Recent advance of erythrocyte-mimicking nanovehicles: From bench to bedside

Journal Pre-proof Recent advance of erythrocyte-mimicking nanovehicles: from bench to bedside ´ Jielai Yang, Fei Wang, Yong Lu, Jin Qi, Lianfu Deng, Flavia Sousa, Bruno Sarmento, Xiangyang Xu, Wenguo Cui

PII:

S0168-3659(19)30596-6

DOI:

https://doi.org/10.1016/j.jconrel.2019.10.032

Reference:

COREL 9981

To appear in: Received Date:

2 September 2019

Revised Date:

11 October 2019

Accepted Date:

16 October 2019

Please cite this article as: Yang J, Wang F, Lu Y, Qi J, Deng L, Sousa F, Sarmento B, Xu X, Cui W, Recent advance of erythrocyte-mimicking nanovehicles: from bench to bedside, Journal of Controlled Release (2019), doi: https://doi.org/10.1016/j.jconrel.2019.10.032

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Recent advance of erythrocyte-mimicking nanovehicles: from bench to bedside Jielai Yanga,b, Fei Wanga, Yong Lua, Jin Qia, Lianfu Denga, Flávia Sousac,d,e, Bruno Sarmentoc,d,e,*, Xiangyang Xua,b,*, Wenguo Cuia,* a

Shanghai Institute of Traumatology and Orthopaedics, Shanghai Key Laboratory for

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Prevention and Treatment of Bone and Joint Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Ruijin 2nd Road, Shanghai 200025, P. R. China

Department of orthopedics, Ruijin Hospital, Shanghai Jiao Tong University School of

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b

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INEB – Instituto de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen

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208, 4200-393 Porto, Portugal d

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Medicine, 197 Ruijin 2nd Road, Shanghai 200025, P. R. China

i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua

e

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Alfredo Allen 208, 4200-393 Porto, Portugal

CESPU – Instituto de Investigação e Formação Avançada em Ciências e Tecnologias

da Saúde & Instituto Universitário de Ciências da Saúde, Rua Central de Gandra, 1317,

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4585-116 Gandra, Portugal

*Corresponding authors. E-mail addresses: [email protected] (B. Sarmento), [email protected] (X. Xu), [email protected] (W. Cui).

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Graphical Abstract

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nanovehicles, which exhibit huge potential for further clinical applications

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Abstract

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and are anticipated to be translated from bench to bedside.

Erythrocyte-mimicking nanovehicles (EM-NVs) are developed by fusing nanoparticle cores with naturally derived erythrocyte membranes. Compared with conventional nanosystems, EM-NVs hold preferable characteristics of prolonged blood circulation

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time and immune evasion. Due to the cell surface mimetic properties, along with tailored core material, EM-NVs have huge application potential in a large variety of biomedical fields, which are anticipated to revolutionize the present theranostic modalities of diseases in clinic. This review focuses on (I) drug carriers, (II) photosensitizers, (III) antidotes, (IV) vaccines and (V) probes, aiming to present an overall summary of the latest advancement in the application of EM-NVs, and highlight

the major challenges and opportunities in this field.

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Keywords: Erythrocyte; Biomimetic; Nanovehicles; Biomedical application

1. Introduction In the past decades, advances in nanotechnology, especially in nanovehicles (NVs), have revolutionized medical research to gain momentum in the theranostic of diseases [1, 2]. However, conventional NVs have been partially limited in clinical application, because these are recognized as foreign substances by the body, thereby causing immune responses and toxic effects [3]. Efforts have been made to overcome this limitation, such as modifying the polyethylene glycol (PEG) on the NV surface to

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decrease the uptake by the reticuloendothelial system (RE) and mononuclear phagocyte system. Unfortunately, although PEG has been proven to be effective at decreasing

nonspecific targeting in a complex environment, PEG-modified NVs have also been

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demonstrated to activate the immune system and lose efficacy over repeated

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administrations [4]. Consequently, NVs with adjustable surface functionalities and good biocompatibility are highly desirable.

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A myriad of NVs have been extensively studied over the years for biomedical application, including those that composed of lipid, polymer, silica and mental [5-9].

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Lipids, as integral part of the cell membranes, present as an ideal material to generate nanostructures. Due to the amphiphilic nature of lipids, these nanostructures can encapsulate both hydrophilic and hydrophobic molecules. However, the major challenge is the lack of structural integrity, which results in the leakage of the content

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and instability during storage [10]. On the other hand, polymeric NVs have overcome the aforementioned problems of lipid NVs, but still have the issues of toxicity and biocompatibility [11]. Metallic NVs tend to agglomerate and have limitations similar to polymeric NVs, which compromise its biological application [12]. In fact, most of these NVs are still in the research stage, and have limited potential to progress from the laboratory bench to clinical transition. The notion that the complexity of nature can

only be attained by the nature in the design of biomimetic constructs may be the optimal choice. Nature-deprived materials provide tremendous options for NV manufacturing [13, 14]. These can be presented in different forms, including the mimicry of physical properties and the direct leveraging of materials [15, 16]. For the latter, the surface property of NVs can be easily engineered by coating NVs with membranes derived from different cells. The receptors, antigens and ligands on the surface membrane are

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adept at performing a variety of functions, such as cell-cell, cell-protein and cellextracellular matrix communication, within a complex environment [17].

Erythrocytes are the most abundant blood cells in the human body, and these have

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been extensively used in clinical transfusion for centuries [18]. According to different

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blood group antigens (mainly ABO and Rh), erythrocytes can be classified into different types (A, B, O, Rh+ and Rh-), which is a vital guide for clinical transfusion

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and disease diagnosis [19]. Biologically, erythrocytes provide oxygen to cells and tissues, and transport carbon dioxide to the lungs. Normal erythrocytes are of typical

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biconcave structure, and are flexible to deform when traveling through blood vessels of different diameters [20]. The unique properties of erythrocyte are closely connected with its membrane, which comprises of a lipid bilayer with multiple transmembrane proteins, and transmembrane proteins are critical for various biological activities,

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including the process of adhesion, transport and integrity maintenance [21]. Under normal conditions, erythrocytes can circulate in the blood vessels for approximately 40 days in mice, and for 120 days in the human body, before immune clearance [22]. The underlying mechanism is partly due to the CD47, which is a transmembrane protein that interacts with signal-regulatory proteins of immune cells to inhibit phagocytosis

[23, 24]. Mature erythrocytes lack a nucleus and other organelles, making the membrane extraction and purification process more convenient. To date, erythrocyte-mimicking nanovehicles (EM-NVs) are the most well-studied in this field, which exerts profound influence on subsequent researches. Importantly, EM-NVs have great potential for translation in the near future, since blood transfusion is common, and the type-matched erythrocyte is an alternative choice to expand membrane source for clinical application. The present study focuses on EM-NVs as

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drug carriers, photosensitizers, antidotes, vaccines and probes, aiming to provide a comprehensive summary of the recent advancement of EM-NVs. The applications,

main features, disease models, loaded drugs and core materials are summarized in

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Table 1. Finally, the major challenges are discussed for the clinical translation of EM-

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NVs.

2. Proof-of-concept study

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In 2011, researchers reported the cell membrane coating approach by coating poly lactic-co-glycolic acid (PLGA) with erythrocyte membranes for the first time [25]. The

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results revealed that EM-NVs had a superior circulation time of 39.6 hours, when compared to PEGylated nanoparticles (NPs, 15.8 hours). Subsequent studies have revealed that the density and orientation of CD47, a “marker-of-self” protein, is similar between EM-NVs and native erythrocytes [23]. Furthermore, studies have revealed that

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EM-NVs does not induce immune responses upon repeated administrations, and reduces RE uptake with no obvious in vivo toxicity, indicating a priority to PEG [24, 26].

3. Nanocarriers

3.1 Basic passive drug delivery After the preliminary investigation of the properties of EM-NVs in a series studies, researchers have started to advance the platform towards drug carriers. In an initial study, the poly(lactic acid) (PLA) core was used to load the doxorubicin (DOX), and this was coated with erythrocyte membranes [27]. Different loading strategies were compared between physical encapsulation and chemical conjugation. The results revealed that the chemical conjugation strategy resulted in a superior drug release

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profile, and that the erythrocyte membrane could prevent the outward diffusion of encapsulated drugs. In addition, the anticancer efficacy was investigated in a leukemia

cell line, and it was demonstrated that DOX-loaded EM-NVs exhibited higher toxicity,

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when compared to the same amount of free DOX. In another similar study was conducted in a lymphoma mouse model, in which DOX was loaded in the PLGA core,

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and the EM-NVs exhibited better tumor growth suppression, when compared with free

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drug treatment [28]. In addition, the toxicity and early mortality were not observed during the long-term administration of DOX in high doses, indicating the excellent immunocompatibility and advantageous safety profile of this system. In another study,

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paclitaxel (PTX) and DOX were co-encapsulated into magnetic O-carboxymethylchitosan NPs and coated with an Arg-Gly-Asp-anchored erythrocyte membrane, and an effective nanocarrier was developed [29]. Compared with the PEG coating method, this

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bio-inspired nanocarrier exhibited better potential in prolonging in vivo circulation time, enhancing tumor uptake and accumulation. In a related study, gambotic acid (GA)-loaded EM-NVs were investigated in colorectal cancer treatment. Compared to free GA, EM-NVs demonstrated enhanced antitumor efficacy with relatively low toxicity [30]. Beyond vehicles as an anticancer drug, EM-NVs were also used to load rapamycin for antiatherosclerotic therapy [31]. The erythrocyte-mimicking technique

remarkably reduced the in vivo clearance and increased the accumulation of NVs in atherosclerotic plaques, contributing to the effective drug delivery system for atherosclerosis (Fig. 1).

3.2 Controlled passive drug release via external stimuli With the advancement of EM-NVs, more sophisticated strategies have been explored to achieve the controlled drug release. In an initial study, SiO2@TiO2 NVs

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with enhanced photocatalytic activity were used for the controlled degradation of coatings of the erythrocyte membrane [32]. The results revealed that the erythrocyte

coatings on the surface of SiO2@TiO2 particles were effectively degraded after 6-9 minutes of ultraviolet treatment. DTX was effectively encapsulated into the SiO2@TiO2

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particles and gradually released under ultraviolet irradiation. Furthermore, the

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anticancer efficacy was determined by evaluating the cytotoxicity of nanoparticles against MCF-7 human breast cancers, and the results revealed that the gradually

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released DOX could efficiently kill cancer cells. In addition, the photocatalytic process could also generate reactive oxygen species (ROS) that can be used for photodynamic

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therapy (PDT). In a similar study, mesoporous silicon NVs (MSNs) were camouflaged with erythrocyte membranes to co-load DOX and chlorin e6 [33]. The erythrocyte membrane limited the DOX within the mesoporous structure until the stimuli of a far red laser, which elicited chlorin e6 to generate ROX and destroy the erythrocyte

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membrane, eliciting the enhanced accumulation of DOX in the tumor site. In another study, PTX was incorporated into polycaprolactone (PCL) cores to fabricate EM-NVs, and a near-infrared (NIR)-responsive lipid was inserted in the erythrocyte membrane shell for controlled drug release [34]. Using a 4T1 orthotopic mammary metastatic tumor model, the system used in vivo tumor imaging with prolonged circulation lifetime and enhanced tumor-specific drug release under NIR stimuli. The following

experiments demonstrated the excellent anticancer efficacy of these NIR-responsive EM-NVs, with complete control of the primary tumor and 98% inhibition of lung metastasis in vivo. Almost at the same time, another team reported PTX-loaded EMNVs for inhibiting breast cancer [35]. In the present study, by co-administrating with a tumor penetrating peptide, the EM-NVs exhibited a synergism effect of enhanced perfusion into the primary and metastases, resulting in a more effective anti-metastasis

3.3 Controlled passive drug release via internal stimuli

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efficacy (Fig. 2).

In addition to external stimuli, the controlled drug release could be obtained on the basis of environmental changes around the targeted sites. In 2017, erythrocyte

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membrane-wrapped pH sensitive NVs were reported in a lung cancer model [36]. Based

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on the low pH in tumor sites, the group synthesized pH-sensitive poly (L-γ-glutamyl carbocistein)-PTX NVs. The release of PTX from this system was remarkably

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increased at pH 6.5, when compared to that for a normal body (pH 7.4). The subsequent biological experiments indicated that the erythrocyte membrane that wrapped the pH

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sensitive NVs have excellent biological stability and a remarkable tumor growth inhibiting effect. In a similar study, a combinatorial anticancer effect was achieved by using a pH-responsive nanogel [37]. PTX was entrapped in the nanogels, which comprised of two oppositely charged chitosan derivatives and hydroxypropyl-β-

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cyclodextrin acrylate. In addition, interleukin-2 was wrapped in the nanogels to exert an immunotherapeutic effect. By controlling the drug release in these tumor sites, the red blood cell membrane-coated nanoparticles (RBC-NPs) had improved antitumor efficacy through the enhanced drug uptake and accumulation, as well as anticancer immunity. In fact, the tumor microenvironment was subsequently modified by the utility of low dose PTX. In order to realize the remote delivery of small molecules to

the targeted sites, a novel strategy was reported [38]. Using a stable pH gradient, vancomycin and DOX could be remotely loaded into EM-NVs and released in acidic conditions. This delivery system has been confirmed to promote the therapeutic efficacy of vancomycin in a methicillin-resistant staphylococcus aureas (MRSA)infected murine skin model and DOX in a 4T1 mouse breast cancer model. In a separate study, EM-NVs were designed for glucose-responsive delivery [39]. Under normal conditions, pancreatic β-cells are responsible for the meticulous control of blood

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glucose (BG). However, once this suffers from diabetes, an alternative of glucoseresponsive delivery is desperately needed. Based on the interactions between glucose

derivative-modified insulin and the glucose transporters of erythrocyte membrane, the

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investigators designed a novel glucose-responsive insulin delivery system. After the conjugation with glucosamine, the binding efficiency of insulin to the erythrocyte

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membrane was significantly enhanced. However, the binding was reversible under the

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condition of hyperglycemia, thereby inducing the fast release of insulin and subsequent drop in blood glucose. Importantly, this work was an initial attempt of employing an erythrocyte membrane to obtain a controlled insulin delivery with rapid response,

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which may revolutionize the future of diabetes treatment (Fig. 3).

3.4 Active targeting delivery of chemotherapeutics By adding special surface functionalities to EM-NVs, it was possible to make the

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active targeting drug delivery. However, conventional functionalization based on chemical conjugation was not suitable for the modification of the biological membrane, which was partly because some essential components on the membrane surface were prone to denaturation. In order to preserve the membrane’s biological activity, nondisruptive functionalization strategies are urgently needed. In 2013, the lipid insertion approach was first used to functionalize NVs [40]. In this study, with the aid of lipid

tethers and the dynamic conformation of membrane bilayers, the insertion of both folate and nucleolin-targeting aptamer AS1411 could be successfully obtained, thereby ensuring effective targeting against different tumors. The lipid insertion approach was also utilized in a colorectal cancer model [41]. In this study, bispecific targeting EMNVs were built by incorporating anti-EGFR-iRGD protein to the surface membranes. After loading with GA, the system exhibited improved targeting and better anti-cancer efficacy, when compared to bare GA. In order to further expand the application of EM-

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NVs, the modification of live cell membranes was achieved by using maleimideterminal PEG linkers [42]. This approach was capable of binding macromolecules on cell membranes due to the stable anchoring and strong control over the linker length.

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In this study, human recombinant hyaluronidase PH20 was conjugated on erythrocyte, and the results indicated that the long linker outdone the short linker in retaining

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enzyme activity and reducing cell membrane changes. Similarly, another study reported

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a method of using neurotoxin-derived peptide to function EM-NVs with brain targeting capability [43]. In order to break the limitation of the lipid insertion method confined to the modification of neutral molecules and molecules with a weak positive charge,

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the avidin-biotin system was employed to incorporate molecules with a strong positive charge. As a large protein, avidin was introduced into the erythrocyte membrane to shield the electrostatic interaction between the positive charge of the peptide and

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negative charge of the cell membrane. After loading DOX, these NVs could selectively release the drug at brain tumor sites, resulting in the significantly improved median survival of glioma-bearing mice.

3.5 Active targeting delivery of the antibiotic and antibody

Although the chemotherapeutic drugs were mostly loaded in EM-NVs, the system can be employed for loading various other substances. Multidrug-resistant bacterial infection remains as a great concern due to the excessive use of antibiotics. In order to address these problems, researchers have attempted to develop a delivery system based on core-shell gelatin NVs for the efficient release of antibiotics at the site of infection [44]. By coating these erythrocyte membranes, this delivery system exhibited the great capability for toxin-clearance and immune escape, enabling antibiotics with biomimetic

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and detoxifying characteristics. Furthermore, the degradation of NVs at the site of infection bestowed this system with a superior killing effect for bacteria, but with no

obvious systematic toxicity (Fig. 4). Similarly, researchers have reported vancomycin-

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loaded nanogels for intracellular antibacterial treatment [45]. In this study, the nanogel was coated with an erythrocyte membrane, and a disulfide bond-based crosslinker was

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used to achieve redox-responsiveness. When exposed in extracellular conditions, the

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erythrocyte-nanogels could effectively neutralize MRSA-associated toxins, promoting the uptake by macrophages. Once inside these cells, these erythrocyte-nanogels rapidly release their loaded antibiotics to kill the effectively bacteria. Based on the specific

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affinity between pathogenic fungi and erythrocytes, researchers have devised amphotericin B-loaded EM-NVs for antifungal therapy [46]. By anchoring P4.2derived peptide onto the liposome surface, the camouflaging affinity of the erythrocyte

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membrane is strengthened. In another study, an effective intracellular antibody delivery system was developed [47]. When this was coated with an erythrocyte membrane, the system was able to deliver the antibody into the cell, specifically blocking the human telomerase reverse transcriptase (hTERT) expressed only in the cytoplasm. This work provides a promising method for binding intercellular targets that are difficult to obtain by traditional antibody administration.

4. Nano-photosensitizers

4.1 Imaging In 2013, gold NVs were presented for the first time, which could be successfully functionalized with an erythrocyte membrane [48]. Through novel coating methods, these gold NVs were fully enclosed by membranes derived from natural erythrocyte

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membranes. The EM-NVs possessed a right-side-out erythrocyte membrane, enabling NVs to evaded the uptake from macrophages. In addition, the erythrocyte membrane reduced the interactions between NVs and thiolated compounds. In another study, the

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upconversion nanoparticles (UCNPs) were wrapped by erythrocyte membranes for tumor imaging [49]. It was revealed that the cell membrane could prevent the protein

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corona formation around NVs. By inserting folic acid onto the erythrocyte membrane, EM-NVs were able to specifically target tumors. The superior tumor imaging was

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testified using fluorescent NVs. Furthermore, it was demonstrated that no obvious systematic toxicity was observed upon the injection of EM-NVs. In a recent study,

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erythrocyte membranes were used for wrapping bacteria as effective imaging agents in a mice tumor model [50]. With reduced inflammatory response, prolonged in vivo reservation, enhanced accumulation in the disease site and unchanged inherent

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bioactivities, the erythrocyte-camouflaging bacteria revealed great potential for bioimaging, diagnosis and therapy.

4.2 Photothermal therapy After pioneering the research of gold NVs in imaging areas, the first cell membrane-functionalized NVs for photothermal therapy (PTT) were devised [51]. The

fusion of erythrocyte membranes onto gold NVs bestowed good colloidal stability to EM-NVs without altering the unique structure of gold NVs. After irradiation, EM-NVs could selectively kill the cancerous cells in the irradiation area. Furthermore, these EMNVs exhibited enhanced circulation time, when compared with non-functionalized NVs. Upon in vivo administration, EM-NVs exhibited significantly enhanced therapeutic efficacy without noticeable systemic cytotoxicity. In another study, erythrocyte membrane-coated iron oxide magnetic NVs were employed for PTT [52].

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By introducing a simply “ultra-stealth” biomimetic coating, the nanoclusters retained the photothermal functionalities of Fe3O4 while reducing the nonspecific uptake of macrophage. In a breast cancer mice model, EM-NVs exhibited a prolonged circulation

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time and reduced RE capture, thereby enhancing tumor accumulation and PTT efficacy.

In addition, the system exhibited a good in vivo biodistribution, which was suitable for

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the imaging purpose of various tumors. Another recent study reported that Fe3O4

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magnetic NPs were coated with erythrocyte membranes through the microfluidic electroporation approach [53]. Using the microfluidic electroporation strategy, EMNVs exhibited a completer membrane coating and better therapeutic efficacy, when

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compared to those prepared by conventional methods (Fig. 5). In another study, by using natural melanin as an effective photothermal agent, EM-NVs were developed for anti-tumor PTT [54]. In some studies, the photothermal agents triggered the drug

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release and served as part of the synergistic photothermal-chemotherapy. In one example, the erythrocyte membrane camouflaged DOX-loaded hollow prussian blue NVs, which were fabricated for the synergistic photothermal/chemotherapy of cancer [55]. This system exhibited enhanced stability, immune evading capacity, and blood retention time, when compared to those of bare NVs. Similarly, Prussian blue/manganese dioxide EM-NVs were used to increase the DOX loading capacity in

another study [56]. Through the administration of these EM-NVs, the hypoxia condition inside tumors was relieved through the activation of H2O2 to generate oxygen, which in turn disrupted the erythrocyte membranes on the surface and accelerated the release of DOX. Another example reported the application of EM-NVs for the targeted photothermal/chemotherapy of cancer [57]. The PTX was encapsulated into gold NVs to form the core part, and coated with modified erythrocyte membranes on the surface. The results revealed the selective tumor targeting capability of this combined system

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via exposure to near-infrared irradiation. The accumulation capability of photothermal agents to the targeted sites is a critical factor for PTT, and this interacts with certain factors in the microenvironment, such as

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low blood perfusion and hypoxia. In a another study, tumor microenvironment

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modulation improved the PTT of gold NVs for pancreatic ductal adenocarcinoma, which is a challenging cancer without effective therapeutic agents [58]. Cyclopamine

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can disrupt the ECM of pancreatic ductal adenocarcinomas and enhance tumor blood perfusion. Furthermore, the cyclopamine modification was able to remarkably increase

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the accumulation of EM-NVs in tumors, producing a higher photothermal effect, when compared to bare NVs. Another similar study reported the strategy of dilating the tumor vasculature to improve PTT [59]. The endothelin A receptor antagonist can relax tumor vessels and increase blood perfusion, without altering the vessel perfusion of normal

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tissues. By jointly employing the endothelin A receptor antagonist with EM-NVs, the low-dose administration of photothermal agents can achieve excellent PTT efficacy.

4.3 Photodynamic therapy In addition to PTT, another major phototherapy is photodynamic therapy (PDT). For both approaches, increased agents to targeted sites promote therapeutic efficacy. In

a study, a new PDT strategy was developed [60]. UCNPs were incorporated with photosensitizers and coated with an erythrocyte membrane modified with dual targeting moieties. With the unique function of erythrocytes as oxygen carriers, the erythrocyte membrane facilitated the permeation of ground-state oxygen and singlet oxygen to kill cancer cells. In addition, another feature of this system is the decoration of the erythrocyte membrane with dual targeting moieties for the selective recognition of cancer cells and mitochondria. In order to enhance PDT efficacy in the hypoxia tumor

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microenvironment, oxygen self-enriched EM-NVs were developed [61]. Albumin NVs were incorporated with indocyanine green and perfluorocarbon, and cloaked with erythrocyte membranes. Due to the perfluorocarbon, these EM-NVs can enhance the

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PDT by generating more singlet oxygen.

4.4 Radiotherapy

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Radiotherapy is an important strategy for the clinical treatment of malignant cancers. In an example, folate-inserted, erythrocyte membrane-modified bismuth NVs

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were used for X-ray radiotherapy in breast cancer [62]. The erythrocyte membrane provided an immune-evading folate that acted as a tumor targeting agent, and bismuth generated free radicals upon X-ray to kill the cancer cells. Significant tumor inhibition

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was confirmed when this system was used for X-ray radiation. The in vivo histological and biodistribution analysis revealed that EM-NVs were eliminated from the body after two weeks, with no evident damage in major organs. Another similar study reported highly hemocompatible EM-NVs for simultaneous cancer radiosensitization and precise antiangiogenesis [63]. When irradiated with X-rays, these EM-NVs exhibited potent anticancer and antiangiogenic responses, without causing any obvious

histological damage to the non-targeted major organs. In order to relieve the hypoxia condition in tumors, researchers have developed an artificial erythrocyte system for oxygen loading and delivery [64]. Perfluorocarbon, which is a commonly used artificial blood substitute, was encapsulated within the PLGA, and was further coated with the erythrocyte membrane. Due to the nanoscale sizes, these perfluorocarbon-loaded EMNVs efficiently delivered oxygen into tumors, and relieved the tumor hypoxia, thereby

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improving treatment efficacy during radiotherapy.

5. Nano-antidotes

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5.1 Bacterial toxins

Inspired by the notion that most toxins interact with cell membranes to inflict a

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virulence effect, the EM-NVs were first used to neutralize α-hemoly sin from

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Staphylococcus aureus (S. aureus) [65]. In this study, a nanosponge was prepared with a polymeric core wrapped in the erythrocyte membrane. The erythrocyte membrane

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shell was able to absorb a variety of pore-forming toxins. Meanwhile, the PLGA core stabilized the erythrocyte membrane shell for prolonged blood circulation. In order to determine the detoxification ability, α-toxin was mixed with the nanosponge and cultured with mouse erythrocytes. Polymeric NVs, liposomes and erythrocyte

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membrane vesicles were used as the control. Compared with the other samples, the nanosponge sample exhibited superior detoxification capability. Similar results were conformed in the subsequent in vivo study. In addition, experiments with StreptolysinO [65, 66] and melittin [65] also demonstrated reduced erythrocyte hemolysis through the nanosponges, indicating the versatility of this membrane-based platform. The ability of erythrocyte nanosponges against bacterial infection was further studied in

group A streptococcus (GAS) [67]. In this study, the researchers reported that these EM-NVs could sequester streptolysin O and block GAS, thereby increasing bacterial clearance and maintaining immune function. In a skin infection murine model, nanosponges could significantly reduce bacteria and decrease lesion size. In a more recent study, the broad-spectrum detoxification of human erythrocytes was investigated [68]. In this study, membranes for nanosponges were first derived from human erythrocytes and four representative toxins, including melittin, listeriolysin O of listeria

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monocytogenes, streptolysin O of Group A Streptococcus, and α-hemolysin of methicillin-resistant staphylococcus aureus (MRSA), were selected. The results revealed that human erythrocyte nanosponges could effectively inhibit hemolysis in a

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concentration-dependent manner. In addition, this system demonstrated no cytotoxicity when determined on human umbilical vein endothelial cells (HUVEC), and no lethality

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when injected into mice.

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In addition to the most investigated nanosponge formats, there are several different formats. In 2015, hydrogel retaining erythrocyte nanosponges were designed for the

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local treatment of MRSA infection [69]. These nanosponges were constructed by wrapping PLGA@hydrogel NVs with the erythrocyte membrane. The hydrogel composition was optimized to effectively retain the nanosponge in its matrix, without compromising toxin neutralization. Then, the nanosponge retained at the injection sites

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after subcutaneous injection. In a mouse infection model, the hybrid system revealed significantly reduced skin lesions. Interesting, in another study, the hybrid system could also be prepared by 3D-printing through photopolymerization [70]. PEG hydrogel was used as the supporting matrix for EM-NVs, and this was created through a light-based, rapid 3D bio-printing process. Various shapes through different channels were printed for enhanced interactions between these EM-NVs and toxins. A similar nanogel system,

which was described in the drug delivery section, was used for combinatorial antivirulence and responsive antimicrobial delivery [45]. In another study, an erythrocyte membrane-coated nanowire was devised as a novel motor sponge [71]. This new system was developed by coating the erythrocyte membrane onto the ultrasoundpowered gold nanowire motors. The advantage of employing ultrasound-propelled motor sponges to neutralize toxins was 60% more effective than PBS and bare motors, and 30% better than regular motor sponges without the ultrasound field. In another

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study, by mixing two oppositely charged NVs, the self-assembled nanosponges were achieved [72]. The approach that used EM-NVs to fabricate the colloidal gel was based

on the physical self-assembly without chemical crosslinking. The rheological test

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demonstrated that this nanogel has an excellent shear-thinning property, which is suitable for injection. When subcutaneously injected into mouse tissue, the gel

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formulation exhibited a pronounced detoxification capability and prolonged retention

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time. Using a mouse subcutaneous infection model, the nanogel exhibited remarkable antibacterial efficacy by inhibiting skin lesion advancement (Fig. 6).

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5.2 Organophosphate, doxorubicin and antibody Nanosponges are also employed to neutralize other harmful molecules. In a study, organophosphate was intercepted by erythrocyte nanosponges [73]. Organophosphorus compounds can inactivate acetylcholinesterase (AchE), which results in the significant

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accumulation of acetylcholine (ACh) in the body, thereby causing organophosphate poisoning. Based on the fact that AChE is present in red blood cell (RBC) membranes, RBC nanosponges were used as anti-organophosphate agents. These RBC nanosponges maintained the enzymatic activity of AChE, effectively binding dichlorvos to protect other enzymatic substrates. These NVs could enhance the AChE activity in the body and significantly improve the survival of organophosphate poisoning mice. Similarly,

erythrocyte-mimicking oil nanosponges were established for the dual-model detoxification of organophosphates [74]. The biomimetic system neutralized toxicants through both the nonspecific physical partition from the oil core and specific biological binding from the erythrocyte membrane shell (Fig. 7). In another study, EM-NVs were used as a nanoabsorbent for the cellular detoxification of chemotherapeutics [75]. The detoxification effect was charge-dependent, since these could bind positively charged DOX, but not negatively charged methotrexate. Furthermore, by optimizing the internal

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surface modification, this nanosystem could be an ideal platform for detoxification. In another study, erythrocyte nanosponges were used to remove the pathological

antibodies in type-II immune hypersensitivity reactions [76]. In this study, the

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investigators investigated the ability of EM-NVs in eliminating the effects of

pathological antibodies, in order to minimize the burden of the disease without

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immunosuppression. By neutralizing the anti-erythrocyte polycolnal IgG, erythrocyte

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6. Nanovaccines

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nansponges could effectively preserve these circulating erythrocytes.

6.1 Bacteria

In 2013, erythrocyte nanosponges were first used as a safe and effective vaccination

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[77]. In this study, pore-forming toxins (PFTs) were effectively delivered for immune processing through the toxin detaining approach. EM-NVs were used for anchoring PFTs, aiming to inactivate the PFTs without compromising their structural integrity. The staphylococcal α-haemolysin (H1α) was used for the preparation of H1α-loaded nanotoxoids. The release kinetics study revealed no toxins release from nanotoxoids, indicating the safely incorporation of H1α into EM-NVs. Furthermore, immunization

studies have demonstrated that the erythrocyte membrane retained the toxins without causing an immunogenic response, enabling the toxins to be safely processed. When compared with heat-denatured toxins, EM-NV-detained toxins exhibited superior protective immunity against adverse effects. Overall, this novel inactivating approach was beneficial for improving the efficacy of toxoid vaccines. In another study, the method was successfully used as a prophylactic strategy against live MRSA skin infection, which opened the door for the further development of similar platforms

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against many other common, yet deadly, bacterial pathogens [78]. However, the potency of conventional vaccines remains limited, since most vaccines focus on a single antigen, while most bacteria secrete a variety of toxins. In order to address this hurdle,

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on-demand vaccines were fabricated against pathogenic bacteria in a recent study [79] (Fig. 8). Multiple toxins were entrapped in erythrocyte nanosponges to form

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multivalent nanotoxoids. In a mouse model, this multivalent nanotoxoids could

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effectively induce functional immunity against bacterial infection. When compared with vaccines from a denatured method, this nanotoxoid formulation exhibited superior immunogenic efficacy, highlighting the advantage of biomimetic neutralization and

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delivery.

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6.2 Cancers

Beyond antibacterial vaccines, EM-NVs were also investigated in cancer vaccines

[80]. In order to enhance the antigen presenting efficiency of dendritic cells (DCs), EMNVs were used as agonists of toll-like receptor 4 and antigenic peptide. Redox-sensitive peptide was conjugated with PLGA NVs to cleave in the intracellular milieu, and mannose was inserted into the erythrocyte membrane to target the antigen presenting

cells (APCs) in the lymphatic organ. Compared with other vaccines, the biomimetic nanovaccines significantly inhibited tumor growth and metastasis in three different melanoma models. In addition, the nanovaccine could effectively promote CD8+ T cell response and interferon gamma (IFN-γ) secretion.

7. Nanoprobes Cellular surface attachment is a major process for infectious pathogens to elicit

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diseases. Due to the specific binding between pathogens and cells, EM-NVs were designed for the detection of viral pathogens [81]. In an influenza virus model, EMNVs were constructed for virus targeting and isolation. EM-NVs were endowed with

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magnetic functionality by loading iron-oxide particles, which enriched the influenza virus via magnetic extraction. The extracted virus remained active, and could be

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analyzed through conventional methods. This work exhibited the unique function of EM-NVs, providing a novel strategy for the theranostics of infectious diseases.

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Similarly, in another study, erythrocyte nanosponges were used in combination with mass spectrometry for the specific identification of pathogen-associated proteins [82].

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The pathogen GAS and pathogen S. mansoni were used to investigate the detection capability of this approach. Importantly, this work provides a new platform for optimizing the identification of both known or unknown virulence factors in an

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effective and precise manner. In addition to virulence pathogens, antigens on immune cells can also be detected via this biomimetic platform. In a recent study, the targeting of specific immune cell populations was achieved by using EM-NVs that expressed the cognate antigen [83]. It was revealed that EM-NVs exhibited enhanced affinity, when compared with control NVs, in an erythrocyte-specific B cell model. Furthermore, biomimetic NVs could effectively label erythrocyte-induced B cells in both

alloimmunity and autoimmunity murine models.

8. Conclusion and perspective EM-NVs are built through the top-down engineering approach, which overcomes some challenges confronted in the bottom-up models of manufacturing. From the perspective of practical application, these EM-NVs can be applied for patients with minimal risk of immunogenicity by using their own erythrocytes. To date, a variety of

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core materials have been wrapped with erythrocyte membranes to construct core-shell nanovehicles with unique functions. The core materials described in the present study

include polymer, liposome, silicon, hydrogel, mesoporous silica, gold, magnetic

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particle, antibody, bacteria, etc. The present study summarizes the recent advances in EM-NVs for a variety of biomedical applications, including drug delivery, imaging and

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phototherapy, immune modulation, sensing and detection. Undoubtedly, there would be

future.

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a rapid advancement of EM-NVs for other novel biomedical applications in the near

At present, the approach of fabricating EM-NVs, including the preparation of core

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materials, EM isolation and coating process, is relatively mature, and each step can be independently conducted without interfering with each other. Consequently, the largescale production of EM-NVs is possible to achieve. However, several challenges needs

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to be addressed before the clinical utility of EM-NVs can be realized. Among these, the major challenge is to reduce variations in different batches and optimize the fusion process for maximized efficiency. Another major challenge is quality control. It must be ensured that EM-NVs do not contain biological or chemical contaminants. Each step should be carried out aseptically, and performed in compliance with standardized manufacturing procedures. In addition, increasing evidence has indicated the

association of blood types (antigens) with disease risks. Thus, future work may throw light upon the blood-type dependent biological applications of this system, and problems of these regulatory issues should be addressed before clinical applications. Finally, as the biomimetic strategy matures, these challenges would be overcome one by one. In the near future, the success of EM-NVs from bench to bedside may be

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foreseen.

Acknowledgements

The authors gratefully acknowledge the support of the National Key R&D Program of

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China (2018YFC1106200 and 2018YFC1106204,), National Natural Science Foundation of China (81772372 and 51873107), Key subject Construction project of

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Shanghai Municipal Commission of Health and Family Planning (201540102), Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support

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(20171906), Shanghai Jiao Tong University “Medical and Research” Program (ZH2018ZDA04). This paper is a result of the project NORTE-01-0145-FEDER-

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000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was also financed by FEDER - Fundo Europeu de Desenvolvimento Regional Funds through the COMPETE 2020-

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Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia / Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project "Institute for Research and Innovation in Health Sciences" (POCI-01-0145-FEDER007274).

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Lu, R.H. Fang, L. Zhang, In Situ Capture of Bacterial Toxins for Antivirulence

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Vaccination, Advanced materials (Deerfield Beach, Fla.), 29 (2017). [80] Y. Guo, D. Wang, Q. Song, T. Wu, X. Zhuang, Y. Bao, M. Kong, Y. Qi, S. Tan, Z.

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Figure Captions Fig. 1. Illustrations displaying the preparation of RBC/RAP@PLGA for the treatment of atherosclerosis [31]. Adapted with permission. Copyright 2019, Wiley-VCH. Fig. 2. The RBC-mimetic NPs with elongated blood circulation and enhanced tumor penetration for treating metastatic breast cancer [35]. Adapted with permission. Copyright 2016, Wiley-VCH.

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Fig. 3. Schematic of the glucose-responsive insulin delivery system based on red blood cells. Synthesized Glc-Insulin was attached to erythrocytes by interacting with glucose

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receptor/transporter on plasma membranes [39]. Adapted with permission. Copyright 2017, Wiley-VCH.

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Fig. 4. (a) Preparation of vancomycin encapsulated supramolecular gelatin

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nanoparticles with RBC membrane coating layer (Van⊂SGNPs@RBC). (b) Schematic representation of adaptive and multifunctional Van⊂SGNPs@RBC in the treatment of

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a bacterial infection [44]. Adapted with permission. Copyright 2014, American Chemical Society.

Fig. 5. Microfluidic electroporation-facilitated synthesis of RBC-MNs for enhanced imaging-guided cancer therapy. (a) Microfluidic electroporation facilitates the

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synthesis of RBC-MNs. (b) Subsequently, the RBC-MNs, which are collected from the microfluidic chip, enrich in the tumor site after the blood circulation. (c) Biomimetic RBC-MNs are further used for enhanced in vivo tumor MRI and PTT [53]. Adapted with permission. Copyright 2017, American Chemical Society.

Fig. 6. Preparation of nanosponge colloidal gel. (a) Schematic illustration of NC-gel formulation by mixing red blood cell membrane-coated nanoparticles (RBC-NPs), which possess a negative surface charge, with chitosan-modified nanoparticles (ChiNPs) as positively charged nanoparticle counterparts. (b) Images of RBC-NPs, ChiNPs, and NC-gel samples when they were placed onto a flat substrate. Scale bar = 5 mm [72] . Adapted with permission. Copyright 2017, American Chemical Society.

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Fig. 7. Schematic representation of Oil-NS, consisting of an oil droplet surrounded and stabilized by a naturally derived RBC membrane. Oil-NS are designed as a bimodal scavenger for removing OP compounds through concurrently capturing OPs with the

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inner oil phase and the AChE enzyme present on the RBC membrane [74]. Adapted with permission. Copyright 2019, American Chemical Society.

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Fig. 8. Schematic depicting on-demand fabrication of a pathogen-specific nanotoxoid

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and its vaccination benefits.(a) Pathogens secrete virulence factors, which are capable of inserting into target cells and causing their destruction. (b) Using nanosponges

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prepared with the membrane of target cells and incubating the particles with a bacterial supernatant-derived protein fraction, it is possible to generate a nanotoxoid carrying pathogen-specific virulence factors. (c) After vaccination using the nanotoxoid,

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antibodies against the incorporated virulence factors are elicited and can prevent their toxic effects, leaving the intended targets unharmed [79]. Adapted with permission. Copyright 2017, Wiley-VCH.

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Figures

Fig. 1. Illustrations displaying the preparation of RBC/RAP@PLGA for the treatment

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of atherosclerosis [31]. Adapted with permission. Copyright 2019, Wiley-VCH.

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Fig. 2. The RBC-mimetic NPs with elongated blood circulation and enhanced tumor penetration for treating metastatic breast cancer [35]. Adapted with permission.

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Copyright 2016, Wiley-VCH.

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Fig. 3. Schematic of the glucose-responsive insulin delivery system based on red blood cells. Synthesized Glc-Insulin was attached to erythrocytes by interacting with glucose

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2017, Wiley-VCH.

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receptor/transporter on plasma membranes [39]. Adapted with permission. Copyright

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Fig. 4. (a) Preparation of vancomycin encapsulated supramolecular gelatin

nanoparticles with RBC membrane coating layer (Van⊂SGNPs@RBC). (b) Schematic

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representation of adaptive and multifunctional Van⊂SGNPs@RBC in the treatment of

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Chemical Society.

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a bacterial infection [44]. Adapted with permission. Copyright 2014, American

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Fig. 5. Microfluidic electroporation-facilitated synthesis of RBC-MNs for enhanced imaging-guided cancer therapy. (a) Microfluidic electroporation facilitates the

synthesis of RBC-MNs. (b) Subsequently, the RBC-MNs, which are collected from the

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microfluidic chip, enrich in the tumor site after the blood circulation. (c) Biomimetic

RBC-MNs are further used for enhanced in vivo tumor MRI and PTT [53]. Adapted

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with permission. Copyright 2017, American Chemical Society.

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Fig. 6. Preparation of nanosponge colloidal gel. (a) Schematic illustration of NC-gel formulation by mixing red blood cell membrane-coated nanoparticles (RBC-NPs),

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which possess a negative surface charge, with chitosan-modified nanoparticles (ChiNPs) as positively charged nanoparticle counterparts. (b) Images of RBC-NPs, Chi-

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NPs, and NC-gel samples when they were placed onto a flat substrate. Scale bar = 5

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mm [72] . Adapted with permission. Copyright 2017, American Chemical Society.

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Fig. 7. Schematic representation of Oil-NS, consisting of an oil droplet surrounded and

stabilized by a naturally derived RBC membrane. Oil-NS are designed as a bimodal

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scavenger for removing OP compounds through concurrently capturing OPs with the

inner oil phase and the AChE enzyme present on the RBC membrane [74]. Adapted

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with permission. Copyright 2019, American Chemical Society.

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Fig. 8. Schematic depicting on-demand fabrication of a pathogen-specific nanotoxoid and its vaccination benefits. (a) Pathogens secrete virulence factors, which are capable

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of inserting into target cells and causing their destruction. (b) Using nanosponges

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prepared with the membrane of target cells and incubating the particles with a bacterial supernatant-derived protein fraction, it is possible to generate a nanotoxoid carrying

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pathogen-specific virulence factors. (c) After vaccination using the nanotoxoid, antibodies against the incorporated virulence factors are elicited and can prevent their toxic effects, leaving the intended targets unharmed [79]. Adapted with permission.

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Copyright 2017, Wiley-VCH.

Table 1. Erythrocyte-membrane-coated nanovehicles for different applications Disease models

Loaded drugs

Nanocarriers

superior drug release profile, excellent immunocompati bility, advantageous safety profile

Cancer, Atheroscleros is, Bacterial infection, Diabetes

Chemotherape utics, Antibiotics, Antibodies

Nanophotosensiti zers

prolonged in vivo Cancer circulation, enhanced accumulation in the disease site, enhanced phototherapy

Nanoantidotes

prolonged blood circulation, pronounced detoxification capability, superior systemic biosafety

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Chemotherape utics, Perfluorocarbo n,

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Enhanced immunogenic efficacy, excellent safety profile, superior protective immunity against

Material s of inner core PLA, PLGA, SiO2@Ti O2, MSNs, PCL, Hydrogel , Liposom e Gold, UCNPs, Fe3O4, Melanin, Hollow prussian blue, Albumin, Bismuth, PPy, Selenium PLGA, Hydrogel , Gold

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PLGA

7780

2747

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Application Main Features s

Bacterial Antibiotics infection, Organophosp hate poisoning, chemotherap y related toxicity, Type II immune hypersensitiv ity reactions Bacterial / infection, Cancer

4864

6576

influenza virus, bacterial infection, autoimmune hemolytic anemia

/

PLGA

8183

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Nanoprobes

adverse effects Prolonged retention time, enhanced immune evading superior targeting and isolation