The present invention relates to novel sulfated glycopolymers and to the use of these glycopolymers. The novel sulfated glycopolymers may be used in the treatment and/or modulation of inflammation, or for the treatment and/or prevention of ischaemia, including treatment and/or prevention of ischaemia reperfusion injury or ischaemic stroke.

An increasingly large number of lectins have been found to play a major role in both innate and adaptive immunity. One of the most notable of these lectins is the mannose receptor, a 175kDa type 1 transmembrane receptor, also referred to as CD206 (Martinez-Pomares, L., 2012). The mannose receptor is expressed in selected populations of key immune cells such as macrophages and other cells (for example, Kaposi's sarcoma spindle cells, dendritic cells and lymphatic endothelial cells and kidney mesangial cells).

CD206 consists of the following 5 domains: an amino terminal cysteine-rich region (CR domain), a fibronectin type II repeat (FNII), a series of eight tandem C-type lectin-like carbohydrate recognition domains, a transmembrane domain and an intracellular carboxy -terminal tail.

CD206 has been associated with a range of diseases and conditions. For example, previous studies have shown a role of CD206 in the development of kidney crescentic glomerulonephritis in mice whilst other studies have shown that CD206 is overexpressed on alveolar macrophages in lungs of patients with severe chronic obstructive pulmonary disease and suggested its involvement in the pathogenesis of this condition.

Selective targeting of specific lectins in vivo is often very challenging due to the plethora of different lectins with overlapping sugar ligand specificities. In view of this challenge, CD206 has not been widely investigated as a clinical target.

In some embodiments the present invention uses novel sulfated glycopolymers specific for CD206 which selectively bind to the CR domain and modulate, or even suppress, the endocytic activity of selected CD206 expressing cells. According to a first aspect the invention provides a sulfated glycopolymer comprising a polymeric backbone wherein at least one of the monomeric units which form the backbone have a pendant galactose and/or N-acetyl galactosamine group, wherein one or more of the pendant galactose groups are sulfated at one or more of positions 2, 3, 4 and 6 and/or one or more of the N-acetyl galactosamine groups are sulfated at one or more of positions 3, 4 and 6.

In an embodiment, one or more of the pendant galactose and/or N-acetyl galactosamine groups are sulfated at position 3 and/or 4. In another embodiment, one or more of the pendant N-acetyl galactosamine groups are sulfated at position 3 and/or 6. The one or more of the pendant galactose and/or N-acetyl galactosamine groups may be sulfated at only one position, preferably at position 3 or 4. In addition to the sulfated galactose and/or N-acetyl galactosamine groups the sulfated glycopolymer may comprise further pendant groups, these groups may include non- sulfated galactose and/or N-acetyl galactosamine groups or other groups.

Further pendant groups may include fluorescent molecules such as fluorescein, rhodamine, coumarin and Bodipy analogues; residues that can react with amine (such as activated (e.g. N-hydroxysuccinimide and pentafluorophenol) esters; residues that can react with thiol such as maleimide, pyridine disulphide, and Michael acceptors e.g. vinyl sulfone, derivatives; hydrophobic groups such as aromatic and aliphatic groups; or other hydrophilic molecules such as glycerol, other sugars like glucose, and poly(ethylene glycol).

In an embodiment at least about 60%, 70%, 75%, 80%, 85%, 90%, 95% or more of the pendant groups are sulfated galactose and/or N-acetyl galactosamine groups. According to another aspect the invention provides a sulfated glycopolymer consisting of a polymeric backbone wherein at least one of the monomeric units which form the backbone have a pendant galactose and/or N-acetyl galactosamine group, wherein one or more of the pendant galactose groups are sulfated at one or more of positions 2, 3, 4 and 6 and/or one or more of the N-acetyl galactosamine groups are sulfated at one or more of positions 3, 4 and 6. Sulfated galactose or sulfated N-acetyl galactosamine groups which are sulfated at positions 3 or 4 may also be referred to as (S0 ₄ -3/4)-Gal and (S0 ₄ -3/4)-GalNAc sulfated groups respectively.

In an embodiment at least about 20% of the monomeric units in the glycopolymer have a pendant galactose and/or N-acetyl galactosamine group, wherein the pendant galactose group is sulfated at one or more of positions 2, 3, 4 and 6 and the N-acetyl galactosamine group is sulfated at one or more of positions 3, 4 and 6. In another embodiment at least about 30% of the monomeric units in the glycopolymer have a pendant galactose and/or N-acetyl galactosamine group, wherein the pendant galactose group is sulfated at one or more of positions 2, 3, 4 and 6 and the N-acetyl galactosamine group is sulfated at one or more of positions 3, 4 and 6. In a further embodiment at least 40%, 50%, 60%, 70%, 80%, 90% or more of the monomeric units have a pendant galactose and/or N-acetyl galactosamine group, wherein the pendant galactose group is sulfated at one or more of positions 2, 3, 4 and 6 and the N-acetyl galactosamine group is sulfated at one or more of positions 3, 4 and 6.

In an embodiment at least about 20% of the monomeric units in the glycopolymer have a pendant galactose and/or N-acetyl galactosamine group which is sulfated at position 3 and/or 4. In another embodiment at least about 30% of the monomeric units in the glycopolymer have a pendant galactose and/or N-acetyl galactosamine group which is sulfated at position 3 and/or 4. In a further embodiment at least 40%, 50%, 60%, 70%, 80%, 90% or more of the monomeric units have a pendant galactose and/or N- acetyl galactosamine group which are sulfated at position 3 and/or 4.

In another embodiment, the invention provides a glycopolymer wherein at least 20 monomeric units carry a pendant galactose and/or N-acetyl galactosamine group wherein the pendant galactose group is sulfated at one or more of positions 2, 3, 4 and 6 and the N-acetyl galactosamine group is sulfated at one or more of positions 3, 4 and 6. More preferably, at least 30 monomeric units carry a pendant galactose and/or N- acetyl galactosamine group, wherein the pendant galactose group is sulfated at one or more of positions 2, 3 , 4 and 6 and the N-acetyl galactosamine group is sulfated at one or more of positions 3, 4 and 6. The glycopolymer may contain 40, 50, 60, 70, 80, 90, 100 or more monomeric units which carry a pendant galactose and/or N-acetyl galactosamine group, wherein the pendant galactose group is sulfated at one or more of positions 2, 3, 4 and 6 and the N-acetyl galactosamine group is sulfated at one or more of positions 3, 4 and 6.

In another embodiment, the invention provides a glycopolymer wherein at least 20 monomeric units carry a pendant galactose and/or N-acetyl galactosamine group which is sulfated at position 3 and/or 4. More preferably, at least 30 monomeric units carry a pendant galactose and/or N-acetyl galactosamine group which is sulfated at position 3 and/or 4. The glycopolymer may contain 40, 50, 60, 70, 80, 90, 100 or more monomeric units which carry a pendant galactose and/or N-acetyl galactosamine group which is sulfated at position 3 and/or 4. The monomeric units carrying a sulfated galactose and/or sulfated N-acetyl galactosamine group may be located consecutively in the glycopolymer.

In another embodiment the glycopolymer includes a region wherein at least about 20% of the monomeric units in that region carry a pendant galactose and/or N-acetyl galactosamine group, wherein the pendant galactose group is sulfated at one or more of positions 2, 3, 4 and 6 and the N-acetyl galactosamine group is sulfated at one or more of positions 3, 4 and 6. In another embodiment the glycopolymer includes a region wherein at least about 30% of the monomeric units in that region carry a pendant galactose and/or N-acetyl galactosamine group, wherein the pendant galactose group is sulfated at one or more of positions 2, 3, 4 and 6 and the N-acetyl galactosamine group is sulfated at one or more of positions 3, 4 and 6. In a further embodiment a region of the glycopolymer has at least 40%, 50%, 60%, 70%, 80%, 90% or more of the monomeric units carrying a pendant galactose and/or N-acetyl galactosamine groups, wherein the pendant galactose group is sulfated at one or more of positions 2, 3, 4 and 6 and the N-acetyl galactosamine group is sulfated at one or more of positions 3, 4 and 6.

In another embodiment the glycopolymer includes a region wherein at least about 20% of the monomeric units in that region carry a pendant galactose and/or N-acetyl galactosamine group which is sulfated at position 3 and/or 4. In another embodiment the glycopolymer includes a region wherein at least about 30% of the monomeric units in that region carry a pendant galactose and/or N-acetyl galactosamine group which is sulfated at position 3 and/or 4. In a further embodiment a region of the glycopolymer has at least 40%, 50%, 60%, 70%, 80%, 90% or more of the monomeric units carrying a pendant galactose and/or N-acetyl galactosamine groups which are sulfated at position 3 and/or 4.

In a yet further embodiment, the invention provides a glycopolymer which includes a region wherein at least 20 of the monomeric units in that region carry a pendant galactose which is sulfated at one or more of positions 2, 3, 4 and 6 and/or a pendant N-acetyl galactosamine group which is sulfated at one or more of positions 3, 4 and 6, preferably the region comprises at least 30, 40, 50, 60, 70, 80, 90, 100 or more monomeric units which carry a pendant galactose and/or N-acetyl galactosamine group, wherein the pendant galactose group is sulfated at position 2, 3 , 4 and/or 6 and the N-acetyl galactosamine group is sulfated at position 3, 4 or 6.

In a yet further embodiment, the invention provides a glycopolymer which includes a region wherein at least 20 of the monomeric units in that region carry a pendant galactose and/or N-acetyl galactosamine group which is sulfated at position 3 and/or 4, preferably the region comprises at least 30, 40, 50, 60, 70, 80, 90, 100 or more monomeric units which carry a pendant galactose and/or N-acetyl galactosamine group which is sulfated at position 3 and/or 4.

The region may comprise at least 20 consecutive monomer units, preferably at least 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more monomeric units. Preferably the region comprises between about 60 and about 180 consecutive monomeric units, preferably between about 80 and about 180 consecutive monomeric units. In an embodiment the glycopolymer may comprise sufficient pendant galactose and/or N-acetyl galactosamine group, wherein the pendant galactose group is sulfated at one or more of positions 2, 3, 4 and 6 and the N-acetylgalactosamine group is sulfated at one or more of positions 2, 4 and 6 so that the polymer binds to CD206 with an affinity sufficient to inhibit the activity of CD206. In an embodiment the glycopolymer may comprise sufficient pendant galactose and/or N-acetyl galactosamine group which are sulfated at position 3 or 4 so that the polymer binds to CD206 with an affinity sufficient to inhibit the activity of CD206. Preferably the activity inhibited is inhibited by at least 10%, more preferably by at least about 25 %, 50% or more. In cells, the activity inhibited may be the ability of CD206 to be recycled, which may reduce cell uptake of CD206 ligands and could have consequences in the biological activity of the cells such as response to stimulation and migration. Preferably the inhibition is reversible, minimising potential adverse effects of CD206 inhibition. The inhibition may last for at least 12 hours, or for at least 24 hours or for at least 36 hours or more. The inhibition may last no more than 36 hours, no more than 24 hours or no more than 12 hours.

In an embodiment of the invention, the sulfated glycopolymer comprises a polymeric backbone wherein at least 90% of the monomeric units which form the backbone have a pendant galactose and/or N-acetyl galactosamine group, wherein the pendant galactose group and/or N-acetyl galactosamine group is sulfated at position 3 and/or 4.

The percentage of pendant groups in the glycopolymer can be determined by ¾ NMR.

The polymeric backbone may comprise at least 20 repeating monomeric units, preferably at least 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more wherein reference to repeating monomeric units means a monomeric unit of the backbone, for example, acrylamide or methacrylate unit. In some embodiments the backbone may comprise 30- 100 repeating monomeric units. In addition to sulfated galactose and/or sulfated N-acetyl galactosamine groups, the glycopolymer may also comprise galactose and/or N-acetyl galactosamine groups which are not sulfated.

The polymeric backbone may comprise any suitable monomeric units, this may include carbohydrates such as a sugar (polysaccharide), poly(vinyl monomer) including poly(meth)acrylate and poly(meth)acrylamide, polyester, polycarbonate, polyurethane, and polyamides. The polymeric backbone may include blocks which do not include carbohydrate pendant units, such as PEG-glycopolymers and other block copolymers. The sulfated glycopolymer may be at least about 50kDa, at least about 60kDa, at least about 70kDa, at least about 80kDa, at least about 90kDa. The sulfated glycopolymer may be less than about l OOkDa, less than about 90kDa, less than about 80kDa, less than about 70kDa, or less than about 60kDa. Alternatively, in some embodiments the sulfated glycopolymer may have an M _n of between about 1 and about 50kDa, or between about 5 and about 25kDa. In some embodiments, the sulfated glycopolymer may have a M _n of at least 60kDa.

Sulfated glycopolymers having a smaller M _n may be appropriate for instances where it is intended that the sulfated glycopolymer will cross the blood-brain barrier.

The M _n of sulfated glycopolymers according to the invention may be calculated using any standard method, this may include by determining the number of repeat units by ^l NMR or by using size exclusion chromatography (SEC).

In some embodiments, the pendant sulfated galactose and/or sulfated N-acetyl galactosamine groups are attached to the monomeric units of the polymeric backbone via a linker. Where a linker in used, all pendant groups may be attached using the same linker type, or more than one type of linker may be used.

In some embodiments, the linker is a glycoside linker. In some embodiments, the linker is an amino-terminated linker. In some embodiments, the linker includes a 1 ,2,3-triazole group. In some embodiments, the linkers may comprise - 0(CH ₂ )3 S(CH ₂ ) ₂ NH-. In some embodiments, the linker may be a chain of from 1 to 20 member atoms selected from carbon, oxygen, sulfur, nitrogen and phosphorus. The linker may be a straight chain or branched. The linker may also be substituted with one or more substituents including, but not limited to, halo groups, perfluoroalkyl groups, perfluoroalkoxy groups, alkyl groups, such Ci_ ₄ alkyl, alkenyl groups, such as Ci-4 alkenyl, alkynyl groups, such as Ci_ ₄ alkynyl, hydroxy groups, oxo groups, mercapto groups, alkylthio groups, alkoxy groups, nitro groups, azidealkyl groups, aryl or heteroaryl groups, aryloxy or heteroaryloxy groups, aralkyl or heteroaralkyl groups, aralkoxy or heteroaralkoxy groups, HO— (C=0)— groups, heterocylic groups, cycloalkyl groups, amino groups, alkyl - and dialkylamino groups, carbamoyl groups, alkylcarbonyl groups, alkylcarbonyloxy groups, alkoxycarbonyl groups, alkylaminocarbonyl groups, dialkylamino carbonyl groups, arylcarbonyl groups, aryloxycarbonyl groups, alkylsulfonyl groups, arylsulfonyl groups, -NH-NH ₂ ; =N-H; =N-alkyl; -SH; -S-alkyl; -NH-C(O)-; -NH-C(=N)- and the like. Other suitable linkers would be known to one of ordinary skill in the art. In some embodiments the pendant sulfated galactose and/or sulfated N-acetyl galactosamine groups may be attached to the backbone of the glycopolymer via a glycosylic linker connected to the anomeric carbon (C I) of the galactose and/or N- acetyl galactosamine molecule. The sulfated glycopolymer of the invention may specifically bind to CD206 also known as the mannose receptor. Avidity for CD206 can be estimated by surface plasmon resonance (SPR) analysis, to provide K _D values in the micromolar-nanomolar range. The sulfated glycopolymer may be specific for the CR domain of the CD206 receptor.

The sulfated glycopolymers of the invention may prevent or delay mannose receptor recycling. Sulfated glycopolymers of the invention may be used to treat, prevent, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduced incidence of one or more symptoms or features of ischaemia reperfusion injury (IRI), acute kidney injury (AKI), or ischaemic stroke. It will be appreciated that the terms "treat", "treatment" and "treating" as used herein means the management and care of a subject for the purpose of combating a condition, such as a disease or a disorder. The term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, such as administration of the sulfated glycopolymer to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of a subject for the purpose of combating the disease, condition, or disorder and includes the administration of the mannose receptor (CD206) modulators to prevent the onset of the symptoms or complications. The subject to be treated is preferably a mammal, in particular a human, but it may also include animals, such as dogs, cats, horses, cows, sheep and pigs. Ischaemia reperfusion injury (IRI) refers to tissue damage caused when blood supply returns to a tissue after a period of ischemia or lack of oxygen. Paradoxically whilst timely restoration of blood flow to an ischaemic organ (reperfusion) limits tissue destruction, reperfusion per se can also trigger adverse biological responses and promote tissue injury. IRI underlies various clinical diseases that result in significant morbidity and mortality, and which consume vast healthcare resources. Many of the acute kidney injury (AKI) cases recorded each year fall into this category. IRI can lead to Acute Tubular Injury (ATI) which manifests as a sudden decline in kidney function and in some cases progressive kidney failure . Outside of the kidney, IRI can lead to organ damage following myocardial infarction and embolic cerebrovascular accidents.

IRI is of particular concern after transplantation or acute ischaemic episodes in a range of organs including the kidney, liver and heart. Whilst a range of potential treatments have been investigated in preclinical and clinical trials, including drugs such as cyclosporine and hydrogen sulphide, and strategies based on hyperthermia or stem cell infusions, to date, no consistent clinically acceptable means of attenuating IRI is available. Thus devising a means to attenuate or minimise IRI represents a significant unmet clinical need that could produce substantial healthcare and economic benefits across many diverse medical specialities.

According to another aspect the invention provides the use of sulfated glycopolymers according to the invention in the manufacture of a medicament for the treatment and/or prevention of IRI, acute kidney injury, myocardial ischaemia, ischaemic stroke, cancer or autoimmune disease.

According to a further aspect the invention provides sulfated glycopolymers according to the invention for use in the treatment of IRI, acute kidney injury, myocardial ischaemia, ischaemic stroke, cancer or autoimmune disease. According to a further aspect the invention provides a method of treating IRI, acute kidney injury, myocardial ischaemia, ischaemic stroke, cancer or autoimmune disease comprising administering to a subject in need thereof a therapeutically effective amount of a sulfated glycopolymer according to the invention.

According to a further aspect the invention provides the use of sulfated glycopolymers according to the invention in the manufacture of a medicament for the treatment of a macrophage-related or mannose-binding C-type lectin receptor high expressing cell related disease.

According to a further aspect the invention provides sulfated glycopolymers according to the invention for use in the treatment of a macrophage-related or mannose-binding C-type lectin receptor high expressing cell related disease . According to a further aspect the invention provides a method of treating a macrophage-related or a mannose-binding C-type lectin receptor high expressing cell related disease comprising administering to a subject in need thereof a therapeutically effective amount of a sulfated glycopolymer according to the invention. Macrophage-related and other mannose-binding C-type lectin receptor high expressing cell-related diseases for which the glycopolymers and/or compositions herein may be used include, but are not limited to: acquired immune deficiency syndrome (AIDS), acute disseminated encephalomyelitis (AD EM), Addison's disease, agammaglobulinemia, allergic diseases, alopecia areata, Alzheimer's disease, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, arterial plaque disorder, asthma, atherosclerosis, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hypothyroidism, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticarial, autoimmune uveitis, Balo disease/Balo concentric sclerosis, Beliefs disease, Berger's disease, Bickerstaff s encephalitis, Blau syndrome, bullous pemphigoid, Castleman's disease, cardiac conditions, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal osteomyelitis, chronic obstructive pulmonary disease, chronic venous stasis ulcers, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency, contact dermatitis, cranial arteritis, CREST syndrome, Crohn's disease, Cushing's Syndrome, cutaneous leukocytoclastic angiitis, Dego's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, Diabetes mellitus type I, Diabetes mellitus type II diffuse cutaneous systemic sclerosis, Dressler's syndrome, drug-induced lupus, discoid lupus erythematosus, eczema, emphysema, endometriosis, arthritis, enthesitis-related arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic pneumonia, epidermolysis bullosa acquisita, erythema nodosum, erythroblastosis fetalis, essential mixed cryoglobulinemia, Evan's syndrome, fibrodysplasia ossificans progressive, fibrosing alveolitis (or idiopathic pulmonary fibrosis), gastritis, gastrointestinal pemphigoid, Gaucher' s disease, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's encephalopathy, Hashimoto's thyroiditis, heart disease, Henoch-Schonlein purpura, herpes gestationis (aka gestational pemphigoid), hidradenitis suppurativa, HIV infection, Hughes-Stovin syndrome, hypogammaglobulinemia, infectious diseases (including bacterial infectious diseases), idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, inclusion body myositis, ischaemia reperfusion injury, inflammatory arthritis, inflammatory bowel disease, inflammatory dementia, interstitial cystitis, interstitial pneumonitis, juvenile idiopathic arthritis (aka juvenile rheumatoid arthritis), Kawasaki's disease, Lambert- Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), lupoid hepatitis (aka autoimmune hepatitis), lupus erythematosus, lymphomatoid granulomatosis, Majeed syndrome, malignancies including cancers (e.g., sarcoma, Kaposi's sarcoma, lymphoma, leukemia, carcinoma and melanoma), Meniere's disease, microscopic polyangiitis, Miller-Fisher syndrome, mixed connective tissue disease, morphea, Mucha-Habermann disease (aka Pityriasis lichenoides et varioliformis acuta), multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (aka Devic's disease), neuromyotonia, occular cicatricial pemphigoid, opsoclonus myoclonus syndrome, Ord's thyroiditis, palindromic rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus), paraneoplastic cerebellar degeneration, Parkinsonian disorders, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonage-Turner syndrome, pars planitis, pemphigus vulgaris, peripheral artery disease, pernicious anaemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatic, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen's encephalitis, Raynaud phenomenon, relapsing polychondritis, Reiter's syndrome, restenosis, restless leg syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis, schizophrenia, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, sepsis, serum Sickness, Sjogren's syndrome, spondyloarthropathy, Still's disease (adult onset), stiff person syndrome, stroke, subacute bacterial endocarditis (SBE), Susac's syndrome, Sweet's syndrome, Sydenham chorea, sympathetic ophthalmia, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis (aka "giant cell arteritis"), thrombocytopenia, Tolosa-Hunt syndrome,) transplant (e.g., heart/lung transplants) rejection reactions, transverse myelitis, tuberculosis, ulcerative colitis, undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, urticarial vasculitis, vasculitis, vitiligo, and Wegener's granulomatosis.

The sulfated glycopolymer may be used to treat arthritis including enthesitis-related arthritis, inflammatory arthritis, rheumatoid arthritis inflammatory bowel disease, inflammatory dementia, interstitial cystitis, interstitial pneumonitis, juvenile idiopathic arthritis (aka juvenile rheumatoid arthritis), psoriatic arthritis; cancer; cardiac conditions; ischaemia reperfusion injury; stroke; transplantation; infection; and autoimmunity.

According to another aspect of the invention, a sulfated glycopolymer according to the invention may be used in organ transplantation. The sulfated glycopolymer may be used to perfuse the organ prior to transplantation, this may minimise activation of resident macrophages. The sulfated glycopolymer may be used to treat the organ recipient, it may modulate activation of macrophages recruited to the transplanted organ. The transplanted organ may be a heart or liver. According to a further aspect the invention provides the use of sulfated glycopolymers according to the invention in the manufacture of a medicament for targeting tumor- associated macrophages, such as for the treatment of cancer. According to a further aspect the invention provides sulfated glycopolymers according to the invention for use in targeting tumor-associated macrophages, such as for the treatment of cancer.

According to a further aspect the invention provides a method of treating a cancer comprising administering to a subject in need thereof a therapeutically effective amount of a sulfated glycopolymer according to the invention.

The invention also provides a pharmaceutical composition comprising a sulfated glycopolymer according to the invention and a pharmaceutically acceptable excipient.

Pharmaceutical compositions to be used comprise a therapeutically effective amount of a sulfated glycopolymer according to the invention together with one or more pharmaceutically acceptable excipients, such as carriers, diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives, antioxidants, and/or solubility enhancers. The pharmaceutical compositions can be formulated by techniques known in the art, such as the techniques published in Remington's Pharmaceutical Sciences, 20th Edition.

The glycopolymers and pharmaceutical compositions of the invention may be formulated as for oral, parenteral, such as intramuscular, intravenous, subcutaneous, intradermal, intraarterial, intracardial, intracavity, intraperitoneal, transdermal, rectal, nasal, topical, aerosol or vaginal administration. The pharmaceutical composition may be formulated as a dosage form for oral administration. The disclosed glycopolymers or compounds can be administered via any suitable method. The disclosed glycopolymers or compounds can be administered parenterally into the parenchyma or into the circulation so that the disclosed glycopolymers or compounds reach target tissues. The disclosed glycopolymers or compounds can be administered directly into or adjacent to a tumour mass. Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular individual subject may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual subject undergoing therapy.

In an embodiment, for the treatment of IRI, the sulfated glycopolymer according to the present invention may be administered systemically, i.e. intravenously.

In another embodiment, for transplantation, the sulfated glycopolymer according to the present invention may be administered directly to the organ and/or systemically. It will be appreciated that optional features applicable to one aspect or embodiment of the invention can be used in any combination, and in any number. Moreover, they can also be used with any other aspects or embodiments of the invention in any combination and in any number. This includes, but is not limited to, the dependent claims from any claim being used as dependent claims for any other claims of this application.

The invention will be further described, by means of non-limiting example only, with reference to the following figures and experimental examples. Figures 1A-E - demonstrates the structure and ligand-binding modalities of the mannose receptor/CD206 and the structure of the glycopolymers of the present invention.

Figure 1A - shows a schematic representation of the CD206 receptor. CR: Cysteine-rich domain which binds 3/4-O-sulfate galactose (S0 ₄ -3/4)-Gal) and

N-(acetyl galactosamine) (S0 ₄ -3/4)-GalNAc); FNII: collagen-binding Fibronectin type II domain; CTLD: C-type lectin-like domain which binds mannose (Man), fucose (Fuc), N-(acetyl glucosamine) (GlcNAc). Figure IB - shows the chemical structure, composition, and molecular weight of sulfated glycopolymers ( 1 ^st generation). M _n (kDa) is calculated from the number of repeating units as estimated by ^: H NMR, while D was obtained by SEC analysis. All glycopolymers were fluorescently labelled with 2,4,5,7,7'- pentafluorofluorescein (Oregon Green, l _ex/em 488/530 nm) to enable detection in in vitro assays.

Figure 1C - shows details of the polymers specifically tested with human monocyte-derived macrophages.

Figure ID - demonstrates that sulfated polymers on the invention bind to the CR domain of the mouse mannose receptor (moMR-CR or CD206-CR) . Hydrophobic S0 ₄ -3 -Gal sulfated glycopolymers known to interact specifically with the CR domain (2 μg/mL) were immobilized on wells of a 96 well ELISA plate . Fc-chimeras comprising the CR-domain, FNII domain and CTLD l -3 of mouse MR fused to the Fc region of human IgG l were added at a concentration of 2 μg/mL with or without pre-incubation with 3-O-sulfo-galactosylated polymers in a concentration range of 0.08-250 μΜ - sugar units - for inhibition assay. Binding of CD206 fragments were detected with anti-human IgG Fc- specific, alkaline phosphatase conjugate. After 30 minutes of incubation with the specific substrate plates were read at 405 nm. Data are representative of three independent experiments performed in triplicate (N=3) .

Figure IE - demonstrates that sulfated polymers on the invention, exemplified by S 100-DP I 87 bind to the CR domain of the human mannose receptor (huMR-CR). MAXISORP 96-well plates were coated with polymers (5 μg/ml in PBS) and incubated overnight at 4 °C. Wells were washed 3x with TBS buffer and huCR-Fc chimeric protein consisting of the CR-domain of human MR fused to the Fc portion of mouse IgG (Martinez-Pomares et al., 2005) was added at different concentrations (0.2, 1 , 5, 10 μg/ml in TBS) and incubated for 2 hours. Washing step was repeated 3x with TBS buffer. To detect binding anti-mouse IgG Fc-specific antibody (alkaline phosphatase conjugated and diluted 1 : 1000) was added and incubated for 1 hour. Wells were washed again 3x with TBS buffer and 2x with AP buffer. Alkaline phosphatase activity was detected using SIGMAFAST p-nitrophenyl phosphate diluted in AP buffer. OD405 was measured in a Multiskan FC (Thermo Scientific) . Data was processed in Excel and Graphpad Prism 6.

Figures 2A-D - demonstrates the internalisation of sulfated glycopolymers by CD206-presenting cells.

Figure 2A - shows flow cytometry analysis of the uptake of 3-O-sulfo- galactosylated glycopolymers (GPs) with different sugars epitope densities. CD206 ⁺ -CHO and CD206 -CHO cells were incubated with the glycopolymers ( 1.0 μg mL ^"1 ) for 30 minutes at 37°C. After washing, cells were collected and analysed by FACS . The data presented shows a reduced uptake with increasing density of S0 ₄ -3-Gal by CHO-MR cells.

Figure 2B - shows confocal microscopy analysis of CD206 ⁺ -CHO following incubation with Oregon Green (OG) tagged glycopolymers ( 100 μg mL ^"1 ) for 1 h at 37°C. Cells were stained with HOECHST (H) for nuclei and imaged under bright field (BF) and fluorescence (FL) mode.

Figure 2C - shows the uptake of glycopolymers by WT and CD206-KO macrophages quantified by FACS after incubation with 1.0 μg mL ^"1 glycopolymers solutions for 30 minutes at 37°C.

Figure 2D - shows time-dependent uptake of glycopolymers ( 1.0 μg mL ^"1 ) by CD206 ⁺ -CHO cells. After treatment at various incubation times, cells were washed and uptake of Oregon Green-tagged glycopolymers was quantified by FACS .

Figures 3A-C - demonstrate the mechanism of modulation of CD206 endocytic activity in vitro, and CD206 activity inhibition in vivo.

Figure 3A - shows the quantification of CD206 receptor at the cell surface. Cells were incubated for 120 min with 480 μΜ (S0 ₄ -3-Galioo%) 32 glycopolymers (concentration refers to individual sugar repeating units), or cell medium only (positive control cells), then CD206 was immunostained and membrane-bound CD206 quantified by FACS . Data are the means ± SD of duplicates of two independent experiments.

Figure 3B - shows the quantification of total cell CD206 (MR). Blot analysis of cell lysates from MR ⁺ -CHO cells treated with (S0 ₄ -3-Galioo%)32 solutions

480 μΜ (in sugar repeating units) for 30 or 120 minutes. Untreated MR ⁺ -CHO cells were used as controls. MR expression was estimated by optical densitometry of western blots gels. Data are representative of two independent experiments performed in triplicate.

Figure 3C - shows the pH does not affect binding of sulfated glycopolymers to the MR-CR domain. The 7.4-6.0 pH range was chosen to simulate the conditions which GP-CD206 complexes encounter by going from cell membrane to endosomes. Dissociation constant K _D values were estimated by surface plasmon resonance (SPR) analysis, using immobilized CD206, in 10 mM HEPES, 5 mM CaCl ₂ , 0.005% tween-20, 150 mM NaCI, pH 7.4, 6.5, 6. Higher values indicate more favorable dissociation of GP-CD206 (MR) complexes, which would facilitate the recycling of CD206 receptor to the cell membrane.

Figures 4A-E - demonstrate the sulfated glycopolymers of the invention inhibit CD206 endocytic activity in vitro.

Figure 4A - shows glycopolymers of the invention can direct cell uptake of gelatine mediated by CD206 in MR ⁺ -CHO through its FN II domain. Cells were pre-treated for (A) 30 minutes or (B) 2 hours with a range of glycopolymers with different sugar epitopes and molecular weight (480 μΜ in sugar binding units). Control cells were incubated over the same period with cell medium. Cells were then incubated for further 2 hours with fluorescently- tagged gelatin (texas red gelatin, 80 μg mL ^"1 ) in the presence (co-incubation) or in the absence of glycopolymers. Data is expressed as a percentage of gelatin uptake of untreated MR ⁺ -CHO control cells (right column of each panel, TR-Gelatin), as quantified by flow cytometry. Figure 4B - shows glycopolymers of the invention can reduce cell uptake of gelatine mediated by CD206 in human IL-4 treated macrophages. The IL4 treated macrophages (M-CSF+IL-4) display elevated levels of expression of the mannose receptor (MR) compared to the control macrophages (M-CSF).

Human CD 14+-monocyte-derived macrophages were grown on low adherence 24-well plates for 7 days in the presence of M-CSF or M-CSF+IL-4. Polymers were added to a final concentration of 48 μΜ, media with M-CSF (50 ng/ml) and IL-4 (25 ng/ml) was used to give each well a final volume of 1.1ml and incubated at 37 °C for 6 hours. Polymers S 100-DP 187, M 100-DP 187, G100-

DP 187, S 100-DP240 and G100-DP240 were tested (see Figure 1 C). Cells were then incubated in the presence of gelatin ( 10 μg/ml) for 1 hour at 37 °C in total volume of 1.2 ml per well. Plates were placed on ice for 20 minutes for the cells to detach. Macrophages were harvested by gently pipetting, collected into 15 ml Falcon tubes and centrifuged at 350 x g at 4 °C for 5 minutes. The number of cells was adjusted to 200.000 per 100 μΐ in X-Vivo 15. Cells were transferred to 1.5 ml Eppendorf tubes (200 μΐ/tube). Cells were washed with X-Vivo 15 and centrifuged at 1000 rpm (model Prism R), 4 °C, for 5 minutes. Another wash was done with PBS and cells were resuspended in 200 μΐ of PBS and transferred to FACS tubes containing 1 % formaldehyde in PBS .

Fluorescence quantification was done in a MoFlo Astrios (Beckman Coulter) and analysed using Kaluza software. Oregon Green channel (GPs): Laser 488nm, emission spectrum 500-526nm. Texas Red channel (Gelatine): Laser 561nm, emission spectrum 604-624nm.

The data shows that the polymers S 100-DP240 and S 100-DP I 87 inhibit uptake of collagen by IL-4-treated macrophages. Collagen is a MR-ligand so it is clear these polymers inhibit MR-mediated endocytosis in human macrophages. The effect of the S I 00 polymers is less prominent in control macrophages because these cells also express the collagen receptor Endo l 80 which is downregulated by IL4.

Figure 4C - shows inhibition of CD206 endocytic activity elicited by a single initial treatment with sulfated glycopolymers of the invention. MR ⁺ -CHO cells were incubated for 2 hours with (A) (SO ₄ -3 -Gal l 00%) ₃₂ or (B) (S0 ₄ -3 - Gal l 00%) i ₈₇ (480 μΜ in sugar binding units). Control cells were incubated over the same period with cell medium. Cells were then washed and incubated with cell medium. At a scheduled time the medium was replaced with a 80 μg ml/ ¹ of TR-Gelatin solution and incubated for 1 hour. Data is expressed as a percentage of gelatin uptake of untreated CD206 ⁺ -CHO control cells (right column of each panel, TR-Gelatin), as quantified by flow cytometry. Data were analyzed by two-way ANOVA with Bonferroni post-tests, *p<0.05, * *p<0.01.

Figure 4D - shows fluorescent microscopy of kidney and liver sections from animals injected with glycopolymer (GP) followed by TR-Gelatin and control TR-Gelatin only injected animal. In the kidney glomerulus, CD206 is expressed on mesangial cells only. The figures show in the left panel a glomerulus from a mouse injected with sulfated GP followed by TR- Gelatin, demonstrating little (red) gelatin glomerular uptake, in contrast to the galactosylated GP-treated and control mice, in which there is significant TR-Gelatin uptake into the mesangial cells of the glomerulus. In the second row, liver sections from the same animals show attenuated TR-Gelatin uptake in the sulfated GP-treated animals, in contrast to the other two control animals in which there is more substantial uptake. These data confirm that in vivo, sulfated GP are capable of inhibiting TR-Gelatin uptake into CD206 expressing cells.

Figure 4E - shows that in vivo in mice polymers of the invention co-localise with the mannose receptor in at least the liver. Wt mice were injected with OG-labelled polymer S 100-DP 187 (green) and sacrificed 1 h post injection.

Liver was collected, processed for immunolabelling for Mannose receptor (MR, grey) and analysed by confocal microscopy. Cell nucleus was labelled with DAPI (blue). The DAPI image shows cells in the liver section by staining the nucleus. MR (CD206) staining shows the location of the mannose receptor in Kupffer cells and liver sinusoidal endothelial cells. S0 ₄ -3-Gal-DP 187 fluorescence shows where the polymer has bound. The composite image overlays the S0 ₄ -3-Gal-DP 187 staining with the MR staining and shows the two co-localise. Figure 5 - shows (SO ₄ -3 -Gal l 00%) i ₈₇ reduces Acute Tubular Injury after IRI. Acute Tubular Injury (ATI) scores after IRI in mice untreated (no GP) or treated with control non-binding (Gal l 00%) ₃₂ and (Gal l 00%) i ₈₇ , or (S0 ₄ -3- Gal l 00%) i ₈₇ .

A: 200 μΜ, iv, 2 h prior IRI and 24 h after IRI.

B : 400 μΜ iv, 24 h and 2 h prior IRI and 24 h after IRI.

Dose volume in A and B : 200 μΐ

Figure 6 - demonstrates the internalisation of polymers of the invention by human macrophages. Furthermore the data demonstrates that human macrophages are not killed in the process, that the uptake occurs in a dose dependent manner and that there is more uptake in IL-4-treated macrophages (IL-4 upregulates expression of MR). This data demonstrates that the polymers of the invention may be used to target macrophages and to modulate macrophages.

Human CD 14+-monocyte-derived macrophages grown on low adherence 24- well plate for 7 days in the presence of M-CSF or M-CSF+IL-4 were placed on ice for 20 minutes to allow cells to detach. Macrophages were harvested by gently pipetting and collected into 15 ml Falcon tubes and centrifuged at 350 x g, 4 °C for 5 minutes. The number of cells was adjusted to 100.000 per 200 μΐ in X-Vivo 15 (Lonza). Cells were transferred to 1.5 ml Eppendorf tubes (200 μΐ/tube) and 200 μΐ of polymers (S 100-DP 187, M 100-DP 187 and G100-DP 187, see Figure 1 C) were added to each tube . The tubes were incubated at 37 °C for one hour. After incubation, cells were washed with 400 μΐ of X-Vivo 15 and centrifuged at 1000 rpm (model Prism R), 4 °C, for 5 minutes. The pellet was washed in 400 μΐ of ice-cold PBS and centrifuged at 1000 rpm, at 4 °C for 5 minutes. 200 μΐ of PBS was added to each tube and the content was transferred to FACS tubes containing 200 μΐ of 1 % formaldehyde in PBS . Fluorescence quantification was done in FC 500 (Beckman Coulter) and analysed using Kaluza software.

Figure 7 - demonstrates that the internalisation, as previously shown in Figure 6, is increased in the presence of IL4, which increases the expression of the mannose receptor, and is temperature dependent. That is, a temperature of 37 ^° C is needed for the internalisation of the polymers, demonstrating active uptake process. The data demonstrates that the polymers are able to target intracellular compartments of the macrophages and thus are able to change their phenotype and modulate inflammation.

Human CD 14+-monocyte-derived macrophages grown on low adherence 24- well plate for 7 days in the presence of M-CSF or M-CSF+IL-4 were placed on ice for 20 minutes to allow cells to detach. Macrophages were harvested by gently pipetting and collected into 15 ml Falcon tubes and centrifuged at 350 x g, 4 °C for 5 minutes. The number of cells was adjusted to 100.000 per 200 μΐ in X-Vivo 15 (Lonza). Cells were transferred to 1.5 ml Eppendorf tubes (200 μΐ/tube) and 200 μΐ of polymers (S 100-DP 187, M 100-DP 187 and G100-DP 187) were added to each tube. The tubes were incubated at 37 °C or 4 °C for one hour. After incubation, cells were washed with 400 μΐ of X-Vivo 15 and centrifuged at 1000 rpm (model Prism R), 4 °C, for 5 minutes. The pellet was washed in 400 μΐ of ice-cold PBS and centrifuged at 1000 rpm, at 4 °C for 5 minutes. 200 μΐ of PBS was added to each tube and the content was transferred to FACS tubes containing 200 μΐ of 1 % formaldehyde in PBS . Fluorescence quantification was done in FC 500 (Beckman Coulter) and analysed using Kaluza software.

Figure 8 - demonstrates that both 1 ^st and 2 ^nd generation sulfated polymers of the invention act on the same receptor, as demonstrated by unlabelled S I 00- DP240 inhibiting uptake of OG-labelled S 100-DP I 87 by human macrophages.

2 ^nd generation glycopolymers: synthesis described in Typical polymerization conditions (B): synthesis of (SO4-3-Gal) 180

Human CD 14+-monocyte-derived macrophages grown on low adherence 24- well plate for 7 days in the presence of M-CSF were pre-treated with DP-240 series GPs (2 ^nd generation, S 100-DP240 or G100-DP240; 20 μΜ, 40 μΜ, 80 μΜ) for 1 hour and some were left untreated. After one hour pre-treatment the Oregon Green-labelled polymer S 100-DP I 87 ( 1 ^st generation) was added at 10 μΜ to the appropriate wells. Samples were incubated for 2 h or 6 h. The plates were placed on ice for 20 min and macrophages were collected in 1.5 ml Eppendorf tubes, centrifuged at 400 x g, 4oC, 5 min. Pellets were washed with lml of ice cold X-Vivo 15 to each tube and centrifuged at 350 x g, 4°C, 5 min. Cells were washed again. Supernatant was poured off and 200 μΐ cold X-Vivo 15 was added to each tube. Cells were transferred to FACS tubes containing 200 μΐ of 1 % formaldehyde in PBS . Cells were analysed on FACS500.

As can be seen from the graphs, polymer S 100-DP240 (which is unlabelled) inhibits association of S 100-DP I 87 (which is labelled with Oregon green) with human macrophages. This effect is not seen with the control G100-DP240. These results demonstrate that both S 100-DP I 87 and S 100-DP240 compete for the same receptor.

Figure 9 - demonstrates the effect of polymers of the invention of macrophage activation. Figure 9A summarises the hypothesis of the involvement of macrophages and IRI. Figure 9B shows the effect of polymers of the invention in an in vitro system intended to mimic macrophage activation.

To test if S 100-DP I 87 polymers could modulate activation of human macrophages in response to IFN-γ (M l -inducer), human macrophages grown on low adherence 24-well plate for 6 days in the presence of M-CSF were treated to give six conditions: Untreated; S 100-DP 187-treated; G100-DP 187- treated; IFN-γ treated; IFN-γ + S 100-DP 187-treated; IFN-γ + G100-DP 187- treated. Polymers were added to a final concentration of 40μΜ and IFN-γ at l Ong/ml, media with M-CSF (50ng/ml) was used to give each well a final volume of 1. 1ml and incubated at 37 °C for a further 24 hours. Cells were then harvested on day seven and analysed by flow cytometry for surface expression of MR, CD l ib and MHCII. S 100-DP I 87 polymers reduced surface expression of MR and CD l ib in untreated and IFN-y-treated macrophages and inhibit upregulation of MHCII surface expression in response to IFN-γ.

The data presented shows that in the presence of the sulfated polymers the concentration of mannose receptor on the surface of macrophages decreases. Whereas, with a non-sulfated polymer the level of mannose receptor is unchanged. The sulfated polymers are changing the macrophage phenotype by trapping the mannose receptors within the cell. The polymers also change the surface expression levels of CD l ib and MHCII. Normally, in the presence of IFNy macrophages increase the level of MHCII expression on their surface . However, in the presence of the sulfated polymers of the invention no increase in MHCII expression is observed. This further demonstrates that sulfated polymers of the invention prevent the normal activation of macrophages and that the polymers may be used to modulate macrophages and inflammation. For example, the polymers could be used to treat inflammation driven diseases, perhaps by preventing the activation of M l macrophages, this may be useful in the treatment of IRI.

Materials and Methods

Synthesis of sulfated glycopolymers

Trimethylsilyl propargyl methacrylate (1)

Trimethylsilyl propargyl methacrylate was synthesized starting from 3 -(trimethylsilyl) propargyl alcohol following the method described by Ladmiral, V. et al., Journal of the American Chemical Society 2006, 128 ( 14), 4823-4830. Synthesis of N-(Ethyl)-2-pyridylmethanimide (2)

N-(Ethyl)-2-pyridylmethanimide was prepared as described by Haddleton, D.

al, Macromolecules 1999, 32 (7), 21 10-21 19.

Maleimide-protected initiator (3)

The maleimide-protected initiator was synthesized as previously reported by Mantovani, G. et al, Journal of the American Chemical Society 2005, 127 (9), 2966- 2973.

Synthesis of 6-azido-2,4,5,7,7'-pentafluorofluorescein (4)

2,4,5 ,7,7'-hexafluorofluorescein was prepared as reported by Sun, W.-C. et al. , The Journal of Organic Chemistry 1997, 62 ( 19), 6469-6475.

A solution of 2,4,5,6,7,7'-hexafluorofluorescein (490 mg, 1. 1 1 mmol) in acetone was added to a solution of NaN ₃ (86.9 mg, 1.33 mmol) in water ( 1 inL) under stirring. The reaction mixture was heated under reflux at 50°C for 20 hours. The progress of the reaction was monitored by TLC. When the reaction resulted almost complete, acetone was removed under reduced pressure and the aqueous phase freeze-dried. The crude was purified by flash chromatography (silicagel 60, 35-70 μιη) using 100% EtOAc and EtOAc/MeOH 1 : 1 as mobile phases. Fractions containing the desired product were combined and concentrated under vacuum to obtain an orange powder (230 mg, 0.50 mmol, 45%).

ESI-TOF Mass Spectrometry: expected m/z [M-H] ⁺ 464.02, found 464.0279; 435.018 1 (-N ₂ ). FT-IR: v 3416, 3228, 2361 , 2140, 1617, 1557, 1497, 1339, 1300, 1 150, 992, 843, 599, cm ^"1 . *H NMR (400 MHz, MeOD, 298 K) δ = 6.78 (s, 1H, Ar); 6.80 (s, 1H, Ar); 6.93 (s, 1H, Ar); 6.95 (s, 1H, Ar). ¹⁹ F NMR (400 MHz, MeOD, 298 K) δ = - 130.82 (bs , 1 F); - 136.09 (bs, 2F); - 143.64 (m, IF); - 144.03 (m, IF) . ¹ C { ¾} NMR (400 MHz, MeOD, 298 K) δ = 167.03, 166.50, 155.71 , 155.60, 153.22, 150.07, 147.64, 146.67, 145.1 1 , 144.96, 135.79, 134.74, 126.86, 121.22, 1 17.61 , 1 13.93, 1 13.86, 1 13.08, 1 12.86, 105.96. Synthesis of 2'- Azidoethyl -O— D-galactopyranoside (6)

The synthesis of 2-azido ethyl galactose was performed as first reported by Chernyak, A. Y., et al, Carbohydrate Research 1992, 223 (0), 303-309.

Synthesis of 3-0-sulfo-2'- azidoethyl -0~D-galactopyranoside (7)

3-O-sulfo-galactose (7) was synthesized adopting a modified version of the method reported by Manning, D. D., et al , Journal of the American Chemical Society 1997, 119 ( 13), 3 161 -3 162 and Uzawa, H., et al , Chemical Communications 1998, (21), 23 1 1 -23 12. To a solution of 2 '-azidoethyl-0--D-galactopyranoside ( 1.00 g, 4.02 mmol) in MeOH/EtOAc 2: 1 ( 15 mL) and phenylboronic acid (490 mg, 4.02 mmol) were added. A Dean-Stark apparatus, pre-filled with EtOAc, was set up and the reaction mixture was stirred under reflux. After 2 h, the solution was concentrated to 5 mL, a fresh portion of MeOH/EtOAc ( 10 mL) and dibutiltyn oxide ( 1.26 g, 4.42 mmol) were added and the reaction was carried on with the Dean-Stark apparatus under reflux for further 2 h. The solvent was then completely removed under reduced pressure. The intermediate was dissolved in 5 mL of anhydrous DMF and added of trimethylamine sulfur trioxide (700 mg, 5.03 mmol). After ON stirring, MeOH (5 mL) was added to quench the reaction. The solvent was reduced under vacuum, the residue added of a second portion of MeOH (5 mL) and stirred. After 10 minutes, the crude product was precipitated in Et ₂ 0, redissolved in methanol and precipitated again in Et ₂ 0 to completely remove DMF. The obtained precipitate was added of MeOH (5mL), diluted with water ( 10 mL) and extracted with Et ₂ 0 (3xl 0mL). The aqueous layer was then added of anionic exchange resin (Dowex Na ⁺ , previously washed with methanol until clear solution). The mixture was stirred for 15 minutes and then the resin was removed by filtration. The aqueous solution was freeze-dried. The crude product was purified by flash chromatography (silicagel 60, 35-70 μιη, gradient elution 9:1 to 7:3 EtOAc/MeOH). Fractions were analyzed by TLC and the appropriate were concentrated to give the 3-0-sulfo-2'-azidoethyl-0-a,p-D-galactopyranoside (1.17 g, 3.33 mmol, 83%). ESI-TOF Mass Spectrometry: expected m/z [M] ^~ 328.05, found 328.0246. FT-IR: v 3434, 2118, 1635, 1235, 1151, 1063, 990 cm ^"1 . Ή NMR (400 MHz, D ₂ 0) δ 3.4-3.58 (m, 2H, CH ₂ N ₃ ); 3.58-3.74 (m, 2Η, CH ₂ CH ₂ N ₃ ); 3.88-3.98 (m, 2H, CH ₂ OH); 4.10-4.32 (m, 4H, 4xCH); 4.36 (m, 1H, CH); 5.02 (d, 1H, J = 3.9 Hz, CH). "C^H] NMR (100.59 MHz, D ₂ 0, 298 K) δ = a 50.4 (1C, CH ₂ N ₃ ); 66.1 (1C, CH ₂ OH); 66.9 (1C, CH); 67.4 (1C, OCH ₂ CH ₂ Br); 67.6 (1C, CH); 68.8 (1C, CH); 77.4 (1C, CH); 98.3 (1C, Canomeric). β = 50.6 (1C, CH ₂ N ₃ ); 60.9 (1C, CH ₂ OH); 61.7 (1C, CH); 66.9 (1C, OCH ₂ CH ₂ Br); 67.1 (1C, CH); 72.4 (1C, CH); 80.0 (1C, CH); 102.6 (1C, anomeric).

Synthesis of poly(propargyl methacrylate): General polymerization procedure (A) and removal of Si(CH ₃ ) ₃ protecting group

In a typical polymerization trimethylsilylpropargylmethacrylate (1) (2.00 g, 10.2 mmol), N-(ethyl)-2-pyridylmethanimine ligand (2) (4.5 mg, 0.34 mmol) and maleimide-protected initiator (3) (30.4 mg, 0.849 mmol) were dissolved in anhydrous toluene (6 mL) and transferred in a Schlenk tube. The tube was sealed with rubber septum and subjected to five freeze-pump-thaw cycles. A second Schlenk tube containing Cu(I)Br (24 mg, 0.17 mmol) was evacuated and filled with nitrogen. The toluene solution was cannulated into the second tube then placed into an oil bath and maintained at 30°C under stirring. The polymerization kinetic was monitored by scheduled withdraw of samples analyzed by ^: H NMR and GPC. The polymerization was stopped at 70% of conversion by opening the tube to the air. At the end of the polymerization the polymer solution was passed through a short pad of alumina, the column was washed several times with toluene and the solvent removed under reduced pressure .

In case of the backbone with DP32 (m = 32) the trimethylsylyl group deprotection was performed as follows: the residue (2.0 g, 10 mmol of alkyne units) was dissolved in THF (20 mL), added of acetic acid (0.876 mL, 15.3 mmol) and degassed under N ₂ bubbling for 15 minutes. The reaction mixture was cooled to -20 °C and a 1 M TBAF solution in THF ( 15.3 mL, 15.3 mmol) was added dropwise . The reaction was stirred overnight at RT and the deprotection was monitored by ^: H NMR in CDC1 ₃ (disappearance of trimethylsilyl signal at 0.3 ppm). When the reaction was gone to completion the solvent was reduced under vacuum and the polymer precipitated in a mixture of MeOH/water 1 : 1 to remove TBAF. The procedure was repeated twice . The polymer residue was dried under reduced pressure . In case of the backbone with DP 187 (m = 187) the trimethylsylyl group deprotection was performed as follows : the residue (2.0 g, 10 mmol of alkyne units) was dissolved in THF (20 mL), added of acetic acid (0.876 mL, 15.3 mmol) and degassed under N ₂ bubbling for 20 minutes. The mixture was cooled to -20°C and a 1 M TBAF solution in THF ( 15.3 mL, 15.3 mmol) was added dropwise by syringe. The reaction solution was maintained under stirring overnight and allowed to warm up to RT. The reaction mixture was then washed three times in water-EDTA/Et ₂ 0 (400 mL) to remove copper. The organic layers were collected, dried over MgS0 ₄ and the volatile removed under reduced pressure. Amberlite IR- 120 ion-exchange resin (previously washed several times in methanol until clear solution) was then added and the resulting slurry and stirred for 30 minutes. After resin removal by filtration, the organic solvent was concentrated under reduced pressure and the deprotected polymer precipitated into petroleum ether.

Synthesis of Oregon green labeled sulfated glycopolymers.

Fluorescent dye and sugars were attached to the polymer backbone by clicking the required components to the poly (propargyl methacrylate) polymeric precursor. As an example the preparation of Oregon green 100% galactose sulfate glycopolymer (S0 ₄ - 3-Galioo%) 32 is described below.

In a typical procedure, a solution of deprotected polymer scaffold (DP=32, 300 mg, 2.42 mmol of clickable alkyne units), Oregon green azide ( 1 1.2 mg, 0.0242 mmol) and bipyridine ( 152 mg, 0.967 mmol) in DMF ( 15 mL) were degassed by nitrogen bubbling for 15 minutes. CuBr (I) (69.5 mg, 0.484 mmol) was then added to the reaction mixture and the solution bubbled under nitrogen for further 15 minutes. The deep purple solution was stirred at room temperature for 3 days. The reaction was monitored by SEC with visible (λ 496 nm) and RI detection. A 5 mL aliquot was withdrawn, and to this 3-0-sulfo-2 '- Azidoethyl azidoethyl -O -galactopyranoside (7) (3 19 mg, 0.968 mmol) and degassed for 15 minutes under nitrogen. A solution of sodium ascorbate (32 mg, 0. 16 mmol) in water ( 100 μί) was added to the mixture by syringe and the reaction was stirred at room temperature for 2 days. The glycopolymer was precipitated in THF, isolated by centrifugation, re-dissolved in water, transferred into a dialysis membrane (MWCO 3.5 kDa) and dialyzed in the dark against water for 3 days. The glycopolymer water solution was then freeze-dried to give (S0 ₄ -3- Galioo%) 32 glycopolymer as a pink solid. Preparation of the gelatin-Texas Red conjugate (8)

Gelatin-Texas Red conjugate was prepared following the procedure described by Hummert, E., et al , Cellulose 2013, 20 (2), 919-93 1 . A 10 mg mL ^-1 gelatin solution was prepared by dispersing 100 mg of gelatin in 10 mL of 0. 1 M sodium bicarbonate buffer, pH 9, at RT. The suspension was left under mild stirring for 3 hours . Then the mixture was gently heated under stirring in a water bath at 45 °C, until gelatin complete dissolution. A stock solution of Sulforhodamine 101 acid chloride (Texas Red) was prepared dissolving 2.5 mg of the fluorescent dye in 250 μΐ. of anhydrous MeCN.

2 mL aliquots of mg mL ^"1 gelatin solution were cooled down to 4° in ice/water bath. Under moderate stirring, 180 of Sulforhodamine 10 1 acid chloride (Texas Red) stock solution were added. The solutions were stirred overnight in the dark and then dialyzed against 4 L of 10 mM sodium phosphate, 154 mM NaCl, pH 7.4, for 2 days, with at least 4 buffer exchanges . After complete removal of the unreacted Texas Red, the solutions were freeze-dried and stored at -20°C until use .

Synthesis of 2,3-dihydroxypropyl (l-oxo-l -(prop-2-yn-l -ylamino)propan-2-yl) carbonotrithioate Chain Transfer Agent (9)

Synthesis of 2-bromo-N-(prop-2-yn-l -yl)propanamide (9a). Propargylamine (2.00 g, 35.8 mmol) and triethylamine (5.44 g, 53.8 mmol) were dissolved in 100 mL of anhydrous THF and cooled down in an ice bath. Bromopropionyl bromide (8.5 g, 39 mmol) was added dropwise to the solution under stirring . The reaction mixture was stirred at room temperature for 18 hours, then the precipitate of triethylammonium bromide was filtered off and the solvent removed under reduced pressure . The crude product was re-dissolved in Et ₂ 0 ( 100 mL), extracted with DI water (3x 100 mL), acidified to pH 3 with 2M H ₂ S0 ₄ and finally washed with water (2x 100 mL) . The organic layer was dried over MgS0 ₄ and after the solvent was evaporated 9a was isolated as colorless oil (4.7 g, 25 mol, 69%) . ESI-TOF Mass Spectrometry: expected m/z [M-Na] 211.9706, found 211.9681. FT- IR: v 3278, 3064, 2927, 2851, 1749, 1626, 1540, 1446, 1374, 1310, 1243, 1187, 1131, 1076, 1052, 1007, 974, 890 cm ^"1 . ¾ NMR (400 MHz, CDC1 ₃ ) δ 1.88 (d, 3H, J = 7.1 Hz, CH ₃ CH); 2.27 (t, 1H, J = 2.6 Hz, CHCCH ₂ ); 4.04-4.09 (m, 2H, CH ₂ ); 4.42 (q, 1Η, J = 7.1 Hz, CHCH ₃ ); 6.62 (bs, 1H, NH). "C^H} NMR (100.59 MHz, CDC1 ₃ , 298 K) δ = 22.88 (IC, CH ₃ CH); 29.97 (IC, CH ₂ CCH); 44.27 (IC, CHCH ₃ ); 72.08 (IC, CHCCH ₂ ); 78.90 (IC, CCH ₂ ); 169.20 (IC, CONH).

Synthesis of sodium 2, 3-dihydroxypropyl carbonotrithioate (9b).

1-thyoglycerol (10.0 g, 92.5 mmol) was dissolved in anhydrous THF (70 mL), the solution was cooled to 10°C in a water bath and a 60% w/w dispersion of NaH in mineral oil (3.7 g, 92 mmol) was added portion-wise. After each addition the suspension was degassed under N ₂ for 10 minutes. The reaction mixture was left under stirring for 1 hour, then CS ₂ (14.1 g, 185 mmol) was slowly added drop-wise. After 18 hour the organic solvent was removed under reduced pressure and the excess CS ₂ was removed under high vacuum. The crude product was re-dissolved in methanol and precipitated in Et ₂ 0 to obtain 9b as a yellow viscous oil (15 g, 73 mmol, 79%).

FT-IR: v 3248, 2932, 1626, 1421, 1331, 1274, 1058, 990, 819, 920, 878, 819 cm ^"1 . Ή NMR (400 MHz, DMSO-d ₆ ) δ 2.89 (dd, 1H, J = 13.3, 6.6 Hz, CH ₂ S); 2.89 (dd, 1Η, J = 13.3, 4.6 Hz, CH ₂ S) 3.81 (m, 2Η, CH ₂ OH); 3.93 (m, 1H, CHOH); 5.01 (t, 1H, J = 5.7, CH ₂ OH); 5.26 (d, J = 1Η, J= 5.2 CHOH). "C^H} NMR (100.59 MHz, DMSO-d ₆ , 298 K) δ = 44.23 (IC, CH ₂ S); 64.95 (IC, CHOH); 71.06 (IC, CH ₂ OH); 184.96 (IC, CS).

Synthesis of 2, 3-dihydroxypropyl (l-oxo-l-(prop-2-yn-l-ylamino)propan-2-yl) carbonotrithioate RAFT Agent (9).

A solution 2-bromo-N-(prop-2-yn-l-yl)propanamide (9a, 1.00 g, 5.26 mmol) in 2.5 mL of anhydrous THF was added drop-wise to a suspension of sodium 2,3- dihydroxypropyl carbonotrithioate (9b, 910 mg, 4.44 mmol) in anhydrous THF (2.5 mL). The reaction mixture was left under stirring at RT for 72 hour. The solvent was evaporated under reduced pressure and the crude product was purified by flash chromatography (silicagel 60, 35-70 μιη, gradient elution 7:3 to 3:7 Petroleum Ether/EtOAc). Fractions were analyzed by TLC and the appropriate were concentrated to give the desired product as yellow sticky solid (330 mg, 1.13 mmol, 25.5% mol/mol).

ESI-TOF Mass Spectrometry: expected m/z [M-Na] ⁺ 316.01, found 316.01. FT-IR: v 3274, 3071, 2923, 2119, 1644, 1545, 1448, 1420, 1329, 1241, 1065, 1039, 923, 881, 821 cm ^"1 . Rf 0.22 (Petroleum ether/EtOAc 3:7). ¾ NMR (400 MHz, DMSO-d ₆ ) δ 1.49 (d, 3H, J = 7.1 Hz, CH ₃ CH); 3.19 (t, 1H, J = 2.5 Hz, CCHCH ₂ NH); 3.33 (m, 2H, CH ₂ OH); 3.63 (m, 1H, CHOH); 3.66 (m, 2H, CH ₂ CHOH); 3.88 (dt, 2H, J = 5.5, 2.8, NHCH ₂ C); 4.72 (q, 1Η, J = 7.1 Hz, CHCH ₃ ); 4.83 (bs, 1H, OH); 5.25 (bs, 1Η, OH); 8.8 (t, 1Η, J = 5.4, ΝΗ). ¹ 0{Ή} NMR (100.59 MHz, DMSO-d ₆ , 298 K) δ = 18.33 (CH ₃ CH); 29.84 (1C, CH ₂ NH); 41.34 (1C, CH ₂ S); 50.67 (CHCH ₃ ); 66.16 (1C, CHOH); 71.15 (1C, CH ₂ OH); 72.42 (1C, CHCCH ₂ ); 80.13 (1C, CCH); 172.57 (1C, CO); 223.95 (1C, CS).

Synthesis of D-galactosyloxyethyl acrylamide (10)

Synthesis of 2,3,4, 6-O-Tetraacetyl-D-galactosyloxyethyl acrylamide (10a). β-D-Galactose pentaacetate (50.0 g, 128 mmol) was dissolved in anhydrous CH ₂ C1 ₂ (100 mL) and N-hydroxyethyl acrylamide (22.1 g, 192 mmol) was added under stirring. BF ₃ Et ₂ 0 (27.3 g, 192 mmol) was slowly added to reaction mixture drop-wise. The reaction was left under stirring at RT for 48 hours. The reaction mixture was then poured into sat. aq. NaHC0 ₃ (250 mL), washed with sat. NaHC0 ₃ (2x250 mL), water (2x250mL), dried over MgS0 ₄ and evaporated under reduced pressure. The crude product was purified by flash chromatography (silicagel 60, 35-70 μιη, gradient elution 100% Et ₂ 0 to 10% EtOAc in Et ₂ 0) to yield compound 10a as white crystals (13.5 g, 30.3 mmol, 23.7%).

ESI-TOF Mass Spectrometry: expected m/z [M-H] ⁺ 446.16, found 446. FT-IR: v 3258, 1744, 1645, 1610, 1545, 1499, 1363, 1267, 1247, 1171, 1133, 1092, 1038, 998,

933, 901, 812737, 707 cm ^"1 . *H NMR (400 MHz, DMSO-d ₆ ) δ 1.91 (s, 3H, CH ₃ CO);

1.99 (s, 3Η, CH ₃ CO); 2.00 (s, 3Η, CH ₃ CO); 2.11 (s, 3Η, CH ₃ CO); 3.27 (dt, 2Η, J =

9.3, 6.6 Hz, OCH ₂ CH ₂ NH); 3.66 (m, 2H, OCH ₂ CH ₂ NH); 4.04 (m, 2H, CH ₂ OCOCH ₃ );

4.19 (dd, 1H, J = 7.2, 6.3 Hz, CH); 4.72 (d, 1Η, J = 8 Hz, CH _ammeiic ); 4.93 (dd, 1H, J = 10.4, 8 Hz, CH); 5.14 (dd, 1Η, J = 10.4, 3.6 Hz, CH); 5.25 (dd, 1Η, J = 3.5, 0.8 Hz,

CH); 5.57 (dd, 1Η, J = 10.1, 2.3 Hz, CH ₂ CH); 6.07 (dd, 1H, J = 17.1, 2.3 Hz, CH ₂ CH);

6.22 (dd, 1H, J = 17.1, 10.1 Hz, CHCH ₂ ); 8.11 (t, 1H, J = 5.5, NH). "C^H} NMR

(100.59 MHz, DMSO-d ₆ , 298 K) δ = 20.32 (IC, CH ₃ ); 20.39 (IC, CH ₃ ); 20.47 (2C,

CH ₃ ); 38.63 (IC, CH ₂ NH); 61.28 (IC, CH ₂ OCOCH ₃ ); 67.34 (IC, CH ₂ CH ₂ NH); 67.66 (IC, CHOCOCH ₃ ); 68.53 (IC, CHOCOCH ₃ ); 69.89 (IC, CHOCOCH ₃ ); 70.27 (IC,

CHOCOCH ₃ ); 99.98 (IC, C _anomeric ); 125.09 (IC, CH ₂ CH); 131.62 (IC, CHCH ₂ );

164.73 (IC, NHCO); 169.13 (IC, COCH ₃ ); 169.48 (IC, COCH ₃ ); 169.88 (IC,

COCH ₃ ); 169.92 (IC, COCH ₃ ). Synthesis of D-galactosyloxyethyl acrylamide (10).

2,3,4,6-O-Tetraacetyl-D-galactosyloxyethyl acrylamide 10a (13.5 g, 30.3 mmol) was dissolved in a KOH (170 mg, 3.03 mmol) solution in MeOH (150 mL). The mixture was left under stirring at RT for 18 hours, then was passed through a short pad of Si0 ₂ , the solvent was removed under reduced pressure and compound 10 was isolated as white powder (8.32 g, 30.0 mmol, 99%).

ESI-TOF Mass Spectrometry: expected m/z [M-Na] ⁺ 300.1054, found 300.1021. FT- IR: v 3292,2928, 2885,1654, 1621, 1542, 1408, 1317, 1247, 1038981, 891, 777, 698 cm ^"1 . *H NMR (400 MHz, DMSO-d ₆ ) δ 3.42 (m, 8H, CH ₂ NH, CH ₂ OH, CHCH ₂ OH, CHOH and OCH ₂ CH ₂ NH); 3.62 (bs, 1H, CHOH); 3.74 (m, 1H, CHOH); 4.09 (d, 1H, J = 7.1 Hz, CH _anomeric ); 4.37 (d, 1Η, J = 4.4 Hz, OH); 4.61 (t, 1Η, J = 5.5 Hz, OH); 4.73 (bs, 1Η, OH); 4.86 (d, 1Η, J = 3.0 Hz, OH); 5.58 (dd, 1Η, J = 10.1, 2.2 Hz, CH ₂ CH); 6.08 (dd, 1H, J = 17.1, 2.2 Hz, CH ₂ CH); 6.24 (dd, 1H, J = 17.1, 10.1 Hz, CHCH ₂ ); 8.11 (t, 1H, J = 5.5 Hz, NH). "C^H} NMR (100.59 MHz, DMSO-d ₆ , 298 K) δ = 38.95 (1C, CH ₂ NH); 60.53 (1C, CH ₂ OH); 67.75 (1C, CH ₂ CH ₂ NH); 68.18 (1C, CHOH ₃ ); 70.60 (1C, CHOH); 73.32 (1C, CHOH ₃ ); 75.30 (1C, CHOH); 103.78 (1C, Canomeric); 125.26 (1C, CH ₂ CH); 131.71 (1C, CHCH ₂ ); 164.74 (1C, NHCO).

Synthesis of 3-0-sulfo-D-galactosyloxyethyl acrylamide (11)

D-galactosyloxyethyl acrylamide 10 (5.00 g, 18.0 mmol) was dissolved in extra-dry MeOH (30 mL) and phenylboronic acid (2.2 g, 18 mmol) was added to the flask followed by the addition of EtOAc (15 mL). A Dean-Stark apparatus was attached to the reaction vessel and filled with EtOAc. The reaction mixture was heated to reflux and maintained under stirring for 3 hours. The mixture was then cooled down to RT and the volume was reduced to 10 mL. A fresh portion of EtOAc (15 mL) was added together with dibutyltyn oxide (5.66 g, 19.9 mmol) and the mixture was heated to reflux for further 2 hours under stirring. The reaction mixture was then cooled to RT, the solvent was evaporated under reduced pressure, a fresh portion of EtOAc (20 mL) was added to the dry mixture and then completely removed under reduced pressure. The crude product was dissolved in anhydrous DMF (15 mL) and sulfur trioxide trimethylamine complex (3.14 g, 22.6 mmol) was added in one portion under stirring. The suspension was left under stirring at RT for 18 hours, then MeOH (15 mL) was added and the reaction was stirred for further 20 minutes. The volatile solvent was removed under reduced pressure, 20 mL of MeOH were added again and the solution was precipitated in Et ₂ 0. The crude product was dissolved in DI water (100 mL), filtered, and washed with Et ₂ 0 (3xl00mL). The aqueous layer was then added of NaHC0 ₃ (1.52 g, 18.0 mmol) and degassed for 1 hour by bubbling N ₂ . The solution was added of Si0 ₂ (10 g) and freeze-dried. The crude product was purified by flash chromatography (silicagel 60, 35-70 μιη, gradient elution 1% DI water in MeOH to 2.5% DI water in MeOH) to yield compound 11 as white powder (2.7 g, 6.6 mmol 36.7%). Rf 0.96 in MeOH 1% water. ESI-TOF Mass Spectrometry: expected m/z [M] ^~ 356.07, found 356.06. FT-IR: v 3333, 1655, 1554, 1478, 1045, 981, 797cm ^"1 . ¾ NMR (400 MHz, D ₂ 0) δ 3.54 (dd, 2H, J = 10.6, 3.8 Hz, CH ₂ NH); 3.68 (dd, 2H, J = 9.6, 8.0 Hz, C _an CHOH); 3.75 (m, 1H, CHCH ₂ OH); 3.78 (m, 2H, CH ₂ OH); 3.95 (m, 1H, OCH ₂ CH ₂ NH); 4.30 (d, 1H, J = 3.3 Hz, CHOH); 4.33 (d, 1H, J = 9.7, 3.3 Hz, CHOH); 4.53 (d, 1H, J = 7.9 Hz, CH _ammeiic ); 5.77 (dd, 1H, J = 10.1, 1.3 Hz, CH ₂ CH); 6.08 (dd, 1H, J = 17.1, 1.3 Hz, CH ₂ CH); 6.24 (dd, 1H, J = 17.1, 10.0 Hz, CHCH ₂ ); 8.11 (t, 1H, J = 5.5 Hz, NH). NMR (100.59 MHz, D ₂ 0, 298 K) δ = 40.05 (1C, CH ₂ NH); 61.48 (1C, CH ₂ OH); 67.48 (1C, CH ₂ CH ₂ NH); 68.98 (1C, CHOH); 69.48 (1C, CHOH); 75.73 (1C, CHOH); 80.87 (1C, CHOS0 ₃ ^" Na ⁺ ); 103.20 (1C, C _anomeric ); 127.98 (1C, CH ₂ CH); 130.52 (1C, CHCH ₂ ); 169.30 (1C, NHCO).

Typical polymerization conditions (B): synthesis of (S0 ₄ -3-Gal)i ₈ o

3-O-sulfo-D-galactosyloxyethyl acrylamide (1.65 g, 3.97 mmol) was placed in a Schlenk tube equipped with a magnetic bar and dissolved in 1.52 mL of milliQ water. 64.7 μΐ _^ (6.47 mg, 22.1 μιηοΐ) of a freshly prepared 100 mg mL ^"1 dioxane stock solution of 2,3-dihydroxypropyl (l-oxo-l-(prop-2-yn-l-ylamino)propan-2-yl) carbonotrithioate RAFT Agent CTA (9) were added to the reaction vessel. The tube was sealed with a rubber septum and degassed by bubbling argon at RT for 20 minutes. In a separate vial, a freshly made 1 mg mL ^"1 stock solution of VA-044 in milliQ water was degassed by bubbling argon at RT. After 20 minutes, 71.4 of VA-044 stock solution (71.4 μg, 0.221 μιηοΐ) were added to the Schlenk tube via gastight syringe to yield a molar ratio of monomer: CTA: initiator of 180: 1: 0.01. The polymerisation was started by immersing the tube in an oil bath pre-heated at 60°C. After 2 hours the monomer conversion was checked by ^: H NMR analysis in DMSO-d ₆ of aliquots withdrawn from the reaction vessel. When required, consecutive addition of VA-044 stock solution aliquots, prepared as described above, were performed until at least 90% of monomer conversion was reached. At the end of the polymerisation reaction, the mixture was cooled down to RT and diluted to 10 mL with milliQ water. Ethanolamine (27 mg, 27. 1 μί, 448 μιηοΐ) was added to the mixture maintained under stirring and after 5 minutes it was followed by the addition of iodoacetic acid (250 mg, 1.34 mmol). The solution was stirred at RT for 18 hours, and then further diluted with water (final volume 15 mL) and dialysed with a 1 kDa MWCO regenerated cellulose membrane against 5 L of DI water for 3 days, with at least 2 water exchange per day. The aqueous solution inside the dialysis bag was then freeze-dried to yield (S0 ₄ -3-Gal) i8o as white powder (912 mg, 13.4 μιηοΐ, 61.2% mol/mol).

Polymer labelling with Oregon green azide

Glycopolymers were fluorescently labelled via the CuAAC click reaction. In a typical procedure, (Gal) i ₈₀ (630 mg, 13.9 μιηοΐ) was dissolved in 8 mL of milliQ water, added of CuS0 ₄ pentahydrate ( 1.74 mg, 6.97 μιηοΐ) and degassed by bubbling nitrogen for 20 minutes. A stock solution of Oregon Green Azide ( 16.8 mg mL ^"1 ) was prepared in MeOH/Acetone 1 : 1 and 768 μΐ _^ ( 12.9 mg, 27.9 μιηοΐ) were added to the polymer solution. In a separate vial, a freshly made 10 mg mL ^"1 stock solution of sodium ascorbate in milliQ water was degassed by bubbling argon for 20 minutes. 400 μΐ _^ of sodium ascorbate degassed solution (4.2 mg, 21 μιηοΐ) were finally added via gastight syringe to the reaction mixture . The reaction was left under stirring at RT and monitored every 24 hours by size exclusion chromatography using PBS as the mobile phase, with simultaneous UV-vis (λ = 488 nm) and RI detection.

When the reaction reached completion the solution was further diluted with 5 mL of water, transferred into a dialysis bag ( 1 kDa MWCO) and dialysed against 5 L of water for 6 days to remove the excess of the dye. The polymer solution was then freeze-dried to give (Gal) i ₈₀ as an orange powder (600 mg, 13.3 μηιοΐ, 96.1% mol/mol).

In case of sulfated glycopolymers the conditions adopted were slightly different, mixture of milliQ watenDMF 1 : 1 was used as solvent for the reaction and a mol ratio alkyne : CuS0 ₄ pentahydrate : sodium ascorbate of 1 :2:4 was used.

Table I Glycopolymer main features.

Code Mn _theor (kDa) M _n;SEC (kDa) M _W;SEC (kDa) PDI

1 ^st generation a

(Galioo% )32 12.3 14.6 ^b 18.7 ^b 1.30 ^b

(Galioo% ) i87 70.2 29.2 ^b 44.8 ^b 1.54 ^b

(S0 ₄ -3-Gal ₃₃ % )32 13.4 1 1.6 ^c 14.2 ^b 1.22 ^c

(S0 ₄ -3-Gal ₆₆ o _/o ) ₃ 2 14.5 12.8 ^c 15.3 ^C 1.20 ^c

(S04-3-Galioo% )32 15.6 14.2 ^C 16.8 ^C 1.18 ^c

(SO4-3-Gali00% ) l87 89.3 26.0 ^C 35.5 ^C 1.36 ^c

Code

2 ^nd generation

(Gal) ₃₂ 8.5 10.0 ^C 12.6 ^C 1.26 ^c

(Gal) ₁₂₀ 29.7 26.2 ^C 33.4 ^C 1.28 ^c

(Gal) ₁₈₀ 44.7 43.5 ^c 56. T 1.24 ^c

(Gal) ₂₄₀ 60.2 57.3 ^C 68.8 ^C 1.20 ^c

(Gal) ₃₀ o 80.6 74.3 ^C 94.4 ^C 1.27 ^c

(S0 ₄ -3-Gal) ₃₂ 1 1.6 8.8 ^C 10. l ^c 1.23 ^c

(SO ₄ -3-Gal) ₁₂₀ 39.4 46.2 ^C 48. l ^c 1.04 ^c

(SO ₄ -3-Gal) ₂₄₀ 80.3 82.3 ^C 88.7 ^C 1.08 ^c

"Calculated from ¾ NMR.

"Obtained from SEC analysis in DMF+0.1% LiBr (PMMA standards).

Obtained from SEC analysis in PBS (PEG standards).

Polymer codes:

(Galioo%)32 : glycopolymer with average DP (degree of polymerisation) = 32, and 100 % of polymer galactose (Gal) repeating units. (S0 ₄ -3-Gal / ₀ ) ₃ 2 glycopolymer with average DP (degree of polymerisation) = 32, and 66 % of polymer 3-O-sulfated galactose (S0 ₄ -3-Gal) repeating units. The remaining 34% of total repeating units are galactose (Gal) moieties. In (SO^-Ga ^ the remaining 67% of total repeating units are galactose (Gal) moieties.

1st generation glycopolymers: synthesis described in Synthesis of Oregon green labeled sulfated glycopolymers

2 ^nd generation glycopolymers: synthesis described in Typical polymerization conditions (B): synthesis of (S0 ₄ -3-Gal) ₁₈₀

In vivo studies

WT and MR ^_/" C57BL/6J (Charles River laboratory and kindly provided by Dr. M. Nussenzweig, Rockefeller University) mice were kept under specific, pathogen-free conditions and used at 8- 12 weeks of age . Mice were housed at Queen Medical Centre, Central Animal House, University of Nottingham Medical School. All animals were handled in accordance with institutional guidelines issued by the Home Office, United Kingdom.

Cell lines

Chinese Hamster Ovary (CHO and CHO-MR) cell lines were maintained in Dulbecco's modified Eagle 's medium/Ham's F- 12 nutrients (Gibco, Eggenstein, Germany) containing 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100 U mL ^" penicillin and 100 μg mL ^"1 streptomycin, at 37°C, 5% C0 ₂ and 95 % relative umidity. Stable transductants for CHO-MR were selected using 0.6 mg mL ^"1 geneticin.

Bone marrow derived macrophages (BMDM) were obtained from WT and MR ^~ ' ^~ C57BL/6 femurs and tibias bone marrow cells using L929 cell conditioning media (LCCM) or M-CSF as source of granulocyte/macrophage colony stimulating factor. Cells were cultured in 20 cm bacteriologic plastic (BP) petri dishes by re-suspension in 25 mL of R10 media (RMPI supplemented with 10% Fetal bovine serum, 15 % LCCM or 50 ng mL ^"1 M-CSF, 2 mM L-glutamine, 100 U mL^penicillin and 100 μg mL ^_1 streptomycin). 3 days after the seeding, an additional 15 mL of R10 fresh media were added to the plate. On day 6, the media was collected and centrifuged, the supernatant discharged and the pellets re-suspended in 25 mL of fresh R10 and returned to the original plate. After 7 days in culture the attached cells were used for the experiments described below. For cell collection, the media was discharged and the attached cells were washed with 10 mL of sterile phosphate saline buffer (PBS). 10 mL of ice-cold PBS containing 10 mM EDTA were added to each plate and incubated on ice for 10 minutes. The macrophages were detached by gently pipetting the PBS across the dish. The cells were centrifuged at 1000 rpm for 5 minutes, washed with 40 and 10 mL of opti-MEM® and re-suspended in 10 mL of opti-MEM® at a concentration of 1.5xl 0 ⁶ cells mL ^"1 for seeding.

Uptake

CHO or CHO-MR cells were seeded in 24 wells/plate (400 μίΛνεΙΙ, 6.25xl 0 ⁵ cells mL ^"1 ) and grown overnight at 37°C, 5% C0 ₂ . The medium was then removed, the wells were washed with 2x500 of PBS and incubate for 30 minutes with opti- MEM®; the media was aspirated and low and high molecular weight Oregon Green labeled glycopolymers solutions ( 1 μg mL ^"1 , 400 μίΛνεΙΙ) diluted in opti-MEM® supplemented with 2 mM L-glutamine, 100 U mL^penicillin and 100 μg mL ^" Streptomycin were added to the wells. After 30 or 60 minutes of incubation at 37°C the glycopolymers solutions were discharged, the wells rinsed 3x1 mL PBS, the cells harvested using trypsin/EDTA solution diluted 1 : 1 in PBS and fixed in 2% paraformaldehyde in PBS. Untreated cells were considered as negative control. The same study was performed using WT and MR ^" ' ^" BMDM. 200 μΐ, of 1.5xl ⁶ cells suspension in opti-MEM® were transferred into a sterile plastic tube (BD Falcon) and incubated at 37°C for 30 minutes. 200 μΐ _^ of 2 μg mL ^"1 low molecular weight glycopolymers solutions in opti-MEM® were added to the tubes and incubated for further 30 minutes at 37°C. Cells were washed with 2x2 mL ice-cold opti-MEM®, re- suspended in 200 μΐ _^ of the same media and fixed in 2% paraformaldehyde in PBS .

Samples were analyzed using a Beckman Coulter FC500 Series, with tetraCXP SYSTEM Software. The mean fluorescence intensity (MFI) was detected on channel 1 (Oregon green) and at least 2xl 0 ⁴ cells were acquired for single staining.

All experiments were performed in duplicates and repeated at least 2 times.

Competition-inhibition studies

CHO and CHO-MR cells were seeded in 24 wells/plate (400 μίΛνεΙΙ, 6.25xl 0 ⁵ cells mL ^"1 ) and grown overnight at 37°C, 5% C0 ₂ . The media was then discharged, the wells washed with 2x500 μΐ _^ of PBS, and incubate for 30 minutes with opti-MEM®; the media was aspirated and 100% sulfated or galactosylated Oregon Green labeled glycopolymers low (DP=32) and high (DP= 187) molecular weight were added to the wells (400 μΙ,ΛνεΙΙ, 20.6 or 480 μΜ sugar units concentration diluted in opti-MEM®) and incubated for 30 minutes or 2 h. For inhibition experiments the cells were washed with 3xlmL PBS, Gelatin-Texas Red was added to the we lis (400 μΐ,, 80 μ mL ^"1 ); for competition experiments Gelatin-Texas Red was added to the glycopolymers solution on the wells (4.66 μί, 6.95 mg mL ^_1 solution). Cells were then incubated for 2 h at 37°C, in the dark. After the incubation, cells were rinsed with 3x1 mL PBS and trypsinized, harvested and fixed with 2% paraformaldehyde in PBS before FACS analysis. Fluorescence was detected on channel 1 (Oregon green) and channel 3 (Texas Red). A minimum of lxl O ⁴ cells were acquired per sample. Untreated cells were analyzed to eliminate possibility of auto-fluorescent phenomena produced by the cells; cells treated with only Gelatin-Texas Red were set as 100% of ligand uptake; cells treated with only glycopolymer solutions were analyzed to allow for compensation, when required.

Time course experiments

CHO and CHO-MR cells were seeded in 24 wells/plate (400 μίΛνεΙΙ, 3.75xl 0 ⁵ cells mL ^"1 ) and grown overnight at 37°C, 5% C0 ₂ . The media was then discharged, the wells washed with 2x500 μΐ, of PBS, and incubated with opti-MEM®. After 30 minutes, the medium was removed and low and high molecular weight 100% sulfated Oregon Green labeled glycopolymers were added to the wells (400 480 μΜ sugar units concentration diluted in opti-MEM®). After 2 h of incubation at 37°C the cells were washed with 3x lmL PBS, and 400 μΐ _^ of opti-MEM® were added to each well. At scheduled time the media was substituted with Gelatin-Texas Red solution (400 μΐ,, 80 μg mL ^"1 ) and the cells were further incubated for 1 h. Wells were then rinsed with 3x1 mL PBS and trypsinized, harvested and fixed with 2% paraformaldehyde in PBS before FACS analysis. The same FACS conditions and control samples reported in the competition assay were used. MR total and superficial quantification

Western blot - total quantification. CHO-MR cells were seeded in 24 wells/plate (400 μίΛνεΙΙ, 6.25xl0 ⁵ cells mL ^"1 ) and grown overnight at 37°C, 5% C0 ₂ . The media was then discharged, the wells washed with 2x500 μί _^ of PBS, and incubated for 30 minutes with opti-MEM®. The medium was then replaced with low or high molecular weight 100% sulfated Oregon Green labeled glycopolymers (400 μίΛνεΙΙ, 480 μΜ sugar units concentration, diluted in opti-MEM ^® ). After 30 minutes or 2 h of incubation at 37°C the cells were washed with 3xlmL PBS and lysed in 200 uL of ice-cold lysis buffer (2% Triton X-100, 10 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, pH 7.4), containing protease inhibitors, for 30 min at 4°C. Lysates were harvested and centrifuged at 13000 rpm to eliminate nuclei. Supernatants were stored at -20°C. Samples were analyzed for protein concentration by bicinchoninic acid assay. Proteins from cell lysates (2.5 μg protein content) were separated on 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels under non-reducing conditions and then transferred to nitrocellulose membrane using a Trans Blot® Cell apparatus overnight at 200 mA. After blocking with blocking buffer (0.1% tween 20, 5% low fat milk in PBS) for 1 h, membranes were incubated with anti-MR mAb (MR5D3, 2 μg mL ^_1 in blocking buffer) for 1 h. Membranes were washed 3 times with 0.1% tween 20 in PBS and MR was detected using an HRP- conjugated goat-anti-Rat IgG (1 : 1000 dilution in blocking buffer, 1 h at RT). Bound Abs were visualized by enhanced chemiluminescence reagent (ECL system, Amersham Pharmacia Biotech, Bucks, UK) and recorded on a film.

FACS analysis - superficial quantification. CHO-MR cells were seeded in 12 wells/plate ( 1 mL/well, 650xl 0 ³ cells mL ^"1 ) and grown overnight at 37°C, 5% C0 ₂ . The media was then discharged, the wells washed with 2x500 of PBS, and incubated with opti-MEM®. After 30 minutes the medium was replaced with low or high molecular weight sulfated Oregon Green labeled glycopolymers ( 1.00 mL/well, 480 μΜ sugar units concentration diluted in opti-MEM®). After 2 h of incubation at 37°C the cells were washed 3 times with washing buffer (PBS containing 0.5% BSA, 5 mM EDTA, and 2 mM NaN ₃ ), harvested with non-enzymatic cell dissociation buffer on ice, dissociated by gentle pipetting and transferred to a sterile tube. Cell were washed with 2xlmL PBS, re-suspended in 200 μΐ _^ of PBS and fixed with 2% paraformaldehyde in PBS for 30 minutes at 4°C. Non specific binding sites were blocked by incubation with 400 μΐ _^ of blocking buffer (5% heat-inactivated goat serum, 0.5% BSA and 5mM EDTA in PBS) for 30 minutes at 4°C. Cells were then incubated with primary Ab (MR5D3 or IgG2A isotype control, 5 μg mL ^"1 diluted in blocking buffer) for 40 minutes at RT. After incubation cells were washed 3 times in washing buffer and labeled secondary Ab (Alexa fluor® 647-conjugate goat anti-Rat IgG, diluted 1 : 1000 in blocking buffer) was added. After 45 minutes of incubation at RT the samples were washed as described above. Cells were re-suspended in 400 μΐ _^ of PBS containing 2% paraformaldehyde and analyzed by FACS . Untreated cells were taken as 100% of mannose receptor superficial expression. Confocal analysis

To confirm glycopolymers internalization 2x l 0 ⁵ CHO-MR cells were allowed to adhere to glass slide (pre-treated with HC1 1 N and extensively rinsed with RPMI) overnight. Cells were subsequently incubated for 1 h at 37°C with 100 mg mL ^"1 solutions of 100% sulfated glycopolymers, diluted in opti-MEM®. After gentle washing with PBS (3x lmL) cells were fixed with 4% paraformaldehyde in PBS for 10 minutes at RT. Cells were washed as described above and nuclei were stained with 1 μg/mL HOECHST solution in PBS for 15 minutes at RT. After the last cycle of washing, coverslips were mounted with DAKO fluorescent mounting medium and viewed by confocal microscopy using a Zeiss LSM 700 microscope and ZEN201 1 software for images elaboration. Unstained control samples or samples treated with 100% galactosylated glycopolymers were analyzed to check for cell autofluorescence (data not shown).

A 63x oil objective was used for acquiring all images. Section images in the z- dimension were collected.

Glycopolymer affinity for CD206 - Surface Plasmon Resonance (SPR)

Binding experiments were performed by Surface Plasmon Resonance (SPR) on a BIAcore 3000. Approximately 1300-2000 response units (RU) sMR were immobilized on a CM-5 sensor chip surface by amine coupling, which according to formula ( 1 ) would result in an R _max of approximately 100 RU during kinetic binding experiments:

R _max = analyte MW _x immobilized ligand level (1)

ligand MW

After surface activation by injection of 140 EDC NHS mixture in water, 70 of 30 μg mL ^" 'sMR solution in 10 mM HEPES, 5 mM CaCl ₂ , 0.005% Tween-20, 150 mM NaCl, pH 7.4 (running buffer) were covalently coupled to the CM-5 sensor chip. Then, 190 μΐ _^ of ethanolamine was applied to block the sensor chip surface, followed by extensive wash with 1 M NaCl. Data are shown after subtraction from a channel activated identically and then blocked with ethanolamine, which remained untreated throughout the experiments. All experiments were carried out at 25°C. Various concentration (32 to 2400 μΜ referred to sugar units) of glycopolymers solutions in running buffer at pH 7.4, 6.5 or 6 were injected for 120 s at 20 μί min ^"1 flow rate . For sulfated glycopolymers regeneration was achieved by setting 20 minutes as dissociation time . Sensorgrams were analyzed using the software BIAevaluation 4. 1. Data to obtain the binding curves shown were globally fitted to a simple Langmuir model for a 1 : 1 ligand-binding model, simultaneously fitting association and dissociation in the experimental curves at several glycopolymers concentrations.

In vivo studies: IRI model.

C57BL/6 mice underwent a unilateral renal ischaemia reperfusion injury (IRI), by clamping the left renal artery for 35 minutes, followed by 48-hour recovery. Glycopolymers were injected according to different regimens which included one intravenous injection prior to IRI, one IV injection before and one afterwards and a three dose protocol with one injection prior to IRI, 2 hours later and 24 hours later. All injections were through the tail vein. Urine was collected 12 hours before sacrifice, which was performed after 48 hours. The ischaemic left kidney and control right kidney were scored on histology using a tubular injury score, assessing four variables, tubular dilatation, cast formation, tubular necrosis and loss of tubular brush border (each scored from 0-5). Urine was used for biomarker studies including assessment of KIM- 1 and NGAL. A significant decrease in tubular injury scores was observed in ischaemic kidneys from animals treated with the sulfated but galactosylated controlglycopolymers , with the greatest inhibition observed using the three dose regimen.

Results and Discussion

CD206: binding modalities and glycosylated multivalent ligands

Figure 1A shows the structure of the CD206 endocytic receptor, which has three distinct extracellular binding domains: / ^' . C Lectin-Type Domain (CLTD) which recognizes Man, Fuc, and GluNAc carbohydrates in a Ca ²⁺ -dependent manner, / ^' / ^' . collagen-binding Fibronectin type II (FN II) domain, and / ^' / ^' / ^' . Cysteine-Rich (CR) lectin domain which recognizes (S0 ₄ -3/4)-Gal and (S0 ₄ -3/4)-GalNAc sulfated sugars. A tyrosine-based motif in the cytoplasmic intracellular tail directs the delivery of mannosylated ligands to early endosomes (Figure 1A). Two families of multivalent glycoligands for selective targeting of the different lectin- type domains of CD206 were prepared as shown in Figure I B. Macroligands displaying D-galactopyranose-3 -O-sulfate (S0 ₄ -3 -Gal) repeating units were expected to provide selective binding to CR domain. All polymers had a mucin-like structure, with the sugar binding units grafted to the polymer backbone through a glycoside linker, leaving groups at C3 and C4 of the sugar rings, available for lectin binding. The avidity of binding of carbohydrate-containing polymers to lectin receptors often depends on the polymer chain length, which also directly affects the ability of these multivalent ligands to span over multiple copies of receptors at cell membranes. To isolate the contribution of the nature of the carbohydrate repeating units on binding, it was therefore critical that all glycopolymers investigated have the same average chain length. This was achieved by post-polymerization modification of a polymeric intermediate which incorporated chemoselective handles that are inert towards the polymerization conditions, but can be quantitatively converted into the required carbohydrate binding groups. The protocol described in the materials and methods was followed and involved sequential Cu(I)-catalyzed ATRP, to prepare the required polymer reactive precursor, and Huisgen cycloaddition to introduce S0 ₄ -3 -Gal and Man carbohydrate binding units and a fluorescent tag, either Oregon Green or Nile Blue, to facilitate the detection of glycopolymers in subsequent in vitro assays. In this library of multivalent ligands three parameters were systematically varied, namely i) nature of CD206-binding sugar (S04-3-Gal vs. Man), ii) density of a CD206-binding sugar grafted on each polymer chain - 33, 66, and 100% of the polymer repeating units functionalised with the required sugar ligand, the remaining being occupied by non- CD206 binding galactose (Gal) molecules, and iii) length of glycopolymer chain - 32 or 187 average number of polymer repeating units. The latter were chosen to generate glycopolymers with average molecular weight Mn of 12- 17 and 71 -89 kDa, respectively below and above the 40-60 kDa threshold for glomerular kidney filtration which were expected could behave very differently in vivo due to their potentially different half-life in systemic circulation. Glycopolymers with 100% non-CD206 binding units, (Gal l 00%)32 and (Gal 100%) 187 were also prepared and utilised as negative controls for subsequent in vitro and in vivo studies.

CD206-dependent cellular uptake of glycopolymers (GPs)

The initial hypothesis that with appropriate choice of the sugar recognition elements in the glycopolymer ligands, the two distinct lectin-type domains of CD206, CR and CTLD, could be individually targeted with high degree of specificity was validated by ELISA tests using isolated CR Fc and CTLD4-7 Fc sub-fragments. The results show that the CR can be targeted selectively with sulfated glycopolymers, and the CTLD can be targeted with macroligands containing Man binding units. CD206-presenting cells used were CD206 ⁺ -CHO, which serve as a robust platform to investigate the mechanistic aspects of CD206 binding and uptake, and wild-type murine bone-marrow derived macrophages BMDM. Efficient cell uptake was observed by in CD206 ⁺ -CHO Oregon Green-tagged galactose-3 -O-sulfated glycopolymers as shown in Figure 2 A. No cell uptake was detected in CD206 -CHO, or when (Galioo%)32, which is unable to bind CD206, was employed, confirming that uptake of sulfated Gal glycopolymers was mediated by CD206. Confocal microscopy analysis confirmed that the observed increase in fluorescence of -CHO was due to cell internalization, rather than adhesion of glycopolymers to CD206 ⁺ at the cell membrane (as shown in Figure 2B). The effect on cell uptake of the density of polymer recognition elements was investigated by the use of glycopolymers with variable proportions of 33%, 66 % and 100%, of S0 ₄ -3-Gal repeating units. With S0 ₄ -3-Gal multivalent ligands cell uptake by CD206 ⁺ -CHO slightly decreased with the density of sulfated S0 ₄ -3 -Gal glycopolymers (Figure 2A). An analogous trend was observed in WT murine macrophages (Figure 2D).

In contrast, total uptake of sulfated glycopolymers remained virtually unchanged from 30 to 60 min, indicating that after the 30 min time point S0 ₄ -3-Gal glycopolymers could no longer be internalised (Figure 2C). Importantly, at the range of concentrations employed in this study all glycopolymers were found to be virtually non-toxic upon incubation of at least 24 h, as assessed by an LDH assay.

Mechanism of S0 ₄ -3-Gal glycopolymers-induced inhibition of CD206 endocytic activity in vitro

CD206 mediates clathrin-dependent endocytosis of CD206 ligands to endosomal compartments. Once there the ligand-receptor complexes dissociate and CD206 recycles back to the cell membrane as shown in Figure IB. At every given time most of CD206 is intracellular, with recycling turnover typically occurring in ca. 20 min. Results suggested that after initial cell uptake S0 ₄ -3 -Gal multivalent ligands prevented further CD206-mediated endocytosis. This phenomenon could be explained with i) an alteration of cell trafficking of CD206 which would eventually lead to its degradation, or ii) the formation of very stable S0 ₄ -3-Gal GPs-CD206 complexes unable to eventually dissociate and allow receptor recycling, either at the cell membrane or within endosomal compartments.

To verify whether reduced CD206-mediated ligand uptake was due to intracellular receptor degradation, following incubation of CD206 ⁺ -CHO with GPs for 30 and 120 min, total cellular CD206 was estimated by western blotting using 100% Gal, and S0 ₄ -3-Gal glycopolymers with low (DP 32) and high (DP 187) molecular weight. After incubation with the chosen glycopolymers at 15 μΜ of sugar repeating units, corresponding to 470 and 80 nM of polymer chains, respectively, for 30 and 120 min, Western Blot analysis of cell lysates indicated that the total amount of cell CD206 remained virtually unchanged compared to untreated CD206 ⁺ -CHO cells, for all polymers investigated (as shown in Figure 3B).

However, sulfated multivalent ligands (S0 ₄ -3-Galioo%) 32 and (S0 -3 -Galioo%) i 87 significantly decreased the proportion of CD206 at the surface of the cell membrane compared to untreated CD206 ⁺ -CHO cells. These results demonstrated that blocking of CD206-mediated endocytosis was due to trapping of the receptor into stable ligand- receptor complexes from which the latter could no longer dissociate. Confocal microscopy showed that these complexes were not localized at the cell membrane but rather spatially confined within recycling endosomal compartments (Figure 2B).

S0 -3-Gal multivalent ligands were found to bind very strongly to CD206 and induce a significant decrease in the amount of receptor at the cell membrane. The strength of binding of glycopolymers to CD206 was investigated under a range of conditions which simulated the change in environmental conditions which occurs when the ligands bind the receptor at the cell membrane and are subsequently internalised into relatively acidic endosomal compartments. SPR analysis showed that the avidity of binding of S0 -3-Gal GPs for CD206 slightly decreased from pH 7.4, typical of extracellular environment, to more acidic pH (6.5 and 6.0), with dissociation constants K _D in the μΜ range (Figure 3D). Modulation of CD206 (MR) endocytic activity in vitro and in vivo.

The results demonstrate that the sulfated glycopolymers of the present invention can be used to inhibit CD206 mediated endocytosis and to serve as a means to treat a number of clinical diseases or conditions.

In addition to inhibition of CD206 mediated endocytosis, the ability of sulfated glycopolymers to inhibit the uptake of other CD206 ligands was also investigated. To do this collagen was used as collagen binds to the FNII domain of CD206 in a carbohydrate independent manner. Denatured collagen (gelatin) was fluorescently tagged with Texas Red (TR, l _ex/em 596/615 nm) to facilitate the quantification of its CD206-mediated cell uptake . CD206 ⁺ -CHO cells were chosen as they are more robust than WT macrophages, which facilitated prolonged time-course experiments to be carried out. Initially, CD206 ⁺ -CHO were pre-treated with either (S0 ₄ -3-Galioo%) 32 or (S0 -3 - Galioo%) i87 ( 15 μΜ in sugar binding units) for 30 or 120 min, then Gelatin-TR was added to a final concentration of 80 μg ml/ ¹ . After 2 hours of co-incubation, cell uptake was quantified by FACS . Data clearly showed that internalisation of Gelatin- TR was significantly reduced as compared with untreated cell incubated with Gelatin- TR for 2 h (positive control) . CD206 ⁺ -CHO pre-incubated with analogous non CD206- binding Gal-glycopolymers gave a Gelatin-TR uptake similar to that observed with untreated cells (as shown in Figure 5A). The uptake patterns did not significantly change by increasing the duration of polymer pre-incubation from 30 to 120 min. Similar trends were also observed when after the initial pre-incubation glycopolymers were removed from the culture media, and cells were treated with Gelatin-TR in the absence of glycopolymers, suggesting that after initial polymer pre-incubation, sustained inhibition of CD206-mediated endocytosis could be attained even in the absence of sulfated glycopolymers. The duration of the inhibition of CD206 endocytic activity was investigated via time course inhibition experiments. Cells were initially incubated with either (S0 -3- Galioo%) 32 or (S0 ₄ -3-Galioo%) i87 for 2 h, then glycopolymers were removed from the culture medium. Gelatin-TR was then added and uptake over time was monitored (Figure 4C). For both sulfated glycopolymers full recovery of CD206 endocytic was observed after 48 hours. (S0 -3 -Galioo%)32 produced the best inhibition profile, with Gelatin-TR uptake only re-starting to increase after 24-30 hours. From a therapeutic viewpoint it may be important that not only the uptake inhibitory effect is sustained, so as to reduce the frequency of the required drug intakes, but also that this phenomenon is fully reversible, ensuring that CD206 activity is fully recovered at the end of the treatment.

In addition to CD206 (MR), a range of other lectins are known to recognise mannose- rich molecular patterns in vivo - e.g. Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209) and Mannose-Binding Lectin (MBL), which can activate the lectin pathway of the complement system. Conversely, lectins able to selectively recognise S0 ₄ -3-Gal motifs with sufficient avidity are much less known. Targeting the CR domain therefore offers a potent route for selective binding of CD206 in vivo. Inhibition of CD206 endocytic activity by sulfated glycopolymers was assessed in vivo, in a murine model. Mice were first treated with (S0 ₄ -3-Galioo%)32, [200 μί, 400 μΜ of glycopolymer] then with TR-gelatin [ 16 μg, in 200μί], and uptake of the latter in selected organs was visualized using fluorescent microscopy] . Mice not pre-treated with sulfated glycopolymers, and others pre-treated with non CD206-binding (Gali ₀ o%) 32 and (Galioo%) i87 were used as controls. In the control and TR-gelatin uptake was clearly detectable in the liver - Kupffer cells, the resident liver macrophages, constituting >80% of the body macrophage population (Gronbsek H, et al , J Hepatol (2016), http://dx.doi.org/10.1016/j jhep.2015. l l .021) and TR-gelatin co-localized with CD206 ⁺ cells. No detectable uptake was observed in mice previously pre-treated with (S0 ₄ -3-Galioo%) i87 (Figure 4D), suggesting that this glycopolymer effectively acted as a CD206 blocker in vivo.

In summary, the data demonstrates how the endocytic activity of the CD206 mannose receptor can be modulated both in vitro and in vivo by selectively targeting its distinct lectin-type domains with appropriately designed synthetic glycopolymers according to the invention.

Further results and analysis relating to the use and efficacy of the polymers according to the invention in humans is provided with reference to the figures and figure legends. Under the conditions investigated sulfated glycopolymers displaying galactose 3-0- sulfate units, designed to bind the CR domain acted as efficient CD206 blockers both in vitro and in vivo. The results obtained demonstrate the unexpected activity of 3-0- sulfate Gal glycopolymers which offer a novel category of therapeutics for CD206 inhibition.

Other embodiments are intentionally within the scope of the invention as defined by the appended claims.