Functionalized branched poly-lysine compounds and uses thereof

Abstract

Disclosed herein are functionalized branched poly-lysine compounds and uses thereof, as well as delivery systems in which polymeric nanocarriers are functionalized with branched poly-lysine compounds. Also provided herein are uses of the functionalized branched poly-lysine compounds and the delivery systems for treating joint diseases such as osteoarthritis, and in delivering pharmaceutically active compounds to cartilage and subchondral bone.

Description

[0001]DUKE-43487.601 FUNCTIONALIZED BRANCHED POLY-LYSINE COMPOUNDS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No.63 / 534,131, filed on August 23, 2023, which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under ARPA-H Agreement No. 1AY2AX000013-01 awarded by the Advanced Research Projects Agency for Health, and under AR079189, AR082809, and AG074491 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD Provided herein are functionalized branched poly-lysine compounds and uses thereof, as well as delivery systems in which polymeric nanocarriers are functionalized with branched poly-lysine compounds. Also provided herein are uses of the functionalized branched poly- lysine compounds and the delivery systems for treating joint diseases such as osteoarthritis, and in delivering pharmaceutically active compounds to cartilage and subchondral bone. BACKGROUND Joint diseases that involve the entire joint, such as osteoarthritis (OA), often require delivery of drugs to chondrocytes residing within the cartilage. Although intra-articular drug delivery has been proposed as a more effective strategy compared to systemic administration, the intra-articular administration of drugs remains a challenge due to their rapid clearance within the joint. While nanocarrier-assisted delivery typically increases the half-life of therapeutic molecules within the joint, the diffusion of nanocarriers into the cartilage tissue is hindered by its dense and negatively charged extracellular matrix (ECM). Drug delivery is further complicated for treatments that target the whole cartilage and / or subchondral bone that require deeper drug penetration. Chondrocytes, situated in the lacunae, are sparsely distributed within the anionic ECM, which is comprised of high-density collagen fibers intertwined with negatively charged proteoglycans. The challenges associated with delivering drugs to chondrocytes residing within the dense cartilage tissue are considered to be a key bottleneck that contributes to the unsuccessful outcomes of OA treatments. DUKE-43487.601 Cationic nanocarriers that form weak electrostatic interactions with the anionic cartilage ECM can be an effective approach to overcome the electrostatic barrier. For an effective therapeutic outcome, the nanocarriers first need to adhere to the cartilage tissue, and then penetrate and be retained throughout the full-thickness cartilage. It is therefore important to develop nanomaterials that can rapidly adhere upon intra-articular administration to prevent clearance in the joint and promote transport across the full-thickness cartilage tissue while maintaining adequate retention within the tissue. Transport of molecules, including nanocarriers, within the cartilage tissue is governed by properties such as size, shape, and surface charge, where the latter strongly influences the cartilage-binding properties of the nanocarriers and their retention within the tissue. An optimal net positive charge that enables weak, reversible interactions with the cartilage tissue is necessary for the nanocarriers to penetrate through the full-thickness cartilage tissue, which is ~2-3 mm for human articular joint cartilage. Additionally, the nanocarriers should be able to support high drug loading such that adequate amounts of drug can be delivered with a lower concentration of cationic materials, which are often cytotoxic. Large amounts of cationic polymers can also alter intra- tissue osmotic pressure which could have detrimental effects to cartilage tissue. Research over the years has led to a number of cationic nanocarriers composed of peptides and proteins, amino acids, liposomes, lipid molecules, or polymers to deliver biomolecules to cartilage tissue and also gene editing, with varying levels of success. SUMMARY In one aspect, disclosed herein is a functionalized branched poly-lysine compound, wherein at least one primary amine group of the branched poly-lysine is functionalized with a pharmaceutically active moiety, a polyalkylene oxide moiety, cholesterol, a monosaccharide, an oligosaccharide, or a zwitterionic molecule. In some embodiments, the branched poly-lysine is a branched poly(L-lysine) compound. In some embodiments, at least one primary amine group of the branched poly- lysine is functionalized with a pharmaceutically active moiety. In some embodiments, at least one primary amine group of the branched poly-lysine is functionalized with a hydrophobic drug moiety. In some embodiments, the pharmaceutically active moiety is selected from glucocorticoids, corticosteroids, analgesics, non-steroidal anti-inflammatories, pain drugs, QSRSHPSRFP FRXNGSINJW% =>&*f FRXFLSRNWXW% D@: NRMNGNXSVW% =>&*f NRMNGNXSVW% TVSXJFWJ inhibitors, eptotermin alfa, sprifermin, insulin growth factors, cell-based therapeutics, gene DUKE-43487.601 therapies, GLP-1 based drugs, antibiotics, and metformin drugs. In some embodiments, the pharmaceutically active moiety is selected from methylprednisolone and adenosine. In some embodiments, the pharmaceutically active moiety is attached to the primary amine group of the branched poly-lysine via a direct bond. In some embodiments, the pharmaceutically active moiety is attached to the primary amine group of the branched poly- lysine via a linker. In some embodiments, about 1% to about 50% of the primary amine groups of the branched poly-lysine are functionalized with the pharmaceutically active moiety. In some embodiments, at least one primary amine group of the branched poly-lysine is functionalized with a polyethylene oxide moiety. In some embodiments, polyethylene oxide moiety is a moiety of formula: wherein m is an integer from 4 to 50. In some embodiments, m is 8. In some embodiments, 10% to about 50% of the primary amine groups of the branched poly-lysine are functionalized with the polyethylene oxide moiety. In some embodiments, the functionalized branched poly-lysine compound has a structure of formula (I): or a salt thereof, wherein: R1is an optionally substituted C1-C12alkyl group; each R2is independently -CH2CH2CH2CH2NHRa, wherein each Rais independently hydrogen, a nitrogen protecting group, a polyalkylene oxide moiety, cholesterol, a monosaccharide, an oligosaccharide, a zwitterionic molecule, or a pharmaceutically active moiety, wherein at least one Rais not hydrogen; each R3is independently hydrogen or a group of formula (A): DUKE-43487.601 each R4is independently hydrogen or a group of formula (B): n1, n2, n3, n4, n5, n6, and n7 are each independently 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, R1is an unsubstituted C2-C8alkyl group. In some embodiments, R1is a group of formula: wherein q is 2, 3, 4, 5, 6, 7, or 8, and X is selected from a group comprising a reactive moiety, a polyalkylene glycol moiety, a sugar, and a peptide. In some embodiments, X is a reactive moiety selected from a thiol, an amine, an azide, an alkyne, an alkene, a dibenzocyclooctyne, a maleimide, and a succinimidyl ester. In some embodiments, each R3is hydrogen. In some embodiments, each R3is a group of formula (A). In some embodiments, each R4is hydrogen. In some embodiments, each R4is a group of formula (B). In some embodiments, each R2is independently -CH2CH2CH2CH2NHRa, wherein each Rais independently hydrogen, a nitrogen protecting group, a polyalkylene oxide moiety, or a pharmaceutically active moiety, wherein at least one Rais not hydrogen. In some embodiments, R2is independently -CH2CH2CH2CH2NHRa, wherein each Rais independently hydrogen, a benzyloxycarbonyl group, a polyethylene oxide moiety, or a hydrophobic drug moiety, wherein at least one Rais not hydrogen. DUKE-43487.601 In some embodiments, at least one Rais a pharmaceutically active moiety. In some embodiments, at least one Rais a hydrophobic drug moiety. In some embodiments, the pharmaceutically active moiety is selected from glucocorticoids, corticosteroids, analgesics, RSR&WXJVSNIFP FRXN&NRKPFQQFXSVNJW% TFNR IVYLW% QSRSHPSRFP FRXNGSINJW% =>&*f FRXFLSRNWXW% D@: NRMNGNXSVW% =>&*f NRMNGNXSVW% TVSXJFWJ NRMNGNXSVW% JTXSXJVQNR FPKF% WTVNKJVQNR% NRWYPNR growth factors, cell-based therapeutics, gene therapies, GLP-1 based drugs, antibiotics, and metformin drugs. In some embodiments, the pharmaceutically active moiety is selected from methylprednisolone and adenosine. In some embodiments, pharmaceutically active moiety is attached via a direct bond. In some embodiments, the pharmaceutically active moiety is attached via a linker. In some embodiments, about 1% to about 50% of the Ragroups are a pharmaceutically active moiety. In some embodiments, at least one Ragroup comprises a polyethylene oxide moiety. In some embodiments, the polyethylene oxide moiety is a moiety of formula: wherein m is an integer from 4 to 50. In some embodiments, m is 8. In some embodiments, about 10% to about 50% of the Ragroups comprise the polyethylene oxide moiety. In another aspect, disclosed herein is 35. A delivery system comprising: a polymeric nanocarrier; a pharmaceutically active compound; and a branched poly-lysine moiety covalently attached to the polymeric nanocarrier. In some embodiments, the branched poly-lysine moiety is a branched poly(L-lysine) moiety. In some embodiments, the polymeric nanocarrier comprises a polyester. In some JQGSINQJRXW% XMJ TSP]JWXJV NW TSP]#PFHXNHhHShLP]HSPNH$ FHNI' =R WSQJ JQGSINQJRXW% XMJ polymeric nanocarrier comprises a copolymer of a polyester and a polyalkylene oxide. In WSQJ JQGSINQJRXW% XMJ HSTSP]QJV NW F GPSHO HSTSP]QJV SK TSP]#PFHXNHhHShLP]HSPNH$ FHNI FRI polyethylene glycol. In some embodiments, the pharmaceutically active compound is selected from glucocorticoids, corticosteroids, analgesics, non-steroidal anti-inflammatories, pain drugs, QSRSHPSRFP FRXNGSINJW% =>&*f FRXFLSRNWXW% D@: NRMNGNXSVW% =>&*f NRMNGNXSVW% TVSXJFWJ DUKE-43487.601 inhibitors, eptotermin alfa, sprifermin, insulin growth factors, cell-based therapeutics, gene therapies, GLP-1 based drugs, antibiotics, and metformin drugs. In some embodiments, the pharmaceutically active compound is selected from methylprednisolone and adenosine. In some embodiments, the pharmaceutically active compound is encapsulated within the polymeric nanocarrier. In some embodiments, the branched poly-lysine moiety has a structure of formula (II): or a salt thereof, wherein: p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each R2is independently -CH2CH2CH2CH2NHRa, wherein each Rais independently hydrogen or a nitrogen protecting group; each R3is independently hydrogen or a group of formula (A): each R4is independently hydrogen or a group of formula (B): DUKE-43487.601 n1, n2, n3, n4, n5, n6, and n7 are each independently 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, p is 2, 3, 4, 5, 6, 7, or 8. In some embodiments, each R2is independently -CH2CH2CH2CH2NHRa, wherein each Rais hydrogen. In some embodiments, each R3is hydrogen. In some embodiments, each R3is a group of formula (A). In some embodiments, each R4is hydrogen. In some embodiments, each R4is a group of formula (B). In one aspect, disclosed herein is a method of treating a joint disease in a subject in need thereof, comprising administering to a joint of the subject a therapeutically effective amount of a functionalized branched poly-lysine compound disclosed herein, or a delivery system disclosed herein. In some embodiments, the joint disease is osteoarthritis. In some embodiments, the administering step comprises intra-articular injection of the functionalized branched poly-lysine compound or the delivery system. In one aspect, disclosed herein is a method of delivering a pharmaceutically active compound to cartilage and / or subchondral bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a functionalized branched poly-lysine compound disclosed herein, or a delivery system disclosed herein. In some embodiments, the subject has a joint disease. In some embodiments, the joint disease is osteoarthritis. In some embodiments, the administering step comprises intra-articular injection of the functionalized branched poly-lysine compound or the delivery system. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 shows a reaction scheme for the synthesis of branched poly (L-lysine) molecules with varying degrees of branching and functional amine groups. FIG.2 shows a histogram of DLS measurements of G0, G1 and G2 BPL molecules. FIGS.3A-3E show data demonstrating uptake of BPL molecules by cartilage explants. FIG.3A: Schematic illustration of the BPL uptake experiment using cartilage DUKE-43487.601 explants. FIG.3B: The uptake percentage of BPL molecules in cartilage explants after 24 hours. FIGS.3C-3E: The uptake of various BPL molecules as a function of time up to 24 hours. FIGS.4A-4C show data demonstrating penetration of BPL molecules within the cartilage explant. FIG.4A: Fluorescence images showing intra-cartilage penetration of various BPL molecules from superficial zone to deep zone (indicated by the arrow) after 6 and 24 hours. Scale bar: 200µm. FIGS.4B and 4C: Quantification of the depth of penetration of different BPL molecules at 6 and 24 hours. FIGS.5A-5F show data demonstrating desorption of BPL molecules from the cartilage explant. FIGS.5A and 5D: Desorption of BPL molecules from the cartilage explants FKXJV - IF]W NR *E B6C SV *)E B6C FX ,0 i' :=;C' .6 FRI .93 DMJ 6B> HSRHJRXVFXNSR NR *E and 10X PBS as a function of time. FIGS.5C and 5F: Comparison of the desorption of G0, G1 and G2 BPL molecules in 1X and 10X PBS as a function of time. FIGS.6A-6D show data demonstrating desorption of BPL molecules from the cartilage explant. FIGS.6A-6B: Comparison of desorption of the BPL molecules based on PEGylation in 1X PBS. FIGS.6C-6D: Comparison of desorption of the BPL molecules based on PEGylation in 10X PBS. FIGS.7A-7B show data demonstrating intra-cartilage equilibrium uptake of BPL molecules. FIG.7A: Representative figure showing the cartilage uptake ratio of G0 BPL at varying concentrations. FIG.7B: Equilibrium uptake ratio (Ru) for all the BPL molecules. FIGS.8A-8D show data demonstrating chondrocyte toxicity of BPL molecules. FIG. 8A: Fluorescence images of the explant following 24 hours of culture in chondrocyte medium supplemented with 11.5µM of BPL molecules. Green: live cells. Red: dead cells. Scale bar: 100 µm. FIG.8B: Quantification of cell viability from the live / dead images. FIG.8C: Safranin-O stained cartilage explants cultured in chondrocyte media (CM) with or without G2HP BPL molecules at a concentration of 11.5 µM. Scale bar: 100 µm. FIG.8D: Glycosaminoglycan (GAG) content of the cartilage explants. FIG.9 shows a graph of the percentage of viable cells in cartilage explants after 24 hours of incubation with BPL molecules in chondrocyte media. C: Control (i.e., Cartilage explants cultured in chondrocyte medium without the presence of BPL molecules). FIGS.10A-10G show data demonstrating BPL uptake by the healthy and cytokine challenged cartilage explants. FIGS.10A-10B: BPL uptake by the healthy and OA-mimetic cartilage explants at 6 and 24 hours post-incubation. FIG.10C: Total BPL uptake by the OA- mimetic cartilage explants at 6 and 24 hours post-incubation. FIGS.10D-10E: Penetration DUKE-43487.601 depth of BPL molecules within the healthy and OA-mimetic cartilage explants after 6 and 24 hours. FIG.10F: Penetration depth of BPL molecules within the OA-mimetic cartilage explants after 6 and 24 hours. FIG.10G: Fluorescent images of G2 and G2HP BPL molecules within the OA-mimetic cartilage explants (Blue: DAPI, Purple: Cy5; Scale bar: 200 µm). FIGS.11A-11C show data comparing PEGylated and non-PEGylated BPL molecules. FIGS.11A and 11C: Non-PEGylated G2-BPL molecules showed aggregation in 1X PBS supplemented with synovial fluid. FIG.11B: PEGylation limited the aggregation of the nanocarriers. Scale bar: 50 µm. FIGS.12A-12D show data demonstrating that PEGylated BPL molecules show joint residence and cartilage penetration in rodents. FIGS.12A-12B: Fluorescence images of the mouse knee joint following intra-articular injection of Cy5-G2HP BPL molecules with magnified images shown in FIG.12B. Scale bars: 200 µm. FIG.12C: Representative IVIS images of the rat knee joints over 28 days following intra-articular injection of Cy7- conjugated G2HP BPL molecules. FIG.12D: Time course of fluorescent radiant efficiency of the BPL within the joints. Data are means ± 95% CIs of nonlinear fit, n = 4 joints per formulation. FIGS.13A-13C show standard calibration curves for: (FIG.13A) methylprednisolone, and (FIG.13B) G2HP conjugated with methylprednisolone in PBS at concentrations of 2.5-50 µg / mL and 15.625-500 µg / mL respectively obtained via UV / Vis absorbance spectroscopy (pmax ^255 nm). FIG.13C: standard calibration curve for methylprednisolone in PBS with 10% FBS. At concentrations of 2.5-50 µg / mL obtained via UV / Vis absorbance spectroscopy (pmax^260 nm). FIGS.14A-14B show data for release of methylprednisolone. FIG.14A: Release of methylprednisolone from the G2HP BPL molecules in PBS. FIG.14B: Release of methylprednisolone from the G2HP BPL molecules in PBS and PBS with 10% FBS. FIG.15 shows data demonstrating penetration of PLGA-BPL nanoparticles within cartilage explants. Fluorescence images showing intra-cartilage penetration of various PLGA- BPL nanoparticles from the superficial zone to the deep zone (indicated by the arrow) after +- FRI -1 MSYVW' CHFPJ GFV3.)) gQ' FIG.16 shows data demonstrating penetration of PLGA nanoparticle within the human cartilage explant. Fluorescence images showing intra-cartilage penetration of various PLGA-BPL nanoparticles from the superficial zone to the deep zone (indicated by the arrow) FKXJV +-% -1 M% FRI 0+ MSYVW' CHFPJ GFV3.)) gQ' DUKE-43487.601 FIGS.17A-17B show hydrodynamic size (FIG.17A) and zeta potential (FIG.17B) of the PLGA-BPL and PLGA-BPL loaded with hydrophobic drugs. FIG.18 shows data demonstrating penetration of BPL-PBA nanoparticles within the cartilage explant. Fluorescence images show intra-cartilage penetration of various BPL-PBA nanoparticles from the superficial zone to the deep zone (indicated by the arrow) after 12 MSYVW' CHFPJ GFV3 +)) gQ' FIG.19 shows data demonstrating a dose-dependent effect of adenosine on chondrogenesis of primary human osteoarthritic (OA) chondrocytes. FIGS.20A-20C show characterization data for therapeutic loaded sugar glass micelles. Hydrodynamic diameter of: (FIG.20A) PTH-loaded sugar glass micelles; (FIG. 20B) protein 2-loaded sugar glass micelles; (FIG.20C) Protein 3-loaded sugar glass micelles. FIG.21 shows PTH content in sugar glass micelles determined via PTH (1-34) enzyme-linked immunosorbent assay. FIG.22 shows data from determination of PTH bioactivity via the measurement of intracellular cAMP content. DETAILED DESCRIPTION Disclosed herein are branched poly-lysine (e.g., branched poly(L-lysine)) based cationic nanocarriers of different generations with varying numbers of amine groups and charge density. The branched poly-lysine (BPL) molecules (e.g., branched poly(L-lysine)) have a hydrodynamic diameter of ~40^20 nm and a dense surface functionality ranging from 30 to 150 primary amines. For example, a second generation (G2) BPL molecule provides ~150 functional amine groups. Employing an ex vivo explant culture, the effect of surface charge and molecular architecture (e.g., number of branches) on BPL uptake, penetration, retention, and desorption into and from cartilage have been evaluated, using both healthy and cytokine challenged cartilage explants. Furthermore, drug conjugation to the BPL molecule and release therefrom are disclosed herein. Additionally, in vivo retention of BPL molecules using both mouse knee joint explants and a rat knee injury model suggest that the molecules localize to the cartilage within 24 hours and can be retained within the rat knee joint for at least 14 days. The primary amine groups of the BPL molecules disclosed herein can be functionalized with a variety of groups, including polymers such as polyalkylene oxides (e.g., polyethylene glycol), cholesterol, monosaccharides, oligosaccharides, and zwitterionic molecules. Such modification can modulate the surface charge, density, charge screening, DUKE-43487.601 shape, and size of the compounds, which can improve the biocompatibilities and cytotoxicities of the compounds. The primary amine groups can also be functionalized with a variety of pharmaceutically active moieties, and such functionalized BPL molecules can release the pharmaceutically active moieties following administration to a subject (e.g., via intraarticular injection). Additionally, the BPL molecules can be attached to other drug delivery vehicles, such as polymeric nanocarriers, to facilitate their uptake into cartilage and subchondral bone. Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. For the purpose of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates. Articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element. “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result. The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. As used herein, “and / or” refers to and encompasses any and all DUKE-43487.601 possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”). As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.” Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. As used herein, “treatment,” “therapy” and / or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and / or the remission of the disease, disorder or condition. As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disease, disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder or condition. The term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results. As used herein, the term “administering” an agent, such as a therapeutic entity to an animal or cell, is intended to refer to dispensing, delivering or applying the substance to the intended DUKE-43487.601 target. In terms of the therapeutic agent, the term “administering” is intended to refer to contacting or dispensing, delivering or applying the therapeutic agent to a subject by any suitable route for delivery of the therapeutic agent to the desired location in the animal, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, and administration by the intranasal or respiratory tract route. The term “disease” as used herein includes, but is not limited to, any abnormal condition and / or disorder of a structure or a function that affects a part of an organism. It may be caused by an external factor, such as an infectious disease, injury, etc. or by internal dysfunctions, such as cancer, cancer metastasis, and the like. In some embodiments, the disease comprises joint disease. Examples of joint disease include, but are not limited to, osteoarthritis, rheumatoid arthritis, bursitis, and the like. As used herein, the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals. The term “nonhuman animals” of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. The methods and compositions disclosed herein can be used on a sample either in vitro (for example, on isolated cells or tissues) or in vivo in a subject (i.e., living organism, such as a patient). In some embodiments, the subject comprises a human who is undergoing treatment using an approach as prescribed herein. Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Sorrell, Organic Chemistry, 2ndedition, University Science Books, Sausalito, 2006; Smith, March’s Advanced Organic Chemistry: Reactions, Mechanism, and Structure, 7thEdition, John Wiley & Sons, Inc., New York, 2013; Larock, Comprehensive Organic Transformations, 3rdEdition, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference. DUKE-43487.601 As used herein, the term “alkyl” refers to a radical of a straight or branched saturated hydrocarbon chain. The alkyl chain can include, e.g., from 1 to 24 carbon atoms (C1-C24 alkyl), 1 to 16 carbon atoms (C1-C16 alkyl), 1 to 14 carbon atoms (C1-C14 alkyl), 1 to 12 carbon atoms (C1-C12alkyl), 1 to 10 carbon atoms (C1-C10alkyl), 1 to 8 carbon atoms (C1-C8alkyl), 1 to 6 carbon atoms (C1-C6alkyl), 1 to 4 carbon atoms (C1-C4alkyl), 1 to 3 carbon atoms (C1-C3alkyl), or 1 to 2 carbon atoms (C1-C2alkyl). Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso- butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl. As used herein, the term “nitrogen protecting group” refers to a group intended to protect an amino group against undesirable reactions during synthetic procedures. Common nitrogen protecting groups include acyl groups such as acetyl, benzoyl, 2-bromoacetyl, 4- bromobenzoyl, tert&GYX]PFHJX]P% HFVGS\FPIJM]IJ% +&HMPSVSFHJX]P% -&HMPSVSGJR^S]P% c& chlorobutyryl, 4-nitrobenzoyl, o-nitrophenoxyacetyl, phthalyl, pivaloyl, propionyl, trichloroacetyl, and trifluoroacetyl; sulfonyl groups such as benzenesulfonyl and p- toluenesulfonyl; carbamate-forming groups such as benzyloxycarbonyl (Cbz), tert- butyloxycarbonyl (Boc), p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, fluorenylmethyloxycarbonyl (Fmoc); and the like. Nitrogen protecting groups are well- known in the art and include those described in detail in Greene’s Protective Groups in Organic Synthesis, P. G. M. Wuts, 5th edition, John Wiley & Sons, Inc., 2014, which is incorporated herein by reference. As used herein, the term “pharmaceutically active moiety” refers to a radical of a pharmaceutically active compound, such as a drug compound. When a group or moiety can be substituted, the term “substituted” indicates that one or more (e.g., 1, 2, 3, 4, 5, or 6; in some embodiments 1, 2, or 3; and in other embodiments 1 or 2) hydrogens on the group indicated in the expression using “substituted” can be replaced with a selection of recited indicated groups or with a suitable substituent group known to those of skill in the art (e.g., one or more of the groups recited below), provided that the designated atom’s normal valence is not exceeded. Substituent groups include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, cycloalkyl, cycloalkenyl, guanidino, halo, haloalkyl, haloalkoxy, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, phosphate, phosphonate, sulfonic acid, thiol, thione, or combinations thereof. As used herein, in chemical structures the indication: DUKE-43487.601 represents a point of attachment of one moiety to another moiety (e.g., a substituent group to the rest of the compound). When groups are specified by their conventional chemical formulae, written from left to right, such indication also encompass substituent groups resulting from writing the structure from right to left. For example, if a bivalent group is shown as -CH2O-, such indication also encompasses -OCH2-; similarly, -OC(O)NH- also encompasses -NHC(O)O-. When linker moieties are shown, the linkers can be attached to other moieties of the compound in either direction. Branched Poly-lysine Compounds Disclosed herein are branched poly-lysine compounds, including branched poly(L- lysine) compounds, that are functionalized on their primary amine groups with a variety of moieties such as pharmaceutically active compounds, polyalkylene oxide moieties, cholesterol, monosaccharides, oligosaccharides, and zwitterionic molecules. The pharmaceutically active compounds can be released from the branched poly-lysine compounds following administration to a subject to allow for, e.g., local delivery of the pharmaceutically active compound. Other functional groups, such as polyalkylene oxide moieties, cholesterol, monosaccharides, oligosaccharides, and zwitterionic molecules, can modulate the surface charge, density, charge screening, shape, and size of the compounds. Such modifications can improve the biocompatibilities and cytotoxicities of the compounds. In some embodiments, at least one primary amine group of the branched poly-lysine is functionalized with a pharmaceutically active compound. For example, in some embodiments, at least one primary amine group of the branched poly-lysine is functionalized with a hydrophobic drug moiety. The pharmaceutically active moiety can be selected from, e.g., glucocorticoids, corticosteroids, analgesics, non-steroidal anti-inflammatories, pain IVYLW% QSRSHPSRFP FRXNGSINJW% =>&*f FRXFLSRNWXW% D@: NRMNGNXSVW% =>&*f NRMNGNXSVW% TVSXJFWJ inhibitors, eptotermin alfa, sprifermin, insulin growth factors, cell-based therapeutics, gene therapies, GLP-1 based drugs, antibiotics, and metformin drugs, or any combination thereof. For example, in some embodiments, the pharmaceutically active compound is a glucocorticoid, a corticosteroid, or a non-steroidal anti-inflammatory. In some embodiments, the pharmaceutically active compound is a glucocorticoid such as methylprednisolone. In other embodiments, the pharmaceutically active compound is adenosine. DUKE-43487.601 In some embodiments, about 1% to about 50% of the primary amine groups of the branched poly-lysine are functionalized with the pharmaceutically active compound. For example, in some embodiments, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50% of the primary amine groups of the branched poly-lysine are functionalized with the pharmaceutically active compound, or any range therebetween. In some embodiments, at least one primary amine group of the branched poly-lysine is functionalized with a charge-screening moiety, which can be any group that effectively reduces the overall net positive charge associated with the primary amine groups on the branched poly-lysine when they are in their protonated forms. Exemplary charge-screening moieties include a polyalkylene oxide moiety, cholesterol, a monosaccharide, an oligosaccharide, or a zwitterionic molecule. For example, in some embodiments, at least one primary amine group of the branched poly-lysine is functionalized with a polyalkylene oxide moiety, such as a polyethylene oxide moiety. In exemplary embodiments, the polyethylene oxide moiety is a moiety of formula: wherein m is an integer from 4 to 50, e.g., m is 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, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In an exemplary embodiment, m is 8. In some embodiments, about 10% to about 50% of the primary amine groups of the branched poly-lysine are functionalized with the charge-screening moiety. For example, in some embodiments, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50% of the primary amine groups of DUKE-43487.601 the branched poly-lysine are functionalized with the charge-screening moiety, or any range therebetween. The pharmaceutically active compound and the charge-screening moiety, when present, can be attached to the primary amine groups of the branched poly-lysine compound either via a direct bond or via a linker. For example, if the pharmaceutically active compound or the charge-screening moiety comprises a -COOH group, a straightforward amide coupling reaction can be performed to link the compound to the branched poly-lysine via an amide bond. In other embodiments, the pharmaceutically active compound or the charge-screening moiety can be attached to the branched poly-lysine compound via a linker. In such embodiments, the linker can react on one side with a primary amine group of the branched poly-lysine compound, and on the other side with an appropriate functional group on the pharmaceutically active compound. Such methods of conjugation will be well-understood by those skilled in the art. An exemplary embodiment is illustrated in the Examples, in which 6- alpha-methylprednisolone is functionalized with succinic acid as shown in Scheme 1. The carboxylic acid moiety of the succinic acid group is then available for an amide coupling reaction with a free amine group of the branched poly-lysine. Similar reactions can be used for other pharmaceutically active compounds. Scheme 1 In embodiments in which a linker is used to link the pharmaceutically active compound to the branched poly-lysine, any suitable linker can be used. In embodiments, the linker comprises any combination of -CH2-, -CH(CH3)-, -C(CH3)2-, -CH=CH-, -Cd7-, -O-, - NR'-, -S-, -C(O)-, -C(S)-, -C(NR')-, -S(O)-, -S(O)2-, arylene, heteroarylene, cycloalkylene, and heterocyclylene moieties, wherein the arylene, heteroarylene, cycloalkylene, and heterocyclylene moieties are independently unsubstituted or substituted with 1, 2, or 3 substituents. In some embodiments, the linker comprises any combination of -CH2-, -O-, - C(O)-, and -NH- moieties. In some embodiments, the linker comprises a group that results DUKE-43487.601 from the reaction of two complementary reactive groups. For example, reaction of a thiol and a maleimide produces a group shown below, and such a group is accordingly part of the linker: . The branched poly-lysine compound can be prepared by methods known in the art for synthesizing such polymers. In certain embodiments, the branched poly-lysine compound can be prepared according to a general method known in the art (see Rodríguez-Hernández et al. Biomacromolecules 2003, 4(2), 249-258). In some embodiments, the branched poly-lysine compound, prior to conjugation of any pharmaceutically active compound or charge- screening moiety, has a structure of formula (III): or a salt thereof, wherein: R1is an optionally substituted C1-C12 alkyl group; each R2is independently -CH2CH2CH2CH2NHRa, wherein each Rais independently hydrogen or a nitrogen protecting group; each R3is independently hydrogen or a group of formula (A): each R4is independently hydrogen or a group of formula (B): DUKE-43487.601 n1, n2, n3, n4, n5, n6, and n7 are each independently 5, 6, 7, 8, 9, 10, 11, or 12. As illustrated in more detail in the Examples, branched poly(L-lysine) compounds of formula (III) can be synthesized by ring-opening polymerization reaction using Ni- benzyloxycarbonyl-L-lysine-N-carboxyanhydride (Cbz-Lys-NCA) as a monomer and an initiator with primary amine group(s). First, a lysine oligomer containing a certain number of lysine units, such as 10 lysine units (i.e., n1 = 10), termed as the core peptide, can be synthesized using Cbz-Lys-NCA and an alkylamine, such as hexylamine (i.e., R1is n-hexyl). Three generations of BPL molecules— generation 0 (G0) with two branches (i.e., each R3is hydrogen), generation 1 (G1) with four branches (i.e., each R3is a group of formula (A) and each R4is hydrogen), and generation 2 (G2) with eight branches (i.e., each R3is a group of formula (A) and each R4is a group of formula (B)), can then be synthesized by reacting the Cbz-Lys-NCA with either the core peptide, G0, or G1 as initiators to generate G0, G1, or G2 BPL molecules, respectively (FIG.1). Increasing the number of branches provides multivalent BPL nanocarriers with increasing functional groups and charge density without significantly increasing their hydrodynamic diameter. A theoretical calculation, based on the monomer-to-initiator feed ratio, suggests that the G0, G1, and G2 BPL molecules possess approximately 30, 70, and 150 functional amine groups, respectively (Table 1). Table 1. Theoretical characterization of branched poly(L-lysine) molecules assuming each arm contains 10 lysine repeating units. DUKE-43487.601 In the compounds of formula (III), complete deprotection of the nitrogen protecting groups (Cbz in this example) results in a compound in which each R2is - CH2CH2CH2CH2NHRaand Rais hydrogen. In the event that deprotection is incomplete, however, the compound of formula (III) may include a certain number of remaining nitrogen protecting groups at Ra(e.g., carboxybenzyl groups). The branched poly-lysine compound of formula (III) can be further functionalized with a pharmaceutically active compound and / or charge-screening moieties as discussed above. In such embodiments, the branched poly-lysine compound will be a functionalized branched poly-lysine compound having a structure of formula (I): or a salt thereof, wherein: R1is an optionally substituted C1-C12alkyl group; each R2is independently -CH2CH2CH2CH2NHRa, wherein each Rais independently hydrogen, a nitrogen protecting group, a polyalkylene oxide moiety, cholesterol, a monosaccharide, an oligosaccharide, a zwitterionic molecule, or a pharmaceutically active moiety, wherein at least one Rais not hydrogen; each R3is independently hydrogen or a group of formula (A): each R4is independently hydrogen or a group of formula (B): DUKE-43487.601 n1, n2, n3, n4, n5, n6, and n7 are each independently 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, R1is an unsubstituted C2-C8 alkyl group. In some embodiments, R1is a group of formula: wherein q is 2, 3, 4, 5, 6, 7, or 8, and X is selected from a group comprising a reactive moiety, a polyalkylene glycol moiety, a sugar, and a peptide. In some embodiments, X is a reactive moiety is selected from a thiol, an amine, an azide, an alkyne, an alkene, a dibenzocyclooctyne, a maleimide, and a succinimidyl ester. In such embodiments, the functionalized branched poly-lysine compound of formula (I) will include a reactive group that can be used to covalently attach the compound to another compound, such as a drug nanocarrier as further described below. In some embodiments, R1is a C2-C8alkyl group that allows for non-covalent interaction with another compound (e.g., a drug nanocarrier), such as via an electrostatic interaction, hydrogen bonding, or a hydrophobic interaction. In some embodiments, when R1is a C1-C12 alkyl group (e.g., a C2-C8 alkyl group) that is substituted with a thiol, the thiol groups of two molecules of formula (I) may be taken together to form a disulfide bond. In such embodiments, the compound of formula (I) would be a compound of formula (Ia): wherein the variables are described as above for formula (I), and y is 1-12 (e.g., 2-8). DUKE-43487.601 In some embodiments, in the functionalized branched poly-lysine compound of formula (I), each R3is hydrogen (i.e., the functionalized branched poly-lysine compound is a G0 BPL compound). In other embodiments, in the functionalized branched poly-lysine compound of formula (I), each R3is a group of formula (A) and each R4is hydrogen (i.e., the functionalized branched poly-lysine compound is a G1 BPL compound). In other embodiments, in the functionalized branched poly-lysine compound of formula (I), each R3is a group of formula (A) and each R4is a group of formula (B) (i.e., the functionalized branched poly-lysine compound is a G2 BPL compound). In some embodiments, in the functionalized branched poly-lysine compound of formula (I), each R2is independently -CH2CH2CH2CH2NHRa, wherein each Rais independently hydrogen, a nitrogen protecting group, a polyalkylene oxide moiety, or a pharmaceutically active moiety, wherein at least one Rais not hydrogen. In some embodiments, in the functionalized branched poly-lysine compound of formula (I), each R2is independently -CH2CH2CH2CH2NHRa, wherein each Rais independently hydrogen, a benzyloxycarbonyl group, a polyethylene oxide moiety, or a hydrophobic drug moiety, wherein at least one Rais not hydrogen. For example, in some embodiments, at least one Rais a pharmaceutically active moiety, such as a hydrophobic drug moiety. In such embodiments, the compounds of formula (I) can be used to deliver the pharmaceutically active compounds to a site of interest (e.g., a joint), as the pharmaceutically active compounds can be released via hydrolysis or enzymatic cleavage. The pharmaceutically active moiety can be a moiety derived from any one of a number of pharmaceutically active compounds including, for example, glucocorticoids, corticosteroids, analgesics, non-steroidal anti-inflammatories, pain drugs, monoclonal FRXNGSINJW% =>&*f FRXFLSRNWXW% D@: NRMNGNXSVW% =>&*f NRMNGNXSVW% TVSXJFWJ NRMNGNXSVW% eptotermin alfa, sprifermin, insulin growth factors, cell-based therapeutics, gene therapies, GLP-1 based drugs, antibiotics, and metformin drugs. In some embodiments, the pharmaceutically active moiety is methylprednisolone or adenosine. In some embodiments, about 1% to about 50% of the Ragroups in the compound of formula (I) are a pharmaceutically active moiety. For example, in some embodiments, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, DUKE-43487.601 about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50% of the Ragroups are a pharmaceutically active moiety, or any range therebetween. In some embodiments, at least one is a charge-screening moiety, which can be any group that effectively reduces the overall net positive charge associated with the primary amine groups on the branched poly-lysine when they are in their protonated forms. Exemplary charge-screening moieties include a polyalkylene oxide moiety, cholesterol, a monosaccharide, an oligosaccharide, or a zwitterionic molecule. For example, in some embodiments, at least one Rais a polyalkylene oxide moiety, such as a polyethylene oxide moiety. In exemplary embodiments, the polyethylene oxide moiety is a moiety of formula: wherein m is an integer from 4 to 50, e.g., m is 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, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In an exemplary embodiment, m is 8. In some embodiments, about 10% to about 50% of the Ragroups are a charge- screening moiety. For example, in some embodiments, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50% of the Ragroups are a charge-screening moiety such as a polyalkylene oxide moiety, or any range therebetween. The pharmaceutically active compound and the charge-screening moiety can be attached to the primary amine groups of the branched poly-lysine compound either via a direct bond or via a linker, as discussed above. The present disclosure also includes isotopically-labeled compounds, which are identical to those recited in the formulae disclosed herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds of the invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, but not limited to2H,3H,13C,14C,15N, DUKE-43487.60118O,17O,31P,32P,35S,18F, and36Cl, respectively. Substitution with heavier isotopes such as deuterium (2H) can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using appropriate isotopically- labeled reagent in place of non-isotopically-labeled reagent. Compounds disclosed herein can exist in solvated as well as unsolvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the disclosure encompass both solvated and unsolvated forms. In one embodiment, the compound is amorphous. In one embodiment, the compound is a single polymorph. In another embodiment, the compound is a mixture of polymorphs. In another embodiment, the compound is in a crystalline form. Compounds and intermediates may be isolated and purified by methods well-known to those skilled in the art of organic synthesis. Examples of conventional methods for isolating and purifying compounds can include, but are not limited to, chromatography on solid supports such as silica gel, alumina, or silica derivatized with alkylsilane groups, by recrystallization at high or low temperature with an optional pretreatment with activated carbon, thin-layer chromatography, distillation at various pressures, sublimation under vacuum, and trituration, as described for instance in “Vogel's Textbook of Practical Organic Chemistry,” 5th edition (1989), by Furniss, Hannaford, Smith, and Tatchell, pub. Longman Scientific & Technical, Essex CM202JE, England. Reaction conditions and reaction times for each individual step can vary depending on the particular reactants employed and substituents present in the reactants used. Reactions can be worked up in a conventional manner, e.g., by eliminating the solvent from the residue and further purified according to methodologies generally known in the art such as, but not limited to, crystallization, distillation, extraction, trituration and chromatography. Unless otherwise described, the starting materials and reagents are either commercially available or can be prepared by one skilled in the art from commercially available materials using methods described in the chemical literature. Standard experimentation, including appropriate manipulation of the reaction conditions, reagents and sequence of the synthetic route, protection of any chemical functionality that cannot be compatible with the reaction conditions, and deprotection at a suitable point in the reaction sequence of the method are included in the scope of the DUKE-43487.601 disclosure. Suitable protecting groups and the methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which can be found in PGM Wuts and TW Greene, in Greene's book titled Protective Groups in Organic Synthesis (4thed.), John Wiley & Sons, NY (2006). The synthetic schemes and specific examples as described are illustrative and are not to be read as limiting the scope of the disclosure or the claims. Alternatives, modifications, and equivalents of the synthetic methods and specific examples are contemplated. The branched poly-lysine compounds disclosed herein may exist as pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” refers to salts or zwitterions of the compounds which are water or oil-soluble or dispersible, suitable for treatment of disorders without undue toxicity, irritation, and allergic response, commensurate with a reasonable benefit / risk ratio and effective for their intended use. The salts may be prepared during the final isolation and purification of the compounds or separately by reacting an amino group of the compound with a suitable acid. For example, a compound may be dissolved in a suitable solvent, such as but not limited to methanol and water, and treated with at least one equivalent of an acid, such as hydrochloric acid. The resulting salt may precipitate out and be isolated by filtration and dried under reduced pressure. Alternatively, the solvent and excess acid may be removed under reduced pressure to provide a salt. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, glutamate, para-toluenesulfonate, undecanoate, hydrochloric, hydrobromic, sulfuric, phosphoric, and the like. Amino groups of the compounds may also be quaternized with alkyl chlorides, bromides and iodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl and the like. Delivery Systems The branched poly-lysine compounds (e.g., branched poly(L-lysine) compounds) disclosed herein can also be used in delivery systems, e.g., systems for delivery of pharmaceutically active compounds. In such embodiments, the branched poly-lysine compounds themselves need not be functionalized with a pharmaceutically active moiety or a charge-screening moiety, although such functionalized branched poly-lysine compounds DUKE-43487.601 could be used in such embodiments in desired. However, in certain embodiments, the branched poly-lysine compounds can be used directly to modify drug nanocarriers. In some embodiments, such modification can facilitate the adhesion of the drug nanocarriers to cartilage tissue and penetration across full-thickness cartilage. Accordingly, disclosed herein is a delivery system, comprising: a polymeric nanocarrier; a pharmaceutically active compound; and a branched poly-lysine moiety covalently attached to the polymeric nanocarrier. The polymeric nanocarrier can be any polymer used in the art for drug nanocarriers. For example, in some embodiments, the polymeric nanocarrier comprises a polyester. In WSQJ JQGSINQJRXW% XMJ TSP]JWXJV NW TSP]#PFHXNHhHShLP]HSPNH$ FHNI #B>;5$% [MNHM NW [JPP& known in the art as a nanocarrier. In other embodiments, copolymers can be used as drug nanocarriers. For example, in some embodiments, the polymeric nanocarrier comprises a copolymer of a polyester and a polyalkylene oxide. In some embodiments, the polymeric RFRSHFVVNJV HSQTVNWJW F HSTSP]QJV #J'L'% F GPSHO HSTSP]QJV$ TSP]#PFHXNHhHShLP]HSPNH$ FHNI and polyethylene glycol. In some embodiments, the delivery system includes a pharmaceutically active compound selected from glucocorticoids, corticosteroids, analgesics, non-steroidal anti- NRKPFQQFXSVNJW% TFNR IVYLW% QSRSHPSRFP FRXNGSINJW% =>&*f FRXFLSRNWXW% D@: NRMNGNXSVW% =>&*f inhibitors, protease inhibitors, eptotermin alfa, sprifermin, insulin growth factors, cell-based therapeutics, gene therapies, GLP-1 based drugs, antibiotics, and metformin drugs. In some embodiments, the pharmaceutically active compound is selected from methylprednisolone and adenosine. In some embodiments, the pharmaceutically active compound is encapsulated within the polymeric nanocarrier. In the delivery systems, the branched poly-lysine moiety can have a structure of formula (II): DUKE-43487.601 or a salt thereof, wherein: p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each R2is independently -CH2CH2CH2CH2NHRa, wherein each Rais independently hydrogen or a nitrogen protecting group; each R3is independently hydrogen or a group of formula (A): each R4is independently hydrogen or a group of formula (B): n1, n2, n3, n4, n5, n6, and n7 are each independently 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, p is 4, 5, 6, 7, or 8. In some embodiments, each R2is -CH2CH2CH2CH2NHRa, wherein each Rais hydrogen. DUKE-43487.601 In some embodiments, each R3 is hydrogen (i.e., the branched poly-lysine compound is a G0 branched poly-lysine compound). In some embodiments, in the branched poly-lysine moiety of formula (II), each R3is hydrogen (i.e., the branched poly-lysine moiety is a G0 BPL moiety). In other embodiments, in the branched poly-lysine moiety of formula (II), each R3is a group of formula (A) and each R4is hydrogen (i.e., the branched poly-lysine moiety is a G1 BPL moiety). In other embodiments, in the branched poly-lysine moiety of formula (II), each R3is a group of formula (A) and each R4is a group of formula (B) (i.e., the branched poly-lysine moiety is a G2 BPL moiety). The branched poly-lysine moiety of formula (II) can be covalently attached to the polymeric nanocarrier via a direct bond or via a linker, such as any linker described herein. For example, the branched poly-lysine moiety of formula (II) can be covalently attached to the polymeric nanocarrier by reacting a polymer of the polymeric nanocarrier (e.g., PLGA) having a reactive moiety, with a precursor compound to the moiety of formula (II) that comprises a complementary reactive moiety. Such reactions and linking groups have been described herein; any such linkers and reactive groups can be used in this context as well. The polymeric nanocarrier can be further functionalized which additional functional groups, in addition to the branched poly-lysine moiety. For example, in some embodiments, the polymeric nanocarrier can be further functionalized with a compound that binds to bone, such as alendronate. In some embodiments, the delivery system further comprises additional components for facilitating loading of pharmaceutically active compounds into the nanocarrier. For example, in the case of protein-based pharmaceuticals, the proteins can be incorporated into sugar glass micelles for loading into the nanoparticles. C. Pharmaceutical Compositions and Modes of Administration In another aspect, the present disclosure further provides compositions comprising a compound or a nanocarrier as described herein and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered DUKE-43487.601 tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, phosphate buffers (e.g., monobasic sodium phosphate and / or dibasic sodium phosphate), magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The exact nature of the carrier will depend upon the desired use for the composition, and may range from being suitable or acceptable for veterinary uses to being suitable or acceptable for human use. Techniques and formulations may generally be found in “Remington’s Pharmaceutical Sciences,” (Meade Publishing Co., Easton, Pa.). In some embodiments, the compound or delivery system is formulated for intra- articular injection. For example, in some embodiments, useful injectable preparations include sterile suspensions, solutions or emulsions of the compounds or delivery systems in aqueous or oily vehicles. The compounds and delivery systems, and / or pharmaceutical compositions thereof as provided herein, may also contain formulating agents, such as a suspending, stabilizing and / or dispersing agent. The formulations for injection may be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives. Alternatively, the injectable formulation may be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer (e.g., phosphate-buffered saline), dextrose solution, etc., before use. The amount of compounds and / or pharmaceutical compositions thereof as provided herein administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular compounds and / or pharmaceutical compositions thereof as provided herein, the conversation rate and efficiency into active drug compounds and / or pharmaceutical compositions thereof as provided herein under the selected route of administration, etc. Determination of an effective dosage of compounds and / or pharmaceutical compositions thereof as provided herein for a particular use and mode of administration is DUKE-43487.601 well within the capabilities of those skilled in the art. Effective dosages may be estimated initially from in vitro activity and metabolism assays. For example, an initial dosage of compounds and / or pharmaceutical compositions thereof as provided herein for use in animals may be formulated to achieve a circulating blood or serum concentration of the metabolite active compound that is at or above an IC50of the particular compound as measured in as in vitro assay. Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular compounds and / or pharmaceutical compositions thereof as provided herein via the desired route of administration is well within the capabilities of skilled artisans. Initial dosages of compounds and / or pharmaceutical compositions thereof as provided herein can also be estimated from in vivo data, such as animal models. Animal models useful for testing the efficacy of the active metabolites to treat or prevent the various diseases, disorders or physiological conditions described above are well-known in the art. Animal models suitable for testing the bioavailability and / or metabolism of compounds and / or pharmaceutical compositions thereof as provided herein into active metabolites are also well-known. Ordinarily skilled artisans can routinely adapt such information to determine dosages of particular compounds suitable for human administration. E. Methods of Use The compounds, delivery systems, and pharmaceutical compositions disclosed herein have many features and uses, including but not limited to, uses in treating joint diseases such as osteoarthritis. In an aspect, the disclosure provides a method of treating a joint disease in a subject, the method comprising administering to the joint of the subject a therapeutically effective amount of a compound or delivery system disclosed herein. In some embodiments, the joint disease is selected from osteoarthritis, gout, rheumatoid arthritis, bursitis, tendonitis, infectious arthritis, bone injury, bone degeneration, bone infection, and ankylosing spondylitis. In some embodiments, the joint disease is osteoarthritis. Additionally, as disclosed herein, the presently claimed compounds and delivery systems facilitate uptake into cartilage and subchondral bone. The compounds and delivery systems can therefore be used in a method of delivering a pharmaceutically active compound to cartilage and / or subchondral bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound or delivery system disclosed herein. In some such embodiments, the subject has a joint disease selected from osteoarthritis, DUKE-43487.601 gout, rheumatoid arthritis, bursitis, tendonitis, infectious arthritis, bone injury, bone degeneration, bone infection, and ankylosing spondylitis. In some embodiments, the subject has osteoarthritis. The following Examples are provided by way of illustration and not by way of limitation. Examples All in vitro experiments were repeated at least twice and reproduced, where each individual experiment had a sample size of at least 3. All numerical data are expressed as means plus or minus standard error of the mean. Data were subjected to one-way analysis of variance (ANOVA with post hoc Tukey-Kramer test for multiple comparisons), or two-way analysis of variance (ANOVA with post hoc Bonferroni test for multiple comparisons). P-values of less than 0.05 were considered statistically significant and indicated with an asterisk. For each figure, a table corresponding to their P values and corresponding significance are provided in the supplementary information. All statistical analyses were performed with GraphPad Prism 10.0.0. Example 1 Synthesis and Characterization of Branched Poly(L-Lysine) Molecules The branched poly(L-lysine) (BPL) molecules were synthesized via ring-opening polymerization of Ni-benzyloxycarbonyl-L-lysine-N-carboxyanhydride (Cbz-Lys-NCA) following a series of reactions as shown in the reaction scheme (FIG.1) (Rodríguez-Hernández et al. Biomacromolecules 2003, 4(2), 249-258). Synthesis of each compound / intermediate product are described in detail below. Synthesis of N -benzyloxycarbonyl-L-lysine-N-carboxyanhydride (Cbz-Lys-NCA): Cbz-Lys-NCA was synthesized by reacting Ni-benzyloxycarbonyl-L-lysine (Cbz-Lys) #7FX' RS' 2 / 1-)4 CNLQF&5PIVNHM$ [NXM XVNTMSWLJRJ #7FX' RS' D*- / 04 D7= 7MJQNHFPW$ FX .) i in THF (Cat. no. 186562; Sigma-Aldrich) for 3 hours. The reaction mixture was precipitated and washed in hexane (Cat. no.197360025; Acros Organics) and vacuum dried overnight prior to the following experiments. Synthesis of oligo(Cbz-L-lysine) core peptide: A solution of Cbz-Lys-NCA in dry DMF (0.2 g / mL; Cat. no.9227056; Sigma-Aldrich) was introduced into a Schlenk flask fitted with a drying tube. To this, n-hexylamine (Cat. no. 219703; Sigma-Aldrich) (1:10::n-hexylamine: Cbz-Lys-NCA) was added and the reaction was DUKE-43487.601 carried out at room temperature for 5 days under stirring. The reaction mixture was slowly added to a ~20-fold excess of water, and the precipitated product (termed as core peptide hereafter) was filtered using a Büchner funnel fitted with Whatman grade 1 filter paper (Cat. no. 1001-070; Whatman), washed with DI water, flash frozen in liquid nitrogen, and freeze dried. The core peptide was subsequently reacted with Ne,Ni-di(9-fluorenylmethoxycarbonyl L-lysine) (Ne,Ni-diFmoc; Cat. no.8520410025; Sigma-Aldrich). Ne,Ni-diFmoc coupling to the core peptide: A round-bottom flask containing the core peptide in DMF was reacted with 4 equivalents of Ne,Ni-diFmoc, 4 equivalents of 2-(1H-benzo-triazole-1-yl)-oxy-1,1,3,3- tetramethyluronium hexafluorophosphate (HBTU; Cat. no.8510060025; Sigma-Aldrich), and 12 equivalents of 1-hydroxybenzotriazole (HOBt; Cat. no. 157260; Sigma-Aldrich). To this, 10 equivalents of N,N-diisopropylethylamine (DIPEA; Cat. no.496219; Sigma-Aldrich) were added and the reaction was carried out at room temperature for 3 days. The reaction mixture was precipitated in water, flash frozen in liquid nitrogen, and freeze-dried. The resulting reaction product was washed several times with diethyl ether (Cat. no.32203; Sigma-Aldrich), vacuum dried, and used for the following reaction. Selective removal of Fmoc-protected groups of the end-functionalized polypeptides: A solution of Ne,Ni-diFmoc-Lys end-functionalized oligopeptide in DMF was reacted with piperidine (~ 20% (v / v)) (Cat. no. 104094; Sigma-Aldrich) in a round bottom flask at room temperature for 1 hour and precipitated in water. The precipitated poly(L-lysine) was flash frozen in liquid nitrogen and freeze-dried. The freeze-dried product was washed several times with diethyl ether, and vacuum dried. Generation of branched poly(L-lysine) (G0): The poly(L-lysine) molecules with active amine functional groups, generated by the removal of Fmoc groups, were dissolved in DMF and reacted with Cbz-Lys-NCA (1:10::terminal amine group: Cbz-Lys-NCA). After 5 days, the solution was slowly added to a 20-fold excess of water. The precipitated polymers were filtered using a Büchner funnel fitted with Whatman grade 1 filter paper, flash frozen in liquid nitrogen, and freeze-dried. This reaction results in a branched poly(L-lysine) containing ^30 lysine units. Generation of G1 and G2 BPL molecules: The above reaction steps were repeated to synthesize lysine oligomers with higher branched structures to achieve next generation of BPL molecules (G1 and G2). Briefly, an DUKE-43487.601 Fmoc-protected lysine was conjugated to the terminal amine group of G0 BPL molecules, and subsequently deprotected to obtain two free amine groups at the end of each arm of G0. Then, as in reactions 1 and 3, lysine end-functionalized G0 peptide and Cbz-Lys-NCA monomer (1:10::terminal amine group: Cbz-Lys-NCA) were reacted in DMF for 5 days at 37 °C. The reaction mixture was then slowly added to a 20-fold excess of water to precipitate. The precipitate was filtered using a Büchner funnel fitted with a Whatman grade 1 filter paper, flash frozen in liquid nitrogen, and freeze-dried to obtain G1 BPL molecules. G2 BPL molecules was similarly generated by repeating the above reaction steps using G1 BPL as the starting material and Cbz-Lys-NCA as the monomer. Complete deprotection of BPL molecules: A round-bottom flask with a solution of the branched poly(L-lysine) in trifluoroacetic acid (TFA, ^100 mg / 3 mL) (Cat. no. A12198-36; Alfa Aesar) was reacted with a 4-fold molar excess of a 33 wt. % hydrobromic acid (Cat. no.18735; Sigma-Aldrich) in acetic acid (Cat. no. 695092; Sigma-Aldrich) for 1 hour at room temperature. The reaction mixture was precipitated in diethyl ether and the product in quantitative yield after filtration and vacuum-drying was stored at 1+) i' Characterization data: The successful synthesis of various BPL molecules and their immediate precursors was validated by NMR). For instance, NMR spectra were obtained the G0 BPL with and without the benzyloxycarbonyl (Cbz) groups. Comparison of the peak areas at ^0.83 ppm for the terminal 1CH3 group of the hexylamine unit to that of the aromatic protons of the Cbz groups at ^7.20-7.33 ppm indicated successful polymerization of the lysine monomers. Furthermore, the NMR spectra following the deprotection showed almost complete removal of the Cbz groups as only a negligible signal from the aromatic protons of the Cbz group at 7.20-7.33 ppm could be detected in the NMR spectrum. NMR spectra were also used to estimate the average number of lysine units in each branch as well as the total degrees of polymerization (DP) in different generations of the BPL molecules (Table 2). The average DP and chain length estimations based on the NMR showed slight deviations from the theoretical predictions, which suggests an incomplete polymerization of BPL molecules at G1 and G2 generations. Dynamic light scattering (DLS) and zeta potential measurements were used to further characterize the BPL molecules. The DLS measurements yielded a mean hydrodynamic diameter of ~40 nm for all BPL molecules (i.e., G0, G1, and G2), with overlapping DUKE-43487.601 histograms (FIG.2). The minimal increase in hydrodynamic diameter, observed as the generation increases, can be attributed to the intentional branching of the BPL structure. Zeta potential measurements revealed that the surface charge doubled for the BPL molecules from generation G0 to G1, but further increase in branching from G1 to G2 entailed only a slight increase in surface charge (Table 2). We attribute this observation to effective charge screening by the branches at higher BPL generations. Specifically, the G0 BPL molecules had a zeta potential of about 14.9 ^ 2.3 mV, which increased to 34.9 ^ 1.0 mV for G1 and 52.0 ^ 0.2 mV for G2 BPL molecules (Table 2). The increase in surface charge is associated with the increase in the number of functional amine groups from the lysine units presented at the surface of higher generations of BPL molecules. Table 2. Yields and molecular characteristics of branched poly(L-lysine) molecules of different generations. ND = Not determined Example 2 Functionalization of BPL Molecules Functionalization of BPL molecules with poly(ethylene glycol): The BPL molecules were PEGylated by reacting with N-hydroxysuccinimide (NHS) methyl-PEG8 (mPEG8-SCM; Cat. no. PJK-209; Creative PEGworks). Briefly, BPL molecules were dissolved in 1X PBS and reacted with a solution of mPEG8-SCM in DMSO (Cat. no. 276855; Sigma-Aldrich) for 4 hours at room temperature. The solutions were dialyzed for three days using Spectra / Por 3 Dialysis Tubing with a MWCO 3.5kDa (Cat. no.132724; Repligen) in DI water, flash frozen in liquid nitrogen, and freeze-dried. Two different extents of modification were used: low PEGylation (LP; ^20% of surface functionalization) and high PEGylation (HP; ^35% of surface functionalization). The extent of PEGylation was determined via NMR spectra by comparing the peaks at 3.62 ppm corresponding to the 1OCH2CH2O1 protons of PEG to the peaks at 0.85 ppm corresponding to the terminal 1CH3groups of hexylamine initiator. As anticipated, the PEGylation reduced the DUKE-43487.601 zeta potential of the BPL molecules, which was determined for G1 and G2 generations. For G1 BPL molecules with ~20% and ~35% PEGylation, the zeta potential values were found to be 24.7 ^ 0.9 mV and 25.1 ^ 0.8 mV, respectively. A similar trend was also observed with G2 BPL molecules, where the zeta potential has decreased to 42.5 ^ 0.2 mV and 40.6 ^ 0.9 mV with ~20% and ~35% PEGylation, respectively. The PEGylation reduced the surface charge of the G1 and G2 BPL molecules as some of the free amine groups of the lysine units were utilized for the chemical conjugation of PEG molecules. Functionalization of BPL molecules with cyanine dyes: The cyanine dyes (Cy5 or Cy7) were conjugated to BPL molecules by reacting with N- hydroxysuccinimide (NHS) Cy5 or Cy7 esters (NHS-Cy5 or NHS-Cy7; Cat. no. 43310 and 45020; Lumiprobe). Briefly, BPL molecules were dissolved in 1X PBS and reacted with a solution of NHS-Cy5 or NHS-Cy7 in DMSO for 12 hours at room temperature. The solutions were dialyzed for five days using Spectra / Por 3 Dialysis Tubing, 3.5 kDa MWCO, flash frozen in liquid nitrogen, and freeze-dried. Conjugation of succinic acid linker to 6-alpha-methylprednisolone: To a suspension of 6-alpha-methylprednisolone (1.38 mmol, Cat. no. 338800050; Acros Organics) in dry DCM, (2 mg / mL, Cat. no.270997; Sigma Aldrich), succinic anhydride (2.67 mmol, Cat. no. 239690; Sigma Aldrich) and triethylamine (Cat. no. T0886; Sigma Aldrich) were added under stirring (Carli et al. Chem. Eur. J. 2018, 24,(41), 10300-10305). The mixture was stirred for 10 minutes and the catalytic agent 4-dimethyaminopyridine (0.1 mmol, DMAP, Cat. no. 107700; Sigma Aldrich) was added to it. The reaction was then continued overnight at room temperature. The reaction mixture was precipitated in 1 N HCl and solid precipitate was filtered, washed with water, and vacuum dried to get the white powder of 6-alpha-methylprednisolone conjugated with succinic acid in 85% yield. Conjugation of the modified methylprednisolone to G2HP BPL molecules: The methylprednisolone functionalized with succinic acid was conjugated into the BPL molecules by using amide coupling method. Briefly, the methylprednisolone functionalized with succinic acid (0.084 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; 0.34 mmol, Cat. no. D1601; TCI Chemicals), and NHS (0.34 mmol, Cat. no. 130672; Sigma Aldrich) were dissolved in dry DMF and stirred overnight. The reaction mixture was transferred into a solution of G2HP BPL molecules (0.5 µmol) dissolved in PBS and further reacted for 24 hours. The reaction mixture was placed in a 3.5-kDa membrane and dialyzed against 10% DMSO / water for 24 hours, followed by dialysis in DI water for 2 days. The DUKE-43487.601 reaction mixture was flash frozen in liquid nitrogen and freeze dried to get the solid product, methylprednisolone conjugated to G2HP BPL. Successful conjugation of 6- methylprednisolone was determined by NMR analysis. Example 3 Characterization of BPL Molecules Proton Nuclear Magnetic Resonance Spectroscopy (1H NMR): Successful synthesis of BPL molecules was characterized by NMR measurements, the products were dissolved in deuterated water (Cat. no. 151882; Sigma Aldrich), dimethyl sulfoxide-d6 (Cat. no.364650500; Acros Organic) or methanol-d4 (Cat. no. 343803; Sigma Aldrich) at a concentration of ~1 wt. %. The spectra were recorded with a 500 MHz Agilent / Varian VNMRS spectrometer at room temperature. Zeta Potential characterization: The BPL molecules were dissolved in DI water at concentrations of 3.5-7.5 µM, filtered through a 0.22 µm filter (Cat. no.431229; Corning) and used for zeta potential measurements. A Malvern Zetasizer Nano Series was used with disposable folded capillary cells (Cat. no. DTS1070; Malvern Panalytical) for the measurements. The zeta potential value was determined by averaging three independent measurements, where each measurements involved 100 runs, and the Smoluchowski method was used to calculate the zeta potential value. Dynamic Light Scattering measurements: For dynamic light scattering (DLS) measurements, the polymers were dissolved at 125 µg / mL in PBS and filtered through a 0.22 µm filter. A DynaPro Plate Reader was used with black Corning 96-well plate with clear bottom (Cat. no. CLS3631; Sigma-Aldrich) for DLS measurements. Aggregation of G2 and G2HP BPL molecules in synovial fluid: The G2 and G2HP molecules were dissolved at 625 µg / mL in PBS, and to this bovine synovial fluid (Cat. no. BOV00SYNFL-0110555; BioIVT) was added until the G2 and G2HP molecules were at a concentration of 500 µg / mL, and the synovial fluid was at a concentration of 20% (v / v) (Brown et al. Acta Biomaterialia 2019, 93, 239-257). After the solutions were made, the BPL and synovial fluid solutions were placed in an incubator at 37 °C, shaking at 100 rpm for 30 minutes. After the 30-minute incubation, digital images were taken of the solutions. The aggregates were also imaged with a microscope. DUKE-43487.601 Bovine cartilage explants: Two-week-old bovine knee joints were obtained from Research 87 (Boylston, MA), and cartilage discs of ~ 4 mm diameter and thickness were harvested from the femur and stored in chondrocyte media (DMEM (Cat. no. 11965092; Gibco), 10% (v / v) FBS (Cat. no. 16000044; Gibco), 1% (v / v) ascorbic acid (Cat. no. A4544; Sigma-Aldrich), 1% (v / v) HEPES (Cat. No. 15630080; Gibco), 1% (v / v) L-proline (Cat. no. P5607; Sigma-Aldrich), 1% (v / v) sodium pyruvate (Cat. no. 11360070; Gibco), 1% (v / v) MEM NEAA (Cat. no. 11140050; Gibco) and 1% (v / v) penicillin-streptomycin (Cat. no. 15140122; Gibco) at 37 ^C in an incubator (~24 hours) prior to use. Adhesion and penetration of the BPL molecules across cartilage explants: Bovine cartilage explants of 4 mm in diameter and 4 mm thickness were equilibrated in chondrocyte media 24 hours prior to use. A custom designed device that enables selective exposure of the superficial zone of the cartilage to the BPL solution was used for the experiments. The device (FIG. 3A) was generated from polydimethylsiloxane (PDMS; Ellsworth Adhesives) and the master molds with the required features were 3D printed using polylactic acid (PLA) filaments. Specifically, the PLA master mold was designed to create a cylindrical cartilage holder with an outer diameter of 12 mm and 15 mm thick and an inner compartment of three segments of equal length (5 mm) but varying diameters (upper diameter: 10 mm, middle diameter: 3.95 mm, and lower diameter: 2 mm). Cartilage explants were press fitted into the middle chamber of the PDMS mold and exposed to the upper compartment, which was designed to accommodate 200 ql of BPL solutions. The lower compartment was used to access the PBS when the device was placed in a 24-well plate containing 500 µL of PBS to maintain humidity and limit dehydration of the cartilage samples. Cyanine 5 (Cy5)-conjugated BPL (Cy5-BPL) molecules were dissolved in 1X PBS at a concentration of 500 µg / mL and used for the experiments. The superficial zone of cartilage explants was exposed to 200 µL of the Cy5-BPL solution and incubated at 37 ^C. Adhesion and penetration of BPL molecules to the cartilage explants was determined as a function of time (0-24 hours). The BPL solutions were collected at predetermined time intervals, and fluorescence intensities were measured with excitation and emission wavelengths of 515 nm and 580 nm, respectively. The difference between the fluorescence intensities of the Cy5-BPL molecules before and after incubation was used to determine the amount of BPL molecule taken up by the explant. The concentration of BPL molecules in the solution was estimated from a standard curve and the % uptake of the BPL molecules was calculated: percent of BPL DUKE-43487.601 uptake = ^(BPL uptake / Initial BPL amount) ^ 100^%. The cartilage explants were further analyzed to assess the penetration depth of the BPL molecules across the cartilage by using the same experimental setup. After 6 hours and 24 hours, the cartilage explants were removed from the device, fixed, embedded in OCT (Cat. no. 27050; Ted Pella), sectioned into 15-µm thick sections, and stained with DAPI to mark nuclei (Cat. no. P36971; Invitrogen). The sections were imaged with a Keyence BZ-X710 fluorescence microscope. The penetration experiments [JVJ FPWS VJTJFXJI [NXM GSZNRJ HFVXNPFLJ J\TPFRXW [MNHM [JVJ IJLVFIJI [NXM =>&*f #7FX' RS' 201-LB-005; R&D Systems) (Clutterbuck et al. Ann. NY Acad. Sci. 2009, 1171(1), 428-435). DMJ =>&*f [FW INWWSPZJI NR HMSRIVSH]XJ QJINF FX F HSRHJRXVFXNSR SK +. RL(Q> FRI RJ[ QJINF HSRXFNRNRL =>&*f [FW FIIJI JZJV] SXMJV IF]' 5KXJV - IF]W SK NRHYGFXNSR% XMJ HFVXNPFLJ J\TPFRXW were placed in the PDMS molds, with G2 and G2HP BPL solutions at concentrations of 500 µg / mL. The uptake and penetration of the G2 and G2HP BPL solutions into the degraded cartilage was measured using the previously described methods. Desorption of the BPL molecules from the cartilage explants: The desorption experiments were carried out using cartilage explants following the adsorption of Cy5-conjugated BPL (Cy5-BPL) molecules. After 24 hours of incubation with the Cy5-BPL solution, the cartilage explants were washed with 1X PBS, moved into a 96-well TPFXJ% FRI NRHYGFXJI NR +)) g> SK JNXMJV *E B6C SV *)E B6C HSRXFNRNRL TVSXJFWJ NRMNGNXSVW (Cat. no. P8340; Sigma Aldrich). The protease inhibitors were used to prevent any explant degradation. The PBS solutions were collected following every 24 hours for 4 days and replaced with fresh PBS. The fluorescent intensity of the collected PBS solutions was measured to determine the extent of BPL desorption over the 4 days of incubation time. Equilibrium uptake assay of the BPL molecules into cartilage explants: Cartilage explants (4 mm diameter ^ ^.)) gQ XMNHORJWW$ [JVJ NRHYGFXJI NR +)) g> Cy5- tagged BPL solution with varying initial concentrations (Cinitial) of 150, 300, 750, 1000 FRI *.)) g? #HSRXFNRNRL TVSXJFWJ NRMNGNXSVW$ NR F 2 / &[JPP TPFXJ FX ,0 i' DMJ KPYSVJWHJRHJ intensity of the free solution, following 24 hours, was quantified by the plate reader to obtain the final BPL concentration (Cfinal). In addition, the cartilage explants were removed, washed with PBS, wiped, and weighed to obtain the wet weight. These cartilage explants were then lyophilized and weighed to obtain the dry weight. The uptake ratio (Ru) is defined as the concentration of the BPL molecules within the cartilage disc, which includes both bound (CB) and free (CF) BPL molecules, normalized by the concentration of BPL molecules in the free solution (Cfinal). The equilibrium uptake assay DUKE-43487.601 was used to estimate the binding properties of the BPL molecules (i.e., equilibrium partition coefficient K, and equilibrium dissociation constant KD, and intra-cartilage binding site concentration N) with the cartilage tissue following a first-order, reversible, bimolecular interaction model as reported (Vedadghavami et al. Acta Biomater. 2019, 93, 258-269; Bajpayee et al. Biomaterials 2014, 35, (1), 538-549). The Rucan be described as follows (Equation1): Ru= (CB+ CF) / Cfinal(1) The equilibrium dissociation constant, KD,can be related to CF, CB, and N to the following equation: KD = ^ CF ^ (N1CB)^ / CB (2) The equilibrium partition coefficient (K) of BPL is defined as: K = CF / Cfinal (3) By combining the Equations 1, 2, and 3, the value of Ru can be written as: Ru = K^1 + N / (KD + KCfinal)^ (4) Fitting the Equation 4 with the experimentally obtained values of Ru and Cfinal, the parameters K, N, and KD were obtained. Cytotoxicity / viability assay after culturing bovine explants with BPL molecules: Bovine cartilage explants incubated in chondrocyte medium containing different concentrations of BPL molecules (11.5 µM and 23 µM). The explants were incubated for 24 hours at 37 °C and 5% CO2. After incubation, cartilage explants were manually sliced longitudinally into 100-200 µm thick sections, incubated with 2 µM calcein AM and 4 µM ethidium homodimer-1 (Cat. no. L3224; Invitrogen) and imaged with a Keyence BZ-X710 fluorescence microscope. Fluorescence images was quantified to determine the live (green) and dead (red) cells (Zhang et al. Acta Biomaterialia 2011, 7(9), 3362-3369). Biosynthesis of bovine cartilage explants cultured with G2HP BPL molecules: Bovine cartilage explants were cultured in chondrocyte medium containing G2HP BPL molecules at a concentration of 11.5 µM. At the initial time point, the explants were incubated with either chondrocyte medium or chondrocyte medium with G2HP BPL molecules at 37 °C and 5% CO2. The medium was changed every other day. After 14 days of culture, cartilage explants (n = 3) from each group were fixed with 4% paraformaldehyde overnight. These explants were then dehydrated in 20% sucrose, embedded in OCT medium, and sectioned into 15 µm-thick sections. After sectioning, the cartilage explants were stained with Safranin-O, Fast Green, hematoxylin, and imaged with a Keyence BZ-X710 fluorescence microscope DUKE-43487.601 (Varghese et al. Stem Cells 2010, 28(4), 765-774). Cartilage explants at the initial time point were used as a control. Additionally, biochemical analyses of the cartilage explants via 1,9-dimethylmethylene blue (DMMB; Cat. no.341088; MilliporeSigma) assay were used to quantify the proteoglycan content in the cartilage explants (Varghese et al. Matrix Biol. 2008, 27, (1), 12-21). Three cartilage explants were lyophilized, weighed for their dry weight, and digested in papain (Cat. no. LS003124; Worthington Biochem) overnight at 60 °C. The papain digests of the cartilage were then diluted, mixed with DMMB dye and the absorbance of the solution at 525 nm was measured with a Tecan Infinite F200 Plate Reader. The chondroitin sulfate in each sample was quantified by comparison to a standard curve of chondroitin sulfate of varying concentrations and normalized to the dry weight of the cartilage explants. Localization of BPL molecules in ex vivo mouse knee joints: Following euthanasia, 12-month-old C57BL / 6 male mouse knee joint explants were injected with saline, free Cy5 dye, or Cy5-conjugated BPL molecules (G2HP BPL) in sterile saline (50 µL, 50 µg / mL; 1.9 µM). The concentration of the Cy5 in the free Cy5 group was calculated to be the same as the Cy5 content in the 50 µg / mL G2HP BPL solution. The knee joint explants were left in an incubator at 37 °C and 5% CO2 for 24 hours. After this, the explants were fixed, dehydrated in 30% sucrose, and embedded in OCT medium. Undecalcified joints were then cryosectioned using a Cryojane Tape-Transfer system, counterstained with DAPI, and imaged to visualize the localization and distribution of the injected molecules. In vivo retention of BPL molecules in rat joint: All animal procedures were carried out in accordance with protocols approved by Duke University Institutional Animal Care and Use Committee (IACUC) in compliance with NIH guidelines for laboratory animal care (A151-20-07). Unilateral destabilization of the medial meniscus (DMM) surgery, which is often used to induce post-traumatic osteoarthritis (PTOA), was performed in rats as previously described.47Briefly, twelve-week-old female Lewis rats (n = 4, Charles River) were anesthetized under isoflurane, shaved on both legs, and disinfected with three scrubbing routines of povidone iodine and 70% ethanol. Buprenorphine SR (1 mg / mL, ZooPharm) was injected subcutaneously at a dose of 0.5 mg / kg. The skin and joint capsule were incised, and the medial meniscotibial ligament was transected. Following transection, bupivacaine (0.5%, Hospira) was applied topically, and Vicryl 6-0 sutures were used to close the skin incision. One week after surgery, cyanine 7 (Cy7)-conjugated PEGylated BPL (G2HP BPL) molecules in sterile saline were administered (50 µL, 500 µg / mL; 19 µM) DUKE-43487.601 into injured and uninjured knees of the rat by intra-articular injection. The fluorescent signal in the uninjured joint and in the DMM joint injury model were compared to examine the effect of injury-mediated changes on the retention and penetration of the BPL molecules following intraarticular injection. An IVIS Kinetic System and Living Image software were used to serially acquire (excitation filter: 745 nm, emission filter: indocyanine green, ICG) and quantify fluorescence intensity within each joint over 28 days (Gilpin et al. Adv. Healthc. Mater. 2021, 10(23), 2100777). Radiant efficiency data within a fixed anatomical region of interest (ROI) over time for each group were fit to a single-phase exponential decay with a common plateau compared to untreated animal background fluorescence. Methylprednisolone release kinetics: The release kinetics of 6-alpha-methylprednisolone from the BPL molecules was carried out in PBS as a function of time over 14 days (Newman et al. Biomaterials Sci. 2022, 10(18), 5340-5355). The G2HP BPL molecules conjugated with the drug were dissolved in PBS at a concentration of 10 mg / mL. The solutions (10 mg / mL) were loaded into dialysis bags (MWCO = 2 kDa; Cat. no. D2272; Sigma Aldrich), which were then sealed and placed in a 15- mL centrifuge tube with 5 mL of PBS. At predetermined time intervals (0, 2, 6, 12, 24, 48, 72, 96, 120, 144, 168, 240, 336, and 504 hours), 1 mL of the solution from the outside of the dialysis bag was removed and supplemented with an equal amount of fresh PBS. The drug release from the BPL molecules was calculated by quantifying the methylprednisolone content in the collected solution via UV / Vis absorption spectroscopy (Genesys 10S UV-Vis Spectrophotometer and Semi-Micro Cuvette, Cat. no. 9700-590; VWR) as a function of time. A standard calibration curve of absorbance (at ~255 nm) as a function of methylprednisolone concentration (2.5-50 µg / mL in PBS) was used to calculate the concentration of the released methylprednisolone. Two different standard calibration curves of absorbance (at ~255 nm) as a function of the concentration of G2HP BPL molecules (2.5-50 µg / mL in PBS) and G2HP BPL molecules conjugated with methylprednisolone (15.625-500 µg / mL in PBS) were used to calculate methylprednisolone conjugation to the G2HP BPL molecules. To examine the effect of esterases on methylprednisolone release, we also used PBS containing 10% of FBS as an incubating medium. The G2HP conjugated with the drug was dissolved in PBS containing 10% FBS at a concentration of 10 mg / mL. The solution mixture was loaded into a dialysis bag with a MWCO of 2 kDa, sealed, and placed in a 15-mL centrifuge tube with 5 mL of PBS containing 10% FBS. The release profile up to three days post-incubation was determined using UV / Vis as described above (at ~260 nm). DUKE-43487.601 Results and Discussion a. Cartilage uptake, penetration, and retention of BPL molecules The adhesion and transport of BPL molecules across the cartilage explants were assessed using a custom-designed device that allowed selective exposure of the BPL solution to the superficial zone of the cartilage explant (~4 mm thickness) and allowed diffusion only in one direction (i.e., from top to bottom) (FIG.3A). Cartilage uptake of different generations of BPL molecules tagged with cyanine 5 (Cy5) were determined by measuring relative changes in the fluorescence intensity of the solution (i.e., depletion of BPL molecules in the solution) as a function of time. The cartilage uptake and depth of penetration was further confirmed by image analysis of the cartilage explants. These experiments revealed a rapid initial uptake in the first 5 minutes, followed by a steady plateau for about 60 minutes. A further continuous increase in uptake was observed from about 2 hours to 6 hours. By 24 hours, ~ 80% of the BPL molecules were taken up by the cartilage explant in all different generations of BPL molecules (FIG. 3B). The statistical analysis of data shown in FIG. 3B is provided in Table 3. While all the different BPL molecules showed high affinity for cartilage, there were notable differences in the amount taken up within the first 5 minutes of exposure. Specifically, G0 BPL molecules showed a slightly higher uptake (29 ^ 2%) compared to G1 and G2 generations, which showed similar levels of initial uptake, 21 ^ 4% for G1 and 19 ^ 2% for G2 within the first 5 minutes (FIG.3C). We attribute the higher initial uptake of the G0 BPL molecules to their lower surface charge and slightly smaller size, both of which facilitate faster transport into the cartilage, compared to G1 and G2 BPL molecules. Interestingly, PEGylation of G1 and G2 BPL molecules did not significantly affect their uptake by cartilage (FIGS. 3B, 3D, 3E), which suggests that the PEGylated BPL molecules possess the optimal surface charge required for cartilage adhesion and penetration. These observations are consistent with previous studies which suggested that the transport of cationic nanocarriers across the cartilage tissue is associated with an optimal surface net charge.11, 15, 20, 24, 33Table 3. Multiple comparisons from one-way ANOVA with Tukey post-hoc test from uptake of BPL molecules to cartilage after 24 hours (FIG.3B). DUKE-43487.601 To characterize the depth of penetration, the cartilage explants were removed from the device after 6 and 24 hours of exposure to different BPL molecules, sectioned and imaged (FIG.4A). All the BPL molecules penetrated the cartilage explant by 6 hours of exposure (FIG. 4A). However, G0 BPL molecules penetrated deeper (590 ^ 30 µm) compared to G1 and G2 BPL molecules, which penetrated 500 ^ 50 µm and 520 ^ 25 µm, respectively (FIG.4B). The deeper penetration by the G0 BPL molecules can be attributed toto their higher mobility within the cartilage tissue, aided by their lower surface charge, compared to G1 and G2 BPL molecules. These findings are consistent with the results from the cartilage uptake measurements (FIGS. 3B-3E). The penetration depth, measured after 6 hours of incubation time, increased with increasing PEGylation and was similar to the penetration of G0 BPL molecules (FIG. 4B). These observations further underscore the importance of weak interactions between the BPL molecules and cartilage in promoting their transport across a thick cartilage tissue. Fluorescent images in FIG.4A show that after 24 hours of exposure, the BPL molecules penetrated further into the cartilage tissue; i.e., we found a penetration depth for G0, G1, and G2 BPL molecules of 1550 ^ 50 µm, 1710 ^ 30 µm, and 1620 ^ 25 µm, respectively (FIG.4C). These results suggest that despite the changes in molecular architecture (i.e., branching, PEGylation) and surface charge, all of the different BPL molecules significantly penetrated into the cartilage explant by 24 hours; i.e., to depths that are on the same order of magnitude as human cartilage tissue thickness (Schneider et al. Sci. Rep. 2022, DUKE-43487.601 12(1), 11707; Gau et al. Pediatr. Rheumatol. 2021, 19(1), 71; Hunziker et al. Osteoarthr. Cartilage 2002, 10(7), 564-572). The statistical analyses for FIGS 4B and 4C are provided in Tables 4 and 5, respectively. Table 4. Multiple comparisons from one-way ANOVA with Tukey post-hoc test from penetration of BPL molecules to cartilage after 6 hours (FIG.4B). Table 5. Multiple comparisons from one-way ANOVA with Tukey post-hoc test from penetration of BPL molecules to cartilage after 24 hours (FIG.4C). DUKE-43487.601 To further characterize the electrostatic interactions between the negatively charged cartilage ECM and the positively charged BPL molecules, a desorption assay was performed by incubating the BPL-absorbed cartilage explants in 1X or 10X phosphate buffered saline (PBS) and measuring the amount of BPL molecules released into the solution as a function of time for up to 4 days. The results showed that around ~20% of G0 BPL molecules and 10-15% of all other BPL molecules, including the PEGylated G1 and G2 molecules, were desorbed from the cartilage explants in 1X PBS after 4 days (FIGS. 5A-5C). The effect of branching (G0, G1, G2) and PEGylation on desorption is shown in FIG. 5C and in FIGS. 6A-6B, respectively. In contrast, nearly all BPL molecules were desorbed (75-95%) from the cartilage explants in 10X PBS, a solution with higher ionic strength (FIGS. 5D-5F; FIGS. 6C-6D). PEGylation of G2 BPL molecules promoted their desorption in 1X PBS compared to the non- PEGylated G2 BPL molecules (FIG.6B). The higher desorption of G0 BPL and PEGylated G2 BPL molecules in 1X PBS could be attributed to the reduced surface charge along with the likely screening effect from the PEG chains, resulting in weaker electrostatic interactions with the negatively charged cartilage matrix. The statistical analyses for FIGS.5A and 5D are provided in Tables 6 and 7, respectively. Table 6. Multiple comparisons from one-way ANOVA with Tukey post-hoc test from desorption of BPL molecules from cartilage after 4 days in 1x PBS (FIG.5A). DUKE-43487.601 Table 7. Multiple comparisons from one-way ANOVA with Tukey post-hoc test from desorption of BPL molecules from cartilage after 4 days in 10x PBS (FIG.5D). DUKE-43487.601 By adapting an established protocol, we further examined the binding properties of the various BPL molecules with the cartilage matrix (Lin et al. Chem. Eng. J.2022, 430, 133018; Vedadghavami et al. Acta Biomater. 2019, 93, 258-269; Bajpayee et al. Biomaterials 2014, 35(1), 538-549). An equilibrium uptake assay was performed by incubating a thin cartilage tissue with varying concentrations of BPL molecules (Cinitial) followed by measuring the fluorescence intensity after 24 hours of incubation (Cfinal). We found that with increasing initial BPL concentration (Cinitial), the uptake ratio of the Cy5-labeled BPL molecules also increased; a representative uptake curve for G0 BPL is shown in FIG. 7A. The cartilage uptake equilibrium (Ru) of the BPL molecules was calculated and correlated to Cfinalas described in the methods (FIG.7B). The estimated reversible binding properties of the BPL molecules with the cartilage tissue is given in Table 8. The partition coefficient (K) for BPL molecules decreased with increasing surface charge, and was ~2.8, ~1.5 and ~1.1 for G0, G1, and G2, respectively. The binding site concentration (N) of the BPL molecules showed a similar trend with G0, G1 and G2 having ~708 µM, ~284 µM and ~77 µM, respectively. The values for the equilibrium dissociation constant KD for G0, G1 and G2 were ~221 µM, ~1301 µM and ~6574 µM, respectively, which indicates an increase in KD with increasing surface charge. These trends further suggest the importance of surface charge of the BPL molecules on cartilage binding and transport. PEGylation was found to increase the equilibrium partition coefficient (K) and the binding site density (N) for the G2HP molecules to ~2.2 and ~384 µM, respectively. The equilibrium dissociation constant (KD) of the PEGylated G2 BPL molecules decreased, and it is estimated to be ~247 µM for G2HP. The observed decrease with PEGylation could be due to their weaker reversible electrostatic interactions with the cartilage tissue. More binding sites and reversible binding to cartilage ECM is necessary to maintain an enhanced intra-tissue concentration for sustained delivery. The reversible binding also ensures the effective transport of the nanocarriers across a full-thickness cartilage tissue. Table 8. Estimated equilibrium partition coefficient K, intra-cartilage equilibrium dissociation constant KD, and binding site concentration N from the equilibrium uptake assay. DUKE-43487.601 b. Cytotoxicity of BPL molecules: To determine the cytotoxicity of BPL molecules, cartilage explants were incubated with various BPL molecules at two different concentrations, ^11.5 and 23 µM, for 24 hours and analyzed for live and dead cells (FIGS. 8A-8B and FIG. 9) (Zhang 2011). While both the concentrations of non-PEGylated (G0, G1 and G2) BPL molecules showed some level of toxicity, cartilage explants exposed to 23 µM BPL showed higher cell death (FIG. 9). The cytotoxicity was more prominent with G2 BPL molecules likely because of the higher positive charge (FIGS. 8A-8B; FIG. 9). Cationic biomaterials including nanocarriers are known to be cytotoxic and the toxicity is dependent on their positive surface charges (Lin et al. Chem. Eng. J.2022, 430, 133018; Kim et al. Biomacromolecules 2018, 19, (7), 2483-2495). PEGylation is widely used to reduce the cytotoxicity where it provides a screening effect to the positively charged cationic molecules, and thus reducing the overall surface charge. In agreement with this widely adapted strategy, PEGylated G2 BPL molecules showed less toxicity compared to the bare G2 BPL molecules. All additional characterization involving cartilage tissues and the in vivo studies were carried out using either G2 and / or G2HP BPL molecules. Histological and biochemical analyses for sulfated proteoglycans were carried out to further determine the effect of the BPL molecules on the biosynthetic activity of the cartilage explants cultured in chondrocyte medium supplemented with G2HP BPL molecules for 14 days (Varghese et al. Stem Cells 2010, 28(4), 765-774; Varghese et al. Matrix Biol.2008, 27(1), 12- 21). Safranin-O staining of the cartilage explants showed similar levels of staining among all the groups, as well as explants at the initial time point (FIG. 8C). Additionally, biochemical analyses for the proteoglycan content of the explants from various experimental groups showed similar levels of glycosaminoglycan (GAG) content. Specifically, explants cultured in medium with and without G2HP BPL molecules had a GAG content of 331 ± 16 µg / mg and 327 ± 24 µg / mg, respectively, which is similar to the value for cartilage explants at the initial time point 345 ± 25 µg / mg (FIG.8D). Together these results suggest minimal-to-no detrimental effect of G2HP BPL molecules on chondrocyte viability and function. The statistical analyses for FIG. 8B and 8D are provided in Tables 9 and 10, respectively. DUKE-43487.601 Table 9. Multiple comparisons from one-way ANOVA with Tukey post-hoc test from live- dead viability / cytotoxicity assay after 24 hours of incubation of cartilage explants with BPL molecules at a concentration of 11.5 µM (FIG.8B). Table 10. Multiple comparisons from one-way ANOVA with Tukey post-hoc test from proteoglycan biosynthesis assay, after incubation of BPL molecules and cartilage explants for 14 days (FIG.8D). DMJ TJRJXVFXNSR SK ;+ FRI ;+ QSPJHYPJW FHVSWW FR =>&*f&HMFPPJRLJI HFVXNPFLJ J\TPFRX FW F KYRHXNSR SK XNQJ [FW FPWS J\FQNRJI' 9\TSWYVJ XS =>&*f NW [NIJP] YWJI XS QNQNH XMJ TVSXJSLP]HFR PSWW FWWSHNFXJI [NXM JFVP] WXFLJW SK A5 #7PYXXJVGYHO +))2$' DMJ =>&*f& conditioned cartilage explants exhibited higher uptake of G2 and G2HP BPL molecules compared to healthy cartilage, with approximately 20% higher uptake occurring after 6 hours #:=;' *)5$' <S[JZJV% XMJVJ [FW RS WYHM HMFRLJ NR 6B> YTXFOJ FQSRL XMJ MJFPXM] FRI =>&*f challenged cartilage explants at 24 hours (FIG.10B). The BPL molecules also showed higher TJRJXVFXNSR IJTXM NR =>&*f HSRINXNSRJI J\TPFRXW HSQTFVJI XS XMJ MJFPXM] J\TPFRXW #:=;C' *)8& G). There was no significant difference in the uptake or depth of penetration between the G2 and G2HP BPL molecules (FIGS. 10C and 10F). Since synovial fluid can influence the function of nanocarrier by inducing their aggregation, the behavior of G2 and G2HP BPL molecules in presence of synovial fluid was also examined (Brown 2019). Samples containing G2 BPL molecules showed macroscopic aggregation of the nanocarriers in the presence of synovial fluid and precipitated out of the solution while no such phase separation / aggregation was observed in any of the samples containing G2HP BPL molecules (FIG.11). Statistical analyses for FIGS.10A-10F are provided in Tables 11-16, respectively DUKE-43487.601 Table 11. Multiple comparisons from two-way ANOVA with Bonferroni's post-hoc test from BPL uptake into healthy and cytokine challenged cartilage explants after 6 hours (FIG.10A). Table 12. Multiple comparisons from two-way ANOVA with Bonferroni's post-hoc test from BPL uptake into healthy and cytokine challenged cartilage explants after 24 hours (FIG.10B). Table 13. Multiple comparisons from two-way ANOVA with Bonferroni’s post-hoc test from BPL uptake into cytokine challenged cartilage explants (FIG.10C). Table 14. Multiple comparisons from two-way ANOVA with Bonferroni’s post-hoc test from BPL penetration into healthy and cytokine challenged cartilage explants ay 6 hours (FIG.10D). Table 15. Multiple comparisons from two-way ANOVA with Bonferroni’s post-hoc test from BPL penetration into healthy and cytokine challenged cartilage explants at 24 hours (FIG.10E). DUKE-43487.601 Table 16. Multiple comparisons from two-way ANOVA with Bonferroni’s post-hoc test from BPL penetration into cytokine challenged cartilage explants at 6 and 24 hours (FIG.10F). c. Joint retention of G2HP BPL molecules: To further assess penetration of the BPL molecules into the cartilage following their intra-articular injection, we examined the distribution of the injected Cy5-labeled G2HP BPL within a mouse knee joint explant. Following 24 hours of injection, the mouse knee joint explants were sectioned and imaged via fluorescence microscopy. The fluorescence images showed the presence of Cy5-labelled G2HP BPL molecules throughout the cartilage (FIGS. 12A-12B). Joints injected with the same volume of Cy5 showed minimal to no Cy5 signal at the same exposure time. The retention of the G2HP BPL molecules as a function of time was also examined, following their intra-articular administration to rat knee joints by using Cy7-conjugated BPL molecules (Cy7-G2HP) (Gilpin 2021). To address the effect of joint injury on BPL retention, a surgical destabilization of the medial meniscus (DMM) model was used and compared against the uninjured knee joint. One week following the surgical procedure, the Cy7-G2HP was administered into the joint by intra-articular injection. Both the uninjured and injured joints that received Cy7-G2HP were monitored for 28 days using IVIS imaging. The fluorescent signal of Cy7-G2HP BPL molecules was detected for at least 14 days in both knee joints, with the signal being about 50% of the initial fluorescence intensity (FIG.12C). Residual signal was detected in both the uninjured and injured joints until 28 days (maximum experimental time used). The fluorescent intensity of the region of interest, shown in FIG.12D, suggests that the Cy7-G2HP BPL molecules’ signal attenuated at a slower rate in the uninjured knee joint than in the injured joint. The statistical analysis for FIG.12D is provided in Table 17. Table 17. Multiple comparisons from two-way ANOVA with Bonferroni’s post-hoc test to compare the uninjured and DMM groups from longitudinal IVIS study over 28 days (FIG. 12D). DUKE-43487.601 Further analysis of the data indicated that the half-life of Cy7-G2HP BPL molecules was 12 and 3.4 days in uninjured and injured knee joints, respectively. The lower retention of the BPL molecules in the injured knee could be attributed to the increased inflammation or the changes caused by surgery leading to a faster clearance from the knee joint. Together, the results demonstrate that G2HP BPL molecules can be retained in the knee joint for days to weeks while molecules with similar molecular weight are reported to be cleared in hours (Larsen et al. J. Pharm. Sci. 2008, 97(11), 4622-4654; Gerwin, et al. Adv. Drug Deliver. Rev 2006, 58(2), 226-242). d. G2HP BPL molecules as nanocarriers for methylprednisolone: To further validate the potential of the BPL molecules to be used as a drug carrier, methylprednisolone, a steroid which is used to treat osteoarthritis, was conjugated to the G2HP BPL molecules using an amide coupling reaction. Methylprednisolone was functionalized with succinic acid and then reacted with BPL molecules containing amine functional group. The successful conjugation of the methylprednisolone to the BPL molecules was validated with NMR and the methylprednisolone incorporation to the G2HP BPL molecules was calculated to be ~17% of the total mass (FIG. 13A-13C). Methylprednisolone release from the BPL molecules into PBS as a function of time was evaluated over a period of 14 days (Newman 2022). The release profile was steady and sustained. After 1 day, ~9.3% of the methylprednisolone was released in PBS, and this value was increased to ~14.3% by day 3, and by 14 days, 32.4% of the drug was released from the BPL molecules (FIG.14A). Since the drug was conjugated via an ester bond, the drug release in PBS containing 10% FBS was also examined, as FBS contains esterase, which can be a trigger for the methylprednisolone release. As anticipated, a significantly higher amount of methylprednisolone release from the BPL molecules was observed in presence of FBS; by day 3 (maximum experimental time measured) ~42.3% of methylprednisolone was released from the carrier (FIG. 14B). The statistical analysis for FIG.14B is provided in Tables 18 and 19. DUKE-43487.601 Table 18. Two-way ANOVA with Bonferroni’s post-hoc test from methylprednisolone release from G2HP BPL molecules in PBS and PBS in 10% FBS over 3 days (FIG.14B). Example 4 BPL functionalized nanocarriers for intra-articular delivery of drugs Modification of nanocarriers or nanoparticles with BPL moieties make them ideal carriers for cartilage and subchondral bone. In this example, widely used poly lactic-co- glycolic acid (PLGA) nanoparticles were functionalized. Synthesis of cartilage targeting poly lactic-co-glycolic acid (PLGA) nanoparticles: In order to synthesize cartilage targeting nanoparticles, PLGA was modified with a cartilage- binding branched poly-L-lysine (BPL) peptide to obtain PLGA-PEG-BPL. First, BPL molecules with a disulfide linker at the center were synthesized using ring-opening polymerization (ROP) of Ni-benzyloxycarbonyl-L-lysine-N-carboxyanhydrides (Cbz-Lys- DUKE-43487.601 NCAs) with 2,2'-dithiobis ethanamine as initiator. The disulfide group at the center of the BPL molecules was then selectively cleaved to generate BPL molecules with terminal thiol groups, which in turn reacted with maleimide end-functionalized PLGA via the Michael addition reaction. To prevent non-specific protein adsorption, polyethylene glycol (PEG) was also conjugated to PLGA to obtain PLGA-PEG. PLGA nanoparticles were then prepared via w / o emulsification method by adding an organic solution of PLGA, PLGA-PEG, and PLGA-BPL to an aqueous solution of PBS containing polyvinyl alcohol as an emulsifier. The overall synthesis and characterization method of the nanoparticle is as follows. Synthesis of BPL with disulfide core. Cbz-Lys-NCA (20 equivalents) was dissolved in dry N,N-dimethylformamide (DMF, 100 mg / mL) in a round-bottomed flask fitted with a drying tube. 2,2'-Dithiobis ethanamine (1 equivalent) was added to the reaction mixture and the reaction was continued at room temperature for about 5 days under continuous stirring. The reaction mixture was added to a ^20-fold excess of cold water to precipitate the oligo(Cbz-L- lysine) core peptide. The precipitate was filtered, washed with water, and freeze-dried. A solution of the oligo(Cbz-L-lysine) peptide in DMF was then reacted with Ne,Ni-Di(9- fluorenylmethoxycarbonyl-L-lysine) (Ne,Ni-diFmoc-Lys, 4 equivalents) using 2-(1H-benzo- triazole-1-yl)-oxy-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 4 equivalents), and 1-hydroxy benzotriazole (HOBt, 12 equivalents) and N,N-diisopropylethylamine (DIPEA, 10 equivalents) at room temperature for 3 days. The product was precipitated in excess of cold water, filtered, washed three times with water, and freeze-dried. The freeze-dried powder was washed 4 times with diethyl ether, and vacuum-dried to obtain Ne,Ni-diFmoc-Lys conjugated core peptide. A solution of the Ne,Ni-diFmoc-Lys end-functionalized core peptide in DMF (^200 mg / mL) was reacted with 20% (v / v) piperidine under continuous stirring for an hour at room temperature and subsequently precipitated in water. The precipitate was filtered, washed repeatedly with water, and freeze-dried. The freeze-dried product was washed 4 times with diethyl ether, and vacuum-dried to obtain lysine end-functionalized core peptide. Finally, the lysine end-functionalized core peptide was subsequently reacted with the Cbz-Lys-NCA monomer to obtain next-generation branched peptides (G0). Higher generation of the branched peptides (G1) was then obtained by repeating the above steps of ring-opening polymerization, end functionalization, and subsequent removal of Fmoc protecting groups. The Cbz protecting group was completely removed from BPL molecules to generate free amine groups in BPL molecules. DUKE-43487.601 Synthesis of PLGA-PEG-BPL. Disulfide group containing BPL (G1) was dissolved in methanol. Tris(2-carboxyethyl) phosphine (TCEP, 2 equivalents), dissolved in methanol, was then added to the BPL solution. The mixture was stirred for 6 h following the addition of a small amount of water. The BPL solution was added to a solution of PLGA-PEG-maleimide (LA: GA = 65:35 or 75:25), dissolved in a mixture of acetone and methanol. DIPEA (10 equivalent) was added to the reaction mixture and stirred for 48 h. After the reaction, the solvent was evaporated at reduced pressure and the reaction mixture was washed repeatedly with methanol to remove unreacted BPL and other reagents. The degree of BPL conjugation to PLGA-PEG-BPL was determined by proton NMR spectroscopy (more than 85 % BPL conjugation was observed). Synthesis of PLGA-PEG. PLGA was dissolved in dichloromethane (DCM) and N-(3- dimethylaminopropyl)-Ne&JXM]P HFVGSINNQNIJ M]IVSHMPSVNIJ #987% *) JUYNZFPJRX$ [FW added to the solution and the mixture was stirred for 15 minutes. N-Hydroxysuccinimide (NHS, 10 equivalent) was added to the reaction mixture. After 15 minutes, mPEG-NH2 (Mol. Wt. of PEG = 2 kDa; 5 equivalent) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 24 h following the addition of DIPEA (10 equivalent). The product was precipitated in a mixture of diethyl ether and methanol (1:1). The precipitate was washed repeatedly in the same solvent mixture and dried under vacuum. The percentage of PEG modification was quantified by proton NMR, and over 95% of the PEG modification into the PLGA was observed. Synthesis of cartilage adhering and penetrating PLGA nanoparticles. Cartilage binding PLGA nanoparticles were prepared via w / o emulsification method using a mixture of PLGA, PLGA-PEG, and PLGA-PEG-BPL in the weight ratio of 4: 3: 3. Briefly, PLGA and PLGA-PEG were dissolved in chloroform, and PLGA-PEG-BPL was dissolved in DMSO. The organic solutions (chloroform: DMSO = 9:1) were combined to get a final polymer concentration of 5 mg / mL. The organic solution (2 mL) was added to phosphate buffer saline (PBS, 10 mL) containing 0.4% (w / v) polyvinyl alcohol. The mixture was then emulsified using a probe sonicator for 180 s at 4 ^C and 15-18 kHz output. Following emulsification, the mixture was stirred for 6 h overnight, and centrifuged at 3000 rpm for 10 min to remove large aggregates. The supernatant was collected, and centrifuged using a high-speed ultracentrifuge (100k × g) for 45 min. The pellet of the PLGA nanoparticles was washed repeatedly in deionized water. Finally, the particles were suspended in DI water and stored at 4 ^C. Hereafter, DUKE-43487.601 nanoparticles with and without BPL were termed as PLGA-BPL and PLGA nanoparticles respectively. Characterization of the PLGA nanoparticle. The particle size was measured using dynamic light scattering (DLS), and the average size of the particles was found to be ^ 140 ^ 10 nm. The zeta potential measurements indicate positive surface charges with a zeta potential of ^ +44 ^ 6 mV. The high positive zeta potential is due to the presence of the highly cationic BPL molecules on the PLGA nanoparticles. Notably, the zeta potential of PLGA nanoparticles devoid of any BPL was found to be 128 mV due to the negatively charged carboxylic acid groups of PLGA. Cartilage Penetration of the Nanoparticles. To determine the cartilage binding and penetration ability of the PLGA nanoparticles, a one-directional cartilage penetration was carried out with healthy cartilage explants. Cartilage explants of 4 mm in diameter and 4 mm in height were isolated from the femoral condyles. A custom-designed device that enables selective exposure of the superficial zone of the cartilage to the nanoparticle suspension was used for the penetration study. The device was generated from polydimethylsiloxane (PDMS) and 3D-printed master molds using polylactic acid (PLA) filaments. The PLA master mold was designed to create a cylindrical cartilage holder with an outer diameter of 12 and 15 mm thick and an inner compartment of three segments of equal length (5 mm) but varying diameters (upper diameter: 10 mm, middle diameter: 3.95 mm, and lower diameter: 2 mm). Cartilage explants equilibrated in chondrocyte media were press-fitted into the middle chamber of the PDMS mold and exposed to the upper compartment, which was designed to accommodate nanoparticle suspensions. The lower compartment was used to access the PBS when the device was placed in a 24-well plate to maintain humidity and limit dehydration of the cartilage samples. Cyanine 5 (Cy5) fluorophore-labeled PLGA nanoparticle (prepared using a mixture of PLGA, PLGA-Cy5, PLGA-PEG, and PLGA-PEG-BPL) was added into the middle chamber of the device and incubated for 24 and 48 h. After exposure to the nanoparticles, the cartilage explants were washed with PBS, fixed with 4% PFA, dehydrated in sucrose, embedded in OCT sectioning medium, sectioned into 15 µm thick sections, mounted using a DAPI mounting medium, and imaged with a Keyence BZ- X710 fluorescence microscope. PLGA-BPL nanoparticles showed cartilage penetration within 24 h (FIG. 15). Almost full-thickness penetration (^ 4mm) was observed in just 48 h of incubation (FIG.15). The high penetration depth of the nanoparticle is attributed to its cationic BPL component and its dynamic binding with the negatively charged extracellular matrix. DUKE-43487.601 Cartilage Penetration of the nanoparticles using OA explant. The cartilage penetration ability of the nanoparticle was also evaluated using human OA cartilage explants following the same method as described above. Cartilage plugs of 4 mm diameter and ~2 mm height were punched out using a biopsy punch. The fresh cartilage plugs were equilibrated at ,0 i FRI ." 7A2in chondrocyte media for 24 hours before using for penetration studies. After exposure to the nanoparticles, the cartilage explants were washed with PBS, fixed with 4% PFA, dehydrated in sucrose, embedded in OCT sectioning medium, sectioned into 15 µm thick sections, mounted using a DAPI mounting medium, and imaged with a Keyence BZ-X710 fluorescence microscope. PLGA-BPL nanoparticles showed cartilage penetration within 24 h (FIG.16). The depth of the penetration was found to increase over time while a full-thickness penetration was observed in just 48 h (FIG.16). Notably, PLGA nanoparticles without BPL (labeled with Cy5) did not show any signal for Cy5 in the cartilage which indicated that the PLGA-BPL penetrates cartilage due to the presence of the highly cationic BPL on the surface of the nanoparticles. Synthesis of drug-loaded cartilage binding PLGA nanoparticles. Drug-loaded cartilage targeting PLGA nanoparticles was prepared via the emulsification method as described previously using the PLGA polymers along with the drug molecules (e.g., hydrophobic drug) or drug-loaded sugar-glass micelles (protein-loaded SG NPs). Briefly, PLGA and PLGA-PEG were dissolved in chloroform. PLGA-PEG-BPL was dissolved in DMSO. Hydrophobic drug or protein-loaded SGNPs were then added to the DMSO solution. The organic solutions (chloroform: DMSO = 9:1) were combined to get a final polymer concentration of 5 mg / mL. The mixture (2 mL) was added to phosphate buffer saline (PBS, 10 mL) containing 0.4% (w / v) polyvinyl alcohol and then emulsified using a probe sonicator for a total of 180 s at 4 ^C and 15-18 kHz output. Following emulsification, the mixture was stirred for 6 h, and centrifuged at 3000 rpm for 10 min to remove large aggregates. The supernatant was collected, and centrifuged using a high-speed ultracentrifuge (100k × g) for 45 min. The pellet of PLGA nanoparticles was washed repeatedly in deionized water. Finally, the particles were suspended in DI water and stored at 4 ^C. Drug loading has minimal effect on the particle size and zeta potential as no significant changes in diameter or surface charge were observed following drug loading (FIGS.17A-17B). DUKE-43487.601 Example 5 Generation of Adenosine-Loaded BPL Nanoparticles To deliver adenosine, 3-(acrylamido)phenylboronic acid (3-APBA) containing adenosine molecules have been directly conjugated to the modified BPL molecules, BPL acrylate. Synthesis of BPL acrylate (BPL-AA): Branched poly-l-lysine (BPL, G1, 200 mg) was dissolved in DI water (10 mL) and placed in an ice bath. Acryloyl chloride (127 qL, 0.75 equivalent to the primary amine groups of BPL) was then added dropwise to the BPL solution. The reaction mixture was stirred for 12 h and the pH of the mixture was maintained at 7.5 to 9.0 using the 1 N NaOH solution. The mixture was then dialyzed against DI water for 2-3 days and freeze-dried. The product, BPL-AA was characterized by1HNMR spectroscopy. The spectrum clearly showed the presence of acrylate protons at 5.65-6.17 ppm in addition to the presence of various lysine protons such as 1.44-1.80 ppm characteristics of 1NHCH2(CH2)3CH1 and 4.32 ppm characteristics of 1NHCH2(CH2)3CH1 protons respectively. Synthesis and characterization of adenosine-loaded cartilage targeting nanoparticles: The adenosine-loaded cartilage targeting BPL nanoparticles was synthesized via an inverse emulsion suspension polymerization method. First, adenosine was conjugated to 3-(acrylamido)phenylboronic acid (3-APBA) at ^pH 10-11 to obtain an PBA-adenosine complex. Briefly, 3-APBA (0.25 M, 1 equivalent) was dissolved in water (200 µL) by adjusting the pH to approximately 10-11 via the addition of 5 N NaOH. Adenosine (0.25 M, 1 equivalent) was added to the PBA solution and dissolved to obtain a clear solution. Separately, BPL-AA (1.2-2.4 equivalent) was dissolved in water (200 µL) and PEG-acrylamide (0.03-0.06 M) was added the BPL-AA solution. Both 3-APBA and BPL solutions were mixed and 2,4,6- trimethylbenzoylphosphinate (LAP) was added (final concentration of LAP: 0.4%). The mixture was added in a continuous organic phase of 5% (w / v) span 80 in cyclohexane (16 mL). The mixture was then vortexed extensively for about 2 min and then ultrasonicated for about 90 seconds (probe sonicator, 15–18 kW output). The nanodroplets were exposed to UV irradiation for 10 minutes to obtain the nanoparticles. The particles were then precipitated in acetone, washed with a mixture of 1:1 hexane and acetone (4^20 mL), and then with acetone (2^20 mL). The nanoparticles were then suspended in a minimum amount of DI water and freeze-dried. Nanoparticles without adenosine were synthesized similarly using BPL-AA and 3-APBA devoid of any adenosine. DUKE-43487.601 Characterization of the nanoparticles: The nanoparticles were suspended in DI water at concentrations of 50-150 qg / mL used for zeta potential measurements. A Malvern Zetasizer Nano Series was used with disposable folded capillary cells (Cat. no. DTS1070; Malvern Panalytical) for the measurements. The zeta potential value was determined by averaging three independent measurements, where each measurement involved 100 runs. The nanoparticle made up of BPL-AA and 3-APBA without adenosine (BPL to PBA molar ratio = 0.8) showed high positive zeta potential (45-50 mV) due to the presence of the free amine groups of lysine units of BPL. Notably, the zeta potential of the BPL-PBA nanoparticles loaded with adenosine varied depending on the amount of BPL used. For example, when the molar ratio of BPL to PBA was maintained at 1.2, the nanoparticles showed a zeta potential of 1.5^0.5 mV but when the BPL to PBA ratio was kept at 2.4, the zeta potential increased to 11^1 mV. The changes in the zeta potential are associated with the introduction of the negatively charged hydroxylated PBA during the adenosine loading process, and the molar ratio of positively charged BPL and the negatively charged hydroxylated PBA determined the overall zeta potential values of the BPL-PBA-Adenosine (BPL-PBA-ADO) nanoparticles. The size of the nanoparticles was measured via dynamic light scattering (DLS). For DLS measurements, the nanoparticles were INWWSPZJI FX .)&*.) gL(Q> NR B6C FRI XMJ M]IVSI]RFQNH INFQJXJV [FW QJFWYVJI YWNRL XMJ Malvern Zetasizer Nano Series used with disposable folded capillary cells. The hydrodynamic diameter was determined by averaging three independent measurements. The nanoparticle made up of BPL-AA and 3-APBA without adenosine showed particle size (150-200 nm) similar to the size of the nanoparticle made up of BPL-AA and 3-APBA with adenosine. Adenosine content in the nanoparticles: The adenosine loading capacity in the nanoparticles was measured using UV / visible spectroscopy. An adenosine standard curve was created by using a range (3.12-125 µg / mL) of known adenosine concentrations in 0.1 M acetate buffer of pH 3.5. Adenosine-loaded nanoparticles (~2-5 mg) were suspended in 1 mL of 0.1 M acetate buffer (pH ^3.5). The suspension was then added to a dialysis bag of MWCO 50 kDa. The bag was placed in a 15 mL falcon tube containing 4 mL of the buffer. The solution was OJTX FX ,0 a7 KSV +- M YRIJV HSRWXFRX WMFONRL' DMJ WYTJVRFXFRX [FW HSPPJHXJI FRI FGWSVTXNSR was measured through UV / vis spectroscopy at 260 nm. Adenosine concentration in the supernatant was determined by fitting the absorbance in the standard calibration curve. The amount of adenosine in the nanoparticles was varied by varying the BPL / PBA ratio. For example, when the molar ratio of BPL to PBA was maintained at 1.2, the nanoparticles DUKE-43487.601 contained ~330^25 µg of adenosine per mg of the nanoparticle while 250^20 µg of adenosine per mg of the nanoparticle was observed when the BPL to PBA was kept at 2.4. Cartilage penetration study: Human cartilage explants of 3 mm in diameter and 4 mm thickness were equilibrated in chondrocyte media 24 hours before use. A custom-designed device that enables selective exposure of the superficial zone of the cartilage to the nanoparticle suspension was used for the experiments. The device was generated from polydimethylsiloxane (PDMS) and the master molds with the required features were 3D printed using polylactic acid (PLA) filaments. Specifically, the PLA master mold was designed to create a cylindrical cartilage holder with an outer diameter of 12 and 15 mm thick and an inner compartment of three segments of equal length (5 mm) but varying diameters (upper diameter: 10 mm, middle diameter: 2.95 mm, and lower diameter: 2 mm). Cartilage explants were press-fitted into the middle chamber of the PDMS mold and exposed to the upper compartment, which was IJWNLRJI XS FHHSQQSIFXJ +)) gP SK 6B> WSPYXNSRW' DMJ PS[JV HSQTFVXQJRX [FW YWJI XS FHHJWW XMJ B6C [MJR XMJ IJZNHJ [FW TPFHJI NR F +-&[JPP TPFXJ HSRXFNRNRL .)) g> SK B6C XS QFNRXFNR humidity and limit dehydration of the nanoparticle suspension. Cyanine 5 (Cy5)-conjugated RFRSTFVXNHPJW [JVJ INWWSPZJI NR B6C FX F HSRHJRXVFXNSR SK .)) gL(Q> FRI YWJI KSV XMJ J\TJVNQJRXW' DMJ WYTJVKNHNFP ^SRJ SK HFVXNPFLJ J\TPFRXW [FW J\TSWJI XS +)) g> SK XMJ 7]. labeled suspension and incubated at 37 °C. Adhesion and penetration of the nanoparticles to the cartilage explants were determined by sectioning and imaging the cartilage explant. After a predetermined time interval, the cartilage explants were removed from the device, fixed, JQGJIIJI NR A7D% WJHXNSRJI NRXS *) gQ XMNHO WJHXNSRW% FRI WXFNRJI [NXM 85B=' DMJ WJHXNSRW were imaged with a Keyence BZ-X710 fluorescence microscope. The BPL-PBA-ADO nanoparticles showed cartilage penetration. Both the nanoparticles with lower BPL and higher BPL content (with a zeta potential of 1.5 mV and 11 mV respectively) showed cartilage penetration. Only a slightly higher depth of penetration was observed with higher BPL content (FIG.18). Dose dependent effect of adenosine on chondrogenesis of primary human osteoarthritic (OA) chondrocytes: Human osteoarthritic (OA) chondrocytes were centrifuged to form pellets (250k cells / pellet) and cultured in chondrocyte medium and treated with various concentrations of adenosine (0-60 qg / mL) and cultured for 10 days. The cell pellets were processed and gene expression analysis was carried out by qPCR. Aggrecan and Collagen type II are the two major extracellular matrix components of cartilage. The qPCR analysis shows DUKE-43487.601 significant upregulation of these two markers following adenosine treatment suggesting the anabolic effect of adenosine on arthritic chondrocytes. See FIG.19. Example 6 Incorporation of Protein or Antibody-Based Compounds into the Cartilage Penetrating Nanoparticles Synthesis: Drug-loaded nanoparticles were prepared via the emulsification method using the polymer precursors along with the therapeutic molecule (e.g., PTH, FGF18) -loaded sugar-glass micelles. For example, PLGA and PLGA-PEG, PLGA-DBCO, and therapeutic- loaded sugar glass micelles were dissolved / suspended in chloroform. The mixture (2 mL) was added to phosphate buffer saline (PBS, 10 mL) containing 0.4% (w / v) polyvinyl alcohol and then emulsified using a probe sonicator for a total of 180 s at 4 ^C and 15-18 kHz output. Following emulsification, the mixture was stirred for 6 h, and centrifuged at 3000 rpm for 10 min to remove large aggregates. The supernatant was collected, and centrifuged using a high- speed ultracentrifuge (100k ^ g) for 45 min. The biomolecule-loaded nanoparticles were washed repeatedly in deionized water. Next, the nanoparticles were suspended in DI water and reacted with terminal azide functionalized branched poly-l-lysine (BPL, G1) in water for 3 hours. Following BPL modification, the nanoparticles were washed repeatedly in deionized water and stored at 4 ^C. Characterization of the drug loaded nanoparticles: The particle size was measured using dynamic light scattering (DLS), and the average size of the particles was found to be ^ 180 ^ 40 nm. The zeta potential measurements indicate positive surface charges with a zeta potential of ^ +22 ^ 8 mV. The positive zeta potential is due to the presence of the BPL molecules on the PLGA nanoparticles. Synthesis of cartilage penetrating and bone targeting poly lactic-co-glycolic acid (PLGA) nanoparticles: To synthesize both cartilage and bone binding nanoparticles, in addition to functionalizing the nanoparticle or nanocarriers with BPL molecules, we also functionalized them with bone binding moiety alendronate (Aln) moieties. Again, we use PLGA nanoparticle as a model system. The overall synthesis and characterization of the nanoparticles is as follows. Synthesis of PLGA-PEG. PLGA was dissolved in dichloromethane (DCM) and N-(3- dimethylaminopropyl)-Ne&JXM]P HFVGSINNQNIJ M]IVSHMPSVNIJ #987% *) JUYNZFPJRX$ [FW added to the solution and the mixture was stirred for 15 minutes. N-Hydroxysuccinimide (NHS, DUKE-43487.601 10 equivalent) was added to the reaction mixture. After 15 minutes, mPEG-NH2 (Mol. Wt. of PEG = 2 kDa; 5 equivalent) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 24 h following the addition of DIPEA (10 equivalent). The product was precipitated in a mixture of diethyl ether and methanol (1:1). The precipitate was washed repeatedly in the same solvent mixture and dried under vacuum. The percentage of PEG modification was quantified by proton NMR, and over 95% of the PEG modification into the PLGA was observed. Synthesis of PLGA-DBCO. PLGA was dissolved in dichloromethane (DCM) and N- (3-dimethylaminopropyl)-Ne&JXM]P HFVGSINNQNIJ M]IVSHMPSVNIJ #987% +) JUYNZFPJRX$ [FW added to the solution and the mixture was stirred for 15 minutes. N-Hydroxysuccinimide (NHS, 20 equivalent) was added to the reaction mixture. After 15 minutes, DBCO-amine (10 equivalent) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 24 h following the addition of DIPEA (20 equivalent). The product was precipitated in a mixture of diethyl ether and methanol (1:1). The precipitate was washed repeatedly in the same solvent mixture and dried under vacuum. The percentage of DBCO modification was quantified by proton NMR, and over 98% of the PEG modification into the PLGA was observed. Synthesis of PLGA-PEG-Aln. Azide-PEG-Azide (1g, MW 3.4k) was dissolved in water and N-(3-dimethylaminopropyl)-Ne&JXM]P HFVGSINNQNIJ M]IVSHMPSVNIJ #987% *) equivalent) was added to the solution and the mixture was stirred for 15 minutes. N- Hydroxysuccinimide (NHS, 10 equivalent) was added to the reaction mixture. After 15 minutes, alendronate (10 equivalent) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 24 h. Following the reaction, the mixture was dialyzed against water for 4-5 days and freeze-dried to obtain Azide-PEG-Aln. Next, PLGA-DBCO was dissolved in dichloromethane (DCM), and Azide-PEG-Aln (1.5 equivalent) was added to the solution. The reaction mixture was stirred for about 6 h. The product was precipitated in a mixture of diethyl ether and methanol (1:1). The precipitate was washed repeatedly in diethyl ether and methanol (1:1) mixture and dried under vacuum. Synthesis of BPL molecules with terminal azide group. Cbz-Lys-NCA (20 equivalents) was dissolved in dry N,N-dimethylformamide (DMF, 100 mg / mL) in a round- bottomed flask fitted with a drying tube.11-Azido-3,6,9-trioxaundecan-1-amine (1 equivalent) was added to the reaction mixture and the reaction was continued at room temperature for about 5 days under continuous stirring. The reaction mixture was added to a ^20-fold excess of cold DUKE-43487.601 water to precipitate the oligo(Cbz-L-lysine) core peptide with a terminal azide functional group at one end. The precipitate was filtered, washed with water, and freeze-dried. A solution of the oligo(Cbz-L-lysine) peptide in DMF was then reacted with Ne,Ni-Di(9- fluorenylmethoxycarbonyl-L-lysine) (Ne,Ni-diFmoc-Lys, 4 equivalents) using 2-(1H-benzo- triazole-1-yl)-oxy-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 4 equivalents), and 1-hydroxy benzotriazole (HOBt, 12 equivalents) and N,N-diisopropylethylamine (DIPEA, 10 equivalents) at room temperature for 3 days. The product was precipitated in excess of cold water, filtered, washed three times with water, and freeze-dried. The freeze-dried powder was washed 4 times with diethyl ether, and vacuum-dried to obtain Ne,Ni-diFmoc-Lys conjugated core peptide. A solution of the Ne,Ni-diFmoc-Lys end-functionalized core peptide in DMF (^200 mg / mL) was reacted with 20% (v / v) piperidine under continuous stirring for an hour at room temperature and subsequently precipitated in water. The precipitate was filtered, washed repeatedly with water, and freeze-dried. The freeze-dried product was washed 4 times with diethyl ether, and vacuum-dried to obtain lysine end-functionalized core peptide containing azide functional group on the other end. Next, the core peptide was reacted with the Cbz-Lys- NCA monomer to obtain next-generation branched peptides (G0). Higher generation of the branched peptides (G1) was then obtained by repeating the above steps of ring-opening polymerization, end functionalization, and subsequent removal of Fmoc protecting groups. The Cbz protecting group was completely removed from BPL molecules to generate free amine groups in BPL molecules. Drug Loading: The biomolecules can be loaded as described in the previous examples via encapsulation or by direct conjugation. Example 7 Therapeutic-Loaded Sugar Glass Micelles Synthesis of therapeutic-loaded sugar glass micelles (SG): Dioctyl sulfosuccinate (AOT, 0.004 mol) was dissolved in isooctane (12 mL) in a 50 mL centrifuged tube. An aqueous solution containing therapeutics (Cytokines, Proteins such as PTH, BMP-2, Hormones, Antibodies, Enzymes, etc.) and trehalose sugar was then added dropwise to the isooctane solution. The mass ratio of therapeutics to trehalose was maintained at 1:200 (e.g., 0.5 mg of therapeutics in 100 mg of trehalose) (mass ratio of therapeutics to trehalose can be varied from 1:20 to 1:2000). The water / AOT molar ratio ([H2O] / [AOT]) was set to ^15 (This can be varied from 1:5 to 1:50). After the addition of the aqueous solution, the mixture was continuously vortexed for 2 min until a clear suspension was observed. The resulting inverse DUKE-43487.601 micelles were flash-frozen in liquid nitrogen. The frozen micelles were then lyophilized, followed by washing multiple times with isooctane. The resulting sugar-glass micellar nanoparticles were resuspended in isooctane and stored at b+) _7 YRXNP KYVXMJV Yse. Characterization of therapeutic loaded SG micelles: The size of the various therapeutic-loaded sugar glass micelles was determined via dynamic light scattering. The SG micelles were suspended in isooctane or chloroform at 50-150 qg / mL and the size were measured via a Malvern Zetasizer Nano Series instrument. While the size of the reverse micelles before freeze-drying was found to be 10^2 nm, the hydrodynamic diameter increased to 25-55 nm following freeze-drying (FIGS. 20A-20C). A very few agglomerated particles (>200 nm) were also obtained which were removed by centrifugation (at 100^g, 3 min). These large agglomerates were associated with sample temperature excursions during the freeze- drying process, which can be controlled by providing the liquid nitrogen to keep the samples frozen during the drying process. Therapeutic encapsulation efficiency in SG (Example by PTH encapsulation): PTH encapsulation efficiency was measured via PTH 1-34 enzyme-linked immunosorbent assay following the manufacturer protocol. Briefly, the 20X assay buffer, supplied with the kit, was diluted with DI water to obtain 1X assay buffer and used to dilute or reconstitute all other reagents in this kit and samples. The peptide standard was diluted with 1 mL of 1X assay buffer. The concentration of this stock solution was 1,000 ng / mL. The standard solution was then diluted to obtain PTH concentrations of 0.01-100 ng / mL. 50 qL of the peptide standard solutions (0.01-100 ng / mL) was then added to a secondary antibody-coated immunoplate along with other test samples. To determine the PTH content in sugar glass micelles, the micelles were resuspended in PBS and incubated at 37 ^C for about 48-96 h, and subsequently used for PTH assay. Primary antibody (20 qL), supplied with the kit, was then added to the wells. A biotinylated peptide solution (20 qL) was then added to each well. The immunoplate was sealed with acetate plate sealer (APS) and incubated for 2 hours at room temperature on an orbital shaker (at 300 rpm). Following incubation, the wells were washed repeatedly (4 ^ 350 qL) with 1X assay buffer. Streptavidin-horseradish peroxidase (SA-HRP) solution (100 qL) and the plate was sealed with APS and incubated for 1 hour at room temperature at 300 rpm on an orbital shaker. The plate was washed again repeatedly (4 ^ 350 qL) with 1X assay buffer. TMB solution (100 qL) was then added to the wells and incubated for 1 hour at room temperature on an orbital shaker (300 rpm). 2N HCl (100 qL) was then added to each and the optical density (OD) values were recorded at 450 nm using a Tecan plate reader instrument. The standard DUKE-43487.601 curve was then plotted using 4 parameter logistics in GraphPad Prism and used to calculate the PTH concentration of testing samples. The content in the micelles was found to be 2.2^0.2 qg per mg of the micelles (FIG.21). Retention of bioactivity of therapeutics following encapsulation (Bioactivity of PTH-loaded sugar glass via intracellular cAMP measurements): PTH-sugar glass micelles were freeze-dried to remove isooctane. PBS (1X) was added to the dried samples and incubated at 37 ^C for about 24 h. The PBS solution was then used to measure the bioactivity of released PTH. MC3T3-E1 cells were cultured in e-MEM containing 10% fetal bovine serum (FBS) at 37 °C in 5% CO2 in an incubator. For experiments, 5 x104cells were plated in wells of a 24- well plate in 0.5 mL of e-MEM supplemented with 10% serum, cultured for 3 days, and then transferred to serum-free medium with 0.3% bovine serum albumin (BSA). After 24 h, cells were preincubated in e-MEM containing 0.3% BSA plus 0.1 mM isobutylmethyl xanthine (IBMX) for 60 min at 37 °C. Then various doses of PTH or PTH sugar glass micelles dissolved in e-MEM containing 0.3% BSA and 0.1 mM IBMX were added to the preincubated cells which were further incubated at 37 °C for 10 min. After incubation, the medium was discarded, and intracellular cAMP was determined via a cAMP direct immunoassays kit (Abcam, FG*,111)$' 6VNJKP]% +)) g> SK HJPP P]WNW GYKKJV TVSZNIJI [NXM XMJ ONX [FW FIIJI XS JFHM [JPP and incubated at room temperature for 10 minutes. The cell lysis buffer was then collected in Eppendorf tubes and centrifuged for 5 minutes at 4 °C at 13000 rpm using a cold microcentrifuge to remove any insoluble material. The supernatant was collected and transferred to new tubes and stored on ice. The cAMP content in the cell lysates was then measured by cAMP direct immunoassays kit following the manufacturer’s protocol. The assay is based on the competition between HRP-labeled cAMP and free cAMP present in the sample for cAMP antibody binding sites. In the absence of cAMP, HRP-cAMP conjugate is bound to anti-cAMP antibody exclusively. Free cAMP (unlabeled) present in the test sample competes with HRP-cAMP for anti-cAMP antibody, displacing HRP-cAMP and inhibiting its binding to the antibody. The fluorescence observed (Ex / Em = 540 / 590 nm) is proportional to the activity of HRP-cAMP conjugate: the more cAMP present in the test samples, the lower the fluorescence detected. Treatment of MC3T3-E1 cells with PTH encapsulated in the sugar glass micelles increased the intracellular cAMP level. The encapsulated PTH showed similar cAMP production as the free PTH, indicating bioactivity retention following encapsulation (FIG.22).

Claims

DUKE-43487.601 CLAIMS 1. A functionalized branched poly-lysine compound, wherein at least one primary amine group of the branched poly-lysine is functionalized with a pharmaceutically active moiety, a polyalkylene oxide moiety, cholesterol, a monosaccharide, an oligosaccharide, or a zwitterionic molecule.

2. The functionalized branched poly-lysine compound of claim 1, wherein the branched poly-lysine is a branched poly(L-lysine) compound.

3. The functionalized branched poly-lysine compound of claim 1 or claim 2, wherein at least one primary amine group of the branched poly-lysine is functionalized with a pharmaceutically active moiety.

4. The functionalized branched poly-lysine compound of any one of claims 1-3, wherein at least one primary amine group of the branched poly-lysine is functionalized with a hydrophobic drug moiety.

5. The functionalized branched poly-lysine compound of any one of claims 1-4, wherein the pharmaceutically active moiety is selected from glucocorticoids, corticosteroids, FRFPLJWNHW% RSR&WXJVSNIFP FRXN&NRKPFQQFXSVNJW% TFNR IVYLW% QSRSHPSRFP FRXNGSINJW% =>&*f FRXFLSRNWXW% D@: NRMNGNXSVW% =>&*f NRMNGNXSVW% TVSXJFWJ NRMNGNXSVW% JTXSXJVQNR FPKF% WTVNKJVQNR% insulin growth factors, cell-based therapeutics, gene therapies, GLP-1 based drugs, antibiotics, and metformin drugs.

6. The functionalized branched poly-lysine compound of any one of claims 1-4, wherein the pharmaceutically active moiety is selected from methylprednisolone and adenosine.

7. The functionalized branched poly-lysine compound of any one of claims 1-6, wherein the pharmaceutically active moiety is attached to the primary amine group of the branched poly-lysine via a direct bond.DUKE-43487.601 8. The functionalized branched poly-lysine compound of any one of claims 1-6, wherein the pharmaceutically active moiety is attached to the primary amine group of the branched poly-lysine via a linker.

9. The functionalized branched poly-lysine compound of any one of claims 1-5, wherein about 1% to about 50% of the primary amine groups of the branched poly-lysine are functionalized with the pharmaceutically active moiety.

10. The functionalized branched poly-lysine compound of any one of claims 1-9, wherein at least one primary amine group of the branched poly-lysine is functionalized with a polyethylene oxide moiety.

11. The functionalized branched poly-lysine compound of claim 10, wherein the polyethylene oxide moiety is a moiety of formula:wherein m is an integer from 4 to 50.

12. The functionalized branched poly-lysine compound of claim 11, wherein m is 8.

13. The functionalized branched poly-lysine compound of any one of claims 10-12 wherein about 10% to about 50% of the primary amine groups of the branched poly-lysine are functionalized with the polyethylene oxide moiety.

14. The functionalized branched poly-lysine compound of claim 1, having a structure of formula (I):DUKE-43487.601 or a salt thereof, wherein: R1is an optionally substituted C1-C12 alkyl group; each R2is independently -CH2CH2CH2CH2NHRa, wherein each Rais independently hydrogen, a nitrogen protecting group, a polyalkylene oxide moiety, cholesterol, a monosaccharide, an oligosaccharide, a zwitterionic molecule, or a pharmaceutically active moiety, wherein at least one Rais not hydrogen; each R3is independently hydrogen or a group of formula (A):each R4is independently hydrogen or a group of formula (B):n1, n2, n3, n4, n5, n6, and n7 are each independently 5, 6, 7, 8, 9, 10, 11, or 12.

15. The functionalized branched poly-lysine compound of claim 14, wherein R1is an unsubstituted C2-C8 alkyl group.

16. The functionalized branched poly-lysine compound of claim 14, wherein R1is a group of formula:wherein q is 2, 3, 4, 5, 6, 7, or 8, and X is selected from a group comprising a reactive moiety, a polyalkylene glycol moiety, a sugar, and a peptide.DUKE-43487.601 17. The functionalized branched poly-lysine compound of claim 16, wherein X is a reactive moiety selected from a thiol, an amine, an azide, an alkyne, an alkene, a dibenzocyclooctyne, a maleimide, and a succinimidyl ester.

18. The functionalized branched poly-lysine compound of any one of claims 14-17, wherein each R3is hydrogen.

19. The functionalized branched poly-lysine compound of any one of claims 14-17, wherein each R3is a group of formula (A).

20. The functionalized branched poly-lysine compound of any one of claims 14-19, wherein each R4is hydrogen.

21. The functionalized branched poly-lysine compound of any one of claims 14-19, wherein each R4is a group of formula (B).

22. The functionalized branched poly-lysine compound of any one of claims 14-21, wherein each R2is independently -CH2CH2CH2CH2NHRa, wherein each Rais independently hydrogen, a nitrogen protecting group, a polyalkylene oxide moiety, or a pharmaceutically active moiety, wherein at least one Rais not hydrogen.

23. The functionalized branched poly-lysine compound of any one of claims 14-21, wherein each R2is independently -CH2CH2CH2CH2NHRa, wherein each Rais independently hydrogen, a benzyloxycarbonyl group, a polyethylene oxide moiety, or a hydrophobic drug moiety, wherein at least one Rais not hydrogen.

24. The functionalized branched poly-lysine compound of any one of claims 14-23, wherein at least one Rais a pharmaceutically active moiety.

25. The functionalized branched poly-lysine compound of any one of claims 14-25, wherein at least one Rais a hydrophobic drug moiety.DUKE-43487.601 26. The functionalized branched poly-lysine compound of any one of claims 14-25, wherein the pharmaceutically active moiety is selected from glucocorticoids, corticosteroids, FRFPLJWNHW% RSR&WXJVSNIFP FRXN&NRKPFQQFXSVNJW% TFNR IVYLW% QSRSHPSRFP FRXNGSINJW% =>&*f FRXFLSRNWXW% D@: NRMNGNXSVW% =>&*f NRMNGNXSVW% TVSXJFWJ NRMNGNXSVW% JTXSXJVQNR FPKF% WTVNKJVQNR% insulin growth factors, cell-based therapeutics, gene therapies, GLP-1 based drugs, antibiotics, and metformin drugs.

27. The functionalized branched poly-lysine compound of any one of claims 14-25, wherein the pharmaceutically active moiety is selected from methylprednisolone and adenosine.

28. The functionalized branched poly-lysine compound of any one of claims 14-27, wherein the pharmaceutically active moiety is attached via a direct bond.

29. The functionalized branched poly-lysine compound of any one of claims 14-27, wherein the pharmaceutically active moiety is attached via a linker.

30. The functionalized branched poly-lysine compound of any one of claims 14-29, wherein about 1% to about 50% of the Ragroups are a pharmaceutically active moiety.

31. The functionalized branched poly-lysine compound of any one of claims 14-31, wherein at least one Ragroup comprises a polyethylene oxide moiety.

32. The functionalized branched poly-lysine compound of claim 31, wherein the polyethylene oxide moiety is a moiety of formula:wherein m is an integer from 4 to 50.

33. The functionalized branched poly-lysine compound of claim 32, wherein m is 8.

34. The functionalized branched poly-lysine compound of any one of claims 14-33 wherein about 10% to about 50% of the Ragroups comprise the polyethylene oxide moiety.DUKE-43487.601 35. A delivery system comprising: a polymeric nanocarrier; a pharmaceutically active compound; and a branched poly-lysine moiety covalently attached to the polymeric nanocarrier.

36. The delivery system of claim 35, wherein the branched poly-lysine moiety is a branched poly(L-lysine) moiety.

37. The delivery system of claim 35, wherein the polymeric nanocarrier comprises a polyester. ,1' DMJ IJPNZJV] W]WXJQ SK HPFNQ ,0% [MJVJNR XMJ TSP]JWXJV NW TSP]#PFHXNHhHShLP]HSPNH$ acid.

39. The delivery system of claim 35, wherein the polymeric nanocarrier comprises a copolymer of a polyester and a polyalkylene oxide.

40. The delivery system of claim 39, wherein the copolymer is a block copolymer of TSP]#PFHXNHhHShLP]HSPNH$ FHNI FRI TSP]JXM]PJRJ LP]HSP' 41. The delivery system of any one of claims 35-40, wherein the pharmaceutically active compound is selected from glucocorticoids, corticosteroids, analgesics, non-steroidal anti- NRKPFQQFXSVNJW% TFNR IVYLW% QSRSHPSRFP FRXNGSINJW% =>&*f FRXFLSRNWXW% D@: NRMNGNXSVW% =>&*f inhibitors, protease inhibitors, eptotermin alfa, sprifermin, insulin growth factors, cell-based therapeutics, gene therapies, GLP-1 based drugs, antibiotics, and metformin drugs.

42. The delivery system of any one of claims 35-40, wherein the pharmaceutically active compound is selected from methylprednisolone and adenosine.

43. The delivery system of any one of claims 35-42, wherein the pharmaceutically active compound is encapsulated within the polymeric nanocarrier.DUKE-43487.601 44. The delivery system of any one of claims 35-43, wherein the branched poly-lysine moiety has a structure of formula (II):or a salt thereof, wherein: p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each R2is independently -CH2CH2CH2CH2NHRa, wherein each Rais independently hydrogen or a nitrogen protecting group; each R3is independently hydrogen or a group of formula (A):each R4is independently hydrogen or a group of formula (B):n1, n2, n3, n4, n5, n6, and n7 are each independently 5, 6, 7, 8, 9, 10, 11, or 12.

45. The delivery system of claim 44, wherein p is 2, 3, 4, 5, 6, 7, or 8.DUKE-43487.601 46. The delivery system of claim 44 or claim 45, wherein each R2is independently - CH2CH2CH2CH2NHRa, wherein each Rais hydrogen.

47. The delivery system of any one of claims 44-46, wherein each R3is hydrogen.

48. The delivery system of any one of claims 44-46, wherein each R3is a group of formula (A).

49. The delivery system of any one of claims 44-48, wherein each R4is hydrogen.

50. The delivery system of any one of claims 44-48, wherein each R4is a group of formula (B).

51. A method of treating a joint disease in a subject in need thereof, comprising administering to a joint of the subject a therapeutically effective amount of the functionalized branched poly-lysine compound of any one of claims 1-34, or the delivery system of any one of claims 35-50.

52. The method of claim 51, wherein the joint disease is osteoarthritis.

53. The method of claim 51 or claim 52, wherein the administering step comprises intra- articular injection of the functionalized branched poly-lysine compound or the delivery system.

54. A method of delivering a pharmaceutically active compound to cartilage and / or subchondral bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the functionalized branched poly-lysine compound of any one of claims 1-34, or the delivery system of any one of claims 35-50.

55. The method of claim 54, wherein the subject has a joint disease.

56. The method of claim 55, wherein the joint disease is osteoarthritis.DUKE-43487.601 57. The method of any one of claims 54-56, wherein the administering step comprises intra-articular injection of the functionalized branched poly-lysine compound or the delivery system.

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