Engineered janus nanoparticles for field coverage chemoprevention of lung cancer

Abstract

A lung cancer chemoprevention drug delivery platform comprises a Janus nanoparticle comprising at least two chemopreventive agents for preventing / minimizing lung cancer or preventing recurrence of treated lung cancer. Further, an aerosolized composition is provided that comprises (a) at least one Janus nanoparticle configured to be aerosolized; (b) a bioactive protein inhibitor; (c)a modulator of at least one proinflammatory mediator; (d) an immune checkpoint inhibitor; and (e) short interfering sequence of double stranded RNA (siRNA) to modify proteins aberrantly upregulated in premalignant lung lesions. In another variation, methods for generating a lung cancer chemoprevention drug delivery platform for inhalation by a subject diagnosed with premalignant lung lesion is provided, where the drug delivery platform comprising: (a) a Janus nanoparticle, (b) a bioactive protein inhibitor; (c) a modulator of at least one proinflammatory mediator; (d) an immune checkpoint inhibitor; and (e) siRNA to modify proteins aberrantly upregulated in premalignant lung lesions.

Description

ENGINEERED JANUS NANOPARTICLES FOR FIELD COVERAGE CHEMOPREVENTION OF LUNG CANCERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 560,1 10, filed on March 1 , 2024 and U.S. Provisional Application No. 63 / 708,064, filed on October 16, 2024. The entire disclosures of the above applications are incorporated herein by reference.FIELD

[0002] The present disclosure relates to preventing, minimizing of a cancer, or preventing recurrence of cancer in a subject, such as for chemoprevention, and more particularly to a novel delivery system and compositions therefor.BACKGROUND

[0003] This section provides background information related to the present disclosure which is not necessarily prior art.

[0004] Lung cancer, which represents the top cancer killer worldwide, is projected to kill over 100,000 Americans annually. Many lung cancers (approximately 85 - 90%) are non-small cell lung carcinoma (NSCLC) and are comprised of either adenocarcinomas (adenoCA, predominant) or squamous cell carcinomas (SCCs, approximately 25-35% of all NSCLC). The etiology of lung cancer is often complex and regularly reflects a multitude of factors, such as, for example, exposure to inhaled carcinogens, e.g., burnt tobacco, asbestos, aerosolized hydrocarbons; the local pulmonary milieu, e.g., sustained inflammation or immunosuppression; and the individual's genetics. Despite improved surveillance techniques, e.g., CT imaging and use of high-definition white light / auto- fluorescent bronchoscopy, lung cancers are frequently diagnosed at late clinical stages, which can negatively impact patient mortality and morbidity.

[0005] This delay in diagnosis coupled with the fact that lung cancer is often multifocal renders curative lung resections for upper airway lung cancers (squamous cell and small cell carcinomas) impossible and conveys an abysmal prognosis. Notably, even when premalignant lung lesions are accurately diagnosed, their management ischallenging. Because even high-grade premalignant lung lesions (including the precursor lesions to lung SCC (PML) and adenocarcinoma (atypical adenomatous hyperplasia)) do not always progress to overt cancer, the benefits of local resection and associated morbidity are questionable. In addition, the realistic probability that additional synchronous or metachronous premalignant lesions will develop at other sites also reduces local resection benefits. The fact that NSCLC arises over time from recognized premalignant lesions, however, provides an opportunity for chemoprevention to disrupt the carcinogenic cascade. Notably, the underlying cellular and molecular perturbations have been well- characterized and include telomerase dysregulation, increased -catenin signaling and Wnt expression, and altered metabolic profiles that rely on oxidative phosphorylation and mitochondrial protein and electron transport. In addition, two endogenously produced cytokines, interleukins IL-6 and IL-8, also facilitate lung tumorigenesis via their angiogenic and pro-proliferative qualities. Also, pulmonary epithelial and stromal released IL-8 can establish a local immunosuppressive, tumor-enabling environment via its chemoattraction for myeloid-derived suppressor cells.

[0006] Traditional lung cancer chemoprevention focuses on systemically administered repurposed drugs designed to manage other conditions. This mismatch has been largely unsuccessful and corresponds to undesired side effects due to the non- selective / untargeted nature of administration. And even if the drugs are aerosolized for delivery to airways, it is no guarantee of a better prognosis. For example, a clinical trial employing aerosolized delivery of the anti-hypertensive iloprost demonstrated aerosolized delivery was tolerated, but failed to show any premalignant lung lesions histologic regression.

[0007] However, combinations of drugs selected for their abilities to modulate lung cancer promoting pathways delivered in an appropriate manner can lead to promising results. For instance, local delivery of the synthetic vitamin A derivative, fenretinide (4HPR), in conjunction with the humanized IL-6R inhibitor, tocilizumab (TCZ) inhibits tumorigenesis in vivo. Further, the triple combination of 4HPR, TCZ, and allosteric inhibitor of IL-8’s CXCR1 / 2 receptor, reparixin (REP), cooperatively suppresses cancer invasion.

[0008] While the mechanism of action of the receptor antagonist is straightforward, i.e., suppression of autocrine-paracrine signaling, reduced inflammation andangiogenesis, preservation of the local immune response, 4HPR’s chemopreventive effects are more complex. 4HPR not only possesses growth modulatory effects, but also demonstrates high affinity binding / inactivation of signaling kinases (e.g., FAK, Pyk2, STAT3, Wnt-p-catenin) and interference with cytoskeletal components necessary for invasion and migration.

[0009] Other studies have shown that 4HPR is a redox-active molecule and effectively disrupts mitochondrial electron transport. Another investigation, which assessed the effects of systemically administered 4HPR on expression of the human telomerase reverse transcriptase catalytic subunit (hTERT) in bronchial biopsy specimens of smokers, showed 4HPR significantly reduced hTERT expression.

[0010] Provided the extensive molecular perturbations in premalignant lung lesions, delivery of personalized combination chemoprevention to the target site would be advantageous to augment efficacy and reduce systemic drug-related side effects. Accordingly, a versatile technology that allows for preparation of novel types of drug carriers, such as nanoparticles like Janus nanoparticles (JNPs) that may be readily internalized by epithelial cells from a variety of sites, including the lungs, while simultaneously providing a robust platform for targeted delivery of chemopreventives, would be advantageous.SUMMARY

[0011] This section provides a general summary of the disclosure, and is not a comprehensive disclosure of its full scope or all of its features.

[0012] In certain aspects, the present disclosure relates to a multicompartmental or Janus nanoparticle (JNP)-based lung cancer chemoprevention drug delivery platform comprising: a Janus nanoparticle comprising at least two chemopreventive agents for preventing, minimizing, or treating lung cancer.

[0013] In one aspect, the Janus nanoparticle is morphologically anisotropic and the Janus nanoparticle comprises at least two chemically distinct compartments, wherein a first chemically distinct compartment comprises a first chemopreventive agent and a second chemically distinct compartment comprises a second chemopreventive agent.

[0014] In one aspect, the Janus nanoparticle comprises a protein.

[0015] In one further aspect, the Janus nanoparticle further comprises at least one protein in which at least one chemopreventive agent of the at least two chemopreventive agents is distributed.

[0016] In one further aspect, the at least one protein comprises albumin.

[0017] In one further aspect, the at least chemopreventive agent comprises fen retin ide.

[0018] In one aspect, the Janus nanoparticle comprises a polysaccharide.

[0019] In one further aspect, at least one chemopreventive agent of the at least two chemopreventive agents is distributed in the polysaccharide.

[0020] In one further aspect, the at least polysaccharide comprises chitosan.

[0021] In one further aspect, the at least chemopreventive agent comprises tocilizumab.

[0022] In one aspect, the Janus nanoparticle comprises a lipophilic drug.

[0023] In one aspect, the Janus nanoparticle comprises RNAi.

[0024] In one aspect, the Janus nanoparticle comprises an immune modulatory agent.

[0025] In one further aspect, the immune modulatory agent may be selected from a group consisting of: a checkpoint inhibitor, an immune-augmenting tyrosine kinase inhibitor, an immunostimulatory antibody, an activator of CD40, a histone deacetylase (HDAC) inhibitor, an immunostimulatory cytokine, and combinations thereof.

[0026] In one aspect, the immune modulatory agent may be selected from the group consisting of: avelumab, sunitinib, sorafenib, ipilimumab, interferon alpha, and combinations thereof.

[0027] In one aspect, at least one of the at least two chemopreventive agents is selected from the group consisting of: growth regulatory or anti-proliferative agents, antineoplastic / anti-miotic agents, chemotherapy agents, immune modulatory agents, transcription / translation modifier agents, growth factor and cytokine modulatory agents, biomolecules, nucleic acids, DNA, RNA, plasmids, short interfering sequence of double stranded RNA (siRNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, small nuclear RNA, single stranded DNA, CRISPR CAS-9, aptamers, antibodies, proteins, peptides, amino acids, sugars, targeting molecules, and combinations thereof.

[0028] In one aspect, the Janus nanoparticle (JNP) is configured to be aerosolized for delivery to at least one lung or a target region of a respiratory system of a subject.

[0029] In certain further aspects, the present disclosure relates to an aerosolized composition comprising:(a) at least one Janus nanoparticle;(b) a bioactive protein inhibitor;(c) a modulator of proinflammatory mediators;(d) an immune modulatory agent; and(e) siRNA to modify proteins aberrantly upregulated in premalignant lung lesions.

[0030] In one aspect, the at least one Janus nanoparticle has a zeta potential of greater than ±60 mV.

[0031] In one aspect, the at least one Janus nanoparticle has a nanoparticle aerodynamic diameter of at least 1 nm.

[0032] In one aspect, the at least one Janus nanoparticle has a nanoparticle capacity of at least 1 %.

[0033] In one aspect, the at least one Janus nanoparticle has a nanoparticle maintenance of the drug in a bioavailable / bioactive state during aerosolization of at least 1 %.

[0034] In one aspect, the immune modulatory agent may be selected from a group consisting of: a checkpoint inhibitor, an immune-augmenting tyrosine kinase inhibitor, an immunostimulatory antibody, an activator of CD40, a histone deacetylase (HDAC) inhibitor, an immunostimulatory cytokine, and combinations thereof.

[0035] In one aspect, the immune modulatory agent may be selected from the group consisting of: avelumab, sunitinib, sorafenib, ipilimumab, interferon alpha, and combinations thereof.

[0036] In one aspect, (a) the bioactive protein inhibitor comprises fenretinide, (b) the modulator of proinflammatory mediators comprises tocilizumab, (c) the immune modulatory agent is an immune checkpoint inhibitor comprising avelumab and / or an immunostimulatory antibody (anti-CTLA-4 agent) comprising ipilimumab, and (d) the siRNA comprises STAT3L

[0037] In yet further aspects, the present disclosure relates to a method for generating a Janus nanoparticle (JNP)-based lung cancer chemoprevention drug delivery platform for inhalation by a patient diagnosed with premalignant lung lesions. The JNP- based lung cancer chemoprevention drug delivery platform comprises:(a) Janus nanoparticles;(b) a bioactive protein inhibitor;(c) a modulator of proinflammatory mediators;(d) an immune modulatory agent; and(e) an siRNA to modify proteins aberrantly upregulated in premalignant lung lesions.The method may thus comprise combining the components (a) to (e) of the JNP-based lung cancer chemoprevention drug delivery platform together.

[0038] In one aspect, the Janus nanoparticles are delivered by an ultrasonic nebulizer unit.

[0039] In one aspect, the Janus nanoparticles withstand aerosolized shear forces as determined by post-aerosolized structural integrity bioactive drug retention of at least 1 %.

[0040] In one aspect, the Janus nanoparticles penetrate pulmonary surfactant delivered to target cells.

[0041] In one aspect, (a) the bioactive protein inhibitor comprises fenretinide, (b) the modulator of proinflammatory mediators comprises tocilizumab, (c) the immune modulatory agent comprises avelumab and / or ipilimumab, and (d) the siRNA comprises STAT3.

[0042] In one aspect, the immune modulatory agent may be selected from a group consisting of: a checkpoint inhibitor, an immune-augmenting tyrosine kinase inhibitor, an immunostimulatory antibody, an activator of CD40, a histone deacetylase (HDAC) inhibitor, an immunostimulatory cytokine, and combinations thereof.

[0043] In one aspect, the immune modulatory agent may be selected from the group consisting of: avelumab, sunitinib, sorafenib, ipilimumab, interferon alpha, and combinations thereof.

[0044] Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.DRAWINGS

[0045] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure.

[0046] FIGS. 1A-1 C show a comparative view of three oral squamous cell carcinoma (OSCC) treatment options: (1 ) Janus nanoparticle (JNP) control group lacking any active agent, (2) JNP combined with tocilizumab (TCZ) in accordance with certain aspects of the present disclosure, and (3) TCZ bolus injections. FIG. 1A shows comparative histological cross-sectional views of mitoses (scale bars of 50 micrometers), tumor keratinization (scale bars of 500 micrometers), and ERG transcription factor expression (scale bars of 200 micrometers) for treatment options (1 ) to (3) in a subject. FIG. 1 B shows an average mitotic count comparing treatment options (1 ) to (3) above under a 10 times high power field (HPF). FIG. 1 C shows an ERG staining intensity count for treatment options (1 ) to (3) above.

[0047] FIGS. 2A-2B show graphs demonstrating the in-use stability of Janus nanoparticles (JNP) loaded with fenretinide and tocilizumab (TCZ) according to certain aspects of the present disclosure. More specifically, FIG. 2A shows absorbance spectrum of fenretinide (4HPR)-synthetic protein nanoparticle (sPNP) prepared in accordance with certain aspects of the present disclosure. This spectrum was compared to 15 ug / mL of free 4HPR relative to 4HPR encapsulated high pressure homogenized human serum albumin (HPH-4HPR). FIG. 2B demonstrates successful encapsulation of 4HPR according to certain aspects of the present disclosure. Acetic acid was added to dissemble nanoparticles through the unfolding of protein and to reveal available 4HPR (designated 4HPR-sPNPs pre-acid and 4HPR-sPNPs post-acid).

[0048] FIGS. 3A-3C. FIG. 3A shows a two-phase Janus nanoparticle (JNP) formed in accordance with certain aspects of the present disclosure, where a first phase of the JNP comprises human serum albumin (HSA) and 4HPR active ingredient and asecond phase comprises chitosan (in glycol) and TCZ active ingredient. Structural illumination microscopy was used to confirm the 2-sided Janus nanoparticle configuration and to characterize the individual components of the 400 nm fluorescent labeled 4HPR- TCZ JNP, in FIGS. 3B and 3C. Chitosan: AF488 (green, TCZ) and HSA (magenta, 4HPR), top. Fluorescent intensity is normalized by the max intensity along the x-y profile and z- stack (bottom). JNP is oriented in z-direction. As shown in FIG. 3C, the full width at half maximum (FWHM) is shifted by only 45 nm along the yellow line in the x-y plane, while FMHW is shifted by 200 nm along the z-stack intensity.

[0049] FIG. 4 compares the ability of bolus-delivered 5 micromoles (pM), 4HPR and 4HPR HSA protein nanoparticles (4HPRscPNP), and a control to activate the executioner phase caspase-3 in premalignant and malignant human epithelial cell lines. Blank scPNP particles at the same dosing concentration (6.18x109particles / mL) were applied to the control and bolus-delivered 4HPR groups to account for the impact of nanoparticle addition. FIG. 4 shows units of activity per microgram (pg) of protein versus each comparative example.

[0050] FIGS. 5A-5E show characterization of Janus nanoparticles (JNPs) prepared in accordance with certain aspects of the present teachings having a first phase with HSA and 4HPR and a second phase with chitosan and TCZ. FIG. 5A shows an SEM image of 4HPR-TCZ JNPs where the scale bar is 5 micrometers. The inset of FIG. 5A (a1 ) shows a size determination of 4HPR-TCZ JNP, as assessed by dynamic light scattering. FIG. 5B shows a size distribution of 4HPR-TCZ (using ImageJ™ software). FIG. 5C shows geometric factor analysis, as assessed by SEM, assessing nanoparticle circularities (1 =perfect circle), roundness (1=perfect sphere) and anisotropy (low=more spherical shape) of the 4HPR-TCZ JNPs (ImageJ). FIG. 5D shows mean geometric factor values for 4HPR-TCZ JNPs. FIG. 5E includes (1 , showing nanoparticle diameter over 7 days in different liquids) and (2, showing nanoparticle polydispersity index (PDI) over 7 days in different liquids), showing JNP stability required for drug delivery to a moist location, such as the lungs. Dynamic light measurement stability studies revealed consistency in JNP diameter and consistent polydispersity indices < 0.3 of 4HPR-TCZ JNP over 1 week in H2O and PBS. These data confirm excellent control of size and secondary parameters, such as roundness, circularity and low anisotropy. The low PDI values and sizeconsistency in water and PBS demonstrate JNP stability compare favorably to clinically used nanoparticles such as lipid nanoparticles.

[0051] FIG. 6 shows a combination of 4HPR and TCZ induces immune enhancing with proliferation EMT-suppressive effects in human PML cells. More specifically, FIG. 6 shows canonical suppression pathways (1 ) to (6) associated with cell cycle progression and proliferation, with a plot of z-scores for each.

[0052] FIG. 7 shows a release profile over 72 hours of active ingredients from a multiphasic Janus nanoparticle comprising fenretinide (4-HPR) at 2.3 wt. % and tocilizumab (TCZ) at 23.1 wt. % (4HPR-TCZ JNP) prepared in accordance with certain aspects of the present disclosure. The dashed line represents the curve best fitted to the release data, red=TCZ release and black-4HPR release. The release for TCZ was best fitted to a two-phase exponential decay function (R2= 0.99), and the 4HPR release was found best fitted to the Weibull cumulative distribution function.

[0053] FIGS. 8A-8D show in vivo tumorigenesis of LLISC H520 cells are significantly inhibited by locally delivered multiphasic Janus nanoparticles comprising fenretinide (JNP-4HPR-JNP) and multiphasic Janus nanoparticles comprising fenretinide and tocilizumab (JNP-4HPR-TCZ). FIG. 8A shows comparative histological cross- sectional views of hematoxylin and eosin staining (H & E), proliferation of cells (Ki-67), and programmed cell death (caspase-3 positive) (scale bars of 100 micrometers) for tumor groups: 1 (control, drug-free JNP designated JNP-CTR), 2 (designated JNP-4HPR), 3 (JNP-4HPR-TCZ). FIG. 8B shows histogram images for hemorrhage, FIG. 8C shows proliferation indices (Ki-67) and FIG. 8D shows programmed cell death (caspase-3 positive) each depicting average findings per 10 high power fields. The 4HPR-TCZ JNP provided the higher tumor suppressive impact. 4HPR-TCZ JNP uniquely perturbed tumor vascular integrity, which resulted in increased necrosis in combination treated tumors.

[0054] Corresponding reference numerals indicate corresponding parts throughout the several views of the drawings. The drawings will be described in more detail below.DETAILED DESCRIPTION

[0055] Example embodiments are provided so that this disclosure will be thorough, and will fully convey the scope to those who are skilled in the art. Numerousspecific details are set forth such as examples of specific compositions, components, devices, and methods, to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that example embodiments may be embodied in many different forms and that neither should be construed to limit the scope of the disclosure. In some example embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail.

[0056] The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of stated features, elements, compositions, steps, integers, operations, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Although the open-ended term “comprising,” is to be understood as a non-restrictive term used to describe and claim various embodiments set forth herein, in certain aspects, the term may alternatively be understood to instead be a more limiting and restrictive term, such as “consisting of” or “consisting essentially of.” Thus, for any given embodiment reciting compositions, materials, components, elements, features, integers, operations, and / or process steps, the present disclosure also specifically includes embodiments consisting of, or consisting essentially of, such recited compositions, materials, components, elements, features, integers, operations, and / or process steps. In the case of “consisting of,” the alternative embodiment excludes any additional compositions, materials, components, elements, features, integers, operations, and / or process steps, while in the case of “consisting essentially of,” any additional compositions, materials, components, elements, features, integers, operations, and / or process steps that materially affect the basic and novel characteristics are excluded from such an embodiment, but any compositions, materials, components, elements, features, integers, operations, and / or process steps that do not materially affect the basic and novel characteristics can be included in the embodiment.

[0057] Any method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussedor illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed, unless otherwise indicated.

[0058] When a component, element, or layer is referred to as being “on,” “engaged to,” “connected to,” or “coupled to” another element or layer, it may be directly on, engaged, connected or coupled to the other component, element, or layer, or intervening elements or layers may be present. In contrast, when an element is referred to as being “directly on,” “directly engaged to,” “directly connected to,” or “directly coupled to” another element or layer, there may be no intervening elements or layers present. Other words used to describe the relationship between elements should be interpreted in a like fashion (e.g., “between” versus “directly between,” “adjacent” versus “directly adjacent,” etc.). As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0059] Although the terms first, second, third, etc. may be used herein to describe various steps, elements, components, regions, layers and / or sections, these steps, elements, components, regions, layers and / or sections should not be limited by these terms, unless otherwise indicated. These terms may be only used to distinguish one step, element, component, region, layer or section from another step, element, component, region, layer or section. Terms such as “first,” “second,” and other numerical terms when used herein do not imply a sequence or order unless clearly indicated by the context. Thus, a first step, element, component, region, layer or section discussed below could be termed a second step, element, component, region, layer or section without departing from the teachings of the example embodiments.

[0060] Spatially or temporally relative terms, such as “before,” “after,” “inner,” “outer,” “beneath,” “below,” “lower,” “above,” “upper,” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. Spatially or temporally relative terms may be intended to encompass different orientations of the device or system in use or operation in addition to the orientation depicted in the figures.

[0061] Throughout this disclosure, the numerical values represent approximate measures or limits to ranges to encompass minor deviations from the given values and embodiments having about the value mentioned as well as those having exactly the valuementioned. Other than in the working examples provided at the end of the detailed description, all numerical values of parameters (e.g., of quantities or conditions) in this specification, including the appended claims, are to be understood as being modified in all instances by the term “about” whether or not “about” actually appears before the numerical value. “About” indicates that the stated numerical value allows some slight imprecision (with some approach to exactness in the value; approximately or reasonably close to the value; nearly). If the imprecision provided by “about” is not otherwise understood in the art with this ordinary meaning, then “about” as used herein indicates at least variations that may arise from ordinary methods of measuring and using such parameters. For example, “about” may comprise a variation of less than or equal to 5%, optionally less than or equal to 4%, optionally less than or equal to 3%, optionally less than or equal to 2%, optionally less than or equal to 1%, optionally less than or equal to 0.5%, and in certain aspects, optionally less than or equal to 0.1%.

[0062] In addition, disclosure of ranges includes disclosure of all values and further divided ranges within the entire range, including endpoints and sub-ranges given for the ranges.

[0063] As used herein, the terms “composition” and “material” are used interchangeably to refer broadly to a substance containing at least the preferred chemical constituents, elements, or compounds, but which may also comprise additional elements, compounds, or substances, including trace amounts of impurities, unless otherwise indicated.

[0064] As used herein, unless otherwise indicated, amounts expressed in weight and mass are used interchangeably, but should be understood to reflect a mass of a given component.

[0065] It is understood that while general attributes of certain categories of components, compositions, or compounds may differ, there may be some common attributes and any given material may serve multiple purposes within two or more of such listed classes or categories.

[0066] Example embodiments will now be described more fully with reference to the accompanying drawings.

[0067] The citation of references herein does not constitute an admission that those references are prior art or have any relevance to the patentability of this descriptiondisclosed herein. All references cited in the Detailed Description section of this specification are hereby incorporated by reference in their entirety.

[0068] In various aspects, the present disclosure provides nanoparticle-based drug delivery for preventing and / or minimizing cancer, or preventing recurrence of a treated cancer, and more particularly, lung cancer in a subject, such as for chemoprevention. As used herein, the term “patient” or “subject” can refer to living organisms, such as animals, including, but not limited to humans, livestock, dogs, cats, and other mammals. In certain embodiments, the subject is a human.

[0069] Administration of the therapeutic agents can be carried out at dosages and for periods of time effective for prevention or treatment of a disorder or condition, such as cancer, in a subject. In certain variations, nanoparticles provided by the present technology can be used for immunochemoprevention and thus used as part of a method of preventing or minimizing development or progression of, or preventing recurrence of, a treated lung cancer in a subject. For example, the subject may have a premalignant state or condition, where a cancer could develop or re-develop, e.g., a cancer characterized as having one or more pulmonary lesions, nodules, or tumors, e.g., non-small cell lung cancers (NSCLC), including squamous cell carcinoma of the lung, also known as squamous cell lung cancer (LUSC), and adenocarcinoma (adeno CA), or small-cell lung cancers (SCLC), such as small cell carcinoma, mesothelioma, and the like. In alternative aspects, the cancer may involve other epithelial cells than those found in the lung, such as oral cancers. Examples of oral cancers include, but are not limited to, cancer of the lips, tongue, cheeks, floor of the mouth, hard and soft palate, sinuses, and pharynx (throat). By way of example, such an oral cancer may be oral squamous cell carcinoma (OSCC). Additionally, or alternatively, such nanoparticles can be used as part of a method of treatment, augmentation, or induction in a subject having a pre-cancer or cancer e.g., a cancer of the lung) in combination with an additional therapeutic regimen, such as concurrent radiotherapy. By way of non-limiting example, a course of immune stimulation to reduce the tumor burden in a subject may be used prior to radiation or surgery. The methods may involve administering an effective amount of a nanoparticle to a subject having precancer or cancer. An “effective” amount of a nanoparticle means that it delivers at least one active ingredient in an amount that has a detectable effect for its intended purpose and / or benefit. For example, for a method of chemoprevention, a compositioncomprising a nanoparticle having at least one chemopreventive agent may be administered in an amount sufficient to inhibit (prevent) the progression of a precancerous lesion to a cancer (or induce regression of the precancerous lesion) in a subject.

[0070] As used herein, “administration” of an agent to a subject includes any route of introducing or delivering to a subject an agent to perform the agent's intended function(s). Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), topically, and the like. “Systemic administration” refers to the introducing or delivering to a subject an active agent via a route which introduces or delivers the agent to extensive areas of the subject's body (e.g., greater than about 50% of the body), for example through introduction into the circulatory, gastrointestinal, or lymphatic systems. By contrast, “local administration” refers to the introducing or delivery of an active agent to a subject via a route which introduces or delivers the agent to a target area or areas within the local vicinity of the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration, but are undetectable or detectable at negligible amounts in distal parts of the subject's body. Administration includes self-administration and the administration by another.

[0071] As used herein, the term “chemopreventive” and “chemopreventive agent” are used interchangeably and refer to any chemical or composition comprising one or more agents that, when administered to a subject, prevents a disease or disorder (e.g., cancer and more specifically, lung cancer) from progressing or recurring. A chemopreventive agent can slow, mitigate, block, suppress, or reverse the effects of cancer causing factors or symptoms thereof. Chemopreventive agents may interfere with initiation, promotion, progression, or all stages of multistage carcinogenesis. They may also direct cell growth state via induction of preprogrammed cell death or terminal differentiation. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “chemopreventive” is used or when a particular active agent is specifically identified, it is to be understood that the term includes the agent itself, as well as pharmaceutically acceptable, pharmacologically active salts,esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, and the like.

[0072] The term “preventing” a disorder or unwanted physiological event in a subject refers specifically to the prevention of the progression or recurrence of symptoms and / or their underlying cause, where the subject may or may not exhibit heightened susceptibility to the disorder or event (for example, either inducing regression or preventing progression). As used herein, “secondary chemoprevention of lung cancer"” includes inducing regression of precancerous lesions or nodules, preventing or delaying the progression of premalignant lesions or recurrence of previously treated lung cancer (for example, non-small cell or small cell lung cancers) or signs or symptoms thereof. “Tertiary chemoprevention” entails prevention of recurrence of a previously treated lung cancer. A current standard of care for lung cancer patients is to receive immune stimulating agents after chemotherapy. For localized disease, aerosolized nanoparticle delivery is contemplated as being much easier for a subject, for example, such compositions could be administered at home with a nebulizer, and are associated with markedly lower systemic side effects.

[0073] For example, in certain aspects, an aerosolized nanoparticle-based drug delivery platform is provided for lung cancer chemoprevention. Lung cancer brings a devastating death toll with no effective lung cancer chemoprevention strategy, despite extensive research efforts. However, previous lung cancer chemoprevention strategies, which employ systemic administration of re-purposed drugs developed for other applications, result in toxicities and were not effective for localized delivery. In contrast, the combination of a novel delivery vehicle provided in accordance with various aspects of the present disclosure, namely multi-phasic / compartmental nanoparticles, also referred to herein as Janus nanoparticles, can enable delivery of a lung cancer specific chemoprevention drug payload at the lung target tissue site. Furthermore, concurrent delivery of combinations of at least two chemically diverse agents can provide a multimodal approach needed to minimize or prevent lung cancer. Nanoparticles provide enhance bioavailability and offer stabilization of the drug cargo-including biologies, until a target site is reached in vivo. Poor bioavailability is a common issue with chemopreventive agents. Further, such nanoparticles provide an ability to modify both the particle composition and the surface for optimized target site specific uptake. Lastly, nanoparticlesprovided by the present disclosure are readily internalized by their target cells, which provides focused treatment and prevention of cancer.

[0074] Due to extensive signaling redundancies, effective chemoprevention is desirably multimodal to circumvent resistance. And effective chemoprevention is desirably standardized to adequately help patients. Such consistency can be achieved by utilization of multiphasic particles, also referred to herein as “Janus” particles. Multi-phasic particles have a plurality of physically and / or compositionally distinct phases, where a “phase” may be considered to be a portion of that particle that is chemically and / or physically distinct from another portion of the component. The multi-phasic particles for use in accordance with certain aspects of the disclosure may include a first phase and at least one additional phase that is distinct from the first phase. While traditional Janus particles are often understood to have only two phases or compartments, it should be appreciated that the nanoparticles of the present disclosure may have two, three, or more phases or compartments. In certain aspects, at least one phase comprises a structural or matrix material, such as a protein, a polysaccharide, or a polymer or polymer precursor. Further, the first phase of the at least one additional phase comprises an active ingredient that is a compound or composition that diagnoses, prevents, or treats a physiological disorder. In particular, the active ingredient may be an active ingredient that minimizes, treats, or prevents cancer, such as a chemo-preventive and / or anti-proliferative active ingredient. In various aspects, the active ingredient may minimize or prevent development of lung cancer in a subject. In certain other aspects, the multiphasic or Janus nanoparticle may comprise a plurality of active ingredients, including a first active ingredient in a first phase of compartment and a second active ingredient in the at least one additional phase or compartment. In certain aspects, an active ingredient may be delivered via the multiphasic particle to a particular target within an organism, such as organs, tissues, respiratory system, mouth, eyes, circulatory system, and the like. For example, the composition for preventing, minimizing, or preventing recurrence of a treated lung cancer that includes the multiphasic nanoparticles with active ingredients may be delivered to a subject’s lungs via the subject’s respiratory system, for example, via the mouth, nose, respiratory tract or system, and the like. Combined with chemopreventive mixtures capable of nebulization, a unique lung cancer chemoprevention strategy is provided by various aspects of the present disclosure. Importantly, local aerosolized delivery of drugformulations can leverage a pharmacologic advantage via an ability to deliver therapeutically relevant drug levels to the target site without deleterious drug-related systemic effects.

[0075] As noted above, Janus nanoparticles are special types of multiphasic nanoparticles whose two distinct phases or compartments / chambers can enable encapsulation and delivery of chemically distinct molecules. In addition, it is possible to selectively treat or coat (e.g., to uniquely decorate) regions of the Janus nanoparticle surfaces. The simplest case of a Janus particle is achieved by dividing the particle into two distinct parts, each of them either made of a compositionally distinct / different material, or bearing different functional groups. For example, a Janus particle may have two phases and thus have one half of its surface comprising hydrophilic groups and the other half of its surface having hydrophobic groups. As will be appreciated, the surfaces of the Janus nanoparticles may be treated to have different charges, different targeting ligands or moieties, immune system cloaking agents, or other different properties, such as distinct fluorescence or magnetic properties or the like.

[0076] For example, the nanoparticle can contain additional surface-associated molecules which alter the properties or functions of the nanoparticles. Addition of surface-associated molecules may also be referred to as surface decoration. For instance, cellular uptake or tissue penetration in the respiratory tract epithelia by nanoparticles can be modified by attaching one or more mucoadhesive molecules to the surface of the nanoparticle. In some embodiments, the mucoadhesive molecule is a lectin, thiolated polymer (thiomer), alginate polyethylene glycol acrylate (alginate-PEGAc), poloxamer, or any combination thereof. Optionally, a nanoparticle may contain a mucous penetrating ligand. In some embodiments, the mucous penetrating ligand comprises low molecular weight polyethylene glycol (PEG), for example having a molecular weight of less than or equal to about 50 kDa. One or more molecules or moieties may be attached to the nanoparticle alone or in combination with other molecules or moieties. Molecules for surface decoration can be attached to a nanoparticle via surface binding chemistries of the nanoparticle or alternatively, can be attached to a surface by ligands which serve as linkers between the nanoparticle and the surface decoration molecule. As an example, a nanoparticle can be formed from carboxylic acid-functionalized PLGA, to which ligands may be attached by carbodiimide conjugation chemistries.

[0077] In various aspects, these particles have unique properties related to their asymmetric structure and / or functionalization. As used herein, “JNP” is used interchangeably with Janus nanoparticle, as well as with multiphasic or multicompartmental nanoparticles. A “nano-particle” is a particle that has at least one spatial dimension that is less than or equal to about 2 micrometers (i.e., 2,000 nm), optionally less than or equal to about 1 micrometers ( / .e., less than about 1 ,000 nm), optionally less than or equal to about 0.5 micrometers ( / .e., 500 nm), optionally less than or equal to about 0.25 micrometers ( / .e., 250 nm), optionally less than or equal to about 0.2 micrometers (i.e., 200 nm), and in certain aspects, optionally less than or equal to about 0.1 micrometers (i.e., 100 nm).

[0078] In certain variations, the nanoparticle may have an average diameter of greater than or equal to about 10 nm to less than or equal to about 500 nm, optionally greater than or equal to about 20 nm to less than or equal to about 400 nm, optionally greater than or equal to about 25 nm to less than or equal to about 300 nm, optionally greater than or equal to about 25 nm to less than or equal to about 250 nm, and in certain aspects, optionally greater than or equal to about 25 nm to less than or equal to about 125 nm.

[0079] The present disclosure contemplates in certain variations a JNP-based lung cancer chemoprevention drug delivery platform that comprises at least one type of multiphasic / Janus nanoparticle. The Janus nanoparticles may be respectively morphologically anisotropic and the Janus nanoparticles include at least two chemically distinct compartments or phases, as discussed above. One or more phases or compartments may be formed from materials that are pharmaceutically acceptable or biocompatible materials, in other words, substantially non-toxic to cells and tissue of living organisms. At least one phase of the nanoparticle comprises an active ingredient. The nanoparticle may include two or more active ingredients. An active ingredient is a compound or composition that diagnoses, prevents, or treats a physiological or psychological disease, disorder, or condition of hard or soft tissue in an organism, such as a mammal, as discussed above. A pharmaceutical active ingredient is a drug or other compound operable for the prevention or treatment of a condition or disorder in a human or other animal, the prevention or treatment of a physiological disorder or condition, or to provide a benefit that outweighs potential detrimental impact in a conventional risk-benefitassessment. In certain aspects, the active ingredient may be used for the prevention or treatment of systemic disorders, such as cancer, in particular, lung cancer, including nonsmall cell lung cancers, small lung cell cancers, and mesothelioma, but also may be conventionally used to treat other disorders, such as inflammation, autoimmune diseases, cardiovascular disease, stroke, diabetes, severe respiratory infection, pain control, and the like.

[0080] The ensuing description of suitable active ingredients is merely exemplary and should not be considered as limiting as to the scope of active ingredients which can be introduced into the nanoparticles according to the present disclosure. In certain alternative variations, other suitable active ingredients known or to be discovered by those of skill in the art for these various types of compositions, especially to treat lung cancers, are contemplated. Suitable active ingredients for use in such pharmaceutically and / or cosmetically acceptable compositions are well known to those of skill in the art and include, by way of example, pharmaceutical active ingredients found in the Merck Index, An Encyclopedia of Chemicals, Drugs, and Biologicals, Fourteenth Edition (2006) by Merck Research Laboratories and the International Cosmetic Ingredient Dictionary and Handbook, Eleventh Edition (2006) by Cosmetic Toiletry and Fragrance Association, each incorporated herein by reference. Each additional reference cited or described herein is hereby expressly incorporated by reference in its respective entirety.

[0081] Suitable therapeutic active ingredients may include growth regulatory or anti-proliferative agents; antineoplastic / anti-miotic agents; chemotherapy agents; immunomodulatory agents; transcription / translation modifier agents; growth factor and cytokine modulator agents; anti-inflammatory agents; anti-rejection drugs; anti-thrombotic agents; anti-coagulants; antioxidants; free radical scavengers; nutrients; nucleic acids; saccharides; sugars; nutrients; hormones; cytotoxin; hormonal agonists; hormonal antagonists; inhibitors of hormone biosynthesis and processing; antiestrogens; antiandrogens; non-steroidal anti-inflammatory agents (NSAIDs); antimicrobial agents; antiviral agents; antifungal agents; antibiotics; anesthetic, analgesic or pain-killing agents; antipyretic agents, prostaglandin inhibitors; platelet inhibitors; biologic agents; nucleic acids; DNA de-methylating agents; cholesterol-lowering agents; vasodilating agents; endogenous vasoactive interference agents; angiogenic agents; cardiac failure agents;targeting moieties, including targeting toxin agents; vitamins; nutraceuticals; and combinations thereof.

[0082] Certain suitable active ingredients, or pharmaceutically active ingredients or drugs, are known to those of skill in the art and include, but are not limited to, low- molecular weight molecules, for example, having a molecular weight of less than about 10,000, optionally less than about 1 ,000, and optionally less than about 500, as well as natural and artificial macromolecules, such as proteins, sugars, peptides, nucleic acids, such as DNA, RNA, RNAi (RNA interference), and the like, polymers, biomarkers, including quantum dots, dyes and colorants, inorganic ingredients, and combinations thereof.

[0083] As discussed above, the one or more active agents in the nanoparticle may be a chemopreventive agent used to for preventing, minimizing, or preventing recurrence of a treated lung cancer, and may include growth regulatory or anti-proliferative agents; antineoplastic / anti-miotic agents; chemotherapy agents; immunomodulator agents; transcription / translation modifier agents; growth factor and cytokine modulator agents; biomolecules, including polynucleotides and / or nucleic acids, like DNA, RNA, plasmids, short interfering sequence of double stranded RNA (siRNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, small nuclear RNA, single stranded DNA, CRISPR CAS-9, aptamers, antibodies, polymers, peptides, amino acids, targeting molecules, and any combinations thereof.

[0084] In other variations, the active ingredient of the nanoparticle may be used for diagnostic purposes, such as in various diagnostic medical imaging procedures (for example, radiographic imaging (x-ray), fluorescence spectroscopy, Forster / fluorescent resonance energy-transfer (FRET), computed tomography (CT scan), magnetic resonance imaging (MRI), positron emission tomography (PET), other nuclear imaging, and the like). Active ingredients for use with diagnostic imaging include contrast agents, such as barium sulfate for use with MRI, for example or for example fluorescein isothiocyanate (FITC).

[0085] In various aspects, a Janus nanoparticle according to the present disclosure delivers an effective amount of the active ingredient to a target region within an organism, such as the lungs, as described above in the context of effective amounts of nanoparticles. An “effective” amount of an active ingredient is an amount that whendelivered to the subject has a detectable effect for its intended purpose and / or benefit. For example, the effective amount is sufficient to have the desired prophylactic, therapeutic, and / or diagnostic effect on the target region of a subject / organism (e.g., a mammal) to whom and / or to which the composition comprising the nanoparticles administered. The specific effective amount of the active ingredient, including appropriate dosages and concentrations, will vary with such factors as the composition (nanoparticle composition) in which the active ingredient is provided, the site of intended delivery, the route of administration, the particular condition or subject being treated, the nature of concurrent therapy (if any), the specific active used, the specific dosage form, and the carrier employed, all of which are well known to those of skill in the art. Further, the amount of active ingredient that is “effective” will vary from subject to subject, depending on many factors such as the age, sex, weight, and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be readily determined by one of ordinary skill in the art. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. Dosage regimens can be adjusted to provide the optimum therapeutic response, for example, as determined by quantifiable clinical data. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0086] In certain aspects, a safe and effective amount of an active ingredient in a phase of Janus nanoparticle may be greater than or equal to about 0.0001 to about 95 weight % of the total weight of phase (on a dry basis). It should be noted that where the Janus nanoparticle is distributed in a carrier or composition / delivery platform, the overall concentration will be significantly less than in the nanoparticle itself. In certain aspects, the active ingredient is present in a phase of a Janus nanoparticle at a concentration of greater than or equal to about 0.001% to about 75% by weight of the total weight of the phase or compartment. In other aspects, the active ingredient is present at from of greater than or equal to about 0.01 to less than or equal to about 20%; optionally greater than or equal to about 1% to less than or equal to about 20%; and optionally greater than or equal to about 5% to less than or equal to about 20% by weight. However, as discussed above,the concentration of active ingredient is highly dependent on various factors well known to those of skill in the art, including required dosage for the target region, bioavailability of the active ingredient and the release kinetics of the phase in which the active ingredient is located, among others.

[0087] Thus, the Janus nanoparticles have at least one active ingredient and optionally at least two active ingredients, distributed within one or more phases. For example, such active ingredients can be suspended in protein-containing liquid, a polymer solution, or a polymer melt that will form a semi-solid or solid phase after formation, as will be described further herein. A first phase or compartment can be loaded with an active ingredient or multiple active ingredients. Likewise, a second phase or compartment can be loaded with a distinct active ingredient or multiple active ingredients. In some embodiments, the plurality of phases may each contain one or more distinct active ingredients.

[0088] Therefore, Janus nanoparticles can comprise as active agents or ingredients, proteins, lipophilic drugs, and RNA, among others. In certain aspects, the Janus nanoparticle comprises at least one active ingredient to minimize, prevent, or treat lung cancer in a subject, namely a chemopreventive agent.

[0089] The Janus nanoparticles of the present disclosure may be administered as a pharmaceutical composition in conventional administration routes, such as, for example, nasal and buccal / oral administration, especially intranasal, inhalation, or alternatively, intravenous, topical, subcutaneous, transcutaneous, intramuscular, oral, intra-joint, parenteral, peritoneal, or the like. As discussed above, where the Janus nanoparticles include agents for preventing, minimizing, or preventing recurrence of a treated lung cancer, a pharmaceutical composition for delivering the Janus nanoparticles may be in the form of solid, semi-solid, powders, lyophilized powder, aerosols, or alternatively, in forms such as tablets, pills, capsules, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, or the like, in unit dosage forms suitable for administration of precise dosages.

[0090] In certain aspects, nanoparticles comprising chemopreventive agents are delivered to the subject locally, as compared to systemic delivery of an agent, which can permit use of appreciably lower levels or concentrations (for example, over 1 ,000 fold less) of the agent while providing therapeutic target levels and eliminating deleterious sideeffects. Nanoparticles may be formulated in a delivery platform or vehicle in a range of concentrations. Typically, the concentration of nanoparticles in a vehicle, such as an aerosolized vapor, is a therapeutic amount when administered to a subject. In some embodiments, by way of non-limiting example, a concentration of nanoparticles in a delivery platform can range from greater than or equal to about 1 ppm to less than or equal to about 500,000 ppm, for example, from greater than or equal to about 1 ppm to less than or equal to about 500,000 ppm, from greater than or equal to about 10 ppm to less than or equal to about 100,000 ppm, or from greater than or equal to about 100 ppm to less than or equal to about 10,000 ppm.

[0091] In certain variations, such a localized delivery may be by an aerosolized nanoparticle-based drug delivery platform for lung cancer chemoprevention, which in certain variations, may be administered nasally or orally. An aerosol is generally considered to be a suspension of small solid particles and / or liquid droplets in a gas, such as air. Generally, an aerosol droplet (whether solid or liquid) has a maximum size or diameter of less than or equal to about 5 micrometers, optionally less than or equal to about 2 micrometers, and optionally less than or equal to about 1 micrometer. Thus, the compositions comprising the nanoparticles may be suitable for pulmonary delivery, for example, via intranasal delivery or oral delivery of the pharmaceutical composition.

[0092] In various aspects, the nanoparticles may have an average particle size that reflects a targeted area, in the subject for example, a target area of the lung. By way of example, during LUSC chemoprevention, nanoparticles may have a larger particle size because the target site is upper, larger airways. In contrast, lung adeno CA arises in the small terminal alveoli, so to facilitate delivery further into the lung, smaller, more aerodynamic particles capable of distribution to the terminal alveoli are used. In certain aspects, the nanoparticles are of a size that they are configured to be aerosolized as small aerosol droplets. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle size from greater than or equal to about 0.2 micrometers to less than or equal to about 5 micrometers. Such a composition may be administered by rapid inhalation through the nasal passage from a container of the powder held close to the nose or by an inhaler placed into the mouth for inhalation. Alternately, pharmaceutical compositions suitable for oral or buccal administration may comprise a powder, an aerosolized, and / or atomized solution and / orsuspension comprising Janus nanoparticles with active agents described above. Such powdered and / or aerosolized compositions may have nanoparticles with an average particle and / or droplet size upon dispersion ranging from greater than or equal to about 0.1 nm to less than or equal to about 200 nm.

[0093] The pharmaceutical composition suitable for nasal or oral administration may, for example, comprise greater than or equal to about 0.1% by weight to less than or equal to about 100% by weight of the nanoparticles comprising active ingredient(s), but may comprise one or more of the additional ingredients or additives known in the art for pharmaceutical compositions.

[0094] As discussed above, due to extensive signaling redundancies, effective chemoprevention is ideally multimodal and involves at least two active agents to circumvent resistance. In certain variations, the nanoparticles of the present disclosure comprise at least two chemopreventive agents. For example, local delivery of the synthetic vitamin A derivative, fenretinide (4HPR), in conjunction with a second active ingredient, such as the humanized IL6R inhibitor monoclonal antibody biologic, tocilizumab (TCZ) (e.g., Actemra®) inhibits tumorigenesis in vivo. Further, the triple combination of 4HPR, TCZ and allosteric inhibitor of IL8’s CXCR1 / 2 receptor, reparixin (REP), cooperatively suppresses cancer invasion. While the mechanism of action of the receptor antagonists' is straightforward, for example, suppression of autocrine-paracrine signaling, reduced inflammation and angiogenesis, preservation of the local immune response, 4HPR’s chemopreventive effects are more complex. 4HPR not only possesses growth modulatory effects, but also demonstrates high affinity binding / inactivation of signaling kinases (FAK, Pyk2, STAT3, Wnt-|3-catenin) and interference with cytoskeletal components necessary for invasion and migration. Further, 4HPR is a redox-active molecule and effectively disrupts mitochondrial electron transport. Additionally, systemically administered 4HPR significantly reduced expression of the human telomerase reverse transcriptase catalytic subunit (hTERT) in bronchial biopsy specimens of smokers. In addition to the humanized anti-IL6R monoclonal antibody biologic, tocilizumab (Actemra®), an additional FDA- approved antibody is situximab (Sylvant®). Additional anti-interleukin-6 agents include, but are not limited to, sarilumab, olokizumab, elsilimomab, BMS-945429, sirukumab, and CPSI-2364. Initiation of the angiogenic switch and increased tumor-associatedangiogenesis are important aspects for the progression of premalignant disease to lung cancer growth.

[0095] As local immunosuppression is contributory to lung cancer development, immune modulating agents, such as those that are immune augmenting agents, may be integrated into the nanoparticles, including checkpoint inhibitors. Collectively, the immunochemoprevention contemplated by certain aspects of the present teachings will serve to regulate cell growth state and proliferation, suppress inappropriately sustained inflammation and circumvent the local immune exhaustion.

[0096] Thus, the present technology can deliver effective chemopreventive agents for optimal cancer immunoprevention, whether by minimizing and / or preventing lung cancer or by preventing recurrence of a treated lung cancer, by integrating various inhibitors of advantageous pathways to minimize undesirable cell growth. For example, the nanoparticles can include active agents that can target re-activation of the host’s immune system, so that the host’s body recognizes the neoantigens on the premalignant cells and destroys them. Both oral cancer and lung SCC arise in an immunosuppressed environment, thus the nanoparticles provided herein can advantageously enhance the host’s immune system response. As noted above, an immune modulatory agent / checkpoint inhibitor-tocilizumab-fenretinide formulation (or any dual or multiagent combination) is beneficial to the patient for chemoprevention.

[0097] In one aspect, the immune modulatory agent may be selected from checkpoint inhibitors, such as avelumab (monoclonal antibody), immune-augmenting tyrosine kinase inhibitors, such as sunitinib and sorafenib, immunostimulatory antibodies, such as ipilimumab (antagonizes the immune inhibitory T lymphocyte antigen 4 (anti- CTLA-4 agent), commercially available as Yervoy™) and activators of CD40, which function as stimulators of antigen presenting cells, which are essential for T cell priming and activation. Other variations can incorporate HDAC inhibitors, which upregulate NK cell receptors on target premalignant or malignant cells, or immunostimulatory cytokines such as interferon alpha (can serve to stimulate proliferation, survival and function of tumor inhibiting T and NK cells).

[0098] In one aspect, the nanoparticle may comprise an immune modulatory agent selected from a group consisting of: a checkpoint inhibitor, an immune-augmenting tyrosine kinase inhibitor, an immunostimulatory antibody, an activator of CD40, a histonedeacetylase (HDAC) inhibitor, an immunostimulatory cytokine, and combinations thereof. In a further aspect, the immune modulatory agent may be selected from the group consisting of: avelumab, sunitinib, sorafenib, ipilimumab, interferon alpha, and combinations thereof. In certain variations, the nanoparticle may comprise an immune checkpoint inhibitor comprising avelumab and / or an immunostimulatory antibody (anti- CTLA-4 agent) comprising ipilimumab.

[0099] Thus, the present disclosure contemplates a versatile technology that allows for preparation of novel types of drug carriers, Janus nanoparticles (JNPs), that are a robust platform for targeted delivery of chemopreventives. Nanoparticle formulations are readily internalized by epithelial cells from a variety of sites, including lungs. Importantly, local delivery formulations provide a pharmacologic advantage via their ability to deliver therapeutically relevant drug levels to the target site without deleterious drug-related systemic effects.

[0100] By way of example, in certain variations, an aerosolized composition disclosed herein is a mixture of Janus nanoparticles of synthetic vitamin A derivative, fenretinide (4HPR) and tocilizumab, which may be delivered to target cells in the lungs. In certain variations, a single Janus nanoparticle may comprise a first phase or compartment comprising a first active ingredient, such as fenretinide, and a second phase or compartment comprising a second active ingredient, such as tocilizumab. As noted above, additional active ingredients may also be incorporated into the first and second phase, or alternatively, into a third phase of the nanoparticles. In other aspects, mixtures of distinct Janus nanoparticles may be used concurrently. By way of non-limiting example, while both fenretinide and tocilizumab can be provided in different phases of a single nanoparticle and thus delivered concurrently, in other variations, a first nanoparticle may comprise surface receptor interactive agents or drugs, such as tocilizumab and avelumab (or alternatively or in addition, ipilimumab) together in distinct phases, while a second nanoparticle may comprise agents or drugs that function intracellularly, such as fenretinide and siRNA in distinct phases.

[0101] An aerosolized nanoparticle-based drug delivery platform for lung cancer chemoprevention is thus contemplated. As discussed above, lung cancer brings a devastating death toll with no effective lung cancer chemoprevention strategy, despite extensive research efforts. However, previous lung cancer chemoprevention strategies,which employ systemic administration of re-purposed drugs developed for other applications, result in toxicities and were not effective for localized delivery. In contrast, the combination of a novel delivery vehicle (Janus nanoparticles) enables delivery of a bioavailable and bioactive chemoprevention drug payload at the lung target tissue site. Furthermore, concurrent delivery of combinations of at least two chemically diverse active ingredients / agents will address the need for intervention at multiple targets necessary to fight lung cancer. Janus nanoparticles offer stabilization of the drug cargo-including biologies until a target site is reached and the ability to modify both the particle composition and the surface for optimized target site specific uptake. Lastly, nanoparticles are either readily internalized by their target cells, or bind to selected target cell receptors, which provides focused chemoprevention of cancer.

[0102] In certain aspects, the present disclosure thus contemplates methods of preventing or minimizing cancer, such as lung cancer or oral cancer in a subject, by administering a chemoprevention drug delivery platform that comprises at least two chemopreventive agents, as described in the present disclosure. The chemoprevention drug delivery platform may deliver an aerosolized composition in accordance with certain variations of the present disclosure. For example, an aerosolized composition may comprise at least one Janus nanoparticle configured to be aerosolized, which comprises at least two chemopreventive agents. The at least two chemopreventive agents may be any of those described herein, including by way of example, a bioactive protein inhibitor; a modulator of at least one proinflammatory mediator, an immune modulatory agent; and / or a genetic manipulation agent, such as polynucleotides or transcription / translation modifiers, like short interfering sequence of double stranded RNA (siRNA) that serve to modify proteins aberrantly upregulated in premalignant lung lesions.

[0103] Those of ordinary skill in the art can measure chemopreventative outcomes by several quantifiable parameters including: histologic regression of the premalignant lesion, reduction in premalignant lesional size, suppression of local tumor recurrence, reduction of loss of heterozygosity indices at tumor suppressor gene loci, suppression of angiogenesis / neovascularization and / or induction of terminal differentiation relative to bolus drug delivery, for example. The key step for conversion of premalignant surface epithelial lesions to overt carcinoma is invasion of the basement membrane. Recently generated fenretinide data have shown its high affinity proteinbinding to signaling kinases and extracellular matrix proteins that are essential for directed migration and invasion. A mucoadhesive fenretinide patch formulation, enabling a hydrophobic drug to be functional in a saliva-rich environment and also permeate into keratinized epithelia, was generated in response to the need for chemoprevention of oral premalignant lesions. . Therefore, a second-generation adaptation of the Janus nanoparticle technology, amenable for aerosolized delivery would provide similar affinity to premalignant lung lesions (premalignant lung lesions), given their similarity with premalignant oral epithelial lesions. By way of example, suitable delivery can be accomplished using an ultrasonic nebulizer unit, such as an Aerogen Solo™ which has a central aperture plate being 5 mm perforated with 1 ,000 precision formed holes, where the plate vibrates at 128,000 times per second to produce consistently fine droplets of 1 to 5 micrometers the comprise the Janus nanoparticles (see 30-354 REV II Aerogen Solo Instruction Manual, Aerogen Pharma Corporation, Galway Business Park, Dangan, Galway H91 HE94).

[0104] Also, multiple active agents with complementary mechanisms of action may be employed. Further, the formulations provided by the present disclosure may employ aerosolized Janus nanoparticles for agent delivery. The Janus nanoparticles of the present disclosure provide advantages including: (1 ) concurrent delivery of combinations of two chemically diverse agents, (2) stabilization of the drug cargo-including biologies until target site is reached, and (3) the ability to modify both the particle composition and the surface of the particle for optimized target site specific uptake. A final consideration is (4) that nanoparticles can interact with their target cells either by internalization or cell membrane interactions, contingent on agent delivered.

[0105] JNPs, which are structurally distinct from standard nanoparticles, can provide targeted delivery of chemopreventative mixtures, for example, via aerosol to the lung. The use of JNPs enables one of ordinary skill in the art to deliver two chemically distinct agents, beneficial with additive or synergistic compounds. Protein can be a particularly suitable JNP constituent material because, relative to synthetic polymers, protein formulated JNPs are chemically crosslinked and thus stable. Another key feature is that selective proteins such as human serum albumin exhibit very low toxicity, are non- immunogenic, biocompatible and biodegradable. Stability is necessary to withstand the shear forces attendant during nebulization. Aerosolized proteins are already recognizedas safe and effective, as the U.S. Food and Drug Administration has approved an aerosolized enzyme (dornase alfa) in adult and pediatric patients with cystic fibrosis. Also, protein JNP preparation can be prepared without the use of toxic chemicals or organic solvents, which helps prevent lung irritation.

[0106] Electrohydrodynamic co-jetting creates JNPs, capable of carrying drugs in two chemically distinct phases or compartments that are morphologically anisotropic and, therefore, amenable to deliver two chemically distinct compounds. Cojetting technology lends itself to fabrication of diverse compartment geometries. For instance, bi-compartmental particles with equally sized compartments can be prepared from a range of different biocompatible materials, including proteins, polysaccharides, and the like. In one variation, the material forming at least one phase or compartment of the JNP may comprise biodegradable albumin. Non-limiting examples of suitable proteins for electrohydrodynamic jetting to form a matrix of a phase include those selected from the group consisting of: albumin, human serum albumin, bovine serum albumin, ovalbumin, mucin, transferrin, insulin, lysozyme, hemoglobin, collagen, IgG, enzymes, transport proteins, storage proteins, antibodies, aptamers, chemokines, hormonal proteins, polypeptides, and combinations thereof.

[0107] In certain variations, the material used to form a portion of the JNP may be a biocompatible and biodegradable polymer that is selected from the group consisting of: a polysaccharide, including a mono-, oligo-, or polysaccharide, carboxymethylcellulose, a polymeric starch, chitosan, dextran, cellulose, gelatin, polyethyleneimine (PEI), poly(L-lysine) (PLL), poly(L-arginine), poly(amidoamine) (PAA), poly (amino-co-ester) (PAE), poly(2-N,N-dimethylaminoethylmethacrylate) (PDMAEMA), poly(4-vinylpyridine) (P4VP), polyesters, poly(acrylic acid), poly(methacrylic acid), polyalkylene glycol, such as polyethylene glycol, a methyl vinyl ether / maleic anhydride copolymer, any combinations and equivalents thereof. In certain variations, the polymer may be a polysaccharide, such as chitosan.

[0108] Particles with more than two compartments can be fabricated with this technology for independently controlled release. “Controlled release” refers to release of an active agent from a compartment or phase of the nanoparticle in a controlled fashion so as to achieve a desired pharmacokinetic profile in vivo. An aspect of “controlled release”of an active agent is an ability to manipulate the composition of the phase or compartment to establish the desired kinetics of active agent’s release.

[0109] For each multicompartmental nanoparticle formulation, compartments can be independently loaded with cancer prevention drugs, such as fenretinide. Compartments will be made of different matrix materials, which enable independent surface modification, decoupled release rates, responsiveness, or differential color, as described above. One ordinarily skilled in the art would understand that the disclosed embodiment would also function with other imaging agents, MRI, PET, etc.

[0110] One or more phases of the nanoparticles may be formed of a matrix material selected from a protein, such as an albumin (human serum albumin (HSA)), a polysaccharide, such as chitosan, or alternatively, one or more polymers. By way of example, the Janus nanoparticles may include fluorescent albumin derivatives, as well as fluorescent peptides as imaging agents to enable particle fate analysis. In addition, one ordinarily skilled in the art would understand that many other relevant proteins, in addition to albumin, polysaccharides, polymers, and mixtures thereof could also be included in the nanoparticles. Alternatively, radiolabeled nanoparticles can be formed, for example, for biodistribution studies. For example, in accordance with certain aspects of the present disclosure, electrohydrodynamic co-jetting technology can be used in conjunction with radiolabeling strategies to load nanoparticles, such as JNPs, with radiotracers for PET / SPECT imaging. The electrohydrodynamic co-jetting procedure enables incorporation, stabilization, and delivery of a diverse array of therapeutic agents that include bioactive functional proteins, lipophilic drugs, and RNAH 8-22. While the adaptability of JNP-mediated drug delivery is an advantage for a personalized lung cancer chemoprevention strategy, the JNPs also should have an ability to successfully reach their target sites.

[0111] In certain aspects, the JNP nanoparticles suitable for use in an aerosolized delivery platform according to the present disclosure may be selected to have specific parameters including: (1 ) nanoparticle size, (2) nanoparticle zeta potential, (3) nanoparticle aerodynamic diameter (radius), (4) nanoparticle drug loading, (5) nanoparticle drug combinations, (6) nanoparticle capacity to withstand shear forces generated during aerosolization, (7) nanoparticle preservation of the drug in abioavailable / bioactive state during aerosolization, and (8) drug distribution (e.g., compartmentalization).

[0112] For example, a size of nanoparticles formed via electrohydrodynamic jetting may be controlled and selected based upon the targeted sites of delivery. For example, for upper airway lesions, precursors to lung SCC and small cell carcinoma, particles would be larger as the aerosolized path is shorter. In contrast, adenocarcinoma and its precursors arise in the terminal alveoli, so a smaller particle with greater aerodynamic capacity would be desired.

[0113] Nanoparticle zeta potential, which is the particle’s surface charge in solution, can be controlled in the nanoparticles formed during electrohydrodynamic jetting. Zeta potential of nanoparticles is the best indicator for the stability of a colloidal dispersion, e.g., particles with zeta potential > + 60 mV have excellent stability and reduced tendency toward aggregation. Further, when the JNPs reach the lung, they will again be exposed to a liquid interface (pulmonary surfactant), where stability and reduced aggregation are once again important.

[0114] The nanoparticles can be formed to have a preselected aerodynamic diameter. Nanoparticle aerodynamic diameter (radius) is defined as a sphere with the density of water (1 g cm-3) with properties that enable it to settle in still air. Cascade impactors enable determination of the diameter and its impact on aerosolization properties. In certain aspects, an aerodynamic diameter of a Janus nanoparticle may be greater than or equal to about 1 nm.

[0115] Control over nanoparticle drug loading of chemopreventative agents is a further aspect of the present teachings. As described above, a concentration of active agents or drug loading level in each phase can be highly controlled during electrohydrodynamic jetting to form nanoparticles. For effective chemoprevention, levels of drug / agent at the target site are carefully regulated. Further, for aerosolized delivery, estimated loading based on only about a 10% uptake of the particles is a consideration.

[0116] In certain aspects, a synergy of nanoparticle drug combinations is an important aspect of an ability to combat premalignant lung lesions. One benefit of JNPs is their capacity to concurrently deliver two chemically distinct agents. This property is particularly beneficial when the combination agents have complementary mechanisms of action. The Janus nanoparticles prepared in accordance with the present disclosure canbe tailored to deliver multiple distinct agents that are stable in their respective phases in the nanoparticle and can be successfully delivered to the target cells. Another consideration is where in the target cell the agents should ideally be located when delivered to the target cell. For example, both tocilizumab and avelumab are cell membrane-surface active molecules. In contrast, fenretinide and siRNA molecules need to be internalized within a cell.

[0117] Another property is the particles' robustness as categorized by nanoparticle capacity. This capacity quantifies a nanoparticle’s ability to withstand shear forces generated during aerosolization. JNP may be modified, for example, by chemical cross-linking, to enhance shear force tolerability and thus, capacity. In certain aspects, the Janus nanoparticle has a capacity of greater than or equal to about 1%

[0118] The nanoparticles provided by the present disclosure are designed to maintain the drug in a bioavailable / bioactive state during aerosolization. Such maintenance is paramount during this process, as compounds, like proteins, have a very delicate tertiary structure. For example, the electrohydrodynamic co-jetting process serves to preserve the drug’s tertiary structure in its bioactive state. While not limiting to any particular theory, likely mechanisms for this stabilization include prevention of chemical denaturation and protein folding. Further, hydrophobic compounds, like fenretinide, are not exposed to water and therefore do not auto-crystalize and inactivate during electrohydrodynamic jetting. In certain aspects, the at least one Janus nanoparticle has a nanoparticle maintenance of the drug in a bioavailable / bioactive state during aerosolization of at least 1%.

[0119] As will be appreciated by those of skill in the art, the lung microenvironment is unique and can pose particular challenges as a target region within a subject. The pervasive and essential pulmonary surfactants, which are comprised of the unique phospholipid dipalmitoylphosphatidylcholine and the four surfactant-associated proteins: SP-A, SP-B, SP-C, SP-D, must also be considered for aerosolized Janus nanoparticle delivery. Janus nanoparticles are designed to retain their drug cargo and exhibit the capacity to penetrate pulmonary surfactant(s) to deliver the drug cargo / active agents to the target cells. For example, mucus coated surface epithelia have been observed to readily internalize JNPs, for example, by either by active phagocytosis or passive diffusion.

[0120] As discussed above, suitable active ingredients include pharmacologic agents that encompass likely chemopreventative targets and address premalignant lung lesions pathway disruptions, including gratuitous signaling and immortalization, cytokine signaling, modulation of gene expression, and immune evasion, among others. One particular siRNA against Signal Transducer and Activation of Transcription 3 factor (STAT3i) results in in vitro and in vivo downregulation of STAT3, a central hub associated with progression of certain cancers. Multiple growth factors and cytokines are frequently overexpressed in cancer, such as EGF, FGF, and IL-6, which activate STAT3 via tyrosine phosphorylation. Activated STAT3 (pSTAT3) translocates to the nucleus and participates in the transcription of genes that inhibit apoptosis and promote tumor cell proliferation and metastasis. Nanoparticles have been developed that deliver therapeutically active gene expression modifiers, such as STAT3i and cytokine signaling inhibitors (e.g., IL-6 receptor inhibitor; tocilizumab).

[0121] Regarding active agent / drug categories of interest, one of ordinary skill in the art would appreciate four main categories: (1) growth regulatory or antiproliferative agents, e.g., fenretinide; (2) immune modulation agents, a checkpoint inhibitor, e.g., avelumab, immune-augmenting tyrosine kinase inhibitors; immunostimulatory antibodies, e.g., ipilimumab, activators of CD40, HDAC inhibitors and immunostimulatory cytokines; (3) transcription / translation modifiers, e.g., siRNA-STAT3; and (4) tumor microenvironment-growth factor and cytokine modulators, e.g., tocilizumab, sarilumab, ustekinumab, benralizumab, and the like.

[0122] In one aspect, the immune modulatory agent may be selected from the group consisting of: avelumab, sunitinib, sorafenib, ipilimumab, interferon alpha, and combinations thereof.

[0123] By way of non-limiting example, in certain variations, suitable nanoparticles for use with the present disclosure comprise fenretinide (4-HPR) that may be distributed in an albumin (e.g., human serum albumin (HSA)) matrix material. In certain other variations, suitable nanoparticles may comprise tocilizumab (TCZ) distributed in a matrix material comprising chitosan. In yet other variations, a suitable JNP may have at least two phases, where a first phase comprises albumin (e.g., human serum albumin (HSA)) and 4HPR and a second phase comprises chitosan and TCZ.

[0124] JNP morphology and releasing profile can be assessed by utilizing scanning electron microscopy (SEM), confocal microscopy, Nanosight and dynamic light scattering. Furthermore, fluorescently tagged serum albumin in each phase / compartment will allow visualization of the multicompartmental design (e.g., at least 2 compartments) and monitor of the particles’ fate in vivo, which is essential for development studies.

[0125] JNP-delivered chemopreventive agents can thus suppress procarcinogenic pathways in human premalignant lung lesions cell lines and deliver a pharmacologic advantage, for example, provide chemopreventive relevant drug levels to the target site with negligible systemic drug uptake. In summary, in certain variations, the present disclosure contemplates aerosolized delivery of JNP formulations to deliver selected agents that target lung cancer enabling pathways from both the delivery vehicle and immunochemopreventive perspectives. Moreover, while secondary chemoprevention has been discussed herein as an example, one of ordinary skill in the art would appreciate that the JNP technology could be used for tertiary lung cancer chemoprevention, e.g., JNPs with PD-1 inhibitors.

[0126] Referring initially to FIGS. 1 A-1C, a comparison view of three treatment options are shown. JNP refers to a Janus nanoparticle (JNP) control group lacking any active agent. JNP-TCZ refers to a JNP with tocilizumab (TCZ) in accordance with certain aspects of the present disclosure, while TCZ refers to bolus injections of TCZ. As demonstrated by JNP-TCZ, in comparison with JNP-CTR or TCZ, JNP released TCZ to significantly inhibit OSCC tumor growth in vivo. Notably, OSCC is similar to LUSC for the following reasons: 1 ) upregulation of many of the same molecular pathways, 2) presence of a local milieu characterized by inappropriately sustained inflammation coupled with immunosuppression, 3) tobacco smoking represents the major risk factor for both of these cancers (ergo tumors have many of the same tobacco-initiated signature mutations). JNP-TCZ treated OSCC tumors exhibit increased differentiation in conjunction with significantly reduced mitotic activity and vascular density. In FIG. 1 A, mitotic figures are highlighted by black arrows, hematoxylin and eosin stain. Areas of tumor keratinization are identified with black arrows, hematoxylin and eosin stain. ERG positive cells (Brown chromogen) highlight the endothelial cells of the vascular structures in the tumors. In FIG. 1 B, treatment with JNP-TCZ led to a statistically significant reduction in mitotic activity (p < 0.05) and a decrease in vascular density (p < 0.001 ) (One Way ANOVA, Tukey’s Posthoc test). Treatment with TCZ bolus injections also led to a decrease in vascular density, but the mitotic rate was similar to that measured in OSCC tumors treated with control JNP. FIG. 1 C shows an ERG staining intensity count for each of the treatment options.

[0127] Referring now to FIGS. 2A-2B, absorbance spectrum of fenretinide (4HPR)-synthetic protein nanoparticle (sPNP) are shown to demonstrate the successful encapsulation of 4HPR. Acetic acid was added to dissemble nanoparticles through the unfolding of protein and to reveal available 4HPR. The spectrum was compared to 15 micrograms / mL of free 4HPR relative to 4HPR encapsulated high pressure homogenized human serum albumin (HPH-4HPR). Additional studies confirmed the 4HPR-sPNP formulation increased the solubility and stability of 4HPR in aqueous solutions. Furthermore, the 4HPR-sPNPs retained drug bioavailability, as 4HPR inactivating recrystallization did not occur.

[0128] Relative to synthetic polymers, biologic-based JNPs are safer, biocompatible, biodegradable, nontoxic, nonimmunogenic and can be prepared without the use of toxic chemicals or organic solvents. All these benefits are optimal to reduce lung irritation. As shown in FIG. 3A, the JNPs used in this example have two phases, a first phase comprises human serum albumin (HSA) and 4HPR and a second phase comprises chitosan (in glycol) and TCZ. FIG. 4 demonstrates that HSA JNPs deliver bioactive chemopreventives, and are chemically crosslinked and thus stable, making them capable of withstanding the shear forces exerted during nebulization. More specifically, the data in FIG. 4 show human serum albumin (HSA)-released 4HPR nanoparticles (4HPRscPNP) retain bioactivity. In FIG. 4, 5 iM 4HPR and blank scPNP (n =5), and 4HPRscPNP (5pM 4HPR sustained release over 24 hour) (n= 9) significantly increased caspase-3 activity relative to control cells (n=5). ***p<0.001 and *p<0.05, Kruskal Wallis, Dunns multiple comparison test. No significant difference was detected between the free 4HPR-treated and the 4HPRscPNP-treated cells. Due to 4HPR degradation (half-life of 4HPR approximately 13 hours in vivo), the 4HPRscPNP sustained release treated cells were not exposed to an abrupt 5 pM 4HPR challenge. While apoptosis is desired over uncontrolled growth, terminal differentiation such as is inducible by sustained 4HPR release is the preferable outcome. Further, TCZ chitosan JNPs demonstrate chemopreventive activity in vivo.

[0129] Chitosan contributes mucoadhesive and penetration-enhancing properties that facilitate retention and uptake at the targeted mucus-covered upper airway Type I epithelial cells. Further, as chitosan also exhibits anti-microbial, anti-inflammatory, and anti-oxidative properties, chitosan-based aerosolized formulations have been well- investigated. Finally, the safe and effective use of an FDA-approved aerosolized enzyme, namely Pulmozyme™ (dornase alfa), in adults and pediatric patients with cystic fibrosis- affected lungs demonstrates aerosolized protein safety and efficacy. More extensive characterization studies of the JNPs employed for the in vivo efficacy studies can be seen in FIGS. 5A-5C.

[0130] For example, FIG. 5A shows an SEM image of 4HPR-TCZ JNPs, while the inset labeled a1 shows a size determination of 4HPR-TCZ JNP, as assessed by dynamic light scattering. FIG. 5B shows a size distribution of 4HPR-TCZ (determined with ImageJ™ software). FIG. 5C shows geometric factor analysis, as assessed by SEM, revealed high circularities (1 =perfect circle), excellent roundness (1 =perfect sphere) and low anisotropy (low=more spherical shape) of 4HPR-TCZ (ImageJ). FIG. 5D shows mean geometric factor values for 4HPR-TCZ JNPs. FIG. 5E includes (e1 , showing nanoparticle diameter over 7 days in different liquids) and (e2, showing nanoparticle polydispersity index (PDI) over 7 days in different liquids), showing JNP stability required for drug delivery to a moist location, such as the lungs. Dynamic light measurement stability studies revealed consistency in JNP diameter and consistent polydispersity indices < 0.3 of 4HPR- TCZ JNP over 1 week in H2O and PBS. These data confirm excellent control of size and secondary parameters, such as roundness, circularity and low anisotropy. The low PDI values and size consistency in water and PBS demonstrate JNP stability compare favorably to clinically used nanoparticles such as lipid nanoparticles.

[0131] Combined 4HPR and TCZ delivery induce immune-enhancing with proliferation EMT-suppressive effects in human PML cells. Recent combination RNA sequencing combined with Al have demonstrated promise to predict agent efficacy in patients. The premalignant epithelial cell line, HBEC-KTRL53, was treated with varying doses of 4HPR and TCZ, singularly and in combination for 24 hours. RNA was extracted and RNA sequencing was conducted at the Ohio State Genomics Core facility. Differential gene expression data (Qiagen Ingenuity Pathway Analysis software) revealed that the most profound effects were achieved by the higher combination doses [4HPR (1 pM) andTCZ (2.5 pg / ml)-levels that are readily achievable by local delivery. As depicted in FIG. 6, 4HPR-TCZ uniformly suppressed canonical pathways associated with cell cycle progression, and proliferation, namely pathways (3) to (6), while TREM1 signaling pathway (1 ) and Immunogenic cell death signaling pathway (2) were activated. The 4HPR- TCZ expression inhibition also extended to RHO GTPases, which also contribute to cell cycle progression in addition to actin-microtubule cytoskeleton interactions, cell migration and EMT. 4HPR-TCZ also upregulated expression of immune response pathways. Immunogenic cell death pathways modulate cell surface composition, resulting in release of soluble mediators that activate dendritic cell antigen processing to T cells. TREM1 activation has been reported to exhibit both pro or anti tumorigenic effects. TREM1 immunostimulatory signaling entails innate immune response activation, dendritic cell maturation, antigen presentation and recruitment of NK and CD8+ T cells.

[0132] Local delivery of TCZ-HPR JNPs inhibits LUSC tumorigenesis in vivo. These studies employed a 100% tumorigenic (ATCC NCI H520), STR-validated LLISC cell line. Tumor cells (1 x107suspended in 100 pl Matrigel™) were injected into flanks of female BALB / c mice (Charles River). 24 hours post cell implantation, intratumor treatments (all in 50 pl PBS) were: 1 ) control (drug free JNP - designated JNP-CTR), 2) 4HPR JNP (5 micromoles (pM) release / 96h - designated JNP-4HPR), 3) 4HPR+TCZ JNP (5 micromoles (pM) 4HPR+1.2 micrograms (pg) TCZ release / 96h - designated JNP- 4HPR / TCZ) (See FIG. 8A). Intratumor injections were given every 96 hours (q 96h), for a total of 6 treatments. Mice were euthanized 24 hours following the last treatment. Tumor tissues were excised, formalin fixed, processed, paraffin embedded and sectioned for HE and IHC. Slides were scanned (Leica Dmi8 microscope, Leica Application Suite X) followed by quantitative image analyses (Image Pro software (Media Cybernetics, Rockville, MD.).

[0133] Consistent with H520 cell line aggressiveness, all mice formed tumors. Both 4HPR JNPs and to a greater extent 4HPR and TCZ JNPs significantly affected tumorigenesis. Control tumor cells exhibited very high proliferation indices (Ki67 labeling average 80%), were highly apoptosis resistant [1 % cleaved caspase-3 (casp-3)] and uniquely exhibited infiltrative growth and vascular invasion. JNP-4HPR and JNP- 4HPR / TCZ significantly increased casp-3 (p<0.01 ) and significantly decreased proliferation [(p<0.01 ) Ki67 average 40% & 25%, 4HPR, 4HPR / TCZ, respectively). JNP-4HPR / TCZ also significantly increased vascular instability (p<0.01 ) (See FIGS. 8A-8D). STAT3 signaling is integral for endothelial cell homeostasis and survival during inflammation. These vascular perturbation data may reflect TCZ-STAT3 inhibition. [n=10 for all groups, mean+ / -s.e.m., ANOVA, Tukey's post hoc test (hemorrhage, Ki67), Kruskal Wallis, Dunns post hoc test (caspase-3)].

[0134] While the apparatus, system, and method have been described with reference to various embodiments, those skilled in the art will understand that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope and essence of the disclosure. In addition, many modifications may be made to adapt a particular situation or material in accordance with the teachings of the disclosure without departing from the essential scope thereof. Therefore, it is intended that the disclosure not be limited to the particular embodiments disclosed, but that the disclosure will include all embodiments falling within the scope of the appended claims. In this application all units are in the metric system and all amounts and percentages are by weight, unless otherwise expressly indicated. Also, all citations referred herein are expressly incorporated herein by reference.

Claims

CLAIMS1. A lung cancer chemoprevention drug delivery platform comprising a Janus nanoparticle (JNP) comprising at least two chemopreventive agents for preventing or minimizing lung cancer or preventing recurrence of a treated lung cancer.

2. The lung cancer chemoprevention drug delivery platform of claim 1 , wherein the Janus nanoparticle is morphologically anisotropic and the Janus nanoparticle comprises at least two chemically distinct compartments, wherein a first chemically distinct compartment comprises a first chemopreventive agent and a second chemically distinct compartment comprises a second chemopreventive agent.

3. The lung cancer chemoprevention drug delivery platform of claim 1 , wherein the Janus nanoparticle further comprises at least one protein in which at least one chemopreventive agent of the at least two chemopreventive agents is distributed.

4. The lung cancer chemoprevention drug delivery platform of claim 3, wherein the at least one protein comprises albumin.

5. The lung cancer chemoprevention drug delivery platform of claim 3, wherein the at least chemopreventive agent comprises fenretinide.

6. The lung cancer chemoprevention drug delivery platform of claim 1 , wherein the Janus nanoparticle further comprises at least one polysaccharide in which at least one chemopreventive agent of the at least two chemopreventive agents is distributed.

7. The lung cancer chemoprevention drug delivery platform of claim 6, wherein the at least polysaccharide comprises chitosan.

8. The lung cancer chemoprevention drug delivery platform of claim 6, wherein the at least chemopreventive agent comprises tocilizumab.

9. The lung cancer chemoprevention drug delivery platform of claim 1 , wherein the Janus nanoparticle further comprises a lipophilic drug.

10. The lung cancer chemoprevention drug delivery platform of claim 1 , wherein the Janus nanoparticle further comprises RNAi.1 1. The lung cancer chemoprevention drug delivery platform of claim 1 , wherein at least one of the at least two chemopreventive agents is selected from the group consisting of: growth regulatory or anti-proliferative agents, antineoplastic / anti- miotic agents, chemotherapy agents, immune modulatory agents,transcription / translation modifier agents, growth factor and cytokine modulatory agents, biomolecules, nucleic acids, DNA, RNA, plasmids, short interfering sequence of double stranded RNA (siRNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, small nuclear RNA, single stranded DNA, CRISPR CAS-9, aptamers, antibodies, proteins, peptides, amino acids, sugars, targeting molecules, and combinations thereof.

12. The lung cancer chemoprevention drug delivery platform of claim 1 , wherein the Janus nanoparticle (JNP) is configured to be aerosolized for delivery to at least one lung or a target region of a respiratory system of a subject.

13. An aerosolized composition comprising:(a) at least one Janus nanoparticle configured to be aerosolized;(b) a bioactive protein inhibitor;(c) a modulator of at least one proinflammatory mediator;(d) an immune modulatory agent; and(e) short interfering sequence of double stranded RNA (siRNA) to modify proteins aberrantly upregulated in premalignant lung lesions.

14. The aerosolized composition of claim 13, wherein the at least one Janus nanoparticle has a zeta potential of greater than or equal to about ±60 mV.

15. The aerosolized composition of claim 13, wherein the at least one Janus nanoparticle has an aerodynamic diameter of greater than or equal to about 1 nm.

16. The aerosolized composition of claim 13, wherein the at least one Janus nanoparticle has a nanoparticle capacity of greater than or equal to about 1%.

17. The aerosolized composition of claim 13, wherein the at least one Janus nanoparticle has a nanoparticle maintenance of at least one of:(b) the bioactive protein inhibitor;(c) the modulator of at least one proinflammatory mediator;(d) the immune modulatory agent; and(e) short interfering sequence of double stranded RNA (siRNA), in a bioavailable / bioactive state during aerosolization of at least 1%.

18. The aerosolized composition of claim 13, wherein (a) the bioactive protein inhibitor comprises fenretinide, (b) the modulator of proinflammatory mediators comprises tocilizumab, (c) the immune modulatory agent comprises avelumab and / oripilimumab, and (d) the siRNA comprises siRNA against Signal Transducer and Activation of Transcription 3 factor (STAT3) to modify proteins aberrantly upregulated in a premalignant lung lesion.

19. A method for generating a lung cancer chemoprevention drug delivery platform for inhalation by a patient diagnosed with premalignant lung lesions, the method comprising combining components for the lung cancer chemoprevention drug delivery platform comprising:(a) a Janus nanoparticle;(b) a bioactive protein inhibitor;(c) a modulator of at least one proinflammatory mediator;(d) an immune modulatory agent; and(e) a short interfering sequence of double stranded RNA (siRNA) to modify proteins aberrantly upregulated in premalignant lung lesions.

20. The method of claim 19, wherein the Janus nanoparticle is delivered by an ultrasonic nebulizer unit.

21. The method of claim 19, wherein the Janus nanoparticle withstands aerosolized shear forces as determined by post-aerosolized structural integrity bioactive drug retention of at least 1%.

22. The method of claim 19, whereas the Janus nanoparticle penetrates pulmonary surfactant when delivered to target cells.

23. The method of claim 19, wherein (a) the bioactive protein inhibitor comprises fenretinide, (b) the modulator of at least one proinflammatory mediator comprises tocilizumab, (c) the immune modulatory agent comprises avelumab and / or ipilimumab, and (d) the siRNA comprises siRNA against Signal Transducer and Activation of Transcription 3 factor (STAT3).

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