosis have demonstrated the high capture specificity and purity of hybrid CNPs in detecting and isolating crucial cells from peripheral blood.The continuous development of hybrid CNPs for this purpose is expected to extend to other diseases in the near future.

Summary and Outlook
Coating NPs with HMs gives rise to a diverse array of hybrid CNPs characterized by intriguing properties and therapeutic potentials distinct from their counterparts coated with monotypic cell membranes.This review concentrates on four areas where hybrid CNPs exhibit significant promise.In drug targeting, some hybrid CNPs combine immune stealth with active targeting, facilitating receptor-ligand interactions.Some hybrid CNPs demonstrate improved performance by integrating dualtargeting abilities from distinct membrane components.In immune modulation, hybrid CNPs combine the immune characteristics derived from membranes of cancer cells, DCs, or bacteria.This synergistic approach coordinates antigen, adjuvant, and immune targeting, outclassing counterparts coated with single-cell membranes.In biological neutralization, hybrid CNPs leverage the collective biofunctions of natural protein receptors present on diverse cell membranes.Through hybridization, these receptors operate collaboratively, enabling effective toxin scavenging in complex biological systems.In disease .Reproduced with permission. [73]Copyright 2020, RSC Publisher.
diagnosis, hybrid CNPs excel in detecting and isolating rare cells from peripheral blood samples, concurrently enhancing specificity and efficiency.Examples reviewed in this article are summarized in Table 1, highlighting the rationale behind the hybrid CNP membrane selection and mechanisms of action.
The progress achieved by hybrid CNPs in the above applications is attributable to the continual advancement and accumulated knowledge in cell membrane coating nanotechnology.Toward future development, it has become imperative to delve deeper into understanding the in vivo compatibility and safety of hybrid CNPs.While current hybrid CNP formulations have generally exhibited good biocompatibility, there is a pressing need to conduct comprehensive investigations into their long-term biological effects for future clinical translation.Critical parameters affecting safety, such as size, surface properties, morphology, and substrate chemical composition, must be thoroughly elucidated. [77]In this perspective, developing formulations aimed at reducing substrate toxicity has emerged as a promising avenue for ensuring safer payload delivery.Design strategies, as discussed in this review, offer viable solutions to enhance the efficacy and safety of therapeutic hybrid CNPs, likely contributing to their successful translation. [78]The rational design of the next generation of hybrid CNPs with fine-tuned membrane properties and compositions may benefit from cutting-edge technologies that overcome biological barriers for more effective therapeutic delivery. [79]eanwhile, the distribution of each membrane component on hybrid CNPs may be influenced by membrane miscibility, thereby impacting their overall properties.Understanding how cells modulate the lipid content of their plasma membranes in response to environmental changes provides an avenue to tailor the properties of hybrid CNPs more precisely. [80]As research and development continue, we anticipate that hybrid CNPs will unveil and demonstrate additional unique strengths for diverse biomedical applications, thereby contributing significantly to various disease diagnosis, treatments, and prevention.F) Representative images of chromosome aneuploidy detection using fluorescence in situ hybridization (FISH) analysis of isolated fNRBCs from maternal peripheral blood (red: chromosome 21/Y, green: chromosome 13/X, and cyan: chromosome 18; scale bars = 5 μm).Reproduced with permission. [76]opyright 2021, American Chemical Society.Targets inflammation Targets ECM of the injured tissue Neutrophile membrane PLT membrane [30,31]   Targets CTCs Targets tumor

CSC membrane
PLT membrane [32]   Targets cancer through homotypic recognition Evades immune clearance Calreticulin-expressing L929 cell membrane PLT membrane [34]   Triggers programmed neutrophil removal Targets activated neutrophils RBC membrane Cancer cell membrane [38]   Evades immune clearance Targets cancer through homotypic recognition

Macrophage membrane
Cancer cell membrane [42,43]   Evades immune clearance, targets the metastatic cancer cells Targets cancer through homotypic recognition Retinal endotheliocyte membrane RBC membrane [44]   Targets retinal endotheliocytes through homotypic recognition Evades immune clearance Glioblastoma cancer cell membrane Mitochondria membrane [46]   Permeates across the BBB Targets mitochondria through homotypic recognition after NP internalization

Immune modulation
Cancer cell membrane DC membrane [51,52]   Targets cancer through homotypic recognition T cell immune activation

Cancer cell membrane
Bacterial OM [53]   Targets cancer through homotypic recognition Acts as an immune adjuvant to boost immune activation

Cancer cell membrane
Macrophage membrane [55]   Acts as the secondary immune adjuvant to boost immune activation

Targets the infection sites
Biological neutralization RBC membrane PLT membrane [66]   Neutralizes bacterial PFTs Targets and neutralizes bacteria

Disease diagnosis
Cancer cell membrane Leukocyte membrane [73]   Targets cancer through homotypic recognition Minimizes background leukocyte adsorption

Macrophage membrane
Cancer cell membrane [74]   Reduces interference from background WBCs Targets cancer through homotypic recognition

Figure 1 .
Figure 1.A schematic of the process for creating hybrid CNPs.The process involves the fusion of membranes originating from distinct cell types and their subsequent coating onto NP cores, forming hybrid CNPs.

Figure 4 .
Figure 4. Cytomembrane nanovaccines show therapeutic effects by mimicking tumor cells and antigen-presenting cells.A) A schematic illustration of cancer cell-DC hybrid cell membrane-coated MOF NPs (MOF@FM) for tumor prevention.B) Fusion of DCs and 4T1 cells observed with confocal laser scanning microscopy (red: anti-CD44-APC-labeled 4T1cells; green: anti-MHC II-FITC-labeled DCs; scale bar = 10 μm).C) A transmission electron microscope (TEM) image of MOF@FM (scale bar = 100 nm).D) In vivo fluorescence imaging at the indicated time points after the subcutaneous injection of NPs coated with the cancer cell membrane (MOF@CM), NPs coated with DC membrane (MOF@DM), and MOF@FM.E) Illustration of the experiment design.F) Photos of harvested tumors on day 36 after the tumor challenge.G) Levels of secreted IFN-γ and IL-6 in mice serum measured by ELISA kit.The mean values and s.d. were presented (statistical analysis was performed with one-way ANOVA; ns: not significant; ***p < 0.001; n = 3).H) Flow cytometric quantification of CD3 and CD8 expression on splenic lymphocytes on day 0 after the two immunizations.I) Immunofluorescence observation of CD8 expression in the draining lymph node (scale bar = 50 μm).Reproduced with permission.[51]Copyright 2019, Springer Nature.

Figure 5 .
Figure 5.A hybrid eukaryotic-prokaryotic nanoplatform with a photothermal modality for enhanced antitumor vaccination.A) A schematic illustration of fabricating the eukaryotic-prokaryotic vesicle-coated PI@EPV nanovaccine (OMV: bacterial OM vesicle; CMV: cancer cell membrane vesicle; PI: PLGA-ICG; EPV: eukaryotic-prokaryotic vesicle).B) A pair of fluorescence resonance energy transfer dyes DiO and DiI is chosen to examine the fusion of the two membranes.The OMVs were doped with two fluorescence dyes and fused with increasing amounts of B16F10 CMVs.C) Hydrodynamic size and a TEM image of EPV (CMVs: OMVs = 2:1).D) Hydrodynamic size and E) a TEM image of the resulting PI@EPV nanovaccine.F) Study protocol for the tumor recurrence assay.G) Tumor growth curves of B16F10 or 4T1 after immunization with the nanovaccines (error bar: mean AE SD, n = 5).H) Percentage of tumor-free mice after tumor challenge.Reproduced with permission.[53]Copyright 2020, John Wiley and Sons.

Figure 7 .
Figure 7. Cancer-leukocyte hybrid magnetic CNPs for the ultrasensitive isolation, purification, and nondestructive release of CTCs.A) A schematic illustration of the preparation of CM-LM-MBs (CM: MCF-7 cell membrane; LM: leukocyte membrane; MB: magnetic bead).B) The capture efficiency of various NPs from whole blood spiked with MCF-7, MCF-10A, or HCT 116 cells.C) The capture efficiency of MBs-Ab, CM HeLa -LM-MBs, and CM HeLa -MBs in various cancer cell-spiked human blood samples (Ab: antibody; CM HeLa : HeLa cell membrane).D) The isolation yield of MCF-7 cells from human blood samples spiked with various MCF-7 cells.E) The release efficiency of the captured MCF-7 cells after coincubation with trypsin-EDTA at different incubation times.F) The number and purity of the CTCs separated from 1.0 mL blood samples by the CM 4T1 -LM-MBs and MBs-Ab in 4T1 tumorbearing mice and healthy mice (CM 4T1 : CM4T1 cell membrane).Reproduced with permission.[73]Copyright 2020, RSC Publisher.

Figure 8 .
Figure 8. Enhanced fNRBCs isolation with RBC-leukocyte magnetic CNPs for noninvasive pregnant diagnostics.A) A schematic illustration of NP design and the isolation principle.B) The capture efficiency and purity with the HM-coated NPs (right: an image of HM-coated NPs in a maternal blood sample).Nonspecifically captured WBCs are marked with a red circle (scale bar = 50 μM).C) The detection of fNRBC by NPs from maternal blood samples.The cells captured by NPs were verified as fNRBCs with immunostaining of ε-globin, CD71, and DAPI (ε-globin þ /CD71 þ /DAPI þ cells).D) Fetal origin verification of the captured fNRBCs (scale bar = 5 μm).E) Captured fNRBC counts from 20 maternal blood samples of early gestational age.F) Representative images of chromosome aneuploidy detection using fluorescence in situ hybridization (FISH) analysis of isolated fNRBCs from maternal peripheral blood (red: chromosome 21/Y, green: chromosome 13/X, and cyan: chromosome 18; scale bars = 5 μm).Reproduced with permission.[76]Copyright 2021, American Chemical Society.

Table 1 .
Hybrid cell membrane-coated NPs for biomedical applications.