Lipid polymers for intra-articular administration

Abstract

The present invention provides a composition comprising an anionic lipid composition and a polycationic aggregating agent for use in the intra-articular treatment of a joint disease or disorder, such as osteoarthritis. In addition, a kit for preparing the composition and a novel composition comprising the anionic lipid composition and a polycationic aggregating agent are also provided.

Description

[0001] This invention relates to a composition comprising an anionic lipid composition and a polycationic aggregator for intra-articular treatment of joint diseases or disorders, such as osteoarthritis. Where appropriate, the composition comprises aggregates of anionic lipid composition and a polycationic aggregator. The invention also relates to kits for preparing the composition, and novel compositions comprising anionic lipid compositions and polycationic aggregators. Background of the Invention Osteoarthritis (OA) is a stubborn joint disease affecting more than 500 million people worldwide and imposing enormous costs on healthcare systems. The pathogenesis of OA involves mechanical, inflammatory, fibrotic, and metabolic factors, ultimately leading to joint failure. As the disease progresses, changes in cartilage composition cause erosion, making cartilage more susceptible to mechanical damage and forming cartilage wear particles. These particles help activate synovial macrophages and synovial fibroblasts, leading to synovial fibrosis, joint stiffness, and chronic pain in OA patients. The presence of wear particles is also closely associated with increased cartilage surface roughness, resulting in increased friction and further exacerbating cartilage degeneration.1 Current management strategies for OA include lifestyle modifications, small-molecule analgesics, large-molecule viscoelastic replacement therapy, cell therapy, and surgery. However, these treatments are insufficient to reverse disease progression; therefore, the development of disease-modifying drugs (DMOADs) for osteoarthritis has become a promising strategy. Long-term systemic administration of DMOADs may be accompanied by potential side effects, thus diminishing their therapeutic benefits, prompting increasing attention to intra-articular (IA) administration of lipid-based drug delivery systems (DDSs). 2. This method can reduce systemic exposure, decrease side effects, and improve local bioavailability. Combining cartilage lubrication with drug delivery shows promise in the treatment of OA because DDSs can improve drug retention, achieve on-demand drug release, and reduce cartilage wear. 3. Liposomes have shown promise in improving the efficacy of glucocorticoid therapy in the treatment of osteoarthritis (OA).4 However, some limitations remain to be addressed. One major concern is their small particle size, which may lead to rapid clearance from the joint cavity, thus reducing their therapeutic efficacy.5 Particles smaller than 300 nm are associated with rapid clearance from the joint, requiring frequent administration and increasing the risk of infection. This rapid clearance is partly attributed to phagocytosis by macrophages, which more effectively engulf and clear smaller particles. In contrast, particles larger than 10 μm avoid phagocytosis and can remain in both normal and inflamed joints for more than six weeks.5,6 Furthermore, although small monolayer liposomes exhibit excellent lubricating properties in vitro,7 in vitro studies on cartilage show a lack of lubricating effect.8,9 The excellent lubricating properties of liposomes in vitro are related to the hydration properties of the phospholipids that form the hydration shell, which is pressure-resistant and effectively reduces friction. 7 However, this difference in in vitro studies may be attributed to the infiltration of liposomes into porous cartilage structures, resulting in insufficient retention of them on the surfaces required for efficient lubrication. 8 Recent reports have described the development of calcium-aggregating liposomes (ALs), which enhance drug diffusion barriers by stacking and aggregating multiple phospholipid membranes, thereby enabling sustained drug release.10,11 It has also been reported that using zinc as an aggregating agent can form irreversible aggregates with a diameter of nearly 100 µm,9 and zinc possesses anti-inflammatory activity.12 Data show that the release of the potential DMOAD rapamycin can be prolonged beyond that of conventional liposomes. These ALs effectively and significantly reduce friction in vitro; in vitro, they protect cartilage from increased friction caused by aggregating agents. However, studies on human osteoarthritis synovial fibroblasts (OASFs) have shown that aggregating agents have significant toxicity, thus limiting the system's ability to deliver drugs at therapeutic doses.9 Therefore, there is still a need for an improved alternative delivery system for OA management that is non-toxic to human synovial cells and preferably also has anti-inflammatory and / or anti-fibrotic activity.

[0003] Dendritic polymers have a branched core-shell structure with a precisely defined number of branching foci between the core and shell, thus determining their generation (G1, G2, G3, etc.). As described herein, the inventors have unexpectedly discovered that aggregates formed between polycationic aggregaters and anionic lipid compositions (e.g., dendritic polymer-liposome complexes) can significantly suppress friction and improve particle retention on cartilage surfaces. Therefore, it has been unexpectedly found that the compositions of the present invention can be used for intra-articular delivery of OA therapeutic agents.

[0004] Brief overview of the disclosure According to the present invention, a composition for treating joint diseases or disorders is provided, wherein the composition comprises: Anionic lipid compositions; and Polycationic aggregates, The composition is administered intra-articularly.

[0005] In some cases, the composition may further comprise a pharmaceutically active agent. Therefore, in some embodiments, the composition comprises: Anionic lipid compositions; Polycationic aggregators; and Pharmaceutical active agent, The composition is administered intra-articularly. The pharmaceutically active agent may be incorporated into the anionic lipid composition. The pharmaceutically active agent may be incorporated into a polycationic aggregater, or the pharmaceutically active agent may be composed of a polycationic aggregater.

[0006] A composition comprising: Anionic liposomes; and Polycationic aggregates, The polycationic aggregator is: (i) A cationic dendritic polymer aggregator comprising one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 87 to 90). Composed of SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82); or (ii) A polycationic aggregator selected from: inverse poly(amidoamine) (i-PAMAM), polyethyleneimine (branched or linear), tobramycin, glatiramer acetate, polymyxin B, polylysine, apidaecin 1b, LL-37, polyarginine and Walk11.3.

[0007] In some embodiments, the composition further comprises a pharmaceutically active agent. Therefore, in some embodiments, the composition includes: Anionic liposomes; Polycationic aggregators; and Pharmaceutical active agent, The polycationic aggregator is: (i) A cationic dendritic polymer aggregator comprising one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 87 to 90). Composed of SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82); or (ii) A polycationic aggregator selected from: inverse poly(amidoamine) (i-PAMAM), polyethyleneimine (branched or linear), tobramycin, glatiramer acetate, polymyxin B, polylysine, apidaecin 1b, LL-37, polyarginine and Walk11.3.

[0008] Pharmaceutical active agents can be encapsulated in anionic liposomes.

[0009] A composition comprising: Anionic liposomes; and Cationic dendritic polymer aggregator, Among them, cationic dendritic polymer aggregators include the following linear symbols. (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (consisting of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (consisting of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (consisting of SEQ ID NO: 79 to 82).

[0010] In some embodiments, the composition comprises: Anionic liposomes; and Cationic dendritic polymer aggregator, The cationic dendritic polymer aggregator includes one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4) or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8).

[0011] In some cases, the composition may further comprise a pharmaceutically active agent. Therefore, in some embodiments, the composition comprises: Anionic liposomes; Cationic dendritic polymer aggregators; and Pharmaceutical active agent, Among them, cationic dendritic polymer aggregators include the following linear symbols. (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (consisting of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (consisting of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (consisting of SEQ ID NO: 79 to 82).

[0012] The composition may include: Anionic liposomes; Cationic dendritic polymer aggregators; and Pharmaceutical active agent, The cationic dendritic polymer aggregates include one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4) or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8). The pharmaceutically active agent can be encapsulated in anionic liposomes.

[0013] In some embodiments, the composition is an aggregate composition. The aggregate composition comprises aggregates; the aggregates are composed of an anionic lipid composition (e.g., liposomes) and a polycationic aggregator (e.g., a cationic dendritic polymer aggregator). In some embodiments, the anionic lipid composition and the polycationic aggregator form aggregates prior to intra-articular administration to a subject. In some embodiments, when the anionic lipid composition is in the form of liposomes or particles (e.g., lipid nanoparticles), the aggregate comprises one molecule of polycationic aggregator and a plurality (two or more) of liposomes or lipid particles.

[0014] In some embodiments, the aggregate composition comprises a pharmaceutically active agent; the aggregate composition enables sustained release of the pharmaceutically active agent after intra-articular administration to a subject.

[0015] In addition, aggregate compositions of the present invention are provided for use as pharmaceuticals.

[0016] In addition, aggregate compositions of the present invention are provided for the treatment of joint diseases or disorders.

[0017] A method for treating a subject's joint disease or disorder is also provided, comprising administering an effective amount of the aggregated composition of the present invention to the subject. The aggregated composition may be administered intra-articularly to the subject's joint.

[0018] Furthermore, the use of the aggregated composition of the present invention is provided for the manufacture of a medicament for treating a subject’s joint disease or disorder.

[0019] In addition, aggregate compositions of the present invention are provided for treating joint diseases or disorders, wherein the composition is applied intra-articularly to the joints of a subject.

[0020] In addition, a kit is provided, comprising: a) A first container containing an anionic lipid composition; b) A second container containing a polycationic aggregator; and c) Instructions for mixing a) with b) to provide a composition for the purposes defined herein.

[0021] The first container may further contain a pharmaceutically active agent. Therefore, in some embodiments, the kit includes: a) A first container comprising anionic lipid composition and pharmaceutically active agent; b) A second container containing a polycationic aggregator; and c) Instructions for mixing a) with b) to provide a composition for the purposes defined herein.

[0022] Further aspects and features of the invention are set forth in the detailed description below.

[0023] Brief description of the attached figures Figure 1—Gathering curves of DendriXALs (Dendri1 (De1) and Dendri4 (De4)): showing the optical density variation at λ = 450 nm and the zeta potential as a function of charge ratio (where the positive charge comes from DendriXs and the negative charge comes from DSPG molecules on the liposomes). Each data point in the figure represents the result of a single replicate measurement, and the connecting lines represent the mean of the replicate measurements. Each measurement was performed in triplicate, except for measurements with charge ratios of 500 and 1000, which were performed only once.

[0024] Figure 2 – Aggregation in the presence of different concentrations of NaCl. After mixing DendriXs with liposomes for 5 min, the optical density was measured at 450 nm using a microplate reader. The concentration of DendriXs was maintained at 50 µg / mL, and the lipid content was adjusted (Dendri1: 0.66 mM, Dendri4: 0.73 mM). NaCl was mixed with DendriXs before mixing with the liposomes.

[0025] Figure 3 – Expression of profibrosis genes a) αSMA, b) Col1A1, and c) Col3A1 in OASF cells stimulated with 10 ng / mL TGFβ and treated with DendriXs for 24 h. One-way ANOVA and Tukey's multiple comparison test were used. Statistical significance is indicated as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0026] Figure 4 – Virulence of DendriXs and DendriXALs as determined by the CCK8 cell viability assay. OASF cells were seeded in 96-well plates at a density of 5 × 10^3 cells per well. Cells were then treated with different dendritic polymer concentrations for 48 h, and cell viability was normalized to the fluorescence signal measured in the DMEM treatment group (100%).

[0027] Figure 5 – Cryo-electron microscopy images of liposomes and DendriXALs. The total lipid concentration of all samples was 2 mM, and the DendriX concentrations were adjusted (152 µg / mL Dendri1 and 139 µg / mL Dendri4).

[0028] Figure 6 – Release of rapamycin from a custom dialysis unit using 10% ethanol in ultrapure water as the release medium. In all cases, the total lipid concentration was 5 mM and the L / D ratio was 30 / 1. Results are expressed as mean ± standard deviation of three replicates.

[0029] Figure 7 – Macroscopic morphology of DendriXALs. Liposomes were labeled with a DiD fluorescent probe, and after aggregation with DendriXs, imaging was performed using a fluorescence microscope with a Tx red filter and bright field mode. The total lipid concentration was 5 mM, and the DendriX concentrations were adjusted (Dendri1: 381 µg / mL, Dendri4: 347 µg / mL).

[0030] Figure 8 – Lubrication of isolated porcine cartilage by liposomes, DendriXs, and DendriXALs. DendriX concentrations were kept constant at 50 mg / mL for all samples, while liposome concentrations were adjusted for Dendri4ALs (0.73 mM) and Dendri1ALs (0.66 mM). One-way ANOVA and Tukey's multiple comparison test were performed. Statistical significance is indicated as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Mean COF values ​​for each condition are indicated in the corresponding bars.

[0031] Figure 9 – Retention on the cartilage surface. A) Comparison of fluorescence intensity of the region of interest, which remained consistent across all samples. Each condition was performed in triplicate. Statistical analysis was performed using a t-test to compare two groups; statistical significance was indicated by *p<0.05. B) Representative images of Dendri1ALs, liposomes, and buffer samples at 50× magnification. C) Representative images of Dendri4ALs, liposomes, and buffer samples at 50× magnification. For all samples, the DendriX concentration was kept constant at 50 mg / mL, while the liposome concentration was adjusted for Dendri4ALs (0.73 mM) and Dendri1ALs (0.66 mM).

[0032] Figure 10 – Macrophage uptake of DiD-labeled Dendri4ALs after 3 h of incubation. A) DiD intensity was normalized by cell count and liposome positive control to correct for variability among independently prepared DiD-labeled liposomes. B) Representative histograms of DiD intensity in live macrophages under DMEM negative control, liposome positive control, and Dendri4AL conditions. C) Representative cell images obtained using Image Stream fluorescence imaging flow cytometry. In statistical analysis, unpaired t-tests were used to compare the two groups; statistical significance was indicated by *p<0.05.

[0033] Figures 11A and 11B show aggregation curves of aggregated liposomes prepared using different polycationic aggregaters (as listed in Table 3) at DPPC-DSPG liposome standard concentrations (1 mM) and under different positive / negative charge ratios, with changes in turbidity (optical density) measured at λ = 450 nm at t = 0 h and t = 1 h. Reporting techniques are repeated, n = 4.

[0034] Figure 12 – Uptake of DPPC-DSPG liposomes and aggregated liposomes prepared using different multi-cationic aggregaters (Dendri1 (D1), glatiramer acetate (GA), i-PAMAM, LL-37, polymyxin B (PMB), and polyarginine (Poly-Arg)) by RAW264.7 cells. Statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparison test. Statistical significance was indicated as *p<0.05, **p<0.01, and ****p<0.0001.

[0035] Figure 13 – Comparison of OA synovial fibroblast viability between zinc-aggregated liposomes containing rapamycin (ZnAL) (RAPA-ZnAL) and polycationic aggregated liposomes containing rapamycin (Dendri1AL and Dendri4AL) (RAPA-Dendri1AL and RAPA-Dendri4AL, respectively). The aggregated liposomes were compared with a liposome control without RAPA (Liposome ctrl) and a RAPA control (RAPA ctrl). *CCK-8 assay; **Live / Dead assay.

[0036] Figure 14 – Comparison of cytotoxicity of zinc-aggregated liposomes (Zn2+) with multi-cationic aggregated liposomes prepared using Dendri1, Dendri4, and LL-37 (D1, D4, and LL-37, respectively) against RAW264.7 macrophages. Aggregated liposomes are compared with liposome controls (free liposomes).

[0037] Detailed description In the description and claims of this specification, "comprising," "including," and variations thereof mean "including but not limited to," and are not intended to exclude other parts, additives, components, elements, or steps. In the description and claims of this specification, the singular includes the plural unless the context requires otherwise. In particular, when the indefinite article is used, unless the context requires otherwise, this specification should be understood to cover both the plural and the singular.

[0038] Features, elements, properties, compounds, chemical parts, or groups described in conjunction with a particular aspect, embodiment, or example of the invention should be understood to be applicable to any other aspect, embodiment, or example described herein, unless incompatible with it. All features disclosed in this specification (including any appended claims, abstract, and drawings) and / or all steps of any disclosed method or process may be combined in any manner, except for combinations of at least some of such features and / or steps that are mutually exclusive. The invention is not limited to the details of any of the embodiments described above. The invention applies to any novel one or any novel combination of features disclosed in this specification (including any appended claims, abstract, and drawings), or any novel one or any novel combination of steps applicable to any disclosed method or process.

[0039] To avoid any doubt, the information previously disclosed in this specification under the heading "Background Art" is relevant to this invention and should be read as part of the disclosure of this invention.

[0040] Readers are advised that all documents and files related to this application and submitted at the same time as or prior to this specification, and made publicly available together with this specification, are incorporated herein by reference.

[0041] definition Unless otherwise stated, the following terms used in this specification and claims have the following meanings.

[0042] "The composition of the present invention" refers to a composition comprising anionic lipid compositions, polycationic aggregates, and optionally pharmaceutically active agents.

[0043] The term "treatment" and the therapies covered by this invention include the following and combinations thereof: (1) reducing the risk or inhibiting (e.g., delaying) the occurrence and / or progression of a state, disorder, or condition; (2) preventing (e.g., reducing the risk or delaying the onset of clinical symptoms) the development of a state, disorder, or condition in a patient (e.g., a person or animal) who may have or be susceptible to the state, disorder, or condition but has not yet experienced or exhibited clinical or subclinical symptoms of the state, disorder, or condition; (3) inhibiting the state, disorder, or condition (e.g., preventing, reducing, or delaying the progression of the disease; or, in the case of maintenance therapy, preventing, reducing, or delaying its recurrence; and / or preventing, reducing, or delaying the development of at least one of its clinical or subclinical symptoms); and / or (4) alleviating the condition (e.g., causing the state, disorder, or condition or at least one of its clinical or subclinical symptoms to subside). When the compositions of this invention are used to treat a patient, "treatment" may mean any one or more of the following: maintaining the patient's health; restoring or improving the patient's health; and delaying the progression of the disorder. For patients receiving treatment, the benefit may be statistically significant, or at least perceptible to the patient or physician. It should be understood that a drug does not necessarily produce a clinical effect in every patient administered the drug, and this paragraph should be interpreted accordingly. The compositions and methods described herein may be used for the treatment and / or prevention of disease. The compositions and methods described herein may be used to inhibit or prevent disease progression.

[0044] This treatment may also include maintenance therapy for patients who have had the disorder and whose condition has subsequently improved, for example, due to treatment. These patients may or may not have a symptomatic disorder. Maintenance therapy aims to prevent, reduce, or delay the (re)occurrence or progression of the disorder.

[0045] As used herein, “therapeutic effective dose” means a dose sufficient to: reduce or completely alleviate the symptoms or other adverse effects of a disorder; reverse, completely stop or slow the progression of the disorder; or reduce the risk of the disorder worsening; for example, a dose sufficient to induce remission of osteoarthritis, or a dose sufficient to maintain remission of osteoarthritis. Those skilled in the art can also determine the appropriate duration of treatment, appropriate dosage, and any potential combination therapies based on an assessment of the treatment or preventive response.

[0046] As used herein, “sustained release” includes compositions capable of altering the release of a drug from the composition, particularly including compositions that provide controlled release, prolonged release, modified release, or delayed release, or any combination thereof; for example, enabling delayed and controlled release of a drug from the composition after administration (e.g., after intra-articular administration to a subject’s joint cavity).

[0047] In the context of this invention, the term "antibody" refers to "immunoglobulin" (Ig), which is defined as a protein belonging to the IgG, IgM, IgE, IgA, or IgD class (or any subclass thereof), and includes all conventionally known antibodies and their functional fragments. In the context of this invention, a "functional fragment" of an antibody / immunoglobulin is defined as an antigen-binding fragment or other derivative of a parent antibody that substantially retains the properties of that parent antibody. An "antigen-binding fragment" of an antibody / immunoglobulin is defined as a fragment that retains an antigen-binding region (e.g., the variable region of IgG). The "antigen-binding region" of an antibody is typically located in one or more hypervariable regions of the antibody, namely CDR-1, CDR-2, and / or CDR-3 regions. According to the invention, an "antigen-binding fragment" includes a domain of the F(ab′)2 fragment and a domain of the Fab fragment. The "functional fragments" of this invention include Fab fragments, F(ab′)2 fragments, Fab′ fragments, scFv, dsFv, VHH, bivalent antibodies, trivalent antibodies, tetravalent antibodies, Fc fusion proteins, and mini antibodies. The F(ab′)2 or Fab domains can be engineered to minimize or completely eliminate intermolecular disulfide interactions occurring between the CH1 and CL domains. The antibodies or functional fragments used in this invention can be part of bifunctional or multifunctional constructs.

[0048] The term "dendritic polymer" refers to a polymer compound having a self-centrally branched structure. Therefore, a dendritic polymer consists of a central core and side-chain portions called dendritic units. The term "generation" indicates the number of branches of the dendritic unit portion, such as G1, G2, G3, etc. Therefore, a G3 dendritic polymer (i.e., a third-generation dendritic polymer) means that the dendritic unit has branched four times.

[0049] In some embodiments, the compositions of the present invention comprise a dendritic polymer as a polycationic aggregator. Therefore, in these embodiments, the compositions of the present invention are also referred to as “dendritic polymer aggregated liposomes” or “DendriXALs”. The dendritic polymer used in the compositions of the present invention is also referred to as “DendriXs”. The polycationic aggregator (or cationic dendritic polymer aggregator) may include the following linear symbol: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4), and may also be referred to as “Dendri1”, “De1”, or “D1”. The polycationic aggregator (or cationic dendritic polymer aggregator) may include the following linear symbol: (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8), and may also be referred to as “Dendri4”, “De4”, or “D4”. Polycationic agglomerates (or cationic dendritic polymer agglomerates) may include the following linear symbols: (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86), also known as "SOY041". Polycationic agglomerates (or cationic dendritic polymer agglomerates) may include the following linear symbols: (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90), and also known as "SOY031". Polycationic agglomerates (or cationic dendritic polymer agglomerates) may include the following linear symbols: (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70), and also known as "G3KL". Polycationic agglomerates (or cationic dendritic polymer agglomerates) may include the following linear symbols: (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 71 to 74), and may also be referred to as "DMH13". Polycationic agglomerates (or cationic dendritic polymer agglomerates) may include the following linear symbols: (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78), and may also be referred to as "DMH18". Polycationic agglomerates (or cationic dendritic polymer agglomerates) may include the following linear symbols: (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82), and may also be referred to as "Z34".

[0050] In some embodiments, the compositions of the present invention are substantially free of organic solvents. Therefore, in some embodiments, the organic solvent content in the compositions disclosed herein is less than 10%, less than 5%, less than 1%, suitably less than 0.01%, or preferably less than 0.001%. The compositions of the present invention preferably do not contain detectable organic solvents.

[0051] The term "amino sugar" refers to a sugar molecule in which the hydroxyl group is replaced by an amino group.

[0052] The term "anionic lipid composition" refers to a composition containing lipids, such as compositions containing liposomes, lipid nanoparticles, solid lipid nanoparticles and / or nanostructured lipid carriers, which have a negative surface charge.

[0053] The terms "negative surface charge" or "negative surface potential" refer to the surface potential of an anionic lipid composition (e.g., anionic liposomes) in the pH range of 6.0 to 8.0 (preferably at pH 7.4). The surface potential (z-potential) is measured by electrophoresis. Such methods are well known to those skilled in the art. For example, see "Dynamic Light Scattering (DLS) and Zeta Potential" in the Embodiments section below.

[0054] The term "liposome" refers to a spherical vesicle having at least one lipid bilayer. Examples of liposomes covered by this invention include, but are not limited to, monolayer vesicles (having one lipid bilayer, such as small monolayer vesicles or large monolayer vesicles), multilayer vesicles (having more than one layer of lamellar phase lipid bilayer), and multivesicular vesicles (one of which contains one or more smaller vesicles).

[0055] The term "lipid nanoparticle" refers to nanoparticles composed of lipids and without a solid lipid core. In contrast, the term "solid lipid nanoparticle" (SLN) refers to nanoparticles composed of lipids and containing a solid lipid core matrix.

[0056] The term “nanostructured lipid carrier” (NLC) refers to nanoparticles composed of lipids and containing a mixture of solid lipids and liquid crystal lipids as a core matrix. As is well known in the art, the term NLC includes three morphological models: i) Type I NLC (incomplete crystal model); ii) Type II NLC (multiplex model); and iii) Type III NLC (amorphous model) (as described in Adv Pharm Bull. 2020; 10(2): 150–165, which is incorporated herein by reference).

[0057] The term "polyamine" refers to an organic compound that contains two or more amino groups.

[0058] The term "peptide" refers to a short chain of amino acids (typically 2 to 50) linked together by peptide bonds. Longer chains of amino acids (typically 51 or more) are called "polypeptides".

[0059] The term "amino acid" refers to a molecule containing both an amino and a carboxyl group. Suitable amino acids include, but are not limited to, D- and L-isomers of naturally occurring amino acids, as well as non-natural amino acids prepared through organic synthesis or other metabolic pathways. As used herein, the term "amino acid" includes, but is not limited to, α-amino acids, β-amino acids, protein-derived amino acids, non-protein-derived amino acids, and amino acid analogs.

[0060] As those skilled in the art will know, D-isomers of amino acids can be represented by lowercase letters. For example, dendritic polymer DMH18 (including the following linear symbols: (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78)) contains the D-isomers of the listed amino acids. L-isomers of amino acids can be represented by uppercase letters. For example, dendritic polymer G3KL (including the following linear symbols: (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70)) contains the L-isomers of the listed amino acids. Dendritic polymer SOY031 (including the linear symbols: (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90)) contains a mixture of L-isomers and D-isomers of the listed amino acids. However, it should be understood that when a dendritic polymer is represented herein by linear symbols, the dendritic polymer may include D- and / or L-isomers of the listed amino acids. For example, the dendritic polymer DMH18 (including the following linear symbols: (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78)) may also encompass dendritic polymers comprising all L-isomers of the listed amino acids, or dendritic polymers comprising a mixture of L- and D-isomers of the listed amino acids.

[0061] The term "α-amino acid" refers to a molecule that contains both an amino group and a carboxyl group, both of which are attached to a carbon atom called an α-carbon.

[0062] The term "β-amino acid" refers to a molecule that contains both amino and carboxyl groups in the β configuration.

[0063] The term "protein-derived amino acid" refers to any one of the 20 amino acids commonly found in naturally synthesized peptides, with abbreviations of A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. It also includes selenocysteine ​​and pyrrolidone.

[0064] Non-limiting examples of amino acid analogs include homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium salt.

[0065] Amino acids in this article may be represented by their names, commonly used three-letter symbols, or single-letter symbols recommended by the IUPAC-IUB Biochemistry Nomenclature Committee.

[0066] The term "non-protein amino acid" refers to an amino acid that is not one of the 22 protein-derived amino acids naturally encoded in the genome of an organism. Non-protein amino acids include, but are not limited to, hydroxyproline (Hyp), β-alanine, citrulline (Cit), ornithine (Orn), leucine (Nle, also referred to as {NLE} in this article), 3-nitrotyrosine, nitroarginine, pyroglutamic acid (Pyr), γ-aminobutyric acid (GABA), δ-aminolevulinic acid, cystathionine, thioether amino acids, kernicotinic acid, and diaminopimelic acid.

[0067] The term "polyquaternium," also known as polyquaternium or polyquat, is the international cosmetic ingredient nomenclature for several polycationic polymers used, for example, in the personal care industry. "Polyquaternium" is a neologism used to emphasize the presence of quaternary ammonium centers in the polymer. Different polymers are distinguished by the numerical value following "polyquaternium." For example, polyquaternium-5, polyquaternium-7, and polyquaternium-47.

[0068] The term "polycationic aggregater" refers to an agent that has two or more independent positively charged sites in the pH range of 6.0 to 8.0 (preferably at pH 7.4) and is capable of complexing with one or more molecules (i.e., anionic lipid compositions) with opposite charges through non-covalent interactions (preferably electrostatic interactions).

[0069] The term "aggregation composition" refers to a composition comprising aggregates of a polycationic aggregator that complexes with an anionic lipid composition (e.g., the anionic lipid composition is in the form of anionic liposomes, anionic lipid nanoparticles, or anionic solid lipid nanoparticles). For example, in some embodiments, the aggregation composition may be a composition comprising a dendritic polymer-liposome complex, wherein the polycationic dendritic polymer forms a complex with one or more anionic liposomes (preferably multiple anionic liposomes). Aggregation between the anionic lipid composition having a negative surface potential and the polycationic aggregator is due to non-covalent interactions, including electrostatic interactions, hydrogen bonds, van der Waals forces, or combinations thereof. The aggregation between the polycationic aggregator and the anionic lipid composition described herein can be monitored using analytical techniques well known in the art. For example, aggregation can be observed by measuring changes in optical density (turbidity) and / or zeta potential, as described in the Embodiments section herein.

[0070] "Intra-articular" compositions refer to compositions injected into a subject's joint (e.g., joint cavity). Therefore, the compositions claimed in this invention are administered intra-articularly to a subject to treat joint diseases or disorders.

[0071] The components and excipients of the composition are suitable for its intended use. For example, a pharmaceutical composition contains pharmaceutically acceptable ingredients.

[0072] Unless otherwise stated, the ingredients, components, excipients, etc. of the compositions of the present invention are suited to one or more intended uses as described in other parts of this document.

[0073] In numerical expressions, "about" is intended to cover ±10% of the value. For example, "about 20%" includes a range of 18% to 22%.

[0074] composition This article provides a composition for treating joint diseases or disorders, wherein the composition comprises: Anionic lipid compositions; and Polycationic aggregates, The composition is administered intra-articularly.

[0075] In some embodiments, the composition for this purpose further includes a pharmaceutically active agent. Therefore, the composition for this purpose may include: Anionic lipid compositions; Polycationic aggregators; and Pharmaceutical active agent, The composition is administered intra-articularly.

[0076] In some embodiments, the pharmaceutical active agent is incorporated into an anionic lipid composition. For example, the anionic lipid composition may be a liposome composition, and the pharmaceutical active agent is encapsulated within the liposomes. The pharmaceutical active agent may be a hydrophilic pharmaceutical active agent, encapsulated within the aqueous core of the liposome during lipid bilayer formation. The pharmaceutical active agent may be a hydrophobic pharmaceutical active agent, enriched within the hydrophobic lipid bilayer of the liposome. It is also contemplated to encapsulate water-poorly soluble pharmaceutical active agents within the aqueous core of the liposomes. Such water-poorly soluble active agents may be encapsulated, for example, by a solvent-assisted loading method, wherein a small amount of water-miscible solvent is used during liposome loading to improve solubility (see, for example, Wei-Lun Tang et al.: Systemic study of solvent-assisted active loading of gambogic acid into liposomes and its formulation optimization for improved delivery; Biomaterials, Vol 166, 2018, 13-26).

[0077] An anionic lipid composition, wherein the anionic lipid composition comprises a pharmaceutically active agent; and Polycationic aggregates, The composition is administered intra-articularly.

[0078] In some embodiments, the pharmaceutical active agent is incorporated into the anionic lipid composition at a lipid-to-drug molar ratio of about 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 2.5:1, 1:1, 1:2, 1:3, 1:5, or 1:10. The pharmaceutical active agent may be incorporated into the anionic lipid composition at a lipid-to-drug molar ratio of about 50:1 to about 1:10, for example, about 40:1 to about 5:1, or about 60:1 to about 20:1, or about 35:1 to about 25:1. The pharmaceutical active agent may be incorporated into the anionic lipid composition at a lipid-to-drug molar ratio of about 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, or 10:1. The pharmaceutical active agent may be incorporated into the anionic lipid composition at a lipid-to-drug molar ratio of about 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, or 25:1. Preferably, the pharmaceutical active agent is incorporated into the anionic lipid composition at a lipid-to-drug molar ratio of about 30:1.

[0079] In other embodiments, the composition used for this purpose does not contain a pharmaceutically active agent.

[0080] In some embodiments, the anionic lipid composition forms aggregates with a polycationic aggregator. Therefore, in some embodiments, the composition described herein for this purpose is an aggregate composition comprising an anionic lipid composition (e.g., liposomes) and a polycationic aggregator.

[0081] In some embodiments, the average particle size of the aggregates in the composition ranges from about 0.3 µm to 500 µm. The particle size of the aggregates in the composition can range from about 0.5 µm to 500 µm. The particle size of the aggregates in the composition can also range from about 0.3 µm to 300 µm, for example, from about 1 µm to about 300 µm. The particle size of the aggregates in the composition can range from about 1 µm to 200 µm. The average particle size of the aggregates in the composition can range from 10 µm to 100 µm. For example, the average particle size of the aggregates in the composition can be about 10 µm, 20 µm, 30 µm, 40 µm, 50 µm, 60 µm, 70 µm, 80 µm, 90 µm, or 100 µm. The average particle size of the aggregates in the composition can be determined by analytical methods known in the art. The average aggregate size of the aggregates in the composition can be measured using methods well-known in the art, such as dynamic light scattering (DLS) (suitable for aggregates up to about 1 µm in size), laser diffraction-based particle size analyzer (PSA), cryo-electron microscopy, or optical microscopy (e.g., fluorescence microscopy using suitable dyes to stain lipids). Suitable methods for determining and characterizing lipid aggregates are described in G. Bordon et al. 9 In some embodiments, the composition for this purpose is administered to a subject intra-articularly. Thus, the composition for this purpose (e.g., an aggregated composition) is an injectable composition and is administered intra-articularly to the joint (e.g., joint cavity) of the subject in need.

[0082] In some embodiments, the composition for this purpose comprises a pharmaceutically active agent not incorporated into the anionic lipid composition. Therefore, the anionic lipid composition can form aggregates with the polycationic aggregater and the pharmaceutically active agent.

[0083] In some embodiments, the polycationic aggregater is a pharmaceutically active agent as described herein (e.g., an anti-inflammatory, antimicrobial, or antifibrotic agent). Therefore, anionic lipid compositions can also form aggregates with pharmaceutically active agents.

[0084] In some embodiments, the amount of the polycationic aggregator in the composition is the amount required to achieve the desired pharmaceutical active agent release rate.

[0085] The molar ratio of the anionic lipid composition to the polycationic agglomerator in this composition can vary depending on the properties of the polycationic agglomerator. Generally, the anionic lipid composition is present in a molar excess relative to the polycationic agglomerator. The molar excess of the anionic lipid composition relative to the polycationic agglomerator increases with the increase of the number of cationic groups in the polycationic agglomerator. In some embodiments, the molar ratio of the anionic lipid composition to the polycationic agglomerator in the composition is 2:1 to 2000:1, for example 10:1 to 1500:1, 10:1 to 1200:1, 10:1 to 200:1, 100:1 to 1500:1, or 150:1 to 1200:1. The molar ratio of the anionic lipid composition to the polycationic agglomerator in this composition can be 20:1 to 180:1. The molar ratio of the anionic lipid composition to the polycationic agglomerator in this composition can be 25:1 to 160:1.

[0086] In some embodiments, the anionic lipid composition and the polycationic aggregator form aggregates prior to intra-articular administration to the subject. The aggregate composition may be prepared shortly before administration to the subject. For example, the aggregate composition may be prepared by mixing the anionic lipid composition with the polycationic aggregator to form an aggregate comprising the anionic lipid composition (e.g., liposomes) and the polycationic aggregator. Thus, the aggregate composition described herein can be administered intra-articularly to the subject.

[0087] In other embodiments, the anionic lipid composition and the polycationic aggregater form aggregates in situ after intra-articular administration to the subject. Therefore, the compositions described herein can be administered intra-articularly to the subject and form aggregates in situ.

[0088] In some embodiments, the compositions described herein form an in-situ sustained-release reservoir after intra-articular administration to a subject. In some embodiments, the aggregated compositions described herein form a sustained-release reservoir after intra-articular administration to a subject, from which the pharmaceutically active agent is released.

[0089] In some embodiments, the pH of the composition is in the range of about 6.0 to about 8.0. Where appropriate, the pH of the composition is about 7.0 to about 8.0; preferably, the pH is about 7.1 to about 7.7; more preferably, the pH is about 7.4. The composition may also contain a buffer to maintain the pH within the desired range.

[0090] As illustrated in the embodiments, the ionic strength of the composition can affect the degree of aggregation between the polycationic aggregater and the anionic lipid. Therefore, in some embodiments, any of the compositions described herein may further comprise a salt, such as sodium chloride. The concentration of the salt (e.g., sodium chloride) in the composition can be greater than about 0.1 mM and less than about 300 mM. The concentration of the salt (e.g., sodium chloride) in the composition can be from about 1 mM to about 250 mM. The concentration of the salt (e.g., sodium chloride) in the composition can be from about 10 mM to about 200 mM. In a preferred embodiment, the concentration of the salt (e.g., sodium chloride) in the composition is less than about 150 mM. Therefore, the concentration of sodium chloride can be from about 100 mM to about 150 mM. For example, the composition may contain sodium chloride at a concentration of from about 135 mM to about 145 mM.

[0091] In other embodiments, the compositions described herein may not further contain salts. Specifically, the compositions described herein may not further contain sodium chloride.

[0092] In some embodiments, initiating aggregation between the anionic liposomes described herein and the polycationic aggregator may require a positive / negative charge ratio greater than about 0.5. Therefore, this positive / negative charge ratio can be greater than about 1, for example, from about 1 to about 100. The positive / negative charge ratio can be from about 5 to about 50, for example, about 10. This positive / negative charge ratio can be evaluated as described in the embodiments herein (see, for example, embodiments 2 and 7).

[0093] Compositions containing polycationic aggregators (such as cationic dendritic polymer aggregators). This article also provides a composition comprising: an anionic lipid composition; and a polycationic aggregator, wherein the polycationic aggregator is: (i) A cationic dendritic polymer aggregator comprising one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 87 to 90). Composed of SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82); or (ii) A polycationic aggregator selected from: inverse poly(amidoamine) (i-PAMAM), polyethyleneimine (branched or linear), tobramycin, glatiramer acetate, polymyxin B, polylysine, apidaecin 1b, LL-37, polyarginine and Walk11.3.

[0094] Polycationic aggregators can be: (i) A cationic dendritic polymer aggregator comprising one of the following linear formulas: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); or (ii) A polycationic aggregator selected from the group consisting of inverse poly(amidoamine) (i-PAMAM), LL-37 and polyarginine.

[0095] Therefore, the polycationic aggregator can be a cationic dendritic polymer aggregator with the following linear symbol: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4) (i.e., Dendri1). The polycationic aggregator can be a cationic dendritic polymer aggregator with the following linear symbol: (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8) (i.e., Dendri4). The polycationic aggregator can be i-PAMAM. The polycationic aggregator can be LL-37. The polycationic aggregator can be polyarginine.

[0096] This article also provides a composition comprising an anionic lipid composition and a cationic dendritic polymer aggregater. The cationic dendritic polymer aggregators include one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 71 to 74) Composition); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82). Cationic dendritic polymer aggregators may include one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8).

[0097] In some embodiments, the anionic lipid composition comprises anionic liposomes. In some embodiments, the anionic lipid composition comprises anionic lipid nanoparticles. In some embodiments, the anionic lipid composition comprises solid lipid nanoparticles.

[0098] In some embodiments, the composition further comprises a pharmaceutically active agent. Therefore, in some embodiments, the composition comprises an anionic lipid composition; a polycationic aggregator; and a pharmaceutically active agent, wherein the polycationic aggregator is: (i) A cationic dendritic polymer aggregator comprising one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 87 to 90). Composed of SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82); or (ii) A polycationic aggregator selected from: inverse poly(amidoamine) (i-PAMAM), polyethyleneimine (branched or linear), tobramycin, glatiramer acetate, polymyxin B, polylysine, apidaecin 1b, LL-37, polyarginine and Walk11.3.

[0099] In other embodiments, the composition includes anionic lipid compositions, cationic dendritic polymer aggregates, and pharmaceutically active agents. The cationic dendritic polymer aggregators include one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 71 to 74) Composition); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82). Cationic dendritic polymer aggregators may include one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8).

[0100] In some embodiments, the anionic lipid composition comprises anionic liposomes. Therefore, this document also provides a composition comprising: anionic liposomes; and a polycationic aggregator, wherein the polycationic aggregator is: (i) A cationic dendritic polymer aggregator comprising one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 87 to 90). Composed of SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82); or (ii) A polycationic aggregator selected from: inverse poly(amidoamine) (i-PAMAM), polyethyleneimine (branched or linear), tobramycin, glatiramer acetate, polymyxin B, polylysine, apidaecin 1b, LL-37, polyarginine and Walk11.3.

[0101] In some embodiments, the composition comprises: anionic liposomes, a polycationic aggregator, and a pharmaceutically active agent, wherein the polycationic aggregator is: (i) A cationic dendritic polymer aggregator comprising one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 87 to 90). Composed of SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82); or (ii) A polycationic aggregator selected from: inverse poly(amidoamine) (i-PAMAM), polyethyleneimine (branched or linear), tobramycin, glatiramer acetate, polymyxin B, polylysine, apidaecin 1b, LL-37, polyarginine and Walk11.3.

[0102] This article also provides a composition comprising: • Anionic liposomes; and • Cationic dendritic polymer aggregator, The cationic dendritic polymer aggregators include one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 71 to 74) Composition); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82). Cationic dendritic polymer aggregators may include one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8).

[0103] In some embodiments, the composition comprises: Anionic liposomes; Cationic dendritic polymer aggregators; and Pharmaceutical active agent, The cationic dendritic polymer aggregators include one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 71 to 74) Composition); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82). Cationic dendritic polymer aggregators may include one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8).

[0104] In some embodiments, the anionic liposome comprises a pharmaceutically active agent. For example, the pharmaceutically active agent may be encapsulated within the anionic liposome. The pharmaceutically active agent may be a hydrophilic pharmaceutically active agent and encapsulated within the aqueous core of the anionic liposome during lipid bilayer formation. The pharmaceutically active agent may be a hydrophobic pharmaceutically active agent and enriched within the hydrophobic lipid bilayer of the anionic liposome. The pharmaceutically active agent may also be a water-poorly soluble active agent encapsulated within the aqueous core of the liposome (e.g., using the solvent-assisted liposome loading method described above by Wei-Lun Tang et al.). Therefore, in some embodiments, the composition comprises: an anionic liposome, wherein the anionic liposome comprises a pharmaceutically active agent; and a polycationic aggregator, wherein the polycationic aggregator is: (i) A cationic dendritic polymer aggregator comprising one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 87 to 90). Composed of SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82); or (ii) A polycationic aggregator selected from: inverse poly(amidoamine) (i-PAMAM), polyethyleneimine (branched or linear), tobramycin, glatiramer acetate, polymyxin B, polylysine, apidaecin 1b, LL-37, polyarginine and Walk11.3.

[0105] In other embodiments, the composition comprises: anionic liposomes, wherein the anionic liposomes contain a pharmaceutically active agent; and Cationic dendritic polymer aggregator, The cationic dendritic polymer aggregators include one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 71 to 74) Composition); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82). Cationic dendritic polymer aggregators may include one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8).

[0106] In some embodiments, the pharmaceutical active agent is incorporated into the anionic liposomes at a lipid-to-drug molar ratio of about 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 2.5:1, 1:1, 1:2, 1:3, 1:5, or 1:10. The pharmaceutical active agent may be incorporated into the anionic lipid composition at a lipid-to-drug molar ratio of about 50:1 to about 1:10, for example, a lipid-to-drug molar ratio of about 40:1 to about 5:1, or about 60:1 to about 20:1, or about 35:1 to about 25:1. The pharmaceutical active agent can be incorporated into anionic liposomes at a lipid-to-drug molar ratio of about 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, or 10:1. The pharmaceutical active agent can also be incorporated into anionic liposomes at a lipid-to-drug molar ratio of about 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, or 25:1. In a preferred embodiment, the pharmaceutical active agent is incorporated into anionic liposomes at a lipid-to-drug molar ratio of about 30:1.

[0107] In other embodiments, the composition does not contain a pharmaceutically active agent.

[0108] In some embodiments, the polycationic agglomerant is a cationic dendritic polymer agglomerant. In some embodiments, the cationic dendritic polymer agglomerant includes the linear symbol: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4). In some embodiments, the cationic dendritic polymer agglomerant includes the linear symbol: (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8). In some embodiments, the cationic dendritic polymer agglomerant includes the linear symbol: (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86). In some embodiments, the cationic dendritic polymer agglomerant includes the linear symbol: (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90). In some embodiments, the cationic dendritic polymer aggregator comprises the linear symbol: (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70). In some embodiments, the cationic dendritic polymer aggregator comprises the linear symbol: (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 71 to 74). In some embodiments, the cationic dendritic polymer aggregator comprises the linear symbol: (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78). In some embodiments, the cationic dendritic polymer aggregator comprises the linear symbol: (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82). In a preferred embodiment, the cationic dendritic polymer aggregator comprises one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4) (i.e., Dendri1); or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8) (i.e., Dendri4).

[0109] In some embodiments, the polycationic aggregator is selected from the group consisting of: inverse poly(amidoamine) (i-PAMAM), polyethyleneimine (branched or linear), tobramycin, glatiramer acetate, polymyxin B, polylysine, apidaecin 1b, LL-37, polyarginine, or Walk11.3. In a preferred embodiment, the polycationic aggregator is selected from the group consisting of i-PAMAM, LL-37, and polyarginine. Therefore, the polycationic aggregator can be i-PAMAM. The polycationic aggregator can be LL-37. The polycationic aggregator can be polyarginine.

[0110] In some embodiments, the polycationic agglomerant includes one or more polycationic agglomerants described herein (e.g., one or more cationic dendritic polymer agglomerants).

[0111] Anionic liposomes are composed of negatively charged lipids. In some embodiments, anionic liposomes further include one or more neutral or zwitterionic lipids. Anionic liposomes may also include zwitterionic lipids. Therefore, anionic liposomes can include negatively charged lipids and zwitterionic lipids. In some embodiments, anionic liposomes also include cholesterol. Anionic liposomes can include negatively charged lipids, zwitterionic lipids, and cholesterol. Anionic liposomes may be composed of negatively charged lipids, oligoesters, and cholesterol. In some embodiments, anionic liposomes contain about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% mol / mol of negatively charged lipids, based on the remaining lipids in the liposome. For example, the anionic liposome may contain approximately 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% mol / mol of negatively charged lipids, based on the remaining lipids of the liposome. In a preferred embodiment, the anionic liposome may contain approximately 25% mol / mol of negatively charged lipids, based on the remaining lipids of the liposome. The remaining lipids of the anionic liposome may include zwitterionic lipids and / or cholesterol.

[0112] In some embodiments, the anionic liposomes comprise negatively charged lipids selected from the group consisting of: 1,2-dipalmitoyl-sn-glycerol-3-phosphate (DPPA), 1,2-dioleoyl-sn-glycerol-3-phosphate (DOPA), 1,2-distearateoyl-sn-glycerol-3-phosphate-(1′-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1-rac-glycerol) (DPPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1-rac-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-L-serine (DPPS), and 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1′-inositol). (DPPI), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1′-inositol-3′-4′-5′-triphosphate) (DPPI-P3), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1′-inositol) (DOPI) or 1,2-dioleoyl-sn-glycerol-3-phosphate-(1′-inositol-3′-4′-5′-triphosphate) (DOPI-P3), or combinations thereof.

[0113] In some embodiments, the anionic liposomes further include cholesterol and / or phosphocholine lipids, or combinations thereof. In some embodiments, the anionic liposomes further include cholesterol and / or phosphocholine lipids, such as 1,2-dispalmitoyl-sn-glycerol-3-phosphocholine (DPPC) and 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), or combinations thereof. In some embodiments, the anionic liposomes further include cholesterol, 1,2-dispalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), phosphatidylcholine (PC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), or combinations thereof. In some embodiments, the anionic liposomes further include cholesterol, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC) and / or 1,2-distearatel-sn-glycerol-3-phosphate choline (DSPC), or combinations thereof.

[0114] Therefore, anionic liposomes may include DPPC. Anionic liposomes may include DSPG. Anionic liposomes may include cholesterol. Anionic liposomes may include both DSPG and DPPC. Anionic liposomes may include both DSPG and cholesterol. Anionic liposomes may include DSPG, DPPC, and cholesterol.

[0115] In some embodiments, the composition is an injectable composition. Therefore, the composition can be a subcutaneous, intra-articular, intramuscular, or intradermal injection composition. In some embodiments, the composition is administered to the subject subcutaneously, intra-articularly, intramuscularly, or intradermally. In a preferred embodiment, the aggregated composition is an intra-articular injection composition and is therefore administered to the subject intra-articularly.

[0116] In some embodiments, anionic lipid compositions (e.g., anionic liposomes) form aggregates with polycationic aggregators (e.g., cationic dendritic polymer aggregators). Therefore, in some embodiments, the composition is an aggregate composition comprising aggregates of a polycationic aggregator and anionic lipid compositions (e.g., anionic liposomes). In a preferred embodiment, the composition is an aggregate composition comprising aggregates of a cationic dendritic polymer aggregator and anionic lipid compositions (e.g., anionic liposomes). In some embodiments, the aggregates comprise a polycationic aggregator (e.g., a cationic dendritic polymer aggregator) and one or more anionic liposomes, preferably multiple anionic liposomes. In some embodiments, the average particle size of the aggregates in the aggregate composition ranges from about 0.3 µm to 500 µm. In some embodiments, the average particle size of the aggregates in the aggregate composition ranges from about 0.5 µm to 500 µm. The average particle size of the aggregates in the composition can be in the range of about 1 µm to 200 µm. The average particle size of the aggregates in the composition can be in the range of 10 µm to 100 µm. For example, the average particle size of the aggregates in the composition may be about 10 µm, 20 µm, 30 µm, 40 µm, 50 µm, 60 µm, 70 µm, 80 µm, 90 µm, or 100 µm. The average particle size of the aggregates in the composition can be determined by analytical methods known in the art. The average particle size of the aggregates in the composition can be determined using the known methods described above in conjunction with the composition.

[0117] In some embodiments, the aggregation composition comprises a pharmaceutically active agent not encapsulated in anionic liposomes. Therefore, the anionic liposomes can form aggregates with a polycationic aggregater and the pharmaceutically active agent. The anionic liposomes can also form aggregates with a cationic dendritic polymer aggregater and the pharmaceutically active agent.

[0118] In some embodiments, the amount of the polycationic aggregator (e.g., a cationic dendritic polymer aggregator) in the composition is sufficient to form aggregates with the anionic lipid composition (e.g., anionic liposomes). Therefore, the amount of the cationic dendritic polymer aggregator in the composition can be sufficient to form aggregates with the anionic lipid composition (e.g., anionic liposomes). The amount of the polycationic aggregator (e.g., a cationic dendritic polymer aggregator) can also be used to adjust the release rate of the pharmaceutically active agent after administration of the composition to a subject.

[0119] The molar ratio of anionic liposomes to a polycationic aggregator (e.g., a cationic dendritic polymer aggregator) in the composition can vary depending on the number of cationic groups in the polycationic aggregator (e.g., the cationic dendritic polymer aggregator). In some embodiments, the molar ratio of anionic liposomes to a polycationic aggregator (e.g., a cationic dendritic polymer aggregator) in the composition is from 2:1 to 2000:1, for example, 10:1 to 1500:1, 10:1 to 200:1, 100:1 to 1500:1, or 150:1 to 1200:1. The molar ratio of anionic liposomes to a polycationic aggregator (e.g., a cationic dendritic polymer aggregator) in the composition can be from 20:1 to 180:1. The molar ratio of anionic liposomes to a polycationic aggregator (e.g., a cationic dendritic polymer aggregator) in the composition can be from 25:1 to 160:1.

[0120] In a preferred embodiment, the anionic lipid composition (e.g., anionic liposomes) and the polycationic aggregater (e.g., a cationic dendritic polymer aggregater) form aggregates prior to administration to the subject. In some embodiments, the aggregate composition is administered to the subject subcutaneously, intra-articularly, intramuscularly, or intradermally. Therefore, the anionic liposomes and the polycationic aggregater (e.g., a cationic dendritic polymer aggregater) can form aggregates prior to intra-articular administration to the subject.

[0121] In other embodiments, anionic liposomes and a polycationic aggregator (e.g., a cationic dendritic polymer aggregator) form aggregates in situ after administration to a subject. In some embodiments, the aggregation composition is administered to the subject subcutaneously, intra-articularly, intramuscularly, or intradermally. In a preferred embodiment, the composition is administered to the subject intra-articularly.

[0122] In some embodiments, the aggregated composition comprising a pharmaceutically active agent described herein, when administered to a subject via an intra-articular manner, enables sustained release of the pharmaceutically active agent. In some embodiments, the aggregated composition forms a sustained-release reservoir upon intra-articular administration, thereby releasing the pharmaceutically active agent.

[0123] In some embodiments, the aggregated compositions of the present invention are used to treat the joint diseases or disorders described herein.

[0124] Anionic lipid compositions In some embodiments, the anionic lipid composition comprises liposomes, lipid nanoparticles, solid lipid nanoparticles, or nanostructured lipid carriers, or combinations thereof. In some embodiments, the anionic lipid composition comprises liposomes. In some embodiments, the anionic lipid composition comprises lipid nanoparticles. In some embodiments, the anionic lipid composition comprises solid lipid nanoparticles. In some embodiments, the anionic lipid composition comprises nanostructured lipid carriers. In a preferred embodiment, the anionic lipid composition comprises liposomes. Therefore, the anionic lipid composition can be an anionic liposome composition. In some embodiments, the anionic lipid composition is an anionic monolayer liposome composition. In some embodiments, the anionic lipid composition is an anionic multilayer liposome composition. In some embodiments, the anionic lipid composition is an anionic multivesicular liposome composition.

[0125] Liposomes offer several advantages due to their high biocompatibility (consisting of a phospholipid / cholesterol bilayer and an aqueous phase) and their ability to simultaneously load both hydrophilic and lipophilic drugs. Water-soluble drugs can be incorporated into the aqueous phase of liposomes, while lipophilic drugs can be incorporated into their lipid phase. As mentioned above, water-insoluble drugs can also be incorporated into the aqueous core of liposomes by using solvents during liposome loading. The preparation of liposomes is well known to those skilled in the art, for example, through the thin-film hydration techniques described in the Examples section of this document. It should be understood that the scope of protection of this invention also covers other liposome preparation methods, such as those described in Pharmaceutics. 2022 Mar; 14(3): 543, the contents of which are incorporated herein by reference.

[0126] Liposomes comprise negatively charged lipids (i.e., lipids with negatively charged head groups, also referred to herein as "anionic liposomes"). Introducing a hydrophilic lipid head group onto the outer surface of the liposome structure imparts a negative surface potential.

[0127] In some embodiments, the anionic lipid composition comprises anionic liposomes with an average hydrodynamic diameter of less than about 200 nm. A liposome size of less than 200 nm facilitates aseptic filtration sterilization of the composition. In some embodiments, the anionic lipid composition comprises anionic liposomes with an average hydrodynamic diameter of about 100 nm to about 200 nm. For example, the anionic liposomes have an average hydrodynamic diameter of about 120 nm to about 180 nm, about 130 nm to about 170 nm, or about 140 nm to about 150 nm. In some embodiments, the anionic liposomes have an average hydrodynamic diameter of about 145 nm.

[0128] In some embodiments, the anionic lipid composition comprises anionic liposomes with a polydispersity index of less than 0.5 (preferably less than 0.2). For example, the polydispersity index is from about 0.05 to about 0.2. Therefore, the anionic lipid composition may comprise anionic liposomes with a polydispersity index of about 0.05 to about 0.15.

[0129] Hydrodynamic diameter and polydispersity index can be determined by dynamic light scattering, for example using a Litesizer 500 instrument (Anton Paar®, Austria) with a backscattering angle of 175°, a 658 nm laser, and a measurement temperature of 25 °C.

[0130] In some embodiments, the anionic lipid composition comprises anionic liposomes with a zeta potential of approximately −20 mV to −100 mV. For example, the zeta potential of the anionic liposomes may be −30 mV to −65 mV, or approximately −55 mV to approximately −60 mV. This zeta potential can be determined using conventional techniques, such as electrophoretic light scattering (ELS). For example, a Litesizer 500 instrument (Anton Paar®, Austria) with a backscatter angle of 175°, a 658 nm laser, a measurement temperature of 25 °C, and laser Doppler microelectrophoresis measurements using Omega cuvettes (Anton Paar®, Austria) can be performed.

[0131] In some embodiments, the anionic lipid composition comprises a pharmaceutically active agent. In some embodiments, the anionic lipid composition is a liposome composition, and the liposome composition comprises a pharmaceutically active agent. For example, the pharmaceutically active agent may be encapsulated in liposomes. The pharmaceutically active agent may be a hydrophilic pharmaceutically active agent and encapsulated in the aqueous core of the liposome during lipid bilayer formation. The pharmaceutically active agent may be a hydrophobic pharmaceutically active agent and enriched in the hydrophobic lipid bilayer of the liposome.

[0132] In some embodiments, the pharmaceutical active agent is incorporated into the anionic lipid composition (e.g., a liposome composition) at a lipid-to-drug ratio of about 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 2.5:1, 1:1, 1:2, 1:3, 1:5, or 1:10. The pharmaceutical active agent may be incorporated into the anionic lipid composition at a lipid-to-drug ratio of about 50:1 to about 1:10, for example, about 40:1 to about 5:1, or about 60:1 to about 20:1, or about 35:1 to about 25:1. The pharmaceutical active agent can be incorporated into anionic lipid compositions (e.g., liposome compositions) at a lipid-to-drug ratio of about 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, or 10:1. The pharmaceutical active agent can also be incorporated into anionic lipid compositions (e.g., liposome compositions) at a lipid-to-drug ratio (lipid:drug) of about 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, or 25:1. In a preferred embodiment, the pharmaceutical active agent is incorporated into anionic lipid compositions (e.g., liposome compositions) at a lipid-to-drug ratio of about 30:1.

[0133] Where appropriate, pharmaceutical active agents are incorporated into anionic lipid compositions (e.g., liposome compositions) with high encapsulation efficiency. Encapsulation efficiency (EE %) can be determined according to the following formula: In some embodiments, the encapsulation efficiency (EE %) of the pharmaceutical active agent is at least 20%. For example, the EE % is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, the EE % is from 50% to 99%. For example, the EE % is from 70% to 95%. Therefore, the EE % may be from 70% to 85%.

[0134] In other embodiments, the anionic lipid composition does not contain a pharmaceutically active agent.

[0135] Anionic lipid compositions (such as liposome compositions) comprise negatively charged lipids (anionic lipids). In some embodiments, the anionic lipid composition (such as the liposome composition) further comprises one or more neutral lipids or zwitterionic lipids. In some embodiments, the anionic lipid composition (such as the liposome composition) further comprises zwitterionic lipids. Therefore, the anionic lipid composition (such as the liposome composition) may consist of negatively charged lipids and oligomerized lipids. In some embodiments, the anionic lipid composition (such as the liposome composition) further comprises cholesterol. Therefore, the anionic lipid composition (such as the liposome composition) may comprise negatively charged lipids and zwitterionic lipids. The anionic lipid composition (such as the liposome composition) may comprise negatively charged lipids, zwitterionic lipids, and cholesterol. In some embodiments, the content of negatively charged lipids in the anionic lipid composition (e.g., a liposome composition) can be up to 100 mol%; for example, the content of negatively charged lipids relative to the remaining lipids in the anionic lipid composition can be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99 mol%. For example, the content of negatively charged lipids relative to the remaining lipids in the anionic lipid composition (e.g., a liposome composition) can be about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mol%. In a preferred embodiment, the content of negatively charged lipids in the anionic lipid composition (e.g., a liposome composition) is approximately 25% mol / mol relative to the remaining lipids in the anionic lipid composition. The content of negatively charged lipids in the anionic lipid composition (e.g., a liposome composition) is approximately 25 mol% relative to the remaining lipids in the anionic lipid composition, wherein the remaining lipids include zwitterionic lipids and / or cholesterol.

[0136] In some embodiments, the negatively charged lipids contained in the anionic lipid composition (e.g., the liposome composition) are selected from the group consisting of: 1,2-dipalmitoyl-sn-glycerol-3-phosphate (DPPA), 1,2-dioleoyl-sn-glycerol-3-phosphate (DOPA), 1,2-distearateoyl-sn-glycerol-3-phosphate-(1′-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1-rac-glycerol) (DPPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1-rac-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-L-serine (DPPS), and 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1′-inositol). (DPPI), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1′-inositol-3′-4′-5′-triphosphate) (DPPI-P3), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1′-inositol) (DOPI) or 1,2-dioleoyl-sn-glycerol-3-phosphate-(1′-inositol-3′-4′-5′-triphosphate) (DOPI-P3), or combinations thereof.

[0137] In some embodiments, the anionic lipid composition further comprises one or more neutral or zwitterionic lipids, such as phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, myristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, myristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, diphyranoyl phosphatidylethanolamine, linoleoyl phosphatidylethanolamine, egg yolk phosphatidylcholine, or cholesterol, or combinations thereof. In some embodiments, the anionic lipid composition further comprises cholesterol and / or phosphatidylcholine lipids, or combinations thereof. In principle, the anionic lipid composition may further comprise any suitable phosphatidylcholine lipid, such as neutral phosphatidylcholine lipids or zwitterionic phosphatidylcholine lipids. In some embodiments, the anionic lipid composition further comprises cholesterol and / or phosphatidylcholine lipids (e.g., 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC) and 1,2-distearyl-sn-glycerol-3-phosphatidylcholine (DPPC)), or combinations thereof. In some embodiments, the anionic lipid composition further comprises cholesterol, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), phosphatidylcholine (PC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), or combinations thereof. In some embodiments, the anionic lipid composition further comprises cholesterol, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) or 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), or combinations thereof.

[0138] In some embodiments, the anionic lipid composition (e.g., a liposome composition) comprises DPPC. In some embodiments, the anionic lipid composition (e.g., a liposome composition) comprises DSPG. In some embodiments, the anionic lipid composition (e.g., a liposome composition) comprises cholesterol. In some embodiments, the anionic lipid composition (e.g., a liposome composition) comprises both DSPG and DPPC. In some embodiments, the anionic lipid composition (e.g., a liposome composition) comprises both DSPG and cholesterol. Therefore, the anionic lipid composition (e.g., a liposome composition) may comprise DSPG, DPPC, and cholesterol.

[0139] In some embodiments, the anionic lipid composition (e.g., a liposome composition) comprises 20–50 mol% anionic lipid (e.g., DSPG) and 50–80 mol% neutral or zwitterionic lipids. In some embodiments, the anionic lipid composition (e.g., a liposome composition) comprises 20–50 mol% DSPG and 50–80 mol% DPPC. In some embodiments, the anionic lipid composition (e.g., a liposome composition) comprises about 25% DSPG and about 75% DPPC. In some embodiments, the anionic lipid composition (e.g., a liposome composition) comprises 20–50 mol% anionic lipid (e.g., DSPG) and 50–80 mol% neutral or zwitterionic lipids; wherein the neutral or zwitterionic lipids are selected from DPPC and cholesterol. In some embodiments, the anionic lipid composition (e.g., a liposome composition) comprises 20–30 mol% of anionic lipids (e.g., DSPG) and 70–80 mol% of neutral or zwitterionic lipids; wherein the neutral or zwitterionic lipids are selected from DPPC and cholesterol. In some embodiments, the anionic lipid composition comprises 20–50 mol% of DSPG, 40–50 mol% of DPPC, and 15–35 mol% of cholesterol, wherein the sum of the mol% of DPPC, DSPG, and cholesterol is 100 mol%. In some embodiments, the anionic lipid composition comprises 20–30 mol% of DSPG, 40–50 mol% of DPPC, and 25–35 mol% of cholesterol, wherein the sum of the mol% of DPPC, DSPG, and cholesterol is 100 mol%. In a particular embodiment, the molar ratio of DPPC:cholesterol:DSPG in the anionic lipid composition (e.g., a liposome composition) is approximately 45:25:30.

[0140] Polycationic aggregate In some embodiments, the polycationic aggregator has two or more positively charged sites that are separated from each other under pH 6.0–8.0 (e.g., pH 7.4). The polycationic aggregator may have five or more positively charged sites under pH 6.0–8.0 (e.g., pH 7.4). The polycationic aggregator may have ten or more positively charged sites under pH 6.0–8.0 (e.g., pH 7.4). For example, the polycationic aggregator may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more positively charged sites under pH 6.0–8.0 (e.g., pH 7.4). In some embodiments, the polycationic aggregator is a polycationic organic compound. For example, a polycationic agglomerant can be a polymer containing two or more basic centers (i.e., groups that are partially or fully protonated at physiological pH 6.0–8.0). The positively charged sites in the polycationic agglomerant are electrically separated sites, such as amine groups in diamine, triamine, or polyamine compounds. Those skilled in the art will understand that polycationic agglomerants are not inorganic ions such as Ca2+, Zn2+, or Mg2+.

[0141] In some embodiments, the polycationic aggregator is capable of complexing with one or more oppositely charged molecules in the anionic lipid composition through non-covalent interactions, thereby forming aggregates. Thus, the polycationic aggregator forms aggregates with the anionic lipid composition. The anionic lipid composition may include liposomes. Therefore, the polycationic aggregator can form aggregates with liposomes.

[0142] In preferred cases, the polycationic aggregator is soluble in water at a physiological pH (i.e., approximately pH 7.4).

[0143] In some embodiments, the composition comprises one or more of the polycationic aggregators described herein. For example, the composition may comprise two different polycationic aggregators. The composition may also comprise three different polycationic aggregators.

[0144] In some embodiments, the polycationic aggregater is a pharmaceutically active agent. Thus, the polycationic aggregater may be a pharmaceutically active agent as defined herein, having two or more positively charged sites separated from each other in a pH range of 6.0 to 8.0 (e.g., pH 7.4).

[0145] In some embodiments, the polycationic aggregator is a polyamine. The polyamine may be linear or branched.

[0146] In some embodiments, the polyamine comprises a peptide or polypeptide. The peptide or polypeptide may contain only one type of amino acid. For example, the polyamine may include polylysine, polyhistidine, polyornithine, or polyarginine. Alternatively, the peptide or polypeptide may contain at least two, at least three, at least four, at least five, or at least six different amino acids. In some embodiments, the polyamine or polycationic aggregate is not a protein. Polyarginine is preferred.

[0147] In some embodiments, the polycationic aggregator is a polycationic peptide. The polycationic aggregator may be a polycationic anti-inflammatory peptide. The polycationic aggregator may be a polycationic antifibrotic peptide. The polycationic aggregator may be a polycationic antimicrobial peptide.

[0148] In some embodiments, the polycationic aggregater is an anti-inflammatory agent. In some embodiments, the anti-inflammatory agent is a polycationic peptide. Therefore, the anti-inflammatory agent may be a random copolymer containing one or more amino acid groups. For example, the random copolymer may contain one or more amino acid groups selected from lysine, alanine, glutamic acid, and tyrosine, or combinations thereof. In some embodiments, the anti-inflammatory agent is a polymer containing 40 to 100 amino acid residues, comprising four amino acids selected from L-alanine, L-lysine, L-glutamic acid, and L-tyrosine. In some embodiments, the anti-inflammatory agent is a polymer containing 40 to 100 amino acid residues, comprising four amino acids selected from L-alanine, L-lysine, L-glutamic acid, and L-tyrosine, with a molar ratio of 4.2:3.4:1.4:1.0. Therefore, the anti-inflammatory agent may be glatiramer or a pharmaceutically acceptable salt thereof (e.g., glatiramer acetate). Therefore, in some embodiments, the polycationic aggregater is glatiramer or a pharmaceutically acceptable salt thereof (e.g., glatiramer acetate (GA)).

[0149] In some embodiments, the polycationic aggregater is a polycationic antimicrobial agent. In some embodiments, the antimicrobial agent is a polycationic peptide. The antimicrobial agent may be selected from: cathelicidin, LL-37, polymyxin B (PMB), magainin, aminoglycoside antibiotics (e.g., tobramycin), apidaecin 1b, Walk11.3, and murapavadin. Preferably, the antimicrobial agent is LL-37.

[0150] In a further embodiment, the polycationic aggregator is a polycationic anti-inflammatory agent; the anti-inflammatory agent is selected from glatiramer or a pharmaceutically acceptable salt thereof (e.g., glatiramer acetate).

[0151] In other embodiments, the polycationic agglomerant is a polycationic anti-fibrotic agent.

[0152] In some embodiments, the polycationic aggregater is a polyamine. The polyamine may include one or more amino sugar residues. The polyamine may include polymers containing one or more amino sugar residues, such as chitin or chitosan.

[0153] In some embodiments, the polyamine comprises an aminoglycoside. The aminoglycoside may be selected from the group consisting of: streptomycin, gentamicin, tobramycin, amikacin, dibekacin, sisomicin, netilmicin, neomycin (such as neomycin B, neomycin C, or neomycin E), prazomycin, spectromycin, kanamycin, tobramycin, alpramycin, oxytetracycline B, formalin, and combinations thereof. In some embodiments, the aminoglycoside is tobramycin.

[0154] In some embodiments, the polycationic aggregator includes chitosan, chitin, aminoglycoside, poly(dimethyldiallylammonium chloride), poly(amidoamine) (PAMAM), inverse poly(amidoamine) (i-PAMAM), poly(propyleneimine) (PPI), triazine polymers, polyallylamine, polyethyleneimine (branched or linear), poly(dimethylaminoethyl acrylate), peptides or polypeptides (e.g., polylysine, polyhistidine, polyornithine, polyarginine), polyquaternary ammonium salts (e.g., starch derivatives containing amino or ammonium groups), or any combination thereof.

[0155] In some embodiments, the average molecular weight (e.g., weight-average molecular weight) of the polycationic aggregator is about kDa to about 30 kDa, for example about 3 kDa to about 15 kDa or about 4 kDa to about 12 kDa.

[0156] In some embodiments, the polycationic agglomerant is a polyamine containing 2 to 6 basic nitrogen atoms, such as a polyamine containing 2 to 6 amino, alkylamine, or dialkylamine groups. In some embodiments, the polycationic agglomerant is selected from putrescine, cadaverine, spermidine, spermine, thermospermine, thermophilic pentamine, and thermophilic hexamine.

[0157] In some embodiments, the polycationic aggregator is a dendritic polymer. The dendritic polymer has a branched core-shell structure with a precisely defined number of branch foci between the core and shell, thus determining its generation (G1, G2, G3, etc.). The dendritic polymers described herein are positively charged due to the presence of cationic terminal groups (e.g., lysine residues) at the branch ends.

[0158] Therefore, in some embodiments, the polycationic agglomerator may be a poly(amidoamine) (PAMAM) dendrimer, an inverse poly(amidoamine) (i-PAMAM) dendrimer, a poly(propyleneimine) (PPI) dendrimer, a triazine dendrimer, a phosphorus dendrimer, or a peptide dendrimer. In a preferred embodiment, the polycationic agglomerator may be a peptide dendrimer. In a preferred embodiment, the polycationic agglomerator may be i-PAMAM. In some embodiments, the polycationic agglomerator is a G1, G2, G3, or G4 dendrimer. In some embodiments, the dendrimer is a G2 or G3 dendrimer. Therefore, the dendrimer may be a G2 dendrimer. The dendrimer may also be a G3 dendrimer. In a preferred embodiment, the polycationic agglomerator is a G3 dendrimer.

[0159] In some embodiments, the polycationic aggregator is a dendritic polymer (such as any of the dendritic polymers described herein) with an average molecular weight (e.g., weight average molecular weight) of about 2 kDa to about 20 kDa, for example about 3 kDa to about 15 kDa, or about 4 kDa to about 12 kDa.

[0160] In some embodiments, the polycationic aggregator is a dendritic polymer containing one or more amino acids selected from lysine, leucine, arginine, asparagine, alanine, glutamic acid, tyrosine, and cysteine. The dendritic polymer may also contain one or more amino acids selected from lysine, alanine, glutamic acid, tyrosine, and cysteine.

[0161] In some embodiments, the polycationic agglomerant is a dendritic polymer conforming to the general formula (A)8-(B)4-(C)2-Z, wherein: A is X1AX2X3; B is X1BX4X5X6X7; C is X1BX8X5X7X6X7; where: X1A is selected from lysine and arginine; X1B is lysine; X2 is selected from leucine, oroleucine, and alanine; X3 is either empty or tyrosine; X4 is selected from lysine, arginine, and alanine; Each X5 is independently selected from lysine, leucine, and alanine; Each X6 is either empty or lysine independently; Each X7 can be empty, glutamic acid, or alanine independently; X8 is selected from lysine, tyrosine, leucine, arginine, and glutamic acid; and Z is the core group.

[0162] In some implementations, A is selected from: KL, kl, rl, KA and k{NLE}y (SEQ ID NO: 91).

[0163] In some implementations, B is selected from: KKL, kkl, krl, KAK, and KKAKE (SEQ ID NO: 92).

[0164] In some implementations, C is selected from: KKL, kll, krl, KEKA (SEQ ID NO: 93) and KYKAKA (SEQ ID NO: 94).

[0165] In some embodiments, Z comprises 1 to 10 amino acids; optionally, Z also comprises one or more C2-C30 alkyl groups.

[0166] In some embodiments, the polycationic agglomerant is the dendritic polymer described in Angew. Chem. Int. Ed. 2021, 60, 26403–2640813, which is incorporated herein by reference. In some embodiments, the polycationic agglomerant is a dendritic polymer comprising one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4), (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8), (AcKA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 9 to 12), (KAEKAYA)4(KEKYAKA)2KEKYKA-NH2 (composed of SEQ ID NO: 13 to 15), (AKA)8(KYEK)4(KEKA)2KAKY-OH (composed of SEQ ID NO: 16 to 19), (AcKA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 20 to 23). Composition), (AK)8(KAKAKY)4(KAKEYEY)2KAKEYEY-NH2 (composed of SEQ ID NO: 24 to 27), (KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 28 to 30), (AK)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 31 to 34), (KK)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 35 to 38), (KA)8(KKKAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 39 to 42), (AcKA)8(KKKAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 43) Composed of SEQ ID NO: 47 to 50), ((KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 47 to 50), ((KA)8(KAK)4(KEKA)2KAKEAYCA-NH2)2 (composed of SEQ ID NO: 51 to 54), or (KA)8(KAK)4(KEKA)2KAKEAYC(Fl)A-NH2 (composed of SEQ ID NO: 55 to 58).In some embodiments, the polycationic aggregator is a dendritic polymer comprising one of the following sequences listed in Table 1.

[0167] Table 1—Linear symbols for the dendritic polymers of the present invention, and the decomposition of the dendritic polymers into their corresponding linear amino acid chain core and branch sequences.

[0168] In some embodiments, the polycationic agglomerant is a dendritic polymer comprising one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4), (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8), (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86), (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90), (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70), (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 87 to 90). Composed of SEQ ID NO: 71 to 74), (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78), or (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82). In some embodiments, the polycationic agglomerant is a dendritic polymer comprising the following linear symbol: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4). In some embodiments, the polycationic agglomerant is a dendritic polymer comprising the following linear symbol: (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8). In some embodiments, the polycationic agglomerator is a dendritic polymer comprising the following linear symbols: (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86). In some embodiments, the polycationic agglomerator is a dendritic polymer comprising the following linear symbols: (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90). In some embodiments, the polycationic agglomerator is a dendritic polymer comprising the following linear symbols: (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70). In some embodiments, the polycationic agglomerator is a dendritic polymer comprising the following linear symbols: (KL)8(KKL)4(KLL)2KK(C16)K(C16) (composed of SEQ ID NO: 71 to 74).In some embodiments, the polycationic agglomerant is a dendritic polymer comprising the following linear symbols: (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78). In some embodiments, the polycationic agglomerant is a dendritic polymer comprising the following linear symbols: (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82).

[0169] In some embodiments, the polycationic agglomerator is selected from the following members: reverse poly(amidoamine) (i-PAMAM), polyethyleneimine (branched or linear), tobramycin, glatiramer acetate, polymyxin B, polylysine, apidaecin1b, LL-37, polyarginine, Walk11.3, or dendritic polymers including one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4), (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8), (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86), (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90). (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70), (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 71 to 74), (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78), and (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82). The polycationic agent can be: (i) A cationic dendritic polymer aggregator comprising one of the following linear formulas: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); or (ii) A polycationic aggregator selected from the group consisting of inverse poly(amidoamine) (i-PAMAM), LL-37 and polyarginine.

[0170] In a preferred embodiment, the polycationic aggregater is substantially non-toxic to human osteoarthritis synovial fibroblasts (OASF). The toxicity of the polycationic aggregater can be assessed in vitro using the cell viability assay described in the embodiments herein. In some embodiments, after incubating OASF with 50 µg / mL of the polycationic aggregater for 48 hours, the cell viability is at least 60%, preferably at least 70%, and more preferably at least 75%.

[0171] Pharmaceutical active agent In some embodiments, the composition comprises at least one pharmaceutically active agent. Therefore, the composition may comprise one pharmaceutically active agent. The composition may also comprise more than one pharmaceutically active agent. The composition may also comprise two or more pharmaceutically active agents; for example, the composition may comprise two, three, or four pharmaceutically active agents.

[0172] In some embodiments, the pharmaceutical active agent is selected from hydrophilic or hydrophobic pharmaceutical active agents. Therefore, the pharmaceutical active agent may be hydrophilic and water-soluble. In other embodiments, the pharmaceutical active agent may be hydrophobic and lipophilic.

[0173] In some embodiments, the pharmaceutical active agent is water-soluble. As used herein, "soluble" means that, under given pH conditions (25.0 ± 0.5 °C), the amount of solvent required to produce 1 g of the pharmaceutical active agent is less than 10,000 mL, preferably less than 1,000 mL, more preferably less than 100 mL, and even more preferably less than 30 mL or 10 mL. "Soluble" as used herein may also mean that the substance has a negative logP value. In some embodiments, the pharmaceutical active agent is soluble in water at pH 7.0 and 25.0 ± 0.5 °C.

[0174] In other embodiments, the pharmaceutical active agent is insoluble in water. As used herein, "insoluble" means that, under given pH conditions (e.g., pH 7.0, 25.0 ± 0.5 °C), more than 10,000 mL of solvent is required to produce 1 g of the pharmaceutical active agent. "Insoluble" as used herein may also mean that the substance has a positive logP value.

[0175] The active pharmaceutical ingredient is not particularly limited, and those skilled in the art can select it as needed.

[0176] In some embodiments, the composition can be used to treat joint diseases or disorders. In some embodiments, the composition can be used to inhibit joint diseases or disorders, or to prevent the progression of such diseases or disorders (e.g., osteoarthritis). Therefore, the composition may comprise at least one pharmaceutically active agent; the pharmaceutically active agent is selected from anti-inflammatory agents, antifibrotic agents, antimicrobial agents, nonsteroidal anti-inflammatory agents, corticosteroids, and biological agents (e.g., peptides, proteins, stem cells, antibodies, and antibody fragments).

[0177] In some embodiments, the pharmaceutically active agent is an anti-inflammatory agent. This anti-inflammatory agent may be a corticosteroid, such as hydrocortisone. Corticosteroids are a class of steroids that help reduce inflammatory and immune responses. The composition may contain a corticosteroid. The pharmaceutically active agent may be a corticosteroid selected from hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, triamcinolone, betamethasone, beclomethasone, flunisolone, and fluticasone.

[0178] In some embodiments, the pharmaceutically active agent is an anti-inflammatory agent selected from quercetin, sinomenine, glycyrrhizin, glatiramer acetate, rhein, and Resolvin D1.

[0179] In some embodiments, the pharmaceutically active agent may be an antifibrotic agent. Therefore, the composition may contain an antifibrotic agent selected from pirfenidone or nintedanib.

[0180] In some embodiments, the pharmaceutically active agent may be an antimicrobial agent. Therefore, the composition may comprise an antimicrobial agent selected from cathelicidin, LL-37, polymyxin B, magainin, murapavadin, G3KL, aminoglycoside antibiotics (e.g., tobramycin), apidaecin 1b, and Walk11.3.

[0181] In some embodiments, the pharmaceutically active agent is a nonsteroidal anti-inflammatory agent (NSAID), such as naproxen. NSAIDs can relieve pain, reduce inflammation, reduce fever, and prevent thrombosis. The composition may contain an NSAID selected from naproxen, ibuprofen, diclofenac, celecoxib, meloxicam, or lonoxicam.

[0182] In some embodiments, the pharmaceutically active agent is a chondrogenesis inducer, such as angiopoietin-like protein 3 and its derivatives (e.g., LNA043); for example, Gerwin et al. reported a randomized phase I trial of the angiopoietin-like protein 3 derivative LNA043 for cartilage regeneration in osteoarthritis. Nat Med 28, 2633–2645 (2022). https: / / doi.org / 10.1038 / s41591-022-02059-9. In some embodiments, the pharmaceutically active agent is a biological agent, such as a peptide, protein, antibody, or antibody fragment. In some embodiments, the pharmaceutically active agent is an antibody or a functional fragment thereof. In some embodiments, the pharmaceutically active agent is a peptide.

[0183] In some embodiments, the pharmaceutically active agent is an antibody or a functional fragment thereof, and is suitable for treating joint diseases or disorders, such as osteoarthritis.

[0184] There are no particular limitations on the antibody or functional fragment thereof used in this composition. In one embodiment, the antibody or functional fragment thereof is an antibody. In another embodiment, the antibody or functional fragment thereof is a functional fragment as defined herein. The antibody or functional fragment thereof may also contain one or more modifications, such as by adding or substituting residues, to improve stability, specificity, or targeting. These modifications may include any such modifications known in the art.

[0185] There are no restrictions on the antigens targeted by antibodies or their functional fragments; the antigen may be an immunogen or a peptide, protein, or other molecular structure that the antibody or its functional fragment can specifically bind to. In the most general sense (and unless a specific reference is mentioned), "specificity" or "specific binding" refers to the ability of an antibody or its functional fragment to distinguish a target from irrelevant biomolecules, which can be determined, for example, by specificity assays known in the art.

[0186] In some embodiments, the antibody or its functional fragment is selected from the following: antibodies and functional fragments specific to tumor necrosis factor α (TNFα); antibodies and functional fragments specific to CD20; antibodies and functional fragments specific to interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-1β (IL-1β), or their receptors; antibodies and functional fragments specific to B cell activating factor (BAFF) or its receptors; and antibodies and functional fragments specific to Janus kinase (JAK). In some embodiments, the antibody or its functional fragment is selected from infliximab, adalimumab, etanercept, pecelizumab polyethylene glycol derivative, golimumab, canatumab, belimumab, rituximab, anakinin, and their functional fragments.

[0187] In some embodiments, the active pharmaceutical ingredient (API) is an anti-tumor necrosis factor α inhibitor (TNF inhibitor). TNF inhibitors may be selected from adalimumab, pecelizumab, etanercept, golimumab, and infliximab. In some embodiments, the API is an anti-interleukin-1 agent or an anti-interleukin-1β agent, such as canatumab, anakinin, ileonaseppull, or gevogitizumab. In some embodiments, the API is a JAK inhibitor, such as tofacitinib, filgortinib, utpatinib, TD-1473, brepocitinib (PF-06700841), or PF-06651600.

[0188] In some embodiments, the pharmaceutically active agent is a NOD-like receptor protein 3 (NLRP3) antagonist, such as DFV890, aminoterasone, 3,4-methylenedioxy-β-nitrostyrene (MNS), parthenolide, INF39, CY-09, MCC950 (PubChem CID 91826093), OLT1177 (dapansutrile), or oridonin. In some embodiments, the pharmaceutically active agent is DFV890.

[0189] In some implementations, the pharmaceutically active agent is an immunosuppressant, such as rapamycin.

[0190] In some implementations, the pharmaceutically active agent is an mTOR inhibitor, such as torin-1 or torin-2.

[0191] In some implementations, the pharmaceutically active agent is a Bcr-Abl tyrosine kinase inhibitor, such as dasatinib.

[0192] In some embodiments, the pharmaceutically active agent is a disease-modifying osteoarthritis drug (DMOAD). DMOAD may be selected from the following group: teriparatide, zoledronic acid, denosumab, vitamin D, methotrexate, hydroxychloroquine, etanercept, tucizumab, metformin, liraglutide, MIV-711, TPX-100, XT-150, diacerein, GLPG1972 / S201086, sprifermin, TissueGene-C, lorecivivint, UBX0101, LNA043, LRX712, clodronate, kartogenin, dasatinib, quercetin, rhein, sinomenine, MK-8722, glycyrrhizin, and rapamycin.

[0193] In some embodiments, the pharmaceutically active agent is selected from the group consisting of: triamcinolone acetonide, prednisone, hydrocortisone, methylprednisolone, dexamethasone, betamethasone, hyaluronic acid, cetrozumab pegal, golimumab, belimumab, rituximab, rapamycin, tolin-1, tolin-2, retinoic acid metabolism blockers (e.g., talarozole, liarozole), naproxen, ibuprofen, diclofenac, celecoxib, meloxicam, lonoxicam, kartogenin, dasatinib, quercetin, rhein, sinomenine, glycyrrhizin, glatiramer acetate, insulin-like growth factor 1 (IGF 1), Resolvin D1, fibroblast growth factor 18 (FGF 18), bone morphogenetic protein 7 (BMP-7), anti-IL-1 agents (e.g., canatumab, anaerobicin, ileonaseppull, gevojizumab), and anti-TNF agents. Agents (e.g., infliximab, adalimumab, etanercept), botulinum toxin type A, miRNA, mRNA, JAK inhibitors (e.g., tofacitinib, filogrinib, utpatinib, TD-1473, Brepocitinib (PF-06700841), PF-06651600), pirfenidone, nintinib, cathelicidin, LL-37, polymyxin B, magainin, murapavadin, MK-8722, or combinations thereof.

[0194] Other additives In some embodiments, the compositions of the present invention may further comprise one or more imaging agents. Anionic lipid compositions may further comprise one or more imaging agents. For example, imaging agents may be trapped or encapsulated in anionic lipid compositions (e.g., anionic liposomes) to study their biodistribution after intra-articular administration to patients. Examples of such imaging agents include, but are not limited to: calcein, fluorescein isothiocyanate-labeled dextran (molecular weight 4000) (FITC-dextran), 8-hydroxy-1,3,6-pyrene trisulfonic acid (HPTS), rhodamine, rhodamine-labeled lipids, indocyanine green, long-chain dialkylcarbocyanine dyes (e.g.) 1,1′-Di(octadecyl)-3,3,3′,3′-tetramethylindole carbocyanine perchlorate, 1,1′-Di(dodecyl)-3,3,3′,3′-tetramethylindole carbocyanine perchlorate, 1,1′-Di(hexadecyl)-3,3,3′,3′-tetramethylindole carbocyanine perchlorate, 1,1′-Di(octadecyl)-3,3,3′,3′-tetramethylindole carbocyanine perchlorate, 1,1′-Di(octadecyl)-3,3,3′,3′-tetramethylindole carbocyanine-5,5′-disulfonic acid, 1,1′-Di(octadecyl)-6,6′-di(4-sulfonic phenyl)-3,3,3′,3′-tetramethylindole carbocyanine, 1,1′-Di(octadecyl)-3,3,3′,3′-tetramethylindole carbocyanine, 1,1′-Di(octadecyl)-3,3,3′ ,3′-tetramethylindoledicarbocyanine-5,5′-disulfonic acid), 1,1′-di(octadecyl)-3,3,3′,3′-tetramethylindoledicarbocyanine perchlorate, 4,4′-diisothiocyanate-stilbene-2,2′-disulfonic acid disodium salt, or 1,1′-di(octadecyl)-3,3,3′,3′-tetramethylindoledicarbocyanine 4-chlorobenzenesulfonate; or long-chain dialkylaminostyryl dyes (e.g., 4-(4-(di(hexadecyl)amino)styryl)-N-methylpyridinium iodide, 4-(4-(di(decyl)amino)styryl)-N-methylpyridinium iodide, or 3,3′-di(octadecyl)-5,5′-di(4-sulfonicophenyl)oxacarbocyanine sodium salt). In some embodiments, the anionic lipid composition further includes an electrophoretic additive, such as an additive that imparts a negative charge to the lipid carrier, like dihexadecyl phosphate (DCP). Therefore, the anionic lipid composition may further include DCP.

[0195] In some embodiments, the composition may further comprise one or more additional additives selected from buffers, antioxidants, preservatives, viscosity modifiers or surfactants, or combinations thereof.

[0196] use This document also provides a use of a composition comprising an anionic lipid composition and a polycationic aggregater, used as a carrier of a pharmaceutically active agent, wherein the composition is administered intra-articularly. In some embodiments, the pharmaceutically active agent may be dispersed or dissolved in the anionic lipid composition.

[0197] Furthermore, a use of the aggregation composition is provided: the aggregation composition comprises anionic liposomes and a cationic dendritic polymer aggregater, used as a carrier of a pharmaceutically active agent. In some embodiments, the pharmaceutically active agent may be encapsulated in anionic liposomes.

[0198] The compositions described herein enable sustained and controlled release of the pharmaceutically active agent, with adjustable release duration. Therefore, these compositions are ideally suited for forming a reservoir composition after intra-articular administration, and being composed of lipids, they may offer inherent benefits beyond simply forming an efficient carrier and reservoir for the active agent. For example, the compositions described herein can lubricate cartilage and improve retention on cartilage surfaces.

[0199] Therefore, one advantage of the controlled-release drug library of the present invention is that the pharmaceutical active agent can be gradually released over a longer period of time without the need for repeated administration.

[0200] The compositions of the present invention can form a reservoir for the slow release of pharmaceutically active agents onto the cartilage surface. Of particular importance is that the compositions of the present invention are aggregated compositions, as the size of the aggregates helps reduce clearance from the joint space. Therefore, compared to intra-articular administration of liposomes alone, the compositions of the present invention exhibit stronger cartilage retention and / or stronger cartilage lubrication.

[0201] This article also provides a preformulated composition consisting of an ionic lipid composition (such as anionic liposomes) and a pharmaceutically active agent for manufacturing the compositions of the present invention.

[0202] In some implementations, the pharmaceutical active agent in the preformulation is a hydrophilic pharmaceutical active agent, as described herein.

[0203] Therapeutic uses and applications The compositions of this invention can be used to treat, alleviate, delay, prevent, manage, or cure any joint disease or symptoms associated with joint dysfunction. For the purposes of this disclosure, the term "joint dysfunction" should be considered as any ailment (congenital, autoimmune, or other), injury, or disease in the joint region that leads to joint degeneration, pain, decreased mobility, inflammation, or physiological disorders and functional impairment. This condition may be associated with reduced joint secretion and lubrication, as well as complications from knee and hip replacement surgery.

[0204] According to the present invention, a "joint" can be any one of the following: knee joint, hip joint, ankle joint, shoulder joint, elbow joint, tarsal joint, wrist joint, interphalangeal joint, and intervertebral joint.

[0205] The compositions of the present invention comprise an aggregation composition containing an anionic composition, a polycationic aggregater, and a pharmaceutically active agent for targeted release of the pharmaceutically active agent into the joints of a subject, particularly the synovium, synovial fluid, and / or cartilage. The compositions of the present invention can be effectively used for intra-articular administration by forming a sustained-release reservoir of the pharmaceutically active agent in situ (i.e., on the cartilage surface).

[0206] Therefore, according to the present invention, compositions comprising pharmaceutically active agents for local treatment of joints are expected to be used for the treatment or prevention of joint diseases or disorders. In some embodiments, the compositions of the present invention are used to treat or prevent conditions affecting joints, including the synovium, synovial fluid, cartilage, tendons, ligaments, and bones within the joint. In some embodiments, the compositions of the present invention are used to treat or prevent conditions affecting joints (including the synovium). In some embodiments, the compositions of the present invention are used to treat or prevent conditions affecting synovial fluid. In some embodiments, the compositions of the present invention are used to treat or prevent conditions affecting cartilage.

[0207] The composition may not contain a pharmaceutically active agent. Therefore, in some embodiments, the composition may comprise anionic lipid compositions and polycationic aggregates, and may be used for the prevention or treatment of joint diseases or disorders, particularly those affecting cartilage.

[0208] In other embodiments, the composition may contain a pharmaceutically active agent. Therefore, in some embodiments, the composition may contain anionic lipid compositions, polycationic aggregates, and pharmaceutically active agents, and may be used for the prevention or treatment of joint diseases or disorders, particularly those affecting cartilage.

[0209] This invention provides a composition (e.g., an aggregate composition) for the treatment or prevention of joint diseases or disorders. In some embodiments, the composition of this invention is used to inhibit or prevent the worsening of joint diseases or disorders. Joint diseases or disorders may be selected from the group consisting of: arthritis, osteoarthritis, joint fibrosis, rheumatoid arthritis, gout, bursitis, bacterial arthritis, juvenile idiopathic arthritis, psoriatic arthritis, reactive arthritis, ankylosing spondylitis, tenosynovitis, polymyalgia rheumatica, Paget's disease, lupus, Sjögren's syndrome, osteonecrosis, neuropathic arthropathy, or Lyme disease.

[0210] Therefore, in some embodiments, the compositions of the present invention can be used to treat or prevent arthritis, osteoarthritis, joint fibrosis, rheumatoid arthritis, gout, bursitis, bacterial arthritis, juvenile idiopathic arthritis, psoriatic arthritis, reactive arthritis, ankylosing spondylitis, tendinopathy, polymyalgia rheumatica, Paget's disease, lupus, Sjögren's syndrome, osteonecrosis, neuropathic arthropathy, or Lyme disease. The most common types of arthritis are osteoarthritis and rheumatoid arthritis. Osteoarthritis can affect any joint in the body. It most commonly affects the fingers, toes, wrists, knees, and hips, but can also involve other joints, including the spinal joints. After trauma, it can also affect the shoulder and ankle joints, and in rare cases, the elbow joint. Rheumatoid arthritis typically affects the joints of the hands and feet. Therefore, in addition to treating osteoarthritis in patients with rheumatoid arthritis, the compositions of this invention can also be used to treat both of these conditions.

[0211] Osteoarthritis (OA) is a disabling chronic joint disease characterized by articular cartilage degradation, synovial fibrosis, and low-grade inflammation. OA also disrupts the function of synovial fibroblasts (SFs) within the synovium. SFs are a significant factor contributing to cartilage damage in OA, and synovial fibrosis is associated with chronic joint pain.

[0212] Therefore, in some embodiments, the compositions of the present invention can be used to treat osteoarthritis. In some embodiments, the compositions of the present invention are used to inhibit or prevent the progression of osteoarthritis, such as the degenerative process of cartilage tissue. In some embodiments, the compositions of the present invention are used to treat synovial fibrosis. In some embodiments, the compositions of the present invention can be used to reduce or inhibit cartilage degradation. In some embodiments, the compositions of the present invention are used to reduce or inhibit synovial inflammation. In some embodiments, the compositions of the present invention are used to reduce or inhibit osteophyte formation. In some embodiments, the compositions of the present invention can be used to reduce or inhibit joint swelling. In some embodiments, the compositions of the present invention are used to reduce or inhibit pain associated with joint diseases such as OA. In further embodiments, the compositions of the present invention can be used to treat traumatic joint injuries, sports injuries, joint locking (such as temporomandibular joint (TMJ)), and postoperative status interventions such as joint aspiration, arthroscopic surgery, arthroplasty, knee and hip replacement surgery.

[0213] In a preferred embodiment, the condition to be treated or prevented is primary or secondary osteoarthritis.

[0214] In a further embodiment, the composition of the present invention is used to lubricate the joints of a subject. In a further embodiment, the composition of the present invention is used to lubricate cartilage. The composition is administered to the subject via intra-articular administration. In some embodiments, the composition is administered intra-articularly into the joint cavity. In some embodiments, the composition does not contain a pharmaceutically active agent. Therefore, in some embodiments, the composition comprises an anionic lipid composition and a polycationic aggregater, and can be used to lubricate the joints of a subject. In other embodiments, the composition contains a pharmaceutically active agent. Therefore, in some embodiments, the composition comprises an anionic lipid composition, a polycationic aggregater, and a pharmaceutically active agent, and can be used to lubricate the joints of a subject.

[0215] In further embodiments, the compositions of the present invention are used to enhance joint lubrication and / or prevent joint wear in subjects. In some embodiments, the compositions are administered intra-articularly into the joint cavity. The compositions of the present invention can be used to enhance joint lubrication in subjects. The compositions of the present invention can be used to prevent joint wear in subjects. In some embodiments, the compositions do not contain a pharmaceutically active agent. Therefore, in some embodiments, the compositions comprise anionic lipid compositions and polycationic aggregates, and can be used to enhance joint lubrication and / or prevent joint wear in subjects. In other embodiments, the compositions contain a pharmaceutically active agent. Therefore, in some embodiments, the compositions comprise anionic lipid compositions, polycationic aggregates, and pharmaceutically active agents, and can be used to enhance joint lubrication and / or prevent joint wear in subjects.

[0216] The compositions of the present invention can be used alone or in combination with anti-inflammatory agents, analgesics, muscle relaxants, antidepressants, or agents that promote joint lubrication; said agents are generally used to treat diseases or disorders associated with joint stiffness, such as arthritis. Combination therapy may help reduce agent-related side effects, such as those associated with nonsteroidal anti-inflammatory drugs (NSAIDs); said agents are generally used to prevent, manage, or treat diseases or disorders associated with reduced joint lubrication, such as osteoarthritis. In addition to improving safety, combination therapy may also help improve efficacy.

[0217] It should be understood that any treatment method corresponding to any use of the compositions described herein for treating osteoarthritis is intended to be included within the scope of this invention. Similarly, any use described herein can also be expressed as: the composition being used to prepare a medicament for any osteoarthritis treatment described herein. This invention covers all the corresponding uses described above for preparing a medicament.

[0218] It should also be understood that the compositions of the present invention also include the aggregate compositions of the present invention described herein. In a preferred embodiment of any use and treatment method described herein, the composition administered to the subject is an aggregate composition described herein, i.e., a composition comprising aggregates formed between anionic lipid compositions (e.g., liposomes) and polycationic aggregaters.

[0219] Dosage and administration regimen The dosage of the pharmaceutically active agent prepared in conjunction with the compositions of the present invention depends on the functional dose and the duration of sustained release provided by the reservoir composition formed after administration. Typically, the dose prepared for a particular pharmaceutically active agent is approximately the product of a normal single dose and a multiple of the expected duration of action provided by the formulation. Obviously, adjustments to this dosage are necessary to account for potential adverse reactions from high doses at the beginning of treatment; therefore, this dosage is typically the maximum dose employed. In any case, a suitable specific dosage can be determined through appropriate experimentation.

[0220] The compositions of the present invention are administered intra-articularly. The duration of treatment depends on the nature of the disease or disorder being treated. Where appropriate, intra-articular administration should continue until the condition is cured and / or the symptoms are reduced or eliminated. The upper limit of the treatment period can be determined by a physician. For example, the compositions can be administered intra-articularly once a week to once a year, preferably once every two weeks to once every six months, and more preferably once a month to once a quarter.

[0221] The frequency of administration of the compositions of the present invention depends on several factors that can be determined by a physician, such as the severity of the condition, the responsiveness to initial treatment, and the specific condition being treated. For example, the composition may be administered intra-articularly once a week. In some embodiments, the composition may be administered intra-articularly more than once a week. For example, the composition may be administered intra-articularly once or twice daily. In some embodiments, the composition may be administered intra-articularly once daily. In some embodiments, the composition may be administered intra-articularly every other day. In some embodiments, the composition may be administered intra-articularly once a month. In some embodiments, the composition may be administered intra-articularly once every six weeks. In some embodiments, the composition may be administered intra-articularly once every three months. In some embodiments, the composition may be administered intra-articularly once every four months. In some embodiments, the composition may be administered intra-articularly twice a year. In some embodiments, the composition may be administered intra-articularly once a year.

[0222] The dosage of the pharmaceutically active agent administered with the composition of the present invention may vary due to a variety of factors, including, for example, the age, weight and sex of the animal or human suffering from the disease, the severity of the disease, and the selected frequency of administration.

[0223] The appropriate dosage for intra-articular administration can be determined by a physician. The dosage should effectively improve the lubrication of the treated joint, i.e., reduce friction between the cartilages that make up the joint; such improvement can be demonstrated through clinical trials and / or manifested as an improvement in the health status of the subject receiving the treatment (e.g., reduced pain and improved range of motion in the affected joint). Effective doses are typically determined in appropriately designed clinical trials (dose range studies), and those skilled in the art know how to properly conduct such trials to determine effective doses.

[0224] route of administration The compositions of the present invention can be administered to a subject via any suitable route of administration, whichever is appropriate for the condition to be treated and the pharmaceutically active agent used.

[0225] Routes of administration include, but are not limited to, parenteral administration, such as injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrasheathal, spinal, intracapsular, subcapsular, orbital, intraperitoneal, intratracheal, subcutaneous (intradermal suture layer), intra-articular, subarachnoid, and intrasternal administration. The composition administered via the above routes of administration can be any composition described herein. In some embodiments, the composition comprises: a composition comprising anionic liposomes; and a cationic dendritic polymer aggregator, wherein the cationic dendritic polymer aggregator comprises one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of any sequence in SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of any sequence in SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of any sequence in SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of any sequence in SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (composed of any sequence in SEQ ID NO: 67). (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of any sequence from SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (composed of any sequence from SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (composed of any sequence from SEQ ID NO: 79 to 82), and the aggregated composition may also contain a pharmaceutically active agent. In a preferred embodiment, the composition comprises: a composition comprising anionic liposomes; and a cationic dendritic polymer aggregater, wherein the cationic dendritic polymer aggregater comprises one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of any sequence in SEQ ID NO: 1 to 4); or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of any sequence in SEQ ID NO: 5 to 8), wherein the aggregate composition may further comprise a pharmaceutically active agent. Preferably, the composition comprises aggregates formed by the cationic dendritic polymer aggregater and two or more of the anionic liposomes.

[0226] In some embodiments, the compositions of the present invention are parenteral administration compositions. Therefore, in some embodiments, the compositions of the present invention are injectable formulations and can be administered to subjects subcutaneously, intramuscularly, intra-articularly, or intradermally. Preferably, the compositions are administered to subjects intra-articularly.

[0227] In some embodiments, the compositions of the present invention are administered arthroscopically.

[0228] Therefore, the compositions of the present invention are liquid formulations, suitable as injectable formulations, and particularly suitable as intra-articular injection formulations. Thus, in some embodiments, the compositions of the present invention are liquid formulations, such as gels, or sterile aqueous solutions or sterile oily solutions.

[0229] In some embodiments, the compositions of the present invention are suitable for intra-articular administration, and therefore the viscosity of the composition is at least about 0.01 Pa·s or at least about 0.1 Pa·s, preferably at least about 1 Pa·s, and more preferably at most about 10 Pa·s. For example, the viscosity of the composition is at least about 0.5 Pa·s to about 8 Pa·s. The viscosity can be measured using a viscometer at a shear rate of 0.1 / s.

[0230] In some embodiments, the compositions of the present invention are suitable for intra-articular injection using needles of gauges 20 to 30. For example, needles of gauges 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0231] The advantages of intra-articular administration are that the compositions of the present invention can be directly administered into the synovial fluid near the articular cartilage, and the compositions can also diffuse into the cartilage tissue therefrom. Therefore, the compositions of the present invention can also be directly injected into the joint space, thereby exerting their effects directly at the intended site of action, such as on the articular cartilage.

[0232] The compositions of the present invention can be prepared for administration via an intra-articular route. For example, the compositions may further comprise aqueous and non-aqueous sterile injectable solutions containing antioxidants, buffers, antibacterial agents, and solutes, making the compositions isotonic with the synovial fluid of the subject being treated; and may also comprise aqueous and non-aqueous sterile suspensions, which may contain a suspending medium and a thickener. The compositions may be provided in single-dose or multi-dose containers, such as sealed ampoules or vials, and may be stored in a lyophilized state, requiring only the addition of a sterile carrier fluid (e.g., water for injection) immediately before use. Injectable solutions and suspensions prepared according to the compositions can be formulated from sterile powders, granules, or tablets.

[0233] Subjects The compositions of the present invention are suitable for use in treating subjects affected by any of the diseases or conditions described herein. The compositions of the present invention are preferably suitable for intra-articular treatment of subjects affected by the diseases or conditions described herein, wherein the composition is applied intra-articularly to the subject's joint.

[0234] In some embodiments, the subject may be a warm-blooded mammal. In some specific embodiments, the subject is a human. The subject may be an adult (18 years or older). The subject may be a child under 18 years of age. The age of a pediatric subject may be between 2 and 4 years of age. The age of a pediatric subject may be between 5 and 10 years of age. The age of a pediatric subject may be between 11 and 18 years of age.

[0235] In some embodiments, the subject may be an animal. In some embodiments, the compositions of the present invention can be used as veterinary products for topical treatment of animals. In some embodiments, the compositions of the present invention are used for topical treatment of diseases and conditions in commercial animals such as livestock (e.g., cattle, sheep, etc.). In other embodiments, the compositions of the present invention can be used for topical treatment of diseases or conditions in companion animals such as cats, dogs, and horses.

[0236] Preparation method The compositions of the present invention can be prepared by mixing an anionic lipid composition (such as liposomes) with a polycationic aggregator, as shown in the examples. In some embodiments, the compositions of the present invention can be prepared by mixing an aqueous solution of anionic liposomes with a solution of a polycationic aggregator (e.g., dendritic polymers such as Dendri1 or Dendri4). Mixing is preferably carried out at a pH of about 7.0 to about 8.0 (e.g., about 7.4). Mixing may be carried out in the presence of a suitable buffer (e.g., HEPES) to maintain the pH at the desired level during mixing and aggregation of the anionic lipids with the polycationic aggregator. Mixing may be carried out in the presence of sodium chloride to promote aggregation. The concentration of sodium chloride in the mixture is preferably about 100 mM to about 150 mM, for example, about 135 mM to about 145 mM. Mixing of the anionic lipids and the polycationic aggregator is preferably carried out at ambient temperature, for example, about 20 °C to about 25 °C. The aggregation process can be monitored by assessing the turbidity of the mixture and / or by optical microscopy.

[0237] In a preferred embodiment, the composition of the present invention is sterile. After the anionic lipid composition (such as liposomes) is mixed with the polycationic aggregator solution to form an aggregate solution, the composition can be sterilized using conventional methods, such as terminal sterilization using radiation (such as gamma rays, electron beam sterilization, or ultraviolet light). The aggregate composition can be added to a suitable container (such as a glass bottle) and sealed and sterilized. Alternatively, a sterile solution can be prepared before mixing the anionic lipid composition (such as liposomes) and the polycationic aggregator. The anionic lipid composition solution and the polycationic aggregator solution are then mixed under sterile conditions to obtain the final sterile aggregate composition. The anionic lipid composition solution and the polycationic aggregator solution can be prepared into sterile solutions by recognized methods, such as by sterile filtration of each solution.

[0238] Reagent test kit The kits provided in this article include: a) A first container containing an anionic lipid composition; b) A second container containing a polycationic aggregator; and c) Instructions for mixing a) with b) to provide a composition for the purposes defined herein.

[0239] In some embodiments, the first container also includes a pharmaceutically active agent bound to the anionic lipid composition. Therefore, the kit may include... a) A first container containing an anionic lipid composition, wherein the anionic lipid composition contains a pharmaceutically active agent; b) A second container containing a polycationic aggregator; and c) Instructions for mixing a) with b) to provide a composition for the purposes defined herein.

[0240] In some embodiments, the anionic lipid composition is selected from any of the anionic lipid compositions described herein. For example, the anionic lipid composition may include liposomes. Thus, the first container may contain an anionic liposome composition, wherein the liposomes encapsulate a pharmaceutically active agent.

[0241] In some embodiments, the polycationic agglomerator is selected from any of the polycationic agglomerators described herein. For example, the polycationic agglomerator may be a pharmaceutically active agent. In other embodiments, the polycationic agglomerator may be a dendritic polymer as described herein, such as a peptide dendritic polymer. In some embodiments, the polycationic agglomerator may be a dendritic polymer comprising one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 67 to 70). Composed of SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82). In a preferred embodiment, the polycationic agglomerator may be a dendritic polymer containing one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4) or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8). The polycationic agglomerant may be selected from the following group: i-PAMAM (reverse polyamide amine), polyethyleneimine (branched or linear), tobramycin, glatiramer acetate, polymyxin B, polylysine, apidaecin 1b, LL-37, polyarginine, or Walk11.3. Therefore, the polycationic agglomerant may be: (i) a cationic dendritic polymer agglomerant containing one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); or (ii) A polycationic aggregator selected from the group consisting of inverse poly(amidoamine) (i-PAMAM), LL-37 and polyarginine.

[0242] In some embodiments, the anionic lipid composition in the first container is provided as a lyophilized mixture. In some embodiments, the anionic lipid composition and the pharmaceutically active agent in the first container are provided as a lyophilized mixture. Similarly, the polycationic aggregate can also be lyophilized. The lyophilized mixture can be obtained by methods known to those skilled in the art. Therefore, the kit may contain instructions for hydrating the lyophilized mixture to obtain the composition of the present invention. The lyophilized components in the kit are preferably hydrated with sterile water or physiological saline (such as sterile phosphate-buffered saline).

[0243] In a further embodiment, a kit is also provided, which includes a) A first container containing anionic liposomes; (b) A second container comprising a cationic dendritic polymer aggregator, wherein the cationic dendritic polymer aggregator comprises one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70). Composition); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82); and c) Combine a) with b) to provide a description of the aggregated compositions as defined herein.

[0244] In some embodiments, the first container further includes a pharmaceutically active agent incorporated into anionic liposomes, for example, the pharmaceutically active agent being encapsulated in anionic liposomes.

[0245] In some embodiments, the anionic liposomes are selected from any anionic liposomes described herein. In some embodiments, the cationic dendritic polymer aggregator comprises one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4) or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8). In some embodiments, the cationic dendritic polymer aggregator comprises the following linear symbol: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4). In some embodiments, the cationic dendritic polymer aggregator comprises the following linear symbol: (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8). In some embodiments, the cationic dendritic polymer aggregator comprises the following linear notation: (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86). In some embodiments, the cationic dendritic polymer aggregator comprises the following linear notation: (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90). In some embodiments, the cationic dendritic polymer aggregator comprises the following linear notation: (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70). In some embodiments, the cationic dendritic polymer aggregator comprises the following linear notation: (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 71 to 74). In some embodiments, the cationic dendritic polymer aggregator comprises the following linear notation: (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78). In some embodiments, the cationic dendritic polymer aggregator comprises the following linear notation: (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82).

[0246] In some embodiments, the anionic liposomes in the first container are provided as a lyophilized mixture. In some embodiments, the anionic liposomes and the pharmaceutically active agent in the first container are provided as a lyophilized mixture. The lyophilized mixture can be obtained by methods known to those skilled in the art. Therefore, the kit may contain instructions for hydrating the lyophilized mixture to obtain the aggregated composition of the present invention.

[0247] In some embodiments, the compositions of the present invention can be prepared by connecting a first container and a second container to mix the anionic lipid composition and the polycationic aggregator together to form an aggregated composition. For example, the first container may include a first syringe containing an aqueous solution of the anionic lipid composition, and the second container may include a second syringe containing an aqueous solution of the polycationic aggregator. The mixing of the two solutions can be achieved by connecting the first and second syringes and circulating the mixture from one syringe to the other, thereby forming an aggregated composition. Alternatively, in a dual-barrel syringe system, the first and second containers can be different barrels. The mixing method involves injecting the contents of each barrel into a common outlet channel that is fluidly connected to both barrels, causing the contents of each barrel to mix with each other in the outlet channel to form an aggregated mixture.

[0248] Example Unless otherwise specified, solvents, reagents, and starting materials were purchased from commercial suppliers and used exactly as received. Unless otherwise specified, all reactions were carried out at room temperature. Starting materials were purchased commercially or synthesized according to the methods described herein or literature procedures.

[0249] Abbreviations ALs: aggregated liposomes COF: Coefficient of friction DendriXs - Dendri1 and / or Dendri4 DendriXAL - Dendritic Polymer Aggregates of Liposomes DIC: N,N'-Diisopropylcarbodiimide DiD: 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindoledicarbocyanine, 4-Chlorobenzenesulfonate DLS: Dynamic Light Scattering DMEM: Duchenne Modified Eagle Culture Medium DMF: N,N-Dimethylformamide DODT: 2,2'-(ethylenedioxy)diethylthiol DPBS: Duchenne phosphate-buffered saline DPPC: 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine DSPG: Sodium salt of 1,2-distearyl-sn-glycerol-3-phosphate-(10-rac-glycerol) EDTA: Ethylenediaminetetraacetic acid FBS: Fetal bovine serum FCS: Fetal bovine serum HEPES: 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid min: minutes HPLC: High Performance Liquid Chromatography LC-MS: Liquid Chromatography-Mass Spectrometry MeCN: Acetonitrile OA: Osteoarthritis OASFs: Synovial fibroblasts in osteoarthritis PBS: Phosphate-buffered saline PDI: Polydispersity PI: Propidium iodide RP-HPLC: Reversed-phase high-performance liquid chromatography SF: Synovial fibroblasts TIS: Triisopropylsilane TEM: Transmission electron microscopy TFA: Trifluoroacetic acid Material Phospholipids 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) and sodium 1,2-distearyl-sn-glycerol-3-phosphate (10-rac-glycerol) (DSPG) were supplied by Lipoid (Ludwigshafen, Germany). HEPES solution (1 M) was obtained from Carl Roth® (Karlsruhe, Germany). 1,1'-bis(octadecyl)-3,3,3',3'-tetramethylindole dicarbocyanine, 4-chlorobenzenesulfonate (DiD, catalog number: D7757) was supplied by Thermo Fisher Scientific™ (Waltham, Massachusetts, USA). DMF (N,N-dimethylformamide) was purchased from Thommen-Furler AG (Switzerland), Oxyma Pure (ethyl hydroxyiminocyanocyanate) from SENN AG (Switzerland), DIC (N,N'-diisopropylcarbodiimide) from Iris BIOTECH GMBH (Germany), and piperidine from Acros Organics (Thermo Fisher Scientific™, USA). DODT (2,2'-(ethylenedioxy)diethylthiol) was purchased from Merck® (Germany), and triisopropylsilane and trifluoroacetic acid were purchased from Fluorochem Ltd (UK). All amino acids were supplied by Shanghai Shengpu Zetai Pharmaceutical Technology Co., Ltd. Chemical reagents were used according to the provided standards, and solvents were industrial grade. Amino acids were used as the following derivatives: Fmoc-Lys(Boc)-OH, Fmoc-Lys(Fmoc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Cys(Trt)-OH. Rink Amide AM LL resin was purchased from Novabiochem® (Merck, Germany). Ala-Wang resin was purchased from Iris BIOTECH GMBH. Porcine knee cartilage was obtained from a regional slaughterhouse in München-Buchssee, Switzerland. Chloroform and methanol were purchased from Fisher Scientific (Schwert, Germany). Water with a resistivity of 18.2 MΩ·cm was produced using the Barnstead Smart2 pure water system from Thermo Scientific™ (Pittsburgh, USA).

[0250] Analytical methods Reversed-phase high-performance liquid chromatography (RP-HPLC) Quantification of dendritic polymers was performed using RP-HPLC. Analytical RP-HPLC was conducted using an Ultimate 3000 Fast Separation LC-MS system (DAD-3000RS diode array detector) and a Dionex Acclaim® RSLC 120 C18 column (2.2 µm, 120 Å, 3 × 50 mm, flow rate 1.2 mL / min). Data recording and processing were performed using Dionex™ Chromeleon™ Management System version 6.80 (analytical RP-HPLC). All RP-HPLC analyses used HPLC-grade acetonitrile and Milli-Q deionized water. The eluent solutions were: A: Milli-Q deionized water containing 0.05% trans fatty acids; D: Milli-Q deionized water / acetonitrile containing 0.05% trans fatty acids (10:90, v / v). Preparative RP-HPLC was performed using a Waters® automated Prep LC control system, which includes four modules: a Waters 2489 UV / Vis detector, a Waters 2545 pump, a Waters® FractionCollector III, and a Waters® 2707 autosampler. A Dr. Maisch® GmbH Reprospher® column (C18-DE, 100 × 30 mm, 5 μm particle size, 100 Å pore size, 40 mL / min) was used. Compounds were detected by UV absorption at 214 nm using a Waters® 248 TunableAbsorbance Detector. Data logging and processing were performed using Waters® ChromScope™ version 1.40 from Waters Corporation®. Elution solutions were: A: MilliQ deionized water containing 0.1% trans fatty acids; D: MilliQ deionized water / acetonitrile (10:90, v / v) containing 0.1% trans fatty acids. Mass spectra recorded on a Thermo Scientific™ LTQ OrbitrapXL™.

[0251] Dynamic light scattering (DLS) and zeta potential Liposomes were characterized using DLS and zeta potential measurements. The mean hydrodynamic diameter and polydispersity index (PDI) were measured using a Littizer 500 dynamic light scattering (DLS) analyzer (Anton Paar, Austria) at 25 °C, a 175° backscattering angle, and a 658 nm laser. The zeta potential was assessed using laser Doppler microelectrophoresis with the same instrument and Omega cuvettes (Anton Paar, Austria). The stability of the liposomes was evaluated over 16 weeks at 4 °C. All tests were performed within 24 hours of extrusion and followed by formulation.

[0252] Microscopic imaging of liposomes and DendriXAL To assess morphology, liposomes and DendriXALs were imaged using fluorescence and cryo-TEM (cryo-TEM). For fluorescence microscopy preparation, the lipid films were stained with the non-exchangeable lipophilic dye DiD, and liposomes and DendriXALs were prepared using the same method described previously, while being shielded from light. Then, 20 µL of the formulation was placed on a glass slide, covered with a glass coverslip, and images were captured using an inverted fluorescence microscope (Nikon Eclipse-Ti, Canada) through a Tx red filter. For cryo-TEM imaging, 6–8 µL of each sample was added to a gold grid covered with a porous gold membrane (UltrAuFoil® 2 / 1, Quantifoil® MicroTools GmbH, Jena, Germany). Excess liquid was automatically aspirated between two sheets of filter paper or simply removed from the back of the grid. The samples were then rapidly frozen in liquid ethane (cooled to 180 °C) in a Cryobox™ (Carl Zeiss NTS® GmbH, Oberkochen, Germany). Excess ethane was removed with filter paper, and the samples were immediately transferred to a pre-cooled cryo-electron microscope (Philips CM 120, Eindhoven, Netherlands) using a Gatan™ 626 cryo-transfer holder (Gatan®, Pleasantton, USA), which operates at 120 kV under low-dose conditions. Images were recorded using a 2k CMOS camera (F216, TVIPS, Gowting, Germany), with four images recorded and averaged into one image to minimize noise.

[0253] Flow cytometry The uptake of aggregated liposomes (such as Dendri4Als) and liposomes by RAW264.7 cells was analyzed using an imaging flow cytometer (ImageStream® X Mark II, Cytek Biosciences®, USA).

[0254] First, the cell suspension was filtered to remove aggregates. Dead cells were then labeled with 100 ng / mL propidium iodide (PI, BioLegend®, USA), followed by labeling of all cells with 5 µg / mL Hoechst reagent (Thermofisher Scientific™, USA). The mixture was then incubated at 37 °C for 3 minutes. The suspension was then analyzed using flow cytometry, measuring at least 5000 cells per sample. A screening process was then performed to select single cells, focused cells, and live cells, ensuring that the number of live cells in all samples remained above 500. DiD intensity was analyzed using IDEAS® 6.3 software (Amnis Corporation®, USA), generating histograms and statistical data. Cell fluorescence intensity was normalized to count values. The signal from DMEM-treated cells was subtracted from liposome and aggregated liposome (e.g., Dendri4AL) samples, and the aggregated liposome (e.g., Dendri4AL) samples were further normalized to the corresponding liposome positive controls.

[0255] Fluorescence stereomicroscopy imaging Fluorescence stereomicroscopy imaging (Zeiss AxioZoom® V16, Germany) was used to analyze the cartilage surface in cartilage preservation studies. Images were captured at 10x and 50x magnification using GFP and Cy5 filters, respectively. Fluorescence intensity was analyzed using Zeiss ZEN2.5 software, focusing on fixed areas on the cartilage surface.

[0256] tribology The frictional behavior of self-made cartilage was studied using a UMT-2 tribometer (Bruker®, USA) in linear reciprocating mode, with the presence of a measurement sample. The corresponding components consisted of two segments of porcine cartilage, which were bonded to the upper and lower parts of the tribometer shortly before testing. In each experiment, the upper sample (10x10 mm) was in contact with the lower sample (30x15 mm) under a 1 N load and moved for 10 minutes at a frequency of 1 Hz with a stroke length of 2 mm. During the test, the sample pairs were fully immersed in lubricant. All tests were conducted at a constant temperature of 20 °C and a data acquisition frequency of 500 Hz. The representative coefficient of friction (COF) for each test was determined based on the raw data of lateral and normal forces, taking the average of each friction loop, considering only the middle 90% of the friction loop to avoid transient effects associated with the ends of the stroke. Furthermore, the steady-state frictional behavior of the self-made contact was considered, ignoring the first 20% of the loop to eliminate potential break-in transients.

[0257] Statistical analysis Unless otherwise stated, all experiments were performed at least three times. The values ​​listed are mean ± standard deviation. Microsoft Excel was generally used for calculations, while GraphPad Prism 9.5 was used for generating graphs, performing one-way ANOVA, and conducting Tukey's test (multiple comparisons) and unpaired t-tests (single comparisons) (unless otherwise stated).

[0258] Methods - Preparation of dendritic polymers The linear notation for Dendri1 is: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (consisting of SEQ ID NO: 1 to 4).

[0259] The linear notation for Dendri4 is: (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (consisting of SEQ ID NO: 5 to 8).

[0260] The dendri1 and Dendri4 (DendriXs) dendritic polymers were synthesized using the standard 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis method at 60°C under nitrogen flow, identical to previously reported methods.13 The branch points consist of Fmoc-Lys(Fmoc)-OH, and deprotection of Fmoc yields two free amino groups (main chain and side chain).

[0261] synthesis First, 400 mg of resin (Dendri1: Rink amide, 0.29 mmol / g; Dendri4: Ala-Wang, 0.32 mmol / g) was swelled in DMF for 10 min. The Fmoc group was then double-deprotected in DMF using 20% ​​v / v piperidine solution for 1 min and 4 min respectively. After deprotection, the resin was washed five times with DMF (5 x 8 mL). Bicoupling was performed on first-generation G0 and G1 (8 min each time, 2 times); tetracoupling was performed on second-generation G2 (8 min each time, 4 times); and heptacoupling was performed on third-generation G3 (8 min each time, 7 times). Each coupling reaction was carried out in DMF using 3 mL of 0.2 M amino acid, 2 mL of 0.8 M DIC, and 1.5 mL of 0.8 M Oxyma solution. Between coupling reactions, the resin was washed twice with 8 mL of DMF, and three times after the final coupling. Deprotection of all Fmoc groups was performed as described above. After the final Fmoc deprotection, the resin was washed three times with DMF at room temperature, followed by three times with methanol.

[0262] break away Dendri1 was then removed from the resin using 7 mL of a TFA / TIS / DODT / H2O mixture (94 / 2.5 / 2.5 / 1, v / v / v / v), and Dendri4 was removed using TFA / TIS / H2O (94 / 5 / 1, v / v / v). Subsequently, the peptide solution was mixed with 25 mL of cold tert-butyl methyl ether, centrifuged for 10 minutes at 3500 rpm, evaporated, and dried under argon for purification.

[0263] purification The dried crude product was dissolved in a water / MeCN mixture, filtered (pore size 0.22 μm), and then purified by preparative RP-HPLC using the 60-minute gradient described above, from 100% solvent A to 100% solvent D. The collected fractions were analyzed by analytical LC-MS. The peptides were lyophilized as a foamy white solid. Yields were calculated as TFA salts.

[0264] Quantitative analysis of dendritic polymers The quantification of the obtained dendritic polymer was performed as described above using analytical RP-HPLC combined with LC-MS.

[0265] Methods - Liposome Preparation synthesis Liposomes containing 25 mol% DSPG and varying amounts of DPPC / cholesterol were prepared using a thin-film hydration technique. The lipid stock solution in a chloroform / MeOH mixture (75 / 25 v / v) was appropriately measured and dried in a glass vial under a gentle nitrogen flow, followed by overnight vacuum incubation in a desiccator to remove residual solvent. The lipids were hydrated with 20 mM HEPES buffer (pH 7.4), heated to 70°C, and mixed to prepare vesicles with a final concentration of 20 mM. These vesicles were then subjected to six freeze-thaw cycles and extruded ten times at 70°C using a LIPEX® extruder (Evonik®, Canada) through a 200 nm polycarbonate membrane (Sterlitech® Corporation, USA). Characterization The resulting liposomes were characterized as described above. Briefly, the mean hydrodynamic diameter and polydispersity (PDI) of the formed vesicles were determined using dynamic light scattering (DLS) with a Litesizer 500 instrument (Anton Paar®, Austria) at a backscattering angle of 175°, a laser wavelength of 658 nm, and a temperature of 25°C. Furthermore, the zeta potential was determined by laser Doppler microelectrophoresis using the same equipment and Omega cuvettes (Anton Paar®, Austria).

[0266] Methods - In vivo studies Cell culture Synovial fibroblasts (SFs) were obtained from four OA patients and cultured in 25 cm² flasks on 6-well clear Corning® plates (USA) or 96-well black clear plates (Thermo Fisher Scientific™, USA). Cells were cultured at 37 °C in a 5% CO2-enriched humidified atmosphere using DuPont Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FCS), 50 U / mL penicillin / streptomycin, 2 mM L-glutamine, 10 mM HEPES, and 0.2% amphotericin B (all from Thermo Fisher Scientific™, USA), according to standard protocols. After confluence, OASFs from passages 4 to 6 were used for experiments. Simultaneously, semi-attached mouse mononuclear macrophages (RAW264.7) were cultured under the same conditions as the OASFs. As previously reported, 1% v / v penicillin / streptomycin mixture, 1% v / v L-glutamine (200 nM), and 10% v / v FCS were added to DMEM (4.5 g / L glucose and phenol red). When the cells reached 70-80% confluence, they were harvested with a cell scraper and then passaged for further culture.

[0267] Toxicity of dendritic polymers OASFSs were quantified using Countess™ 3 FL (Thermo Fisher Scientific™, USA) and trypan blue, and then seeded into 96-well plates at a density of 5000 cells per well. After overnight incubation, cells were cultured for 48 hours under various conditions in 200 μL of medium. After incubation, cell viability was determined using the Cell Counting Kit-8 (CCK-8) according to the manufacturer's (Merck®, Germany) instructions. Briefly, medium was aspirated, cells were washed with DPBS (Thermo Fisher Scientific™, USA), and then stained with 100 μL of 10-fold diluted CCK-8. Cells were incubated at 37 °C for 2 hours, and then absorbance was measured at 450 nm using a microplate reader (BioTek Instruments®, USA).

[0268] Gene expression In the gene expression study, cells were seeded as described in the toxicity assay. To stimulate the fibrosis response, 10 ng / mL TGFβ was added to the culture medium along with the test conditions. After 48 hours of incubation, cells were lysed, and RNA was extracted using the Quick-RNA™ Microprep Kit (Zymo Research®, USA) and digested with DNase I on a column according to the manufacturer's instructions. RNA purity and quantity were assessed by measuring the OD ratio at 260 and 280 nm using a Nanodrop (Thermo Fisher Scientific™). Subsequently, the RNA was reverse transcribed and subjected to SYBR® Green Real-Time PCR (primers: αSMA forward primer: 5' GAC AAT GGC TCTGGG CTC TGT AA 3' (SEQ ID NO: 59), reverse primer: 5' ATG CCA TGT TCT ATC GGG TAC TT 3' (SEQ ID NO: 60); Col1A1 forward primer: 5' CAG CCG CTT CACCTA CAG C 3' (SEQ ID NO: 61), reverse primer: 5' TTT TGT ATTCAA TCA CTG TCT TGC C 3' (SEQ ID NO: 62); Col3A1 forward primer: 5' GGA CCT CCT GGTGCT ATA GGT 3' (SEQ ID NO: 63), reverse primer: 5' CGG GTC TAC CTGATT CTC CAT 3' (SEQ ID NO: 64)). Data were analyzed using the comparative CT method and expressed as 2−ΔΔCT (i.e., x times). As previously described, RPLP0 was used as an internal reference gene for sample normalization (forward primer: 5'-GCG TCC TCG TGG AAGTGA CATCG 3' (SEQ ID NO: 65), reverse primer: 5'-TCA GGG ATT GCCACG CAG GG 3' (SEQ ID NO: 66)).

[0269] Table 2 - Primer Table Liposome aggregation and liposome uptake RAW264.7 cells (2.4 million) were initially incubated in T25 flasks and left to stand overnight. The next day, the cell culture medium was removed, and the cells were washed with PBS. They were then incubated for 3 hours with freshly prepared fluorescently labeled DiD liposomes as positive controls and DiD aggregated liposomes (such as DiD-Denri4ALs). DMEM was used as a negative control. Samples were prepared in phenol red-free medium with fetal bovine serum (FCS) content reduced to 2% (v / v), following previously reported protocols. After incubation, the cells were washed twice with PBS and then collected using a cell scraper. Subsequently, the cells were centrifuged at 500 g for 3 min and resuspended in 150 µL buffer containing 0.02% EDTA, 2% v / v FCS, 1% v / v L-glutamine (200 nM), and 1 mM pyruvate, and incubated at 4°C. The resuspended cells were stored on ice until analysis was performed using flow cytometry, as described above.

[0270] Cartilage preservation Porcine cartilage explants were cut into 0.5 x 0.5 cm pieces, washed with PBS, and placed in 12-well microplates. The samples were then immersed in 1.5 mL of 20 mM HEPES, Dendri1AL (50 mg / mL Dendri1), Dendri4AL (50 mg / mL Dendri4), and liposome solutions containing the same concentrations as DendriXALs. All liposome formulations were labeled with 0.05 mol% lipophilic DiD dye. After incubation at 37°C for 24 hours, the samples were washed with 1.5 mL of PBS. The cartilage surface was analyzed using fluorescence stereomicroscopy, as described above.

[0271] LD50 assessment Cell Culture: Raw264.7 cells were cultured in Dulbecco revised Eagle medium (DMEM, high glucose) (11965084; Gibco) supplemented with 10% fetal bovine serum (FCS) (F7524-500 mL; Sigma Aldrich), penicillin 100 U / mL, and streptomycin 0.1 mg / mL (15140122; Gibco). Cells were passaged using a cell scraper at their exponential growth phase, when cell confluence was 70-90%, and all cells were passages 9-19. Composite toxicity assays were performed in three biological replicates, each replicate three times. Specific LD50 values ​​of the composite were determined in all three replicates.

[0272] LD50 Assessment: Cells were seeded into 96-well plates one day prior to treatment (5000 cells per well, 100 µL of medium) and cultured overnight for attachment. Cells were then treated using a tandem dilution method in DMEM (phenol red-free, 2% FBS-free) at decreasing doses of the compound (stock solution 2 mM, Hepes buffer), from 200 µg / mL to 1.5 µg / mL for compound treatment, and for Zn²⁺ concentrations from 10 mM to 1.2 nM, or with liposome concentrations from 5 mM to 0.1 mM, followed by adjustment of the compound concentration. After 24 hours of incubation, the medium was removed, cells were washed once with PBS, and 100 µL of 10% AlamarBlue solution (in DMEM, phenol red-free, 2% FBS-free) was added to the wells. The plates were then incubated for another 2.5 hours. Fluorescence was measured at 590 nm using a Molecular Devices SpectraMax i3x microplate reader with an excitation wavelength of 545 nm to evaluate the plates. The results were then normalized to the fluorescence of the carrier to determine the percentage of viable cells. If necessary, these results were further evaluated in Prism to determine the LD50 of the compound.

[0273] Example 1 – Preparation and Characterization of Dendritic Polymer Aggregates (DendriXAL) Aggregated liposomes (ALs) were prepared by quadrupling 0.4 mM liposomes with dendritic polymer solutions (DendriXs - Dendri1 and Dendri4) at different concentrations in 20 mM HEPES, followed by gentle stirring for 5 minutes. The generated DendriXALs were evaluated using a microplate reader as previously reported. Briefly, 50 µL of liposomes at an initial concentration of 0.4 mM were mixed with 150 µL of DendriXs at different concentrations and placed in 96-well microplates with clear, flat quartz bottoms (Hellma® GmbH & Co. KG, Germany). After gently stirring the mixture for 5 minutes, the optical density was measured at 450 nm using an Infinite M Pro200F-PlexNano microplate reader (Tecan®, Switzerland). The zeta potential of DendriXALs was measured using a Litesizer® 500 instrument, following the same method as for liposomes. In the process involving the aggregation of salts, DendriXs were first mixed with different concentrations of NaCl, and then DendriXALs were prepared using the same method as described above. Subsequently, the optical density was measured according to the aforementioned method.

[0274] To investigate whether the aggregation process induces a burst release of the encapsulated model drug, rapamycin was added to liposomes at a lipid-to-drug ratio of 30:1, following the previously described method. The in vitro release profile was evaluated over seven days using a custom-designed dialysis apparatus and a 10% ethanol solution (in ultrapure water). This method ensured drug stability and maintained sedimentation conditions without affecting the liposome composition.

[0275] Example 2 – Analysis of polycationic aggregates Aggregation studies were conducted on peptide dendritic polymers Dendri1 and Dendri4 (DendriXs) by measuring changes in optical density and zeta potential under different charge ratios. To calculate these charge ratios, the surface area of ​​the liposomes was first determined based on their average diameter. Then, the number of phospholipid head groups on the surface was estimated by utilizing the average surface area of ​​DPPC molecules and weighting it according to the proportion of DSPG molecules in the formulation (i.e., 25 mol%). The following equation was used to calculate the charge ratios in Figure 1: (where N = quantity).

[0276] Although the Dendri1 and Dendri4 molecules have different numbers of positive charges (12 for Dendri1 and 17 for Dendri4), the polymerization curves depicted in Figure 1 are very similar. A sharp increase in optical density was observed at a charge ratio of 1, which is closely related to the change in zeta potential across the x-axis. Similar phenomena have been observed with divalent cations in previous studies. The observed phenomena can be explained as follows: as the negative charge is neutralized on the anionic liposomes, the increase in positively charged dendritic polymers gradually reduces the repulsive force between colloidal particles until the total charge reaches zero, at which point the system becomes unstable. The decrease in optical density after the aggregation peak may be due to the formation of water-soluble "dendritic structures" caused by the increased charge, a phenomenon previously observed in poly(amidoamine) (PAMAM) dendritic polymers and anionic liposomes. Figure 2 shows the aggregation in the presence of salt, indicating that aggregation is most pronounced at physiological concentrations of NaCl. Conversely, salt concentrations exceeding 150 mM NaCl lead to reduced aggregation.

[0277] DendriXs were then evaluated in an in vitro fibrosis model using human OASFSs. The results in Figure 3 show that DendriXs significantly reduced the expression of α-smooth muscle actin (αSMA) gene, while the reduction in type I collagen (Col1A1) expression was significant only in Dendri4.

[0278] To assess the toxicity of DendriXs and DendriXALs, OASFs were incubated with DendriXs and DendriXALs containing 50 µg / mL DendriXs and corresponding concentrations of liposomes for 48 hours. Cell viability was then assessed using a CCK8 assay kit.

[0279] The results in Figure 4 show that no significant toxicity was induced under the test conditions, and the lowest cell viability was observed in Dendri4AL containing 50 µg / mL Dendri4. Overall, these results indicate that DendriXs are effective aggregators capable of reducing the expression of fibrosis markers in human OASFSs and alleviating the inflammatory response previously reported in monocytes, both of which are associated with the treatment of pathologies such as osteoarthritis. Furthermore, the low toxicity of this system unlocks the previously limited potential of aggregated liposomes in the treatment of osteoarthritis.

[0280] Example 3: Visualization of DendriXAL The aggregates were imaged using cryo-electron microscopy (cryoTEM), and representative images are shown in Figure 5. The images show that the liposomes are mainly monolayered and nanoscale, consistent with DLS data. Upon addition of DendriXs to the system, clear aggregation was observed, with the membranes tightly aggregated and bound by DendriXs. This is evident from the occasional bends in the minimum distance between the membranes, which corresponds to the size of the DendriXs, particularly noticeable in the Dendri1AL images. No significant penetration of DendriXs into the liposome core was observed, which could otherwise induce a sudden release of the encapsulated material. This was confirmed by in vitro rapamycin release studies, in which the drug was encapsulated in liposomes at a lipid-to-drug molar ratio of 30:1. Figure 6 shows no significant difference in release rates among ordinary liposomes, Dendri1ALs, and Dendri4ALs. The interaction between dendritic polymers and the phospholipid bilayer has been thoroughly investigated, and studies have shown that the generation of dendritic polymers plays a crucial role, with higher-generation dendritic polymers causing more significant membrane perturbations than lower-generation dendritic polymers. Since Dendri1 and Dendri4 are both G3 dendritic polymers, their effect on the surface is limited, which further confirms the results of the cryo-electron microscopy images and rapamycin release studies.

[0281] To further elucidate the macroscopic morphology of the system, DendriXAL was imaged using fluorescence microscopy. The images in Figure 7 depict its complex morphological features and micron-scale structure. This evidence suggests that using different aggregating agents can reduce toxicity and impart anti-fibrotic and anti-inflammatory properties to the drug delivery system, while the overall structure of the dosage form remains largely unchanged. Since it is non-toxic, post-aggregation purification is also unnecessary. This simplification of the preparation process subsequently reduces associated costs and complexity.

[0282] Example 4 - Cartilage Lubrication Activation of synovial fibroblasts is closely associated with pro-inflammatory and pro-fibrotic factors, and is also closely related to the presence of cartilage wear particles generated during cartilage erosion. In osteoarthritis (OA), increased cartilage surface roughness further exacerbates this process, leading to higher friction and mechanical wear. Natural biological lubricants prevent cartilage wear in healthy joints by providing effective lubrication, but these lubricants are reduced in OA, impairing the lubricating effect of synovial fluid and leading to increased friction and cartilage degeneration. Small liposomes initially showed promise in reducing friction in vitro, but their performance in in vitro studies has been less than satisfactory compared to larger particles due to their small size. Recently, it has been reported that liposome aggregates can protect cartilage from friction induced by aggregates and form a protective layer on porcine cartilage in vitro. However, the coefficient of friction (COF) was not significantly reduced compared to liposomes.

[0283] To determine whether DendriXALs could more effectively reduce COF, we tested their macromolecular effects on the same in vitro porcine model, as shown in Figure 8. The results indicated that Dendri1AL (COF = 0.02) performed better than conventional liposomes (COF = 0.08) in reducing COF and successfully protected cartilage from the negative effects of free Dendri1 (COF = 0.12) on the cartilage surface. Similar observations were observed with Dendri4AL, which reduced friction more effectively than all other control groups. The increased friction of DendriXs is likely due to the strong electrostatic interaction between the highly cationic peptides and the negatively charged cartilage surface, which could potentially damage the cartilage. The liposomes in the DendriXAL system encapsulate cations and bind them tightly to the surface, preventing surface deformation. It is speculated that the lubricating effect of the liposomes is due to the formation of a hydrated shell in the phospholipid head region, which has strong compressive strength and reduces friction. In the case of DendriXAL, it is assumed that the large particles observed in Figure 7 form a broad protective layer on the cartilage surface, preventing excessive friction or wear of the cartilage surface.

[0284] Example 5 - DendriXAL Preservation on Cartilage A crucial aspect of cartilage lubrication and abrasion prevention is material retention on the cartilage surface, as lubrication is only possible when the material is present at the interface. Small nanoparticles are known to be rapidly removed from joints via lymphatic drainage systems and phagocytosis.5,6 To increase retention time in joints, researchers have developed cartilage-targeting systems, typically designed to penetrate cartilage pores2, but these do not necessarily provide lubrication because they are not adequately retained on the cartilage surface.8 To test whether DendriXALs could improve retention on the cartilage surface, researchers cut porcine cartilage, placed it in 12-well plates, and incubated it for 24 hours with buffer, DiD-labeled plain liposomes, and DendriXALs. Figure 9A reports a comparison of strength measurements of liposomes and DendriXALs, while Figures 9B and 9C show cartilage surfaces treated with Dendri1AL and Dendri4AL, respectively, at 50x magnification. There was no difference in retention rates between conventional liposomes and Dendri1ALs, but Dendri4ALs exhibited significantly higher retention rates. The observed increase in Dendri4ALs retention can be attributed to the approximately 2 kDa increase in molecular weight and the increased number of positive charges in the Dendri4 dendrimer relative to the Dendri1 dendrimer (9.4 kDa and 17 positive charges, 7.4 kDa and 12 positive charges, respectively). This difference in molecular weight and charge density likely promotes stronger interactions between Dendri4ALs and the cartilage surface, thereby enhancing retention. This enhanced interaction may result from increased electrostatic attraction between the Dendri4 dendrimer and the anionic liposomes, as well as the negatively charged cartilage surface. Furthermore, the larger size of the Dendri4 dendrimer may also contribute to increased retention through steric effects, promoting more stable binding to the cartilage surface. These results explain the superior performance of Dendri4ALs in the lubrication study shown in Figure 8, where COF was significantly reduced compared to conventional liposomes. The lack of increased retention of Dendri1ALs is quite interesting, given that this system already outperforms free liposomes in reducing friction. A possible explanation for this result is that increased surface retention is not the sole mechanism for system lubrication. In fact, during macro-tribological measurements, the cartilage was co-incubated with DendriXALs without any washing steps, which preserved the aggregates on the cartilage surface.

[0285] Example 6 - Cellular uptake of Dendri4ALs Given the crucial role of particle size and retention within the joint cavity, addressing the uptake of drugs by immune cells is a key consideration in developing efficient drug delivery systems. The rate of drug uptake by these immune cells significantly influences the clearance rate of the delivered drug, thus largely determining its efficacy. Regarding liposomes, while their effectiveness in enhancing drug retention within the joint space has been documented, their relatively small size often makes them susceptible to accelerated clearance.19 This rapid clearance is primarily mediated by phagocytosis, a biological process particularly influenced by vesicle size.20 Studies have shown that particles larger than 10 µm can significantly evade immune cell uptake, thereby enhancing their retention in the target region.5,6 However, particle size is only one factor; the composition of these vesicles also plays an equally important role. Specifically, although liposomes containing cationic lipids are associated with toxicity issues, they exhibit a tendency to form protein coronas. This leads to stronger adsorption of surrounding proteins to their surface, enhancing the phagocytic process and accelerating their clearance from the joint cavity. In contrast, neutral nanoparticles, due to their lower protein adsorption capacity, have a longer persistence time in plasma. 21 Therefore, both particle size and composition are crucial for developing the best drug delivery system for the treatment of osteoarthritis.

[0286] To assess this aspect, Dendri4AL was selected to test its uptake in RAW264.7 macrophages. This selection was based on its excellent performance in joint lubrication and its ability to improve cartilage preservation. The DiD-labeled system was co-incubated with cells and compared with negative control DMEM and positive control liposomes. After 3 hours of treatment, cells were harvested and analyzed using Image Stream® fluorescence imaging flow cytometry. The results shown in Figure 10 show a significant reduction in DiD intensity in live cells. These findings may be related to the presence of large macroscopic Dendri4AL structures shown in Figure 7, which may hinder phagocytosis through stereotactic effects. A representative image in Figure 10C shows that the uptaken particles are integrated into the cytoplasm and distributed around the nucleus. Dendri4ALs can still be phagocytosed by macrophages, albeit to a lesser extent, which can be explained by the softness of the liposome material, which is associated with promoting cellular uptake.

[0287] Example 7 – Aggregation curves of DPPC:DSPG liposomes and polycationic aggregates Other polycationic aggregators listed in Table 3 were used to test and evaluate their ability to induce aggregate formation when interacting with negatively charged liposomes. Experiments involved co-incubating fixed concentrations of liposomes (1 mM, DSPG:DPPC ratio 1:3 mol / mol) with different concentrations of aggregators to achieve the desired charge ratio (the ratio of positive to negative charge). The following equations were used to calculate the charge ratios in Figures 11A and 11B: (where N = quantity).

[0288] The mixture was prepared in 96-well microplates with a smooth, transparent quartz bottom (Hellma® GmbH & Co., Germany). After gently stirring the mixture for 5 minutes, the optical density was measured at 450 nm using an Infinite M Pro 200F-PlexNano microplate reader (Tecan®, Switzerland). The results in Figures 11A and 11B demonstrate that all tested polycationic aggregators were capable of inducing aggregation, and that the method is generally applicable to them.

[0289] Table 3 – Application of polycationic aggregaters in aggregation studies (Example 7) and macrophage uptake studies with RAW264.7 cells (Example 8) Example 8 – Macrophage Uptake Study To evaluate the ability of the liposomes and certain aggregates described in Example 7 to reduce joint immune clearance, a key factor in the persistence of intra-articular injections at the injection site, a macrophage uptake assay was performed using the protocol described in the "Aggregated Liposomes and Liposome Uptake" section and analyzed using imaging flow cytometry as described in the "Flow Cytometry" section above. Aggregates were prepared using Dendri1 (D1), glatiramer acetate (GA), i-PAMAM, LL-37, polymyxin B (PMB), and polyarginine (Poly-Arg) (see Table 3, which lists these polycationic aggregates and their respective charge-to-weight ratios), and their ability to evade immune clearance was evaluated.

[0290] Figure 12 shows the results of this study, indicating that macrophage uptake of the aggregates was reduced compared to the liposome control group. This suggests that combining polycationic aggregates with liposomes is an effective strategy to reduce clearance in the joint, making it suitable for intra-articular injection.

[0291] Example 9 – Toxicity Study of Synovial Fibroblasts in Osteoarthritis – Comparison of aggregated liposomes prepared using the following aggregaters: polycationic aggregaters Dendri1 and Dendri4; and divalent Zn2+. Previous studies on the use of aggregated liposomes for intra-articular (IA) injection have employed divalent aggregaters, such as zinc (Zn2+), in formulations. These formulations require large amounts of cations to deliver therapeutic drugs like rapamycin (RAPA) at effective concentrations, leading to high toxicity in relevant cell cultures, including synovial fibroblasts from osteoarthritis patients. Attempts to reduce this toxicity by dialysis of excess Zn2+ (formulations recommended for intra-articular administration contain 150 mM Zn2+ per 5 mM liposome before dialysis, which is essential) have been ineffective, as the final formulations still exhibit significant toxicity. Therefore, novel aggregaters are crucial for the successful application of liposome aggregates in intra-articular injection. The toxicity of RAPA-loaded aggregated liposomes (ZnAL, Denri1AL, and Dendri4AL) was determined using the aforementioned "Toxicity of Dendritic Polymers" method. However, cell viability was determined using the *Cell Counting Kit-8 (CCK-8, Merck®, Germany) or the **LIVE / DEAD™ Activity Assay Kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions, as shown in Figure 13. Figure 13 shows that, compared to the free liposome control group, higher doses of RAPA can be delivered using polyvalent cations (e.g., Dendri1 with a 12+ charge and Dendri4 with a 17+ charge) without inducing significant toxicity. The principle is that polyvalent cations aggregate liposomes more efficiently than divalent cations, requiring fewer molecules for aggregation and maintaining a favorable LD50. This results in a lower overall cation concentration and reduced toxicity, as shown in Table 4. This substitution simplifies the preparation process and enhances the clinical convertibility of the formulation, as no further processing is required after aggregation, unlike Zn2+ aggregates.

[0292] Table 4 - Composition of ZnALs, Dendri1ALs, and Dendri4ALs at the maximum effective RAPA dose Example 10 - Toxicity Study of Immune Cell Lines Direct comparison of aggregated liposomes prepared using the following aggregating agents: polycationic aggregators: LL-37, Dendri1, and Dendri4; and divalent Zn2+. To further demonstrate the biocompatibility of the compositions of the present invention, direct cytotoxicity studies were conducted, comparing i) the effects of aggregated liposomes prepared using several polycationic aggregaters (Dendri1 (D1), Dendri4 (D4), and LL-37) with ii) divalent Zn2+ aggregates on RAW264.7 macrophages (Figure 14). Macrophages play a crucial role in joint inflammation and the clearance of substances from the synovium, and are therefore essential for osteoarthritis (OA). These aggregates consist of liposomes (DPPC:DSPG = 3:1 molar ratio) with standard concentrations and required amounts of aggregaters (see Table 5). The Zn2+ aggregates were prepared according to the protocol described by Bordon et al.

[0293] The results of this study demonstrate that Zn2+ aggregated liposomes exhibit strong cytotoxicity, killing almost all cells at the lowest liposome concentration (0.1 mM). In contrast, aggregated liposomes prepared using polycationic aggregaters (Dendri1 (D1), Dendri4 (D4), and LL-37) did not exhibit cytotoxicity at the same concentration. These results are consistent with previous findings using OASFs and highlight the significant advantages of using polycationic aggregaters instead of divalent Zn2+ in forming intra-articular liposome aggregates.

[0294] Table 5 lists the composition of the aggregates used in this study, which is essential for the formation of stable aggregates. Compared to the polycationic aggregators (D1, D4, and LL-37), the composition of Zn2+ requires a significantly higher molar concentration of aggregator. This further illustrates that Zn2+ requires more molecules to achieve liposome aggregation compared to polyvalent cations.

[0295] Table 5 - Aggregate composition at 0.5 mM liposome concentration. The composition was selected based on the aggregation curves of the polycationic aggregator used and the reported optimal concentration of divalent Zn²⁺ aggregates, with ZnCl₂ used for aggregate formation, as described by Bordon et al. 9 LD50 assessment Table 6 below shows the comparative toxicity of ZnCl2 (Zn2+) to a series of polycationic aggregates on macrophage cell lines. The LD50 of ZnCl2 was 5.87 µg / mL, lower than the LD50 of the other tested polycationic aggregates, indicating that it has higher cytotoxicity. This study was conducted according to the "LD50 assessment" method described above.

[0296] Table 6 - LD50 values ​​for different aggregators. All concentrations are in µg / mL.

[0297] Other implementation plans The present invention is further illustrated by the following numbered embodiments: 1. A composition for treating joint diseases or disorders, wherein the composition comprises an anionic lipid composition and a polycationic aggregater. The composition is administered intra-articularly.

[0298] 2. The therapeutic composition according to claim 1, wherein the composition further comprises a pharmaceutically active agent, and the pharmaceutically active agent may be incorporated into an anionic lipid composition.

[0299] 3. The therapeutic composition according to claim 1 or claim 2, wherein the anionic lipid composition comprises liposomes, lipid nanoparticles, solid lipid nanoparticles, and nanostructured lipid carriers.

[0300] 4. The therapeutic composition according to claim 3, wherein the anionic lipid composition comprises liposomes.

[0301] 5. The therapeutic composition according to claim 4, wherein the liposomes comprise negatively charged lipids selected from the group consisting of: 1,2-dipalmitoyl-sn-glycerol-3-phosphate (DPPA), 1,2-dioleoyl-sn-glycerol-3-phosphate (DOPA), 1,2-distearateoyl-sn-glycerol-3-phosphate-(1′-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-l-serine (DOPS), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1-rac-glycerol) (DPPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1-rac-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-l-serine (DPPS), and 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1′-inositol). (DPPI), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1′-inositol-3′-4′-5′-triphosphate) (DPPI-P3), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1′-inositol) (DOPI), or 1,2-dioleoyl-sn-glycerol-3-phosphate-(1′-inositol-3′-4′-5′-triphosphate) (DOPI-P3), or combinations thereof.

[0302] 6. The therapeutic composition according to claim 5, wherein the liposomes further comprise one or more neutral or amphoteric lipids, such as cholesterol, or phosphatidylcholine lipids (e.g., 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC) or 1,2-distearate-sn-glycerol-3-phosphatidylcholine (DSPC)), or combinations thereof.

[0303] 7. The composition for treatment according to any one of claims 1 to 6, wherein the polycationic aggregator comprises two or more individual positively charged sites and has a pH value in the range of 6.0 to 8.0.

[0304] 8. The composition for treatment according to claim 7, wherein the polycationic agglomerator is a pharmaceutically active agent, and the polycationic agglomerator may be a polycationic anti-inflammatory drug or a polycationic anti-fibrotic drug.

[0305] 9. The composition for treatment according to claim 8, wherein the polycationic aggregater is an anti-inflammatory drug comprising an amino acid selected from the group consisting of lysine, alanine, glutamic acid, and tyrosine, or a combination thereof, wherein the anti-inflammatory drug comprises a polymer consisting of 40 to 100 amino acids, comprising four amino acids selected from L-alanine, L-lysine, L-glutamic acid, and L-tyrosine, for example, the anti-inflammatory drug being glatiramer or a pharmaceutically acceptable salt thereof.

[0306] 10. The composition for treatment according to claim 8, wherein the anti-inflammatory agent is glatiramer acetate.

[0307] 11. The composition for treatment according to claim 8, wherein the polycationic aggregator is a polycationic antifibrotic agent.

[0308] 12. The therapeutic composition according to claim 7, wherein the polycationic aggregator comprises chitosan, poly(dimethyldiallylammonium chloride), polyamine, polyallylamine, polyethyleneimine, poly(dimethylaminoethyl acrylate), polylysine, polyhistidine, polyornithine, polyarginine, polyveratrol (a starch derivative having an amino or ammonium group), or combinations thereof.

[0309] 13. The composition for treatment according to claim 7, wherein the polycationic aggregator is a polycationic dendritic polymer.

[0310] 14. The therapeutic composition according to claim 13, wherein the dendritic polymer is a poly(amidoamine) (PAMAM) dendritic polymer, a poly(propyleneimine) (PPI) dendritic polymer, a triazine dendritic polymer, a phosphorus dendritic polymer, or a peptide dendritic polymer.

[0311] 15. The composition for treatment according to claim 13 or claim 14, wherein the dendritic polymer is a G2 or G3 dendritic polymer. The dendritic polymer is preferably G3 dendritic polymer.

[0312] 16. The therapeutic composition according to any one of claims 13 to 15, wherein the dendritic polymer comprises an amino acid selected from the group consisting of lysine, alanine, glutamic acid, tyrosine, and cysteine, or a combination thereof.

[0313] 17. A therapeutic composition according to any one of claims 13 to 16, wherein the dendritic polymer comprises one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4) or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8).

[0314] 18. A therapeutic composition according to any one of claims 2 to 17, wherein the pharmaceutically active ingredient is selected from the group consisting of: anti-inflammatory drugs, antifibrotic drugs, antimicrobial drugs, nonsteroidal anti-inflammatory drugs, corticosteroids, and biological agents (e.g., peptides, proteins, stem cells, antibodies, and antibody fragments).

[0315] 19. A therapeutic composition according to any one of claims 2 to 18, wherein the pharmaceutically active ingredient is selected from the group consisting of: trifluorochlorothiazide, prednisone, hydrocortisone, methylprednisolone, dexamethasone, beclothiazide, hyaluronic acid, cetrus zumab, golimumab, belimumab, rituximab, rapamycin, tolin-1, tolin-2, retinoic acid metabolism blockers (e.g., tapazole, rialozide), naproxen, ibuprofen, diclofenac, celecoxib, meloxicam, lonoxicam, captogan, dasatinib, quercetin, thymol, sinazole, liquiline, glatiramer acetate, insulin-like growth factor 1 (IGF1), rosufen D1, fibroblast growth factor 18 (FGF18), bone morphogenetic protein (BMP) 7, and anti-IL-1. Preparations (e.g., canacuumab, anacumumab), anti-TNF drugs (e.g., infliximab, adalimumab, etanercept), botulinum toxin A, JAK inhibitors (e.g., tofacitinib, felotinib, utpatinib, TD-1473, Brepocitinib (PF-06700841), PF-06651600), pirfenidone or nidatenil, feline peptide, LL-37, polymyxin B, maganine, murapavadine, MK-8722, or combinations thereof.

[0316] 20. A composition for treatment according to any one of claims 1 to 19, wherein the joint disease or disorder is selected from arthritis, osteoarthritis, joint fibrosis, rheumatoid arthritis, gout, bursitis, bacterial arthritis, juvenile idiopathic arthritis, psoriatic arthritis, reactive arthritis, ankylosing spondylitis, tendinopathy, polymyalgia rheumatica, Paget's disease, lupus, Sjögren's syndrome, osteonecrosis, neuropathic arthropathy, or Lyme disease.

[0317] 21. The composition for treatment according to claim 20, wherein the joint disease or disorder is osteoarthritis.

[0318] 22. A therapeutic composition according to any one of claims 1 to 21, wherein the composition comprises an aggregate comprising an anionic lipid composition (e.g., liposomes) and a polycationic aggregator.

[0319] 23. A therapeutic composition according to any one of claims 1 to 22, wherein the composition forms a sustained-release reservoir after intra-articular administration for use by a subject.

[0320] 24. A composition comprising • Anionic liposomes; and • Cationic dendritic polymer aggregator, The cationic dendritic polymer aggregator includes one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4) or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8).

[0321] 25. The composition according to claim 24, further comprising a pharmaceutically active agent, wherein the pharmaceutically active agent is encapsulated in anionic liposomes.

[0322] 26. The composition according to claim 24 or claim 25, wherein the anionic liposome comprises a negatively charged lipid selected from the group consisting of: 1,2-dipalmitoyl-sn-glycerol-3-phosphate (DPPA), 1,2-dioleoyl-sn-glycerol-3-phosphate (DOPA), 1,2-distearateoyl-sn-glycerol-3-phosphate-(1′-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-l-serine (DOPS), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1-rac-glycerol) (DPPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1-rac-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-l-serine (DPPS), and 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1′-inositol). (DPPI), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1′-inositol-3′-4′-5′-triphosphate) (DPPI-P3), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1′-inositol) (DOPI), or 1,2-dioleoyl-sn-glycerol-3-phosphate-(1′-inositol-3′-4′-5′-triphosphate) (DOPI-P3), or combinations thereof.

[0323] 27. The composition according to claim 26, wherein the anionic liposome further comprises one or more neutral or amphoteric lipids, such as cholesterol, or phosphatidylcholine lipids (e.g., 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC) or 1,2-distearate-sn-glycerol-3-phosphatidylcholine (DSPC)), or combinations thereof.

[0324] 28. The composition according to any one of claims 24 to 27, wherein the composition comprises an aggregate consisting of a cationic dendritic polymer aggregater and a plurality of anionic liposomes.

[0325] 29. The kit includes: a) A first container containing an anionic lipid composition; b) A second container containing a polycationic aggregator; and c) Combining a) with b) to provide a description of a composition for treatment according to any one of claims 1 to 23.

[0326] 30. The kit according to claim 29, wherein the first container further comprises a pharmaceutically active agent incorporated into an anionic lipid composition.

[0327] Further implementation plan The present invention is further illustrated by the following numbered embodiments: P1. A composition for treating joint diseases or disorders, wherein the composition comprises an anionic lipid composition and a polycationic aggregater. The composition is administered intra-articularly.

[0328] P2. The therapeutic composition according to P1, wherein the composition further comprises a pharmaceutically active agent that can be bound to an anionic lipid composition.

[0329] P3. The therapeutic composition according to P1 or P2, wherein the anionic lipid composition comprises liposomes, lipid nanoparticles, solid lipid nanoparticles, and nanostructured lipid carriers.

[0330] P4. The therapeutic composition according to P3, wherein the anionic lipid composition comprises liposomes.

[0331] P5. The therapeutic composition according to P4, wherein the liposomes comprise negatively charged lipids selected from the group consisting of: 1,2-dipalmitoyl-sn-glycerol-3-phosphate (DPPA), 1,2-dioleoyl-sn-glycerol-3-phosphate (DOPA), 1,2-distearateoyl-sn-glycerol-3-phosphate-(1′-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-l-serine (DOPS), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1-rac-glycerol) (DPPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1-rac-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-l-serine (DPPS), and 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1′-inositol). (DPPI), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1′-inositol-3′-4′-5′-triphosphate) (DPPI-P3), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1′-inositol) (DOPI), or 1,2-dioleoyl-sn-glycerol-3-phosphate-(1′-inositol-3′-4′-5′-triphosphate) (DOPI-P3), or combinations thereof.

[0332] P6. The composition for treatment according to P5, wherein the liposomes further comprise one or more neutral or amphoteric lipids, such as cholesterol, or phosphatidylcholine lipids (e.g., 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC) or 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC)), or combinations thereof.

[0333] P7. A therapeutic composition according to any one of P1 to P6, wherein the polycationic aggregator comprises two or more individual positively charged sites and has a pH value in the range of 6.0 to 8.0.

[0334] P8. The composition for treatment according to P7, wherein the polycationic agglomerant is a pharmaceutically active agent, and the polycationic agglomerant may be a polycationic anti-inflammatory agent, a polycationic antimicrobial agent, or a polycationic antifibrotic agent.

[0335] P9. The composition for treatment according to P8, wherein the polycationic aggregater is an anti-inflammatory drug comprising an amino acid selected from the group consisting of lysine, alanine, glutamic acid, and tyrosine, or a combination thereof, wherein the anti-inflammatory drug comprises a polymer consisting of 40 to 100 amino acids, comprising four amino acids selected from L-alanine, L-lysine, L-glutamic acid, and L-tyrosine, for example, the anti-inflammatory drug being glatiramer or a pharmaceutically acceptable salt thereof.

[0336] P10. The composition for treatment according to P8, wherein the anti-inflammatory agent is glatiramer acetate.

[0337] P11. The composition for treatment according to P8, wherein the polycationic aggregator is a polycationic antifibrotic agent.

[0338] P12. The composition for treatment according to P7, wherein the polycationic aggregator comprises a polyamine.

[0339] P13. The composition for treatment according to P12, wherein the polyamine comprises a peptide or polypeptide.

[0340] P14. The therapeutic composition according to P13, wherein the peptide or polypeptide comprises at least two, at least three, at least four, at least five, or at least six different amino acids.

[0341] P15. The therapeutic composition according to P13, wherein the peptide or polypeptide includes polylysine, polyhistidine, polyornithine, or polyarginine.

[0342] P16. The composition for treatment according to P12, wherein the polyamine comprises one or more amino sugar residues.

[0343] P17. The therapeutic composition according to P16, comprising a polymer of one or more amino sugar residues, wherein the polymer may be chitosan or chitin.

[0344] P18. The composition for treatment according to P16, wherein the polyamine includes aminoglycosides.

[0345] P19. A therapeutic composition according to any one of claims P12 to P17, wherein the polyamine comprises chitosan, chitin, aminoglycosides, poly(dimethyldiallylammonium chloride), poly(amidoamine) (PAMAM), inverse poly(amidoamine) (i-PAMAM), poly(propyleneimine) (PPI), triazine-based polymers, polyallylamine, polyethyleneimine, poly(dimethylaminoethyl acrylate), peptides or polypeptides (e.g., polylysine, polyhistidine, polyornithine, polyarginine), polyquaternary ammonium salts, or any combination thereof.

[0346] P20. The therapeutic composition according to P18, wherein the aminoglycoside is selected from streptomycin, gentamicin, tobramycin, amikacin, dibekacin, sisomicin, netilmicin, neomycin (e.g., neomycin B, neomycin C or neomycin E), prazomycin, spectromycin, kanamycin, tobramycin, alpramycin, oxytetracycline B, formalin, and combinations thereof.

[0347] P21. A therapeutic composition according to any one of P12 to P19, wherein the polyamine is branched.

[0348] P22. The composition for treatment according to P7, wherein the polycationic aggregator is a polycationic dendritic polymer.

[0349] P23. The composition for treatment according to P13, wherein the dendritic polymer is a poly(amidoamine) (PAMAM) dendritic polymer, an inverse poly(amidoamine) (i-PAMAM) dendritic polymer, a poly(propyleneimine) (PPI) dendritic polymer, a triazine dendritic polymer, a phosphorus dendritic polymer, or a peptide dendritic polymer.

[0350] P24. The composition for treatment according to P13 or P14, wherein the dendritic polymer is a G2 or G3 dendritic polymer. The dendritic polymer is preferably G3 dendritic polymer.

[0351] P25. A therapeutic composition according to any one of P13 to P15, wherein the dendritic polymer comprises an amino acid selected from the group consisting of lysine, leucine, arginine, aspartic acid, alanine, glutamic acid, tyrosine, and cysteine, or a combination thereof. The dendritic polymer may contain amino acids selected from the group consisting of lysine, alanine, glutamic acid, tyrosine, and cysteine, or combinations thereof.

[0352] P26. The therapeutic composition according to P25, wherein the dendritic polymer is represented by the following general formula: (A)8-(B)4-(C)2-Z, wherein: A is X1AX2X3; B is X1BX4X5X6X7; C is X1BX8X5X7X6X7; where: X1A is selected from lysine and arginine; X1B is lysine; X2 is selected from leucine, oroleucine, and alanine; X3 is either empty or tyrosine; X4 is selected from lysine, arginine, and alanine; Each X5 is independently selected from lysine, leucine, and alanine; Each X6 is either empty or lysine independently; Each X7 can be empty, glutamic acid, or alanine independently; X8 is selected from lysine, tyrosine, leucine, arginine, and glutamic acid; and Z is the core group.

[0353] P27. The composition for treatment according to P26, wherein A is selected from: KL, kl, rl, KA and k{NLE}y (SEQ ID NO: 91).

[0354] P28. The therapeutic composition according to P26 or P27, wherein B is selected from: KKL, kkl, krl, KAK and KKAKE (SEQ ID NO: 92).

[0355] P29. The composition for treatment according to any one of P26 to P28, wherein C is selected from: KKL, kll, krl, KEKA (SEQ ID NO: 93) and KYKAKA (SEQ ID NO: 94).

[0356] P30. A therapeutic composition according to any one of P26 to P29, wherein Z comprises 1 to 10 amino acids, wherein Z may comprise one or more C2-C30 alkyl groups.

[0357] P31. The composition for treatment according to any one of P22 to P30, wherein the dendritic polymer comprises one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (consisting of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (consisting of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (consisting of SEQ ID NO: 79 to 82).

[0358] P32. The therapeutic composition according to claim P31, wherein the dendritic polymer comprises one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4) or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8).

[0359] P33. A therapeutic composition according to any one of P1 to P7, wherein the polycationic aggregator is selected from the group consisting of: inverse poly(amidoamine) (i-PAMAM), polyethyleneimine (branched or linear), tobramycin, glatiramer acetate, polymyxin B, polylysine, apidaecin 1b, LL-37, polyarginine, Walk11.3, or (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87) (kl)8(kkl)4(kkl)2KKL (composed of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82).

[0360] P34. A therapeutic composition according to any one of P2 to P33, wherein the pharmaceutically active ingredient is selected from the group consisting of: anti-inflammatory drugs, antifibrotic drugs, antimicrobial drugs, nonsteroidal anti-inflammatory drugs, corticosteroids and biological agents (e.g., peptides, proteins, stem cells, antibodies and antibody fragments).

[0361] P35. A therapeutic composition according to any one of P2 to P34, wherein the pharmaceutically active ingredient is selected from the group consisting of: trifluorochlorothiazide, prednisone, hydrocortisone, methylprednisolone, dexamethasone, beclothiazide, hyaluronic acid, cetrus zumab, golimumab, belimumab, rituximab, rapamycin, tolin-1, tolin-2, retinoic acid metabolism blockers (e.g., tapazole, rialozide), naproxen, ibuprofen, diclofenac, celecoxib, meloxicam, lonoxicam, captogan, dasatinib, quercetin, thymol, sinazole, liquiline, glatiramer acetate, insulin-like growth factor 1 (IGF1), rosufen D1, fibroblast growth factor 18 (FGF18), bone morphogenetic protein (BMP) 7, anti-IL-1 Preparations (e.g., canacuumab, anacumumab), anti-TNF drugs (e.g., infliximab, adalimumab, etanercept), botulinum toxin A, JAK inhibitors (e.g., tofacitinib, felotinib, utpatinib, TD-1473, Brepocitinib (PF-06700841), PF-06651600), pirfenidone or nidatenil, feline peptide, LL-37, polymyxin B, maganine, murapavadine, MK-8722, or combinations thereof.

[0362] P36. A composition for treatment according to any one of P1 to P35, wherein the joint disease or disorder is selected from arthritis, osteoarthritis, joint fibrosis, rheumatoid arthritis, gout, bursitis, bacterial arthritis, juvenile idiopathic arthritis, psoriatic arthritis, reactive arthritis, ankylosing spondylitis, tendinopathy, polymyalgia rheumatica, Paget's disease, lupus, Sjögren's syndrome, osteonecrosis, neuropathic arthropathy, or Lyme disease.

[0363] P37. The composition for treatment according to P36, wherein the joint disease or disorder is osteoarthritis.

[0364] P38. A therapeutic composition according to any one of P1 to P37, wherein the composition comprises an aggregate containing an anionic lipid composition (e.g., liposomes) and a polycationic aggregator.

[0365] P39. A therapeutic composition according to any one of P1 to P38, wherein the composition forms a sustained-release reservoir after intra-articular administration for use by a subject.

[0366] P40. A composition comprising • Anionic liposomes; and • Polycationic aggregates, The polycationic aggregator is: (i) A cationic dendritic polymer aggregator comprising one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (composed of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 87 to 90). Composed of SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (composed of SEQ ID NO: 79 to 82); or (ii) A polycationic aggregator selected from: inverse poly(amidoamine) (i-PAMAM), polyethyleneimine (branched or linear), tobramycin, glatiramer acetate, polymyxin B, polylysine, apidaecin 1b, LL-37, polyarginine and Walk11.3.

[0367] P41. The composition according to P40, wherein the polycationic aggregator is a cationic dendritic polymer aggregator, comprising one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C18) (consisting of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (consisting of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C16)k(C16) (composed of SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C18) (consisting of SEQ ID NO: 79 to 82).

[0368] P42. The composition according to P41, wherein the cationic dendritic polymer aggregator comprises one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4) or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8).

[0369] P43. The composition according to any one of claims P40 to P42 further comprises a pharmaceutically active agent, said pharmaceutically active agent being encapsulated in anionic liposomes.

[0370] P44. The composition according to any one of claims P40 to P43, wherein the anionic liposome comprises a negatively charged lipid selected from the group consisting of: 1,2-dipalmitoyl-sn-glycerol-3-phosphate (DPPA), 1,2-dioleoyl-sn-glycerol-3-phosphate (DOPA), 1,2-distearateoyl-sn-glycerol-3-phosphate-(1′-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-l-serine (DOPS), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1-rac-glycerol) (DPPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1-rac-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-l-serine (DPPS), and 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1′-inositol). (DPPI), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1′-inositol-3′-4′-5′-triphosphate) (DPPI-P3), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1′-inositol) (DOPI), or 1,2-dioleoyl-sn-glycerol-3-phosphate-(1′-inositol-3′-4′-5′-triphosphate) (DOPI-P3), or combinations thereof.

[0371] P45. The composition according to P44, wherein the anionic liposome further comprises one or more neutral or amphoteric lipids, such as cholesterol, or phosphatidylcholine lipids (e.g., 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC) or 1,2-distearate-sn-glycerol-3-phosphatidylcholine (DSPC)), or combinations thereof.

[0372] P46. The composition according to any one of P40 to P45, wherein the composition comprises an aggregate consisting of a cationic dendritic polymer aggregater and a plurality of anionic liposomes.

[0373] P47. The kit includes: a) A first container containing an anionic lipid composition; b) A second container containing a polycationic aggregator; and c) Combining a) with b) to provide a description of a composition for treatment according to any one of P1 to P39.

[0374] P48. The kit according to P47, wherein the first container further comprises a pharmaceutically active agent incorporated into the anionic lipid composition.

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Claims

1. A composition for treating joint diseases or disorders, wherein the composition comprises an anionic lipid composition and a polycationic aggregater, in, The composition is administered intra-articularly.

2. The composition for use according to claim 1, wherein the composition further comprises a pharmaceutically active agent, optionally wherein the pharmaceutically active agent is incorporated into the anionic lipid composition.

3. The composition for use according to claim 1 or claim 2, wherein the anionic lipid composition comprises liposomes, lipid nanoparticles, solid lipid nanoparticles, and nanostructured lipid carriers.

4. The composition for use according to claim 3, wherein the anionic lipid composition comprises anionic liposomes.

5. The composition for use according to claim 4, wherein the liposomes comprise negatively charged lipids selected from the group consisting of: 1,2-dipalmitoyl- sn - glycerin 3-Phosphate (DPPA), 1,2-dioleoyl- sn - glycerin -3-phosphate (DOPA), 1,2-distearate- sn - glycerin -3-phosphate-(1′- rac 1,2-Dioleoyl- glycerol) (DSPG), 1,2-dioleoyl- sn -Glyceryl-3-phosphate-1-serine (DOPS), 1,2-dipalmitoyl- sn -Glyceryl-3-phosphate-(1-rac-glycerol) (DPPG), 1,2-dioleoyl- sn -Glyceryl-3-phosphate-(1-rac-glycerol) (DOPG), 1,2-dipalmitoyl- sn 1,2-Dipalmitoyl-sn-glycerol-3-phosphate-(1′-myo-inositol) (DPPI), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1′-myo-inositol-3′-4′-5′-triphosphate) (DPPI-P3), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1′-myo-inositol) (DOPI), or 1,2-dioleoyl-sn-glycerol-3-phosphate-(1′-myo-inositol-3′-4′-5′-triphosphate) (DOPI-P3), or combinations thereof; Optionally, the liposomes further comprise one or more neutral or zwitterionic lipids, such as cholesterol, or phosphatidylcholine lipids (e.g., 1,2-dipalmitoyl- sn -glycerol-3-phosphatidylcholine (DPPC) or 1,2-distearate-sn-glycerol-3-phosphatidylcholine (DSPC)), or combinations thereof.

6. The composition for use according to any one of claims 1 to 5, wherein: (i) The polycationic aggregator contains two or more independent positively charged sites in a pH range of 6.0 to 8.0; and / or (ii) The polycationic aggregator is a polycationic pharmaceutical active agent, optionally wherein the polycationic aggregator is a polycationic anti-inflammatory agent, a polycationic antimicrobial agent or a polycationic antifibrotic agent; Optionally, the polycationic aggregator is an anti-inflammatory agent comprising an amino acid selected from the group consisting of lysine, alanine, glutamic acid, and tyrosine, or a combination thereof. Optionally, the anti-inflammatory agent comprises a polymer containing 40 to 100 amino acid polymers, said polymer comprising four amino acids selected from L-alanine, L-lysine, L-glutamic acid, and L-tyrosine, for example, said anti-inflammatory agent is glatiramer or a pharmaceutically acceptable salt thereof (e.g., glatiramer acetate); or (iii) The polycationic agglomerant is a polycationic anti-fibrotic agent; or (iv) The polycationic aggregator comprises a polyamine, optionally wherein the polyamine comprises chitosan, chitin, aminoglycosides, poly(dimethyldiallylammonium chloride), poly(amidoamine) (PAMAM), inverse poly(amidoamine) (i-PAMAM), poly(propyleneimine) (PPI), triazine-based polymers, polyallylamine, polyethyleneimine, poly(dimethylaminoethyl acrylate), peptides or polypeptides (e.g., polylysine, polyhistidine, polyornithine, polyarginine), polyquaternary ammonium salts or any combination thereof.

7. The composition for use according to any one of claims 1 to 5, wherein the polycationic aggregator is a polycationic dendritic polymer; optionally wherein: (i) The dendritic polymer is a poly(amidoamine) (PAMAM) dendritic polymer, an inverse poly(amidoamine) (i-PAMAM) dendritic polymer, a poly(propyleneimine) (PPI) dendritic polymer, a triazine dendritic polymer, a phosphorus dendritic polymer, or a peptide dendritic polymer; (ii) The dendritic polymer is a G2 or G3 dendritic polymer. Preferably, the dendritic polymer is a G3 dendritic polymer; and / or (iii) The dendritic polymer comprises amino acids selected from the group consisting of lysine, leucine, arginine, aspartic acid, alanine, glutamic acid, tyrosine, and cysteine, or combinations thereof.

8. The composition for use according to any one of claims 1 to 5, wherein the polycationic aggregator is a dendritic polymer selected from one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C 18 (Composed of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (consisting of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C 16 )k(C 16 (consisting of SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (composed of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C 18 (consisting of SEQ ID NO: 79 to 82) Optionally, the dendritic polymer is selected from the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4) and (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8).

9. The composition for use according to any one of claims 2 to 8, wherein the pharmaceutically active agent is selected from the group consisting of: anti-inflammatory agents, anti-fibrotic agents, antimicrobial agents, nonsteroidal anti-inflammatory agents, corticosteroids, and biological agents (e.g., peptides, proteins, stem cells, antibodies, and antibody fragments). Optionally, the pharmaceutically active agent described herein is selected from the following group: triamcinolone, prednisone, hydrocortisone, methylprednisolone, dexamethasone, betamethasone, hyaluronic acid, sertozumab, golimumab, belimumab, rituximab, rapamycin, torin-1, torin-2, retinoic acid metabolism blockers (e.g., talarozol, riazol), naproxen, ibuprofen, diclofenac, celecoxib, meloxicam, lornoxicam, kartogenin, dasatinib, quercetin, rhein, sinomethoxazole, liquiline, glatiramer acetate, insulin-like growth factor 1 (IGF1), regressor D1, fibroblast growth factor 18 (FGF18), bone morphogenetic protein (BMP), anti-IL-1 agents (e.g., canatumab, anaerobicin, linaccept, givozumab), anti-TNF agents. Drugs (e.g., infliximab, adalimumab, etanercept), botulinum toxin A, JAK inhibitors (e.g., tofacitinib, fenlotinib, utpatinib, TD-1473, brepocitinib (PF-06700841), PF-06651600), pirfenidone or nintedanib, cathelicidin, LL-37, polymyxin B, magainin, murapavadin, MK-8722, or combinations thereof.

10. The composition for use according to any one of claims 1 to 9, wherein the joint disease or disorder is selected from arthritis, osteoarthritis, joint fibrosis, rheumatoid arthritis, gout, bursitis, bacterial arthritis, juvenile idiopathic arthritis, psoriatic arthritis, reactive arthritis, ankylosing spondylitis, tendinopathy, polymyalgia rheumatica, Paget's disease, lupus, Sjögren's syndrome, osteonecrosis, neuropathic arthropathy, or Lyme disease; Optionally, the joint disease or disorder described therein is osteoarthritis.

11. The composition for use according to any one of claims 1 to 10, wherein the composition comprises an aggregate containing an anionic lipid composition (e.g., liposomes) and a polycationic aggregator.

12. The composition for use according to any one of claims 1 to 11, wherein the composition forms a sustained-release reservoir after administration into the joint of a subject.

13. A composition comprising Anionic liposomes; and Polycationic aggregates, The polycationic aggregator is: (i) Cationic dendritic polymer aggregators comprising one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4); (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (k{NLE}y)8(krl)4(krl)2kk(C 18 (consisting of SEQ ID NO: 83 to 86); (k{NLE}y)8(kkl)4(kll)2kllll (consisting of SEQ ID NO: 87 to 90); (KL)8(KKL)4(KKL)2KKL (consisting of SEQ ID NO: 67 to 70); (kl)8(kkl)4(kll)2kk(C 16 )k(C 16 (consisting of SEQ ID NO: 71 to 74); (kl)8(kkl)4(kll)2kllll (consisting of SEQ ID NO: 75 to 78); or (rl)8(krl)4(krl)2kk(C 18 (consisting of SEQ ID NO: 79 to 82); or (ii) A polycationic aggregator selected from: inverse poly(amidoamine) (i-PAMAM), polyethyleneimine (branched or linear), tobramycin, glatiramer acetate, polymyxin B, polylysine, apidaecin 1b, LL-37, polyarginine, or Walk11.

3. Choose one of them: (i) The composition comprises Anionic liposomes; and Cationic dendritic polymer aggregator, The cationic dendritic polymer aggregator comprises one of the following linear symbols: (KA)8(KAK)4(KEKA)2KAKEAYCA-NH2 (composed of SEQ ID NO: 1 to 4) or (KA)8(KKAKE)4(KYKAKA)2KAYKKA-OH (composed of SEQ ID NO: 5 to 8); (ii) The composition further comprises a pharmaceutically active agent, wherein the pharmaceutically active agent is optionally encapsulated in anionic liposomes; (iii) The anionic liposomes comprise negatively charged lipids selected from the group consisting of: 1,2-dipalmitoyl- sn - glycerin 3-Phosphate (DPPA), 1,2-dioleoyl- sn - glycerin -3-phosphate (DOPA), 1,2-distearate- sn - glycerin -3-phosphate-(1′- rac 1,2-Dioleoyl-sn-glycerol-3-phosphate-l-serine (DOPS), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1-rac-glycerol) (DPPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1-rac-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-l-serine (DPPS), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1′-myo-inositol) (DPPI), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1′-myo-inositol-3′-4′-5′-triphosphate) (DPPI-P3), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1′-myo-inositol) (DOPI) Or 1,2-dioleoyl-sn-glycerol-3-phosphate-(1′-myo-inositol-3′-4′-5′-triphosphate) (DOPI-P3), or combinations thereof; and / or (iv) The anionic liposome further comprises one or more neutral or zwitterionic lipids, such as cholesterol, or phosphatidylcholine lipids (e.g., 1,2-dipalmitoyl- sn -glycerol-3-phosphatidylcholine (DPPC) or 1,2-distearate-sn-glycerol-3-phosphatidylcholine (DSPC)), or combinations thereof.

14. The composition of claim 13, wherein the composition comprises an aggregate containing a polycationic aggregator (e.g., a cationic dendritic polymer aggregator) and a plurality of anionic liposomes.

15. A kit comprising: a) A first container containing an anionic lipid composition; b) A second container containing a polycationic aggregator; as well as c) Combining a) with b) to provide a description of the composition for use according to any one of claims 1 to 12; Optionally, the first container further includes a pharmaceutically active agent incorporated into the anionic lipid composition.

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