Red blood cells drug delivery systems for biomedical applications

Red blood cells drug delivery systems for biomedical applications | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 13 March 2025 V1 Latest version Share on Red blood cells drug delivery systems for biomedical applications Author : Huizi Deng Authors Info & Affiliations https://doi.org/10.22541/au.174188580.03172875/v1 552 views 198 downloads Contents Abstract Acknowledgements Conflict of Interest Data availability Ethics approval statement History of RBCs as drug carriers Characteristics of RBCs Drug delivery methods of RBCs Application of RBCs drug delivery systems in diseases Conclusions and perspectives CRediT authorship contribution statement Tables Figures References Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The characteristics of red blood cells (RBCs), such as low immunogenicity, long circulation time and targeting ability, make them excellent drug delivery carriers. Currently, some new advancements have been achieved in the research on the delivery systems of RBCs and their derivatives, and they have found applications in diverse biomedicine areas. This article conducts a detailed analysis on the characteristics of RBCs, the historical development of their application as drug delivery systems, the practical application as well as the advantages and disadvantages of different delivery methods, and their applications in various diseases, aiming to provide references for the research and development of RBCs in the sphere of drug delivery. Red blood cells drug delivery systems for biomedical applications Huizi Deng, Dr. a , Xiaobei Cheng, Dr. a , Yi Li, Dr. a , Yameng Ling, Ms. a,b , Yuli Wang, Prof. a , Yang Yang, Prof. a,* , Chunsheng Gao, Prof. a,* a State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China b Henan University, Kaifeng, Henan 475004, China * Corresponding authors. E-mail addresses: [email protected] (Y. Yang), [email protected] (C. Gao). Acknowledgements Not applicable. Conflict of Interest The authors declare no conflict of interest. Data availability No data was used for the research described in the article. Ethics approval statement Not applicable. Abstract : The characteristics of red blood cells (RBCs), such as low immunogenicity, long circulation time and targeting ability, make them excellent drug delivery carriers. Currently, some new advancements have been achieved in the research on the delivery systems of RBCs and their derivatives, and they have found applications in diverse biomedicine areas. This article conducts a detailed analysis on the characteristics of RBCs, the historical development of their application as drug delivery systems, the practical application as well as the advantages and disadvantages of different delivery methods, and their applications in various diseases, aiming to provide references for the research and development of RBCs in the sphere of drug delivery. Keywords : red blood cell; erythrocyte; red blood cell membrane; engineered red blood cell; nanocarrier; drug delivery system An ideal drug delivery system should have high drug loading efficiency, good bioavailability, long circulation time with low immunogenicity, and precise targeting ability, so as to improve drug efficacy and reduce toxic and side effects 1 . Since nanotechnology has been applied to drug delivery systems, liposomes, micelles, polymers, inorganic nanocarriers and nanogels, etc. have been developed to meet different delivery requirements and have shown good therapeutic effects. However, problems such as low targeting ability, poor stability and easy clearance by the immune system still exist. 2 To overcome these problems, researchers have explored biomimetic nanodrug delivery systems based on autologous cells, such as tumor cells, blood cells, stem cells and immune cells delivery systems. These biomimetic nanodrug delivery systems have been proven to have the advantages of good biocompatibility, abundant sources and strong targeting ability. 3 Among them, RBCs is the most common cell in the blood, and has unique biological characteristics, such as large specific surface area, no nucleus, specific targeting liver and spleen, long circulation time and good biocompatibility, which is regarded as an ideal drug delivery carrier. Currently, a series of studies have explored the use of red blood cells to deliver various drugs, enzymes, antigens, nucleic acids and nanoparticles, etc. for the treatment of different diseases, and remarkable results have been achieved. 4 This article reviews the various ways in which red blood cells are used in drug delivery systems and their applications in different diseases. History of RBCs as drug carriers The term ”red blood cell” was first put forward by Lee Van Hock in the 17th century when he observed human blood samples. In the 18th century, Howson further determined the morphology of red blood cells as flat disc. In 1953, Gardos loaded adenosine triphosphate (ATP) into the ”RBCs ghosts”, which laid the foundation for subsequent RBCs membrane loading of active ingredients. In 1959, Marsden and Ostling achieved the entrapment of dextran in RBCs for the first time, 5 this was a further attempt to load drugs with RBCs. In the 1970s, some researchers began to use RBCs for drug delivery. For example, Ihler et al. loaded β-glucosidase and β-galactosidase into RBCs for the treatment of Gaucher disease and other diseases requiring enzyme replacement therapy 6 . In 1979, the concept of RBCs as drug carriers was formally introduced, 7 which initiated a boom in preclinical research on RBCs as drug carriers. In 1994, Lejeune et al. prepared nanoscale RBCs by membrane extrusion technology to treat leukemia, successfully achieving the nanoscale of RBCs. 8 In 2013, Zhang et al. constructed a bionic toxin nanosponge to absorb harmful toxins as bait, which facilitated the biomimetic research of RBCs. 9 In 2014, the American company Rubius Therapeutics was established and proposed the concept of ”red blood cell therapy” for the first time. That was, through stem cell engineering technology, human hematopoietic stem cells were transformed into RBCs that could express specific drugs, cultured in vitro and then returned to patients to treat diseases such as immune system diseases and tumors. However, after the failure of the phenylketonuria syndrome drug candidate RTX-134 and the solid tumor drug candidate RTX-240 in clinical trials, Rubius Therapeutics announced its dissolution in 2023 ( Figure 1) . Nevertheless, there are some companies such as EryDel, Anokion, Westlake Therapeutics and Carcell Biopharma that are still exploring commercialization, and the formal application of RBCs drug delivery systems in clinical practice still needs efforts. Characteristics of RBCs Red blood cells are the most copious cell variety in the blood, accounting for approximately 45% of all total blood cells. 10 As carriers of oxygen and carbon dioxide, RBCs have developed some unique biological features (Figure 2) . Mature RBCs showed a biconcave disk-like structure, about 7 μm in diameter, and about 2 μm in the middle of the depression, 11 and the surface area as high as 140 μm, 2 which provides a unique opportunity for the loading and binding of drugs. 4 Red blood cell membrane (RBCm) is composed of 39.5% protein, 35.1% lipids, 19.5% water and 5.8% carbohydrates, 12 and its structure is based on a lipid bilayer, with proteins covering the surface or embedded inside the lipid bilayer, which can be described by a flow-through Mosaic model. The major components of lipids include phospholipids and cholesterol, in which the distribution of phospholipids is asymmetric. The outer monolayer is composed of phosphatidylcholine (PC), which forms a mobile lipid region, and sphingomyelin (SM), which plays a stationary role. 13 The inner monolayer is composed of phosphatidylethanolamine (PE), phosphatidylserine (PS) and a small amount of phosphoinositide (PI). These three components are negatively charged, but under physiological conditions, most of the phospholipids are electrically neutral. 14 The unsaturation of the phospholipid molecules, the ratio of cholesterol to phospholipids, and the ratio of PC to SM all affect the fluidity of RBCm. Alternatively, lipid transporters are able to regulate the composition of phospholipids, and external lipid molecules can alter the arrangement and distribution of phospholipids to expose PS to the external surface, thereby allowing RBCs to be recognized by macrophages. 15 Red blood cell membrane contains more than 50 membrane proteins. The peripheral proteins covering the membrane surface can be easily extracted under a specific pH environment, while the integrated proteins embedded in the phospholipid bilayer can only be removed by specific chemical reagents, but this method will cause great damage to the membrane structure. 16 RBCs can circulate for up to 120 days in humans and 40 days in mice. 17 A series of membrane proteins regulate the long circulation of red blood cells, among which CD47 plays a crucial role. CD47 is a complete membrane protein with five transmembrane regions, which enables it to be firmly embedded in RBCm. Meanwhile, it also has an extracellular domain similar to an immunoglobulin V structure, which helps RBCm maintain stability in the blood circulation and prolong the lifespan of RBCs. Additionally, CD47, as a self-marking signal expressed on the surface of healthy RBCs, when interacting with signal regulatory protein alpha (SIRPα) expressed on macrophages, releases a ”don’t eat me” signal, thereby avoiding being phagocytosed by macrophages. 18 The expression of CD47 on the surface of aged or damaged RBCm decreases, allowing macrophages to recognize and phagocytize these RBCs, leading to their complete biodegradation. Other membrane proteins on the surface of RBCm, such as C8 binding protein (C8bp), homologous restriction protein (HRP), decay accelerating factor (DAF), membrane cofactor protein (MCP), complement receptor 1 (CR1), and CD59, also play important roles in defending against complement system attacks. 19 Additionally, the Rh and ABO blood group systems expressed on RBCm pose a risk of hemolysis when blood is transfused between individuals with different blood types. 20 Red blood cells originate from hematopoietic stem cells in the bone marrow, which have the ability to self-renew and differentiate into various blood cells. In a specific hematopoietic microenvironment, hematopoietic stem cells begin to differentiate into erythroid progenitor cells. Erythroid progenitor cells continuously proliferate and differentiate in the bone marrow, successively forming proerythroblasts, normoblasts, and reticulocytes. During this stage, the cells undergo a series of morphological and structural changes, one of the most important of which is the expulsion of the nucleus. After the nucleus is expelled, the red blood cell becomes a mature, enucleated red blood cell. Enucleation provides the red blood cell with more internal space to accommodate more hemoglobin and carry more drugs. At the same time, oxygen is not consumed by the red blood cell’s own metabolism, making it a more efficient oxygen carrier. RBCs can pass through narrow capillaries without being restricted by the nucleus and organelles, allowing them to deform more flexibly for the transport of related substances. 21 Additionally, the differentiation process of RBCs can be utilized to genetically engineer hematopoietic stem cells. 22 Red blood cells have excellent deformability, enabling them to pass through capillaries less than half their diameter and transport oxygen from lungs to all tissues in the body and carbon dioxide from tissues to lungs. Under normal physiological conditions, the shape changes of RBCs are reversible. In some pathological conditions, RBCm may undergo plastic changes and become permanently deformed due to excessive shear force. 23 Additionally, as RBCs age normally, their deformability gradually decreases, and they are isolated by the endothelial spaces in the spleen (0.5-1 μm) and eventually phagocytized by splenic macrophages. 24 The deformability of RBCs mainly depends on the intracellular viscosity and the flexibility of RBCm. Intracellular viscosity is mainly determined by the concentration and structure of hemoglobin. As hemoglobin concentrations rise, not only does the viscosity of RBCs increase and their elasticity decline, but also the regional boundaries of the membrane might be modified because of imperfect lateral packing, which in turn enhances membrane permeability. 4 The flexibility of RBCm depends on the intracellular calcium concentration. The maintenance of normal mechanical behavior depends on the low intracellular calcium level maintained by the active ATP-dependent calcium pump within RBCm. Meanwhile, the degree of hydration of the cell is an important factor affecting the relationship between the surface area and volume of RBCs. When RBCs are overhydrated, their volume increases while the surface area remains unchanged, resulting in a decrease in deformability. When RBCs are underhydrated, the concentration of hemoglobin increases, leading to a decrease in deformability. 25 Cell lysis might occur when the surface area of RBCs experiences a 3% -4% increase. Therefore, we should pay attention to the red blood cell’s stability and deformability to avoid premature clearance by the immune system when handling RBCs. 26 Drug delivery methods of RBCs Red blood cells have great advantages as drug delivery systems, including ⅰ) The large specific surface area and anucleate structure provide sufficient space for drug loading. ⅱ) The abundant membrane proteins not only help the drug delivery system evade phagocytosis by the immune system but also enable specific targeting of the liver and spleen, facilitating precise drug delivery. ⅲ) The long circulation life in the body, up to 120 days, provides a basis for the sustained release of drugs. ⅳ) The excellent rheological properties allow RBCs to freely pass through capillaries, which is beneficial for the targeting and accumulation of drug delivery systems. ⅴ) RBCs are derived from human and animals, which are abundant in quantity and have good biocompatibility and low toxic side effects. Currently, a large number of studies have used RBCs as drug delivery carriers, and the drugs delivered included small molecule substances, nucleic acids, proteins, polypeptides, nanoparticles, etc. 27 According to the different ways of using RBCs, the delivery methods can be divided into intracellular delivery system of RBCs, drug delivery systems based on RBCm, gene engineered RBCs delivery system and RBCs derived extracellular vesicle delivery system. This section elaborated on various methods of drug delivery by RBCs ( Figure 3) and explored the advantages and disadvantages of different delivery methods ( Table 1 ). Intracellular delivery system of RBCs The intracellular delivery system of red blood cells is encapsulating drugs inside RBCs to prevent the drugs from being cleared by the reticuloendothelial system, thereby prolonging their circulation time. At present, the methods for intracellular drug delivery in RBCs mainly include osmotic method, electroporation method, chemical substance method, transmembrane peptide method, microfluidic extrusion method, etc. The osmotic method refers to a technique that adjusts different osmotic pressures to create transient pores in the red blood cell membrane, so as to encapsulate drugs inside red blood cells. 55 At present, the relatively mature penetration methods include the low osmotic pre-swelling method and the low osmotic dialysis method. The low osmotic pre-swelling method involves placing RBCs in a relatively hypotonic solution to cause the cells to swell, opening the tight junctions, and creating multiple transient pores of 20-50 nm on the surface of RBCm. After the drug enters the interior of the RBCs through these pores, the hypotonic solution is replaced with an isotonic one, and the pores on RBCm close, trapping the drug inside the RBCs. 56 For instance, Harisa et al. used this method to encapsulate pravastatin chitosan nanogels into RBCs for liver-targeted therapy of liver cancer. 28 The low osmotic dialysis method utilizes a dialysis bag with a hypotonic solution to reduce the osmotic pressure of red blood cells or increases the contact area between red blood cells and buffer solution in a special dialyzer under flow dialysis conditions, thereby enhancing the loading efficiency of red blood cells. 29 For example, Biagiotti et al. used this method to load cyclosporine A and tacrolimus to improve the bioavailability and reduce the toxicity of these two drugs. 30 The osmotic method is simple to operate and is the most common way to load drugs into RBCs, suitable for small molecule drugs, biological agents, and nanoparticles with smaller particle sizes. However, it has disadvantages such as significant differences in loading efficiency for different drugs and the inability to control the release speed and extent of the drugs. The electroporation method refers to the process of applying pulsed electric fields to red blood cell membrane, creating pores in the membrane due to the transmembrane potential difference, thus allowing drugs to enter the interior of red blood cells through these pores. 57 For instance, Saulis et al. used this method to load ascorbic acid and mannitol into RBCs. 31 The electroporation method is not affected by osmotic pressure differences and can load drugs relatively uniformly into RBCs. However, it requires sophisticated equipment and precise operation. If the voltage is too low, RBCm cannot form sufficient pores, and if it is too high, RBCm may rupture, leading to cell death. The chemical substance method involves using special structured chemical substances to induce endocytosis in red blood cells, thereby loading drugs into the interior of red blood cells. 58 For example, Harisa et al. used this method to load pravastatin into red blood cells, achieving an encapsulation efficiency of 94% and a red blood cell recovery rate of 87% to 93%. 32 The chemical substance method causes less damage to red blood cell membrane, largely maintaining its integrity, and the intracellular substances are less likely to leak out. However, it is only applicable to drugs with both hydrophobic and hydrophilic groups, such as cationic or anionic drugs. The transmembrane peptide method refers to the process of loading drugs into red blood cells using cell-penetrating peptides. Cell-penetrating peptides are short peptides with special structures and functions that can penetrate cell membranes. After combining drugs with these peptides, the peptides mediate the passage of drugs through red blood cell membrane into the interior of red blood cells. 59 For instance, He et al. used a low molecular weight protamine cell-penetrating peptide to deliver L-asparaginase into RBCs, increasing the survival period of lymphoma mice by 44%. 33 The transmembrane peptide method does not require the formation of pores in RBCm, reducing damage to RBCs and effectively delivering drugs into the interior of RBCs. However, the drug loading capacity is limited by the binding ability of the cell-penetrating peptide to the drug. Therefore, when using this method for drug loading, it is necessary to select appropriate cell-penetrating peptides. 60 The microfluidic extrusion method involves red blood cells passing through a microfluidic device with a size smaller than their diameter, where they are mechanically extruded, temporarily opening red blood cell membrane to load drugs into the interior of red blood cells. 61 For example, Raposo et al. used this technology to encapsulate antigens to generate tolerogenic antigen carriers for restoring immune tolerance in the body. 34 Microfluidic extrusion has strong versatility and flexibility, can load various drugs well, and meanwhile maintain the integrity of RBCs. At the same time, microfluidic devices have multiple parallel channels, which are convenient for large scale RBCs processing. However, after mechanical extrusion, most red blood cells will age, which may lead to their easy clearance by the immune system. 62 Drug delivery systems based on RBCm The surface area of a single red blood cell membrane can reach 140 μm 2, and it contains various membrane proteins, making it a good location for drug loading. Moreover, there is no need to puncture the red blood cell membrane surface, which to some extent reduces the damage to the structure and function of red blood cells. According to different drug loading mechanisms, drug delivery systems based on red blood cell membranes mainly include chemical conjugation method, receptor mediated method, hitchhiking method, red blood cell membrane coating method, and hybrid membrane fusion method. Chemical conjugation refers to the method of drug loading by chemically connecting drug molecules to groups on the red blood cell membrane surface through covalent bonds, biotin-avidin coupling, etc. For example, Ataullakhanov et al. used a non-specific chemical crosslinker glutaraldehyde to connect the amino groups of anthraquinone antibiotics to the amino groups on the RBCm to increase the circulation time of antibiotics in the body. 35 Müller et al. used a bifunctional coupling reagent to connect thiolated heparin to the RBCm, exerting a long-lasting anticoagulant and thrombosis prevention effect. 36 Additionally, Wang et al. introduced avidin to the RBCm surface and used the biotin-avidin coupling to connect cyclic peptides to RBCs. This chemical conjugation has stronger specificity than covalent bonds, significantly extending the circulation time of drugs in the body and having good targeting properties. 37 Chemical conjugation method has strong specificity, but excessive modification may affect the biocompatibility and deformability of RBCs, damage CD47 or other protective proteins, and promote the generation of reactive oxygen species. 63 The receptor mediated method utilizes the receptor-ligand interaction between proteins on the red blood cell membrane surface and specific drugs for drug delivery. For example, glycophorin A is a sialic acid glycoprotein with receptor function on the RBCm surface, which has high affinity and specificity and can be used as a target for ligand binding. The Sahoo team modified the surface of nanoparticles loaded with bovine serum albumin with the ERY1 amino acid peptide that has high affinity for glycophorin A, allowing the nanoparticles to attach to the RBCm through receptor-ligand interaction and thereby increasing the circulation half-life of the nanoparticles in the body. At the same time, the study found that at high concentrations, the strong affinity of nanoparticles for glycophorin A reduced the deformability of RBCs. 38 The receptor mediated method causes less damage to RBCs compared to the chemical conjugation method, but excessive drug loading may affect the surface proteins of RBCm, thereby affecting the deformability and long circulation of RBCs. In addition, since the surfaces of various cells in the body have the same or similar receptors, it may lead to the binding of drugs to other non-specific cells in the body, thus affecting the therapeutic efficiency of the drugs. 64 The red blood cells hitchhiking method is a technique of drug delivery through non-covalent interactions such as hydrophobic interactions, electrostatic interactions, and hydrogen bonds between drugs and red blood cell membrane. 40, 65 For example, Zhao team directly connected polylactic-co-glycolic acid (PLGA) nanoparticles encapsulating the chemokine CXCL10 to the surface of RBCs through non-covalent interactions. After entering the blood circulation, the nanoparticles desorbed from the surface of RBCs due to the shear force of pulmonary capillaries and aggregated at the metastatic sites in the lungs. Meanwhile, CXCL10 can attract effector immune cells to trigger local cytotoxic immune responses, thereby inducing systemic immunity. 40 The hitchhiking method can effectively improve the accumulation of drugs in the liver and spleen, enhancing the organ-specific targeting of drugs. However, there are also problems such as the weak connection between nanoparticles and RBCs, which makes desorption likely to occur. Moreover, nanoparticles of different sizes and shapes have different pharmacokinetics and biodistribution. A large number of detached nanoparticles are cleared by the reticuloendothelial system, resulting in insufficient accumulation at the target site. 66, 67 Therefore, it is necessary to select appropriate nanoparticles for hitchhiking to improve therapeutic efficacy. Additionally, when using this method to achieve targeting of organs such as the heart and brain, it mostly requires the assistance of arterial intubation, and the operation is relatively complicated. 67 The red blood cell membrane coating method is a way that does not utilize intact red blood cells but separately extracts red blood cell membranes to coat drug loaded nanoparticles so as to achieve drug delivery. Intact RBCs are of micron size, directly using RBCs to load drugs may limit the in vitro diffusion ability of drugs and reduce their targeting ability. Extracting RBCm for drug delivery combines the advantages of nanoparticles in controlling drug release with the low immunogenicity and long circulation ability of RBCm. 43, 68 Zhu et al. developed a biomimetic multifunctional nanoreactor by encapsulating glucose oxidase and gold nanorods in RBCm coated metal organic framework nanoparticles. By combining glucose oxidase based starvation therapy with gold nanorod based photothermal therapy, it was used for the synergistic treatment of colon cancer. The coating of RBCm endowed the nanoparticles with longer circulation time and enhanced immune evasion ability. 43 Additionally, aged or damaged RBCs are phagocytosed by macrophages in the spleen, and this property can be utilized to deliver tumor associated antigens to antigen presenting cells and elicit a strong immune response. 69 The innate targeting of RBCs is mainly towards the reticuloendothelial system, liver, and spleen cells. If targeting other tissues and organs is required, RBCm needs to be functionally modified and engineered. Common targeting ligands include folic acid, arginine-glycine-aspartic acid peptide (RGD), and triphenylphosphine (TPP), etc. 44, 70 Gao et al. constructed an albumin nanocarrier system coated with RBCm and modified with TPP and T807, which not only could penetrate the blood-brain barrier but also targeted neural cells and further localized in mitochondria to protect neurons. 44 The RBCm coating method provides more possibilities for drug delivery, but during the extraction of RBCm or its fusion with nanoparticles, conformational changes in membrane anchored fragments may occur, leading to enhanced immunogenicity and activation of the immune response in vivo. 71 The hybrid membrane fusion method is a way that fuses red blood cell membrane with other biological membranes to synergistically leverage their advantages and improve the immune evasion ability and targeting ability of drug delivery systems. Currently, various biological membranes have been developed for fusion with RBCm, such as tumor cell membranes, 46 macrophage membranes, 47 platelet membranes, 48 retinal endothelial cell membranes 72 and some other biological membranes. 73 And these membranes’ inherent properties are utilized to target different tissues and diseases. Zheng et al. fused human HCT116 colon cancer cell membranes with RBCm to prepare RBCs-cancer cell hybrid membranes and coated them on the reactive oxygen species (ROS) responsive nanoparticles to achieve homologous targeting of colon cancer and prolonged blood circulation. 46 You’s team fused macrophage membranes with RBCm and inserted hyaluronic acid into the hybrid membranes, which were then coated on the surface of atorvastatin loaded graphene quantum dots to enhance the activation of macrophages within atherosclerotic plaques. 47 Li et al. constructed a multifunctional biomimetic nanomedicine delivery system assembled from platelet membranes and RBCm for targeted delivery of BRD4 inhibitor JQ1 to treat cardiac fibrosis. The platelet membranes endowed the nanoparticles with the ability to target cardiac fibroblasts and collagen, while the participation of RBCm enhanced the long time circulation ability of the nanoparticles. 48 Li’s team fused retinal endothelial cell membranes with RBCm and coated them on the surface of PLGA nanoparticles to achieve homotypic targeting of retinal endothelial cells, providing an effective method for the treatment of blinding diseases. 72 Besides natural cell membranes, liposomes can also be fused with RBCm. For example, Jiang’s team proposed a nanodelivery system of polymyxin B-modified liposomes fused with RBCm, which could effectively target Escherichia coli membranes and neutralize endotoxins and exotoxins from toxin sources. 73 The hybrid membrane fusion method bypasses the cumbersome modification process and can be used to design precise biomimetic nanodelivery systems for various delivery needs under different pathological conditions. However, there are also problems that the fusion process is prone to cause hemolysis of RBCs and the fusion efficiency is affected by multiple factors. 74 Gene engineered RBCs delivery system Compared with mature red blood cell modification techniques, creating new red blood cells for drug delivery using stem cells and gene editing technology is a new approach. This method takes advantage of the characteristic that erythroid progenitor cells gradually lose their nuclei during differentiation, which eliminates the risk of allogeneic rejection in drug loaded red blood cells. 75 Currently, several research teams have utilized this method to modify hematopoietic stem cells (HSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs) to generate new RBCs, 49 and these new RBCs have been applied in the treatment of diseases such as thalassemia, malaria, autoimmune diseases, and tumors. 50 Zhang et al. transformed RBCs into artificial antigen presenting cells, enabling them to present peptides segments bound to the major histocompatibility complex I, the co-stimulatory ligand 4-1BBL, and interleukin IL-12. The research outcomes indicated that this alteration decreased the circulating toxicity of 4-1BBL and IL-12 and instigated memory immunity along with antigen spreading, making it an effective in vivo cellular immunotherapy. 51 In clinical applications, Rubius Therapeutics company once used gene engineered RBCs method to treat phenylketonuria. However, in the phase Ib clinical trial of gene engineered RBCs expressing phenylalanine ammonialyase (RTX-134), unexplained data emerged. Researchers attributed this phenomenon to the low cell dose usage and the insufficient sensitivity of flow cytometry for detecting circulating cells. 75 After efficient modification, gene engineered RBCs can utilize the transcription and translation systems of stem cells themselves to express corresponding drugs, meanwhile possessing good biosafety. However, there are still some problems such as high modification costs and complicated operations. RBCs derived extracellular vesicle delivery system Extracellular vesicles are nanovesicles secreted by cells, with a size ranging from 30 nm to 1000 nm. 76 Erythrocyte membranes nanovesicles (EMNVs) share similar characteristics with red blood cells and carry proteins, lipids, and microRNAs (miRNAs) from the parent red blood cells. 77 However, there are also some differences, such as EMNVs carrying more tumor susceptibility gene 101 (TSG101), 78 and expressing less hemoglobin. 4 The main methods for generating EMNVs include the chemical substance method and the extrusion method. The chemical substance method is a way to induce cells to release extracellular vesicles in the culture medium by adding chemical reagents, and it is the most commonly used approach for generating EMNVs at present. The chemical substance method is based on the mechanism of triggering the influx of calcium ions to activate phospholipid transferases and thus induce the vesiculation of RBCs. 79 Previous studies have shown that calcium ion modulators such as ionomycin, 80 calcium chloride/ethylenediaminetetraacetic acid 81 and lysophosphatidic acid 82 can promote the generation of EMNV. The extrusion method involves first rupturing RBCs in a hypotonic solution to release their contents, followed by repeated washing to obtain a membrane-like structure, and then repeatedly extruding through a polycarbonate membrane or by ultrasound to form vesicular structures. 52 For instance, Biagiotti et al. prepared EMNVs carrying dextran and small interfering RNA (siRNA) via the extrusion method, and the characterization showed the particle size was between 100 nm and 300 nm, with high specificity, and each red blood cell could produce up to 450 nanovesicles. 53 The electroporation method is an emerging method for loading nucleic acids into EMNVs. Firstly, calcium ionophores are used to stimulate the generation of EMNVs, and then nucleic acids are loaded into EMNVs through an electroporation instrument. For example, Usman et al. studied the loading of miRNA, Cas9 RNA, and guide RNA (gRNA) into EMNVs via this method to achieve specific RNA delivery. 54 The RBCs derived extracellular vesicle method retains the advantages of RBCs targeting and long circulation time, while overcoming the disadvantage of large size in the RBCs delivery system. However, the extrusion process may lead to the displacement or loss of proteins on the membrane surface. Moreover, the entry of calcium ion modulators and the exposure of phosphatidylserine caused by the rearrangement of cell membranes will make EMNVs more likely to be recognized and cleared by the reticuloendothelial system. 82 In addition, the yield and loading efficiency of EMNVs are also challenging issues. Application of RBCs drug delivery systems in diseases Red blood cells drug delivery systems are widely used in cancer, central nervous system diseases, infectious diseases, metabolic diseases, toxin clearance and other fields due to its development maturity, richness of delivery methods, biological feature such as long circulation, targeting and biocompatibility ( Figure 4 ). Application in cancer In the treatment of tumors, problems such as non-targeted toxicity caused by drug damage to normal tissues, drug’s short half-life, and easy clearance by the reticuloendothelial system are quite thorny issues. However, the advantages of long circulation and targeting ability of red blood cells bring hope for solving these problems. Red blood cell drug delivery system has been applied in a variety of tumors such as head and neck cancer, cervical cancer, colon cancer, gastric cancer, breast cancer, ovarian cancer, melanoma, lung cancer, liver cancer and leukemia, and has been used for the delivery of small molecule drugs, peptides, photosensitizers, viruses and nanoparticles (Table 2) . 41, 45, 83-89 Bai et al. constructed a RBCs nanovesicle modified with the peptide iRGD-TRP-PK1, which was loaded with the chemotherapy drug doxorubicin. iRGD was a tumor targeting peptide that could bind to head and neck cancer cells with high expression of integrin αVβ3, achieving tumor targeting of the nanodelivery system. Meanwhile, TRP-PK1 was inserted into the lipid bilayer of the RBCm, forming a potassium ion leakage channel on the RBCs vesicle membrane. In the normal tissue environment, the extracellular fluid and the internal potassium ion concentration of the vesicle were similar, and the RBCm remains stable, preventing drug release. However, in the tumor cell environment, the high intracellular potassium concentration formed a concentration gradient with the internal vesicle, causing the RBCm to rupture and release the drug specifically into the tumor cells, reducing the toxic side effects on non-tumor tissues. This delivery system utilized the dual functions of TRP-PK1-iRGD peptides in tumor targeting and intracellular release, as well as the modifiability and long circulation of RBCs, to improve the targeted delivery efficiency of doxorubicin, effectively killing tumor cells while reducing side effects on normal organs. 83 Apart from the surface modification of red blood cells, 84 the hitchhiking function of red blood cells can also be utilized to target drugs to the lungs. Liu et al. developed a red blood cell delivery system for oncolytic viruses (OV) to treat tumor lung metastasis (Figure 5) . OV was adsorbed on the surface of RBCs through electrostatic interaction to avoid phagocytosis by the immune system and prolong its circulation time in the blood circulation. When passing through the narrow pulmonary capillaries and tumor metastasis nodules, OV was sheared and retained in the lungs, enhancing the targeting of lung metastasis. Moreover, the positively charged polyethyleneimine (PEI) modified on the surface of OV could promote the uptake of OV by tumor cells, enhance transfection efficiency, and improve the anti-tumor lung metastasis and immune efficacy. This method reduced the toxicity of OV to normal liver tissue and systemic cytokine storms, prolonged the circulation time of OV, and could reach deep into the lungs for treatment, providing a new approach for tumor lung metastasis. 41 Furthermore, inspired by the excellent properties of red blood cells, Li’s team constructed a red blood cell like drug delivery system. The main component of RBCs is hemoglobin, which is composed of heme and globin. Heme contains a porphyrin structure that can form stable complexes with various metal ions. Additionally, the porphyrin structure is a common photosensitizer that can generate ROS under light irradiation, damaging mitochondrial DNA. Globin is easily cleared by macrophages with high expression of CD163, a typical characteristic of M2 type tumor associated macrophages (TAMs), enabling the targeting of M2 type macrophages. Based on these characteristics of RBCs, the research team constructed a manganese porphyrin complex, externally wrapped with RBCm and internally encapsulated with globin and perfluorohexane (PFH). After accumulation at the tumor site through the enhanced permeability and retention (EPR) effect, targeted ultrasound caused PFH to vaporize, leading to the rupture of the RBCm and the release of the contents. Globin targeted M2 type TAMs, while porphyrin generated ROS under light exposure, damaging mtDNA and activating the cGAS-STING pathway. Manganese ions colud enhance the enzymatic activity of cGAS, synergistically activating the cGAS-STING pathway, promoting the repolarization of TAMs and the secretion of pro-inflammatory cytokines, inducing the maturation of dendritic cells and the differentiation of T cells, thereby achieving a powerful anti-tumor immune response. 45 Application in central nervous system diseases The presence of the blood brain barrier (BBB) is crucial for preventing harmful substances from entering the brain, but it also restricts the delivery of most small molecules, peptides, proteins, and nucleic acids, thereby limiting the treatment of neurodegenerative diseases, brain tumors, brain infections, and strokes. 90 Developing a method to cross the BBB and target the central nervous system (CNS) is essential, and red blood cells offer a promising approach. At present, red blood cell drug delivery systems have been applied in a variety of central nervous system diseases such as glioblastoma, encephalomyelitis, ischemic stroke, Alzheimer’s disease, traumatic brain injury, and drug addiction (Table 3) . 91-96 Song et al. developed a tetrahedral DNA based biomimetic nanomedicine delivery system encapsulated by a hybrid cell membrane composed of RBCm, macrophage membranes, and tumor cell membranes for treating glioblastoma (GBM) (Figure 6) . The hybrid cell membrane integrated the specific biological functions of these three cell types. The angiopep-2 targeted peptide modified RBCm served as a biomimetic shell with targeting ligands, extending nanoparticle circulation time while facilitating BBB crossing and GBM targeting. The macrophage membrane retained the surface protein profile and biological interface characteristics of its source cells, protecting nanoparticles from immune cell phagocytosis. The folic acid modified tumor cell membrane enabled homologous targeting of tumor cells. This hybrid cell membrane enveloped a tetrahedral DNA nanostructure loaded with the hydrophobic chemotherapy drug LMP, protecting the DNA from enzymatic degradation, prolonging its half-life, and addressing issues such as short circulation times, difficulty crossing the BBB, and low drug permeability. Research results demonstrated that this biomimetic nanodelivery system successfully crossed the BBB and targeted tumor cells, achieving precise GBM treatment and improving therapeutic efficacy. 91 In addition to the biomimetic targeting effect of red blood cells, the hemoglobin within red blood cells has inspired novel delivery systems for CNS diseases. Hemoglobin released extracellularly after intracerebral hemorrhage or from dead cells can induce upregulation of heme oxygenase-1 (HO-1), which metabolizes hemoglobin into carbon monoxide and bilirubin, producing ROS scavenging and anti-inflammatory effects. Given the rich hemoglobin content in RBCs, they may serve as an exogenous source to induce HO-1 upregulation. Based on this hypothesis, Yin et al. constructed a red blood cell nanovesicle dual modified with RVG29 targeting peptide for brain vascular endothelial cells and MG1 targeting peptide for M1 type microglia. The RVG29 peptide directed the nanovesicles to the brain, and then crossed the BBB. In the inflammatory environment, the disulfide bond linked RVG29 peptide responded to high ROS concentrations, exposing the MG1 peptide and targeting M1-type microglia. Subsequently, microglia engulfed the nanovesicles and released the internal hemoglobin. Hemoglobin induced M2 polarization of microglia via HO-1 upregulation, promoting endogenous anti-inflammatory cytokine expression and antioxidant production of carbon monoxide and bilirubin, alleviating brain inflammation and demonstrating therapeutic benefits in middle cerebral artery occlusion (MCAO) and experimental autoimmune encephalomyelitis (EAE) models. 92 Furthermore, hemoglobin in red blood cells acts as a natural oxygen regulator, reversibly binding or releasing oxygen. Liu et al. utilized this mechanism to construct a polymer based nano RBCs to reshape the metabolic microenvironment during acute ischemic stroke (AIS). AIS involves two phases: hypoxia ischemia and reperfusion. During hypoxia ischemia, HIF-1α responds to hypoxic conditions, initiating VEGF expression, leading to BBB disruption and microcirculation damage. During reperfusion, excess oxygen levels produce ROS, causing mitochondrial damage and blocking ATP generation. Moreover, dysregulation of key glucose metabolism signaling molecules Akt/GSK-3β reduces glucose uptake and glycolysis. Liu designed a microthrombus targeted CREKA peptide modified polydopamine nanocage loaded with hemoglobin and moxifloxacin. CREKA targeted the nano RBCs to ischemic sites. During hypoxia ischemia, the nano RBCs released oxygen in response to hypoxic conditions. During reperfusion, they bound excess oxygen to block ROS production and promoted microglial polarization. In the recovery phase, the nano RBCs regulated the Akt/GSK-3β pathway by releasing moxifloxacin, activating glucose metabolism and protecting the BBB. Research results showed that this approach positively regulated key metabolic factors, including oxygen balance and glucose metabolism, achieving favorable short-term and long-term therapeutic outcomes in both permanent middle cerebral artery occlusion (pMCAO) and transient middle cerebral artery occlusion (tMCAO) mouse models. 93 Application in infectious diseases Delivering drugs to the infection site is crucial for treating infectious diseases. However, current drug delivery systems can only transport a small portion of the drugs to the infection site after systemic administration, and most of them cannot penetrate the biofilm and are easily cleared by the immune system, resulting in unsatisfactory clinical outcomes. 97 Therefore, developing an efficient drug delivery system for infectious diseases is of great significance. Currently, red blood cell drug delivery systems have been used in bacterial infections, viral infections and parasitic infections, and various methods have been adopted to treat or prevent these diseases, such as RBCs hitchhiking method, RBCs derived extracellular vesicles, RBCm coating method and low osmotic pre-swelling method （Table 4） . 42, 98-105 Yu et al. constructed a zwitterionic polymer (poly(2-(N-oxide-N,N-diethylamino)ethyl methacrylate), OPDEA) micelle, which adsorbed onto the RBCm through phospholipid affinity and encapsulated the commonly used antibiotic clarithromycin inside. After intravenous injection, the OPDEA micelles hitched a ride on RBCs to ensure prolonged circulation. Due to the stronger interaction between OPDEA and N-acetylmuramic acid, the main component of the Gram-positive bacterial cell wall, compared to the phospholipids of RBCs, the micelles detached from RBCs and penetrated deep into the biofilm upon reaching the infection site, releasing clarithromycin to exert its antibacterial effect. This nano-delivery system demonstrated excellent anti-inflammatory effects in peritonitis and pneumonia models. Compared to free clarithromycin, which only retained 5% of the initial dose after 2 hours of circulation, the delivery system still had over 16% of the initial dose after 10 hours of circulation. Additionally, compared to PEG-modified fluorescein, the OPDEA-modified delivery system showed significantly enhanced accumulation at the peritonitis and pneumonia sites. 42 Although some success has been achieved in improving the targeted drug delivery efficiency through red blood cell hitchhiking, current strategies for coupling nanodrugs to the cell surface mainly rely on covalent bonds, physical adsorption, or specific ligand-receptor interactions, which often involve complex synthetic processes and cannot achieve controlled drug release. 106 Based on this, Li et al. designed an inflammation responsive bio-orthogonal supramolecular coupling strategy by taking advantage of the weakened binding affinity between β-cyclodextrin (β-CD) and ferrocene (Fc) under oxidative conditions and the high ROS levels in the inflammatory microenvironment. Specifically, β-CD was linked to DSPE-PEG and inserted into the phospholipid bilayer of RBCs, while Fc was modified on liposomes and encapsulated with the anti-inflammatory drug curcumin. Through the host-guest behavior of β-CD and Fc, Fc-modified liposomes were coupled to the surface of RBCs. This delivery system utilized the red blood cell hitchhiking effect to prolong the circulation time of liposomes, and selectively dissociated to release the anti-inflammatory drug in the high ROS environment of pneumonia, promoting high accumulation of the drug at the inflammatory site and effectively treating pneumonia by down-regulating inflammatory cytokines and improving pulmonary edema and repolarizing pro-inflammatory macrophages. 98 Red blood cells not only own drug delivery functions but their immune functions have also been recently exploited by researchers for disease prevention and treatment. 107 Studies have shown that the phosphatidylserine receptor was a PS-binding protein that mediated the uptake of apoptotic bodies, and many enveloped viruses used the mechanism of PS binding to its receptor to adhere and internalize into cells. As the cell surface receptor of SARS-CoV-2, the PS receptor enhanced the binding and infection toward human lung cells by the virus 108 RBCm contained abundant PS, which might be an effective mean to competitively suppress the entry of SARS-CoV-2 into cells. Migara et al. created a delivery system based on extracelluar vesicles derived from RBCs that internally contained Antisense oligonucleotides (ASOs) targeting the key SARS-CoV-2 gene ( Figure 7 ). In vitro and in vivo experiments, EMNVs loaded with ASOs showed better antiviral effects than free ASOs, significantly improving the survival rate of infected mice. Mechanism studies demonstrated that the antiviral effect of EMNVs was not through direct binding with the virus or blocking the interaction of S1-ACE2, but through competition with the virus for PS receptors, in which T cell immunoglobulin mucin domain-1 (TIM-1) played an important role in EMNV S -mediated viral neutralization, and its expression level affected EMNVs binding, viral infection and neutralization ability. In addition, EMNVs, as a carrier, was superior to synthetic nanoparticles in terms of immunogenicity, toxicity and biocompatibility. 99 Applications in metabolic diseases Enzymes are catalysts for reactions in living organisms and play a significant role in maintaining the normal physiological functions. The deficiency or loss of function of key enzymes can disrupt metabolism and subsequently lead to metabolic diseases. Clinically, therapeutic enzymes are used to address these issues. However, free enzymes have drawbacks such as poor stability in vivo and strong immunogenicity, which limit the therapeutic effects. Red blood cells have a long circulation time and low immunogenicity. As carriers for enzymes, they can protect enzymes from being cleared by the immune system and release enzymes slowly, enabling the enzymes to function in the body. 27, 109 At present, RBCs drug delivery systems have been used in a variety of metabolic disorders such as glucose metabolism, purine metabolism, lipid metabolism and amino acid metabolism, and various methods have been adopted for the treatment of metabolic diseases, such as RBCs hitchhiking, receptor mediated method, RBCm membrane coating method, and low osmotic dialysis method (Table 5) . 39, 110-114 Diabetes is a systemic metabolic disease caused by the inability of the pancreas to secrete insulin or insufficient insulin secretion. Patients must rely on exogenous insulin for treatment. However, frequent insulin injections multiple times a day can lead to subcutaneous fat atrophy, allergic reactions, and local hypoglycemic symptoms. Therefore, the design of more precise and long-acting insulin formulations is of great significance. Although most of the formulations developed in recent years have intelligent responsive release functions, they cannot achieve long circulation. 115 Xu et al. designed a red blood cell mimicking glucose-responsive release system that could significantly improve the long circulation effect of insulin. They used acid-responsive materials to simultaneously load glucose oxidase, catalase and insulin. And the acid-responsive property was transformed into glucose- responsiveness through enzymatic reactions, allowing insulin to be released more precisely according to changes in blood glucose levels. Most importantly, the glucose- responsive nanoparticles were innovatively coupled to RBCs using electrostatic adsorption, thereby obtaining the natural long circulation characteristics of RBCs. The results showed that this system could keep the blood glucose of diabetic mice within the normal range for 48 hours and below 380 mg/dL for 72 hours. 110 Hyperuricemia is a metabolic disease caused by purine metabolism disorders or impaired uric acid (UA) excretion. 116 Currently, the measures for treating hyperuricemia include using xanthine oxidase inhibitors to inhibit UA production, using renal tubular reabsorption blockers to promote UA excretion, or using urate oxidase (UOX) to metabolize UA into allantoin for excretion. However, these methods have problems such as significant side effects, cumulative liver and kidney toxicity, insufficient enzyme activity, and low bioavailability. 117 Some researchers have attempted to reduce the immunogenicity of UOX by PEGylation, 118 encapsulate UOX in albumin hydrogels to extend circulation time, 119 and use catalase (CAT) like nano-enzymes to accelerate UA degradation. 120 However, the continuous UA degradation effect still depends on the enzyme activity of UOX, the removal rate of by-products, circulation time, and in vivo stability. Li et al. developed ZIF-8 nanoparticles encapsulating UOX-CAT cascade enzymes, which were assembled on the surface of RBCs through tannic acid ligands to integrate and solve the above problems ( Figure 8) . After intravenous injection of this RBCs delivery system, UA molecules in the blood were degraded in the UOX-CAT cascade catalytic reaction, and the high oxygen environment around the RBCs further accelerated the degradation of UA. Meanwhile, the encapsulation effect of ZIF-8 and the RBCs hitchhiking effect protected UOX from being degraded by enzymes in the body and being cleared systemically. The research results showed that this RBCs delivery system could normalize the UA level in acute hyperuricemia model mice within 2 hours, and the elimination half-life of UOX in the nano RBCs group was more than three times that of the free group, demonstrating good short-term and long-term therapeutic effects for hyperuricemia. 39 n addition, Ban et al. developed a more direct method for delivering uricase to red blood cells for the treatment of hyperuricemia. Unlike surface modification techniques, this team directly encapsulated UOX into the internal space of RBCs through a simple low osmotic pre-swelling method. The RBCm protected the intracellular UOX from degradation by proteases while not affecting the permeability of small molecules such as UA. The hydrogen peroxide produced during the UA oxidation process could be rapidly decomposed in the presence of hemoglobin that mimics CAT, avoiding unnecessary oxidative damage. Importantly, RBCs hided the immunogenicity of uricase by blocking antibody mediated macrophage clearance, making long-term repeated administration also effective. In a monosodium urate (MSU) induced acute gout model, this delivery method significantly reduced joint edema and inflammation, with low systemic toxicity, making it a safe and effective treatment strategy. 121 Application in toxin clearance In the field of poisoning treatment, the most common approach currently is to rapidly reduce the level of toxins in the body through various means. The current broad spectrum purification strategies mostly rely on porous materials like activated carbon to adsorb toxins, but this method is passive adsorption, lacks selectivity, and various substances in the circulatory system can interfere, reducing the adsorption rate and affecting the treatment effect. 122 Red blood cells offer a possible solution to the above problems. Researchers have developed a series of RBCs detoxification strategies for the detoxification of various types of toxins, including small molecule toxins, harmful enzymes, bacterial toxins, and heavy metals, and have explored new preparation methods such as lipid insertion, ultrasonic, and extrusion (Table 6) . 123-128 For instance, paraquat circulated through the body tissues with the blood, and its strong positive charge made it easy to accumulate in tissues and undergo redox reactions, generating ROS and damaging tissues. 129 The blood purification techniques currently used in clinical practice could only remove a small amount of paraquat from the blood. After blood purification, paraquat could still be released from stored tissues, resulting in unsatisfactory treatment effects. 130 Li et al. designed a nanoparticle modified RBCs detoxifier based on the molecular structure and accumulation characteristics of paraquat. Carboxylic acid functionalized pillar [6] arene (WP6) molecules were encapsulated in Janus dendrimer amphiphile (JDA) molecules and modified on the surface of RBCs through lipid insertion technology to achieve long circulation. When positively charged paraquat molecules contacted with negatively charged detoxifiers, a non-covalent guest exchange reaction occurred in the blood for continuous paraquat detoxification. The research results showed that this detoxifier exhibited excellent active toxin capture ability in vitro and in vivo and could provide good protection for the lungs and kidneys. 123 The biomimetic drug delivery platforms that combine nanomaterials with different functions and cell membranes from different sources can capture chemical reagents, bacterial toxins, autoantibodies, inflammatory factors and viruses. 131 However, research on directly inhibiting the delivery of harmful enzymes is still relatively limited, as the bait in enzyme reactions has a sacrificial nature rather than an inhibitory nature. To overcame this limitation, Zhang et al. developed a RBCm coated phospholipase A2 (PLA2) enzyme inhibitor delivery platform. PLA2 enzymes catalyze the cleavage of glycerophospholipids, generating free fatty acids and lysophospholipids, and play a crucial role in phospholipid digestion, host defense, and signal transduction. However, under pathological conditions, elevated PLA2 activity can lead to poisoning and autoimmune diseases. Many small molecule compounds and antibody inhibitors have been developed for the treatment of these diseases, but these inhibitors have side effects such as poor solubility and off-target toxicity. Zhang et al. utilized the biomimetic effect of RBCm, coated on the surface of PLGA, and simultaneously inserted melittin and oleyl-oxyethyl-phosphorylcholine (OOPC) into RBCm. Melittin acted as a PLA2 attractant, attracting PLA2 together with membrane lipids. OOPC served as an enzyme inhibitor to kill PLA2 activity. The research results demonstrated that this RBCs delivery system effectively inhibited PLA2-induced hemolysis in mice, showing significant survival benefits and no obvious toxicity, making it a safe and effective enzyme inhibitor delivery technology. 124 The isolation mechanism of the detoxification effect of cell membranes on nanoparticles is the stoichiometric binding between toxins and cell membranes. 132 However, this stoichiometric binding mechanism is inefficient and cannot achieve high detoxification of lethal organophosphorus compounds (OPs) and other chemicals. 133 To achieve in vivo interception of toxic OPs, Qin et al. developed a RBCs nano-cleaner ( Figure 9 ). That is, RBCm were extracted by osmotic method and then covered on the surface of PLGA through co-extrusion technology. Organophosphorus hydrolase (OPH) was modified by DSPE-PEG and inserted into RBCm to construct a novel OPH modified nano-cleaner. The surface of RBCm contained abundant acetylcholinesterase (AChE), which, in synergy with OPH, protected AChE from OPs damage through targeted binding and catalytic degradation. At the same time, the encapsulation effect of RBCs increased the circulation half-life of OPH, reducing the need for high doses and frequent administration. Experimental results using methyl paraoxon (MPO) as a model OPs showed that this nano-cleaner protected the AChE on RBCm produced by itself from OPs attack, reduced the symptoms of poisoning in mice, increased survival rate, and did not cause obvious toxicity, making it a novel and effective dual-mode antidote. 125 Applications in other diseases After the implantation of cardiac devices, monocyte driven inflammatory responses often lead to cardiac remodeling and fibrosis, which in turn cause serious complications such as arrhythmia and heart failure. 134 In recent years, N6-methyladenosine (m6A) RNA methylation, as an important post-transcriptional regulatory mechanism, has been confirmed to play a significant role in a variety of cellular processes, including cell proliferation, differentiation and immune responses. 135 However, the specific role and mechanism of m6A methylation in monocyte mediated cardiac fibrosis remain unclear. Li et al. investigated the effects of inhibiting m6A modification on cardiac fibrosis and function by using RBCs vesicle nanodrug delivery system with the m6A methylation modification enzyme inhibitor STM2457 (Figure 10). The research results showed that the expression of m6A methylation modification enzymes in peripheral blood mononuclear cells was significantly upregulated in patients with persistent conduction block after cardiac device implantation. By taking advantage of the natural phagocytic ability of monocytes towards RBCs vesicles, the nanodrug delivery system successfully targeted STM2457 to monocytes, significantly reducing the m6A modification level in cardiac macrophages, effectively inhibiting cardiac fibrosis and inflammatory responses, and showing no obvious toxic effects on the liver, lungs, spleen, and kidneys after long-term use. 136 Osteoporosis is caused by an imbalance in the activity of osteoblasts and osteoclasts. Inhibiting the formation and activity of osteoclasts can reduce bone resorption and reverse osteoporosis. 137 Studies have shown that inhibiting miR-214 in osteoclasts and osteoclast precursors is an effective method for treating osteoporosis. 138 However, nucleic acid drugs are unstable in physiological environments and are easily degraded by nucleases. The classic delivery of nucleic acid drugs is achieved through adenoviruses or liposomes, but these methods have the disadvantages of immunogenicity, cytotoxicity, and lack of targeting. 139 Moreover, the RBCs drug delivery system has the merits of low immunogenicity, low toxicity and high engineerability, and is expected to improve the above problems. Xu et al. developed a strategy for delivering anti-miR-214 miRNA using extracellular vesicles derived from RBCs. EMNVs were generated by calcium ion stimulation, and the surface of EMNVs was modified with CP05 peptide, which specifically bound to transmembrane protein CD63, and osteoclast targeting peptide TBP through non-covalent protein peptide interactions. CP05 peptide endowed the surface modification ability of EMNVs through specific binding, and the non-covalent modification did not affect the function of surface receptors of EMNVs. TBP peptide targeted osteoclasts, improving the therapeutic efficiency of miRNA and reducing damage to other cells. 140 Conventional red blood cell drug delivery systems involve extracting red blood cells from animals or humans, engineering them in vitro, and then reinfusing them into the body for the treatment of related diseases. Recently, a new study has developed a method that actively utilized the body’s own RBCs for drug delivery. Specifically, researchers constructed a nanoparticle based on chitosan and sodium heparin, modified with RGD peptides and 2-(N-oxide-N,N-diethylamino)ethyl methacrylate (ODE), and loaded with urokinase type plasminogen activator (uPA) for the treatment of thrombosis. 141 Chitosan was a natural cationic polysaccharide that was pH-sensitive and could simultaneously respond to the acidic and tense microenvironment of the thrombus site, enabling drug-responsive release. 142 Sodium heparin, a frequently utilized anionic polysaccharide anticoagulant in clinical practice, could prevent thrombus recurrence by effectively inhibiting the activity of thrombin and prothrombin and enhancing the activity of antithrombin. 143 RGD peptides could link with the αIIbβ3 protein presented on the surface of activated platelets, enabling the nanoparticles to target the thrombus site and prevent negative influences on normal tissues. 144 ODE was a linker, and the N-oxide part could interact with phosphatidylcholine or phosphatidylethanolamine on the RBCm through the agency of numerous weak hydrogen bonds and electrical charges, actively binding to RBCs and resisting non-specific protein adsorption during the conveyance of drugs in the blood. 141 uPA was a thrombolytic agent that could energetically dissolve existing thrombi and trigger the fibrinolytic system, but it had problems of low bioavailability, short half-life, and narrow therapeutic window. 145 The constructed nanoparticles could actively adhere to RBCs in vivo and convey uPA concealed by RBCs, thereby lengthening their blood circulation time and half-life. The thrombosis mouse models of the tail vein and abdominal aortic regions confirmed the excellent targeting and thrombolytic capabilities of these nanoparticles. 141 Utilizing the body’s own RBCs for drug delivery overcame the influence of different RBCs sources and blood types, providing the possibility for large scale production of the product, but the in vivo RBC adhesion efficiency still needed further research. In addition to delivering therapeutic drugs, contrast agents can also be attached to the surface of RBCs delivery systems or encapsulated within them for imaging or for the integration of diagnosis and treatment. 146 Compared with free contrast agents, RBCs delivery systems usually improve the efficiency of imaging procedures. On one hand, they enhance the biocompatibility and stability of contrast agents and extend their half-life in the body. On the other hand, they manipulate the distribution of imaging agents to achieve more effective imaging of target tissues. 147 Zheng et al. prepared a photothermal diagnostic platform based on semiconductor polymer nanoparticles, whose surface was camouflaged with RBCm for near-infrared light guided photoacoustic imaging and photothermal therapy. The prepared nanoparticles had good near-infrared light absorbance, providing enhanced photoacoustic imaging signals for tumor imaging. At the same time, they had excellent high photothermal conversion ability, providing enhanced photothermal killing effects for tumor treatment. The RBCm camouflage improved the biocompatibility of the nanoparticles, reduced retention in the reticuloendothelial system, prolonged blood circulation, and improved tumor accumulation (Figure 11) . 148 Srivastava et al. constructed a dual modal plasmonic nanoparticle based on gold nanoparticles for simultaneous surface enhanced Raman scattering and photoacoustic imaging of deep tumor models. The encapsulation of RBCm could reduce protein adsorption and cellular uptake, increased tissue penetration depth, and did not affect imaging signals. The tumor targeting peptide RGD was inserted into the RBCm to target tumor tissues. In the image-guided resection of simulated tumor models, the dual imaging agent demonstrated excellent functionality. Photoacoustic imaging was used to determine the precise tumor location, while surface enhanced Raman scattering spectroscopy signals were used for tumor identification and differentiation, providing an effective approach for intraoperative tumor detection and resection. 149 In addition to single drug treatment methods, combined treatment approaches involving multiple novel modalities such as photothermal, photodynamic, microwave, magnetic, gas, and immunotherapy are increasingly attracting attention. Multifunctional nanomaterials with unique physicochemical properties offer the possibility of integrating various treatment methods, 150 and RBCs preparations also play an important role in this context. 151 Cao et al. constructed a multifunctional nanomedicine for chemotherapy, photothermal therapy, photodynamic therapy, and immunotherapy by encapsulating ultrasmall photothermal agents (black phosphorus quantum dots), the chemotherapeutic drug paclitaxel, and the immunomodulator polydopamine in a hybrid membrane camouflaged liposome formed by the fusion of RBCm and tumor cell membranes to treat breast cancer and inhibit lung metastasis. 152 Chen et al. developed a gallium ion based metal organic framework loaded with the thymidylate synthase inhibitor 5-fluorouracil and covered with a hybrid membrane formed by the fusion of RBCm and platelet membranes for the treatment of in situ breast tumors and lung metastases. The hybrid cell membrane targeted the nanoplatform to the tumor site, gallium ions possessed microwave responsiveness and induced immunogenic cell death, and 5-fluorouracil inhibited the synthesis of DNA and RNA in tumor cells. This nanoplatform was a comprehensive platform combining microwave therapy and immunotherapy, overcoming the limitations of single microwave therapy in controlling tumor metastasis and residual tumors. 153 Weng et al. proposed a combination of magnetic hyperthermia, which utilized the innate magnetic responsiveness of RBCs containing ferrous ions, and active nanotherapy using sperm cells loaded with thrombolytic agent urokinase and anticoagulant dipyridamole for the treatment of thrombosis (Figure 12) . 154 Sperm cells, due to their microtubule flagella and rheological properties, 155 could navigate to high viscosity thrombus sites and penetrate into the blood clot. Meanwhile, the high density of RBCs at the thrombus site generated heat under an external alternating magnetic field, improving the thrombus environment and extending the effective therapeutic window for thrombolysis. 156 Additionally, magnetic hyperthermia promoted the release of therapeutic drugs from nanoparticles and the production of extracellular vesicles and anti-inflammatory factors by M2 macrophages. 154 This combined effect reduced the neurotoxicity of traditional iron oxide nanoparticles, 157 and offered advantages in immune regulation, anti-inflammation, and neuroprotection. Wang et al. designed a photothermal agent prussian blue, loaded with the nitric oxide donor BNN6 and camouflaged with RBCm to achieve the synergistic treatment of tumors through photothermal therapy and gas therapy. Prussian blue was not only a mesoporous nanocarrier but also had excellent photothermal effects. BNN6 was photothermal sensitive and generated nitric oxide under near-infrared light irradiation to induce tumor cell apoptosis. The RBCm prevented premature drug leakage and strengthened the delivery of nanoparticles to the tumor site, augmenting the efficiency of the combined therapy. 158 In brief, red blood cell drug delivery systems have been widely applied in various diseases. This article hopes to provide some reference for the subsequent research on designing personalized red blood cell delivery systems for precise disease treatment, as well as for choosing appropriate drug delivery methods and combined strategies. Conclusions and perspectives Red blood cells, being the most plentiful blood cells in the body, exhibit unique physiological characteristics such as large surface area, long circulation time, high targeting ability, and good biocompatibility. Utilizing them as drug delivery carriers can achieve extended circulation time of drugs in the body, avoid phagocytosis by the immune system, and target specific sites. However, during the preparation of RBCs based drug delivery systems, there may be displacement or loss of surface proteins on the RBCm, which can lead to the loss of long circulation ability, reduced deformability, and potential risks of hemolysis and thrombosis. Currently, many studies have explored the application of RBCs drug delivery systems in various diseases, but their successful translation into clinical practice is rare. The limitations mainly lie in the difficulty of large-scale production and strict storage and transportation conditions. Due to the existence of different blood types in humans, delivery systems from different blood types can easily induce coagulation and immune reactions. In research, individual samples from different individuals are often processed separately, making large-scale production difficult. 159 Using O-type donor blood as the source of RBCs may help produce ready-to-use RBCs drug delivery products. Additionally, due to the complex production process and significant individual differences, there are still many challenges in producing RBCs products with high consistency and reproducibility. 75 Studies have shown that the shelf life of RBCs carriers prepared from autologous blood was 30 minutes, while that of those prepared from allogeneic blood was 72 hours. Both of these times were very short, posing significant difficulties for clinical use. 160 In the future, continuous improvement of preparation techniques and optimization of extraction and purification processes are needed to increase the production rate of RBCs and address batch-to-batch variations. Moreover, to maintain the integrity of the RBCm, fresh or properly stored RBCs are required, as they cannot be preserved for long periods through low temperature freezing or liquid nitrogen, as this would affect the quality of the RBCm and the loading efficiency of drugs. Additionally, aged or damaged RBCs carry risks such as hemoglobin leakage and exposure of phosphatidylserine, which can easily lead to recognition by the immune system and activation of immune responses. 11 Studies have confirmed that methods such as liposome encapsulation, 161 ultra low temperature storage, 162 trehalose treatment, 163 and pre-freezing oxidation method 164 have good protective effects on the storage of RBCs. In conclusion, red blood cells as drug delivery systems have significant advantages and provide new methods and ideas for the field of drug delivery. Despite facing challenges in production, storage, and clinical application, with continuous research and technological advancements, red blood cells still hold considerable potential in drug delivery and disease treatment. CRediT authorship contribution statement Huizi Deng : Investigation, Formal analysis, Writing-original draft, Software, Visualization. Xiaobei Cheng : Investigation, Formal analysis, Writing-review & editing. Yi Li: Investigation, Formal analysis. Yameng Ling : Investigation. Yuli Wang : Investigation. Yang Yang : Writing-review & editing, Supervision, Conceptualization. Chunsheng Gao : Validation, Supervision, Conceptualization. Tables Table 1 Advantages and disadvantages of different red blood cell delivery methods Intracellular delivery system of RBCs Osmotic method Adjust different osmotic pressures to create pores in the RBCm Easy to operate and suitable for small molecule, biological agents, and smaller particle sizes nanoparticles Inability to control the release speed and extent of the drug 31-33 Electroporation method Apply pulsed electric fields to create pores in the RBCm Load drugs uniformly into RBCs Require sophisticated equipment and precise operation 34 Chemical substance method Use chemical substances to induce endocytosis in RBCs Less damage to RBCm and the intracellular substances are less likely to leak out Only applicable to drugs with both hydrophobic and hydrophilic groups 35 Transmembrane peptide method Use cell-penetrating peptides to load drugs Less damage to RBCm Drug loading capacity is limited by the binding ability of the cell-penetrating peptide to the drug 36 Microfluidic extrusion method Use a microfluidic device to load drugs Strong versatility and flexibility to load various drugs and convenient for large scale RBCs processing RBCs are easy to age after mechanical extrusion and the long-term circulation ability decreases 37 Drug delivery systems based on RBCm Chemical conjugation method Use chemical bond between drugs and RBCm Strong specificity to load drugs Might affect the biocompatibility and deformability of RBCs 38-40 Receptor mediated method Use receptor-ligand interaction between drugs and RBCm Less damage to RBCm Might lead to the binding of drugs to other non-specific cells and affect therapeutic efficiency 41,42 RBCs hitchhiking method Use non-covalent interactions between drugs and RBCm Suitable for nanoparticles and improve its accumulation in specific organs Weak connection between nanoparticles and RBCs might cause desorption 43-45 RBCm coating method Extract RBCm to coat drug loaded nanoparticles Strong flexibility and applicability Conformational changes in membrane anchored fragments may occur 46-48 Hybrid membrane fusion method Fuse RBCm with other biological membranes to load drugs Combine the advantages of multiple biological membranes Fusion process is prone to cause hemolysis of RBCs and the fusion efficiency is affected by multiple factors 49-51 Gene engineered RBCs delivery system Use stem cells and gene editing technology to create new RBCs Good biosafety High modification costs and complicated operations 52-54 RBCs derived extracellular vesicle delivery system Use extracellular vesicles secreted by RBCs to load drugs Retain the advantages of RBCs and overcome the disadvantage of large size in the RBCs delivery system Preparation process might affect the surface proteins and the yield and loading efficiency is challenging 55-57 Table 2 Application of red blood cell drug delivery system in cancer Head and neck cancer Doxorubicin RBCm coating method The targeted delivery efficiency of doxorubicin was improved, and the side effects on normal organs were reduced while the tumor cells were killed efficiently. 90 Cervical cancer HPV16 oncoprotein E6/E7 Chemical conjugation method It activated CD8+ T cells and reduced suppressor myeloid cells in the spleen, resulting in systemic antitumor activity. 91 Colon cancer Oncolytic Verus RBCs hitchhiking method It reduced the toxicity of OV to normal liver tissue and systemic cytokine storm, prolonged the circulation time of OV, and could be treated deeply in the lungs. 44 Gastric cancer Globin and perfluorohexane RBCm coating method Globin targeted M2-type TAMs, while porphyrins generated ROS upon illumination, destroyed mtDNA and activated the cGAS-STING pathway to achieve antitumor immunity. 48 Breast cancer Silybin RBCm coating method Based on fibroblast targeting, the delivery system reversed cisplatin resistance, reshaped the tumor microenvironment, and inhibited invasion and metastasis. 92 Ovarian cancer Indocyanine green Osmotic method Red blood cell nanoplatform containing cholesterol and folate improved fluorescence imaging of ovarian tumors. 93 Melanoma Docetaxel RBCm coating method Malaria mimetic red blood cell nanoparticles loaded with docetaxel retained the selectivity of naturally infected red blood cells and demonstrated tumor targeting and therapeutic effects in a melanoma model. 94 Lung/Liver cancer and melanoma Paclitaxel RBCm coating method The nanodrug delivery system showed good biocompatibility, prolonged the circulation time of paclitaxel, and enhanced its anti-tumor effect on lung cancer, liver cancer and melanoma. 95 Leukemia Meclofenamic acid, CM 272 and MOF RBCm coating method This nanosystem inhibited the growth of leukemia cells by targeting DNA and histone methylation, and activated cytotoxic and memory T cells by increasing the antigenicity of leukemia cells. 96 Table 3 Application of red blood cell drug delivery system in central nervous system diseases Glioblastoma LMP Hybrid membrane fusion method The hybrid cell membrane protected DNA from enzymatic degradation and solved the problems of short blood circulation time of hydrophobic drugs in vivo, difficulty in crossing the blood-brain barrier, and low drug permeability. 101 Encephalomyelitis and artery occlusion Endogenous hemoglobin Extrusion method RVG29 and MG1 peptides targeted cerebrovascular endothelial cells and M1 microglia cells, respectively. Hemoglobin initiated the M2 polarization of microglia, and promoted the production of antioxidants carbon monoxide and bilirubin, thereby alleviating the inflammatory environment in the brain. 102 Ischemic stroke Hemoglobin and methoxacin Non-covalent interaction Nano red blood cell could accumulate in the ischemic core, and cellular metabolic homeostasis and responsiveness were restored through regulation of oxygen balance, microglial polarization, activation of glucose metabolism, and neovascularization. 103 Alzheimer’s disease Carbon quantum dots and polydopamine RBCm coating method Red blood cell membrane, quantum dots and polydopamine, acting as long circulation, metal ion chelator and enzyme mimetics, respectively, alleviated neuroinflammation and improved behavioral defects in mice. 104 Traumatic brain injury Olaparib RBCm coating method Red blood cell membrane coated nanoparticles with C3 and SS31 peptides could effectively improve mitochondrial function and reduce neuronal cell death by targeted delivery of olaparib to brain mitochondria. 105 Methamphetamine addiction Resveratrol RBCm coating method Angiopep-2 modified red blood cell delivery system successfully delivered resveratrol to the brain and improved synaptic plasticity impaired by methamphetamine, which had good anti-addiction and neuroprotective effects. 106 Table 4 Application of red blood cell drug delivery system in infectious diseases Peritonitis and pneumonia Clarithromycin RBCs hitchhiking Red blood cell hitchhiking prolonged the circulation time of clarithromycin, and the accumulation of clarithromycin was greatly enhanced in the sites of peritonitis and pneumonia, showing a good anti-inflammatory effect. 45 Pneumonia Curcumin RBCs hitchhiking The red blood cell delivery system prolonged the circulation time of curcumin, allowing its responsive release at sites of inflammation and enhancing its efficacy in the treatment of pneumonia. 110 SARS-CoV-2 Antisense oligonucleotides RBCs derived extracellular vesicle Red blood cell derived extracellular vesicles could inhibit SARS-CoV-2 infection in a phosphatidylserine dependent manner, and the therapeutic efficacy of this antiviral therapy was further extended by antisense oligonucleotides targeting conserved regions of key SARS-CoV-2 genes. 111 Staphylococcus aureus infections Naftifine and hemoglobin RBCm coating method Naftifen disrupted staphylococcal xanthin biosynthesis. Hemoglobin reduced bacterial hydrogen sulfide levels, triggered lipid peroxidation, promoted neutrophil chemotaxis and respiratory burst. Red blood cell membranes altered bacterial lipid composition. This nanoplatform was highly effective in treating mice infected with antimicrobial resistant Staphylococcus aureus . 112 Multidrug resistant bacteria Perfluorocarbons and IR780 RBCm coating method The pore-forming toxins of methicillin-resistant Staphylococcus aureus , group A Streptococcus and Listeria monocytogenes were absorbed by the nanobubbles and enhanced bactericidal effect was achieved under gas therapy and photodynamic therapy. 113 Porphyromonas gingivalis Gallium Porphyrin RBCm coating method Red blood cells targeted nanoparticles to Porphyromonas gingivalis , then released gallium porphyrin, and produced reactive oxygen species to kill Porphyromonas gingivalis and alleviate periodontitis. 114 SARS-CoV-2 vaccine SARS-CoV-2 spike protein RBCm coating method This virus-like particle took advantage of the clearance of red blood cells by the liver and spleen to activate the immune system and induce antibody production. 115 African swine fever virus vaccine Viral antigen p54 RBCm coating method The splenic homing ability of red blood cells and the dendritic cell-targeting ability of mannose facilitated targeted delivery of antigens to splenic dendritic cells, activating both humoral and cellular immune responses. 116 Malaria Artesunate Low osmotic pre-swelling method The liver targeting of red blood cells promoted the aggregation of artesunate in the liver. And the embedding of red blood cells improved the sustained release of artesunate, achieving a better anti-malarial effect than the free drug. 117 Table 5 Application of red blood cell drug delivery system in metabolic diseases Diabetes Glucose oxidase, catalase and insulin RBCs hitchhiking Insulin had a longer circulation time and could be accurately released according to the glucose level to exert long-term blood glucose control. 126 Hyperuricemia Urate oxidase Receptor mediated method The red blood cell delivery system protected urate oxidase from enzymatic degradation and systemic clearance, and could rapidly normalize uric acid levels in mice with acute hyperuricemia. 42 Glucose/lipid metabolism disorder Blackberry polysaccharide selenium nanoparticles RBCm coating method The camouflage of the Red blood cell membrane improved the biocompatibility and bioabsorption of the nanoparticles, and the nanoparticles improved glucose and lipid metabolism through PI 3K/AKT and AMPK signaling pathways, respectively. 127 Atherosclerosis Probucol RBCm coating method Red blood cells increased the bioavailability of probucol. After being internalized by cells, probucol effectively reduced the levels of lipids and related metabolic enzymes in mice, thus alleviating atherosclerosis. 128 Phenylketonuria Phenylalanine ammonia lyase Low osmotic dialysis method Red blood cells improve the biocompatibility and circulation time of phenylalanine hydroxylase, reduced its biodegradability and immunogenicity, and effectively decreased the concentration of phenylalanine in mice. 129 Hyperammonemia Glutamine synthetase Low osmotic dialysis method The red blood cell delivery strategy retained glutaminase activity for at least 48 hours in the hyperammonemic mice and reduced blood ammonia concentrations by approximately 50%. 130 Table 6 Application of red blood cell drug delivery system in toxin clearance Paraquat poisoning WP6 Lipid insertion method The antidote exhibited excellent active toxin capture ability both in vitro and in vivo, and achieved good lung and kidney protection. 142 Phospholipase A2 poisoning Melittin and OOPC Ultrasonic method Melittin and membrane lipids attracted PLA2, and OOPC was used to kill PLA2 activity, effectively inhibiting PLA2-induced hemolysis in mice. 143 Organophosphorus poisoning Organophosphorus hydrolase Lipid insertion method Red blood cells and organophosphorus hydrolases acted synergistically to successfully detoxize methyl paraoxon through targeted binding and catalytic degradation. 144 Bacterial toxins poisoning None Extrusion method Red blood cell nanodiscs provided natural membrane lipids and proteins that could absorb and trap bacterial toxins, effectively inhibiting toxin-induced hemolysis and cytotoxicity. 145 Heavy metal poisoning Dimercaptosuccinic acid RBCm coating method Red blood cells improved the bioavailability of dimercaptosuccinic acid and enabled its long-term sustained release, thereby improving the survival of mice with chronic lead poisoning. 146 Pathogenic bacteria and toxins poisoning Gold nanowire Hybrid membrane fusion method Hybrid cell membrane delivery systems that combined the functions of red blood cells to target toxins and platelets to adhere to pathogens achieved the simultaneous removal of pathogens and toxins. 147 Figures Figure 1 History of Red blood cells as drug carriers. Created in https://BioRender.com. Figure 2 Red blood cell membrane skeleton and protein distribution. Created in https://BioRender.com. Figure 3 Drug delivery methods of RBCs. A) Intracellular delivery system of RBCs. B) Drug delivery systems based on RBCm. C) Gene engineered RBCs delivery system. D) RBCs derived extracellular vesicle delivery system. Created in https://BioRender.com. Figure 4 Application of red blood cells drug delivery systems in different diseases. Created in https://BioRender.com. Figure 5 Schematic illustration of the preparation of ELeOVt and the mechanism of ELeOVt to improve the anti-lung metastatic tumor effect and the biocompatibility of OVs. A) The construction of ELeOVt. B) The mechanism of ELeOVt to improve the anti-lung metastatic tumor effect of OVs. C) The mechanism of ELeOVt to improve the biocompatibility of OVs. Reproduced under the terms of the Creative Commons CCBY license. 44 Copyright 2023, The Authors. Advanced Science published by Wiley-VCH GmbH. Figure 6 Preparation and characterization of the hybrid cell membrane coated tetrahedral DNA nanostructure. Reproduced under the terms of the Creative Commons CCBY license. 101 Copyright 2024 Wiley-VCH GmbH. Figure 7 Schematic illustrating EMNVs-mediated inhibition of viral entry and replication. Reproduced under the terms of the Creative Commons CCBY license. 111 Copyright 2023, The Authors. Published by American Chemical Society. Figure 8 Schematic illustration of super-assembly of UOX-CAT@ZIF-8 nanoparticles-based building blocks on the RBCs surface to form armored RBCs biohybrids for hyperuricemia treatment. Reproduced under the terms of the Creative Commons CCBY license. 42 Copyright 2023, The Authors. Advanced Science published by Wiley-VCH GmbH. Figure 9 Schematic illustration of OPH enzyme-armed nano-cleaner for bimodal detoxification of MPO. a) Design and fabrication of the nano-cleaner. b) Mechanisms of detoxification of the nano-cleaner. Reproduced under the terms of the Creative Commons CCBY license. 144 Copyright 2024, The Authors. Figure 10 Schematic of general mechanisms and therapeutic strategies for modulating monocyte m6A modification in cardiac fibrosis and remodeling processes. A) Standard regulation model of m6A modification. B) Local infiltration of leukocytes, mainly monocytes, following occluder implantation for VSD interventional closure. C) Three-step assembly of the RBCs vesicle biohybrid system. D)Treating processes in a mouse model of cardiac remodeling. E) Mechanism of the RBCs vesicle delivery system. Reproduced under the terms of the Creative Commons CCBY license. 158 Copyright 2024, The Authors. Figure 11 Schematic illustration for the preparation of SPN@RBCM nanoparticles and their applications in photoacoustic imaging and photothermal therapy. Reproduced under the terms of the Creative Commons CCBY license. 171 Copyright 2020, The Authors. Figure 12 Diagram illustrating the step-by-step process for fabricating dipyridamole spermatozoon propelled cellular submarine hirudin peptide/urokinase/protamine nanoparticles and depicts their thrombolytic mechanism. Reproduced under the terms of the Creative Commons CCBY license. 179 Copyright 2024, The Authors. Biography Huizi Deng received her B.S. degree from China Pharmaceutical University in 2018 and M.S. degree from Shanghai Jiao Tong University in 2021. Currently, she is pursuing her Ph.D. degree at Beijing Institute of Pharmacology and Toxicology under the supervision of Prof. Chunsheng Gao. Her research interests focus on the development of nano drug delivery system based on red blood cell carriers. Xiaobei Cheng received her B.S. degree and M.S. degree from China Pharmaceutical University in 2018 and 2021, separately. Currently, she is pursuing her Ph.D. degree at Beijing Institute of Pharmacology and Toxicology under the supervision of Prof. Chunsheng Gao. Her research interests focus on the application of genetically engineered red blood cells. Chunsheng Gao received his B.S. degree from The Second Military Medical University in 1992. He received his M.S. degree and Ph.D. degree from Beijing Institute of Pharmacology and Toxicology in 1999 and 2006, separately. His current research focus on the development and clinical applications of red blood cell carriers. References 1. (1) Wang, X.; Mao, K. R.; Zhang, X. N.; Zhang, Y. N.; Yang, Y. G.; Sun, T. M. Red blood cell derived nanocarrier drug delivery system: A promising strategy for tumor therapy. IMed 2024 , 2 (3). (2) Awad, R.; Avital, A.; Sosnik, A. 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