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WO2024206627A2 - Methods and compositions for treating aortic valve disease - Google Patents

Methods and compositions for treating aortic valve disease Download PDF

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Publication number
WO2024206627A2
WO2024206627A2 PCT/US2024/021962 US2024021962W WO2024206627A2 WO 2024206627 A2 WO2024206627 A2 WO 2024206627A2 US 2024021962 W US2024021962 W US 2024021962W WO 2024206627 A2 WO2024206627 A2 WO 2024206627A2
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WO
WIPO (PCT)
Prior art keywords
nanoparticle
nanoparticles
cad
cavd
bay
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PCT/US2024/021962
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French (fr)
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WO2024206627A3 (en
Inventor
Jonathan T. BUTCHER
Alexander D. CRUZ
Clare Kelley HALL
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Cornell University
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Publication of WO2024206627A2 publication Critical patent/WO2024206627A2/en
Publication of WO2024206627A3 publication Critical patent/WO2024206627A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/275—Nitriles; Isonitriles
    • A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

Definitions

  • the present disclosure relates to a nanoparticle for treating and/or diagnosing calcific aortic valve disease (“CAVD”), a therapeutic composition comprising the nanoparticle, and a method of treating and/or diagnosing calcific aortic valve disease.
  • CAVD calcific aortic valve disease
  • Calcific Aortic Valve Disease is the most prevalent valvular heart disease in the world, affecting up to 25% of adults over the age of 65 (Alushi et al., “Calcific Aortic Valve Disease-Natural History and Future Therapeutic Strategies,” Frontiers in Pharmacology 11 :685 (2020)).
  • CAVD begins as aortic sclerosis which is characterized by calcification and thickening of the three leaflets that comprise the aortic valve (Prasad and Bhalodkar, “Aortic Sclerosis-a Marker of Coronary Atherosclerosis,” Clinical Cardiology 27:671-673 (2004)).
  • Aortic sclerosis progresses into aortic stenosis when large calcific nodules on the aortic valve leaflets hinder the leaflets’ mobility and obstruct blood flow through the heart (Alushi et al., “Calcific Aortic Valve Disease-Natural History and Future Therapeutic Strategies,” Frontiers in Pharmacology 11 :685 (2020)).
  • This later stage in CAVD is associated with approximately a 50% increase in the risk of myocardial infarction and the risk of death from a cardiovascular cause (Prasad and Bhalodkar, “Aortic Sclerosis-a Marker of Coronary Atherosclerosis,” Clinical Cardiology 27:671-673 (2004)).
  • statins have been experimented with in the cardiac field to investigate if they are a potential therapy as patients with CAVD experience lipid accumulation and deposits on their Aortic Valve.
  • statins have proved to be unreliable and ineffective as a CAVD therapy (Lee and Choi, “Involvement of Inflammatory Responses in the Early Development of Calcific Aortic Valve Disease: Lessons from Statin Therapy,” Anim. Cells Syst. (Seoul) 22:390-399 (2016)).
  • Nanoparticle drug delivery has been a massive triumph in biomedical research, expansively studied in cancer therapies (van der Meel et al., “Smart Cancer Nanomedicine,” Nat. Nanotechnol. 14: 1007-1017 (2019); Wadajkar et al., “Dual-Imaging Enabled Cancer-Targeting Nanoparticles,” Adv. Healthc. Mater. 1 :450-456 (2012)). They are an excellent tool to deliver treatment due to their site-specific targeting ability, making them a promising candidate to target proteins implicated in CAVD (Yetisgin et al., “Therapeutic Nanoparticles and Their Targeted Delivery Applications,” Molecules 25:2193 (2020)).
  • Cadherin-11 is a type II classical cadherin protein that is of particular interest in the study of CAVD. Cad-11 mediates cell migration and differentiation of mesenchymal cells into osteo- and chondro- lineages, and when overexpressed, disrupts proper homeostasis of the extracellular matrix that is critical for aortic valve integrity (Sung et al., “Cadherin-11 Overexpression Induces Extracellular Matrix Remodeling and Calcification in Mature Aortic Valves,” Arteriosclerosis, Thrombosis, and Vascular Biology 36 : 1627-1637 (2016)). This overexpression affects both aortic valve endothelial cells and aortic valve interstitial cells.
  • Cad-11 is endogenously found in healthy human aortic valve endothelial cells (HAVECs) (Zhou et al., “Cadherin-11 Expression Patterns in Heart Valves Associate With Key Functions During Embryonic Cushion Formation, Valve Maturation and Calcification,” Cells, Tissues, Organs 198:300-10 (2013); Butcher et al., Transcriptional Profiles of Valvular and Vascular Endothelial Cells Reveal Phenotypic Differences: Influence of Shear Stress,” Arteriosclerosis, Thrombosis, and Vascular Biology 26:69-77 (2006)).
  • HAVECs human aortic valve endothelial cells
  • RhoA Rho protein family GTPase
  • Sox9 a transcription factor
  • Tumor necrosis factor a is an inflammatory cytokine that has been found to significantly upregulate cad-11 expression, induce EndMT in aortic valve endothelial cells, and accelerate calcification in aortic valve interstitial cells (Vandooren et al., “Tumor Necrosis Factor a Drives Cadherin 11 Expression in Rheumatoid Inflammation,” Arthritis & Rheumatism 58:3051-3062 (2008); Mahler et al., “Inflammatory Cytokines Promote Mesenchymal Transformation in Embryonic and Adult Valve Endothelial Cells,” Arteriosclerosis, Thrombosis, and Vascular Biology 33: 121-130 (2013); Yu et al., “Tumor Necrosis Factor-a Accelerates the Calcification of Human Aortic Valve Interstitial Cells Obtained from Patients with Calcific Aortic Valve Stenosis via the BMP2-
  • TNFcr also activates the canonical nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-KB) pathway (Gee et al., “NFKB (Nuclear Factor K-Light-Chain Enhancer of Activated B Cells) Activity Regulates Cell-Type- Specific and Context-Specific Susceptibility to Calcification in the Aortic Valve,” Arterioscler. Thromb. Vase. Biol. 40:638-655 (2020)).
  • NF-KB canonical nuclear factor-kappa-light-chain-enhancer of activated B cells
  • the canonical pathway is activated when pro- inflammatory molecules, like TNFcr, bind to cell surface receptors and cause the activation of the IKB kinase complex which allows NF-KB dimers to translocate to the nucleus and activate DNA transcription (“The NF-KB Signaling Pathway.” The NF-KB Signaling Pathway - Creative Diagnostics (2020)). Recent findings indicate that the activation of the NF-KB canonical pathway can coordinate pathogenic calcification in aortic valve endothelial and interstitial cells (Gee et al., “NFKB (Nuclear Factor K-Light-Chain Enhancer of Activated B Cells) Activity Regulates Cell-
  • BAY 11-7082 has been shown to significantly inhibit the NF-KB pathway which reduced proteinuria and inhibited renal dysfunction in lupus-prone mice (Zhao et al., “Bayl 1-7082 Attenuates Murine Lupus Nephritis Via Inhibiting Nlrp3 Inflammasome and NF-KB Activation,” International Immunopharmacology 17:116-122 (2013)).
  • One aspect of the present disclosure relates to a nanoparticle for treating and/or diagnosing calcific aortic valve disease (CAVD).
  • the nanoparticle comprises a nanoparticle core and a targeting molecule associated with an exterior surface of the nanoparticle core.
  • the therapeutic composition comprises a nanoparticle for treating CAVD and a pharmaceutically acceptable carrier.
  • a further aspect of the present disclosure relates to a method of treating and/or diagnosing CAVD.
  • the method comprises contacting a nanoparticle with endothelial tissue of a subject at risk for CAVD.
  • Calcific Aortic Valve Disease is an increasingly prevalent disease defined by calcification and thickening of the aortic valve, resulting in improper heart functioning.
  • CAVD Calcific Aortic Valve Disease
  • PLGA lactic-co-glycolic acid
  • the parallel upregulation of cad-11 expression and activation of the NF-KB pathway by TNFcr provides an opportunity to deliver a therapeutic (e.g., BAY 11-7082) to cells actively expressing the NF-KB pathway by encapsulating the drug in biodegradable poly (lactic- co-glycolic acid) (PLGA) nanoparticles that bind to cad-11.
  • a therapeutic e.g., BAY 11-7082
  • PLGA biodegradable poly (lactic- co-glycolic acid)
  • Cad-11 antibodies can be conjugated, or associated, to the outside of nanoparticles to direct the nanoparticles to sites exhibiting cad-11 expression.
  • cad-11 antibody-conjugated PLGA nanoparticle contacts the cad-11 expressing cell
  • the nanoparticle is endocytosed and the contents inside the nanoparticle are released inside the cell after incubation for several hours.
  • the ability to conjugate cad-11 antibodies to the nanoparticles and to control the release of BAY 11-7082 suggests that PLGA nanoparticles have the potential to increase site-specific targeting of the drug and decrease systemic toxicity.
  • the nanoparticles home to the VECs, and when adhered/internalized release their payload, which in this case is an NF-KB inhibitor, which then blocks NF-KB activity.
  • the main advantage over the free drug (e.g. circulating) is that the residence time next to the valves is low given the very high fluid shear stresses occurring during ejection.
  • the selective binding by VECs via Cad-11 improves specificity to VECs, thus likely requiring less dose and/or fewer off target effects.
  • the benefit of blocking VEC NF-KB activity includes: reduction of EndMT, inhibition of endogenous reprogramming, reduction of osteogenic differentiation, and improved quiescent behavior through maintained NO secretion.
  • the nanoparticles can also home to VICs through/around the VECs at later disease stages, which would convey similar benefits to the VICs, with the exception of EndMT as they are already mesenchymal cells.
  • FIGS. 2A-2C are illustrations showing cross-sections of three different embodiments of cad-11 antibody-conjugated nanoparticles.
  • FIG. 2A is an illustration showing a cad-11 nanoparticle with coumarin 6 (C6) dye encapsulated in the nanoparticle core (cad-11 C6 nanoparticle).
  • FIG. 2B is an illustration showing a cad-11 nanoparticle with BAY 11-7082, an IKK inhibitor encapsulated in the nanoparticle core (cad-11 BAY nanoparticle).
  • FIG. 2C is an illustration showing a cad-11 nanoparticle with Cy5.5 dye encapsulated in the nanoparticle core (cad-11 Cy5.5 nanoparticle).
  • FIGS. 3A-3B shows PLGA nanoparticle properties and the release profile of cad- 11 BAY nanoparticles.
  • FIG. 3 A is a summary showing the size, poly dispersity, and zeta potential of the PLGA, BAY PLGA, and cad-11 BAY PLGA nanoparticles.
  • FIG. 3B is a graph showing the amount of BAY 11-7082 released per BAY PLGA nanoparticle over the span of 14 days.
  • FIG. 4 shows a schematic of the experimental procedures used to evaluate cad-11 nanoparticle binding specificity.
  • Human aortic valve endothelial cells (HA VECs) and human umbilical vein endothelial cells (HUVECs) were cultured and passaged into microwell plates. Following, cad-11 C6 nanoparticles were added to the microwell and incubated with the HAVECs and HUVECs for 3.5 hours. The cells were washed and then imaged to examine the fluorescence exhibited by the respective cells.
  • FIGS. 5A-5E demonstrate cad-11 C6 nanoparticle targeting of various cell types. Cells were incubated with 50 pg/mL of cad-11 C6 nanoparticles for 3.5 hours.
  • FIG. 5 A is a fluorescent image showing HAVECs after incubation with the cad-11 C6 nanoparticles.
  • FIG. 5B is a fluorescent image showing HUVECs after incubation with the cad-11 C6 nanoparticles.
  • FIG. 5C is a fluorescent image showing PAVICs after incubation with the cad-11 C6 nanoparticles.
  • FIG. 5D is a fluorescent image showing PAVECs after incubation with the cad-11 C6 nanoparticles.
  • FIG. 5E is a graph showing the proportion of HAVECs, HUVECs, PAVICs, and PAVECs that display fluorescent intensity above the background fluorescence threshold.
  • FIG. 6 shows a schematic of experimental procedures used to evaluate the effect of cad-11 BAY nanoparticles in PAVEC-PAVIC hydrogels.
  • 3D PAVEC-PAVIC collagen hydrogels were cultured for 5 days and then incubated with different doses of cad-11 BAY nanoparticles.
  • Various endpoint assays were used to assess disease progression in the hydrogels.
  • FIGS. 7A-7C demonstrate the effects of BAY 11-7082 treatment on PAVEC- PAVIC hydrogels cultured in osteogenic media (OGM).
  • FIG. 7A is a series of images showing PAVEC-PAVIC hydrogels cultured for 7 days in basal control media (GM) (DI -7), OGM (Dl- 7), or OGM (Dl-3) then OGM + lOuM BAY 11-7082 (D3-7). Images of the PAVEC-PAVIC hydrogels were acquired at days 1, 3, and 5. On day 7, the PAVEC-PAVIC hydrogels were stained with Alumblen Red Calcium Binding Stain (ARS) to evaluate calcific nodule formation.
  • FIG. 7B are images showing ARS staining of PAVEC-PAVIC hydrogels cultured in GM, OGM, or OGM with 10 pM BAY 11-7082 (OGMB) after 7 days.
  • FIGS. 8A-8B demonstrate the cellular compaction of the PAVEC-PAVIC hydrogels.
  • FIG. 8A is a series of images showing PAVEC-PAVIC hydrogels cultured for seven days in GM with no nanoparticles or OGM with 0 pM, 5 pM, 10 pM, and 20 pM BAY 11-7082 delivered via cad-11 BAY nanoparticles. The periphery of the PAVEC-PAVIC hydrogels is outlined in the images. Scale bars indicate 1 mm.
  • FIG. 9 is a series of images showing a-smooth muscle actin (a-SMA) staining in the PAVEC-PAVIC hydrogels treated with cad-11 BAY nanoparticles. Sections were stained for a-SMA to visualize the interstitial cells and diseased endothelial cells that had transitioned to mesenchymal phenotype. Sections were additionally stained with nuclear blue to label the nuclei.
  • FIG. 10 is a series of images showing VE-cadherin staining in PAVEC-PAVIC hydrogels treated with cad-11 BAY nanoparticles. Sections were stained for VE-cadherin to label PAVECs. Sections were additionally stained with nuclear blue to label the nuclei.
  • FIG. 11 is a series of images showing hematoxylin and eosin (H&E) staining of the hydrogels treated with different doses of cad-11 BAY nanoparticles. Scale bars indicate 0.5 mm.
  • FIG. 12 shows a schematic of experimental procedures to evaluate the efficacy of the cad-11 BAY nanoparticles in vivo.
  • An initial biodistribution study would be used to determine the cad-11 nanoparticle targeting sites in vivo.
  • a dose-response curve would be generated to determine the ECso of the cad-11 BAY nanoparticles.
  • the ECso dose would then be tested in a larger cohort of mice to determine the effects of the cad-11 BAY nanoparticles in vivo.
  • FIGS. 13A-13B demonstrate BAY 11-7082 treatment decreases cellular remodeling and calcific nodule density.
  • FIG. 13A is a graph showing area compaction in the Bayl 1@D1, Bayl 1@CAVD, GM, and OGM conditions.
  • FIG. 13B is a graph showing nodule number in the GM, OGM, OGM+Bay at CAVD Diagnosis, and OGM+Bay- Day 1 (preventative) conditions.
  • FIG. 14 demonstrates the effect of BAY 11-7082 on co-cultured spring gels. Images were taken after 7 days of incubation of co-culture PAVEC/PAVIC spring gels.
  • the term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, such as within 50%, or within 20%, or within 10%, or within 5% (or any amount or range within 5-50%) of a given value or range. The allowable variation encompassed by the term “about” or “approximately” may depend on the context.
  • the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
  • the foregoing also applies to words having similar meanings such as the terms, “including”, “involving”, “having”, and their derivatives.
  • the term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
  • the second component as used herein is different from the other components or first component.
  • a “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.
  • polypeptide “peptide”, or “protein” are used interchangeably and to refer to a polymer of amino acid residues.
  • the terms encompass all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP- ribosylation, pegylation, biotinylation, etc.).
  • subject means any animal, preferably a mammal, most preferably a human.
  • mammal encompasses any mammal. Examples of a subject as described herein include but are not limited to fish, birds, reptiles, or mammals, e.g., human, rabbit, cow, pig, sheep, chicken, rat, or mouse.
  • aortic valve refers to the heart valve that divides the left ventricle and the aorta.
  • the aortic valve opens during left ventricular contraction and then closes to prohibit the backwash of oxygenated blood from the aorta into the ventricle.
  • the aortic valve typically contains 3 valve leaflets in most individuals, but may contain 2 valve leaflets in some individuals.
  • calcific aortic valve disease refers to a disease state in which there is calcification and fibrosis of the aortic valve, encompassing aortic sclerosis and aortic stenosis.
  • aortic sclerosis refers to the thickening and calcification of the aortic valve leaflets in the absence of obstruction to left ventricular outflow.
  • aortic stenosis refers to a condition of valvular pathology in which left ventricular outflow is obstructed.
  • inhibiting refers to the reduction or suppression of a given condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.
  • NF -KB -inhibitor means a compound that inhibits the cell transcription factor nuclear kappa-B (NF-KB).
  • Examples of delivery vehicles described herein include but are not limited to nanoparticles and liposomes.
  • the present disclosure describes a nanoparticle delivery vehicle that can be used with a variety of subjects including warm blooded animals, particularly mammals, including humans, dogs, cats and other small animals, and farm animals. Additionally, the nanoparticles of the present disclosure can be used with prokaryotic and eukaryotic microorganisms and with in vitro cultures. The nanoparticle delivery vehicle of the present disclosure can be used as a diagnostic agent in all the above subjects, as well as in the capacity of a therapeutic agent.
  • Nanoparticle or “nanoparticle core” as used herein denotes a carrier structure which is biocompatible with and sufficiently resistant to chemical and/or physical destruction by the environment of use such that a sufficient amount of the carrier structure remains substantially intact so as to be able to reach the target site.
  • Nanoparticles can be solid colloidal particles ranging in size from 1 to 1000 nm. Nanoparticles can have any diameter less than or equal to 1000 nm, including 5, 10, 15, 20, 25, 30, 50, 100, 500 and 750 nm.
  • the nanoparticle is formed of a polymer.
  • polymer as used herein, is given its ordinary meaning as used in the art, z.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds.
  • the repeat units may all be identical, or in some cases, there may be more than one type of repeat unit present within the polymer.
  • the polymer can be biologically derived, i.e., a biopolymer. Non-limiting examples include peptides or proteins. In some cases, additional moieties may also be present in the polymer, for example biological moieties such as those described below.
  • the polymer is said to be a “copolymer.” It is to be understood that in any embodiment employing a polymer, the polymer being employed may be a copolymer in some cases.
  • the repeat units forming the copolymer may be arranged in any fashion. For example, the repeat units may be arranged in a random order, in an alternating order, or as a block copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc.
  • Block copolymers may have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.
  • Nanoparticles can include copolymers, which, in some embodiments, describes two or more polymers (such as those described herein) that have been associated with each other, usually by covalent bonding of the two or more polymers together.
  • a copolymer may comprise a first polymer and a second polymer, which have been conjugated together to form a block copolymer where the first polymer can be a first block of the block copolymer and the second polymer can be a second block of the block copolymer.
  • a block copolymer may, in some cases, contain multiple blocks of polymer, and that a “block copolymer,” as used herein, is not limited to only block copolymers having only a single first block and a single second block.
  • a block copolymer may comprise a first block comprising a first polymer, a second block comprising a second polymer, and a third block comprising a third polymer or the first polymer, etc.
  • block copolymers can contain any number of first blocks of a first polymer and second blocks of a second polymer (and in certain cases, third blocks, fourth blocks, etc.).
  • block copolymers can also be formed, in some instances, from other block copolymers.
  • a first block copolymer may be conjugated to another polymer (which may be a homopolymer, a biopolymer, another block copolymer, etc.), to form a new block copolymer containing multiple types of blocks, and/or to other moieties (e.g., to non-polymeric moieties).
  • a polymer e.g., copolymer, e.g., block copolymer
  • a biocompatible polymer i.e., the polymer that does not typically induce an adverse response when inserted or injected into a living subject, for example, without significant inflammation and/or acute rejection of the polymer by the immune system, for instance, via a T-cell response.
  • the nanoparticles contemplated herein can be non- immunogenic.
  • Biocompatibility typically refers to the acute rejection of material by at least a portion of the immune system, i.e., a nonbiocompatible material implanted into a subject provokes an immune response in the subject that can be severe enough such that the rejection of the material by the immune system cannot be adequately controlled, and often is of a degree such that the material must be removed from the subject.
  • One simple test to determine biocompatibility can be to expose a polymer to cells in vitro.
  • biocompatible polymers are polymers that typically will not result in significant cell death at moderate concentrations. For instance, a biocompatible polymer may cause less than about 20% cell death when exposed to such cells.
  • contemplated biocompatible polymers may be biodegradable, i.e., the polymer is able to degrade, chemically and/or biologically, within a physiological environment, such as within the body.
  • biodegradable polymers are those that, when introduced into cells, are broken down by the cellular machinery (biologically degradable) and/or by a chemical process, such as hydrolysis, (chemically degradable) into components that the cells can either reuse or dispose of without significant toxic effect on the cells.
  • the biodegradable polymer and their degradation byproducts can be biocompatible.
  • the nanoparticle core comprises a biodegradable polymer.
  • the nanoparticle is designed to only degrade once internalized by the cell which may in some instances occur via compartments that exhibit unique biological or biochemical environments.
  • the unique biological environment may be but is not limited to low pH.
  • Contemplated nanoparticle polyesters include, for example, copolymers and/or block copolymers comprising lactic acid and/or glycolic acid units, such as poly(lactic acid-co- glycolic acid) and poly(lactide-co-glycolide), collectively referred to herein as “PLGA”; and homopolymers comprising glycolic acid units, referred to herein as “PGA,” and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D- lactide, and poly-D,L-lactide, collectively referred to herein as “PLA.”
  • exemplary polyesters include, for example, polyhydroxyacids; PEGylated polymers and copolymers of lactide and glycolide (e.g., PEGylated PLA (PLA-PEG), PEGylated PGA, PEGylated PLGA, and derivatives thereof.
  • a contemplated nanoparticle may include PLGA.
  • PLGA is a biocompatible and biodegradable co-polymer of lactic acid and glycolic acid, and various forms of PLGA can be characterized by the ratio of lactic acid:gly colic acid.
  • Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lactic acid.
  • the degradation rate of PLGA can be adjusted by altering the lactic acid-glycolic acid ratio.
  • PLGA to be used in accordance with the present invention can be characterized by a lactic acid:glycolic acid ratio of approximately 85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60, approximately 25:75, or approximately 15:85.
  • the ratio of lactic acid to glycolic acid monomers in the polymer of the particle may be selected to optimize for various parameters such as therapeutic agent release and/or polymer degradation kinetics.
  • the nanoparticle core comprises poly(lactic-co-glycolic acid).
  • the delivery vehicle is a liposome.
  • liposome means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
  • liposomes may include the following: their biocompatibility and biodegradability, incorporation of a wide range of water and lipid soluble drugs; and they afford protection to encapsulated drugs from metabolism and degradation. Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size, and the aqueous volume of the liposomes.
  • Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
  • Methods for preparing liposomes for use in the present disclosure include those disclosed in Bangham et al., “Diffusion of Univalent Ions Across the Lamellae of Swollen Phospholipids,” J. Mol. Biol. 13:238-52 (1965); U.S. Patent No. 5,653,996 to Hsu; U.S. Patent No. 5,643,599 to Lee et al.; U.S. Patent No. 5,885,613 to Holland et al.; U.S. Patent No.
  • a “targeting molecule” is a molecule that is able to bind to or otherwise associate with a molecular target, for example, a membrane component, a cell surface receptor, cadherin-11 (cad-11) or the like.
  • a delivery vehicle comprising the targeting molecule may become localized or converge at a particular targeted site, for instance, endothelial tissue, a disease site, a tissue, an organ, a type of cell, etc. As such, the delivery vehicle may be “targetspecific.”
  • contemplated targeting molecules include, without limitation, a peptide, polypeptide, protein, glycoprotein, carbohydrate, or lipid.
  • a targeting molecule may be a naturally occurring or synthetic ligand for a cell surface receptor.
  • a targeting molecule can be an antibody, which term is intended to include antibody fragments, characteristic portions of antibodies, single chain targeting moieties which can be identified, for example, using procedures such as phage display.
  • Targeting molecules may also be a targeting peptide, targeting peptidomimetic, or a small molecule, whether naturally-occurring or artificially created (e.g., via chemical synthesis).
  • the targeting molecule comprises a molecule that targets an endothelial molecule that is indicative of CAVD.
  • the targeting component is selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein, a peptide, and aptamer, and a small molecule.
  • the targeting molecule is an antibody against molecular targets on endothelial tissue and, for example, specifically bind markers associated with CAVD.
  • the targeting molecule comprises a molecule that targets a portion of Cadherin 11 protein.
  • the targeting molecule comprises an anti -Cadherin 11 antibody.
  • Antibodies that may be used as targeting molecules in the nanoparticles of the present disclosure may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intrabodies”), antibody fragments (e.g. Fv, Fab and F(ab)2), half-antibodies, hybrid derivatives, as well as single chain antibodies (scFv), chimeric antibodies and de-immunized or humanized antibodies (Ed Harlow and David Lane, USING ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 1999);
  • Antibodies may also be generated using recombinant DNA technology, such as, for example, an antibody or fragment thereof expressed by a bacteriophage.
  • the synthetic antibody is generated by the synthesis of a DNA molecule encoding and expressing the antibody of the present disclosure or the synthesis of an amino acid sequence specifying the antibody, where the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
  • Methods for monoclonal antibody production may be carried out using the techniques described herein or are well-known in the art (MONOCLONAL ANTIBODIES - PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS (Mary A. Ritter and Heather M. Ladyman eds., 1995), which is hereby incorporated by reference in its entirety).
  • the process involves obtaining immune cells (lymphocytes) from the spleen of a mammal which has been previously immunized with the antigen of interest either in vivo or in vitro.
  • monoclonal antibodies can be made using recombinant DNA methods as described in U.S. Patent No. 4,816,567 to Cabilly et al, which is hereby incorporated by reference in its entirety.
  • the polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cells, for example, by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody.
  • the isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E.
  • coli cells simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein
  • monoclonal antibodies are generated by the host cells.
  • recombinant monoclonal antibodies or fragments thereof of the desired species can be isolated from phage display libraries (McCafferty et al., “Phage Antibodies: Filamentous Phage Displaying Antibody Variable Domains,” Nature 348:552-554 (1990); Clackson et al., “Making Antibody Fragments using Phage Display Libraries,” Nature 352:624-628 (1991); and Marks et al., “By-Passing Immunization. Human Antibodies from V-Gene Libraries Displayed on Phage,” J. Mol. Biol. 222:581-597 (1991), all of which are hereby incorporated by reference in their entirety).
  • the polynucleotide(s) encoding a monoclonal antibody can further be modified using recombinant DNA technology to generate alternative antibodies or derivatives.
  • the constant domains of the light and heavy chains of a mouse monoclonal antibody can be substituted by those regions derived from a human antibody to generate a chimeric antibody.
  • the constant domains of the light and/or heavy chains of a monoclonal antibody can be substituted by a non-immunoglobulin polypeptide to generate a fusion antibody.
  • the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody.
  • site-directed or high-density mutagenesis of the variable region can be used to optimize specificity and affinity of a monoclonal antibody.
  • the monoclonal antibody of the present disclosure can be a humanized antibody.
  • Humanized antibodies are antibodies that contain minimal sequences from non-human (e.g., murine) antibodies within the variable regions. Such antibodies are used therapeutically to reduce antigenicity and human anti-mouse antibody responses when administered to a human subject.
  • humanized antibodies are typically human antibodies with minimal to no non-human sequences.
  • a human antibody is an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human.
  • binding portions include the monovalent Fab fragments, Fv fragments (e.g., single-chain antibody, scFv), and single variable VH and VL domains, and F(ab’)2 fragments, Bis-scFv, diabodies, triabodies, minibodies, etc.
  • antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in James Goding, MONOCLONAL ANTIBODIES PRINCIPLES AND PRACTICE 98-118 (Academic Press, 1983) and Ed Harlow and David Lane, ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Laboratory, 1988), both of which are hereby incorporated by reference in their entirety, or other methods known in the art.
  • Antibody mimics are also suitable for use in accordance with the present disclosure.
  • a number of antibody mimics are known in the art including, without limitation, those known as monobodies, which are derived from the tenth human fibronectin type III domain ( 10 Fn3) (Koide et al., “The Fibronectin Type III Domain as a Scaffold for Novel Binding Proteins,” J. Mol. Biol. 284: 1141-1151 (1998); Koide et al., “Probing Protein Conformational Changes in Living Cells by Using Designer Binding Proteins: Application to the Estrogen Receptor,” Proc. Natl. Acad. Sci.
  • the targeting molecule is a small molecule against molecular targets on endothelial tissue and, for example, specifically bind markers associated with CAVD.
  • small molecules that bind cad-11 are used.
  • An exemplary small molecule against cad-11 includes SD-133.
  • the targeting molecule is a peptide, protein fragment, truncated peptide, or partial fragment of a biomarker of CAVD, for example cad-11.
  • the nanoparticle comprises more than one type of targeting molecule.
  • the nanoparticle core comprises one or more types of therapeutic molecules.
  • the terms “linked”, “joined”, “grafted”, “tethered”, “associated”, and “conjugated” in the context of the delivery vehicles disclosed herein, are used interchangeably to refer to any method known in the art for functionally connecting targeting molecules, including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, and electrostatic bonding.
  • a “therapeutic molecule” is an agent, or combination of agents, that treats a cell, tissue, or subject having a condition requiring therapy, when contacted with the cell, tissue or subject.
  • the therapeutic molecule is an NFKB inhibitor.
  • NFKB is a transcription factor that plays an important role in many cellular processes.
  • Vertebrate NFKB transcription complexes can be any of a variety of homo- and heterodimers formed by the subunits p50, p52, c-Rel, RelA (p65), and RelB.
  • IKB IKB
  • IKK IKB kinase
  • the IKK complex contains two kinase subunits, IKKa and IKK/?, and an associated scaffold-like regulatory protein called NEMO (IKKy).
  • IKKy an associated scaffold-like regulatory protein called NEMO
  • TNFa tumor necrosis factor a
  • IL-1 interleukin-1
  • the IKK complex is activated in part by phosphorylation of specific serine residues in the activation loop of each IKK subunit.
  • the activated IKK complex can then phosphorylate IKB on two serine residues in human IKB.
  • IKK Phosphorylation of the IKB by IKK signals it for ubiquitination at specific lysine residues by the SCF-/?-TrCP E3 ubiquitin ligase complex, which targets the IKB for degradation by the 26S proteasome
  • the freed NFKB may then translocate into the nucleus, and along with other transcription factors, activate transcription of target genes (see e.g., Karin et al., “The IKK NFkB System: a Treasure Trove for Drug Development,” Nat. Rev. 3 : 17-26 (2004); Gilmore et al., “Inhibitors of NF-kB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006), both of which are hereby incorporated by reference in their entirety).
  • NFKB activation refers to a state of the NFKB molecule that is capable of participating in transcription activation.
  • Inhibitors of NFKB activation generally refer to an agent that either partially or completely blocks NFKB participation in the activation of many its target genes.
  • a large number of NFKB target genes have been reported in the literature. The mRNAs of these target genes are normally present at low levels and their levels increase dramatically when NFKB and other transcription factors bind to regulatory elements of these genes and activate their transcription.
  • NFKB inhibitors may inhibit NFKB at any step in the NFKB activation process.
  • the inhibitor may be a general inhibitor of NFKB activation, or the inhibitor may inhibit specific pathways of induction. Inhibitors that target multiple steps in the NF/cB pathway are also contemplated for use herein.
  • inhibition of NF-KB activation can occur by three mechanisms: (1) blockage of the incoming activation signal at an early stage (e.g., binding of ligand to its receptor) resulting in complete abrogation of the signal's effect; (2) interference with a cytoplasmic step in the NFKB activation pathway by blockage of a specific component of the cascade (e.g., the activation of the IKK complex or degradation of IKB); or (3) blockage of NFKB nuclear activity, that is, inhibiting its translocation to the nucleus, its binding to DNA, a nuclear modification of NF-KB that affects its activity or specificity (e.g. protein acetylation and/or methylation), or an interaction of NFKB on DNA with specific or basal transcription machinery.
  • blockage of the incoming activation signal at an early stage e.g., binding of ligand to its receptor
  • interference with a cytoplasmic step in the NFKB activation pathway by blockage of a specific component of the cascade e.g.
  • the NFKB inhibitor may be, without limitation, an upstream NFKB target inhibitor, an IKK phosphorylation inhibitor, an IKB phosphorylation inhibitor, an IKB degradation inhibitor, a proteasome inhibitor, a protease inhibitor, an IKB upregulation inhibitor, and NFKB nuclear translocation inhibitor, an NFKB expression inhibitor, and an NFKB transactivation inhibitor.
  • the therapeutic molecule is an inhibitor of the NFKB signaling pathway and is selected from the group consisting of a protein kinase inhibitor, a protein phosphatase inhibitor, an inhibitor of protein acetylation, a protein methyltransferase inhibitor, a proteasome inhibitor, an inhibitor of protein ubiquitination, an NFKB nuclear translocation inhibitor, an inhibitor of NFKB DNA binding activity.
  • NFKB neuropeptide-binding protein
  • Specific inhibitors of NFKB include, without limitation, those identified in Gilmore et al., “Inhibitors of NF-kB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006), which is hereby incorporated by reference in its entirety.
  • the inhibitor may be, without limitation, a small molecule, a peptide, a nucleic acid, an antioxidant, a microbial protein, a viral protein, or an anti-inflammatory agent.
  • Small molecule NFKB inhibitors are well known in the art and are described in, for example, Gilmore et al., “Inhibitors of NF-KB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006) and Ramadass et al., “Small Molecule Nf-KB Pathway Inhibitors in Clinic,” Int. J. Mol. Sci. 21 (14): 5164 (2020), both of which are hereby incorporated by reference in their entirety.
  • small molecules are typically organic, peptide or non-peptide molecules, having a molecular weight less than 10,000 Da, less than 5,000 Da, less than 1,000 Da, and less than 500 Da.
  • This class of modulators includes chemically synthesized molecules, for instance, compounds from combinatorial chemical libraries.
  • Peptides are also contemplated for use as NFKB inhibitors.
  • cell- permeable peptides such as SN-50, that contain nuclear localization sequences of NFKB can saturate the process involved in the uptake of NFKB into the nucleus (Gilmore et al., “Inhibitors of NF-kB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006), which is hereby incorporated by reference in its entirety).
  • Peptides can be obtained by known isolation and purification protocols from natural sources, can be synthesized by standard solid or solution phase peptide synthesis methods according to the known peptide sequence of the peptide, or can be obtained from commercially available preparations or peptide libraries. Included herein are peptides that exhibit the biological binding properties of the native peptide and retain the specific binding characteristics of the native peptide. Derivatives and analogs of the peptide, as used herein, include modifications in the composition, identity, and derivatization of the individual amino acids of the peptide provided that the peptide retains the specific binding properties of the native peptide.
  • modifications would include modification of any of the amino acids to include the D-stereoisomer, substitution in the aromatic side chain of an aromatic amino acid, derivatization of the amino or carboxyl groups in the side chains of an amino acid containing such a group in a side chain, substitutions in the amino or carboxy terminus of the peptide, linkage of the peptide to a second peptide or biologically active moiety, and cyclization of the peptide (G. Van Binst and D. Tourwe, “Backbone Modifications in Somatostatin Analogues: Relation Between Conformation and Activity,” Peptide Research 5:8-13 (1992), which is hereby incorporated by reference in its entirety).
  • Nucleic Acids may also be used to inhibit NFKB activity.
  • specific NFKB DNA binding can be blocked by the use of decoy oligonucleotides that have NFKB binding sites. These decoy oligonucleotides function by competing with NFKB binding to specific gene promoters (Gilmore et al., “Inhibitors of NF-kB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006), which is hereby incorporated by reference in its entirety).
  • Antioxidants are also contemplated for use in the methods described herein.
  • antioxidants are known in the art to inhibit NFKB and are described in Gilmore et al., “Inhibitors of NF-kB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006), which is hereby incorporated by reference in its entirety.
  • Viruses can produce certain proteins that have mechanisms to inhibit NFKB signaling, including, without limitation, the encoding of IxB-like inhibitors of NFKB, the cleaving of p65, and the targeting of IKK (Powell et al., J Virol 70:8527-8533 (1996); Camus- Bouclainville et al., J Virol 78:2510-2516 (2004); Thoetkiattikul et al., Proc Natl Acad Sci USA 102: 11426-11431 (2005), all of which are hereby incorporated by reference in their entirety). Therefore, use of such viral proteins is also contemplated. [0100] Microbial proteins also produce proteins capable of inhibiting NFKB.
  • the YopJ protein encoded by the enteropathogen Yersinia pseudotuberculosis inhibits NFKB activation by deubiquitinating I/cB, which prevents its degradation (Gilmore et al., “Inhibitors of NF-kB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006), which is hereby incorporated by reference in its entirety).
  • Anti-inflammatory agents are also contemplated for use as NF-KB inhibitors.
  • Anti-inflammatory agents that inhibit NF-KB are well known in the art.
  • nonsteroidal anti-inflammatory drugs such as aspirin, ibuprofen, sulindac, indomethacin are known in the art to inhibit NF-KB activation (Gilmore et al., “Inhibitors of NF-kB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006), which is hereby incorporated by reference in its entirety).
  • Glucocorticoids such as dexamethasone, prednisone and methylprednisolone are also known to inhibit NF-KB (Gilmore et al., “Inhibitors of NF-kB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006), which is hereby incorporated by reference in its entirety).
  • the therapeutic molecules may comprise an antiinflammatory drug.
  • the anti-inflammatory drug comprises BAY 11-7082.
  • the inhibitor of the NF-KB signaling pathway is selected from the group consisting of ATP analogs, BMS-34554, parthenolide, arsenite, epoxyquinoids, gene-based inhibitors, SB203580, denbinobin, tyrosine kinase inhibitors, rhein, TNAP, betaine, epoxyquinol B, M2L, CCK-8, KSR2, golli BG21, BAY11-7082, protein phosphatase 2A, cytosine arabinoside, OspF, gallic acid, Daxx, anacardic acid, Set9 inhibitor, bortezomib, ALLnL, LLM, Z-LLnV, Z-LLL, lactacystine, N-cbz-Leu-Leu-leucinal (MG132), MG115, ubiquitin ligase inhibitors, salinosporamide A (NPI-0052), DCIC, TPCK, TL
  • the inhibitor of the NF-KB signaling pathway is BAY11- 7082.
  • NF-KB inhibitors include but are not limited to Rac-1 inhibitors and RhoA inhibitors (Vaidya et al., “Rael Mediates Cadherin-11 Induced Cellular Pathogenic Processes in Aortic Valve Calcification,” Cardiovasc. Pathol. 58: 107414 (2022); Farrar et al. “Valve Interstitial Cell Tensional Homeostasis Directs Calcification and Extracellular Matrix Remodeling Processes Via RhoA Signaling.” Biomalerials 105:25-37 (2016), both of which are hereby incorporated by reference in their entirety).
  • the nanoparticle of the present disclosure may comprise an imaging component. Suitable imaging components include, without limitation, a diagnostic dye.
  • a “diagnostic molecule” is an agent utilized to detect and diagnose CAVD in vivo. This is achieved by encapsulating the diagnostic molecule within the delivery vehicle component, administering the delivery vehicle component, and then imaging the subject.
  • imaging can include any one or more of: planar radionuclide imaging, positron emission tomography (PET), echo-planar imaging (EPI), single photon emission computed tomography (SPECT), sonographic imaging (e.g., radiation-free, contrastspecific, high frequency, two-dimensional), magnetic resonance imaging (MRI, also referred to as magnetic resonance tomography or MRT), X-ray, computed tomographic (CT) scans, fluorescence imaging, near-infrared imaging and other medically useful or adaptable imaging techniques.
  • PET positron emission tomography
  • EPI echo-planar imaging
  • SPECT single photon emission computed tomography
  • sonographic imaging e.g., radiation-free, contrastspecific, high frequency, two-dimensional
  • MRI magnetic resonance imaging
  • MRT magnetic resonance tomography
  • CT computed tomographic
  • a “pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutically acceptable 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.
  • Examples of pharmaceutically acceptable carriers include water, e.g., buffered with phosphate, citrate and another organic acid.
  • antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counterions such as sodium; and/or nonionic surfactants.
  • antioxidants such as ascorbic acid
  • proteins such as serum albumin, gelatin, or immunoglobulins
  • hydrophilic polymers such as polyvinylpyrrolidone
  • amino acids such as glycine, glutamine, asparagine, arginine or lysine
  • aqueous solution saline solutions and aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions.
  • the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant.
  • Suitable pharmaceutically acceptable carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin.
  • the terms “pharmaceutically acceptable carrier” e.g., additives such as diluents, immunostimulants, adjuvants, antioxidants, preservatives, and solubilizing agents
  • pharmaceutically acceptable carrier e.g., additives such as diluents, immunostimulants, adjuvants, antioxidants, preservatives, and solubilizing agents
  • compositions comprising the delivery vehicle described herein may comprise buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline, and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannito
  • adjuvants include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and aluminum oxide, including nanoparticles comprising alum or nanoalum formulations), calcium phosphate (e.g., Masson JD et al, Expert Rev Vaccines 16: 289-299 (2017), which is hereby incorporated by reference in its entirety), monophosphoryl lipid A (MPL) or 3-de-O-acylated monophosphoryl lipid A (3D-MPL) (see e.g., United Kingdom Patent GB2220211, EP0971739, EPl 194166, US6491919, all of which are hereby incorporated by reference in their entirety), AS01, AS02, AS03 and AS04 (see e.g.
  • alum such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and aluminum oxide, including nanoparticles comprising alum or nanoalum formulations
  • calcium phosphate
  • the adjuvant comprises Quil-A, such as for instance commercially obtainable from Brenntag (now Croda) or Invivogen.
  • QuilA contains the water-extractable fraction of saponins from the Quillaja saponaria Molina tree. These saponins belong to the group of triterpenoid saponins, that have a common triterpenoid backbone structure. Saponins are known to induce a strong adjuvant response to T-dependent as well as T-independent antigens, as well as strong cytotoxic CD8+ lymphocyte responses and potentiating the response to mucosal antigens.
  • the adjuvant is AS01, for example AS01B.
  • AS01 is an adjuvant system containing MPL (3-O-desacyl-4'-monophosphoryl lipid A), QS21 (Quillaja saponaria Molina, fraction 21), and liposomes.
  • the AS01 is commercially available or can be made as described in WO 96/33739, which is hereby incorporated by reference in its entirety.
  • Certain adjuvants comprise emulsions, which are mixtures of two immiscible fluids, e.g. oil and water, one of which is suspended as small drops inside the other and are stabilized by surface-active agents.
  • Oil-in-water emulsions have water forming the continuous phase, surrounding small droplets of oil, while water-in-oil emulsions have oil forming the continuous phase.
  • Certain oil-in-water emulsions comprise squalene (a metabolizable oil).
  • Certain adjuvants comprise block copolymers, which are copolymers formed when two monomers cluster together and form blocks of repeating units.
  • An example of a water in oil emulsion comprising a block copolymer, squalene and a microparticulate stabilizer is TiterMax®, which can be commercially obtained from Sigma-Aldrich.
  • composition according to the present disclosure may be administered to a subject.
  • another aspect of the present disclosure relates to a method of treating and/or diagnosing CAVD.
  • This method involves contacting a nanoparticle disclosed herein with endothelial tissue of a subject at risk for CAVD.
  • the term “treat” or “treating” refers to the application or administration of the nanoparticle of the invention to a subject, e.g., a patient.
  • the treatment can be to cure, heal, alleviate, relieve, alter, remedy, ameliorate, palliate, improve or affect CAVD or the symptoms of CAVD.
  • the ability to detect or diagnose CAVD may include determining whether the patient is in an earlier stage of CAVD, or has developed early, moderate, or severe forms of CAVD.
  • the method further comprises detecting a presence of the nanoparti cle(s) on the subject’s endothelial tissue.
  • the method further comprises determining a CAVD disease state based on presence of the nanoparti cle(s) on the subject’s endothelial tissue.
  • contacting may be carried out using methods known in the art including by administering parenterally, topically, intravenously, orally, subcutaneously, intraperitoneally, intranasally, or by intramuscular means.
  • the nanoparticles or pharmaceutical compositions according to the present disclosure are formulated for subcutaneous administration.
  • the nanoparticles or pharmaceutical compositions according to the present disclosure are formulated for intramuscular injection. In some embodiments, this type of injection is performed in the arm or leg muscles.
  • Intravenous injections as well as intraperitoneal injections, intraarterial, intracranial, or intradermal injections may also be effective in generating an immune response.
  • the contacting is carried out parenterally, topically, intravenously, orally, subcutaneously, intraperitoneally, intranasally, or by intramuscular means.
  • the delivery vehicle (nanoparticle) or pharmaceutical compositions according to the present disclosure are formulated for parenteral administration.
  • Solutions or suspensions of the nanoparticles or pharmaceutical compositions can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils.
  • Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil.
  • water, saline, aqueous dextrose and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • compositions suitable for injectable use include sterile aqueous solutions or dispersions and/or sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g, glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
  • the delivery vehicle (nanoparticle) or pharmaceutical compositions of the present disclosure may be formulated for parenteral administration by injection, e.g, by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.
  • Intraperitoneal or intrathecal administration of the delivery vehicle or pharmaceutical compositions of the present disclosure can also be achieved using infusion pump devices such as those described by Medtronic, Northridge, CA. Such devices allow continuous infusion of desired compounds avoiding multiple injections and multiple manipulations.
  • the delivery vehicle or pharmaceutical compositions of the present disclosure may also be formulated as a depot preparation.
  • Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the composition as described herein is a solid formulation, e.g., a freeze-dried or spray-dried composition, which can be used as is, or whereto the physician or the subject adds solvents, and/or diluents prior to use.
  • Solid dosage forms can include tablets, such as compressed tablets, and/or coated tablets, and capsules (e.g., hard or soft gelatin capsules).
  • the composition can also be in the form of sachets, dragees, powders, granules, lozenges, or powders for reconstitution, for example.
  • the methods of the present disclosure involve administering any one of the compositions described supra.
  • a suitable subject for treatment in accordance with these aspects of the present disclosure is a subject at risk of developing CAVD and/or a subject at risk of developing conditions associated with CAVD.
  • a prophylactically effective amount of the composition is administered to the subject to prevent or mitigate CAVD.
  • a prophylactically effective amount is the amount necessary to prevent or mitigate CAVD.
  • the target “subject” encompasses any animal, preferably a mammal, more preferably a human.
  • the target subject encompasses any subject that is at risk of developing CAVD. Any, adult, or elderly adult at risk for, or having, CAVD can be treated in accordance with the methods and compositions described herein.
  • Particularly suitable subjects include those at risk of developing CAVD.
  • Other suitable subjects include those subjects which may have or are at risk for developing a condition associated with or resulting from CAVD.
  • compositions may also be accounted for when administering the composition under conditions effective to induce a treatment and/or diagnosis for CAVD.
  • factors include, for example and without limitation, the concentration of the active agents in the composition, the mode and frequency of administration, and the subject details, such as age, weight and overall health and immune condition.
  • concentration of the active agents in the composition includes, for example and without limitation, the concentration of the active agents in the composition, the mode and frequency of administration, and the subject details, such as age, weight and overall health and immune condition.
  • a clinician may administer a composition as described herein until a dosage is reached that provides the desired or required prophylactic effect. The progress of the prophylactic response can be monitored by conventional assays.
  • the composition as described herein is administered prophylactically to prevent, delay, or inhibit the development of CAVD in a subject at risk of developing CAVD or at risk of developing an associated condition.
  • prophylactic administration of the composition is effective to fully prevent CAVD in an individual.
  • prophylactic administration is effective to prevent the full extent of a condition that would otherwise develop in the absence of such administration, z.e., substantially prevent or inhibit CAVD in an individual.
  • the dosage of the composition is one that is adequate to prevent onset of CAVD, and is capable of achieving a reduction in a number of symptoms, a decrease in the severity of at least one symptom, or a delay in the further progression of at least one symptom, or even a total alleviation of CAVD.
  • Prophylactically effective amounts of the compositions described herein will depend on whether an adjuvant is co-administered, with higher dosages being required in the absence of adjuvant.
  • the amount of the composition useful for administration can vary from 1 pg-500 pg per patient. In some embodiments, 5, 10, 20, 25, 50 or 100 pg is used for each human injection. Occasionally, a higher dose of 1-50 mg per injection is used. In some embodiments, about 10, 20, 30, 40 or 50 mg is used for each human injection.
  • the timing of injections can vary significantly from once a year to once a decade.
  • HAVECs and HUVECs were cultured in T75 flasks and then 8-well p-slides coated with 50 pg/mL of rat tail collagen I at 37 °C and 5% CO2.
  • HAVECs and HUVECs were cultured in Endothelial Growth Media-2 (Sigma- Aldrich) with 1% penicillin-streptomycin and 10% fetal bovine serum. HUVECs were used from passage four to five and HAVECs were used from passage three to four.
  • PAVICs and PAVECs were cultured in T75 flasks and then 8-well p- slides at 37 °C and 5% CO2.
  • PAVICs and PAVECs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) with 1% penicillin-streptomycin and 10% fetal bovine serum.
  • DMEM Modified Eagle Medium
  • the PAVEC flasks were coated with 50 pg/mL of rat tail collagen I.
  • PAVICs were used from passage four to six and PAVECs were used from passage three to five. Cells were passaged from a T75 into 8-well p-slides at a seeding density of 5 * 10 4 cells/well.
  • Circular springs were placed in molded wells made of polydimethylsiloxane (PDMS).
  • PAVICs were encapsulated within a neutralized collagen gel suspension (2 mg/mL) in the PDMS wells at a density of 1,000,000 cells/mL.
  • a neutralized collagen gel suspension was made with rat tail collagen I, sterile 18 MQ water, fetal bovine serum, 5 x DMEM, and 0.1 M HC1.
  • PAVECs were topically seeded onto the PAVICs at a density of 50,000 cells/cm 2 .
  • Hydrogels were incubated in DMEM with 1% penicillin-streptomycin and 10% fetal bovine serum for 24 hours at 37 °C and 5% CO2 before osteogenic media (OGM) was applied.
  • OGM osteogenic media
  • DMEM was supplemented with 1% penicillin-streptomycin, 10% fetal bovine serum, lOmmol/L P-glycerophosphate, 50 pg/mL L-ascorbic acid, and 100 nM dexamethasone.
  • the hydrogels were cultured for up to seven days.
  • PLGA nanoparticles containing either coumarin 6 (C6) dye, BAY11-7082, or Cy5.5 dye were made according to the methods outlined in (Menon et al., “Dual -Drug Containing Core-Shell Nanoparticles for Lung Cancer Therapy,” Scientific Reports 7: 13249 (2017); Yaman et al., “Melanoma Peptide MHC Specific TCR Expressing T-Cell Membrane Camouflaged PLGA Nanoparticles for Treatment of Melanoma Skin Cancer,” Frontiers in Bioengineering and Biotechnology 8:943 (2020); Menon et al., “Polymeric Nanoparticles for Pulmonary Protein and DNA Delivery,” Acta Biomaterialia 10: 2643-2652 (2014), all of which are hereby incorporated by reference in their entirety) Synthesis of dye-loaded or drug-loaded PLGA nanoparticles requires dissolving PLGA 50:50 and dye or BAY11-7082 in various solvents, like
  • PLGA nanoparticles After isolating the PLGA nanoparticles, they are freeze-dried for 24 hours (Menon et al., “Polymeric Nanoparticles for Pulmonary Protein and DNA Delivery,” Acta Biomaterialia 10: 2643-2652 (2014), which is hereby incorporated by reference in its entirety).
  • 3 mg of nanoparticles were dissolved in 500 pL of pH 8.2 NaHCCL and incubated at room temperature for two hours with 10 pg of cad-11 antibody (Invitrogen). 500 pL of Tris-NH2 was subsequently added to the nanoparticle suspension and the nanoparticles were centrifuged at 15,000 rpm for 30 min.
  • the supernatant was discarded and the nanoparticle pellet was resuspended in 1 mL of pH 7.4 IX PBS.
  • the nanoparticles were centrifuged again at 15,000 rpm for 30 min.
  • the supernatant was discarded and the cad-11 nanoparticles were resuspended in the desired amount of IX PBS or cell media.
  • the cad-11 antibody was conjugated to the nanoparticles 24 hours before the first use of the nanoparticles.
  • the cad-11 nanoparticles were stored at -20°C for up to two weeks.
  • the doses of BAY11-7082 released by the cad-11 BAY nanoparticles was calculated using the release curve provided in FIG. 3B.
  • 3D PAVEC-PAVIC collagen hydrogels were fixed in methanol for 30 minutes and the hydrogels were rinsed three times for 15 minutes in IX PBS with calcium and magnesium. The hydrogels were then stained with 2% Alizarin Red Stain (ARS) for 30 minutes. The ARS was aspirated and the hydrogels were rinsed three times for 15 minutes in 18 M water. Each hydrogel was imaged using a Zeiss Discovery V.20 Stereoscope. ImageJ was used to identify calcific nodules and to measure the compaction of the area of the hydrogels.
  • ARS Alizarin Red Stain
  • Paraffin-embedded 3D PAVEC-PAVIC hydrogels were sectioned (10pm thick). The sections were deparaffinized by performing three, three-minute washes in D-limonene. The sections were subsequently rehydrated by placing them in 100% ethanol, followed by 95% ethanol, 70% ethanol, then tap water for three minutes each. Sections were stained in Harris’ Alum Hematoxylin (Sigma- Aldrich) for ten minutes and afterward, washed in tap water for three minutes.
  • the sections were placed in 1% acid alcohol for 20 seconds followed by a 90-second wash in tap water, a minute wash in 0.2% ammonia water, a 90-second wash in tap water, a minute wash in 70% ethanol, and a minute wash in 1% alcoholic Eosin Y (Sigma-Aldrich). The sections were then placed in 95% ethanol for two minutes, 100% ethanol for two minutes, and D-limonene for three minutes. One drop of Permount Mounting Medium (Invitrogen) was placed onto the stained sections and the slides were covered with a coverslip. The slides were imaged using a Zeiss Discovery V.20 Stereoscope.
  • ApoE mice Jackson Laboratory- stock no. 002052
  • C57BL/6J-congenic Apoe tmlUnc mice are homozygous, mutant knockouts (002052 - B6.129P2- Apoe J, which is hereby incorporated by reference in its entirety).
  • ApoE -/mice were fed a high- fat diet (Harlan Teklad No.
  • TD88137 consisting of 42% of calories from fat and 0.25% cholesterol, to drive atherosclerotic plaque formation
  • Sasso et al. “The Apoe-/- Mouse Model: a Suitable Model to Study Cardiovascular and Respiratory Diseases in the Context of Cigarette Smoke Exposure and Harm Reduction,” Journal of Translational Medicine 14: 146 (2016); (Meir and Leitersdorf, “Atherosclerosis in the Apolipoprotein E-Deficient Mouse: a Decade of Progress,” Arteriosclerosis, Thrombosis, and Vascular Biology 24: 1006-1014 (2004), all of which are hereby incorporated by reference in their entirety). Genotyping
  • a tissue sample was collected from the ear of each ApoE -/- mouse. To isolate the DNA, the ear tissue samples were submerged in 50 pL of Extracta DNA Prep reagent (QuantaBio) and the samples were incubated at 90°C for 30 minutes. 50 pL of the stabilizing reagent (QuantaBio) was then applied to each sample. GoTaq Green Master Mix (Promega), nuclease-free water, a forward and reverse primer, and the DNA sample were combined. The samples underwent PCR according to the settings outlined by The Jackson Laboratory in their ApoE -/- genotyping protocol (Protocol 22364-Apoe, which is hereby incorporated by reference in its entirety). The samples were ran through a 1% agarose gel with SYBR Safe DNA Gel Stain (Invitrogen). The gel was imaged using the Bio-Rad ChemiDoc Imaging System.
  • Paraffin-embedded 3D PAVEC-PAVIC hydrogels were sectioned (10pm thick). The sections were deparaffinized by performing three, three-minute washes in D-limonene. The sections were then rehydrated by placing them in 100% ethanol, followed by 95% ethanol, 70% ethanol, then deionized water for three minutes each. The rehydrated sections were placed in a Coplin with boiling IX citrate buffer solution and heated in the Coplin on high for two minutes in a pressure cooker. The Coplin was cooled to room temperature and the samples were permeabilized with 0.3% Triton-X for 15 minutes.
  • the slides were washed three times for 10 minutes in PBST (IX PBS with 0.05% Tween 20) and the samples were blocked in IX PBS with 1% bovine serum albumin (BSA), 0.3 M glycine, and 5% goat serum. After one hour of incubation at room temperature and three, 10-minute washes in PBST, the samples were incubated overnight at 4°C with a primary antibody diluted in PBST, 1% BSA, and 0.3 M glycine. The samples were washed three times for 15 minutes in PBST.
  • the secondary antibodies were species-specific, raised in goat, and conjugated to an Alexa Fluor dye.
  • the secondary antibody was added to the sample in a 1 :500 (4 pg/mL) dilution and the samples were incubated for one hour at room temperature. Finally, the samples were washed three times for 15 minutes in PBST and counterstained using Nuclear Blue (Invitrogen). ProLong Gold Antifade Mountant (Invitrogen) was placed onto each slide with a coverslip. I imaged the slides using a Zeiss Confocal 710.
  • mice were anesthetized by setting the isoflurane vaporizer at 3.5%. Following, 5.6 mg of cad-11 Cy5.5 nanoparticles were retro-orbitally injected into the mouse. Two hours and four hours after injection, the mouse was imaged using the IVIS Spectrum. To image the mouse using the IVIS Spectrum, the mouse was anesthetized by setting the isoflurane vaporizer at 3.5%, eye ointment was applied to the mouse, and then the mouse was quickly transferred to the induction chamber within the IVIS Spectrum. Once the mouse was in the induction chamber, the isoflurane vaporizer was reduced to 1.5% and an induction flow of 500 cc/min was used. Each IVIS Spectrum image was taken using the fluorescence and epi-illumination settings. Following, the mouse was euthanized using CO2 and the heart and liver were dissected. All Georgia IACUC and CARE guidelines for animal handling and procedures were adhered to.
  • the cad-11 BAY nanoparticle release profile (FIG. 3B) was used to determine the amount of cad-11 BAY nanoparticles needed to achieve a particular dose of BAY 11-7082 and the incubation time of the cad-11 BAY nanoparticles with the hydrogels.
  • the cad-11 BAY nanoparticles released 82 pg of BAY 11-7082 per 1 mg of nanoparticles.
  • the majority of the drug was released by the 48-hour time point.
  • the hydrogels in OGM tear off the springs around day eight, with calcific nodule formation observed around day four or five. Therefore, applying the cad-11 BAY nanoparticles on day five for 48 hours until day seven maximized the amount of drug released from the nanoparticles while ensuring the majority of the hydrogels had not tom off the springs and that visible calcification had occurred.
  • cad-11 antibody-conjugated PLGA nanoparticles loaded with coumarin 6 dye (cad-11 C6 nanoparticles) was assessed (which are shown in FIG. 2A).
  • FIG. 4 A methodological overview of this objective is pictured in FIG. 4. Since cad-11 is endogenously expressed at higher levels in the aortic valve endothelium compared to other endothelial populations, it was hypothesized that the cad-11 C6 nanoparticles would target aortic valve endothelial cells more specifically than endothelial cells originating from other tissue.
  • cad-11 nanoparticles The targeting specificity of cad-11 nanoparticles was assessed by administering 50 pg/mL of cad-11 C6 nanoparticles suspended in IX PBS to HAVECs, HUVECs, PAVICs, and PAVECs. After 3.5 hours of incubation with the cells, each well was washed with IX PBS before imaging. This incubation time was selected based on the release kinetics of the nanoparticles, determined in FIG. 3B. All cell types displayed fluorescence on imaging. To analyze each image, the cell counting function in ImageJ was used to count the total number of cells in each image. This first required enhancing the contrast of the image significantly so that the border of all objects in the image was visible and could be detected by the cell counting function.
  • the threshold function in ImageJ was used to sort the pixels in each image into either the background or the foreground of the image.
  • the cells in each image that displayed enough fluorescence to be sorted into the foreground were counted.
  • the proportion of cells above the fluorescence threshold was calculated by dividing the number of cells above the fluorescence threshold by the total number of cells in that image.
  • There was no significant difference among the proportion of HAVECs, PAVICs, and PAVECs above the fluorescence threshold which indicates no significant difference in cad- 11 C6 nanoparticle targeting among these cell types.
  • FIGS. 5A-5E illustrates that there was a greater proportion of HAVECs exhibiting more intense fluorescence than HUVECs. This means that the HAVECs were targeted more by the cad- 11 C6 nanoparticles than the HUVECs, as hypothesized.
  • cad- 11 expression is not restricted to the aortic valve. However, this could raise concerns about off- target effects in vivo. While this is a valid worry, this experiment illustrates that cad-11 nanoparticles target undesired cell types significantly less than the desired cell type.
  • cad-11 C6 nanoparticles were administered to PAVECs and PAVICs to validate that PAVECs and PAVICs would be sufficiently targeted by cad-11 nanoparticles.
  • Porcine cells were to be used in subsequent 3D cell culture models since this cell type has previously been shown to be a robust model for studying calcification in vitro (Gee et al., “NFKB (Nuclear Factor K-Light-Chain Enhancer of Activated B Cells) Activity Regulates Cell-Type-Specific and Context-Specific Susceptibility to Calcification in the Aortic Valve,” Arterioscler. Thromb. Vase. Biol.
  • FIG. 6 shows a schematic of this objective. It was hypothesized that diseased 3D PAVEC-PAVIC collagen hydrogels treated with cad-11 BAY nanoparticles would exhibit less calcification than untreated, diseased hydrogels.
  • the PAVECs and PAVICs within the untreated hydrogels were expected to display inflammatory phenotypes indicative of EndMT and changes in the levels of primary cell type markers like VE-cadherin and a-smooth muscle actin (a- SMA).
  • 3D PAVEC-PAVIC collagen hydrogels were cultured for seven days in either GM, OGM, or OGM for DI -3 and then 0GM+ BAY 11-7082 for D3-7 (OGM+BAY) (FIG. 7 A).
  • hydrogels were stained with ARS to evaluate calcific nodule formation.
  • a marked visible reduction in calcific nodule number was observed in the OGM+BAY treated group compared to the OGM group. This shows the efficacy and rescue effect of BAY 11-7082.
  • 3D PAVEC-PAVIC collagen hydrogels were cultured for seven days in either general GM or OGM. After treatment with OGM for five days, the hydrogels displayed calcific nodule formation. At day 5, an interventional lOpM dose of BAY11-7082 was applied to the hydrogels via cad-11 BAY nanoparticles. The cad-11 BAY nanoparticles were incubated with the hydrogels for 48 hours. These cad-11 BAY nanoparticle-treated hydrogels are referred to as the OGMB group. On day seven, the hydrogels were stained with ARS (FIG. 7B). The small, dark spots homogeneously spread throughout the magnified image of the GM hydrogel are likely endothelial cells.
  • ARS FIG. 7B
  • the large, heterogenous regions of darker staining are calcific nodules in the OGM and OGMB groups.
  • the amount of calcium in each hydrogel was quantified using a plate reader assay. There was not a significant reduction in calcium concentration between the OGM and OGMB groups.
  • FIG. 8A The hydrogels treated with 0 pM, 5 pM, 10 pM, and 20 pM of BAY1 1-7082 all displayed cellular compaction (FIG. 8A).
  • the compaction proportion in FIG. 8B is the ratio between the circumference of the hydrogel on day seven and the circumference of the hydrogel on day one.
  • the positive control was the 0 pM group and the negative control was the GM group.
  • the positive control showed significantly more compaction than the negative control.
  • Tissue compaction is an essential morphogenetic process characterized by mechanical interactions among cells that cause shrinkage of the tissue (Turlier and Maitre, “Mechanics of Tissue Compaction,” Seminars in Cell & Developmental Biology 47- 48: 110-117 (2015), which is hereby incorporated by reference in its entirety).
  • PAVEC-PAVIC hydrogels treated with OGM have been shown to compact significantly (Gee et al., “Valve Endothelial-Interstitial Interactions Drive Emergent Complex Calcific Lesion Formation In Vitro,” Biomaterials 269: 120669 (2021), which is hereby incorporated by reference in its entirety).
  • the amount of cr-SMA expression was expected to decrease as the dose of BAY11- 7082 increased since fewer PAVECs would be transitioning to a mesenchymal phenotype. If the integrity of the hydrogels had been preserved, it was expected to see more distinct regions of a- SMA and VE-cadherin expression to mirror where the PAVECs and PAVICs were seeded in each hydrogel.
  • Area compaction is a measurement of cellular tissue remodeling. Greater collagen and cellular remodeling occurs to a greater degree under osteogenic disease conditions.
  • BAY 11- 7082 shows promising effects with significantly decreased tissue compaction when BAY 11- 7082 is given from Day 1 vs at CAVD, showing similar results to the General media, the healthy control group (FIG. 13 A).
  • the number of calcific nodules can be significantly reduced and compared to healthy conditions when treatment is given Day 1.
  • Nodule number in the OGM + Bay 11 given at CAVD diagnosis shows a decreased mean number of nodules compared to OGM conditions. For FIG.
  • images were taken after 7 days of incubation of co-culture PAVEC/PAVIC spring gels.
  • Row one shows general media healthy control.
  • Row two shows osteogenic media conditions given Day 1.
  • Row 3 shows OGM + Bayl 1 given at Day 1 - shows no presence of CAVD calcific nodules in the matrix.
  • Row 4 shows OGM + Bay 11 given at the diagnosis of CAVD calcific nodule presentation.
  • the efficacy of cad-11 BAY nanoparticles could be evaluated in vivo using atherosclerosis-prone apolipoprotein E-deficient (ApoE -/-) mouse models.
  • ApoE mice fed a high-fat diet develop hypercholesterolemia which drives atherosclerotic plaque formation by 32 weeks (Sasso et al., “The Apoe-/- Mouse Model: a Suitable Model to Study Cardiovascular and Respiratory Diseases in the Context of Cigarette Smoke Exposure and Harm Reduction,” Journal of Translational Medicine 14: 146 (2016), which is hereby incorporated by reference in its entirety).
  • ApoE mice on a high-fat diet show increased total plasma cholesterol levels and moderately increased triglyceride levels, providing a systemic proinflammatory environment that is ideal for simulating CAVD (Sasso et al., “The Apoe-/- Mouse Model: a Suitable Model to Study Cardiovascular and Respiratory Diseases in the Context of Cigarette Smoke Exposure and Harm Reduction,” Journal of Translational Medicine 14: 146 (2016); Meir and Leitersdorf, “Atherosclerosis in the Apolipoprotein E-Deficient Mouse: a Decade of Progress, " Arteriosclerosis, Thrombosis, and Vascular Biology 24: 1006-1014 (2004); 002052 - B6.129P2-Apoe/J, all of which are hereby incorporated by reference in their entirety).
  • FIG. 12 illustrates a methodological overview of a biodistribution study to determine whether cad-11 antibody-conjugated PLGA nanoparticles loaded with Cy5.5 (cad-11 Cy5.5 nanoparticles) hone to the aortic valve in vivo.
  • BAY 11-7082 Since cad-11 nanoparticles showed cellspecific targeting in vitro, a lower dose of BAY 11-7082 is expected to be more potent than BAY 11-7082 given systemically.
  • cardiac ultrasounds will be performed on Axe ApoE mice to obtain left ventricle ejection velocity readings before and after treatment. The hearts will then be disected and stained for protein markers to evaluate the extent of EndMT progression, valve calcification, and cellular toxicity.
  • the corresponding dose would be administered to a larger cohort of ApoE mice that are at least 32 weeks old, on a high-fat diet, and show signs of elevated left ventricle outflow blood ejection velocity on ultrasounds. The resulting posttreatment heart health of these mice would be assessed.
  • These in vivo studies will offer greater insight into the potential therapeutic uses for cad-11 nanoparticles. Moreover, these in vivo studies will likely raise many questions about the off-target effects of the nanoparticles, how to scale down the dose of BAY11-7082 when it is delivered via cad-11 nanoparticles, and other potential limitations of this novel therapy.

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Abstract

The present disclosure relates to a nanoparticle for treating and/or diagnosing calcific aortic valve disease (CAVD). The nanoparticle comprises a nanoparticle core and a targeting molecule associated with an exterior surface of the nanoparticle core. Also disclosed is a therapeutic composition comprising the nanoparticle disclosed herein and a method of treating and/or diagnosing CAVD.

Description

METHODS AND COMPOSITIONS FOR TREATING AORTIC VALVE DISEASE
[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63/455,156, filed March 28, 2023, which is hereby incorporated by reference in its entirety.
[0002] This invention was made with government support under R01 HL143247 awarded by National Institutes of Health. The government has certain rights in the invention.
FIELD
[0003] The present disclosure relates to a nanoparticle for treating and/or diagnosing calcific aortic valve disease (“CAVD”), a therapeutic composition comprising the nanoparticle, and a method of treating and/or diagnosing calcific aortic valve disease.
BACKGROUND
[0004] Calcific Aortic Valve Disease (CAVD) is the most prevalent valvular heart disease in the world, affecting up to 25% of adults over the age of 65 (Alushi et al., “Calcific Aortic Valve Disease-Natural History and Future Therapeutic Strategies,” Frontiers in Pharmacology 11 :685 (2020)). CAVD begins as aortic sclerosis which is characterized by calcification and thickening of the three leaflets that comprise the aortic valve (Prasad and Bhalodkar, “Aortic Sclerosis-a Marker of Coronary Atherosclerosis,” Clinical Cardiology 27:671-673 (2004)). Aortic sclerosis progresses into aortic stenosis when large calcific nodules on the aortic valve leaflets hinder the leaflets’ mobility and obstruct blood flow through the heart (Alushi et al., “Calcific Aortic Valve Disease-Natural History and Future Therapeutic Strategies,” Frontiers in Pharmacology 11 :685 (2020)). This later stage in CAVD is associated with approximately a 50% increase in the risk of myocardial infarction and the risk of death from a cardiovascular cause (Prasad and Bhalodkar, “Aortic Sclerosis-a Marker of Coronary Atherosclerosis,” Clinical Cardiology 27:671-673 (2004)). The current gold standard for diagnosis is echocardiography, highlighted by valve regurgitation (Rajamannan et al., “Calcific Aortic Valve Disease: Not Simply a Degenerative Process: A Review and Agenda for Research from the National Heart and Lung and Blood Institute Aortic Stenosis Working Group,” Circulation 124: 1783-91 (2011)). Currently, aortic valve replacement surgery is the most effective treatment option for advanced aortic stenosis (Sung et al., “Cadherin-11 Overexpression Induces Extracellular Matrix Remodeling and Calcification in Mature Aortic Valves,” Arteriosclerosis, Thrombosis, and Vascular Biology 36: 1627-1637 (2016)). However, up to one-third of symptomatic aortic stenosis patients cannot safely undergo this major surgery due to age, co-morbidities, and high- risk cardiac disease (Cribier, “Percutaneous Implantation of Aortic Valve Prostheses in Patients with Calcific Aortic Stenosis - Technical Advances,” Interventional Cardiology Review 2:68-69 (2007)). This highlights the need to study non-invasive therapies to prevent the progression and development of CAVD (Alushi et al., “Calcific Aortic Valve Disease-Natural History and Future Therapeutic Strategies,” Frontiers in Pharmacology 11 :685 (2020)).
[0005] Statins have been experimented with in the cardiac field to investigate if they are a potential therapy as patients with CAVD experience lipid accumulation and deposits on their Aortic Valve. However, statins have proved to be unreliable and ineffective as a CAVD therapy (Lee and Choi, “Involvement of Inflammatory Responses in the Early Development of Calcific Aortic Valve Disease: Lessons from Statin Therapy,” Anim. Cells Syst. (Seoul) 22:390-399 (2018)).
[0006] Nanoparticle drug delivery has been a massive triumph in biomedical research, expansively studied in cancer therapies (van der Meel et al., “Smart Cancer Nanomedicine,” Nat. Nanotechnol. 14: 1007-1017 (2019); Wadajkar et al., “Dual-Imaging Enabled Cancer-Targeting Nanoparticles,” Adv. Healthc. Mater. 1 :450-456 (2012)). They are an excellent tool to deliver treatment due to their site-specific targeting ability, making them a promising candidate to target proteins implicated in CAVD (Yetisgin et al., “Therapeutic Nanoparticles and Their Targeted Delivery Applications,” Molecules 25:2193 (2020)).
[0007] Cadherin-11 (cad-11) is a type II classical cadherin protein that is of particular interest in the study of CAVD. Cad-11 mediates cell migration and differentiation of mesenchymal cells into osteo- and chondro- lineages, and when overexpressed, disrupts proper homeostasis of the extracellular matrix that is critical for aortic valve integrity (Sung et al., “Cadherin-11 Overexpression Induces Extracellular Matrix Remodeling and Calcification in Mature Aortic Valves,” Arteriosclerosis, Thrombosis, and Vascular Biology 36 : 1627-1637 (2016)). This overexpression affects both aortic valve endothelial cells and aortic valve interstitial cells. Cad-11 is endogenously found in healthy human aortic valve endothelial cells (HAVECs) (Zhou et al., “Cadherin-11 Expression Patterns in Heart Valves Associate With Key Functions During Embryonic Cushion Formation, Valve Maturation and Calcification,” Cells, Tissues, Organs 198:300-10 (2013); Butcher et al., Transcriptional Profiles of Valvular and Vascular Endothelial Cells Reveal Phenotypic Differences: Influence of Shear Stress,” Arteriosclerosis, Thrombosis, and Vascular Biology 26:69-77 (2006)). Recent work has indicated cad-11 overexpression in aortic valve endothelial cells causes the activation of GTP-Racl, a GTPase, and /?-catenin translocation to the nucleus (Vaidya et al., “Rael Mediates Cadherin-11 Induced Cellular Pathogenic Processes in Aortic Valve Calcification,” Cardiovasc. Pathol. 58: 107414 (2022)). This pathway drives an endothelial to mesenchymal transition (EndMT) where endothelial cells adopt a mesenchymal cell phenotype, degrade their surrounding extracellular matrix, and invade the valve interstitium. This process diminishes the integrity of the aortic valve endothelial cell layer and promotes calcification (Gee et al., “NFKB (Nuclear Factor K-Light-Chain Enhancer of Activated B Cells) Activity Regulates Cell-Type-Specific and Context-Specific Susceptibility to Calcification in the Aortic Valve,” Arterioscler. Thromb. Vase. Biol. 40:638-655 (2020)). It has been found that in aortic valve interstitial cells cad-11 overexpression activates RhoA, a Rho protein family GTPase, and Sox9, a transcription factor, which results in extracellular matrix remodeling that hinders aortic valve functioning (Sung et al., “Cadherin-11 Overexpression Induces Extracellular Matrix Remodeling and Calcification in Mature Aortic Valves,” Arteriosclerosis, Thrombosis, and Vascular Biology 36 : 1627-1637 (2016)). Elucidating the molecular pathways that coordinate CAVD development and progression in aortic valve endothelial and interstitial cells is essential for developing valve-specific therapies.
[0008] Tumor necrosis factor a (TNFcr) is an inflammatory cytokine that has been found to significantly upregulate cad-11 expression, induce EndMT in aortic valve endothelial cells, and accelerate calcification in aortic valve interstitial cells (Vandooren et al., “Tumor Necrosis Factor a Drives Cadherin 11 Expression in Rheumatoid Inflammation,” Arthritis & Rheumatism 58:3051-3062 (2008); Mahler et al., “Inflammatory Cytokines Promote Mesenchymal Transformation in Embryonic and Adult Valve Endothelial Cells,” Arteriosclerosis, Thrombosis, and Vascular Biology 33: 121-130 (2013); Yu et al., “Tumor Necrosis Factor-a Accelerates the Calcification of Human Aortic Valve Interstitial Cells Obtained from Patients with Calcific Aortic Valve Stenosis via the BMP2-Dlx5 Pathway,” Journal of Pharmacology and Experimental Therapeutics 337: 16-23 (2010)). Importantly, TNFcr also activates the canonical nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-KB) pathway (Gee et al., “NFKB (Nuclear Factor K-Light-Chain Enhancer of Activated B Cells) Activity Regulates Cell-Type- Specific and Context-Specific Susceptibility to Calcification in the Aortic Valve,” Arterioscler. Thromb. Vase. Biol. 40:638-655 (2020)). The canonical pathway is activated when pro- inflammatory molecules, like TNFcr, bind to cell surface receptors and cause the activation of the IKB kinase complex which allows NF-KB dimers to translocate to the nucleus and activate DNA transcription (“The NF-KB Signaling Pathway.” The NF-KB Signaling Pathway - Creative Diagnostics (2020)). Recent findings indicate that the activation of the NF-KB canonical pathway can coordinate pathogenic calcification in aortic valve endothelial and interstitial cells (Gee et al., “NFKB (Nuclear Factor K-Light-Chain Enhancer of Activated B Cells) Activity Regulates Cell-
Type-Specific and Context-Specific Susceptibility to Calcification in the Aortic Valve,” Arterioscler. Thromb. Vase. Biol. 40:638-655 (2020)). Specifically, the nuclear translocation of the )65 IRelA subunit dimer in the NF-KB pathway has been found to induce aortic valve endothelial cell differentiation into osteogenic cell lineages and is active in pro-osteogenic aortic valve interstitial cell differentiation (Gee et al., “NFKB (Nuclear Factor K-Light-Chain Enhancer of Activated B Cells) Activity Regulates Cell-Type-Specific and Context-Specific Susceptibility to Calcification in the Aortic Valve,” Arterioscler. Thromb. Vase. Biol. 40:638-655 (2020)). [0009] BAY 11-7082 is a potent anti-inflammatory, small-molecule drug that functions as an irreversible inhibitor of the NF-KB canonical pathway by preventing the phosphorylation of the IK-BU protein by the IKK complex (Pierce et al., “Novel Inhibitors of Cytokine-Induced IKBO. Phosphorylation and Endothelial Cell Adhesion Molecule Expression Show Anti-Inflammatory Effects in Vivo,” Journal of Biological Chemistry 272:21096-21103 (1997)). Consequently, BAY 11-7082 prevents the subunit dimer from reaching the nucleus. Previous in vitro studies showed BAY 11-7082 significantly disrupts the translocation of the p65 subunit to the nucleus, preventing the transcription of genes that promote inflammation and cellular proliferation (Gee et al., “NFKB (Nuclear Factor K-Light-Chain Enhancer of Activated B Cells) Activity Regulates Cell-Type-Specific and Context-Specific Susceptibility to Calcification in the Aortic Valve,” Arterioscler. Thromb. Vase. Biol. 40:638-655 (2020); Rauert-Wunderlich et al., “The IKK Inhibitor Bay 11-7082 Induces Cell Death Independent from Inhibition of Activation of NFKB Transcription Factors,” PLoS ONE 8:e59292 (2013); Lee et al., “BAY 11-7082 Is a Broad- Spectrum Inhibitor with Anti-Inflammatory Activity against Multiple Targets,” Mediators of Inflammation (2012)). In vivo, BAY 11-7082 has been shown to significantly inhibit the NF-KB pathway which reduced proteinuria and inhibited renal dysfunction in lupus-prone mice (Zhao et al., “Bayl 1-7082 Attenuates Murine Lupus Nephritis Via Inhibiting Nlrp3 Inflammasome and NF-KB Activation,” International Immunopharmacology 17:116-122 (2013)). Additionally, BAY 11-7082 reduced tumor growth and caused apoptosis of cancerous cells in mice with adult T- cell leukemia (Ohsugi et al., “In Vivo Antitumor Activity of the NF-KB Inhibitor Dehydroxymethylepoxy quinomicin in a Mouse Model of Adult T-Cell Leukemia,” Carcinogenesis 26: 1382-1388 (2005)). Despite the promising therapeutic potential of BAY 11- 7082, the effect of this drug has not been evaluated in the heart and warrants further investigation as a potential CAVD therapy.
[0010] The present disclosure is directed to overcoming these and other deficiencies in the art. SUMMARY
[0011] One aspect of the present disclosure relates to a nanoparticle for treating and/or diagnosing calcific aortic valve disease (CAVD). The nanoparticle comprises a nanoparticle core and a targeting molecule associated with an exterior surface of the nanoparticle core.
[0012] Another aspect of the present disclosure relates to a therapeutic composition. The therapeutic composition comprises a nanoparticle for treating CAVD and a pharmaceutically acceptable carrier.
[0013] A further aspect of the present disclosure relates to a method of treating and/or diagnosing CAVD. The method comprises contacting a nanoparticle with endothelial tissue of a subject at risk for CAVD.
[0014] Calcific Aortic Valve Disease (CAVD) is an increasingly prevalent disease defined by calcification and thickening of the aortic valve, resulting in improper heart functioning. Unfortunately, the only treatment option for CAVD requires invasive surgery which is unsafe for a large percentage of patients. This highlights a need for non-invasive CAVD treatments. This study aims to elucidate the potential therapeutic uses of cadherin-11 (cad-11) antibody-conjugated poly (lactic-co-glycolic acid) (PLGA) nanoparticles (cad-11 nanoparticles) to deliver BAY 11-7082, a potent anti-inflammatory drug, to diseased aortic valves. 2D in vitro models were used to illustrate that cad-11 nanoparticles show cell-specific targeting. The diseased 3D in vitro models constructed and treated with various doses of BAY 11-7082 delivered via cad-11 nanoparticles showed no reduction in calcific nodule formation but did show a decrease in cellular compaction in a dose-dependent manner. These results, in combination with preliminary and future in vivo experiments, offer insight into the potential therapeutic role of cad-11 nanoparticles and the molecular pathways responsible for the development and progression of CAVD.
[0015] The parallel upregulation of cad-11 expression and activation of the NF-KB pathway by TNFcr provides an opportunity to deliver a therapeutic (e.g., BAY 11-7082) to cells actively expressing the NF-KB pathway by encapsulating the drug in biodegradable poly (lactic- co-glycolic acid) (PLGA) nanoparticles that bind to cad-11. Cad-11 antibodies can be conjugated, or associated, to the outside of nanoparticles to direct the nanoparticles to sites exhibiting cad-11 expression. Once the cad-11 antibody-conjugated PLGA nanoparticle (cad-11 nanoparticle) contacts the cad-11 expressing cell, the nanoparticle is endocytosed and the contents inside the nanoparticle are released inside the cell after incubation for several hours. The ability to conjugate cad-11 antibodies to the nanoparticles and to control the release of BAY 11-7082 suggests that PLGA nanoparticles have the potential to increase site-specific targeting of the drug and decrease systemic toxicity.
[0016] The nanoparticles home to the VECs, and when adhered/internalized release their payload, which in this case is an NF-KB inhibitor, which then blocks NF-KB activity. The main advantage over the free drug (e.g. circulating) is that the residence time next to the valves is low given the very high fluid shear stresses occurring during ejection. The selective binding by VECs via Cad-11 improves specificity to VECs, thus likely requiring less dose and/or fewer off target effects. The benefit of blocking VEC NF-KB activity includes: reduction of EndMT, inhibition of endogenous reprogramming, reduction of osteogenic differentiation, and improved quiescent behavior through maintained NO secretion. The nanoparticles can also home to VICs through/around the VECs at later disease stages, which would convey similar benefits to the VICs, with the exception of EndMT as they are already mesenchymal cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is an illustration showing a conceptual diagram of the alterations that occur in CAVD, the resulting downstream inflammatory signaling, and cad-11 nanoparticles with cad- 11 conjugated antibodies targeting inflammatory valve tissue. Endothelial cells are specifically targeted by the cad-11 nanoparticles, preventing endothelial to mesenchymal transition and osteoblast like differentiation through inhibition of the NF-KB pathway.
[0018] FIGS. 2A-2C are illustrations showing cross-sections of three different embodiments of cad-11 antibody-conjugated nanoparticles. FIG. 2A is an illustration showing a cad-11 nanoparticle with coumarin 6 (C6) dye encapsulated in the nanoparticle core (cad-11 C6 nanoparticle). FIG. 2B is an illustration showing a cad-11 nanoparticle with BAY 11-7082, an IKK inhibitor encapsulated in the nanoparticle core (cad-11 BAY nanoparticle). FIG. 2C is an illustration showing a cad-11 nanoparticle with Cy5.5 dye encapsulated in the nanoparticle core (cad-11 Cy5.5 nanoparticle).
[0019] FIGS. 3A-3B shows PLGA nanoparticle properties and the release profile of cad- 11 BAY nanoparticles. FIG. 3 A is a summary showing the size, poly dispersity, and zeta potential of the PLGA, BAY PLGA, and cad-11 BAY PLGA nanoparticles. FIG. 3B is a graph showing the amount of BAY 11-7082 released per BAY PLGA nanoparticle over the span of 14 days.
[0020] FIG. 4 shows a schematic of the experimental procedures used to evaluate cad-11 nanoparticle binding specificity. Human aortic valve endothelial cells (HA VECs) and human umbilical vein endothelial cells (HUVECs) were cultured and passaged into microwell plates. Following, cad-11 C6 nanoparticles were added to the microwell and incubated with the HAVECs and HUVECs for 3.5 hours. The cells were washed and then imaged to examine the fluorescence exhibited by the respective cells.
[0021] FIGS. 5A-5E demonstrate cad-11 C6 nanoparticle targeting of various cell types. Cells were incubated with 50 pg/mL of cad-11 C6 nanoparticles for 3.5 hours. FIG. 5 A is a fluorescent image showing HAVECs after incubation with the cad-11 C6 nanoparticles. FIG. 5B is a fluorescent image showing HUVECs after incubation with the cad-11 C6 nanoparticles. FIG. 5C is a fluorescent image showing PAVICs after incubation with the cad-11 C6 nanoparticles. FIG. 5D is a fluorescent image showing PAVECs after incubation with the cad-11 C6 nanoparticles. FIG. 5E is a graph showing the proportion of HAVECs, HUVECs, PAVICs, and PAVECs that display fluorescent intensity above the background fluorescence threshold.
[0022] FIG. 6 shows a schematic of experimental procedures used to evaluate the effect of cad-11 BAY nanoparticles in PAVEC-PAVIC hydrogels. 3D PAVEC-PAVIC collagen hydrogels were cultured for 5 days and then incubated with different doses of cad-11 BAY nanoparticles. Various endpoint assays were used to assess disease progression in the hydrogels. [0023] FIGS. 7A-7C demonstrate the effects of BAY 11-7082 treatment on PAVEC- PAVIC hydrogels cultured in osteogenic media (OGM). FIG. 7A is a series of images showing PAVEC-PAVIC hydrogels cultured for 7 days in basal control media (GM) (DI -7), OGM (Dl- 7), or OGM (Dl-3) then OGM + lOuM BAY 11-7082 (D3-7). Images of the PAVEC-PAVIC hydrogels were acquired at days 1, 3, and 5. On day 7, the PAVEC-PAVIC hydrogels were stained with Alizaren Red Calcium Binding Stain (ARS) to evaluate calcific nodule formation. FIG. 7B are images showing ARS staining of PAVEC-PAVIC hydrogels cultured in GM, OGM, or OGM with 10 pM BAY 11-7082 (OGMB) after 7 days. The bottom series of images is showing a magnified version of the top series of images. FIG. 7C is a graph showing a plate reader assay to quantify calcium concentrations in hydrogels. There was significantly more calcium in the GM group compared to the OGM and OGMB groups. A Kruskal -Wallis test followed by a Dunn’s test determined there was no significant difference in calcium concentration between the OGM and OGMB groups. Significant p-values are displayed on the graph (a = 0.05). N = 6 for each hydrogel group.
[0024] FIGS. 8A-8B demonstrate the cellular compaction of the PAVEC-PAVIC hydrogels. FIG. 8A is a series of images showing PAVEC-PAVIC hydrogels cultured for seven days in GM with no nanoparticles or OGM with 0 pM, 5 pM, 10 pM, and 20 pM BAY 11-7082 delivered via cad-11 BAY nanoparticles. The periphery of the PAVEC-PAVIC hydrogels is outlined in the images. Scale bars indicate 1 mm. FIG. 8B is a graph showing the compaction proportion among the PAVEC-PAVIC hydrogels treated with various doses of BAY 11-7082 delivered via cad-11 BAY nanoparticles. A Kruskal -Wallis test followed by a Dunn’s test was used to identify significant pairwise comparisons. Significant p-values are displayed on the graph (<z = 0.05). N = 6 for each treatment group.
[0025] FIG. 9 is a series of images showing a-smooth muscle actin (a-SMA) staining in the PAVEC-PAVIC hydrogels treated with cad-11 BAY nanoparticles. Sections were stained for a-SMA to visualize the interstitial cells and diseased endothelial cells that had transitioned to mesenchymal phenotype. Sections were additionally stained with nuclear blue to label the nuclei. [0026] FIG. 10 is a series of images showing VE-cadherin staining in PAVEC-PAVIC hydrogels treated with cad-11 BAY nanoparticles. Sections were stained for VE-cadherin to label PAVECs. Sections were additionally stained with nuclear blue to label the nuclei.
[0027] FIG. 11 is a series of images showing hematoxylin and eosin (H&E) staining of the hydrogels treated with different doses of cad-11 BAY nanoparticles. Scale bars indicate 0.5 mm.
[0028] FIG. 12 shows a schematic of experimental procedures to evaluate the efficacy of the cad-11 BAY nanoparticles in vivo. An initial biodistribution study would be used to determine the cad-11 nanoparticle targeting sites in vivo. Following, a dose-response curve would be generated to determine the ECso of the cad-11 BAY nanoparticles. The ECso dose would then be tested in a larger cohort of mice to determine the effects of the cad-11 BAY nanoparticles in vivo.
[0029] FIGS. 13A-13B demonstrate BAY 11-7082 treatment decreases cellular remodeling and calcific nodule density. FIG. 13A is a graph showing area compaction in the Bayl 1@D1, Bayl 1@CAVD, GM, and OGM conditions. FIG. 13B is a graph showing nodule number in the GM, OGM, OGM+Bay at CAVD Diagnosis, and OGM+Bay- Day 1 (preventative) conditions.
[0030] FIG. 14 demonstrates the effect of BAY 11-7082 on co-cultured spring gels. Images were taken after 7 days of incubation of co-culture PAVEC/PAVIC spring gels.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0031] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present application herein described for which they are suitable as would be understood by a person skilled in the art. Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular compositions or methodologies described, as these may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of embodiments herein which will be limited only by the appended claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of embodiments herein, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that embodiments herein are not entitled to antedate such disclosure by virtue of prior invention. [0032] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure.
[0033] The term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, such as within 50%, or within 20%, or within 10%, or within 5% (or any amount or range within 5-50%) of a given value or range. The allowable variation encompassed by the term “about” or “approximately” may depend on the context.
[0034] The term “and/or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed items is used or present.
[0035] As will be understood by a person of ordinary skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof, as well as any value within a range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so on. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and so on. As will also be understood by a person of ordinary skill in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges or specific values therein as discussed above. Finally, as will be understood by a person of ordinary skill in the art, and as discussed above, a range includes each individual value.
[0036] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “involving”, “having”, and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and/or steps. In embodiments or claims where the term comprising (or the like) is used as the transition phrase, such embodiments can also be envisioned with replacement of the term “comprising” with the terms “consisting of’ or “consisting essentially of.” The methods, kits, systems, and/or compositions of the present disclosure can comprise, consist essentially of, or consist of, the components disclosed.
[0037] In embodiments comprising an “additional” or “second” component, the second component as used herein is different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.
[0038] The term “polypeptide,” “peptide”, or “protein” are used interchangeably and to refer to a polymer of amino acid residues. The terms encompass all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP- ribosylation, pegylation, biotinylation, etc.).
[0039] As used herein, “subject” means any animal, preferably a mammal, most preferably a human. The term “mammal” as used herein, encompasses any mammal. Examples of a subject as described herein include but are not limited to fish, birds, reptiles, or mammals, e.g., human, rabbit, cow, pig, sheep, chicken, rat, or mouse.
[0040] As used herein, the term “aortic valve” refers to the heart valve that divides the left ventricle and the aorta. The aortic valve opens during left ventricular contraction and then closes to prohibit the backwash of oxygenated blood from the aorta into the ventricle. The aortic valve typically contains 3 valve leaflets in most individuals, but may contain 2 valve leaflets in some individuals.
[0041] As used herein, “calcific aortic valve disease” refers to a disease state in which there is calcification and fibrosis of the aortic valve, encompassing aortic sclerosis and aortic stenosis.
[0042] As used herein, “aortic sclerosis” refers to the thickening and calcification of the aortic valve leaflets in the absence of obstruction to left ventricular outflow.
[0043] As used herein, “aortic stenosis” refers to a condition of valvular pathology in which left ventricular outflow is obstructed.
[0044] As used herein, “inhibiting” refers to the reduction or suppression of a given condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.
[0045] As used herein, “NF -KB -inhibitor” means a compound that inhibits the cell transcription factor nuclear kappa-B (NF-KB).
[0046] Certain terms employed in the specification, examples, and claims are collected herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, some embodiments of the methods and materials are now described.
[0047] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Delivery Vehicles
[0048] Examples of delivery vehicles described herein include but are not limited to nanoparticles and liposomes.
[0049] The present disclosure describes a nanoparticle delivery vehicle that can be used with a variety of subjects including warm blooded animals, particularly mammals, including humans, dogs, cats and other small animals, and farm animals. Additionally, the nanoparticles of the present disclosure can be used with prokaryotic and eukaryotic microorganisms and with in vitro cultures. The nanoparticle delivery vehicle of the present disclosure can be used as a diagnostic agent in all the above subjects, as well as in the capacity of a therapeutic agent.
[0050] The term “nanoparticle” or “nanoparticle core” as used herein denotes a carrier structure which is biocompatible with and sufficiently resistant to chemical and/or physical destruction by the environment of use such that a sufficient amount of the carrier structure remains substantially intact so as to be able to reach the target site. Nanoparticles can be solid colloidal particles ranging in size from 1 to 1000 nm. Nanoparticles can have any diameter less than or equal to 1000 nm, including 5, 10, 15, 20, 25, 30, 50, 100, 500 and 750 nm.
[0051] In some embodiments, the nanoparticle is formed of a polymer. The term “polymer,” as used herein, is given its ordinary meaning as used in the art, z.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. The repeat units may all be identical, or in some cases, there may be more than one type of repeat unit present within the polymer. In some cases, the polymer can be biologically derived, i.e., a biopolymer. Non-limiting examples include peptides or proteins. In some cases, additional moieties may also be present in the polymer, for example biological moieties such as those described below. If more than one type of repeat unit is present within the polymer, then the polymer is said to be a “copolymer.” It is to be understood that in any embodiment employing a polymer, the polymer being employed may be a copolymer in some cases. The repeat units forming the copolymer may be arranged in any fashion. For example, the repeat units may be arranged in a random order, in an alternating order, or as a block copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers may have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.
[0052] Nanoparticles can include copolymers, which, in some embodiments, describes two or more polymers (such as those described herein) that have been associated with each other, usually by covalent bonding of the two or more polymers together. Thus, a copolymer may comprise a first polymer and a second polymer, which have been conjugated together to form a block copolymer where the first polymer can be a first block of the block copolymer and the second polymer can be a second block of the block copolymer. Of course, those of ordinary skill in the art will understand that a block copolymer may, in some cases, contain multiple blocks of polymer, and that a “block copolymer,” as used herein, is not limited to only block copolymers having only a single first block and a single second block. For instance, a block copolymer may comprise a first block comprising a first polymer, a second block comprising a second polymer, and a third block comprising a third polymer or the first polymer, etc. In some cases, block copolymers can contain any number of first blocks of a first polymer and second blocks of a second polymer (and in certain cases, third blocks, fourth blocks, etc.). In addition, it should be noted that block copolymers can also be formed, in some instances, from other block copolymers. For example, a first block copolymer may be conjugated to another polymer (which may be a homopolymer, a biopolymer, another block copolymer, etc.), to form a new block copolymer containing multiple types of blocks, and/or to other moieties (e.g., to non-polymeric moieties).
[0053] In some embodiments, a polymer (e.g., copolymer, e.g., block copolymer) contemplated herein includes a biocompatible polymer, i.e., the polymer that does not typically induce an adverse response when inserted or injected into a living subject, for example, without significant inflammation and/or acute rejection of the polymer by the immune system, for instance, via a T-cell response. Accordingly, the nanoparticles contemplated herein can be non- immunogenic.
[0054] Biocompatibility typically refers to the acute rejection of material by at least a portion of the immune system, i.e., a nonbiocompatible material implanted into a subject provokes an immune response in the subject that can be severe enough such that the rejection of the material by the immune system cannot be adequately controlled, and often is of a degree such that the material must be removed from the subject. One simple test to determine biocompatibility can be to expose a polymer to cells in vitro., biocompatible polymers are polymers that typically will not result in significant cell death at moderate concentrations. For instance, a biocompatible polymer may cause less than about 20% cell death when exposed to such cells.
[0055] In some embodiments, contemplated biocompatible polymers may be biodegradable, i.e., the polymer is able to degrade, chemically and/or biologically, within a physiological environment, such as within the body. As used herein, “biodegradable” polymers are those that, when introduced into cells, are broken down by the cellular machinery (biologically degradable) and/or by a chemical process, such as hydrolysis, (chemically degradable) into components that the cells can either reuse or dispose of without significant toxic effect on the cells. In one embodiment, the biodegradable polymer and their degradation byproducts can be biocompatible.
[0056] In some embodiments, the nanoparticle core comprises a biodegradable polymer.
[0057] In some embodiments, the nanoparticle is designed to only degrade once internalized by the cell which may in some instances occur via compartments that exhibit unique biological or biochemical environments. In accordance with this embodiment, the unique biological environment may be but is not limited to low pH.
[0058] Contemplated nanoparticle polyesters include, for example, copolymers and/or block copolymers comprising lactic acid and/or glycolic acid units, such as poly(lactic acid-co- glycolic acid) and poly(lactide-co-glycolide), collectively referred to herein as “PLGA”; and homopolymers comprising glycolic acid units, referred to herein as “PGA,” and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D- lactide, and poly-D,L-lactide, collectively referred to herein as “PLA.” In some embodiments, exemplary polyesters include, for example, polyhydroxyacids; PEGylated polymers and copolymers of lactide and glycolide (e.g., PEGylated PLA (PLA-PEG), PEGylated PGA, PEGylated PLGA, and derivatives thereof.
[0059] In some embodiments, a contemplated nanoparticle may include PLGA. PLGA is a biocompatible and biodegradable co-polymer of lactic acid and glycolic acid, and various forms of PLGA can be characterized by the ratio of lactic acid:gly colic acid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lactic acid. The degradation rate of PLGA can be adjusted by altering the lactic acid-glycolic acid ratio. In some embodiments, PLGA to be used in accordance with the present invention can be characterized by a lactic acid:glycolic acid ratio of approximately 85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60, approximately 25:75, or approximately 15:85. In some embodiments, the ratio of lactic acid to glycolic acid monomers in the polymer of the particle (e.g., the PLGA block copolymer or PLGA-PEG block copolymer), may be selected to optimize for various parameters such as therapeutic agent release and/or polymer degradation kinetics.
[0060] In some embodiments, the nanoparticle core comprises poly(lactic-co-glycolic acid).
[0061] In some embodiments of the present disclosure, the delivery vehicle is a liposome. The term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
[0062] Several advantages of liposomes may include the following: their biocompatibility and biodegradability, incorporation of a wide range of water and lipid soluble drugs; and they afford protection to encapsulated drugs from metabolism and degradation. Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size, and the aqueous volume of the liposomes.
[0063] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
[0064] Methods for preparing liposomes for use in the present disclosure include those disclosed in Bangham et al., “Diffusion of Univalent Ions Across the Lamellae of Swollen Phospholipids,” J. Mol. Biol. 13:238-52 (1965); U.S. Patent No. 5,653,996 to Hsu; U.S. Patent No. 5,643,599 to Lee et al.; U.S. Patent No. 5,885,613 to Holland et al.; U.S. Patent No.
5,631,237 to Dzau & Kaneda, and U.S. Patent No. 5,059,421 to Loughrey et al., all of which are hereby incorporated by reference in their entirety.
Targeting molecules
[0065] As used herein, a “targeting molecule” is a molecule that is able to bind to or otherwise associate with a molecular target, for example, a membrane component, a cell surface receptor, cadherin-11 (cad-11) or the like. A delivery vehicle comprising the targeting molecule may become localized or converge at a particular targeted site, for instance, endothelial tissue, a disease site, a tissue, an organ, a type of cell, etc. As such, the delivery vehicle may be “targetspecific.”
[0066] For example, contemplated targeting molecules include, without limitation, a peptide, polypeptide, protein, glycoprotein, carbohydrate, or lipid. A targeting molecule may be a naturally occurring or synthetic ligand for a cell surface receptor. A targeting molecule can be an antibody, which term is intended to include antibody fragments, characteristic portions of antibodies, single chain targeting moieties which can be identified, for example, using procedures such as phage display. Targeting molecules may also be a targeting peptide, targeting peptidomimetic, or a small molecule, whether naturally-occurring or artificially created (e.g., via chemical synthesis).
[0067] In some embodiments, the targeting molecule comprises a molecule that targets an endothelial molecule that is indicative of CAVD.
[0068] In some embodiments, the targeting component is selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein, a peptide, and aptamer, and a small molecule. [0069] In some embodiments, the targeting molecule is an antibody against molecular targets on endothelial tissue and, for example, specifically bind markers associated with CAVD.
[0070] In some embodiments, the targeting molecule comprises a molecule that targets a portion of Cadherin 11 protein.
[0071] In some embodiments, the targeting molecule comprises an anti -Cadherin 11 antibody.
[0072] Antibodies that may be used as targeting molecules in the nanoparticles of the present disclosure may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intrabodies”), antibody fragments (e.g. Fv, Fab and F(ab)2), half-antibodies, hybrid derivatives, as well as single chain antibodies (scFv), chimeric antibodies and de-immunized or humanized antibodies (Ed Harlow and David Lane, USING ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 1999);
Houston et al., “Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti -Digoxin Single-Chain Fv Analogue Produced in Escherichia coli," Proc. Natl. Acad.
Sci. USA 85:5879-5883 (1988); Bird et al, “Single-Chain Antigen-Binding Proteins,” Science 242:423-426 (1988), each of which is hereby incorporated by reference in its entirety).
[0073] Antibodies may also be generated using recombinant DNA technology, such as, for example, an antibody or fragment thereof expressed by a bacteriophage. Alternatively, the synthetic antibody is generated by the synthesis of a DNA molecule encoding and expressing the antibody of the present disclosure or the synthesis of an amino acid sequence specifying the antibody, where the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
[0074] Methods for monoclonal antibody production may be carried out using the techniques described herein or are well-known in the art (MONOCLONAL ANTIBODIES - PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS (Mary A. Ritter and Heather M. Ladyman eds., 1995), which is hereby incorporated by reference in its entirety). Generally, the process involves obtaining immune cells (lymphocytes) from the spleen of a mammal which has been previously immunized with the antigen of interest either in vivo or in vitro.
[0075] Alternatively monoclonal antibodies can be made using recombinant DNA methods as described in U.S. Patent No. 4,816,567 to Cabilly et al, which is hereby incorporated by reference in its entirety. The polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cells, for example, by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, monoclonal antibodies are generated by the host cells. Also, recombinant monoclonal antibodies or fragments thereof of the desired species can be isolated from phage display libraries (McCafferty et al., “Phage Antibodies: Filamentous Phage Displaying Antibody Variable Domains,” Nature 348:552-554 (1990); Clackson et al., “Making Antibody Fragments using Phage Display Libraries,” Nature 352:624-628 (1991); and Marks et al., “By-Passing Immunization. Human Antibodies from V-Gene Libraries Displayed on Phage,” J. Mol. Biol. 222:581-597 (1991), all of which are hereby incorporated by reference in their entirety).
[0076] The polynucleotide(s) encoding a monoclonal antibody can further be modified using recombinant DNA technology to generate alternative antibodies or derivatives. For example, the constant domains of the light and heavy chains of a mouse monoclonal antibody can be substituted by those regions derived from a human antibody to generate a chimeric antibody. Alternatively, the constant domains of the light and/or heavy chains of a monoclonal antibody can be substituted by a non-immunoglobulin polypeptide to generate a fusion antibody. In other embodiments, the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Furthermore, site-directed or high-density mutagenesis of the variable region can be used to optimize specificity and affinity of a monoclonal antibody.
[0077] The monoclonal antibody of the present disclosure can be a humanized antibody. Humanized antibodies are antibodies that contain minimal sequences from non-human (e.g., murine) antibodies within the variable regions. Such antibodies are used therapeutically to reduce antigenicity and human anti-mouse antibody responses when administered to a human subject. In practice, humanized antibodies are typically human antibodies with minimal to no non-human sequences. A human antibody is an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human.
[0078] In addition to whole antibodies, the present disclosure encompasses antigen binding portions of such antibodies. Such binding portions include the monovalent Fab fragments, Fv fragments (e.g., single-chain antibody, scFv), and single variable VH and VL domains, and F(ab’)2 fragments, Bis-scFv, diabodies, triabodies, minibodies, etc. These antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in James Goding, MONOCLONAL ANTIBODIES PRINCIPLES AND PRACTICE 98-118 (Academic Press, 1983) and Ed Harlow and David Lane, ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Laboratory, 1988), both of which are hereby incorporated by reference in their entirety, or other methods known in the art.
[0079] It may further be desirable, especially in the case of antibody fragments, to modify the antibody in order to increase its serum half-life. This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle (e.g., by DNA or peptide synthesis).
[0080] Antibody mimics are also suitable for use in accordance with the present disclosure. A number of antibody mimics are known in the art including, without limitation, those known as monobodies, which are derived from the tenth human fibronectin type III domain (10Fn3) (Koide et al., “The Fibronectin Type III Domain as a Scaffold for Novel Binding Proteins,” J. Mol. Biol. 284: 1141-1151 (1998); Koide et al., “Probing Protein Conformational Changes in Living Cells by Using Designer Binding Proteins: Application to the Estrogen Receptor,” Proc. Natl. Acad. Sci. USA 99: 1253-1258 (2002), each of which is hereby incorporated by reference in its entirety); and those known as affibodies, which are derived from the stable alpha-helical bacterial receptor domain Z of staphylococcal protein A (Nord et al., “Binding Proteins Selected from Combinatorial Libraries of an alpha-helical Bacterial Receptor Domain,” Nature BiotechnoL 15(8):772-777 (1997), which is hereby incorporated by reference in its entirety).
[0081] In another embodiment, the targeting molecule is a small molecule against molecular targets on endothelial tissue and, for example, specifically bind markers associated with CAVD. In particular, small molecules that bind cad-11 are used. An exemplary small molecule against cad-11 includes SD-133.
[0082] In another embodiment, the targeting molecule is a peptide, protein fragment, truncated peptide, or partial fragment of a biomarker of CAVD, for example cad-11.
[0083] In some embodiments, the nanoparticle comprises more than one type of targeting molecule.
[0084] In some embodiments, the nanoparticle core comprises one or more types of therapeutic molecules.
[0085] The terms “linked”, “joined”, “grafted”, “tethered”, “associated”, and “conjugated” in the context of the delivery vehicles disclosed herein, are used interchangeably to refer to any method known in the art for functionally connecting targeting molecules, including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, and electrostatic bonding.
Therapeutic and diagnostic molecules
[0086] As used herein, a “therapeutic molecule” is an agent, or combination of agents, that treats a cell, tissue, or subject having a condition requiring therapy, when contacted with the cell, tissue or subject.
[0087] In some embodiments, the therapeutic molecule is an NFKB inhibitor. NFKB is a transcription factor that plays an important role in many cellular processes. Vertebrate NFKB transcription complexes can be any of a variety of homo- and heterodimers formed by the subunits p50, p52, c-Rel, RelA (p65), and RelB. In its inactive state, NFKB resides in the cytoplasm and is bound to another protein called IKB. Upon cell activation, IKB may be modified and targeted for degradation by IKB (IKK) kinase. The IKK complex contains two kinase subunits, IKKa and IKK/?, and an associated scaffold-like regulatory protein called NEMO (IKKy). After stimulation of cells by agents such as tumor necrosis factor a (TNFa), interleukin-1 (IL-1) or various pathogens, the IKK complex is activated in part by phosphorylation of specific serine residues in the activation loop of each IKK subunit. The activated IKK complex can then phosphorylate IKB on two serine residues in human IKB. Phosphorylation of the IKB by IKK signals it for ubiquitination at specific lysine residues by the SCF-/?-TrCP E3 ubiquitin ligase complex, which targets the IKB for degradation by the 26S proteasome The freed NFKB may then translocate into the nucleus, and along with other transcription factors, activate transcription of target genes (see e.g., Karin et al., “The IKK NFkB System: a Treasure Trove for Drug Development,” Nat. Rev. 3 : 17-26 (2004); Gilmore et al., “Inhibitors of NF-kB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006), both of which are hereby incorporated by reference in their entirety).
[0088] NFKB activation refers to a state of the NFKB molecule that is capable of participating in transcription activation. Inhibitors of NFKB activation generally refer to an agent that either partially or completely blocks NFKB participation in the activation of many its target genes. A large number of NFKB target genes have been reported in the literature. The mRNAs of these target genes are normally present at low levels and their levels increase dramatically when NFKB and other transcription factors bind to regulatory elements of these genes and activate their transcription.
[0089] NFKB inhibitors may inhibit NFKB at any step in the NFKB activation process. The inhibitor may be a general inhibitor of NFKB activation, or the inhibitor may inhibit specific pathways of induction. Inhibitors that target multiple steps in the NF/cB pathway are also contemplated for use herein. Generally, inhibition of NF-KB activation can occur by three mechanisms: (1) blockage of the incoming activation signal at an early stage (e.g., binding of ligand to its receptor) resulting in complete abrogation of the signal's effect; (2) interference with a cytoplasmic step in the NFKB activation pathway by blockage of a specific component of the cascade (e.g., the activation of the IKK complex or degradation of IKB); or (3) blockage of NFKB nuclear activity, that is, inhibiting its translocation to the nucleus, its binding to DNA, a nuclear modification of NF-KB that affects its activity or specificity (e.g. protein acetylation and/or methylation), or an interaction of NFKB on DNA with specific or basal transcription machinery. [0090] Thus, the NFKB inhibitor may be, without limitation, an upstream NFKB target inhibitor, an IKK phosphorylation inhibitor, an IKB phosphorylation inhibitor, an IKB degradation inhibitor, a proteasome inhibitor, a protease inhibitor, an IKB upregulation inhibitor, and NFKB nuclear translocation inhibitor, an NFKB expression inhibitor, and an NFKB transactivation inhibitor.
[0091] In some embodiments, the therapeutic molecule is an inhibitor of the NFKB signaling pathway and is selected from the group consisting of a protein kinase inhibitor, a protein phosphatase inhibitor, an inhibitor of protein acetylation, a protein methyltransferase inhibitor, a proteasome inhibitor, an inhibitor of protein ubiquitination, an NFKB nuclear translocation inhibitor, an inhibitor of NFKB DNA binding activity.
[0092] Specific inhibitors of NFKB are well known in the art and include, without limitation, those identified in Gilmore et al., “Inhibitors of NF-kB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006), which is hereby incorporated by reference in its entirety.
[0093] The inhibitor may be, without limitation, a small molecule, a peptide, a nucleic acid, an antioxidant, a microbial protein, a viral protein, or an anti-inflammatory agent.
[0094] Small molecule NFKB inhibitors are well known in the art and are described in, for example, Gilmore et al., “Inhibitors of NF-KB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006) and Ramadass et al., “Small Molecule Nf-KB Pathway Inhibitors in Clinic,” Int. J. Mol. Sci. 21 (14): 5164 (2020), both of which are hereby incorporated by reference in their entirety. As used herein, "small molecules" are typically organic, peptide or non-peptide molecules, having a molecular weight less than 10,000 Da, less than 5,000 Da, less than 1,000 Da, and less than 500 Da. This class of modulators includes chemically synthesized molecules, for instance, compounds from combinatorial chemical libraries.
[0095] Peptides are also contemplated for use as NFKB inhibitors. For example, cell- permeable peptides, such as SN-50, that contain nuclear localization sequences of NFKB can saturate the process involved in the uptake of NFKB into the nucleus (Gilmore et al., “Inhibitors of NF-kB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006), which is hereby incorporated by reference in its entirety).
[0096] Peptides can be obtained by known isolation and purification protocols from natural sources, can be synthesized by standard solid or solution phase peptide synthesis methods according to the known peptide sequence of the peptide, or can be obtained from commercially available preparations or peptide libraries. Included herein are peptides that exhibit the biological binding properties of the native peptide and retain the specific binding characteristics of the native peptide. Derivatives and analogs of the peptide, as used herein, include modifications in the composition, identity, and derivatization of the individual amino acids of the peptide provided that the peptide retains the specific binding properties of the native peptide. Examples of such modifications would include modification of any of the amino acids to include the D-stereoisomer, substitution in the aromatic side chain of an aromatic amino acid, derivatization of the amino or carboxyl groups in the side chains of an amino acid containing such a group in a side chain, substitutions in the amino or carboxy terminus of the peptide, linkage of the peptide to a second peptide or biologically active moiety, and cyclization of the peptide (G. Van Binst and D. Tourwe, “Backbone Modifications in Somatostatin Analogues: Relation Between Conformation and Activity,” Peptide Research 5:8-13 (1992), which is hereby incorporated by reference in its entirety).
[0097] Nucleic Acids may also be used to inhibit NFKB activity. By way of example, specific NFKB DNA binding can be blocked by the use of decoy oligonucleotides that have NFKB binding sites. These decoy oligonucleotides function by competing with NFKB binding to specific gene promoters (Gilmore et al., “Inhibitors of NF-kB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006), which is hereby incorporated by reference in its entirety). [0098] Antioxidants are also contemplated for use in the methods described herein. Several antioxidants are known in the art to inhibit NFKB and are described in Gilmore et al., “Inhibitors of NF-kB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006), which is hereby incorporated by reference in its entirety.
[0099] Viruses can produce certain proteins that have mechanisms to inhibit NFKB signaling, including, without limitation, the encoding of IxB-like inhibitors of NFKB, the cleaving of p65, and the targeting of IKK (Powell et al., J Virol 70:8527-8533 (1996); Camus- Bouclainville et al., J Virol 78:2510-2516 (2004); Thoetkiattikul et al., Proc Natl Acad Sci USA 102: 11426-11431 (2005), all of which are hereby incorporated by reference in their entirety). Therefore, use of such viral proteins is also contemplated. [0100] Microbial proteins also produce proteins capable of inhibiting NFKB. By way of example, the YopJ protein encoded by the enteropathogen Yersinia pseudotuberculosis inhibits NFKB activation by deubiquitinating I/cB, which prevents its degradation (Gilmore et al., “Inhibitors of NF-kB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006), which is hereby incorporated by reference in its entirety).
[0101] Anti-inflammatory agents are also contemplated for use as NF-KB inhibitors. Anti-inflammatory agents that inhibit NF-KB are well known in the art. For example, nonsteroidal anti-inflammatory drugs, such as aspirin, ibuprofen, sulindac, indomethacin are known in the art to inhibit NF-KB activation (Gilmore et al., “Inhibitors of NF-kB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006), which is hereby incorporated by reference in its entirety). Glucocorticoids, such as dexamethasone, prednisone and methylprednisolone are also known to inhibit NF-KB (Gilmore et al., “Inhibitors of NF-kB Signaling: 785 and Counting,” Oncogene 25:6887-6899 (2006), which is hereby incorporated by reference in its entirety).
[0102] In some embodiments, the therapeutic molecules may comprise an antiinflammatory drug.
[0103] In some embodiments the anti-inflammatory drug comprises BAY 11-7082.
[0104] In some embodiments, the inhibitor of the NF-KB signaling pathway is selected from the group consisting of ATP analogs, BMS-34554, parthenolide, arsenite, epoxyquinoids, gene-based inhibitors, SB203580, denbinobin, tyrosine kinase inhibitors, rhein, TNAP, betaine, epoxyquinol B, M2L, CCK-8, KSR2, golli BG21, BAY11-7082, protein phosphatase 2A, cytosine arabinoside, OspF, gallic acid, Daxx, anacardic acid, Set9 inhibitor, bortezomib, ALLnL, LLM, Z-LLnV, Z-LLL, lactacystine, N-cbz-Leu-Leu-leucinal (MG132), MG115, ubiquitin ligase inhibitors, salinosporamide A (NPI-0052), DCIC, TPCK, TLCK, BTEE, APNE, YopJ, R0196-9920, A20 (TNFAIP3), SN50, dehydroxymethylepoxyquinomicin, sesquiterpene lactones, decoy oligodeoxynucleotides, BH4, and combinations thereof.
[0105] In some embodiments, the inhibitor of the NF-KB signaling pathway is BAY11- 7082.
[0106] Alternative therapeutic molecules beyond NF-KB inhibitors that may be useful according to the present disclosure include but are not limited to Rac-1 inhibitors and RhoA inhibitors (Vaidya et al., “Rael Mediates Cadherin-11 Induced Cellular Pathogenic Processes in Aortic Valve Calcification,” Cardiovasc. Pathol. 58: 107414 (2022); Farrar et al. “Valve Interstitial Cell Tensional Homeostasis Directs Calcification and Extracellular Matrix Remodeling Processes Via RhoA Signaling.” Biomalerials 105:25-37 (2016), both of which are hereby incorporated by reference in their entirety). [0107] The nanoparticle of the present disclosure may comprise an imaging component. Suitable imaging components include, without limitation, a diagnostic dye.
[0108] As used herein, a “diagnostic molecule” is an agent utilized to detect and diagnose CAVD in vivo. This is achieved by encapsulating the diagnostic molecule within the delivery vehicle component, administering the delivery vehicle component, and then imaging the subject.
[0109] Examples of diagnostic molecules in accordance with the present application are radiolabels such as Ga68, F18, Cu67, 1311, U lin, 1231, 99mTc, 32P, 1251, 3H, 14C and 188Rh, fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, positron emitting isotopes detectable by a positron emission tomography (“PET”) scanner, chemiluminescers such as luciferin, and enzymatic markers such as peroxidase or phosphatase. [0110] In some embodiments, the nanoparticle core comprises one or more types of diagnostic molecules. In accordance with such embodiments, the one or more types of diagnostic molecules comprise one or more diagnostic dyes.
[oni] In the case of a radiolabeled agent, the agent is localized to the antigen with which the targeting molecule reacts and is detected or “imaged” in vivo using known techniques such as radionuclear scanning using e.g., a gamma camera or emission tomography. See e.g., A. R. Bradwell et al., “Developments in Antibody Imaging,” Monoclonal Antibodies for Cancer Detection and Therapy, R. W. Baldwin et al., (eds.), pp. 65-85 (Academic Press 1985), which is hereby incorporated by reference in its entirety. Alternatively, a positron emission transaxial tomography scanner, such as designated Pet VI located at Brookhaven National Laboratory, can be used where the radiolabel emits positrons (e.g., 11C, 18F, 150, and 13N).
[0112] As used herein, imaging can include any one or more of: planar radionuclide imaging, positron emission tomography (PET), echo-planar imaging (EPI), single photon emission computed tomography (SPECT), sonographic imaging (e.g., radiation-free, contrastspecific, high frequency, two-dimensional), magnetic resonance imaging (MRI, also referred to as magnetic resonance tomography or MRT), X-ray, computed tomographic (CT) scans, fluorescence imaging, near-infrared imaging and other medically useful or adaptable imaging techniques.
[0113] The delivery vehicle may incorporate any known fluorescent compound, such as fluorescent organic compounds, dyes, pigments, or combinations thereof. Non-limiting fluorescent molecules that may be used in the present disclosure include, Coumarin 6, Cy5, Cy5.5, Cy2, fluorescein isothiocyanate (FITC), Cy7, fluorescein, Cy3, Cy3.5, Texas Red, rhodamine, and Alexa Fluorescent Dyes (such as Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 633, Alexa Fluor 555, and Alexa Fluor 647). Fluorescent molecules that can be used also include fluorescent proteins, such as GFP (green fluorescent protein), enhanced GFP (EGFP), blue fluorescent protein and derivatives (BFP, EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein and derivatives (CFP, ECFP, Cerulean, CyPet) and yellow fluorescent protein and derivatives (YFP, Citrine, Venus, YPet).
[0114] In some embodiments, the nanoparticle core comprises one or more types of diagnostic molecules. In accordance with such embodiments, the one or more types of diagnostic molecules comprise one or more diagnostic dyes.
[0115] In some embodiments, the diagnostic dyes may be selected from the group consisting of C6 fluorescent dye and Cy 5.5 fluorescent dye.
[0116] In some embodiments, the nanoparticle core comprises one or more types of diagnostic and/or therapeutic molecules.
[0117] In some embodiments, the nanoparticle core encapsulates one or more types of diagnostic and/or therapeutic molecules.
Pharmaceutical Composition
[0118] Another aspect of the present disclosure relates to a pharmaceutical composition comprising a delivery vehicle as described herein and a pharmaceutically acceptable carrier.
[0119] The phrase “pharmaceutically acceptable”, as used in connection with compositions described herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). In some embodiments, the term “pharmaceutically acceptable” means approved by a regulatory agency or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.
[0120] A “pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutically acceptable 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. Examples of pharmaceutically acceptable carriers include water, e.g., buffered with phosphate, citrate and another organic acid. Representative examples of pharmaceutically acceptable excipients that may be useful in the present disclosure include antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counterions such as sodium; and/or nonionic surfactants. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutically acceptable carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin. As used herein, the terms “pharmaceutically acceptable carrier” (e.g., additives such as diluents, immunostimulants, adjuvants, antioxidants, preservatives, and solubilizing agents) are non-toxic to the subject administered the composition at the dosages and concentrations employed.
[0121] Pharmaceutical compositions comprising the delivery vehicle described herein may comprise buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
[0122] Specific examples of adjuvants include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and aluminum oxide, including nanoparticles comprising alum or nanoalum formulations), calcium phosphate (e.g., Masson JD et al, Expert Rev Vaccines 16: 289-299 (2017), which is hereby incorporated by reference in its entirety), monophosphoryl lipid A (MPL) or 3-de-O-acylated monophosphoryl lipid A (3D-MPL) (see e.g., United Kingdom Patent GB2220211, EP0971739, EPl 194166, US6491919, all of which are hereby incorporated by reference in their entirety), AS01, AS02, AS03 and AS04 (see e.g. EPl 126876, US7357936 for AS04, EP0671948, EP0761231, US5750110 for AS02, all of which are hereby incorporated by reference in their entirety), imidazopyridine compounds (see W02007/109812, which is hereby incorporated by reference in its entirety), imidazoquinoxaline compounds (see W02007/109813, which is hereby incorporated by reference in its entirety), delta-inulin (e.g. Petrovsky N and PD Cooper, Vaccine 33: 5920-5926 (2015), which is hereby incorporated by reference in its entirety), STING- activating synthetic cyclic-di-nucleotides (e.g. US20150056224, which is hereby incorporated by reference in its entirety), combinations of lecithin and carbomer homopolymers (e.g.
US6676958), and saponins, such as Quil A and QS21 (see e.g. Zhu D and W Tuo, 2016, Nat Prod Chem Res 3: el 13 (doi: 10.4172/2329-6836. lOOOel 13), which is hereby incorporated by reference in its entirety), optionally in combination with QS7 (see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Patent No, 5,057,540, all of which are hereby incorporated by reference in their entirety). In any embodiment, the adjuvant is Freund’s adjuvant (complete or incomplete). In any embodiment, the adjuvant comprises Quil-A, such as for instance commercially obtainable from Brenntag (now Croda) or Invivogen. QuilA contains the water-extractable fraction of saponins from the Quillaja saponaria Molina tree. These saponins belong to the group of triterpenoid saponins, that have a common triterpenoid backbone structure. Saponins are known to induce a strong adjuvant response to T-dependent as well as T-independent antigens, as well as strong cytotoxic CD8+ lymphocyte responses and potentiating the response to mucosal antigens. They can also be combined with cholesterol and phospholipids, to form immunostimulatory complexes (ISCOMs), wherein QuilA adjuvant can activate both antibody- mediated and cell-mediated immune responses to a broad range of antigens from different origins. In certain embodiments, the adjuvant is AS01, for example AS01B. AS01 is an adjuvant system containing MPL (3-O-desacyl-4'-monophosphoryl lipid A), QS21 (Quillaja saponaria Molina, fraction 21), and liposomes. In certain embodiments, the AS01 is commercially available or can be made as described in WO 96/33739, which is hereby incorporated by reference in its entirety. Certain adjuvants comprise emulsions, which are mixtures of two immiscible fluids, e.g. oil and water, one of which is suspended as small drops inside the other and are stabilized by surface-active agents. Oil-in-water emulsions have water forming the continuous phase, surrounding small droplets of oil, while water-in-oil emulsions have oil forming the continuous phase. Certain oil-in-water emulsions comprise squalene (a metabolizable oil). Certain adjuvants comprise block copolymers, which are copolymers formed when two monomers cluster together and form blocks of repeating units. An example of a water in oil emulsion comprising a block copolymer, squalene and a microparticulate stabilizer is TiterMax®, which can be commercially obtained from Sigma-Aldrich.
Treatment Methods
[0123] The pharmaceutical composition according to the present disclosure may be administered to a subject.
[0124] Thus, another aspect of the present disclosure relates to a method of treating and/or diagnosing CAVD. This method involves contacting a nanoparticle disclosed herein with endothelial tissue of a subject at risk for CAVD. [0125] As used herein, the term “treat” or “treating” refers to the application or administration of the nanoparticle of the invention to a subject, e.g., a patient. The treatment can be to cure, heal, alleviate, relieve, alter, remedy, ameliorate, palliate, improve or affect CAVD or the symptoms of CAVD.
[0126] As used herein, the ability to detect or diagnose CAVD may include determining whether the patient is in an earlier stage of CAVD, or has developed early, moderate, or severe forms of CAVD.
[0127] In some embodiments, the method further comprises detecting a presence of the nanoparti cle(s) on the subject’s endothelial tissue.
[0128] In some embodiments, the method further comprises determining a CAVD disease state based on presence of the nanoparti cle(s) on the subject’s endothelial tissue.
[0129] In carrying out this and other aspects of the present disclosure, contacting may be carried out using methods known in the art including by administering parenterally, topically, intravenously, orally, subcutaneously, intraperitoneally, intranasally, or by intramuscular means. In some embodiments, the nanoparticles or pharmaceutical compositions according to the present disclosure are formulated for subcutaneous administration. In some embodiments, the nanoparticles or pharmaceutical compositions according to the present disclosure are formulated for intramuscular injection. In some embodiments, this type of injection is performed in the arm or leg muscles. Intravenous injections as well as intraperitoneal injections, intraarterial, intracranial, or intradermal injections may also be effective in generating an immune response. [0130] In some embodiments, the contacting is carried out parenterally, topically, intravenously, orally, subcutaneously, intraperitoneally, intranasally, or by intramuscular means. [0131] In some embodiments, the delivery vehicle (nanoparticle) or pharmaceutical compositions according to the present disclosure are formulated for parenteral administration.
Solutions or suspensions of the nanoparticles or pharmaceutical compositions can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
[0132] Pharmaceutical formulations suitable for injectable use include sterile aqueous solutions or dispersions and/or sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g, glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
[0133] When it is desirable to deliver the delivery vehicle (nanoparticle) or pharmaceutical compositions of the present disclosure systemically, they may be formulated for parenteral administration by injection, e.g, by bolus injection or continuous infusion.
Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers, with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.
[0134] Intraperitoneal or intrathecal administration of the delivery vehicle or pharmaceutical compositions of the present disclosure can also be achieved using infusion pump devices such as those described by Medtronic, Northridge, CA. Such devices allow continuous infusion of desired compounds avoiding multiple injections and multiple manipulations.
[0135] In addition to the formulations described previously, the delivery vehicle or pharmaceutical compositions of the present disclosure may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
[0136] In any embodiment, the composition as described herein is a solid formulation, e.g., a freeze-dried or spray-dried composition, which can be used as is, or whereto the physician or the subject adds solvents, and/or diluents prior to use. Solid dosage forms can include tablets, such as compressed tablets, and/or coated tablets, and capsules (e.g., hard or soft gelatin capsules). The composition can also be in the form of sachets, dragees, powders, granules, lozenges, or powders for reconstitution, for example.
[0137] The methods of the present disclosure involve administering any one of the compositions described supra. A suitable subject for treatment in accordance with these aspects of the present disclosure is a subject at risk of developing CAVD and/or a subject at risk of developing conditions associated with CAVD. [0138] In accordance with this aspect of the present disclosure, a prophylactically effective amount of the composition is administered to the subject to prevent or mitigate CAVD. A prophylactically effective amount is the amount necessary to prevent or mitigate CAVD.
[0139] For purposes of this aspect of the disclosure, the target “subject” encompasses any animal, preferably a mammal, more preferably a human. In the context of administering a composition for purposes of preventing, inhibiting, or reducing the severity of a CAVD in a subject, the target subject encompasses any subject that is at risk of developing CAVD. Any, adult, or elderly adult at risk for, or having, CAVD can be treated in accordance with the methods and compositions described herein. Particularly suitable subjects include those at risk of developing CAVD. Other suitable subjects include those subjects which may have or are at risk for developing a condition associated with or resulting from CAVD.
[0140] Numerous other factors may also be accounted for when administering the composition under conditions effective to induce a treatment and/or diagnosis for CAVD. These factors include, for example and without limitation, the concentration of the active agents in the composition, the mode and frequency of administration, and the subject details, such as age, weight and overall health and immune condition. General guidance can be found, for example, in the publications of the International Conference on Harmonization and in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Company 1990), which is hereby incorporated by reference in its entirety. A clinician may administer a composition as described herein until a dosage is reached that provides the desired or required prophylactic effect. The progress of the prophylactic response can be monitored by conventional assays.
[0141] In one embodiment of the present disclosure, the composition as described herein is administered prophylactically to prevent, delay, or inhibit the development of CAVD in a subject at risk of developing CAVD or at risk of developing an associated condition. In some embodiments of the present disclosure, prophylactic administration of the composition is effective to fully prevent CAVD in an individual. In other embodiments, prophylactic administration is effective to prevent the full extent of a condition that would otherwise develop in the absence of such administration, z.e., substantially prevent or inhibit CAVD in an individual.
[0142] In the context of using prophylactic compositions to prevent CAVD, the dosage of the composition is one that is adequate to prevent onset of CAVD, and is capable of achieving a reduction in a number of symptoms, a decrease in the severity of at least one symptom, or a delay in the further progression of at least one symptom, or even a total alleviation of CAVD. [0143] Prophylactically effective amounts of the compositions described herein will depend on whether an adjuvant is co-administered, with higher dosages being required in the absence of adjuvant. The amount of the composition useful for administration can vary from 1 pg-500 pg per patient. In some embodiments, 5, 10, 20, 25, 50 or 100 pg is used for each human injection. Occasionally, a higher dose of 1-50 mg per injection is used. In some embodiments, about 10, 20, 30, 40 or 50 mg is used for each human injection. The timing of injections can vary significantly from once a year to once a decade.
EXAMPLES
[0144] The examples below are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof.
Materials and Methods
2D Cell Culture
[0145] HAVECs and HUVECs were cultured in T75 flasks and then 8-well p-slides coated with 50 pg/mL of rat tail collagen I at 37 °C and 5% CO2. HAVECs and HUVECs were cultured in Endothelial Growth Media-2 (Sigma- Aldrich) with 1% penicillin-streptomycin and 10% fetal bovine serum. HUVECs were used from passage four to five and HAVECs were used from passage three to four. PAVICs and PAVECs were cultured in T75 flasks and then 8-well p- slides at 37 °C and 5% CO2. PAVICs and PAVECs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) with 1% penicillin-streptomycin and 10% fetal bovine serum. The PAVEC flasks were coated with 50 pg/mL of rat tail collagen I. PAVICs were used from passage four to six and PAVECs were used from passage three to five. Cells were passaged from a T75 into 8-well p-slides at a seeding density of 5 * 104 cells/well.
3D Hydrogel Cell Culture
[0146] Circular springs were placed in molded wells made of polydimethylsiloxane (PDMS). PAVICs were encapsulated within a neutralized collagen gel suspension (2 mg/mL) in the PDMS wells at a density of 1,000,000 cells/mL. A neutralized collagen gel suspension was made with rat tail collagen I, sterile 18 MQ water, fetal bovine serum, 5 x DMEM, and 0.1 M HC1. PAVECs were topically seeded onto the PAVICs at a density of 50,000 cells/cm2. Hydrogels were incubated in DMEM with 1% penicillin-streptomycin and 10% fetal bovine serum for 24 hours at 37 °C and 5% CO2 before osteogenic media (OGM) was applied. To create OGM, DMEM was supplemented with 1% penicillin-streptomycin, 10% fetal bovine serum, lOmmol/L P-glycerophosphate, 50 pg/mL L-ascorbic acid, and 100 nM dexamethasone. The hydrogels were cultured for up to seven days.
Cadherin-11 Antibody Conjugation to PLGA Nanoparticles
[0147] PLGA nanoparticles containing either coumarin 6 (C6) dye, BAY11-7082, or Cy5.5 dye were made according to the methods outlined in (Menon et al., “Dual -Drug Containing Core-Shell Nanoparticles for Lung Cancer Therapy,” Scientific Reports 7: 13249 (2017); Yaman et al., “Melanoma Peptide MHC Specific TCR Expressing T-Cell Membrane Camouflaged PLGA Nanoparticles for Treatment of Melanoma Skin Cancer,” Frontiers in Bioengineering and Biotechnology 8:943 (2020); Menon et al., “Polymeric Nanoparticles for Pulmonary Protein and DNA Delivery,” Acta Biomaterialia 10: 2643-2652 (2014), all of which are hereby incorporated by reference in their entirety) Synthesis of dye-loaded or drug-loaded PLGA nanoparticles requires dissolving PLGA 50:50 and dye or BAY11-7082 in various solvents, like dichloromethane and poly (vinyl alcohol), then sonicating the solution (Menon et al., “Polymeric Nanoparticles for Pulmonary Protein and DNA Delivery,” Acta Biomaterialia 10: 2643-2652 (2014), which is hereby incorporated by reference in its entirety). After isolating the PLGA nanoparticles, they are freeze-dried for 24 hours (Menon et al., “Polymeric Nanoparticles for Pulmonary Protein and DNA Delivery,” Acta Biomaterialia 10: 2643-2652 (2014), which is hereby incorporated by reference in its entirety). To conjugate antibodies to the nanoparticles, 3 mg of nanoparticles were dissolved in 500 pL of pH 8.2 NaHCCL and incubated at room temperature for two hours with 10 pg of cad-11 antibody (Invitrogen). 500 pL of Tris-NH2 was subsequently added to the nanoparticle suspension and the nanoparticles were centrifuged at 15,000 rpm for 30 min. The supernatant was discarded and the nanoparticle pellet was resuspended in 1 mL of pH 7.4 IX PBS. The nanoparticles were centrifuged again at 15,000 rpm for 30 min. The supernatant was discarded and the cad-11 nanoparticles were resuspended in the desired amount of IX PBS or cell media. The cad-11 antibody was conjugated to the nanoparticles 24 hours before the first use of the nanoparticles. The cad-11 nanoparticles were stored at -20°C for up to two weeks. The doses of BAY11-7082 released by the cad-11 BAY nanoparticles was calculated using the release curve provided in FIG. 3B.
Alizarin Red Staining
[0148] 3D PAVEC-PAVIC collagen hydrogels were fixed in methanol for 30 minutes and the hydrogels were rinsed three times for 15 minutes in IX PBS with calcium and magnesium. The hydrogels were then stained with 2% Alizarin Red Stain (ARS) for 30 minutes. The ARS was aspirated and the hydrogels were rinsed three times for 15 minutes in 18 M water. Each hydrogel was imaged using a Zeiss Discovery V.20 Stereoscope. ImageJ was used to identify calcific nodules and to measure the compaction of the area of the hydrogels.
Hematoxylin and Eosin Staining
[0149] Paraffin-embedded 3D PAVEC-PAVIC hydrogels were sectioned (10pm thick). The sections were deparaffinized by performing three, three-minute washes in D-limonene. The sections were subsequently rehydrated by placing them in 100% ethanol, followed by 95% ethanol, 70% ethanol, then tap water for three minutes each. Sections were stained in Harris’ Alum Hematoxylin (Sigma- Aldrich) for ten minutes and afterward, washed in tap water for three minutes. The sections were placed in 1% acid alcohol for 20 seconds followed by a 90-second wash in tap water, a minute wash in 0.2% ammonia water, a 90-second wash in tap water, a minute wash in 70% ethanol, and a minute wash in 1% alcoholic Eosin Y (Sigma-Aldrich). The sections were then placed in 95% ethanol for two minutes, 100% ethanol for two minutes, and D-limonene for three minutes. One drop of Permount Mounting Medium (Invitrogen) was placed onto the stained sections and the slides were covered with a coverslip. The slides were imaged using a Zeiss Discovery V.20 Stereoscope.
Calcium Plate Reader Assay
[0150] The 3D PAVEC-PAVIC collagen hydrogels stained with ARS were cut out from the PDMS wells using a 4 mm biopsy punch and each sample was incubated for 24 hours at 60 °C in 210pL of 5 N hydrochloric acid. The hydrogels were centrifuged at 10,000 rpm for 15 minutes. To create a melting curve, a serial dilution of ARS was pipetted into a 96-well plate. Then, 100 pL of each hydrogel gel supernatant was pipetted into two wells in the 96-well plate. A plate reader was used to measure the absorbance of the samples at 490 nm and the results were analyzed using R.
Mouse Models
[0151] ApoE mice (Jackson Laboratory- stock no. 002052), alternatively known as C57BL/6J-congenic ApoetmlUnc mice, are homozygous, mutant knockouts (002052 - B6.129P2- Apoe J, which is hereby incorporated by reference in its entirety). ApoE -/mice were fed a high- fat diet (Harlan Teklad No. TD88137), consisting of 42% of calories from fat and 0.25% cholesterol, to drive atherosclerotic plaque formation (Sasso et al., “The Apoe-/- Mouse Model: a Suitable Model to Study Cardiovascular and Respiratory Diseases in the Context of Cigarette Smoke Exposure and Harm Reduction,” Journal of Translational Medicine 14: 146 (2016); (Meir and Leitersdorf, “Atherosclerosis in the Apolipoprotein E-Deficient Mouse: a Decade of Progress,” Arteriosclerosis, Thrombosis, and Vascular Biology 24: 1006-1014 (2004), all of which are hereby incorporated by reference in their entirety). Genotyping
[0152] A tissue sample was collected from the ear of each ApoE -/- mouse. To isolate the DNA, the ear tissue samples were submerged in 50 pL of Extracta DNA Prep reagent (QuantaBio) and the samples were incubated at 90°C for 30 minutes. 50 pL of the stabilizing reagent (QuantaBio) was then applied to each sample. GoTaq Green Master Mix (Promega), nuclease-free water, a forward and reverse primer, and the DNA sample were combined. The samples underwent PCR according to the settings outlined by The Jackson Laboratory in their ApoE -/- genotyping protocol (Protocol 22364-Apoe, which is hereby incorporated by reference in its entirety). The samples were ran through a 1% agarose gel with SYBR Safe DNA Gel Stain (Invitrogen). The gel was imaged using the Bio-Rad ChemiDoc Imaging System.
Immunohistochemistry
[0153] Paraffin-embedded 3D PAVEC-PAVIC hydrogels were sectioned (10pm thick). The sections were deparaffinized by performing three, three-minute washes in D-limonene. The sections were then rehydrated by placing them in 100% ethanol, followed by 95% ethanol, 70% ethanol, then deionized water for three minutes each. The rehydrated sections were placed in a Coplin with boiling IX citrate buffer solution and heated in the Coplin on high for two minutes in a pressure cooker. The Coplin was cooled to room temperature and the samples were permeabilized with 0.3% Triton-X for 15 minutes. The slides were washed three times for 10 minutes in PBST (IX PBS with 0.05% Tween 20) and the samples were blocked in IX PBS with 1% bovine serum albumin (BSA), 0.3 M glycine, and 5% goat serum. After one hour of incubation at room temperature and three, 10-minute washes in PBST, the samples were incubated overnight at 4°C with a primary antibody diluted in PBST, 1% BSA, and 0.3 M glycine. The samples were washed three times for 15 minutes in PBST. The secondary antibodies were species-specific, raised in goat, and conjugated to an Alexa Fluor dye. The secondary antibody was added to the sample in a 1 :500 (4 pg/mL) dilution and the samples were incubated for one hour at room temperature. Finally, the samples were washed three times for 15 minutes in PBST and counterstained using Nuclear Blue (Invitrogen). ProLong Gold Antifade Mountant (Invitrogen) was placed onto each slide with a coverslip. I imaged the slides using a Zeiss Confocal 710.
Table 1: Antibodies
Figure imgf000035_0001
Figure imgf000036_0001
In Vivo Biodistribution Study
[0154] Mice were anesthetized by setting the isoflurane vaporizer at 3.5%. Following, 5.6 mg of cad-11 Cy5.5 nanoparticles were retro-orbitally injected into the mouse. Two hours and four hours after injection, the mouse was imaged using the IVIS Spectrum. To image the mouse using the IVIS Spectrum, the mouse was anesthetized by setting the isoflurane vaporizer at 3.5%, eye ointment was applied to the mouse, and then the mouse was quickly transferred to the induction chamber within the IVIS Spectrum. Once the mouse was in the induction chamber, the isoflurane vaporizer was reduced to 1.5% and an induction flow of 500 cc/min was used. Each IVIS Spectrum image was taken using the fluorescence and epi-illumination settings. Following, the mouse was euthanized using CO2 and the heart and liver were dissected. All Cornell IACUC and CARE guidelines for animal handling and procedures were adhered to.
Statistical Methods
[0155] The data was first assessed to determine whether it met the assumptions of a oneway ANOVA. A Shapiro-Wilks test and Levene’s test for assessing normality and variance, respectively, indicated that the data in these figures did not meet the assumptions of a one-way ANOVA. Therefore, the non-parametric Kruskal-Wallis test was used to determine if there were significant differences among groups. To identify significant pairwise comparisons, a Dunn’s test was used with a Bonferroni correction. For all statistical tests, a = 0.05. R software was used for all statistical analysis.
Example 1 - Cad -11 BAY Nanoparticle Release Profile
[0156] The cad-11 BAY nanoparticle release profile (FIG. 3B) was used to determine the amount of cad-11 BAY nanoparticles needed to achieve a particular dose of BAY 11-7082 and the incubation time of the cad-11 BAY nanoparticles with the hydrogels. Within 48 hours, the cad-11 BAY nanoparticles released 82 pg of BAY 11-7082 per 1 mg of nanoparticles. The majority of the drug was released by the 48-hour time point. The hydrogels in OGM tear off the springs around day eight, with calcific nodule formation observed around day four or five. Therefore, applying the cad-11 BAY nanoparticles on day five for 48 hours until day seven maximized the amount of drug released from the nanoparticles while ensuring the majority of the hydrogels had not tom off the springs and that visible calcification had occurred.
Example 2 - Cad-11 Nanoparticles Show Cell-Specific Targeting
[0157] First, the binding specificity of cad-11 antibody-conjugated PLGA nanoparticles loaded with coumarin 6 dye (cad-11 C6 nanoparticles) was assessed (which are shown in FIG. 2A). A methodological overview of this objective is pictured in FIG. 4. Since cad-11 is endogenously expressed at higher levels in the aortic valve endothelium compared to other endothelial populations, it was hypothesized that the cad-11 C6 nanoparticles would target aortic valve endothelial cells more specifically than endothelial cells originating from other tissue.
[0158] The targeting specificity of cad-11 nanoparticles was assessed by administering 50 pg/mL of cad-11 C6 nanoparticles suspended in IX PBS to HAVECs, HUVECs, PAVICs, and PAVECs. After 3.5 hours of incubation with the cells, each well was washed with IX PBS before imaging. This incubation time was selected based on the release kinetics of the nanoparticles, determined in FIG. 3B. All cell types displayed fluorescence on imaging. To analyze each image, the cell counting function in ImageJ was used to count the total number of cells in each image. This first required enhancing the contrast of the image significantly so that the border of all objects in the image was visible and could be detected by the cell counting function. Then, on the original image, the threshold function in ImageJ was used to sort the pixels in each image into either the background or the foreground of the image. The cells in each image that displayed enough fluorescence to be sorted into the foreground were counted. Lastly, the proportion of cells above the fluorescence threshold was calculated by dividing the number of cells above the fluorescence threshold by the total number of cells in that image. There was a significantly lower proportion of HUVECs above the fluorescence threshold than all other cell types tested, indicating that the HUVECs were targeted less by the cad-11 C6 nanoparticles. There was no significant difference among the proportion of HAVECs, PAVICs, and PAVECs above the fluorescence threshold which indicates no significant difference in cad- 11 C6 nanoparticle targeting among these cell types.
[0159] This experiment addressed whether cad-11 nanoparticles targeted the desired cell type. FIGS. 5A-5E illustrates that there was a greater proportion of HAVECs exhibiting more intense fluorescence than HUVECs. This means that the HAVECs were targeted more by the cad- 11 C6 nanoparticles than the HUVECs, as hypothesized. As previously mentioned, healthy humans have a significantly higher level of cad-11 in the aortic valve endothelium compared to other endothelial populations which is why the HAVECs were expected to be targeted by the cad-11 C6 nanoparticles more than the HUVECs (Zhou et al., “Cadherin-11 Expression Patterns in Heart Valves Associate With Key Functions During Embryonic Cushion Formation, Valve Maturation and Calcification,” Cells, tissues, organs 198:300-10 (2013), which is hereby incorporated by reference in its entirety). It is worth noting that a small proportion of the HUVECs displayed a significant amount of fluorescence in each image, indicating that an undesired cell population was targeted by these nanoparticles. This is not unexpected since cad- 11 expression is not restricted to the aortic valve. However, this could raise concerns about off- target effects in vivo. While this is a valid worry, this experiment illustrates that cad-11 nanoparticles target undesired cell types significantly less than the desired cell type.
[0160] Additionally, cad-11 C6 nanoparticles were administered to PAVECs and PAVICs to validate that PAVECs and PAVICs would be sufficiently targeted by cad-11 nanoparticles. Porcine cells were to be used in subsequent 3D cell culture models since this cell type has previously been shown to be a robust model for studying calcification in vitro (Gee et al., “NFKB (Nuclear Factor K-Light-Chain Enhancer of Activated B Cells) Activity Regulates Cell-Type-Specific and Context-Specific Susceptibility to Calcification in the Aortic Valve,” Arterioscler. Thromb. Vase. Biol. 40:638-655 (2020), which is hereby incorporated by reference in its entirety). Both porcine cell types displayed similar proportions of cells above the fluorescence threshold when compared to HAVECs (FIGS. 5C-5E). The comparable targeting of PAVECs and PAVICs by the cad-11 C6 nanoparticles was expected since previous findings suggest that normal levels of cad-11 expression in aortic valve endothelial cells and aortic valve interstitial cells plays an essential role in maintaining aortic valve health (Sung et al., “Cadherin- 11 Overexpression Induces Extracellular Matrix Remodeling and Calcification in Mature Aortic Valves,” Arteriosclerosis, Thrombosis, and Vascular Biology 36: 1627-1637 (2016); Gee et al., “NFKB (Nuclear Factor K-Light-Chain Enhancer of Activated B Cells) Activity Regulates Cell- Type-Specific and Context-Specific Susceptibility to Calcification in the Aortic Valve,” Arterioscler. Thromb. Vase. Biol. 40:638-655 (2020), all of which are hereby incorporated by reference in their entirety). Together, these data provide evidence that PAVECs and PAVICs would be sufficiently targeted by cad-11 nanoparticles in more complex 3D cell culture models. Example 3- Evaluating the Effect of Cad-11 BAY Nanoparticles in PAVEC-PAVIC Hydrogels
[0161] Next, the effect of cad-11 antibody-conjugated PLGA nanoparticles loaded with BAY 11-7082 (cad-11 BAY nanoparticles) (FIG. 2B), on porcine aortic valve endothelial (PAVECs) and porcine aortic valve interstitial cells (PAVICs) exhibiting calcific nodule formation was evaluated. FIG. 6 shows a schematic of this objective. It was hypothesized that diseased 3D PAVEC-PAVIC collagen hydrogels treated with cad-11 BAY nanoparticles would exhibit less calcification than untreated, diseased hydrogels. Additionally, the PAVECs and PAVICs within the untreated hydrogels were expected to display inflammatory phenotypes indicative of EndMT and changes in the levels of primary cell type markers like VE-cadherin and a-smooth muscle actin (a- SMA).
[0162] 3D PAVEC-PAVIC collagen hydrogels were cultured for seven days in either GM, OGM, or OGM for DI -3 and then 0GM+ BAY 11-7082 for D3-7 (OGM+BAY) (FIG. 7 A). At day 7, hydrogels were stained with ARS to evaluate calcific nodule formation. A marked visible reduction in calcific nodule number was observed in the OGM+BAY treated group compared to the OGM group. This shows the efficacy and rescue effect of BAY 11-7082.
[0163] Following, 3D PAVEC-PAVIC collagen hydrogels were cultured for seven days in either general GM or OGM. After treatment with OGM for five days, the hydrogels displayed calcific nodule formation. At day 5, an interventional lOpM dose of BAY11-7082 was applied to the hydrogels via cad-11 BAY nanoparticles. The cad-11 BAY nanoparticles were incubated with the hydrogels for 48 hours. These cad-11 BAY nanoparticle-treated hydrogels are referred to as the OGMB group. On day seven, the hydrogels were stained with ARS (FIG. 7B). The small, dark spots homogeneously spread throughout the magnified image of the GM hydrogel are likely endothelial cells. The large, heterogenous regions of darker staining are calcific nodules in the OGM and OGMB groups. The amount of calcium in each hydrogel was quantified using a plate reader assay. There was not a significant reduction in calcium concentration between the OGM and OGMB groups.
[0164] The interventional effectiveness of cad-11 BAY nanoparticles in diseased 3D PAVEC-PAVIC collagen hydrogels was assessed. Previous studies have shown a range of BAY1 1-7082 doses to significantly reduce NF-KB expression without reducing cell viability. Lee et al. found that in macrophage cell lines, a 20 pM dose of BAY11-7082 was the highest dose that significantly reduced NF-KB expression without significantly reducing cell viability (Lee et al., “BAY 11-7082 Is a Broad-Spectrum Inhibitor with Anti-Inflammatory Activity against Multiple Targets,” Mediators of Inflammation (2012), which is hereby incorporated by reference in its entirety). Alternatively, Wang et al. determined that doses of BAY 11-7082 6.5pM and above significantly decreased cell viability in a dose-dependent manner in healthy liver cells (Wang et al., “Nf-Kb Inhibition Alleviates Carbon Tetrachloride-Induced Liver Fibrosis Via Suppression of Activated Hepatic Stellate Cells,” Experimental and Therapeutic Medicine 8:95- 99 (2014), which is hereby incorporated by reference in its entirety). Hu et al. found a 2.5 pM dose of BAY11-7082 was sufficient to significantly reduce the viability of four different uveal melanoma cell lines : (Hu et al., “The Pharmacological NF-KB Inhibitor BAY11-7082 Induces Cell Apoptosis and Inhibits the Migration of Human Uveal Melanoma Cells,” International Journal of Molecular Sciences 13: 15653-15667 (2012), which is hereby incorporated by reference in its entirety). Since the cad-11 BAY nanoparticles release BAY 11-7082 once endocytosed by the cell, meaning more of the drug makes it to the target cells, it was anticipated a smaller dose of the drug would have a greater effect on cell viability and NF-KB activity compared to systemically delivered BAY 11-7082. Based on this information, an initial dose of 10 pM BAY11-7082 was used. This dose was expected to be high enough to see a reduction in disease progression without being significantly detrimental to cell viability.
[0165] Interestingly, an interventional 10 pM dose of BAY11-7082 delivered via cad-11 BAY nanoparticles did not significantly reduce calcific nodule formation in the hydrogels treated with OGM for seven days, as indicated by the calcium concentration in the OGMB group (FIG. 7C). One plausible explanation for this finding is that the diseased hydrogels had reached a ‘point of no return’ at which therapies are unlikely to significantly stop the progression of calcification (Shuvy et al., “Hyperphosphatemia Is Required for Initiation but Not Propagation of Kidney Failure-Induced Calcific Aortic Valve Disease,” American Journal of Physiology-Heart and Circulatory Physiology 317:H395-H704 (2019); Porras et al., “Creation of Disease-Inspired Biomaterial Environments to Mimic Pathological Events in Early Calcific Aortic Valve Disease,” Proceedings of the National Academy of Sciences 115: E363-E371 (2018), all of which are hereby incorporated by reference in their entirety). Although it is unknown whether a true ‘point of no return’ exists in CAVD or what molecular pathways might contribute to this phenomenon, these diseased hydrogels may have reached a point in disease progression at which a high dose of BAY11-7082 could not slow down or prevent further calcific nodule formation. Additionally, the OGM and OGMB groups showed a large variance in calcium concentration within each group and the negative control, the GM group, showed significantly more calcification than both the OGM group and the OGMB group. The seemingly high calcium concentration in the GM group, which should have shown no calcium, could indicate that the pH in the hydrogel is not ideal. A basic pH would darken the color of the hydrogel, altering the reading from the plate reader. Moreover, it is unlikely that the hydrogels treated with GM actually calcified as the plate reader assay suggests since there were no visual signs of calcific nodule formation like there were in the OGM and OGMB groups.
Example 4- Evaluating the Effect of Cad-11 BAY Nanoparticles on Cellular Compaction and EndMT Progression
[0166] By day seven, hydrogels treated with GM and 0 pM, 5 pM, 10 pM, and 20 pM of BAY1 1-7082 all displayed cellular compaction (FIG. 8A). The hydrogels treated with 0 pM, 5 pM, 10 pM, and 20 pM of BAY11-7082 via cad-11 BAY nanoparticles showed increasingly less compaction as the dose increased. The compaction proportion in FIG. 8B is the ratio between the circumference of the hydrogel on day seven and the circumference of the hydrogel on day one. The positive control was the 0 pM group and the negative control was the GM group. The positive control showed significantly more compaction than the negative control.
Cellular compaction was used as an additional metric of disease progression in 3D PAVEC- PAVIC collagen hydrogels. Tissue compaction is an essential morphogenetic process characterized by mechanical interactions among cells that cause shrinkage of the tissue (Turlier and Maitre, “Mechanics of Tissue Compaction,” Seminars in Cell & Developmental Biology 47- 48: 110-117 (2015), which is hereby incorporated by reference in its entirety). PAVEC-PAVIC hydrogels treated with OGM have been shown to compact significantly (Gee et al., “Valve Endothelial-Interstitial Interactions Drive Emergent Complex Calcific Lesion Formation In Vitro,” Biomaterials 269: 120669 (2021), which is hereby incorporated by reference in its entirety). The results from this study are in line with these previous findings. Hydrogels treated with OGM and 0 pM, 5 pM, 10 pM, and 20 pM of BAY11-7082 or GM showed a decrease in compaction in a dose-dependent manner (FIG. 8B), however, there was no significant difference in compaction between the treatment groups and the controls. While this is not strong evidence, these findings could indicate a reduction in disease progression. IHC staining of these hydrogels was largely inconclusive due to the fragility of the hydrogels during the embedding process (FIGS. 9 and 10), but staining for protein markers, like cr-SMA and VE-cadherin, could indicate the extent to which different doses of BAY11-7082 affected the progression of EndMT and consequently disease progression. Alternatively, and more plausibly, the decrease in compaction of hydrogels treated with higher doses could be explained by increased cell toxicity.
Hematoxylin & Eosin staining visually showed a decrease in cell density as the dose of BAY11- 7082 increased (FIG. 11). This could indicate that high doses of BAY11-7082 decreased cellular proliferation which resulted in decreased compaction. [0167] The integrity of the hydrogels was comprised which makes it difficult to know where the PAVECs and PAVICs are within each sample. However, each dose group shows a relatively heterogeneous spread of cells. There are noticeably longer, stringier cells and more rounded cells which match the morphology of PAVICs and PAVECs, respectively. The long, dark purple strands in the 0 pM and 5 pM sections are potentially large regions of collagen that have folded in on itself during the embedding process. The cell density visually decreases as the dose of BAY1 1-7082 increases. This potentially suggests that higher doses of BAY11-7082 decrease rates of cell division. This staining was done before IHC staining to generally assess the state of the hydrogel, cell morphology, and cell density.
[0168] Immunostaining of 3D PAVEC-PAVIC collagen hydrogel sections indicated a- SMA and VE-cadherin expression which are protein markers of PAVICs and PAVECs respectively (FIGS. 9 and 10) (Ma et al., “Endothelial-to-Mesenchymal Transition in Calcific Aortic Valve Disease,” Acta. Cardiol. Sin. 36: 183-194 (2020), which is hereby incorporated by reference in its entirety). Hydrogel sections were irregularly shaped despite fixation in 4% paraformaldehyde (PF A) overnight.
[0169] Unfortunately, the hydrogels lost their structural integrity during the paraffin embedding processes, making it difficult to identify where the PAVECs and PAVICs were originally located within the hydrogels and what protein markers they expressed or lacked after treatment with different doses of BAY11-7082. The PAVECs in the hydrogels were expected to show increasing levels of VE-cadherin as the dose of BAY11-7082 increased. A higher amount of VE-cadherin expression in PAVECs would have indicated that those cells had not lost their endothelial phenotype, begun undergoing EndMT, and contributed to disease progression. Conversely, the amount of cr-SMA expression was expected to decrease as the dose of BAY11- 7082 increased since fewer PAVECs would be transitioning to a mesenchymal phenotype. If the integrity of the hydrogels had been preserved, it was expected to see more distinct regions of a- SMA and VE-cadherin expression to mirror where the PAVECs and PAVICs were seeded in each hydrogel.
Example 5- Effects of BAY 11-7082 on Cellular Remodeling, Calcific Nodule Formation, and Co-Cultured Spring Gels
[0170] Area compaction is a measurement of cellular tissue remodeling. Greater collagen and cellular remodeling occurs to a greater degree under osteogenic disease conditions. BAY 11- 7082 shows promising effects with significantly decreased tissue compaction when BAY 11- 7082 is given from Day 1 vs at CAVD, showing similar results to the General media, the healthy control group (FIG. 13 A). In line with the decreased cellular remodeling/ compaction that occurs when BAY 11-7082 treatment is given at Day 1, the number of calcific nodules can be significantly reduced and compared to healthy conditions when treatment is given Day 1. Nodule number in the OGM + Bay 11 given at CAVD diagnosis shows a decreased mean number of nodules compared to OGM conditions. For FIG. 14, images were taken after 7 days of incubation of co-culture PAVEC/PAVIC spring gels. Row one shows general media healthy control. Row two shows osteogenic media conditions given Day 1. Row 3 shows OGM + Bayl 1 given at Day 1 - shows no presence of CAVD calcific nodules in the matrix. Row 4 shows OGM + Bay 11 given at the diagnosis of CAVD calcific nodule presentation.
Prophetic Example 1- Effect of Cad-11 BAY Nanoparticles In Vivo
[0171] The efficacy of cad-11 BAY nanoparticles could be evaluated in vivo using atherosclerosis-prone apolipoprotein E-deficient (ApoE -/-) mouse models. ApoE mice fed a high-fat diet develop hypercholesterolemia which drives atherosclerotic plaque formation by 32 weeks (Sasso et al., “The Apoe-/- Mouse Model: a Suitable Model to Study Cardiovascular and Respiratory Diseases in the Context of Cigarette Smoke Exposure and Harm Reduction,” Journal of Translational Medicine 14: 146 (2016), which is hereby incorporated by reference in its entirety). Additionally, ApoE mice on a high-fat diet show increased total plasma cholesterol levels and moderately increased triglyceride levels, providing a systemic proinflammatory environment that is ideal for simulating CAVD (Sasso et al., “The Apoe-/- Mouse Model: a Suitable Model to Study Cardiovascular and Respiratory Diseases in the Context of Cigarette Smoke Exposure and Harm Reduction,” Journal of Translational Medicine 14: 146 (2016); Meir and Leitersdorf, “Atherosclerosis in the Apolipoprotein E-Deficient Mouse: a Decade of Progress, " Arteriosclerosis, Thrombosis, and Vascular Biology 24: 1006-1014 (2004); 002052 - B6.129P2-Apoe/J, all of which are hereby incorporated by reference in their entirety). FIG. 12 illustrates a methodological overview of a biodistribution study to determine whether cad-11 antibody-conjugated PLGA nanoparticles loaded with Cy5.5 (cad-11 Cy5.5 nanoparticles) hone to the aortic valve in vivo.
[0172] If the cad-11 Cy5.5 nanoparticles sufficiently target the aortic valve, increasing doses of BAY 11-7082 delivered via cad-11 BAY nanoparticles would be administered to ApoE - /- mice to determine a half-maximal effective concentration dose (ECso). Then, the ECso dose would be administered to ApoE -I- mice and their heart health would be assessed using ultrasound imaging and immunohistochemistry (IHC). It was hypothesized that the ApoE -/- mice treated with the ECso dose of BAY 11-7082 via cad-11 BAY nanoparticles would have smaller calcific lesions, less inflammation, and reduced left ventricle blood ejection velocity compared to untreated mice. Ultimately, these objectives could be used to elucidate the potential of cad-11 BAY NPs as a novel therapeutic to slow CAVD development and progression.
[0173] Previous studies that have used BAY11-7082 in vivo have used a 5mg/kg dose (Zhao et al., “Bayl 1-7082 Attenuates Murine Lupus Nephritis Via Inhibiting Nlrp3 Inflammasome and NF-KB Activation,” International Immunopharmacology 17: 116-122 (2013); Chen et al., “BAY 11-7082, a Nuclear Factor-KB Inhibitor, Induces Apoptosis and S Phase Arrest in Gastric Cancer Cells,” Journal of Gastroenterology 49:864-874 (2014), all of which are hereby incorporated by reference in their entirety). Since cad-11 nanoparticles showed cellspecific targeting in vitro, a lower dose of BAY 11-7082 is expected to be more potent than BAY 11-7082 given systemically. To assess the heart health of the mice given different doses, cardiac ultrasounds will be performed on Axe ApoE mice to obtain left ventricle ejection velocity readings before and after treatment. The hearts will then be disected and stained for protein markers to evaluate the extent of EndMT progression, valve calcification, and cellular toxicity.
[0174] Once the ECso was determined, the corresponding dose would be administered to a larger cohort of ApoE mice that are at least 32 weeks old, on a high-fat diet, and show signs of elevated left ventricle outflow blood ejection velocity on ultrasounds. The resulting posttreatment heart health of these mice would be assessed. These in vivo studies will offer greater insight into the potential therapeutic uses for cad-11 nanoparticles. Moreover, these in vivo studies will likely raise many questions about the off-target effects of the nanoparticles, how to scale down the dose of BAY11-7082 when it is delivered via cad-11 nanoparticles, and other potential limitations of this novel therapy.
Discussion of Examples 1-5
[0175] Interpreting the calcium plate reader assay and compaction data together illustrates a potential sequence of diseased cellular states in CAVD progression. On the one hand, a lack of significant reduction in calcific nodule formation in hydrogels treated with a 10 pM dose of BAY1 1-7082 suggested no significant reduction in disease progression. On the other hand, hydrogels treated with various doses of BAY11-7082 showed a decrease in compaction in a dosedependent manner, potentially suggesting a reduction in disease progression. Specifically, the hydrogels treated with a 10 pM dose of BAY11-7082 showed cellular compaction yet there was no reduction in calcific nodule formation in these hydrogels. These findings can be reconciled by supposing that a reduction in hydrogel compaction precedes a reduction in calcific nodule formation. Sung et al. found that cell-matrix compaction and cell-cell adhesivity, both of which contribute to hydrogel compaction, are conducive to calcific nodule formation (Sung et al., “Cadherin-11 Overexpression Induces Extracellular Matrix Remodeling and Calcification in Mature Aortic Valves,” Arteriosclerosis, Thrombosis, and Vascular Biology 36: 1627-1637 (2016), which is hereby incorporated by reference in its entirety). So, it is plausible that a 10 pM dose of BAY11-7082 decreased cell-matrix compaction and cell-cell adhesivity but not enough to decrease subsequent calcific nodule formation.
[0176] Overall, these experiments have established the groundwork for future experiments to further determine the potential therapeutic use of cad-11 BAY nanoparticles to slow the progression and development of CAVD in vivo. The use of cad-11 C6 nanoparticles in vitro suggested that cad-11 nanoparticles show cell-specific targeting. The experiments evaluating the effect of cad-11 BAY nanoparticles in diseased 3D PAVEC-PAVIC collagen hydrogels suggested repetitions of this experiment with the changes I proposed would greatly clarify the potential role of this therapy in slowing the progression of CAVD in vitro.
Completion of the in vivo studies outlined would further elucidate the potential therapeutic role of cad-11 BAY nanoparticles while likely raising many more questions that warrant further investigation. In sum, this project explores a novel CAVD therapy that could one day provide a non-invasive treatment option for patients who cannot safely undergo major surgery.
[0177] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED IS:
1. A nanoparticle for treating and/or diagnosing calcific aortic valve disease (CAVD), the nanoparticle comprising: a nanoparticle core; and a targeting molecule associated with an exterior surface of the nanoparticle core.
2. The nanoparticle of claim 1, wherein the nanoparticle core comprises one or more types of diagnostic and/or therapeutic molecules.
3. The nanoparticle of claim 2, wherein the nanoparticle core encapsulates one or more types of diagnostic and/or therapeutic molecules.
4. The nanoparticle of claim 2 or claim 3, wherein the nanoparticle core comprises one or more types of diagnostic molecules.
5. The nanoparticle of claim 4, wherein the one or more types of diagnostic molecules comprise one or more diagnostic dyes.
6. The nanoparticle of claim 5, wherein the diagnostic dyes are selected from the group consisting of C6 fluorescent dye and Cy 5.5 fluorescent dye.
7. The nanoparticle of claim 2 or claim 3, wherein the nanoparticle core comprises one or more types of therapeutic molecules.
8. The nanoparticle of claim 7, wherein the one or more types of therapeutic molecules comprise a drug for treating CAVD.
9. The nanoparticle of claim 7 or claim 8, wherein the one or more types of therapeutic molecules comprise an anti-inflammatory drug.
10. The nanoparticle of claim 9, wherein the anti-inflammatory drug comprises Bay 11-7082.
11. The nanoparticle of any one of the preceding claims, wherein the targeting molecule comprises a molecule that targets an endothelial molecule that is indicative of CAVD.
12. The nanoparticle of any one of the preceding claims, wherein the targeting molecule comprises a molecule that targets a portion of Cadherin 11 protein.
13. The nanoparticle of any one of the preceding claims, wherein the targeting molecule comprises an anti -Cadherin 11 antibody.
14. The nanoparticle of any one of the preceding claims, wherein the nanoparticle comprises more than one type of targeting molecule.
15. The nanoparticle of any one of the preceding claims, wherein the nanoparticle core comprises a biodegradable polymer.
16. The nanoparticle of any one of the preceding claims, wherein the nanoparticle core comprises polylactic-co-glycolic acid.
17. A therapeutic composition comprising: a nanoparticle according to any one of the preceding claims; and a pharmaceutically acceptable carrier.
18. A method of treating and/or diagnosing CAVD, the method comprising: contacting a nanoparticle of any one of claims 1-17 with endothelial tissue of a subject at risk for CAVD.
19. The method of claim 18, further comprising: detecting a presence of the nanoparti cl e(s) on the subject’s endothelial tissue.
20. The method of claim 19, further comprising: determining a CAVD disease state based on presence of the nanoparticle(s) on the subject’s endothelial tissue.
21. The method according to claim any one of claims 18-20, wherein said contacting is carried out parenterally, topically, intravenously, orally, subcutaneously, intraperitoneally, intranasally, or by intramuscular means.
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