Artificial Cells, Nanomedicine, and Biotechnology An International Journal ISSN: 2169-1401 (Print) 2169-141X (Online) Journal homepage: https://www.tandfonline.com/loi/ianb20 Preclinical evaluation of antigen-specific nanotherapy based on phosphatidylserine- liposomes for type 1 diabetes Adrian Villalba, Silvia Rodriguez-Fernandez, Rosa-Maria Ampudia, Mary Cano-Sarabia, David Perna-Barrull, Cesc Bertran-Cobo, Clara Ehrenberg, Daniel Maspoch & Marta Vives-Pi To cite this article: Adrian Villalba, Silvia Rodriguez-Fernandez, Rosa-Maria Ampudia, Mary Cano- Sarabia, David Perna-Barrull, Cesc Bertran-Cobo, Clara Ehrenberg, Daniel Maspoch & Marta Vives-Pi (2020) Preclinical evaluation of antigen-specific nanotherapy based on phosphatidylserine- liposomes for type 1 diabetes, Artificial Cells, Nanomedicine, and Biotechnology, 48:1, 77-83, DOI: 10.1080/21691401.2019.1699812 To link to this article: https://doi.org/10.1080/21691401.2019.1699812 © 2019 The Author(s). Published by Informa View supplementary material UK Limited, trading as Taylor & Francis Group Published online: 18 Dec 2019. Submit your article to this journal Article views: 1032 View related articles View Crossmark data Citing articles: 1 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=ianb20 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 2020, VOL. 48, NO. 1, 77–83 https://doi.org/10.1080/21691401.2019.1699812 Preclinical evaluation of antigen-specific nanotherapy based on phosphatidylserine-liposomes for type 1 diabetes Adrian Villalbaa, Silvia Rodriguez-Fernandeza, Rosa-Maria Ampudiaa, Mary Cano-Sarabiab, David Perna-Barrulla, Cesc Bertran-Coboa , Clara Ehrenberga, Daniel Maspochb,c and Marta Vives-Pia,d a Immunology Section, Germans Trias i Pujol Research Institute, Autonomous University of Barcelona, Badalona, Spain; bCatalan Institute of Nanoscience and Nanotechnology, Bellaterra, Spain; cInstitucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain; dCIBER of Diabetes and Associated Metabolic Disease (CIBERDEM). ISCIII, Barcelona, Spain ABSTRACT ARTICLE HISTORY Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of insulin-producing cells. Received 3 July 2019 Due to the ability of apoptotic cells clearance to induce tolerance, we previously generated liposomes Revised 27 September 2019 rich in phophatidylserine (PS) –a feature of apoptotic cells– loaded with insulin peptides to mimic Accepted 27 September 2019 apoptotic beta-cells. PS-liposomes arrested autoimmunity in experimental T1D through the induction KEYWORDS of tolerance. The aim of this study was to investigate the potential of several peptides from different Nanovesicles; immunother- T1D autoantigens encapsulated in (PS)-liposomes for T1D prevention and to assess its safety. T1D auto- apy; autoimmunity antigens (Insulin, C-peptide, GAD65 and IA2) were encapsulated in PS-liposomes. Liposomes were administered to the ’gold-standard’ model for the study of autoimmune T1D, the Non-Obese Diabetic mouse, that spontaneously develop the disease. Safety and toxicity of liposomes were also determined. Only PS-liposomes encapsulating insulin peptides decrease T1D incidence in the Non-Obese Diabetic mouse model. Disease prevention correlates with a decrease in the severity of the autoimmune islet destruction driven by leukocytes. PS-liposomes neither showed toxic effect nor secondary complica- tions. Among the here referred autoantigens, insulin peptides are the best candidates to be encapsu- lated in liposomes, like an artificial apoptotic cell, for the arrest of autoimmunity in T1D in a safe manner. Introduction efferocytosis, or phagocytosis of apoptotic cells. These dying cells exhibit surface phosphatidylserine (PS) to be distin- An essential goal to both prevent and cure type 1 diabetes guished from living cells and to allow for their recognition by (T1D) is to arrest the autoimmune reaction against b-cells. phagocytes [5,6]. Following these principles, an antigen-spe- Insulin is a key autoantigen in T1D and the only b-cell-spe- cific cell immunotherapy using apoptotic b-cells was demon- cific autoantigen, as well as a major target of the auto- strated efficacious in re-establishing specific immunological immune attack mediated by T lymphocytes [1]. Moreover, tolerance, which was mediated by dendritic cells (DCs) ren- there is also a genetic association between polymorphisms of dered tolerogenic after efferocytosis [7]. Because autologous the insulin gene and the risk to develop T1D [2]. However, in apoptotic b-cells for T1D treatment cannot be easily addition to insulin, other autoantigens such as the glutamic obtained, artificial cells (PS-liposomes) were generated to acid decarboxylase 65 (GAD65), islet-antigen 2 (IA2) and zinc mimic tolerogenic apoptotic cell function [8]. A previous transporter 8 (ZnT8) have been described in T1D. They are work demonstrated the therapeutic potential of PS-liposomes known to induce autoantibodies and T cell responses. encapsulating insulin peptides – as a main autoantigen in Autoantibodies against insulin or GAD isoforms are the most T1D – to prevent T1D in an experimental model of the dis- common in the early phases of T1D and, occasionally, mul- ease, the Non-Obese Diabetic (NOD) mouse [8]. The NOD tiple auto-antibodies arise during advanced stages of the mouse, developed in 1980 [9] is considered the ‘gold-stand- pathology [3]. Although insulin is a primary autoantigen in ard’ model for the study of T1D [10]. These mice develop T1D, other autoantigens are involved in the progression of T1D spontaneously around 12 weeks of age, after the the disease after epitope spreading [4]. immune-mediated destruction of insulin-producing b cells, Furthermore, in order to arrest the autoimmune reaction commonly called insulitis. NOD mice have been extensively against b-cells, immunological tolerance to T1D autoantigens utilised to study the ethiopathogenesis of the disease and to must be restored. A process known to promote tolerance is test new immunotherapies. This model mouse closely CONTACT Marta Vives-Pi mvives@igtp.cat Immunology Section, Germans Trias i Pujol Research Institute. Carretera Canyet s/n. 08916 Badalona, Spain Supplemental data for this article is available online at https://doi.org/10.1080/21691401.2019.1699812. ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 78 A. VILLALBA ET AL. resembles human disease, albeit the obvious differences with apoptotic bodies as described, under sterile conditions and at human populations. a final concentration of 30 mM [11]. Encapsulation efficiencies In order to better mimic the apoptotic b-cell, and to try to (EE) were calculated according to the equation EE (%) ¼ [(pep- increase the efficacy of the nanotherapy, the present study tide, total–peptide, out)/peptide, total]  100, where peptide, aimed at encapsulating other peptides from several T1D total is the initial peptide concentration and peptide, out is autoantigens in PS-liposomes and to test its therapeutic the concentration of non-encapsulated peptide. To measure effect in arresting autoimmunity. In addition, and in order to the peptide, out, liposome suspensions were centrifuged at confirm the safety of the nanotherapy, the effects of a severe 110.000  G at 10  C for 30 min. The concentration of non- administration strategy were assessed in NOD mice. encapsulated peptide was assessed in the supernatants by PIERCE BCA protein assay kit (Thermo Fisher Scientific Inc., IL, USA). For toxicity experiments, empty PS-liposomes were gen- Material and methods erated. The distribution of the particle size and the stability – Mice given as f potential – were measured by dynamic light scatter- ing using Malvern Zetasizer (Malvern Instruments, Malvern, Wild-type non-obese diabetic (NOD) mice were purchased UK) in undiluted samples. The morphology of liposomes was from Charles River (Calco, Lecco, Italy) and kept under spe- examined by cryogenic transmission electron microscopy with cific pathogen-free conditions at the Animal House facility of a JEOL-JEM 1400 device (Jeol Ltd., Tokyo, Japan). the Germans Trias i Pujol Research Institute. Mice were in a temperature- and humidity-controlled room with 12 h light/ 12h dark cycle, provided with standard chow diet (Teklad T1D prevention and assessment of islet leukocytic Global 14% Protein Rodent Diet, Envigo, Sant Feliu de infiltration Codines, Spain) and water ad libitum. To detect spontaneous Normoglycemic 8-week-old NOD mice were treated with a development of T1D, NOD mice were monitored daily for single dose of liposomes. Briefly, 3.5 mg of the different pep- urine glucose using Glucocard strips (Menarini, Barcelona, tide-filled PS-liposomes in 200 lL saline solution were Spain), starting at 10 weeks of age and until 25 weeks of age. injected i.p. Thus, PSAB-lipo group received 200 lL of a mix Mice with glycosuria were confirmed diabetic when the of PSA and PSB liposomes, PSGAD65-lipo group received blood glucose level was >300 mg/dL. Mice were anesthetised 200 lL of a mix of PSG1 and PSG2 liposomes, PSIA2-lipo with isofluorane inhalation to obtain serum and euthanized group received 200 lL of PSI1 and PSI2 liposomes and by cervical dislocation to harvest the pancreas at 25 weeks of PSCpep-lipo group received 200 lL of PSC liposomes. A age or at T1D onset. The study was approved by the group containing 200 lL of a mix of all the different peptide- Committee on the Ethics of Animal Experimentation of the loaded PS-liposomes (Cock-lipo) was included, in which each Germans Trias i Pujol Research Institute and carried out in liposome fraction was present in an equal amount. A group strict accordance with the recommendations in the Guide for containing the mix of all the different peptides (200 lL) was the Care and Use of Laboratory Animals of the Generalitat de also included (Cock-Pep), the amount of peptide given being Catalunya, Catalan Government. in accordance to encapsulation efficiency inside the lipo- somes. Sham group received saline solution. Each group con- Peptides and liposomes tained 5–9 animals. Mice were monitored daily for glycosuria (Glucocard strips, Menarini, Barcelona, Spain) until 25 weeks Peptides were selected because they are target epitopes in of age or detection of T1D. To determine the destructive islet T1D [4]. Peptides from Insulin, C-peptide, GAD65 and IA2 leukocytic infiltration, insulitis score was assessed at 25 weeks (Table 1) were chosen (GL Biochem, Shanghai, China). in all non-diabetic mice. Pancreases were snap frozen in an Peptides were >95% pure and Trifluoroacetic acid was isopentane/cold acetone bath. Non-overlapping cryosections removed. Liposomes were composed of 1,2-dioleoyl-sn-glyc- (5 lm) were stained with haematoxylin and eosin (H/E). A ero3-phospho-L-serine (sodium salt) (DOPS, Lipoid, blind analysis was performed by two independent observers, Steinhausen, Switzerland) and 1,2-didodecanoyl-sn-glycero-3- analysing a minimum of 40 islets per animal. Insulitis was phosphocholine (DLPC, Lipoid), and cholesterol (CH, Sigma scored as previously described: 0, no insulitis; 1, peri-insular; Aldrich, MO, USA). Liposomes were prepared using the thin- 2, mild insulitis (<25% of the infiltrated islet); 3, 25–75% of film hydration method from a lipid mixture, to mimic the islet infiltrated; 4, >75% islet infiltration [12]. Table 1. Sequences of peptides encapsulated in PS-liposomes. Peptide Sequence (N0 –C0 ) Length Aminoacids C-peptide57–87 EVEDPQVAQLELGGGPGAGDLQTLALEVAQQ 31 GAD65216–236 EYVTLKKMREIIGWPGGSGD 20 GAD65290–309 AALGIGTDSVILIKCDERGK 20 IA2816–846 GVKQCDRYWPDEGSSLYHVYEVNLVSEHIWC 31 IA2870–898 FHFLSWPAEGTPASTRPLLDFRRKVNKCY 29 Insulin90–110 (A chain) GIVDQCCTSICSLYQLENYCN 21 Insulin25–54 (B chain) FVKQHLCGSHLVEALYLVCGERGFFYTPMS 30 Subindex refers to the position of the query sequence to the protein. GAD65: glutamic acid decarboxylase 65 ; IA2: islet-antigen 2. ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 79 Table 2. Features of the liposomes used in the study. Liposome Encapsulated peptide Diameter (nm) Polydispersity index f-potential (mV) Encapsulation efficiency (%) PSI1 IA2816–846 459 0.236 30.8 98.69 PSI2 IA2870–898 436 0.275 31.4 72.61 PSG1 GAD65216–236 618 1.000 36.7 39.20 PSG2 GAD65290–309 440 0.285 32 31.00 PSC C peptide57–87 437 0.126 34 16.80 PSA Insulin90–110 (A chain) 712 0.364 46.6 32.29 PSB Insulin25–54 (B chain) 628 0.325 44.9 89.63 PS Empty 645 0.638 –35.9 – Subindex of peptides refers to the position of the query sequence to the protein. PS: Phosphatidylserine-rich liposomes; GAD65: glutamic acid decarboxylase 65 ; IA2: islet-antigen 2. Evaluation of the toxicity of PS-liposomes Table 3. Amount of peptide encapsulated in a dose (200 mL) of PS-liposomes (3.5 mg lipid). In order to determine the safety of PS-liposomes, a severe Treatment Peptide Peptide per dose (mg) administration strategy was tested in NOD mice. Six normo- PSG GAD65216–236 39,2 glycemic mice (8–12-week-old, 3 males and 3 females) were GAD65290–309 31 PSI IA2816–846 98,69 daily injected i.p. with 200 mL of 3.5 mg 30 mM-lipid PS-lipo- IA2870–898 72,61 somes during 14 consecutive days. Simultaneously, PBS PSC C-peptide57–87 16,8 administration was used as control (sham) in 6 mice (3 males PSAB Insulin (A-chain)90–110 32,29 Insulin (B chain)25–54 89,63 and 3 females). Mice health and welfare was monitored Cock-Pep GAD65216–236 14,28 (Supplementary Table S1) during 21 days in terms of weight, GAD65290–309 14,28 IA2816–846 14,28 glucosuria, physical appearance (fur, presence of secretions IA2870–898 14,28 or bruises) and behaviour (barbering, physical activity, C-peptide57–87 14,28 aggressiveness, stereotyped activities and pain). Mice were Insulin90–110 14,28 Insulin25–54 14,28 euthanised at the end of the follow-up or if the total score Cock-lipo GAD65216–236 5,59 reached a maximum value of 3. Serum from 4 mice of each GAD65290–309 4,42 group was analysed for biochemical parameters, such as total IA2816–846 14,09 IA2870–898 10,37 cholesterol (Total CH), high-density lipoprotein (HDL) CH, tri- C-peptide57–87 2,40 glycerides, creatinine, urea and alanine transferase (ALT). Insulin90–110 4,61 Insulin25–54 13,26 Cock-Pep: mixture of all non-encapsulated peptides; Cock-lipo: mixture of all Statistical analysis PS-liposomes encapsulating different peptides. The statistical analysis was performed using the Prism 7.0 software (GraphPad software Inc., CA, USA). Mantel-Cox Log- filled liposomes did not show a decrease in the incidence Rank was used to T1D incidence analysis. Mann–Whitney test either (80%) (Figure 1). In order to confirm that mice at the was used for comparisons of nonparametric and unpaired end of follow-up did not develop T1D, urine glucose concen- data. A p-value  .05 was considered significant. tration (glycosuria) and serum C-peptide concentration –as indicators of b-cell function– were assessed. All mice showed Results negative glycosuria and detectable C-peptide concentration (range 900–3000 pg/mL). No statistical differences were found PS-liposomes encapsulating insulin, but not C-peptide, between groups (data not shown). GAD65 or IA2 peptides decrease T1D incidence Liposomes were characterised in terms of diameter, polydis- PS-liposomes encapsulating insulin peptides reduce the persity index (PdI), surface charge (f-potential) and efficiency severity of the autoimmune islet destruction of peptide encapsulation (Table 2). All liposomes had a final lipid concentration of 30 mM. NOD mice were treated with a Given the reduced incidence of T1D in both PSAB-lipo and single dose of liposomes before T1D onset (8 weeks old) Pep-Cock treated mice, islet leukocytic infiltration was deter- (Table 3). At the end of the follow-up period, T1D incidence mined at the end of the follow-up period showing a bio- tended to be lower in both PSAB-lipo and Cock-Pep groups logical reduction of insulitis, although non-significant, in both (33.3% and 42.8%, respectively) when compared with the treated groups (PSAB-lipo: 1.45 ± 0.57 and Cock-Pep: sham group (66.6%) (Figure 1). Hence, and as expected, a 1.32 ± 0.34) when compared to sham mice (1.85 ± 0.60) 50%-fold reduction in T1D incidence was achieved in the (Figure 2(A)). Furthermore, the analysis of the percentages of PSAB-lipo group. The groups treated with liposomes encap- islets classified in each infiltration category revealed that in sulating IA2, GAD65 or C-peptide did not show any reduction both treated groups more than 64% of the islets remained in the incidence of the disease (80%, 65% and 83.3% respect- insulitis-free (type 0) or with benign insulitis (type 1); in the ively) when compared with sham-treated animals (Figure 1). control group, 48% of the islets were scored with destructive Moreover, the group treated with the mix of the peptide- insulitis (type 2, 3 and 4) (Figure 2(B,C)). 80 A. VILLALBA ET AL. Figure 1. T1D incidence in NOD mice treated with PS-liposomes filled with different autoantigenic peptides. Cumulative incidence (percentage) of T1D in NOD mice treated with IA2 liposomes (PSIA2-lipo, blue, n ¼ 5), GAD65 liposomes (PSGAD65-lipo, red, n ¼ 6), C-peptide liposomes (PSCpep-lipo, yellow, n ¼ 6), insulin lip- osomes (PSAB-lipo, grey, n ¼ 6), a mixture of all liposomes (Cock-lipo, green, n ¼ 5) and a mixture of all non-encapsulated peptides (Cock-Pep, black, n ¼ 7). A sham group was also included (violet, n ¼ 9). The arrow indicates the time-point of the single-dose administration (at 8 weeks). No significant differences in T1D incidence were found between groups (Mantel-Cox Log-Rank). PSIA2-lipo: IA2 liposomes; PSGAD65-lipo: GAD65 liposomes; Cock-lipo: a mixture of all liposomes; PSCpep-lipo: C-peptide liposomes; Cock-Pep: a mixture of all non-encapsulated peptides; PSAB-lipo: insulin liposomes. Figure 2. Islet leukocytic infiltrate (insulitis) following administration of nanotherapy. (A) Insulitis score from non-diabetic mice at the end of the follow-up period at 25 weeks, including the PSAB-lipo (n ¼ 3), Cock-Pep (n ¼ 3) and sham (n ¼ 3) groups. Results are mean ± Standard Deviation (SD). No statistical differences were found between groups (Mann-Whitney test). (B) Percentage of islets in each of the infiltration categories: White ¼ 0, no insulitis; Dotted ¼ 1, peri-insular; Striped ¼ 2, mild insulitis (<25% of the infiltrated islet); Squared ¼ 3, 25–75% of the islet infiltrated; Black ¼ 4, >75% islet infiltration. (C) Representative images of insulitis in islets from H/E-pancreatic cryostat sections (5 mm) from the three different groups. Scale bar represents 100 mm. PSAB-lipo: insulin PS-liposomes; Cock-Pep: mix- ture of all non-encapsulated peptides. PS-liposomes treatment demonstrated optimal safety complications were observed both in daily PS-liposomes- and tolerability in NOD mice treated and sham group (Table 4) and no animal had to be The observational study, taking into account the criteria used euthanised due to severe complications of the treatment. All in welfare monitoring of mice used for toxicity and tolerabil- mice were negative for glucosuria during the 14 days of treat- ity assays (Supplementary Table S1), demonstrated the safety ment and 7 days after withdrawal. The analysis of the serum of daily PS-liposomes administration in NOD mice. Only mild showed no significant differences between PS-liposomes and ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 81 sham groups at the end of the study (Figure 3). Total CH, the differences here reported can be due to the biological HDL CH, triglycerides and ALT concentrations remained features of the encapsulated autoantigens. The role of the unaltered and within reference values for NOD mice [13]. different autoantigens in the stages of autoimmunity in T1D Urea concentration in both the groups was found above ref- is also relevant. On the one hand, insulin is the most erence values. Creatinine concentration was similar in both expressed specific autoantigen in the target b-cells on which groups, but reference values were not available. the autoimmune reaction is focussed and, on the other hand, single positivity for autoantibodies against insulin but not for the rest of autoantigens is associated with the greatest risk Discussion to develop T1D [15]. Moreover, it has been described that This study shows that the administration of PS-liposomes insulin is an early autoantigen in T1D whereas the other encapsulating insulin peptides is the best strategy to reduce autoantigens seem to be the consequence of epitope spread- the incidence of T1D, spontaneously developed in NOD mice, ing, after the activation of autoimmunity [15]. IA2 expression when compared to other encapsulated autoantigen peptides is not exclusive of pancreatic b-cells in humans nor rodents, (GAD65, IA2 and C-peptide). Liposomes are vesicles com- being found expressed also in many neuroendocrine cells posed of lipid bilayers that can be used as a vehicle for drug [16–18]. This lack of specificity would explain the unsuccess- delivery. Considering that the physicochemical features of ful therapeutic use by its single encapsulation in PS-lipo- the liposomes are homogeneous, and that empty liposomes somes. GAD65 expression is also present in other tissues due to its role in the biosynthesis of gamma-aminobutyric acid have no effect on T1D and other autoimmune diseases [8,14], (GABA) like GABAergic cells [19]. Furthermore, it has been Table 4. NOD mice daily treated with PS-liposomes (14 days) with recently reported that GAD65 has no capacity to prevent T1D disturbances. in wild type NOD mice [20]. Finally, the role of C-peptide as Mice with disturbances autoantigen in T1D remained controversial despite recent Parameter Sham PS-liposomes work hints at its action as an autoantigen [21–23]. This work Loss of 10% of body weight 1/6 (16%) 2/6 (33%) validates the use of PS-liposomes encapsulating insulin pepti- Piloerection 0/6 (0%) 1/6 (16%) des to prevent the development of autoimmune T1D and Mild bruises on the abdomen 1/6 (16%) 0/6 (0%) tends to reduce the insulitis in the experimental NOD Mild bruises on the tail 1/6 (16%) 0/6 (0%) Mild aggressive behaviour 0/6 (0%) 1/6 (16%) model [8]. Data presented as number of mice with disturbances in each group and per- A different encapsulation efficiency has been observed for centage (%). the different peptides and this fact must be taken into Figure 3. Effect of daily administration of PS-liposomes on blood biochemical parameters of NOD mice at the end of the study (Day 21). In vivo toxicity assay by measuring the levels of (A) Total CH, mg/dL, (B) HDL CH, mg/dL), (C) triglycerides (mg/dL), (D) ALT, U/L, (E) urea, mg/dL and (F) creatinine, mg/dL in serum of PBS- treated mice (n ¼ 4, squares) and PS-liposomes-treated mice (n ¼ 4, circles), at the end of the study (Day 21). Continuous lines represent the mean of each group. The grey area depicts normal ranges of parameters and the median (discontinuous line) of the different analytes for NOD mice. Significant differences were not found between groups (Mann-Whitney test). CH: cholesterol; ALT: Alanine transferase. 82 A. VILLALBA ET AL. account to both understand the results in this study and to Disclosure statement design future nanotherapies. The lowest percentage of MVP, MCS and DM hold patents that relate to liposomes and are co- encapsulation corresponds to liposomes encapsulating C- founder of Ahead Therapeutics S.L. The other authors report no conflicts peptide and, as mentioned above, the role of this molecule of interest in this work. as autoantigen remains unclear. Both facts may be the reason of the lack of effect in T1D prevention. Moreover, the mixture of liposomes (Cock-lipo) contains a 7-fold reduction of the Funding total insulin peptide amount carried in the PSAB-lipo, result- This work was suported by the Foundation La Marato  de TV3 under ing in a loss of therapeutic action. Grant 201632_10. CIBER of Diabetes and Associated Metabolic Diseases Apart from the nanotherapy described here, a mixture of (CIBERDEM) is an initiative from Instituto de Salud Carlos III (Spain). SRF is supported by the Agency for Management of University and Research the selected autoantigens (Cock-Pep) was found to reduce Grants (AGAUR) of the Generalitat de Catalunya. the incidence of the disease when compared with the sham group but with a minor effect than the observed with PS-lip- osomes encapsulating insulin. The synergistic effect of PS in the liposomes and the encapsulated peptide has been ORCID described in previous work showing a tolerogenic effect Cesc Bertran-Cobo http://orcid.org/0000-0001-9286-9595 mediated by DCs [8]. Many therapies have been tested both for prevention and treatment of T1D in the NOD mice based on peptides [24]. The here reported effect of combined pep- References tides fits well with previous data showing a preventive effect [1] Michels AW, Landry LG, McDaniel KA, et al. Islet-Derived CD4 T of T1D after peptide administration of insulin, GAD65 and cells targeting proinsulin in human autoimmune diabetes. 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