Cytomembrane Infused Polymer Accelerating Delivery of Myelin

Sep 28, 2018 - Institute of Immunology, PLA, Third Military Medical University (Army Medical University), Chongqing 400038 , China. ‡ Institute of B...
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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,† Lixin Zheng,∥ Gaoxing Luo,*,§ Malcolm Xing,*,‡,§ and Yuzhang Wu*,† ACS Nano 2018.12:11579-11590. Downloaded from pubs.acs.org by UNIV OF NEW ENGLAND on 01/14/19. For personal use only.

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Institute of Immunology, PLA, Third Military Medical University (Army Medical University), Chongqing 400038, China Institute of Burn Research, State Key Laboratory of Trauma, Burn and Combined Injury, Key Laboratory of Disease Proteomics of Chongqing, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, China § Department of Mechanical Engineering, University of Manitoba, Winnipeg, MB R3T 2N2, Canada ∥ Molecular Development of the Immune System Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States ‡

S Supporting Information *

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 promising results in preclinical animal models. Here we developed functional amphiphilic hyperbranched (HB) polymers with different grafting degrees of hydrophobic chains as antigen myelin antigen oligodendrocyte glycoprotein (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 dendritic cells (BMDCs) was correlated with the hydrophobicity of polymers. We observe that these polymers have a higher ability to activate BMDCs and a higher efficacy to induce antigen-specific T cell apoptosis than soluble peptides, irrespective of hydrophobicity. We show that intravenous injection of polymer-conjugated MOG peptide, but not soluble peptide, markedly treats the clinical symptoms of experimental autoimmune encephalomyelitis in mice. Together, these results demonstrate the potential for using amphiphilic HB polymers as antigen carriers to deliver peptides for pathogenic autoreactive T cell deletion/tolerance strategies to treat autoimmune disorders. KEYWORDS: peptide delivery, autoimmune diseases, immune tolerance, restimulation-induced cell death, experimental autoimmune encephalomyelitis

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utoimmune diseases such as multiple sclerosis (MS) and type 1 diabetes occur when the autoreactive cells, especially T cells, are aberrantly activated and expanded, resulting in collateral damage of self-tissues. Current therapeutics for autoimmune diseases mainly rely on nonspecific immune suppression and general control of inflammation, with risk of compromising the host immune system.1 To solve this 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 restimulation-induced cell death (RICD).3−6 © 2018 American Chemical Society

However, attempts to use a 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 a myelin basic protein (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 and thus raise a safety concern for soluble peptide strategies.8,9 Myelin peptides coupled to autologous antigen-presenting cells (APCs) have shown beneficial Received: September 5, 2018 Accepted: September 28, 2018 Published: September 28, 2018 11579

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Scheme 1. Illustration showing the mechanisms of HB polymer−peptide-induced tolerance and the potential for autoimmune disease 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.

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 autoantigen 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 experimental autoimmune encephalomyelitis (EAE).13−16 To date, nanoparticle-based carriers such as polymeric nanoparticles, liposomes made by noncytotoxic polymers, and others have been tested for delivery of antigen peptides into APCs.17,18 Among different carrier platforms, polymers with hyperbranched (HB) or dendritic side architectures appear to have exceptional structural features including high-content ancillary branches, irregular shapes, and 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 immunotherapy by self-assembling the hydrophobic chain with a 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 myelin antigen oligodendrocyte glycoprotein (MOG)35−55 peptides derived from myelin oligodendrocyte glycoprotein or control OVA323−339 peptides for functional assessments. We examined their ability to deliver antigen peptide into bone marrow derived dendritic cells (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 that show potent application to treat autoimmune diseases through inducing antigen-specific T cell tolerance.

RESULTS AND DISCUSSION Synthesis of Amphiphilic HB Polymer via RAFT Polymerization. Compared 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, peptide, and protein delivery in biomedical applications. The synthetic route of amphiphilic hyperbranched polymers is given in Figure 1, in which acrylate-containing trithiocarbonate chain transfer agent (DMATC-acrylate) monomers were used to copolymerize 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 an excessive amount of PEG-diacrylate (PEGDA) was added to obtain acrylate-containing hyperbranched polythioether via a thiol−ene addition reaction, as shown in Figure S1 (Supporting Information). Further, functional peptides containing cysteine (free thiol moieties) on the 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 polymer-peptides. To evaluate the hydrophobicity, water contact angles on amphiphilic HB polymers 1 and 2 and linear hydrophilic 11580

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

flow cytometry analysis (Figure 2a). The fluorescence intensity of HB polymer-1 (HB-MOG-1) with higher hydrophobicity was 2 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 was a key factor 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, BMDC cells were stimulated by the rhodamine B-labeled polymer at 37 °C for 3 h, followed by observation under a confocal scanning microscope (Figure 2c). Cells treated with HB-MOG-1, which has a higher hydrophobicity, showed localization of red fluorescence of rhodamine B-labeled polymer in the endosome as well as cytosol. When cells were treated with HB-MOG-2, which has less hydrophobicity, dotted fluorescence was observed, with less diffused fluorescence, in the cytosol as compared to the

polymer PEG (Mn 5000 Da, control group) coating were compared (Supporting Information Figure S3). With more hydrophobic octadecyl pendants, HB polymer 1 showed a higher hydrophobicity, with a water contact angle of 77.3 ± 2.6°, relative to HB polymer 2, with a contact angle of 62.8 ± 2.8°. Without octadecyl moieties, the water contact angle of the linear PEG polymer coating dropped drastically to 31.1 ± 4.3°, indicating that the presence of octadecyl groups clearly improved the overall hydrophobicity of HB polymers. 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. MOG35−55 can be used as a model peptide for functional analyses of antigen-presenting cells such as DCs. Murine BMDCs were prepared and incubated with rhodamine B (RDB)-labeled HB-MOG of different hydrophobicity at 37 °C for 3 h. The uptake amount was quantified by measuring the mean fluorescence intensity (MFI) using 11581

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Figure 2. Uptake efficacy of HB polymer conjugated MOG peptide. The cells were stimulated by free MOG peptide (20 μg/mL), rhodamine B-labeled HB-MOG-1 (20 μg/mL MOG, 1 mg/mL RDB-HB-1), and HB-MOG-2 (20 μg/mL MOG, 0.8 mg/mL RDB-HB-2) for 3 h. The uptake efficacy was evaluated by (a) histograms and (b) mean fluorescence intensity (MFI) of RDB through flow cytometry. Data are representative of three independent experiments. *p < 0.05, **p < 0.01 (by Student’s t test). (c) Confocal laser scanning microscopy of BMDCs treated with rhodamine B-labeled HB-MOG of different hydrophobicity. BMDCs were incubated with rhodamine B-labeled HB-MOG-1 (20 μg/mL MOG, 1 mg/mL RDB-HB-1) and HB-MOG-2 (20 μg/mL MOG, 0.8 mg/mL RDB-HB-2) for 3 h at 37 °C. Images were captured with a Leica scanning microscope (Leica TCS-SP5) with a 40× oil objective. RB-HB panel, rhodamine B-labeled cells’ fluorescent images; DAPI panel, BMDCs nuclear stained with DAPI; merged panel, merged images; right panel, magnification of merged images. Data are representative of three independent experiments. *p < 0.05, **p < 0.01 (by Student’s t test).

(Figure 3). We obtained similar results for HB-OVAp-induced activation of bone marrow derived macrophages. As shown in Figure S4 (Supporting Information), we observed a higher frequency of MHC class II and CD86 coexpression from either HB-OVA-1- or HB-OVA-2-treated cells as compared to the free OVA peptide treatment. Together, these data suggest that HB-polymer-modified peptides can enhance APC activation for antigen presentation, whereas the underlying mechanism appears to be irrespective of the polymer’s hydrophobicity. ̈ HB Polymer Enhanced the Activation of Naive + DO11.10 OVA-Specific CD4 T cells through Effective OVA Peptide Presentation. To investigate the effect of ̈ T cell activation, HB-polymer-peptide on antigen-specific naive we analyzed T cell activation markers CD44 and CD69 after stimulation of splenocytes from DO11.10 mice (Tg (DO11.10) TCR/OVA p323−339 specific) in the presence of HB-OVA-1, HB-OVA-2, or phosphate-buffered saline (PBS) vehicle control for 3 days at 37 °C. The results show that both HB-OVA-1 and HB-OVA-2 cultured with DO11.10 splenocytes can enhance the expression of CD44 and CD69 in activated T cells compared with the vehicle treatment group (Figure 4), indicating these two HB-polymer-conjugated OVA can be efficiently ̈ DO11.10 presented by the splenic APCs for activating naive + CD4 T cells. The frequency of activated DO11.10 CD4+ T cells was slightly higher when stimulated with HB-OVA-1 than with HB-OVA-2, of less hydrophobicity, which indicates more efficient T cell activation ability of peptide with HB polymer-1. Nevertheless, these results suggest that OVA peptide conjugation with HB polymers strongly enhances antigen presentation

HB-MOG-1-treated cells. Thus, HB polymer with a higher hydrophobicity chain is preferable for fusing with the endosome and liposome membrane, resulting in the destabilization of the membrane and efficient release of MOG into 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 were retained in the endosomes. On the basis of this finding, we speculate that HB polymer-1, having higher hydrophobicity, favors antigen peptide presentation through MHC class I. Activation of BMDCs by HB Polymer-MOG. As described above, APCs can efficiently capture the HB polymer complex. This sets a platform for further investigations of the antigen complex on essential APC functions, including upregulation of MHC class II and costimulatory molecules such as CD86 on APCs during the immune response. BMDCs were prepared and stimulated with free MOGp or HB-MOG that have different hydrophobicity at 37 °C overnight. Using HB-polymermodified MOGp induced 65−70% of MHC class II and CD86 coexpression in loaded BMDCs, though irrespective of the differences in hydrophobicity for the polymers examined, which indicates a significant increase in APC activation as compared to the free MOGp or vehicle alone treated BMDCs, which gave only 40% coexpression of the two activation markers 11582

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Figure 3. Activation of BMDCs by HB-MOG-1 and HB-MOG-2 or MOG peptide at 20 μg/mL peptide concentration for overnight stimulation. Cells were stained with CD86 and MHCII antibody and measured by flow cytometry. (a) Values in the upper right corner represent the percentage of CD86 and MHCII double positive cells. (b) Percentage of cells expressing the indicated markers.

Figure 4. Activation of DO11.10 T cells by polymer-conjugated OVA peptide. Splenocytes from DO11.10 mice were in vitro stimulated with 15 μg/mL HB-OVA-1, HB-OVA-2 (normalized to peptide mass), or PBS vehicle control for 3 days at 37 °C. (a, c) T cells were then analyzed by flow cytometry for T cell activation markers CD44 and CD69. (b, d) Percentage of CD4+ T cells with indicated markers. *p < 0.05, **p < 0.01; ***p < 0.001 (by Student’s t test).

finding of antigen-specific depletion of effector T cells through restimulation-induced cell death holds great promise for the treatment of T-cell-mediated autoimmune diseases such as multiple sclerosis. Studies have shown that activated antigenspecific T cells are extremely sensitive to RICD.26 To understand the potent therapeutic effect of HB polymer conjugated MOG peptide, we investigated its ability to induce antigenspecific 2D2 T cell apoptosis by RICD. In vitro activated CD4+ T cells were prepared from MOG35−55-specific TCR-transgenic 2D2 mice and incubated with peritoneal macrophages in the presence of HB-MOG-1, HB-MOG-2, or PBS. We used a flowcytometry-based annexin V/propidium iodide (PI) staining method to determine apoptotic T cells. We observed that

from APCs, resulting in efficient activation of specific CD4+ T cells, as evidenced from the observed increase in the level of T cell activation marker CD44 and CD69 expressions. HB Polymer-MOGp Induces RICD of Activated Specific T Cells. Multiple sclerosis, a common autoimmune disorder in the northern hemisphere, is believed to be caused by T-cellmediated inflammatory damage of the CNS (central nervous system) tissues. Approaches to induce antigen-specific tolerance could ameliorate the pathogenic autoimmune response and prevent the disease (Jian et al., unpublished data). It is well accepted that T cells in MS patients recognize myelin protein antigens including myelin basic protein, proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein.25 The 11583

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Figure 5. Apoptosis of in vitro activated antigen-specific CD4+ T cells as shown by annexin V and PI staining. MOG-specifc 2D2 T cells were in vitro activated by 15 μg/mL MOG peptide and then with IL-2 (200 U/mL) for 2 days. Cells were then cocultured with mouse peritoneal macophages in the presence of MOG peptide or hyperbranched conjugated MOG overnight. T cells were then analyzed by flow cytometry for annexin V and PI staining. *p < 0.05 (by Student’s t test).

Figure 6. In vivo assessment of CNS tissue penetrating ability of HB-MOG in EAE mice. (a) Ex vivo images of normal organs (heart, liver, spleen, lung, and kidney) and CNS tissues (meninges, brain, and spinal cord) removed from EAE mice with a clinical scoring of 3 that were sacrificed 3 h after i.v. injection. Bright-field (left panel), fluorescent images (middle panel), and merged images (right panel). (b) Quantified fluorescence intensity for tissues in (a) (n = 3). *p < 0.05 (by Student’s t test).

system. At 3 h postinjection, as shown in Figure 6a, fluorescence signals accumulated in the CNS tissues including the meninges, brain, and spinal cord and also in the normal organs except the spleen (Figure 6b), with the signal intensity presentation irrespective of the polymer’s hydrophobicity. We then prepared spinal cord tissue slides from EAE mice 3 h after intravenous injection of rhodamine B-labeled HB-MOG-1 or HB-MOG-2. We imaged blood vessels through staining CD31 expressed on blood vessel endothelial cells. As shown in Figure S5 (Supporting Information), rhodamine B-labeled HB-MOG-1 or HB-MOG-2 distribute around blood vessels. We further examined the blood circulation of HB polymers in normal mice. Rhodamine B-labeled HB-MOG-1 or HB-MOG-2 were intravenously injected, and sera were collected at different time points to measure the fluorescence intensity of rhodamine B. As shown in Figure 7, no significant difference was found between rhodamine B-labeled HB-MOG-1 and HB-MOG-2: both exhibit prolonged half-lives (being 1.598 and 1.983 h, respectively), in comparison to several minutes reported for dye-labeled peptides.27,28 The results indicate that HB polymer conjugated MOG have prolonged blood circulation time and can efficiently pass across blood brain barrier and get into the CNS tissue, rendering the CNS local restimulation and depletion of effector T cells plausible. HB Polymer Has Few Side Effects Both in Vitro and in Vivo. In vitro cytotoxicity of HB-1 and HB-2 was evaluated

CD4+ T cells stimulated with HB-MOG-1- and HB-MOG-2loaded macrophages showed a substantially higher frequency of annexin V/PI staining (38.2% and 29.9%, respectively) compared to vehicle controls (12.5%) (Figure 5). By contrast, free MOG peptide stimulation caused little apoptosis of T cells (18.4%). The results indicate that HB polymer conjugated peptides have a higher efficacy to induce apoptosis of antigenspecific effector T cells, indicating that HB polymers are potent carriers for high-efficient mounting of tolerogenic peptides on APCs, and thus to induce immune tolerance in vivo for treating T-cell-mediated autoimmune diseases. In Vivo Assessment of the CNS-Penetrating Capability of HB-MOG of Different Hydrophobicity. To investigate whether HB-MOG can penetrate the blood brain barrier, enter the CNS, and induce local restimulation of infiltrated effector T cells, we chose the most widely used EAE mouse model. We initiated EAE by subcutaneously injecting MOG p35−55 emulsified in complete Freund’s adjuvant, to induce immunemediated demyelinating disease in C57BL/6 mice. The mice showed an onset of disease signs at day 12, with the peak of observed motor deficits at day 15. A 100 μg amount of rhodamine B-labeled HB-MOG-1 or HB-MOG-2 was injected intravenously to EAE mice on day 15 after immunization. Normal organs and CNS tissues were harvested 3 h after the injection, and the subsequent fluorescence distribution was examined using a custom-made whole-body optical imaging 11584

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settings. Thus, HB-1 and HB-2 appear to be safe for use both in vitro and in vivo. Therapeutic Effects. Encouraged by the results showing that HB-MOG could induce RICD of MOG-specific T cells in vitro (Figure 5), we next investigated the therapeutic effects of HB-MOG in treating MOG-induced EAE, speculating that the polymer-conjugated MOG peptide would induce antigenspecific T cell depletion in the affected CNS. We induced the EAE model using MOG35−55 peptides as aforementioned, with the disease characterized by CNS inflammation and hind limb paralysis. The immunization gave an onset of clinical disease signs at day 12 with the peak of clinical scores 3−4 at day 15. To model the clinical situation for treating MS patients with acute disease attack, EAE mice on day 15 of EAE induction with clinical scores of 3−3.5 were chosen for the treatment groups. On days 15, 17, and 19 (Figure 12a), these mice received a single dose of 40 μg (normalized to peptide mass) of HB-MOG-1 (40 μg of MOG and 2 mg of HB polymer-1), HB-MOG-2 (40 μg of MOG and 1.6 mg of HB polymer-2), MOG peptide (40 μg), or PBS, by tail vein intravenous (i.v.) injection. PBS injection has no impact on the disease, uniformly maintaining an average clinical score of 3.5−4 during days 15 to 23 (Figure 12a). By contrast, both the HB-MOG-1and HB-MOG-2-treated mice showed a rapid recovery of motor function and progressive resolution of the disease, which appears to be superior to the limited therapeutic effect obtained from i.v. injection of the nonconjugated MOG peptide in EAE mice. Histopathological analysis showed that HB-MOG-1 and HB-MOG-2 treatment significantly reduced infiltrating inflammatory cells in the spinal cord (SC) as compared to the MOG peptide group at day 25 post EAE induction, indicating that the disease amelioration correlates with a decrease in accumulation of immune cells in the spinal cord (Figure 12b). Thus, HB-MOG-1 and HB-MOG-2 achieved therapeutic effects on the disease. We then investigated HB-MOG’s treatment effects in a relapsing−remitting model of EAE, which recapitulates the most common clinical disease pattern of relapsing−remitting (RR) MS. A mixture of MOGp35−55 and PLPp139−151 in CFA was used to induce RR-EAE in (B6xSJL) F1 mice. F1 mice developed the first wave of the disease from day 9, with the peak clinical score usually on day 13. In our experiment, most mice recovered from the first wave after 4−5 days of disease development. Then a severe relapse stage followed in most of the mice. We tested the ability of HB-MOG-1 and HB-MOG-2 to prevent relapse by treating during the first wave of the disease (therapeutic treatment: on days 13, 15, and 17). We observed that both HB-MOG-1- and HB-MOG-2-treated mice showed reduced clinical scores from day 13 followed by earlier recovery from the first wave of the disease as compared to the empty polymer HB-1- or HB-2-treated mice. HB-MOGtreated mice also developed a milder relapse stage compared to the vehicle, empty HB-1- or HB-2-treated mice (Supporting Information Figure S6). To further investigate the treatment mechanisms in vivo, we analyzed the T cell kinetics after HB-MOG-1 or HB-MOG-2 injection in the MOG-induced EAE model. After one dose of HB-MOG-1 or HB-MOG-2 on day 15 of disease-initiating immunization, both the frequency and amount of spinal cord infiltrating CD3+ T cells decreased (Figure 12c−e). We further evaluated whether these spinal cord T cells were undergoing apoptosis induced by polymer-conjugated peptide administration. We observed that CD3+CD45.2+ T cells from EAE

Figure 7. Blood clearance curves of rhodamine B-labeled HB-MOG-1 and HB-MOG-2 in mice.

using the MTT assay. Cell proliferation assays showed that the proliferation rate of RAW 264.7 cells was not affected when treated with HB-1 or HB-2 at a low concentration of 5 μg/mL to a high concentration of 100 μg/mL (Figure 8). There is no

Figure 8. In vitro cytotoxicity evaluation of HB-1 and HB-2. Effect of HB-1 and HB-2 on cell viability of RAW 264.7 cells using the MTT assay.

significant difference between HB-1 and HB-2 in terms of cytotoxicity to RAW 264.7 cells, indicating that both HB-1 and HB-2 are not cytotoxic to cultured RAW 264.7 cells in vitro. To evaluate potential side effects of HB-1 and HB-2 in vivo, blood samples were collected from the mice or rats 48 h after tail vein injection of HB-1 or HB-2 for hemanalysis and biochemical analyses (serum collected from SD rats). As shown in Figure 9, there were no significant differences in the blood hemanalysis parameters (white blood cells, red blood cells, hemoglobin, hemotocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and blood platelets) among HB-1-, HB-2-, and vehicle-treated mice. Hepatic and renal function parameters (urea nitrogen, albumin, aspartate aminotransferase, alanine aminotransferase, creatinine, uric acid, and total bilirubin) were not affected after HB-1 and HB-2 administration (Figure 10). The results suggested that in vivo administration of HB-1 or HB-2 has few side effects. To detect the immunogenicity of HB polymer, 200 μg HB-1 or HB-2 was intraperitoneally (i.p.) injected into normal mice. OVA protein injection was used as the control. Sera were harvested 15 days after injection, and ELISA was used to detect levels of anti-HB antibodies. The results show that the anti-HB-1 or anti-HB-2 antibody levels were barely detectable when compared to the OVA-injected group correlating to serum dilutions (Figure 11). The results indicate that HB elicits no detectable immune response under our experimental 11585

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Figure 9. Evaluation of the side effects of HB-MOG-1 and HB-MOG-2 in vivo: blood hemanalysis analysis of white blood cells, red blood cells, hemoglobin, hemotocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and blood platelets. n = 5.

Figure 10. Evaluation of the side effects of HB-MOG-1 and HB-MOG-2 on hepatic and renal function in vivo. Biochemical analyses of hepatic and renal function parameters (including urea nitrogen, albumin, aspartate aminotransferase, alanine aminotransferase, creatinine, uric acid, and total bilirubin). n = 4 for each group. SD rats were used for biochemical analyses. Blood serum samples were collected from SD rats 48 h after tail vein injection of 2 mg/mL HB-MOG-1 or HB-MOG-2.

mice spinal cord treated with one dose of HB-MOG-1 or HBMOG-2 showed substantially greater annexin V/PI double staining compared to those from vehicle-treated mice, indicating induction of apoptosis (Figure 12c,f). These data show that HB-MOG-1 and HB-MOG-2 induce apoptosis of the spinal cord infiltrating T cells in association with disease reduction; this coincides with the in vitro RICD results in Figure 5. In summary, we show that HB-MOG potently induces the

death of encephalitogenic T cells through improved antigen presentation in the diseased CNS, thereby reversing experimental allergic encephalomyelitis and ameliorating the disease in more complicated RR EAE in mice.

CONCLUSION This study demonstrates the ability of amphiphilic HB polymers as an antigen delivery system that has several key features. 11586

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attributed to the adjustable hydrophobic moieties within the polymer’s stem structure. Another feature is its effect on activation of antigen-loaded APCs, capable of greatly enhancing the potency of antigen presentation by APCs, which appears to be essential for inducing RICD of pathogenic effector T cells. These HB polymers are superior to free antigenic peptides for delivery and activation efficiency into BMDCs. BMDCs pulsed with polymer-conjugated peptides induced a higher frequency of apoptosis in activated antigen-specific CD4+ T cells than free peptides. Finally, results from in vivo experiments show that administration of HB polymer-MOGp to the established EAE mice results in significant therapeutic effects on the diseases. Further treatment mechanism analysis indicates that HB polymer-MOGp induces apoptosis of the spinal cord infiltrating T cells and reduces CNS inflammatory infiltration. Together, our results demonstrate that amphiphilic HB polymers are efficient antigen carriers for immune tolerance induction, especially promising for targeting and deleting defined pathogenic T cells for treating T-cell-mediated autoimmune disorders.

Figure 11. Immunogenicity of HB-polymer in C57BL/6 mice. Mice were injected i.p. with HB-1 (200 μg), HB-2 (200 μg), or OVA protein (200 μg). The serum anti-HB-1, anti-HB-2, and antiOVA antibody levels were assessed on day 15 after injection. Serum was diluted as 1:500, 1:1000, and 1:2000. OVA protein was used as a positive control. n = 3 for each group.

The polymer has a high efficiency for antigen peptide uptake by macrophages, dendritic cells, and likely other APCs,

Figure 12. HB-MOG treatment reduces the severity of MOG-induced EAE. (a) Therapy of EAE on days 15, 17, and 19 with i.v. injection of 40 μg of MOG (normalizing to peptide mass) conjugated with HB polymer-1, HB polymer-2, or free peptide. PBS was used as vehicle control. One representative of three independent experiments is shown; n = 3 for each group. Solid graphs show means and SEM of clinical disease scores of EAE mice from day 11 post the EAE immunization. (b) Histopathological analyses of HE-stained spinal cord tissues from different groups of treated mice in (a). Spinal cords were harvested 25 days after induction. The images are a representative of three mice. (c) Decrease and apoptosis of infiltrated T cells in the spinal cord of EAE mice after one dose of HB-MOG-1 or HB-MOG-2 treatment on day 15 post MOG immunization. Spinal cord infiltrating cells were isolated 1 day after treatment. (d, e) Frequency and cell counts of total spinal cord infiltrating CD3+CD45+ T cells of EAE mice treated with PBS, HB-MOG-1, and HB-MOG-2 as measured by FACS. (f) Apoptosis frequency of CD3+ T cells in the spinal cord as shown by annexin V and PI double staining after one dose of treatment. SC is the abbreviation for spinal cord. *p < 0.05, **p < 0.01 (by Student’s t test). 11587

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transferred into the flask through a cannula to precipitate the product out of solution. After 15 min, the colorless/light yellow product was sticky on the bottom, and most of the precipitant solvent was removed out of the reactor through a cannula under a nitrogen flow. Sequentially, 2 mL of degassed DCM was injected into the reactor to redissolve the obtained polythiols, followed by injections of 0.57 mL of PEGDA (0.63 g, 258 Da) and 0.01 mL of 1-butylamine. After 4 h, the reaction was stopped and the product was precipitated out of hexane, and the acrylate-containing HB polythioethers were recovered after drying under vacuum. Conjugation of HB Polythioether Acrylate with Polypeptides. Both MOG and OVA peptide sequences used for conjugation contain a cysteine unit on one side of the molecular chain. An 80 mg amount of HB polymer, 2 mg of peptide (OVA), and 0.2 mg of 1-butylamine were mixed in 5 mL of water and stirred overnight, and then the solution was dialyzed against doubly distilled (DD) water in a dialysis tube (MWCO: 3500 Da) for 2 days. With the presence of 0.5 mg of 1-butylamine, the conjugation protocol of 5 mg of peptide (MOG) with 200 mg of HB polymer was similar in 12.5 mL of water. Finally, the purified product was recovered by lyophilization. Rhodamine B Labeling. Fluorescent agent rhodamine B was labeled onto HB polymer-MOG via amidation with free amines on lysine or arginine sites. In summary, 100 mg of HB polymer-MOG, 2 mg of rhodamine B, 0.8 mg of NHS, and 0.48 mg of EDC were shielded from light and stirred in 10 mL of water for 24 h at room temperature. After reaction, the solution was dialyzed against DD water in a dialysis tube (MWCO: 3500 Da) and shielded from light for 2 days. The product was recovered by freeze-drying. NMR Characterization. All 1H NMR spectra were performed on a Bruker Advance 300 MHz spectrometer with CDCl3 as the solvent, at the concentration of 10 mg/mL. Water Contact Angle Analysis. Generally, 50 mg of test sample was dissolved in 0.5 mL of chloroform completely and then coated onto a glass slide by solution casting. For water contact angle measurement, water droplets of 5 μL were dropped onto the test coating layers, and photos were captured after 2 s. Flow Cytometry. All flow cytometry antibodies were purchased from eBioscience (San Diego, CA, USA) or Biolegend. Single-cell suspensions from in vitro expanded T cells, bone marrow-derived DCs, or peritoneal macrophages were washed with PBS and incubated with surface-staining antibodies (usually 1:200 dilution) at 4 °C for 30 min in the dark. The cells were then washed with PBS once and then analyzed using a FACsAria cytometer (BD, Franklin Lakes, NJ, USA). Generation of Murine Bone Marrow-Derived DCs. Bone marrow cells were prepared from 6-week-old C57BL/6 or BALB/C mice and in RPMI 1640 containing 5% FBS, 10 ng/mL recombinant murine granulocyte-macrophage colony-stimulating factor, and 100 U/mL of penicillin and streptomycin. On day 3 and day 5, cells were washed and the medium was replenished. Nonadherent cells were harvested on day 6 as immature BMDCs. In Vitro Uptake of HB Polymer Conjugated Peptides by BMDCs. Immature BMDCs were incubated with 20 μg/mL (normalizing to peptide mass) rhodamine B labeled HB-MOG-1, HB-MOG-2, or free MOG peptide for 3 h at 37 °C. The cells were then washed with PBS, and the fluorescence uptake was acquired on a BD FACsAria cytometer. To assess the uptake of rhodamine B-labeled HB-MOG-1 and HB-MOG-2 using confocal microscopy, BMDCs cells were seeded into 35 mm glass-bottom cell culture dishes at a concentration of 1 × 105/mL. HB-MOG-1 and HB-MOG2 at a concentration of 20 μg/mL (normalizing to peptide mass) were added to the wells in triplicate and incubated for 3 h. Uptake of RDB in cells was visualized by a Leica scanning microscope (Leica TCS-SP5) with a 40× oil objective. The images were analyzed using Leica LAS AF Lite 2.6 software. Stimulation of BMDCs by HB Polymer Conjugated Peptides. To assess the activation of BMDCs after incubation with HB polymer conjugated peptides, the DCs were pulsed with HB-MOG-1, HB-MOG-2, or free MOG peptide at 20 μg/mL peptide concentration for 3 h, washed, and incubated further for 24 h at 37 °C. After 24 h,

MATERIALS AND METHODS Materials. 2-Hydroxylethyl acrylate (97%, Alfa Aesar), poly(ethylene glycol) methyl ether acrylate (480 Da, Aldrich), poly(ethylene glycol) diacrylate (258 Da, Aldrich), octadecyl acrylate (97%, Aldrich), and dichloromethane (DCM) were predried by activated 3 Å molecular sieves for 72 h. Azobisisobutyronitrile (AIBN) was purified by recrystallization in methanol. The MOG and OVA peptides were synthesized by Chinese Peptide Company (Hangzhou). All other chemicals were ordered from Sigma-Aldrich and used as received. Mice and EAE Induction. 2D2 TCR (MOG35−55 specific TCR) transgenic mice, DO11.10 TCR transgenic (OVA-peptide-specific TCR, H-2d) mice, and C57/BL6 and BALB/c mice were from Jackson Laboratories. Mice were maintained in a specific pathogenfree facility and used under protocols approved by the Army Medical University Animal Care and Use Committee. EAE was induced by immunization of female C57BL/6 mice (10−12 weeks old) with an emulsion containing 100 μg of MOG p35−55 peptide and 400 μg of M. tuberculosis extract H37 Ra (Difco) in CFA. Mice then received 200 ng of pertussis toxin (List Biological Laboratories) i.p. at 4 and 24 h after immunization. To induce the RR-EAE model in (B6xSJL) F1 mice, we immunized F1 mice with 200 μL of an emulsion containing 200 μg of MOG35−55 peptide, 40 μg of PLP139−151 peptide, and 400 μg of M. tuberculosis extract H37 Ra (Difco) in CFA. At 4 and 24 h after immunization, mice received 75 ng of pertussis toxin i.p. EAE was scored by the methods described previously.29 Synthesis of S-Dodecyl-S′-(α,α′-dimethyl-α″-acetic acid)trithiocarbonate. Synthesis of DMATC chain transfer agent was performed according to a protocol published previously.30 Synthesis of DMATC-Acrylate. The DMATC chain transfer agent was conjugated with acrylate via esterification of DMATC and hydroxyethyl acrylate with DMAP/EDC catalysis. To a 25 mL roundbottom flask were added 1.46 g of DMATC, 1.15 g of EDC hydrochloride salt, and 0.073 g of DMAP, and the flask was predried under vacuum at room temperature overnight. Then 1.4 g of hydroxyethyl acrylate was added, and the flask was degassed with nitrogen for 20 min. Then 15 mL of anhydrous DCM was transferred into the vessel with a syringe under nitrogen protection. The solution was stirred at 40 °C for 48 h. The DCM solution was washed with brine in a separation funnel six times, and the organic layer was collected and dried with anhydrous sodium sulfate. The DCM was removed by rotational evaporation, and the final product was dried under vacuum and recovered as a yellow oil. Synthesis of HB PEG-r-PODA-r-PTTC Polytrithiocarbonates. The hydrophilicity and hydrophobicity of HB polymer were tailored by adjusting the feeding ratio of the hydrophilic monomer PEGMEA and ODA. The molar ratio of AIBN:DMATC-acrylate:PEGMEA: ODA is 1:10:40:20 or 1:10:40:10 for HB random polymer-1 and -2, respectively. Briefly, 0.011 g of AIBN, 0.3 g of DMATC-acrylate, 1.25 g of PEGMEA, 0.422 or 0.211 g of ODA, and 2 mL of toluene were added into a 10 mL round-bottom flask sealed with a rubber septa cap. The solution was degassed by sparging with nitrogen gas for 20 min and then stirred at 60 °C in an oil bath for 18 h. After reaction, the reaction was quenched in cold water, and the product was precipitated out of hexane. To remove unreacted monomers completely, the product was stirred in hexane for 6 h and the solvent was changed every 2 h. Finally, hexane was poured out, and the final product was recovered by vacuum drying. Modification of HB Polythioether-Acrylate. The obtained polytrithiocarbonates were further cleaved to form polythiols through the aminolysis reaction conducted with 1-butylamine under nitrogen protection. To prevent obtained HB polythiols from cross-linking through oxidation of thiol moieties, the thiol−ene addition was carried out sequentially to convert thiols to thioether under a nitrogen environment. Briefly, 0.6 g of HB polytrithiocarbonates was dissolved in 1.5 mL of DCM in a 10 mL sealed flask, followed by addition of 0.5 mL of 1-butylamine (∼20-fold) under nitrogen protection. The yellow solution faded to colorless or light yellow after 30 min, and then nitrogen gas was sparged into the solution to remove most of the DCM and 1-butylamine. Nitrogen-degassed hexane (∼10 mL) was 11588

DOI: 10.1021/acsnano.8b06575 ACS Nano 2018, 12, 11579−11590

Article

ACS Nano cells were harvested and incubated with staining antibody (antiCD11C, anti-CD86, and anti-mouse I-A/I-E) and then applied to flow cytometry. In Vitro Activation/Restimulation and Apoptosis Assay of T Cells. For activation and proliferation of TCR-transgenic T cells, splenocytes from 2D2 mice or DO11.10 mice were stimulated in vitro in RPMI 1640 medium containing 10% FBS, 2 mM glutamine, 100 U/mL penicillin and streptomycin, and 15 μg/mL MOG or OVA peptide. After 3 days, activated T cells were washed and then cultured in complete RPMI1640 medium containing 100 U/mL recombinant mouse IL-2. For apoptosis assay, after 2 days, dead cells were removed by gradient centrifugation using Ficoll-paque, and then the activated T cells were cocultured with BMDCs stimulated with 30 μg/mL HB conjugated peptide or free peptide overnight. Cells were harvested and apoptosis was determined by staining with FITC-conjugated annexin V (eBioscience) and 5 μg/mL propidium iodide, then analyzed by flow cytometry. ̈ For detecting the effects of HB polymer conjugated OVA on naive T cell activation, splenocytes from DO11.10 mice were cultured in RPMI 1640 medium containing 10% FBS, 2 mM glutamine, and 100 U/mL of penicillin and streptomycin with 15 μg/mL (normalizing to peptide mass) of the following antigens: HB-OVA-1, HB-OVA-2, or PBS control for 3 days. To measure the expression of activation markers on the T cell surface, cells were incubated for 30 min on ice using fluorescent antibodies (anti-CD4, anti-CD44, and anti-CD69). Fluorescence Images of the Tissues. Organs (heart, liver, spleen, lung, and kidney) and CNS tissues (meninges, brain, and spinal cord) from EAE mice 3 h after rhodamine B-labeled HB-MOG-1 or HB-MOG-2 i.v. injection were harvested; then the fluorescence images were acquired using an IVIS Spectrum in vivo optical imaging system (PerkinElmer) with a filter (excitation: 535 nm, emission: 620 nm). The data were analyzed using Living Image 4.5.2 software. Histological Analysis. EAE mice at day 25 after treatment were perfused with PBS, and spinal cords were collected and fixed in 10% formalin. Thoracic spinal cord sections were stained with hematoxylin and eosin for visualization of leukocyte infiltration. Statistical Analysis. Prism 6.0 (GraphPad Prism) software was used for statistical analyses using Student’s t test. Significance was defined as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

Key Research and Development Program of China (2016YFA0502204), the National Natural Science Foundation of China (31201081), and the major research plan of the National Natural Science Foundation of China (91442203). M.X. thanks the of National Science and Engineering Research Council of Canadian (NSERC) Discovery Grant, NSERC Discovery Accelerator Supplements Award, and Canada Foundation of Innovation for support.

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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.8b06575. Additional information (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Malcolm Xing: 0000-0002-3547-0462 Author Contributions #

J. Li, D. Qiu, and Y. Liu contributed equally to this paper.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank Z. Zhang and L. Lu for technical assistance (MoE Key Laboratory for Biomedical Photonics, Huazhong University of Science and Technology, China). We thank L. Zou, J. Wang, S. He, and X. Tang (Institute of Immunology, Army Medical University, China) for technical assistance and support. This research work was supported by the National 11589

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DOI: 10.1021/acsnano.8b06575 ACS Nano 2018, 12, 11579−11590