WO2020240445A1

WO2020240445A1 - Phosphatidylcholine liposomes for use in autoimmune diseases

Info

Publication number: WO2020240445A1
Application number: PCT/IB2020/055040
Authority: WO
Inventors: Ana Cláudia FERNANDES LIMA; Rui Luís GONÇALVES DOS REIS; Helena Susana DA COSTA MACHADO FERREIRA; Nuno João MELEIRO ALVES DAS NEVES
Original assignee: Association for the Advancement of Tissue Engineering and Cell Based Technologies and Therapies A4TEC
Current assignee: Association for the Advancement of Tissue Engineering and Cell Based Technologies and Therapies A4TEC
Priority date: 2019-05-27
Filing date: 2020-05-27
Publication date: 2020-12-03
Anticipated expiration: 2021-11-27

Abstract

The present disclosure relates to new liposomes for use in medicine, in particular a therapy for autoimmune diseases relying on the (i) enhanced vascular permeability of inflamed synovial tissues for liposomes accumulation; (ii) antioxidant and antiangiogenic effects as well as disease monitoring and/or liposomes biodistribution conferred by gold nanoparticles; (iii) IL-23 neutralization, by antibodies immobilized at the surface of the liposomes, to inhibit Th17 cells differentiation and, consequently, to reduce the production of IL-17. This strategy presents a great potential to increase anti-IL-23 bioavailability, while decreasing adverse off-target effects.

Description

D E S C R I P T I O N

PHOSPHATIDYLCHOLINE LIPOSOMES FOR USE IN AUTOIMMUNE DISEASES

TECHNICAL FIELD

[0001] The present disclosure relates to new liposomes for use in medicine, namely in the field of autoimmune diseases (ADs) and therapeutic treatments of these diseases based on Nanotechnology.

[0002] The present invention presents a therapy for ADs based on a nanotherapeutic approach relying on the: enhanced vascular permeability of inflamed synovial tissues for liposome accumulation; antioxidant and antiangiogenic effects as well as disease monitoring and/or liposomes biodistribution conferred by gold nanoparticles; IL-23 neutralization by antibodies immobilized at the surface of the liposomes to inhibit Thl7 cells differentiation and, consequently, to reduce the production of IL-17. The combination of these approaches is innovative and presents a great potential to increase anti-IL-23 antibodies bioavailability, while decreasing adverse off-target effects.

BACKGROUND

[0003] Autoimmune diseases (ADs) are a complex class of diseases resulting from the immune system failure to differentiate between self and non-self-antigens.¹ This failure directs the immune system to attack self-antigens as foreign entities, which leads to tissue damage. The world prevalence of autoimmunity is approximately 3-5% and its incidence is rising dramatically.² There are currently more than one hundred identified ADs. Among them, autoimmune arthritis is one of the major categories of rheumatic diseases.³ The most common forms are rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA). Despite these diseases have distinct clinical phenotypes, they share some similarities including the joint inflammation and cartilage and bone degradation.⁴ The majority of the patients exhibit joint swelling, pain, tenderness and stiffness. However, being these diseases systemic, they can affect the entire body and many other symptoms may occur, including skin rashes, eye inflammation, hair loss, dry mouth and fever.

[0004] Cytokines, the proteins that enable the communication in the immune system, regulate a broad range of the inflammatory processes that are implicated in the pathogenesis of autoimmune arthritis.^{5 7} Therefore, pro-inflammatory cytokines, such as interleukins (IL; IL-Ib, IL-6, IL-17, IL-23), tumour necrosis factor-a (TNF-a) and interferon-g (IFN-y), are attractive therapeutic targets in these conditions.

[0005] In the past decade, the development of new biological agents to inhibit cytokines activity revolutionized the ADs treatment. The outstanding therapeutic outcomes of infliximab, a chimeric antibody specific for TNF-a, led to the development and clinical use of further anti-TNF agents and other cytokine inhibitors. In RA these agents not only transformed the patients' treatment, but also yielded important insights into its pathophysiology. One of those important findings was related with the crucial role of IL- 23/Thl7 pathway in autoimmunity.^8-9 IL-23 plays a central role in T cell-mediated responses and has been considered a key promoter of several immune-mediated conditions, such as colitis, psoriasis and arthritis.¹⁰ It is a heterodimeric cytokine and a member of the IL-12 cytokine family, having a specific pl9 subunit and sharing the subunit IL-12p40 with the IL-12. Nevertheless, despite a strong structural relationship, these two cytokines present different biologic roles, with IL-12 and IL-23 driving Thl and Thl7 cell responses, respectively.¹¹ Therefore, in the presence of other factors, such as IL-6 and transforming growth factor (TGF)- , IL-23 induces the differentiation of naive CD 4⁺ T cells into Thl7 cells. Thl7 cells are crucial to incite local tissue inflammation, since they stimulate the production of inflammatory cytokines, including IL-17, IL-22, TNF-a and granulocyte-macrophage colony-stimulating factor (GMCSF). IL-17 also stimulates the upregulation of pro-inflammatory cytokines (IL-6, IL-8, TNF-a) as well as of chemokines and metalloproteinases. Moreover, IL-17 and TNF-a act synergistically to promote increased expression of endothelial cell adhesion molecules, thereby further increasing granulocyte recruitment to sites of inflammation. Therefore, IL-23 is an early factor of the immune cascade. [0006] Monoclonal antibodies that inhibit IL-23 have been studied to treat ADs.¹⁰ Ustekinumab, a human monoclonal antibody that inhibits both IL-12 and IL-23 is approved by the US Food and Drug Administration (FDA) to use in PsA. Guselkumab is a human monoclonal antibody that binds to the pl9 subunit of IL-23 to inhibit its downstream signalling. It is currently approved for the treatment of adults with moderate to severe plaque psoriasis, and it is in clinical trials for pustular psoriasis (phase III, NCT02343744), PsA (phase III, NCT0315828, NCT03162796) and RA (phase II, NCT01645280). Many other IL-23 anti-antibodies are under clinical evaluation for several immune-mediated conditions. Nonetheless, the therapeutic efficacy of these biological agents is limited, due to their short half-life. They are rapidly cleared from the body and, consequently, repeated drug administration and prolonged time of treatment are required. In addition, antibodies, as many other drugs, have serious side effects and safety concerns.

[0007] Liposomes have been used to improve drug efficacy and safety in several pharmaceutical and medical applications.¹² Indeed, after the first liposomal formulation was introduced in the market, Doxif in 1995, many others are following the same trend. They are spherical vesicles of phospholipids bilayers enclosing an aqueous cavity. These carriers can be designed with specific characteristics adjusted to the requirements of the therapeutic agent chemical properties and mode of action as well as to the specificity of the disease. Furthermore, the lipidic carriers can incorporate drugs and/or imaging agents.

[0008] Gold nanoparticles (AuNPs) are characterized by good biocompatibility, easy synthesis, facile surface modification and tunable optical properties, and were used for various biomedical applications.¹³ The binding of gold nanoparticles (AuNPs) to the vascular endothelial growth factor (VEGF) confers them an antiangiogenic effect.¹⁴ Moreover, the antioxidant effects of these imaging agents have been well- demonstrated, for instance, after intraarticular injection in arthritic rats.¹⁵ Indeed, by quenching reactive oxygen species (ROS), AuNPs inhibit the receptor activator of nuclear factor KB ligand (RANKL)-induced osteoclast formation that increases bone and cartilage erosion. Therefore, AuNPs can be use in the treatment of autoimmune arthritis (especially RA), as VEGF, osteoclasts and ROS are the main contributors to their pathogenesis.¹⁶ Furthermore, AuNPs have optical properties that allow their use as a contrast agent to monitorthe biodistribution of the liposomes by e.g. Computer Assisted Tomography scanning or X-Ray.

GENERAL DESCRIPTION

[0009] The present invention discloses a therapy for autoimmune diseases (ADs) based on a nanotherapeutic approach relying on the: enhanced vascular permeability of inflamed synovial tissues for unilamellar liposomes (LUVs) accumulation; antioxidant and antiangiogenic effects as well as disease monitoring and liposomes biodistribution conferred by gold nanoparticles (AuNPs); IL-23 neutralization by antibodies immobilized at the surface of the liposomes to inhibit Thl7 cells differentiation and, consequently, to reduce the production of IL-17. The combination of these strategies is innovative and presents a great potential to increase anti-IL-23 antibodies bioavailability, while decreasing adverse off-target effects.

[0010] An aspect of the present disclosure comprises a liposome for targeted delivery comprising cholesterol, phosphatidylcholine, a PEG-lipid molecule and a vitamin; wherein the liposome wall comprises cholesterol, phosphatidylcholine, 1,2-distearoyl- sn-glycero-3-phosphoethanolamine (DSPE) and the vitamin; wherein the PEG molecule is bounded to the liposome wall surface through DSPE (namely at the liposome external wall); and at least one antibody bounded to the liposome wall surface or at least one antibody bounded to the PEG-lipid molecule, wherein the antibody is able to bind and/or neutralize an antigen.

[0011] In an embodiment, the vitamin is a-tocopherol.

[0012] In an embodiment, the liposome encapsulates a nanoparticle, an active substance, or mixtures thereof.

[0013] In another embodiment, the liposome further comprises at least a nanoparticle encapsulated by the liposomes.

[0014] In an embodiment, the nanoparticle is a gold nanoparticle. [0015] In an embodiment, the PEG-lipid molecule and the antibody are bounded by a covalent bond.

[0016] In an embodiment, the PEG-lipid molecule is 1,2-distearoyl-sn-glycero-B- phosphoethanolamineN-[maleimide(polyethyleneglycol)-2000.

[0017] In an embodiment, the antibody is an antibody against a pro-inflammatory cytokine.

[0018] In an embodiment, the antibody is an anti-interleukin, preferably anti-interleukin 23.

[0019] In an embodiment, said phosphatidylcholine is phosphatidylcholine from egg- yolk.

[0020] In an embodiment, the liposome is for use in medicine or veterinary.

[0021] In an embodiment, the liposome is for use in the treatment or therapy of autoimmune diseases and its symptoms, namely joint inflammation, joint swelling, cartilage and bone degradation, skin rashes, eye inflammation, hair loss, dry mouth or fever.

[0022] In an embodiment, the liposome is for use in the treatment or therapy of autoimmune arthritis, namely rheumatic diseases.

[0023] In an embodiment, the liposome is for use in the treatment or therapy of rheumatoid arthritis, ankylosing spondylitis, colitis or psoriatic arthritis.

[0024] In an embodiment, the liposome is for use as an antioxidant or as an antiangiogenic agent.

[0025] In an embodiment, the liposome is a large unilamellar liposome.

[0026] In an embodiment, the large unilamellar liposomes has a size between around 100 nm and 200 nm, particularly between 120 nm and 150 nm.

[0027] In an embodiment, the size of the large unilamellar functionalized liposomes ranges between 120-140 nm; preferably 124 nm -130 nm.

[0028] In another embodiment, the large unilamellar liposomes have a size around 124 nm when empty and around 130 nm when functionalized/encapsulate with/a substance/nanoparticle.

[0029] In an embodiment, the liposome shape is spherical. [0030] In an aspect of the present disclosure, the method for the production of the liposomes comprises the steps of:

• preparing unilamellar liposomes, comprising:

o lipid film formation,

o addition of a buffer with gold nanoparticles (AuNPs),

o mixing and formation of the multilamellar liposomes (MLVs), o formation of LUVs using extrusion;

• activating the selected antibody, comprising:

o Thiol insertion into the antibodies through the reaction with the Traut's reagent (2-iminothiolane) and EDTA, Dialysis to obtain purified activated antibodies,

• Antibodies immobilization at LUVs surface, comprising:

o Thiol groups of the antibodies covalently linked to the terminal maleimide groups present at the LUVs surface.

[0031] In an embodiment, the method for the production of unilamellar liposomes comprises the steps of:

obtaining a lipid film,

adding a buffer, preferably a buffered gold nanoparticles suspension (i.e. a suspension prepared from a buffer solution and gold nanoparticles),

mixing and forming multilamellar liposomes, extruding the multilamellar liposomes to obtain unilamellar liposomes.

[0032] In an embodiment, the method for the production of unilamellar liposomes further comprises a step for antibody immobilization at unilamellar liposomes surface by covalent link between the terminal maleimide group of the LUVs surface and the thiol group of the antibody.

[0033] In an embodiment, the method for the production of unilamellar liposomes further comprises an antibody activation by previously inserting a thiol group into the antibody, preferably through the reaction with the Traut's reagent (2-iminothiolane) and EDTA. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of the present disclosure.

[0035] Figure 1: Size distribution of the LUVs+AuNPs (A). Stability evaluation of LUVs+AuNPs kept in Heppes buffer at 4 °C for 6 months (B). TEM micrographs of the AuNPs (C) and LUVs+AuNPs (D).

[0036] Figure 2: Biological performance of the endothelial cell line (EA.hy926)(A) and human articular chondrocytes (hACs) (B) cultured with different concentrations of LUVs: cell viability (I), cell proliferation (II) and total protein synthesis (III) after 1, 3, and 7 days of culture. Asterisk (*) denotes significant differences (p < 0.01) compared to the control (0 mM).

[0037] Figure 3: SEM micrographs of LUVs cultured with EA cell line (A) and hACs (B) in the absence (control, I) and in the presence of liposomes at different concentrations: 1000 (II) and 2000 pM (III). Scale bar 10 pm.

[0038] Figure 4: Percentage of THP-1 cell line viability after cultured with different concentrations of LUVs (A). IL-23 quantification on the conditioned cultured medium of activated (LPS+INF) THP-1 cell line without treatment and with treatment with biofunctionalized liposomes (LUVs+Abs) (B). Asterisk (*) denotes significant differences (p < 0.05).

[0039] Figure 5: Biological effects of the PBMCs of healthy donors (A) and RA patients (B) with neutral activation (Ctr) and Thl7 activation without treatment (No treat), treatment with LUVs (LUVs) and treatment with the biofunctionalized LUVs with anti-IL- 23 antibodies (LUVs+Abs) and analysed regarding metabolic activity (I) and IL-17A production (II) a denotes significant difference compared to the Ctr, and b compared to No treat group, being * p < 0.05, ** p < 0.01, *** p<0.001 and **** p < 0.0001.

[0040] Figure 6: Shows a schematic representation of an embodiment of a liposomes production and functionalization. DETAILED DESCRIPTION

[0041] The present disclosure relates to new liposomes for use in medicine, in particular a therapy for autoimmune diseases relying on the (i) enhanced vascular permeability of inflamed synovial tissues for liposomes accumulation; (ii) antioxidant and antiangiogenic effects as well as disease monitoring and/or liposomes biodistribution conferred by gold nanoparticles; (iii) IL-23 neutralization, by antibodies immobilized at the surface of the liposomes, to inhibit Thl7 cells differentiation and, consequently, to reduce the production of IL-17. This strategy presents a great potential to increase anti-IL-23 bioavailability, while decreasing adverse off-target effects.

[0042] In an embodiment, the liposomes of the present invention comprise cholesterol, egg-yolk phosphatidylcholine (EPC), l,2-distearoyl-sn-glycero-3- phosphoethanolamineN-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG-Mal) and a- tocopherol.

[0043] In an embodiment, the liposomes are prepared from phospholipids, the main components of cells membranes. They act as non-irritating moisturizing agents and they are biocompatible, biodegradable and nontoxic.¹⁷ The PEG present in the DSPE-PEG-Mal increases the circulation time by reducing the opsonisation process. Moreover, a- tocopherol is a vitamin with antioxidant properties. Gold nanoparticles (AuNPs) were also incorporated into the liposomes to potentiate the antioxidant and antiangiogenic effects, as well as, for disease monitoring and/or liposomes biodistribution.

[0044] In an embodiment, the present invention is intended to provide the following technical effects: a) to increase the therapeutic index of biological agents through their binding to the liposomes; b) dual synergistic effect of the biological agents and the AuNPs, c) to monitor the biodistribution of the liposomes by the AuNPs; d) validate the therapeutic potential of the proposed therapy after its systemic injection; e) exceed the performance of the currently used therapies;// increase future treatment modalities, by including other therapeutic agents into the liposomes.

[0045] In an embodiment, after physicochemical characterization of unilamellar liposomes (LUVs), including particle size, polydispersity index (PDI), zeta-potential and morphology, antibodies were immobilized at the liposomes surface through thiol chemistry.

[0046] In an embodiment, the LUVs cytocompatibility was assessed in the presence of (1) an endothelial cell line (EA.hy926), (2) a human monocyte-like cell line (THP-1); and (3) human articular chondrocytes (hACs). Then, the capture ability of the immobilized antibodies was assessed using stimulated macrophages.

[0047] In a further embodiment, in order to ensure the capture and neutralization of the IL-23 by the antibodies immobilized at the LUVs surface, peripheral blood mononuclear cell (PBMCs) from healthy and RA patients were activated through Thl7 differentiation, which was achieved by stimulation with anti-CD3/anti-CD28 beads, 10 ng/ml of IL-Ib and 10 ng of IL-23. Then, the inhibition of Thl7 differentiation and, consequently, IL-17A production was assessed.

[0048] The safety and efficacy of the new therapy is evaluated in established RA animal models, and show to have activity and effect. Indeed, RA are selected and utilized as a 'proof of concept' of the innovative therapy due to its high prevalence. Indeed, RA affects up to 1% of the worldwide population.¹⁸

[0049] In what concerns characterization and functionalization of liposomes, LUVs comprising cholesterol/EPC/DSPE-PEG-Mal at 1:0.85:0.15 (n/n) and a-tocopherol at 1:200 (M/M) were successfully produced and characterized in terms of size, PDI and zeta potential (Table 1). The size was ¾124 nm for empty liposomes and ¾130 nm for liposomes encapsulating AuNPs. Both formulations revealed low PDI values (< 0.2), which indicates the homogeneity of the populations. Additionally, zeta potential measurements revealed that the surface charge is around -20 mV. Storage stability was also assessed, and as shown in Figure IB, the liposomes were stable for 6 months (at this time the size increased 10%).

[0050] From TEM analyses, it was possible to analyse that AuNPs presented a spherical shape with dimensions around 20 nm (Figure 1C), which is in agreement with the manufacturer specifications. The size of the LUVs+AuNPs of 130 nm is in agreement to the DLS measurements (Figure ID). In addition, the microphotograph confirms the encapsulation of the gold nanoparticles into the liposomes and also shows its spherical shape.

[0051] In an embodiment, to quantify the amount of the anti-IL-23 antibodies that were covalently immobilized at the liposomes surface, an indirect method was used, namely the measurement of the fluorescence of the unbound secondary antibody. The difference of the fluorescence obtained in the presence of the total amount and unbound of the secondary antibody demonstrated that 82.1 ± 8.4% of anti-IL-23 antibodies were efficiently immobilized at the liposomes surface.

[0052] The size distribution and zeta potential of the biofunctionalized liposomes remained approximately the same (Table 1).

[0053] Table 1. Size distribution and zeta potential of the developed liposomes incorporating or not AuNPs.

[0054] Concerning liposomes cytocompatibility, to use the proposed formulation in the RA treatment, the potential toxicity of the liposomes was assessed using: (1) an endothelial cell line (EA.hy926), (2) a human monocyte-like cell line (THP-1); and (3) hACs.

[0055] Endothelial cells and hACs are suitable models to assess any toxic interaction between the developed formulation with the blood vessels and the cells from the targeted tissue. Indeed, hACs isolated from diseased knee arthroplasties have a phenotype associated with arthritic diseases. Additionally, since the immune system has an important role in these diseases, the cytocompatibility with macrophages was assessed. [0056] Different biological assays were conducted to assess cell viability (Alamar Blue assay), proliferation (DNA quantification), total protein synthesis and morphology (SEM) after 1, 3, and 7 days of culture.

[0057] In an embodiment, for EA and hACs, the results presented on Figure 2 revealed that LUVs+AuNPs are biocompatible, since the cell viability and proliferation were not significantly affected. The culture of LUVs+AuNPs with the EA cell line showed no differences in the metabolic activity for all the tested concentrations. Even though, lower values of DNA and higher quantification of protein were found for the maximum concentration of liposomes, in general it can be assumed the non-toxicity of the produced LUVs. For the hACs, although there is statically difference after 1 day of culture at the concentration of 2000 mM in comparison with the control, after 3 and 7 days the LUVs+AuNPs were not cytotoxic for those cells. Moreover, no differences were observed in the DNA and total protein quantification. These results were corroborated by SEM analyses (Figure 3), which shown no changes in the cells morphology.

[0058] In an embodiment, concerning IL-23 capture by functionalized LUVs. To assess the cytokine capture capacity of the biofunctionalized liposomes, a conditioned cultured medium of activated macrophages was used. After 24 h of incubation with 2000 pM of biofunctionalized liposomes, no cytotoxicity was observed (Figure 4A) and 53.5 ± 9.6% of the produced cytokine was captured (Figure 4B).

[0059] In an embodiment, the biologic effects from IL-23 neutralization by functionalized liposomes were studied. To confirm the inhibition of the IL-17A production by the anti-IL-23 antibodies immobilized at the LUVs' surface, PBMCs from healthy and RA donors were activated for the Thl7 phenotype (stimulation with anti- CD3/anti-CD28 beads, 10 ng/mL of IL-Ib and 10 ng of IL-23) for 24 h. Then, three different conditions were tested: (1) no treatment (no treat), (2) treatment with LUVs (LUVs) and (3) treatment with biofunctionalized LUVs (LUVs+Abs). Non activated PBMCs were used as controls (Ctr).

[0060] In an embodiment, regarding the cell viability, the Thl7 differentiation reduced the metabolic activity in healthy donors (Figure 5A), since there was a significant decrease in the stimulation without treatment (p < 0.05) in comparison with the control. This reduction was prevented by the LUVs. In the RA donors (Figure 5B), the capture and inactivation of the IL-23 conferred by the LUVs significantly increased the metabolic activity.

[0061] In an embodiment, from the quantification of I L-17A, there is a significantly increase (p < 0.0001) in the amount of IL-17A without treatment, in comparison with the control. Moreover, while the treatment with LUVs had little impact on the IL-17A production, the treatment with the biofunctionalized LUVs significantly reduced the IL- 17A production. PBMCs from RA patients produced more IL-17A than PBMCs from healthy donors when activated to the Thl7 phenotype. This shown the high susceptibility to immune cells activation of the diseased patients. Moreover, the IL-17A production was reduced following a similar trend to that of healthy donors. Therefore, these results clearly indicate the positive impact of the IL-23 neutralization by the liposomes in the TH17 differentiation of the PBMCs.

[0062] In an embodiment, the method of production of the liposomes is also an object of the present invention.

[0063] In an embodiment, liposomes production and functionalization may be performed as described in the diagram of Figure 6.

[0001] Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.

[0002] Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known i n the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0003] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0004] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0005] The above described embodiments are combinable.

[0006] The following claims further set out particular embodiments of the disclosure.

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Claims

C L A I M S

1. A liposome for targeted delivery comprising cholesterol, phosphatidylcholine, a PEG- lipid molecule, and a vitamin;

wherein the liposome wall comprises cholesterol, phosphatidylcholine, 1,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and the vitamin;

wherein the PEG molecule is bounded to the liposome wall surface through DSPE;

and at least one antibody bounded to the liposome wall surface or at least one antibody bounded to the PEG-lipid molecule, wherein the antibody is able to bind and/or neutralize an antigen.

2. The liposome according to any of the previous claim wherein the vitamin is a- tocopherol.

3. The liposome according to any of the previous claims wherein the liposome encapsulates a nanoparticle, an active substance, or mixtures thereof.

4. The liposome according to any of the previous claims wherein the nanoparticle is a gold nanoparticle.

5. The liposome according to any of the previous claims wherein the PEG-lipid molecule and the antibody are bounded by a covalent bond.

6. The liposome according to any of the previous claims wherein the PEG-lipid molecule is l,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[maleimide(polyethyleneglycol)- 2000.

7. The liposome according to any of the previous claims wherein the antibody is an antibody against a pro-inflammatory cytokine.

8. The liposome according to any of the previous claims wherein the antibody is an anti interleukin, preferably anti-interleukin 23.

9. The liposome according to any of the previous claims wherein said phosphatidylcholine is phosphatidylcholine from egg-yolk.

10. The liposome according to any of the previous claims for use in medicine or veterinary.

11. The liposome according to any of the previous claims for use in the treatment or therapy of autoimmune diseases and its symptoms, namely joint inflammation, joint swelling, cartilage and bone degradation, skin rashes, eye inflammation, hair loss, dry mouth or fever.

12. The liposome according to any of the previous claims for use in the treatment or therapy of autoimmune arthritis, namely rheumatic diseases.

13. The liposome according to any of the previous claims for use as an antioxidant or as an antiangiogenic agent.

14. The liposome according to any of the previous claims wherein the liposome is a large unilamellar liposome.

15. The liposome according to any of the previous claims wherein the large unilamellar liposomes has a size between around 100 nm and 200 nm, particularly between 120 nm and 150 nm.

16. The liposome according to any of the previous claims wherein the size of the large unilamellar functionalized liposomes ranges between 120-140 nm; preferably 124 nm -130 nm.

17. The liposome according to any of the previous claims wherein the liposome shape is spherical.

18. A method for the production of unilamellar liposomes described in any of the previous claims comprising the steps of:

obtaining a lipid film, adding a buffer, preferably a buffered gold nanoparticles suspension, mixing and forming multilamellar liposomes, extruding the multilamellar liposomes to obtain unilamellar liposomes.

19. The method according to the previous claim further comprising a step for antibody immobilization at unilamellar liposomes surface by covalent link between the terminal maleimide group of the LUVs surface and the thiol group of the antibody.

20. The method according to the previous claim comprising an antibody activation by previously inserting a thiol group into the antibody, preferably through the reaction with the Traut's reagent (2-iminothiolane) and EDTA.