Liposomes with cyclic RGD peptide motif triggers acute immune response in mice

Accepted Manuscript Liposomes with cyclic RGD peptide motif triggers acute immune response in mice

Xiaoyi Wang, Huan Wang, Kuan Jiang, Yanyu Zhang, Changyou Zhan, Man Ying, Mingfei Zhang, Linwei Lu, Ruifeng Wang, Songli Wang, Diane J. Burgess, Hao Wang, Weiyue Lu PII: DOI: Reference:

S0168-3659(18)30698-9 https://doi.org/10.1016/j.jconrel.2018.12.003 COREL 9553

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

4 September 2018 18 November 2018 3 December 2018

Please cite this article as: Xiaoyi Wang, Huan Wang, Kuan Jiang, Yanyu Zhang, Changyou Zhan, Man Ying, Mingfei Zhang, Linwei Lu, Ruifeng Wang, Songli Wang, Diane J. Burgess, Hao Wang, Weiyue Lu , Liposomes with cyclic RGD peptide motif triggers acute immune response in mice. Corel (2018), https://doi.org/10.1016/j.jconrel.2018.12.003

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ACCEPTED MANUSCRIPT Liposomes with Cyclic RGD Peptide Motif Triggers Acute Immune Response in Mice Xiaoyi Wang a,b, Huan Wang a, Kuan Jiang a, Yanyu Zhang a, Changyou Zhan a,c,d, Man Ying a, Mingfei a e a a d,g b, Zhang , Linwei Lu , Ruifeng Wang , Songli Wang , Diane J. Burgess , Hao Wang *, Weiyue Lu a,e,f , *

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a Department of Pharmaceutics, School of Pharmacy, Fudan University & Key Laboratory of Smart Drug Delivery (Fudan University), Ministry of Education and PLA, Shanghai 201203, China b States National Pharmaceutical Engineering and Research Center, Shanghai 201203, China c Department of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China d State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, China e The Department of Integrative Medicine, Huashan Hospital, Fudan University, and The Institutes of Integrative Medicine of Fudan University, Shanghai 200041, China f Minhang Branch, Zhongshan Hospital and Institute of Fudan-Minghang Academic Health System, Minghang Hospital, Fudan University, Shanghai 201199, China g School of Pharmacy, University of Connecticut, Storrs, CT 06269, United State s of America

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ABSTRACT

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Liposomes with peptides motifs have been widely applied for targeted delivery of anticancer drugs. However, few studies have questioned whether peptide modification on liposomes may induce serious toxicity associated with immune stimulation. Here, we report that display of a tumor targeting cyclic RGD peptide (e.g. c(RGDyK) and c(RGDfK) on the surface of liposomes can be a potent inducer of lethal hypersensitivity-like reactions in mice upon re-administration, with the main symptom a sudden drop in body temperature. The hypothermia usually abates within 4 hours but is sometimes lethal with death happening within 30 min post injection. This reaction has been proven to be IgE-independent acute systemic anaphylaxis, which may due to IgG immune complex triggered complement activation, anaphylatoxin and cytokine release, etc., leading to acute conspicuous organ damage. Results from an exploration of influence factors showed that the immunotoxicity of c(RGDyK)-liposomes could not be eliminated by minimizing the c(RGDyK) motif ratio, or by decreasing injection doses in the normal dose range, or by increasing the mPEG-DSPE motif ratio. However, encapsulation of a strong cytotoxic drug completely shut off this unwanted immune response. Investigation with a series of peptides containing the RGD sequence suggested that the lethal immunotoxicity of the cyclic RGD peptide was RGD sequence and peptide cyclization dependent. This study provides a valuable alert for the utilization of peptide modified liposomes in drug delivery, especially when carrying low-toxicity drugs.

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KEY WORDS Cyclic RGD peptide, c(RGDyK), c(RGDfK), liposomes, hypothermia, immunotoxicity

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1. Introduction

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Liposomes are biocompatible, non-toxic vesicular nanocarriers, which are widely used to encapsulate therapeutic agents to provide sustained release, improved biodistribution and enhanced anticancer efficacy [1]. Several liposomes have been approved by the FDA for clinical use and more effort is being focused on further avoidance of off target effects of entrapped drugs by engineering liposomes via a number of diverse strategies. One of the most commonly used strategies is modification with targeting peptides [2,3]. Cyclic arginine-glycine-aspartic acid (RGD) peptides are ligands of the adhesion receptor αv β3 integrin. Owing to the fact that the αv β3 integrin is over-expressed in cancer cells and the neovasculature [4], modification of drug delivery systems with cyclic RGD peptides imparts a tumordirecting property [5,6]. Many researchers have reported that liposomal cytotoxic anticancer drug formulations with cyclic RGD peptide modification display an aggregate survival benefit in glioma bearing mice, owing to the enhanced blood-brain tumor barrier (BBTB) transport and glioma cellular uptake [7-12]. Accordingly, in the present work, c(RGDyK) decorated liposomes were used to encapsulate a low-toxic molecular targeted anticancer drug for glioma treatment. However, an unexpected serious side effect occurred during investigation of the in vivo anti-glioma effect in intracranial glioma bearing BALB/c nude mice. A sharp drop in body temperature and even death was noted exclusively in the mice in c(RGDyK) containing liposomal drug treatment group at the time of repeated intravenous administration, while other groups (free drug and liposomal drug without peptide motif) remained normal. The in vivo anti-glioma experiment was repeated thrice, and every time hypothermia occurred within 15 min of repeated injection with the c(RGDyK) containing liposomal drug formulation at all doses investigated. After excluding the hypothesis of contamination, this acute and reproducible toxicity suggests the possibility that liposomes with the c(RGDyK) motif (c(RGDyK)-liposomes) possess the capacity of triggering allergic reactions. In support of this, a series of histological and immunological investigations were conducted using ICR mice. Factors influencing c(RGDyK)-liposomes’ immunotoxicity and the structural basis that determines the immunotoxic properties of RGD peptides were investigated. These results appear to have important implications for the evaluation and therapeutic use of peptide modified liposomal drug formulations.

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2. Materials and Methods

2.1. Reagents and mice HSPC was purchased from AVT Bio. Inc. (China). Cholesterol, citric acid, Sephadex G50 and DiD were purchased from Sigma-Aldrich (USA). MPEG 20 00-DSPE and mal-PEG3400-DSPE were purchased from Laysan Bio. Inc (USA). C(RGDyK), RGD, RGDC, RGDyK, c(RGDfK) and c( GRDyK) were customized from KareBay Biochem, Inc (China). Some batches of c(RGDyK) were obtained from GL Biochem Ltd. (China). Percoll was purchased from Yeasen Bio. Inc. (China). Peroxidase Conjugated AffiniPure Goat Anti-Mouse IgG (H+L) (ZB-2305) was bought from ZSGB-BIO (China). Goat Anti-Mouse IgG1 heavy chain (HRP) (ab97240), Goat Anti-Mouse IgG2a heavy chain (HRP) (ab97245), Goat Anti-Mouse IgG2b heavy chain (HRP) (ab97250), Goat Anti-Mouse IgG2c heavy chain (HRP) (ab97255), Goat Anti-Mouse IgG3 heavy chain (HRP) (ab97260), Rat monoclonal 23G3 Anti-Mouse IgE epsilon chain (HRP) (ab99574), Goat Anti-Mouse IgM mu chain (HRP) (ab97230)

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2.2. Preparation of liposomes and other formulations

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and Mouse Complement C5a ELISA Kit (ab193718) were purchased from Abcam (UK). Interleukin (IL)-2, IL-6, IL-8, interferon-γ (IFN-γ), IFN-α and tumor necrosis factor-α (TNF-α) ELISA kits were purchased from MultiSciences (China). Red blood cell lysis buffer and TMB Horseradish Peroxidase Color Development Solution for ELISA were obtained from Beyotime Biotechnology (China). Cell Proliferation Dye eFluor ® 670 was purchased from eBioscience (USA). Sterile Cell Strainer was purchased from Fisher Scientific (USA). 15% sheep red blood cells SRBC and SRBC haemolysin (1:4000) were purchased from Solarbio (China). All other chemicals were purchased from China National Pharmaceutical Group Corporation. Male ICR mice of 4~6 weeks of age were obtained from Shanghai Super-B&K laboratory animal Corp. Ltd. (China) and kept under SPF conditions in a temperature-controlled room (22ºC) on a 12-h light/dark cycle in filter-top cages and with ad libitum access to food and water. For some experiments, BALB/c nude mice obtained from the same company were used. All animal studies were conducted in accordance with guidelines from the ethics committee of Fudan University.

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Liposomes with or without motif of different peptide ligands were prepared using the published thin-film hydration method [7]. Peptide c(RGDyK) modified nanodisks [13] and micelles [14] were also prepared using a similar method in accordance with previous reports. Labeling of liposomes with DiD was achieved by mixing of DiD with lipid compositions in chloroform. To prepare doxorubicin loaded liposomes, a traditional ammonium sulfate gradient loading method was used [15].Replacement of the external water phase and the removal of free drug were achieved using a Sephadex G50 column. Particle size of the resultant formulations was determined using a Malvern Zetasizer. Drug quantification was conducted using HPLC. To confirm morphology of some of the formulations, CryoTEM imaging was conducted using cryo-electron microscopy (FEI, Hillsboro, USA). Details of the composition and particle size of different formulations are summarized in Supplementary Table 1.

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2.3. Measurement of mouse body surface temperature

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Body surface temperature of each mouse was measured three times using OMRON infrared forehead thermometers (MC-872K, measurement range: 0~50°C), from which average values were obtained. The use of OMRON infrared forehead thermometers has been shown in our preliminary studies to be a fast and accurate method to monitor change in body surface temperature of mice.

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2.4. Histopathology examination and detection of immune complex depositions To evaluate the pathological lesions induced by various liposomes and investigate possible immune complex depositions in main organs, one mouse from each treatment group was sacrificed under deep anesthesia at 1 hour post the third treatment. The heart, liver, kidney, spleen, lung and brain of each mouse were collected, fixed in 4% formalin for 24 h and embedded in paraffin for HE and immunohistochemistry staining. For one mouse in c(RGDyK)-liposomes 3rd group (the mouse died 30 min post injection), organs were collected immediately after death. HE staining was performed by staining tissue sections using hematoxylin and eosin. For staining with IgG and IgM immune complex depositions, slides described as above were deparaffinized, rehydrated and incubated overnight with goat anti-mouse IgG or IgM HRP-conjugated antibody at a dilution of 1:100. The slides were then counterstained with hematoxylin and images were captured using a Nikon Diaphot 300 microscope (Nikon).

ACCEPTED MANUSCRIPT 2.5. Determination of immunoglobulins in mouse serum

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2.6. In vivo cytokine release assays

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Sera liposome/peptide-liposome specific IgG, IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgM and IgE antibodies were determined using indirect-ELISA [16] with liposomes or peptide modified liposomes as the antigen. Briefly, high binding ELISA 96-well plates were coated with solutions of 100 μL ethanol containing various antigens with a final concentration of peptide of 0.4 μM or mPEG-DSPE 1 μM, equals to HSPC 10 μM. Alternatively, plates were directly coated with mPEG-DSPE ethanol solution (100 μM) for effective determination of anti-PEG antibody. Lipid-coated plates were allowed to air dry for 4 h before being blocked for 60 min with pH 7.0 PBS containing 1% BSA and subsequently washed 3 times with wash solution (pH 7.0 PBS containing 0.05% Tween 20). Serum samples of mice were diluted in serial ratios using PBS (1:30, 1:450, 1:1800, 1:7200, 1:28800, 1:115200), each dilution was applied in appointed corresponding antigen coated wells for 1 h at 37°C and washed 3 times as described before. 100 μL HRP-conjugated Goat Anti-mouse IgG (1:5000, final concentration 0.08 μg/mL), IgG1 (1:12500, final concentration 0.08 μg/mL), IgG2a (1:12500, final concentration 0.08 μg/mL 0.08 μg/mL), IgG2b (1:12500, final concentration 0.08 μg/mL), IgG2c (1:12500, final concentration 0.08 μg/mL), IgG3 (1:12500, final concentration 0.08 μg/mL), IgM (1:10000, final concentration 0.1 μg/mL) or IgE (1:5000, final concentration 0.07 μg/mL) in PBS containing 0.1% BSA was added to each well. After incubation for 60 min at 37°C, wells were washed 3 times with the wash solution. Coloration was initiated by adding 150 μL of TMB Chromogen Solution for ELISA. After 15 min-incubation at room temperature, the reaction was stopped by adding 50 μL of 2 M H2SO4 and optical density (OD) reflecting the IgG and IgM titers was measured at 450 nm using BIO-TEC Plate Reader. For calculation of antibody titer, OD values were adjusted to sigmoid dose-response curves and determined as the last dilution which give an OD value of 0.2.

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Three groups of ICR mice (n=9) received saline, liposomes or c(RGDyK)-liposomes respectively at a dose of 75 μmol HSPC/kg/administered three times at one day intervals. 15 min after the third injection (when mice treated with c(RGDyK)-liposomes begin to experience hypothermia), mice were bled from their orbital sinus and the serum samples were processed for cytokine analysis. IL-2, IL-6, IL-8, IF N-γ, IF N-α and TNF-α were quantified using ELISA kits. Briefly, serum samples were appropriately diluted. Serial dilution of cytokine standards were also prepared in the same diluent. Controls, standards, and serum samples were added to the wells of ELISA kits, and tested according to the manufacturers’ instructions for each of the ELISA kits. 2.7. In vitro lymphocyte proliferation assays Lymphocyte proliferation in response to c(RGDyK)-liposomes was tested. ICR mice or BALB/c nude mice were immunized three times with c(RGDyK)-liposomes at one day intervals. 7 days after immunization, spleens isolated from these mice were processed for immunologic evaluation according to a reported protocol [17,18]. The spleens were first squeezed through a 100-μm strainer for the purpose of disruption. Erythrocytes were removed with lysing buffer and the remaining cells were gently washed twice in ice-cold PBS and filtered through a 40-μm strainer. To reduce biological variation, splenocytes from three mice were pooled. Lymphocytes as well as monocytes were isolated from whole splenocytes by a standard Percoll density centrifugation technique. Cells (at 2 × 7 10 /mL) were then labeled by mixing with an equal volume of eFluor 670 cell proliferation dye in PBS to obtain a final concentration of 15 μM and were incubated at 37 °C in the dark for 10 min. Labeling was arrested by adding 5 volumes of cold complete media (containing 15% FBS) and incubat ing in

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ice for 5 minutes. Cells were washed three times with cold complete media and resuspended in complete media at a density of 2 × 107 cells/100 μL before being added to 96-well plate. To each well of cells (100 μL), liposomes with or without the modification of c(RGDyK) were added to stimulate lymphocytes. The final concentration of HSPC was 0.08 μmol/mL. Mitogen concanavalin A (Con A, final concentration 4 μg/mL) was added to appropriate wells as positive controls. Controls with no mitogen were also plated. The plates were incubated at 37°C in the CO2 incubator. 5 days later, cells were processed for flow cytometry analysis. Medium supernatants were centrifuged for 10 min at 13,000 rpm. Cytokines in supernatants were then quantified as described in “In vivo cytokine release assays”. In addition, cultured cells were collected on day 3. Viability of cells was measured by trypan blue exclusion assay. 2.8.Biodistribution study

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ICR mice received liposome or c(RGDyK)-liposome stimulation twice at one day intervals (starting at day 0). At day 4, mice were re-stimulated with liposomes/DiD or c(RGDyK)-liposomes/DiD (liposomes/DiD3rd and c(RGDyK)-liposomes3rd). Mice without any treatment were directly injected with liposomes/DiD or c(RGDyK)-liposomes/DiD (liposomes/DiD1st and c(RGDyK)-liposomes1st ). The injection dose of DiD labelled or unlabeled liposomes and c(RGDyK)-liposomes was 75 μmol HSPC/kg/administration. 4 h after injection of DiD labelled liposomes, mice were bled and perfused under deep anesthesia. Pictures of mice were captured to show leakage of liposomes/DiD from vessels. Heart, liver, spleen, lung, kidney, brain, paws and ears were collected and weighed. All samples, except for blood samples, were further processed by homogenization. Fluorescence intensity of DiD in 100 μL of each blood or homogenate sample was assayed on Microplate Reader infinite M200 PRO (TECAN，USA). Results were expressed as microgram of DiD per gram of tissue.

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2.9. Immunizations

Table 1. Immunization protocols of mice used in experiments with various purposes.

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Groups, formulations and injection doses 6 groups: (1) saline, (2) liposomes, (3) c(RGDyK) dissolved in saline, (4) c(RGDyK)PEG3400-DSPE dissolved in saline, (5) liposomes+ c(RGDyK)-PEG3400-DSPE mixture and (6) c(RGDyK)-liposomes. (n=3, dose: 75 μmol HSPC/kg/administration or 3 μmol c(RGDyK)/kg/administration). 6 groups: mice received the third c(RGD yK)liposomes stimulation at (1) day 4, (2) da y 7, (3) day 10, (4) day 15, (5) day 20, (6) day 30. (n=6, dose: 75 μmol HSPC/kg/administration).

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Purpose 1. Identification of components accounting for the c(RGD yK)liposome induced hypothermia or death

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2. In vivo antic(RGDyK)-liposomes IgG and IgM responses and incidence of hypothermia

3. Influence of peptide modification ratios on the immunotoxicity of c(RGDyK)-liposomes 4. Influence of administration dos es on the immunotoxicity of c(RGDyK)-liposomes

5 groups: (1) saline, (2) liposomes, (3) 2% c(RGDyK)-liposomes, (4) 1% c(RGDyK)liposomes, (5) 0.5% c(RGDyK)-liposomes. (n=3, dose: 75 μmol HSPC/kg/administration). 7 groups: (1) saline, (2) liposomes, (3-4) 2% c(RGDyK)-liposomes 75, 12.5 μmol HSPC/kg/administration, (5-7) 1% c(RGDyK)liposomes 150, 75, 12.5 μmol HSPC/kg/administration (n=3).

Immunizations ICR mice were intravenously injected with 100 μL of various formulations five times at one day interval, all treatment groups were re-stimulated with c(RGDyK)-liposomes 48 hours after the 5th stimulation. 36 ICR mice received an intravenous injection of c(RGDyK)-liposomes twice at one day intervals and were divided into 6 groups to receive the third injection at different time points (day 4, 7, 10, 15, 20 and 30). ICR mice were immunized intravenously with 100 μL of corresponding formulations six times at one day intervals. ICR mice were immunized intravenously with 100 μL of corresponding formulations six times at one day intervals.

ACCEPTED MANUSCRIPT ICR mice were immunized intravenously with 100 μL of corresponding formulations six times at one day intervals.

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ICR mice were immunized intravenously with 100 μL of corresponding formulations six times at one day intervals. ICR mice were immunized intravenously with 100 μL of corresponding formulations six times at one day intervals. For the test of cross response between other RGD peptide-liposome induced antibodies and c(RGDyK)-liposomes, another 7 groups of mice were immunized with various formulations for the first two stimulations, and all injected with c(RGDyK)-liposomes at the third stimulation.

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6 groups: (1) saline, (2) liposomes, (3) c(RGDyK)-liposomes (3% mPEG-DSPE), (4) c(RGDyK)-liposomes (10% mPEG-DSPE), (5) c(RGDyK)-Disk (20% mPEG-DSPE), (6) c(RGDyK)-Micell (98% mPEG-DSPE) (n=3, dose: 12.5 μmol HSPC/kg/administration). 4 groups: (1) saline, (2) liposomes, (3) c(RGDyK)-liposomes, (4) c(RGDyK)liposomes/DOX. (n=3, dose: 75 μmol HSPC/kg/administration). 7 groups: (1) saline, (2) liposomes, (3) c(RGDyK)-liposomes, (4) RGDC-liposomes, (5) RGDyK-liposomes, (6) c(RGDfK)-liposomes and (7) c(GRDyK)-liposomes. (n=3, dose: 75 μmol HSPC/kg/administration).

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5. Influence of mPEGDSPE modification ratios on the immunotoxicity of c(RGDyK) modified nanoformulations 6. Influence of encapsulated drugs on the immunotoxicity of c(RGDyK)-liposomes 7. In vestigation of immunotoxicity of RGD derived peptides

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Mice used in different experiments were immunized with various formulations following different protocols as listed in Table 1. All mice received the first stimulation at day 0. Blood was withdrawn from orbital sinus 48 hours after the second immunization at day 4 (before the third stimulation) with mice under isoflurane anesthesia, processed to serum and frozen at -80°C for later IgG and IgM antibody analysis via ELISA as described in “2.5. Determination of immunoglobulins in mouse serum”. Besides, surface body temperature and mortality of mice in each group were recorded after 30 min of each stimulation. For “2. in vivo anti-c(RGDyK)-liposome IgG and IgM responses and incidence of hypothermia”, serum samples of the 30 mice were collected before the first and second immunizations. 2.10. Determination of complement activation product C5a

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The complement activation product, C5a, was determined in serum using Mouse Complement C5a ELISA Kit. Serum samples were collected from ICR mice at 1 hour before the first (control serum) and the third injection (antiserum) for each formulation. Serum aliquots were then incubated with liposomes, c(RGDyK)-liposomes (HSPC 20 mM) or positive control LPS (0.1 mg/ml) (10:1 by volume) for 1 hour at 37 °C to test the sera C5a production following the first ( control serum+each formulation) and the third exposure (antiserum+corresponding formulation) of each formulation. 2.11. Hemolytic complement activity assay The hemolytic complement activity assay was performed using standard procedures [19,20]. Briefly, SRBC haemolysin (1:400) was used to sensitize 5% sheep erythrocytes (1:1 by volume) through 30-min waterbath incubation at 37°C. Then, 50 µl of five serial dilutions of ICR mouse serum (from 1/2) were mixed with 50 µl of sensitized sheep erythrocytes and incubated for 30 min at 37°C. Reactions were stopped by centrifugation at 2000 rpm for 5 min. The supernatants were read at 540 nm. PBS was used as a negative control and distilled water as 100% lysis control. 2.12. In vitro and in vivo reaction between c(RGDyK) and anti-c(RGDyK)-liposome IgG

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Reaction between c(RGDyK) and anti-c(RGDyK)-liposome IgG antibodies in vitro and in vivo were first conducted for the purpose of validation of using c(RGDyK) as an αv β3 integrin inhibitor. For in vitro testing, PBS, c(RGDyK), liposomes or c(RGDyK)-liposomes containing c(RGDyK) 1 mg/kg or HSPC 4 mg/kg were mixed with serum from c(RGDyK)-liposome immunized mice (1:1 v/v), incubated at 37°C for 1 hour. The change in the IgG titer was determined by indirect-ELISA as described above. For the in vivo test, mice were pre-dosed with saline (n=3), liposomes (n=3), c(RGDyK) (n=3) or c(RGDyK)-liposomes (n=9) at day 0 and day 2. Blood samples from each mouse were collected at 1 hour before the third stimulation at day 4. After then, mice receiving saline, liposomes, and c(RGDyK) were injected with the same formulation as the third stimulation. ICR mice immunized with c(RGDyK)-liposomes were divided into three groups, 1 hour post the third stimulation with c(RGDyK), liposomes and c(RGDyK)-liposomes respectively, mice were bled again. Serum samples from these three groups were described as c(RGDyK) -liposome immunized mice “+c(RGDyK) 3rd after”, “+liposomes3rd after” and “+c(RGDyK)-liposomes3rd after”. Body temperatures were measured at 30 min after each injection. 2.13. In vivo αvβ 3 integrin inhibition

2.14. Statistical analysis

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For exploration of impact of in vivo inhibition of αv β3 integrin on anti-c(RGDyK)-liposomes antibody production and onset of hypothermia, c(RGDyK) were intravenously pre-injected as a αv β3 integrin inhibitor. Briefly, ICR mice were pre-dosed with c(RGDyK) or liposomes 0.5/1 hour before injection of c(RGDyK)-liposomes at days 0, 2 and 4 (described as “c(RGDyK) 0.5h pre-dose”, “c(RGDyK) 1h pre-dose”, “liposome 0.5h pre-dose”, “liposome 1h pre-dose” +c(RGDyK)-liposomes). Mice receiving c(RGDyK)-liposomes only (described as “without pre-dose” +c(RGDyK)-liposomes) were used as positive controls and the ones receiving saline, liposomes or c(RGDyK) twice with the same protocol were included as negative controls. Blood samples were collected for determination of IgG production at 1 hour before the third injection. Body temperature was recorded at 30 min post each stimulation.

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3. Results

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All statistical analyses were performed using GraphPad Prism Version 6. The data were analyzed by one-way ANOVA for multiple comparisons. The significance level was set at 0.05 .

3.1. Liposomal dependence of c(RGDyK)-liposome induced lethal hypothermia A pilot study was performed to determine whether the c(RGDyK)-liposomes without drug loading causes similar toxicity in ICR mice to those with drug before any further experimentation. A similar pattern of toxicity was reproduced in the ICR mice after repeated injection with c(RGDyK)-liposomes without drug at one day intervals (the same administration schedule used in the in vivo anti-glioma experiment). Since hypothermia induced by c(RGDyK)-liposomes mostly happened following the third injection, the body temperature of ICR mice recorded after third injection were shown to determine the toxicity of formulations tested in all following experimentations. To anchor c(RGDyK) on the surface of liposomes, c(RGDyK) was first conjugated with mal PEG3400-DSPE through a mercapto-maleimide reaction. To assess whether free c(RGDyK)/c(RGDyK)-PEG3400-DSPE alone was sufficient to trigger hypothermia or whether

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modification on the liposomes was necessary to induce toxicity, c(RGDyK), c(RGDyK)-PEG3400DSPE and liposomes were separately prepared and injected intravenously into ICR mice at one day intervals. The body surface temperature were recorded following each injection. As shown in Figure 1, the mice that received c(RGDyK)-liposomes experienced severe hypothermia within 30 min following the third injection, with their body temperature dropping to around 32°C. One third of the mice suffered acute death and the remainder survived from the hypothermia and their temperature returned to normal within 4 hours. By contrast, free c(RGDyK), c(RGDyK)-PEG3400-DSPE or its mixture with liposomes (featuring no insertion of c(RGDyK)-PEG3400-DSPE in the liposome membrane) failed to induce hypothermia at the third or other later injections. However, a 6th injection which consisted of c(RGDyK)-liposomes triggered hypothermia in the c(RGDyK)-PEG3400-DSPE and the liposomes+c(RGDyK)-PEG3400-DSPE treatment groups (data not shown). Thus, modification of c(RGDyK) on liposomes appears to be important for the onset of hypothermia.

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Figure 1. Identification of components contributing to the c(RGDyK)-liposome induced hypothermia or death. ICR mice received various formulations via tail veil injection 5 times at one day intervals and all treatment groups were re-stimulated with c(RGDyK)-liposomes at the 6th stimulation (dose: 75 μmol HSPC/kg/administration or 3 μmol c(RGDyK)/kg/administration). Body surface temperature and mortality of mice at 30 min post each stimulation of various formulations were recorded. The graph shows body temperature and mortality following the third stimulation. For each group, n=3. 3.2. Multi-organ lesions and biodistribution characteristics of c(RGDyK)-liposomes Histopathology examinations were performed to investigate any pathological changes in the main organs of mice that experienced lethal hypothermia. As expected, there were indeed obvious lesions in mice that underwent acute death. The most affected organs were the liver, lung and kidney. Proteinosis (collagen) and inflammatory cell infiltration in pulmonary alveoli along with hyperaemia in the liver and kidney was observed when acute death occurred (Figure 2, a). Hepatic damage was especially extensive (Figure 2, b). In addition, as demonstrated by a biodistribution study, in mice that experienced hypothermia, there was a significantly elevated distribution of DiD in acromegaly and ear skin indicating capillary leakage (Supplementary Figure 1). The blue color of DiD labelled

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c(RGDyK)-liposomes in mice skin was even visible to the naked eye (Figure 2, c). Moreover, compared with liposomes/DiD3 rd, c(RGDyK)-liposomes/DiD3rd showed improved distribution in liver, spleen and lung. Even following the first injection, c(RGDyK)-liposomes/DiD1st showed rapid clearance from the blood and elevated accumulation in these three organs (Supplementary Figure 1). Taken together, mice suffering from acute hypothermia elicited by c(RGDyK)-liposomes had the complications of capillary leakage, pulmonary alveolar proteinosis and inflammatory cell infiltration, as well as nephritic and hepatic hyperaemia. Based on these observed systemic complications and the repeated evidence that hypothermia caused by c(RGDyK)-liposomes is acute, lethal, reproducible and follows a similar pattern to allergic reactions, it is suspected that c(RGDyK)liposomes induces immunogenicity or a severe case of complement activation-related pseudoallergy (CARPA), a hypersensitivity reaction caused by many nanomedicines, including PEGylated liposomes.

Figure 2. Examination of pathological changes in mice experiencing hypothermia or death. (a) H&E staining to explore histopathological changes in the main organs at 1 hour post the third injection with the different formulations. (b) Visible hepatic damage of mouse that experienced acute death post the third c(RGDyK)-liposome injection. In (a) and (b), ICR mice were intravenously

ACCEPTED MANUSCRIPT injected with saline, liposomes or c(RGDyK)-liposomes thrice at one day intervals (dose: 75 μmol HSPC/kg/administration). (c) Appearance of c(RGDyK)-liposomes/DiD and visible accumulation of c(RGDyK)-liposomes/DiD in mouse skin at 4 hours post the third injection with c(RGDyK)liposomes/DiD (as indicated by arrows). ICR mice were injected with liposomes or c(RGDyK)liposomes at days 0, 2 and liposomes/DiD or c(RGDyK)-liposomes/DiD at day 4 (dose: 75 μmol HSPC/kg/administration). Blue color in skin was observed in mouse experiencing hypother mia following c(RGDyK)-liposome/DiD injection, indicating capillary leakage.

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3.3. Mechanistic insight into lethal hypothermia induced by c(RGDyK) -liposomes

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In support of our hypothesis, a series of immunological evaluations were conducted, including investigation of serum IgG, IgM and IgE antibody production at 1 hour before the third stimulation of c(RGDyK)-liposomes, in vitro lymphocyte proliferation assays, immune complex formation/depositions, complement activation and cytokine release following the third injection. As expected, robust production of anti-c(RGDyK)-liposome IgG and IgM antibodies comparing the liposome treatment groups (P<0.0001 and P<0.001 for IgG and IgM respectively) were observed exclusively in mice that experienced hypothermia following the third injection (Figure 3, a, b). Results from the IgE assay (Figure 3, c) show no production of IgE in mice treated with the c(RGDyK)liposomes. These results suggest that the life-threatening hypothermia is a result of the immunogenicity of c(RGDyK)-liposomes and mainly involves IgG and/or IgM. In vitro lymphocyte proliferation assays further confirmed this immunogenicity as lymphocytes from the c(RGDyK)liposome immunized mice showed c(RGDyK)-liposome specific proliferation and secretion of proliferation related cytokines (Supplementary Figure 2, a, b). The immunohistochemical staining (Figure 3, e, f) showed extensive presence of both IgG and IgM immune complex deposition in the liver, lung and kidney of the mice that suffered severe hypothermia and death. The IgG immune complex deposition was more serious compared to the IgM complex deposition. The formation of IgG and IgM immune complexes were further confirmed by the plunge in the IgG and IgM titer one hour post administration of c(RGDyK) -liposomes (Figure 3, d).

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Figure 3. Immunological evaluations on c(RGDyK)-liposomes. ICR mice were immunized at days 0, 2 and 4 with saline, liposomes or c(RGDyK)-liposomes (dose: 75 μmol HSPC/kg/administration). (a-c) Detection of anti-c(RGDyK)-liposome IgG, IgM and IgE antibodies in sera at day 4 before the third c(RGDyK)-liposome injection. n=3 for IgG and IgM, n=6 for IgE. (d) Changes in IgG and IgM antibody titers after the third c(RGDyK)-liposome injection (n=6). (e, f) Immunohistochemical analysis of IgG (e) and IgM (f) immune complex depositions af ter the third c(RGDyK)-liposome injection. NS P>0.05, *P<0.05, *** P<0.001 and **** P<0.0001.

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Since the formation and deposition of immune complex may activate the complement system through a classical pathway, resulting in the release of anaphylatoxin C3a, C5a, pro -inflammatory cytokines, etc., which have the potential to cause immunologic injuries, complement activation and cytokine release were investigated. Complement activation induced by c(RGDyK)-liposomes was assessed by analyzing the haemolytic complement function of the classical pathway and one of the activation products, C5a, following the third exposure of c(RGDyK)-liposomes [21]. C5a was determined using an ELISA Kit. Serum samples were collected at 1 hour before the first ( control serum) and the third injection (antiserum) for each formulation, incubated with either liposomes, c(RGDyK)-liposomes or the positive control (LPS) for one 1 hour. Co-incubation of control serum with each formulation represented the first exposure while co-incubation of antiserum with each formulation represented the third. As shown in Figure 4 (a), compared to control serum, the first exposure of liposomes or c(RGDyK)-liposomes only induced a slightly elevated production of C5a (P>0.05), implying a minor activation of the complement system. However, the C5a level was significantly increased following the exposure of both formulation s with their corresponding antiserums (liposomes: P<0.05, c(RGDyK)-liposomes: P<0.001). Given the fact that antiserums contain antibodies, the complement activation might be triggered by the newly formed immune complex during incubation. LPS as a potent alternative pathway complement activator [22]

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significantly enhanced the C5a production (P<0.0001) at the first exposure. Correspondingly, the first intravenous injection of LPS triggered a toxicity syndrome characterized by a drop in body temperature within 1 hour (Figure 4, b) and reduced activity, similar to that induced by repeated injection of c(RGDyK)-liposomes, however the LPS triggered toxicity syndrome was more serious and it took longer for the mice to recover. Haemolytic complement activity of serum was assayed using standard procedures for further evaluation of complement activation [20]. Test serum was collected at 1 hour following the first injection of saline, LPS, liposomes and c(RGDyK)-liposomes, and also collected at 1 hour before and after the third injection of liposomes and c(RGDyK)-liposomes. Haemolysin sensitized SRBC was incubated with serial dilution of test serums for 30 min at 37°C. The fold of serum dilution inducing 50% haemolysis (CH50) represents the function of classical complement pathway. Decrease in CH50 indicates the depletion of components of the classical pathway. According to Figure 4 (b) and (c), repeated injection of c(RGDyK)-liposomes significantly depleted complement activity of serum, thereby reducing the 50% lysis of SRBC to less than 2 fold dilution while that of saline treated serum is 10.5, indicating a massive activation and depletion of complement components. In contrast, the first c(RGDyK)-liposome injection minimally affected complement activation, hence preserving most residual complement activity (CH50 8.97). The differe nce in complement activity between the first and third injection was consistent with C5a production, suggesting that extensive complement activation was triggered by immune complexes through the classical pathway. Owing to the fact that LPS activates an alternative complement pathway [22,23], C1q which is necessary for the activation of classical complement pathways was not depleted. Serum at one hour post LPS injection still possessed the ability to induced haemolysis and showed a mild fall in CH50 (5.74). In addition, although liposomes also significantly activated the complement system at repeated injection, due to the existence of anti-PEG IgM (Supplementary Figure 8), the C5a production is significantly lower (P<0.01) and the CH50 is higher than c(RGDyK)-liposomes. Accordingly, the extent of complement activation was not as severe with c(RGDyK)-liposomes. Circulating levels of cytokines are indicative of immune responses and over-secretion of cytokines also has the potential to trigger dangerous syndrome [24-26]. Accordingly, the secretion of representative cytokines IFN-γ, IL-6, IL-8, IL-2 and TNF-α was analyzed at 15 min after the third stimulation with c(RGDyK)-liposomes (the time when hypothermia occurred). The results showed that in this short time, the third injection of c(RGDyK)-liposomes had resulted in significantly elevated secretion of IFN-γ, IF N-α and IL-8 (Figure 4, d). Among the released cytokines, IL-8 is the most likely cytokine to cause organ lesions. As it is a potent neutrophil attractant and activator and has also been shown to play a significant role in acute lung injury/acute respiratory distress syndrome (ALI/ARDS) [27,28]. Overstimulation and dysfunction of these recruited neutrophils results in the release of a number of pro-inflammatory molecules and proteases resulting in further damage to organs. IFN-γ has been shown to correlate with protective immunity. Several studies have reported that IFN-γ production is important for TB vaccine efficacy [29,30]. IFN-α is involved in promoting an adaptive immune response. The cytokine release reported here was able to serve as additional evidence of complement activation and inflammatory responses triggered by immune complex depositions.

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Figure 4. Complement activation induced by repeated c(RGDyK)-liposome injection. (a) ELISA results of complement activation product C5a in mouse serum. Control serum is serum from untreated ICR mice. Control serum was incubated with LPS (0.1 mg/ml), liposomes or c(RGDyK)liposomes (HSPC 20 mM) for 1 hour at 37 °C. For anti-liposome and anti-c(RGDyK)-liposome serum, ICR mice were immunized with liposomes or c(RGDyK)-liposomes twice (75 μmol HSPC/kg/administration) at one day intervals, 48 hours post the second injection, blood was collected to produce antiserum. Antiserum was then incubated with the corresponding formulation at 10:1 (V/V) for 1 hour at 37 °C. (b) Body temperature at 1 hour post the first injection with LPS, liposomes, c(RGDyK)-liposomes and post the third injection with liposomes and c(RGDyK)liposomes. (c) Haemolytic complement activity of mouse serum. Serum of ICR mice was collected at 1 hour following the first injection of saline, LPS, liposomes and c(RGDyK) -liposomes and also at 1 hour before and after the third injection of liposomes and c(RGDyK)-liposomes. The serum was then diluted and incubated with haemolysin sensitized SRBC for 30 min at 37°C. (d) In vivo cytokine production at 15 min following the third c(RGDyK)-liposome injection. For (a-c), n=3; d, n=9. Values are mean ± SD.*P<0.05, **P<0.01, *** P<0.001 and **** P<0.0001. CARPA represents a novel subcategory of acute type I hypersensitivity reactions that can be caused by many nanomedicines including PEGylated liposomes. It is normally mild, transient and preventable by appropriate precautions [31,32] and arises mostly at the first treatment [33-35]. Although no studies have focused on the potential of c(RGDyK) modified liposomes in triggering

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CARPA, it has been reported that modification of another cyclic RGD peptide c(RGDfC) on the surface of microbubbles can trigger CARPA in human serum in vitro [36]. This coupled with the evidence reported here (slightly increased C5a and decreased CH50 following the first administration of c(RGDyK)-liposomes) implies that c(RGDyK)-liposomes should also be able to trigger CAPPA. However, based on the features of CARPA and our reported acute immune response (triggered by repeated injection of c(RGDyK)-liposomes and dependent on antibody production) the hypersensitivity syndrome reported here, (although accompanied by complement activation), is unlikely CAPPA, but rather a type III hypersensitivity, or more precisely IgEindependent systemic anaphylaxis. Anaphylaxis is an acute, severe, and potentially fatal systemic allergic reaction. In mice, immediate hypothermia is a common characteristic of systemic anaphylaxis and has been used as an indicator of the onset of anaphylaxis in many studies [37-40]. IgE, mast cells, and histamine have long been associated with anaphylaxis, but more and more evidence ha s shown that IgG antibodies are also important in elicitation of passive and active systemic anaphylaxis in mice [40-42]. Cells other than mast cells, including macrophages and basophils are thought to play pivotal roles in IgG mediated anaphylaxis upon capture of IgG-allergen complexes through FcγRs and release of platelet-activating factor (PAF), leading to increased vascular permeability [39,40,43-45]. In addition, complement activation triggered by IgG immune complex has been reported to trigger anaphylaxis, through the release of anaphylatoxins and other subsequent reactions [44]. The antibody responses of all formulations used in “2.9” (Purpose 1) were systematic ally investigated in order to identify components contributing to the c(RGDyK)-liposome induced immunotoxicity. Free c(RGDyK) did not induce detectable c(RGDyK)-specific IgG or IgM antibody production. However, elevated IgG and IgM antibody levels were detected in both c(RGDyK)PEG3400-DSPE-treated and liposomes+c(RGDyK)-PEG3400-DSPE mixture-treated groups (Supplementary Figure 3). Interestingly, according to Figure 1 (a), preformation of IgG and IgM antibody in these two groups did not lead to any drop in body temperature at repeated injection of c(RGDyK)-PEG3400-DSPE or its liposome mixture, but rechallenge with c(RGDyK)-liposomes evoked hypothermia. This phenomenon indicates that antibodies induced by c(RGDyK)-PEG3400-DSPE could also interact with c(RGDyK)-liposomes. However, considering that c(RGDyK)-liposomes and c(RGDyK)-PEG3400-DSPE are morphologically distinct, c(RGDyK)-liposome-antibody immune complexes will be different from c(RGDyK)-PEG3400-DSPE-antibody in molecular size. As mentioned above, the IgG immune complex is an important pathogenetic mechanism in systemic anaphylaxis . Furthermore, the molecular size of immune complexes is considered to determine immune responses and outcomes, including their deposition and precise sites of tissue localization, as well as their removal by the monocyte-macrophage system, and their efficiency in activation of the serum complement pathways [46]. Thus, molecular size of immune complexes may account for the difference between the immune reactions induced by rechallenge of c(RGDyK)-PEG3400-DSPE and c(RGDyK)-liposomes in the same mice. It can be concluded that the immunogenicity of c(RGDyK) was conferred by the conjugation of mal-PEG3400-DSPE, and insertion of c(RGDyK)-PEG3400-DSPE into the liposomal membrane further potentiated its immunogenicity as well as the capacity to induce life-threatening immunotoxicity. Since hypothermia induced by c(RGDyK)-liposomes was first discovered in BALB/c congenitally athymic nude mice, the immune response of c(RGDyK)-liposomes in nude mice was tested. As expected, BALB/c nude mice produced even stronger anti-c(RGDyK)-liposome IgG and IgM antibodies compared to the ICR mice and they also experienced hypothermia (Supplementary Figure 2, c, e, f). Lymphocytes from spleen of c(RGDyK)-liposome immunized nude mice proliferated

ACCEPTED MANUSCRIPT under c(RGDyK)-liposome stimulation as well (Supplementary Figure 2, d). Importantly, antibody production and the onset of hypothermia was reproducible in both ICR mice and BALB/c nude mice in different experiments using different batches of c(RGDyK) or c(RGDyK) from different biological companies. 3.4.

Anti-c(RGDyK)-liposome

IgG and IgM

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The time course of anti-c(RGDyK)-liposome IgG and IgM production and the relationship between antibody titers and the incidence of hypothermia were investigated. As demonstrated in Figure 5 (a), production of both IgG and IgM antibodies soared after the second injection and peaked at days 4-7, after that the IgM antibody rapidly decreased and disappeared by 20 days. In contrast, the IgG antibody was long-lasting, even at 30 days post the second stimulation, the average antibody titer was still around 300. The incidence of systemic anaphylaxis (hypothermia) at the third injection declined with the decrease of the IgG and IgM titers. Furthermore, according to the fact that anaphylaxis still happened after the disappearance of IgM antibody, anaphylaxis appeared to be dependent on the existence of IgG rather than IgM. Figure 5 (b) clarifies the relationship between antibody titers and hypothermia at the third injection, using the data from Figure 5 (a) and some other experiments. Hypothermia did not occur when the IgG antibody titer is less than 235, but 100% of the mice went through hypothermia when the titer was more than 1450. c(RGDyK)-liposome stimulation possesses the potential to trigger allergic reaction in mice (but not 100%) when the preformed IgG antibody titer ranges from 235 to 1450. This IgG dependent phenomenon of hypothermia provided additional evidence that c(RGDyK)-liposome induced anaphylaxis was IgE-independent. In clinical settings, anticancer drugs are generally injected with one or two week intervals. Thus, the time course of antibody production and the incidence of anaphylaxis following repeated injection at one and two-week intervals were investigated. As shown in Supplementary Figure 4, prolonging the injection interval declined the incidence of anaphylaxis and the occurrence of anaphylaxis was still dependent on IgG antibody titers. For repeated stimulations at one-week intervals, due to the presence of IgG antibodies before the second injection, 66% mice experienced hypothermia post injection. Afterwards, with the decline in the IgG antibody titers, the incidence of anaphylaxis following repeated injections decreased in general. For repeated injections at two -week intervals, the incidence further declined.

Figure 5. Anti-c(RGDyK)-liposome antibody production and its influence on the onset of hypothermia. (a) Time course of anti-c(RGDyK)-liposome IgG and IgM production and the

ACCEPTED MANUSCRIPT incidence of hypothermia post the third stimulation with c(RGDyK)-liposomes at indicated days (as indicated by arrows, n=6). 36 ICR mice received an intravenous injection of c(RGDyK)-liposomes twice (first injection at day 0 and second injection at day 2) and were divided into 6 groups to receive the third injection at different time points (days 4, 7, 10, 15, 20 and 30). Injection dose: 75 μmol HSPC/kg/administration. Blood samples were collected at 1 hour before each stimulation. Body temperatures were recorded at 30 min after each stimulation. Percentage indicates the incidence of hypothermia. (b) Relationship between the onset of hypothermia and IgG and IgM productio n.

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3.5. Characterizing c(RGDyK)-liposomes as TI antigens and mechanisms of c(RGDyK)liposomes interaction with immune cells

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Most peptides, if immunogenic, are T cell dependent (TD) antigens and require T -cell help for Bcell activation and antibody production. When peptides are packed on the liposomal surface, special repetitive biochemical structures which resemble T cell independent type 2 (TI-2) antigens can be formed. Determination of antibody isotype confirmed c(RGDyK)-liposomes as a kind of TI-2 antigen. As shown in Figure 6 (a), IgG3 was the dominant subclass of c(RGDyK)-liposome specific IgG antibodies. This result plus the rapid appearance of IgG antibodies demonstrated in Figure 4 (a) suggest a T cell independent (TI) antibody production [47,48]. This explains why c(RGDyK)liposomes induced a potent IgG response in T cell defective BALB/c nude mice. However, TI antigens typically stimulate transient IgM antibody production with little or no IgG, IgA, or IgE production. Thus, for development of peptide vaccines in the form of liposomes, a second signal is needed to allow efficient antibody transformation. Usually, adjuvant, e.g. lipid A which targets TLR4 receptors on B cells, is integrated into liposomes to serve as the second signal. As has been shown here, c(RGDyK)-liposomes induced a potent IgG response in the absence of any other adjuvant, indicating that they might react with immune system through some special signaling pathways. For exploration of the underlying mechanisms, we investigated the possible role of c(RGDyK)-αv β3 integrin interaction, in the innate function of c(RGDyK). After confirming that c(RGDyK) does not interact with anti-c(RGDyK)-liposome IgG antibodies both in vitro (Figure 6, b) and in vivo (Figure 6, c, Supplementary Figure 5, a), c(RGDyK) was utilized as a αv β3 integrin inhibitor. The influence of αv β3 integrin inhibition on anti-c(RGDyK)-liposome IgG production (Figure 6, d) and the onset of hypothermia (Supplementary Figure 5, b) was investigated. Pre-dosing with c(RGDyK) had little influence on the production of IgG compared to no pre-treatment, indicating that interaction of c(RGDyK)-liposomes with the immune system may not only rely on c(RGDyK)-αv β3 integrin interaction. However, pre-dosing with blank liposomes decreased the incidence of hypothermia from 100% to 67%, implying that immune cells may recognize c(RGDyK)liposomes through at least one common pathway with liposomes.

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Figure 6. Identification of c(RGDyK)-liposomes as TI antigens and mechanisms of interaction of c(RGDyK)-liposomes with immune cells. (a) Determination of antibody isotype suggested c(RGDyK)-liposomes TI antigens (n=3). (b) In vitro and (c) in vivo test of reaction between c(RGDyK)/liposomes with anti-c(RGDyK)-liposome IgG antibody for validation of using c(RGDyK) as αvβ3 integrin inhibitor. In (b), antiserum refers to anti-c(RGDyK)-liposome serum. In (c), “saline”, “liposomes” and “c(RGDyK)”: ICR mice were injected with saline, liposomes or c(RGDyK) three times at one day intervals; c(RGDyK)-liposome immunized mice “+c(RGDyK) 3rd”, “+liposomes3rd” and “+c(RGDyK)-liposomes3rd”: ICR mice were immunized with c(RGDyK)-liposomes twice and injected with c(RGDyK), liposomes or c(RGDyK)-liposomes as the third stimulation (dose: 75 μmol HSPC/kg/administration). Blood samples were collected at 1 hour before and 1 hour after the third injection. (d) Effect of pre-treatment of c(RGDyK) and liposomes on in vivo anti-c(RGDyK)-liposome IgG production in ICR mice. “c(RGDyK) 0.5h pre-dose”, “c(RGDyK) 1h pre-dose”, “liposome 0.5h pre-dose” and “liposome 1h pre-dose” +c(RGDyK)-liposomes: ICR mice were pre-injected with c(RGDyK) or liposomes 0.5 or 1 hour before the injection of c(RGDyK)-liposomes at days 0, 2 and 4; “without pre-dose” +c(RGDyK)-liposomes: ICR mice received c(RGDyK)-liposomes three times as positive control; “saline”, “liposomes” and “c(RGDyK)”: ICR mice received saline, liposomes or c(RGDyK) three times as a negative control. For each group, n=3. NS P>0.05, *P<0.05 and *** P<0.001.

ACCEPTED MANUSCRIPT 3.6. Exploration of factors influencing the immunotoxicity of c(RGDyK)-liposomes and the structural basis that determines the immunotoxic properties of RGD peptides

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Although a severe immunotoxicity of c(RGDyK)-liposomes was shown, this result was puzzling and contradictory, as using the same targeting ligand and carrier, early experiments did not identify any safety concerns [7,8]. Accordingly, the differences between the present and previous research was investigated, and four factors were identified: c(RGDyK) modification ratio, injection dosage, mPEG-DSPE modification ratio and entrapped drug. These factors were investigated in an effort to understand the immunotoxicity of the c(RGDyK)-liposomes. According to Figure 7 (a) and supplementary Figure 6 (a), although the production of antic(RGDyK)-liposome IgG was significantly reduced by decreasing c(RGDyK) modification ratio to 1% (the ratio used in previous study [7]), the incidence of hypothermia did not change. No acute death was observed at the third injection when the ratio was as low as 0.5%, but there were delayed effects more than 12 hours following the fourth and fifth injection (data not shown) and the IgG antibody titer induced by 0.5% c(RGDyK)-liposomes was in the range of potentially causing anaphylaxis. Thus, decreasing the c(RGDyK) modification ratio could not prevent the onset of hypersensitivity or death. The dosage administered in the previous studies were about 6 times lower than the present study, and accordingly the effect of reducing the injection dose of c(RGDyK)-liposomes was investigated. IgG production appeared to be negatively correlated with both 1% and 2% c(RGDyK)-liposomes injection (Figure 7, b). This inverse dose response may be due to a high-dose induced B cell tolerance, which is a common phenomenon for TI antigens [49-51]. In addition, acute hypothermia occurred in almost all the mice receiving 1% or 2% c(RGDyK)-liposomes refer to supplementary Figure 6 (b). Thus, lowering the injection dosage has no significant positive improvement on alleviating c(RGDyK)-liposome induced immunotoxic reaction. Efforts were then made on changing the modification ratio of mPEG-DSPE. According to Supplementary Figure 7 and the particle size in Supplementary Table 1, from 3% mPEG-DSPE to 98% mPEG-DSPE, the morphology of the carriers was transformed from liposomes to micelles. During the process of transformation, attenuated risk of acute systemic anaphylaxis occurred (Supplementary Figure 6, c). No hypothermia was observed and the level of IgG production was significantly reduced (P <0.0001) when the motif ratio of mPEG-DSPE was maximized to the extent that micelles formed (Figure 7, c). This observation indicates that mPEG-DSPE modification plays a protective role in preventing the onset of lethal acute systemic anaphylaxis. But due to the previously reported immunogenicity of PEG [49,52], minimizing the severity of c(RGDyK) modification induced immunotoxicity through maximizing the ratio of mPEG-DSPE in the carrier may also increase the incidence of immunotoxicity induced by PEG. However, in the normally used range for liposomes (less than 10% PEG), acute hypothermia occurred, indicating that the mPEG-DSPE modification ratio may play a minor role in the discrepancy between past and present studies. In addition, it is important to note that, despite incapability of liposomes (5% mPEG-DSPE) to induce an anti-PEG IgM response, anti-PEG IgM antibody was indeed produced and was detectable using a literature reported ELISA plate antigen coating method (Supplementary Figure 8). Further, the influence of loaded drugs on immunotoxicity of c(RGDyK) -liposomes was compared, as both of the previous studies used doxorubicin as the model drug, whereas the present study involved a safe low-toxicity drug. Surprisingly, no hypothermia was observed when the c(RGDyK)liposomes were loaded with doxorubicin and the IgG response were completely abrogated (Figure 7, d and Supplementary Figure 6, d). According to literature reports, doxorubicin is immunosuppressive as it can affect antibody production and various aspects of immunity [18]. Thus, the absence of

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immunotoxicity of c(RGDyK)-liposomes after doxorubicin loading may be due to the high cytotoxicity of doxorubicin, as relevant immune cells would be killed by doxorubicin. Importantly, these observations may indicate that c(RGDyK)-liposomes containing strong cytotoxic agents such as doxorubicin are relatively safe for clinical use even if repeated injections are required. To evaluate whether the observed immunotoxicity of c(RGDyK) -liposomes was dependent on the peptide amino acid sequence or cyclization, liposomes containing various RGD derived peptides were prepared, including RGDC-liposomes, RGDyK-liposomes, c(RGDfK)-liposomes and c(GRDyK)-liposomes. Among which RGDC is the shortest form of RGD related peptide that can be anchored on liposomes; RGDyK is the linear form of c(RGDyK), c(GRDyK) a pseudopeptide and c(RGDfK) another commonly used cyclized RGD peptide [5,11,53-55]. All the peptides were modified on liposomes at a molar ratio of 2% (Supplementary Table 1), and administrated at the dose of 75 μmol HSPC/kg/administration to ICR mice at one day intervals. As demonstrated in Figure 7 (e), c(RGDfK)-liposomes showed the same pattern of toxicity as c(RGDyK)-liposomes, with respect to elevated antibody production and the onset of hypothermia or acute death (Supplementary Figure 6 (e), one third of the mice died post the third injection). This observation plus the evidence of no observed toxicity in the two linear RGD peptides modified liposomes (RGDC-liposomes and RGDyK-liposomes) underline the importance of cyclization in RGD peptide modified liposome induced immunotoxicity. Moreover, levels of IgG antibodies against pseudopeptide c(GRDyK) modified liposomes (c(GRDyK)-liposomes) were significantly reduced comparing that of c(RGDyK)-liposomes (P<0.05). No mouse in the c(GRDyK)-liposome treatment group experienced hypothermia. To some extent, these results indicate the requirement of RGD sequence in triggering lethal immunotoxicity by RGD peptide liposomes. Furthermore, according to the Supplementary Figure 6 (f), mice immunized with c(RGDfK)liposomes twice experienced varying degrees of hypothermia after being challenged with c(RGDyK) liposomes at the third stimulation while mice immunized with c(GRDyK)-liposomes remained normal after the same treatment. Thus, the anti-c(RGDfK) antibody also responded to c(RGDyK)-liposomes while the anti-c(GRDyK) antibody did not, suggesting a common epitope exists between c(RGDyK) and c(RGDfK), which both anti-c(RGDyK)-liposome and anti-c(RGDfK)-liposome IgG could recognize. The common epitope is apparently RGD and robust production of the anti-RGD antibody seems to be able to put mice at risk of lethal systemic anaphylaxis. This result provides additional evidence of the importance of the RGD sequence in triggering lethal immunotoxicity by RGD peptide liposomes.

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Figure 7. Investigation of impact factors on immunogenicity of c(RGDyK) -liposomes and other RGD peptide modified liposomes. Influence of: (a) liposomal c(RGDyK) modification ratio, (b) injection dosage of c(RGDyK)-liposomes, (c) mPEG-DSPE modification ratio and (d) loaded drug on anti-c(RGDyK)-liposome IgG antibody production at 1 hour before the third injection in ICR mice. (e) IgG production induced by other RGD sequences containing peptide modified liposomes. ICR mice were immunized with various formulations for six times at one day intervals starting at day 0. IgG production at 1 hour before the third injection was determined. Dose for (a, d, e): 75 μmol HSPC/kg/administration; Dose for (b): 150 μmol HSPC/kg/administration (2×1% c(RGDyK)liposomes); 75 μmol HSPC/kg/administration (liposomes, 2% and 1% c(RGDyK)-liposomes), 12.5 μmol HSPC/kg/administration (1/6 2% and 1% c(RGDyK)-liposomes); dose for (c): 12.5 μmol HSPC/kg/administration. For each group, n=3. NS P>0.05, *P<0.05, **P<0.01, *** P<0.001 and **** P<0.0001. 4. Discussion

With the increasing use of nanotechnology, safety is a most important concern. Nanoparticles have been known to be immunostimulatory [56-58], that is, the immune system efficiently recognizes them as foreign substances and mounts a multilevel immune response, including allergic reactions against them. More researchers focus on the immnotoxicity of novel polymeric nanomaterials, inorganic materials, graphene oxide, etc. Although studies have shown that PEGylated liposomes are able to induce production of anti-PEG IgM [16,59] or severe CARPA in a few patients [60,61], limited reports exist on significantly enhanced life-threating consequences by modification of the peptide ligands on them. Here it is reported that anchoring cyclized RGD peptide ligands on PEGylated liposomes can elicit severe and even lethal immune response in a T cell independent manner, manifested by the robust

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and long-lasting production of IgG antibodies, IgG-antigen immune complex formation, complement activation and cytokine release at the rechallenge. Figure 8 concludes the process and tentative mechanisms explaining c(RGDyK)-liposome induced lethal toxicity: the first few stimulations with c(RGDyK)-liposomes allows the production of sufficient IgG and IgM antibodies. When c(RGDyK)liposomes are rechallenged in the presence of antibodies, IgG-c(RGDyK)-liposome complexes are formed and deposit, leading to the instantaneous activation of the complement system, release of anaphylatoxins, cytokines and perhaps other unexplored reactions. All the reactions following the rechallenge may work together and cause systemic pathological changes, including capillary leakage, pulmonary alveolar proteinosis and inflammatory cell infiltration, nephritic and hepatic hyperaemia, and finally hypothermia or death. This toxicity can be masked by encapsulation of strong cytotoxic drugs. The fact that most reported c(RGDyK)-liposomes were utilized to encapsulate cytotoxic drugs explains why the immunotoxicity of c(RGDyK)-liposomes has not been discovered previously. According to the present study, when c(RGDyK)-liposomes serve as a delivery vehicle for low-toxicity drugs, the immunotoxicity may emerge. Some peptides were also shown to have this problem, although the severity was lower than c(RGDyK)-liposomes. This current study serves as a valuable warning for the development of peptide modified liposomes, especially when they are used for encapsulation of low-toxicity drugs. It was not previously anticipated that c(RGDyK)-liposomes would present such a strong immunotoxicity, since c(RGDyK) is a small peptide consisting of just five amino acids. Moreover, one of the cyclic RGD peptide, Cilengitide (cyclo(RGDfV)) is well tolerated in patients with recurrent glioblastoma, and no safety concerns have been reported, further denying the likelihood of its immunotoxicity [62]. However, studies have shown that unconjugated peptides themselves are weakly immunogenic, and when attached to larger scaffolds potent antibody response may be induced [47,63]. Additionally, liposomes were the first particulate drug delivery system to be shown to offer adjuvant action [64]. Consistent with these reports, the present results are reasonable. It is shown here that c(RGDyK) failed to stimulate antibody responses and c(RGDyK)-PEG3400-DSPE triggered antibody responses, although not as strong as c(RGDyK)-liposomes. Conversely, c(RGDyK)-liposome induced antibodies could be most effectively detected by ELISA with c(RGDyK)liposomes as the coating antigen, while mildly detectable using c(RGDyK)-PEG3400-DSPE and completely undetectable using c(RGDyK). This also served as the rationale for naming the antibodies, anti-c(RGDyK)-liposome rather than anti-c(RGDyK) antibody (Supplementary Figure 9). According to the present results, liposomes enable non-immunotoxic c(RGDyK) to induce a potent immune response below lipid concentrations routinely used in cancer treatment studies. This lifethreatening toxicity of c(RGDyK)-liposomes is to some extent dependent on the RGD sequence and cyclization. This requirement of liposomes and cyclization is possibly due to the altered conformation and physical characteristics that facilitate immune recognition. Previous studies indicate that integrin is widely expressed on various immune and epithelial cells, and thus plays an important role in boosting immune responses [65-67] and triggering allergic reactions [68]. Serving as an effective ligand to αv β3 integrin, c(RGDyK) possibly enables efficient immune recognition through cross-linking of c(RGDyK)-liposomes with αv β3 integrin on immune cells, and thereby, promotes IgG responses. However, inhibition of αv β3 integrin failed to prevent IgG production or onset of allergic reactions while pre-dosing with liposomes caused reduced incidence of hypothermia. Although this experiment did not clarify the crutial role of c(RGDyK)-αv β3 integrin interaction in c(RGDyK)-liposome induced IgG response, it is postulated that c(RGDyK)-liposomes, as TI-2 antigens, could efficiently cross-link with immune cells at least through one common pathway with peptide unmodified PEGylated liposomes. Moreover, due to the enhanced uptake of c(RGDyK)-

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liposomes by the spleen (a major site of immune response) at both the first and repeated injections (Supplementary Figure 1), the chance for c(RGDyK)-liposomes to interact with splenic immune cells is enhanced. In addition, according to the cytokine release (Figure 4, d), IFN-γ secretion might also assist in the antibody transformation from IgM to IgG, as IFN-γ is also an important second signal for inducing TI-2 antigen to produce IgG antibody [29,30,69-71]. While engineering of liposomes through modification of peptides promises a strategy for targeted drug delivery, it is important to ensure the safety of such products by elucidating how they interact with critical biological systems before they can be safely utilized in humans. The present study provides valuable evidence of the potential of certain peptide modified liposomes in triggering lethal allergic reactions. However, additional research is needed to identify more putative immunogenic motifs within peptides and to elucidate the underlying mechanism of how c(RGDyK)-liposomes induces IgG response in more detail. Such studies will help to provide a basis for the rational design of peptide modified liposomes that could avoid activation of the immune system.

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Figure 8. Schematic representation of the mechanisms by which c(RGDyK) -liposome induce hypothermia or death. 5. Conclusions

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The first experimental evidence regarding a reproducible life-threating immunotoxicty in mice of liposomes with cyclized RGD peptide motifs is reported. This toxicity is an IgG immune complexdriven acute systemic anaphylaxis and happens in a T cell independent manner. This research suggests that liposomes augment the immunotoxicity of certain peptide ligands and therefore immunotoxicity must be a most important consideration in the development of peptide modified liposomal drug formulations, as serious consequences may result, especially when are used to encapsulate low-toxicity drugs. Acknowledgements This work was supported by the National Natural Science Foundation of China (No.81773657, No.81690263 and No.81473149), the Shanghai Education Commission Major Project (2017-01-0700-07-E00052), the Shanghai International Science and Technology Cooperation Project (No.16430723800) and the National Basic Research Program of China (973 Program, No.2013CB932500).

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