Cytomembrane Infused Polymer Accelerating Delivery of Myelin

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Cytomembrane Infused Polymer Accelerating Delivery of Myelin Antigen Peptide to Treat Experimental Autoimmune Encephalomyelitis Jian Li, Ding Qiu, Yuqing Liu, Jian Xiong, Ying Wang, Xia Yang, Xiaolan Fu, Lixing Zheng, Gaoxing Luo, Malcolm M.Q. Xing, and Yuzhang Wu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b06575 • Publication Date (Web): 28 Sep 2018 Downloaded from http://pubs.acs.org on September 29, 2018

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Cytomembrane Infused Polymer Accelerating Delivery of Myelin Antigen Peptide to Treat Experimental Autoimmune Encephalomyelitis

Jian Li,1,# Ding Qiu,1,# Yuqing Liu,2,3,# Jian Xiong,1 Ying Wang,3 Xia Yang,1 Xiaolan Fu,1 Lixin Zheng,4 Gaoxing Luo,*,3 Malcolm Xing,*,2,3 Yuzhang Wu,*,1

1

Institute of Immunology, PLA, Army Medical University, Chongqing, China. Department of Mechanical Engineering, University of Manitoba, Winnipeg, MB, Canada. 3 Institute of Burn Research, Southwest Hospital, Army Medical University, Chongqing, China. 4 Molecular Development of the Immune System Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA. J.L., D.Q. and Y.L. contribute equally to the paper. 2

*Correspondence authors: M.X. ([email protected]), G.L. ([email protected]) and Y.W. ([email protected])

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Abstract While there has been extensive development of soluble epitope-specific peptides to induce immune tolerance for the treatment of autoimmune diseases, the clinical efficacy of soluble peptides-based immunotherapy was still uncertain. Recent strategies to develop antigen carriers coupled with peptides have shown promise results in preclinical animal models. Here we developed functional amphiphilic hyperbranched (HB) polymers with different grafting degrees of hydrophobic chains as antigen MOG peptide carriers and evaluated their ability to induce immune tolerance. We show that these polymers could efficiently deliver antigen peptide, and the uptake amount by bone marrow DCs (BMDCs) was correlated with the hydrophobicity of polymers. We observe that these polymers have higher ability to activate BMDCs and higher efficacy to induce antigen-specific T cell apoptosis than soluble peptide, irrespective of hydrophobicity. We show that intravenous injection of polymers conjugated MOG peptide, but not soluble peptide, markedly treat the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in mice. Together, these results demonstrate the potential for using amphiphilic HB polymers as antigen carriers to deliver peptides for pathogenic auto-reactive T cells deletion/tolerance strategies to treat autoimmune disorders.

Keywords: peptide delivery; autoimmune diseases; immune tolerance; re-stimulation induced cell death; experimental autoimmune encephalomyelitis

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Autoimmune diseases such as multiple sclerosis (MS) and type 1 diabetes occur when the auto-reactive cells, especially T cells are aberrantly activated and expanded, result in collateral damage of self-tissues. Current therapeutics for autoimmune diseases are mainly rely on non-specific immune suppression and general control of inflammation, with risk of compromising the host immune system.1 To solve the problem, the induction of antigen-specific immune tolerance has long been pursued as an ideal strategy for treating autoimmune disorders.2 Using soluble T cell-specific peptide/protein has been demonstrated to be a promising method for tolerance induction with consequential prevention and cure of diseases in animal models of autoimmune diseases. The underlying mechanism seems to rely on the induction of T cell anergic status or re-stimulation induced cell death (RICD).3-6 However, attempts to use T cell specific epitope-peptide for the treatment of autoimmune diseases have failed in clinical trial. A phase III clinical trial showed no beneficial effect of an MBP-derived 17 amino-acid synthetic peptide, in SPMS patients who are HLA DR2(+) or DR4(+).7 It has been observed that systematic delivery of soluble peptide toleragens might induce anaphylactic responses in mouse and primate animal models thus raise the safety concern for soluble peptides strategies.8-9 Myelin peptides coupled to autologous antigen-presenting cells (APCs) has shown beneficial therapeutic effects in MS patients in a phase I clinical trial.10 The method, however, faces challenges from isolating autologous APCs, selecting and coupling of appropriate peptides to the cells, setting limits for its clinical application. Therefore, we sought to develop an antigen carrier that can efficiently deliver auto-antigen peptides into APCs such as dendritic cells (DCs) and macrophages. These tolerogen loaded APCs interact with local infiltrated effector T cells and efficiently induce immune tolerance. Most recently, nanoparticle-based approaches have shown promising technical advances

to modulate immune

responses.11-12

Nanoparticles coupled

with

epitope-specific peptides induced immune tolerance for prevention and treatment of EAE.13-16 To date, nanoparticle-based carriers such as polymeric nanoparticles, liposomes made by non-cytotoxic polymers and others, have been tested for delivery of antigen peptides into APCs.17-18 Among different carrier platforms, polymers with

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hyperbranched (HB) or dendritic siding architectures appear to have exceptional structural features including high-content ancillary branches, irregular shapes, large numbers of chain-end functional groups, all preferable for drug delivery.19-21 Herein, we provided a facile method to prepare functional HB amphiphilic polythioethers that are capable of conjugating functional peptides and can be used to deliver antigens for immuno therapy by self-assembling the hydrophobic chain with cellular membrane (Scheme 1 and Figure 1). In order to evaluate the effects of hydrophobicity on antigen specific immune response, we developed two amphiphilic HB polymers with different grafting degrees of hydrophobic octadecyl acrylate side chains, and conjugated with encephalitogenic MOG35-55 peptides derived from myelin oligodendrocyte glycoprotein or control OVA323-339 peptides for functional assessments. We examined their ability to deliver antigen peptide into BMDCs, to activate BMDCs and to induce immune tolerance through inducing in vitro activated antigen-specific T cell death, and investigated their potential applications as an antigen delivery carrier for autoimmune disease therapy. The potential side effects of HB polymers and their immunogenicity were examined in vivo. The results demonstrate that amphiphilic HB polymers are efficient antigen/peptide carriers which show potent application to treat autoimmune diseases through inducing antigen-specific T cell tolerance.

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Scheme 1. Illustration shows the mechanisms of HB polymer-peptide-induced tolerance and the potential for autoimmune diseases treatment. First, HB polymer-peptide was taken up by APC after tail vein injection. Second, APC presents peptide to antigen-specific effector CD4+ T cells, leading to the restimulation of TCR and the induction of antigen-specific CD4+ T cell death through RICD.

Results and discussion Synthesis of amphiphilic HB polymer via RAFT polymerization Comparing to the traditional strategies for preparing HB polymers, the reversible addition-fragmentation chain transfer (RAFT) polymerization technique is much more versatile, and has the great advantage of high tolerances to various functional groups. The chain transfer functional groups could further be converted to highly reactive and biocompatible thiol moieties, indicating its huge potential for drug, peptides, and proteins delivery in biomedical applications. The synthetic route of amphiphilic hyperbranched polymers was given in Figure 1, in which acrylate containing trithiocarbonate chain transfer agent (DMATC-acrylate) monomers were used to be copolymerized with hydrophilic poly(ethylene glycol) methyl ether acrylate (PEGMEA) oligomers and hydrophobic octadecyl acrylate (ODA) monomers to achieve amphiphilic hyperbranched polymers. The hydrophobicity was tuned by adjusting the feeding ratio of DMATC-acrylate: PEGMEA: ODA as 1:4:2 or 1:4:1 for hyperbranched polymer 1 or 2. The trithiocarbonate groups on hyperbranched polymers were further cleaved to form free thiols via aminolysis of 1-butylamine, and sequentially much excessive amount of PEG-diacrylate (PEGDA) were added to obtain acrylate containing hyperbranched polythioether via thiol-ene addition reaction, as shown in Figure S1 (Supporting information). Further, functional peptides containing cysteine (free thiol moieties) on N terminus, OVA or MOG peptide sequences, were conjugated onto hyperbranched polymers through thiol-ene addition to obtain hyperbranched polymer loaded peptide sequences. Rhodamine B was further linked to free amines of lysine and arginine units in peptide sequences via EDC/NHS catalyzed amidation reactions to make fluorescent labels on the obtained HB polymerpeptides. To evaluate the hydrophobicity, water contact angles on amphiphilic HB polymer 1,

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2 and linear hydrophilic polymer PEG (Mn 5000Da, control group) coating were compared (Supporting information Figure S3). With more hydrophobic octadecyl pendants, HB polymer 1 showed with higher hydrophobicity with the water contact angle of 77.3 ± 2.6°, relative to HB polymer 2 with the contact angle of 62.8 ± 2.8°. Without octadecyl moieties, the water contact angle of linear PEG polymer coating dropped drastically to 31.1 ± 4.3°, indicating the presence of octadecyl groups improved the overall hydrophobicity of HB polymers obviously.

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Figure 1. Design of HB polymer-peptide for efficient antigen presentation and induction of immune tolerance for the treatment of autoimmune diseases. The facile synthetic approach

of

HB polymers

through

RAFT polymerization, and

post-polymerization modification of HB poly(thiolester) as functional peptide carriers for efficient antigen presentation and induction of immune tolerance for the treatment of autoimmune diseases

Uptake of HB polymer conjugated MOG peptides by BMDCs We evaluated the influence of HB polymer with different hydrophobicity on the uptake efficiency of MOG35-55 peptide, a dominant encephalitogenic peptide derived from myelin antigen oligodendrocyte glycoprotein (MOG). MOG35-55

can be used

as a model peptide for functional analyses of antigen presenting cells (APCs) such as DCs. Murine bone marrow derived dendritic cells (BMDCs) were prepared and incubated with rhodamine B (RDB) labeled HB-MOG of different hydrophobicity at 37 °C for 3 hours. The uptake amount was quantified by measuring the median fluorescence intensity (MFI) using flow cytometry analysis (Figure 2a). The fluorescence intensity of HB polymer-1 (HB-MOG-1) with higher hydrophobicity was two times higher than that of the HB polymer-2 （HB-MOG-2）with less hydrophobicity, which indicates more efficient uptake of peptide with HB polymer-1 (Figure 2b). Previous studies have shown that the hydrophobicity of polymer adjuvants were key factors affecting the uptake and subsequent immune responses of conjugated antigen.22-23 In this study, the results suggest that the loading intensities of HB polymers in target cells can be regulated by altering the hydrophobicity of the HB chain. To monitor the intracellular distribution of HB-MOG, BMDCs cells were stimulated by the rhodamine B labeled polymer at 37 °C for 3 hours, followed with observation under a confocal scanning microscope (Figure 2c). Cells treated with HB-MOG-1 that has higher hydrophobicity showed localization of red fluorescence of rhodamine B labeled polymer in the endosome as well as cytosol. When cells were

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treated with HB-MOG-2 that has less hydrophobicity, dotted fluorescence were observed, with less diffused fluorescence, in the cytosol as compare to the HB-MOG-1 treated cells. Thus, HB polymer with higher hydrophobicity chain is preferable for fusing with endosome and liposome membrane, resulting in the destabilization of the membrane and efficient release of MOG in to the cytosol.24 This also suggests that HB polymer-1 conjugated peptides might be taken up by the cells through endocytosis, a classical format for antigen peptide presentation by MHC class II molecules, meanwhile some of the peptides released into the cytosol might be cross-presented through MHC class I molecules. HB polymer-2 with less hydrophobicity might be taken up by the cells and presented more efficiently through MHC class II molecules because most of the peptides retained in the endosomes. Based on this finding, we speculate that HB polymer-1 having higher hydrophobicity favors antigen peptide presentation through MHC class I.

Figure 2. Uptake efficacy of HB polymer conjugated MOG peptide. The cells were stimulated by free MOG peptide (20ug/ml), rhodamine B labeled HB-MOG-1 (20ug/ml MOG, 1mg/ml RDB-HB-1), and HB-MOG-2 (20ug/ml MOG, 0.8mg/ml RDB-HB-2) for 3 hours. The uptake efficacy was evaluated by (a) histograms and (b)

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mean fluorescence intensity (MFI) of RDB through flow cytometry. Data are representative of 3 independent experiments. * p