WO2025250344A1 - Polypeptide nanotubes linked by disulfide bonds for delivery - Google Patents

Abstract

A C-terminal fragment of insulin-like growth factor binding protein 2 (IGFBP2) containing 3 cysteine residues has been shown to spontaneously self-assemble into nanotube (NT) structures. These NTs can encapsulate drugs during the assembly process and then deliver their cargo intracellularly following binding to interns and endocytosis. The integrin-dependence of this process is mediated by the presence of a naturally occurring RGD motif within the peptide sequence. A particular advantage of such NTs is that by encapsulating small molecule drugs, tissues are protected from their adverse effects. Here, the use of NTs to deliver small drugs and biologics across the blood-brain barrier (BBB) by penetrating the CNS and delivering their cargo is described.

Description

DESCRIPTION

POLYPEPTIDE NANOTUBES LINKED BY DISULFIDE BONDS FOR DELIVERY

FEDERAL FUNDING SUPPORT CLAUSE

[0001] The invention was made with government support under grant nos. R01 CA78887, R01 CA134845 and AG079748 awarded by the National Institutes of Health. The government has certain rights in the invention.

PRIORITY CLAIM

[0002] This application claims benefit of priority to U.S. Provisional Application Serial No. 63/652,939, filed May 29, 2024, the entire contents of which are hereby incorporated by reference.

SEQUENCE LISTING

[0003] Pursuant to 37 C.F.R. § 1.821(c), a sequence listing is submitted herewith as an XML file named “MESCP0143WO.xml”, created on February 24, 2025, and having a size of 9,031 bytes. The content of the aforementioned file is hereby incorporated by reference in its entirety.

BACKGROUND

1. Field

[0004] The present disclosure relates generally to the field of molecular biology, neurology and oncology. More particularly, it concerns structured nanotube delivery vehicles for therapeutic agents to the CNS/across the BBB.

2. Description of Related Art

[0005] A frequent limitation in treating Alzheimer’s disease (AD) and AD-related dementias (ADRD), as well as other CNS diseases, is the inability of small drugs and biologicals to cross the blood-brain barrier (BBB) and access the CNS. The incidence of dementia, characterized by memory loss and reduced cognition, is rapidly rising worldwide as the aging population expands. AD is the predominant form of dementia (60-70% of cases). Other dementias fall under the umbrella of ADRD (Beishon & Panerai, 2021). AD is a neurodegenerative disorder estimated to affect over 50 million people worldwide and ~6.2 million in the US (2021 Alzheimer's Disease Facts and Figures, 2021); these numbers are expected to double in 20 years (2021 Alzheimer’s Disease Facts and Figures, 2021). The brains of AD sufferers are defined by brain atrophy, the presence of senile plaques containing aggregates of the Amyloid ^-protein (Ap) and neurofibrillary tangles (NFTs) comprised of hyperphosphorylated tau protein (Zlokovic, 2011).

[0006] Currently, there is no cure for AD. In the early stages, patients are often managed with 1 of 3 cholinesterase inhibitors - galantamine (GAL), rivastigmine or donepezil, and the methyl -D-aspartate receptor antagonist, memantine. Small molecule drugs, biologicals and monoclonal antibody (mAh) drugs have variable efficiencies crossing the BBB to enter the CNS (Pardridge, 2020). Unfortunately, the ability of unmodified mAbs (Schneider, 2020; Restifo, 2021) to penetrate the CNS is controversial, underscoring the unmet need for targeted drug delivery across the BBB. In addition, oral GAL treatment, among other drugs, often elicits adverse systemic effects impacting patient compliance, further accentuating the need for encapsulation of drugs meant for CNS delivery. This has led to the use of lipophilic molecules, fusion proteins, liposomes, and nanoparticles for drug delivery to the CNS (Pardridge, 2007; Li et al., 2021). Many have employed carbon or gold nano-formulations which are often toxic, reducing their therapeutic value (Kavosi et al., 2018). Thus, there remains a need for improved delivery of drugs to the CNS.

SUMMARY

[0007] In a first embodiment, the present disclosure a method of delivering an agent to the central nervous system pf a subject comprising:

(a) providing composition comprising a nanotube composed of plurality of self-assembling polypeptide subunits with an amino acid sequence at least 95% identical to

CVNPNTGKLIQGAPTIRGDPECHLFYNEQQEACGVHTQRMT (SEQ ID NO: 2) or

GPLGSPG1RGSCVNPNTGKL1QGAPT1RGDPECHLFYNEQQEACGVHT QRMT (SEQ ID NO: 1) linked by cysteine disulfide bonds and comprising an agent encapsulated in said nanotube; and

(b) delivering said composition to said subject.

[0008] The polypeptide subunits may each have identical sequences. The polypeptide subunits may be about 10 nm to about 500 nm in length, or about 100 nm to about 500 nm in length, or about 100 nm to about 250 nm in length. The agent may be a therapeutic agent, such as a chemo- or radiotherapeutic suitable for treating a brain cancer, a therapeutic agent for treating dementia (Alzheimer’s Disease/AD-related dementias), Parkinson’s disease, multiple sclerosis, vascular dementia, or stroke. The agent may be a diagnostic agent. The polypeptide subunits may comprise at least 3 cysteine residues, such as wherein an intramolecular disulfide bond is present between the first and second cysteine of the same subunit and an intermolecular bond between the third cysteine of the same subunit and a cysteine from a different subunit. The polypeptide subunits may comprise an amino acid sequence at least 96%, 97%, 98%, 99% or 100% identical to CVNPNTGKLIQGAPTIRGDPECHLFYNEQQEACGVHTQRMT (SEQ ID NO: 2) or

GPLGSPGIRGSCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEACGVHTQRMT (SEQ ID NO: 1). The polypeptide subunits may comprise an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2. The polypeptide subunits may comprise an amino acid sequence identical to SEQ ID NO: 2. The polypeptide subunits may comprise an amino acid sequence at least 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. The polypeptide subunits may comprise an amino acid sequence identical to SEQ ID NO: 1. The polypeptide may be PEGylated. [0009] The composition is administered daily, such as more than once, such as on a continuous basis. The method may further comprise administering an additional therapeutic agent, such as an anti-cancer agent selected from the group consisting of chemotherapy, radiotherapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy and immunotherapy. The additional therapeutic agent may be an anti- Alzheimer’s Disease agent. The composition may be administered intravenously, subcutaneously, orally, transdermally, in sustained release, in controlled release, in delayed release, as a suppository, or sublingually. Administering the composition may comprise administration locally or regionally to the CNS/brain.

[0010] In another embodiment, there is provided a composition comprising (a) nanotube composed of plurality of self-assembling polypeptide subunits with an amino acid sequence at least 95% identical to

CVNPNTGKLIQGAPTIRGDPECHLFYNEQQEACGVHTQRMT (SEQ ID NO: 2) or GPLGSPGIRGSCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEACGVHTQRMT (SEQ ID NO: 1) linked by cysteine disulfide bonds and comprising an agent encapsulated in said nanotube; and (b) an anti- Alzheimer’s Disease drug, an anti-Parkinson’ s disease drug, an antimultiple sclerosis drug, a vascular dementia drug, or stroke drug (e.g., TPA or a derivative thereof). In a further embodiment, there is a kit comprising such composition, optionally further comprising instructions for the use of said composition.

[0011] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0012] As used herein in the specification and claims, “a” or “an” may mean one or more. As used herein in the specification and claims, when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein, in the specification and claim, “another” or “a further” may mean at least a second or more. [0013] As used herein in the specification and claims, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0014] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating certain embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0016] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0017] FIG. 1. Structure of the BBB. An SOP for generating SMC-101 NTs, 100 nm in length, and their efficient loading with galantamine (GAL) or IgG will be defined. Uptake and transcytosis of NTs by brain endothelial cells in vitro and their RGD-dependence will be established. Mouse studies will evaluate NT toxicity in mice and their ability to cross the BBB and enter the brain. Top: shows a cross-section of a brain capillary which is the neurovascular unit of the BBB. Bottom: illustrates integrin-dependent transcytosis and adsorptive transcytosis of SMC-101 NTs from the blood to the brain by a BBB endothelial cell.

[0018] FIG. 2. SMC-101-104. Changes to SMC-101 are shown in italics; the three Cysteine residues are bolded; RGD is underlined. SMC- 102 is the negative control replacing RGD with RGE (italics in SMC- 102). SMC- 103 has an insertion of the RGDKGPD (SEQ ID NO: 3), or iRGD peptide (bold and underlines). SMC- 104 contains an LXXL (LQVL; in italics) flanking sequence to enhance RGD binding to integrins, shown in italics. SMC-101 has a mass of 5,565 Da. (Creative Peptides, >95% pure) (SEQ ID NOs: 6-9).

[0019] FIG. 3. Transmission electron micrograph of SMC-101 NTs. SMC-101 NTs average 1-10 pm long and 35 nm wide. The inventors will fine-tune assembly to yield NTs that are 100 nm x 35 nm (1 x w; (Asampille et al., 2018).

[0020] FIG. 4. Binding of SMC-101 NTs to cells. Top: FITC-SMC-101 NTs, (green), were bound to HeLa cells (shown at 4 hr). Bottom: Control- excess -RGDS peptide added to compete with SMC-101. Nuclei stained with DAPI (blue). SMC-101 uptake was decreased by inhibitor peptide addition (Asampille et al., 2018). [0021] FIG. 5. Loss of cell viability is DOX dose dependent. HeLa cells were treated with DOX, NTs or DOX-loaded NTs and cell viability was evaluated using the MTT assay (Asampille et al., 2018).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0022] Nanotubular structures have been formed by self-assembly of a polypeptide fragment at the C-terminal end (residues 249-289) of human insulin-like growth factor binding protein-2 (hIGFBP-2249-289) (Swain et al., 2010). Wild-type hIGFBP-2249-289 has two cysteines in its primary sequence. An Arg to Cys substitution at residue 281, due to a cloning error, fortuitously led the inventor to create a version of the 249-289 peptide that surprisingly was capable of self-assembly. However, the polypeptide fragment considered had an additional cysteine due to a mutation at R281. The polypeptide (hIGFBP-2249-289 (R281C)) thus had an odd number of cysteines, which resulted in spontaneous self-assembly to form soluble nanotubular structures via intermolecular disulfide bonds. Further, the polypeptide fragment contains an RGD motif in its sequence. Upon formation of nanotubes, an array of RGD is displayed on the surface providing a unique feature for active targeting of cancer cells through integrin binding. Therefore, cellular imaging and drug delivery using these nanotubes is an attractive option for targeting cancer cells.

[0023] As discussed above, there remains an urgent need for improved compositions and methods for delivery of agents to the central nervous system (CNS). Here, the inventors propose using their NTs as first-in-class peptide-based CNS targeting agents capable of safely passing through the body, targeting the BBB and delivering drugs to the CNS. These NTs are natural, biodegradable peptides that spontaneously assemble into NTs. Previous in vitro studies show that these NTs are stable, non-toxic, and capable of binding to and delivering their cargo into cells (Swain et al., 2010; Asampille et al., 2018).

[0024] The NTs of this disclosure provide a compositional advantage over current nanomaterials with respect to crossing the BBB/CNS targeting. As already mentioned, they are peptide-based agents derived from a serum/extracellular matrix (ECM) protein (Habibi et al. , 2016). They are efficiently prepared from a parent peptide, and drug loading occurs during NT assembly in a single step. Furthermore, they lack a heparin-binding domain and are less “sticky” than intact IGFBP2 (Rosenzweig, 2004) enhancing movement within the CNS and their intrinsic RGD motifs will enhance CNS penetration via integrin-mediated transcytosis. As such, they have distinct advantages over chemically based nanomaterials and can deliver loaded drug cargo for CNS delivery (hippocampal neurons/amyloid plaque).

[0025] These and other aspects of the disclosure are discussed further below. I. Definitions

[0026] As used herein, the term “administering” or “administration” typically refers to the administration of a composition to a subject to achieve delivery of an agent that is, or is included in, a composition to a target site or a site to be treated. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be parenteral. In some embodiments, administration may be intravenous. In some embodiments, administration may be oral. In some embodiments, administration may be via injection. In some embodiments, administration may be systemic. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., infusion, perfusion) for at least a selected period of time.

[0027] In general, the term “agent”, as used herein, is used to refer to an entity (e.g., for example, a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc., or complex, combination, mixture or system (e.g., cell, tissue, organism) thereof), or phenomenon (e.g., heat, electric current or field, magnetic force or field, etc.). In appropriate circumstances, as will be clear from context to those skilled in the art, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively, or additionally, as context will make clear, the term may be used to refer to a natural product in that it is found in and/or is obtained from nature. In some instances, again as will be clear from context, the term may be used to refer to one or more entities that is manmade in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, for example, that may be screened to identify or characterize active agents within them. In some cases, the term “agent” may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound or entity that is not a polymer and/or is substantially free of any polymer and/or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.

[0028] As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish and/or worms. In some embodiments, an animal may be a transgenic animal, a genetically engineered animal and/or a clone.

[0029] The term “cancer” is used herein to generally refer to a disease or condition in which cells of a tissue of interest exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, cancer may comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic.

[0030] As used herein, the term “in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (e.g., animal, plant and/or microbe). As used herein, the term “in vivo" refers to events that occur within an organism (e.g., animal, plant and/or microbe).

[0031] As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, an active agent is present in unit dose amounts appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0032] As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or poly anhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0033] As used herein, the term “subject” or “test subject” refers to any organism to which a compound (e.g., an oligonucleotide) or composition is administered in accordance with the present disclosure e.g., for experimental, diagnostic, prophylactic and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, nonhuman primates, and humans; insects; worms; etc.) and plants. In some embodiments, a subject is a human. In some embodiments, a subject may be suffering from and/or susceptible to a disease, disorder, and/or condition. In some embodiments, a subject may be suffering from and/or susceptible to a cancer. In some embodiments, a subject displays one or more symptoms of a disease, disorder, and/or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.

[0034] An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with and/or displays one or more symptoms of a disease, disorder, and/or condition. An individual who is “susceptible to” a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

[0035] As used herein, the term “therapeutic agent” in general refers to any agent that elicits a desired effect (e.g., a desired biological, clinical, or pharmacological effect) when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, an appropriate population is a population of subjects suffering from and/or susceptible to a disease, disorder or condition. In some embodiments, an appropriate population is a population of model organisms. In some embodiments, an appropriate population may be defined by one or more criterion such as age group, gender, genetic background, preexisting clinical conditions, prior exposure to therapy. In some embodiments, a therapeutic agent is a substance that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms or features of a disease, disorder, and/or condition in a subject when administered to the subject in an effective amount. In some embodiments, a “therapeutic agent” is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a “therapeutic agent” is an agent for which a medical prescription is required for administration to humans. [0036] As used herein, the term “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0037] As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who exhibits a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who is suffering from a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who is suffering from a cancer or a central nervous system disease or disorder.

IL Self- Assembling IGFBP2-Derived Nanotubes

[0038] NTs of the present disclosure are derived from a naturally occurring serum/ECM localized protein, IGFBP2, that can be loaded with and deliver drugs to cells in vitro (Swain et al., 2010; Asampille et al., 2018). The present disclosure relates to the use of NTs as delivering drugs across the BBB and into the CNS. The intrinsic RGD motif present in the described NTs targets integrins on the BBB endothelium. This can facilitate NT transcytosis into the CNS and diffusion to the hippocampus for drug delivery. The described experiments will provide critical information regarding NT efficacy in targeting the BBB in vitro and in vivo (Fig. 1) with the objective of using the described NTs is to increase drug delivery to the CNS while avoiding toxic systemic effects.

[0039] In an example, SMC- 101 is a peptide derived from the 41 residue C-terminal domain of IGFBP2 (Fig. 2) which spontaneously assembles into NTs (35 nm wide x 10 nm - 10 pm long (Fig. 3), driven and stabilized by forming of disulfide bonds (Kibbey et al., 2006). Each peptide contains an integrin binding RGD motif (Humphries et al. , 2006). SMC-101 NTs are stable in vitro (Swain et al., 2010; Asampille et al., 2018) suggesting they will have long half-lives that will endure binding, uptake, and transcytosis (Figs. 4-5). Being derived from a serum/ECM protein, SMC-101 NTs are biocompatible and biodegradable (Asampille et al., 2018) natural products, having a functional advantage over other nanomaterials (Mahajan et al., 2018). These NTs hold great therapeutic promise as non-toxic drug delivery agents. Information on these reagents is also provided in U.S. Patent Publication No. 20200206313 and Asampille et al. (2018).

III. Therapeutic and Diagnostic Payloads

A. Therapeutic Payloads

[0040] In some aspects, therapeutic agents for use according to the embodiments include chemotherapeutic agents, such as for treating hyperproliferative diseases including cancers. For example, in some aspects the chemotherapeutic agent is a protein kinase inhibitor such as a EGFR, VEGFR, AKT, Erbl, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor or BRAF inhibitors. Nonlimiting examples of protein kinase inhibitors include Afatinib, Axitinib, Bevacizumab, Bosutinib, Cetuximab, Crizotinib, Dasatinib, Erlotinib, Fostamatinib, Gefitinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Panitumumab, Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib, Saracatinib, Sorafenib, Sunitinib, Trastuzumab, Vandetanib, AP23451, Vemurafenib, MK-2206, GSK690693, A-443654, VQD-002, Miltefosine, Perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridaforolimus, Alvocidib, Genistein, Selumetinib, AZD-6244, Vatalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016 or a mixture thereof. [0041] Yet further chemotherapeutic agents include, for example, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, famesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids or derivatives of any of the above.

B. Diagnostic Payloads

[0042] Many appropriate imaging agents are known in the art. The imaging moieties used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents.

[0043] In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).

[0044] In the case of radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine²¹¹, ¹⁴carbon, ⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen, iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹ , ⁵⁹iron, ³²phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur, technicium^99m and/or yttrium⁹⁰. ^12SI is often preferred for use in certain embodiments, and techniciiim⁹⁹'" and/or indium¹¹¹ are also often preferred due to their low energy and suitability for long range detection. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetracetic acid (EDTA). [0045] Among the fluorescent labels contemplated for use include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY- TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6- JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Near IR Red, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.

IV. Disease States

A. Central Nervous System Cancers

Primary CNS Tumors. A variety of cancers arise with primary tumors in the brain/central nervous system and are thus candidates for treatment using the compositions and methods disclosed here. Examples include tumors arising from the glia, including gliomas (e.g., diffuse intrinsic pontine gliomas, ganglogliomas), astrocytomas, oligodendrogliomas, and ependymomas. Tumors also arise from neurons, including medulloblastoma and embryonal tumors, such as neuroectodermal tumors (PNET), embryonal tumors with multilayer rosettes (ETMR), and aytpical teratoid/rhabdoid tumors (ATRT). Sadly, many of these cancers arise in pediatric patients.

Another type of primary central nervous system tumor is CNS lymphoma, a rare nonHodgkin lymphoma in which cancer cells from lymph tissue form in the brain and/or spinal cord (primary CNS). Because the eye is so close to the brain, primary CNS lymphoma can also start in the eye (called ocular lymphoma). The cancer can also involve the spinal fluid that bathes the spinal cord and brain. This is called leptomeningeal lymphoma.

Metastatic Brain/CNS Tumors. In theory, any cancer can metastasize to the brain, so it is possible that a brain metastasis may arise from cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, pancreas, testis, tongue, cervix, or uterus. Non-limiting examples of cancer histological types include neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo- alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; and myeloid sarcoma. Particularly common brain metastases include those arising from breast, lung, prostate, colon and renal cancers, as well as melanomas.

B. Non-Cancer Central Nervous System Diseases

[0046] The present disclosure also is directed to the treatment of central nervous system (CNS) disease. Such diseases include Alzheimer’s Disease, Alzheimer’ s-related dementias (ARD), Parkinson’s disease, multiple sclerosis, vascular dementia, and stroke.

[0047] Alzheimer's disease/ARD. Alzheimer's disease (AD) is a neurodegenerative disease that usually starts slowly and progressively worsens, and is the cause of 60-70% of cases of dementia. The most common early symptom is difficulty in remembering recent events. As the disease advances, symptoms can include problems with language, disorientation (including easily getting lost), mood swings, loss of motivation, selfneglect, and behavioral issues. As a person's condition declines, they often withdraw from family and society. Gradually, bodily functions are lost, ultimately leading to death. Although the speed of progression can vary, the average life expectancy following diagnosis is three to twelve years.

[0048] There are many environmental and genetic risk factors associated with development of AD. The strongest genetic risk factor is from an allele of apolipoprotein E. Other risk factors include a history of head injury, clinical depression, and high blood pressure. The progress of the protein misfolding disease is largely associated with amyloid plaques, neurofibrillary tangles, and loss of neuronal connections in the brain. A probable diagnosis is based on the history of the illness and cognitive testing, with medical imaging and blood tests to rule out other possible causes. Initial symptoms are often mistaken for normal brain aging. Examination of brain tissue is needed for a definite diagnosis, but this can only take place after death.

[0049] No treatments can stop or reverse its progression, though some may temporarily improve symptoms. A healthy diet, physical activity, and social engagement are generally beneficial in ageing, and may help in reducing the risk of cognitive decline and Alzheimer’s. Affected people become increasingly reliant on others for assistance, often placing a burden on caregivers. The pressures can include social, psychological, physical, and economic elements. Exercise programs may be beneficial with respect to activities of daily living and can potentially improve outcomes. Behavioral problems or psychosis due to dementia are sometimes treated with antipsychotics, but this has an increased risk of early death.

[0050] As of 2020, there were approximately 50 million people worldwide with Alzheimer's disease. It most often begins in people over 65 years of age, although up to 10% of cases are early-onset impacting those in their 30s to mid-60s. It affects about 6% of people 65 years and older, and women more often than men. Alzheimer's financial burden on society is large, with an estimated global annual cost of one trillion U.S. dollars. It is ranked as the seventh leading cause of death worldwide.

[0051] Medications used to treat the cognitive symptoms of Alzheimer's disease rather than the underlying cause include four acetylcholinesterase inhibitors (tacrine, rivastigmine, galantamine, and donepezil) and memantine, an NMDA receptor antagonist. The acetylcholinesterase inhibitors are intended for those with mild to severe Alzheimer's, whereas memantine is intended for those with moderate or severe Alzheimer's disease. The benefit from their use is small, but may be the result of poor blood-brain-barrier transport. In addition, biologies being developed that have an even larger size will no doubt face the same challenges in terms of brain accessibility.

[0052] Reduction in the activity of cholinergic neurons is a well-known feature of Alzheimer’s disease. Acetylcholinesterase inhibitors are employed to reduce the rate at which acetylcholine (ACh) is broken down, thereby increasing the concentration of ACh in the brain and combating the loss of ACh caused by the death of cholinergic neurons. There is evidence for the efficacy of these medications in mild to moderate Alzheimer's disease, and some evidence for their use in the advanced stage. The use of these drugs in mild cognitive impairment has not shown any effect in a delay of the onset of Alzheimer's disease. The most common side effects are nausea and vomiting, both of which are linked to cholinergic excess. These side effects arise in approximately 10-20% of users, are mild to moderate in severity, and can be managed by slowly adjusting medication doses. Less common secondary effects include muscle cramps, decreased heart rate (bradycardia), decreased appetite and weight, and increased gastric acid production. [0053] Glutamate is an excitatory neurotransmitter of the nervous system, although excessive amounts in the brain can lead to cell death through a process called excitotoxicity which consists of the overstimulation of glutamate receptors. Excitotoxicity occurs not only in Alzheimer's disease, but also in other neurological diseases such as Parkinson’s disease and multiple sclerosis. Memantine is a noncompetitive NMDA receptor antagonist first used as an anti-influenza agent. It acts on the glutamatergic system by blocking NMDA receptors and inhibiting their overstimulation by glutamate. Memantine has been shown to have a small benefit in the treatment of moderate to severe Alzheimer's disease. Reported adverse events with memantine are infrequent and mild, including hallucinations, confusion, dizziness, headache and fatigue. The combination of memantine and donepezil has been shown to be "of statistically significant but clinically marginal effectiveness.”

[0054] An extract of Ginkgo biloba known as EGb 761 has been used for treating Alzheimer’s and other neuropsychiatric disorders. Its use is approved throughout Europe. The World Federation of Biological Psychiatry guidelines lists EGb 761 with the same weight of evidence (level B) given to acetylcholinesterase inhibitors and memantine. EGb 761 is the only one that showed improvement of symptoms in both Alzheimer's disease and vascular dementia. EGb 761 may have a role either on its own or as an add-on if other therapies prove ineffective. A 2016 review concluded that the quality of evidence from clinical trials on Ginkgo biloba has been insufficient to warrant its use for treating Alzheimer's disease.

[0055] Atypical antipsychotics are modestly useful in reducing aggression and psychosis in people with Alzheimer's disease, but their advantages are offset by serious adverse effects, such as stroke, movement difficulties or cognitive decline. When used in the longterm, they have been shown to associate with increased mortality. Stopping antipsychotic use in this group of people appears to be safe.

[0056] Vascular dementia. Vascular dementia is dementia caused by a series of strokes. Restricted blood flow due to strokes reduces oxygen and glucose delivery to the brain, causing cell injury and neurological deficits in the affected region. Subtypes of vascular dementia include subcortical vascular dementia, multi-infarct dementia, stroke-related dementia, and mixed dementia. [0057] Subcortical vascular dementia occurs from damage to small blood vessels in the brain. Multi-infarct dementia results from a series of small strokes affecting several brain regions. Stroke-related dementia involving successive small strokes causes a more gradual decline in cognition. Dementia may occur when neurodegenerative and cerebrovascular pathologies are mixed, as in susceptible elderly people (75 years and older). Cognitive decline can be traced back to occurrence of successive strokes. ICD- 11 lists vascular dementia as dementia due to cerebrovascular disease. DSM-5 lists vascular dementia as either major or mild vascular neurocognitive disorder.

[0058] Vascular dementia can be caused by ischemic or hemorrhagic infarcts affecting multiple brain areas, including the anterior cerebral artery territory, the parietal lobes, or the cingulate gyrus. On rare occasions, infarcts in the hippocampus or thalamus are the cause of dementia. History of stroke increases the risk of developing dementia by around 70%, and recent stroke increases the risk by around 120%. Brain vascular lesions can also be the result of diffuse cerebrovascular disease, such as small vessel disease.

[0059] Risk factors for vascular dementia include increasing age, hypertension, smoking, hypercholesterolemia, diabetes mellitus, cardiovascular disease, and cerebrovascular disease. Other risk factors include lifestyle, geographic origin, and APOE-e4 genotype. Vascular dementia can sometimes be triggered by cerebral amyloid angiopathy, which involves accumulation of amyloid beta plaques in the walls of the cerebral arteries, leading to breakdown and rupture of the vessels. Since amyloid plaques are a characteristic feature of Alzheimer's disease, vascular dementia may occur as a consequence.

[0060] Parkinson's disease (PD). Parkinson’s disease, or simply Parkinson's, is a long-term neurodegenerative disease of mainly the central nervous system that affects both the motor system and non-motor systems. The symptoms usually emerge slowly, and as the disease progresses, non-motor symptoms become more common. Usual symptoms are tremor, slowness of movement, rigidity, and difficulty with balance, collectively known as parkinsonism. Parkinson's disease dementia, falls, and neuropsychiatric problems such as sleep abnormalities, psychosis, mood swings, or behavioral changes may arise in advanced stages.

[0061] Most of the cases of Parkinson's disease are sporadic and a few contributing factors have been identified. Pathophysiology is characterized by nerve cell death in the locus coeruleus and substantia nigra, latter being a midbrain region that supplies dopamine to the basal ganglia which are involved in voluntary motor control. The cause of this cell death is poorly understood but includes the alpha- synuclein aggregation into Lewy bodies within the neurons. Other possible factors involve genetic and environmental mechanisms as well as medications, lifestyle or previous conditions.

[0062] Diagnosis is mainly based on signs and symptoms, usually motor-related, found via neurological examination, though medical imaging like neuromelanin MRI can support the diagnosis. Usual onset is in people over 60 years of age of whom about one percent are affected. In those younger than 50, it is termed early-onset PD.

[0063] No cure is known, and treatment aims to mitigate symptoms. Initial treatment typically includes L-DOPA, MAO-B inhibitors, or dopamine agonists. As the disease progresses, these medications become less effective and produce a side effect marked by involuntary muscle movements. Diet and certain forms of rehabilitation have shown some effectiveness at improving symptoms. Deep brain stimulation has been used to reduce severe motor symptoms where drugs are ineffective. There is little evidence for treatment of the nonmovement-related symptoms, such as sleep disturbances and mood instability. The average life expectancy is near-normal.

[0064] The underlying cause of PD is unknown, yet is assumed to be influenced primarily by an interaction of genetic and environmental factors. Nonetheless, the most significant risk factor is age with a prevalence of 1 percent in those aged over 65 and approximately 4.3 percent in age over 85. Genetic components comprise SNCA, LRRK2, and PARK2 among others, while environmental risks include exposure to pesticides or heavy metals. Timing of exposure factor may influence the progression or severity of certain stages. However, caffeine and nicotine exhibit neuroprotective features, hence lowering the risk of PD. About 85 percent of cases occur sporadic, meaning that there is no family history.

[0065] Multiple sclerosis (MS). MS is an autoimmune disease in which the insulating covers of nerve cells in the brain and spinal cord are damaged. This damage disrupts the ability of parts of the nervous system to transmit signals, resulting in a range of signs and symptoms, including physical, mental, and sometimes psychiatric problems. Specific symptoms can include double vision, vision loss, eye pain, muscle weakness, and loss of sensation or coordination. MS takes several forms, with new symptoms either occurring in isolated attacks (relapsing forms) or building up over time (progressive forms). In the relapsing forms of MS, between attacks, symptoms may disappear completely, although some permanent neurological problems often remain, especially as the disease advances. In the progressive forms of MS, bodily functions slowly deteriorate, and disability worsens once symptoms manifest and will steadily continue to do so if the disease is left untreated.

[0066] While the cause is unclear, the underlying mechanism is thought to be either destruction by the immune system or failure of the myelin-producing cells. Proposed causes for this include immune dysregulation, genetics, and environmental factors, such as viral infections. MS is usually diagnosed based on the presenting signs and symptoms and the results of supporting medical tests.

[0067] No cure for multiple sclerosis is known. Current treatments are aimed at mitigating inflammation and resulting symptoms from acute flares and prevention of further attacks with disease-modifying medications. Physical therapy and occupational therapy, along with patient-centered symptom management, can help with people's ability to function. The long-term outcome is difficult to predict; better outcomes are more often seen in women, those who develop the disease early in life, those with a relapsing course, and those who initially experienced few attacks.

[0068] Multiple sclerosis is the most common immune-mediated disorder affecting the central nervous system. Nearly one million people in the United States had MS in 2022, and in 2020, about 2.8 million people were affected globally, with rates varying widely in different regions and among different populations. The disease usually begins between the ages of 20 and 50 and is twice as common in women as in men. MS was first described in 1868 by French neurologist Jean-Martin Charcot.

[0069] Stroke. Stroke, also known as a cerebrovascular accident (CVA) or brain attack) is a medical condition in which poor blood flow to the brain causes cell death. There are two main types of strokes: ischemic, due to lack of blood flow, and hemorrhagic, due to bleeding. Both cause parts of the brain to stop functioning properly.

[0070] Signs and symptoms of stroke may include an inability to move or feel on one side of the body, problems understanding or speaking, dizziness, or loss of vision to one side. Signs and symptoms often appear soon after the stroke has occurred. If symptoms last less than one or two hours, the stroke is a transient ischemic attack (TIA), also called a mini- stroke. Hemorrhagic stroke may also be associated with a severe headache. The symptoms of stroke can be permanent. Long-term complications may include pneumonia and loss of bladder control.

[0071] The biggest risk factor for stroke is high blood pressure. Other risk factors include high blood cholesterol, tobacco smoking, obesity, diabetes mellitus, a previous TIA, end-stage kidney disease, and atrial fibrillation. Ischemic stroke is typically caused by blockage of a blood vessel, though there are also less common causes. Hemorrhagic stroke is caused by either bleeding directly into the brain or into the space between the brain's membranes. Bleeding may occur due to a ruptured brain aneurysm. Diagnosis is typically based on a physical exam and supported by medical imaging such as a CT scan or MRI scan. A CT scan can rule out bleeding, but may not necessarily rule out ischemia, which early on typically does not show up on a CT scan. Other tests such as an electrocardiogram (ECG) and blood tests are done to determine risk factors and rule out other possible causes. Low blood sugar may cause similar symptoms.

[0072] Prevention includes decreasing risk factors, surgery to open up the arteries to the brain in those with problematic carotid narrowing, and warfarin in people with atrial fibrillation. Aspirin or statins may be recommended by physicians for prevention. Stroke is a medical emergency. Ischemic strokes, if detected within three to four-and-a-half hours, may be treatable with medication that can break down the clot, while hemorrhagic strokes sometimes benefit from surgery. Treatment to attempt recovery of lost function is called stroke rehabilitation, and ideally takes place in a stroke unit; however, these are not available in much of the world. In one embodiment the drug tissue plasminogen activator (tPA) encapsulation, which is highly useful in treating strokes, may advantageously he included in the nanotuhes to enhance its bioavailability and/or increase safety. This would include recent tPA genetic modifications such as tenecteplase (TNKase or TNK).

V. Pharmaceutical Formulations and Routes of Administration

[0073] The present disclosure provides pharmaceutical compositions including both the NTs of the disclosure and other ancillary agents that may be used in combination with drug or label loaded NTs. Such compositions comprise a prophylactically or therapeutically effective amount of an agent, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0074] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain aprophylactically or therapeutically effective amount of the agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intracranial, intra- spinal, intrabuccal, intranasal, nebulized, bronchial inhalation, intra-rectal, vaginal, topical or delivered by mechanical ventilation.

[0075] Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine , 2-ethylamino ethanol, histidine, procaine, and the like.

[0076] Generally, ingredients of compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

VI. Examples

[0077] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

Example 1

[0078] Nanotubes (NTs). Nanotechnology-based drug delivery platforms still require refinement to reduce their systemic toxicides. An approach to limit toxicity is to use NTs (Swain et al. , 2010; Asampille et al. , 2018) for drug delivery. Graphene-based carbon NTs held strong promise for therapeutic application but are too toxic (John et al., 2015; Bianco et al., 2005) and some liposome-based drug delivery reagents (Liu et al., 2017; Liu et al., 2016) have had success. There is an unmet need for new, non-toxic nanomaterials capable of targeting the BBB.

[0079] Electron microscopy of SMC- 101 NTs revealed they range in length from 10 nm -10 pm and are always 35 nm wide (Fig. 3, (Asampille et al., 2018)). NT length depends on how long SMC- 101 peptides are allowed to assemble. The inventors intend to standardize assembly times to generate NTs of 100 nm in length. This length promotes NT extravasation through leaky endothelial cell junctions via enhanced permeability and retention (Nakamura et al. , 2016), and their uptake by endothelial cells via endocytosis/transcytosis, pinocytosis, and phagocytosis (Fig. 1). NT lengths of 100 - 500 nm are considered optimal for passing through endothelial cell fenestrations and in accessing the transcytotic - endocytic cell machinery (Nel et al., 2017; Kokkinos et al., 2020). NTs of 0.5 - 1 pm in length enter cells by macro- pinocytosis; NTs 1-10 pm long enter by phagocytosis (Akinc & Battaglia, 2013). [0080] To test the RGD-dependence of NT association, SMC-101 NTs loaded with the auto-fluorescent drug doxorubicin (DOX) were incubated with HeLa cells for 4 h in the absence or presence of competing RGDS peptide to inhibit SMC-101 NT RGD binding to integrins (Gleeson et al. , 2001). Confocal microscopy revealed (Fig. 4) significant SMC-101 bound to and was taken up into cells. In toxicity assays, there was a dose-dependent effect of loaded DOX on cell viability (Fig. 5). Note that empty SMC-101 NTs did not decrease cell viability (Fig. 5).

[0081] Preliminary studies showed that NTs can readily be prepared from SMC-101 peptides and incorporate drugs or dye (DOX or FITC (Swain et al., 2010; Asampille et al., 2018). SMC-101 NTs bind to cell surface integrins, are internalized and disassembled, thus releasing their payload and then degraded. The rapid one-step assembly and drug-loading (Swain et al., 2010; Asampille et al., 2018) provide a strong advantage for SMC-101 NT function over available nanomaterials. By combining the RGD motif and its BBB targeting features with GAL and its poor CNS access with SMC-101 NTs, the inventors proposed that this synergistic combination will yield a safe, therapeutic drug delivery strategy that will benefit patients with AD and ADRD.

[0082] SMC-101 NT assembly, drug-loading and transcytosis by endothelial cells in vitro. To optimize SMC-101 NT assembly and drug-loading, the inventors seek to standardize SMC-101 NT production, size (100 nm in length (Swain et al., 2010; Asampille et al. , 2018)) and transcytosis. SMC-101 NT assembly will be carried out at 25°C with synthetic peptides (Fig. 2; 95% pure, Creative Peptides, Shirley, NY; see letter/quote). NTs will be assembled by dissolving peptides/diluting reductants to enable disulfide bond formation. Assembly will be terminated by rapid dialysis/concentration of the reaction using a lO kDa cutoff Amicon filter to remove free parent peptide(s) (5.6 kDa). Assembly time course with SMC- 101 will be tested first; the other SMC peptides (Fig. 2) will also be evaluated and are expected to be similar. SMC-101 NT length will be monitored with a ZetaView® Nanoparticle Analyzer (Particle Metrix, Inc, Mebane, NC), which measures particles ranging from 5 nm to 5 pm. Zeta potential will be determined to verify net surface charge for drug-loading/release (see below). Aliquots collected hourly on glass coverslips over 12 h will be monitored for NT size. SMC- 101 NTs are intrinsically fluorescent, and can be visualized by fluorescence microscopy (Swain et al. , 2010). Alternatively, peptides will be labeled with NHS-Alexa Fluor 488 (ThermoFisher) before assembly (see below for - NHS-Alexa Fluor 633) or they will be loaded with dye (FITC, Fig. 4; (Swain et al. , 2010; Asampille et al. , 2018)). The inventors will verify NT length by transmission electron microscopy. Negative control SMC-102 (non-integrin binding (RGE; Fig. 2) will be compared with SMC-101 in these experiments. They will further test the efficacy of converting the SMC-101 RGD to an iRGD motif: CRGDKGPDC (SEQ ID NO: 4) (SMC- 103; Fig. 2). iRGD peptides provide a tissue-penetrating benefit that may enhance SMC-103 NT delivery across the endothelium (Sugahara et al., 2009; Liu et al., 2017; Ruoslahti, 2017; Nel et al., 2017; Meng & Nel, 2018). This peptide also generates a peptide that activates Neuropilin-1 receptor endocytosis, enhancing SMC-101 NT endocytosis/transcytosis via the bystander effect to reduce proteolysis and nanotube cleavage (Teesalu et al. , 2009). The impact of changing the RGD flanking sequence to RGDLXXL (SEQ ID NO: 5) (SMC- 104) is to stabilize avP6 integrin-RGD complexes and SMC- 104 NT binding/transit across endothelial cells (Dicara et al., 2008; Altmann et al., 2017).

[0083] NT serum half-life. SMC-101 NTs are stable for days in an aqueous buffer at 30°C - 90°C (Asampille et al., 2018). The inventors will test their stability in serum in preparation for in vivo studies. NTs will be biotinylated with NHS-Alexa-biotin, incubated in fetal bovine serum or human serum at 37°C and aliquots will be taken at 1, 30, 60 and 120 min, 12 h and 24 h, heated to inactivate proteases, and mixed 1 : 1 with a slurry of neutravidin-agarose and incubated for 30 min at 25°C. Beads will be collected, SDS sample buffer (reductant-free) will be added and the samples will be resolved on 12% acrylamide SDS gels. Proteins will be transferred to nitrocellulose and NTs detected using avidin-horseradish peroxidase. NT stability will be evaluated by monitoring decreases in size; SMC-101 NTs migrate in the 90- 120 kDa range on SDS-PAGE (Swain et al., 2010). The inventors will map cleavage sites by mass spectrometry and introduce aa substitutions to reduce proteolysis to reduce cleavage. The longer the half-life of the NTs, the more effective they may be in drug delivery. At the same time, the potential for greater toxicity exists.

[0084] SMC-101 NT toxicity in vitro. Cytotoxicity of NTs is a function of their purity, concentration, chemical modifications, immunogenicity, inflammatory responses, oxidative effects, pulmonary and cardiovascular toxicides (Kavosi et al., 2018). The inventors have previously shown in in vitro studies that empty SMC-101 NTs are non-toxic to HeLa cells over a 4 h incubation (and up to 48 h; (Asampille et al., 2018)) based on an MTT cell viability assay. This approach will be repeated with the human brain endothelial cell line hCMEC/D3 in 24, 48 and 72 h incubations at 37°C in triplicate and testing a series of SMC-101 NT doses ranging from 50 pg/ml to 500 pg/ml and control incubations lacking NTs. Analysis of variance (ANOVA) models will be used to compare the nominal variables between treated and nontreated mice. Results will be expressed as mean ± SD. P < 0.05 considered statistically significant.

[0085] SMC-101 NT drug loading and release. Loading efficiency for GAL into SMC-101 NTs will be determined during NT assembly and post NT assembly. Cargo loading into and onto NTs (Hilder & Hill, 2009) as observed for DOX and SMC-101 will be monitored (Swain et al. , 2010; Asampille et al. , 2018). GAL is a small molecule (287 Da), like DOX (263 Da), which the inventors have experience in loading. SMC-101 has an isoelectric point of 6.9 and a low aliphatic index (i.e., is non-hydrophobic). These parameters are of note as chargeinteractions can influence drug-loading/retention. SMC-101 is negatively charged at pH >6.9. Thus, a higher pH may enhance interactions or at pH<6.9, reduce interactions. SMC-101 (100 pM) will be mixed with 2 mM GAL yielding a protein: drug ratio of 1:20 followed by NT assembly and free drug removal by dialysis. GAL encapsulation efficiency and its content will be quantified using standard methods (Asampille et al., 2018; Biabanikhankahdani etal., 2016) to determine surface vs. interior GAL-NT levels (Urey et al. , 2017). The inventors will test GAL release at pH 5 and 7. Note: during transcytosis, acidification may further require NT- containing endosome fusion with lysosomes (Toth et al., 2019).

[0086] SMC-101 cellular uptake and transcytosis in an in vitro model of the BBB. Anatomically, the BBB is comprised of the cerebral microvascular endothelium, which, together with astrocytes, pericytes, neurons and the extracellular matrix (ECM), form the "neurovascular unit" responsible for the function of the BBB/CNS protection (Edwards & Bix, 2019; Helms et al., 2016). Co-culture of human brain microvascular endothelial cells (ECs) along with pericytes, astrocytes and neurons can closely recapitulate BBB barrier function (Helms et al., 2016). The inventors’ goal is to obtain Go/No Go data on the ability of SMC NT preparations to cross the BBB, using hCMEC/D3 cells as a model (Toth et al., 2019). Several BBB model systems have been reported (Helms et al., 2016). They will use hCMEC/D3 monolayers (Weksler et al. , 2005) (Sigma Aldrich St. Louis, MO) grown on transwell™ inserts to mimic BBB-EC barrier function, an ENDOHM-6 EVOM™ chamber, (WPI instruments, Sarasota, FL) and a Trans-endothelial Electrical Resistance (TEER) cut-off value of 30-50 Q cm². This model is well-suited for NT transit. This will allow the inventors to follow SMC-101 NT transcytosis of the BBB in vitro (Helms et al., 2016; Poller et al., 2008). They will monitor empty Alexa-labeled SMC- 101 NT transcytosis to the apical (luminal) compartment and its delivery to the basolateral (abluminal) chamber. Aliquots will be collected and analyzed by HPLC and uv spectrophotometry (Yu et al., 2015). GAL-loaded NTs will be compared to empty NTs and drug alone. Cells will be incubated for 12, 24, 48, 72, 96,120 h at 37°C before analyzing cell viability (Fig. 5; (Lu et al., 2017)). Incubation times/doses will be adjusted, as will testing of additional drugs. Alternatively, fluorescent NT uptake into and across hCMEC/D3 monolayers will be examined by confocal microscopy /live cell imaging in the Cell and Molecular Imaging Shared Resource at Rollings Cancer Center. All experiments will be performed in triplicate.

[0087] RGD-facilitated cellular uptake of NTs. The inventors expect SMC- 101 NTs to bind to hCMEC/D3 cells and undergo transcytosis in a receptor-mediated manner. The alternative pathway is adsorptive transcytosis (Fig. 1) which includes pinocytosis/macropinocytosis and is non-selective. Endocytosis/transcytosis will be determined for SMC-101 NTs and control SMC-102 NTs (RGE; Fig. 2) using hCMEC/D3 cells. Alternatively, the inventors will use excess RGDS peptides to compete with SMC-101 NTs (Fig. 4). Binding and uptake of NTs derived from all SMC NT peptides will be compared to determine whether the RGD modifications improve transcytosis. Fig. 4 shows RGD- dependence of NT binding/uptake based on decreased FITC fluorescence in cells treated with excess -RGDS- peptide. Fluorescent NT transcytosis by hCMEC/D3 cell lines will be measured by using transwell™ inserts and confocal microscopy.

* * *

[0088] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims. VII. References

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

U.S. Patent Publication No. 20200206313

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Claims

WHAT IS CLAIMED IS:

1. A method of delivering an agent to the central nervous system pf a subject comprising:

(a) providing composition comprising nanotube composed of plurality of selfassembling polypeptide subunits with an amino acid sequence at least 95% identical to CVNPNTGKLIQGAPTIRGDPECHLFYNEQQEACGVHTQRMT (SEQ ID NO: 2) or GPLGSPGIRGSCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEACGVHTQRMT (SEQ ID NO: 1) linked by cysteine disulfide bonds and comprising an agent encapsulated in said nanotube; and

(b) delivering said composition to said subject.

2. The method of claim 1 , wherein the polypeptide subunits each have identical sequences.

3. The method of claim 1 or claim 2, wherein the polypeptide subunits are about 10 nm to about 500 nm in length, or about 100 nm to about 500 nm in length, or about 100 nm to about 250 nm in length.

4. The method of any one of claims 1-3, the agent is a therapeutic agent.

5. The method of claim 4, wherein the therapeutic agent is chemo- or radiotherapeutic suitable for treating a brain cancer, a therapeutic agent for treating dementia (Alzheimer’s Disease/ AD-related dementias), Parkinson’s disease, multiple sclerosis, vascular dementia, or stroke.

6. The method of any one of claims 1-4, wherein the agent is a diagnostic agent.

7. The method of any one of claims 1-6, wherein the polypeptide subunits comprise at least 3 cysteine residues, such as wherein an intramolecular disulfide bond is present between the first and second cysteine of the same subunit and an intermolecular bond between the third cysteine of the same subunit and a cysteine from a different subunit.

8. The method of any one of claims 1-7, wherein the polypeptide subunits comprise an amino acid sequence at least 96%, 97%, 98%, 99% or 100% identical to CVNPNTGKLIQGAPTIRGDPECHLFYNEQQEACGVHTQRMT (SEQ ID NO: 2) or GPLGSPGIRGSCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEACGVHTQRMT (SEQ ID NO: 1).

9. The method of claim 8, wherein the polypeptide subunits comprise an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2.

10. The method of claim 9, wherein the polypeptide subunits comprise an amino acid sequence identical to SEQ ID NO: 2.

11. The method of claim 8, wherein the polypeptide subunits comprise an amino acid sequence at least 96%, 97%, 98% or 99% identical to SEQ ID NO: 1.

12. The method of claim 11, wherein the polypeptide subunits comprise an amino acid sequence identical to SEQ ID NO: 1.

13. The method of any one of claims 1-12, wherein the polypeptide is PEGylated.

14. The method of any one of claims 1-13, wherein the composition is administered daily.

15. The method of claim 14, wherein the composition is administered more than once, such as on a continuous basis.

16. The method of any one of claims 1-15, further comprising administering an additional therapeutic agent.

17. The method of claim 16, wherein the additional therapeutic agent is an anti-cancer agent selected from the group consisting of chemotherapy, radiotherapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy and immunotherapy.

18. The method of claim 16, wherein the additional therapeutic agent is an anti- Alzheimer’ s Disease agent.

19. The method of any one of claims 1-18, wherein the composition is administered intravenously, subcutaneously, orally, transdermally, in sustained release, in controlled release, in delayed release, as a suppository, or sublingually.

20. The method of any one of claims 1-18, wherein administering the composition comprises administration locally or regionally to the CNS/brain.

21. A composition comprising (a) nanotube composed of plurality of self-assembling polypeptide subunits with an amino acid sequence at least 95% identical to CVNPNTGKLIQGAPTIRGDPECHLFYNEQQEACGVHTQRMT (SEQ ID NO: 2) or GPLGSPGIRGSCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEACGVHTQRMT (SEQ ID NO: 1) linked by cysteine disulfide bonds and comprising an agent encapsulated in said nanotube; and (b) an anti- Alzheimer’s Disease drug, an anti-Parkinson’ s disease drug, an antimultiple sclerosis drug, a vascular dementia drug, or stroke drug.

22. A kit comprising the composition of claim 21, optionally further comprising instructions for the use of said composition.