Oriented Assembly of Cell-Mimicking Nanoparticles

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Oriented Assembly of Cell-Mimicking Nanoparticles via a Molecular Affinity Strategy for Targeted Drug Delivery Jing Xie, Qing Shen, Kexin Huang, Tingyu Zheng, Liting Cheng, Zhen Zhang, Yang Yu, Guojian Liao, Xiaoyou Wang, and Chong Li ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b09681 • Publication Date (Web): 25 Apr 2019 Downloaded from http://pubs.acs.org on April 26, 2019

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A peptide ligand derived from the cytoplasmic protein P4.2 was adopted to specifically recognize the cytoplasmic domain of Band 3, a key transmembrane receptor of erythrocytes. Once anchored onto the surface of liposomes, the P4.2-derived peptide would interact with the isolated RBC membrane, forming a ‘hidden peptide button,’ which ensures the right-side-out-oriented coating.

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Oriented Assembly of Cell-Mimicking Nanoparticles via a Molecular Affinity Strategy for Targeted Drug Delivery Jing Xie,† Qing Shen, ‡ Kexin Huang,† Tingyu Zheng,† Liting Cheng,† Zhen Zhang,§Yang Yu,† Guojian Liao,† Xiaoyou Wang,† *and Chong Li†*

† Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing, 400715, China. ‡ Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200032, China § Department of Clinical Laboratory, Chongqing General Hospital, Chongqing, 400014, China.

Abstract: Cell membrane-cloaking is an emerging field in drug delivery, in which specific functions of parent cells are conferred to newly-formed biomimetic vehicles. A growing variety of delivery systems with diverse surface properties have been utilized for this strategy, but it is unclear whether the affinity of membrane–core pairs could guarantee effective and proper camouflaging. In this study, we propose a concise and 1


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effective “molecular affinity” strategy, using the intracellular domain of transmembrane receptors as “grippers” during membrane coating. Red blood cell (RBC) membranes and cationic liposomes were adopted for fabrication, and a peptide ligand derived from the cytoplasmic protein P4.2 was prepared to specifically recognize the cytoplasmic domain of band 3, a key transmembrane receptor of erythrocytes. Once anchored onto the liposome surface, the P4.2-derived peptide would interact with the isolated RBC membrane, forming a “hidden peptide button,” which ensures the right-side-out orientation. The membrane-coated liposomes exhibited an appropriate size distribution around 100 nm and high stability, with superior circulation durations compared with those of conventional PEGylated liposomes. Importantly, they possessed the ability to target Candida albicans by the interaction between the pathogenic fungus and host erythrocytes and to neutralize hemotoxin secreted by the pathogenic fungi. The curative effect of the model drug was thus substantially improved. In summary, the “molecular affinity” strategy may provide a powerful and universal approach for the construction of cell membrane-coated biomaterials and nanomedicines at both the laboratory and industrial scales.

Keywords: biomimetics, red blood cell membranes, peptide ligand, targeted drug delivery, fungal infection

Based on the rapid development of biomimetic nano-delivery systems, cell membrane camouflaging has emerged as a promising technical platform with increasing applications,1 enabling preferred nano-bio interactions between foreign nanoparticles and 2


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the complex physiological environment, such as immunocompatibility, binding to the injured vasculature, penetrating inflamed brain tumors, and transport across the endothelium barrier.2-7 This field has seen an expanding library of cell membranes being adopted, and strategies for multi-membrane cloaking as well as enriched options for nanoparticulate cores have further extended its vast applications.8, 9 However, researchers may encounter two vital issues in the fabrication of cell membrane-camouflaged nanocarriers, which, if not properly solved, would impose serious restrictions on the development of this strategy. In particular, it is necessary to confirm (1) that the membranes are successfully coated on the nanoparticulate cores and (2) that they are in the correct orientation, considering the asymmetric biological properties of the membranes. Zhang et al. pioneered a highly-efficient extrusion coating technique for the right-side-out fabrication of several membrane-cloaked poly(lactic-co-glycolic acid) (PLGA) nanoparticles with various cell membrane types,2 along with detailed studies of the coating mechanism based on electrostatic interactions between the cell membrane and negative-charged PLGA nanoparticles. It is worth noting that effective coating was not always automatically achieved by random cell membrane-core pairs. According to their studies, the repulsion between negatively charged PLGA nanoparticles and cell membrane enabled effective coating, while the attraction between positively-charged PLGA nanoparticles and cell membrane led to large aggregations instead.10 Considering the extensive potential applications and the growing variety of cell membranes and nanoparticulate cores, it is necessary to explore additional strategies for construction, especially in the absence of proper affinity between certain cell membrane–core pairs. Therefore, more efforts are needed to explore the controllability of the membrane coating 3


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orientation in order to support their biomimetic functions. A potentially valuable application of membrane-coated nanodelivery systems is in the treatment of fungal infections, which have been largely neglected by the public,11 but are in fact a serious medical issue worldwide and a significant threat to public health.12 Despite the 1.6 million deaths caused by fungi infection annually and the expanding at-risk population, 13, 14 the antifungal pipeline is still insufficient, and toxicity remains a constant concern for existing antifungal agents. 15 Therefore, the development of safe and efficient formulations becomes an important solution. We propose a “molecular affinity” strategy for the construction of a biomimetic drug delivery system with a right-side-out membrane coating. We hypothesize that efficient, right-side-out membrane coating could be realized based on the specific interaction between the intracellular domain of key transmembrane receptors within the cell membrane and the corresponding peptide as a specific ligand, specifically connecting the surface of ligand-modified nanoparticles with the inner side of the cell membrane. Considering the interaction of pathogenic fungi with host erythrocytes, we adopted Band 3, a main transmembrane receptor of the RBC, as the “gripper” and designed a peptide ligand for a “molecular affinity” strategy. We systematically evaluated and characterized the RBC membrane-coated liposome, including analyses of its targeting ability and curative effect against the pathogenic fungus Candida albicans.

Results and Discussion Design and Evaluation of the Peptide Ligand 4


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Band 3, also known as AE1 (anion exchanger 1) or SLC4A1 (solute carrier family 4 member 1), is a classic transmembrane protein specifically distributed in erythrocyte membranes. Its many functions rely on the interaction between its intracellular domain and various cytoplasmic proteins. In other words, these intracellular proteins possess special fragments or domains that could specifically interact with the cytoplasmic tail of the band 3 protein. Protein 4.2 (P4.2, erythrocyte membrane protein band 4.2, or EPB42) is a cytoskeletal protein in erythrocytes consisting of multiple fragments with specific affinity for the intracellular domain of band 3.16, 17 Based on the interaction between P4.2 and band 3, we constructed a potential peptide ligand targeting the cytoplasmic domain of Band 3 (Scheme 1). By homology modeling and molecular docking, we found that the peptide sequence LFVRRGQPFTIILYF derived from human P4.2 could specifically interact with the band 3 protein from humans or mice (Figure 1, Figure S1 & S2). In particular, the positively charged Arg4 and Arg5 of the P4.2 peptide formed an electrostatic interaction with Glu156 and Glu318 in band 3, respectively. Tyr14 formed a hydrogen bond with Glu139 in band 3. His148 and Arg317 in mouse band 3 formed hydrogen bonds with the backbone of the P4.2 peptide. Ile12 stretched into the hydrophobic pocket formed by Tyr155, Cys152, Leu134, and Thr132. Phe2 also formed hydrophobic interactions with Leu134 and Leu137. In addition to XScore calculations, surface plasmon resonance (SPR) was also used to confirm the affinity of the P4.2 peptide to band 3, using a random sequence as a negative control. According to the results shown in Table S1, the experimentally determined binding affinity was in good agreement with the theoretical value, and the random sequence had only marginal affinity towards band 3 (Figure S3), validating the specific affinity of P4.2 peptide towards 5


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mouse band 3. Besides the direct measurement of binding affinity, the effective binding between P4.2 peptide and Band.3 was also verified by a competitive binding experiment. P4.2 protein (1 pmol) was immobilized on a chip, and its binding with Band.3 was significantly inhibited by P4.2 peptide when the peptide concentration reached over 80nM (i.e. the molar ratio of P4.2 peptide/P4.2 protein was above 8:1), also demonstrating the high affinity between P4.2 peptide to Band.3. (Figure S4) Characterization of the Orientation of Coated Liposomes The fabrication process for RBC membrane-coated liposomes (RBC-LIP) is illustrated in Figure 2a. First, conventional cationic liposomes loaded with amphotericin B (AmB) were prepared. Then, the P4.2-derived peptide ligand with a palmitic acid linked to its N-terminal by amido bond (Figure S5), was inserted onto the surface of the liposome membrane under moderate incubation conditions (post-insertion), and the RBC membrane was coated by the interaction between the Band 3 intracellular domain and the peptide on the surface of liposomes. It is noteworthy that the sufficient interaction between P4.2 peptide and the intracellular domain of membrane Band 3 was guaranteed by two methods. Firstly, as detected by quantitative western blot, over 50% of the proteins bound on the RBCm had been removed after the extraction and preparation process of RBCm, leaving a large number of binding sites for P4.2 peptide ligand (Figure S6). Secondly, the molar ratio between P4.2 peptide and phospholipid in the liposomal system was 0.5%, ensuring that the molar ratio between P4.2 peptide and P4.2 protein would reach 8:1, a ratio high enough for competitive binding of P4.2 peptide with Band 3 at the existence of P4.2 protein. Electron microscopy confirmed uniform morphological characteristics with a core-shell structure, indicating a membrane-coating layer 6


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surrounding the liposome (Figure 2b and Figure S7). The AmB-loaded RBC-LIP (RBC-LIP-AmB) was around 100 nm and negatively charged (Figure S8a and S8b), with a high drug-encapsulating efficacy of 90.87 ± 0.69%. No significant change in RBC-LIP particle size was observed over 72 h at room temperature, suggesting its good stability (Figure S8c). Compared with conventional bare LIP (Figure S9), RBC-LIP showed an approximately 15 nm larger particle size and a lower zeta potential, which was relevant for the RBC membrane coating. In addition, as shown in Figure S10, the sustained-release behavior of RBC-LIP-AmB was more favorable than that of bare LIP-AmB. To verify the RBC membrane coverage, the membrane protein content of RBC-LIP and RBC membranes (RBCm) was first examined by western blotting (WB). RBC-LIP gained a parallel protein content to that of treated RBCm after the cloaking process (Figure 2c). According to Figure 2d, successful retention of the characteristic membrane protein band 3 and CD47 in RBC-LIP was confirmed. Confocal laser scanning microscope (CLSM) images also demonstrated complete colocalization of the LIP core and RBCm in RBC-LIP (Figure 2e, 2f and 2g), indicating the successful wrapping of RBCm on the liposomes. The integrity of the coverage was examined by an aggregation assay. Based on the crosslinking ability of streptavidin and biotin, biotinylated LIP would readily aggregate at streptavidin. As shown in Figure S11, for a low RBC membrane/liposome ratio, membrane coverage failed to prevent aggregation, and the aggregation level gradually decreased with an increase in the membrane/liposome ratio. As the membrane/liposome ratio reached 700 μL of blood/mL of liposome or higher, the addition of streptavidin no longer induced a significant size change, indicating complete 7


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RBC membrane shielding over the biotinylated LIP cores. Importantly, the right-side-out coating of RBCm based on the “hidden peptide button” was further examined. Sialic acid is only distributed on the outer surface of the RBC surface, and its asymmetric distribution could serve as a quantitative indicator of the RBC membrane sidedness in RBC-LIP. The sialic acid content was measured by the trypsinization method, and no difference was detected between RBC-LIP and RBCm, indicating the right-side-out coating of the RBC membrane on the liposome core (Figure S12). CD47 is a specific biomarker of erythrocytes with an extracellular domain and intracellular domain, and immunogold staining experiments targeting different domains of CD47 confirmed the right-side-out RBCm coating. As shown in Figure 2h and 2i, TEM images verified the existence of the CD47 extracellular domain on the outer surface of RBC-LIP-AmB, but no significant staining of the CD47 intracellular domain was observed, indicating that the membrane coating was in the correct orientation. In comparison with the designated button peptide, random peptide modification did not lead to the effective coating of RBCm on cationic or neutral liposomes, as both intracellular and extracellular CD47 was present on their outer surfaces, which was proved by immunogold staining (Figure S13). Additionally, according to the fluorescent colocalization assay, only marginal colocalization between core and membrane was observed without the assistance of P4.2 peptide, whether the liposome core was positively charged or electrically neutral. (Figure S14) This proved again that, instead of electrostatic interactions, it was the molecular affinity strategy which truly enabled effective coating. Evaluation of RBC-Inherited Properties of RBC-LIP-AmB 8


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By the right-side-out coating of RBCm, liposomes could gain inherent biomimetic properties from RBCs, including immunocompatibility and prolonged circulation.18 The retention of CD47 on RBC-LIP ensured effective macrophage escape of RBC-LIP-AmB. As shown in Figure 3a&b and Figure S15, the macrophage uptake of RBC-LIP was significantly reduced compared with that in other LIP groups, including ctrl-RBC-LIP, a control group in which liposomes were post-inserted with randomly-sequenced P4.2 peptide and then incubated with RBCm. Interestingly, RBC-LIP even outperformed conventional PEG-LIP in escaping macrophage capture due to the functional proteins on the RBCm. Accordingly, RBC-LIP-AmB also exhibited significantly prolonged in vivo circulation compared with that of conventional 5%-PEGylated liposomes, which is of considerable significance for its application in drug delivery. Specifically, at 24 h after administration, the blood concentration of the RBC-LIP-AmB group was two-fold higher than that of the PEG-LIP-AmB group (Figure 3c). In addition to the pharmacokinetic characteristics, targeting ability was improved by the RBCm coating based on the specific affinity between pathogenic fungi and erythrocytes. Iron is an essential metal for fungal pathogens, while hemoglobin is an important iron resource for various pathogenic fungi. Candida albicans could actively capture erythrocytes for iron uptake, primarily by the active binding of CD21 on C. albicans to C3d on erythrocytes.19, 20 The active binding of RBC-LIP to C. albicans was confirmed by fluorescence microscopy as well as flow cytometry at the cellular level (Figure 3d). The binding mechanism was clarified by a receptor saturation experiment, and the blocking effects of both the antibody against C3d on erythrocytes and the antibody against the CD21 complement receptor were verified separately. No significant 9


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binding was observed after the blocking of either the C3d antibody or CD21complement receptor antibody, verifying that the targeting effect was mediated by the binding of CD21 and C3d; these results were confirmed by quantitative flow cytometry (Figure S16). In the in vivo C. albicans infection model, RBC-LIP exhibited specific accumulation in infected lungs based on live imaging (Figure 4 and S17) and CLSM images of tissue sections indicated that effective colocalization of RBC-LIP and the fungal pathogen (Figure S18), while the in vivo targeting effect towards infected lungs and C. albicans was significantly inhibited when the RBC-LIP was pre-incubated with C3d antibody before injection. A quantitative in vivo tissue distribution analysis also confirmed the targeting function towards infected lungs, providing a foundation for therapeutic applications (Figure S19). Therapeutic Efficacy of RBC-LIP-AmB Amphotericin B possesses a strong antifungal efficacy accompanied by significant toxicity. By modifying its biodistribution, the liposomal formulation of AmB exhibits reduced side effects and has been on the market since the 20th century. Based on the existing AmB liposome preparation, the right-side-out coating of RBCm guaranteed by the post-insertion of a button peptide could enable the convenient and effective cloaking of erythrocyte membranes, further enhancing the therapeutic effect. In vitro antifungal results (Figure 5a) proved that RBC-LIP-AmB had an equivalent effect to that of free AmB, and superior effects to those of LIP-AmB, PEG-LIP-AmB and ctrl-RBC-LIP-AmB. At an AmB concentration of 2 μg/mL, the RBC-LIP-AmB group inhibited the growth of fungi. In contrast, the PEG-LIP-AmB group, ctrl-RBC-LIP-AmB group and LIP-AmB group did not show effective inhibition until the AmB concentration 10


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exceeded 4 μg/mL, which was twice the concentration needed for the RBC-LIP-AmB group. In vivo experiments demonstrated reduced C. albicans infection as well as a prolonged survival time for RBC-LIP-AmB treatment. As summarized in Figure 5b & Figure S20, the colony-forming units (CFU) in the lungs of the model animal were effectively reduced by RBC-LIP-AmB compared with the CFUs in the PEG-LIP-AmB group after 7 d of treatment. MRI (magnetic resonance imaging) was also adopted to visualize the signals of infected lungs in vivo. As shown in Figure 5d and S21, the infected mice with no treatment exhibited increased MRI signals (white) at lung sites, indicating severe fungal inflammation. The RBC-LIP-AmB group exhibited a complete recovery with no remaining infection signals, superior to the results in the PEG-LIP-AmB, ctrl-RBC-LIP-AmB and LIP-AmB groups. According to the survival rates summarized in Figure 5c, 75% of the infected mice treated with RBC-LIP-AmB survived over 15 d, while in the other three treatment groups, the survival rate dropped to 40%, 15% and 10% respectively. In the untreated group, none of the infected mice survived for 8 days. Moreover, the pathogenesis of some pathogenic fungi includes hemolysis toxicity by the secretion of hemotoxin, which could also be neutralized by RBC-LIP-AmB due to its RBC membrane properties. According to Figure S22, RBC-LIP exhibited a significantly increased detoxification effect compared to that of negative control group, while bare LIP showed no significant protection against the hemolysis toxicity of C. albicans. The main physiological indexes and H&E (hematoxylin & eosin) staining results for RBC-LIP-AmB also demonstrated good compatibility (Figure S23 and S24). The concept of targeting is no longer limited to the interaction between therapeutic 11


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agents and their destinations. The “molecular affinity strategy” has expanded the concept of ligand targeting towards the hidden inner side of the cell membrane, ensuring the “targeted fabrication” of a right-side-out membrane-coated drug delivery system itself. Interactions between transmembrane receptors and the corresponding cytoplasmic protein are universal in natural conditions and thus may provide rich natural resources for the development of the required button peptide ligand. In this case, the “hidden peptide button” design would have widespread applications for the development of smarter and biomimetic drug delivery systems. Furthermore, based on the feasibility of oriented coating by targeted fabrication, a “wrong-side-out” coating could also be designated, with alternative effects. The intracellular domain of the membrane peptide could be intentionally exposed on the surface of the “wrong-side-out” coated vehicles, and might serve as a specific vaccine for necrosis sites with membrane eversion for the treatment of cancer and other diseases. 21-25 Conclusion Biological membrane coating has emerged as a highly promising strategy in a wide range of fields, including drug delivery and biomedical materials, and has drawn increasing attention in both academia and industry. We proposed a strategy to construct a right-side-out biomembrane-coated delivery system based on the intracellular domain of a membrane protein; this strategy can contribute to the study of biomembrane-coated drug delivery systems and can expand the applications of various biological membrane-related nanomaterials and nanomedicine. Experimental Section Surface Plasmon Resonance (SPR) Analysis 12


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The interactions between P4.2 peptide and band 3 were examined by SPR (Nicoya Lifescience, Waterloo, Canada).26, 27 To immobilize band 3 onto the NTA sensor chip, the sensor chip was firstly loaded with NiCl2, followed by an injection with a His-tagged band 3 protein according to the manufacturer’s instructions. To validate the affinity

between P4.2 peptide and band 3, P4.2 peptide of varying concentrations was injected to pass over the sensor chip at a constant flow rate of 20 μL/min in the running buffer (10 mM Hepes, 150 mM NaCl, 0.005% Surfactant P20, pH 7.4). The experiment was carried out at 25°C, and all concentrations were measured in triplicate and the sensor surface was regenerated by injecting 200 mM imidazole. For the competitive binding assay, recombinant P4.2 protein with GST tag at its N-terminal was immobilized on the chip obtaining 1000 RUs (response units) or 1ng bound protein/mm2 of sensor surface. The immobilized amount of P4.2 protein can be further calculated as 1 pmol based on the chip surface area radius. P4.2 peptides at various concentrations were co-injected with a fixed concentration of Band.3 protein (20nM) onto the immobilized protein. The inhibition of Band.3 to P4.2 protein was indicated by a loss in total RUs. The random P4.2 peptide served as a negative control. Homology Modeling and Molecular Docking The crystal structure of human band3 protein has been determined at 2.6 Å resolutions (PDB ID: 1hyn), which formed a tight symmetric dimer. The FASTA program (http://www.ebi.ac.uk/Tools/fasta/index.html) was used to identify sequence homology. The mouse band3 protein shares 83% similarity with the human band3 protein (FigureS1). 13


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The homology module of schrodinger program suite was employed to assemble the 3D models of the mouse band3 protein using the X-ray crystal structure of human band3 protein as template. A reasonable conformation was chosen from the top 10 candidates that have the lowest root mean square deviation values and considerable geometrical compatibility. The best model was submitted to energy minimization with schrodinger program. The PROCHECK statistics showed that over 90% residues in the mouse band3 protein model were in either the most favored or in the additionally allowed regions of the Ramachandran map. 28 The 3D structure model of the mouse band3 protein was show in Figure S2. The 3D structure model of P4.2 peptide was predicted by PEP-FOLD program.29 The docking program ZDOCK was used for the preliminary peptide-protein docking, 30

and the flex-peptide docking program in Rosetta suite was further used to refine the

minimum-energy conformations binding to the mouse band3 protein. 31 Binding affinities were predicted by X-Score 1.2 with empirical scoring functions. 32 Isolation of RBC-Membrane The RBC membrane was isolated from red blood cells following published protocols with modifications.

33

In brief, whole blood was freshly collected from female Kunming

mice (6-8 weeks) through cardiac puncture using a syringe preconditioned with heparin. The whole blood was then centrifuged at 1500×g for 10 min at 4℃ to remove the serum and the buffy coat, and the resulting RBCs were washed by ice-cold PBS for three times. The washed RBCs were then subjected to a hypotonic medium treatment for hemolysis to 14


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remove their intracellular contents; briefly, excessive amount of cold hypotonic lysing buffer（1/4×PBS）were mixed with as-washed RBCs, and kept in an ice bath for 30 min. The supernatant was subsequently centrifuged for four times at 20000×g for 30 min at 4 ℃ to collect the light-pink RBC ghost pellets. To partially remove band 4.2, ghosts were incubated in 1 M KC1 at pH 9.0 for 30 minutes on ice. The suspension was centrifuged at 12000 rpm for 30 minutes and the supernatant was discarded. Preparation of RBC-LIP In order to coat the RBC membrane onto the surface of LIP-AmB, the preparation process mainly consisted of two steps: preparing ligand-coupled LIP-AmB by post-insertion method, and coating the ligand-modified LIP-AmB with RBC membrane by direct extrusion method. 3, 34 To prepare ligand-coupled LIP-AmB, the original LIP-AmB were incubated with P4.2 peptide for 30 min at 25 ℃ . Then the RBC-LIP was fabricated bydirectly coating ligand-modified LIP-AmB with RBC membrane via extruding. Briefly, the RBC membranes were sonicated for 5 min using a bath sonicator at a power of 100 W. Then, 1 mL of LIP-AmB was mixed with the purified RBC membrane derived from 1 mL of whole blood and incubated for 30 min, subsequently extruded for 20 times through 400-nm and then 200-nm polycarbonate porous membranes using an Avanti mini extruder to prepare RBC-LIP. Membrane Sidedness Assay of RBC-LIP Membrane sidedness assay of RBC-LIP was evaluated according to the method described 15


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by Hu C M et al. 35 A drop of the RBC-LIP or bare liposome solution was deposited onto a glow-discharged carbon-coated copper grid, and then blocked with 5 wt% bovine serum albumin. For immunogold staining, the sample was incubated with 3 drops of 100 μg/mL anti-CD47 (extracellular or intracellular) antibody solution ， that specifically targets an extracellular/intracellular sequence of CD47. After 1 min incubation, the sample was stained with 3 drops of gold conjugated secondary antibody against rabbit IgG (5 nM) solution and then washed with 10 drops of distilled water. For negative staining, the sample was washed with distilled water and stained with 1% phosphotungstic acid. These samples were then imaged using transmission electron microscopy (TEM). The controlled RBC-LIPs groups, in which either cationic liposomes or neutral liposomes were anchored with a random peptide ligand and incubated with RBC membranes, were detected by the same method. To further confirm the membrane sidedness of RBC-LIP, another method was adopted to quantify the amount of sialic acid, a characteristic carbohydrate terminus on RBC glycans.10 Briefly, 1 mL of prepared RBC-LIP was incubated with 100 units of sialidase at 25℃ for 2h, and the mixture was centrifuged at 200000 g for 45 min. The supernatant was collected and the amount of sialic acid was quantified using a sialic acid assay kit. RBC ghost and bare liposome cores at the same amount were used as a positive and a negative control, respectively. Macrophage Uptake Study To test the stealth ability of as-prepared RBC-LIP, murine RAW 264.7 macrophage cell 16


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was chosen for cell uptake studies. 36, 37 For qualitative and quantitative cellular uptake measurements, liposome was labeled with the hydrophobic fluorescence probe Nile red. Approximately 8 104 RAW264.7 cells were seeded in 24-well plates in DMEM with 10% fetal bovine serum, 1% penicillin/streptomycin and cultured at 37℃ in 5% CO2 for 24 h. Then, NiL-labeled bare liposome, RBC-LIP, ctrl-RBC-LIP and PEG-LIP were added to each well with NiL concentration of 0.2 μg/mL, and incubated for 1h. For fluorescence imaging, the cells were gently washed three times with PBS, fixed with 4% paraformaldehyde solution for 30 min, and then washed with PBS, stained with DAPI for another 10 min, and the cells were observed by a confocal fluorescence microscope. Meanwhile, the cells were detached with trypsin EDTA (Gibco), and collected for quantification by a BD FACSVerseTM system (BD, USA). In Vitro Interaction of RBC-LIP with C. albicans The in vitro interaction of RBC-LIP with C. albicans was evaluated by fluorescence microscope and FACS measurements.

38-40

Briefly, C. albicans was diluted to 1×105

CFU/well with cell culture medium (5% mouse serum) and seeded into 24-well plates, followed by the addition of different DiI-labeled liposomes groups with 10 μg/mL DiI concentration, and incubated for 2 h. To clarify the possible biomimetic function of RBC-LIP, C. albicans were preincubated with 4 μg/mL anti-CD21 mAbs for 30 min at 37℃, and then DiI-labeled RBC-LIP was added. For competitive inhibition assays, 1.0 mL of DiI-labeled RBC-LIP was mixed with 10 μg of anti-C3d mAbs, and then incubated with C. albicans. The fungi were washed with PBS three times after incubation, 17


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and the fungi-associated fluorescence was visualized under a fluorescence microscope (Leica, Germany) and further quantified by BD FACSVerseTM system (BD, USA). In Vivo and ex Vivo Imaging of RBC-LIP in a Lung Infected Model. 50 µL of C. albicans cell suspension were administered to mice at 1 × 108 CFU/mL via intranasal instillation to develop a lung-infected model. 41, 42 LIP, PEG-LIP, RBC-LIP and ctrl-RBC-LIP were labeled with a near-infrared probe DIR (Ex/Em=720/790 nm) for real-time in vivo and ex vivo tracking. In another control group, the DiR-labeled RBC-LIP (1.0 mL) was mixed with 10 μg of anti-C3d mAbs to block the C3d in RBC membrane, which was termed as the blocked RBC-LIP. After 48h infection, the infected mice were randomly separated into five groups and intravenously injected with corresponding formulations at a dose of 0.2 mg/kg. In vivo images were recorded using FX pro in vivo imaging system at a predetermined time points (1, 2, 4 and 8 h). For ex vivo images, mice were sacrificed at 1, 2, 4 and 8 h post administration, the major organs (heart, liver, spleen, lung, kidney and brain) were carefully collected, rinsed with cold PBS and observed. In Vivo Pharmacodynamics Efficacy Tissue burden study: Female BALB/c mice were inoculated with 50 μL of the C. albicans suspension (1×108 CFU/mL) through intranasal instillation to develop a lung-infected model. At 48 h after inoculation, mice were randomly divided into five groups

and injected with saline, LIP-AmB, PEG-LIP-AmB, RBC-LIP-AmB and

ctrl-RBC-LIP-AmB at 2 mg/kg of Amphotericin B, 43- 45 respectively. At day 3 and day 7, 18


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mice were sacrificed and the lung tissues from each group were carefully removed, weighed, and homogenized with sterile saline. The homogenate was serially diluted with sterile saline, and then 100 μL of the suspension was inoculated onto Sabouraud dextrose agar with 0.05% chloramphenicol and incubated at 30°C for 48 h to determine CFU/g lung tissue. Survival rates study: The lung-infected mice model was induced as described above. At 24 h after inoculation, the infected mice were randomly divided to five groups and treated intravenously with saline, LIP-AmB, PEG-LIP-AmB, RBC-LIP-AmB and ctrl-RBC-LIP-AmB, respectively, with a single dose (AmB concentration 2mg/kg). The survival rates of the model mice were monitored for 15 days after treatment. MRI Assay: On the 7th day after treatment, animals were anaesthetized with isoflurane gas and the MRI examinations were carried out. The MRI examinations were detected on a 1.0 T MRI scanner (NM42-040H-I, Niumag, China), and the mean signal intensities of the lung regions were measured using Image J.46 Associated Content Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 

Experiment section (Materials, Cell Culture, Experiment Animals, Preparation of Amphotericin B Liposomes, Optimization of RBC-LIP Preparation, Characterization 19


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of RBC-LIP, Characterization of RBC Membrane Proteins in RBC-LIP, Fluorescent Co-Localization Analysis, Pharmacokinetic Study, Fluorescence Co-Localization of RBC-LIP/DiI and C. albicans in the Lung, Biodistribution Studies, In Vitro Anti-Fungal Activity, In Vivo Toxicity Studies, Detoxification against Fungal Hemolysin, Supporting Figures (S1-S24) and Supporting Tables (S1) (PDF) Author Information Corresponding Authors *Email: [email protected]; [email protected] ORCID Chong Li: 0000-0002-6049-7675 Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81673376, 81803472, 21807071), and Natural Science Foundation of Chongqing (cstc2015jcyjBX0100) .

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Scheme 1. Design of the peptide ligand targeting the intracellular domain of Band 3 based on the interaction between P4.2 and Band 3.

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Figure 1. P4.2 peptide binding mode in the mouse band 3 protein and its affinity. (a) The mouse band 3 protein was evaluated by the electrostatic surface. The P4.2 peptide is shown in a cartoon representation and is colored in green. (b) Detailed interaction between P4.2 peptide and mouse band 3. Residues involved in binding are represented in stick format. The P4.2 peptide is shown in green and residues in mouse band 3 are shown in white. Hydrogen bonds are denoted by black dashed lines. (c) Surface plasmon resonance analysis of the affinity between the peptide ligand and band 3.

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Figure 2. Preparation and characterization of RBC-LIP. (a) Summary of the targeted fabrication of red blood cell membrane (RBCm)-coated liposomes (RBC-LIP). The P4.2-derived peptide ligand was modified on AmB-loaded liposomes using a post-insertion technique. (b) TEM (transmission electron microscope) image of RBC-LIP 30


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(scale bar = 50 nm). (c) Protein content of i: RBC ghost, ii: treated RBCm, iii: RBC-LIP, as determined by SDS-PAGE; protein marker shown in the left column. (d) Western blotting analysis of Band 3 and CD47 in RBC ghost, treated RBCm, and RBC-LIP. (e, f, g) Co-localization of LIP (red) and RBCm (green) by CLSM (scale bar = 5 μm). (h) TEM images of RBC-LIP, (scale bar = 200 nm), and a magnified view of a single RBC-LIP (scale bar = 50 nm), with the extracellular domain of CD47 indicated by immunogold labeling. (i) TEM images of RBC-LIP with the intracellular domain of CD47 stained by immunogold labeling (scale bar=200 nm). No significant staining of CD47 intracellular domain was observed.

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Figure 3. Inherited behaviors of RBC-LIP from RBCs. (a, b) Macrophage uptake of LIP, ctrl-RBC-LIP, PEG-LIP and RBC-LIP by CLSM (a) (scale bar =20 μm) and flow cytometry (b). (c) Pharmacokinetics of RBC-LIP-AmB in mice for 48 h after administration, and AmB blood concentration at 24 h of LIP-AmB, PEG-LIP-AmB, RBC-LIP-AmB and ctrl-RBC-LIP-AmB treatment. (n = 3, *p < 0.05, **p < 0.01) (d) 32


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Interaction of RBC-LIP and Candida albicans (scale bar = 25 μm). The RBCm-coated liposomes could actively target the pathogenic fungi, and this interaction could be competitively blocked using a C3d antibody or CD21 complement receptor antibody.

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Figure 4. In vivo targeting ability of RBC-LIP and other formulations in infected mouse models determined by live imaging.

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Figure 5. Therapeutic effect of RBC-LIP-AmB. (a) In vitro antifungal assay of RBC-LIP-AmB, PEG-LIP-AmB, LIP-AmB, ctrl-RBC-LIP-AmB and free AmB. (n = 3) (b) Colony-forming units (CFU) in the lungs of the infected mouse model after different treatments. (n = 3, *p < 0.05, **p < 0.01) (c) Survival rate of infected mice after different treatments. (*p < 0.05, **p < 0.01) (d) In vivo evaluation of infection signals after different treatments by MRI. The sites of infected lungs are indicated by red rectangles.

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Oriented Assembly of Cell-Mimicking Nanoparticles

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