Liposome Encapsulation of Oncolytic Virus M1 To Reduce

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Liposome encapsulation of oncolytic virus M1 to reduce immunogenicity and immune clearance in vivo Yalong Wang, Huizhi Huang, Haijuan Zou, Xuyan Tian, jun hu, Pengxin Qiu, Haiyan Hu, and Guangmei Yan Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01046 • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 3, 2019

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Molecular Pharmaceutics

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Liposome encapsulation of oncolytic virus M1 to

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reduce immunogenicity and immune clearance in

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vivo

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Yalong Wang,†,# Huizhi Huang,†,# Haijuan Zou,†,§ Xuyan Tian,‡ Jun Hu,‡ Pengxin Qiu,‡ Haiyan

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Hu,†,* and Guangmei Yan‡

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†

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Guangzhou Higher Education Mega Center, Guangzhou 510006, P.R. China

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‡

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Zhongshan II Road 74, Guangzhou 510080, P.R. China

School of Pharmaceutical Sciences, Sun Yat-sen University, Waihuan East Road 132,

Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University,

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§

Department of Pharmacy, the Seventh Affiliated Hospital of Sun Yat-sen University,

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Shenzhen 518107, P.R. China

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KEYWORDS: Oncolytic virus M1; Liposome; Immune clearance; Immunogenicity

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ACS Paragon Plus Environment

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract Graphic

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ABSTRACT: Oncolytic viral therapy is an attractive novel strategy for cancer therapy. As a

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natural alphavirus, oncolytic virus M1 is able to infect and kill various zinc finger antiviral protein

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(ZAP)-deficient tumor cells selectively, while leaving normal cells undamaged. However, M1 can

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trigger the production of neutralizing antibodies that dramatically weaken its antitumor effect. In

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order to attenuate immunogenicity of therapeutic M1 virus, we encapsulated it into liposomes

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(referred to as M-LPO) using the thin-film hydration method. The effect of anti-M1 neutralizing

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antibody on M-LPO was examined in LoVo and Hep 3B cell lines. In the absence of neutralizing

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antibodies, treating cells with naked M1, blank liposomes (LPO), M-LPO or a simple mixture of

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M1 and liposomes (LPO+M1) inhibited cell growth. In the presence of neutralizing antibodies,

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only M-LPO inhibited cell growth. After intravenous administration, M-LPO reduced the

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production of M1-neutralizing antibody and the corresponding immune response. Analysis of M-

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LPO uptake by cells was examined by confocal microscopy using M1 labelled with FITC and

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liposomal shells labelled with RhB. The results suggest that M1 may be released from liposomes

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before or after M-LPO internalization. Taken together, our results suggest that encapsulating

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oncolytic virus M1 in liposomes may reduce intrinsic viral immunogenicity for improved

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anticancer therapy.

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INTRODUCTION

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Viral therapy is quite promising for the treatment of cancer when the virus selectively replicates

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in tumor cells rather than normal cells, which significantly improves the safety and efficacy of

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tumor therapy.1-3 In effect, this amounts to selective targeting of tumor cells. Examples of viruses

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that naturally target cancer cells are autonomously replicating H1 parvovirus,4 reovirus,5, 6 and

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Newcastle disease virus.7, 8 Even more viruses can be adapted or engineered to target cancer cells,

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including adenovirus from which the E1B-55K gene has been knocked out9, 10 herpes simplex virus

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from which the ICP 34.5 gene has been knocked out11, 12 and vesicular stomatitis virus carrying a

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modification in the IFN-β gene.13

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Naturally occurring M1, a Getah-like alphavirus isolated from culicine mosquitoes and abundant

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on the Chinese island of Hainan,14 may be promising for selective targeting of cancer cells. Its

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significant antitumor activity15 is selective for cancer cells deficient in zinc-finger antiviral

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proteins (ZAPs), including Hep 3B cells, LoVo cells, C-33A cells, and Huh-7 cells16. In addition,

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compounds that mimic the mitochondria-derived activator of caspases potentiate the selective

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oncolytic effect of M1 in cancer cells, enhancing M1 replication and the bystander killing effect.17

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However, the use of M1 for anti-cancer therapy is inhibited by its high immunogenicity:

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intravenous administration triggers production of neutralizing antibodies, leading to a strong

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immune response that inactivates the virus and eliminates the therapeutic effect18, 19 To counteract

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this inactivation, researchers have encapsulated oncolytic viruses in fragments of plasma

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membrane.20-23 Alternatively, oncolytic virus can be wrapped in liposomes, which are attractive

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because they offer a large hydrophilic lumen for packaging diverse cargo and protecting it from

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cellular and humoral responses.24-27 To date, studies of liposomal encapsulation of such viruses


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have been limited to non-enveloped adenovirus. It is unclear whether this strategy can work for

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M1, which is already enclosed within a phospholipid layer.

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In this study, liposome-encapsulated M1 (M-LPO) were prepared to protect M1 from neutralizing

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antibodies in vitro as well as reduce M1 immunogenicity in vivo. The physicochemical and

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morphological properties of M-LPO were investigated. Moreover, the effects of anti-M1

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neutralizing antibody on M-LPO and naked M1 were examined in LoVo and Hep 3B cell lines in

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vitro. And the antigenicity of M-LPO, compared to M1, was evaluated after intravenous

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administration. Further, analysis of M-LPO uptake by cells was performed by confocal microscopy

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using M1 labelled with FITC and liposomal shells labelled with RhB.

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MATERIALS AND METHODS

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Materials. Cell lines were purchased from the American Type Culture Collection, the Shanghai

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Institute of Cell Biology, and the Guangzhou Institute of Biomedicine and Health. Oncolytic virus

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M1 was provided by Guangmei Yan research group of Sun Yat-sen University (Guangzhou,

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China). Soybean lecithin (S100) and egg yolk lecithin (PC-98T) was obtained from Lipoid GmbH

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(Germany) and A.V.T. (Shanghai, China) respectively. Cholesterol was purchased from

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Guangzhou QiHua Medical Equipment Co., Ltd (Guangzhou, China). Cells were cultured in

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DMEM supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin (Life Technologies).

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M1 was grown in Vero cells in SFM (Life Technologies). Virus titer in plaque-forming units (pfu)

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was determined by TCID50 assay using BHK-21 cells. Rhodamine DHPE was purchased from

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Life Technologies, and FITC was purchased from MP Biomedicals.


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Preparation of liposome-encapsulated M1. M1 was purified by ultrafiltration in a centrifuge

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column with molecular weight cut-off of 50 kDa with three spins of 20 min at 2500 rpm. Then M1

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was diluted in 0.1M phosphate-buffered saline (PBS, pH 7.4) to a suitable titer.

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Liposome-encapsulated M1 was prepared using the thin-film hydration method.28 Soybean lecithin

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and cholesterol (4:1, w/w) were dissolved in mixed organic solvent [CH2Cl2 : ethyl alcohol (1:2,

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v/v)]. Organic solvent was removed by vacuum rotary evaporation at 100 rpm at 45 ℃, yielding a

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dry lipid film. The film was hydrated using 1  108 pfu/mL M1 in 0.1 M PBS (pH 7.4) and vortexed

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for 5 min at room temperature. The suspension was extruded 15 times through polycarbonate

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membranes of 200 nm pore size (Millipore, Bedford, MA, USA) and then passed through a

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Cellufine Sulfate column to separate out naked M1. The isolated M-LPO was sterile-filtered

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through a 0.45 m membrane (Millipore). In addition, M-LPO prepared from egg yolk lecithin

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was also studied. The separation operation for each sample was repeated three times for the titer

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detection.

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As a negative control, blank liposomes (LPO) were prepared as above, except that the films were

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hydrated in PBS without M1. Besides the thin-film hydration method, two other methods had been

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developed and compared. They are calcium-induced phase change method29 and co-incubation

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method. The detailed preparation methods could been seen in the Supporting Information (Section

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2).

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Characterization. Samples of LPO, M-LPO or M1 were transferred to a clean hydrophobic

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surface, and a copper grid was carefully inverted onto the drop and allowed to soak for 90 s. The

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excess liquid was removed with filter paper, and the grid was negatively stained for 90 s with 1%


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phosphotungstic acid. Grids were air-dried and then observed under a JEM-2100F transmission

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electron microscopy (TEM) (JEOL, Japan).

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Average size and zeta potential of M1, LPO and M-LPO were measured using a laser-based

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analyzer (Zeta sizer Nano ZS90, Malvern, UK).

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Encapsulation efficiency (EE) of M-LPO was calculated using the formula EE% = Wa / Wtotal 

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100%, where Wa and Wtotal are the amount of protein in M-LPO after or before passage through

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the Cellufine Sulfate column, respectively. Protein concentration was estimated using through the

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microBCA kit (120 mL, Cwbiotech, China) based on absorbance at 570 nm. The detailed

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determination process of encapsulation efficiency could be found in Supporting Information

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(Section 3).

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Cell viability assay. Cells were seeded into 96-well plates at a density of 5 × 103 cells per well

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and then incubated for 24 h in an atmosphere of 5% CO2 and 95% air at 37 ℃. The medium was

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replaced with DMEM containing varying concentrations of M1, LPO, a simple mixture of LPO

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and M1 (LPO+M1), or M-LPO. At 4 h later, the medium was replaced with complete DMEM and

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the cells were incubated for another 48 h. Surviving cells were quantified using the colorimetric

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MTT assay based on the formula Survival % = (A490 for the treated cells/A490 for the control cells)

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× 100%, where A490 is the absorbance at 490 nm. Tests were performed in triplicate.

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Preparation of M1-neutralizing antibody. Sprague-Dawley rats were injected intravenously

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with 5  107 pfu M1 once every day for 3 days in the tail, then they received a second intravenous

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injection with 5  107 pfu M1 in the same way. At 7 days after the second immunization, blood

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was taken, allowed to sit for 30 min, and centrifuged at 3000 rpm for 10 min. The serum

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supernatant containing M1-neutralizing antibody was stored at -20 ℃. Besides, the serum was


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repackaged and stored at -20 ℃, thus avoiding the repeatedly freeze-thaw resulting in the

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inactivation of neutralizing antibody.

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Virus neutralization in vitro. Appropriate dilutions of neutralizing antibody were determined

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using the method of fixed virus dilution. Neutralizing antibody was diluted 64, 128, 256, and 512

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times in DMEM. These dilutions were mixed with the same volume of M1 at multiplicity of

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infection (MOI) of 0.1, and the mixture was incubated at 37 ℃ for 1 h. The mixture was added to

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LoVo cells, and viability was measured. The negative control (NCtrl) cells were treated with

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DMEM only; the positive control (PCtrl) cells, with M1 at MOI 0.1; and the treated control (TCtrl)

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cells, with M1 at MOI 0.1 that had been incubated with DMEM at 37 ℃ for 1 h.

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Immune neutralization in vitro. DMEM, M1, LPO, LPO+M1 or M-LPO was mixed with

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neutralizing antibody and incubated at 37 ℃ for 1 h to achieve complete immunity. Then the

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mixtures were added to LoVo or Hep 3B cultures and cell viability was measured. As a negative

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control, cells were exposed to M1, LPO, LPO+M1 or M-LPO without pre-incubation with

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neutralizing antibody. M1 was present at an MOI of 0.1 in cultures exposed to virus.

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Antigenicity of M-LPO in vivo. Six-week-old female Balb/c mice were randomly divided into

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four groups of six mice each, then immunized intravenously via tail injection with saline, M1, LPO

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or M-LPO. One week later, the mice received a second tail injection of the same dose. Two weeks

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after the second injection, blood was taken, allowed to rest for 30 min, and centrifuged at 3000

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rpm for 10 min. The supernatant was mixed with an equal volume of naked M1 and incubated at

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37 ℃ for 1 h. M1 titer was then determined.


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Uptake of M-LPO by LoVo cells. Uptake of M-LPO was analyzed by confocal microscopy using

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doubly labelled M-LPO with M1 labelled with FITC and liposomal shells labelled with Rhodamine

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DHPE (FM1-RhLPO).

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To prepare FITC-labelled M1 (FM1), M1 was mixed with an equal volume of FITC solution (0.1

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mg/ml) in 0.5 M bicarbonate buffer (pH 9.0) and left at room temperature for 1 h in the dark. The

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mixture was passed through a Sephadex G-50 column washed in PBS to remove unconjugated

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FITC. The effluent fraction containing labelled virus was collected and sterile-filtered through a

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0.45 m Millipore filter.30, 31

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During production of the thin film for liposome preparation, the 1% soybean lecithin was replaced

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by Rhodamine DHPE, and the film was hydrated using FM1. In this way, doubly labelled FM1-

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RhLPO was prepared.

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Localization of FM1-RhLPO after uptake into LoVo cells was analyzed using laser scanning

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confocal microscopy (LSM710, Zeiss, Germany). LoVo cells were seeded into confocal dishes

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(catalog no. 801002, 15 mm diameter, Nest) at a density of 1 × 105 cells per well and incubated

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for 24 h. Then cells were treated for 4 h with FM1 or FM1-RhLPO with M1 at an MOI of 0.1.

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Negative control cells were treated with DMEM. Cells were washed six times in PBS, fixed for

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30 min in 4% paraformaldehyde, stained for 25 min with DAPI (10 g /mL) and washed three

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times in PBS. Free DAPI was washed away using PBS, then fluorescence images were observed

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under a laser scanning confocal microscope.

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Statistical analysis. All data are presented as the mean ± SD of at least three determinations.

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Differences between treatment groups were assessed for significance using ANOVA.


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RESULTS AND DISSCUSSIONS

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Selection of M-LPO preparation method. Most studies of virus wrapped in liposome have used

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non-enveloped adenovirus-associated viruses. To encapsulate these viruses into liposomes, a

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method based on calcium-induced phase change was usually used. We speculated that this method

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may not be optimal for the enveloped M1 virus, because Ca2+ affects the stability of its

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phospholipid layer. To our knowledge, liposome encapsulation of M1 has never been reported.

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Prior to screen the preparation method, soybean lecithin and egg yolk lecithin were compared as

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lipid materials to prepare liposomes encapsulating M1. The particle size of M-LPO made from egg

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yolk lecithin significantly increased and the sample turned into opalescent turbid on the third day

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at 4℃. In view of the instability of the liposomes made from egg yolk lecithin, soybean lecithin

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was finally selected for the subsequent research (data not shown).

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For optimization of M-LPO preparation, we tested three methods: thin-film hydration, calcium-

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induced phase change, and co-incubation. As shown in table 1, the highest titer of M-LPO was

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obtained using the thin-film hydration method. M-LPO that was prepared by co-incubation showed

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nearly undetectable antitumor activity, suggesting that naked M1 cannot passively enter the

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liposome. Thin-film hydration is simpler than the method based on calcium-induced phase change,

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and it gives a higher M-LPO titer. Therefore, the thin-film hydration method was used to prepare

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M-LPO for further study.

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Table 1. M-LPO titers obtained with different methods

Method

Titer (pfu/mL)±SD


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Thin-film hydration

1.8×105±2.8×104

Calcium-induced phase change

4.2×104±6.6×103

Co-incubation

8.2×102±0

Page 10 of 30

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Characterization. Abstract graphic shows a schematic representation of how M-LPO is designed

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to evade immune inactivation to act on cancer cells. M-LPO and naked M1 accumulate at the

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tumor site passively due to the enhanced permeability and retention (EPR) effect. After

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intravenous M1 injection, the body produces M1-neutralizing antibodies, but the phospholipid

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bilayer of the liposome of M-LPO prevents the antibodies from binding to epitopes on M1. As a

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result, M1 accumulates in tumors in an active form, resulting in cancer cell apoptosis.

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TEM revealed homogeneous morphology of naked M1, LPO, and M-LPO in Figure 1. Naked M1

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appeared spherical or nearly so, consistent with its known structure. The outline of M1 indicated

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that the average particle size of M1 was about from 65 to 80nm. It can also be seen from Figure

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(M-LPO) that M1 could be encapsulated into liposomes with one virus per liposome in average.

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As seen in Table 2, the particle sizes of M-LPO and LPO were similar, 155.9 nm and 167.4 nm,

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respectively, which may be attributed to 15 extrusions of suspension through polycarbonate

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membranes of 200 nm pore size. It was also found that those particle sizes were about 1.6 times

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larger than that of M1. These sizes suggest that the encapsulated virus can be efficiently transported

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across vessel walls into tumor tissues, where it can accumulate through the EPR effect. Presumably

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the outer phospholipid bilayer of the liposome should protect M1 from inactivation by antibodies

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in the blood.

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Figure 1. TEM images of blank liposome (LPO), naked M1 (M1) and liposome-encapsulated M1

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(M-LPO). White hollow arrows indicate M1 membrane, and white solid arrow indicate liposome

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membrane.

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Table 2. Particle Sizes and Zeta Potentials of LPO, M1 and M-LPO

Sample

Particle Size (nm)

SD (nm)

PDIa

SD

Zeta Potential (mV)

SD (mV)

M1

99.4

1.75

0.188

0.072

-12.3

0.56

LPO

167.4

1.25

0.108

0.022

-10.0

0.43

M-LPO

155.9

2.49

0.101

0.006

-14.1

0.26

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aPDI:

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The EE% of M-LPO was 28.50%±2.85% (n=3) based on assay of viral protein, while the titer of

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M-LPO was 5.65×106 ±1.83×106 pfu/mL (n=3) based on the TCID50 assay. The relatively low

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EE% may be because M1 has a molecular weight in the megadaltons, so it is more difficult to

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encapsulate in liposomes.

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Antibody-mediated inactivation of M1. M1-neutralizing antibody was prepared in rats, then the

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optimal dilution was determined using the fixed virus dilution method with LoVo cells. Survival

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rates were not significantly different between PCtrl and TCtrl cultures (Figure 2), suggesting that

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treating virus for 1 h at 37 ℃ negligibly affected its activity. These data further showed that 64-

polydispersity index.


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fold dilution of antibody nearly entirely inactivated M1, leading to similar virus activity as in NCtrl

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cultures. As expected, higher dilutions resulted in higher viral activity, with 512-fold dilution

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giving viral activity close to the TCtrl. These results indicate that the neutralizing antibody in

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serum affected M1 activity, and 64-fold dilution was used in further experiments to investigate the

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protective effect of liposomes on M1.

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Figure 2. Virus neutralization test. All the groups except positive control group (PCtrl) were

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treated with neutralizing antibody for 1 h at 37 ℃. The sample of negative control group (NCtrl)

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was DMEM; that of PCtrl was M1 untreated; that of treated control group (TCtrl) was M1 treated.

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The value in abscissa was on behalf of the dilution ratio of antibody. ns, no significant difference;

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***, P < 0.001.

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M-LPO protects M1 from immune neutralization in vitro. We compared the effect of M1 or

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M-LPO on the viability of M1-sensitive LoVo and Hep 3B cancer cells. M1 markedly induced cell

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death: viability after treatment with M1 at MOI of 1.0 was 6.9% for LoVo cells and 3.8% for Hep

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3B cells. Next we compared the viability of these cells after exposure to M1 at an MOI of 0.1 in

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the form of naked M1, LPO+M1 or M-LPO.


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In these first experiments, the virus was not previously treated with neutralizing antibody (Non-

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immunized, Figure 3). Control cultures were treated with pure DMEM or LPO. Treating cells with

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non-neutralized M1 led to 38.1% survival in the case of LoVo cells or 43.3% in the case of Hep

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3B cells. This is consistent with the known sensitivity of both cell lines to M1. The survival rates

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when LoVo or Hep 3B cells were incubated with LPO+M1 were lower by 10.9% or 15.7%,

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respectively, than when they were incubated with M1. This may mean that M1 internalization by

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tumor cells may be enhanced in the presence of blank liposomes, which should be examined in

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future work. Compared with control cultures, the survival rates of LoVo or Hep 3B cells incubated

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with M-LPO declined significantly, suggesting that encapsulating M1 in liposomes does not mask

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its antitumor activity. They were a little higher by 9.3% or 11.3%, respectively, than when they

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were incubated with M1, which may be due to the outer phospholipid layer may delay virus

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biosynthesis, assembly and release.

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These experiments were repeated after previously treating the virus with neutralizing antibody

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(Immunized, Figure 3). The survival rate was above 95% with M1, indicating that our conditions

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were adequate to inactivate the naked virus nearly completely. Similar inactivation was observed

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with LPO+M1, suggesting that virus sensitivity to antibody inactivation is unaffected by the

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presence of blank liposomes and that virus does not passively enter into liposomes to become

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protected from inactivation. In contrast, the survival rate was only 62.0% for LoVo cells and 56.8%

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for Hep 3B cells after treatment with neutralized M-LPO, suggesting that the liposomes efficiently

249

blocked antibody inactivation of M1.

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In short, M-LPO showed antitumor efficacy in vitro similar to that of naked M1 in the absence of

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prior antibody neutralization. In the presence of such neutralization, naked M1 was completely

252

inactivated, while M-LPO largely retained its ability to kill M1-sensitive Hep 3B and LoVo cells.


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Figure 3. Antitumor activity of M-LPO in LoVo cells (A) and Hep 3B cells (B) with (Immunized)

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or without (Non-immunized) neutralizing antibody. ns, no significant difference; ***, P < 0.001.

256 257

M-LPO reduces M1 immunogenicity in vivo. Next we examined the immune response of M-

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LPO in vivo. Mice were injected intravenously with naked M1, blank liposomes or M-LPO. Two

259

weeks after the second injection, serum was harvested and titers of M1-neutralizing antibody were

260

determined using an indirect method, since the method of detecting the quantity of M1 virus

261

neutralizing antibody is still in building. In this indirect approach, we mixed the serum from treated

262

mice with a purified preparation of naked M1 of known titer. We reasoned that higher titer of M1-

263

neutralizing antibodies in the serum should reduce the titer of M1. As expected, serum from M1-

264

treated mice led to the lowest titer in the purified M1 preparation in Figure 4. The apparent titer in

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serum from LPO-treated mice was similar to that in serum from control animals, suggesting that

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blank liposomes on their own do not stimulate production of M1-neutralizing antibody. Titer in

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serum from M-LPO-treated mice appeared to be lower than that in serum from M1-treated animals.

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These results are consistent with the idea that the outer phospholipid bilayer in M-LPO can mask

269

epitopes on M1, triggering lower production of M1-neutralizing antibodies.


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270 271

Figure 4. Titer of M1 when incubated with the serum of mice. Those mice were respectively

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immunized by saline (Ctrl), naked M1 (M1), blank liposome (LPO) or liposome-encapsulated M1

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(M-LPO). **, P < 0.01; ***, P < 0.001.

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Analysis of M-LPO uptake into cancer cells. To ensure the most reliable analysis of M-LPO

275

uptake into target cells, we adopted a double labelling strategy in which, for the first time to our

276

knowledge, M1 was conjugated to FITC. This was achieved by incubating M1 in alkaline solution,

277

such that a covalent bond formed between the thiamine moiety in FITC and the amino group of

278

lysines in M1. Free FITC was removed on a Sephadex G-50 column (Figure 5). Free FITC eluted

279

in 10-25 mL: these fractions showed maximum fluorescence but failed to infect BHK cells.

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Fractions eluting at 3-5 mL showed high viral titer and fluorescence, identifying them as FITC-

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labelled M1.


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282 283

Figure 5. Elution profile of FITC-labelled M1. Each drip’s titer and fluorescence intensity were

284

detected.

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FM1 was encapsulated into liposomes previously labelled with Rhodamine DHPE (RhLPO), and

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this formulation was added to LoVo cells for 4 h, after which cells were analyzed for uptake of

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FM1-RhLPO based on green signal from FM1 and red signal from the RhLPO (Figure 6). The

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intact complexes of liposome-encapsulated M1 gave yellow signal due to colocalization of the two

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labelled components. Cell nuclei appeared blue because of DAPI staining. Cells treated with naked

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FM1 showed fluorescence scattered around nuclei, suggesting that FM1 can be taken up by LoVo

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cells in the same manner as M1. Cells treated with FM1-RhLPO showed some green signal in the

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overlay channel, suggesting rupture of some liposomes before their internalization, liberating FM1

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to be internalized into tumor cells. As expected, there was also yellow signal in the overlay

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channel, suggesting that a proportion of the liposomes enter tumor cells intact, subsequently

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releasing FM1 after uptake. In other words, our data suggest that M-LPO may release its M1 cargo

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before or after internalization, and that the liposome coating does not alter M1 internalization.

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Further experiments should examine the specific internalization pathways involved.


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298 299

Figure 6. Confocal microscopy images of LoVo cells. Blue channel: cell nucleus stained by DAPI;

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Green channel: M1 virus labelled by FITC; Red channel: liposome containing Rhodamine DHPE.

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CONCLUSIONS

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While oncolytic viruses show potential as anti-cancer treatments, they can elicit neutralizing

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immune responses. Here we demonstrate that encapsulating the M1 alphavirus in liposomes

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strongly reduces its immunogenicity and may preserve its anti-tumor efficacy in M1-sensitive

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cancers. Our results suggest that the outer phospholipid coating on M1 effectively blocks the

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binding of M1-neutralizing antibodies to the virus without affecting the ability of the virus to be

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internalized into target cells. Our experiments with doubly-labelled M-LPO suggests that M1 can

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be liberated from the liposomes before or after uptake by cancer cells. These experiments are, to


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our knowledge, the first report of liposome encapsulation of M1 and the first report of M1-FITC

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conjugation for post-uptake analysis. Our findings justify further work into liposome-encapsulated

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M1 as a promising anti-cancer therapy. Since liposomes contain hydrophilic and hydrophobic

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regions that can encapsulate small molecules, our platform may be useful for combining M1 and

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small molecules for synergistic effects.

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ASSOCIATED CONTENT

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Supporting Information.

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The following files are available free of charge.

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The separation of naked M1 and liposome-encapsulated M1 using a Cellufine Sulfate column; the

318

preparation of liposome-encapsulated M1 by calcium-induced phase change method and co-

319

incubation method; the determination of encapsulation efficiency by detecting the total protein of

320

M1 (PDF)

321

AUTHOR INFORMATION

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Corresponding Author

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* Tel: +86 20 39336119. E-mail: [email protected].

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Author Contributions

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# These authors contributed equally to this work.

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENT


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We thank Prof. Wenbo Zhu, Dr. Kai Li, Dr. Yuan Lin and Dr. Jiangkai Liang for technical

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assistance with operation of M1. This work was funded by the National Natural Science

331

Foundation of China (Grants 81473154, 81573447 and 81603127); Fundamental Research Funds

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for central universities (Grants 18ykzd08); Natural Science Foundation of Guangdong Province,

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China (Grants 2016A030310160 and 2016A030310146); Science and Technology Planning

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Project of Guangdong Province, China (Grant 20160909); Research and Development Project of

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Applied Science and Technology of Guangdong Province, China (Grant 2016B020237004) and

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Science and Technology Planning Project of Guangdong Province, China (Grant

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2015B020211003).

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ABBREVIATIONS

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ZAP, zinc finger antiviral protein; M-LPO, liposome-encapsulated M1; FM1, FITC-labelled M1;

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FM1-RhLPO, doubly labelled M-LPO with M1 labelled by FITC and liposomal shells labelled by

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Rhodamine DHPE; LPO, blank liposomes; TEM, transmission electron microscopy; EE,

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Encapsulation efficiency; MOI, multiplicity of infection.

343

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Figure 1. TEM images of blank liposome (LPO), naked M1 (M1) and liposome-encapsulated M1 (M-LPO). White hollow arrows indicate M1 membrane, and white solid arrow indicate liposome membrane. 74x27mm (600 x 600 DPI)


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Figure 2. Virus neutralization test. All the groups except positive control group (PCtrl) were treated with neutralizing antibody for 1 h at 37 ℃. The sample of negative control group (NCtrl) was DMEM; that of PCtrl was M1 untreated; that of treated control group (TCtrl) was M1 treated. The value in abscissa was on behalf of the dilution ratio of antibody. ns, no significant difference; ***, P < 0.001. 59x48mm (600 x 600 DPI)



Figure 3. Antitumor activity of M-LPO in LoVo cells (A) and Hep 3B cells (B) with (Immunized) or without (Non-immunized) neutralizing antibody. ns, no significant difference; ***, P < 0.001. 114x40mm (600 x 600 DPI)


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Figure 4. Titer of M1 when incubated with the serum of mice. Those mice were respectively immunized by saline (Ctrl), naked M1 (M1), blank liposome (LPO) or liposome-encapsulated M1 (M-LPO). **, P < 0.01; ***, P < 0.001. 74x59mm (600 x 600 DPI)



Figure 5. Elution profile of FITC-labelled M1. Each drip’s titer and fluorescence intensity were detected.


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Figure 6. Confocal microscopy images of LoVo cells. Blue channel: cell nucleus stained by DAPI; Green channel: M1 virus labelled by FITC; Red channel: liposome containing Rhodamine DHPE.



Table of Contents Graphic/Abstract Graphic


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Liposome Encapsulation of Oncolytic Virus M1 To Reduce

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