Genetically Engineered Cell Membrane Nanovesicles for Oncolytic

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Genetically Engineered Cell Membrane Nanovesicles for Oncolytic Adenovirus Delivery: A Versatile Platform for Cancer Virotherapy Peng Lv, Xuan Liu, Xiaomei Chen, Chao Liu, Yang Zhang, Chengchao Chu, Junqing Wang, Xiaoyong Wang, Xiaoyuan Chen, and Gang Liu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b00145 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 9, 2019

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Genetically Engineered Cell Membrane Nanovesicles for Oncolytic Adenovirus Delivery: A Versatile Platform for Cancer Virotherapy Peng Lv¶, Xuan Liu¶, Xiaomei Chen¶, Chao Liu¶, Yang Zhang¶, Chengchao Chu¶, Junqing Wang¶, Xiaoyong Wang¶, Xiaoyuan Chen§, and Gang Liu¶*

¶State

Key Laboratory of Molecular Vaccinology and Molecular Diagnostics and

Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen, 361102, China

§Laboratory

of Molecular Imaging and Nanomedicine, National Institute of

Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, MD 20892, USA

*Email: [email protected]. Tel: 86-592-2880648

1

ACS Paragon Plus Environment

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ABSTRACT: Currently, various oncolytic adenoviruses (OA) are being explored in both preclinical and clinical virotherapy. However, the pre-existing neutralizing antibodies (nAbs) and poor targeting delivery are major obstacles for systemically administered OA. Therefore, we designed bioengineered cell membrane nanovesicles (BCMNs) that harbor targeting ligands to achieve robust anti-viral immune shielding and targeting capabilities for oncolytic virotherapy. We employed two distinct biomimetic synthetic approaches: the first is based on in-vitro genetic membrane engineering to embed targeting ligands on the cell membrane, and the second is based on in-vivo expression of CRISPR-engineered targeting ligands on red-blood-cell membranes. The results indicate that both bioengineering approaches preserve the infectivity and replication capacity of OA in the presence of nAbs, in vitro and in vivo. Notably, OA@BCMNs demonstrated a significant suppression of the induced innate and adaptive immune responses against OA. Enhanced targeting delivery, viral oncolysis and survival benefits in multiple xenograft models were observed without overt toxicity. These findings reveal that OA@BCMNs may provide a clinical basis for improving oncolytic virotherapy by overcoming undesired anti-viral immunity and enhancing cancer cell selectivity via biomimetic synthesis approaches. KEYWORDS: Oncolytic adenovirus, Bioengineered cell membrane, Targeting delivery, Cancer virotherapy

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Oncolytic virotherapy is a therapeutic approach to cancer treatment that utilizes viruses that selectively replicate in tumor cells. This has shown promising results in both preclinical studies and clinical trials1, 2. Oncolytic adenoviruses (OA) have been extensively explored for cancer virotherapy3, 4. Recently, the US Food and Drug Administration approved talimogene laherparepvec (T-VEC) for the treatment of melanoma lesions in the skin and lymph nodes5. In addition, several human clinical trials have reported the use of oncolytic virus in local cancer gene therapy6. However, there are issues related to oncolytic virus delivery via the bloodstream, such as nonspecific sequestration7, 8, preexisting antivirus immunity and the innate immune response9, 10, which pose major barriers to the development of oncolytic virotherapy11. So far, considerable efforts have been devoted to developing new OA delivery systems, such as endogenous bioengineering of OA12-14, exogenous engineering of PEGlation15, and hybrid vector systems16-18. Nonetheless, major challenges still remain with regard to the poor targeting delivery of systemically administered OA. Extracellular vesicles (EVs) are a heterogeneous collection of membrane-bound carriers with complex cargoes, including proteins, lipids, and nucleic acids, which are released by almost all types of cells19-21. Both preclinical and clinical interest in EVs has increased rapidly as mounting evidence shows that they may constitute an ideal drug

delivery

system.

Multiple

strategies

have

exploited

naturally

or

tumor-cell-derived EVs for the delivery of exogenous therapeutic reagents, such as small-molecule anti-inflammatory drugs22, macromolecular drugs like siRNA23, and 3


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vaccine-like tumor-associated antigens for presentation in the immune system24, 25. EVs have been demonstrated to have multiple advantages over currently available drug delivery systems, such as their ability to protect cargoes from degradation in circulation, overcoming natural barriers, good biocompatibility, and stability in the circulation26-30. More importantly, EVs are easily to be genetically modified for targeted delivery and feature the characteristics of both nano-sized and cell-based drug delivery platforms. EVs are being bioengineered with highly stable "mimics" of targeting ligands via genetic modification procedures31, which have the potential to serve as an ideal OA delivery system to overcome current challenges for cancer therapies by systemic administration. This study proposes a facile and efficient strategy for fabricating a new class of bioengineered cell membrane nanovesicles (BCMNs) that can harbor different targeting ligands via natural biosynthetic procedures. The goal is to achieve robust anti-viral immune shielding and unique targeting capabilities for oncolytic virotherapy (Scheme 1). We employed two distinct biomimetic synthetic approaches: one is based on in-vitro genetic membrane engineering to embed targeting ligands on the cell membrane, and 2) the other involves the in-vivo expression of CRISPR-engineered targeting ligands on red-blood-cell (RBC) membranes. We show that OA can be directly encapsulated into BCMNs and exhibit liposome-like nanostructure, efficient loading, and excellent cancer-targeting specificities. We also show that the systemically injectable system of BCMNs is able to evade preexisting anti-OA immunity. Moreover, systemic administration of the 4


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OA@BCMNs evades the innate immune response and achieves efficient targeted delivery to desired tumor sites. The OA@BCMNs efficiently inhibit tumor cell proliferation, induce apoptosis while sparing normal tissues and cells, and exhibit enhanced anti-tumor efficacy in vitro and in vivo. This OA delivery strategy is promising for developing new concepts and providing new insights for improved cancer virotherapy applications. Recent studies have demonstrated that sodium taurocholate cotransporting polypeptide (NTCP) is a functional receptor for preS1 and multiple peptides derived from preS1 can specifically interact with NTCP32,33. The specific interaction between NTCP and preS1 provides a new strategy for targeted drug delivery. As a proof of principle, the targeting peptide preS132 was selected to design and generate nanovesicles (BCMNs-preS1) to improve the efficacy of targeted delivery with EVs. Firstly, we applied a biomimetic synthesis strategy to prepare BCMNs-preS1 by following a previously reported procedure33. As expected, the results of polyacrylamide gel electrophoresis and Western blotting assay showed that preS1 was found in the BCMNs-preS1 nanovesicles (Figures 1A and S1). For competition experiments, the HepG2-NTCP cells were pre-incubated with free preS1 peptides to block the NTCP sites. The obviously decreased green fluorescence in the cells implies that preS1-mediated site-specific targeting is the mechanism by which BCMNs-preS1 facilitates cellular uptake (Figure S2). A protein pull-down assay showed that beads incubated with BCMNs could capture the preS1 on the BCMNs-preS1 by bridging biotinylated anti-preS1 antibody and streptavidin due to the presence of preS1 on 5


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the exterior of BCMNs-preS1 (Figure S3). To evaluate the targeting ability of BCMNs-preS1, we compared the cellular binding abilities of BCMNs-preS1 and EVs. CLSM images showed that BCMNs-preS1 exhibited good specific cellular binding ability to HepG2-NTCP, whereas weak green light signals were observed in the EVs groups after incubation for 4 h (Figure 1B). These results demonstrate that BCMNs could be surface modified with bioactive ligands and obtain specific targeting ability. Next, the BCMNs were used to encapsulate OA and the successful coating was verified under Transmission electron microscopy (TEM). The TEM images showed that the vesicle-virus composites were spherically shaped and composed of adenovirus at the core and BCMNs as shells (Figure 1C). This suggests that the OA were successfully coated with BCMNs. The size and surface charge of the nanovesicle-virus composites were examined using dynamic light scattering (DLS). The results showed that the average hydrodynamic diameter of BCMNs was 150±10 nm, which slightly increased to 180±5 nm for the vesicle-virus composites after coating the surface of OA (80±10 nm) (Figure 1D). Zeta-potential measurements of the BCMNs, OA and OA@BCMNs revealed negative surface charges of -5±3 mV, -45±2 mV and -11±1 mV, respectively (Figure 1E). The surface charge of the composites was as low as that of the BCMNs, which also confirmed the successful surface coating. Notably, the average size of BCMNs and OA@BCMNs did not change within 10 days at 4 °C, while the size of OA increased after stored 10 days (Figure S4). These findings indicated that OA@BCMNs partly improved the storage stability compared to naked OA. 6


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To evaluate the structure of the OA@BCMNs further, we performed a dot-blot assay to detect the viral surface proteins using OA-specific antibody (anti-Hexon antibody). As a result, the anti-Hexon antibody could detect Hexon protein on the naked OA surface, but it was undetectable on OA@BCMNs (Figure 1F). However, under denature conditions, the protein could be detected in the OA@BCMNs after the naked adenovirus particle was encapsulated by BCMNs, which could block the accessibility of the antibody to the virus surface. The migration of OA@BCMNs was retarded in the gel electrophoresis assays (Figure 1G), indicating the presence of OA inside the BCMNs. The encapsulation efficiency of the nanovesicle-virus composites was evaluated using High-Sensitivity Flow Cytometry (HSFCM)34. To assess the performance of the instrument, BCMNs were stained with CFSE (BCMNs-CFSE), and the nucleic acids of OA were stained with SYTO 62. The nanovesicle-virus composites were passed sequentially through the tightly focused laser beam of the HSFCM, and the fluorescence signals of CFSE and SYTO 62 emitted from each individual particle were detected simultaneously. The cell membrane nanovesicles with CFSE FL signals were detected concurrently with the SYTO 62 FL signals and can be inferred to be the OA-encapsulated BCMNs (Figure 1H, left). This population of nanovesicle-virus composites was present in the bivariate dot plot (Figure 1H, right). All of the results demonstrate the successful preparation of the OA-encapsulated BCMNs nanostructure. We next determined whether the BCMNs covering the surface of OA could influence 7


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the infectivity of the virus. First, we examined the cytopathic effect of OA@BCMNs on HEK 293 cells. The experiment showed that OA@BCMNs had similar infectivity to the original OA after 72 h post infection (Figures 2A and S5). The kinetics of the cytotoxicity of OA@BCMNs were then evaluated by studying a set of human liver cancer cell lines (HepG2 and Huh-7) and human normal cells (HUVEC and LO2). OA@BCMNs and OA showed an apparent and time-dependent cytotoxicity against tumor cells (Figure 2B). OA@BCMNs caused no significant cytotoxicity in human normal cells (HUVEC and LO2), even at a multiplicity of infection (MOI) of up to 1 (Figure S6). Importantly, BCMNs did not exhibit cytotoxicity against tumor cells, which was consistent with the results obtained with human normal cells. These findings indicated that OA@BCMNs could deliver OA to tumor cells and maintained tumor-selective cytotoxicity effects. The presence of OA was next detected by real-time quantitative PCR (qPCR) to better understand the tumor-selective efficacy, non-specificity and corresponding toxicity of OA@BCMNs toward normal cells. This was done using primers targeting the adenoviral E1a gene in tumor cells (HepG2) and normal cells (LO2) at 24 h after infection with OA@BCMNs. The OA@BCMNs-treated HepG2 exhibited more virus accumulation than LO2 (Figure 2C). Moreover, after 24 h of incubation of HepG2 or LO2 with OA@BCMNs, virus particles were found in the intracellular space in HepG2 cells but not in the LO2 cells (Figure 2D). Together, these data suggest that BCMNs could deliver OA into tumor cells, and the replication and assembly only take place in tumor cells, leading to lysis of the tumor cells. 8


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One of the major limitations of the systemic administration of Ad5-based adenovirus vaccine is pre-existing immunity. The adverse impact of pre-existing neutralizing antibodies against Ad5-based adenovirus includes inactivated adenoviruses and a reduced expression of inserted genes. A neutralization assay was performed to evaluate the capability of OA@BCMNs to evade the neutralizing anti-Ad5 serum in vitro, which was monitored using the cellular fluorescence signals expressed with a GFP transgene. In this context, Ad5-GFP (a non-replicating adenovirus) and Ad5-GFP@BCMNs were incubated with serum containing a high titer of neutralizing antibodies. It was expected that the GFP expression of naked Ad5-GFP would be substantially blocked upon exposure with high levels of neutralizing antibodies in HepG2 cells. However, the GFP expression in the Ad5-GFP@BCMNs-treated HepG2 cells remained essentially unaffected in the presence of neutralizing antibodies even at high titers (Figure 3A). Similarly, a flow cytometry assay revealed higher average GFP

fluorescence

intensities

in

the

Ad5-GFP@BCMNs-treated

cells

than

Ad5-GFP-treated cells (Figure 3B). These results imply that BCMNs masked the adenovirus particles from serum neutralization in vitro and suggest the direct ablation of the recognition between adenovirus epitopes and neutralizing serum. Another major barrier to the systemic administration of OA-based vaccine is the host immune response, including innate and adaptive immune responses. We tested whether BCMNs-coated OA could evade anti-Ad5 immunity and subsequently inflammation. IL-6 and TNF-α are two major inflammatory cytokines induced by the systemic delivery of Ad5-based vaccine. In the in vitro assay, the levels of IL-6 in 9


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RAW264.7 media were significantly increased in the OA-treated group. In contrast, the levels of IL-6 in the medium were not significantly increased in the BCMNs and OA@BCMNs groups (Figure 3C). Moreover, systemic administration of OA significantly increased levels of serum IL-6 and TNF-α (Figures 3D-E), but the administration of BCMNs and OA@BCMNs did not. These results suggest that encapsulating OA with BCMNs enabled it to evade the innate response by shielding of the OA surface from the immune system. Next, we examined whether OA still maintains its anti-tumor capacity in the presence of anti-Ad5 antibody in vivo. In the HepG2-NTCP subcutaneous tumor model, anti-Ad5 serum was intraperitoneally injected into nude mice one day before injection of OA or OA@BCMNs-preS1. As expected, the OA group showed a therapeutic effect against tumor growth a week later, but the administration of anti-Ad5 serum significantly blocked this effect. Furthermore, the OA@BCMNs-preS1 treated-group showed equally remarkable inhibition of tumor growth regardless of the presence or absence of anti-Ad5 serum (Figure 3F). Consequently, the anti-tumor capacity of OA-encapsulated BCMNs was not affected by antiviral antibodies in vivo. As hepatic damage results in increased serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), however, in our experiment, systemic administration of OA@BCMNs did not significantly increase ALT and AST levels in serum compared with PBS and BCMNs (Figures 3G-H). In addition, hematoxylin and eosin staining (H&E) of the tissues showed that the systemic administration of OA@BCMNs caused no significant damage to the body 10


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(Figure S7). These results suggest that the systemic delivery of OA@BCMNs is safe. To evaluate the targeting efficacy of the coated OA, the biodistribution of OA@BCMNs-preS1 was examined. Nude mice bearing HepG2-NTCP subcutaneous tumors were treated with Ad5-GFP@BCMNs-preS1 and Ad5-GFP. A fluorescence imaging system was used to monitor the GFP intensity in different tissues to assess the targeted delivery capacity. After four injections, in vivo imaging showed that mice treated with Ad5-GFP@BCMNs-preS1 produced strong GFP signals in the tumor four days after the final treatment, whereas mice treated with naked Ad5-GFP exhibited negligible and mild GFP signals (Figure 4A).

The presence of OA in

different tissues of OA@BCMNs-preS1 treated mice was detected by qPCR using primers targeting the adenoviral E1a gene. The tumor tissues exhibited more virus accumulation than other tissues (Figure 4B). These data indicated that the systemic administration of BCMNs-coated OA could enhance the accumulation of the virus in whatever type of tissue we desired, resulting in efficient specific tissue-targeting delivery in vivo. Simultaneously, we evaluated the anti-tumor efficacy of OA@BCMNs-preS1 via intravenous injection in vivo. The HepG2-NTCP subcutaneous tumor model on nude mice was utilized to evaluate the therapeutic efficacy. When the tumor volumes reached 80-100 mm3, PBS (control), BCMNs-preS1, OA and OA@BCMNs-preS1 were systemically administered once every two days (four times in total). The mice treated with BCMNs-preS1 exhibited rapid tumor growth, which was similar to the PBS group, suggesting that BCMNs-preS1 as the virus-delivery nanovehicles showed no anti-tumor growth efficacy. And the mice treated with 11


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OA@BCMNs-preS1 showed significant anti-tumor growth efficacy compared with the PBS group (Figure 4C), while the mice treated with naked OA exhibited slight anti-tumor growth efficacy. Notably, we also utilized the HepG2-NTCP orthotopic model of liver cancer for a long-term survival analysis. As expected, the OA@BCMNs-preS1 treated group showed a prolonged survival of mice, compared with the naked OA treated group (Figure 4D). The H&E staining of tumor tissues after last treatment from the HepG2-NTCP orthotopic model mice showed that OA@BCMNs-preS1 treated mice exhibited larger necrotic areas than those harvested from PBS, BCMNs-preS1 and OA-treated mice (Figure 4E). These data suggested that OA@BCMNs-preS1 got an enhanced antitumor efficiency compared to OA in vivo. To further validate the BCMNs as a versatile nanoplatform for OA delivery, we examined the in-vivo expression of CRISPR-engineered targeting ligands on RBC membranes. RBCs could potentially be an ideal source for BCMNs production, and in-body genetic engineering strategies to enhance the natural targeting function of RBC-derived BCMNs would find a balance between anti-tumor and anti-viral immunity. Given the fact that, Asn-Gly-Arg (NGR) could recognize a specific isoform of aminopeptidase N (APN) which is a membrane-bound metalloproteinase of tumor cells. Thus, NGR has been identified as a potent targeting ligand for the delivery of drugs35. For the first time, we present NGR tripeptide as a potent tumor-targeting ligand on the exterior of RBC-membrane-derived nanovesicles via in-body genetic engineering to generate large-scale amounts of RBC-derived BCMNs for the 12


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tumor-targeted delivery of OA. The BCMNs-NGR were harvested using erythrocytes from the NGR transgenic mouse strain as shown in Figure 5A (see Supporting Information and methods section for details). As expected, Western blotting assay showed that NGR was found in nanovesicles (BCMNs-NGR) (Figure 5B). We proposed and confirmed that OA@BCMNs-NGR could target both tumor tissue and tumor cells over-expressing the aminopeptidase N (APN) isoform via NGR-ligand35. Human PC3 prostate cancer cells, U87 glioma cancer cells, and HepG2 cells have high levels of APN expression, while human MCF-7 breast cancer cells have low levels of APN expression. These cells were chosen as models of tumor cells. We compared the cellular binding abilities of BCMNs-NGR labeled by CFSE (green) to these tumor cells. The CLSM imaging showed that BCMNs-NGR exhibited good specific cellular binding ability to HepG2, U87, and PC3 cells, whereas weak green light signals were observed in the MCF7 groups after incubation for 4 h (Figure 5C). These results suggest that BCMNs-NGR has a high affinity to tumor cells that express a high level of APN. To evaluate the specific accumulation of OA@BCMNs-NGR in tumor, the biodistribution of OA@BCMNs-NGR was examined in a similar way to that mentioned above. Nude mice bearing HepG2 subcutaneous tumors were treated with

Ad5-GFP@BCMNs-NGR

and

Ad5-GFP.

As

expected,

the

Ad5-GFP@BCMNs-NGR-treated mice elicited strong GFP signals in tumor tissues (Figure 5D), which were significantly higher than the signals in naked Ad5-GFP treated mice. This indicates that the systemic administration of BCMNs-NGR could 13


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enhance the accumulation of OA in tumors. Moreover, the HepG2 subcutaneous tumor model in nude mice was used to evaluate the therapeutic efficacy. The group treated with OA@BCMNs-NGR exhibited significantly reduced tumor growth and complete tumor regression within 15 d without significant body weight loss. However, the mice treated with naked OA exhibited slight anti-tumor growth efficacy (Figures 5E-F), suggesting that OA@BCMNs-NGR also achieved enhanced anti-tumor efficiency compared to OA in vivo. The H&E staining of tumor tissues after the last treatment provided additional evidence that OA@BCMNs-NGR had an enhanced antitumor efficacy when administered systemically. Tumors harvested from the OA@BCMNs-NGR group exhibited larger necrotic areas than those from the PBS and OA-treated groups (Figure 5G). Both the TUNEL and Ki-67 assays showed a remarkable increase in apoptotic cells in the tumors obtained from the OA@BCMNs-NGR treated mice. This suggests that BCMN-NGR effectively delivered OA to the tumor and exhibited enhanced anti-tumor efficacy. Importantly, the BCMNs combine the natural functionalities of donor cell membranes and the bioengineering flexibility of targeting ligands, and such versatility provides a means of designing exciting new OA delivery systems for cancer virotherapy in future applications. In summary, we have presented an agile and versatile OA delivery platform that can be manipulated for precision virotherapy applications. The platform is applicable for the selective targeting of specific cancer cells and minimal antiviral immune response, which limit current clinical virotherapies for cancer. The BCMNs are 14


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endogenous carriers that transport targeting ligands based on in-vitro and in-vivo genetic membrane engineering expression. Ligands can be inserted into the BCMNs and are easily subjected to posttranslational modifications via biosynthesis processes. This method involves the synchronous synthesis and display of targeting ligands with the correct spatial orientation and activity. This leads to excellent targeting capacities and the facile production of the BCMNs. Our strategy of shielding OA with BCMNs was successfully demonstrated to have the potential to maximize the advantages of nanobiotechnology and overcome the disadvantages of OA. The results showed that the BCMNs have a capability of specifically targeting cancer tissue and cells, as well as a stealth effect in the immune system. These features enable the efficient systemic delivery of OA, evasion of host immune responses, and the reduction of side effects. Further investigation and modification are definitely necessary. Examples of future work include the purification of large-scale production, the optimization of the stability with common storage methods, and validations in large animal models. This novel BCMNs nanoplatform could potentially provide a valuable tool for future clinical translation to fabricate smart OA delivery systems based on a patient’s own cells, such as autologous immunocytes, mesenchymal stem cells, and cancer cells. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements The work was supported by the National Key Research and Development Program of 15


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China (2017YFA0205201 and 2018YFA0107301), the National Natural Science Foundation of China (81422023, 81603015, 81871404, U1705281, and U1505221), the Fundamental Research Funds for the Central Universities (20720160065 and 20720150141), and the Program for New Century Excellent Talents in University, China (NCET-13-0502). All animal experiments were approved by the Animal Management and Ethics Committee of Xiamen University. Supporting Information Additional details on the experimental methods, construction and synthesis of plasmid, Proteins Pull-down assay, storage stability, cytotoxicity and H&E-stained major organ samples. REFERENCES (1) Cervantes-Garcia, D.; Ortiz-Lopez, R.; Mayek-Perez, N.; Rojas-Martinez, A. Ann Hepatol 2008, 7, (1), 34-45. (2) Cockle, J. V.; Scott, K. J. Arch Dis Child Educ Pract Ed 2018, 103, (1), 43-45. (3) Choi, J. W.; Lee, J. S.; Kim, S. W.; Yun, C. O. Adv Drug Deliv Rev 2012, 64, (8), 720-9. (4) Uusi-Kerttula, H.; Hulin-Curtis, S.; Davies, J.; Parker, A. L. Viruses 2015, 7, (11), 6009-42. (5) Harrington, K. J.; Puzanov, I.; Hecht, J. R.; Hodi, F. S.; Szabo, Z.; Murugappan, S.; Kaufman, H. L. Expert Rev Anticancer Ther 2015, 15, (12), 1389-403. (6) Pol, J.; Buque, A.; Aranda, F.; Bloy, N.; Cremer, I.; Eggermont, A.; Erbs, P.; Fucikova, J.; Galon, J.; Limacher, J. M.; Preville, X.; Sautes-Fridman, C.; Spisek, R.; Zitvogel, L.; Kroemer, G.; Galluzzi, L. Oncoimmunology 2016, 5, (2), e1117740. (7) Di Paolo, N. C.; Shayakhmetov, D. M. Curr Opin Mol Ther 2009, 11, (5), 523-31. 16


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Scheme 1. Design features and proposed mechanism of OA@BCMNs. (A) Production of BCMNs from bioengineered donor cells. (B) OA encapsulated into BCMNs, forming the bioengineered cell-derived membrane nanovesicle-OA complex (OA@BCMNs). (C) BCMNs protected OA from neutralizing antibodies. (D) BCMNs deliveried OA to the desired tissue or cells and binding to the receptor, leading OA enter the cell. (E) OA infected and amplified in tumor cells, causing the tumor cell lysis. (F) OA was released into the microenvironment and infected tumor cells nearby.

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Figure 1. Characterization of OA-encapsulated BCMNs harboring tissue specific ligands preS1. (A) A schematic illustration for BCMNs and Western blotting analysis to detect the preS1 and marker protein Alix on the OA@BCMNs. (B) CLSM images to detect the binding capacity of BCMNs-preS1 to NTCP by incubated HepG2-NTCP with BCMNs-preS1 or EVs for 4 h. Blue, green, and red colors represented DAPI-stained cell nuclei, CFSE labelled BCMNs-preS1 and EVs, and NTCP, respectively. (C) TEM images of BCMNs, OA, and OA@BCMNs negatively stained with uranyl acetate. Scale bar: 100 nm. (D, E) The size distribution and zeta-potential of BCMNs, OA, and OA@BCMNs measured by dynamic light scattering (DLS). (F) A dot blot assay was 21


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performed to further evaluate the structure of OA@BCMNs by using an Hexon antibody. (G) A gel-retardation assay was performed to preliminary evaluated the encapsulation capability of OA@BCMNs. Arrow indicates the band of OA. (H) The encapsulation efficiency of OA@BCMNs was further evaluated by using a High Sensitivity Flow Cytometry (HSFCM). FITC-CFSE intensity, PC5.5-SYTO62 intensity represented BCMNs and OA, respectively.

Figure 2. Anti-tumor effects of OA@BCMNs in vitro. (A) Cytopathic effect of BCMNs, OA, and OA@BCMNs after 72 h post infection in HEK 293 cells. (B) Cell viability was determined using the MTT assay. Human liver cancer cell lines (HepG2 and Huh7) and normal cells (HUVEC and LO2) were treated with BCMNs, OA and OA@BCMNs at 200 VPs/cell. (C) Viral genome copies were quantified using real time qPCR in HepG2 cells and LO2 cells after 24 h incubation with OA@BCMNs at 200 VPs/cell. (D) Electron microscope images of HepG2 cells and LO2 cells after 24 h incubation with OA@BCMNs at 200 VPs/cell.

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Figure 3. OA@BCMNs evaded preexisting immunity in vitro and in vivo. (A) Neutralization of Ad5-GFP@BCMNs and Ad5-GFP in HEK 293 cells with anti-Ad5 serum at indicated dilutions. (B) Flow cytometry assay of Ad5-GFP@BCMNs and Ad5-GFP in HEK 293 cells with anti-Ad5 serum at indicated dilutions. (C) IL-6 levels in the medium of RAW264.7 macrophage cellsafter 6 h of infection. (D, E) Serum IL-6, TNF-α levels at the indicated level of systemic administration (n = 5). *P < 0.05, ***P < 0.001, NS indicates P > 0.05. (F)Anti-Adenovirus antibodies did not inhibit the antitumor effect of OA@BCMNs in nude mice HepG2-NTCP tumor model at indicated treatments. *P < 0.05; **P < 0.01. Bars represent the mean tumor volume ± SEM. (G, H) Serum ALT and AST levels. 23


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Figure 4. Systemic administration of OA@BCMNs-preS1 showed efficient target delivery and enhanced anti-tumor efficacy in vivo. (A) Fluorescence images in HepG2-NTCP tumor-bearing nude mice at 4 days after the intravenous injection of Ad5-GFP@BCMNs-preS1 or Ad5-GFP (5× 109 VPs, once every day for 4 times). (B) Viral genome copies in excised tumors and organs after the indicated intravenous injection were quantified using real time qPCR. (C, D) Tumor growth curves and percent survival of HepG2-NTCP bearing nude mice in different groups after the indicated treatments. Treatment with BCMNs-preS1 significantly inhibited tumor growth and extended survival. (E) Tumor sections were stained with H&E. All the panels were magnified 400-fold.

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Figure 5. OA@BCMNs-NGR from transgenic mouse erythrocyte showed efficient target delivery and enhanced antitumor efficacy in vivo. (A) A schematic illustration for the synthesis of BCMNs-NGR. (B) Western blotting analysis to detect the NGR and marker protein Alix on the BCMNs-NGR. (C) CLSM images to detect the binding capacity of BCMNs-NGR to HepG2, PC3 and U87 cells (high level of expression of APN) and MCF-7 cells (low level of expression of APN). (D) Fluorescence images of organs from HepG2 tumor-bearing nude mice in different groups. (E, F) Tumor 25


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growth curves and body weight of HepG2 bearing nude mice in different groups after the intravenous injection of OA@BCMNs-NGR or OA (5× 109 VPs, once every two days for 4 times). (G) Tumor sections were stained with H&E, TUNEL, and Ki-67. All the panels were magnified 400-fold.

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Genetically Engineered Cell Membrane Nanovesicles for Oncolytic

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