Liposomal conjugates and uses thereof

Abstract

The present invention relates to liposomes, and in particular to liposome conjugates and methods of use thereof for joint lubrication.

Description

[0001] LIPOSOMAL CONJUGATES AND USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to liposomes, and in particular to liposome conjugates and methods of use thereof for joint lubrication.

[0004] BACKGROUND OF THE INVENTION

[0005] Healthy and functioning joints are essential for the well-being of an individual. Sufficient biolubrication is a prerequisite for proper joint mobility, which is crucial for prevention and amelioration of degradative changes of the joint.

[0006] Articular cartilage forms a smooth, tough, elastic and flexible surface that facilitates bone movement in the joint. The cartilage is composed of chondrocyte cells, which produce large quantities of collagenous extracellular matrix (ECM), rich in proteoglycans (particularly aggrecan and lubricin) and elastin fibers. Different categories of cartilage are classified according to their relative amounts of collagen and proteoglycan. Hyaline articular cartilage contains the highest quantity of collagen (about 60%), being the main protein in the ECM. Several collagen subtypes have been identified in hyaline articular cartilage, the main and most abundant being type II, IX and XI collagens. Additional less abundant collagens include type III to VI, X, XII, XIV, XVI, XXII, and XXVII collagens. All these collagens have been found to play a key role in healthy cartilage, regardless of whether they are more or less abundant (Alcaide-Ruggiero et al., Int J Mol Sci., 22(24): 13329, (2021)).

[0007] The collagen protein is composed of a triple helix, which generally consists of two identical chains (alpha- 1) and an additional chain that differs slightly in its chemical composition (alpha-2) (Brodsky and Persikov, Adv. Prot. Chem., 70: 301-339 (2005)). The amino acid composition of collagen is atypical for proteins, particularly with respect to its high hydroxyproline content. In the articular cartilage, the collagen, mostly collagen type II, functions to constrain the proteoglycans, providing structure and stability to the ECM.

[0008] Due to its predominant presence in the cartilage, collagen is a promising target for directing delivery of a desired element to the cartilage. Hubbell et al. (Nat. Mat., 7:248-254 (2008)), disclose polymer nanoparticles modified with a peptide ligand having amino acid sequence WYRGRL, which was demonstrated to bind to collagen type II, for resisting rapid release of the poly (propylene sulfide) (PPS) nanoparticles and clearance from the cartilage. Peptide- functionalized nanoparticles targeted articular cartilage up to 72-fold more than nanoparticles displaying a scrambled peptide sequence following intra-articular injection in a mouse. Jiang et al., (J. Nanosci. Nanotechnol. 18, 4 (2018)) describes a poly(lactic-co-glycolic acid) (PLGA)-based nanoscale drug delivery system, including maleimide-PEG-PLGA nanoparticles with FITC-tagged WYRGRL peptide linked to the surface thereof through a thioether bond with the maleimide. The delivery system was found to bind with high specificity to cartilage tissue in vitro and ex vivo.

[0009] U.S. 7,592,009 discloses ligands that specifically bind to articular cartilage tissues, including uses for targeting therapeutics towards articular cartilage tissue and new materials for articular cartilage. The ligands are effective in vivo to target therapeutic materials to articular cartilage.

[0010] Boundary lubrication, in which layers of lubricant molecules separate opposing surfaces, occurs under loading of articular joints. Several different substances have been proposed as the native boundary lubricants in articular cartilage, including Hyaluronic acid (HA) and lubricin. Pickard et al. (Biomaterials 19,1807-1812 (1998)) and Schwartz and Hills (Br. J. Rheumatol. 37, 21-26 (1998)) demonstrated that phospholipids defined as surface active phospholipids of lubricin facilitate joint lubrication in articular cartilage. Hills and coworkers (Vecchio et al. Rheumatology 38,1020-1021 (1999)) demonstrated that osteoarthritis (OA) joints have a surface-active phospholipids (SAPL) deficiency, and that injection of the surface-active phospholipid 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) into joints of OA patients resulted in mobility improvement lasting up to 14 weeks without major side effects. In another study, lipidic globular vesicles were observed on the surface of healthy cartilage, which are assumed to play a major role in lubrication (Watanabe et al., Med. Elect. Microsc. 33,16-24 (2000)). Using animal models, Forsey et al. (Biomaterials 27, 4581-4590 (2006)) have shown that use of high molecular weight HA (-2000 kDa) combined with DPPC improved lubricating ability of the latter.

[0011] Osteoarthritis (OA) is the most common form of joint diseases, and is characterized by articular cartilage degradation, osteophyte formation, bone remodeling, joint space narrowing, and joint inflammation. The etiology of OA is unknown, but is believed to be multifactorial, including hereditary, metabolic causes, mechanical damages, and inflammatory in nature. Osteoarthritis of the knees has higher prevalence than any other joints. Clinical manifestations of OA in the knees include pain in and around the joint, particularly on weight-bearing of the knee joint, stiffness of the joint after rest, and limited joint motion due to pain and / or stiffness of the joint. The end result of all forms of OA is often loss of function of the joint or limb, imposing significant economic burden on the individuals as well as the society. Diagnosis of OA is based on signs and symptoms, and is often confirmed with imaging studies or using laboratory tests to rule out concomitant inflammatory causes. Since the pathophysiology of OA is unknown, current recommendations for managing OA focus on relieving pain and stiffness, and improving physical function as important goals of therapy. Non-surgical treatment of OA focuses on reducing overloading of joints, physiotherapy, and alleviation of pain and inflammation, usually by topical, systemic, or intraarticular (IA) administration of drugs.

[0012] Currently available medication regimens for most OA patients include non-opioid analgesics. This is mainly because long-term opioid use for OA is generally discouraged due to lack of information on benefits as well as risks of addiction and other side effects. Accordingly, agents such as acetaminophen / paracetamol, nonsteroidal anti-inflammatory drugs (NSAIDs), counter-irritants, glucosamine or chondroitin, corticosteroids, and injectable hyaluronic acid are typically used. While these pharmaceutical agents can provide transient pain relief quite effectively, long-term use of NSAIDs has been found to be associated with increased risk of gastrointestinal bleeding, hypertension, cardiovascular events, congestive heart failure, and renal insufficiency. NSAIDs in the form of topical application and cyclooxygenase II (COX II) inhibitors are considered somewhat safer than other NSAIDs in terms of gastrointestinal side effects, although COX II inhibitors are contraindicated in patients with a history of coronary artery disease (Mehta et al., Statistics in Medicine. 30(28), 3267-3284 (2011); Zhang et al., Clin. Geriatr. Med. 26:3, 55-369 (2010); Glyn-Jones et al., Lancet. 386, 376-387 (2015)). While corticosteroid injections reduce inflammation and ease pain faster than NSAIDs, their relief is only temporary. Corticosteroid injections are typically given every three months and the number of injections is typically limited to four in the same joint.

[0013] Because of the high incidence of side effects associated with long-term therapy of NSAIDs and corticosteroids, effective and safer alternative treatments for OA are urgently needed.

[0014] HA injections are generally administered every week for 3 to 5 weeks. Even though HA injections were found to be safe when used for prolonged periods, various studies report that they offer only a small benefit. Additionally, injectable HA compositions are known to have various side effects, such as mobility difficulties, muscle pain or stiffness, pain in the joints and swelling or redness in the joint.

[0015] U.S. 6,800,298 discloses dextran-based hydrogel compositions containing lipids, particularly phospholipids, for lubrication of mammalian joints.

[0016] U.S. 2005 / 0123593 is directed to a composition comprising glycosaminoglycans encapsulated in a liposomal delivery system for intraarticular administration for the treatment of osteoarthritis.

[0017] U.S. 8,895,054 describes methods of joint lubrication and / or prevention of cartilage wear making use of liposomes having membranes with at least one phospholipid (PL) of the group consisting of a glycerophospholipid (GPL) having two, being the same or different, C12-C16 hydrocarbon chain and a sphingolipid (SPL) having a C12-C15 hydrocarbon chain, the one or more membranes having a phase transition temperature in which solid ordered (SO) to liquid disordered (LD) phase transition occurs, the phase transition temperature being within a temperature of about 20°C to about 39°C for lubrication of joints.

[0018] WO 2019 / 038763 describes a pharmaceutical composition for the lubrication of joints, the pharmaceutical composition comprising a non-ionic tonicity agent comprising a polyol, and liposomes comprising at least one membrane comprising at least one phospholipid (PL) selected from a glycerophospholipid (GPL), said GPL having two C12-C18 hydrocarbon chains, being the same or different, and sphingomyelin (SM) having a C12-C18 hydrocarbon chain, the pharmaceutical composition being essentially free of an additional pharmaceutically active agent, wherein the at least one membrane has a phase transition temperature in the range of about 20°C to about 39°C and the joint has a joint temperature which is above the phase transition temperature.

[0019] There remains an unmet need for a therapeutically effective pharmaceutical composition for joint lubrication, which would provide a long-lasting effect, while reducing the probability of side effects associated with frequent intra-articular administrations.

[0020] SUMMARY OF THE INVENTION

[0021] The present invention provides compositions comprising a plurality of liposomes and methods of use thereof for joint lubrication and for the treatment of joint dysfunction or injury. In particular, there is provided a composition comprising liposomes conjugated to at least one peptide comprising a collagen-binding sequence, which prolongs the residence time of the liposomes at the cartilage of a treated joint.

[0022] It was found that conjugation of cartilage-binding peptides to liposomes via a state-of-the- art maleimide linkage produced a drastic increase in macrophage uptake of the peptide-conjugated liposomes. While the addition of the cartilage-binding peptide raised the binding-level of the liposomes to the cartilage, the severe surge in macrophage uptake which accompanied the addition of the peptide lead to an overall null or even negative effect on the residence of the liposomes in the cartilage area. The present invention is based, in part, on the unexpected finding that changing the conjugation of the peptide to the liposome reduced macrophage uptake levels. Surprisingly, when direct conjugation between the liposomes and the cartilage-binding peptides was applied, the level of macrophage uptake was almost identical to the level of uptake of liposomes with no conjugated peptide (referred to herein also as “bare liposomes” or “non-conjugated liposomes”). Thus, while direct linking of the peptide to the lipids of the liposome, as opposed to maleimide linkage, did not substantially adversely affect the macrophage uptake as compared to nonconjugated liposomes, it enabled increased cartilage binding levels which would enable prolonging the residence time of the liposomes on the cartilage and meniscus surfaces, allowing an effective and long-standing lubrication of a joint in need thereof, with reduced frequency of administration and enhanced patient compliance.

[0023] According to a first aspect of the present invention, there is provided a liposomal composition comprising a plurality of liposomes, optionally suspended in a liquid medium, wherein the liposomes comprise an internal aqueous phase surrounded by at least one membrane comprising a phospholipid (PL) which is conjugated to a peptide comprising a collagen-binding sequence, wherein a free carboxyl group (COOH) of a side chain or the C-terminus of the peptide is conjugated directly to an amine group of the PL via an amide bond.

[0024] According to another aspect of the present invention, there is provided a liposomal composition comprising a plurality of liposomes suspended in a liquid medium, wherein the liposomes comprise an internal aqueous phase surrounded by at least one membrane comprising a phospholipid (PL) which is conjugated to a peptide comprising a collagen-binding sequence capable of binding a collagen which is present in the cartilage, wherein conjugation of the PL to the peptide is by a free carboxyl group (COOH) of a side chain or the C-terminus of the peptide being conjugated directly to an amine group of the PL via an amide bond, with no intermediate linking moiety.

[0025] According to another aspect of the present invention, there is provided a liposomal composition for use in joint lubrication in a subject in need thereof, comprising a plurality of liposomes, optionally suspended in a liquid medium, wherein the liposomes comprise an internal aqueous phase surrounded by at least one membrane comprising a phospholipid (PL) which is conjugated to a peptide comprising a collagen-binding sequence, wherein a free carboxyl group (COOH) of a side chain or the C-terminus of the peptide is conjugated directly to an amine group of the PL via an amide bond.

[0026] According to yet another aspect of the present invention, there is provided a method for joint lubrication in a subject in need thereof comprising administering a therapeutically effective amount of a liposomal composition into a cavity of the joint, wherein the liposomal composition comprises a plurality of liposomes, optionally suspended in a liquid medium, wherein the liposomes comprise an internal aqueous phase surrounded by at least one membrane comprising a phospholipid (PL) which is conjugated to a peptide comprising a collagen-binding sequence, wherein a free carboxyl group (COOH) of a side chain or the C-terminus of the peptide is conjugated directly to an amine group of the PL via an amide bond. According to some embodiments, the collagen binding sequence binds at least one collagen type selected from collagen type-I, collagen type-II, type-III, collagen type-IV, collagen type-V, type- VI, type-IX, type-X, type-XI, type-XII, type-XIV, type-XVI, type-XXII, and type-XXVII. According to some embodiments, the collagen binding sequence binds at least one collagen type selected from collagen type-II, type-IX and type-XI. According to some embodiments, the at least one collagen-binding sequence is a collagen type-II binding sequence. According to some embodiments, the collagen type-II binding sequence comprises at least one of WYRGRL (SEQ ID NO: 1) and an amino acid sequence comprising GPO. According to some embodiments, the collagen type-II binding sequence comprises the amino acid sequence WYRGRL (SEQ ID NO: 1). According to some embodiments, the collagen type-II binding sequence comprises the amino acid sequence GPO.

[0027] According to some embodiments, the collagen-binding sequence comprises at least of WYRGRL (SEQ ID NO: 1) and an amino acid sequence comprising GPO. According to some particular embodiments, the collagen-binding sequence comprises the amino acid sequence WYRGRL (SEQ ID NO: 1). According to some embodiments, the collagen-binding sequence comprises the amino acid sequence GPO. According to some embodiments, the peptide comprises the collagen-binding sequence once. According to some embodiments, the peptide comprises the collagen-binding sequence at least once. According to some embodiments, the peptide comprises one or more repeats of the collagen-binding sequence. According to some embodiments, the peptide comprises one repeat of the collagen-binding sequence. According to some embodiments, the peptide comprises two or more repeats of the collagen-binding sequence. According to some embodiments, the peptide comprises between 1 and 15 repeats of the collagen-binding sequence. According to some embodiments, the peptide comprises between 1 and 12 repeats of the GPO collagen-binding sequence. According to some embodiments, the peptide comprises between 2 and 8 repeats of the GPO collagen-binding sequence. According to some embodiments, the peptide comprises between 3 and 5 repeats of the GPO collagen-binding sequence. According to some embodiments, the peptide comprises 4 repeats of the GPO collagen-binding sequence.

[0028] According to some embodiments, the peptide comprises a spacer sequence between the collagen-binding sequence and the PL. According to some embodiments, the spacer sequence comprises 1 to 10 amino acids, including each integer within the specified range. According to some specific embodiments, the spacer comprises between 1 to 6 amino acids, including each integer within the specified range. According to further embodiments, the spacer comprises between 3 to 5 amino acids, including each integer within the specified range. According to some specific embodiments, the spacer comprises 3 amino acids. According to some embodiments, the spacer comprises amino acids selected from serine, glycine, proline, alanine, and a combination thereof. According to some particular embodiments, the spacer comprises an amino acid sequence selected from GGS, SGS, and SSS. Each possibility represents a separate embodiment.

[0029] According to some embodiments, the peptide is devoid of a spacer sequence.

[0030] According to some particular embodiments, the peptide comprises an amino acid sequence selected from the group consisting of WYRGRL (SEQ ID NO: 2), WYRGRLSGS (SEQ ID NO: 3), WYRGRLSGSSGS (SEQ ID NO: 4), WYRGRLGDPGD WYRGRLSGS (SEQ ID NO: 5), and GPOGPOGPOGPOGPO (SEQ ID NO: 6). Each possibility represents a separate embodiment. According to some embodiments, the peptide conjugated to the lipid comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and a combination thereof. Each possibility represents a separate embodiment. According to some embodiments, a single liposome comprises a combination of peptide sequences conjugated to lipids thereof, as above. According to some further embodiments, a liposomal composition comprises a mixture of liposomes, each liposome conjugated to peptides having one of the sequences selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. Each possibility represents a separate embodiment. For example, a liposomal composition can comprise 50% of liposomes conjugated to peptides comprising SEQ ID NO: 3 and 50% of liposomes conjugated to peptides comprising SEQ ID NO: 4. Any other relative ratio or combination of peptides is included herein. According to some embodiments, the peptide comprises an acyl group at the N-terminus thereof (acylated). According to further embodiments, the peptide is not acylated at the N-terminus thereof. According to some further embodiments, a portion of the peptides conjugated to the liposomes are acylated and a portion of the peptides conjugated to the liposomes are non-acetylated.

[0031] According to some embodiments, the peptide is connected to a phosphatidylamine lipid. According to other embodiments, the phosphatidylamine lipid is selected from the group consisting of l,2-Dipahmtoyl-sn-glycero-3 -phosphoethanolamine (DPPE), 1,2-dilauroyl-L- phosphatidyl-ethanolamine (DLPE), l,2-Dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-Diphytanoyl-sn-glycero-3 -phosphoethanolamine (DPhPE), l,3-Dipalmitoyl-sn-glycero-2- phosphoethanolamine (1,3-DPPE), l-Palmitoyl-3-oleoyl-sn-glycero-2-phosphoethanolamine (1,3-POPE), Biotin-Phosphatidylethanolamine, l,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), l,2-Distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), and Phosphatidylserine (PS). Each possibility represents a separate embodiment. According to some embodiments, the peptide is activated prior to conjugation with the lipid. According to some embodiments, the phospholipid conjugated to the peptide constitutes between about 1 % to about 20% of lipids in the liposome, including each value within the specified range. According to some embodiments, the phospholipid conjugated to the peptide constitutes between about 2% to about 18% of lipids in the liposome, including each value within the specified range. According to some embodiments, the phospholipid conjugated to the peptide constitutes between about 2% to about 15% of lipids in the liposome, including each value within the specified range. According to some embodiments, the phospholipid conjugated to the peptide constitutes between about 2% to about 12% of lipids in the liposome, including each value within the specified range. According to some embodiments, the phospholipid conjugated to the peptide constitutes between about 2% to about 10% of lipids in the liposome, including each value within the specified range. According to some embodiments, the phospholipid conjugated to the peptide constitutes between about 2% to about 8% of lipids in the liposome, including each value within the specified range.

[0032] According to some embodiments, the liposomes further comprise a PL comprising two acyl chains selected from the group consisting of Ci4, C15, Ci6, and Cis acyl chains. Each possibility represents a separate embodiment. In one embodiment, at least one of said chains is a saturated hydrocarbon chain. In further embodiments, the two hydrocarbon chains are saturated. In some embodiments, the PL is a phosphatidylcholine (PC) selected from the group consisting of 1 ,2-dimyristoyl- n-glycero-3 -phosphocholine (DMPC) ; 1 ,2-dipalmitoyl- n-glycero-3 - phosphocholine (DPPC); l,2-dipentadecanoyl-sn-glycero-3 -phosphocholine (Cl 5); 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC); N-palmitoyl-D-eryt / zro- sphingosylphosphorylcholine (D-erythro C16), and a mixture or combination thereof. Each possibility represents a separate embodiment.

[0033] According to additional embodiments, the liposomes comprise a phosphatidylethanolamine and at least one of l,2-dipalmitoyl- n-glycero-3 -phosphocholine (DPPC) and l,2-dimyristoyl-sn-glycero-3 -phosphocholine (DMPC). Each possibility represents a separate embodiment.

[0034] According to some embodiments, the liposomes comprise a phosphatidylethanolamine and a combination of DMPC and DPPC. According to further embodiments, the liposomes comprise a combination of DPPE, DMPC, and DPPC. In further embodiments, the mole percent of DPPE is in the range of about 1% to about 10%, the mole percent of DMPC is in the range of about 24% to about 70%, and the mole percent of DPPC is in the range of about 75% to about 29%, including all values within the respective specified ranges. In certain embodiments, the mole percent ratio of DMPC to DPPC to DPPE is about 40:57.5:2.5 to about 55:40:5, including all iterations of ratios within the specified range. In particular embodiments, the mole percent ratio of DMPC to DPPC to DPPE is about 45:50:5.

[0035] According to certain embodiments, the phospholipids are present in the liposomal composition at a total concentration that ranges from about 20 to about 500 mM, including each value within the specified range. In various embodiments, the total concentration of the phospholipids in the composition ranges from about 50 to about 300 mM, including each value within the specified range. In further embodiments, the total concentration of the phospholipids in the composition ranges from about 100 to about 200 mM, including each value within the specified range.

[0036] According to some embodiments, the liposomal composition is suspended in a liquid medium. According to certain embodiments, the internal aqueous phase and / or liquid medium comprise a buffer. In various embodiments, the buffer is a histidine buffer. In other embodiments, the buffer is a phosphate buffered saline (PBS). In yet other embodiments, the liposomal liquid medium has a pH in the range of about 5 to about 8, including each value within the specified range. In further embodiments, the liquid medium has a viscosity of about 2 to about 10 cP as measured at 25°C, including each value within the specified range.

[0037] According to additional embodiments, the liquid medium further comprises a tonicity agent. In yet other embodiments, the liquid medium further comprises a non-ionic tonicity agent. In some embodiments, the non-ionic tonicity agent comprises a polyol. In some embodiments, the polyol comprises at least three hydroxyl groups, at least four hydroxyl groups, or at least five hydroxyl groups. Each possibility represents a separate embodiment. According to some embodiments, the polyol is linear. According to some further embodiments, the polyol comprises a linear carbon chain of at least three carbons. In various embodiments, the polyol comprises at least one of mannitol, sorbitol, glycerol, erythritol, maltitol, isomalt, trimethylolpropane, pentaerythritol, dextrose, lactose, and trehalose. Each possibility represents a separate embodiment. In particular embodiments, the polyol is mannitol.

[0038] According to some embodiments, the liposomal composition comprises a non-ionic tonicity agent in a weight percent ranging from about 0.05% to about 10% (w / w) of the total weight of the composition, including each value within the specified range. In other embodiments, the weight percent of the non-ionic tonicity agent in the composition ranges from about 0.1% to about 7% (w / w) of the total weight of the composition, including each value within the specified range. In yet other embodiments, the weight percent of the non-ionic tonicity agent in the composition ranges from about 0.5% (w / w) to about 5% (w / w) of the total weight of the composition, including each value within the specified range. According to some embodiments, the liposomes in the liposomal composition are characterized by a zeta potential in the range of about (-30) to about 10 mV, when measured at a histidine mannitol buffer (HMB) having a pH of 6.5, including each value within the specified range. According to some embodiments, the liposomes in the liposomal composition are characterized by a zeta potential in the range of about (-25) to about 5 mV, including each value within the specified range. According to some embodiments, the zeta potential of the liposomes is negative. According to some embodiments, the liposomes in the liposomal composition are characterized by a zeta potential in the range of about (-20) to about 5 mV, including each value within the specified range. According to some embodiments, the zeta potential of the liposomes is negative.

[0039] According to some embodiments, the liposomes in the liposomal composition have a phase transition onset temperature in the range of about 20°C to about 42°C, including each value within the specified range. According to yet other embodiments, the liposomes in the liposomal composition have a phase transition onset temperature in the range of about 22°C to about 41°C, including each value within the specified range. According to yet other embodiments, the liposomes in the liposomal composition have a phase transition onset temperature in the range of about 22°C to about 39°C, including each value within the specified range. According to yet other embodiments, the liposomes in the liposomal composition have a phase transition onset temperature in the range of about 22°C to about 36°C, including each value within the specified range. According to yet other embodiments, the liposomes in the liposomal composition have a phase transition onset temperature in the range of about 22°C to about 34°C, including each value within the specified range. According to yet other embodiments, the liposomes in the liposomal composition have a phase transition onset temperature in the range of about 22°C to about 32°C, including each value within the specified range. In specific embodiments, the liposomes in the liposomal composition have a phase transition onset temperature in the range of about 24°C to about 31°C, including each value within the specified range.

[0040] According to various embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 50 to about 400 nm, including each value within the specified range. According to other embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 50 to about 300 nm, including each value within the specified range. According to yet other embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 50 to about 200 nm, including each value within the specified range. According to further embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 50 to about 150 nm, including each value within the specified range. According to particular embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 50 to about 120 nm, including each value within the specified range. According to other particular embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 50 to about 100 nm, including each value within the specified range.

[0041] According to some embodiments, the liposomes in the liposomal composition have an average particle size of more than 250 nm. According to some embodiments, the liposomes in the liposomal composition have an average particle size of more than 500 nm. According to some embodiments, the liposomes in the liposomal composition have an average particle size of more than 750 nm. According to some embodiments, the liposomes in the liposomal composition have an average particle size of more than 1,000 nm.

[0042] According to various embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 500 to about 5,000 nm, including each value within the specified range. According to various embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 500 to about 4,000 nm, including each value within the specified range. According to other embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 500 to about 3,000 nm, including each value within the specified range. According to yet other embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 500 to about 2,000 nm, including each value within the specified range. According to further embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 500 to about 1,500 nm, including each value within the specified range. According to other embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 1,000 to about 4,500 nm, including each value within the specified range. According to various embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 1,000 to about 5,000 nm, including each value within the specified range. According to various embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 1,000 to about 4,000 nm, including each value within the specified range. According to other embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 1,000 to about 3,000 nm, including each value within the specified range. According to yet other embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 1,000 to about 2,000 nm, including each value within the specified range. According to further embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 1,000 to about 1,500 nm, including each value within the specified range.

[0043] According to some embodiments, the liposomal composition has a zeta potential in the range of about (-15) to about 5 mV; the liposomes comprise DMPC, DPPC, and DPPE; the liposomes have an average particle size of more than 1,000 nm; and the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

[0044] According to certain embodiments, the liposomes in the liposomal composition are substantially in the form of small unilamellar vesicles (SUVs). According to certain embodiments, the liposomes in the liposomal composition are substantially in the form of multilamellar vesicles (MLVs). According to certain embodiments, the liposomes in the liposomal composition are substantially in the form of large unilamellar vesicles (LUVs).

[0045] According to additional embodiments, the liposomes in the liposomal composition have a unimodal size distribution. According to yet other embodiments, the liposomes in the liposomal composition have a bimodal size distribution. According to some embodiments, the bimodal size distribution comprises MLVs having an average particle size in the range of about 1,000 to about 5,000 nm, and SUVs having an average particle size distribution in the range of about 50 to about 400 nm, or specifically in the range of about 50 to 100 nm, including each value within the specified ranges.

[0046] According to various embodiments, lubricating a joint comprises at least one of treatment of joint disorder, reduction of joint pain, irritation and / or wear, or any combination thereof. Each possibility represents a separate embodiment. In one embodiment, lubricating a joint comprises the treatment, management or prevention of an articular disorder or condition or symptoms arising therefrom. In another embodiment, the articular disorder or condition is selected from the group consisting of rheumatoid arthritis and osteoarthritis. Each possibility represents a separate embodiment. In further embodiments, the liposomal composition of the present invention is useful in traumatic joint injury, locked joint, sports injury, traumatic injury towards osteoarthritis (OA), joint following arthrocentesis, arthroscopic surgery, open joint surgery, joint replacement, and / or psoriatic arthritis. Each possibility represents a separate embodiment.

[0047] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0048] BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 depicts the UV absorption levels at different wavelengths of liposomes of Formulations #1 and #2, dissolved in EtOH.

[0050] Figures 2A-2D depict fluorescent measurements and friction tests of liposomes of Formulations #1 and #2. (2A) Cartilage binding, background fluorescence of untreated cartilage discs was substracted from data; (2B) static coefficient of friction; (2C) dynamic coefficient of friction; (2D) Macrophage uptake of liposomes, background fluorescence of untreated macrophages was substracted from data.

[0051] Figures 3A-3B depict fluorescent measurements of liposomes of Formulations #1 and #3. (3A) Cartilage binding; (3B) Macrophage uptake of liposomes.

[0052] Figures 4A-4B depict fluorescent measurements of liposomes of Formulations #2 and #4. (4A) Cartilage binding; (4B) Macrophage uptake of liposomes.

[0053] Figures 5A-5B depict fluorescent measurements of liposomes of Formulations #5, #6, #7, and #8. (5A) Cartilage binding; (5B) Macrophage uptake of liposomes.

[0054] Figures 6A-6B depict fluorescent measurements of liposomes of Formulations #1, #2, and #9. (6A) Macrophage uptake of liposomes; (6B) Cartilage binding.

[0055] Figures 7A-7B depict fluorescent measurements of liposomes of Formulations #1, #2, #9 and #10. (7A) Macrophage uptake of liposomes; (7B) Cartilage binding.

[0056] Figures 8A-8B depict fluorescent measurements of liposomes of Formulations #9, #10, #11 and #12. (8A) Macrophage uptake of liposomes; (8B) Cartilage binding.

[0057] Figures 9A-9B depict the quotient of cartilage binding of liposomes of Formulations #2 to #12 divided by the macrophage uptake thereof (normalized). (9A) Einear representation of said quotient; (9B) Logarithmic representation of the said quotient.

[0058] Figures 10A-10C depict a comparison between the fluorescent measurements of liposomes of Formulations #4 and #9. (10A) Cartilage binding; (10B) Macrophage uptake of liposomes; (10C) Logarithmic representation of a quotient of the cartilage binding divided by the macrophage uptake (normalized).

[0059] Figures 11A-11C depict a comparison between the fluorescent measurements of liposomes of Formulations #7 and #10. (11A) Cartilage binding; (11B) Macrophage uptake of liposomes; (11C) Logarithmic representation of a quotient of the cartilage binding divided by the macrophage uptake (normalized). Figures 12A-12C depict a comparison between the fluorescent measurements of liposomes of Formulations #13 and #14. (12A) Cartilage binding; (12B) Macrophage uptake of liposomes; (12C) Logarithmic representation of a quotient of the cartilage binding divided by the macrophage uptake (normalized).

[0060] DETAILED DESCRIPTION OF THE INVENTION

[0061] The present invention provides a liposomal composition characterized by liposomes conjugated to at least one collagen-binding peptide via a direct peptide-bond, for use in the lubrication of mammalian joints, said lubrication providing reduced pain, irritation, and wear of the joint, and allowing the improvement or restoration of joint mobility. The liposomal composition can further be used for the treatment, management or prevention of an articular disorder or condition.

[0062] Jiang et al., (J. Nanosci. Nanotechnol. 18, 4 (2018)) discloses a nanoscale drug delivery system including maleimide-PEG-PLGA nanoparticles conjugated to a WYRGRL collagen- binding peptide through a thioether bond with the maleimide. The delivery system was found to bind with high specificity to cartilage tissue in vitro and ex vivo.

[0063] In contrast, the present invention is based on the unexpected discovery that liposomes containing lipids conjugated via a maleimide linker to a peptide comprising a cartilage binding sequence, such as a collagen-binding sequence, displayed extremely high levels of macrophage uptake, which were only reduced when the method of conjugation of the peptide to the liposome was changed to directly linking the peptide to an amine group of the lipid, via an amide bond. Surprisingly, it was consistently found that liposomes which were directly bound to the peptide had a higher combined effect than liposomes which were conjugated to the peptide via the maleimide linker, the combined effect calculated by dividing the cartilage binding of the peptide- conjugated liposomes by the respective macrophage uptake thereof (referred to herein also as the “quotient” of the liposome). It is contemplated that the combined effect of the liposomes is indicative or correlated with the residence time of the peptide-conjugated liposomes within a treated joint.

[0064] Thus, according to the principles of the present invention, the pep tide-liposome conjugate is characterized by at least one PL of the liposome being conjugated to a peptide comprising a collagen-binding sequence. According to some embodiments, the peptide is conjugated directly to the PL. According to some embodiments, the carboxyl group (COOH) at the C-terminus of the peptide or a free carboxyl group of a side chain of the peptide is conjugated directly to the PL. According to some embodiments, the peptide is conjugated directly to an amine group of the PL. According to some embodiments, the carboxyl group (COOH) at the C-terminus or a side chain of the peptide is conjugated directly to an amine group of the PL via an amide bond. According to some embodiments, the plurality of liposomes are suspended in a liquid medium. According to some embodiments, the liposomes comprise an internal aqueous phase surrounded by at least one membrane comprising the PL.

[0065] According to some embodiments, the peptide-lipid conjugate does not include any intermediate moiety, e.g., a linker. According to some embodiments, the peptide-lipid conjugate does not include an intermediate moiety which is not one of the 22 proteinogenic amino acids. According to some embodiments, the PL is not connected to a linker. According to some further embodiments, the peptide is not connected to a linker. According to some embodiments, the peptide-lipid conjugate does not include an intermediate maleimide moiety.

[0066] According to some embodiments, the liposome does not include polyethylene glycol (PEG).

[0067] As used herein, the term “peptide” generally refers to an amino acid sequence which is derived from a particular source, or which performs a specific function. Peptides according to the present invention include collagen-binding sequences, e.g., SEQ ID NO: 1, GPO, and other collagen-binding sequences known in the art.

[0068] Peptides according to the present invention may include amino acid residues including natural and / or non-natural amino acid residues. The terms also include post-expression modifications of the peptide, for example, glycosylation, sialylation, acylation, acetylation, phosphorylation, and the like. According to some embodiments, the peptides of the invention do not comprise post-expression modifications. According to some embodiments, the peptides of the invention are acylated. According to some embodiments, the N-termini of the peptides of the invention are acylated. According to some embodiments, the peptides may contain modifications with respect to a native or natural sequence, as long as the peptide maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0069] In some embodiments, amino acid sequence variants of the peptides provided herein are contemplated. A variant typically differs from a peptide specifically disclosed herein in one or more substitutions, deletions, additions and / or insertions. Such variants can be naturally occurring or can be synthetically generated, for example, by modifying one or more of the above peptide sequences of the invention and evaluating one or more biological activities of the peptide as described herein, and / or using any of a number of known techniques. For example, it may be desirable to improve the binding affinity and / or other biological properties of the peptide. Amino acid sequence variants of a peptide may be prepared by introducing appropriate modifications into the nucleotide sequence encoding it, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the peptide. Any combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final sequence possesses the desired characteristics, e.g., binding collagen present in cartilage, such as collagen type II.

[0070] According to some aspects and embodiments, the isoelectric point (PI) of the peptides within the scope of the present invention is in the range of about 5 to about 9, including each value within the specified range. Thus, for example, the isoelectric point is at pH of about 5.5 to about 8.5, about 6.0 to about 8.0, or about 6.5 to about 7.5, including each value within the specified ranges. Without being bound by any theory or mechanism of action, it is contemplated that the peptides encompassed by the present invention are hydrophilic thereby selectively protruding from the exterior surface of the liposomes.

[0071] The peptides of the present invention may be produced by any method known in the art, including recombinant and synthetic methods. Synthetic methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, or classical solution synthesis, all well known to one skilled in the art. The resulted peptide sequence is confirmed by methods known to one skilled in the art, for example amino acid sequencing.

[0072] In some embodiments, recombinant protein techniques are used to generate the peptides of the present invention. Recombinant techniques are described, for example, by Puetz and Wurm, Processes 7(8), 476, (2019); O' Flaherty et al. Biotech. Adv., Volume 43, (2020); Burnett and Burnett Plants People Planet 2(2), 121-132, (2020); Gnoth et al., Bioproc, and Biosys. Engin. 31, 21-39, (2008); Yang et al., App. Micro, and Biotech. 105, 6607-6626, (2021); and Tripathi and Shrivastava, Front. Bioeng. Biotechnol., Section Synthetic Bio., Volume 7, (2019).

[0073] According to some embodiments, the peptide including the collagen-binding sequence may be activated prior to being reacted with the amine of the PL. For example, activation may include, but is not limited to, acidifying the peptide, adding a crosslinker to the peptide, and the like. In exemplary embodiments, the crosslinker may be a carbodiimide selected from, but not limited to, EDC (EDAC, EDCI, l-ethyl-3 -(3 -dimethylaminopropyl) carbodiimide), DCC (N,N'- dicyclohexylcarbodiimide), and DIC (N,N'-diisopropylcarbodiimide). According to some specific embodiments, the crosslinker is EDC. According to some embodiments, the EDC activating reaction includes the addition of N-hydroxy succinimide (NHS) or sulfo-NHS.

[0074] In some aspects of the present invention, the at least one collagen-binding sequence is operational to bind a collagen which is present in the cartilage. According to some embodiments, the at least one collagen-binding sequence is capable of binding a collagen which is present in the cartilage. The term “cartilage”, as used herein, refers to any type of cartilage present in the body of a treated subject. According to some particular embodiments, the term “cartilage” refers to a cartilage selected from hyaline cartilage (known also as “articular cartilage”), fibrocartilage (including menisci, ligaments and tendons), elastic cartilage, and hypertrophic cartilage. According to some embodiments, the term “cartilage” refers to hyaline cartilage, hypertrophic cartilage, and menisci. According to some embodiments, the term “cartilage” includes both healthy and pathological cartilage, pathological cartilage including, but not limited to, osteoarthritic cartilage, degenerative cartilage, rheumatoid cartilage, crystal laden (gout) cartilage.

[0075] As used herein, the term “present in the cartilage” refers to collagen which is present in a mammalian cartilage, optionally in a human cartilage. According to some embodiments, the term “collagen present in the cartilage” refers to collagen naturally present in a healthy joint. According to some embodiments, the term “collagen present in the cartilage” refers to collagen naturally present in a joint suffering from a disorder (e.g., osteoarthritis, or any other joint disorder). According to some embodiments, the term “collagen present in the cartilage” refers to collagen naturally present in a healthy joint and / or in a joint suffering from a disorder. According to some embodiments, the term “collagen present in the cartilage” refers to collagen naturally present in cartilage of a subject in need thereof.

[0076] According to some embodiments, the term “collagen present in the cartilage” refers to collagen present in hyaline cartilage, which includes, but is not limited to, collagen type-II, collagen type-III, collagen type-IV, collagen type-V, collagen type- VI, collagen type-XII, collagen type-IX, collagen type-X, collagen type-XI, collagen type-XIV, collagen type-XVI, collagen type- XXII, collagen type-XXVI. According to some embodiments, the term “collagen present in the cartilage” refers to collagen present in fibrocartilage (such as the meniscus), which includes, but is not limited to, collagen type-I, collagen type-II, and collagen-type V. According to some embodiments, the term “collagen present in the cartilage” refers to collagen present in hypertrophic cartilage, which includes, but is not limited to, collagen type-X. According to some embodiments, the term “collagen present in the cartilage” refers to collagen present in elastic cartilage, which includes, but is not limited to, collagen type-II, collagen type- VI, collagen type-IX, collagen type- X, and collagen type-XI. According to some embodiments, the term “collagen present in the cartilage” refers to collagen present in any one of hyaline cartilage, fibrocartilage, hypertrophic cartilage, and elastic cartilage. According to some embodiments, the term “collagen present in the cartilage” refers to collagen present in any one of hyaline cartilage, fibrocartilage, and hypertrophic cartilage. According to some embodiments, the term “collagen present in the cartilage” refers to collagen present in any one of hyaline cartilage and fibrocartilage.

[0077] According to some embodiments, the collagen binding sequence binds at least one collagen type selected from collagen type-I, collagen type-II, collagen type-III, collagen type-IV, collagen type-V, collagen type- VI, collagen type-IX, collagen type-X, collagen type-XI, collagen type-XII, collagen type-XIV, collagen type-XVI, collagen type-XXII, and collagen type-XXVII. According to some embodiments, the collagen-binding sequence is operational to bind a collagen which is present in high amounts in the cartilage, i.e., collagen type-II, collagen type-IX, and / or collagen type-XI. According to some specific embodiments, the collagen-binding sequence present in the peptide is a collagen type-II binding sequence. As used herein, the term “collagen-binding sequence” refers to an amino acid sequence which has been demonstrated to bind with high specificity to collagen protein, particularly to collagen protein present in the cartilage.

[0078] Li, Xiaojing, et al. (J. Amer. Chem. Soc. 145.20 (2023)) discloses collagen hybridizing peptides comprising repeats of a GXY amino acid sequence (e.g., GPO) which effectively bound various collagen protein types (such as collagen type I, type II, type III, type IV, and others). Qi, Yingying, et al. (Chem. Sci., 13, 12567-12576 (2022)) further disclosed collagen mimetic peptides comprising repeats of the amino acid sequence POG, and optionally also OGP, which were also found to be effective collagen binders.

[0079] According to some embodiments, the collagen binding sequence comprises a collagen hybridizing peptide (CHP), i.e., a synthetic peptide that can specifically bind collagen strands, particularly through hydrogen bonding. According to some embodiments, the collagen binding sequence comprises a collagen mimetic peptide (CMP), i.e., a synthetic peptide that designed to replicated the structure and function of collagen. According to some embodiments, the collagen binding sequence comprises at least one of a CHP and a CMP. According to some embodiments, the at least one of a CHP and a CMP comprises the amino acid sequence GlyXaaYaa (herein GXY) at least once. According to some embodiments, the GXY sequence comprises GlyProHyp (Glycine-Proline-Hydroxyproline, herein GPO). According to some embodiments, the at least one of a CHP and CMP comprises at least one repeat of the sequence GPO. According to some embodiments, the at least one of a CHP and CMP comprises at least 2 repeats, at least 3 repeats, or at least 4 repeats of the sequence GPO. Each possibility represents a separate embodiment of the invention.

[0080] According to some embodiments, the collagen binding sequence comprises between 1 to 14 repeats of the sequence GPO. According to some embodiments, the collagen binding sequence comprises between 1 and 12 repeats of the sequence GPO, between 2 and 12 repeats, between 3 and 12 repeats, between 4 and 12 repeats, between 5 and 12 repeats, between 1 and 10 repeats, between 2 and 10 repeats, between 3 and 10 repeats, between 4 and 10 repeats, between 5 and 10 repeats, between 6 and 10 repeats, between 2 and 9 repeats, between 3 and 9 repeats, between 4 and 9 repeats, between 5 and 9 repeats, between 6 and 9 repeats, between 7 and 9 repeats, between

[0081] 1 and 8 repeats, between 2 and 8 repeats, between 3 and 8 repeats, between 4 and 8 repeats, between 5 and 8 repeats, between 6 and 8 repeats, between 2 and 7 repeats, between 3 and 7 repeats, between 4 and 7 repeats, between 5 and 7 repeats, between 1 and 6 repeats, between 2 and 6 repeats, between 3 and 6 repeats, between 4 and 6 repeats, between 1 and 5 repeats, between 2 and 5 repeats, between 3 and 5 repeats, between 4 and 5 repeats, between 1 and 4 repeats, between

[0082] 2 and 4 repeats, between 3 and 4 repeats, between 1 and 3 repeats, between 2 and 3 repeats, or between 1 and 2 repeats of the sequence GPO. Each possibility represents a separate embodiment of the invention. According to some embodiments, the collagen binding sequence comprises 4 repeats of the sequence GPO (SEQ ID NO: 6).

[0083] According to some embodiments, the collagen binding sequence comprises a sequence selected from the group consisting of SEQ ID NO: 6, GPOGPO (SEQ ID NO: 10), GPOGPOGPO (SEQ ID NO: 11), GPOGPOGPOGPO (SEQ ID NO: 12), GPOGPOGPOGPOGPOGPO (SEQ ID NO: 13), GPOGPOGPOGPOGPOGPOGPO (SEQ ID NO: 14),

[0084] GPOGPOGPOGPOGPOGPOGPOGPO (SEQ ID NO: 15),

[0085] GPOGPOGPOGPOGPOGPOGPOGPOGPO (SEQ ID NO: 16),

[0086] GPOGPOGPOGPOGPOGPOGPOGPOGPOGPO (SEQ ID NO: 17),

[0087] GPOGPOGPOGPOGPOGPOGPOGPOGPOGPOGPO (SEQ ID NO: 18),

[0088] GPOGPOGPOGPOGPOGPOGPOGPOGPOGPOGPOGPO (SEQ ID NO: 19), GPOGPOGPOGPOGPOGPOGPOGPOGPOGPOGPOGPOGPO (SEQ ID NO: 20), GPOGPOGPOGPOGPOGPOGPOGPOGPOGPOGPOGPOGPOGPO (SEQ ID NO: 21), and GPOGPOGPOGPOGPOGPOGPOGPOGPOGPOGPOGPOGPOGPOGPO (SEQ ID NO: 22).

[0089] According to some embodiments, the collagen binding sequence comprises no more than 15 repeats of the sequence GPO. According to some embodiments, the collagen binding sequence comprises no more than 12 repeats, no more than 10 repeats, no more than 9 repeats, no more than 8 repeats, no more than 7 repeats, no more than 6 repeats, or no more than 5 repeats of the sequence GPO. Each possibility represents a separate embodiment of the invention.

[0090] According to some embodiments, the at least one of a CHP and a CMP comprises at least one of each of the amino acids Gly, Pro, and Hyp. According to some embodiments, the at least one of a CHP and CMP comprises the sequence GPO, POG, and / or of the sequence OGP at least once. According to some embodiments, the at least one of a CHP and CMP comprises at least 1 repeat, at least 2 repeats, at least 3 repeats, or at least 4 repeats of the sequence GPO, POG, and / or of the sequence OGP. Each possibility represents a separate embodiment of the invention.

[0091] According to some embodiments, the collagen binding sequence comprises the sequence POG at least once. According to some embodiments, the collagen binding sequence comprises at least 1 repeat, at least 2 repeats, at least 3 repeats, at least 4 repeats, or at least 5 repeats of the sequence POG. Each possibility represents a separate embodiment of the invention.

[0092] According to some embodiments, the collagen binding sequence comprises no more than 15 repeats of the sequence POG. According to some embodiments, the collagen binding sequence comprises no more than 14 repeats, no more than 13 repeats, no more than 12 repeats, no more than 11 repeats, no more than 10 repeats, no more than 8 repeats, or no more than 5 repeats of the sequence POG. Each possibility represents a separate embodiment of the invention.

[0093] Lin, Chin-Yu, et al. (Nat. Biomed. Engin. 6.10 (2022)) discloses various phage-displayed peptide sequences which share the consensual amino acid sequence WXPXW (SEQ ID NO: 7). Peptides comprising said sequence were found to effectively bind at least collagen type XII, presence of which collagen is characteristic of human cartilage affected by osteoarthritis.

[0094] According to some embodiments, the collagen binding sequence comprises SEQ ID NO: 7. According to some embodiments, the collagen binding sequence binds collagen XII. According to some embodiments, the collagen binding sequence comprises at least one sequence selected from DLQYWYPIWDTH (SEQ ID NO: 8), DAYWHPVWVHDP (SEQ ID NO: 9), and derivatives thereof. According to some embodiments, the collagen binding sequence comprises SEQ ID NO: 8. According to some embodiments, the collagen binding sequence comprises SEQ ID NO: 9.

[0095] According to some embodiments, the collagen-binding sequence comprises at least one amino acid sequence selected from WYRGRL (SEQ ID NO: 1), GPO, POG, OGP, WXPXW (SEQ ID NO: 7), and combinations thereof.

[0096] According to some particular embodiments, the collagen-binding sequence comprises at least one sequence selected from WYRGRL (SEQ ID NO: 2), WYRGRLSGS (SEQ ID NO: 3), WYRGRLSGSSGS (SEQ ID NO: 4), WYRGRLGDPGD WYRGRLSGS (SEQ ID NO: 5), and GPOGPOGPOGPOGPO (SEQ ID NO: 6).

[0097] According to some specific embodiments, the collagen-binding sequence present in the peptide is a collagen type-II binding sequence. According to some embodiments, the collagen type-II binding sequence comprises at least one of WYRGRL (SEQ ID NO: 1) and the amino acid sequence GPO. According to some particular embodiments, the collagen type-II binding sequence comprises the amino acid sequence WYRGRL (SEQ ID NO: 1). According to some embodiments, the collagen type-II binding sequence comprises at least one sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. According to some embodiments, the collagen type-II binding sequence comprises at least one sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

[0098] As used herein, the term “collagen-binding peptide”, often referred to only as “the peptide”, refers to a peptide according to the present invention which comprises a collagen-binding amino acid sequence. According to some embodiments, the peptide comprises the collagen-binding sequence once. According to some embodiments, the peptide comprises one or more repeats of the collagen-biding sequence. As used herein, the term “repeat” refers to an additional use or appearance of an element, e.g., an amino acid sequence, beyond a single or first use or appearance thereof. Thus, “one repeat” refers to two uses or appearances of the repeated element, “two repeats” refers to three uses or appearances of the element, etc. According to some embodiments, the peptide comprises one repeat of the collagen-binding sequence, i.e., includes the collagen- binding sequence twice. According to some further embodiments, the peptide comprises two repeats of the collagen-binding sequence. According to some particular embodiments, the peptide comprises two or more repeats of the collagen-binding sequence.

[0099] According to some embodiments, the peptide consists of the collagen-binding sequence. According to some further embodiments, the peptide consists of the collagen-binding sequence, and an additional amino acid. According to some embodiments, the peptide consists of the collagen-binding sequence, and an additional amino acid at the N- or C-termini thereof. According to some embodiments, the additional amino acid is selected from the group consisting of aspartic acid, and glutamic acid. According to some embodiments, the additional amino acid is aspartic acid.

[0100] In some aspects of the present invention, the peptide comprises a spacer amino acid sequence between the collagen-binding sequence and the lipid. According to some embodiments, the spacer sequence consists of 1 to 15 amino acids, including each integer within the specified range. According to some specific embodiments, the spacer comprises between 1 to 12 amino acids, between 1 to 10 amino acids, between 1 to 6 amino acids, between 1 to 5 amino acids, between 1 to 4 amino acids, between 1 to 3 amino acids, between 2 to 12 amino acids, between 2 to 10 amino acids, between 2 to 8 amino acids, between 2 to 6 amino acids, between 2 to 5 amino acids, between 2 to 4 amino acids, between 2 to 3 amino acids, between 3 to 12 amino acids, between 3 to 9 amino acids, between 3 to 6 amino acids, between 3 to 5 amino acids, or between 3 to 4 amino acids. Each possibility represents a separate embodiment. According to particular embodiments, the spacer comprises between 3 to 5 amino acids. According to some specific embodiments, the spacer comprises 3 amino acids. According to some embodiments, the spacer comprises amino acids selected from serine, glycine, proline, alanine, and a combination thereof. According to some specific embodiments, the spacer comprises amino acids selected from serine, glycine, and a combination thereof. According to some particular embodiments, the spacer comprises an amino acid sequence selected from GGS, SGS, and SSS. Each possibility represents a separate embodiment.

[0101] According to some embodiments, the peptide does not comprise a spacer amino acid between the collagen-binding peptide and the lipid.

[0102] According to some embodiments, the peptide comprises one or more neutralizing amino acids, for balancing the electric charge of the peptide. According to some embodiments, the peptide comprises between 1-3 neutralizing amino acids. According to some embodiments, the peptide comprises a single neutralizing amino acid. According to some embodiments, the neutralizing amino acid(s) is selected from the group consisting of aspartic acid (D), Glutamic acid (E), and arginine (R). According to some embodiments, the neutralizing amino acid is selected from D and R. According to some embodiments, the peptide comprises SEQ ID NO: 1, and the neutralizing amino acid is D. According to some embodiments, the peptide comprises the amino acid GPO at least once, and the neutralizing amino acid is R. According to some embodiments, neutralizing amino acid is located at the C-terminus of the peptide, and the conjugation to the lipid is though the carboxyl group thereof.

[0103] According to some embodiments, the peptide comprises an amino acid sequence selected from the group consisting of WYRGRL (SEQ ID NO: 2), WYRGRLSGS (SEQ ID NO: 3), WYRGRLSGSSGS (SEQ ID NO: 4), WYRGRLGDPGD WYRGRLSGS (SEQ ID NO: 5), and GPOGPOGPOGPOGPO (SEQ ID NO: 6). According to some particular embodiments, the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. According to some embodiments, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. According to some further embodiments, the peptide comprises an amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NO: 4. According to some specific embodiments, the peptide comprises an amino acid sequence as set forth in SEQ ID NO: 3. According to some embodiments, the peptide consists of the amino acid sequence as set forth in SEQ ID NO: 3.

[0104] In some aspects of the invention, the peptides conjugated to the liposomes comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1, GPO, POG, OGP, SEQ ID NO: 7, and a mixture or combination thereof. According to some embodiments, a single liposome comprises a combination of 2 or more different peptide sequences conjugated to lipids thereof, the peptide sequences respectively comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, GPO, POG, OGP, and SEQ ID NO: 7. According to some further embodiments, a liposomal composition comprises a mixture of liposomes, each liposome being conjugated to peptides comprising one of the sequences selected from SEQ ID NO: 1, GPO, POG, OGP, and SEQ ID NO: 7. According to some embodiments, the peptide can comprise the sequence once, or may include several repeats of the sequence. According to some embodiments, particularly regarding sequences GPO, POG, and OGP, the peptide can include between 1 and 14 repeats of the sequence, including each value within the specified range.

[0105] According to some embodiments, the peptides conjugated to the liposomes comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and a mixture or combination thereof. According to some embodiments, a single liposome comprises a combination of 2 or more different peptide sequences conjugated to lipids thereof, selected from SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO:

[0106] 4, SEQ ID NO: 5, and SEQ ID NO: 6. According to some further embodiments, a liposomal composition comprises a mixture of liposomes, each liposome being conjugated to peptides having one of the sequences selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:

[0107] 5, and SEQ ID NO: 6. For example, a liposomal composition can comprise 50% of liposomes conjugated to peptides comprising SEQ ID NO: 3 and 50% of liposomes conjugated to peptides comprising SEQ ID NO: 4. Additional compositions include, but are not limited to, 50% SEQ ID NO: 3 and 50% SEQ ID NO: 5; 50% SEQ ID NO: 2 and 50% SEQ ID NO: 3; 50% SEQ ID NO: 3 and 50% SEQ ID NO: 6; 50% SEQ ID NO: 4 and 50% SEQ ID NO: 6; 50% SEQ ID NO: 3, 25% SEQ ID NO: 4, and 25% SEQ ID NO: 5; 25% SEQ ID NO: 2, 25% SEQ ID NO: 3, 25% SEQ ID NO: 4, and 25% SEQ ID NO: 5. Each possibility represents a separate embodiment. Any other relative ratio or combination of peptides is included herein.

[0108] According to some embodiments, the peptides conjugated to the liposomes comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and a mixture or combination thereof. According to some embodiments, a single liposome comprises a combination of 2 or more peptide sequences conjugated to lipids thereof, selected from SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. According to some further embodiments, a liposomal composition comprises a mixture of liposomes, each liposome being conjugated to peptides having one of the sequences selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. For example, a liposomal composition can comprise 50% of liposomes conjugated to peptides comprising SEQ ID NO: 3 and 50% of liposomes conjugated to peptides comprising SEQ ID NO: 4. Additional compositions include, but are not limited to, 50% SEQ ID NO: 3 and 50% SEQ ID NO: 5; 50% SEQ ID NO: 2 and 50% SEQ ID NO: 3; 50% SEQ ID NO: 3, 25% SEQ ID NO: 4, and 25% SEQ ID NO: 5; 25% SEQ ID NO: 2, 25% SEQ ID NO: 3, 25% SEQ ID NO: 4, and 25% SEQ ID NO: 5. Each possibility represents a separate embodiment. Any other relative ratio or combination of peptides is included herein.

[0109] According to some embodiments, the peptide is connected to a lipid comprising an amine moiety. According to further embodiments, the peptide is connected to a phosphatidylamine lipid. Phosphatidylamine lipids within the scope of the present invention include, but are not limited to, l,2-Dipahmtoyl-sn-glycero-3 -phosphoethanolamine (DPPE), 1,2-dilauroyl-L-phosphatidyl- ethanolamine (DLPE), l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- Diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE), l,3-Dipalmitoyl-sn-glycero-2- phosphoethanolamine (1,3-DPPE), l-Palmitoyl-3-oleoyl-sn-glycero-2-phosphoethanolamine (1,3-POPE), Biotin-Phosphatidylethanolamine, l,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), l,2-Distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), and Phosphatidylserine (PS). Each possibility represents a separate embodiment. According to some embodiments, the peptide is activated prior to conjugation with the lipid.

[0110] In some aspects of the invention, about 1% to about 20% of the PL which make up the liposome are conjugated to peptides comprising a collagen-binding sequence. According to some embodiments, the phospholipid conjugated to the peptide constitutes between about 1% to about 20% of lipids in the liposome, including each value within the specified range. According to some embodiments, the phospholipid conjugated to the peptide constitutes between about 2% to about 18%, between about 2% to about 15%, between about 2% to about 12%, between about 2% to about 10%, or between about 2% to about 8%. Each possibility represents a separate embodiment of the invention. According to some specific embodiments, about 2% to about 5% of the PL which make up the liposome are conjugated to peptides comprising a collagen-binding sequence, according to the present invention.

[0111] Additional phospholipids that may be incorporated within the liposomes disclosed herein include, but are not limited to, glycerophospholipids (GPL) and sphingomyelin (SM). Each possibility represents a separate embodiment. In some embodiments of the present invention, the GPL comprises two acyl chains. In further embodiments, said chains are selected from the group consisting of Ci4, C15, Ci6, and Cis acyl chains. Each possibility represents a separate embodiment. In certain embodiments, at least one of said hydrocarbon chains is a saturated hydrocarbon chain. In further embodiments, the two hydrocarbon chains are saturated. In other embodiments, the GPL is a phosphatidylcholine (PC). According to some further embodiments, the GPL which forms the liposome membrane(s) is a mixture of PC and PE.

[0112] PCs, PEs, and SMs are zwitterionic phospholipids with cationic choline or ethanolamine and anionic diester phosphate moieties (constituting the phosphocholine head group). The hydrophobic part of the PC, PE and PG includes 2 hydrocarbon chains (e.g., acyls and alkyls). The SM also has two hydrophobic hydrocarbon chains of which one is the chain of the sphingoid base itself and the other is N-acyl chain. PC, PE, SM and PG in which the hydrocarbon chains are above 12 carbon atoms are all cylinder-like in shape and their packing parameter is in the range of about 0.74 to about 1.0. They form lipid bilayers which become highly hydrated and vesiculate to form lipid vesicles (liposomes) above the solid ordered (SO) to liquid disordered (LD) phase transition temperature.

[0113] In certain embodiments, the PC comprises at least one of l,2-dimyristoyl- n-glycero-3- phosphocholine (DMPC); l,2-dipah toyl-sn-glycero-3 -phosphocholine (DPPC); 1,2- dipentadecanoyl- n-glycero-3 -phosphocholine (C 15) ; 1 ,2-di stcaroy l-.sn-glyccro-3 -phosphocholine (DSPC); N-palmitoyl-D-eryt / zro-sphingosylphosphorylcholine (D-erythro C16), and a mixture or combination thereof. Each possibility represents a separate embodiment.

[0114] In additional embodiments, the PC is selected from the group consisting of 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC); l,2-dipahmtoyl-sn-glycero-3 -phosphocholine (DPPC),

[0115] 1.2-dipentadecanoyl-sn-glycero-3 -phosphocholine (Cl 5), l,2-distearoyl- n-glycero-3- phosphocholine (DSPC), and N-palmitoyl-D-eryt / zro-sphingosylphosphorylcholine (D-erythro C16). Each possibility represents a separate embodiment. In one embodiment, the PC comprises

[0116] 1.2-dipahmtoyl-sn-glycero-3 -phosphocholine (DPPC). In another embodiment, the PC comprises

[0117] 1.2-dimyristoyl-sn-glycero-3 -phosphocholine (DMPC). According to some embodiments, the PC comprises a combination of DMPC and DPPC

[0118] According to particular embodiments, the liposomes encompassed by the present invention comprises a combination of DMPC, DPPC, and DPPE. In further embodiments, the mole percent of DMPC is in the range of about 24% to about 70%, the mole percent of DPPC is in the range of about 75% to about 29%, and the mole percent of DPPE is in the range of about 1% to about 10%, including all values within the respective specified ranges. In certain embodiments, the mole percent ratio of DMPC to DPPC to DPPE is about 30:58:2 to about 55:37:8, including all iterations of ratios within the specified range. In certain embodiments, the mole percent ratio of DMPC to DPPC to DPPE is in the range of about 40:57.5:2.5 to about 55:40:5, including all iterations of ratios within the specified range. In particular embodiments, the mole percent ratio of DMPC to DPPC to DPPE is about 45:50:5. In further embodiments, the liposomes encompassed by the present invention comprise a combination of DMPC, C15, and DPPE. In further embodiments, the mole percent ratio of DMPC to C15 to DPPE is in the range of about 25:73:2 to about 42:50:8, including all iterations of ratios within the specified range.

[0119] In some embodiments, the liposomes encompassed by the present invention comprise a combination of DMPC, DSPC, and DPPE. In further embodiments, the mole percent ratio of DMPC to DSPC to DPPE is about 88: 10:2 to about 55:37:8, including all iterations of ratios within the specified range. In particular embodiments, the mole percent ratio of DMPC to DSPC to DPPE is about 72:23:5.

[0120] In certain embodiments, the liposomes encompassed by the present invention comprise a combination of DMPC, D-erythro Cl 6, and DPPE. In further embodiments, the mole percent ratio of DMPC to D-erythro C16 to DPPE is in the range of about 10:88:2 to about 24:68:8, including all iterations of ratios within the specified range.

[0121] The liposomal composition according to the principles of the present invention, comprises phospholipids in a concentration that ranges from about 20 to about 500 mM, including each value within the specified range. In various embodiments, the total concentration of the phospholipids in the composition ranges from about 50 to about 300 mM, including each value within the specified range. In certain embodiments, the total concentration of the phospholipids in the composition ranges from about 100 to about 200 mM, including each value within the specified range. In yet further embodiments, the concentration ranges from about 130 to about 170 mM, including each value within the specified range. In certain embodiments, the total PL concentration is about 150 mM.

[0122] In some embodiments, the total PL concentration ranges from about 10 to about 500 mg / ml, including each value within the specified range. In further embodiments, the concentration ranges from about 30 to about 300 mg / ml, including each value within the specified range. In still further embodiments, the concentration ranges from about 50 to about 200 mg / ml, including each value within the specified range. In certain embodiments, the total PL concentration is about 100 mg / ml.

[0123] In various embodiments, the PL is present in the liposomal composition in a weight percent ranging from about 0.5% to about 30% (w / w), including each value within the specified range. In yet other embodiments, the weight percent is from about 3% to about 30% (w / w), including each value within the specified range. In further embodiments, the weight percent is from about 1% to about 20% (w / w) of the total weight of the composition, including each value within the specified range. In particular embodiments, the PL is present in the liposomal composition in a weight percent of about 10% (w / w). In some aspects of the present invention, the liposomes are characterized by a zeta potential which prevents rapid clearance thereof from the cartilage. In some further aspects of the present invention, the liposomes are characterized by a zeta potential which protects the liposomes from forming strong interactions with other molecules present in the articular region. In some additional aspects of the invention, the liposomes of the present invention are characterized by a zeta potential which prevents rapid clearance thereof from the cartilage, while at the same time abstaining from forming strong interactions with other molecules present in the articular region. According to some embodiments, the zeta potential, as measured in HMB having a pH of 6.5, is in a range of about (- 30) to about 10 mV, including each value within the specified range. According to some embodiments, the zeta potential, as measured in HMB having a pH of 6.5, is in a range of about (- 25) to about 10 mV, including each value within the specified range. According to some embodiments, the zeta potential, as measured in HMB having a pH of 6.5, is in a range of about (- 20) to about 10 mV, including each value within the specified range. According to some embodiments, the zeta potential, as measured in HMB having a pH of 6.5, is in a range of about (- 10) to about 10 mV, including each value within the specified range. According to some embodiments, the liposome is characterized by a zeta potential in the range of about (-30) to about 5 mV, about (-30) to about 0 mV, about (-30) to about (-5) mV, about (-30) to about (-10) mV, about (-30) to about (-15) mV, about (-30) to about (-20) mV, about (-30) to about (-25) mV, about (-25) to about 5 mV, about (-25) to about 0 mV, about (-25) to about (-5) mV, about (-25) to about (-10) mV, about (-25) to about (-15) mV, about (-25) to about (-20) mV, about (-20) to about 5 mV, about (-20) to about 0 mV, about (-20) to about (-5) mV, about (-20) to about (-10) mV, about (-20) to about (-15) mV, about (-15) to about 5 mV, about (-15) to about 0 mV, about (-15) to about (-5) mV, about (-15) to about (-10), about (-10) to about 9 mV, about (-10) to about 8 mV, about (-10) to about 7 mV, about (-10) to about 6 mV, about (-10) to about 5 mV, about (-10) to about 4 mV, about (-10) to about (3) mV, about (-10) to about 2 mV, about (-10) to about 1 mV, about (-10) to about 0 mV, about (-10) to about (-1) mV, about (-10) to about (-2) mV, about (-10) to about (-3) mV, about (-10) to about (-4) mV, (-10) to about (-5) mV, about (-10) to about (-6) mV, about (-10) to about (-7) mV, about (-10) to about (-8) mV, or about (-5) to about (-2.5) mV, including each value within the specified ranges. Each possibility represents a separate embodiment. According to some embodiments, the zeta potential of the liposomes is negative.

[0124] According to some embodiments, the liposomal composition of the present invention, including peptide-conjugated liposomes of which are conjugated to the cartilage binding peptide via a direct amide bond (referred to herein also as “directly-bound peptide-conjugated liposomes” or as “zero-chemistry peptide-conjugated liposomes”), is characterized by having a macrophage uptake level which is at least 10% lower than the macrophage uptake of a liposomal compositions containing peptide-conjugated liposomes which are conjugated via a maleimide linker to substantially the same peptide (referred to herein as “corresponding maleimide-linked peptide- conjugated liposomes”). According to some embodiments, the liposomal composition of the present invention, including directly-bound peptide-conjugated liposomes, is characterized by having a macrophage uptake level which is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% lower than the macrophage uptake of liposomal compositions containing corresponding maleimide-linked peptide-conjugated liposomes. Each possibility represents a separate embodiment of the present invention.

[0125] According to some embodiments, the directly-bound peptide-conjugated liposomes according to the present invention, are characterized by having a macrophage uptake level which is at least 10% lower than the macrophage uptake of corresponding maleimide-linked peptide- conjugated liposomes. According to some embodiments, the directly-bound peptide-conjugated liposomes according to the present invention, are characterized by having a macrophage uptake level which is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% lower than the macrophage uptake of corresponding maleimide-linked peptide-conjugated liposomes. Each possibility represents a separate embodiment of the present invention.

[0126] According to some embodiments, the liposomal composition of the present invention, including directly-bound peptide-conjugated liposomes, is characterized by having a macrophage uptake level which is at least 1.5-fold reduced relative to the macrophage uptake of liposomal compositions containing corresponding maleimide-linked peptide-conjugated liposomes. According to some embodiments, the liposomal composition of the present invention, including directly-bound peptide-conjugated liposomes, is characterized by having a macrophage uptake level which is at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 12-fold, at least 15-fold, or at least 20-fold reduced relative to the macrophage uptake of a liposomal compositions containing corresponding maleimide-linked peptide-conjugated liposomes. Each possibility represents a separate embodiment of the present invention.

[0127] According to some embodiments, the directly-bound peptide-conjugated liposomes according to the present invention, are characterized by having a macrophage uptake level which is at least 1.5-fold reduced relative to the macrophage uptake of corresponding maleimide-linked peptide-conjugated liposomes. According to some embodiments, the directly-bound peptide- conjugated liposomes according to the present invention, are characterized by having a macrophage uptake level which is at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8 -fold, at least 9-fold, at least 10-fold, at least 12-fold, at least 15-fold, or at least 20-fold reduced relative to the macrophage uptake of corresponding maleimide- linked peptide-conjugated liposomes. Each possibility represents a separate embodiment of the present invention.

[0128] According to some embodiments, the macrophage uptake level is measured in fluorescence units (FU).

[0129] According to some embodiments, the liposomal composition of the present invention, including directly-bound peptide-conjugated liposomes, is characterized by having a quotient of the cartilage binding level of the liposomes divided by the macrophage uptake level thereof (referred to herein also as “a quotient”, “a combined residence-time effect”, “an overall residencetime effect”, or simply as “a combined effect” or “an overall effect”), which is at least 5% higher than the quotient (i.e., overall residence-time effect) of a liposomal compositions containing corresponding maleimide-linked peptide-conjugated liposomes. According to some embodiments, the liposomal composition of the present invention, including directly-bound peptide-conjugated liposomes, is characterized by having a quotient which is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, or at least 300% higher than the quotient of a liposomal compositions containing corresponding maleimide-linked peptide-conjugated liposomes. Each possibility represents a separate embodiment of the present invention.

[0130] According to some embodiments, the directly-bound peptide-conjugated liposomes according to the present invention, are characterized by having a quotient which is at least 5% higher than the quotient of corresponding maleimide-linked peptide-conjugated liposomes. According to some embodiments, the directly-bound peptide-conjugated liposomes according to the present invention, are characterized by having a quotient which is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% lower than the quotient of corresponding maleimide-linked peptide-conjugated liposomes. Each possibility represents a separate embodiment of the present invention.

[0131] According to some embodiments, the liposomal composition of the present invention, including directly-bound peptide-conjugated liposomes, is characterized by having a quotient which is at least 1.2-fold increased relative to the quotient of liposomal compositions containing corresponding maleimide-linked peptide-conjugated liposomes. According to some embodiments, the liposomal composition of the present invention, including directly-bound peptide-conjugated liposomes, is characterized by having a quotient which is at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8 -fold, at least 9-fold, at least 10-fold, at least 12-fold, at least 15-fold, or at least 20-fold increased relative to the quotient of liposomal compositions containing corresponding maleimide-linked peptide-conjugated liposomes. Each possibility represents a separate embodiment of the present invention.

[0132] According to some embodiments, the directly-bound peptide-conjugated liposomes according to the present invention, are characterized by having a quotient which is at least 1.2-fold increased relative to the quotient of corresponding maleimide-linked peptide-conjugated liposomes. According to some embodiments, the directly-bound peptide-conjugated liposomes according to the present invention, are characterized by having a quotient which is at least 1.25- fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4- fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 12-fold, at least 15-fold, or at least 20-fold increased relative to the quotient of corresponding maleimide-linked peptide-conjugated liposomes. Each possibility represents a separate embodiment of the present invention.

[0133] According to some embodiments, the cartilage binding is in fluorescence units (FU), and the quotient is calculated by dividing the FU measured for the cartilage binding by the FU measured for the macrophage uptake.

[0134] According to some embodiments, the liposomal composition of the present invention, including directly-bound peptide-conjugated liposomes, is characterized by having a macrophage uptake level which is less than 20-fold increased relative to the macrophage uptake level of liposomal compositions containing comparable liposomes with no conjugated peptide. The term “comparable liposomes”, as used herein, refers to liposomes having at least 90% or at least 95% identity in their lipid composition. According to some embodiments, the liposomal composition of the present invention, including directly-bound peptide-conjugated liposomes, is characterized by having a macrophage uptake level which is less than 15-fold, less than 12-fold, less than 10- fold, less than 9-fold, less than 8-fold, less than 7-fold, less than 6-fold, less than 5-fold, less than 4-fold, less than 3-fold, less than 2.5-fold, less than 2-fold, less than 1.75-fold, less than 1.5-fold, or less than 1.25-fold increased relative to the macrophage uptake level of liposomal compositions containing comparable liposomes with no conjugated peptide. Each possibility represents a separate embodiment of the invention. According to some embodiments, the liposomal composition of the present invention, including directly-bound peptide-conjugated liposomes, is characterized by having a macrophage uptake level which is substantially equal to the macrophage uptake level of liposomal compositions containing comparable liposomes with no conjugated peptide.

[0135] According to some embodiments, directly-bound peptide-conjugated liposomes according to the present invention, are characterized by having a macrophage uptake level which is less than 20-fold increased relative to the macrophage uptake level of comparable liposomes with no conjugated peptide. According to some embodiments, directly-bound peptide-conjugated liposomes according to the present invention, are characterized by having a macrophage uptake level which is less than 15-fold, less than 12-fold, less than 10-fold, less than 9-fold, less than 8- fold, less than 7-fold, less than 6-fold, less than 5-fold, less than 4-fold, less than 3-fold, less than 2.5-fold, less than 2-fold, less than 1.75-fold, less than 1.5-fold, or less than 1.25-fold increased relative to the macrophage uptake level of comparable liposomes with no conjugated peptide. Each possibility represents a separate embodiment of the invention.

[0136] According to some embodiments, directly-bound peptide-conjugated liposomes according to the present invention, are characterized by having a macrophage uptake level which is substantially equal to the macrophage uptake level of comparable liposomes with no conjugated peptide.

[0137] According to some embodiments, the liposomal composition of the present invention, including directly-bound peptide-conjugated liposomes, is characterized by having a quotient of the cartilage binding level of the liposomes divided by the macrophage uptake level thereof, which is at least 2.5% higher than the quotient of a liposomal compositions containing comparable liposomes with no conjugated peptide. According to some embodiments, the liposomal composition of the present invention, including directly-bound peptide-conjugated liposomes, is characterized by having a quotient which is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 100% higher than the quotient of a liposomal compositions containing comparable liposomes with no conjugated peptide. Each possibility represents a separate embodiment of the present invention.

[0138] According to some embodiments, directly-bound peptide-conjugated liposomes according to the present invention, are characterized by having a quotient of the cartilage binding level of the liposomes divided by the macrophage uptake level thereof, which is at least 2.5% higher than the quotient of a liposomal compositions containing comparable liposomes with no conjugated peptide. According to some embodiments, the liposomal composition of the present invention, including directly -bound peptide-conjugated liposomes, is characterized by having a quotient which is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 100% higher than the quotient of a comparable liposomes with no conjugated peptide. Each possibility represents a separate embodiment of the present invention. The peptide-conjugated liposomes according to the present invention comprise an internal aqueous phase surrounded by at least one membrane (i.e., the PL bilayer). According to the principles of the present invention, the internal aqueous phase does not include a nanoparticle. Within the scope of the present invention are peptide-conjugated liposomes suspended in a liquid medium. According to some embodiments, the internal aqueous phase and the liquid medium are the same. According to other embodiments, the internal aqueous phase and the liquid medium are different. According to further embodiments, the internal aqueous phase and / or the liquid medium comprise a buffer. In additional embodiments, the buffer is a histidine buffer. In other embodiments, the buffer is a phosphate-buffered saline.

[0139] Typically, the liquid medium in which the peptide-conjugated liposomes are suspended further comprises a tonicity agent. As used herein, the term “tonicity agent” refers to an excipient capable of adjusting the osmotic pressure of a liquid, liquid compositions, and formulations. In some embodiments, the tonicity agent adjusts the osmotic pressure of the liquid medium to be isotonic, making the liposomal composition biologically compatible with the synovial joints of mammals and therefore suitable for use in pharmaceutical compositions for intra-articular injection.

[0140] Tonicity agents, according to the principles of the present invention, may be ionic tonicity agents or non-ionic tonicity agents, with each possibility representing a separate embodiment. Currently preferred is the use of non-ionic tonicity agents, for example polyols. In some embodiments, the polyol comprises at least three hydroxyl groups, at least four hydroxyl groups, or at least five hydroxyl groups. Each possibility represents a separate embodiment. According to some embodiments, the polyol is linear. According to some further embodiments, the polyol comprises a linear carbon chain of at least three carbons. In various embodiments, the polyol comprises at least one of mannitol, sorbitol, glycerol, erythritol, maltitol, isomalt, trimethylolpropane, pentaerythritol, dextrose, lactose, and trehalose. Each possibility represents a separate embodiment. In particular embodiments, the polyol is mannitol. In other embodiments, the polyol is glycerol. In other embodiments, the non-ionic tonicity agent comprises a combination of polyols. In yet other embodiments, the polyol comprises a combination of mannitol and glycerol. In further embodiments, the polyol is sorbitol. In one embodiment, the polyol does not include xylitol. The liposomal composition can further include a combination of a polyol with an additional tonicity agent.

[0141] In some embodiments, the liposomal composition comprises a non-ionic tonicity agent in a weight percent ranging from about 0.05% to about 10% (w / w) of the total weight of the composition, including each value within the specified range. In other embodiments, the weight percent of the non-ionic tonicity agent in the composition ranges from about 0.1% to about 7% (w / w) of the total weight of the composition, including each value within the specified range. In yet other embodiments, the weight percent of the non-ionic tonicity agent in the composition ranges from about 0.5% (w / w) to about 5% (w / w) of the total weight of the composition, including each value within the specified range.

[0142] In some embodiments, the concentration of the non-ionic tonicity agent in the liposomal composition ranges from about 1 mg / ml to about 70 mg / ml, including each value within the specified range. In further embodiments, the concentration of the non-ionic tonicity agent ranges from about 10 mg / ml to about 70 mg / ml, including each value within the specified range. In still further embodiments, the concentration of the non-ionic tonicity agent ranges from about 10 mg / ml to about 50 mg / ml, including each value within the specified range. In certain embodiments, the concentration of the non-ionic tonicity agent is about 40 mg / ml. In additional embodiments, the concentration of the non-ionic tonicity agent is about 20 mg / ml.

[0143] In some embodiments, the liposomal composition has osmolality in the range of about 200 to about 600 mOsm, including each value within the specified range. In further embodiments, the liposomal composition has osmolality in the range of about 250 to about 500 mOsm, including each value within the specified range. In further embodiments, the liposomal composition has osmolality in the range of about 250 to about 400 mOsm, including each value within the specified range. In certain embodiments, the liposomal composition has osmolality of about 300 mOsm. In certain such embodiments, the liposomal composition is isotonic.

[0144] In some embodiments, the weight ratio between the liposomes and the non-ionic tonicity agent ranges from about 40: 1 to about 1:2, including all iterations of ratios within the specified range. In further embodiments, the weight ratio between the liposomes and the non-ionic tonicity agent ranges from about 30: 1 to about 2: 1, including all iterations of ratios within the specified range. In further embodiments, the weight ratio between the liposomes and the non-ionic tonicity agent ranges from about 15: 1 to about 2: 1, including all iterations of ratios within the specified range. In still further embodiments, the weight ratio between the liposomes and the non-ionic tonicity agent ranges from about 10: 1 to about 2: 1, including all iterations of ratios within the specified range. In yet further embodiments, the weight ratio between the liposomes and the non- ionic tonicity agent ranges from about 6: 1 to about 2: 1, including all iterations of ratios within the specified range. In additional embodiments, the weight ratio between the liposomes and the nonionic tonicity agent ranges from about 12: 1 to about 6: 1, including all iterations of ratios within the specified range. In additional embodiments, the weight ratio between the liposomes and the nonionic tonicity agent ranges from about 10: 1 to about 7: 1, including all iterations of ratios within the specified range.

[0145] According to some embodiments, the liposomes in the liposomal composition are characterized by a mean particle size in the range of about 50 to about 500 nm including any range therebetween. Within the scope of the present invention are mean particle sizes in the range of about 50 to about 400 nm, about 50 to about 300 nm, about 50 to about 200 nm, about 50 to about 150 nm, about 50 to about 140 nm, and about 50 to about 120 nm, each possibility represents a separate embodiment. In some embodiments, the liposomes in the liposomal composition are characterized by a mean particle size of less than 100 nm, for example in the range of about 50 to about 90 nm, including any range therebetween.

[0146] According to various embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 500 to about 5,000 nm, including each value within the specified range. According to other embodiments, the liposomes in the liposomal composition have an average particle size in the range of about 500 to about 4,500 nm, about 500 to about 4,200 nm, about 1,000 to about 5,000 nm, about 1,000 to about 4,500 nm, about 1,000 to about 4,200 nm, about 2,000 to about 4,500 nm, about 2,000 to about 4,000 nm, or about 3,000 to about 4,500 nm. Each possibility represents a separate embodiment.

[0147] The terms “average particle size” or “mean particle size” as used herein refer to a statistical average particle size (diameter) in a population of particles. The diameter of a substantially spherical particle can refer to a physical or hydrodynamic diameter. When non-spherical particles are characterized, the average particle size may refer to the largest linear distance between two points on the surface of the particle. Average particle sizes can be determined using various techniques known in the art including, but not limited to, laser diffraction, light scattering, sedimentation field flow fractionation, photon correlation spectroscopy, disc centrifugation, the Coulter Counter method, and the like. Each possibility represents a separate embodiment. In some embodiments, the terms “average particle size” or “mean particle size” refer to the mean diameter of a liposome derived from particle size distribution based on a number distribution model. In other embodiments, said terms refer to the mean diameter of a liposome derived from particle size distribution based on a volume distribution model. In additional embodiments, said terms refer to the mean diameter of a liposome derived from particle size distribution based on a surface area distribution model.

[0148] In some aspects and embodiments, the liposomes in the liposomal composition have a unimodal or monodispersed size distribution. In other aspects and embodiments, the liposomes in the liposomal composition have a bimodal size distribution. The term “unimodal” as used herein refers to a size distribution that comprises a single designated population. In some embodiments, the term “unimodal” refers to a size distribution containing one peak in a size distribution curve. In alternative embodiments, the term refers to the distribution of particles in which at least 50% or more of the particles are within ± 30% of the average particle size. The term “bimodal” as used herein refers to a size distribution that comprises two components designated first and second populations. In some embodiments, the term “bimodal” refers to a size distribution containing two separate peaks in a size distribution curve. In other embodiments, the term “bimodal” refers to a size distribution that can be deconvoluted into two components having varying degrees of separation. In accordance with the latter embodiments, a peak having a shoulder on one tail is regarded as a bimodal size distribution having two components.

[0149] According to some aspects and embodiments, the liposomes are in the form of small unilamellar vesicles (SUVs). The term “small unilamellar vesicles (SUVs)” as used herein refers to a vesicle composed of a single lipid bilayer, i.e., a membrane, which encloses an internal aqueous phase. Said vesicles can be of any shape including, but not limited to, ellipsoids, discoids, pear- shaped vesicles, cup-shaped vesicles, budded vesicles, and spherical vesicles. Each possibility represents a separate embodiment. Preferably, the small unilamellar liposomes of the present invention are substantially spherical in shape. In some embodiments, the SUVs are characterized by having an average particle size in the range of about 50 to about 400 nm, including each value within the specified range. For example, the SUVs may have an average particle size in the range of about 50 to about 300 nm, about 50 to about 200 nm, about 50 to about 150 nm, about 50 to about 140 nm, or about 50 to about 120 nm, including each value within the specified ranges. Each possibility represents a separate embodiment.

[0150] According to the principles of the present invention, a liposomal composition comprising liposomes in the form of SUVs refers to a composition comprising liposomes, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the liposomes are in the form of SUVs. Each possibility represents a separate embodiment.

[0151] In some aspects and embodiments, the liposomes are in the form of large unilamellar vesicles (LUVs). The term “large unilamellar vesicles (LUVs)” as used herein refers to a vesicle composed of a single lipid bilayer which encloses an internal aqueous phase. Similar to SUVs, LUVs may be in any shape including, but not limited to, ellipsoids, discoids, pear-shaped vesicles, cup-shaped vesicles, budded vesicles, and spherical vesicles. Each possibility represents a separate embodiment. Preferably, the LUVs of the present invention are substantially spherical in shape. In some embodiments, the LUVs are characterized by having an average particle size in the range of about 500 to about 5,000 nm, including each value within the specified range. For example, the LUVs may have an average particle size in the range of about 500 to about 4,000 nm, about 500 to about 3,000 nm, about 500 to about 2,000 nm, about 500 to about 1,500 nm, or about 500 to about 1,000 nm, including each value within the specified ranges. Each possibility represents a separate embodiment.

[0152] According to the principles of the present invention, a liposomal composition comprising liposomes in the form of LUVs refers to a composition comprising liposomes, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the liposomes are in the form of LUVs. Each possibility represents a separate embodiment.

[0153] In some aspects and embodiments, the liposomal composition comprises liposomes in the form of multilamellar vesicles (MLVs). The term “multilamellar vesicles (MLVs)” as used herein refers to a vesicle composed of at least two lipid bilayers, i.e., membranes, which enclose an internal aqueous phase. The number of lipid bilayers in MLVs according to the principles of the present invention may vary between 2 to 50, for example between 2 to 40 or between 2 to 30 bilayers, including each integer within the specified ranges. It is to be understood that when the liposomes are in the form of MLVs, the peptide-conjugated lipids are not exclusively present on the surface of the liposomes and may be incorporated therein in the various bilayers constituting the liposomes. In some embodiments, the MLVs are characterized by having an average particle size in the range of about 50 nm to about 10 pm, including each value within the specified range. For example, the LUVs may have an average particle size in the range of about 100 nm to about 10 pm, about 500 nm to about 10 pm, about 1 pm to about 10 pm, or about 1 pm to about 8 pm, including each value within the specified ranges. Each possibility represents a separate embodiment.

[0154] According to the principles of the present invention, a liposomal composition comprising liposomes in the form of MLVs refers to a liposomal composition in which at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the liposomes are in the form of MLVs. Each possibility represents a separate embodiment.

[0155] In one embodiment, the liposomal composition comprises a combination of SUVs and MLVs. In another embodiment, the liposomal composition is substantially devoid of MLVs. In some embodiments, the liposomes have a phase transition onset temperature in the range of about 20°C to about 42°C, including each value within the specified range. Within the scope of the present invention are phase transition onset temperatures in the range of about 21°C to about 42°C, or about 22°C to about 42°C, including each value within the specified ranges. Each possibility represents a separate embodiment.

[0156] The term “phase transition temperature”, as used herein, refers in some embodiments, to a temperature at which solid ordered (SO) to liquid disordered (LD) phase transition of the liposomes occurs. The phase transition temperature of the liposomes can be evaluated by Differential Scanning Calorimetry (DSC). Various parameters of the DSC thermogram which can be examined to assess the phase transition temperature include Ton, which represents the temperature at which the SO-LD phase transition is initiated and Toff, which represents the temperature at which the SO-LD phase transition ends during heating scans, and Tp, and Tm, which represent the temperature at which the maximum change in the heat capacity during the pretransition (Tp) and main transition (Tm) occurs, respectively. In some embodiments, the term “phase transition temperature” refers to the Tm. In other embodiments, the term “phase transition temperature” refers to the temperature range of the SO to LD phase transition.

[0157] According to some aspects and embodiments, the peptide-conjugated liposomes have a phase transition onset temperature that is lower than the physiological temperature of a mammalian synovial joint. In accordance with these embodiments, it is contemplated that the peptide- conjugated liposomes are in the liquid-disordered (LD) phase upon entering the synovial joint while administered to a subject. Typical phase transition onset temperatures within the scope of the present invention are in the range of about 20°C to about 41°C, including each value within the specified range. Exemplary phase transition onset temperatures are in the range of about 20°C to about 39°C, about 20°C to about 38°C, about 20°C to about 36°C, about 20°C to about 34°C, about 22°C to about 39°C, 22°C to about 36°C, 22°C to about 32°C, or about 24°C to about 31°C. Each possibility represents a separate embodiment. In certain embodiments, the peptide-conjugated liposomes in the liposomal compositions of the present invention are characterized by a phase transition onset temperature of about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, or about 41°C. Each possibility represents a separate embodiment.

[0158] Within the scope of the present invention is a liquid medium suspending the liposomes which is characterized by a viscosity ranging from about 0.1 to about 15 cP, including each value within the specified range. In currently preferred embodiments, the viscosity of the liquid medium is in the range of about 2 to about 10 cP, including each value within the specified range. For example, the viscosity of the liposomal composition is about 0.1, about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, or about 15 cP, with each possibility representing a separate embodiment. The viscosity may be measured as is known in the art using a suitable viscometer. For example, viscosity can be measured using a viscometer such as, but not limited to, a Brookfield Viscometer or an Anton Paar Rheoplus viscometer with an appropriate setup. In some embodiments, the viscosity is measured using an RVT Brookfield Viscometer at 25°C.

[0159] The pH of the liposomal composition is typically in the range of about 5 to about 8, including each value within the specified range. For example, the pH of the liposomal composition is about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, or about 8.0, with each possibility representing a separate embodiment. According to some embodiments, The pH of the liposomal composition is in the range of about 6.0 to about 7.0, or about 6.3 to about 6.7. It is to be understood that the pH of the liposomal composition may be adjusted by the addition of acid(s) (e.g., HC1) and / or base(s) (e.g., NaOH) as is known in the art.

[0160] The liposomal compositions are obtainable by any method known in the art. Non-limiting examples for the preparation of the liposomal compositions of the present invention include sonication, extrusion, high-pressure homogenization, microfluidic mixing, and flow focusing. The nanometric liposomes may also be produced by size reduction of larger micrometric liposomes. For example, micrometric liposomes can be extruded through a filter having a pore size <100 nm.

[0161] The liposomal compositions of the invention may be used to treat, alleviate, ameliorate, retard, prevent, manage, or cure any articular disorder or symptoms arising therefrom which is associated with joint dysfunction. The term “articular disorder” as used herein, should be held to mean any affliction (congenital, autoimmune, or otherwise), injury, or disease of the articular region which causes degeneration, pain, reduction in mobility, inflammation, irritation, or physiological disruption and dysfunction of joints. The disorder may be associated with reduced joint secretion and lubrication as well as complications of knee or hip replacement.

[0162] The joint in accordance with the principles of the invention may be any one of the knees, hip, ankle, shoulder, elbow, tarsal, carpal, interphalangeal, and intervertebral. Each possibility represents a separate embodiment of the invention. In certain embodiments, said joint is a knee joint. Specific articular disorders within the scope of the present invention include, but are not limited to, deficiencies of joint secretion and / or lubrication arising from arthritis, including conditions of joint erosion in rheumatoid arthritis, osteoarthritis, osteoarthritis in rheumatoid arthritis patients, traumatic joint injury (including sports injury), locked joint (such as in temporomandibular joint (TMJ)), status post arthrocentesis, arthroscopic surgery, open joint surgery, joint (e.g., knee or hip) replacement in mammals, preferably humans. Each possibility represents a separate embodiment. A preferred disorder to be treated or prevented by the use of the liposomal composition of the invention is osteoarthritis.

[0163] In certain embodiments, the liposomal composition is for the reduction of knee joint pain in osteoarthritis patients.

[0164] The liposomal composition of the present invention could be used as a prophylactic measure to prevent future damage or degeneration. For example, the liposomal composition could be administered intra-articularly to athletes intermittently throughout their careers to minimize the risk of stress-related injury or cartilage degeneration.

[0165] In some embodiments, the liposomal composition is in a form suitable for parenteral administration. The parenteral administration of the liposomal composition of the invention into an articular cavity of a patient can be performed by a method chosen from the group consisting of intra-articular injection, arthroscopic administration, or surgical administration. Each possibility represents a separate embodiment. Accordingly, in some embodiments, the liposomal composition is formulated in a form suitable for administration via a route selected from intra-articular injection, arthroscopic administration, or by surgical administration. Each possibility represents a separate embodiment.

[0166] The liposomal composition according to the various embodiments of the invention may be administered in a volume of from about 0.5 ml to about 10 ml, including each value within the specified range. In further embodiments, the liposomal composition is administered in a volume of from about 1 ml to about 6 ml, including each value within the specified range. In certain embodiments, the liposomal composition is administered in a volume of about 3 ml.

[0167] In some embodiments, one dosage unit of the liposomal composition comprises from about 50 mg to about 1000 mg phospholipids, including each value within the specified range. In some embodiments, one dosage unit of the liposomal composition comprises from about 100 mg to about 800 mg phospholipids, including each value within the specified range. In a certain embodiment, one dosage unit of the liposomal composition comprises about 300 mg phospholipids. In another certain embodiment, one dosage unit of the liposomal composition comprises about 100 mg phospholipids. In additional embodiments, one dosage unit of the liposomal composition comprises about 600 mg phospholipids.

[0168] In some embodiments, one dosage unit of the liposomal composition comprises from about 30 mg to about 500 mg DPPC, including each value within the specified range. In some embodiments, one dosage unit of the liposomal composition comprises from about 50 mg to about 480 mg DPPC, including each value within the specified range. In a certain embodiment, one dosage unit of the liposomal composition comprises about 180 mg DPPC. In another embodiment, one dosage unit of the liposomal composition comprises about 60 mg DPPC. In additional embodiments, one dosage unit of the liposomal composition comprises about 365 mg DPPC.

[0169] In some embodiments, one dosage unit of the liposomal composition comprises from about 20 mg to about 450 mg DMPC, including each value within the specified range. In some embodiments, one dosage unit of the liposomal composition comprises from about 40 mg to about 300 mg DMPC, including each value within the specified range. In a certain embodiment, one dosage unit of the liposomal composition comprises about 140 mg DMPC. In another embodiment, one dosage unit of the liposomal composition comprises about 45 mg DMPC. In a certain embodiment, one dosage unit of the liposomal composition comprises about 275 mg DMPC.

[0170] In some embodiments, one dosage unit of the liposomal composition comprises from about 2 mg to about 50 mg DPPE, at least 20% of which is conjugated to collagen-binding peptide, including each value within the specified range. In some embodiments, one dosage unit of the liposomal composition comprises from about 4 mg to about 30 mg DPPE, including each value within the specified range. In a certain embodiment, one dosage unit of the liposomal composition comprises about 15 mg DPPE. In another embodiment, one dosage unit of the liposomal composition comprises about 5 mg DPPE. In a certain embodiment, one dosage unit of the liposomal composition comprises about 30 mg DPPE, at least 20% of which is conjugated to collagen-binding peptide, or particularly at least 50% of which is conjugated to collagen-binding peptide.

[0171] The liposomal composition can be portioned in vials, in single injections, or in any other convenient way for practical use.

[0172] Subjects to which administration of the liposomal compositions of the invention is contemplated include mammals, such as, but not limited to, humans and other primates.

[0173] Additionally, it should be emphasized that the peptide-conjugated liposomes used in the liposomal composition of the present invention are themselves used as an active ingredient and not as a carrier of a certain pharmaceutically active agent. As such and as mentioned hereinabove, the liposomal compositions according to the principles of the present invention are essentially free of an additional pharmaceutically active agent. The term “essentially free of an additional pharmaceutically active agent”, as used herein, refers in some embodiments to the liposomal composition including less than a therapeutically effective amount of the pharmaceutically active agent, which is known for use in joint lubrication, treatment of joint dysfunction, reduction of joint pain, irritation and / or wear, or any combination thereof. The term “known for use”, as used herein, refers in some embodiments, to pharmaceutically active agents approved for the indicated use at the time of the invention. In further embodiments, the term “known for use” refers to pharmaceutically active agents that are mentioned in scientific literature and / or patents as being suitable for the indicated use.

[0174] In some embodiments, the liposomal composition of the present invention does not include a pharmaceutically active agent which is a lubrication agent, such as, inter alia, glycosaminoglycan or a pharmaceutically acceptable salt, ester, or derivative thereof. In certain embodiments, said glycosaminoglycan is hyaluronic acid or hyaluronan-containing salt or ester. In other embodiments, hyaluronic acid is not encapsulated within the liposomes. Additionally, or alternatively, the hyaluronic acid is not dispersed in the liquid medium. In some currently preferred embodiments, the liposomal composition is essentially free of hyaluronic acid, or a pharmaceutically acceptable salt or ester thereof. The term “essentially free”, as used in connection with hyaluronic acid, refers in some embodiments to the liposomal composition including less than a therapeutically effective amount of hyaluronic acid or its salt or ester. In additional embodiments, the term “essentially free” refers to the liposomal composition including less than a detectable amount of hyaluronic acid or its salt or ester.

[0175] In various embodiments, the liposomal composition of the present invention does not include a pharmaceutically active agent which is a lubrication agent selected from superficial zone protein (SZP), lubricin, proteoglycan 4 and analogs and derivatives thereof.

[0176] In certain embodiments, the liposomal composition of the present invention does not include a pharmaceutically active agent which is an anti-inflammatory agent, such as xylitol, betamethasone, prednisolone, piroxicam, aspirin, flurbiprofen, (+)-N-{4-[3-(4- fluorophenoxy)phenoxy]-2-cyclopenten-l-yl}-N-hydroxyurea salsalate, difhmisal, ibuprofen, fenoprofen, fenamate, ketoprofen, nabumetone, naproxen, diclofenac, indomethacin, sulindac, tolmetin, etodolac, ketorolac, oxaprozin, celecoxib, meclofenamate, mefenamic acid, oxyphenbutazone, phenylbutazone, salicylates, or phyto sphingosine type agents.

[0177] In further embodiments, the liposomal composition of the present invention does not include a pharmaceutically active agent which is an antiviral agent, such as acyclovir, nelfinavir, or virazole. In additional embodiments, the liposomal composition of the present invention does not include a pharmaceutically active agent which is an antibiotic, including antibiotics belonging to the family of penicillines, cephalosporins, aminoglyco sidics, macrolides, carbapenem and penem, beta-lactam monocyclic, inhibitors of beta-lactamases, tetracyclins, polipeptidic antibiotics, chloramphenicol and derivatives, poly-etheric ionophores, and quinolones. Non-limiting examples of such antibiotics include ampicillin, dapsone, chloramphenicol, neomycin, cefaclor, cefadroxil, cephalexin, cephradine, erythromycin, clindamycin, lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, ticarcillin, rifampin, tetracycline, fusidic acid, lincomicyn, novobiocine, and spectinomycin.

[0178] In some embodiments, the liposomal composition of the present invention does not include a pharmaceutically active agent which is an anti-infective agent, such as benzalkonium chloride or chlorhexidine.

[0179] In other embodiments, the liposomal composition of the present invention does not include a pharmaceutically active agent which is a steroid. The term “steroid” as used herein, refers to naturally occurring steroids and their derivatives as well as synthetic or semi- synthetic steroid analogues having steroid-like activity. The steroid can be a glucocorticoid or corticosteroid. Examples of specific natural and synthetic steroids include, but are not limited to, aldosterone, beclomethasone, betamethasone, budesonide, cloprednol, cortisone, cortivazol, deoxycortone, desonide, desoximetasone, dexamethasone, difluorocortolone, fluclorolone, flumethasone, flunisolide, fluocinolone, fluocinonide, fluocortin butyl, fluorocortisone, fluorocortolone, fluorometholone, flurandrenolone, fluticasone, halcinonide, hydrocortisone, icomethasone, meprednisone, methylprednisolone, paramethasone, prednisolone, prednisone, tixocortol or triamcinolone, and their respective pharmaceutically acceptable salts or derivatives.

[0180] According to certain embodiments, the phospholipids are used in the pharmaceutical composition of the present invention as a sole active ingredient.

[0181] According to particular embodiments, the liposomal composition consists essentially of the non-ionic tonicity agent comprising a polyol, the peptide-conjugated liposomes, and the liquid medium, as described herein. In some embodiments, the term “consisting essentially of’ refers to a composition whose only active ingredient is the indicated active ingredient (i.e., peptide- conjugated liposomes), however, other compounds may be included which are for stabilizing, preserving, or controlling osmolarity, viscosity, and / or pH of the composition, but are not involved directly in the therapeutic effect of the peptide-conjugated liposomes. In various embodiments, the term “consisting” refers to a composition, which contains the peptide-conjugated liposomes, the tonicity agent, and a pharmaceutically acceptable vehicle or excipient.

[0182] It is noted for clarification, that the embodiments above excluding additional active agents from the liposomal composition, obviously do not exclude the liposomal composition of the invention being delivered as part of a treatment regimen to a subject in need thereof, which treatment regimen may include additional therapies or medications which are intended to treat the joint dysfunction.

[0183] According to some embodiments, there is provided a method for the preparation of a collagen-binding peptide-conjugated liposome for delivery to a patient in need thereof, the method comprising one or more of the steps of: a) forming a lipid-peptide conjugate connected via an amide-bond, comprising the step of activating the peptide in a liquid solution, adding phospholipids dissolved in an organic solvent at a desired ratio, and incubating to form the lipid-peptide conjugate; and b) generating the liposomes, comprising the steps of evaporating the lipid-peptide conjugate solution, introducing additional PL at a desired ratio, incubating in hot buffer, diluting, and precipitating in a desired buffer.

[0184] In some embodiments, activating the peptide comprises crosslinking thereof, optionally using a carbodiimide selected from, but not limited to: EDC (EDAC, EDCI, l-ethyl-3-(3- dimethylaminopropyl) carbodiimide), DCC (N,N'-dicyclohexylcarbodiimide), and DIC (N,N'- diisopropylcarbodiimide) .

[0185] In some embodiments, the liposomes are suspended in a buffer selected from histidine buffer and phosphate buffered saline.

[0186] According to some embodiments, the method includes the further step of extruding the liposomes through a series of filters to form small liposomes.

[0187] According to some embodiments, the method for the preparation of the liposomes may include various modifications to finely adjust the components of the composition, as well as the ratio between the components, so as to obtain the most effective composition. The modifications may include, for example, such parameters as, but not limited to: the specific lipids used for the formation of the lipid composition, the ratio between the lipids of the lipid compositions, the pH at which reactions are performed, the temperatures at which reactions are performed, the conditions at which the reactions are formed, the time length of various steps, and the like, or any combination thereof. The liposomal compositions according to the various embodiments of the invention can be sterilized and if desired mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, synthetic emulsifiers, additional salts for influencing osmotic pressure, coloring, and / or aromatic substances and the like which do not deleteriously react with the liposomes.

[0188] In a further aspect of the present invention there is provided a method for lubricating a joint of a mammal, the method comprising administering into a cavity of the joint a liposomal composition according to an embodiment disclosed herein-above. According to some embodiments, lubricating a joint comprises the treatment, management or prevention of an articular disorder or condition or a symptom arising therefrom. According to some embodiments, the articular disorder or condition is selected from the group consisting of rheumatoid arthritis and osteoarthritis. According to some embodiments, lubricating a joint comprises use in traumatic joint injury, locked joint, sports injury, traumatic injury towards osteoarthritis (OA), joint following arthrocentesis, arthroscopic surgery, open joint surgery, joint replacement, and / or psoriatic arthritis. According to some embodiments, the joint is selected from the group consisting of knee, hip, ankle, shoulder, elbow, tarsal, carpal, interphalangeal, and intervertebral joint.

[0189] As used herein and in the appended claims, the term “about” refers to ±10%.

[0190] As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a phospholipid” includes a plurality of such phospholipids and combinations thereof known to those skilled in the art, and so forth. It should be noted that the term “and” or the term “or” are generally employed in its sense including “and / or” unless the context clearly dictates otherwise.

[0191] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0192] EXAMPLES

[0193] Methods

[0194] Lipid-peptide conjugate preparation

[0195] A peptide comprising the collagen type 2-binding amino acid sequence WYRGRL (SEQ ID NO: 1), and having a cysteine amino acid on the C-terminus thereof, was mixed with DSPE- maleimide lipid (7.53 mg) (l,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-maleimide), and incubated overnight together with MeOH (3.75 ml) and CHCls (3.75 ml), to achieve a DSPE lipidpeptide conjugate. Liposome preparation

[0196] The lipid-peptide conjugate solutions were evaporated, and appropriate amounts of DMPC, DPPC, and EtOH were added to achieve a desired degree of liposome formation. Specifically, for 361 nano-moles of conjugated lipid-peptide, 2.22mg DMPC, 2.62mg DPPC, and 4.3 microliter of EtOH were added. For analytical purposes, 16:0 PE Liss Rhodamine was also added (9 micrograms). This solution was incubated at 55°C for 30 minutes, and then a histidine mannitol buffer pH 6.5 (HMB) at 55°C was added to invoke the formation of liposomes. The suspension was diluted 10-fold, and precipitated twice in the desired buffer, e.g., HMB or PBS. In some cases, the liposomes were sequentially extruded through a series of filters (e.g. 0.4 pm, 0.2 pm 0.1 pm, 0.05 pm) to obtain smaller liposomes.

[0197] Liposome size measurement

[0198] Particle size and particle size distribution (PSD) were measured using a laser diffraction particle sizer (Mastersizer 3000, (V:3.63) Hydro MV, Malvern Instruments, U.K.). Liposome suspensions (100 pl) were dissolved in 900 pl HMB, and were analyzed using a refractive index (RI) of 1.4 and an absorption of 0.1 5 for the liposomes, a RI of 1.33 for the HMB and obscuration level of 15%.

[0199] Zeta potential

[0200] Zeta potential was measured at 250°C using a Zetasizer Nano Series (Nano-ZS, model ZEN3600, serial no: MAL 1016044, Malvern Instruments) and DTS1060C clear disposable zeta cell-type cuvettes. The applied voltage was 150V. Liposomes were diluted to 1 mg / ml in 4% mannitol and mixed into 10 mM HMB (pH 6.5). The RI used for the liposome sample was 1.45 and the absorption was 0.1. The RI of the dispersant (HMB) was 1.33 and the viscosity was 0.9 centipoise (cP). The zeta potential was calculated using the Smoluchowski equation (F(Ka) =1.5).

[0201] Cartilage binding assay

[0202] Osteochondral cylinders (diameter = 6mm) were punched out from fresh porcine knee joints and used to prepare cartilage discs using a scalpel. The discs were immersed in binding solution containing HMB (35% volume / volume), PBS (35% volume / volume), fresh porcine synovial fluid (30% volume / volume) and rhodamine-labelled liposomes at a concentration of 20 mg / ml. The tubes containing the discs in binding solution were incubated at 34°C for 1 hour under continuous gentle rotation. After thorough washing with PBS, bound lipids were extracted from the discs with hot ethanol (0.15 ml; 500°C, 10 min) and fluorescence was read at 530 / 590 nm. Fluorescence was normalized to that of input (binding reaction) for each liposome type.

[0203] Macrophage uptake assay Liposome preparation was done as described above. RAW 264.7 murine macrophages were plated in 12 well plates in 1ml DMEM full media (10% FBS, 2% L-GLU, 1% P / S) at a concertation of 2X105 cells per well. After O / N incubation at 37°C, cells were treated with liposomes at 0.2 mg / ml (total lipids) at 37°C. Macrophage-containing wells with no added liposomes were used as a negative (or baseline) control. After 3.5 hours the media was removed, and the cells were washed 3 times in PBS (room temperature). The cells were then harvested by scraping and transferred to flow cytometry glass tubes. The cells were analyzed by flow cytometry on a CYTOFLEXS S system using the PE channel (excitation 561 emission 585).

[0204] Comparative Example 1: Preparation and analysis of peptide-conjugated liposomal composition

[0205] A liposomal formulation, including a lipid conjugated to a collagen-binding lipid, was prepared, and characterized for size and zeta potential, as described in the methods section above. As a control, a formulation including liposomes without peptide-conjugated lipids was prepared. The formulations and characteristics thereof are presented in Table 1 below.

[0206] Table 1: Formulations and characteristics of lipid-conjugate and control liposomes

[0207] In order to verify the pep tide-liposome conjugation, i.e., successful incorporation of the collagen-binding peptide-DPPE lipid conjugate into the liposomes, the liposomes were diluted 1 / 20 in EtOH, and UV absorption was measured. The results are presented in Figure 1.

[0208] As can be seen, a first peak appears in the absorption graph at 560 nm in both the control liposome and the lipid-peptide conjugate liposome which is the characteristic absorption of Rhodamine, added into the liposomes for purpose of detection. In the absorption graph of the peptide-conjugated liposome, there appears an additional peak at 280 nm, indicating the presence of the peptide.

[0209] Following ascertaining the presence of the peptide comprising the collagen-binding sequence, the effect of adding the peptide to the liposomes on the cartilage binding ability of the liposomes was tested. Compositions including liposomes of Formulations #1 and #2, respectively, were subjected to a cartilage binding assay, as described in the methods section above.

[0210] As can be seen in Figure 2A, substantially higher levels of cartilage-binding were achieved for the peptide-conjugated liposomes, as compared to the control non-conjugated, or “bare”, liposomes, in the order of almost 3-fold increase.

[0211] To further determine the effect of the increased cartilage binding on the lubricating ability of the liposomes, the formulations were used in a cartilage-on-glass lubrication assay. Briefly, the liposomes, each at lOOmg / ml concentration in HMB were used for the friction study. Fresh porcine articular cartilage from femoral condyles and trochlea was used to prepare cartilage discs (6 mm in diameter). The discs (n=4 for each type of liposomes) were glued on plastic holders via their bony side. Cartilage-on-glass friction tests (N=4) were performed using porcine cartilage discs (diameter=6 mm) held upside-down and submerged into a lubricant chamber set at 37°C on a glass surface. Testing was performed using a customized two-axis tribometer with a lockable selfalignment mechanism to ensure full contact of each disc with the glass surface. Each disc was compressed under a force of 5 N, a dwelling step of 5 seconds and then horizontal movement was initiated at Imm / sec over 20 mm followed by dwelling for 0.5 sec, disconnection from the glass and back to start point. Each disc underwent 10 such cycles. For each cycle, both the static and kinetic coefficients of friction (CoFs) were measured by the tribometer. In order to test the ability of the liposomes to reduce friction of the cartilage surface in a persistent manner, friction was measured sequentially in the presence of PBS (PBS 1st), liposomes and 3 additional tests, each time in the presence of fresh PBS (PBS 2nd, PBS 3rd, PBS 4th).

[0212] Figures 2B-2C show the average k (for n=4 discs) static and kinetic friction coefficients, expressed as % of the values obtained for PBS 1stwhich served as a reference point.

[0213] While Formulation #1 was able to significantly reduce both coefficients (also following their removal from the chamber in PBS 2nd), this formulation failed to keep the coefficients low upon further buffer replacements (PBS 3rdand PBS 4th). In contrast, Formulation #2 though seemingly being less efficient than Formulation #1 as a lubricant when present in excess, was able to keep the coefficients low along the entire study, up to last buffer exchange, suggesting a higher persistence on the cartilage surface thereby leading to persistent lubrication.

[0214] Having established the positive effect of the peptide conjugation on the association of the liposomes with cartilage, indicating enhanced residence time in the synovial fluid, and prior to progression to in vivo experimentation, analysis was performed to better assess the performance of the liposomal formulation in natural biological environment. To this end, Formulations #1 and #2 were subjected to a macrophage uptake assay, as described in the methods section above. A desired outcome for macrophage uptake assay is for the result to be as low macrophage uptake as possible, indicating longer survival of the injected liposomal composition in the joint. The results of the macrophage assay of Formulations #1 and #2 is presented in Figure 2D.

[0215] As can be seen in the figure, the peptide-conjugate liposomes of Formulation #2 showed a dramatic increase in macrophage uptake, as compared to the non-conjugated liposomes of Formulation #1. This result indicated a strong negative effect of macrophages on the residence time of the liposomes at the treated joint, possibly cancelling out any positive effect produced by the enhanced cartilage binding of the peptide-conjugated liposomes. In an attempt to estimate the overall effect of the peptide conjugation on the residence time of liposomes of Formulation #2 at a treated cartilage, the enhanced cartilage-binding levels of Formulation #2 were set-off against the enhanced macrophage uptake levels thereof, by calculating the quotient of the ligand-binding level divided by the macrophage-uptake level. This quotient was compared to the same quotient for the ligand-binding and macrophage uptake levels of nonconjugated liposomes (Formulation #1). The results of the comparison (normalized) are presented in Figure 2E.

[0216] As is clearly shown in the Figure, the estimated overall effect of Formulation #2, despite a substantial enhancement in ligand binding levels, is lower than the overall state of the bare liposomes of Formulation #1. This is indicative of a devastating effect of the enhanced macrophage uptake, which can entirely cancel out any advantage in ligand binding.

[0217] Example 1: Preparation and analysis of peptide-conjugated liposomal composition

[0218] In an attempt to overcome the severe macrophage uptake of the peptide-conjugated liposomes, the size of the liposomes was reduced. The liposomes of Formulation #1 were extruded through a series of filters (0.4 pm, 0.2 pm, 0.1 pm, and 0.05 pm, producing liposomes having an average diameter of 62 nm (Formulation #3). Of note, the size reduction was not performed on liposomes of Formulation #2, as they were not extrudable. The liposomes were subjected to both cartilage binding assay and macrophage uptake assay, as described above.

[0219] As can be seen in Figures 3A and 3B, the size reduction of the liposomes did not have any noticeable effect on the macrophage uptake or on the cartilage binding of the small liposomes.

[0220] Next, in a further attempt to reduce the macrophage uptake, chemical modification of the outward facing end of the collagen-binding peptide was performed. Liposomes as in Formulation #2 (namely, DPPC / DMPC / WYRGRLC-maleimide-DSPE (50% / 45% / 5%)) were acetylated, i.e., an acetyl group was added to the N-terminus of the peptide. Zeta potential of the acetylated liposomes (Formulation #4 herein, DPPC / DMPC / Ac-WYRGRLC-maleimide-DSPE (50% / 45% / 5%)) was measured, and found to have been reduced to (-2.9) mV to levels similar to the zeta potential of the non-conjugated liposomes of Formulation #1 (-2.4 mV). The liposomes of Formulation #4 were then subjected to macrophage uptake assay, according to the method described above. As can be seen in Figure 4A, the macrophage uptake was substantially reduced as compared to Formulation #2 (although still much higher than the microphage uptake of Formulation #1), suggesting slower clearance of the liposomes from a site of treatment. Following this encouraging result, the liposomal composition of formulation #4 was subjected also to a cartilage binding assay as described above. As shown in Figure 4B, Formulation 4# displayed a substantially lower cartilage binding as compared to Formulation #2, being only a little bit higher than the binding levels of the original non-conjugated liposomes of Formulation #1.

[0221] Modifications of the peptide sequence were then tested. Spacers of 1 to 3 amino acids were added at the C-terminus, between the peptide collagen-binding peptide sequence and the cysteine amino acid, which is used to connect with the maleimide linker. The spacer provides improved accessibility to the peptide sequence, which could stabilize the binding of the liposomes to the cartilage collagen. Table 2 below details the formulations including the different spacers, and the measured zeta potentials thereof.

[0222] Table 2: Formulations including different spacers

[0223] Formulations #5 to #8 were then subjected both to macrophage uptake assays and cartilage binding assays, according to the methods described above. The results are presented in Figures 5A and 5B. As can be clearly seen in the figures, formulations which were successful in enhancing the cartilage binding (i.e., Formulations #6 and #7) also suffered from high levels of macrophage uptake, whereas formulations which had relatively low levels of macrophage uptake (mainly Formulation #5, and also #8) were not effective in enhancing cartilage binding.

[0224] In a further attempt to reduce macrophage uptake of the pep tide-liposome conjugates, variation in the conjugation method of the peptide to the liposome, specifically to the DPPE lipid thereof, was performed. An acylated collagen-binding peptide comprising an aspartic acid on its C-terminus (instead of the cysteine) was activated using EDC (N-(3-Dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride) (1.39 mg) and sNHS (N-Hydroxy sulfo succinimide sodium salt) (1.26 mg), mixed in 85uL H2O for 1 hour at RT. DPPE (0.25 mg) was added to the solution at a concentration of 1.515 mg / ml in THF (Tetrahydrofuran) solution (10% H2O), and the reactants were incubated overnight. This resulted in the formation of DPPE-peptide conjugates having a direct amide bond with the amine grouped of DPPE, with no intermediate linking moiety (referred to herein as “zero-chemistry DPPE-peptide” or “non-maleimide DPPE-peptide”).

[0225] Liposomes were then formed with the zero-chemistry DPPE-peptide conjugates as described in the methods section above (Formulation #9, DPPC / DMPC / DPPE / Ac-WYRGRLD- DPPE (50% / 45% / 2% / 3%)). It is noted that the liposomes of Formulation #9 included only 3% of DPPE-peptide conjugated lipids, instead of 5% used in the other formulations. The liposomes were extruded through a series of filters (0.4 pm, 0.2 pm, 0.1 pm, and 0.05 pm, producing liposomes having an average diameter of 65 nm, and a zeta potential measured at (-10.3) mV. The newly formed formulation was then subjected to cartilage binding and macrophage uptake assays. The results of the assays, as compared to Formulations #1 and #2 (i.e., bare liposomes, and the corresponding maleimide-linked peptide-conjugated liposomes, respectively) are presented in Figures 6A and 6B.

[0226] Surprisingly, as can be seen in the figures, liposomes containing directly bound DPPE- peptide conjugates presented a drastic decrease of the macrophage uptake as compared to the liposomes of Formulation #2, while at the same time also achieving enhanced cartilage binding relative to the bare liposomes, albeit not at the same levels as those achieved by the liposomes containing DPPE -peptides conjugated via a maleimide linker.

[0227] Upon establishing the reduced macrophage uptake achieved by EDC / NHS chemistry, further formulations using this approach were tested. Formulation #10, which is based on formulation #9 but includes an amino acid spacer (SGS) between the collagen-binding peptide and the aspartic acid, was prepared according to the methods described above (Formulation #10 - DPPC / DMPC / Ac-WYRGRLSGSD-DPPE (50% / 45% / 5%). Following preparation of Formulation #10, the size and zeta potential of the liposomes were measured at 73 nm and (-9.8) mV, respectively, and the composition was subjected to cartilage binding and macrophage uptake assays. The results of the assays, as compared to Formulations #1, #2, and #9 are presented in Figures 7A and 7B.

[0228] As can be seen in the figures, Formulation #10, including the addition of the 3 amino acid spacer to the conjugated-peptide thereof, had a reduced macrophage uptake compared to Formulation #9, as had been hoped (Figure 7A). However, surprisingly, contrary to the previously tested formulations, Formulation #10 also achieved a highly enhanced cartilage binding, displaying levels which are about 3-fold higher than any of the other tested formulations, even those having high macrophage uptake (Figure 7B).

[0229] Following this unexpected positive result, formulations including two repetitions of the spacer (DPPC / DMPC / Ac- WYRGRLSGSSGSD-DPPE (50% / 45% / 5%)) and two repetitions of the peptide (DPPC / DMPC / Ac-WYRGRLGDPGDWYRGRLSGSD (50% / 45% / 5%)) were prepared (Formulations 11# and 12#, respectively). The formulations underwent macrophage uptake and cartilage binding assays, and the results were compared to Formulations #9 and #10.

[0230] As can be seen in Figures 8A and 8B, the formulation including the double spacer SGSSGS (i.e., Formulation #11) showed a somewhat lower ligand binding as compared to Formulation #10, although still showing high levels compared to all other formulations, and also showing some reduction in the level of macrophage consumption. Formulation #12 showed a favorable substantial decrease in macrophage uptake, yet also a lower level of ligand binding compared to Formulation #10. That being said, the level of ligand binding of Formulation #12 was still substantially higher than the ligand binding of the bare liposomes, indicating an improved residence time in a treated joint also for this formulation.

[0231] To acquire a tentative estimate of the overall (combined) effect of the different formulations of collagen-binding peptide conjugated to liposomes, on the actual residence time of the respective liposomes at the cartilage of a treated joint, the ligand-binding levels of each of the liposomes was set-off against the macrophage-uptake thereof. Figure 9A is a normalized [linear] bar-chart presenting the quotient of the ligand-binding level (“higher-is-better”) of each peptide- conjugated liposome divided by the macrophage-uptake level (“lower-is-better”) thereof. Figure 9B is a logarithmic representation of the quotients shown in Figure 9A.

[0232] As is distinctly shown in Figures 9A-9B, it was surprisingly found that the combined effect of all zero-chemistry formulations (i.e., Formulations #9 to #12), as represented by the respective quotients thereof, was higher than the combined effect of all the maleimide-linker Formulations #2 to #8. Strikingly, the zero-chemistry formulation having the lowest estimated combined effect (i.e., Formulation #9, with only 3% peptide coverage) had a 2-fold higher estimated combined effect (i.e., quotient) than the best performing maleimide formulation (i.e., Formulation #5).

[0233] To further verify the superior combined effect achieved by the zero-chemistry peptide- conjugated liposomal formulations over the combined effect of formulations conjugated via maleimide linker, comparison was made between formulations having the exact same peptide sequence, differing only in the conjugation method, zero-chemistry versus maleimide linker. Specifically, Formulation #4 was compared to Formulation #9, each of the formulations conjugated, via a maleimide linker and direct peptide bond, respectively, to an acylated peptide without an additional spacer (SEQ ID NO. 2 (Ac)). Formulation #7 was compared to Formulation #10, each of the formulations conjugated, via a maleimide linker and direct peptide bond, respectively, to an acylated peptide with an additional SGS spacer (SEQ ID NO. 3 (Ac)). The results of the comparisons are presented in Figures 10A-10C and 11A-11C.

[0234] As can be clearly seen in both comparisons between specific peptide sequences, in formulations which differed only in the method of conjugation (i.e., having the same liposomes and the same peptide sequence) the zero-chemistry conjugation method displayed a substantially improved overall effect than the conjugation via a maleimide linker.

[0235] Example 2: Analysis of liposomal compositions conjugated to additional collagen-binding

[0236] Having established the pronounced effect of the zero-chemistry peptide-conjugation method on the overall estimated residence time at a treated joint of the WYRGRL-liposomal formulations, and, particularly, having established the drastic reduction of macrophage uptake achieved by the zero-chemistry conjugation method as compared to the maleimide conjugation method, conjugation of additional collagen-binding peptides to liposomes was tested.

[0237] To this end, collagen hybridizing peptides (CHP) made up of repeats of the amino acid sequence Glycine-Proline-Hydroxyproline (GPO), which have been found to effectively bind different collagen protein types, were selected. A peptide comprising 5 sequential repeats of GPO amino acids (referred to herein as “5GPO”, SEQ ID NO. 6) was conjugated to liposomes having a similar lipid composition to the liposomes used in Example 1 above. The GPO5 peptides were conjugated to the liposomes via a maleimide linker or a direct peptide bond (i.e., zero-chemistry), forming Formulation #13 and Formulation #14, respectively. The liposomal formulations were subjected to cartilage binding assays and macrophage uptake assays, as described above, and the results of the assays were set off against each other (i.e., the cartilage binding was divided by the macrophage uptake) to arrive at an estimated combined effect of the peptide conjugation on the residence time of the peptides at a treated joint cartilage. The results of the assays and the quotient thereof are presented in Table 3 below, and in Figures 12A-12C.

[0238] Table 3: 5 GPO peptide assays

[0239] As can be clearly seen in the figures and Table above, although the GPO5 conjugated by the maleimide linker (Formulation #13) displayed a larger increase in collagen binding than the directly bound GPO5 (Formulation #14), it also displayed strikingly higher levels of macrophage uptake as compared with formulation #14. Formulation #14, on the other hand, while not achieving quite such high levels of collagen binding, displayed much lower levels of macrophage uptake, such that the combined effect of Formulation #14 was more than 2-fold the combined effect of Formulation #13. This surprising result, taken together with the results of Example 1, further establishes the reduced macrophage uptake and enhanced residence time at a treated joint of liposomes that are directly conjugated to cartilage binding peptides, particularly to collagen binding peptides, as opposed to liposomes that are conjugated to such peptides via a maleimide linker.

[0240] Example 3: In vivo effect of Formulations containing liposomes conjugated to collagen-

[0241] Next, the effect of conjugating collagen-binding peptides to liposomes (via different conjugation methods) on the residence time of the liposomes at a treated osteoarthritis joint in vivo, is examined. Mono-iodoacetate (MIA) OA is induced in 8 weeks old (250 gram) male Wistar rats by intra-articular (IA) injection of 0.6mg MIA in saline into the left knee. Liposomal formulations are prepared comprising liposomes conjugated to peptides comprising SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, the peptides conjugated to the liposomes via a direct amide bond or via a maleimide linker, as described in Examples 1 and 2 above. At 14 days post MIA injection, 30uL of the liposomal formulations comprising the peptide-conjugated liposomes (lOOmg / ml in histidine mannitol buffer), labelled using sulfo- Cy5.5-phosphatidylethanolamine, are injected IA into both knees. At several time points post injection of liposomes (1, 2, 3, 4 and 8 weeks), knees are harvested and fixed in formaldehyde, decalcified in EDTA for 4-6 weeks, and then frozen in optimal cutting temperature (OCT) compound. Cryosections (~ 10 microns thick) are stained with DAPI to visualize cell nuclei. Toluidine blue staining of sulphated glycosaminoglycans is done on successive slides, to visualize cartilage architecture including regions with dead chondrocytes due to MIA. The presence of Cy5.5-labelled liposomes on cartilage was detected by visualization of the tissue sections in a fluorescent microscope using a Cy5.5 filter.

[0242] Whole knees that are not analyzed by histology, are disassembled into femurs, tibia and patella, by transection of ligaments. Femur heads and tibial plateau are further isolated from femurs and tibia. Bound lipids in femur heads, tibial plateaus and patellas, are quantitated by extraction of the knee part in 0.3 ml of hot ethanol (55 °C) for 10 minutes followed by Cy5.5 fluorescence reading at 675 (EX) / 705 (EM)in a fluorimeter (Tecan) against a Cy5.5-liposomes standard curve, which bound lipids are representative of presence of the IA administered liposomal compositions in the treated joint.

[0243] The results of the quantification of the different liposomal formulations are analyzed, and particular comparison is made between formulations containing directly bound pep tide-liposome conjugates, and pep tide-liposome conjugates bound via a maleimide linker.

[0244] Table 4: List of Formulations

[0245] It is appreciated by persons skilled in the art that the present invention is not limited by what has been particularly shown and described hereinabove. Rather the scope of the present invention includes both combinations and sub-combinations of various features described hereinabove as well as variations and modifications. Therefore, the invention is not to be constructed as restricted to the particularly described embodiments, and the scope and concept of the invention will be more readily understood by references to the claims, which follow.

Claims

CLAIMS:

1. A liposomal composition comprising a plurality of liposomes suspended in a liquid medium, wherein the liposomes comprise an internal aqueous phase surrounded by at least one membrane comprising a phospholipid (PL) which is conjugated to a peptide comprising a collagen-binding sequence capable of binding a collagen which is present in the cartilage, wherein conjugation of the PL to the peptide is by a free carboxyl group (COOH) of a side chain or the C-terminus of the peptide being conjugated directly to an amine group of the PL via an amide bond, with no intermediate linking moiety.

2. The liposomal composition of claim 1, wherein the collagen-binding sequence is a collagen type-II binding peptide sequence.

3. The liposomal composition of claim 1 or 2, wherein the collagen-binding sequence comprises at least one of SEQ ID NO: 1 and an amino acid sequence comprising GPO.

4. The liposomal composition of any one of claims 1 to 3, wherein the peptide further comprises a spacer sequence between the collagen-binding sequence and the PL.

5. The liposomal composition of claim 4, wherein the spacer sequence comprises 1 to 10 amino acids.

6. The liposomal composition of claim 5, wherein the spacer sequence comprises 3 amino acids.

7. The liposomal composition of any one of claims 4 to 6, wherein the spacer sequence comprises amino acids selected from glycine, serine, proline, alanine, and a combination thereof.

8. The liposomal composition of any of claims 4 to 7, wherein the spacer comprises a sequence selected from GGS, SGS, and SSS.

9. The liposomal composition of any one of the preceding claims, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

10. The liposomal composition of claim 9, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 3.

11. The liposomal composition of any one of the preceding claims, wherein the N-terminus of the peptide is acylated.

12. The liposomal composition of any one of the preceding claims, wherein the peptide comprises at least one repeat of the collagen-binding sequence.

13. The liposomal composition of any one of the preceding claims, wherein the phospholipid conjugated to the peptide constitutes between about 1% to about 20% of lipids in the liposome.

14. The liposomal composition of any one of the preceding claims, wherein the PL is a phosphatidylamine lipid.

15. The liposomal composition of claim 14, wherein the phosphatidylamine lipid is selected from the group consisting of l,2-Dipalmitoyl- n-glycero-3- phosphoethanolamine (DPPE), 1,2-dilauroyl-L-phosphatidyl-ethanolamine (DLPE), l,2-Dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1,2-Diphytanoyl-sn- glycero-3 -phosphoethanolamine (DPhPE), 1 ,3-Dipalmitoyl-sn-glycero-2- phosphoethanolamine (1,3-DPPE), l-Palmitoyl-3-oleoyl-sn-glycero-2- phosphoethanolamine (1,3-POPE), Biotin-Phosphatidylethanolamine, 1,2- Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-Distearoyl-sn-glycero- 3 -phosphoethanolamine (DSPE), and Phosphatidylserine (PS).

16. The liposomal composition of any one of the preceding claims, wherein the liposomes further comprise a PL selected from a glycerophospholipid (GPL) having two C12- Ci8 hydrocarbon chains, being the same or different, and sphingomyelin (SM) having a C12-C18 hydrocarbon chain.

17. The liposomal composition of claim 16, wherein the GPL is a phosphatidylcholine (PC).

18. The liposomal composition of claim 17, wherein the PC is selected from the group consisting of l,2-dimyristoyl-sn-glycero-3 -phosphocholine (DMPC); 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC); 1 ,2-dipcntadccanoyl-.yn-glyccro-3- phosphocholine (C15); l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); N- palmitoyl-D-eryt / zro-sphingosylphosphorylcholine (D-erythro C16), and a mixture or combination thereof.

19. The liposomal composition of any one claims 16 to 18, wherein the liposomes comprise DMPC, DPPC, and DPPE.

20. The liposomal composition of claim 19, comprising DMPC at a mole percent in the range of about 24% to about 70%, DPPC at a mole percent in the range of about 75% to about 29%, and DPPE at a mole percent in the range of about 1% to about 10%.

21. The liposomal composition of any one of claims 19 or 20, wherein the peptide is conjugated to the DPPE.

22. The liposomal composition of any one of the preceding claims, having a zeta potential in the range of about (-25) to about 5 mV.

23. The liposomal composition of any one of the preceding claims, wherein the liposomes have an average particle size of more than 1,000 nm.

24. The liposomal composition of any one of the preceding claims, having a zeta potential in the range of about (-15) to about 5 mV, wherein the liposomes comprise DMPC, DPPC, and DPPE, wherein the liposomes have an average particle size of more than 1,000 nm, and wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

25. The liposomal composition of any one of the preceding claims, wherein the liposomes have a unimodal size distribution or a bimodal size distribution.

26. The liposomal composition of any one of the preceding claims, wherein the liposomes have a phase transition onset temperature in the range of about 20°C to about 41°C.

27. The liposomal composition of any one of the preceding claims, wherein the at least one PL is at a concentration within the range of about 20 to about 500 mM.

28. The liposomal composition of any one of the preceding claims, wherein the liquid medium comprises a buffer.

29. The liposomal composition of claim 28, wherein the buffer is selected from histidine buffer and phosphate-buffered saline.

30. The liposomal composition of any one of the preceding claims, having a pH in the range of about 5 to about 8.

31. The liposomal composition of claim 30, having a pH of 6.5.

32. The liposomal composition of any one of the preceding claims, wherein the liquid medium further comprises a tonicity agent.

33. The liposomal composition of claim 32, wherein the tonicity agent is a non-ionic tonicity agent.

34. The liposomal composition of claim 33, wherein the non-ionic tonicity agent comprises a polyol.

35. The liposomal composition of claim 34, wherein the polyol comprises at least one of mannitol, sorbitol, glycerol, erythritol, maltitol, isomalt, trimethylolpropane, pentaerythritol, dextrose, lactose, trehalose, or a combination thereof.

36. The liposomal composition of claim 35, wherein the polyol is mannitol.

37. The liposomal composition of any one of claims 32 to 36, wherein the tonicity agent is in a weight percent ranging from about 0.05% to about 10% (w / w) of the total weight of the composition.

38. The liposomal composition of any one of the preceding claims, wherein the liposomal composition is essentially free of a pharmaceutically active agent.

39. The liposomal composition of any one of the preceding claims, for use in lubricating a joint of a subject in need thereof.

40. The liposomal composition for use of claim 39, wherein lubricating a joint comprises the treatment, management or prevention of an articular disorder or condition or a symptom arising therefrom.

41. The liposomal composition for use according to claim 40, wherein the articular disorder or condition is selected from the group consisting of rheumatoid arthritis and osteoarthritis.

42. The liposomal composition for use of any one of claims 39 to 41, wherein lubricating a joint comprises use in traumatic joint injury, locked joint, sports injury, traumatic injury towards osteoarthritis (OA), joint following arthrocentesis, arthroscopic surgery, open joint surgery, joint replacement, and / or psoriatic arthritis.

43. The liposomal composition for use of any one of claims 39 to 42, wherein the joint is selected from the group consisting of knee, hip, ankle, shoulder, elbow, tarsal, carpal, interphalangeal, and intervertebral joint.

44. The liposomal composition for use of any one of claims 39 to 43, wherein the subject is a mammal.

45. The liposomal composition for use of claim 44, wherein the subject is a human.

46. A method for lubricating a joint of a mammal, the method comprising administering into a cavity of the joint a liposomal composition of any one of claims 1 to 38.

47. The method of claim 46, wherein lubricating a joint comprises the treatment, management or prevention of an articular disorder or condition or a symptom arising therefrom.

48. The method of claim 47, wherein the articular disorder or condition is selected from the group consisting of rheumatoid arthritis and osteoarthritis.

49. The method of any one of claims 46 to 48, wherein lubricating a joint comprises use in traumatic joint injury, locked joint, sports injury, traumatic injury towards osteoarthritis (OA), joint following arthrocentesis, arthroscopic surgery, open joint surgery, joint replacement, and / or psoriatic arthritis.

50. The method of any one of claims 46 to 49, wherein the joint is selected from the group consisting of knee, hip, ankle, shoulder, elbow, tarsal, carpal, interphalangeal, and intervertebral joint.

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