Erythroliposomes: Integrated Hybrid Nanovesicles ... - ACS Publications

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Erythroliposomes: Integrated Hybrid Nanovesicles Composed of Erythrocyte Membranes and Artificial Lipid Membranes for Pore-Forming Toxin Clearance Yuwei He, Ruixiang Li, Haichun Li, Shuya Zhang, Wentao Dai, Qian Wu, Lixian Jiang, Zicong Zheng, Shun Shen, Xing Chen, Yuefei Zhu, Jianxin Wang, and Zhiqing Pang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b08964 • Publication Date (Web): 11 Mar 2019 Downloaded from http://pubs.acs.org on March 11, 2019

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Erythroliposomes: Integrated Hybrid Nanovesicles Composed of Erythrocyte Membranes and Artificial Lipid Membranes for Pore-Forming Toxin Clearance Yuwei He1#, Ruixiang Li2#, Haichun Li1, Shuya Zhang1, Wentao Dai3, Qian Wu3, Lixian Jiang2, Zicong Zheng1, Shun Shen1, Xing Chen1, Yuefei Zhu1, Jianxin Wang1,4*, Zhiqing Pang1* Yuwei He, [email protected] Ruixiang Li, [email protected] Haichun Li, [email protected] Shuya Zhang, [email protected] Wentao Dai, [email protected] Qian Wu, [email protected] Lixian Jiang, [email protected] Zicong Zheng, [email protected] Shun Shen, [email protected]

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Xing Chen, [email protected] Yuefei Zhu, [email protected] Jianxin Wang, [email protected] Zhiqing Pang, [email protected] 1

Department of Pharmaceutics, School of Pharmacy, Fudan University & Key Laboratory of

Smart Drug Delivery, Ministry of Education, Shanghai 201203, China 2

Innovation Research Institute of Traditional Chinese Medicine, Shanghai University of

Traditional Chinese Medicine, Shanghai 201203, China 3

Shanghai Center for Bioinformation Technology, Shanghai Industrial Technology Institute,

Shanghai 201203, China 4

Institute of Integrated Chinese and Western Medicine, Fudan University, Shanghai 200040,

China #

These authors contributed equally to this work.


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ABSTRACT

Pore-forming toxins (PFTs) are the most common bacterial virulence proteins and play a significant role in the pathogenesis of bacterial infections; thus, PFTs are an attractive therapeutic target in bacterial infections. Inspired by the pore-forming process and mechanism of PFTs, we designed an integrated hybrid nanovesicle—the erythroliposome (called the RM-PL)— for PFT detoxification by fusing natural red blood cell (RBC) membranes with artificial lipid membranes. The lipid and RBC membranes were mutually beneficial when integrated into a hybrid nanovesicle structure. The RBC membrane endowed RM-PLs with the capacity for detoxification, while the PEGylated lipid membrane stabilized the RM-PLs and greatly improved the detoxification capacity of the RBC membrane. With α-hemolysin (Hlα) as a model PFT, we demonstrated that RM-PLs could not only significantly reduce the toxicity of Hlα to erythrocytes in vitro but also effectively sponge Hlα in vivo and rescue mice from Hlα-induced damage. Moreover, the high detoxification capacity of RM-PLs was shown to be partly related to the expression of the Hlα receptor protein, a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) on the RBC membrane. Consequently, as a component integrating natural and artificial materials, the erythroliposome nanoplatform inspires potential strategies for antivirulence therapy.

KEYWORDS: erythroliposome, detoxification, hybrid nanovesicle, pore-forming toxins, red blood cell membrane, artificial lipid membrane


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Bacterial infections are a prominent cause of morbidity and mortality worldwide,1 and the need for mining novel therapeutic strategies is urgent, especially because of the increase in antimicrobial resistance.2,3 Pore-forming toxins (PFTs) are the largest class of protein toxins and are produced by various pathogenic bacteria, including Streptococcus pneumoniae, group A and B streptococci, Staphylococcus aureus (S. aureus), Escherichia coli and Mycobacterium tuberculosis.4 During bacterial infection, PFTs can act as an accomplice promoting pathogen growth, dissemination, and colonization through the disruption of epithelial barriers and interactions with the immune system.5 Studies have demonstrated that inhibiting PFTs can greatly reduce the severity of bacterial infections.6 Furthermore, for multidrug-resistant bacterial infections, virulence-targeted therapy is much more effective than direct antibacterial therapy and inhibits the development of resistance.7 Therefore, disarming bacterial PFTs is considered a rational and promising approach for the treatment of bacterial infections.8 As their name suggests, PFTs attack the cell membrane, forming transmembrane pores and thus altering the permeability of cells and initiating virulence.9 With inspiration from the principles governing natural toxin-cell membrane interactions, antivirulence strategies have been focused on designing biomimetic cell membranes for PFT neutralization.10 For instance, artificial liposomes containing sphingomyelin and a high cholesterol content were engineered to sequester bacterial PFTs and provide the basic lipid bilayer structure for toxin neutralization.11 The increasingly complete characterization of the structure and function of PFTs has shown that most PFTs recognize their target host cells with high specificity and affinity, largely because of their close interactions with specific receptors, including sugars, lipids and proteins, on the cell membrane.5 For example, phosphatidylcholine (PC), a disintegrin and metalloproteinase domaincontaining protein 10 (ADAM10), and disintegrin were identified as the specific receptors for α-


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hemolysin (Hlα), which is secreted by gram-positive S. aureus.5,12 Hence, in addition to the basic lipid structure, other complex cell membrane components also play a crucial role in the poreforming process of PFTs. In an attempt to retain the components and structure of the plasma membrane, a “nanosponge” was explored for the detoxification of PFTs by coating red blood cell (RBC) membranes onto polymeric nanoparticles.13,14 As RBCs are the target host cells for most PFTs, the cloaked RBC membrane could absorb various PFTs with high affinity.14,15 The results showed that although RBC membrane vesicles (RMVs) alone failed to neutralize the toxins, nanosponges with poly(lactic-co-glycolic acid) (PLGA) nanoparticle cores could detoxify various PFTs and stabilize the toxin-RBC membrane complex for vaccination.14,15 However, the detoxification capacity of nanosponges or artificial liposomes as biodetoxification nanoagents was challenged when the bacterial infection progressed. Moreover, the ease of nanosponge production needs to be considered carefully, because the current cell membrane coating process might be complicated and difficult to scale up. Moreover, the coating process might denature cell membrane proteins and further decrease the detoxification capacity.16 Therefore, biodetoxification nanoagents with high detoxification capacities and easy production processes are urgently needed for antivirulence therapy. According to the mechanism underlying the pore-forming process of PFTs, the recognition and binding of PFTs depend only on a specific small region on the cell membrane,5 suggesting that a small fraction of the cell membrane might effectively detoxify PFTs. Thus, exploiting artificial lipid membranes to replace most of the natural cell membrane might be promising, possibly maintaining or even enhancing the detoxification capacity of the cell membrane and eventually economizing this property. In addition, compared with natural cell membranes, artificial lipid membranes are more easily modified and more feasibly scaled up. Hence, inspired by the pore-


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forming process and mechanism of PFTs, we developed an integrated hybrid nanovesicle—the erythroliposome (called the RM-PL)—to detoxify PFTs by fusing natural RBC membranes and artificial lipid membranes, taking advantage of both biomaterials. The preparation of RM-PLs was carefully characterized. With Hlα as a model PFT, the toxin-neutralizing effect of RM-PLs was investigated in vitro in RBCs and human umbilical vein endothelial cells (HUVECs). Moreover, the detoxification effect of RM-PLs was assessed in vivo in mice subcutaneously and intravenously challenged with Hlα. All animal experiments were performed according to the experimental protocol approved by the Animal Experiment Ethics Committee of Fudan University. RESULTS AND DISCUSSION Preparation of RM-PLs. Hybrid nanovesicles were prepared by the conventional thin filmhydration and extrusion method, as shown in Figure 1A. To manufacture RM-PLs, RBC membranes were first obtained by the hypotonic method. RBCs isolated from whole blood of ICR mice were ruptured in hypotonic medium followed by centrifugation to separate the RBC membranes from the intracellular contents. The purified RBC membranes were preserved at 80 ℃ for further use. Soybean PC with a low phase transition temperature was selected as the main component of the hybrid nanovesicles. The addition of artificial lipid material can modulate the structural fluidity of the natural cell membrane, which particularly benefits the extrusion process. The pure RBC membrane was difficult to extrude through the polycarbonate membranes, but the procedure became much easier as the input of the artificial lipid membrane increased. In the present study, RBC and lipid membranes were fused at an RBC membrane:lipid membrane surface area ratio of 1:16 (Supplementary Discussion), indicating that most of the surface area of the RM-PLs was covered by an artificial lipid membrane. In addition,


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mPEG2000-DSPE was added to enhance the stability of RM-PLs in vitro and in vivo. The mixture of RBC membranes and artificial lipid membranes was physically extruded through polycarbonate membranes to facilitate cell membrane-lipid membrane fusion with little damage to cell membrane components. Easy scale-up production of RM-PLs could conceivably be performed after the parameters are defined.


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Figure 1. Fabrication of RM-PLs. (A) Schematic showing the preparation of RM-PLs. Purified RBC membranes were fused to liposomes through extrusion. (B) Liposomes labeled with a pair of FRET fluorescent dyes were fused with increasing amounts of RBC membranes, and their fluorescence spectra were recorded. Lm:RBCm indicates the weight ratio of liposomes to RBC membrane proteins. (C) Confocal fluorescence images of RM-PLs (right) and the physical


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mixture of liposomes and RBC membranes (left). Red, lipid membrane; green, RBC membrane; scale bar = 1 μm. (D) DSC measurement of PLs, RMVs and RM-PLs revealed the change in the transition temperature (Tm) of liposomes after fusion with RBC membranes. (E) The FT-IR spectra of RM-PLs, PLs and RMVs confirmed the presence of RBC membrane proteins in RMPLs. Verification of membrane fusion. To verify the successful fusion of the natural cell membrane and the artificial lipid membrane, membrane fusion studies were performed. The lipid membrane was labeled with a pair of FRET dyes (C6-NBD and RhB-DHPE) and fused to increasing amounts of RBC membrane. The fluorescence spectra of the samples demonstrated a recovery of the fluorescence signal at 534 nm (C6-NBD) and a decrease in the fluorescence signal at 583 nm (RhB-DHPE) with an increasing amount of RBC membrane (Figure 1B), suggesting that the RBC membrane was inserted into the lipid membrane and weakened the FRET of the dyes. Furthermore, the overlay of the lipid membrane and RBC membrane in RMPLs could be visualized via confocal fluorescence microscopy when the lipid membrane and RBC membrane were labeled with the red fluorescent dye DiD and the green fluorescent dye DiI, respectively. However, the physical mixture of the lipid membrane and RBC membrane exhibited distinct green and red fluorescent puncta (Figures 1C and S1). DSC, a widely used thermoanalytical technique, was used to analyze the changes in the thermal properties of liposomes after fusion with natural cell membranes. Compared with the transition temperature of PLs (Tm=47.5 ℃), the Tm of RM-PLs was increased to 50.3 ℃, which was almost the same as that of RMVs (Tm=50.4 ℃) (Figure 1D), indicating the fusion of the RBC and lipid membranes. In addition, FT-IR spectroscopy revealed that similar typical protein absorption bands were present in the RMV and RM-PL groups relative to those found in the PL


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group, manifesting the incorporation of RBC membrane proteins into the hybrid nanovesicles (Figure 1E).17 Among the absorption bands, 1,700-1,600 cm-1 was associated with C=O stretching vibrations, while 1,600-1,500 cm-1 was due to NH bending with C-N stretching vibrations. Taken together, these results strongly demonstrated the successful fusion of RBC and lipid membranes and the retention of RBC membrane components in the hybrid nanovesicles. Analysis of protein composition. The retention of membrane proteins on RM-PLs was typically determined by SDS-PAGE (Figure 2A). Compared with RMVs, RM-PLs retained almost all of the cell membrane proteins. Additionally, western blot analysis was carried out to analyze key proteins in RM-PLs. CD47 is a typical marker of self with an immunomodulatory effect,18 and ADAM10 is a key protein receptor on the cell membrane that plays an important role in the process of Hlα toxin insertion.5,12 The western blotting results revealed that both proteins were present on RM-PLs without loss during the fusion process (Figures 2B and S2).


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Figure 2. Membrane protein composition of RM-PLs. (A) SDS-PAGE analysis of RM-PLs and RMVs. (B) Western blotting of CD47 and ADAM10 in RM-PLs and RMVs. (C) Numbers of proteins identified in RM-PLs and RMVs. (D) Classification of RM-PL proteins by biological process. (E) Classification of RM-PL proteins by molecular function. (F) Heat maps of the expression of typical membrane proteins on RM-PLs and RMVs. (G) The orientation of glycosylated membrane proteins in RM-PLs, RMVs, and RBCs was determined with Texas RedX-conjugated WGA. * P

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