Biocompatible and biodegradable polymer nanodiscs as lipoprotein-mimicking nanocarriers with high stability and long circulation time for drug delivery

Abstract

Embodiments of the present disclosure pertain to an active agent carrier that includes a disc-shaped membrane with a plurality of self-assembled amphiphilic block copolymers encased by membrane stabilizing agents, where the amphiphilic block copolymers include hydrophilic blocks and hydrophobic blocks, and where at least one active agent is associated with the disc-shaped membrane. Tunable numbers of targeting agents may also be associated with the disc-shaped membrane. Additional embodiments pertain to methods of delivering one or more active agents to a subject by administering to the subject an active agent carrier of the present disclosure. Further embodiments pertain to methods of making an active agent carrier of the present disclosure.

Description

TITLE BIOCOMPATIBLE AND BIODEGRADABLE POLYMER NANODISCS AS LIPOPROTEIN-MIMICKING NANOCARRIERS WITH HIGH STABILITY AND LONG CIRCULATION TIME FOR DRUG DELIVERY STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under 2213969 awarded by the National Science Foundation. The government has certain rights in this invention. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 569,130, filed on March 23, 2024. The entirety of the aforementioned application is incorporated herein by reference. BACKGROUND

[0003] A need exists for more effective active agent delivery carriers. Various embodiments of the present disclosure aim to address the aforementioned need. SUMMARY

[0004] In some embodiments, the present disclosure pertains to an active agent carrier that includes a disc-shaped membrane with a plurality of self-assembled amphiphilic block copolymers and a membrane stabilizing agent. In some embodiments, the amphiphilic block copolymers include hydrophilic blocks and hydrophobic blocks. In some embodiments, at least one active agent is associated with the disc-shaped membrane. In some embodiments, the membrane stabilizing agent encases the disc-shaped membrane.

[0005] Additional embodiments of the present disclosure pertain to methods of delivering one or more active agents to a subject. Such methods generally include administering to the subject an active agent carrier of the present disclosure, where the active agent carrier is associated with the active agent. 1 4823-4406-5785v.313368-42

[0006] Additional embodiments of the present disclosure pertain to methods of making an active agent carrier of the present disclosure. In some embodiments, such methods include: mixing a plurality of amphiphilic block copolymers with a membrane stabilizing agent to form a self- assembled disc-shaped membrane encased by the membrane stabilizing agent; and associating the disc-shaped membrane with at least one active agent. DRAWINGS

[0007] FIG.1 provides a conceptual design of a polymer nanodisc (PND) as a novel nanocarrier for active agent (e.g., anticancer drug) delivery. The amphiphilic block copolymer membrane (i.e., hydrophobic membrane interior in light color; hydrophilic membrane surface in dark color) is encased by membrane scaffold proteins 16 (MSPs). The PND may carry tissue targeting moieties 18 (e.g., tumor-homing moieties) on the membrane surface and active agents 14 (e.g., chemotherapy agents) inside.

[0008] FIG.2 shows a transmission electron microscope (TEM) image of a lipid nanodisc (LND) in aggregated form.

[0009] FIGS.3A-3C provide data illustrating that PNDs show superior stability over LNDs. FIG. 3A shows a size exclusion chromatography (SEC) revealing a monodisperse PND fraction with the co-existence of the protein (e.g., MsbA) and polymer, where the protein is embedded in the PND membrane. The inset shows cryo electron microscopy (CryoEM) of the protein-embedded PND(i.e., MsbA-PND). FIG. 3B shows a schematic view of an MsbA-PND. FIG. 3C shows non-aggregated fractions of LNDs and PNDs at different temperature and time.

[0010] FIGS.4A-4D show the synthesis and characterization of PEG-b-PLA-Dox. FIGS.4A-4B show reaction schemes to synthesize MBC monomer and mPEG-b-P(LA-co-MBC-Dox), respectively. FIG.4C shows a1H-NMR confirming the successful synthesis of MBC. FIG.4D shows SEC traces of the macroinitiator mPEG45(black; Mn / Ɖ: 2kD / 1.03) and four mPEG45-b- P(LA0.9-co-MBC0.1)40 at different reaction times: 30 s (5.6kD / 1.06), 2 min (5.7kD / 1.07), 5 min (5.7kD / 1.07), and 60 min (5.7kD / 1.08), respectively. The polymerization reaches ~100% conversion within 30 seconds.

[0011] FIG.5 shows a1H-NMR spectrum confirming the successful synthesis of urea I-O. 2 4823-4406-5785v.313368-42

[0012] FIG. 6 shows the synthesis and optimization of reaction times for mPEG-b-PLA block polymer under the cooperative catalytic system.

[0013] FIG. 7 shows a1H-NMR spectrum confirming the successful synthesis of mPEG45-b- PLA60.

[0014] FIG. 8 shows a1H-NMR spectrum confirming the successful synthesis of benzyl-2,2- bis(methylol)propionate.

[0015] FIG.9 shows gel permeation chromatography (GPC) dRI traces that confirm the successful synthesis of well-defined mPEG-b-PBC.

[0016] FIG. 10 shows a1H-NMR spectrum that confirms the successful synthesis of mPEG-b- PBC.

[0017] FIG. 11 shows a1H-NMR spectrum that confirms the successful synthesis of mPEG-b- PCC.

[0018] FIG. 12 shows a1H-NMR spectrum that confirms the successful synthesis of mPEG-b- (PCC-g-BA).

[0019] FIG. 13 shows a1H-NMR spectrum that confirms the successful synthesis of mPEG-b- PCC-Dox.

[0020] FIG.14 shows a1H-NMR spectrum that confirms the successful synthesis of HOOC-PEG- b-PLA for FA conjugation.

[0021] FIG.15 shows a DLS trace of the size distribution of polymersomes formed from mPEG45- b-PLA60.

[0022] FIG. 16 shows a TEM confirming polymersome formation of model amphiphilic block copolymers.

[0023] FIGS.17A-C show the characterization of PNDs. FIG.17A provides SEC traces (black: dRI; gray: A280), which shows a major self-assembled structure in the 12.2-17 nm size range that is encased by MSP. FIG.17B provides dynamic light scattering (DLS) of an eluted fraction that shows a diameter that peaked at ~13 nm. FIG. 17C shows a TEM that reveals uniformly-sized PNDs in this fraction (scale bar: 50 nm).

[0024] FIG.18 provides a Dox calibration curve. 3 4823-4406-5785v.313368-42

[0025] FIG.19 shows that the cumulative release of Dox increases from 5% (at pH 7.4) to 25% (at pH 5). The pH-responsive release of Dox supports the proof-of-concept design of the controlled release of PND-carried chemotherapeutics at the tumor sites.

[0026] FIG.20 shows an MTT assay against MCF7 cancer cells, which show that empty PNDs comprised of mPEG-b-PLA do not have an adverse effect on the cancer cells, but the Dox-carrying PNDs kill the cancer cells with a low IC50 (~ 3 μM) of the polymer concentration. DETAILED DESCRIPTION

[0027] It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that include more than one unit unless specifically stated otherwise.

[0028] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls. 4 4823-4406-5785v.313368-42

[0029] Drug delivery in general, and anticancer drug delivery in particular, has been hampered by the difficulty to deliver therapeutic agents across aqueous physiological barriers to reach the deep- seated disease sites with desired pharmacokinetics and biodistribution. Since the discovery of enhanced permeability and retention (EPR) effect of tumors, nanotechnology has been widely anticipated to benefit the diagnostic and treatment of cancers because the EPR effect dictates that nanocarriers should preferentially accumulate at tumor sites. Notwithstanding the significant progress that results in successful FDA approval of a few nanocarriers, efficacious patient responses remain modest compared to conventional drug formulations because of an inability to control the nanoparticle transport inside the body.

[0030] The importance of shape on regulating in vivo transport of nanocarriers is increasingly recognized, and mounting evidence suggests disc-shaped nanocarriers outperform spherical ones with prolonged circulation half-lives and higher cellular internalization rates. In fact, nascent high- density lipoproteins in the blood are lipid nanodiscs (LNDs) and efficient nanocarriers that mediate reverse cholesterol transport from peripheral cells back to the liver.

[0031] Nonetheless, most nanocarriers under development for drug delivery are spherical. Although LNDs are being pursued for drug delivery, as a nanocarrier platform, they suffer from low stability and payload capacity, short shelf life, and difficulty for chemical modifications.

[0032] Furthermore, current nanocarriers in the market, such as liposomes, lipid nanoparticles, or micelles, are of spherical shape with a broad size distribution and limited stability. As a result, they have short circulation time and poor success rate of reaching the tumor site. In fact, despite the high hope of using nanocarriers for drug delivery, it was found that, on average, only 0.7% of administered nanocarriers were actually delivered to the tumor sites.

[0033] As such, a need exists for more effective active agent delivery carriers. Various embodiments of the present disclosure aim to address the aforementioned need.

[0034] In some embodiments, the present disclosure pertains to an active agent carrier that includes a disc-shaped membrane with a plurality of self-assembled amphiphilic block copolymers. In some embodiments, the amphiphilic block copolymers include hydrophilic blocks and hydrophobic blocks. In some embodiments, at least one active agent is associated with the disc-shaped membrane. In some embodiments, the disc-shaped membrane also includes a membrane stabilizing agent that encases the disc-shaped membrane. 5 4823-4406-5785v.313368-42

[0035] FIG.1 provides an example of an active agent carrier of the present disclosure, which is identified as active agent carrier 10. In this example, active agent carrier 10 includes a plurality of amphiphilic block copolymers 12 in the form of a self-assembled disc-shaped membrane 13. In this example, amphiphilic block copolymers 12 consist of hydrophilic blocks and hydrophobic blocks that work synergistically to form the disc-shaped membrane 13, with the hydrophobic and hydrophilic blocks self-assembled into the membrane interior and membrane surface, respectively. In this example, active agents 14 are associated with amphiphilic block copolymers 12 through a pH-cleavable linkage 15. Additionally, active agent carrier 10 includes membrane stabilizing agent 16, which encases disc-shaped membrane 13, and which is operable to maintain the shape and size of disc-shaped membrane 13. In this example, active agent carrier 10 also includes targeting agent 18, which is operable to direct active agent carrier 10 to a specific tissue or cell.

[0036] Additional embodiments of the present disclosure pertain to methods of delivering one or more active agents to a cell. Such methods generally include associating the cell with an active agent carrier of the present disclosure, where the active agent carrier is associated with the active agent. In some embodiments, the association includes incubating the cell with the active agent carriers of the present disclosure. In some embodiments, the association occurs in vitro. In some embodiments, the association occurs in vivo in a subject.

[0037] Additional embodiments of the present disclosure pertain to methods of delivering one or more active agents to a subject. Such methods generally include administering to the subject an active agent carrier of the present disclosure, where the active agent carrier is associated with the active agent.

[0038] Additional embodiments of the present disclosure pertain to methods of making an active agent carrier of the present disclosure. In some embodiments, such methods include: mixing a plurality of amphiphilic block copolymers to form a self-assembled disc-shaped membrane; and associating the disc-shaped membrane with at least one active agent. 6 4823-4406-5785v.313368-42

[0039] In some embodiments, methods of making an active agent carrier of the present disclosure include: mixing a plurality of amphiphilic block copolymers with a membrane stabilizing agent to form a self-assembled disc-shaped membrane encased by the membrane stabilizing agent; and associating the disc-shaped membrane with at least one active agent. In some other embodiments, such methods include: modifying a plurality of amphiphilic block copolymers with at least one active agent; and mixing a plurality of amphiphilic block copolymers, some of which carry at least one active agent, with the membrane stabilizing agent to form a self-assembled disc-shaped membrane.

[0040] As set forth in more detail herein, the active agent carriers and methods of the present disclosure can include numerous embodiments.

[0041] Polymers

[0042] The active agent carriers of the present disclosure can include various polymers. Moreover, the methods of the present disclosure may utilize various polymers to make the active agent carriers of the present disclosure.

[0043] For instance, in some embodiments, the polymers include amphiphilic block copolymers. In some embodiments, the amphiphilic block copolymers include biodegradable polymers (i.e., polymers that are able to degrade completely through microbial degradation). In some embodiments, the amphiphilic block copolymers include biocompatible polymers. In some embodiments, the amphiphilic block copolymers include, without limitation, biocompatible polymers and biodegradable polymers. 7 4823-4406-5785v.313368-42

[0044] In some embodiments, the amphiphilic block copolymers include hydrophilic blocks and hydrophobic blocks. In some embodiments, the hydrophilic blocks of the amphiphilic block copolymers include a variety of polymer types from different polymer families. Examples include, without limitation, polyethylene glycol (PEG), methoxy-polyethylene glycol (mPEG), hydrophilic vinyl polymers such as poly(4-vinyl-N-methylpyridine iodide) (P4MVP), hydrophilic polypeptides including but are not limited to polyserine, poly(glutamic acid) (PGA), poly(aspartic acid) (PASA), and polylysine, hydrophilic polyesters, poly(α-hydroxy acid) (PHA), hydrophilic polycarbonates such as poly(5-methyl-2-oxo-1,3-dioxane-5-carboxylate), hydrophilic polycaprolactone (PCL) such as poly[γ-2-[2-(2-methoxyethoxy)ethoxy]ethoxy-ε-caprolactone] (PME3CL), hydrophilic polyacrylates such as poly(N,N-dimethylamino-2-ethyl methacrylate) (PDMAEMA), poly(carboxybetaine), derivatives thereof, or combinations thereof. In some embodiments, the hydrophilic blocks of the amphiphilic block copolymers are neutral polymers, anionic polymers, cationic polymers, or zwitterionic polymers.

[0045] In some embodiments, the hydrophilic blocks of the amphiphilic block copolymers are neutral, such as PEG. In some embodiments, the hydrophilic blocks of the amphiphilic block copolymers are anionic, such as poly(glutamic acid) (PGA). In some embodiments, the hydrophilic blocks of the amphiphilic block copolymers are cationic, such as polylysine. In some embodiments, the hydrophilic blocks of the amphiphilic block copolymers are zwitterionic, such as poly(carboxybetaine). In some embodiments, the hydrophilic blocks of the amphiphilic block copolymers are combinations of two or more of the aforementioned polymers.

[0046] In some embodiments, the hydrophobic blocks of the amphiphilic block copolymers include a variety of polymer types from different polymer families. Examples include, without limitation, hydrophobic polyesters such as polylactide (PLA), hydrophobic vinyl polymers such as polybutadiene (PBD) or hydroxyl-terminated polybutadiene (PBD-(OH)2), hydrophobic polypeptides such as polyphenylalanine, polyalanine, polyvaline, polyleicine, polymethionine, polyisoleucine, polytyrosine, and polytryptophan, hydrophobic polycarbonates such as poly(5- methyl-5-benzyloxycarbonyl-1,3-dioxane-2-one) (PMBC), hydrophobic polycaprolactone (PCL), hydrophobic polyacrylates such as poly(methyl acrylate) (PMA), derivatives thereof, or combinations thereof. 8 4823-4406-5785v.313368-42

[0047] In some embodiments, the amphiphilic block copolymers include polybutadiene (PBD)-b- (poly(4-vinylpyridine)28)2 (i.e., PBD-b-(P4MVP28)2). In some embodiments illustrated in FIG.1, the amphiphilic block copolymers include methoxy-polyethylene glycol (mPEG) and polylactide (PLA) (mPEG-b-PLA).

[0048] Disc-shaped membranes

[0049] The active agent carriers of the present disclosure can include various types of disc-shaped membranes. For instance, in some embodiments, the disc-shaped membrane is in the form of a two-dimensional disc shaped membrane (e.g., disc-shaped membrane 13 shown in FIG. 1). In some embodiments, the disc-shaped membrane includes a hydrophilic surface and a hydrophobic interior.

[0050] The disc-shaped membranes of the present disclosure can include various sizes. For instance, in some embodiments, the disc-shaped membrane has a diameter ranging from about 5 nm to about 500 nm. In some embodiments, the disc-shaped membrane has a diameter ranging from about 400 nm to about 600 nm. In some embodiments, the disc-shaped membrane has a diameter ranging from about 5 nm to about 20 nm. In some embodiments, the disc-shaped membrane has a diameter ranging from about 7 nm to about 17 nm. In some embodiments, the disc-shaped membrane has a diameter ranging from about 10 nm to about 40 nm. In some embodiments, the disc-shaped membrane has a diameter ranging from about 10 nm to about 50 nm. In some embodiments, the disc-shaped membrane has a diameter ranging from about 10 nm to about 100 nm. In some embodiments, the disc-shaped membrane has a diameter ranging from about 20 nm to about 100 nm. In some embodiments, the disc-shaped membrane has a diameter ranging from about 100 nm to about 500 nm. In some embodiments, the disc-shaped membrane has a diameter ranging from about 50 nm to about 500 nm.

[0051] The disc-shaped membranes of the present disclosure can include various charges. For instance, in some embodiments, the disc-shaped membrane has a net cationic charge. In some embodiments, the disc-shaped membrane has a net anionic charge. In some embodiments, the disc- shaped membrane has zwitterionic charge (i.e., no net charge). In some embodiments, the disc- shaped membrane has no charge (i.e., a neutral charge).

[0052] Active agents 9 4823-4406-5785v.313368-42

[0053] The active agent carriers of the present disclosure can include various active agents. Additionally, the methods of the present disclosure may deliver various active agents into cells and subjects. For instance, in some embodiments, the active agent includes, without limitation, nucleotides, genes, mRNA, miRNA, siRNA, DNA, circular RNA, circular DNA, single stranded RNA, single stranded DNA, double stranded RNA, double stranded DNA, gene-editing systems, proteins, therapeutic agents, chemotherapeutic agents, antibodies, genetically encoded materials, biological materials, tracing agents, drugs, antibiotics, fluorescent dyes, contrast agents, quantum dots, or combinations thereof. In some embodiments, the active agent includes a drug. In some embodiments, the drug is an anti-cancer drug.

[0054] Active agents may be associated with the active agent carriers of the present disclosure in various manners. For instance, in some embodiments, the active agent is encapsulated within a disc-shaped membrane (e.g., active agent 14 encapsulated within disc-shaped membrane 13, as illustrated in FIG. 1). In some embodiments, the active agent is associated on the surface of a disc-shaped membrane. In some embodiments, the active agent is associated on the edge of a disc- shaped membrane. In some embodiments, the active agent carriers of the present disclosure may include a tunable number of active agents.

[0055] In some embodiments, the active agent of the present disclosure may be covalently linked to the polymers of the present disclosure. For instance, in some embodiments, the active agents of the present disclosure may be covalently linked to the polymers through a pH-cleavable linkage (e.g., amphiphilic block copolymers 12 covalently linked to active agent 14 through pH-cleavable linkage 15, as illustrated in FIG.1). In some embodiments, the pH-cleavable linkage includes a pH-sensitive Schiff bond.

[0056] Membrane stabilizing agents 10 4823-4406-5785v.313368-42

[0057] In some embodiments, the active agent carriers of the present disclosure may also include a membrane stabilizing agent (e.g., membrane stabilizing agent 16 illustrated in FIG.1). In some embodiments, the membrane stabilizing agent encases the disc-shaped membrane. In some embodiments, the membrane stabilizing agent is operable to maintain the shape and size of the disc-shaped membrane. In some embodiments, the membrane stabilizing agent includes membrane-scaffold polymers. In some embodiments, the membrane stabilizing agent includes membrane-scaffold proteins including but are not limited to Saposins, Synucleins, and Spycatcher- SppyTag. In some embodiments, the membrane stabilizing agent includes membrane-scaffold proteins derived from human Apo-AI proteins including but are not limited to MSP1, MSP2, or their variants such as MSP1E3 and MSP1E3D1. In some embodiments, the membrane stabilizing agent includes engineered Apo-AI mimetic peptides such as 18A and 22A. In some embodiments, the membrane stabilizing agent includes synthetic peptide analogs.

[0058] Targeting agents

[0059] In some embodiments, the active agent carriers of the present disclosure may also include a targeting agent (e.g., targeting agent 18 illustrated in FIG.1). In some embodiments, the targeting agent is operable to direct the active agent carriers of the present disclosure to a specific tissue or cell (e.g., a cancerous tissue or cell).

[0060] The active agent carriers of the present disclosure may include various types of targeting agents. For instance, in some embodiments, the targeting agent includes, without limitation, an antibody, an antibody fragment, an aptamer, or combinations thereof. In some embodiments, the targeting agent includes cancer cell targeting moieties.

[0061] In some embodiments, the targeting agent includes trastuzumab emtansine that targets the over-expressed human epidermal growth factor receptor-2 (HER2) on cancer cells. In some embodiments, the targeting agent includes cancer cell targeting moieties. In some embodiments, the targeting agent includes folic acid that targets the over-expressed folate receptors on cancer cells. In some embodiments, the active agent carriers of the present disclosure may include a tunable number of targeting agents.

[0062] Uses 11 4823-4406-5785v.313368-42

[0063] The active agent carriers of the present disclosure may be suitable for various uses and applications. For instance, in some embodiments, the active agent carriers of the present disclosure may be suitable for use in delivering one or more active agents to a cell. In some embodiments, the active agent carriers of the present disclosure may be suitable for use in delivering one or more active agents to a subject.

[0064] Active agent administration

[0065] Additional embodiments of the present disclosure pertain to methods of delivering one or more active agents to a subject by administering to the subject an active agent carrier of the present disclosure. The active agent carriers of the present disclosure may be administered in various manners. For instance, in some embodiments, the administration occurs by a method that includes, without limitation, intravenous administration, subcutaneous administration, transdermal administration, topical administration, intraarterial administration, intrathecal administration, intracranial administration, intraperitoneal administration, intraspinal administration, intranasal administration, intraocular administration, oral administration, intratumor administration, local administration, or combinations thereof. In some embodiments, the administration includes local administration to a specific tissue of a subject, such as a cancerous tissue.

[0066] Treatment or prevention of conditions

[0067] In some embodiments, the methods of the present disclosure may be utilized to treat or prevent a condition in a subject. In some embodiments, the condition is cancer, such as breast cancer. In some of such embodiments, the active agent includes an anti-cancer drug.

[0068] Subjects

[0069] The active agent carriers of the present disclosure may be administered to various subjects. For instance, in some embodiments, the subject is a human being. In some embodiments, the subject is a non-human mammal. In some embodiments, the non-human mammal includes, without limitation, a horse, a rabbit, a mouse, a rat, a pig, a sheep, a cow, a dog, or a cat. In some embodiments, the non-human mammal is a domestic animal, such as a dog or a cat.

[0070] In some embodiments, the subject is suffering from a condition that is to be treated or prevented. In some embodiments, the condition is cancer.

[0071] Methods of making active agent carriers 12 4823-4406-5785v.313368-42

[0072] Additional embodiments of the present disclosure pertain to methods of making an active agent carrier of the present disclosure. In some embodiments, such methods include: mixing a plurality of amphiphilic block copolymers to form a self-assembled disc-shaped membrane; and associating the disc-shaped membrane with at least one active agent. In some embodiments, methods of making an active agent carrier of the present disclosure include: mixing a plurality of amphiphilic block copolymers with a membrane stabilizing agent to form a self-assembled disc- shaped membrane encased by the membrane stabilizing agent; and associating the disc-shaped membrane with at least one active agent. In some other embodiments, such methods include: modifying a plurality of amphiphilic block copolymers with at least one active agent; and mixing a plurality of amphiphilic block copolymers, some of which carry at least one active agent, with the membrane stabilizing agent to form a self-assembled disc-shaped membrane.

[0073] In some embodiments, the associating of the disc-shaped membrane with at least one active agent includes covalently linking the amphiphilic block copolymers with at least one active agent, followed by mixing a plurality of amphiphilic block copolymers, some of which carry at least one active agent, with the membrane stabilizing agent to form a self-assembled disc-shaped membrane. In some embodiments, the active agent becomes covalently linked to the amphiphilic block copolymers through a pH-cleavable linkage. In some embodiments, the pH-cleavable linkage includes a pH-sensitive Schiff bond.

[0074] Active agents may become associated with various portions of disc-shaped membranes. For instance, in some embodiments, the active agent becomes encapsulated within the disc-shaped membrane. In some embodiments, the active agent becomes associated with the surface of the disc-shaped membrane. In some embodiments, the active agent becomes associated with the edge of the disc-shaped membrane.

[0075] In some embodiments, the methods of the present disclosure also include a step of mixing the amphiphilic block copolymers of the present disclosure with a membrane stabilizing agent. In some embodiments, the membrane stabilizing agent maintains the shape and size of the disc- shaped membrane. In some embodiments, the membrane stabilizing agent maintains the size of the disc-shaped membrane. 13 4823-4406-5785v.313368-42

[0076] In some embodiments, the methods of the present disclosure also include a step of associating the disc-shaped membrane with a targeting agent. In some embodiments, the targeting agent is operable to direct the active agent carrier to a specific tissue or cell.

[0077] Targeting agents may become associated with various portions of disc-shaped membranes. For instance, in some embodiments, the targeting agent becomes associated with the surface of the disc-shaped membrane. In some embodiments, the targeting agent becomes associated with the edge of the disc-shaped membrane.

[0078] Advantages and Applications

[0079] The methods and active agent carriers of the present disclosure provide numerous advantages and applications. For instance, in some embodiments, the active agent carriers of the present disclosure can be used to deliver anticancer drugs to tumor sites with high stability and long circulation times, thereby providing a more effective and efficient drug delivery method.

[0080] Moreover, methods and active agent carriers of the present disclosure overcome the limitation of current nanocarriers by developing a disc-shaped nanocarrier (i.e., polymer nanodiscs) that mimics the high-density lipoproteins in blood in shape while replacing the fluidic and labile lipids with robust biocompatible polymers. Unlike lipids, the chemically versatile polymers open many opportunities on conjugating active agents (e.g., anticancer drugs) and targeting moieties (e.g., tumor-targeting moieties) to fine tune the pharmacokinetics and pharmacodynamics of the active agent carriers of the present disclosure. 14 4823-4406-5785v.313368-42

[0081] In some embodiments, the methods and active agent carriers of the present disclosure provide at least the following advantages: (1) by being a disc-shaped nanocarrier instead of a spherical one (as currently being used in the market), the active agent carriers of the present disclosure are able to mimic nascent HDL in blood and have better in vivo transport behavior with long circulation time; (2) by having a monodisperse size as defined by the length of membrane- scaffold proteins instead of broad size distributions (as currently being used in the market), the active agent carriers of the present disclosure can help best utilize the enhanced permeability and retention (EPR) effect to reach tumor sites; (3) unlike existing nanocarriers included of lipid or other small molecules that are fluidic and labile such as lipid nanodiscs (LNDs), lipid nanoparticles (LNPs), and liposomes, the active agent carriers of the present disclosure are more stable against disassembly or aggregation, which increases the shelf life of formulated drugs; and (4) the ability to incorporate chemically versatile polymers into the active agent carriers of the present disclosure opens many opportunities on conjugating anticancer drugs and tumor-targeting moieties to fine tune the pharmacokinetics and phamacodynamics of the nanocarriers.

[0082] Additional Embodiments

[0083] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicant notes that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0084] Example 1. Polymer Nanodiscs as Novel Nanocarriers for Anticancer Drug Delivery 15 4823-4406-5785v.313368-42

[0085] In this Example, Applicant illustrates the development of polymer nanodiscs (PNDs) as two-dimensional 2D polymer membranes encased by membrane-scaffold proteins (MSPs), with their well-defined diameters (~7-17 nm) controlled by the size of their belt-like MSP variants. Previously, Applicant used the hydroxyl-terminated polybutadiene (PBD-(OH)2) as the building block to prepare the well-defined triblock copolymer PBD-b-(poly(4-vinylpyridine)28)2 (i.e., PBD- b-(P4VP28)2) that self-assembles in water or buffers into polymer membranes. Applicant demonstrated successfully the formation of PNDs comprised of co-assembled PBD-b-(P4VP28)2and MSPs, and the utility of such PNDs to support the structural and functional understanding of membrane proteins (MPs), as well as their potential for the development of MP-based biotechnology and medical applications.

[0086] In this Example, Applicant demonstrates the feasibility to develop biocompatible and biodegradable nanocarreirs based on the PND platform for drug delivery. As a prototype, Applicant focuses on anticancer drug delivery. Applicant demonstrates the use of methoxy- polyethylene glycol (mPEG) and polylactide (PLA)) block copolymer (mPEG-b-PLA) and its derivatives that carry doxorubicin (Dox), a model chemotherapy drug, via a pH-responsive Schiff bond to self-assemble with MSPs into PNDs with well-defined shape, size, and tunable drug- carrying capacity.

[0087] Cancer is the second largest cause of fatality in the United States and globally. Despite benchtop success of various highly effective anticancer drug formulations, their controlled delivery across aqueous physiological pathways to reach deep-seated cancer cells with desirable pharmacokinetics and biodistribution remains an outstanding challenge.

[0088] Since the discovery of enhanced permeability and retention (EPR) effect of tumors, nanotechnology has been widely anticipated to benefit the diagnostic and treatment of cancers because the EPR effect dictates that nanoparticles should preferentially accumulate at the tumor sites. Numerous nanocarriers, including inorganic nanoparticles, micelles, liposomes, polymersomes, and protein or polymer conjugates, have been tested in the past 40 years, and a handful of which have gained FDA approval. 16 4823-4406-5785v.313368-42

[0089] Despite the significant strides, efficacious patient responses remain modest compared to conventional drug formulations. For instance, a recent study discovered that, on average, only ~0.7% of administered nanoparticles were actually delivered to tumor sites.

[0090] Furthermore, a gap of knowledge exists on how to engineer nanocarriers beyond simply controlling their sizes. In contrast to synthetic nanocarriers that often assume a spherical shape and a broad size distribution, biology offers many fine examples of nanocarrier designs adapted to transport with precisely controlled size, shape, surface chemistry, and spatial organization of chemical compositions, which are driven solely by the self-assembly process that relies on a multitude of inter- and intra-molecular forces.

[0091] For instance, high density lipoprotein nanoparticles (HDL) consist of clusters of lipids encased by apolipoproteins, a family of biologically-derived amphipathic random copolymers. The initial form of HDL (or “nascent HDL”) is a well-known lipid nanodisc (LND), which mediates reverse cholesterol transport from peripheral cells back to the liver, and it can be produced in vitro via self-assembly of lipids with MSPs derived from recombinant apolipoproteins.

[0092] The self-assembled LNDs represent a novel family of 2-dimensional (2D) materials with great potential for drug delivery, as mounting evidences suggest that disc-shaped particles outperform spherical ones with prolonged blood circulation half-lives and higher cellular internalization rates. Adapting LNDs for anticancer drug delivery has attracted lots of attentions in recent years. However, as a nanocarrier platform, LNDs suffer from low stability, short shelf life, limited drug loading capacity, and difficulty for chemical modifications.

[0093] This Example aims to develop novel PNDs with optimal biocompatibility and biodegradability, long-term stability, high drug loading capacity, and facile modification chemistry as a new generation of anticancer nanocarriers (FIG. 1). Applicant hypothesized that amphipathic MSPs will encase and transform amphiphilic block copolymers carrying anticancer drugs and tumor-homing moieties into well-defined lipoprotein-mimicking PNDs that display enhanced stability and circulation time.

[0094] Example 1.1. Background and rationale 17 4823-4406-5785v.313368-42

[0095] Ever since the discovery of the EPR effect of tumors, nanocarriers have become widely anticipated to benefit the diagnosis and treatment of cancers. A preferred nanocarrier should be able to navigate in circulatory systems, recognize the tumor cells with high selectivity, latch on and invade the diseased cells to release the payloads, and leave no residues behind. However, such goals are challenging because nanocarriers face a multitude of biological barriers, such as opsonization and sequestration by the mononuclear phagocyte system (MPS), nonspecific distribution, hemorheological blood flow limitations, elevated intratumoral pressure against extravasation to distal regions in the tumor, cellular internalization, endosomal and lysosomal entrapments, and expulsion by drug efflux pumps.

[0096] Translational success of any nanocarrier depends on its ability to address not only one or few, but all biological barriers simultaneously. The size, shape, and surface chemistry work synergistically to help nanocarriers break the multitude of biological barriers. It is well known that nanocarriers that are 7 nm or smaller are distributed non-specifically into the extracellular space and rapidly cleared by renal filtration. However, nanocarriers in the range of 50-100 nm tend to accumulate in the liver due to the vascular fenestrations of their noncontinuous endothelia.

[0097] Larger nanoparticles are preferentially retained by splenic filtration because of the ~200- 500 nm size range of inter endothelial cell slits in the spleen. Although fenestrations in tumor vasculature range in sizes from ~380-780 nm, the degree of tumor vascularity is ultimately limited by fibrosis and the collagen density, which in many cases prevent penetration of nanoparticles greater than ~40 nm. On top of those important biological pore sizes, larger nanoparticles are cleared more rapidly by MPS than smaller ones.

[0098] Taken together, it is reasonable to expect that a good nanocarrier is better off to have focused sizes preferentially within ~10-40 nm. However, in reality, most of the as-prepared liposomes and polymersomes are polydisperse in nature with sizes ranging from ~50 to 500 nm. Moreover, most conjugated polymers have hydrodynamic diameters smaller than 10 nm. Micelles not only have polydisperse sizes (typical diameters ~10-100 nm), but also undergo rapid assembly- disassembly equilibrium that complicates their pharmacokinetics. 18 4823-4406-5785v.313368-42

[0099] Inorganic nanoparticles can be synthesized with well-defined sizes. However, their own cytotoxicity and the difficulty to upload and release pharmaceuticals are some of the most significant drawbacks. Even taking size control as one simple yardstick, current technologies are still far from optimal to take advantage of the EPR effect.

[0100] Besides size control, the importance of shape on nanocarrier design is increasingly recognized. Additionally, mounting evidence suggests that disc-shaped particles outperform spherical ones with prolonged blood circulation half-lives and higher cellular internalization rates. For example, spherical particles are more readily cleared by MPS than disc-shaped ones.

[0101] Before transvascular transport to reach the tumor sites, nanocarriers should be able to drift laterally towards the vessel walls (i.e., margination) like leukocytes do during an inflammatory process, which allows them to sense the vessel walls for biological signatures, such as the overexpressed antigens used as ‘docking sites’ in vascular targeting, or the presence of openings and fenestrations through which small nanocarriers can cross the endothelial barrier.

[0102] In contrast to spherical nanocarriers, nanodiscs experience torques during circulation, resulting in tumbling and rotation that increase margination toward transvascular transport. Once reaching the tumor sites, nanodiscs show enhanced cell internalization frequency at higher rates owing to their larger surface-to-cell contact areas and adhesion points.

[0103] Since synthetic nanocarriers often assume a spherical shape with a broad size distribution, new strategies to prepare disc-shaped nanocarriers with well-defined sizes are needed to improve the in vivo transport of nanocarriers and their bioavailability. Moreover, the surface chemistry of nanocarriers should help target the cancer cells selectively while evading MPS clearance. 19 4823-4406-5785v.313368-42

[0104] Surface-conjugation with poly(ethylene glycol) (i.e., PEGylation) is still the gold standard in clinical uses to evade MPS clearance. However, the emergence of PEG-specific antibodies and immunotoxicity in patients is worrisome. Alternative hydrophilic “stealth” polymers such as zwitterionic poly(carboxybetaine) have been developed to address such issues. Similarly, surface- conjugation with tumor homing moieties is another powerful approach to target cancer cells because they often overexpress specific receptors known as ‘‘cancer signatures”, such as Her2 / neu, scavenger receptor class B type-1 (SR-B1), epidermal growth factor receptor, somatostatin, folic acid (Fa) receptors, αvβ3 integrins, and low density lipoprotein receptors.

[0105] A preferred nanocarrier platform should be chemically versatile for surface modifications to evade MPS clearance and target cancer cells selectively. In view of such preferences, LNDs have attracted lots of attentions for anticancer drug delivery because their discoidal shape and well- defined sizes (i.e., ~7-17 nm depending on MSPs) are preferred for improved in vivo transport and bioavailability.

[0106] However, LNDs suffer from low stability, short shelf life, limited carrying capacity, and difficulty for chemical modifications. Unlike liposome-based drug delivery systems such as Doxil® and Myocet® in which poorly water-soluble chemotherapeutics are carried in large quantities as crystals in their intraliposomal compartment, LNDs don’t have such a compartment.

[0107] Carrying chemotherapeutics via physisorption is challenging due to the limited bilayer capacity and inevitable premature drug release. Lipids are not amenable to chemical modifications, either. Arduous approaches would need to be improvised for drug conjugation or tumor targeting. Most critically, the fluidic and labile nature of lipid bilayers gives rise to the instability of LNDs.

[0108] LNDs are known to have a short shelf life and tend to aggregate into “rouleaux” (FIG.2). Additionally, dynamic exchange and adsorption of external lipids into LNDs constantly occurs, which is revealed in vivo as the maturation of LNDs into spherical HDLs. This instability greatly hampers translational application of LNDs and calls for new nanodisc design with long-term stability, high drug loading capacity, and facile modification chemistry. 20 4823-4406-5785v.313368-42

[0109] Applicant discovered that polymersome membranes (i.e., the synthetic mimics of lipid bilayers) can be solubilized by detergents and subsequently self-assemble with MSPs into PNDs when the detergents are removed. The MSP variant Applicant used is MSP1E3D1, a frequently used MSP variant in LNDs. The amphiphilic block copolymer consists of hydrophilic P4VP block covalently joined with hydrophobic PBD block, which self-assemble into polymersomes by itself.

[0110] When Applicant reconstituted with MsbA, a model membrane protein, the resultant MsbAPNDs showed a focused size distribution (d~13 nm) by dynamic light scattering (DLS). The UV-vis trace of size exclusion chromatography (SEC) also showed a monodisperse PND fraction with coexistence of protein (A280) and polymer (A259) (FIG.3A). The cryoEM study of the PND fraction clearly revealed the morphology of MsbA-carrying PNDs (FIG. 3A inset), with its schematic illustration shown in FIG.3B.

[0111] Unlike LNDs that aggregate in a few hours at room temperature and in 1-2 days even when stored at 4°C, Applicant’s PNDs are stable at all temperatures (4-37°C) for at least one week (FIG.3C). This pilot study sheds light on a new path to develop robust, scalable, and chemically versatile PNDs as novel nanocarriers to overcome the limitations of LNDs while still retaining their unique advantages.

[0112] Example 1.2. Experimental results

[0113] As a model system, PNDs comprised of FDA approved, biocompatible, and biodegradable copolymers, such as methoxy-polyethylene glycol (mPEG) and polylactide (PLA) blocks that carry doxorubicin (Dox) through a pH-sensitive Schiff bond (mPEG-b-PLA-Dox) were developed in this Example. Applicant developed a methodology to prepare well-defined, polymersome-forming amphiphilic diblock copolymers, mPEG-b-PLA, via ring-opening polymerization (ROP). 21 4823-4406-5785v.313368-42

[0114] Applicant also tested the concept of conjugating Dox, a chemotherapy drug, to the hydrophobic PLA block of the copolymer via a pH-sensitive Schiff bond. Dox is among the "first line" anticancer drugs and is effective against more types of cancer (e.g., leukemias, lymphomas, breast, uterine, ovarian, and lung cancers) than most other chemotherapy agents. Besides its well- known absorption, distribution, metabolism, and excretion behaviors, Dox has distinct optical spectra (both UV-vis absorbance and fluorescence) that allow facile and accurate quantification of its pharmacokinetics and biodistribution.

[0115] The Schiff bond enables tumor-specific Dox release when PNDs reach the low pH microenvironment of tumors. The Dox-carrying PNDs show pH-responsive drug release under conditions mimicking tumor microenvironment and efficient killing of model cancer cells when tested in vitro.

[0116] Applicant prepared the biocompatible and biodegradable amphiphilic block copolymers with controlled payload capacity of Dox. First, Applicant synthesized a new monomer, 5-methyl- 5-benzyloxycarbonyl-1,3-dioxane-2-one (MBC) (FIG. 4A), and confirmed the successful synthesis by1H-NMR (FIG.4C). Applicant tested the ROP of mPEG-b-P(LA-co-MBC) using a dual catalytic system consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and (thio)urea (i.e., urea I-0). Next, Applicant derivatized MBC to carry Dox in the hydrophobic PLA block (FIG. 4B). Applicant confirmed that the ROP completed in seconds with narrow polydispersity indices (Ɖ) (FIG.4D).

[0117] Example 1.3. Materials and methods

[0118] All chemicals were purchased from Sigma Aldrich at ACS grade. The chemical structure of synthesized monomers and polymers is characterized by1H and13C nuclear magnetic resonance (NMR) (400 M and 500M JEOL NMR spectrometers) and Fourier transform infrared (FTIR) spectra (Thermo-Electron Nicolet 4700 spectrometer). The molecular weight was measured by gel permeation chromatography (GPC) on an Agilent 1260 chromatograph system equipped with 1260 VWD UV detector, Wyatt Optilab REX refractive index detector and Wyatt miniDAWN TREOS multi-angle light scattering detectors. 22 4823-4406-5785v.313368-42

[0119] Dimethylformamide (DMF) with ammonium acetate (0.02 M) was used as the mobile phase with a flow rate of 0.5 mL / min and run at 50 °C. Polymersome and PND formation behavior of the model amphiphilic block copolymers was characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal miscoscopy. The DLS measurements were performed at 25 °C on a Zetasizer Nano ZSP using 40- μL disposable micro-cuvettes. Each sample was measured in triplicate, in which each measurement consisted of 10-20 scans.

[0120] The Zetasizer program determined the number of scans necessary by analyzing the autocorrelation function and its fit. The volume-based size distribution, intensity-based size distribution, and the averaged diameters were obtained using Zetasizer software (version 8.02; Malvern Instruments, Westborough, MA). The SEM was measured on a JEOL JSM 7000F Field Emission SEM (Peabody, MA) or a Hitachi S-4300 E / N FESEM, and the TEM was analyzed on a Hitachi H- 7650 TEM system (Hitachi High-Tech in America, Dallas, TX) running the AMT Image Capture Engine V602 (AMT Imaging Direct, Woburn, MA) for data collection. Confocal microscopy was performed on a Nikon T1-E microscope with A1 confocal and STORM super- resolution modules. Other characterization methods are mentioned where they are used.

[0121] Example 1.4. Reaction optimization

[0122] Since ROP is a transesterification reaction at the propagating site, it requires a high degree of selectivity and specificity for the monomers. Lack of these features in the polymerization reaction leads to long reaction times and backbiting within the polymer chains that lead to broad molecular weight distribution. To resolve this problem, it has been suggested that for maximum catalytic activity in dual catalytic systems pKa of (thio)urea should match closely with the pKa of the base used. A catalytic system consisting of (thio)urea and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was proposed, where the (thio)urea activates the monomer and DBU activates the alcohol. This co-catalyst system enhances the polymerization rate by a dual activation mechanism, completes the polymerization within very short times, and provides narrow molecular distribution in polymers. 23 4823-4406-5785v.313368-42

[0123] For ROP, Applicant selected urea I-0 (pKa= 13.8) and DBU (pKa= 13.9) as a co-catalyst system. Synthesis of the urea I-0 was achieved by reacting ((3,5-bis(trifluoromethyl)phenyl) isocyanate and 3,5-bis(trifluoromethyl)aniline in THF with high yield and purity (Scheme 1).24 4823-4406-5785v.313368-42

[0124] The successful synthesis of the urea I-O was confirmed by1H-NMR (FIG.5). The1H- NMR spectra of urea I-O shows a characteristic peak of aromatic protons as 8.3 and 7.6 ppm.

[0125] Polymerization conditions to synthesize well-defined mPEG-b-PLA with the right molecular weight of the PLA block was achieved by keeping the PEG block the same (i.e., 2,000 Da). Such an approach essentially tunes the amphiphilic balance of the block copolymer to favor their self-assembly into polymersome membranes.

[0126] As a general rule, a hydrophilic block fraction (f hydrophilic) of ~35±10% in an amphiphilic diblock copolymer would favor polymersome membrane formation. To obtain the optimal time for the mPEG-b-PLA synthesis, Applicant used a co-operative catalytic system using urea I-O and DBU. Applicant kept a constant ratio of initiator, DBU, and urea as 1:2:2 and selected the target degree of polymerization (DP) of 60 for PLA units using mPEG45as macroinitiator.

[0127] Applicant monitored the DP by taking out a small amount of samples from the reaction mixture at predetermined time points and analyzing the samples by GPC after neutralizing the DBU by the addition of an excess amount of benzoic acid. This control experiment showed that the polymerization reaction is almost completed within 60 seconds, as shown in dRI traces in FIG. 6.

[0128] The dRI traces showed that an increase in time more than 2 min leads to loss of narrow distribution in the molecular weight of the polymer. After1H-NMR analysis, it was found that the conversion rate of lactide to polylactide is almost 90% within 30 sec.1H-NMR spectra of mPEG45- b-PLA60showed the characteristic peaks of polymer and confirm the attachment of almost 60 PLA units. The PLA backbone protons appeared at 5.15-5.13, PEG backbone protons at 3.62, methoxy protons of mPEG at 3.36, and methyl protons of PLA at 1.57-1.55 ppm (FIG.7).

[0129] Example 1.5. Reaction optimization for the synthesis of Dox-carrying amphiphilic block copolymers 25 4823-4406-5785v.313368-42

[0130] Applicant tested two approaches to prepare the amphiphilic block copolymers that carry Dox, a model chemotherapy drug. In the first approach (FIG. 4B), Applicant introduced the 5- methyl-5-benzyloxycarbonyl-1,3-dioxane-2-one (MBC) moieties into the hydrophobic PLG block to carry Dox. To achieve the coupling, Applicant synthesized methoxy poly(ethyleneglycol)-b- poly(lactide-co-carbonate) [mPEG-b-P(LA-co-BC)]. Applicant first synthesized the monomer MBC from 2,2-bis(hydroxymethyl)propionic acid. In brief, 2,2-bis(hydroxymethyl)propionic acid was first reacted with benzyl bromide at 100 °C for 15 h to get the intermediate benzyl-2,2- bis(methylol)propionate. Then, the monomer 5-methyl-5-benzyloxycarbonyl-1,3-dioxane-2-one (MBC) was synthesized by reacting benzyl-2,2-bis(methylol)propionate with triphosgene at −70 °C in the presence of pyridine in dry dichloromethane (FIG.4A).

[0131] Successful synthesis of MBC was confirmed by the1H-NMR of intermediate benzyl-2,2- bis(methylol)propionate (FIG. 8), and the final product MBC (FIG. 4C). The benzyl-2,2 bis(methylol)propionate was synthesized in a high yield of 70% and with high purity. The1H- NMR for intermediate benzyl-2,2-bis(methylol)propionate show characteristic peaks of aromatic protons in the region 7.35-7.24 ppm, benzylic protons at 5.20, methylene protons at 3.95-3.71, hydroxy protons at 2.80, and methyl protons at 1.06 ppm. The successful synthesis of monomer MBC was also achieved with a high yield of 80% and with high purity. The1H-NMR confirms the synthesis of the monomer (FIG.4C) and shows characteristic peaks for aromatic protons at 7.3- 7.34, benzylic protons at 5.22, methylene protons at 4.72-4.19, and methyl protons at 1.33 ppm.

[0132] Shifting of peaks for methylene protons and methyl protons along with the disappearance of bishydroxyl protons confirmed the cyclization through a carbonate bond. After the successful synthesis of the monomer, Applicant optimized the polymerization reaction conditions with individual monomers (i.e., LA and MBC), to synthesize mPEG-b-P(LA-co-BC). 26 4823-4406-5785v.313368-42

[0133] Since the LA and MBC have a significant difference in this polymerization rate, Applicant decided to optimize the reaction time for the synthesis of random polymerization using LA and MBC to synthesize mPEG-b-P(LA-co-BC) polymer under similar reaction conditions and monitored the progress of the reaction by GPC. Here, Applicant found that this polymerization reaction is almost complete in 30 min as shown in the GPC dRI trace and no significant difference was observed after 30 mins of reaction time. Thus, Applicant selected 30 min as an optimal time for this polymerization reaction (FIG.4D).

[0134] Example 1.6. Synthesis and characterization of amphiphilic block copolymers with conjugated Dox

[0135] Besides the synthetic route presented in FIG. 4B, Applicant also synthesized mPEG-b- PBC to be used to carry Dox (i.e., mPEG-b-PCC-Dox) and doped into the PND. Applicant considers this route to be flexible in terms of controlling the payload capacity of Dox, as it can be achieved by simply doping mPEG-b-PLA with mPEG-b-PCC-Dox during PND formation. For mPEG-b-PBC, Applicant found that optimal DBU concentration of 5 mol% to the mPEG provides a narrow distribution of molecular weight in the resultant block copolymer.

[0136] GPC trace of the mPEG-b-PBC has shown narrow molecular weight distribution with a PDI of 1.05 with an expected molecular weight compared with the starting mPEG (FIG.9).1H- NMR analysis also confirmed successful polymerization. The typical aromatic protons from the benzyl protection were found in the range of 7.31-7.21, benzylic protons at 5.14-5.11, methylene protons from the backbone of MBC at 4.25, protons from the PEG backbone at 3.64-3.63, methoxy protons of PEG at 3.36, and methyl protons of MBC at 1.21 ppm (FIG.10).

[0137] For conjugation of DOX to the mPEG-b-PBC polymer through a pH-sensitive linkage, the block polymer, mPEG-b-PBC, was first treated with Pd / C to remove the benzyl protection to access the free carboxylic acid, similar to that described in FIG. 4B. Next, the 4-hydroxy benzaldehyde was conjugated to the free carboxylic acids through the EDC / HOBt coupling reaction in dry DMF. Finally, the free aldehyde group on the polymer backbone was used to form a Schiff base with the free amine of the DOX under basic conditions. 27 4823-4406-5785v.313368-42

[0138] Applicant removed the benzyl protection group from the mPEG-b-PBC polymer using a previously established method with some modifications. Briefly, Applicant used a standard Pd / C hydrogen environment to remove the benzyl protection. In a typical mPEG-b-PCC1H-NMR spectra (FIG.11), carboxylic acid protons were observed at 12.99, PCC backbone protons at 4.15, PEG backbone protons at 3.47, methoxy protons of PEG at 3.20, and methyl protons of PCC at 1.09 ppm. The absence of aromatic protons in the region 7-8 ppm along with the absence of benzylic protons at 5.2 ppm confirm the successful removal of the benzylic protection group from the polymer.

[0139] Next, Applicant conjugated 4-hydroxybenzaldehyde with the carboxylic acids of mPEG- b-PCC via the standard EDC / HOBt coupling reaction in anhydrous DMF. The coupling reaction proceeds smoothly and is completed within 24 h of reaction time at room temperature. The appearance of benzaldehyde proton at 9.95-9.91 and set of aromatic protons at 7.87-7.80 and 7.25- 7.17 ppm confirm the successful conjugation (FIG.12).

[0140] Finally, the pH-sensitive conjugation of Dox with the polymer was achieved by another existing procedure. Under a typical conjugation reaction, the Dox was first converted to a free base by neutralizing the hydrochloride salt by treating the DMSO solution of Dox with 2-3 equivalents of anhydrous triethylamine. Then, the Dox free base was allowed to react with the aldehyde groups of the polymer under a catalytic amount of glacial acetic acid (GAA). The 1HNMR spectra of the Dox conjugated polymer has characteristic peaks that confirm the successful conjugation of the Dox with the polymer. Based on the 1HNMR analysis, a polymer chain has approximately 4-5 Dox molecules conjugated through the Schiff bond (FIG.13).

[0141] Example 1.7. Synthesis and characterization of amphiphilic block copolymers with cancer targeting moieties 28 4823-4406-5785v.313368-42

[0142] As a model system, Applicant introduced folic acid (FA) as a cancer targeting moiety on amphiphilic block copolymers. Applicant chose FA and Dox as the exemplary tumor-targeting moiety and anticancer drug, respectively. However, FA and Dox can be replaceable by other tumor-targeting ligands (e.g., EGF, RGD, TfR, and / or antibodies) or chemotherapy agents (e.g., Paclitaxel, Camptothecin, and / or Gemcitabine), if needed, without breaking the same guiding concepts derived from this Example.

[0143] FA plays a key role in DNA synthesis and replication, cell division, growth, and survival. Three subtypes of folate receptors (FR) have been identified, among which FRα is expressed at very low levels in healthy cells, but very high levels in numerous cancers, including ovarian, head and neck carcinomas, mesothelioma, breast, colon, renal, and lung tumors, in order to meet the high folate demand of those tumor cells.

[0144] For the synthesis of FA-PEG-b- PLA, Applicant followed the synthetic route presented in Scheme 2.Scheme 2. Reaction design for the synthesis of FA-PEG-b-PLA. 29 4823-4406-5785v.313368-42

[0145] Here, Applicant selected the use of a bifunctional PEG polymer (i.e., COOH-PEG-OH) with carboxylic acid and hydroxy functionalized PEG at either end, respectively. The hydroxy end of the polymer will be utilized for the polymer chain growth and the carboxylic acid terminal will be used to conjugate folic acid to the polymer.

[0146] For the COOH-PEG-PLA polymerization reaction, Applicant selected to use Tin (II) 2- ethylhexanoate, Sn(Oct)2 as a catalyst instead of DBU because Applicant tested DBU and found it was ineffective.1H-NMR analysis also confirmed the successful synthesis of HOOC-PEG-b- PLA for FA conjugation. A typical1H-NMR spectrum of COOH-PEG-b-PLA shows PLA backbone protons at 5.23-5.18, PEG backbone protons at 3.51, and methyl protons of PLA at 1.48- 1.46 ppm (FIG. 14). Conjugation of folic acid to produce FA-PEG-b-PLA was successfully achieved via the classic esterification reaction catalyzed by DCC / DMAP.

[0147] Example 1.8. Characterization of polymersome formation

[0148] Applicant tested the polymersome formation behavior of model amphiphilic block copolymers by DLS and TEM characterization. For example, to test whether mPEG45-b-PLA60will self-assemble into polymersomes, Applicant used a dialysis method where a small amount (5- 8 mg) of the copolymer was dissolved in a small volume of acetone (2-3 mL) first. The solution was then dialyzed against Millipore water to remove the acetone over 2 days. After dialysis, the size of the formed polymersomes was measured using the DLS method.

[0149] Applicant found a uniform distribution of polymersomes with an average size of 446.2 nm, which confirms the successful formation of polymersomes (FIG.15). Applicant also studied the polymersome formation under TEM. A typical example of the self-assembled polymersome is shown in FIG.16. The diameters of the polymersomes (d ~65±14 nm) observed under TEM when negatively stained with uranium acetate are smaller than those observed by DLS, possibly due to the shrink of polymersomes under high vacuum. Additionally, the polymersome membranes appear unbroken, underscoring the higher membrane stability of polymersome membranes than lipid bilayers.

[0150] Example 1.9. Characterization of PNDs 30 4823-4406-5785v.313368-42

[0151] Applicant confirmed the successful preparation of PNDs comprised of the amphiphilic copolymers and MSP1E3D1 (FIGS.17A-17C). Since the recombinant MSP1E3D1 carries a His6 tag, the self-assembled products after dialysis (MWCO = 50 kD) to remove detergents and free MSPs were purified by Ni2+-charged NTA resin, and the washed-off solution from the NTA resin was separated by SEC to reveal an MSP component in the 12.2-17 nm range, with its refractive index signal (dRI; black) overlapping with MSP absorbance (A280; green).

[0152] The SEC column was calibrated by thyroglobulin (TG), apoferritin (Apo), alcohol dehydrogenase (ADH), and albumin (Alb) with known Stokes diameters at 17.0, 12.2, 9.2, and 7.1 nm, respectively (FIG. 17A). DLS of this fraction further confirmed its size was peaked at ~13 nm (FIG.17B), and TEM (stained with 1% uranyl acetate) showed that it contains nanoparticles of uniform sizes (d ~12±2.3 nm; FIG.17C), likely PNDs.

[0153] Example 1.10. Characterization of Dox payload capacity

[0154] Using the strong UV-Vis absorption peak of Dox at 480 nm, a calibration curve of Dox in PBS buffer (pH=5) was first built (FIG.18). To find the wt.% loading of Dox in the PNDs, the Schiff bond's hydrolysis was performed to cleave Dox from the polymer backbone in PBS at pH 5.0. The resultant Dox solution was diluted ten times to bring the concentration in the calibration curve range. Based on the calibration curve, Applicant found that about 21.6% wt. / wt. of Dox is carried by the PNDs consisting of the mPEG-b-PCC-Dox polymer. This payload capacity can be tuned further by doping the Dox-carrying polymer chains with the regular mPEG-b-PLA polymer chains during the PND formation.

[0155] Example 1.11. pH-dependent Dox release profiles

[0156] The pH-dependent Dox release from the PNDs were characterized under PBS buffers of pH=5 and 7.4, respectively, that mimic the tumor microenvironment (TME) and healthy tissues by dialyzing the PNDs (~5 mg / mL in 1 mL) in the buffers to measure the cumulative Dox release. 31 4823-4406-5785v.313368-42

[0157] As a proof of-concept demonstration, the Dox was found to be released at a much higher rate under the conditions that mimic TME than that mimic healthy tissues (FIG. 19), indicating the possibility of stimuli-responsive Dox release at the tumor site. Note the cumulative Dox release as measured in the buffer plateaued at ~25% after ~ 3 days, suggesting that most of the released Dox was likely trapped inside the membrane of PNDs, and sustained Dox release is possible while the polymer membranes is gradually degraded.

[0158] Example 1.12. Cytotoxicity assays

[0159] MTT assays of model cancer cells in the presence of PNDs with and without conjugated Dox are shown in FIG. 20. The standard MTT assay was used to determine the IC50 values of empty PNDs comprised of mPEG-b-PLA and Dox-carrying PNDs comprised of mPEG-b-PCC- Dox, respectively, against MCF7, a model breast cancer cell line.

[0160] The empty PNDs show no cytotoxicity, confirming the biocompatibility of the mPEG-b- PLA copolymers. In contrast, the Dox-carrying PNDs comprised of mPEG-b-PCC-Dox show an IC50of ~3 μM in terms of the polymer concentration. This benchtop assay once again confirms the feasibility of developing the Dox-carrying PNDs as novel nanocarriers for cancer therapy.

[0161] Example 1.13. Summary

[0162] This Example pertains to the development of biocompatible and biodegradable polymer nanodiscs (PNDs) for anticancer drug delivery. PNDs are disc-shaped nanocarriers like lipid nanodiscs (LNDs) but with their fluidic and labile lipid bilayer replaced by robust and chemically versatile block copolymer membranes. Applicant’s proof-of-concept data demonstrate that doxorubicin, a chemotherapeutic drug, can be successfully carried by PNDs and released under conditions that mimic the microenvironment of tumor sites. 32 4823-4406-5785v.313368-42

[0163] The descriptions in this Example also describe methods and compositions of PNDs for enhancing the delivery of drugs for disease treatment. The PNDs consist of amphiphilic block copolymers made up of FDA-approved methoxy-polyethylene glycol (mPEG) and polylactide (PLA) that can carry a drug (e.g., chemotherapy) via a pH sensitive Schiff bond. Under acidic conditions common in tumors, the pH-sensitive Schiff bond enables more specific drug delivery at the tumor site. Applicant has generated a prototype of the PNDs with the FDA-approved chemotherapy doxorubicin and tested it within an in vitro assay within the laboratory. Applicant has demonstrated that treatment with Applicant’s doxorubicin-carrying PNDs (“mPEG-PCC-BA- DOX” in FIG. 20) elicits the killing of breast cancer cells compared to empty PNDs (“mPEG- PLA” in FIG.20).

[0164] Overall, these data suggest that the PNDs could be a viable drug carrier. Moreover, the PNDs offer the following key benefits: (1) a novel drug delivery system that uses nanodiscs to encapsulate a drug for improving the targeted treatment of diseases such as cancer; (2) the use of PNDs to deliver a chemotherapy agent to breast cancer cells; and (3) the potential to decrease the toxicity of many drugs while improving efficacy through enhanced delivery kinetics and specific pH targeting.

[0165] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein. 33 4823-4406-5785v.313368-42

Claims

WHAT IS CLAIMED IS:

1. An active agent carrier, wherein the carrier comprises: a disc-shaped membrane comprising a plurality of self-assembled amphiphilic block copolymers and a membrane stabilizing agent, wherein the amphiphilic block copolymers comprise hydrophilic blocks and hydrophobic blocks, and wherein the membrane stabilizing agent encases the disc-shaped membrane; and at least one active agent associated with the disc-shaped membrane.

2. The active agent carrier of claim 1, wherein the hydrophilic blocks of the amphiphilic block copolymers are selected from the group consisting of polyethylene glycol (PEG), methoxy- polyethylene glycol (mPEG), hydrophilic vinyl polymers, poly(4-vinyl-N-methylpyridine iodide) (P4MVP), hydrophilic polypeptides, polyserine, poly(glutamic acid) (PGA), poly(aspartic acid) (PASA), polylysine, hydrophilic polyesters, poly(α-hydroxy acid) (PHA), hydrophilic polycarbonates, poly(5-methyl-2-oxo-1,3-dioxane-5-carboxylate), hydrophilic polycaprolactone (PCL), poly[γ-2-[2-(2-methoxyethoxy)ethoxy]ethoxy-ε-caprolactone] (PME3CL), hydrophilic polyacrylates, poly(N,N-dimethylamino-2-ethyl methacrylate) (PDMAEMA), poly(carboxybetaine), derivatives thereof, or combinations thereof.

3. The active agent carrier of claim 1, wherein the hydrophobic blocks of the amphiphilic block copolymers are selected from the group consisting of hydrophobic polyesters, polylactide (PLA), hydrophobic vinyl polymers, polybutadiene (PBD), hydroxyl-terminated polybutadiene (PBD- (OH)2), hydrophobic polypeptides, polyphenylalanine, polyalanine, polyvaline, polyleicine, polymethionine, polyisoleucine, polytyrosine, polytryptophan, hydrophobic polycarbonates, poly(5-methyl-5-benzyloxycarbonyl-1,3-dioxane-2-one) (PMBC), hydrophobic polycaprolactone (PCL), hydrophobic polyacrylates, poly(methyl acrylate) (PMA), derivatives thereof, or combinations thereof. 34 4823-4406-5785v.313368-424. The active agent carrier of claim 1, wherein the amphiphilic block copolymers comprise methoxy-polyethylene glycol (mPEG) and polylactide (PLA) (mPEG-b-PLA).

5. The active agent carrier of claim 1, wherein the disc-shaped membrane comprises a hydrophilic surface and a hydrophobic interior.

6. The active agent carrier of claim 1, wherein the disc-shaped membrane has a diameter ranging from about 5 nm to about 20 nm.

7. The active agent carrier of claim 1, wherein the disc-shaped membrane has a neutral charge.

8. The active agent carrier of claim 1, wherein the active agent is selected from the group consisting of nucleotides, genes, mRNA, miRNA, siRNA, DNA, circular RNA, circular DNA, single stranded RNA, single stranded DNA, double stranded RNA, double stranded DNA, gene- editing systems, proteins, therapeutic agents, chemotherapeutic agents, antibodies, genetically encoded materials, biological materials, tracing agents, drugs, antibiotics, fluorescent dyes, contrast agents, quantum dots, or combinations thereof.

9. The active agent carrier of claim 1, wherein the active agent comprises an anti-cancer drug.

10. The active agent carrier of claim 1, wherein the active agent is encapsulated within the disc- shaped membrane.

11. The active agent carrier of claim 1, wherein the active agent is covalently linked to the amphiphilic block copolymers.

12. The active agent carrier of claim 11, wherein the active agent is covalently linked to the amphiphilic block copolymers through a pH-cleavable linkage. 35 4823-4406-5785v.313368-4213. The active agent carrier of claim 12, wherein the pH-cleavable linkage comprises a pH- sensitive Schiff bond.

14. The active agent carrier of claim 1, wherein the membrane stabilizing agent is operable to maintain the shape and size of the disc-shaped membrane.

15. The active agent carrier of claim 1, wherein the membrane stabilizing agent comprises membrane-scaffold proteins.

16. The active agent carrier of claim 1, further comprising a targeting agent operable to direct the active agent carrier to a specific tissue or cell.

17. The active agent carrier of claim 16, wherein the targeting agent is selected from the group consisting of an antibody, an antibody fragment, an aptamer, or combinations thereof.

18. The active agent carrier of claim 16, wherein the targeting agent comprises cancer cell targeting moieties.

19. The active agent carrier of claim 1, wherein the active agent carrier is suitable for use in delivering one or more active agents to a subject.

20. A method of delivering one or more active agents to a subject, said method comprising: administering to the subject an active agent carrier comprising: a disc-shaped membrane comprising a plurality of self-assembled amphiphilic block copolymers and a membrane stabilizing agent, wherein the amphiphilic block copolymers comprise hydrophilic blocks and hydrophobic blocks, and wherein the membrane stabilizing agent encases the disc-shaped membrane; and at least one active agent associated with the disc-shaped membrane. 36 4823-4406-5785v.313368-4221. The method of claim 20, wherein the administering occurs by a method selected from the group consisting of intravenous administration, subcutaneous administration, transdermal administration, topical administration, intraarterial administration, intrathecal administration, intracranial administration, intraperitoneal administration, intraspinal administration, intranasal administration, intraocular administration, oral administration, intratumor administration, local administration, or combinations thereof.

22. The method of claim 20, wherein the administering comprises local administration to a specific tissue of a subject.

23. The method of claim 22, wherein the tissue comprises cancerous tissue.

24. The method of claim 20, wherein the method is utilized to treat or prevent a condition in the subject.

25. The method of claim 24, wherein the condition is cancer, and wherein the active agent comprises an anti-cancer drug.

26. The method of claim 20, wherein the subject is a human being.

27. The method of claim 20, wherein the amphiphilic block copolymers comprise methoxy- polyethylene glycol (mPEG) and polylactide (PLA) (mPEG-b-PLA).

28. The method of claim 20, wherein the disc-shaped membrane comprises a hydrophilic surface and a hydrophobic interior.

29. The method of claim 20, wherein the disc-shaped membrane has a diameter ranging from about 5 nm to about 20 nm. 37 4823-4406-5785v.313368-4230. The method of claim 20, wherein the active agent is covalently linked to the amphiphilic block copolymers through a pH-cleavable linkage.

31. The method of claim 30, wherein the pH-cleavable linkage comprises a pH-sensitive Schiff bond.

32. The method of claim 20, wherein the membrane stabilizing agent is operable to maintain the shape and size of the disc-shaped membrane.

33. The method of claim 20, wherein the membrane stabilizing agent comprises membrane- scaffold proteins.

34. The method of claim 20, wherein the disc-shaped membrane further comprises a targeting agent operable to direct the active agent carrier to a specific tissue or cell.

35. The method of claim 34, wherein the targeting agent comprises cancer cell targeting moieties.

36. A method of making an active agent carrier, said method comprising: mixing a plurality of amphiphilic block copolymers and a membrane stabilizing agent to form a self-assembled disc-shaped membrane encased by the membrane stabilizing agent; and associating the disc-shaped membrane with at least one active agent.

37. The method of claim 36, wherein the associating of the disc-shaped membrane with at least one active agent comprises covalently linking the amphiphilic block copolymers with the active agent.

38. The method of claim 37, wherein the active agent becomes covalently linked to the amphiphilic block copolymers through a pH-cleavable linkage. 38 4823-4406-5785v.313368-4239. The method of claim 38, wherein the pH-cleavable linkage comprises a pH-sensitive Schiff bond.

40. The method of claim 36, wherein the membrane stabilizing agent maintains the shape and size of the disc-shaped membrane.

41. The method of claim 36, further comprising a step of associating the disc-shaped membrane with a targeting agent, wherein the targeting agent is operable to direct the active agent carrier to a specific tissue or cell. 39 4823-4406-5785v.313368-42

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